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Recent Articles in Nucleic Acids Research

Mattiacio JL, Read LK
Roles for TbDSS-1 in RNA surveillance and decay of maturation by-products from the 12S rRNA locus.
Nucleic Acids Res. 2007 Nov 21; .
The Trypanosoma brucei exoribonuclease, TbDSS-1, has been implicated in multiple aspects of mitochondrial RNA metabolism. Here, we investigate the role of TbDSS-1 in RNA processing and surveillance by analyzing 12S rRNA processing intermediates in TbDSS-1 RNAi cells. RNA fragments corresponding to leader sequence upstream of 12S rRNA accumulate upon TbDSS-1 depletion. The 5' extremity of 12S rRNA is generated by endonucleolytic cleavage, and TbDSS-1 degrades resulting upstream maturation by-products. RNAs with 5' ends at position -141 and 3' ends adjacent to the mature 5' end of 12S rRNA are common and invariably possess oligo(U) tails. 12S rRNAs with mature 3' ends and unprocessed 5' ends also accumulate in TbDSS-1 depleted cells, suggesting that these RNAs represent dead-end products normally destined for decay by TbDSS-1 in an RNA surveillance pathway. Together, these data indicate dual roles for TbDSS-1 in degradation of 12S rRNA maturation by-products and as part of a mitochondrial RNA surveillance pathway that eliminates stalled 12S processing intermediates. We further provide evidence that TbDSS-1 degrades RNAs originating upstream of the first gene on the minor strand of the mitochondrial maxicircle suggesting that TbDSS-1 also removes non-functional RNAs generated from other regions of the mitochondrial genome. [Abstract/Link to Full Text]

Monteiro PT, Mendes ND, Teixeira MC, d'Orey S, Tenreiro S, Mira NP, Pais H, Francisco AP, Carvalho AM, Louren�o AB, S�-Correia I, Oliveira AL, Freitas AT
YEASTRACT-DISCOVERER: new tools to improve the analysis of transcriptional regulatory associations in Saccharomyces cerevisiae.
Nucleic Acids Res. 2007 Nov 21;
The Yeast search for transcriptional regulators and consensus tracking (YEASTRACT) information system (www.yeastract.com) was developed to support the analysis of transcription regulatory associations in Saccharomyces cerevisiae. Last updated in September 2007, this database contains over 30 990 regulatory associations between Transcription Factors (TFs) and target genes and includes 284 specific DNA binding sites for 108 characterized TFs. Computational tools are also provided to facilitate the exploitation of the gathered data when solving a number of biological questions, in particular the ones that involve the analysis of global gene expression results. In this new release, YEASTRACT includes DISCOVERER, a set of computational tools that can be used to identify complex motifs over-represented in the promoter regions of co-regulated genes. The motifs identified are then clustered in families, represented by a position weight matrix and are automatically compared with the known transcription factor binding sites described in YEASTRACT. Additionally, in this new release, it is possible to generate graphic depictions of transcriptional regulatory networks for documented or potential regulatory associations between TFs and target genes. The visual display of these networks of interactions is instrumental in functional studies. Tutorials are available on the system to exemplify the use of all the available tools. [Abstract/Link to Full Text]

Hsu SD, Chu CH, Tsou AP, Chen SJ, Chen HC, Hsu PW, Wong YH, Chen YH, Chen GH, Huang HD
miRNAMap 2.0: genomic maps of microRNAs in metazoan genomes.
Nucleic Acids Res. 2007 Nov 19;
MicroRNAs (miRNAs) are small non-coding RNA molecules that can negatively regulate gene expression and thus control numerous cellular mechanisms. This work develops a resource, miRNAMap 2.0, for collecting experimentally verified microRNAs and experimentally verified miRNA target genes in human, mouse, rat and other metazoan genomes. Three computational tools, miRanda, RNAhybrid and TargetScan, were employed to identify miRNA targets in 3'-UTR of genes as well as the known miRNA targets. Various criteria for filtering the putative miRNA targets are applied to reduce the false positive prediction rate of miRNA target sites. Additionally, miRNA expression profiles can provide valuable clues on the characteristics of miRNAs, including tissue specificity and differential expression in cancer/normal cell. Therefore, quantitative polymerase chain reaction experiments were performed to monitor the expression profiles of 224 human miRNAs in 18 major normal tissues in human. The negative correlation between the miRNA expression profile and the expression profiles of its target genes typically helps to elucidate the regulatory functions of the miRNA. The interface is also redesigned and enhanced. The miRNAMap 2.0 is now available at http://miRNAMap.mbc.nctu.edu.tw/. [Abstract/Link to Full Text]

Canaran P, Buckler ES, Glaubitz JC, Stein L, Sun Q, Zhao W, Ware D
Panzea: an update on new content and features.
Nucleic Acids Res. 2007 Nov 19;
Panzea (http://www.panzea.org), the public web site of the project 'Molecular and Functional Diversity in the Maize Genome', has expanded over the past two years in data content, display tools and informational sections. The most significant data content expansions occurred for the single nucleotide polymorphism (SNP), sequencing, isozyme and phenotypic data types. We have enhanced our existing web display tools and have launched a number of new tools for data display and analysis. For example, we have implemented one that allows users to find polymorphisms between two accessions, a geographic map tool to visualize the geographic distribution of SNPs, simple sequence repeats (SSRs) and isozyme alleles and a graphical view of the placement of Panzea markers and genes/loci on genetic and physical maps. One goal of the informatics component of our project has been to generate code that can be used by other groups. We have enhanced our existing code base and have made our new tools available. Finally, we have also made available new informational sections as part of our educational and outreach efforts. [Abstract/Link to Full Text]

Gunawardana D, Cheng HC, Gayler KR
Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2).
Nucleic Acids Res. 2007 Nov 19;
The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn(2+)- or Mg(2+)-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B. [Abstract/Link to Full Text]

Tsou JH, Chang KY, Wang WC, Tseng JT, Su WC, Hung LY, Chang WC, Chen BK
Nucleolin regulates c-Jun/Sp1-dependent transcriptional activation of cPLA2{alpha} in phorbol ester-treated non-small cell lung cancer A549 cells.
Nucleic Acids Res. 2007 Dec 11;
The expression of cPLA(2) is critical for transformed growth of non-small cell lung cancer (NSCLC). It is known that phorbol 12-myristate 13-acetate (PMA)-activated signal transduction pathway is thought to be involved in the oncogene action in NSCLC and enzymatic activation of cPLA(2.) However, the transcriptional regulation of cPLA2alpha in PMA-activated NSCLC is not clear. In this study, we found that PMA induced the mRNA level and protein expression of cPLA2alpha. In addition, two Sp1-binding sites of cPLA2alpha promoter were required for response to PMA and c-Jun overexpression. Small interfering RNA (siRNA) of c-Jun and nucleolin inhibited PMA induced the promoter activity and protein expression of cPLA2alpha. Furthermore, PMA stimulated the formation of c-Jun/Sp1 and c-Jun/nucleolin complexes as well as the binding of these transcription factor complexes to the cPLA2alpha promoter. Although Sp1-binding sites were required for the bindings of Sp1 and nucleolin to the promoter, the binding of nucleolin or Sp1 to the promoter was independent of each other. Our results revealed that c-Jun/nucleolin and c-Jun/Sp1 complexes play an important role in PMA-regulated cPLA2alpha gene expression. It is likely that nucleolin binding at place of Sp1 on gene promoter could also mediate the regulation of c-Jun/Sp1-activated genes. [Abstract/Link to Full Text]

Mundt JM, Hah SS, Sumbad RA, Schramm V, Henderson PT
Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells.
Nucleic Acids Res. 2007 Nov 19;
7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [(14)C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 10(6) nucleotides, 5-6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis. [Abstract/Link to Full Text]

Rederstorff M, Allamand V, Guicheney P, Gartioux C, Richard P, Chaigne D, Krol A, Lescure A
Ex vivo correction of selenoprotein N deficiency in rigid spine muscular dystrophy caused by a mutation in the selenocysteine codon.
Nucleic Acids Res. 2007 Nov 19;
Premature termination of translation due to nonsense mutations is a frequent cause of inherited diseases. Therefore, many efforts were invested in the development of strategies or compounds to selectively suppress this default. Selenoproteins are interesting candidates considering the idiosyncrasy of the amino acid selenocysteine (Sec) insertion mechanism. Here, we focused our studies on SEPN1, a selenoprotein gene whose mutations entail genetic disorders resulting in different forms of muscular diseases. Selective correction of a nonsense mutation at the Sec codon (UGA to UAA) was undertaken with a corrector tRNA(Sec) that was engineered to harbor a compensatory mutation in the anticodon. We demonstrated that its expression restored synthesis of a full-length selenoprotein N both in HeLa cells and in skin fibroblasts from a patient carrying the mutated Sec codon. Readthrough of the UAA codon was effectively dependent on the Sec insertion machinery, therefore being highly selective for this gene and unlikely to generate off-target effects. In addition, we observed that expression of the corrector tRNA(Sec) stabilized the mutated SEPN1 transcript that was otherwise more subject to degradation. In conclusion, our data provide interesting evidence that premature termination of translation due to nonsense mutations is amenable to correction, in the context of the specialized selenoprotein synthesis mechanism. [Abstract/Link to Full Text]

Galperin MY
The Molecular Biology Database Collection: 2008 update.
Nucleic Acids Res. 2007 Nov 19;
The Nucleic Acids Research online Molecular Biology Database Collection is a public repository that lists more than 1000 databases described in this and previous Nucleic Acids Research annual database issues, as well as a selection of molecular biology databases described in other journals. All databases included in this Collection are freely available to the public. The 2008 update includes 1078 databases, 110 more than the previous one. The links to more than 80 databases have been updated and 25 obsolete databases have been removed from the list. The complete database list and summaries are available online at the Nucleic Acids Research web site, http://nar.oxfordjournals.org/. [Abstract/Link to Full Text]

Xiang Z, Todd T, Ku KP, Kovacic BL, Larson CB, Chen F, Hodges AP, Tian Y, Olenzek EA, Zhao B, Colby LA, Rush HG, Gilsdorf JR, Jourdian GW, He Y
VIOLIN: vaccine investigation and online information network.
Nucleic Acids Res. 2007 Nov 19;
Vaccines are among the most efficacious and cost-effective tools for reducing morbidity and mortality caused by infectious diseases. The vaccine investigation and online information network (VIOLIN) is a web-based central resource, allowing easy curation, comparison and analysis of vaccine-related research data across various human pathogens (e.g. Haemophilus influenzae, human immunodeficiency virus (HIV) and Plasmodium falciparum) of medical importance and across humans, other natural hosts and laboratory animals. Vaccine-related peer-reviewed literature data have been downloaded into the database from PubMed and are searchable through various literature search programs. Vaccine data are also annotated, edited and submitted to the database through a web-based interactive system that integrates efficient computational literature mining and accurate manual curation. Curated information includes general microbial pathogenesis and host protective immunity, vaccine preparation and characteristics, stimulated host responses after vaccination and protection efficacy after challenge. Vaccine-related pathogen and host genes are also annotated and available for searching through customized BLAST programs. All VIOLIN data are available for download in an eXtensible Markup Language (XML)-based data exchange format. VIOLIN is expected to become a centralized source of vaccine information and to provide investigators in basic and clinical sciences with curated data and bioinformatics tools for vaccine research and development. VIOLIN is publicly available at http://www.violinet.org. [Abstract/Link to Full Text]

Nottebaum S, Tan L, Trzaska D, Carney HC, Weinzierl RO
The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant protein complexes.
Nucleic Acids Res. 2007 Nov 19;
The in-depth structure/function analysis of large protein complexes, such as RNA polymerases (RNAPs), requires an experimental platform capable of assembling variants of such enzymes in large numbers in a reproducible manner under defined in vitro conditions. Here we describe a streamlined and integrated protocol for assembling recombinant archaeal RNAPs in a high-throughput 96-well format. All aspects of the procedure including construction of redesigned expression plasmids, development of automated protein extraction/in vitro assembly methods and activity assays were specifically adapted for implementation on robotic platforms. The optimized strategy allows the parallel assembly and activity assay of 96 recombinant RNAPs (including wild-type and mutant variants) with little or no human intervention within 24 h. We demonstrate the high-throughput potential of this system by evaluating the side-chain requirements of a single amino acid position of the RNAP Bridge Helix using saturation mutagenesis. [Abstract/Link to Full Text]

Chen J, Dupradeau FY, Case DA, Turner CJ, Stubbe J
DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics.
Nucleic Acids Res. 2007 Nov 19;
Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5'-CCA AAG(6) XA(8)C CGG G-3', where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4'-oxidized abasic sites. Biochemistry, 46, 3096-3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3' to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site. [Abstract/Link to Full Text]

Gracy J, Le-Nguyen D, Gelly JC, Kaas Q, Heitz A, Chiche L
KNOTTIN: the knottin or inhibitor cystine knot scaffold in 2007.
Nucleic Acids Res. 2007 Nov 19;
The KNOTTIN database provides standardized information on the small disulfide-rich proteins with a knotted topology called knottins or inhibitor cystine knots. Static pages present the essential historical or recent results about knottin discoveries, sequences, structures, syntheses, folding, functions, applications and bibliography. New tools, KNOTER3D and KNOTER1D, are provided to determine or predict if a user query (3D structure or sequence) is a knottin. These tools are now used to automate the database update. All knottin structures and sequences in the database are now standardized according to the knottin nomenclature based on loop lengths between knotted cysteines, and to the knottin numbering scheme. Therefore, the whole KNOTTIN database (sequences and structures) can now be searched using loop lengths, in addition to keyword and sequence (BLAST, HMMER) searches. Renumbered and structurally fitted knottin PDB files are available for download as well as renumbered sequences, sequence alignments and logos. The knottin numbering scheme is used for automatic drawing of standardized two-dimensional Colliers de Perles of any knottin structure or sequence in the database or provided by the user. The KNOTTIN database is available at http://knottin.cbs.cnrs.fr. [Abstract/Link to Full Text]

Kuiken C, Hraber P, Thurmond J, Yusim K
The hepatitis C sequence database in Los Alamos.
Nucleic Acids Res. 2007 Nov 19;
The hepatitis C virus (HCV) is a significant public health threat worldwide. The virus is highly variable and evolves rapidly, making it an elusive target for the immune system and for vaccine and drug design. Presently, approximately 50 000 HCV sequences have been published. A central website that provides annotated sequences and analysis tools will be helpful to HCV scientists worldwide. The HCV sequence database collects and annotates sequence data, and provides them to the public via a website that contains a user-friendly search interface and a large number of sequence analysis tools, following the model of the highly regarded and widely used Los Alamos HIV database. The HCV website can be accessed via http://hcv.lanl.gov and http://hcv-db.org. [Abstract/Link to Full Text]

Park MA, Seok YJ, Jeong G, Lee JS
SUMO1 negatively regulates BRCA1-mediated transcription, via modulation of promoter occupancy.
Nucleic Acids Res. 2007 Nov 19;
BRCA1, a tumor suppressor gene, is implicated in the repression and activation of transcription via interactions with a diverse range of proteins. The mechanisms regulating the action of BRCA1 are not fully understood. Here, we use the promoters of Gadd45alpha, p27(KIP1) and p21(WAF1/CIP1) to demonstrate that SUMO1 represses transactivation potential of BRCA1 by causing BRCA1 to be released from the promoters and augmenting histone deacetylation via recruitment of histone deacetylase (HDAC) activity. Consistently, silencing of SUMO1 led to recruitment of BRCA1 and release of HDAC1 at the BRCA1 target promoters, and subsequent transcriptional activation of the BRCA1 target genes. Furthermore, a sumoylation-incompetent mutant missing the sumoylation donor site suppressed BRCA1-induced activation of transcription, whereas E2 UBC9 or the dominant-negative mutant UBC9 had no effect, implying that repression of BRCA1-mediated activation of transcription by SUMO1 is independent of sumoylation. Repression of BRCA1-mediated activation of transcription by SUMO1 was reversed by DNA damage by inducing the release of SUMO1 from the Gadd45alpha promoter and the recruitment of BRCA1, along with increased histone acetylation, to enhance activation of transcription. Together, our data provide evidence that SUMO1 plays a role in the activation-repression switch of BRCA1-mediated transcription via modulation of promoter occupancy. [Abstract/Link to Full Text]

Scherr M, Venturini L, Battmer K, Schaller-Schoenitz M, Schaefer D, Dallmann I, Ganser A, Eder M
Lentivirus-mediated antagomir expression for specific inhibition of miRNA function.
Nucleic Acids Res. 2007 Nov 19;
Micro RNAs (miRNA) regulate gene expression by hybridization and recruitment of multi-protein complexes to complementary mRNA target sequences. miRNA function can transiently be antagonized by antagomirs-chemically modified oligonucleotides complementary to individual miRNAs. Here, we describe the induction of stable loss-of-function phenotypes for specific miRNAs by lentivirus-mediated antagomir expression. Lentivirally expressed antagomirs are transcribed from a H1-promoter located within the lentiviral 3'LTR and were directed against miRNAs encoded on the polycistronic miR17-92 transcript. Functional silencing of miR-18a, miR-19b and miR-20a by the corresponding antagomirs specifically relieves miRNA-mediated reporter gene repression. Inhibition of miRNA function correlates to reduction of 'miRNA' amplification by miRNA-specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 antagomirs in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals individual and complementary functions of miR-18a and miR-20a and demonstrates specific miRNA impact on cell proliferation in a cell culture model. [Abstract/Link to Full Text]

Brosh RM, Bohr VA
Human premature aging, DNA repair and RecQ helicases.
Nucleic Acids Res. 2007 Nov 15;
Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects. [Abstract/Link to Full Text]

Pandya GA, Holmes MH, Sunkara S, Sparks A, Bai Y, Verratti K, Saeed K, Venepally P, Jarrahi B, Fleischmann RD, Peterson SN
A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip(R) whole-genome resequencing platform.
Nucleic Acids Res. 2007 Nov 15;
DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip(R) array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%. [Abstract/Link to Full Text]

Bryne JC, Valen E, Tang MH, Marstrand T, Winther O, da Piedade I, Krogh A, Lenhard B, Sandelin A
JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update.
Nucleic Acids Res. 2007 Nov 15;
JASPAR is a popular open-access database for matrix models describing DNA-binding preferences for transcription factors and other DNA patterns. With its third major release, JASPAR has been expanded and equipped with additional functions aimed at both casual and power users. The heart of the JASPAR database-the JASPAR CORE sub-database-has increased by 12% in size, and three new specialized sub-databases have been added. New functions include clustering of matrix models by similarity, generation of random matrices by sampling from selected sets of existing models and a language-independent Web Service applications programming interface for matrix retrieval. JASPAR is available at http://jaspar.genereg.net. [Abstract/Link to Full Text]

Griffith OL, Montgomery SB, Bernier B, Chu B, Kasaian K, Aerts S, Mahony S, Sleumer MC, Bilenky M, Haeussler M, Griffith M, Gallo SM, Giardine B, Hooghe B, Van Loo P, Blanco E, Ticoll A, Lithwick S, Portales-Casamar E, Donaldson IJ, Robertson G, Wadelius C, De Bleser P, Vlieghe D, Halfon MS, Wasserman W, Hardison R, Bergman CM, Jones SJ
ORegAnno: an open-access community-driven resource for regulatory annotation.
Nucleic Acids Res. 2007 Nov 15;
ORegAnno is an open-source, open-access database and literature curation system for community-based annotation of experimentally identified DNA regulatory regions, transcription factor binding sites and regulatory variants. The current release comprises 30 145 records curated from 922 publications and describing regulatory sequences for over 3853 genes and 465 transcription factors from 19 species. A new feature called the 'publication queue' allows users to input relevant papers from scientific literature as targets for annotation. The queue contains 4438 gene regulation papers entered by experts and another 54 351 identified by text-mining methods. Users can enter or 'check out' papers from the queue for manual curation using a series of user-friendly annotation pages. A typical record entry consists of species, sequence type, sequence, target gene, binding factor, experimental outcome and one or more lines of experimental evidence. An evidence ontology was developed to describe and categorize these experiments. Records are cross-referenced to Ensembl or Entrez gene identifiers, PubMed and dbSNP and can be visualized in the Ensembl or UCSC genome browsers. All data are freely available through search pages, XML data dumps or web services at: http://www.oreganno.org. [Abstract/Link to Full Text]

Editorial.
Nucleic Acids Res. 2007 Nov 14; [Abstract/Link to Full Text]

Roy TW, Bhagwat AS
Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethylation.
Nucleic Acids Res. 2007 Nov 14;
The Escherichia coli AlkB protein catalyzes the direct reversal of alkylation damage to DNA; primarily 1-methyladenine (1mA) and 3-methylcytosine (3mC) lesions created by endogenous or environmental alkylating agents. AlkB is a member of the non-heme iron (II) alpha-ketoglutarate-dependent dioxygenase superfamily, which removes the alkyl group through oxidation eliminating a methyl group as formaldehyde. We have developed a fluorescence-based assay for the dealkylation activity of this family of enzymes. It uses formaldehyde dehydrogenase to convert formaldehyde to formic acid and monitors the creation of an NADH analog using fluorescence. This assay is a great improvement over the existing assays for DNA demethylation in that it is continuous, rapid and does not require radioactively labeled material. It may also be used to study other demethylation reactions including demethylation of histones. We used it to determine the kinetic constants for AlkB and found them to be somewhat different than previously reported values. The results show that AlkB demethylates 1mA and 3mC with comparable efficiencies and has only a modest preference for a single-stranded DNA substrate over its double-stranded DNA counterpart. [Abstract/Link to Full Text]

Minoo P, Hu L, Zhu N, Borok Z, Bellusci S, Groffen J, Kardassis D, Li C
SMAD3 prevents binding of NKX2.1 and FOXA1 to the SpB promoter through its MH1 and MH2 domains.
Nucleic Acids Res. 2007 Nov 14;
Mechanisms of gene repression by transforming growth factor-beta (TGF-beta) are not well understood. TGF-beta represses transcription of pulmonary surfactant protein-B gene in lung epithelial cells. Repression is mediated by SMAD3 through interactions with NKX2.1 and FOXA1, two key transcription factors that are positive regulators of SpB transcription. In this study, we found that SMAD3 interacts through its MAD domains, MH1 and MH2 with NKX2.1 and FOXA1 proteins. The sites of interaction on NKX2.1 are located within the NH2 and COOH domains, known to be involved in transactivation function. In comparison, weaker interaction of FOXA1 winged helix, and the NH(2)-terminal domains was documented with SMAD3. Both in vitro studies and in vivo ChIP assays show that interaction of SMAD3 MH1 and MH2 domains with NKX2.1 and FOXA1 results in reduced binding of NKX2.1 and FOXA1 to their cognate DNA-binding sites, and diminished promoter occupancy within the SpB promoter. Thus, these studies reveal for the first time a mechanism of TGF-beta-induced SpB gene repression that involves interactions between specific SMAD3 domains and the corresponding functional sites on NKX2.1 and FOXA1 transcription factors. [Abstract/Link to Full Text]

Harada R, Vadnais C, Sansregret L, Leduy L, B�rub� G, Robert F, Nepveu A
Genome-wide location analysis and expression studies reveal a role for p110 CUX1 in the activation of DNA replication genes.
Nucleic Acids Res. 2007 Nov 14;
Proteolytic processing of the CUX1 transcription factor generates an isoform, p110 that accelerates entry into S phase. To identify targets of p110 CUX1 that are involved in cell cycle progression, we performed genome-wide location analysis using a promoter microarray. Since there are no antibodies that specifically recognize p110, but not the full-length protein, we expressed physiological levels of a p110 isoform with two tags and purified chromatin by tandem affinity purification (ChAP). Conventional ChIP performed on synchronized populations of cells confirmed that p110 CUX1 is recruited to the promoter of cell cycle-related targets preferentially during S phase. Multiple approaches including silencing RNA (siRNA), transient infection with retroviral vectors, constitutive expression and reporter assays demonstrated that most cell cycle targets are activated whereas a few are repressed or not affected by p110 CUX1. Functional classes that were over-represented among targets included DNA replication initiation. Consistent with this finding, constitutive expression of p110 CUX1 led to a premature and more robust induction of replication genes during cell cycle progression, and stimulated the long-term replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV). [Abstract/Link to Full Text]

Gajria B, Bahl A, Brestelli J, Dommer J, Fischer S, Gao X, Heiges M, Iodice J, Kissinger JC, Mackey AJ, Pinney DF, Roos DS, Stoeckert CJ, Wang H, Brunk BP
ToxoDB: an integrated Toxoplasma gondii database resource.
Nucleic Acids Res. 2007 Nov 14;
ToxoDB (http://ToxoDB.org) is a genome and functional genomic database for the protozoan parasite Toxoplasma gondii. It incorporates the sequence and annotation of the T. gondii ME49 strain, as well as genome sequences for the GT1, VEG and RH (Chr Ia, Chr Ib) strains. Sequence information is integrated with various other genomic-scale data, including community annotation, ESTs, gene expression and proteomics data. ToxoDB has matured significantly since its initial release. Here we outline the numerous updates with respect to the data and increased functionality available on the website. [Abstract/Link to Full Text]

Sandin P, B�rjesson K, Li H, M�rtensson J, Brown T, Wilhelmsson LM, Albinsson B
Characterization and use of an unprecedentedly bright and structurally non-perturbing fluorescent DNA base analogue.
Nucleic Acids Res. 2007 Nov 14;
This article presents the first evidence that the DNA base analogue 1,3-diaza-2-oxophenoxazine, tC(O), is highly fluorescent, both as free nucleoside and incorporated in an arbitrary DNA structure. tC(O) is thoroughly characterized with respect to its photophysical properties and structural performance in single- and double-stranded oligonucleotides. The lowest energy absorption band at 360 nm (epsilon = 9000 M(-1) cm(-1)) is dominated by a single in-plane polarized electronic transition and the fluorescence, centred at 465 nm, has a quantum yield of 0.3. When incorporated into double-stranded DNA, tC(O) shows only minor variations in fluorescence intensity and lifetime with neighbouring bases, and the average quantum yield is 0.22. These features make tC(O), on average, the brightest DNA-incorporated base analogue so far reported. Furthermore, it base pairs exclusively with guanine and causes minimal perturbations to the native structure of DNA. These properties make tC(O) a promising base analogue that is perfectly suited for e.g. photophysical studies of DNA interacting with macromolecules (proteins) or for determining size and shape of DNA tertiary structures using techniques such as fluorescence anisotropy and fluorescence resonance energy transfer (FRET). [Abstract/Link to Full Text]

Lewis SM, Cerquozzi S, Graber TE, Ungureanu NH, Andrews M, Holcik M
The eIF4G homolog DAP5/p97 supports the translation of select mRNAs during endoplasmic reticulum stress.
Nucleic Acids Res. 2007 Nov 14;
DAP5/p97 is a member of the eIF4G family of translation initiation factors that has been suggested to play an important role in the translation of select messenger RNA molecules. We have shown previously that the caspase-cleaved form of DAP5/p97, termed p86, is required for the induction of the endoplasmic reticulum (ER)-stress-responsive internal ribosome entry site (IRES) of the caspase inhibitor HIAP2. We show here that expression of DAP5/p97 is enhanced during ER stress by selective recruitment of DAP5/p97 mRNA into polysomes via the DAP5/p97 IRES. Importantly, enhanced translation mediated by the DAP5/p97 IRES is dependent on DAP5/p97 itself, thus providing a positive feedback loop. In addition, we show that activation of DAP5/p97 and HIAP2 IRES during ER stress requires DAP5/p97. Significantly, the induction of DAP5/p97 during ER stress is caspase-independent, whereas the induction of HIAP2 requires proteolytic processing of DAP5/p97. Thus, DAP5/p97 is a translational activator that selectively modulates translation of specific mRNAs during conditions of cellular stress in both a caspase-dependent and caspase-independent manner. [Abstract/Link to Full Text]

Hulo N, Bairoch A, Bulliard V, Cerutti L, Cuche BA, Castro ED, Lachaize C, Langendijk-Genevaux PS, Sigrist CJ
The 20 years of PROSITE.
Nucleic Acids Res. 2007 Nov 14;
PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. In this article, we describe the implementation of a new method to assign a status to pattern matches, the new PROSITE web page and a new approach to improve the specificity and sensitivity of PROSITE methods. The latest version of PROSITE (release 20.19 of 11 September 2007) contains 1319 patterns, 745 profiles and 764 ProRules. Over the past 2 years, about 200 domains have been added, and now 53% of UniProtKB/Swiss-Prot entries (release 54.2 of 11 September 2007) have a PROSITE match. PROSITE is available on the web at: http://www.expasy.org/prosite/. [Abstract/Link to Full Text]

Wilming LG, Gilbert JG, Howe K, Trevanion S, Hubbard T, Harrow JL
The vertebrate genome annotation (Vega) database.
Nucleic Acids Res. 2007 Nov 14;
The Vertebrate Genome Annotation (Vega) database (http://vega.sanger.ac.uk) was first made public in 2004 and has been designed to view manual annotation of human, mouse and zebrafish genomic sequences produced at the Wellcome Trust Sanger Institute. Since its initial release, the number of human annotated loci has more than doubled to close to 33 000 and now contains comprehensive annotation on 20 of the 24 human chromosomes, four whole mouse chromosomes and around 40% of the zebrafish Danio rerio genome. In addition, we offer manual annotation of a number of haplotype regions in mouse and human and regions of comparative interest in pig and dog that are unique to Vega. [Abstract/Link to Full Text]

Vecerek B, Rajkowitsch L, Sonnleitner E, Schroeder R, Bl�si U
The C-terminal domain of Escherichia coli Hfq is required for regulation.
Nucleic Acids Res. 2007 Nov 13;
The Escherichia coli RNA chaperone Hfq is involved in riboregulation of target mRNAs by small trans-encoded non-coding (ncRNAs). Previous structural and genetic studies revealed a RNA-binding surface on either site of the Hfq-hexamer, which suggested that one hexamer can bring together two RNAs in a pairwise fashion. The Hfq proteins of different bacteria consist of an evolutionarily conserved core, whereas there is considerable variation at the C-terminus, with the gamma- and beta-proteobacteria possessing the longest C-terminal extension. Using different model systems, we show that a C-terminally truncated variant of Hfq (Hfq(65)), comprising the conserved hexameric core of Hfq, is defective in auto- and riboregulation. Although Hfq(65) retained the capacity to bind ncRNAs, and, as evidenced by fluorescence resonance energy transfer assays, to induce structural changes in the ncRNA DsrA, the truncated variant was unable to accommodate two non-complementary RNA oligonucleotides, and was defective in mRNA binding. These studies indicate that the C-terminal extension of E. coli Hfq constitutes a hitherto unrecognized RNA interaction surface with specificity for mRNAs. [Abstract/Link to Full Text]

Recent Articles in Genome Research

Suzuki MM, Kerr AR, De Sousa D, Bird A
CpG methylation is targeted to transcription units in an invertebrate genome.
Genome Res. 2007 May;17(5):625-31.
DNA is methylated at the dinucleotide CpG in genomes of a wide range of plants and animals. Among animals, variable patterns of genomic CpG methylation have been described, ranging from undetectable levels (e.g., in Caenorhabditis elegans) to high levels of global methylation in the vertebrates. The most frequent pattern in invertebrate animals, however, is mosaic methylation, comprising domains of methylated DNA interspersed with unmethylated domains. To understand the origin of mosaic DNA methylation patterns, we examined the distribution of DNA methylation in the Ciona intestinalis genome. Bisulfite sequencing and computational analysis revealed methylated domains with sharp boundaries that strongly colocalize with approximately 60% of transcription units. By contrast, promoters, intergenic DNA, and transposons are not preferentially targeted by DNA methylation. Methylated transcription units include evolutionarily conserved genes, whereas the most highly expressed genes preferentially belong to the unmethylated fraction. The results lend support to the hypothesis that CpG methylation functions to suppress spurious transcriptional initiation within infrequently transcribed genes. [Abstract/Link to Full Text]

Zhang L, Jie C, Obie C, Abidi F, Schwartz CE, Stevenson RE, Valle D, Wang T
X chromosome cDNA microarray screening identifies a functional PLP2 promoter polymorphism enriched in patients with X-linked mental retardation.
Genome Res. 2007 May;17(5):641-8.
X-linked Mental Retardation (XLMR) occurs in 1 in 600 males and is highly genetically heterogeneous. We used a novel human X chromosome cDNA microarray (XCA) to survey the expression profile of X-linked genes in lymphoblasts of XLMR males. Genes with altered expression verified by Northern blot and/or quantitative PCR were considered candidates. To validate this approach, we documented the expected changes of expression in samples from a patient with a known X chromosome microdeletion and from patients with multiple copies of the X chromosome. We used our XCA to survey lymphoblast RNA samples from 43 unrelated XLMR males and found 15 genes with significant (>or=1.5-fold) reduction in expression in at least one proband. Of these, subsequent analysis confirmed altered expression in 12. We followed up one, PLP2, at Xp11.23, which exhibits approximately fourfold decreased expression in two patients. Sequencing analysis in both patients revealed a promoter variant, -113C>A, that alters the core-binding site of the transcription factor ELK1. We showed that PLP2-(-113C>A) is sufficient to cause reduced expression using a luciferase reporter system and is enriched in a cohort of males with probable XLMR (14 of 239, 5.85%) as compared to normal males (9 of 577, 1.56%) (chi2=11.07, P<0.001). PLP2 is expressed abundantly in the pyramidal cells of hippocampus and granular cells of the cerebellum in the brain. We conclude that our XCA screening is an efficient strategy to identify genes that show significant changes in transcript abundance as candidate genes for XLMR. [Abstract/Link to Full Text]

Garcia-Perez JL, Doucet AJ, Bucheton A, Moran JV, Gilbert N
Distinct mechanisms for trans-mediated mobilization of cellular RNAs by the LINE-1 reverse transcriptase.
Genome Res. 2007 May;17(5):602-11.
Long Interspersed Element-1 (LINE-1 or L1) sequences comprise approximately 17% of human DNA and ongoing L1 retrotransposition continues to impact genome evolution. The L1-encoded proteins also can mobilize other cellular RNAs (e.g., Alu retrotransposons, SVA retrotransposons, and U6 snRNAs), which comprise approximately 13% of human DNA. Here, we demonstrate that the trans-mediated mobilization of non-L1 RNAs can occur by either template choice or template-switching mechanisms. Remarkably, these mechanisms are not mutually exclusive, as both processes can operate sequentially on the same RNA template. Finally, we provide evidence that efficient U6 snRNA retrotransposition requires both ORF1p and ORF2p, providing indirect evidence for the action of ORF1p in U6 snRNA retrotransposition. Thus, we propose that the LINE-1-encoded reverse transcriptase can mediate the retrotransposition of non-L1 RNAs by distinct mechanisms. [Abstract/Link to Full Text]

Edderkaoui B, Baylink DJ, Beamer WG, Wergedal JE, Porte R, Chaudhuri A, Mohan S
Identification of mouse Duffy antigen receptor for chemokines (Darc) as a BMD QTL gene.
Genome Res. 2007 May;17(5):577-85.
It is now well known that bone mineral density (BMD) variance is determined by both genetic and environmental factors. Accordingly, studies in human and animal models have revealed evidence for the presence of several quantitative trait loci (QTL) that contribute to BMD variations. However, the identification of BMD QTL genes remains a big challenge. In the current study, we focused our efforts to identify the BMD candidate gene in chromosome 1 (Chr 1) QTL that was detected from a cross involving high BMD CAST/EiJ (CAST) and low BMD C57BL/6J (B6) mice. To this end, we have combined several approaches including: (1) fine mapping the BMD QTL in Chr 1 of the B6.CAST F2 female mice using a large number of polymorphic markers; (2) the generation of congenic sublines of mice by repeated backcrossing of CAST with B6 mice and phenotype characterization; (3) expression profiling genes in the QTL region; and (4) SNP analyses to identify the mouse Duffy Antigen Receptor for Chemokines (Darc) as a candidate gene for Chr 1 BMD QTL2. We verified the involvement of the Darc protein in BMD variation by evaluating the skeletal phenotype of Darc-knockout mice and congenic sublines of mice carrying small chromosomal segments from CAST BMD QTL. Based on the findings that Darc-antibody blocked formation of multinucleated osteoclasts in vitro and that Darc from CAST binds chemokines, known to regulate osteoclast formation, with reduced affinity compared with Darc from B6 mice, we conclude that Darc regulates BMD negatively by increasing osteoclast formation, and that the genetic association between Darc gene polymorphism and BMD variations in humans merits investigation. [Abstract/Link to Full Text]

Mank JE, Axelsson E, Ellegren H
Fast-X on the Z: rapid evolution of sex-linked genes in birds.
Genome Res. 2007 May;17(5):618-24.
Theoretical work predicts natural selection to be more efficient in the fixation of beneficial mutations in X-linked genes than in autosomal genes. This "fast-X effect" should be evident by an increased ratio of nonsynonymous to synonymous substitutions (dN/dS) for sex-linked genes; however, recent studies have produced mixed support for this expectation. To make an independent test of the idea of fast-X evolution, we focused on birds, which have female heterogamety (males ZZ, females ZW), where analogous arguments would predict a fast-Z effect. We aligned 2.8 Mb of orthologous protein-coding sequence of zebra finch and chicken from 172 Z-linked and 4848 autosomal genes. Zebra finch data were in the form of EST sequences from brain cDNA libraries, while chicken genes were from the draft genome sequence. The dN/dS ratio was significantly higher for Z-linked (0.110) than for all autosomal genes (0.085; P=0.002), as well as for genes linked to similarly sized autosomes 1-10 (0.0948; P=0.04). This pattern of fast-Z was evident even after we accounted for the nonrandom distribution of male-biased genes. We also examined the nature of standing variation in the chicken protein-coding regions. The ratio of nonsynonymous to synonymous polymorphism (pN/pS) did not differ significantly between genes on the Z chromosome (0.104) and on the autosomes (0.0908). In conjunction, these results suggest that evolution proceeds more quickly on the Z chromosome, where hemizygous exposure of beneficial nondominant mutations increases the rate of fixation. [Abstract/Link to Full Text]

Cuppen E, Gort E, Hazendonk E, Mudde J, van de Belt J, Nijman IJ, Guryev V, Plasterk RH
Efficient target-selected mutagenesis in Caenorhabditis elegans: toward a knockout for every gene.
Genome Res. 2007 May;17(5):649-58.
Reverse genetic or gene-driven knockout approaches have contributed significantly to the success of model organisms for fundamental and biomedical research. Although various technologies are available for C. elegans, none of them scale very well for genome-wide application. To address this, we implemented a target-selected knockout approach that is based on random chemical mutagenesis and detection of single nucleotide mutations in genes of interest using high-throughput resequencing. A clonal library of 6144 EMS-mutagenized worms was established and screened, resulting in the identification of 1044 induced mutations in 109 Mbp, which translates into an average spacing between exonic mutations in the library of only 17 bp. We covered 25% of the open reading frames of 32 genes and identified one or more inactivating mutations (nonsense or splice site) in 84% of them. Extrapolation of our results indicates that nonsense mutations for >90% of all C. elegans genes are present in the library. To identify all of these mutations, one only needs to inspect those positions that--given the known specificity of the mutagen--can result in the introduction of a stop codon. We define these positions as nonsense introducing mutations (NIMs). The genome-wide collection of possible NIMs can be calculated for any organism with a sequenced genome and reduces the screening complexity by 200- to 2000-fold, depending on the organism and mutagen. For EMS-mutagenized C. elegans, there are only approximately 500,000 NIMs. We show that a NIM genotyping approach employing high-density microarrays can, in principle, be used for the genome-wide identification of C. elegans knockouts. [Abstract/Link to Full Text]

Mikawa S, Morozumi T, Shimanuki S, Hayashi T, Uenishi H, Domukai M, Okumura N, Awata T
Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor, germ cell nuclear factor (NR6A1).
Genome Res. 2007 May;17(5):586-93.
The number of vertebrae in pigs varies and is associated with meat productivity. Wild boars, which are ancestors of domestic pigs, have 19 vertebrae. In comparison, European commercial breeds have 21-23 vertebrae, probably owing to selective breeding for enlargement of body size. We previously identified two quantitative trait loci (QTL) for the number of vertebrae on Sus scrofa chromosomes (SSC) 1 and 7. These QTL explained an increase of more than two vertebrae. Here, we performed a map-based study to define the QTL region on SSC1. By using three F2 experimental families, we performed interval mapping and recombination analyses and defined the QTL within a 1.9-cM interval. Then we analyzed the linkage disequilibrium of microsatellite markers in this interval and found that 10 adjacent markers in a 300-kb region were almost fixed in European commercial breeds. Genetic variation of the markers was observed in Asian local breeds or wild boars. This region encoded an orphan nuclear receptor, germ cell nuclear factor (NR6A1, formerly known as GCNF), which contained an amino acid substitution (Pro192Leu) coincident with the QTL. This substitution altered the binding activity of NR6A1 to its corepressors, nuclear receptor-associated protein 80 (RAP80) and nuclear receptor corepressor 1 (NCOR1). In addition, somites of mouse embryos demonstrated expression of NR6A1 protein. Together, these results suggest that NR6A1 is a strong candidate for one of the QTL that influence number of vertebrae in pigs. [Abstract/Link to Full Text]

Zhang R, Peng Y, Wang W, Su B
Rapid evolution of an X-linked microRNA cluster in primates.
Genome Res. 2007 May;17(5):612-7.
MicroRNAs (miRNAs) are a growing class of small RNAs (about 22 nt) that play crucial regulatory roles in the genome by targeting mRNAs for cleavage or translational repression. Most of the identified miRNAs are highly conserved among species, indicating strong functional constraint on miRNA evolution. However, nonconserved miRNAs may contribute to functional novelties during evolution. Recently, an X-linked miRNA cluster was reported with multiple copies in primates but not in rodents or dog. Here we sequenced and compared this miRNA cluster in major primate lineages including human, great ape, lesser ape, Old World monkey, and New World monkey. Our data indicate rapid evolution of this cluster in primates including frequent tandem duplications and nucleotide substitutions. In addition, lineage-specific substitutions were observed in human and chimpanzee, leading to the emergence of potential novel mature miRNAs. The expression analysis in rhesus monkeys revealed a strong correlation between miRNA expression changes and male sexual maturation, suggesting regulatory roles of this miRNA cluster in testis development and spermatogenesis. We propose that, like protein-coding genes, miRNA genes involved in male reproduction are subject to rapid adaptive changes that may contribute to functional novelties during evolution. [Abstract/Link to Full Text]

Chen K, McLellan MD, Ding L, Wendl MC, Kasai Y, Wilson RK, Mardis ER
PolyScan: an automatic indel and SNP detection approach to the analysis of human resequencing data.
Genome Res. 2007 May;17(5):659-66.
Small insertions and deletions (indels) and single nucleotide polymorphisms (SNPs) are common genetic variants that are thought to be associated with a wide variety of human diseases. Owing to the genome's size and complexity, manually characterizing each one of these variations in an individual is not practical. While significant progress has been made in automated single-base mutation discovery from the sequences of diploid PCR products, automated and reliable detection of indels continues to pose difficult challenges. In this paper, we present PolyScan, an algorithm and software implementation designed to provide de novo heterozygous indel detection and improved SNP identification in the context of high-throughput medical resequencing. Tests on a human diploid PCR-based sequence data set, consisting of 90,270 traces from 13 genes, indicate that PolyScan identified approximately 90% of the 151 consensus indel sites and approximately 84% of the 1546 heterozygous indels previously identified by manual inspection. Tests on tumor-derived data show that PolyScan better identifies high-quality, low-level mutations as compared with other mutation detection software. Moreover, SNP identification improves when reprocessing the results of other programs. These results suggest that PolyScan may play a useful role in the post human genome project research era. [Abstract/Link to Full Text]

Hanada K, Zhang X, Borevitz JO, Li WH, Shiu SH
A large number of novel coding small open reading frames in the intergenic regions of the Arabidopsis thaliana genome are transcribed and/or under purifying selection.
Genome Res. 2007 May;17(5):632-40.
Large-scale cDNA sequencing projects and tiling array studies have revealed the presence of many unannotated genes. For protein coding genes, small coding sequences may not be identified by gene finders because of the conservative nature of prediction algorithms. In this study, we identified small open reading frames (sORFs) with high coding potential by a simple gene finding method (Coding Index, CI) based on the nucleotide composition bias found in most coding sequences. Applying this method to 18 Arabidopsis thaliana and 84 yeast sORF genes with evidence of expression at the protein level gives 100% accurate prediction. In the A. thaliana genome, we identified 7159 sORFs that are likely coding sequences (coding sORFs) with the CI measure at the 1% false-positive rate. To determine if these coding sORFs are parts of functional genes, we evaluated each coding sORF for evidence of transcription or evolutionary conservation. At the 5% false-positive rate, we found that 2996 coding sORFs are likely expressed in at least one experimental condition of the A. thaliana tiling array data. In addition, the evolutionary conservation of each A. thaliana sORF was examined within A. thaliana or between A. thaliana and five plants with complete or partial genome sequences. In 3997 coding sORFs with readily identifiable homologous sequences, 2376 are subject to purifying selection at the 1% false-positive rate. After eliminating coding sORFs with similarity to known transposable elements and those that are likely missing exons of known genes, the remaining 3241 coding sORFs with either evidence of transcription or purifying selection likely belong to novel coding genes in the A. thaliana genome. [Abstract/Link to Full Text]

Ponjavic J, Ponting CP, Lunter G
Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs.
Genome Res. 2007 May;17(5):556-65.
Long transcripts that do not encode protein have only rarely been the subject of experimental scrutiny. Presumably, this is owing to the current lack of evidence of their functionality, thereby leaving an impression that, instead, they represent "transcriptional noise." Here, we describe an analysis of 3122 long and full-length, noncoding RNAs ("macroRNAs") from the mouse, and compare their sequences and their promoters with orthologous sequence from human and from rat. We considered three independent signatures of purifying selection related to substitutions, sequence insertions and deletions, and splicing. We find that the evolution of the set of noncoding RNAs is not consistent with neutralist explanations. Rather, our results indicate that purifying selection has acted on the macroRNAs' promoters, primary sequence, and consensus splice site motifs. Promoters have experienced the greatest elimination of nucleotide substitutions, insertions, and deletions. The proportion of conserved sequence (4.1%-5.5%) in these macroRNAs is comparable to the density of exons within protein-coding transcripts (5.2%). These macroRNAs, taken together, thus possess the imprint of purifying selection, thereby indicating their functionality. Our findings should now provide an incentive for the experimental investigation of these macroRNAs' functions. [Abstract/Link to Full Text]

Kikuta H, Laplante M, Navratilova P, Komisarczuk AZ, Engstr�m PG, Fredman D, Akalin A, Caccamo M, Sealy I, Howe K, Ghislain J, Pezeron G, Mourrain P, Ellingsen S, Oates AC, Thisse C, Thisse B, Foucher I, Adolf B, Geling A, Lenhard B, Becker TS
Genomic regulatory blocks encompass multiple neighboring genes and maintain conserved synteny in vertebrates.
Genome Res. 2007 May;17(5):545-55.
We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated "bystander" genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs. [Abstract/Link to Full Text]

Baran AA, Silverman KA, Zeskand J, Koratkar R, Palmer A, McCullen K, Curran WJ, Edmonston TB, Siracusa LD, Buchberg AM
The modifier of Min 2 (Mom2) locus: embryonic lethality of a mutation in the Atp5a1 gene suggests a novel mechanism of polyp suppression.
Genome Res. 2007 May;17(5):566-76.
Inactivation of the APC gene is considered the initiating event in human colorectal cancer. Modifier genes that influence the penetrance of mutations in tumor-suppressor genes hold great potential for preventing the development of cancer. The mechanism by which modifier genes alter adenoma incidence can be readily studied in mice that inherit mutations in the Apc gene. We identified a new modifier locus of ApcMin-induced intestinal tumorigenesis called Modifier of Min 2 (Mom2). The polyp-resistant Mom2R phenotype resulted from a spontaneous mutation and linkage analysis localized Mom2 to distal chromosome 18. To obtain recombinant chromosomes for use in refining the Mom2 interval, we generated congenic DBA.B6 ApcMin/+, Mom2R/+ mice. An intercross revealed that Mom2R encodes a recessive embryonic lethal mutation. We devised an exclusion strategy for mapping the Mom2 locus using embryonic lethality as a method of selection. Expression and sequence analyses of candidate genes identified a duplication of four nucleotides within exon 3 of the alpha subunit of the ATP synthase (Atp5a1) gene. Tumor analyses revealed a novel mechanism of polyp suppression by Mom2R in Min mice. Furthermore, we show that more adenomas progress to carcinomas in Min mice that carry the Mom2R mutation. The absence of loss of heterozygosity (LOH) at the Apc locus, combined with the tendency of adenomas to progress to carcinomas, indicates that the sequence of events leading to tumors in ApcMin/+ Mom2R/+ mice is consistent with the features of human tumor initiation and progression. [Abstract/Link to Full Text]

Shaikh TH, O'Connor RJ, Pierpont ME, McGrath J, Hacker AM, Nimmakayalu M, Geiger E, Emanuel BS, Saitta SC
Low copy repeats mediate distal chromosome 22q11.2 deletions: sequence analysis predicts breakpoint mechanisms.
Genome Res. 2007 Apr;17(4):482-91.
Genomic disorders contribute significantly to genetic disease and, as detection methods improve, greater numbers are being defined. Paralogous low copy repeats (LCRs) mediate many of the chromosomal rearrangements that underlie these disorders, predisposing chromosomes to recombination errors. Deletions of proximal 22q11.2 comprise the most frequently occurring microdeletion syndrome, DiGeorge/Velocardiofacial syndrome (DGS/VCFS), in which most breakpoints have been localized to a 3 Mb region containing four large LCRs. Immediately distal to this region, there are another four related but smaller LCRs that have not been characterized extensively. We used paralog-specific primers and long-range PCR to clone, sequence, and examine the distal deletion breakpoints from two patients with de novo deletions mapping to these distal LCRs. Our results present definitive evidence of the direct involvement of LCRs in 22q11 deletions and map both breakpoints to the BCRL module, common to most 22q11 LCRs, suggesting a potential region for LCR-mediated rearrangement both in the distal LCRs and in the DGS interval. These are the first reported cases of distal 22q11 deletions in which breakpoints have been characterized at the nucleotide level within LCRs, confirming that distal 22q11 LCRs can and do mediate rearrangements leading to genomic disorders. [Abstract/Link to Full Text]

Tenesa A, Navarro P, Hayes BJ, Duffy DL, Clarke GM, Goddard ME, Visscher PM
Recent human effective population size estimated from linkage disequilibrium.
Genome Res. 2007 Apr;17(4):520-6.
Effective population size (N(e)) determines the amount of genetic variation, genetic drift, and linkage disequilibrium (LD) in populations. Here, we present the first genome-wide estimates of human effective population size from LD data. Chromosome-specific effective population size was estimated for all autosomes and the X chromosome from estimated LD between SNP pairs <100 kb apart. We account for variation in recombination rate by using coalescent-based estimates of fine-scale recombination rate from one sample and correlating these with LD in an independent sample. Phase I of the HapMap project produced between 18 and 22 million SNP pairs in samples from four populations: Yoruba from Ibadan (YRI), Nigeria; Japanese from Tokyo (JPT); Han Chinese from Beijing (HCB); and residents from Utah with ancestry from northern and western Europe (CEU). For CEU, JPT, and HCB, the estimate of effective population size, adjusted for SNP ascertainment bias, was approximately 3100, whereas the estimate for the YRI was approximately 7500, consistent with the out-of-Africa theory of ancestral human population expansion and concurrent bottlenecks. We show that the decay in LD over distance between SNPs is consistent with recent population growth. The estimates of N(e) are lower than previously published estimates based on heterozygosity, possibly because they represent one or more bottlenecks in human population size that occurred approximately 10,000 to 200,000 years ago. [Abstract/Link to Full Text]

Zhang Z, Hesselberth JR, Fields S
Genome-wide identification of spliced introns using a tiling microarray.
Genome Res. 2007 Apr;17(4):503-9.
The prediction of gene models from genome sequence remains an unsolved problem. One hallmark of eukaryotic gene structure is the presence of introns, which are spliced out of pre-mRNAs prior to translation. The excised introns are released in the form of lariats, which must be debranched prior to their turnover. In the yeast Saccharomyces cerevisiae, the absence of the debranching enzyme causes these lariat RNAs to accumulate. This accumulation allows a comparison of tiling array signals of RNA from the debranching mutant to the wild-type parent strain, and thus the identification of lariats on a genome-wide scale. This approach identified 141 of 272 known introns, confirmed three previously predicted introns, predicted four novel introns (of which two were experimentally confirmed), and led to the reannotation of four others. In many instances, signals from the tiling array delineated the 5' splice site and branchpoint site, confirming predicted gene structures. Nearly all introns that went undetected are present in mRNAs expressed at low levels. Overall, 97% of the significant probes could be attributed either to spliced introns or to genes up-regulated by deletion of the debranching enzyme. Because the debranching enzyme is conserved among eukaryotes, this approach could be generally applicable for the annotation of eukaryotic genes and the detection of novel and alternative splice forms. [Abstract/Link to Full Text]

Mitrovich QM, Tuch BB, Guthrie C, Johnson AD
Computational and experimental approaches double the number of known introns in the pathogenic yeast Candida albicans.
Genome Res. 2007 Apr;17(4):492-502.
Candida albicans is the most common fungal pathogen of humans. Frequently found as a commensal within the digestive tracts of healthy individuals, C. albicans is an opportunistic pathogen that causes a wide variety of clinical syndromes in immuno-compromised individuals. A comprehensive annotation of the C. albicans genome sequence was recently published. Because many C. albicans coding sequences are interrupted by introns, proper intron annotation is essential for the accurate definition of genes in this pathogen. Intron annotation is also important for identifying potential targets of splicing regulation, a common mechanism of gene control in eukaryotes. In this study, we report an improved annotation of C. albicans introns. In addition to correcting the existing intron annotations, 25% of which were incorrect, we have used novel computational and experimental approaches to identify new introns, bringing the total to 415, almost double the number previously known. Our identification methods focus primarily on intron features rather than protein-coding features, overcoming biases of traditional intron annotation methods. Introns are not randomly distributed in C. albicans, and are over-represented in genes involved in specific cellular processes, such as splicing, translation, and mitochondrial respiration. This nonrandom distribution suggests functional roles for these introns, and we demonstrate that splicing of two transcripts whose introns have unusual sequence features is responsive to environmental factors. [Abstract/Link to Full Text]

Gotter AL, Nimmakayalu MA, Jalali GR, Hacker AM, Vorstman J, Conforto Duffy D, Medne L, Emanuel BS
A palindrome-driven complex rearrangement of 22q11.2 and 8q24.1 elucidated using novel technologies.
Genome Res. 2007 Apr;17(4):470-81.
Constitutional translocations at the same 22q11.21 low copy repeat B (LCR-B) breakpoint involved in the recurrent t(11;22) are relatively abundant. A novel 46,XY,t(8;22)(q24.13;q11.21) rearrangement was investigated to determine whether the recurrent LCR-B breakpoint is involved. Investigations demonstrated an inversion of the 3Mb region typically deleted in patients with the 22q11.2 deletion syndrome. The 22q11.21 inversion appears to be mediated by low copy repeats, and is presumed to have taken place prior to translocation with 8q24.13. Despite predictions based on inversions observed in other chromosomes harboring low copy repeats, this 22q11.2 inversion has not been observed previously. The current studies utilize novel laser microdissection and MLPA (multiplex ligation-dependent probe amplification) approaches, as adjuncts to FISH, to map the breakpoints of the complex rearrangements of 22q11.21 and 8q24.21. The t(8;22) occurs between the recurrent site on 22q11.21 and an AT-rich site at 8q24.13, making it the fifth different chromosomal locus characterized at the nucleotide level engaged in a translocation with the unstable recurrent breakpoint at 22q11.21. Like the others, this breakpoint occurs at the center of a palindromic sequence. This sequence appears capable of forming a perfect 145 bp stem-loop. Remarkably, this site appears to have been involved in a previously reported t(3;8) occurring between 8q24.13 and FRA3B on 3p14.2. Further, the fragile site-like nature of all of the breakpoint sites involved in translocations with the recurrent site on 22q11.21, suggests a mechanism based on delay of DNA replication in the initiation of these chromosomal rearrangements. [Abstract/Link to Full Text]

Bollenbach T, Vetsigian K, Kishony R
Evolution and multilevel optimization of the genetic code.
Genome Res. 2007 Apr;17(4):401-4.
The discovery of the genetic code was one of the most important advances of modern biology. But there is more to a DNA code than protein sequence; DNA carries signals for splicing, localization, folding, and regulation that are often embedded within the protein-coding sequence. In this issue, Itzkovitz and Alon show that the specific 64-to-20 mapping found in the genetic code may have been optimized for permitting protein-coding regions to carry this extra information and suggest that this property may have evolved as a side benefit of selection to minimize the negative effects of frameshift errors. [Abstract/Link to Full Text]

Itzkovitz S, Alon U
The genetic code is nearly optimal for allowing additional information within protein-coding sequences.
Genome Res. 2007 Apr;17(4):405-12.
DNA sequences that code for proteins need to convey, in addition to the protein-coding information, several different signals at the same time. These "parallel codes" include binding sequences for regulatory and structural proteins, signals for splicing, and RNA secondary structure. Here, we show that the universal genetic code can efficiently carry arbitrary parallel codes much better than the vast majority of other possible genetic codes. This property is related to the identity of the stop codons. We find that the ability to support parallel codes is strongly tied to another useful property of the genetic code--minimization of the effects of frame-shift translation errors. Whereas many of the known regulatory codes reside in nontranslated regions of the genome, the present findings suggest that protein-coding regions can readily carry abundant additional information. [Abstract/Link to Full Text]

Yellaboina S, Goyal K, Mande SC
Inferring genome-wide functional linkages in E. coli by combining improved genome context methods: comparison with high-throughput experimental data.
Genome Res. 2007 Apr;17(4):527-35.
Cellular functions are determined by interactions among proteins in the cells. Recognition of these interactions forms an important step in understanding biology at the systems level. Here, we report an interaction network of Escherichia coli, obtained by training a Support Vector Machine on the high quality of interactions in the EcoCyc database, and with the assumption that the periplasmic and cytoplasmic proteins may not interact with each other. The data features included correlation coefficient between bit score phylogenetic profiles, frequency of their co-occurrence in predicted operons, and a new measure--the distance between translational start sites of the genes. The combined genome context methods show a high accuracy of prediction on the test data and predict a total of 78,122 binary interactions. The majority of the interactions identified by high-throughput experimental methods correspond to indirect interaction (interactions through neighbors) in the predicted network. Correlation of the predicted network with the gene essentiality data shows that the essential genes in E. coli exhibit a high linking number, whereas the nonessential genes exhibit a low linking number. Furthermore, our predicted protein-protein interaction network shows that the proteins involved in replication, DNA repair, transcription, translation, and cell wall synthesis are highly connected. We therefore believe that our predicted network will serve as a useful resource in understanding prokaryotic biology. [Abstract/Link to Full Text]

Bundy JG, Papp B, Harmston R, Browne RA, Clayson EM, Burton N, Reece RJ, Oliver SG, Brindle KM
Evaluation of predicted network modules in yeast metabolism using NMR-based metabolite profiling.
Genome Res. 2007 Apr;17(4):510-9.
Genome-scale metabolic models promise important insights into cell function. However, the definition of pathways and functional network modules within these models, and in the biochemical literature in general, is often based on intuitive reasoning. Although mathematical methods have been proposed to identify modules, which are defined as groups of reactions with correlated fluxes, there is a need for experimental verification. We show here that multivariate statistical analysis of the NMR-derived intra- and extracellular metabolite profiles of single-gene deletion mutants in specific metabolic pathways in the yeast Saccharomyces cerevisiae identified outliers whose profiles were markedly different from those of the other mutants in their respective pathways. Application of flux coupling analysis to a metabolic model of this yeast showed that the deleted gene in an outlying mutant encoded an enzyme that was not part of the same functional network module as the other enzymes in the pathway. We suggest that metabolomic methods such as this, which do not require any knowledge of how a gene deletion might perturb the metabolic network, provide an empirical method for validating and ultimately refining the predicted network structure. [Abstract/Link to Full Text]

Pace JK, Feschotte C
The evolutionary history of human DNA transposons: evidence for intense activity in the primate lineage.
Genome Res. 2007 Apr;17(4):422-32.
Class 2, or DNA transposons, make up approximately 3% of the human genome, yet the evolutionary history of these elements has been largely overlooked and remains poorly understood. Here we carried out the first comprehensive analysis of the activity of human DNA transposons over the course of primate evolution using three independent computational methods. First, we conducted an exhaustive search for human DNA transposons nested within L1 and Alu elements known to be primate specific. Second, we assessed the presence/absence of 794 human DNA transposons at orthologous positions in 10 mammalian species using sequence data generated by The ENCODE Project. These two approaches, which do not rely upon sequence divergence, allowed us to classify DNA transposons into three different categories: anthropoid specific (40-63 My), primate specific (64-80 My), and eutherian wide (81-150 My). Finally, we used this data to calculate the substitution rates of DNA transposons for each category and refine the age of each family based on the average percent divergence of individual copies to their consensus. Based on these combined methods, we can confidently estimate that at least 40 human DNA transposon families, representing approximately 98,000 elements ( approximately 33 Mb) in the human genome, have been active in the primate lineage. There was a cessation in the transpositional activity of DNA transposons during the later phase of the primate radiation, with no evidence of elements younger than approximately 37 My. This data points to intense activity of DNA transposons during the mammalian radiation and early primate evolution, followed, apparently, by their mass extinction in an anthropoid primate ancestor. [Abstract/Link to Full Text]

Murphy WJ, Pringle TH, Crider TA, Springer MS, Miller W
Using genomic data to unravel the root of the placental mammal phylogeny.
Genome Res. 2007 Apr;17(4):413-21.
The phylogeny of placental mammals is a critical framework for choosing future genome sequencing targets and for resolving the ancestral mammalian genome at the nucleotide level. Despite considerable recent progress defining superordinal relationships, several branches remain poorly resolved, including the root of the placental tree. Here we analyzed the genome sequence assemblies of human, armadillo, elephant, and opossum to identify informative coding indels that would serve as rare genomic changes to infer early events in placental mammal phylogeny. We also expanded our species sampling by including sequence data from >30 ongoing genome projects, followed by PCR and sequencing validation of each indel in additional taxa. Our data provide support for a sister-group relationship between Afrotheria and Xenarthra (the Atlantogenata hypothesis), which is in turn the sister-taxon to Boreoeutheria. We failed to recover any indels in support of a basal position for Xenarthra (Epitheria), which is suggested by morphology and a recent retroposon analysis, or a hypothesis with Afrotheria basal (Exafricoplacentalia), which is favored by phylogenetic analysis of large nuclear gene data sets. In addition, we identified two retroposon insertions that also support Atlantogenata and none for the alternative hypotheses. A revised molecular timescale based on these phylogenetic inferences suggests Afrotheria and Xenarthra diverged from other placental mammals approximately 103 (95-114) million years ago. We discuss the impacts of this topology on earlier phylogenetic reconstructions and repeat-based inferences of phylogeny. [Abstract/Link to Full Text]

Hu Y, Rolfs A, Bhullar B, Murthy TV, Zhu C, Berger MF, Camargo AA, Kelley F, McCarron S, Jepson D, Richardson A, Raphael J, Moreira D, Taycher E, Zuo D, Mohr S, Kane MF, Williamson J, Simpson A, Bulyk ML, Harlow E, Marsischky G, Kolodner RD, LaBaer J
Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae.
Genome Res. 2007 Apr;17(4):536-43.
The availability of an annotated genome sequence for the yeast Saccharomyces cerevisiae has made possible the proteome-scale study of protein function and protein-protein interactions. These studies rely on availability of cloned open reading frame (ORF) collections that can be used for cell-free or cell-based protein expression. Several yeast ORF collections are available, but their use and data interpretation can be hindered by reliance on now out-of-date annotations, the inflexible presence of N- or C-terminal tags, and/or the unknown presence of mutations introduced during the cloning process. High-throughput biochemical and genetic analyses would benefit from a "gold standard" (fully sequence-verified, high-quality) ORF collection, which allows for high confidence in and reproducibility of experimental results. Here, we describe Yeast FLEXGene, a S. cerevisiae protein-coding clone collection that covers over 5000 predicted protein-coding sequences. The clone set covers 87% of the current S. cerevisiae genome annotation and includes full sequencing of each ORF insert. Availability of this collection makes possible a wide variety of studies from purified proteins to mutation suppression analysis, which should contribute to a global understanding of yeast protein function. [Abstract/Link to Full Text]

Mirabeau O, Perlas E, Severini C, Audero E, Gascuel O, Possenti R, Birney E, Rosenthal N, Gross C
Identification of novel peptide hormones in the human proteome by hidden Markov model screening.
Genome Res. 2007 Mar;17(3):320-7.
Peptide hormones are small, processed, and secreted peptides that signal via membrane receptors and play critical roles in normal and pathological physiology. The search for novel peptide hormones has been hampered by their small size, low or restricted expression, and lack of sequence similarity. To overcome these difficulties, we developed a bioinformatics search tool based on the hidden Markov model formalism that uses several peptide hormone sequence features to estimate the likelihood that a protein contains a processed and secreted peptide of this class. Application of this tool to an alignment of mammalian proteomes ranked 90% of known peptide hormones among the top 300 proteins. An analysis of the top scoring hypothetical and poorly annotated human proteins identified two novel candidate peptide hormones. Biochemical analysis of the two candidates, which we called spexin and augurin, showed that both were localized to secretory granules in a transfected pancreatic cell line and were recovered from the cell supernatant. Spexin was expressed in the submucosal layer of the mouse esophagus and stomach, and a predicted peptide from the spexin precursor induced muscle contraction in a rat stomach explant assay. Augurin was specifically expressed in mouse endocrine tissues, including pituitary and adrenal gland, choroid plexus, and the atrio-ventricular node of the heart. Our findings demonstrate the utility of a bioinformatics approach to identify novel biologically active peptides. Peptide hormones and their receptors are important diagnostic and therapeutic targets, and our results suggest that spexin and augurin are novel peptide hormones likely to be involved in physiological homeostasis. [Abstract/Link to Full Text]

Ling KH, Rajandream MA, Rivailler P, Ivens A, Yap SJ, Madeira AM, Mungall K, Billington K, Yee WY, Bankier AT, Carroll F, Durham AM, Peters N, Loo SS, Isa MN, Novaes J, Quail M, Rosli R, Nor Shamsudin M, Sobreira TJ, Tivey AR, Wai SF, White S, Wu X, Kerhornou A, Blake D, Mohamed R, Shirley M, Gruber A, Berriman M, Tomley F, Dear PH, Wan KL
Sequencing and analysis of chromosome 1 of Eimeria tenella reveals a unique segmental organization.
Genome Res. 2007 Mar;17(3):311-9.
Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome--which appears to be representative of the genome--is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation. [Abstract/Link to Full Text]

Zhu W, Buell CR
Improvement of whole-genome annotation of cereals through comparative analyses.
Genome Res. 2007 Mar;17(3):299-310.
Rice is an important model species for the Poaceae and other monocotyledonous plants. With the availability of a near-complete, finished, and annotated rice genome, we performed genome level comparisons between rice and all plant species in which large genomic or transcriptomic data sets are available to determine the utility of cross-species sequence for structural and functional annotation of the rice genome. Through comparative analyses with four plant genome sequence data sets and transcript assemblies from 185 plant species, we were able to confirm and improve the structural annotation of the rice genome. Support for 38,109 (89.3%) of the total 42,653 nontransposable element-related genes in the rice genome in the form of a rice expressed sequence tag, full-length cDNA, or plant homolog from our comparative analyses could be found. Although the majority of the putative homologs were obtained from Poaceae species, putative homologs were identified in dicotyledonous angiosperms, gymnosperms, and other plants such as algae, moss, and fern. A set of rice genes (7669) lacking a putative homolog was identified which may be lineage-specific genes that evolved after speciation and have a role in species diversity. Improvements to the current rice gene structural annotation could be identified from our comparative alignments and we were able to identify 487 genes which were mostly likely missed in the current rice genome annotation and another 500 genes for structural annotation review. We were able to demonstrate the utility of cross-species comparative alignments in the identification of noncoding sequences and in confirmation of gene nesting in rice. [Abstract/Link to Full Text]

Kukekova AV, Trut LN, Oskina IN, Johnson JL, Temnykh SV, Kharlamova AV, Shepeleva DV, Gulievich RG, Shikhevich SG, Graphodatsky AS, Aguirre GD, Acland GM
A meiotic linkage map of the silver fox, aligned and compared to the canine genome.
Genome Res. 2007 Mar;17(3):387-99.
A meiotic linkage map is essential for mapping traits of interest and is often the first step toward understanding a cryptic genome. Specific strains of silver fox (a variant of the red fox, Vulpes vulpes), which segregate behavioral and morphological phenotypes, create a need for such a map. One such strain, selected for docility, exhibits friendly dog-like responses to humans, in contrast to another strain selected for aggression. Development of a fox map is facilitated by the known cytogenetic homologies between the dog and fox, and by the availability of high resolution canine genome maps and sequence data. Furthermore, the high genomic sequence identity between dog and fox allows adaptation of canine microsatellites for genotyping and meiotic mapping in foxes. Using 320 such markers, we have constructed the first meiotic linkage map of the fox genome. The resulting sex-averaged map covers 16 fox autosomes and the X chromosome with an average inter-marker distance of 7.5 cM. The total map length corresponds to 1480.2 cM. From comparison of sex-averaged meiotic linkage maps of the fox and dog genomes, suppression of recombination in pericentromeric regions of the metacentric fox chromosomes was apparent, relative to the corresponding segments of acrocentric dog chromosomes. Alignment of the fox meiotic map against the 7.6x canine genome sequence revealed high conservation of marker order between homologous regions of the two species. The fox meiotic map provides a critical tool for genetic studies in foxes and identification of genetic loci and genes implicated in fox domestication. [Abstract/Link to Full Text]

Goios A, Pereira L, Bogue M, Macaulay V, Amorim A
mtDNA phylogeny and evolution of laboratory mouse strains.
Genome Res. 2007 Mar;17(3):293-8.
Inbred mouse strains have been maintained for more than 100 years, and they are thought to be a mixture of four different mouse subspecies. Although genealogies have been established, female inbred mouse phylogenies remain unexplored. By a phylogenetic analysis of newly generated complete mitochondrial DNA sequence data in 16 strains, we show here that all common inbred strains descend from the same Mus musculus domesticus female wild ancestor, and suggest that they present a different mitochondrial evolutionary process than their wild relatives with a faster accumulation of replacement substitutions. Our data complement forthcoming results on resequencing of a group of priority strains, and they follow recent efforts of the Mouse Phenome Project to collect and make publicly available information on various strains. [Abstract/Link to Full Text]

Recent Articles in Journal of Applied Genetics

Paluszczak J, Baer-Dubowska W
Epigenetic diagnostics of cancer--the application of DNA methylation markers.
J Appl Genet. 2006;47(4):365-75.
In recent years it has become apparent that epigenetic events are potentially equally responsible for cancer initiation and progression as genetic abnormalities. DNA methylation is the main epigenetic modification in humans. Two DNA methylation lesions coexist in human neoplasms: hypermethylation of promoter regions of specific genes within a context of genomic hypomethylation. Aberrant methylation is found at early stages of carcinogenesis and distinct types of cancer exhibit specific patterns of methylation changes. Tumor specific DNA is readily obtainable from different clinical samples and methylation status analysis often permits sensitive disease detection. Methylation markers may also serve for prognostic and predictive purposes as they often reflect the metastatic potential and sensitivity to therapy. As current findings show a great potential of recently characterised methylation markers, more studies in the field are needed in the future. Large clinical studies of newly developed markers are especially needed. The review describes the diagnostic potential of DNA methylation markers. [Abstract/Link to Full Text]

Hor�k P, Knoll A, Dvor�k J
The retinal fascin gene 2 (FSCN2)--partial structural analysis and polymorphism detection in dogs with progressive retinal atrophy (PRA).
J Appl Genet. 2006;47(4):361-4.
The retinal fascin 2 gene (FSCN2) underwent a molecular analysis, a search for polymorphisms and an evaluation as a candidate gene for retinopathies in dogs. Specific fragments of the gene encompassing partial exon 1 and intron 1, and exons 2-5 with respective introns were sequenced and these data were deposited in the GenBank database. Three distinct polymorphic sites detectable with PCR-RFLP were found--AM050719: g.237G>A, AM050719: g.525A>G, and AM050720: g.1071A>G. No positive associations between these polymorphisms and the PRA-clinical status were observed in the investigated population consisting of Poodles, American Cocker Spaniels, and English Cocker Spaniels. In spite of that, the FSCN2 gene remains an excellent candidate gene for retinopathies in dogs and the results can contribute to further research in this field. [Abstract/Link to Full Text]

Iwa?czyk E, Juras R, Cholewi?ski G, Cothran EG
Genetic structure and phylogenetic relationships of the Polish Heavy horse.
J Appl Genet. 2006;47(4):353-9.
In this study a wide range of genetic markers (12 microsatellites, 7 blood-group loci, 10 blood-protein loci) and mitochondrial DNA (mtDNA) were used to assess genetic diversity in Polish Heavy horses. Three random samples were sequenced for 421 bp of the mitochondrial D-loop region, but no clear phylogenetic patterns were seen in mtDNA variation. Both heterozygosity and diversity levels are fairly high in Polish Heavy horses. In phylogenetic analysis the draught horses form a distinct cluster that pairs with the true pony breeds. Within this 'cold-blooded' group, the Polish Heavy Horse clusters most closely with the Posavina breed from Croatia and the Breton breed from France. From the standpoint of genetic conservation, the Polish Heavy Horse does not appear to be in jeopardy. [Abstract/Link to Full Text]

Oponowicz A, Franczak A, Kurowicka B, Kotwica G
Relative transcript abundance of oxytocin receptor gene in porcine uterus during luteolysis and early pregnancy.
J Appl Genet. 2006;47(4):345-51.
The aim of the present study was to investigate transcript localization of the oxytocin receptor (OTR) gene in different cells of the porcine uterus during luteolysis and early pregnancy (days 14-16) using in situ hybridization (ISH). OTR mRNA was localized in the uterine luminal epithelium (LEC), glandular epithelium (GEC), stromal cells (SC) of the endometrium, in the longitudinal muscle layer (LM) and circular muscle layer (CM) of the myometrium. The OTR transcript was quantified by optical density units of silver grains. The OTR transcript levels in the endometrium and myometrium were statistically higher during luteolysis than during early pregnancy (P<0.05). Besides, during luteolysis, the mRNA level was higher in the LEC, GEC of the endometrium and LM of the myometrium compared to that observed in the SC of endometrium and CM of the myometrium, respectively (P<0.05). In summary: 1) the level of OTR mRNA in uterine tissues is higher during luteolysis compared to early pregnancy, 2) the OTR transcript level in endometrial cells did not correspond to the sensitivity of these cells to oxytocin (OT), 3) the myometrial expression of the OTR gene is appropriate to control contractile activity and secretion of PG during luteolysis. [Abstract/Link to Full Text]

Karaca�ren B, Kadarmideen HN, Janss LL
Investigation of major gene for milk yield, milking speed, dry matter intake, and body weight in dairy cattle.
J Appl Genet. 2006;47(4):337-43.
The main aim of this study was to determine if there exist any major gene for milk yield (MY), milking speed (MS), dry matter intake (DMI), and body weight (BW) recorded at various stages of lactation in first-lactation dairy cows (2543 observations from 320 cows) kept at the research farm of the Swiss Federal Institute of Technology between April 1994 and April 2004. Data were modelled based a simple repeatability covariance structure and analysed by using Bayesian segregation analyses. Gibbs sampling was used to make statistical inferences on posterior distributions; inferences were based on a single run of the Markov chain for each trait with 500,000 samples, with each 10th sample collected because of the high correlation among the samples. The posterior mean (+/-SD) of major gene variance was 2.61 (+/-2.46) for MY, 0.83 (+/-1.26) for MS, 4.37 (+/-2.34) for DMI, and 2056.43 (+/-665.67) for BW. Highest posterior density regions for 3 of the 4 traits did not include 0 (except MS), which supported the evidence for major gene. With additional tests for agreement with Mendelian transmission probabilities, we could only confirm the existence of a major gene for MY, but not for MS, DMI, and BW. Expected Mendelian transmission probabilities and their model fits were also compared. [Abstract/Link to Full Text]

Pahlavani MH, Abolhasani K
Xenia effect on seed and embryo size in cotton (Gossypium hirsutum L.).
J Appl Genet. 2006;47(4):331-5.
The term xenia was coined to describe the effect of foreign pollen on the development and characters of the seed. To study its importance and consequences for various seed traits in cotton (Gossypium hirsutum L.), the effect of pollen genotype on seed and embryo weight was studied with seeds from 15 F1 hybrids. Cross-fertilization changed seed weight by up to 7.0% in relation to self-fertilization. Xenia effect significantly increased embryo weight of cross-fertilized seeds, by up to 14.4% in comparison to self-fertilized seeds. Seeds of some crosses had a lower hull content than corresponding selfed seeds. On average, the xenia effect was greater for embryo weight than for seed weight. However, in some crosses there was no difference between cross- and self-fertilized seeds for seed weight, embryo weight, moisture content and hull content. Positive xenia effects for seed weight and embryo weight may help us to establish uniform stands of vigorous hybrid seedlings, especially under unfavourable conditions. Also, larger seed and embryo weight, along with lower hull content, could result in higher oil yield. Therefore, careful choosing of genotypes as parents and of cross direction in the production of hybrid seed is very important in cotton. [Abstract/Link to Full Text]

Kalinowski A, Rad?owski M, Bocian A
Effects of interaction between pollen coat eluates and pistil at the molecular level in self-compatible and self-incompatible plants of Lolium multiflorum Lam.
J Appl Genet. 2006;47(4):319-29.
Two-dimensional electrophoresis (2-DE) of soluble proteins and enzymes was performed and specific activities of 5 enzymes (esterase, pectinesterase, acid phosphatase, protease and diaphorase) were determined in stigmas of Lolium multiflorum (Italian ryegrass) treated with self or foreign pollen coat eluates (pc). Also, a low-molecular-weight fraction of the treated self-compatible (SC) and self-incompatible (SI) stigmas was analyzed by high-pressure liquid chromatography (HPLC). The treatment of stigmas with foreign pollen induced the loss of 42% of the control sample proteins in SC plants but only of 5.5% in SI plants. In contrast, the treatment of stigmas with foreign pollen induced the loss of 15% proteins in SC plants and of 29% in SI plants. Specific activities of esterase, pectinesterase and diaphorase were higher in SC than in SI stigmas. The 2-DE enzyme patterns indicated qualitative relationships between the presence of some isoforms of acid phosphatase or protease and the treatment with self or foreign pc in SC and SI stigmas. No changes were observed in HPLC profiles of the low-molecular-weight fraction from SC and SI stigmas treated or not with pc. The presented results revealed different reactions of SC and SI stigmas to the treatment with self or foreign pc. Further investigations may explain if any of the observed reactions represent specific reorientations in the style, facilitating cross- or self-pollination. [Abstract/Link to Full Text]

Pniewski T, Kapusta J, P?ucienniczak A
Agrobacterium-mediated transformation of yellow lupin to generate callus tissue producing HBV surface antigen in a long-term culture.
J Appl Genet. 2006;47(4):309-18.
The idea of an oral vaccine administered as a portion of plant tissue requires a high level of antigen production. An improved protocol for the induction of transgenic yellow lupin calli or tumours, reaching 44% of transformation rate, is presented here. It has been developed by using the nptII marker gene and the uidA reporter gene as well as various Agrobacterium strains and plant explants. This method of seedling and hypocotyl transformation was applied to raise calli or tumours producing a small surface antigen of Hepatitis B Virus (S-HBsAg). Lupin tissue lines were long-term cultured on selection media maintaining the growth rate and high expression level of the native form of S-HBs, up to 6 microg per g of fresh tissue. [Abstract/Link to Full Text]

Fukuta S, Mizukami Y, Ishida A, Kanbe M
Development of loop-mediated isothermal amplification (LAMP)-based SNP markers for shelf-life in melon (Cucumis melo L.).
J Appl Genet. 2006;47(4):303-8.
In this study, LAMP markers linked to shelf-life in melon (Cucumis melo L.) were developed by converting a cleaved amplified polymorphic sequences (CAPS) marker (C2). The CAPS-PCR fragments from the long-shelf-life melon (O-3) and short-shelf-life melon (Nat-2) were cloned and sequenced to construct LAMP primers. A single nucleotide polymorphism (SNP) was identified between O-3 and Nat-2. LAMP primers were designed to detect the SNP. In the LAMP reaction to detect long-shelf-life melon, the turbidity of the templates using O-3, F1, homozygous long-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. In contrast, the turbidity of Nat-2 and homozygous short-shelf-life F2 lines did not increase even after 90 min. In the LAMP reaction to detect short-shelf-life melon, the turbidity of the templates using Nat-2, F1, homozygous short-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. But the turbidity of O-3 and homozygous long-shelf-life F2 lines did not increase after 90 min. This attests to the high reliability and usefulness of LAMP for marker-assisted selection. [Abstract/Link to Full Text]

Laudencia-Chingcuanco DL, Stamova BS, Lazo GR, Cui X, Anderson OD
Analysis of the wheat endosperm transcriptome.
J Appl Genet. 2006;47(4):287-302.
Among the cereals, wheat is the most widely grown geographically and is part of the staple diet in much of the world. Understanding how the cereal endosperm develops and functions will help generate better tools to manipulate grain qualities important to end-users. We used a genomics approach to identify and characterize genes that are expressed in the wheat endosperm. We analyzed the 17,949 publicly available wheat endosperm EST sequences to identify genes involved in the biological processes that occur within this tissue. Clustering and assembly of the ESTs resulted in the identification of 6,187 tentative unique genes, 2,358 of which formed contigs and 3,829 remained as singletons. A BLAST similarity search against the NCBI non-redundant sequence database revealed abundant messages for storage proteins, putative defense proteins, and proteins involved in starch and sucrose metabolism. The level of abundance of the putatively identified genes reflects the physiology of the developing endosperm. Half of the identified genes have unknown functions. Approximately 61% of the endosperm ESTs has been tentatively mapped in the hexaploid wheat genome. Using microarrays for global RNA profiling, we identified endosperm genes that are specifically up regulated in the developing grain. [Abstract/Link to Full Text]

Filipecki M, Malepszy S
Unintended consequences of plant transformation: a molecular insight.
J Appl Genet. 2006;47(4):277-86.
Plant genomes are dynamic structures having both the system to maintain and accurately reproduce the information encoded therein and the ability to accept more or less random changes, which is one of the foundations of evolution. Crop improvement and various uncontrolled stress factors can induce unintended genetic and epigenetic variations. In this review it is attempted to summarize factors causing such changes and the molecular nature of these variations in transgenic plants. Unintended effects in transgenic plants can be divided into three main groups: first, pleiotropic effects of integrated DNA on the host plant genome; second, the influence of the integration site and transgene architecture on transgene expression level and stability; and third, the effect of various stresses related to tissue handling, regeneration and clonal propagation. Many of these factors are recently being redefined due to new researches, which apply modern highly sensitive analytical techniques and sequenced model organisms. The ability to inspect large portions of genomes clearly shows that tissue culture contributes to a vast majority of observed genetic and epigenetic changes. Nevertheless, monitoring of thousands transcripts, proteins and metabolites reveals that unintended variation most often falls in the range of natural differences between landraces or varieties. We expect that an increasing amount of evidence on many important crop species will support these observations in the nearest future. [Abstract/Link to Full Text]

Constantinou M, Binka-Kowalska A, Borkowska E, Zajac E, Ja?muzna P, Matych J, Nawrocka A, Ka?uzewski B
Application of multiplex FISH, CGH and MSSCP techniques for cytogenetic and molecular analysis of transitional cell carcinoma (TCC) cells in voided urine specimens.
J Appl Genet. 2006;47(3):273-5.
Multiplex FISH (UroVysion), Comparative Genomic Hybridization (CGH), and Multitemperature Single-Strand Conformation Polymorphism (MSSCP) were applied for non-invasive diagnosis and prognosis of bladder cancer. The UroVysion test was positive in 80% of patients with pT1 and in 100% of patients with either pT2 or pT3 tumours. Tumours with pT3T4 stages were characterized by high numbers of chromosomal imbalances, detected by CGH. The mutation of the p53 gene was detected in 16% of patients, but only in those with pT2 or pT3 tumours. [Abstract/Link to Full Text]

Zi�?kowska I, Mosor M, Nowak J
Regional distribution of heterozygous 657del5 mutation carriers of the NBS1 gene in Wielkopolska province (Poland).
J Appl Genet. 2006;47(3):269-72.
The homozygous 657del5 mutation, called Slavic mutation, of the NBS1 gene, causes the Nijmegen Breakage Syndrome (NBS). This syndrome is connected with a high incidence of malignancies in early childhood. A high frequency of NBS heterozygotes was found among patients with melanoma, breast, ovary and prostate cancer. The aim of our research was to determine the frequency of 657del5 mutation of the NBS1 gene in the population of Wielkopolska province. For this purpose, we analysed blood samples from anonymous Guthrie cards. In a group of 2090 newborns from the whole province, we found 16 heterozygous mutation carriers. The frequency of 1/131 is higher than 1/190 reported for populations from other regions in Poland. We observed differential regional distribution of heterozygous 657del5 mutation carriers within the province: among 464 samples from the eastern part of Wielkopolska we found 6 carriers (1/77), in contrast to the southern part without any carrier among 625 samples analysed. The high mean frequency of heterozygous 657del5 mutation (1/131) in Wielkopolska province may be associated with cancer incidence in this region. [Abstract/Link to Full Text]

Smigiel R, Lebioda A, Patkowski D, Czernik J, Dobosz T, Pesz K, Kaczmarz M, Sasiadek MM
Single nucleotide polymorphisms in the RET gene and their correlations with Hirschsprung disease phenotype.
J Appl Genet. 2006;47(3):261-7.
Hirschsprung disease (HSCR) is a congenital, heterogeneous disorder, characterized by the absence of intestinal ganglion cells. Recent advances show that the RET gene is a major locus involved in the pathogenesis of HSCR. The aim of this study was to analyse if the HSCR phenotype in the Polish population is associated with the presence of polymorphisms in exons 2, 3, 7, 11, 13, 14 and 15 of the RET gene. Molecular results were compared with clinical and long-term follow-up data in 70 Polish patients with HSCR (84.3% with a short segment and 15.7% with a long segment of aganglionic gut). Single-nucleotide polymorphisms were analysed by using the minisequencing SNaPshot multiplex method. The 135G>A polymorphism in RET exon 2 was overrepresented in HSCR patients, compared with a healthy control group. Moreover, the 135G>A variant was shown to be associated with the severe HSCR phenotype. Two other polymorphisms, 2071G>A in exon 11 and 2712C>G in exon 15, were underrepresented in the patients. The results confirm that these RET polymorphisms play a role in the aetiology of HSCR. [Abstract/Link to Full Text]

Kocha?ski A
How to assess the pathogenicity of mutations in Charcot-Marie-Tooth disease and other diseases?
J Appl Genet. 2006;47(3):255-60.
Knowledge whether a certain DNA variant is a pathogenic mutation or a harmless polymorphism is a critical issue in medical genetics, in which results of a molecular analysis may serve as a basis for diagnosis and genetic counseling. Due to its genetic heterogeneity expressed at the levels of loci, genes and mutations, Charcot-Marie-Tooth (CMT) disease can serve as a model group of clinically homogenous diseases for studying the pathogenicity of mutations. Close to a 17p11.2-p12 duplication occurring in 70% of patients with the demyelinating form of CMT disease, numerous mutations have been identified in poorly characterized genes coding for proteins of an unknown function. Functional analyses, segregation analyses of large pedigrees, and inclusion of large control groups are required to assess the potential pathogenicity of CMT mutations. Hence, the pathogenicity of numerous CMT mutations remains unclear. Some variants detected in the CMT genes and originally described as pathogenic mutations have been shown to have a polymorphic character. In contrast, polymorphisms initially considered harmless were later reclassified as pathogenic mutations. However, the process of assessing the pathogenicity of mutations, as presented in this study for CMT disorders, is a more general issue concerning all disorders with a genetic background. Since the number of DNA variants is still growing, in the near future geneticists will increasingly have to cope with the problem of pathogenicity of identified genetic variants. [Abstract/Link to Full Text]

Aranishi F, Okimoto T
A simple and reliable method for DNA extraction from bivalve mantle.
J Appl Genet. 2006;47(3):251-4.
A simple and reliable method was developed for extracting genomic DNA from preserved mantle tissues of Pacific oyster Crassostrea gigas for reproducible PCR amplification. The method was based on destruction of the tissue using Proteinase K, Chelex 100 resin, detergents, and urea, followed by preferential capturing of genomic DNA with silica particles. Approximately 5 mg of mantle tissue provided a sufficient quality and quantity of DNA for several hundreds of PCR reactions amplifying the hypervariable mitochondrial DNA intergenic spacer, which is a useful genetic marker for population structure analysis of Pacific oyster. The method can be applied for DNA preparation from not only fresh and frozen but also ethanol-preserved mantle tissues, so this rapid and economical method can serve for investigating a large number of bivalve specimens collected in the field and next transported in ethanol at ambient temperature. [Abstract/Link to Full Text]

Michalak E, Lipi?ski D, S?omski R
Loop formation by the transgene WAP:6xHishGH in transgenic rabbit fibroblasts, revealed by fluorescence in situ hybridization to nuclear halos.
J Appl Genet. 2006;47(3):247-9.
Using fluorescence in situ hybridization (FISH) to somatic nuclear halos from transgenic rabbits WAP:6xHishGH, we present evidence for stability of transgenesis at the chromatin level. FISH performed on fibroblasts from a homozygous individual showed 2 independent loops from both chromosomes of pair 7. On a heterozygous individual, FISH detected a single loop. According to the concept of chromatin loops and their influence on gene expression, this shows that the human growth hormone transgene, which was actively expressed in mammary gland under the influence of the tissue-specific promoter, was inactive in examined skin fibroblasts. [Abstract/Link to Full Text]

Sazanov AA, Malewski T, Kami?ski S, Zwierzchowski L
Characterization of the CHORI-240 BAC clones containing the bovine CSN1S1, CSN2, STATH, CSN1S2 and CSN3 genes.
J Appl Genet. 2006;47(3):243-5.
Nineteen BAC clones were identified by hybridization of the bovine genomic BAC library CHORI-240 with mixed CSN1S1- and CSN3-specific probes. Two of the clones were shown to contain the genes CSN1S1, CSN1S2, CSN2, STATH and CSN3, and five were proved to include the genes CSN2, STATH, CSN1S2 and CSN3. These data showed that the BAC contig was established for the whole casein cluster, including all known five genes. [Abstract/Link to Full Text]

Patel RK, Singh KM, Soni KJ, Chauhan JB, Sambasiva Rao KR
Lack of carriers of citrullinaemia and DUMPS in Indian Holstein cattle.
J Appl Genet. 2006;47(3):239-42.
The present study investigated the occurrence of 2 autosomal recessive genetic diseases, bovine citrullinaemia and deficiency of uridine monophosphate synthase (DUMPS), in Indian Holstein cattle. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed on a group of 642 animals, mainly HF and HF crossbred cattle, to identify carriers of these diseases. None of the animals were carriers of citrullinaemia or DUMPS. It is possible that with the mounting selection pressure, the international gene pool may diminish, and consequently the risk of dissemination of inherited defects will increase. It is therefore recommended to screen breeding bulls for their breed-specific genetic diseases before they are inducted in artificial insemination programmes, to minimize the risk. [Abstract/Link to Full Text]

G�rny AG, Garczy?ski S, Banaszak Z, ?ugowska B
Genetic variation in the efficiency of nitrogen utilization and photosynthetic activity of flag leaves among the old and modern germplasm of winter wheat.
J Appl Genet. 2006;47(3):231-7.
Genotypic variation in major components of the efficiency of nitrogen utilization and photosynthetic activity of flag leaves among old (released 1881-1963) and modern (released 1969-2003) cultivars of winter wheat was studied in field conditions under varied N fertilization levels (110, 90 and 80 kg N ha-1). Significant genotypic differences were observed for all characters. Their heritabilities ranged from 0.37 to 0.93 and were the lowest for the leaf efficiency of gas exchange, photosynthetic rate, straw N content and the economic index of N utilization efficiency (NUE). Some modern cultivars exhibited an enhanced tolerance to N shortage and several attributes of efficient N utilization (e.g. later senescing and more photosynthetically active flag leaves, increased ability to redistribute N into grains). The genotypes may serve as donors of appropriate characteristics for breeding. The observed cultivar-by-fertilization interactions suggest, however, that evaluations under diverse fertilization regimes may be necessary when searching for improved wheat efficiency and adaptation to less favourable environments. [Abstract/Link to Full Text]

Sm�kal P
Development of an efficient retrotransposon-based fingerprinting method for rapid pea variety identification.
J Appl Genet. 2006;47(3):221-30.
Fast and efficient DNA fingerprinting of crop cultivars and individuals is frequently used in both theoretical population genetics and in practical breeding. Numerous DNA marker technologies exist and the ratio of speed, cost and accuracy are of importance. Therefore even in species where highly accurate and polymorphic marker systems are available, such as microsatellite SSR (simple sequence repeats), also alternative methods may be of interest. Thanks to their high abundance and ubiquity, temporary mobile retrotransposable elements come into recent focus. Their properties, such as genome wide distribution and well-defined origin of individual insertions by descent, predetermine them for use as molecular markers. In this study, several Ty3-gypsy type retrotransposons have been developed and adopted for the inter-retrotransposon amplified polymorphism (IRAP) method, which is suitable for fast and efficient pea cultivar fingerprinting. The method can easily distinguish even between genetically closely related pea cultivars and provide high polymorphic information content (PIC) in a single PCR analysis. [Abstract/Link to Full Text]

Kosmala A, Zwierzykowska E, Zwierzykowski Z
Chromosome pairing in triploid intergeneric hybrids of Festuca pratensis with Lolium multiflorum, revealed by GISH.
J Appl Genet. 2006;47(3):215-20.
Genomic in situ hybridisation (GISH) was used to reveal chromosome pairing in two partly fertile, triploid (2n = 3x = 21) hybrids obtained by crossing the diploid (2n = 2x = 14) Festuca pratensis Huds. (designated FpFp), used as a female parent, with the autotetraploid (2n = 4x = 28) Lolium multiflorum Lam. (designated LmLmLmLm), used as a male parent. The pattern of chromosome pairing calculated on the basis of the mean values of chromosome configurations identified in all 100 PMCs analysed, was: 0.71I Lm + 2.24I Fp + 2.18II Lm/Lm + 0.54II Lm/Fp + 4.18III Lm/Lm/Fp. A relatively high number of Lm/Lm bivalents and Fp univalents, and a low number of Lm/Fp bivalents and Lm univalents indicated that the pairing was preferential between L. multiflorum chromosomes. Other observations regarding chromosome pairing within the Lm/Lm/Fp trivalents also confirmed this preferential pairing in the analysed triploids, as the Fp chromosome was not randomly located in the chain- and frying-pan-shaped trivalents. The similarities and differences in chromosome pairing at metaphase I and the level of preferential pairing between Lolium chromosomes in the different triploid Lolium-Festuca hybrids are discussed. [Abstract/Link to Full Text]

Barnett RD, Blount AR, Pfahler PL, Bruckner PL, Wesenberg DM, Johnson JW
Environmental stability and heritability estimates for grain yield and test weight in triticale.
J Appl Genet. 2006;47(3):207-13.
Hexaploid triticale has many advantages over both parental species for both grain and forage production in certain environments. Additional information on environmental stability and heritability would be desirable to develop appropriate selection strategies in the production of superior widely-adapted cultivars. The grain yield of 22 diverse genotypes grown at four ecologically-distinct geographical locations [Quincy, FL, USA (approximate geographical coordinates (AGC) = 30 degreesN 84 degreesW, approximate elevation (AE) = 58 m), Plains, GA, USA (AGC = 32 degreesN 84 degreesW, AE = 76 m), Bozeman, MT USA (AGC = 45 degreesN 111 degreesW, AE = 1458 m), and Aberdeen, ID, USA (AGC = 42 degreesN 112 degreesW, AE = 1360 m)] was measured in two years with winter and spring planting dates only at Bozeman and Aberdeen. Test weight (grain weight in a given volume) was determined for two years at Bozeman and Aberdeen at both planting dates and one year at Quincy. Stability analyses indicated that significant (P < 0.01) variation in means, regression coefficients, and deviation mean squares of the genotypes were present for both characters. Realized heritability (h2) estimates were as follows: grain yield ranged from -0.02 to 0.80 with a mean of 0.57; test weight ranged from 0.63 to 1.05 with a mean of 0.93. The results indicated that substantial genetic variation is present and selection for widely-adapted cultivars would be effective for both characters especially test weight. [Abstract/Link to Full Text]

Olszewska A
Dental treatment strategies in a 40-year-old patient with cleidocranial dysplasia.
J Appl Genet. 2006;47(2):199-201.
Oral anomalies and dental treatment in a patient with cleidocranial dysplasia (referred to the dental clinic at the age of 40 years) are presented. Five supernumerary teeth were found in the patient: three in the maxilla in the area of molars and two in the mandibula in the area of premolars. Therapy included surgical exposure of impacted teeth in combination with removal of supernumerary teeth. [Abstract/Link to Full Text]

Zapa?owicz K, Wygledowska G, Roszkowski T, Bednarowska A
Harlequin ichthyosis--difficulties in prenatal diagnosis.
J Appl Genet. 2006;47(2):195-7.
Ichthyoses belong to the group of genodermatoses, characterized by hyperkeratosis and desquamation of the epidermis. Clinical manifestation is heterogeneous and depends on the type of the disease. Harlequin foetus is the most severe form of congenital ichtyosis, inherited as an autosomal recessive trait. The disfunction of the epidermis begins prenatally. Neonates are often born prematurely, in severe condition. At present better care and treatment prolong the length and quality of children's life. We report a case of harlequin ichthyosis. Parents were healthy and there was no history of ichthyosis or other congenital anomalies in the family. Sonography at the 26th week of gestation revealed anomalies of the fetal face; however, the diagnosis of harlequin ichthyosis was not established prenatally. The male child was born alive at the 37th week of the third pregnancy, with birth weight of 2900 g. Typical features of harlequin ichthyosis were present at birth. Intensive neonatological care was necessary. The child survived and at the time of the report was 6 months old and in good condition. [Abstract/Link to Full Text]

G�rska-Kot A, B?az W, Pszeniczna E, Rusin J, Materna-Kiryluk A, Homa E, Hejda G, Franus J
Trends in diagnosis and prevalence of critical congenital heart defects in the Podkarpacie province in 2002-2004, based on data from the Polish Registry of Congenital Malformations.
J Appl Genet. 2006;47(2):191-4.
This paper presents results of data collection regarding congenital heart defects (CHD) for the Polish Registry of Congenital Malformations (PRCM) from the Podkarpacie province in 2002-2004. Routine methods (fetal echocardiography, clinical examination) and recently also unique methods (screening echocardiography and pulse oximetry) were used for early detection of critical cardiac malformations. Critical CHD were detected there in 107 newborns in 2002-2004, so the mean prevalence reached 1.71/1000. Within this group, death rate decreased from 8 deaths in 2002 to 2 deaths in 2004, mainly thanks to improved detection and treatment of CHD. [Abstract/Link to Full Text]

P�tzsch S, Hoyer-Schuschke J, Seelig M, Steinbicker V
Knowledge among young people about folic acid and its importance during pregnancy: a survey in the Federal State of Saxony-Anhalt (Germany).
J Appl Genet. 2006;47(2):187-90.
Periconceptional folic acid supplementation is recommended to prevent congenital malformations, mainly neural tube defects, but only 7% of pregnant women in Saxony-Anhalt (Germany) take folic acid at least 4 weeks before conception and in the first 3 months of pregnancy. From March to June 2004, we sent standardized questionnaires about folic acid and its importance during pregnancy to 33 schools in the Federal State of Saxony-Anhalt. A total of 4332 young people aged 1521 years completed the questionnaire, of which 2632 were girls (61%) and 1685 were boys (39%). The majority of them (61%) had heard about folic acid, but only 5% knew that it is a vitamin and 0.7% were aware of the physiological functions of folic acid. Only 22% of the young people answered that folic acid should be taken before and during pregnancy, whereas almost all respondents knew other precautions during pregnancy, e.g. "no smoking" and "no alcohol". Our survey shows that the level of awareness of the importance of folic acid at schools is very low. We suggest that the problem of folic acid should be included in the curricula of biology classes at schools to spread the knowledge of this subject among young people. [Abstract/Link to Full Text]

Sieroszewski P, Perenc M, Ba?-Budecka E, Suzin J
Ultrasound diagnostic schema for the determination of increased risk for chromosomal fetal aneuploidies in the first half of pregnancy.
J Appl Genet. 2006;47(2):177-85.
The aim of the study was to develop an early ultrasound diagnostic schema for the determination of increased risk for fetal chromosomal aneuploidies. The study was conducted on a population of 1318 pregnant women divided into 2 groups: 1255 women with the normal course of pregnancy and 63 women with diagnosed fetal abnormalities. There were 34 cases of chromosomal abnormalities (trisomy 21, 18, 13; triploidy; unbalanced inversion 9; deletion 16) and 29 cases of structural malformations. The estimation of the range of normal values was performed for the nuchal translucency (NT) measurement between 11 and 13 weeks and the nasal bone length (NB) measurement between 12 and 20 week. The results obtained in the collective set of normal pregnancies constituted the basis for the calculation of the range of normal values. The measurements of NB and NT showed a linear value increase with the pregnancy course. The following test characteristics (correlation to CRL) were recorded: NB - sensitivity 60%, specificity 98%, positive predictive value (PPV+) 43%, negative predictive value (NPV-) 98.9%. For the assumption that the test outcome means the presence or absence of the nasal bone in the ultrasound scan the sensitivity was 40%, but specificity 100%; NT - sensitivity 63.6%, specificity 98.2%, PPV+ 38.9%, NPV - 98.2%; NT + NB - presents similar characteristic to the NB or NT alone - sensitivity 55.6%, specificity 98.6%, PPV+ 50%, NPV - 98.9%. The following test characteristics for chromosomal aberration markers (correlation to BPD) were observed: NB - sensitivity 68.4%, specificity 97.4%, PPV+ 56.5%, NPV - 98.4%; NT - sensitivity 73.9%, specificity 97.9%, PPV+ 54.8%, NPV- 99.2%; NT + NB - sensitivity 94.7%, specificity 98.9%, PPV+ 90%, NPV - 99.7%, respectively. The "genetic sonogram" protocol for the structural defect detection was analysed: sensitivity was 80%, specificity 100%, PPV+ 100%, NPV - 99.7%. It is concluded that the new biometric parameter--nasal bone length (NB) and the corrected one--nuchal translucency thickness (NT) are useful markers for fetal abnormalities, especially for chromosomal aberrations. High predictive values of the diagnostic schema for the detection of aneuploidies and structural defects indicate that its application in correlation with the biparietal diameter (BPD) is highly recommended. The proposed schema is an effective algorithm for prenatal diagnostics characterised by high prognostic values. The possible introduction of the schema could result in a decrease of the invasive procedure rates, which could minimise the rate of miscarriages as a complication of amniocenteses. [Abstract/Link to Full Text]

Krzemie? G, Roszkowska-Blaim M, Kostro I, Wojnar J, Karpi?ska M, Sekowska R
Urological anomalies in children with renal agenesis or multicystic dysplastic kidney.
J Appl Genet. 2006;47(2):171-6.
This study aimed to determine the frequency of associated urological abnormalities in children with unilateral renal agenesis (RA) or multicystic dysplastic kidney (MCDK). In total, 38 children (10 girls, 28 boys) were studied: 21 with RA and 17 with MCDK. In 14 children (37%) anomalies of the urinary tract were suspected prenatally in ultrasound studies. In the remaining 24 children the diagnosis of RA/MCDK was made postnatally: in 13 (34%) in the first 7 days of life, in 11 (29%) at the age of 8 days to 34 months, mean 10.6+/-8.05 months. Voiding cystourethrography was done in 36 (95%) children, the isotopic 99mTc-EC/DMSA scan of the kidney in 29 (67%), and urography in 8. Urological anomalies were present in 11 (29%) children: in 7 (33%) with RA and in 4 (24%) with MCDK. Vesicoureteral reflux was diagnosed in 8 children: grade II in 4, III in 3, and IV in 1 (in 1 child to duplicated, in 1 to ectopic kidney); ureterovesical junction obstruction in 2 (9.5%); and ureteropelvic junction obstruction in 1 (4.8%). Among them, 2 children demanded surgery on the contralateral urinary tract: pyeloplasty in 1, antireflux procedure in 1; while 9 children were treated conservatively. Compensatory hypertrophy of the contralateral kidney was found in 90% of children. Thus due to an increased risk of pathological changes in the single functioning kidney, lifelong nephrological care is recommended in patients with unilateral RA/MCDK. [Abstract/Link to Full Text]

Goc B, Walencka Z, W?och A, Wojciechowska E, Wiecek-W?odarska D, Krzystolik-?adzi?ska J, Bober K, Swietli?ski J
Trisomy 18 in neonates: prenatal diagnosis, clinical features, therapeutic dilemmas and outcome.
J Appl Genet. 2006;47(2):165-70.
The study aimed to analyse the clinical courses of aggressively treated neonates with cytogenetically confirmed trisomy 18, with special attention focused on the efficiency of prenatal diagnostics, associated malformations, therapeutic dilemmas and outcomes. We investigated retrospectively the data concerning 20 neonates with trisomy 18, admitted to the Neonatal Intensive Care Unit (NICU) in Katowice between January 2000 and February 2005. Their birth weights ranged from 650 g to 2400 g, mean 1812 g; gestational age ranged from 27 to 42 weeks, median 38 weeks. Intrauterine growth retardation was noticed in 90% of neonates. Trisomy 18 was suspected prenatally in 40% of cases. Most (80%) of newborns were delivered by caesarean section (92% of neonates with prenatally unrecognized chromosomal defects, 62% of neonates with trisomy 18 suspicion) and 70% of infants needed respiratory support immediately after birth. Cardiac defects were present in 95%, central nervous system malformations in 65%, severe anomalies of digestive system or abdominal wall in 25% of patients. Nine surgical operations were performed during hospitalization (4 were palliative cardiac surgeries). Six patients (30%) survived the neonatal period and were discharged from the NICU. The median survival of the neonates who died was 20 days. In 4 cases cardiac problems implicated their death; in others, deaths were attributed to multiorgan failure, prematurity and/or infection. Further improvement of efficiency of prenatal ultrasound screening for diagnosis of trisomy 18 in the fetus is necessary. A lack of prenatal diagnosis of trisomy 18 in the fetus results in a high rate of unnecessary caesarean sections in these pregnancies. Despite the aggressive treatment most neonates with trisomy 18 died during the neonatal period. The majority of deaths were attributed to cardiorespiratory and multiorgan failure. Concerning the poor prognosis, prompt karyotyping (using FISH) of clinically suspected trisomy 18 is very important, because many invasive procedures and surgeries may then be avoided. [Abstract/Link to Full Text]

Recent Articles in Genetics and Molecular Research

Perna SJ, Cardoso GL, Guerreiro JF
Duffy blood group genotypes among African-Brazilian communities of the Amazon region.
Genet Mol Res. 2007;6(1):166-72.
Duffy blood group genotype was studied in 95 unrelated subjects from four African-Brazilian communities of the Amazon region: Trombetas, Pitimandeua, Curia�, and Mazag�o Velho. Genotyping was performed using an allele-specific primer polymerase chain reaction technique for determining the three major alleles at FY blood group, and as expected, FY*O allele was the most common one, with frequencies ranging from 56.4% in Mazag�o Velho to 72.2% in Pitimandeua, whereas the FY*O/FY*O genotype was found with frequencies between 32.3% in Mazag�o Velho and 58.8% in Curia�. Genotype and allele distributions in the four Amazonian communities are consistent with a predominantly African origin with some degree of local differentiation and admixture with people of Caucasian ancestry and/or Amerindians. These results reveal that the impact of the FY*O/FY*O genotype on the transmission and endemicity of the vivax malaria deserves to be investigated in full detail in an attempt to identify the contribution of host biological factors and explain the non-homogeneous prevalence of malaria in the region expressed by its different levels of exposure. [Abstract/Link to Full Text]

Frigieri MC, Thompson GM, Pandolfi JR, Zanelli CF, Valentini SR
Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae.
Genet Mol Res. 2007;6(1):152-65.
The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking. [Abstract/Link to Full Text]

Rinc�n G, Young AE, Bannasch DL, Medrano JF
Characterization of variation in the canine suppressor of cytokine signaling-2 (SOCS2) gene.
Genet Mol Res. 2007;6(1):144-51.
Suppressor of cytokine signaling 2 (SOCS2) is a negative regulator of growth hormone signaling. The deletion of SOCS2 in mice results in a 30-50% increase in post-natal growth. In an effort to identify polymorphisms in the SOCS2 gene that may be associated with body size in dogs, we characterized the canine SOCS2 gene and analyzed its genetic diversity among small and large dog breeds. The study was carried out on a total of 520 dogs from 66 different breeds. Dogs were classified as large or small based on height and weight as determined by their respective American Kennel Club breed standards. The SH2 and SOCS domains of the canine SOCS2 gene were sequenced in 32 dogs from different breeds. Only one non-synonymous sequence variant (DQ415457:g.326G>T) was detected which corresponds to an amino acid change (Asp127Tyr). All samples were genotyped by PCR/RFLP and the allele frequencies were determined for each dog breed. The T allele was distributed primarily among European large dog breeds with a gene frequency ranging from 0.72 to 0.04. The nature of the nucleotide change and the effect on the protein together with the finding of a QTL related to body size in the same CFA15 region by other researchers suggest canine SOCS2 as a potential candidate gene for body size in dogs. Future studies will be needed to clarify the role of the 326G>T polymorphism and its interaction with genes like growth hormone and insulin-like growth factor 1. [Abstract/Link to Full Text]

Ricci GL, Silva N, Pagliarini MS, Scapim CA
B chromosomes in popcorn (Zea mays L.).
Genet Mol Res. 2007;6(1):137-43.
Cytological analysis of microsporogenesis in 72 popcorn plants, comprising nine from the original population (CMS-43, S(0)) and 63 from seven cycles of self-fertilization (S(1) to S(7)), one plant of S(0) generation (plant 2) was identified with B chromosomes. The number of B chromosomes varied from two to three in the same anther. The pattern of chromosome pairing and meiotic behavior of Bs were similar to those found in other plant species. The presence of B chromosomes did not affect chiasma frequency and chiasma distribution in A chromosomes. This is the first report of B chromosomes in popcorn. [Abstract/Link to Full Text]

Appio KT, Weber LI
Temporal genetic differentiation in cultured and natural beds of the brown mussel Perna perna (Mytilidae).
Genet Mol Res. 2007;6(1):127-36.
Perna perna is the most important cultivated mussel of Santa Catarina, Brazil, sustaining an important economic input for many local families. Natural stocks of P. perna are depleted by the extraction of adults and seeds for consumption and culture. The aim of the present study was to use the microsatellite locus pms-2 to study the variation of the genetic composition and diversity between natural and cultured stocks in samples of 2001 and 2005 from Penha, Santa Catarina. DNA was extracted from adductor muscle by Chelex/proteinase-K and phenol/chloroform protocols. Amplification by polymerase chain reaction was performed using specific primers for analyzing the pms-2 locus. Polymerase chain reaction products were submitted to vertical denatured 6% polyacrylamide gel electrophoresis and horizontal 2% agarose gel electrophoresis, and visualized by silver staining and ethidium bromide, respectively. Allele diversity and heterozygote deficiency were higher for samples of 2005 than for those of 2001. No significant genetic differentiation was found between natural and cultured stocks of 2001 by the chi(2) test, but G(2) (likelihood ratio) detected slight differences (I = 0.949; chi(2), P = 0.147; G(2), P = 0.046), while cultured and natural stocks of 2005 were very different (I = 0.798, P = 0.006). Between the years of 2001 and 2005, a large change in genetic composition was observed (I = 0.582; P < 0.001). Although nothing is known about natural changes in the genetic composition of this species with time, the results suggest a strong impact of human activities on natural stocks of P. perna, which is expected to be related to heavy extraction and farming. [Abstract/Link to Full Text]

Maffei EM, Pompolo SG
Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae).
Genet Mol Res. 2007;6(1):122-6.
The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment. [Abstract/Link to Full Text]

Nassar N, Vizzotto CS, Schwartz CA, Pires OR
Cassava diversity in Brazil: the case of carotenoid-rich landraces.
Genet Mol Res. 2007;6(1):116-21.
In Brazil, the center of cassava origin, cassava landraces have acquired through their domestication a large diversity in relation to many economic traits such as high content of carotenoids and excellent palatability among other characters. One of these clones, which has been grown by indigenous Brazilian farmers and is now being maintained in the University of Bras�lia gene bank, showed a high level of lycopene content (5 mg/kg viz. a viz. zero in common cultivars, and 12-20 mg/kg in tomato, a lycopene-rich vegetable). A second landrace called UnB 400 had a high content of beta-carotene, which reached 4 mg/kg. [Abstract/Link to Full Text]

Kashiwabara AY, Vieira DC, Machado-Lima A, Durham AM
Splice site prediction using stochastic regular grammars.
Genet Mol Res. 2007;6(1):105-15.
This paper presents a novel approach to the problem of splice site prediction, by applying stochastic grammar inference. We used four grammar inference algorithms to infer 1465 grammars, and used 10-fold cross-validation to select the best grammar for each algorithm. The corresponding grammars were embedded into a classifier and used to run splice site prediction and compare the results with those of NNSPLICE, the predictor used by the Genie gene finder. We indicate possible paths to improve this performance by using Sakakibara's windowing technique to find probability thresholds that will lower false-positive predictions. [Abstract/Link to Full Text]

Martins CF, Dode MN, B�o SN, Rumpf R
The use of the acridine orange test and the TUNEL assay to assess the integrity of freeze-dried bovine spermatozoa DNA.
Genet Mol Res. 2007;6(1):94-104.
The ability to detect nuclear damage is an important tool for the development of sperm preservation methods. We used the acridine orange test (AOT) and the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) assay to assess the DNA status of sperm cells preserved with different lyophilization media. The AOT did not detect any differences between different lyophilization media. However, differences in DNA integrity were observed among treatments with the TUNEL assay, suggesting that TUNEL is a more sensitive method to evaluate sperm DNA. The use of TCM 199 and 10% FCS as a lyophilization medium resulted in 14% of the cells with DNA fragmentation in TUNEL test. The AOT indicated only 4% of the cells with chromatin damage, with this same treatment, with no significant differences when compared to the other treatments. The degree of DNA fragmentation was negatively related to fertilizing potential, as sperm DNA damage was inversely correlated with pro-nucleus formation. The TUNEL assay was found to be an efficient method to detect DNA damage in sperm, and it could be used as a tool to predict male fertility. [Abstract/Link to Full Text]

Al-Seaf A, Keown JF, Van Vleck LD
Impact of bovine somatotropin on ranking for genetic value of dairy sires for milk yield traits and somatic cell score.
Genet Mol Res. 2007;6(1):79-93.
Records of Holstein cows were used to examine how different models account for the effect of bovine somatotropin (bST) treatment on genetic evaluation of dairy sires for yield traits and somatic cell score. Data set 1 included 65,720 first-lactation records. Set 2 included 50,644 second-lactation records. Set 3 included 45,505 records for lactations three, four and five. Estimated breeding values (EBV) of sires were with three different animal models. With Model 1, bST administration was ignored. With Model 2, bST administration was used as a fixed effect. With Model 3, administration of bST was used to define the contemporary group (herd-year-month of calving-bST). Correlations for EBV of 1,366 sires with treated daughters between pairs of the three models were calculated for milk, fat and protein yields and somatic cell score for the three data sets. Correlations for EBV of sires between pairs of models for all traits ranged from 0.971 to 0.999. Fractions of sires with bST-treated progeny selected in common (top 10 to 15%) were 0.94 and usually greater for all pairs of models for all traits and parities. For this study, the method of statistical adjustment for bST treatment resulted in a negligible effect on genetic evaluations of sires when some daughters were treated with bST and suggests that selection of sires to produce the next generation of sires and cows might not be significantly affected by how the effect of bST is modeled for prediction of breeding values for milk, fat and protein yields and somatic cell score. [Abstract/Link to Full Text]

Al-Seaf A, Keown JF, Van Vleck LD
Estimates of correlations among yield traits and somatic cell score with different models to adjust for bovine somatotropin effects on Holstein dairy cows.
Genet Mol Res. 2007;6(1):67-78.
Records of Holstein cows from the Dairy Records Processing Center at Raleigh, NC were edited to obtain three data sets: 65,720 first, 50,694 second, and 65,445 later lactations. Correlations among yield traits and somatic cell score were estimated with three different models: 1) bovine somatotropin (bST) administration ignored, 2) bST administration as a fixed effect and 3) administration of bST as part of the contemporary group (herd-year-month-bST). Heritability estimates ranged from 0.13 to 0.17 for milk, 0.12 to 0.20 for fat, 0.14 to 0.16 for protein yields, and 0.08 to 0.09 for somatic cell score. Estimates were less for later than first lactations. Estimates of genetic correlations among yields ranged from 0.35 to 0.85 with no important differences between estimates with the 3 models. Estimates for lactation 2 agreed with estimates for lactation 1. Estimates of genetic correlations for later lactations were generally greater than for lactations 1 and 2 except between milk and protein yields. Estimates of genetic correlations between yields and somatic cell score were mostly negative or small (-0.45 to 0.11). Estimates of environmental correlations among yield traits were similar with all models (0.77 to 0.97). Estimates of environmental correlations between yields and somatic cell score were negative (-0.22 to -0.14). Estimates of phenotypic correlations among yield traits ranged from 0.70 to 0.95. Estimates of phenotypic correlations between yields and somatic cell score were small and negative. For all three data sets and all traits, no important differences in estimates of genetic parameters were found for the two models that adjusted for bST and the model that did not. [Abstract/Link to Full Text]

Carvalho FM, Wolfgramm EV, Degasperi I, Verbeno BM, Vianna BA, Chagas FF, Perroni AM, Paula F, Louro ID
Molecular cytogenetic analysis of a ring-Y infertile male patient.
Genet Mol Res. 2007;6(1):59-66.
In the present study, we report on the case of a 43-year-old male patient seeking for fertility assistance, who showed a seminal analysis and testicular biopsy of complete azoospermia. Peripheral blood culture for chromosome studies revealed a karyotype of 46 chromosomes with a ring-Y-chromosome that lost the long arm heterochromatin. Molecular analysis of genomic DNA from the patient detected the presence of the sex-determining region of the Y-chromosome (SRY) but the complete absence of regions involved in spermatogenesis (AZFa, AZFb, AZFc). Several molecular markers distributed along the Y-chromosome were tested through PCR amplification, and a breakpoint was established close to the centromere, predicting the deletion of the growth control region, in agreement with the short stature observed in this patient. All results obtained through molecular cytogenetic characterization are in accordance with the clinical features observed in this patient. [Abstract/Link to Full Text]

Howell WM, Keller GE, Kirkpatrick JD, Jenkins RL, Hunsinger RN, McLaughlin EW
Effects of the plant steroidal hormone, 24-epibrassinolide, on the mitotic index and growth of onion (Allium cepa) root tips.
Genet Mol Res. 2007;6(1):50-8.
The purpose of the present study was to determine the effects of the steroidal plant hormone, 24-epibrassinolide (BL), on the mitotic index and growth of onion (Allium cepa) root tips. The classical Allium test was used to gather and quantify data on the rate of root growth, the stages of mitosis, and the number of mitoses in control and BL-treated groups of onions. Low doses of BL (0.005 ppm) nearly doubled the mean root length and the number of mitoses over that of controls. Intermediate doses of BL (0.05 ppm) also produced mean root lengths and number of mitoses that were significantly greater than those of the controls. The highest dose of BL (0.5 ppm) produced mean root lengths and number of mitoses that were less than control values, but the differences were not statistically significant. Examination of longitudinally sectioned root tips produced relatively similar results. This study confirms the suppositions of previous authors who have claimed that exogenously applied BL can increase the number of mitoses in plants, but failed to show cytogenetic data. This is the first report detailing the effects of BL on chromosomes and the cell cycle. [Abstract/Link to Full Text]

G�m�?-Akay G, Unal AE, Bayar S, Karadayi K, Elhan AH, Sunguro?lu A, T�k�n A
Telomerase activity could be used as a marker for neoplastic transformation in gastric adenocarcinoma: but it does not have a prognostic significance.
Genet Mol Res. 2007;6(1):41-9.
Telomerase activity is responsible for telomere maintenance and is believed to be crucial in most immortal cells and cancer cells; however, its clinicopathological significance in gastric cancer remains to be clarified. The aim of the present study was to assess whether malignant progression of gastric adenocarcinoma correlates with telomerase activity. We also investigated the correlation between telomerase activity and histopathological findings. We examined telomerase activity in tumor specimens and adjacent normal tissues from 43 patients with gastric adenocarcinoma. Telomerase activity was measured quantitatively by the TRAPEZE Gel Based Telomerase Detection Kit. Approximately 98% of the tumor tissues were telomerase positive, but telomerase activity was detected not only in tumor tissues but also in normal gastric mucosa. Although telomerase activity was found to be higher in tumor samples than normal tissue for each subject, we could not find a general cut-off level for telomerase activity in gastric adenocarcinoma. In addition, telomerase activity was not correlated with tumor invasion, lymph node involvement and histological stage. Our results support the idea that telomerase reactivation is a common event in gastric adenocarcinoma and it is not related to histopathological parameters. Since it is difficult to set a cut-off level for this type of cancer, we suggest that the prognostic utility of telomerase assay has not yet reached the clinic in terms of predicting outcome for patients with gastric adenocarcinoma. For the assessment of gastric carcinoma, telomerase activity should be evaluated in both tumor and normal tissues, because normal gastric mucosa samples show appreciable telomerase activity. [Abstract/Link to Full Text]

Souza HV, Bicudo HE, Itoyama MM
Study of chromosomal and nucleolar aspects in testes of Nysius californicus (Heteroptera: Lygaeidae).
Genet Mol Res. 2007;6(1):33-40.
In Nysius californicus (family Lygaeidae, subfamily Orsillinae), a pest commonly known as the seed bug, the chromosome complement is 2n = 16 (12A + 2m + XY), testes are formed by seven seminiferous tubules covered by an orange-colored membrane, and spermatogenesis is cystic. At prophase, sex chromosomes are heteropycnotic and autosomes usually show a chiasma. At metaphase, sex chromosomes along with microchromosomes may be seen located at the center of a ring formed by the remaining autosomes. A characteristic specific of N. californicus was the presence of nucleolar material observed from the cystic cell to the completely differentiated spermatozoon. Variations in size, shape and location of the nucleolar material occur during this process, denoting a variable degree of activity in the different stages. [Abstract/Link to Full Text]

Martins WF, Ayres CF, Lucena WA
Genetic diversity of Brazilian natural populations of Anthonomus grandis Boheman (Coleoptera: Curculionidae), the major cotton pest in the New World.
Genet Mol Res. 2007;6(1):23-32.
Twenty-five RAPD loci and 6 isozyme loci were studied to characterize the genetic variability of natural populations of Anthonomus grandis from two agroecosystems of Brazil. The random-amplified polymorphic DNA data disclosed a polymorphism that varied from 52 to 84% and a heterozygosity of 0.189 to 0.347. The index of genetic differentiation (GST) among the six populations was 0.258. The analysis of isozymes showed a polymorphism and a heterozygosity ranging from 25 to 100% and 0.174 to 0.277, respectively. The genetic differentiation (FST) among the populations obtained by isozyme data was 0.544. It was possible to observe rare alleles in the populations from the Northeast region. The markers examined allowed us to distinguish populations from large-scale, intensive farming region (cotton belts) versus populations from areas of small-scale farming [Abstract/Link to Full Text]

Kawasaki PM, Kano FS, Vidotto O, Vidotto MC
Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paran� State, Brazil.
Genet Mol Res. 2007;6(1):15-22.
Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paran , Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post-immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis. [Abstract/Link to Full Text]

Barni GS, Strapazzon R, Guerra JC, Moretto G
Mitochondrial genome differences between the stingless bees Melipona rufiventris and Melipona mondury (Apidae: Meliponini).
Genet Mol Res. 2007;6(1):8-14.
Within the Meliponini, a widely distributed group of stingless bees, Melipona rufiventris has been considered as a single, cohesive species. Recently, analysis of morphological characters led to the splitting of this species into two species, M. mondury and M. rufiventris. The former occurs in the Atlantic Rain Forest ranging from Santa Catarina to Bahia States, while the latter is found in other parts of Brazil. We used PCR + RFLP to identify genetic marker patterns of the mtDNA between these species. Nine mtDNA regions were amplified and digested with four restriction enzymes (EcoRI, EcoRV, HindIII, and HinfI). Six species-specific restriction sites were identified for M. mondury and M. rufiventris with all enzymes, except for HindIII. The molecular data agree with the morphological classification. [Abstract/Link to Full Text]

Kochi C, Longui CA, Lemos-Marini SH, Guerra-Junior G, Melo MB, Calliari LE, Monte O
The influence of parental origin of X chromosome genes on the stature of patients with 45 X Turner syndrome.
Genet Mol Res. 2007;6(1):1-7.
Thirty-seven 45 X Turner syndrome patients with confirmed peripheral blood lymphocyte karyotype were initially selected to determine the origin of the retained X chromosome and to correlate it with their parents' stature. Blood samples were available in 25 families. The parental origin of the X chromosome was determined in 24 informative families through the analysis of the exon 1-CAG repeat variation of the androgen receptor gene. In 70.8% of the cases, the retained X chromosome was maternal in origin and 29.2% was paternal. When we classified the patients according to maternal (Xm) or paternal (Xp) X chromosome, there was a positive correlation between patients' and maternal heights only in the Xm group. There was no correlation with paternal height in either group, and a significant correlation with target height was only observed in the Xm group. In conclusion, maternal height is the best variable correlating with the height of 45 X Turner syndrome patients who retain the maternal X chromosome, suggesting a strong influence of genes located on the maternal X chromosome on stature. [Abstract/Link to Full Text]

Carvalho PC, Freitas SS, Lima AB, Barros M, Bittencourt I, Degrave W, Cordovil I, Fonseca R, Carvalho MG, Moura Neto RS, Cabello PH
Personalized diagnosis by cached solutions with hypertension as a study model.
Genet Mol Res. 2006;5(4):856-67.
Statistical modeling of links between genetic profiles with environmental and clinical data to aid in medical diagnosis is a challenge. Here, we present a computational approach for rapidly selecting important clinical data to assist in medical decisions based on personalized genetic profiles. What could take hours or days of computing is available on-the-fly, making this strategy feasible to implement as a routine without demanding great computing power. The key to rapidly obtaining an optimal/nearly optimal mathematical function that can evaluate the "disease stage" by combining information of genetic profiles with personal clinical data is done by querying a precomputed solution database. The database is previously generated by a new hybrid feature selection method that makes use of support vector machines, recursive feature elimination and random sub-space search. Here, to evaluate the method, data from polymorphisms in the renin-angiotensin-aldosterone system genes together with clinical data were obtained from patients with hypertension and control subjects. The disease "risk" was determined by classifying the patients' data with a support vector machine model based on the optimized feature; then measuring the Euclidean distance to the hyperplane decision function. Our results showed the association of renin-angiotensin-aldosterone system gene haplotypes with hypertension. The association of polymorphism patterns with different ethnic groups was also tracked by the feature selection process. A demonstration of this method is also available online on the project's web site. [Abstract/Link to Full Text]

Melo SC, Pungartnik C, Cascardo JC, Brendel M
Rapid and efficient protocol for DNA extraction and molecular identification of the basidiomycete Crinipellis perniciosa.
Genet Mol Res. 2006;5(4):851-5.
DNA isolation from some fungal organisms is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. Beginning with a yeast Saccharomyces cerevisiae genomic DNA isolation method, we developed a 30-min DNA isolation protocol for filamentous fungi by combining cell wall digestion with cell disruption by glass beads. High-quality DNA was isolated with good yield from the hyphae of Crinipellis perniciosa, which causes witches' broom disease in cacao, from three other filamentous fungi, Lentinus edodes, Agaricus blazei, Trichoderma stromaticum, and from the yeast S. cerevisiae. Genomic DNA was suitable for PCR of specific actin primers of C. perniciosa, allowing it to be differentiated from fungal contaminants, including its natural competitor, T. stromaticum. [Abstract/Link to Full Text]

Rodrigues FM, Telles MP, Resende LV, Soares TN, Diniz-Filho JA, J�como AT, Silveira L
Transferability of short tandem repeat markers for two wild Canid species inhabiting the Brazilian Cerrado.
Genet Mol Res. 2006;5(4):846-50.
The maned wolf (Chrysocyon brachyurus) and the crab-eating fox (Cerdocyon thous) are two wild-canid species found in the Brazilian Cerrado. We tested cross-amplification and transferability of 29 short tandem repeat primers originally developed for cattle and domestic dogs and cats on 38 individuals of each of these two species, collected in the Emas National Park, which is the largest national park in the Cerrado region. Six of these primers were successfully transferred (CSSM-038, PEZ-05, PEZ-12, LOCO-13, LOCO-15, and PEZ-20); five of which were found to be polymorphic. Genetic parameter values (number of alleles per locus, observed and expected heterozygosities, and fixation indices) were within the expected range reported for canid populations worldwide. [Abstract/Link to Full Text]

Risso-Pascotto C, Pagliarini MS, Valle CB
Microsporogenesis in Brachiaria dictyoneura (Fig. & De Not.) Stapf (Poaceae: Paniceae).
Genet Mol Res. 2006;5(4):837-45.
Microsporogenesis was analyzed in five accessions of Brachiaria dictyoneura presenting x = 6 as the basic chromosome number. All accessions were tetraploid (2n = 4x = 24) with chromosome pairing in bi-, tri-, and quadrivalents. The recorded meiotic abnormalities were those typical of polyploids, including precocious chromosome migration to the poles, laggard chromosomes, and micronucleus formation. The frequency of these abnormalities, however, was lower than those reported for other polyploid accessions previously analyzed for other Brachiaria species. Cell fusion and absence of cytokinesis were also recorded in some accessions, leading to restitutional nucleus formation in some cells. Genetically unbalanced microspores, binucleate, and 2n microspores were found among normal meiotic products as results from these abnormalities. The limitation in using these accessions as pollen donor in interspecific crosses with sexual species with x = 7 or x = 9 in breeding programs is discussed. [Abstract/Link to Full Text]

Horimoto AR, Ferraz JB, Balieiro JC, Eler JP
Estimation of genetic parameters for a new model for defining body structure scores (frame scores) in Nellore cattle.
Genet Mol Res. 2006;5(4):828-36.
Models for estimation of frame scores in Nellore beef cattle (FRAME_GMA) were developed, comparing them with frame scores estimated using equations proposed by the Beef Improvement Federation (FRAME_BIF, USA). Correlation among frame scores obtained by these two methodologies, along with the independent variables considered in the estimation models, were also studied. A data set with 12,728 animals, with ages between 490 and 610 days, was used. The models that best adjusted to FRAME_GMA included hip height, weight and interaction between height and weight. Estimates of heritability for FRAME_GMA and FRAME_BIF were 0.26 +/- 0.03 and 0.23 +/- 0.03, respectively, in single trait analysis, and 0.28 and 0.24, respectively, in multi-trait analysis. Phenotypic Pearson and Spearman correlation coefficients between FRAME_GMA and FRAME_BIF for males were 0.87 and 0.83, respectively, being lower than those found for females (0.92 for both coefficients). Genetic correlation between the frame scores did not differ between genders, with values of 0.92 for the Pearson coefficient and 0.91 for the Spearman coefficient. We concluded that FRAME_GMA was better adapted to this data set than FRAME_BIF. Other studies need to be made to evaluate the applicability of this proposed model to other populations of Nellore beef cattle and for other age groups. [Abstract/Link to Full Text]

Trivedi S, Gehlot HS, Rao SR
Protein thermostability in Archaea and Eubacteria.
Genet Mol Res. 2006;5(4):816-27.
In order to survive at high temperatures, thermophilic prokaryotes (Archaea and Eubacteria) adopt different strategies. Among several important contributing factors for stability of proteins are CG-rich codons, the ratio of charged amino acids compared to uncharged amino acids, ionic interactions, amino acid preferences and their distribution, post-translational modifications, and solute accumulation. However, these factors may differ from taxon to taxon, both within and between species depending upon the composition of proteins found in these organisms. This is exemplified in the case of differences in strategies adopted by soluble proteins and membrane proteins. Therefore, it appears that no single factor or combination of factors together can be universally attributed to the provision of thermal stability in proteins. [Abstract/Link to Full Text]

Moreira PR, Morielle-Versute E
Genetic variability in species of bats revealed by RAPD analysis.
Genet Mol Res. 2006;5(4):804-15.
Random amplified polymorphic DNA molecular marker was utilized as a means of analyzing genetic variability in seven bat species: Molossus molossus, M. rufus, Eumops glaucinus, E. perotis, Myotis nigricans, Eptesicus furinalis, and Artibeus planirostris. The determination of genetic diversity was based on 741 bands produced by a 20-random primer set. Only eight bands were considered monomorphic to one species. The greatest number of bands and the most polymorphic condition were exhibited by M. molossus, followed by M. nigricans, A. planirostris, E. furinalis, E. glaucinus, M. rufus, and E. perotis. Nei's genetic diversity index in the seven species considering the 20 primers was not greater than 0.22, but some primers were capable of detecting values between 0.39 and 0.49. Nei's unbiased genetic distance values and the UPGMA clustering pattern show that M. molossus and M. rufus have a close genetic relationship, unlike that observed between E. perotis and E. glaucinus. The latter was clustered with A. planirostris and E. furinalis. The low values for genetic diversity and distance observed indicate a genetic conservatism in the seven species. The fluorescent in situ hybridization experiments did not confirm a monomorphic condition for the eight bands identified, demonstrating that the monomorphic bands obtained by random amplified polymorphic DNA are insufficient for the identification of bat species. [Abstract/Link to Full Text]

Risso-Pascotto C, Mendes DV, Silva N, Pagliarini MS, Valle CB
Evidence of allopolyploidy in Brachiaria brizantha (Poaceae: Paniceae) through chromosome arrangement at metaphase plate during microsporogenesis.
Genet Mol Res. 2006;5(4):797-803.
In the hexaploid (2n = 6x = 54) accession B176 of Brachiaria brizantha, one cytological characteristic differentiated it from the other accessions previously analyzed with the same ploidy level. Nearly 40% of meiocytes displayed the chromosome set arranged at two metaphase plates at the poles of the cell, close to the membrane. In these cells, both metaphase plates were arranged in an angle to form a typical tripolar spindle. Therefore, cells did not show normal chromosome segregation at anaphase I. Only nine univalent chromosomes migrated from each plate to the opposite pole with the remainder staying immobile on the plate. As a result of such spindle orientation and chromosome behavior, trinucleate telophases I were recorded. After telophase, cytokinesis eliminated the small nuclei into a microcyte. The second division proceeded normally, with the presence of microcytes in all phases. The origin of such an abnormality was explained on the hexaploid level of the accession which could have resulted by chromosome doubling of a triploid derived from species that did not display the same behavior for spindle organization. The high percentage of meiotic abnormalities recorded in this accession compromises fertility and renders it inadequate for the breeding program. [Abstract/Link to Full Text]

Seno LO, Cardoso VL, Tonhati H
Responses to selection for milk traits in dairy buffaloes.
Genet Mol Res. 2006;5(4):790-6.
The aim of the present study was to estimate the index and individual responses to selection for milk (MY), fat (FY) and protein (PY) yields for different breeding goals for two commercial buffalo milk production systems in S�o Paulo State characterized by: 1) all milk produced is sold to the industry (MILK) and 2) all milk produced is used in the mozzarella cheese-making process at the farm (MOZZARELLA). The current payment policy is based exclusively on milk volume. The mozzarella price refers to the wholesale selling price. Index responses to selection (IR) were calculated for three different breeding goals (BG): 1) MY exclusively (BG(1)); 2) FY + PY (BG(2)) and 3) MY + FY + PY (BG(3)). IR for the MILK system were 41.79 US dollars (BG(1)), 5.91 US dollars (BG(2)) and 38.22 US dollars (BG(3)). For the MOZZARELLA system, IR were 179.50 US dollars (BG(1)), 262.85 US dollars (BG(2)) and 402.41 US dollars (BG(3)). The results suggest that for the present circumstances, selection for milk components is not advantageous when milk is produced for sale to the industry. However, when mozzarella making is added to the system, the selection for components and milk volume is the most economically beneficial. [Abstract/Link to Full Text]

Brinkrolf K, Brune I, Tauch A
Transcriptional regulation of catabolic pathways for aromatic compounds in Corynebacterium glutamicum.
Genet Mol Res. 2006;5(4):773-89.
Corynebacterium glutamicum is a gram-positive soil microorganism able to utilize a large variety of aromatic compounds as the sole carbon source. The corresponding catabolic routes are associated with multiple ring-fission dioxygenases and among other channeling reactions, include the gentisate pathway, the protocatechuate and catechol branches of the beta-ketoadipate pathway and two potential hydroxyquinol pathways. Genes encoding the enzymatic machinery for the bioconversion of aromatic compounds are organized in several clusters in the C. glutamicum genome. Expression of the gene clusters is under specific transcriptional control, apparently including eight DNA-binding proteins belonging to the AraC, IclR, LuxR, PadR, and TetR families of transcriptional regulators. Expression of the gentisate pathway involved in the utilization of 3-hydroxybenzoate and gentisate is positively regulated by an IclR-type activator. The metabolic channeling of ferulate, vanillin and vanillate into the protocatechuate branch of the beta-ketoadipate pathway is controlled by a PadR-like repressor. Regulatory proteins of the IclR and LuxR families participate in transcriptional regulation of the branches of the beta-ketoadipate pathway that are involved in the utilization of benzoate, 4-hydroxybenzoate and protocatechuate. The channeling of phenol into this pathway may be under positive transcriptional control by an AraC-type activator. One of the potential hydroxyquinol pathways of C. glutamicum is apparently repressed by a TetR-type regulator. This global analysis revealed that transcriptional regulation of aromatic compound utilization is mainly controlled by single regulatory proteins sensing the presence of aromatic compounds, thus representing single input motifs within the transcriptional regulatory network of C. glutamicum. [Abstract/Link to Full Text]

Trivedi S
Comparison of simple sequence repeats in 19 Archaea.
Genet Mol Res. 2006;5(4):741-72.
All organisms that have been studied until now have been found to have differential distribution of simple sequence repeats (SSRs), with more SSRs in intergenic than in coding sequences. SSR distribution was investigated in Archaea genomes where complete chromosome sequences of 19 Archaea were analyzed with the program SPUTNIK to find di- to penta-nucleotide repeats. The number of repeats was determined for the complete chromosome sequences and for the coding and non-coding sequences. Different from what has been found for other groups of organisms, there is an abundance of SSRs in coding regions of the genome of some Archaea. Dinucleotide repeats were rare and CG repeats were found in only two Archaea. In general, trinucleotide repeats are the most abundant SSR motifs; however, pentanucleotide repeats are abundant in some Archaea. Some of the tetranucleotide and pentanucleotide repeat motifs are organism specific. In general, repeats are short and CG-rich repeats are present in Archaea having a CG-rich genome. Among the 19 Archaea, SSR density was not correlated with genome size or with optimum growth temperature. Pentanucleotide density had an inverse correlation with the CG content of the genome. [Abstract/Link to Full Text]