Intrinsically bent DNA sites in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> third chromosome amplified domain

Bent DNA sites promote the curvature of DNA in both eukaryotic and prokaryotic chromosomes. Here, we investigate the localization and structure of intrinsically bent DNA sites in the extensively characterized Drosophila melanogaster third chromosome DAFC-66D segment (Drosophila amplicon in the follicle cells). This region contains the amplification control element ACE3, which is a replication enhancer that acts in cis to activate the major replication origin ori-β. Through both electrophoretic and in silico analysis, we have identified three major bent DNA sites in DAFC-66D. The bent DNA site (b1) is localized in the ACE3 element, whereas the other two bent DNA sites (b2 and b3) are localized in the ori-β region. Four additional bent DNA sites were identified in the intron of the S18 gene and near the TATA box of the S15, S19, and S16 genes. The identification of DNA bent sites in genomic regions previously characterized as functionally relevant for DNA amplification further supports a function for DNA bent sites in DNA replication in eukaryotes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome-scale reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of the <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> ATCC 824 metabolic network

To understand the metabolic characteristics of Clostridium acetobutylicum and to examine the potential for enhanced butanol production, we reconstructed the genome-scale metabolic network from its annotated genomic sequence and analyzed strategies to improve its butanol production. The generated reconstructed network consists of 502 reactions and 479 metabolites and was used as the basis for an in silico model that could compute metabolic and growth performance for comparison with fermentation data. The in silico model successfully predicted metabolic fluxes during the acidogenic phase using classical flux balance analysis. Nonlinear programming was used to predict metabolic fluxes during the solventogenic phase. In addition, essential genes were predicted via single gene deletion studies. This genome-scale in silico metabolic model of C. acetobutylicum should be useful for genome-wide metabolic analysis as well as strain development for improving production of biochemicals, including butanol. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. L. and H. Y. equally contributed to this work.     

Gene fusion in <Emphasis Type="Italic">Helicobacter pylori</Emphasis>: making the ends meet

Fusion genes have been reported as a means of enabling the development of novel or enhanced functions. In this report, we analyzed fusion genes in the genomes of two Helicobacter pylori strains (26695 and J99) and identified 32 fusion genes that are present as neighbours in one strain (components) and are fused in the second (composite), and vice-versa. The mechanism for each case of gene fusion is explored. 28 out of 32 genes identified as fusion products in this analysis were reported as essential genes in the previously documented transposon mutagenesis of H. pylori strain G27. This observation suggests the potential of the products of fusion genes as putative microbial drug targets. These results underscore the utility of bacterial genomic sequence comparisons for understanding gene evolution and for in silico drug target identification in the post-genomic era. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM-3.

Summary The genetic environment of plasmid-borne bla _TEM mutant genes, encoding nine distinct TEM-type extended-spectrum -lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine bla _TEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes bla _TEM-1 or _–2. A 6.6 kb DNA fragment of pCFF04 containing bla _TEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that bla _TEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was, in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with bla _TEM-3.     

Variability and genetics of spacer DNA sequences between the ribosomal-RNA genes of hexaploid wheat (Triticum aestivum)

Summary Using restriction enzyme digests of genomic DNA extracted from the leaves of 25 hexaploid wheat (Triticum aestivum L. em. Thell.) cultivars and their hybrids, restriction fragment length polymorphisms of the spacer DNA which separates the ribosomal-RNA genes have been examined. (From one to three thousand of these genes are borne on chromosomes 1B and 6B of hexaploid wheat). The data show that there are three distinct alleles of the 1B locus, designated Nor-B1a, Nor-B1b, and Nor-B1c, and at least five allelic variants of the 6B locus, designated Nor-B2a, Nor-B2b, Nor-B2c, Nor-B2d, and Nor-B2e. A further, previously reported allele on 6B has been named Nor-B2f. Chromosome 5D has only one allelic variant, Nor-D3. Whereas the major spacer variants of the 1B alleles apparently differ by the loss or gain of one or two of the 133 bp sub-repeat units within the spacer DNA, the 6B allelic variants show major differences in their compositions and lengths. This may be related to the greater number of rDNA repeat units at this locus. The practical implications of these differences and their application to wheat breeding are discussed.     

Unique pattern of <Emphasis Type="Italic">R-</Emphasis>gene variation within populations in Arabidopsis

An understanding of the variation pattern in disease resistance (R) genes is essential for its use in breeding programs aimed at neutralizing the threat of pathogens. Although the variation between populations is well known, there is little research about R-gene variation patterns within populations. Here, we investigate the polymorphism at three R-gene loci of 39 individual plants from nine populations of Arabidopsis thaliana. Our data suggest that alleles of each locus from individuals within a local population were either nearly identical, or highly diverse as ones between populations. The vast majority (92.5%) of within-population variation was shared globally, with high levels of allelic diversity (up to 11.7%) and abundant diverse-alleles. This unique pattern of within-population variation at R-loci suggests that individual plants within a population had the great potential to maintain a high level of globally-shared polymorphisms, and that the diversifying selection was the major force maintaining such polymorphisms. Consequently, the shared-polymorphism became recyclable for new R-genes, as the corresponding avirulence re-emerges in pathogen populations. Nucleotide sequence data reported are available in the GenBank databases under the accession numbers EF368054-EF368158.     

Strategies for performing genotype–phenotype association studies in nonhuman primates

Anthropoid primate models offer opportunities to study genetic influence on alcohol consumption and alcohol-related intermediate phenotypes in socially and behaviorally complex animal models that are closely related to humans, and in which functionally equivalent or orthologous genetic variants exist. This review will discuss the methods commonly used for performing candidate gene-based studies in rhesus macaques in order to model how functional genetic variation moderates risk for human psychiatric disorders. Various in silico and in vitro approaches to identifying functional genetic variants for performance of these studies will be discussed. Next, I will provide examples of how this approach can be used for performing candidate gene-based studies and for examining gene by environment interactions. Finally, these approaches will then be placed in the context of how function-guided studies can inform us of genetic variants that may be under selection across species, demonstrating how functional genetic variants that may have conferred selective advantage at some point in the evolutionary history of humans could increase risk for addictive disorders in modern society.     

Grain isozyme and ribosomal DNA variability in Hordeum spontaneum populations from Israel

Summary Grain isozyme and ribosomal DNA (rDNA) variability was examined in Hordeum spontaneum populations sampled from 27 geographical sites in Israel. Considerable phenotypic variability was observed with variants of ADH1, EST3, EST10, BMY1 and WSP detected, which are not available in the H. vulgare gene pool. Seven new rDNA phenotypes were detected in the H. spontaneum populations. Shannon's index of diversity was used to partition the total phenotypic variation into between and within population components. Most of the variation occurred between H. spontaneum populations. The distribution of both grain isozyme and rDNA phenotypes was non-random and correlated with a range of ecogeographical factors. In particular, the G phenotype of BMY1 was restricted to the Negev Desert and Dead Sea regions of Israel. Over 78% of the variation in the frequency of this particular phenotype could be explained by the number of rainy days per year and mean temperature in January. This suggests that variation at this locus or at loci linked to it may be of adaptive significance and of value in the introgression of genes controlling abiotic stress tolerance from H. spontaneum into the H. vulgare gene pool.     

Common genetic variation in eight genes of the <Emphasis Type="Italic">GH</Emphasis>/<Emphasis Type="Italic">IGF1</Emphasis> axis does not contribute to adult height variation

Stature (adult height) is one of the most heritable human traits, yet few genes, if any, have been convincingly associated with adult height variation in the general population. Here, we selected 150 tag SNPs from eight candidate genes in the growth hormone (GH)/insulin-like growth factor-1 (IGF1) axis (GHR, GHRH, GHRHR, IGF1, IGFALS, IGFBP3, JAK2, STAT5B), and genotyped them in ∼2,200 individuals ascertained for short or tall stature. Nominally significant tag SNPs were then tested in three additional replication cohorts, including a family-based panel to rule out spurious associations owing to population stratification. Across the four height cohorts (N = 6,075 individuals), we did not observe any consistent associations between stature and common variants (≥5% minor allele frequency) in these eight genes, including a common deletion of the growth hormone receptor gene exon 3. Tests of epistatic interactions between these genes did not yield any results beyond those expected by chance. Although we have not tested all genes in the GH/IGF1 axis, our results indicate that common variation in these GH/IGF1 axis genes is not a major determinant of stature, and suggest that if common variation contributes to adult height variation in the general population, the variants are in other, possibly unanticipated genes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tandem duplication of alkaline phosphatase genes and polymorphism in the intergenic sequence in<Emphasis Type="Italic"> Bombyx mori</Emphasis>

Alkaline phosphatases are ubiquitous in organisms from bacteria to human. Two alkaline phosphatase genes, Alp-m and Alp-s, were independently cloned from the silkworm Bombyx mori. They were mapped to a small DNA region and shown to be organized in tandem. Exon-intron structures of the two genes were highly conserved, with the exception of the second intron in Alp-m, which has no counterpart in Alp-s. The similarity between the nucleotide sequences of the exons of the two genes was strikingly high (60–79%), suggesting that Alp-m and Alp-s originated from a duplication of their common ancestor gene. The intergenic sequence between the two Alp genes shows length polymorphism in different B. mori strains, which can be explained by presence/absence of two putative insertion sequences. This structural variation suggests a possible scenario for the divergence of the two Alp genes after the duplication event.Communicated by G. Reuter     

Genus-specific distribution and pathovar-specific variation of the glycinecin R gene homologs in <Emphasis Type="Italic">Xanthomonas</Emphasis> genomes

Xanthomonas axonopodis pv. glycines produces bacteriocins called glycinecin, and two glycinecin genes, glyA and glyR, were reported previously. In this paper, we describe genomic distribution and variation of the glyR gene revealed by extensive Southern hybridization analysis. In contrast to the glyA gene present only in X. axonopodis pv. glycines, the glyR gene was found to be distributed widely in all the pathovars of Xanthomas genus. It was also found that the glyR gene is a multigene family while the glyA is a single copy gene. Moreover, the copy number and the variation of the glyR multigene are unique to each pathovar of Xanthomonas. The uniqueness can be easily detected by the patterns resulted from Southern hybridization using the genomic digests. Thus, we suggest the glyR gene can serve as a useful genus-specific and pathovar-specific DNA marker for Xanthomonas. One of the glyR homologs was further isolated from X. axonopodis pv. glycines, and analyzed to be functional with strong inhibitory activity against several members of Xanthomonas.     

BAHD superfamily of acyl-CoA dependent acyltransferases in <Emphasis Type="Italic">Populus</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis>: bioinformatics and gene expression

Plant acyl-CoA dependent acyltransferases constitute a large specific protein superfamily, named BAHD. Using the conserved sequence motifs of BAHD members, we searched the genome sequences of Populus and Arabidopsis, and identified, respectively, 94- and 61-putative genes. Subsequently, we analyzed the phylogeny, gene structure, and chromosomal distribution of BAHD members of both species; then, we profiled expression patterns of BAHD genes by “in silico” northern- and microarray-analyses based on public databases, and by RT-PCR. While our genomic- and bioinformatic- analyses provided full sets of BAHD superfamily genes, and cleaned up a few existing annotation errors, importantly it led to our recognizing several unique Arabidopsis BAHD genes that inversely overlapped with their neighboring genes on the genome, and disclosing a potential natural anti-sense regulation for gene expressions. Systemic gene-expression profiling of BAHD members revealed distinct tissue-specific/preferential expression patterns, indicating their diverse biological functions. Our study affords a strong knowledge base for understanding BAHD members’ evolutionary relationships and gene functions implicated in plant growth, development and metabolism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structure,diversity and evolutionary patterns of expressed MHC class II<Emphasis Type="Italic">B</Emphasis> genes in chub (<Emphasis Type="Italic">Squalius cephalus</Emphasis>), a cyprinid fish species from Europe

The polymorphism of exon 2 of the DAB genes (major histocompatibility complex [MHC] class IIB) was investigated for the first time in the freshwater cyprinid fish species, Squalius cephalus, in the wide range of its distribution in Europe. We identified 111 different MHC class IIB variants in 15 chub populations distributed from Finland to Spain. The sequence analysis showed that many structurally important amino acid sites that were conserved among tetrapods were also conserved in chub. The analysis of recombination indicated that it does not play an important role in producing and maintaining the variation of DAB genes analyzed in the present study. The exon 2 was shown to be subjected to intense positive selection. Phylogenetic analysis and sequence identities suggest the presence of two class IIB loci (DAB1-like and DAB3-like) in chub. Nevertheless, the presence of three DAB3-like sequence variants in several individuals indicates the duplication of the DAB3 gene. A contrasting selection pattern was found in DAB1-like and DAB3-like genes, which suggests the potential functional differences between these genes. Some DAB sequence variants were shared among the populations of different mtDNA lineages. The phylogenetic analyses did not confirm any biogeographical pattern of the genetic structure of MHC IIB in chub, which is in line with balancing selection and trans-species polymorphism in MHC genes. Nevertheless, cluster analysis based on the presence/absence of DAB sequence variants in the populations showed the phylogeophraphical pattern corresponding to the mtDNA lineages, which indicates that neutral selection can partially explain the MHC IIB evolution in chub.     

Lessons from the deep: mechanisms behind diversification of eukaryotic protein complexes

Genetic variation is the major mechanism behind adaptation and evolutionary change. As most proteins operate through interactions with other proteins, changes in protein complex composition and subunit sequence provide potentially new functions. Comparative genomics can reveal expansions, losses and sequence divergence within protein-coding genes, but in silico analysis cannot detect subunit substitutions or replacements of entire protein complexes. Insights into these fundamental evolutionary processes require broad and extensive comparative analyses, from both in silico and experimental evidence. Here, we combine data from both approaches and consider the gamut of possible protein complex compositional changes that arise during evolution, citing examples of complete conservation to partial and total replacement by functional analogues. We focus in part on complexes in trypanosomes as they represent one of the better studied non-animal/non-fungal lineages, but extend insights across the eukaryotes by extensive comparative genomic analysis. We argue that gene loss plays an important role in diversification of protein complexes and hence enhancement of eukaryotic diversity.     

Survey of resistance gene analogs in <Emphasis Type="Italic">Solanum caripense</Emphasis>, a relative of potato and tomato,and update on <Emphasis Type="Italic">R</Emphasis> gene genealogy

The resistance (R) proteins of the TIR- and non-TIR (or CC-) superfamilies possess a nucleotide binding site (NBS) domain. Within an R gene, the NBS is the region of highest conservation, suggesting an essential role in triggering R protein activity. We compared the NBS domain of functional R genes and resistance gene analogs (RGA) amplified from S. caripense genomic DNA via PCR using specific and degenerate primers with its counterpart from other plants. An overall high degree of sequence conservation was apparent throughout the P-loop, kinase-2 and kinase-3a motifs of NBS fragments from all plants. Within the non-TIR class of R genes a prominent sub-class similar to the potato R1 gene conferring resistance to late blight, was detected. All non-TIR-R1-like R gene fragments that were sequenced possessed an intact open reading frame, whereas 22% of all non-TIR-non-R1-like fragments and 59% of all TIR-NBS RGA fragments had an interrupted reading frame or contained transposon-specific sequence. The non-TIR-R1-like fragments had high similarity to Solanaceae R genes and low similarity to RGAs of other plant species including A. thaliana and the cereals. It is concluded that appearance of the non-TIR-R1-like NBS domain represents a relatively recent evolutionary development. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Bos indicus introgression into (peri‐)alpine cattle breeds – evidence from the analysis of bovine whey protein variants

The major bovine whey proteins, α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG), exhibit breed‐specific genetic variation. The aim of this study was to identify possible new protein variants and determine the distribution of variants across a variety of 18 taurine and indicine cattle breeds applying a DNA‐based sequencing approach. To this end, the open reading frames of the respective genes (LALBA and LGB) were sequenced in 476 animals. Within the LALBA gene, a previously unknown synonymous and a previously undesignated non‐synonymous nucleotide exchange were identified. Furthermore, two known α‐LA variants (A and B) and four known β‐LG variants (A, B, C and W) were determined. The occurrence of typical indicine variants in some taurine cattle breeds, such as Suisse Eringer, German Hinterwälder and Hungarian Grey Steppe, further supports the hypothesis of ancient Bos indicus introgression into (peri‐)alpine cattle breeds.     

Family‐based genome scan for age at onset of late‐onset Alzheimer's disease in whole exome sequencing data

Alzheimer's disease (AD) is a common and complex neurodegenerative disease. Age at onset (AAO) of AD is an important component phenotype with a genetic basis, and identification of genes in which variation affects AAO would contribute to identification of factors that affect timing of onset. Increase in AAO through prevention or therapeutic measures would have enormous benefits by delaying AD and its associated morbidities. In this paper, we performed a family‐based genome‐wide association study for AAO of late‐onset AD in whole exome sequence data generated in multigenerational families with multiple AD cases. We conducted single marker and gene‐based burden tests for common and rare variants, respectively. We combined association analyses with variance component linkage analysis, and with reference to prior studies, in order to enhance evidence of the identified genes. For variants and genes implicated by the association study, we performed a gene‐set enrichment analysis to identify potential novel pathways associated with AAO of AD. We found statistically significant association with AAO for three genes (WRN, NTN4 and LAMC3) with common associated variants, and for four genes (SLC8A3, SLC19A3, MADD and LRRK2) with multiple rare‐associated variants that have a plausible biological function related to AD. The genes we have identified are in pathways that are strong candidates for involvement in the development of AD pathology and may lead to a better understanding of AD pathogenesis.     

Polymorphism of the IGHA gene in sheep

Genetic variation in immunoglobulin A, the most abundant immunoglobulin in mammalian cells, has not been reported in ruminants. In this study, variation in the immunoglobulin heavy alpha chain constant gene (IGHA) of sheep was investigated by amplification of a fragment that included the hinge coding sequence, followed by single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Three novel sequences, each characterized by unique SSCP banding patterns, were identified. One or two sequences were detected in individual sheep and all the sequences identified shared high homology to the published ovine and bovine IGHA sequences, suggesting that these sequences represent allelic variants of the IGHA gene in sheep. Sequence alignment showed that these sequences differed mainly in the 3′ end of exon 1 and in the coding sequence of the hinge region. There was either a deletion or an insertion of two codons in the hinge coding region in these allelic variants. Codon usage in the hinge coding region was quite different from that in the non-hinge coding regions of the gene, suggesting different evolution of the IGHA hinge sequence. Three novel amino acid sequences of ovine IGHA were also predicted, and variation in these sequences might not only affect antigen recognition but also susceptibility to cleavage by bacterial or parasitic proteases. Nucleotide sequence data reported in this paper have been submitted to the NCBI GenBank nucleotide sequence database and have been assigned the accession nos. AY956424–AY956426.