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Whole chromosomal and segmental uniparental disomy (UPD) is one of the causes of imprinting disorder and other recessive disorders. Most investigations of UPD were performed only using cases with relevant phenotypic features and included few markers. However, the diagnosis of cases with segmental UPD requires a large number of molecular investigations. Currently, the accurate frequency of whole chromosomal and segmental UPD in a normal developing embryo is not well understood. Here, we present whole chromosome and segmental UPD analysis using single nucleotide polymorphism (SNP) microarray data of 173 mother-father-child trios (519 individuals) from six populations (including 170 HapMap trios). For two of these trios, we also investigated the possibility of shorter segmental UPD as a consequence of homologous recombination repair (HR) for DNA double strand breaks (DSBs) during the early developing stage using high-coverage whole-genome sequencing (WGS) data from 1000 Genomes Project. This could be overlooked by SNP microarray. We identified one obvious segmental paternal uniparental isodisomy (iUPD) (8.2 mega bases) in one HapMap sample from 173 trios using Genome-Wide Human SNP Array 6.0 (SNP6.0 array) data. However, we could not identify shorter segmental iUPD in two trios using WGS data. Finally, we estimated the rate of segmental UPD to be one per 173 births (0.578%) based on the UPD screening for 173 trios in general populations. Based on the autosomal chromosome pairs investigated, we estimate the rate of segmental UPD to be one per 3806 chromosome pairs (0.026%). These data imply the possibility of hidden segmental UPD in normal individuals., Gene, 512(2), pp.267-274; 2013

Gene conversion, the unidirectional exchange of genetic material between homologous sequences, is thought to strongly influence patterns of genetic diversity. The high diversity of major histocompatibility complex (MHC) genes in many species is thought to reflect a long history of gene conversion events both within and among loci. Theoretical work suggests that intra- and interlocus gene conversion leave characteristic signatures of nucleotide diversity, but empirical studies of MHC variation have rarely been able to analyze the effects of conversion events in isolation, due to the presence of multiple gene copies in most species. The potbellied seahorse (Hippocampus abdominalis), a species with a single copy of the MH class II beta-chain gene (MHIIb), provides an ideal system in which to explore predictions on the effects of intralocus gene conversion on patterns of genetic diversity. The genetic diversity of the MHIIb peptide binding region (PBR) is high in the seahorse, similar to other vertebrate species. In contrast, the remainder of the gene shows a total absence of synonymous variation and low levels of intronic sequence diversity, concentrated in 3 short repetitive regions and 1–12 SNPs per intron. The distribution of substitutions across the gene results in a patchwork pattern of shared polymorphism between otherwise divergent sequences. The pattern of nucleotide diversity observed in the seahorse MHIIb gene is congruent with theoretical expectations for intralocus gene conversion, indicating that this evolutionary mechanism has played an important role in MHC gene evolution, contributing to both the high diversity in the PBR and the low diversity outside this region. Neutral variation at this locus may be further reduced due to biases in nucleotide composition and functional constraints.

article i nfo Human Fetuin-A (FETUA, also termed AHSG) is a serum protein composed of two cystatin-like domains D1 and D2 of together 235 amino acids (aa) and an unrelated domain D3 of 114 aa. Though the protein plays a role in diverse physiological and pathological processes, comparably little is known about sequence evolution of FETUA. We therefore analyzed its molecular evolution on the basis of coding sequences of 16 primate species. Ratios of non-synonymous to synonymous substitution rates (dn/ds= ω) suggest that a previously reported acceleration of sequence evolution of exon 7, which encodes domain D3, is driven by positive selection instead of neutral evolution. Irrespective of the maximum likelihood model used, a remarkable 40% of codon sites is estimated to be positively selected in domain D3, with an average ω of 4.3. Analyses of GC/AT- and AT/GC-substitutions reveal that GC-biased gene conversion (gBGC) cannot explain the observed pattern. We thus reject the extended null model of molecular evolution (neutral evolution and gBGC) and conclude that positive Darwinian selection contributes to the sequence evolution of FETUA domain D3. Remarkably, domain D3 of human FETUA is characterized by a significant accumulation of proteolytic cleavage sites. Though speculative at present, the accelerated evolution of domain D3 might be due to the evolution of differential cleavage site patterns across primates reflecting different paths to achieve one goal, the decomposition of FETUA under pathological conditions. Present analyses use a PERL-script ("Substitution Bias Indicator", SBI) for the extraction of the numbers of GC/AT- and AT/GC-substitutions from supplemental results files of PAML analyses. The script is available upon request and the presented approach of testing the extended null hypothesis of molecular evolution can be transferred to every other protein coding locus.

Protobothrops flavoviridis (Crotalinae) venom gland phospholipase A 2 (PLA 2 ) isozyme genes have evolved in an accelerated manner to acquire diverse physiological activities in their products. For elucidation of the multiplication mechanism of PLA 2 genes, a 25,026 bp genome segment harboring five PLA 2 isozyme genes was obtained from Amami-Oshima P. flavoviridis liver and sequenced. The gene PfPLA 2 encoded [Lys 49 ]PLA 2 called BPII, the gene PfPLA 4 neurotoxic [Asp 49 ]PLA 2 called PLA-N, the gene PfPLA 5 basic [Asp 49 ]PLA 2 called PLA-B, and PfPLA 1(ψ) and PfPLA 3(ψ) were the inactivated genes. The 5′ truncated reverse transcriptase (RT) elements, whose intact forms constitute long interspersed nuclear elements (LINEs), were found in close proximity to the 3′ end of PLA 2 genes and named PLA 2 gene-coupled RT fragments (PcRTFs). The facts that PcRTFs have the stem–loop and repetitive sequence in the 3′ untranslated region (UTR) which is characteristic of CR1 LINEs suggest that PcRTFs are the debris of P. flavoviridis ancestral CR1 LINEs, denoted as Pf CR1s. Since the associated pairs of PLA 2 genes and PcRTFs are arranged in tandem in the 25,026 bp segment, it is thought that an ancestral PLA 2 gene- Pf CR1 unit ( PfPLA - Pf CR1) which was produced by retrotransposition of Pf CR1 by itself to the 3′ end of PLA 2 gene duplicated several times to form a multimer of PfPLA - Pf CR1, a cluster of PLA 2 genes, in the period after Crotalinae and Viperinae snakes branched off. Recombinational hot spot of a 37 bp segment, named Scomb , was found in the region 548 bp upstream from the TATA box of PLA 2 genes. Thus, it could be assumed that multiplication of PfPLA - Pf CR1 occurred by unequal crossing over of the segment, - Scomb - PfPLA - Pf CR1- Scomb- . The Pf CR1 moieties were afterward disrupted in the 5′ portion to PcRTFs. The detection of two types of PcRTFs different in length which were produced by elimination of two definitive sequences in Pf CR1 moiety possibly by gene conversion clearly supports such process but not multiplication of the PLA 2 gene-PcRTF unit.

The present study identified 12 MLO genes in rice that were located on chromosomes 1, 2, 3, 4, 5, 6, 10, and 11 respectively without any obvious clustering. On a genome scale we showed that the expansion of rice MLO gene family was primarily attributed to segmental duplication produced by polyploidy, rather than through tandem amplification. Gene conversion events should also play important roles in the evolution of MLO genes. The results of relative rate ratio test and maximum likelihood analysis suggested that positive selection should have occurred after gene duplication and/or speciation, prompting the formation of distinct MLO subfamilies. Functional divergence analysis provided statistical evidence for shifted evolutionary rate after gene duplication. Compared to extracellular loop 3 and Ca 2+ -binding domain, much stronger functional constraints should impose on intracellular loop 2, although all of the three regions might be under purifying selection. The sliding window analysis of d N / d S ratio values identified one sequence region where strong functional constraints must impose on, and consequently should be crucial for functionality of MLO genes.

Interferon alpha (IFNA) genes code for proteins with important signaling roles during the innate immune response. Phylogenetically, IFNA family members in eutherians (placental mammals) cluster together in a species-specific manner except for closely related species (i.e. Homo sapiens and Pan troglodytes) where gene-specific clustering is evident. Previous research has been unable to clarify whether gene conversion or recent gene duplication accounts for gene-specific clustering, partly because the similarity of members of the IFNA family within species has made it historically difficult to identify the exact composition of IFNA gene families. IFNA gene families were fully characterized in recently available genomes from Canis familiaris, Macaca mulatta, P. troglodytes and Rattus norvegicus, and combined with previously characterized IFNA gene families from H. sapiens and Mus musculus, for the analysis of both whole and partial gene conversion events using a variety of statistical methods. Gene conversion was inferred in every eutherian species analyzed and comparison of the IFNA gene family locus between primate species revealed independent gene duplication in M. mulatta. Thus, both gene conversion and gene duplication have shaped the evolution of the IFNA gene family in eutherian species. Scenarios may be envisaged whereby the increased production of a specific IFN-alpha protein would be beneficial against a particular pathogenic infection. Gene conversion, similar to duplication, provides a mechanism by which the protein product of a specific IFNA gene can be increased.

Humans have seven alcohol dehydrogenase genes ( ADH ) falling into five classes. Three out of the seven genes ( ADH1A , ADH1B and ADH1C ) belonging to Class I are expressed primarily in liver and code the main enzymes catalyzing ethanol oxidization. The three genes are tandemly arrayed within the ADH cluster on chromosome 4 and have very high nucleotide similarity to each other (exons: > 90%; introns: > 70%), suggesting the genes have been generated by duplication event(s). One explanation for maintaining similarity of such clustered genes is homogenization via gene conversion(s). Alternatively, recency of the duplications or some other functional constraints might explain the high similarities among the genes. To test for gene conversion, we sequenced introns 2, 3, and 8 of all three Class I genes (total > 15.0 kb) for five non-human primates – four great apes and one Old World Monkey (OWM) – and compared them with those of humans. The phylogenetic analysis shows each intron sequence clusters strongly within each gene, giving no evidence for gene conversion(s). Several lines of evidence indicate that the first split was between ADH1C and the gene that gave rise to ADH1A and ADH1B . We also analyzed cDNA sequences of the three genes that have been previously reported in mouse and Catarrhines (OWMs, chimpanzee, and humans) and found that the synonymous and non-synonymous substitution ( d N / d S ) ratios in all pairs are less than 1 representing purifying selection. This suggests that purifying selection is more important than gene conversion(s) in maintaining the overall sequence similarity among the Class I genes. We speculate that the highly conserved sequences on the three duplicated genes in primates have been achieved essentially by maintaining stability of the hetero-dimer formation that might have been related to dietary adaptation in primate evolution.

The complete mouse mitochondrial genome (16.3 kb) was directly cloned into a Bacillus subtilis genome (BGM) vector. Two DNA segments of 2.06 and 2.14 kb that flank the internal 12 kb of the mitochondrial DNA (mtDNA) were subcloned into an Escherichia coli plasmid. Subsequent integration of the plasmid at the cloning locus of the BGM vector yielded a derivative specific for the targeted cloning of the internal 12-kb mtDNA region. The BGM vector took up mtDNA purified from mouse liver and integrated it by homologous recombination at the two preinstalled mtDNA-flanking sequences. The complete cloned mtDNA in the BGM vector was converted to a covalently closed circular (ccc) plasmid form via gene conversion in B. subtilis. The mtDNA carried on this plasmid was then isolated and transferred to E. coli. DNA sequence fidelity and stability through the BGM vector-mediated cloning process were confirmed.

Several studies of nucleotide substitution patterns in mammalian species suggested that GC-rich isochores might be vanishing in mammalian genomes. However, the number of genes and the number of genomes included in these studies might not have given a reliable broad view of the trend in GC change in mammals. It is therefore worth exploiting this issue with a broader coverage of mammalian genomes using a reliable approach, the maximum likelihood approach. We have applied two maximum likelihood methods to infer the ancestral GC contents of 176 mammalian genes from representative eutherian species and at least one marsupial species. Except for a large GC decrease in marsupial genes, we found no general decreasing trend in GC content in GC-rich genes or in other genes among eutherian mammals; indeed, the GC content of GC-rich genes appears to have increased in recent times in some genomes, e.g., the rabbit. For the large GC decrease in marsupials, it could be mainly due to the great reduction in chromosome number, which could lead to a large reduction in recombination rate and thus also a large reduction in the rate of gene conversion. Since many eutherian mammals still maintain a fairly large number of chromosomes, it is unlikely that GC-rich isochores are vanishing in these mammals.

A comprehensive dataset of 62 beta globin gene sequences from various vertebrates was compiled to test the molecular clock and to estimate dates of gene duplications. We found that evolution of the beta globin family of genes is not clock-like, a result that is at odds with the common use of this family as an example of a constant rate of evolution over time. Divergence dates were estimated either with or without assuming the molecular clock, and both analyses produced similar date estimates, which are also in general agreement with estimates reported previously. In addition we report date estimates for seven previously unexamined duplication events within the beta globin family. Despite multiple sources of rate variation, the average rate across the beta globin phylogeny yielded reasonable estimates of divergence dates in most cases. Exceptions were cases of gene conversion, where it appears to have led to underestimates of divergence dates. Our results suggest (i) the major duplications giving rise to the paralogous beta globin genes are associated with significant evolutionary rate variation among gene lineages; and (ii) genes arising from more recent gene duplications (e.g., tandem duplications within lineages) do not appear to differ greatly in rate. We believe this pattern reflects a complex interplay of evolutionary forces where natural selection for diversifying paralogous functions and lineage-specific effects contribute to rate variation on a long-term basis, while gene conversion tends to increase sequence similarity. Gene conversion effects appear to be stronger on recent gene duplicates, as their sequences are highly similar. Lastly, phylogenetic analyses do not support a previous report that avian globins are members of a relic lineage of omega globins.

Alu elements are the most active and predominant type of short interspersed elements (SINEs) in the human genome. Recently inserted polymorphic (for presence/absence) Alu elements contribute to genome diversity among different human populations, and they are useful genetic markers for population genetic studies. The objective of this study is to identify polymorphic Alu insertions through an in silico comparative genomics approach and to analyze their distribution pattern throughout the human genome. By computationally comparing the public and Celera sequence assemblies of the human genome, we identified a total of 800 polymorphic Alu elements. We used polymerase chain reaction-based assays to screen a randomly selected set of 16 of these 800 Alu insertion polymorphisms using a human diversity panel to demonstrate the efficiency of our approach. Based on sequence analysis of the 800 Alu polymorphisms, we report three new Alu subfamilies, Ya3, Ya4b, and Yb11, with Yb11 being the smallest known Alu subfamily. Analysis of retrotransposition activity revealed Yb11, Ya8, Ya5, Yb9, and Yb8 as the most active Alu subfamilies and the maintenance of a very low level of retrotransposition activity or recent gene conversion events involving S subfamilies. The 800 polymorphic Alu insertions are characterized by the presence of target site duplications (TSDs) and longer than average polyA-tail length. Their pre-integration sites largely follow an extended “NT-AARA” motif. Among chromosomes, the density of Alu insertion polymorphisms is positively correlated with the Alu-site availability and is inversely correlated with the densities of older Alu elements and genes.

Lifelong persistent infection of cattle is a hallmark of the tick transmitted pathogen Anaplasma marginale. Antigenic variation of Major Surface Protein 2 (MSP2) plays an important role in evasion of the host immune response to allow persistence. Antigenic variation of MSP2 is achieved by gene conversion of pseudogenes into the single operon linked expression site and the diversity of variants is defined by the pseudogene repertoire. Once an animal is persistently infected with one strain of A. marginale, infection with a second strain (superinfection) is rare. However, we recently detected animals superinfected with different strains of A. marginale and hypothesized that the msp2 pseudogene repertoire would be distinct in these superinfecting strains, consistent with encoding different sets of surface variants. Five strains of A. marginale were selected in order to identify and compare msp2 pseudogene content; these included strains with similar and different msp1α genotypes, and genotypes that were representative of the strains that were found in the superinfected animals. Southern blot analysis of strains associated with superinfection revealed distinctly different msp2 banding patterns, in contrast to a pattern suggesting identical pseudogene content among related strains not associated with superinfection. Indeed, targeted sequence analysis of msp2 pseudogenes showed identical pseudogene repertoires in genotypically closely related strains and varying amounts of dissimilarity in the pseudogene repertoire in strains with distinctly different msp1α genotypes, but totally different msp2 pseudogene repertoires between the strains that were found in superinfected animals. This finding supports the hypothesis that the occurrence of superinfection reflects the differences in the msp2 repertoire and corresponding diversity of variants.

We previously discovered Y-chromosomal red-green opsin genes in two types of owl monkeys with different chromosomal characteristics. In one type, the Y-linked opsin gene is a single-copy intact gene and in the other, the genes exist as multiple pseudogenes on a Y/autosome fusion chromosome. In the present study, we first distinguished the two types of monkeys as distinct allopatric species on the basis of karyotypic characteristics: Aotus lemurinus griseimembra (Karyotype III, diploid chromosome number [2n] = 53) and Aotus azarae boliviensis (Karyotype VI; male 2n = 49; female 2n = 50), belonging to the northern and southern species groups, respectively, separated by the Amazon River system. Our sequence analysis revealed a common L1-Alu-Alu insertion between the two species in the 3′-flanking region of the X-linked opsin genes. The insertion was absent in the Y-linked opsin genes and in the human red and green opsin genes, indicating that it occurred in the X copy before the split into northern and southern species and after the X to Y duplication, i.e. duplication preceded speciation. We also show that in the northern species, the Y-linked opsin gene has evolved concomitantly with the X-linked copy whereas in the southern species, the Y-autosome fusion possibly led to decoupling evolutionary processes between X- and Y-linked copies and subsequent degeneration and duplications of the Y-linked opsin gene.

We investigated the overall variability of the S1a satellite DNA repeats in ten European populations of Rana temporaria by a new procedure that determines the average sequence of the repeats in a genome. The average genomic sequences show that only 17% of the S1a repeat sequence (494 bp) is variable. The variable positions contain the same major and minor bases in all or many of the population samples tested, but the percentages of these bases can greatly vary among populations. This indicates the presence in the species of an enormous number of repeats having a different distribution of bases in these variable positions. Individual genomes contain thousands of repeat variants, but these mixtures have very similar characteristics in all populations because they present the same type of restricted and species-specific variability. Southern blots analyses and sequences of cloned S1a repeats fully support this conclusion. The S1 satellite DNA of other European brown frog species also presents properties indicating the same type of variability. This first characterisation of the overall repeat variability of a satellite DNA in a species has revealed features that cannot be determined by gene conversion and crossing over. Our results suggest that a specific directional process based on rolling circle amplification should play a relevant role in the evolution of satellite DNA.

The genomic features of the small subunit ADP-glucose pyrophosphorylase (AGPase) isoforms are different in barley and maize. The two isoforms found in barley originated from one single gene through alternative splicing, while two independent genes encode the two isoforms in maize. To ascertain the genomic organizations of two small subunit AGPase isoforms in sweetpotato (ibAGP1 and ibAGP2), we isolated genomic DNAs containing the entire coding regions of two genes. Complete genomic structures of ibAGP1 and ibAGP2 were ascertained by the sequencing of these genomic regions. The transcribed regions of ibAGP1 and ibAGP2, comprising nine exons and eight introns, were distributed over 3.9 and 4.0 kb, respectively. The eight introns differed in length, from 76 to 946 bp in ibAGP1, and from 76 to 811 bp in ibAGP2, while the locations of introns in ibAGP1 and ibAGP2 were identical. There was 46–58% sequence identity between the intron sequences of the two genes. Intron sequence analyses suggested that either duplication in each intron, or gene conversion between introns of two isoforms, might cause major intron size differences between the two genes. Altogether, these results indicate that two small subunit AGPase isoforms in sweetpotato are encoded by two independent genes, in a fashion similar to that of maize small subunit AGPase genes. D 2004 Elsevier B.V. All rights reserved.

Recent studies have demonstrated that the Echinococcus granulosus antigen B (AgB) interferes with the intermediate hosts' immune response and is encoded by a multigene family. The number of members within the family is still uncertain, but there are several evidences of a large genetic variability. The E. granulosus AgB genomic sequences available in nucleotide databases can be grouped into four clades, corresponding to genes EgAgB1, EgAgB2, EgAgB3 and EgAgB4. In the present study, we use PCR amplifications followed by cloning and sequencing to evaluate the genetic variability for AgB isoforms. Two pairs of primers were independently used for PCR amplification. Both PCR reactions from each of three isolated protoscolex (larvae) were cloned in a plasmid vector and the plasmid inserts of 30 colonies from each cloning experiment were sequenced. Using phylogenetic tools, the 113 EgAgB clones are classified as follows: 25 are related to EgAgB1, 24 to EgAgB2, 9 to EgAgB3 and 39 to EgAgB4. The remaining 16 clones form a separate cluster, which we name EgAgB5, more closely related to EgAgB3 than to any of the other genes. Within each gene group, a number of variant sequences occur, which differ from one another by one or few nucleotides. One EgAgB3 clone has a premature stop codon (pseudogene) and an EgAgB2 clone lacks the region corresponding to the intron. The overall variation cannot be explained by differences among the asexual protoscoleces, or by experimental artifacts. Using Echinococcuss AgB genes from other species/strains as outgroups, neutrality is rejected for EgAgB2, and balancing selection is detected for EgAgB5, which also seems to be involved in gene conversion. We suggest that EgAgB1-EgAgB5 represent a family of contingency genes, that is, genes that are variably expressed, so that some but not others are expressed in each individual parasite. Contingency genes are common in parasitic protozoa and other microparasites, but the EgAgB family is the first set identified in a multicellular parasite. © 2004 Elsevier B.V. All rights reserved.

The satellite DNA of ants Messor barbarus and Messor bouvieri is analysed. The results are compared with the satellite DNA data from Messor structor previously reported and with new data obtained from the genome of geographically distinct M. structor population, which have shown that this satellite DNA is highly conserved within the species. The satellite DNA is organized as tandemly repeated 79 bp monomers in all species. The sampled sequences of the three species show a high similarity and all belong to the same family of satellite DNA. Sequence comparisons suggested the occurrence of highly effective homogenization mechanism acting upon the ant genomes. In accordance with this hypothesis, putative gene conversion tracts are identified when the different monomers of the same species are compared. The highest sequence conservation in all species corresponds to a single region with inverted repeats. A CENP-B-like motif was found in this region. The possibility that it may be involved in the homogenization of satellite DNA is discussed.

The somatic recombination of different germline-encoded gene segments constitutes a principal source of antibody diversity. In order to investigate the diversity in recombined gene segments encoding the immunoglobulin heavy chain of the Antarctic teleost Trematomus bernacchii, a VH library was constructed by 5'-RACE (rapid amplification of cDNA ends) using RNA isolated from the spleen of an individual specimen. Analysis of cDNA sequences of 45 rearranged VH/D/JH segments revealed specific features, such as: high number of repeats, up to 8 bp long, and palindromic sequences, especially in CDRs (complementary determining regions); occurrence of the RGYW consensus, known as mutational hot spot, higher than in other species. Sixty-four percent of single base substitutions was found within this motif. In addition, the usage of serine codons showed a clear bias for AGY in CDRs, particularly in CDR2, and for TCN in FRs (framework regions). In CDRs, the frequency of non-synonymous changes was higher than that of synonymous changes. Diversity generated by insertions/deletions occurred more often than in other species; inserted bases were often repeats of adjacent bases. In particular the CDR2 showed the highest length variability as compared to other species. Alignment of VH sequences indicated that also the gene conversion mechanism may contribute to generating diversity. These data indicate a CDR mutability higher than in other species and provide some insights into the hypermutational events that may also occur in teleosts.

Genomic analysis was performed for the murine odorant receptor (OR) genes. The MOR28 cluster on chromosome 14 was extensively studied. It contains six OR genes, MOR28, 10, 83, 29A, 29B and 30. The human homolog of this cluster is located on the human chromosome 14, and contains five OR genes, HOR28/10, 83, 29A, 29B and 30. Sequence comparison of these OR gene paralogs and orthologs suggests that the coding homologies are accounted for not only by recent gene duplication, but also by gene conversion among the coding sequences within the cluster. A possible role of gene conversion in the olfactory system is discussed in the context of the olfactory map.

Staphylococcal Panton-Valentine leukocidin (PVL) is an important virulence factor, which causes leukocytolysis and tissue necrosis. Our previous report on the existence of the PVL genes (lukS-PV and lukF-PV) on the genome of prophage phiPVL in the Staphylococcus aureus strain V8 suggested the horizontal transmission of PVL genes by temperate bacteriophage among S. aureus (Kaneko, et al., 1998. Gene 215, 57-67). Here, we demonstrated the phage conversion of S. aureus leading to the production of PVL by discovery of a novel PVL-carrying phage, phiSLT (Staphylococcal Leukocytolytic Toxin) from a clinical isolate of S. aureus. phiSLT was able to lysogenize several clinical isolates of PVL-negative S. aureus strains as well as strain RN4220 at the conserved 29-bp sequence (attB) and all the lysogenized S. aureus strains had the ability to produce PVL. phiSLT had an elongated head of about 100x50 nm and a flexible tail of 400 nm long, that was quite different from phiPVL which had an isometric hexagonal head of about 60 nm diameter. The linear double-stranded phiSLT genome comprised 42,942 bp with 29-bp attachment core sequences and contained 62 open reading frames. Only 6.4 kbp region containing lysis cassette, PVL genes, attP, integrase, and orf204 of phiSLT was identical to that of phiPVL, while other regions were different from those of phiPVL. Thus, it can be concluded that PVL genes are carried by different temperate phages, which have the same attachment site.

It has been proposed that proteins which are involved in host defence and susceptibility undergo accelerated evolution. Chemokine receptors have roles as pro-inflammatory agents acting in response to infection, and in addition are receptors for entry of viruses and other pathogens into cells. Consistent with this, their rate of evolution is higher than that for other members of the seven-transmembrane domain receptor family. The pattern of evolution of the chemokine receptors was examined in detail. Both chromosomal clusters of chemokine receptors (CC and CXC) showed evidence of a number of gene conversions. These are likely to have resulted in protein sequence changes, which could possibly alter function. 45% of a control group of clustered genes also showed evidence of conversion. Thus, the fixation of a gene conversion is not in itself sufficiently unusual in tandemly repeated genes and cannot be taken as strong evidence of a selection for a novel function. However, the degree of amino acid difference between the chemokine receptors CCR1 and CCR3 was greater than that for any of the control genes. Such changes could have functional implications for inter-species differences in chemokine receptor interactions with pathogens.

Telomeres in the dipteran Chironomus pallidivittatus terminate with 340 bp tandem DNA repeats belonging to different subfamilies with characteristic intertelomeric distribution. We have now found, interspersed between such repeats, a composite element of approx. 1400 bp present in two similar size variants, with several components of nontelomeric origin. There were about 50 copies of the element, predominantly or exclusively present in a previously defined group of telomeres, characterized by a unique set of telomeric tandem repeat subfamilies. Elements were integrated at irregular distances from each other, and intervening telomeric tandem repeat DNA was variable in composition. Nevertheless, the flanks immediately surrounding the elements were identical for different elements; in other words, there was a site-specific insertion. We suggest that this selective invasion of a small part of the genome by an interspersed, probably rapidly evolving element is best explained by repeated gene conversions.

We have isolated two alpha-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two alpha-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo alpha-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech alpha-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod alpha-tubulins. The protein sequences of the leech alpha-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.

The intron positions found in globin genes of plants, protozoa and invertebrates have been interpreted as evidence for a three-intron-four-exon structure of the ancestral globin gene. In particular, the so-called 'central' introns, which are not found in vertebrate globin genes but are present in a variety of invertebrate and plant species, have been used as an argument for an ancestral gene structure featuring three introns. We have analyzed the presence or absence of central introns in the Gb genes 2beta, 9 and 7A of various European and Australasian species of the insect Chironomus. We find unrelated central introns at different positions in some of the species investigated, while other species completely lack introns in these genes. This variable distribution of introns is parsimoniously explained by independent intron additions. Such a gain of introns may occur convergently at identical positions in unrelated taxa. Insertion by gene conversion may be a viable mechanism to explain intron gain.

The nucleotide (nt) sequences of the 5′-flanking regions of two Brassica self-incompatibility genes, SLG 9 and SRK 9 , were determined. Their sequences were highly conserved: a region spanning 1.9 kb in the 5′-flanking region was completely identical except for a 1319-bp segment in SLG 9 . These observations strongly suggest that SLG 9 and SRK 9 together with their promoter regions were involved in a gene duplication or conversion event which occurred before the 1319-bp SLG 9 -specific sequence was inserted in SLG 9 or deleted in SRK 9 .

The coding region of the involucrin gene of Tupaia glis has been cloned and sequenced. It resembles the involucrin coding region of other non-anthropoid mammals in possessing a segment of related, short tandem repeats at a defined location, but in Tupaia, there has been recent serial duplication of a repeat into which a cysteine codon had earlier been introduced. As a result of the duplication, there is a total of as many as six cysteine codons in the segment of repeats, a number larger than for any other species yet examined. In Rattus there has been a comparable but independent addition of cysteine codons, and both Tupaia and Rattus have eliminated an otherwise conserved cysteine codon 75 located close to but outside the segment of repeats. In Tupaia, this elimination probably occurred by gene conversion. Also independently, the gene of Canis has added cysteine codons to the segment of repeats but has not yet lost cysteine 75. It is proposed that the gain and the loss of cysteine codons are parts of a multi-stage program of cysteine relocation.

We have investigated the requirements necessary for high-level production of the hepatitis B virus (HBV strain ayw) large surface glycoprotein preS1 with vaccinia virus (VV) recombinants. In earlier studies, only nanogram amounts of preS1 could be obtained from cells infected with an appropriate recombinant VV carrying the preS1 gene under the transcriptional control of a conventional VV promoter (p7.5). Here, we report that the use of an improved promoter system, i.e., the bacteriophage T7 polymerase/VV hybrid expression system (T7/EMC system) in combination with a G-C conversion at position 5 of the preS1 open reading frame, deleting the myristylation motif of the polypeptide, results in an at least 12-fold increase in preS1 expression compared to the wild-type preS1 expressed with the strongest homologous VV promoter system known so far. Although the T7/EMC promoter system was most effective, improved expression of the modified preS1 (preS1dMyr) is independent from the promoter system used, from the insertion locus of the modified preS1 within the VV genome and also from the cell line used for expression studies.

A highly repetitive sequence in the genomic DNA of the bivalve mollusc Donax trunculus (Dt) has been identified upon restriction with EcoRV . During the time-course of DNA digestion, genomic fragments resolved electrophoretically into a ladder-like banding pattern revealing a tandem arrangement of the repeated elements, thus representing satellite DNA sequences. Cloning and sequence analysis unraveled the presence of two groups of monomer units which can be considered distinctive satellite subfamilies. Each subclass is distinguishable by the presence of 17 evenly spread diagnostic nucleotides (nt). The respective consensus sequences are 155 bp in length and differ by 11%, while relevant internal substructures were not observed. The two satellite subfamilies constitute 0.23 and 0.09% of the Dt genome, corresponding to 20 000 and 7600 copies per haploid complement, respectively. Sequence mutations often appear to be shared between two or more monomer variants, indicating a high degree of homogenization as opposed to that of random mutational events. Shared mutations among variants appear either as single changes or in long stretches. This pattern may arise from gene conversion mechanisms acting at different levels, such as the spread of nt sequences of a similar length to the monomer repeat itself, and the diffusion of short tracts a few bp long. Subfamilies might have evolved from the occasional amplification and spreading of a monomer variant effected by gene conversion events