Analysis of a Gene Conversion Gradient at the His4 Locus in Saccharomyces Cerevisiae

Heteroduplexes formed between genes on homologous chromosomes are intermediates in meiotic recombination. In the HIS4 gene of Saccharomyces cerevisiae, most mutant alleles at the 5' end of the gene have a higher rate of meiotic recombination (gene conversion) than mutant alleles at the 3' end of the gene. Such gradients are usually interpreted as indicating a higher frequency of heteroduplex formation at the high conversion end of the gene. We present evidence indicating that the gradient of conversion at HIS4 primarily reflects the direction of mismatch repair rather than the frequency of heteroduplex formation. We also identify a site located between the 5' end of HIS4 and the 3' end of BIK1 that stimulates heteroduplex formation at HIS4 and BIK1.     

Molecular genetic characteristics of Duchenne-Becker muscular dystrophy in the Republic of Moldova

Solution to some problems of clinical genealogical and molecular genetic study of Duchenne muscular dystrophy (DMD) in the Republic of Moldova and prenatal diagnosis aimed at preventing the birth of infants with this disease is proposed. An integrated clinical and molecular genetic study of families with a high risk of DMD has allowed its specific characteristics in the Moldovan population to be identified. The spectrum of mutations at the gene level in DMD patients and their role in prenatal and clinical diagnosis has been determined. RFLP analysis and PCR have been used to estimate the informativeness of families with a high DMD risk; prenatal diagnosis has been performed in some of them. Population analysis of the frequencies of polymorphic restriction sites have been carried out for loci pERT87-8/Tag1, pERT87-15/BamH1, and 16intron/Tag1. The results of analysis of deletion frequencies in the dystrophin gene and the frequencies of the pERT87-8, pERT87-15, and 16intron intragenic polymorphic loci have served as a basis for a strategy of molecular diagnosis. The new strategy allows the informativeness to be evaluated and, hence, clinical, preclinical, and prenatal diagnosis to be performed in approximately 94% of cases. A modified PCR method (MPCR) using the system of primers pERT87-8/Tag1 and 16intron/Tag1 has been developed for direct search for deletions. The method makes it possible to avoid diagnostic errors and decrease both the duration and the cost of the analysis.     

Microsatellite Allelic Homoplasy Due to Variable Flanking Sequences

Microsatellite DNA sequences have become the dominant source of nuclear genetic markers for most applications. It is important to investigate the basis of variation between alleles and to know if current assumptions about the mechanisms of microsatellite mutation (that is to say, variations involving simple changes in the number of repeat) are correct. We have characterized, by DNA sequencing, the human alleles of a new highly informative (CA)n repeat localized approximately 20 kb centromeric to the HLA-B gene. Although 12 alleles were identified based on conventional length criteria, sequencing of the alleles demonstrated that differences between alleles were found to be more complex than previously assumed: A high degree of microsatellite variability is due to variation in the region immediately flanking the repeat. These data indicate that the mutational process which generates polymorphism in this region has involved not only simple changes in the number of dinucleotide CA repeats but also perturbations in the nonrepeated 5′ and 3′ flanking sequences. Three families of alleles (not visible from the overall length of the alleles), with presumably separate evolutionary histories, exist and can yield to homoplasy of size. Effectively, we can observe alleles of the same size with different internal structures which are separated by a significant amount of variation. Although allelic homoplasy for noninterrupted microsatellite loci has been suggested between different species, it has not been unequivocally demonstrated within species. A strong association is noted between alleles defined at the sequence level and HLA-B alleles. The observation of several families of alleles at the population level provides information about the evolutionary history and mutation processes of microsatellites and may have implications for the use of these markers in phylogenetic, linkage disequilibrium studies, and gene mapping. Received: 14 May 1996 / Accepted: 9 September 1996     

Isolation of a further anonymous informative DNA sequence from chromosome seven closely linked to cystic fibrosis.

A library prepared from flow-sorted chromosomes was used to isolate single-copy sequences from chromosome seven. One such sequence 7C22 has been shown to be polymorphic for an EcoRI restriction site and to be informative for the study of CF in approximately 35% of matings. The segregation of the 7C22 alleles was followed through nineteen informative families with more than one child affected by cystic fibrosis. We report that the locus for 7C22 is linked to the locus for cystic fibrosis at a recombination fraction of 0.045. This marker will prove useful in improving the accuracy and informativeness of prenatal diagnosis and in constructing a fine genetic map around the cystic fibrosis gene.     

Analysis of VH gene replacement events in a B cell lymphoma

We have analyzed a series of recombinational events at the IgH chain locus of the B cell lymphoma, NFS-5. Each of these recombinational events results in the replacement of the VH gene segment of the rearranged H chain gene (VhDJh) with that of an upstream germline gene segment. Replacements on the productive and nonproductive alleles have been observed. In each case, the recombination occurs in close proximity to a highly conserved heptameric sequence (5'TACTGTG3') which is located at the 3' end of the VH coding region. In the two examples of recombination on the productive allele that have been analyzed, the initial VHQ52 gene is replaced by different VH7183 genes. On the non-productive allele, sequential replacement events have been analyzed: the initial VHQ52 rearrangement is first replaced by a closely related VHQ52 gene, followed by a second replacement using a VHQ52 pseudogene. Southern blot analysis using VH probes indicates that these recombinations may be accompanied by the deletion of germline VH genes belonging to both the VHQ52 and VH7183 families, suggesting that these gene families are interspersed in the NFS/N mouse.     

The nature of introns 4-7 largely reflects the lineage specificity of HLA-A alleles

For most HLA-A alleles the phylogeny of the 3' non-coding regions has not yet been studied systematically. In this study, we have determined the sequences of introns 4-7 in 50 HLA-A variants, and have computed nucleotide substitution rates and phylogenetic relationships. The A2/A28, A9, and A10 groups were characterized by clear lineage specificity. For the A19 group, lineage specificity was weaker. A*3001 clustered together with the alleles of the A1/A3/A11/A36 serological family, but not with the A19 group alleles. Reduced lineage specificity was also observed for the alleles of the A1/A3/A11/A36groups. The 3' intron sequences of A*8001 were clearly distinct from all other alleles studied. In several cases two allelic groups shared identical intron sequences, whereby the patterns varied with the introns. A similar situation has been previously described for the 5' introns. Since recombination is the major mechanism of HLA diversification, the intronic lineage specificity corresponds to the comparatively lower recombination rate of the HLA-A 3' exons. The low level of recombination within the 3' region of HLA-A is supported by the low CpG content with a maximum of 3.0% in this region compared with up to 10.7% in the 5' region. Apart from phylogenetic studies of HLA diversity and diversification, the sequence data obtained in our study may prove valuable for the development of a haplotype-specific sequencing strategy for the HLA-A3' exons and for the explanation of recombination events in newly described HLA class I alleles.     

Germline deletion of Igh 3' regulatory region elements hs 5, 6, 7 (hs5-7) affects B cell-specific regulation, rearrangement, and insulation of the Igh locus

Regulatory elements located within an ～28-kb region 3' of the Igh gene cluster (3' regulatory region) are required for class switch recombination and for high levels of IgH expression in plasma cells. We previously defined novel DNase I hypersensitive sites (hs) 5, 6, 7 immediately downstream of this region. The hs 5-7 region (hs5-7) contains a high density of binding sites for CCCTC-binding factor (CTCF), a zinc finger protein associated with mammalian insulator activity, and is an anchor for interactions with CTCF sites flanking the D(H) region. To test the function of hs5-7, we generated mice with an 8-kb deletion encompassing all three hs elements. B cells from hs5-7 knockout (KO) (hs5-7KO) mice showed a modest increase in expression of the nearest downstream gene. In addition, Igh alleles in hs5-7KO mice were in a less contracted configuration compared with wild-type Igh alleles and showed a 2-fold increase in the usage of proximal V(H)7183 gene families. Hs5-7KO mice were essentially indistinguishable from wild-type mice in B cell development, allelic regulation, class switch recombination, and chromosomal looping. We conclude that hs5-7, a high-density CTCF-binding region at the 3' end of the Igh locus, impacts usage of V(H) regions as far as 500 kb away.     

Nucleotide sequences of heat shock activated genes in Drosophila melanogaster. I. Sequences in the regions of the 5' and 3' ends of the hsp 70 gene in the hybrid plasmid 56H8.

We present the sequences at the 5' and 3' ends of one hsp 70 gene variant which is derived from the chromosomal locus 87A7. The 5' end of the hsp 70 mRNA has also been determined. 550 bp upstream from the 5' end of the hsp 70 mRNA, there is a very A+T rich region shown by heteroduplex analysis to be also present at the same position in other hsp 70 genes9. The 5' end of the hsp 70 mRNA was found 26 bp after a characteristic "Hogness box". The first ATG codon was found 250 bp downstream from the 5' end of the hsp 70 mRNA. We also determined the termination codon at the 3' end of the hsp 70 gene. Comparisons with other genes are discussed.     

Linkage and association mapping of the LRP5 locus on chromosome 11q13 in type 1 diabetes

Linkage of chromosome 11q13 to type 1 diabetes (T1D) was first reported from genome scans (Davies et al. 1994; Hashimoto et al. 1994) resulting in P <2.2 x 10(-5) (Luo et al. 1996) and designated IDDM4 ( insulin dependent diabetes mellitus 4). Association mapping under the linkage peak using 12 polymorphic microsatellite markers suggested some evidence of association with a two-marker haplotype, D11S1917*03-H0570POLYA*02, which was under-transmitted to affected siblings and over-transmitted to unaffected siblings ( P=1.5 x 10(-6)) (Nakagawa et al. 1998). Others have reported evidence for T1D association of the microsatellite marker D11S987, which is approximately 100 kb proximal to D11S1917 (Eckenrode et al. 2000). We have sequenced a 400-kb interval surrounding these loci and identified four genes, including the low-density lipoprotein receptor related protein (LRP5) gene, which has been considered as a functional candidate gene for T1D (Hey et al. 1998; Twells et al. 2001). Consequently, we have developed a comprehensive SNP map of the LRP5 gene region, and identified 95 SNPs encompassing 269 kb of genomic DNA, characterised the LD in the region and haplotypes (Twells et al. 2003). Here, we present our refined linkage curve of the IDDM4 region, comprising 32 microsatellite markers and 12 SNPs, providing a peak MLS=2.58, P=5 x 10(-4), at LRP5 g.17646G>T. The disease association data, largely focused in the LRP5 region with 1,106 T1D families, provided no further evidence for disease association at LRP5 or at D11S987. A second dataset, comprising 1,569 families from Finland, failed to replicate our previous findings at LRP5. The continued search for the variants of the putative IDDM4 locus will greatly benefit from the future development of a haplotype map of the genome.     

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction.     

The Maize Transposable Element Ac Induces Recombination between the Donor Site and an Homologous Ectopic Sequence

The prominent repair mechanism of DNA double-strand breaks formed upon excision of the maize Ac transposable element is via nonhomologous end joining. In this work we have studied the role of homologous recombination as an additional repair pathway. To this end, we developed an assay whereby β-Glucuronidase (GUS) activity is restored upon recombination between two homologous ectopic (nonallelic) sequences in transgenic tobacco plants. One of the recombination partners carried a deletion at the 5' end of GUS and an Ac or a Ds element inserted at the deletion site. The other partner carried an intact 5' end of the GUS open reading frame and had a deletion at the 3' end of the gene. Based on GUS reactivation data, we found that the excision of Ac induced recombination between ectopic sequences by at least two orders of magnitude. Recombination events, visualized by blue staining, were detected in seedlings, in pollen and in protoplasts. DNA fragments corresponding to recombination events were recovered exclusively in crosses with Ac-carrying plants, providing physical evidence for Ac-induced ectopic recombination. The occurrence of ectopic recombination following double-strand breaks is a potentially important factor in plant genome evolution.     

Pronounced differences of recombination activity at the sex determination locus of the honeybee, a locus under strong balancing selection

Recombination decreases the association of linked nucleotide sites and can influence levels of polymorphism in natural populations. When coupled with selection, recombination may relax potential conflict among linked genes, a concept that has played a central role in research on the evolution of recombination. The sex determination locus (SDL) of the honeybee is an informative example for exploring the combined forces of recombination, selection, and linkage on sequence evolution. Balancing selection at SDL is very strong and homozygous individuals at SDL are eliminated by worker bees. The recombination rate is increased up to four times that of the genomewide average in the region surrounding SDL. Analysis of nucleotide diversity (pi) reveals a sevenfold increase of polymorphism within the sex determination gene complementary sex determiner (csd) that rapidly declines within 45 kb to levels of genomewide estimates. Although no recombination was observed within SDL, which contains csd, analyses of heterogeneity, shared polymorphic sites, and linkage disequilibrium (LD) show that recombination has contributed to the evolution of the 5' part of some csd sequences. Gene conversion, however, has not obviously contributed to the evolution of csd sequences. The local control of recombination appears to be related to SDL function and mode of selection. The homogenizing force of recombination is reduced within SDL, which preserves allelic differences and specificity, while the increase of recombination activity around SDL relaxes conflict between SDL and linked genes.     

De novo rearrangements found in 2% of index patients with spinal muscular atrophy: mutational mechanisms, parental origin, mutation rate, and implications for genetic counseling.

Spinal muscular atrophy (SMA) is a relatively common autosomal recessive neuromuscular disorder. We have identified de novo rearrangements in 7 (approximately 2%) index patients from 340 informative SMA families. In each, the rearrangements resulted in the absence of the telomeric copy of the survival motor neuron (SMN) gene (telSMN), in two cases accompanied by the loss of the neuronal apoptosis-inhibitory protein gene . Haplotype analysis revealed unequal recombination in four cases, with loss of markers Ag1-CA and C212, which are near the 5' ends of the SMN genes. In one case, an interchromosomal rearrangement involving both the SMN genes and a regrouping of Ag1-CA and C212 alleles must have occurred, suggesting either interchromosomal gene conversion or double recombination. In two cases, no such rearrangement was observed, but loss of telSMN plus Ag1-CA and C212 alleles in one case suggested intrachromosomal deletion or gene conversion. In six of the seven cases, the de novo rearrangement had occurred during paternal meiosis. Direct detection of de novo SMA mutations by molecular genetic means has allowed us to estimate for the first time the mutation rate for a recessive disorder in humans. The sex-averaged rate of 1.1 x 10(-4), arrived at in a proband-based approach, compares well with the rate of 0.9 x 10(-4) expected under a mutation-selection equilibrium for SMA. These findings have important implications for genetic counseling and prenatal diagnosis in that they emphasize the relevance of indirect genotype analysis in combination with direct SMN-gene deletion testing in SMA families.     

RNA recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus A59 and fusion-negative mouse hepatitis virus 2.

It has previously been shown that the murine coronavirus mouse hepatitis virus (MHV) undergoes RNA recombination at a relatively high frequency in both tissue culture and infected animals. Thus far, all of the recombination sites had been localized at the 5' half of the RNA genome. We have now performed a cross between MHV-2, a fusion-negative murine coronavirus, and a temperature-sensitive mutant of the A59 strain of MHV, which is fusion positive at the permissive temperature. By selecting fusion-positive viruses at the nonpermissive temperature, we isolated several recombinants containing multiple crossovers in a single genome. Some of the recombinants became fusion negative during the plaque purification. The fusion ability of the recombinants parallels the presence or absence of the A59 genomic sequences encoding peplomers. Several of the recombinants have crossovers within 3' end genes which encode viral structural proteins, N and E1. These recombination sites were not specifically selected with the selection markers used. This finding, together with results of previous recombination studies, indicates that RNA recombination can occur almost anywhere from the 5' end to the 3' end along the entire genome. The data also show that the replacement of A59 genetic sequences at the 5' end of gene C, which encodes the peplomer protein, with the fusion-negative MHV-2 sequences do not affect the fusion ability of the recombinant viruses. Thus, the crucial determinant for the fusion-inducing capability appears to reside in the more carboxyl portion of the peplomer protein.     

Segregation and linkage studies of plasma dopamine-beta-hydroxylase (DBH), erythrocyte catechol-O-methyltransferase (COMT), and platelet monoamine oxidase (MAO): possible linkage between the ABO locus and a gene controlling DBH activity.

Measurements of dopamine-beta-hydroxylase (DBH), catechol-O-methyltransferase (COMT), and monoamine oxidase (MAO) along with 27 polymorphic marker phenotypes were available for 162 patients with major affective disorders and 1,125 of their relatives. Levels of enzymes were previously found not to be associated with illness. Pedigree analysis methods for quantitative traits are used to test single-gene hypotheses for segregation of DBH in 32 families with 411 individuals. COMT in 30 families with 351 individuals, and MAO in 50 families with 309 individuals. The familial distribution of both DBH and COMT are consistent with two codominant alleles at the same locus that account for 56% and 59% of the total variance, respectively. MAO activity cannot be shown to be segregating as a single major gene, but a purely nongenetic hypothesis is also rejected. A possible linkage of a locus for DBH to the ABO locus is indicated by a maximum lod score of 1.82 at 0% and 10% recombination fractions for males and females, respectively. A lod score of 0.61 at 0% recombination for a similar analysis in a single large pedigree was reported by Elston et al., making the combined lod score for the two studies equal to 2.32 at 0% recombination.