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Gene conversion, linkage, and the evolution of multigene families.

The evolution of the probabilities of genetic identity within and between the loci of a multigene family is investigated. Unbiased gene conversion, equal crossing over, random genetic drift, and mutation to new alleles are incorporated. Generations are discrete and nonoverlapping; the diploid, monoecious population mates at random. The linkage map is arbitrary, and the location dependence of the probabilities of identity is formulated exactly. The greatest of the rates of gene conversion, random drift, and mutation is epsilon much less than 1. For interchromosomal conversion, the equilibrium probabilities of identity are within order epsilon [i.e., O(epsilon)] of those in a simple model that has no location dependence and, at equilibrium, no linkage disequilibrium. At equilibrium, the linkage disequilibria are of O(epsilon); they are evaluated explicitly with an error of O(epsilon 2); they may be negative if symmetric heteroduplexes occur. The ultimate rate and pattern of convergence to equilibrium are within O(epsilon 2) and O(epsilon), respectively, of that of the same simple model. If linkage is loose (i.e., all the crossover rates greatly exceed epsilon, though they may still be much less than 1/2), the linkage disequilibria are reduced to O(epsilon) in a time of O(-ln epsilon). If intrachromosomal conversion is incorporated, the same results hold for loose linkage, except that, if the crossover rates are much less than 1/2, then the linkage disequilibria generally exceed those for pure interchromosomal conversion.

Biological Evolution↗

R339H and P453S: CYP21 mutations associated with nonclassic steroid 21-hydroxylase deficiency that are not apparent gene conversions.

Steroid 21-hydroxylase deficiency is the most common enzymatic defect causing congenital adrenal hyperplasia, an inherited disorder of cortisol biosynthesis. All mutations thus far characterized that cause this disorder appear to result from recombinations between the gene encoding the enzyme, CYP21B (CYP21), and the adjacent pseudogene, CYP21A (CYP21P). These are either deletions caused by unequal crossing-over during meiosis or apparent transfers of deleterious sequences from CYP21A to CYP21B, a phenomenon termed gene conversion. However, a small percentage of alleles do not carry such a mutation. We analyzed DNA from a patient with the mild, nonclassic form of 21-hydroxylase deficiency, who carried one allele that had no gene conversions detectable by hybridization with oligonucleotide probes. Sequence analysis revealed that this allele carried two missense mutations, R339H and P453S, neither of which has been previously observed in CYP21A or CYP21B. Each of these mutations was introduced into CYP21 cDNA which was then expressed in COS1 cells using a vaccinia virus system. Each mutation reduced the ability of the enzyme to 21-hydroxylate 17-hydroxyprogesterone to 50% of normal and the ability to metabolize progesterone to 20% of normal. Thus, each of these mutations represents a potential nonclassic 21-hydroxylase deficiency allele that is not the result of an apparent gene conversion.

Adrenal Hyperplasia, Congenital↗

Activation-induced cytidine deaminase initiates immunoglobulin gene conversion and hypermutation by a common intermediate.

Depending on the species and the lymphoid organ, activation-induced cytidine deaminase (AID) expression triggers diversification of the rearranged immunoglobulin (Ig) genes by pseudo V (psiV) gene- templated gene conversion or somatic hypermutation. To investigate how AID can alternatively induce recombination or hypermutation, psiV gene deletions were introduced into the rearranged light chain locus of the DT40 B-cell line. We show that the stepwise removal of the psiV donors not only reduces and eventually abolishes Ig gene conversion, but also activates AID-dependent Ig hypermutation. This strongly supports a model in which AID induces a common modification in the rearranged V(D)J segment, leading to a conversion tract in the presence of nearby donor sequences and to a point mutation in their absence.

Animals↗

Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer sequences.

It is thought that gene conversion (GCV) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes occur in two steps: the generation of uracils in DNA by activation-induced cytidine deaminase, followed by their subsequent repair by various DNA repair pathways to generate sequence-diversified products. It is not known how either of the two steps is targeted specifically to Ig loci. Because of the tight link between transcription and SHM, we have investigated the role of endogenous Ig light chain (IgL) transcriptional control elements in GCV/SHM in the chicken B cell line DT40. Promoter substitution experiments led to identification of a strong RNA polymerase II promoter incapable of supporting efficient GCV/SHM. This surprising finding indicates that high levels of transcription are not sufficient for robust GCV/SHM in Ig loci. Deletion of the IgL enhancer in a context in which high-level transcription was not compromised showed that the enhancer is not necessary for GCV/SHM. Our results indicate that cis-acting elements are important for Ig gene diversification, and we propose that targeting specificity is achieved through the combined action of several Ig locus elements that include the promoter.

Actins↗

Serological confirmation in Ia mutant B6.C-H-2bm12 of gene conversion.

Recently it has been suggested that a short segment of the DNA sequence of the Ia beta gene in the mutant B6.C-H-2bm12 (bm12) is derived from the Eb beta gene by a gene-conversion-like mechanism, and that the same segment is also seen in the Ek beta gene. To obtain serological evidence for this idea, we produced an antibody against the "new" determinant on the I-Abm12 molecule by immunizing A.BY mice with bm12 cells. After absorption with B6 lymphocytes to remove antibodies against background antigens, the antisera lysed bm12 cells. Typical Ia peaks were obtained by immunoprecipitation. The absorbed antiserum reacted with B10.A (Ek beta), B10.A(5R) (Eb beta) and B10.S(9R) (Es beta), but not B10. The unabsorbed antiserum is specific for Ia when tested on A background mice. The antiserum lysed spleen cells of Ik strains (A/Sn, A.AL, A.TBR1, and A.TFR1) but not Ak strain, A.TBR13 (KsAkEbSbDb), confirming the presence of antibodies against the I-Ek molecule. This anti-serum also lysed the cells from (A.BY X A.TFR5)F1, which expresses the transcomplementing Eb beta Ek alpha molecule and from the (A.SW X A.TFR5)F1 which expresses the transcomplementing Es beta Ek alpha molecule. These data are consistent with DNA sequence analyses, and show the existence of a determinant (Ia.51) generated in the bm12 mutant by a gene-conversion-like event that is also present in the I-Ek, I-Eb, and I-Es beta polypeptide chains.

Animals↗

Statistical methods of DNA sequence analysis: detection of intragenic recombination or gene conversion.

Simple but exact statistical tests for detecting a cluster of associated nucleotide changes in DNA are presented. The tests are based on the linear distribution of a set of s sites among a total of n sites, where the s sites may be the variable sites, sites of insertion/deletion, or categorized in some other way. These tests are especially useful for detecting gene conversion and intragenic recombination in a sample of DNA sequences. In this case, the sites of interest are those that correspond to particular ways of splitting the sequences into two groups (e.g., sequences A and D vs. sequences B, C, and E-J). Each such split is termed a phylogenetic partition. Application of these methods to a well-documented case of gene conversion in human gamma-globin genes shows that sites corresponding to two of the three observed partitions are significantly clustered, whereas application to hominoid mitochondrial DNA sequences--among which no recombination is expected to occur--shows no evidence of such clustering. This indicates that clustering of partition-specific sites is largely due to intragenic recombination or gene conversion. Alternative hypotheses explaining the observed clustering of sites, such as biased selection or mutation, are discussed.

Animals↗

Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: a novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease.

OBJECTIVE: To analyse the mutational spectrum, the associated haplotypes and the genotype-phenotype correlation, and to design a reliable and rational approach for CYP21 mutation detection in Slovenian congenital adrenal hyperplasia (CAH) patients. DESIGN: Molecular analysis of the CYP21 gene was performed in 36 CAH patients and 79 family members. METHODS: Southern blotting, sequence-specific PCR amplification (PCR-SSP), sequence-specific oligonucleotide hybridisation (PCR-SSO) and sequencing were used to detect CYP21 gene deletions, conversions and point mutations. RESULTS: CYP21 gene deletion was the most frequent mutation (36.4%). Large gene conversions detectable only by Southern blotting represented 12.1%, and gene conversions involving the promoter region represented 7.6% of the mutated alleles. The most frequent point mutations were: intron 2 splice mutation 16.7%, Ile172Asn mutation 7.6%, Gln318Stop 7.5% and Pro30Leu 12.2% of alleles. A correlation between the genotype and the clinical phenotype similar to those described for large populations was observed. The finding of Pro30Leu mutation linked to a gene conversion could explain the simple virilising (SV) phenotype in compound heterozygotes for the Pro30Leu and a severe mutation. In two siblings with a salt wasting form of CAH (SW-CAH), a novel mutation Ala15Thr was found on the allele characterised by Pro30Leu mutation and gene conversion involving the promoter region. CONCLUSIONS: Our genotyping approach allowed reliable diagnosis of CAH in the Slovenian population. The high frequency of CYP21 gene aberrations on Pro30Leu positive alleles justified systematic searching for a gene conversion in the promoter region using the PCR-SSP reaction.

Adrenal Hyperplasia, Congenital↗

Biased intrachromosomal gene conversion in a chromosome lineage.

A model for the evolution of a family of tandemly repeated genes in a single chromosome lineage under intrachromosomal gene conversion [43] is analyzed further and extended. Direct and diffusion approximations are derived for the exact fixation probabilities, mean time to fixation or loss, and mean conditional fixation time of Nagylaki and Petes [43]. The distribution of the number of variant repeats under the joint action of gene conversion and reversible mutation is investigated; exact and approximate expressions are derived for the stationary distribution. It is shown that conversional bias greatly increases the amount of sequence homogeneity at equilibrium. The diffusion processes studied here also apply to selection and mutation in a finite population, and some new results are established for that classical problem.

Alleles↗

Cytochrome P450 and actin genes expressed in Helicoverpa zea and Helicoverpa armigera: paralogy/orthology identification, gene conversion and evolution.

Molecular phylogenetic analysis was conducted using conserved cytoplasmic actin and diversified cytochrome P450 (P450) sequences isolated from Helicoverpa zea and Helicoverpa armigera, two species thought to be closely related based on allozyme analyses. These sequences were compared in turn with published sequences from other insects to gain insight into how different gene families evolve. In Bombyx mori and these Helicoverpa species, cytoplasmic actin genes are present as a pair of tandemly duplicated paralogs with coding sequence identities as high as 95.5% (B. mori), 98.9% (H. zea) and 98.5% (H. armigera) due to recent 5'-polar gene conversions. Phylogeny and interspecies comparisons assign the six actin genes into two orthologous groups: HaA3a/HzA3a/BmA3 and HaA3b/HzA3b/BmA4, which exhibit more similarities between H. zea and H. armigera than between Helicoverpa species and B. mori. Like the actin genes in H. zea, four CYP6B genes exist as two pairs of duplicated paralogs with recent 5'-polar gene conversions. Interspecific comparisons and phylogeny analysis identified three groups of orthologous CYP6B genes: H. zea CYP6B8 or CYP6B28/H. armigera CYP6B7, H. zea CYP6B27/H. armigera CYP6B6, and H. zea CYP6B9/H. armigera CYP6B2/Heliothis virescens CYP6B10. The low degree of divergence in the first two of these groups is comparable to allelic variation within a single species. These orthologous relationships and the high degrees of similarity in both actin and P450 genes strongly indicate that these Helicoverpa species are extremely closely related.

Actins↗

Evolution of Rh blood group genes have experienced gene conversions and positive selection.

There are two tightly linked loci (D and CE) for the human Rh blood group. Their gene products are membrane proteins having 12 transmembrane domains and form a complex with Rh50 glycoprotein on erythrocytes. We constructed phylogenetic networks of human and nonhuman primate Rh genes, and the network patterns suggested the occurrences of gene conversions. We therefore used a modified site-by-site reconstruction method by using two assumed gene trees and detected 9 or 11 converted regions. After eliminating the effect of gene conversions, we estimated numbers of nonsynonymous and synonymous substitutions for each branch of both trees. Whichever gene tree we selected the branch connecting hominoids and Old World monkeys showed significantly higher nonsynonymous than synonymous substitutions, an indication of positive selection. Many other branches also showed higher nonsynonymous than synonymous substitutions; this suggests that the Rh genes have experienced some kind of positive selection.

Animals↗

Gene conversions within the skeletal myosin multigene family.

Comparisons of the nucleotide sequences of the light meromyosin (LMM) region of developmentally regulated fast chicken myosin heavy chain (MHC) isoforms indicates that chicken MHC isoforms are more similar to each other than to MHC isoforms in other species. The sequence data provide evidence that gene conversion events have occurred recently among the isoforms. An embryonic (Cemb1) isoform and neonatal isoform have the most extensive regions of sequence identity. Similar gene conversion events are present in the rat alpha- and beta-cardiac MHCs, but were not obvious in the LMM of developmentally regulated fast human MHC isoforms. The data suggest that gene conversion events can play a significant role in the evolution of the MHC multigene families and that concerted evolution of the chicken multigene family occurred after the divergence of mammals and avians.

Animals↗

Gene conversion and different population histories may explain the contrast between polymorphism and linkage disequilibrium levels.

To characterize linkage disequilibrium (LD) levels in human populations, we have analyzed 10 independent noncoding segments in three population samples from the major ethnic groups--that is, Africans, Asians, and Europeans. Descriptive statistics show that LD decays much faster in the African samples than in the non-African ones. With the assumption of an equilibrium model, we estimated the population crossing-over parameter (4N(e)r(bp), where N(e) is the effective population size and r(bp) is the crossing-over rate per generation between adjacent base pairs) in the presence of gene conversion. In the African sample, LD and polymorphism levels lead to similar estimates of effective population size, as expected under an equilibrium model. Conversely, in both non-African samples, LD levels suggest a smaller effective population size than that implied by polymorphism levels. This observation is paralleled by significant departures from an equilibrium model in the spectrum of allele frequencies of the non-African samples. Besides ruling out the possibility that non-African populations are at equilibrium, these results suggest different demographic history (temporal and spatial) of these groups. Interestingly, the African sample fits the expectations of an equilibrium model based on polymorphism and divergence levels and on frequency spectrum. For this sample, the estimated ratio of gene conversion to crossing-over rates is 7.3 for a mean tract length of 500 bp, suggesting that gene conversion may be more frequent than previously thought. These findings imply that disease-association studies will require a much denser map of polymorphic sites in African than in non-African populations.

Africa↗

A role for reverse transcripts in gene conversion.

Recombination between a diffusible reverse transcript and its homologous chromosomal allele has been proposed as a mechanism for the precise removal of introns from DNA and gene conversion of dispersed repeated sequences. We have reported that RNA-mediated recombination occurs in the yeast Saccharomyces cerevisiae. This recombination requires expression of the retrotransposon Ty, and results in intron loss from a plasmid-borne marker gene and the formation of pseudogenes. Because the pseudogenes are embedded in Ty sequences, chromosomal insertion could have been mediated by Ty integrase or by homologous recombination with endogenous Ty sequences. The structure of the chromosomal recombinants and the fact that plasmid and chromosomal recombination can have different requirements demanded a direct demonstration of RNA-mediated gene conversion of a chromosomal allele. Here we report the first demonstration, to our knowledge, of recombination between a reverse transcript and its chromosomal homologue and describe an assay that specifically detects this novel recombination pathway.

Base Sequence↗

Molecular models accounting for the gene conversion reactions mediating gonococcal pilin antigenic variation.

The pilus antigenic variation (Av) system of Neisseria gonorrhoeae is one of several high-frequency variation systems that utilize gene conversion to switch between numerous forms of an antigen on the cell surface. We have tested three predictions of the first models that explain the movement of DNA during pilin Av: (i) Av requires two recombinations at short regions of identity, (ii) circular intermediates exist that carry pilE/pilS hybrid loci and (iii) these pilE/pilS hybrid loci target the pilS sequences to a recipient pilE gene. We confirm that normal pilin Av utilizes recombination at very short regions of DNA sequence identity and that these recombination events can occur independent of homologous recombination functions. We have isolated covalently closed circular DNA molecules carrying hybrid pilin loci, but propose that an alternative hybrid molecule is the intermediate of pilin Av. Our most striking finding is that transformation of isolated pilE/pilS hybrid loci targets the pilS sequences of the hybrid to a recipient pilE at frequencies much higher than normal recombination frequencies. These results show that the different steps of a model that explains pilin Av can be separately tested to support the validity of these novel models that account for the high-frequency gene conversions that mediate pilin Av.

Antigenic Variation↗

The estimation of the number and the length distribution of gene conversion tracts from population DNA sequence data.

DNA sequence variation studies report the transfer of small segments of DNA among different sequences caused by gene conversion events. Here, we provide an algorithm to detect gene conversion tracts and a statistical model to estimate the number and the length distribution of conversion tracts for population DNA sequence data. Two length distributions are defined in the model: (1) that of the observed tract lengths and (2) that of the true tract lengths. If the latter follows a geometric distribution, the relationship between both distributions depends on two basic parameters: psi, which measures the probability of detecting a converted site, and phi, the parameter of the geometric distribution, from which the average true tract length, 1/(1-phi), can be estimated. Expressions are provided for estimating phi by the method of the moments and that of the maximum likelihood. The robustness of the model is examined by computer simulation. The present methods have been applied to the published rp49 sequences of Drosophila subobscura. Maximum likelihood estimate of phi for this data set is 0.9918, which represents an average conversion tract length of 122 bp. Only a small percentage of extant conversion events is detected.

Animals↗

Eliminating gene conversion improves high-throughput genetics in Saccharomyces cerevisiae.

Synthetic genetic analysis was improved by eliminating leaky expression of the HIS3 reporter and gene conversion between the HIS3 reporter and his3Delta1. Leaky expression was eliminated using 3-aminotriazole and gene conversion was eliminated by using the Schizosaccharomyces pombe his5+ gene, resulting in a 5- to 10-fold improvement in the efficiency of SGA.

Amitrole↗

Possible involvement of the yeast POLIII DNA polymerase in induced gene conversion.

In Saccharomyces cerevisiae, three different DNA polymerase complexes, POLI, POLII and POLIII, are known to be involved in DNA replication. The catalytic subunit of POLIII is encoded by the essential CDC2 gene. The existence of different thermosensitive noncomplementing mutants of CDC2 offers the possibility of using a genetic approach to investigate the involvement of POLIII in induced gene conversion. When cdc2 heteroallelic cells were irradiated and incubated under restrictive conditions, almost no induction of thermoresistant cells could be detected, suggesting an essential role for POLIII in mitotic gene conversion events.

DNA Polymerase III↗

Overexpression of Rad51 inhibits double-strand break-induced homologous recombination but does not affect gene conversion tract lengths.

DNA double-strand breaks (DSBs) in yeast are repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad51 forms nucleoprotein filaments at processed broken ends that effect strand exchange, forming heteroduplex DNA (hDNA) that gives rise to a gene conversion tract. We hypothesized that excess Rad51 would increase gene conversion tract lengths. We found that excess Rad51 reduced DSB-induced HR but did not alter tract lengths or other outcomes including rates of crossovers, break-induced replication, or chromosome loss. Thus, excess Rad51 appears to influence DSB-induced HR at an early stage. MAT heterozygosity largely mitigated the inhibitory effect of excess Rad51 on allelic HR, but not direct repeat HR. Excess Rad52 had no effect on DSB-induced HR efficiency or outcome, nor did it mitigate the dominant negative effects of excess Rad51. Excess Rad51 had little effect on DSB-induced lethality in wild-type cells, but it did enhance lethality in yku70Delta mutants. Interestingly, dnl4Delta showed marked DSB-induced lethality but this was not further enhanced by excess Rad51. The differential effects of yku70Delta and dnl4Delta indicate that the enhanced killing with excess Rad51 in yku70Delta is not due to its NHEJ defect, but may reflect its defect in end-protection and/or its inability to escape from checkpoint arrest. Srs2 displaces Rad51 from nucleoprotein filaments in vitro, suggesting that excess Rad51 might antagonize Srs2. We show that excess Rad51 does not reduce survival of wild-type cells treated with methylmethane sulfonate (MMS), or cells suffering a single DSB. In contrast, excess Rad51 sensitized srs2Delta cells to both MMS and a single DSB. These results support the idea that excess Rad51 antagonizes Srs2, and underscores the importance of displacing Rad51 from nucleoprotein filaments to achieve optimum repair efficiency.

Cell Survival↗