Gene Conversion-like Events Research Articles

Diversity and evolution of the DRB1*03 family: description of DRB1*03022,*0307,*0308.

Three previously unreported DRB1*03 alleles are described, adding to the diversity of the DRB1 family of alleles. DRB1*03022 contains a silent substitution at codon 77. DRB1*0307 differs from DRB1*03011 by a substitution at codon 26 resulting in a predicted change from tyrosine to phenylalanine. DRB1*0308 is almost identical to DRB1*03011 differing at codon 58 which specifies the glutamic acid residue commonly found in DRB1*11 alleles. The new alleles (DRB1*03022,*0307,*0308) may have arisen by gene conversion-like events and add to the increasing complexity of the HLA system.

A new HLA-DQB1*0306 allele sharing motifs from DQB1*03032 and DQB1*04 sequences.

We have discovered a new HLA-DQB1 allele in a Japanese family, MAT. In the family the new allele segregates in three generations and demonstrates the positive association with DRB1*0901. We observed a novel RFLP pattern in the course of examining the modified PCR-RFLP method for HLA-DQB1 genotyping. The PCR-SSOP analysis also showed a new hybridized pattern. Sequence analysis of the allele indicates that it was generated by a gene conversion-like event between the HLADQB1*03032 and one of DQB1*04 contemporary alleles. This new allelic product did not react with all of allosera and monoclonal antibodies against DQ1, DQ2, DQ3, DQ4 and DQ7. The HLA molecule encoded by the allele is not defined by serology. This new allele was officially recognized and named DQB1*0306 by the WHO Nomenclature Committee in November 1995.

Molecular Genetic Analysis of Variant Phenotypes of the ABO Blood Group System

ABO is clinically the most important blood group system in transfusion medicine and includes many variant phenotypes. To understand the molecular genetic basis of this polymorphic system, we have analyzed genomic DNAs obtained from Japanese individuals possessing variant ABO phenotypes including A2, Ax, Ael, cis-AB, Bx, and Bel. By polymerase chain reaction-single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses, we identified 11 different alleles. These alleles had nucleotide sequences different from those of the previously described 13 different alleles responsible for the common ABO phenotypes. Analysis of the nucleotide sequences of the alleles responsible for those variant phenotypes showed that the amino acid residues at position 266 and 268 may be crucial for transferase specificity, whereas those at positions 214, 216, 223, 291, and 352 may be critical for the activity level. Nine of the 11 alleles, responsible for the A2, Ax, Ael, cis-AB, Bx, and Bel phenotypes, were presumed to be generated from common ABO alleles by single nucleotide mutations such as nonsynonymous substitution, deletion, or insertion. Two other alleles, responsible for the A2 and Ael phenotypes, may have originated by recombination, gene conversionlike events or accumulation of nucleotide substitutions. Our data indicate that different alleles could cause the same ABO variant phenotypes, and that these alleles do not necessarily belong to a single evolutionary lineage.

A determination of the frequency of gene conversion in unmanipulated mouse sperm.

Gene conversion, sometimes also called micro gene conversion or gene conversion-like events, has been proposed to act on a number of genes in higher eukaryotes, such as gamma-globin, beta-tubulin, major urinary protein, and amyloid A genes. In the immune system, immunoglobulin genes and major histocompatibility complex class I and class II genes have been implicated. The notion that integral segments of DNA have been transferred from one gene to another in these cases has, however, met considerable resistance. We have devised a PCR assay detecting only the molecule that results if the E beta d-derived fragment analogous to that introduced in the bm12 mutation is transferred to the A beta k gene. We have proceeded to analyze sperm from the F1 cross C3H/HeJ (haplotype k) x BALB/c (haplotype d). In our assay, we find that the frequency for conversion of this particular DNA segment is 2 x 10(-6). This frequency is relevant only in the germ line; when liver cells were tested as an example of somatic cells, no events were observed, implying a frequency of < 2 x 10(-8) in liver. Fragments > 100 bp seem to be possible to transfer in this conversion.

Serologic and nucleotide sequencing analyses of a novel DR52-associated DRB1 allele with the DR ‘NJ25’ specificity, designated DRB1 ∗1307

A novel DR52-associated DRB1 ∗ allele, designated DRB1 ∗1307, was encountered in the course of our HLA-DRB1 genotyping study in a Japanese population by PCR-RFLP. Comparison of the nucleotide sequence of its second exon with those of the other known DRB1 alleles revealed that DRB1 ∗1307 was most similar to DRB1 ∗1101, differing by two amino acid substitutions. From a family study, DRB1 ∗1307 was found to segregate with a haplotype of DRB3 ∗0202-DQA1 ∗0501-DQB1 ∗0301, which was also observed with DRB1 ∗1101 in a Japanese population. DRB1 ∗1307 was recognized in three of 652 healthy Japanese controls (gene frequency: 0.24%) with the same DR-DQ haplotype, indicating that DRB1 ∗1307 arose from DRB1 ∗1101 by a gene conversionlike event(s) and/or point mutations. Further, it was also observed that this allele had a strong linkage disequilibrium with HLA-B70 ( p < 0.001). This new DRB1 ∗1307 allele was serologically defined as DR ‘NJ25,’ and it gave an almost identical serologic pattern to DRB1 ∗1406. On sequence comparison, however, no unique amino acid residues conserved in DRB1 ∗1406 and DRB1 ∗1307 but absent in all the other DRB1 alleles could be found, indicating that two amino acid changes at positions 47 and 58 abolished the reactivity against the DR11 antisera.

Glycophorin He(Sta) of the human red blood cell membrane is encoded by a complex hybrid gene resulting from two recombinational events

A complex glycophorin (GP) variant of the human red blood cell membrane exhibiting both He and Sta antigens was characterized at the molecular level. Restriction mapping identified two novel Msp I fragments derived from the 5′ and 3′ portions of the GPHe(Sta) gene, respectively. Genomic DNA, including exons II-V and their splice junctions, was amplified by polymerase chain reaction, and the nucleotide sequences were determined. Comparison with the GPA and GPB sequences showed the presence in GPHe(Sta) of multiple recombinational breakpoints. In the 5′ region of the variant gene, a sequence covering a portion of exon II to intron 2 had been transferred from GPA to GPB, resulting in a B-A-B hybrid structure. Such a gene conversion-like event introduced a number of templated and untemplated nucleotide replacements and was the direct cause for the expression of the He antigen. In the 3′ region of the variant gene, an unequal crossover from GPB to GPA took place in the third intron at a recombination site apparently identical to that observed in the B-A hybrid GPSta type A gene. These results indicated that GPHe(Sta) occurs as a B-A-B-A hybrid gene, most likely originating from a two-step mechanism of homologous recombination. Transcript analysis showed the maturation from the GPHe(Sta) pre-mRNA of two shortened mRNAs of which the exon III-deleted species encodes both the He and Sta antigens.

Glycophorin He(Sta) of the Human Red Blood Cell Membrane Is Encoded by a Complex Hybrid Gene Resulting From Two Recombinational Events

A complex glycophorin (GP) variant of the human red blood cell membrane exhibiting both He and Sta antigens was characterized at the molecular level. Restriction mapping identified two novel Msp I fragments derived from the 5' and 3' portions of the GPHe(Sta) gene, respectively. Genomic DNA, including exons II-V and their splice junctions, was amplified by polymerase chain reaction, and the nucleotide sequences were determined. Comparison with the GPA and GPB sequences showed the presence in GPHe(Sta) of multiple recombinational breakpoints. In the 5' region of the variant gene, a sequence covering a portion of exon II to intron 2 had been transferred from GPA to GPB, resulting in a B-A-B hybrid structure. Such a gene conversion-like event introduced a number of templated and untemplated nucleotide replacements and was the direct cause for the expression of the He antigen. In the 3' region of the variant gene, an unequal crossover from GPB to GPA took place in the third intron at a recombination site apparently identical to that observed in the B-A hybrid GPSta type A gene. These results indicated that GPHe(Sta) occurs as a B-A-B-A hybrid gene, most likely originating from a two-step mechanism of homologous recombination. Transcript analysis showed the maturation from the GPHe(Sta) pre-mRNA of two shortened mRNAs of which the exon III-deleted species encodes both the He and Sta antigens.

Tandemly repeated transgenes of the human minisatellite MS32 (D1S8), with novel mouse gamma satellite integration.

The human hypervariable minisatellite MS32 has a well characterised internal repeat unit array and high mutation rates have been observed at this locus. Analysis of MS32 mutants has shown that male germline mutations are polarised to one end of the array and frequently involve complex gene conversion-like events, suggesting that tandem repeat instability may be modulated by cis-acting sequences flanking the locus. In order to investigate the processes affecting MS32 mutation rate and mechanism, we have created transgenic mice harbouring an MS32 allele. Here we describe the organisation of eight transgenic insertions. Analysis of these transgenic loci by MVR-PCR and structural analysis of the junctions between mouse flanking DNA and the transgenic loci has shed light on mechanisms of integration and rearrangement of the tandem repeated transgenes. Sequence analysis of the mouse DNA flanking these transgenes has shown that 5 of the 8 insertions have integrated into mouse gamma satellite repeated sequence. This suggests a non-random integration of the MS32 transgene construct into the mouse genome.

The Vχ genes of the L regions and the repertoire of Vχ gene sequences in the human germ line

Only 14 of the 25 V kappa genes and pseudogenes had been found before as parts of the L regions. The cloning and linking described in the accompanying report allowed us now to assign to Lp or Ld some V kappa genes which had been found before on scattered clones. In addition the sequences of several still unknown genes are reported here, thus completing the publication of the V kappa genes of the kappa locus as far as they are potentially functional or have only one or two 1-bp defects. Of the V kappa genes of the kappa locus, 32 are potentially functional, 16 have minor defects, 3 have both potentially functional and slightly defective alleles and 25 are pseudogenes which amounts to a repertoire of 76 V kappa-related gene sequences. The V kappa genes of the L regions are, within the subgroups, particularly similar to each other, which is in part due to common evolutionary origins and in part caused by gene conversion-like events. One donor-acceptor pair could be clearly identified, since converted and not-converted alleles of the acceptor gene were found. In other cases the duplicates of the converted genes served as non-converted controls.

MOLECULAR PATHOLOGY OF 21-HYDROXYLASE DEFICIENCY

Pathological mutations in steroid 21-hydroxylase deficiency appear to derive exclusively from sequence exchange between the 21-hydroxylase gene (CYP21B) and a closely related pseudogene (CYP21A), located 30 kb telomeric to CYP21B and functionally inactive because of several mutations distributed along its length. Three basic classes of disease chromosome are seen: (a) deletion chromosomes where about 30 kb of DNA has been eliminated and a single 21-hydroxylase gene, often a CYP21A/CYP21B fusion gene, remains; (b) chromosomes which have 2 or more CYP21 genes, but without an apparent evidence for CYP21B-specifisce quences; (c) chromosomes which have 2 or more CYP21 genes, in which there is at least one gene with CYP21B-specific sequences, either a fusion gene or a mosaic, so-called “converted” gene. In the latter type of gene a central segment which may be very small or substantial shows CYP21A-specifics equences. Data will be presented on de novo mutations, showing how disease chromosomes can arise byunequal crossover and gene conversion-like events. The frequencies of different pathological mutations and genotype-phenotype correlations will also be discussed.

A de novo pathological point mutation at the 21–hydroxylase locus: implications for gene conversion in the human genome

More than two hundred characterized 21-hydroxylase deficiency alleles appear to result exclusively from sequence exchanges involving the 21-hydroxylase gene (CYP21B) and a closely related pseudogene (CYP21A). Gene conversion-like events have also been reported in many other human gene clusters, but in the absence of a de novo mutation, the alternative explanation of a multiple recombination is possible. We now report a de novo pathological mutation at the 21-hydroxylase locus. DNA sequence analysis suggests that the mutation arose by a microconversion event involving exchange of up to 390 nucleotides between maternal CYP21A and CYP21B genes. This putative de novo gene conversion event appears to be the first characterized in humans.

Donation: a new, facile method of gene replacement in yeast.

We describe here a new method for the introduction of non-selectable alleles into Saccharomyces cerevisiae, gene replacement by donation. This method only requires the availability of an autonomously replicating, selectable plasmid containing the allele to be introduced into yeast. The plasmid is digested at a restriction site (or sites) within this allele, and introduced into yeast by transformation. In the course of double-strand break repair, the entering plasmid donates genetic information to the chromosome, replacing the chromosomal allele in a gene conversion-like event. Gene replacement events are identified by a phenotypic screen of the transformants. When necessary, the transforming plasmid may be subsequently lost by segregation during permissive growth. We have studied several parameters affecting the utility of this protocol as a method of gene replacement. Together with our previous results, the results show gene replacement by donation to be a useful, facile method, yielding gene replacement in up to 1.5% of transformants.

DNA typing of HLA class II genes in B-lymphoblastoid cell lines homozygous for HLA.

The HLA class II genotypes were determined in the B-lymphoblastoid cell lines selected for the Tenth International Histocompatibility Workshop. The HLA class II genes were determined by the PCR-SSOP method using the reagents provided by the Eleventh Histocompatibility Workshop. Additional studies have been performed for further characterization of HLA class II polymorphism on these cell lines. It is observed that several cell lines have HLA class II haplotypes with the same DRB1, DQA1 and DQB1 alleles on both haplotypes but different alleles at the other class II loci, confirming that these cell lines are not truly HLA class II-homozygous. Other cell lines carried HLA class II haplotypes which were only different at the DRB1 gene. These results suggest double recombination events or gene conversion-like events in generation of HLA DR, DQ haplotypes. These cell lines provide an important tool as references for HLA DNA typing.

Identification of molecular defects causing congenital adrenal hyperplasia by cloning and differential hybridization of polymerase chain reaction-amplified 21-hydroxylase (CYP21) genes.

Congenital adrenal hyperplasia (CAH), one of the most common autosomal recessive disorders, is caused primarily by defects in the gene encoding steroid 21-hydroxylase, CYP21B. The molecular diagnosis of CAH, important for prenatal diagnosis, carrier detection, and a better understanding of the various clinical CAH forms, is complicated by the close proximity of a highly similar pseudogene, CYP21A, containing (and probably donating, by gene conversion-like events) most of the defects underlying CAH. In this study, we describe an efficient strategy to identify molecular defects causing CAH: polymerase chain reaction-amplified CYP21 loci are cloned and hybridized to a set of oligonucleotides, allowing rapid and allele-specific identification of all known CYP21B mutations relevant to 21-hydroxylase function. Possible new mutations can be identified by subsequent nucleic acid sequencing provided they reside within the cloned CYP21B fragment (from the TATA box to the 8th of the 10 CYP21B gene exons). Using this method, the CYP21B gene mutations of a heterozygous carrier and 25 CAH patients have been identified by oligonucleotide hybridization. All disease haplotypes seem to have been generated by recombinational events involving the CYP21A pseudogene. In 5 individuals, these data were subsequently verified by nucleic acid sequencing. The procedure can be used for diagnostic applications and may facilitate identification of new CYP21B defects.

Exon encoding the antigen-binding site of MHC class II beta-chains is divided into two subregions with different evolutionary histories.

The evolution of the Ag-binding site of polymorphic class II molecules was investigated by comparing the pattern of silent and replacement substitutions in the first domain exon of DQB and DRB alleles in two distantly related mammalian species, man and cattle. We show that the first domain can be subdivided into two regions, corresponding to the beta-strand and alpha-helical regions, with distinct evolutionary histories. The data for the alpha-helical region are in conflict with the standard phylogeny of mammalian class II genes. In this region, there is only weak locus divergence at the protein level and the frequency of silent substitutions is extremely low between nonorthologous genes (i.e., DQB-DRB) within species. We propose that the major underlying cause for the observed sequence similarity in the alpha-helical region is due to a selective constraint restricting the sequence divergence at the protein level. This selective constraint may be related to the interaction between different isotypic forms of polymorphic class II beta-chains and a common ligand. The extremely low frequencies of silent substitutions between nonorthologous genes within species are most likely due to the transfer of sequence information between loci by the occurrence of gene conversion-like events in the particular gene segment.

DRw11 haplotypes: Continuum of DRB1 diversity augmented by unique DQ/DRw52 associations

cDNA sequencing of the first domains of DRB1, DRB3, DQA1, and DQB1 alleles was used to examine the extent of diversity in American black individuals expressing several DRw11 haplotypes. In addition to previously described DRw11 alleles, DRB1∗1102 and DRB1∗1103, two new DRB1 alleles, DRB1∗11012 and DRB1∗11042, were identified which differ from previously described alleles at the nucleic acid but not at the protein level. Gene conversion-like events have likely generated the DRw11 microvariation resulting in the merging of DRw11 with the DRw13 allele family. The DRw11 alleles are associated with various DQ alleles DQw1 (DQw5 and DQw6), DQw7, and a serologically undefined DQ allele. This undefined DQ molecule, comprised of a DQα/β combination encoded by a DQw7α gene (DQA1∗0301) and a DQw2β gene (DQB1∗0201), was previously observed in some DR7 and DR9 haplotypes. DRw11 haplotype diversity is augmented by the association of one of the DRw11 alleles with the DRw52c allele in contrast to the more common DRw11, DRw52b association. The extensive diversity exhibited by the DRw11 and DRw13 family of haplotypes coupled with their high frequency in populations of African ancestry suggest that the DRw11/w13 allele family may be very old and/or that these haplotypes carry some selective advantage.

Exposed epitopes on a Trypanosoma equiperdum variant surface glycoprotein altered by point mutations.

African trypanosomes are covered by a dense protein layer that is immunologically distinct on different trypanosome isolates and is termed the variant surface glycoprotein (VSG). The different VSGs are expressed in a general order, where some VSGs appear preferentially early in infection and others only later. The exposed epitopes on a late antigen, VSG 78, of T.equiperdum were studied by the technique of monoclonal antibody (MAb) escape selection. MAbs that neutralize trypanosomes bearing VSG 78 reacted with the VSG only when it was attached to the trypanosome surface, suggesting that the most immunogenic surface epitopes are conformational. Trypanosome clones resistant to one of the MAbs yet still expressing VSG 78 or 78(20) were isolated in vitro. Two independent variants resistant to MAb H3 changed Ser192 to Arg by a single base change in the VSG gene and a variant resistant to MAb H21 had a single base change that converted Gln172 to Glu. A variant resistant to MAb H7 had several changes in the VSG gene, a gene conversion in the 5' region and an isolated mutation in codon 220 that is proposed to be responsible for the resistance phenotype. The isotypic bias of the MAbs against VSG 78 and an analysis of the natural variants that are resistant to MAb 78H21 suggest that glycosylation plays a role in the immunogenicity of these proteins. The analysis defines some of the exposed amino acid residues and demonstrates that VSG genes are altered by mutations and small gene conversions as well as replaced by large gene conversion-like events. The results provide biological data supporting the model of VSG structure obtained by crystallographic studies.

Effects of Individual Mutations in the P-450(C21) Pseudogene on the P-450(C21) Activity and Their Distribution in the Patient Genomes of Congenital Steroid 21-Hydroxylase Deficiency1

Recent observations have suggested that the pathological mutations in human P-450(C21) deficiency are generated through gene conversion-like events between the functional gene [P-450(21)B] and the pseudogene [P-450(C21)A]. To address this point more extensively, we investigated the effects of the base changes in the A pseudogene on the P-450(21) activity by using the COS cell expression system. In addition to the defective mutations found previously in the pseudogene, four single base changes with amino acid substitutions of Pro(30), Ile(172), Val(282), or Arg(356) were further identified as causing complete [Arg(356)] or partial [Pro(30), Ile(172), and Val(282)] inactivation of P-450(C21). Blot hybridization analysis of patient DNAs using oligonucleotide probes specific for these mutations revealed that the splicing mutation in the 2nd intron was distributed most frequently in both simple-virilizing and salt-wasting forms. The mutation Ile(172) seemed to be frequent in patients with the less severe simple-virilizing form, whereas the mutation Arg(356), together with other most serious mutations reported previously, was preferentially associated with salt-wasting, the most severe form of the disease. In combination with the present results of the effects of various mutations on the P-50(C21) activity, a survey of the distribution of the various mutations in the patient genomes so far reported suggests that the heterogeneous clinical symptoms of this genetic disease are somehow related to the degree of attenuation of the activities of the mutated gene products.

Evolution of the HLA-DR1 gene family. Structural and functional analysis of the new allele "DR-BON".

The molecules of the MHC are highly polymorphic and involve in Ag presentation; their striking genetic polymorphism allows probable interactions with a large variety of antigenic fragments when these are presented to the TCR. It is therefore of interest to explore the extent of this polymorphism and the mechanisms of its generation. We have studied the class II HLA-DR blank allele DR-BON that has been previously defined by MLR, restriction fragment length polymorphism, and two-dimensional gel electrophoresis. A cDNA library was constructed from a DR-BON homozygous typing cell and cDNA corresponding to DR alpha- and DR beta-chains were sequenced. By comparison with other known alpha- and beta-chain sequences it is shown that the alpha-chain is invariant and the beta-chain differs from DR1 by only six nucleotides, clustered in the third variable region with three amino acid changes at position 67, 70, and 71. The short DNA stretch of sequence encoding the 67-71 region is also present in other DR alleles: DR4/Dw10, DRw13, and some DRw11 specificities. Therefore we propose that a gene conversion-like event has occurred between the DRB1 *0101 (DR1/Dw1) gene and one of these three DRB1 genes. Extensive typing has been performed with a DR-BON-specific 17-mer oligonucleotide. Cross-hybridization with other genes than the ones expected from DNA sequence comparison was not observed. A selected panel of DR-BON reactive T cell clones shows three patterns of reactivity. Some clones are strictly DR-BON specific; some cross-reacted with DRw13 and a few cross-reacted with other haplotypes. The role of different epitopes of the third variable region of HLA-DR beta chain in allo-reaction is discussed.

Molecular analysis of the serologically defined HLA-Aw19 antigens. A genetically distinct family of HLA-A antigens comprising A29, A31, A32, and Aw33, but probably not A30.

The HLA-Aw19 complex consists of a number of serologically cross-reactive Ag (i.e., A29, A30, A31, A32, and Aw33) which exhibit an epitope shared by HLA-B and -C proteins. To investigate the structural basis for these serologic cross-reactivities, we have cloned and determined the nucleotide sequences for A30, A31, and Aw33, and compared the predicted amino acid sequences with those already available for A29, A32, and other class I allelic products. All alleles of the Aw19 group contained A-locus-specific sequences, exhibiting "A-ness." The structural similarities between Aw19 polypeptides were found in the alpha 1 and alpha 2 domains, where shared amino acid residues were identified that correlated with observed serological reactivity patterns. Seven Aw19-specific nucleotides were found. Two of these were silent substitutions, but the remaining five resulted in Aw19-specific amino acid residues. Each of the HLA-A alleles can be classified into one of the five serologically cross-reacting groups. In the Aw19 group, the alleles A29, A31, A32, and Aw33 are closely related serologically as well as genetically whereas A30 probably belongs to the A1/A3/A11 group. The similarity between A30 and the other Aw19 alleles may have resulted from two independent gene conversions affecting exons 2 and 3. Additional mutations or gene conversion-like events in A30 were also noted. It is postulated that gene conversions have played a significant role in the divergence of the Aw19 alleles. However, each serologically cross-reactive Aw19 allotype appears to have arisen directly from a common ancestral allele. A30 was the only exception, and this allele may represent an unusual allotype, which is subject to a high rate of genetic changes, as is seen in the H-2Kb gene of the mouse.