A de novo recombination in the ABO blood group gene and evidence for the occurrence of recombination products

We have encountered a paternity case where exclusion of the putative father was only observed in the ABO blood group (mother, B; child, A1; putative father, O), among the many polymorphic markers tested, including DNA fingerprints and microsatellite markers. Cloning a part of the ABO gene, PCR-amplified from the trio’s genomes, followed by sequencing the cloned fragments, showed that one allele of the child had a hybrid nature, comprising exon 6 of the B allele and exon 7 of the O1 allele. Based on the evidence that exon 7 is crucial for the sugar-nucleotide specificity of A1 and B transferases and that the O1 allele is only specified by the 261G deletion in exon 6 of the consensus sequence of the A1 allele, we concluded that the hybrid allele encodes a transferase with A1 specificity, resulting, presumably, from de novo recombination between the B and O1 alleles of the mother during meiosis. Screening of random populations demonstrated the occurrence of four other hybrid alleles. Sequencing of intron VI from the five hybrid alleles showed that the junctions of the hybrid alleles were located within intron VI, the intron VI-exon 7 boundaries, or exon 7. Recombinational events seem to be partly involved in the genesis of sequence diversities of the ABO gene. Received: 25 October 1996     

Recombination and gene conversion-like events may contribute to ABO gene diversity causing various phenotypes

We identified five different alleles, tentatively named ABO*O301, *0302, *R102, *R103, and *A110, in Japanese individuals possessing the blood group O phenotype. These alleles lack the guanine deletion at nucleotide position 261 which is shared by a majority of O alleles. Nucleotide sequence analysis revealed that *0301 and *0302 had single nonsynonymous substitutions compared with *A101 or *A102 responsible for the A1 phenotype. Analysis of intron 6 at the ABO gene by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing revealed that *R102 and *R103 had chimeric sequences of A-02 and B-02, respectively, from exons 6 to 7. In the analysis of five other chimeric alleles detected in the same manner, we identified a total of four different recombination-breakpoints within or near intron 6. When 510 unrelated Japanese were examined, the frequency of the chimeric alleles generated by recombination in intron 6 or exon 7 was estimated to be 1.7%. In addition, we found that *O301, *A110, *C101, *A111, and 35% of *A102 had a unique A-B-A chimeric sequence at intron 6, presumed to originate from a gene conversion-like event. We had previously established that *A110 also had an A-O2-A chimeric sequence around nucleotide position 646 in exon 7. Thus this allele has an A-B-A-O2-A chimeric sequence from intron 6 to exon 7 probably generated by two different gene conversions. Similar patchwork sequences around nucleotide position 646 in exon 7 were observed in two other new alleles responsible for the Ax and B3 phenotypes. Thus, the site is presumably a hotspot for gene conversion. These results indicate that both recombination and gene conversion-like events play important roles in generating ABO gene diversity.     

Acquisition of the rfb-gnd cluster in evolution of Escherichia coli O55 and O157

The rfb region specifies the structure of lipopolysaccharide side chains that comprise the diverse gram-negative bacterial somatic (O) antigens. The rfb locus is adjacent to gnd, which is a polymorphic gene encoding 6-phosphogluconate dehydrogenase. To determine if rfb and gnd cotransfer, we sequenced gnd in five O55 and 13 O157 strains of Escherichia coli. E. coli O157:H7 has a gnd allele (allele A) that is only 82% identical to the gnd allele (allele D) of closely related E. coli O55:H7. In contrast, gnd alleles of E. coli O55 in distant lineages are >99.9% identical to gnd allele D. Though gnd alleles B and C in E. coli O157 that are distantly related to E. coli O157:H7 are more similar to allele A than to allele D, there are nucleotide differences at 4 to 6% of their sites. Alleles B and C can be found in E. coli O157 in different lineages, but we have found allele A only in E. coli O157 belonging to the DEC5 lineage. DNA 3' to the O55 gnd allele in diverse E. coli lineages has sequences homologous to tnpA of the Salmonella enterica serovar Typhimurium IS200 element, E. coli Rhs elements (including an H-rpt gene), and portions of the O111 and O157 rfb regions. We conclude that rfb and gnd cotransferred into E. coli O55 and O157 in widely separated lineages and that recombination was responsible for recent antigenic shifts in the emergence of pathogenic E. coli O55 and O157.     

凹耳蛙MHCII类B基因第二外显子多态性分析

两栖类正经历全球范围内的种群衰退,很多两栖动物集群灭绝事件与环境病原体(如壶菌(Batrachochytrium dendrobatidis)的侵扰有关。MHC基因的表达产物在有颌脊椎动物免疫应答过程中起关键作用,其多态性通常与动物对疾病的抗性或易感性密切相关,因而被认为是研究动物适应性进化的最佳候选基因之一。本文对中国特有的无尾两栖动物凹耳蛙(Odorrana tormota)MHC II类B基因多态性进行初步研究。首先,利用1对通用引物扩增出凹耳蛙MHC II类B基因exon2长约180bp的DNA片段。在此基础上,利用ligation-mediated PCR进一步获取侧翼未知序列,序列拼接后长2,030bp,包含exon2以及intron1和intron2的部分序列。基于上述序列设计出凹耳蛙B基因exon2特异性引物(IIQ1BU/IIQ1BD),对该物种黄山种群32个样品进行PCR扩增和克隆测序,共获得34个不同的等位基因,等位基因序列核苷酸和氨基酸变异位点的比例分别为16.17%(33/204)和26.87%(18/67),大多数氨基酸变异位点位于推测的抗原结合位点(antigen binding sites,ABS)。每个样品包含2-5个等位基因,结合等位基因序列特征以及cDNA表达分析结果,推测凹耳蛙至少拥有3个可表达的B基因座位。与文献报道的蛙科其他物种比较后发现,尽管凹耳蛙目前的分布区非常狭窄,但其MHC II类B基因多态性明显高于蛙科其他动物。等位基因碱基替换模式提示凹耳蛙MHC II类B基因曾经历过强烈的正选择作用,ABS区的dN值显著大于dS(P<0.05),PAML软件包CODEML程序中不同模型的似然比检测(likelihood rate test)结果同样支持上述推论,贝叶斯经验贝叶斯路径(Bayesian Em-pirical Bayes)共检测出5个显著受正选择作用的氨基酸位点。贝叶斯系统树的拓扑结构显示,无尾两栖类不同科的等位基因分别形成单系群,但蛙科不同属的等位基因未能形成单系群,蛙属绿池蛙(Rana clamitans)的1个等位基因与臭蛙属凹耳蛙的部分等位基因享有共同的谱系关系,提示蛙科不同属间的B基因存在跨种多态性。     

中国人罕见的cisAB变异型分子机制分析

为研究中国人群ABO血型系统中罕见的cisAB变异型的分子遗传背景, 用血型血清学方法鉴定12例ABO血型疑难样本和116例随机无血缘关系样本, 应用聚合酶链反应和DNA序列分析等方法对样本ABO基因酶活性编码区外显子6、7和侧翼内含子进行突变筛选和检测, 并对不同基因型的扩增片段进行单倍体序列分析。血清学检测发现12个样本均为(A强B弱)或(A弱B强), PCR产物直接序列分析表明这些样本均为cisAB变异型, 并存在4种基因型。通过单倍体序列分析, 从中发现2种cisAB等位基因, 其中cisAB01不仅存在外显子7的467C>T和803G>C变异, 还在第6内含子存在未经报道的163T>C和179C>T变异; 另一种等位基因是在B101基础上保留了A101的803G位点, 编码一条176G、235S、266M和268G组合的多肽链。经检索, 未发现该组合的ABO等位基因, 该等位基因已被国际血型抗原基因突变数据库命名为cisAB05等位基因。文章系统研究了中国人群cisAB变异型分子机制, 发现了一种新的cisAB05等位基因, 同时根据内含子序列推测cisAB01等位基因可能是通过A101和B101等位基因间的同源交换而形成的。     

Full-length sequence analysis of the HLA-DRB1 locus suggests a recent origin of alleles

The HLA region harbors some of the most polymorphic loci in the human genome. Among them is the class II locus HLA-DRB1, with more than 400 known alleles. The age of the polymorphism and the rate at which new alleles are generated at HLA loci has caused much controversy over the years. Previous studies have mostly been restricted to the 270 base pairs that constitute the second exon and represent the most variable part of the gene. Here, we investigate the evolutionary history of the HLA-DRB1 locus on the basis of an analysis of 15 genomic full-length alleles (10-15 kb). In addition, the variation in 49 complete coding sequences and 322 exon 2 sequences were analyzed. When excluding exon 2 from the analysis, the diversity at the synonymous sites was found to be similar to the intron diversity. The overall diversity in noncoding region was also similar to the genome average. The DRB1*03 lineage has been found in human, chimpanzee, bonobo, gorilla, and orangutan. An ancestral "proto HLA-DRB1*03 lineage" appeared to have diverged in the last 5 million years into the human-specific lineages *08, *11, *13, and *14. With exception to exon 2, both the coding- and the noncoding diversity suggests a recent origin (<1 million years ago) for most of the alleles at the HLA-DRB1 locus. Sites encoding for amino acids involved in antigen binding [antigen recognizing sites (ARS)] appear to have a more ancient origin. Taken together, the recent origin of most alleles, the high diversity between allelic lineages, and the ancient origin of sequence motifs in exon 2, is consistent with a relatively rapid generation of novel alleles by gene conversion like events.     

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A¹ (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *13101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.     

Comparison of allele O sequences of the human and non-human primate ABO system

 Like humans, non-human primates express the antigens A and B of the ABO histoblood group system. In chimpanzees, only A and O types are found, while the types A, B, AB, and O are found in macaques. The sequences of exons 6 and 7 of two chimpanzee O alleles (O ^del and O ^x ), two macaque species O alleles (rhesus monkey and crab-eating macaque), and sequences of exon 7 of two major chimpanzee A alleles (A ^1ch and A ^2ch ) were established. The sequences of cDNAs corresponding to the chimpanzee and rhesus monkey O alleles were characterized from exon 1 to 7 and from exon 4 to 7, respectively. A comparison of our results with ABO gene sequences already published by others demonstrates that human and non-human primate O alleles are species-specific and result from independent silencing mutations. These observations reinforce the hypothesis that the maintenance of the ABO gene polymorphism in primates reflects convergent evolution more than transpecies inheritance of ancestor alleles. Received: 30 July 1998 / Revised: 12 December 1998     

Genomic divergence of Escherichia coli strains: evidence for horizontal transfer and variation in mutation rates.

This report describes the sequencing in the Escherichia coli B genome of 36 randomly chosen regions that are present in most or all of the fully sequenced E. coli genomes. The phylogenetic relationships among E. coli strains were examined, and evidence for the horizontal gene transfer and variation in mutation rates was determined. The overall phylogenetic tree indicated that E. coli B and K-12 are the most closely related strains, with E. coli O157:H7 being more distantly related, Shigella flexneri 2a even more, and E. coli CFT073 the most distant strain. Within the B, K-12, and O157:H7 clusters, several regions supported alternative topologies. While horizontal transfer may explain these phylogenetic incongruities, faster evolution at synonymous sites along the O157:H7 lineage was also identified. Further interpretation of these results is confounded by an association among genes showing more rapid evolution and results supporting horizontal transfer. Using genes supporting the B and K-12 clusters, an estimate of the genomic mutation rate from a long-term experiment with E. coli B, and an estimate of 200 generations per year, it was estimated that B and K-12 diverged several hundred thousand years ago, while O157:H7 split off from their common ancestor about 1.5-2 million years ago.     

Molecular basis for subtypic differences of the “a” subunit of coagulation factor XIII with description of the genesis of the subtypes

The “a” subunit of human coagulation factor XIII (F13A) exhibits genetic polymorphism defined by four common alleles, F13A*1A, *1B, *2A, and *2B. We have previously suggested on the basis of the isoelectric focusing patterns of the four allele products that point mutations at two separate sites and one intragenic crossing over might be involved in the genes of F13A polymorphism. Here, we report nucleotide substitutions associated with F13A polymorphism. A C/T transition of the second nucelotide of codon 564 in exon 12 is responsible for the difference between F13A*1A and *1B and that between F13A*2A and *2B, and a set of two base changes in codons 650 and 651 in exon 14 leads to the differences between F13A*1A and *2A and those between F13A*1B and *2B. The four combinations of the point mutations at the two exons thus correspond to the four alleles, two of which were generated by the point mutations from ancestral monomorphic gene. The results suggest strongly that intragenic crossing over must be involved in the genesis of the fourth allele. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods discriminating these base changes in exons 12 and 14 are also presented.     

Class I MHC polymorphism and evolution in endangered California Chinook and other Pacific salmon

Twelve MHC class I exon 2 sequences were uncovered in a sample from the endangered Sacramento River winter-run Chinook salmon in the central valley of California. Phylogenetic analysis of the 12 sequences indicates that the alleles descend from two of six major allelic lineages found among four Pacific salmon species. Nine of the 12 alleles belong to an allelic lineage that began diversifying 8 million years ago, just prior to the estimated time of Chinook speciation. The most recent common ancestor of all 12 winter-run alleles is estimated to be 15 million years ago, approximately 5 million years before the radiation of the Pacific salmon species. The average nonsynonymous distance among the peptide binding-region codons of exon 2 for the 12 alleles is significantly higher than the average synonymous distance in these codons. We estimate the symmetrical overdominant selection coefficient against homozygotes for this exon to be 0.038. Thus, strong positive and balancing selection has maintained functional diversity in the peptide-binding region of the exon over millions of years and this variation has not yet been substantially eliminated by increased genetic drift due to the recent dramatic decline in abundance of this Chinook salmon population.     

Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C) and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo.     

Molecular genetics of the apolipoprotein B gene in pigs in relation to atherosclerosis

Immunologically defined alleles of the pig apolipoprotein B (ApoB) locus (apoB) are correlated with different blood cholesterol levels and predisposition towards premature coronary heart disease. We show here that these alleles are associated with differences in the apoB gene by identifying six restriction fragment length polymorphisms at the pig apoB locus. We have sequenced a 2.4-kb fragment encompassing exons 11 through 14 of one allele, and 7.1 kb from the 3' one-third of exon 26 to about 1 kb past the last exon from another allele. The decoded amino acids of the pig and human ApoB proteins are identical at 70% of these positions. One region close to the C-terminus of the protein is surprisingly different in pigs and humans (57% identity) but the C-terminal region is relatively well conserved (74% identity). Neither of the two putative low-density lipoprotein (LDL) receptor-binding sites is completely conserved in pigs and humans, but identical stretches of amino acids occur near these sites more frequently than in the other sequenced regions. We compare the nucleotide sequences of the region encompassing the putative LDL receptor-binding sites from four pig alleles, including one implicated directly in atherosclerosis. None of the differences appears to account for the hypercholesterolemic phenotype. We conclude that significant differences in the physiology of LDL particles result from changes outside the putative receptor-binding region.     

The Drosophila mus101 gene, which links DNA repair, replication and condensation of heterochromatin in mitosis, encodes a protein with seven BRCA1 C-terminus domains

The mutagen-sensitive-101 (mus101) gene of Drosophila melanogaster was first identified 25 years ago through mutations conferring larval hypersensitivity to DNA-damaging agents. Other alleles of mus101 causing different phenotypes were later isolated: a female sterile allele results in a defect in a tissue-specific form of DNA synthesis (chorion gene amplification) and lethal alleles cause mitotic chromosome instability that can be observed genetically and cytologically. The latter phenotype presents as a striking failure of mitotic chromosomes of larval neuroblasts to undergo condensation of pericentric heterochromatic regions, as we show for a newly described mutant carrying lethal allele mus101(lcd). To gain further insight into the function of the Mus101 protein we have molecularly cloned the gene using a positional cloning strategy. We report here that mus101 encodes a member of the BRCT (BRCA1 C terminus) domain superfamily of proteins implicated in DNA repair and cell cycle checkpoint control. Mus101, which contains seven BRCT domains distributed throughout its length, is most similar to human TopBP1, a protein identified through its in vitro association with DNA topoisomerase IIbeta. Mus101 also shares sequence similarity with the fission yeast Rad4/Cut5 protein required for repair, replication, and checkpoint control, suggesting that the two proteins may be functional homologs.     

Evolution of the ABO blood group gene in Japanese macaque

We determined 5 sequences of Japanese macaque ABO blood group gene exon 7 (ca. 0.5 kb) and 2 sequences for exon 5 and intron 6 (ca. 1.7 kb). We compared those data with published sequences of other Old World monkey species, and the results suggest that alleles A and B were polymorphic in the ancestral species of macaques, and that B type allele evolved independently in macaque and baboon lineages.     

Association of seven polymorphisms of the D2 dopamine receptor gene with brain receptor-binding characteristics

Association of alleles at the Taql A, Taql B, intron 6, Taql D, exon 7, exon 8, and promoter-141C sites of the D2 dopamine receptor gene with D2 dopamine receptor binding characteristics in the caudate nucleus of Caucasian alcoholic and nonalcoholic subjects was determined. For the Taql D, exon 7, exon 8, and promoter-141C sites there were no significant allelic differences in Bmax (number of binding sites) or Kd (binding affinity) of the D2 dopamine receptors. However, subjects having the minor alleles at the Taql A, Taql B, and intron 6 sites had significantly lower Bmax than subjects not having them. None of these three polymorphisms had any significant effect on Kd. Highly significant linkage disequilibria were observed among the Taql A, Taql B, and intron 6 polymorphic sites, but linkage disequilibria between these three sites and each of the Taql D, exon 7, exon 8, and promoter-141C sites were of lesser or of no significance. Taken together, these findings suggest that the Taql A, Taql B, and intron 6 polymorphisms, but not the Taql D, exon 7, exon 8, and promoter-141C polymorphisms, are in linkage disequilibrium with a functional allelic variant that affects D2 dopamine receptor expression.     

Molecular evidence for compound heterozygosity in hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is an inborn error of metabolism, inherited as an autosomal recessive disorder and caused by a decrease in the activity of fructose-1-phosphate aldolase (aldolase B) in affected individuals. Investigation of the molecular basis of HFI is reported here by the identification of two molecular lesions in the aldolase B gene of the HFI individual. Using polymerase chain reaction to specifically amplify exons at this locus and T7 polymerase for the sequence determination of these double-stranded fragments, we show the mutational heterogeneity of the proband. One allele, previously indicated by restriction analysis, was confirmed as A149P (Ala 149 to Pro in exon 5). The other allele was identified as a 4-bp deletion found in exon 4, a deletion which causes a frameshift at codon 118, resulting in a truncated protein of 132 amino acids. Segregation of these mutant alleles in the proband's family was shown by using allele-specific oligodeoxynucleotides to probe blots of amplified DNA. The techniques employed here represent a rapid and efficient method for detection of other mutations in families with this disease. In addition, the ability to detect mutant alleles by allele-specific hybridization offers a new method for definitive diagnosis, a method which avoids a fructose loading or liver-biopsy examination.     

Recessive allelic variations of three microsatellite sites within the<Emphasis Type="Italic">O2</Emphasis> gene in maize

Recessive allelic variations were investigated at 3 microsatellite (SSR) sites within theO2 gene by using 14 inbredo2 lines and a wild-type line in maize. Among the 15 lines, allelic variations were observed at umc1066, phi057, and phi112 sites. Two alleles were found at the umc1066 site—a recessive allele with 2 perfect GCCAGA repeats and a dominant allele with 3 perfect repeats. Three alleles were found at the phi057 site—2 recessive alleles with 3 and 5 perfect GCC repeats, respectively, and another with 4 perfect repeats consistent with a dominant allele. At least 4 alleles exist at the phi112 site—among which 1 recessive allele has a 1-bp deletion, another has a 15-bp deletion, and other has no PCR products compared to the dominant allele; all the alleles have unchanged AG repeats. The phi057 site in exon 6 was identified to be a hypervariable region in the coding sequence of the02 gene, in addition to the 2 hypervariable regions in exon 1 previously reported. The primary mechanisms underlying the variations in repeat numbers and regions flanking the SSR within theO2 gene appear to be unequal crossing over and replication slippage. Furthermore, base substitution of SSR motif can create heteroalleles and modify the repeat number of SSR. The lysine content of kernel in theO2 ando2 lines correlates to a considerable extent with nucleotide variations at the umc1066, phi057, and phi112 sites. Our study suggests that it is best to use the 3 markers together in molecular marker-assisted selection for high-lysine maize materials.