The structural basis of the multiple forms of human complement component C4

cDNA clones of human complement components C4A and C4B alleles were prepared from mRNA obtained from the liver of a donor heterozygous at both loci. cDNA from one C4A allele was sequenced to give the derived complete amino acid sequence of 1722 amino acid residues of the C4 single chain precursor molecule and the estimated sequences of the three peptide chains of secreted C4. Comparison with partial sequences of a second C4A allele and a C4B allele has led to the tentative identification of some class differences in nucleotide sequences between C4A and C4B and of allelic differences between C4A alleles in this highly polymorphic system.     

Characterization of Mhc-DRB allelic diversity in white-tailed deer (Odocoileus virginianus) provides insight into Mhc-DRB allelic evolution within Cervidae

 Although white-tailed deer (Odocoileus virginianus) are one of North America's best studied mammals, no information is available concerning allelic diversity at any locus of the major histocompatibility complex in this taxon. Using the polymerase chain reaction, single-stranded conformation polymorphism analysis, and DNA sequencing techniques, 15 DRB exon 2 alleles were identified among 150 white-tailed deer from a single population in southeastern Oklahoma. These alleles represent a single locus and exhibit a high degree of nucleotide and amino acid polymorphism, with most amino acid variation occurring at positions forming the peptide binding sites. Furthermore, twenty-seven amino acid residues unique to white-tailed deer DRB alleles were detected, with 19 of these occurring at residues forming contact points of the peptide binding region. Significantly higher rates of nonsynonymous than synonymous substitutions were detected among these DRB alleles. In contrast to other studies of Artiodactyla DRB sequences, interallelic recombination does not appear to be playing a significant role in the generation of allelic diversity at this locus in white-tailed deer. To examine evolution of white-tailed deer (Odvi-DRB) alleles within Cervidae, we performed a phylogenetic analysis of all published red deer (Ceel-DRB), roe deer (Caca-DRB), and moose (Alal-DRB) DRB alleles. The phylogenetic tree clearly shows a trans-species persistence of DRB lineages among these taxa. Moreover, this phylogenetic tree provides insight into evolution of DRB allelic lineages within Cervidae and may aid in assignment of red deer DRB alleles to specific loci. Received: 25 June 1998 / Revised: 2 September 1998     

Haplotypic polymorphisms of the TNFB gene

The NTFB genes from two major histocomptibility complex (MHC) ancestral haplotypes have been compared. The genes carried by the ancestral haplotypes 8.1 (A1,B8,BfS,C4AQ0, C4B1,DR3) and 57.1 (A1,B57, BfS,C4A6,C4B1,DR7) were cloned and sequenced to determine the degree of polymorphism. In this report we show that the r e spective TNF genes are allelic and have unique nucleotide sequences. The data demonstrate the presence of three nucleotide differences between the TNFB alleles of 8.1 and 57.1. Two of the differences occur in untranslated regions of the gene but the third nucleotide change results in amino acid differences in the mature TNFB protein. These polymorphisms may have implications with respect to differential regulation in disease-and nondisease-associated haplotypes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M55913.     

Isolation, characterization and evolution of ovine major histocompatibility complex class II DRA and DQA genes

Four full-length ovine major histocompatibility complex (MHC) class II A cDNA clones coding for new alleles of DRA, DQA1 and DQA2 genes were isolated from two ovine Λgt10 cDNA libraries. The derived amino acid sequences of these clones resemble class II A molecules from other species in both size and structure. Restriction fragment length polymorphism analysis, using an Ovar-DRA probe on DNA from Merino and Romney sheep revealed only limited polymorphism in contrast to the high levels of polymorphism revealed by Ovar-DQA probes. Comparison of the predicted amino acid sequences for the three ovine A genes with class II A genes from five other species revealed that the most variable region of the molecule is the signal peptide. Although virtually every amino acid site shows variation, within or between species, there are some blocks of highly conserved residues. Within gene comparisons of nucleotide differences reveal that the greatest number of changes is found between the alleles of Ovar-DQA1 and -DQA2 genes and the least between Ovar-DRA1 alleles. Phylogenetic analysis of class IIA sequences from several species place DRA and DQA genes on two distinct branches, with Ovar-DRA1 and BOLA-DRA, and Ovar-DQA1 and BOLA-DQA being most similar on their respective branches.     

Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis

We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis. A 163 by DNA segment from a 5 portion of the gyrA gene was amplified from the DNA of each mutant strain. After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis). Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains. The patterns were classified into 13 types, and the DNA sequence of each type was determined. A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles. Eight were single base pair substitutions, four were substitutions of two consecutive base pairs, and one was a substitution of three consecutive base pairs. Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes. We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait.     

C4 gene polymorphism in primates: evolution,generation, and Chido and Rodgers antigenicity

Eleven new C4d genomic primate sequences of the fourth complement factor (C4) have been obtained. Seven of them belong to five species not yet explored for this gene: Pan paniscus (pygmy chimpanzee), Cercopithecus aethiops (green monkey), Macaca mulatta (rhesus monkey), Macaca fascicularis (cynomolgus), and Saguinus oedipus (cotton top tamarin). The New World monkeys (tamarins, four individuals) sequenced for C4 have a single C4d sequence only, which shows a B isotypic specificity and a Rodgers 3 (Rg3), Chido 1 (Ch1) antigenicity. Rg3 and Ch1 could thus be the oldest Rg/Ch specificity (at least 50 million years old) and Rg1, Rg2, Ch3, and Ch6 could be more recent human-specific antigens. Mechanisms of C4d polymorphism generation were analyzed by compiling all the presently available sequences. Examples of both point mutations and crossing-over events among C4d primate sequences could be detected. The problem of a possible trans-species inheritance of C4d polymorphism was addressed and two apparently contradicting dendrograms were obtained. One of them, constructed by using both exon and intron sequences, does not support trans-species evolution, but supports the proposed theory of extensive homogenization of the C4 genes occurring within each species, because alleles from each primate species cluster together. Another completely different dendrogram, obtained by using exon sequences only, suggests the existence of trans-species evolution for C4d polymorphism, because alleles belonging to different species cluster together in a way similar to that found for HLA class I or II alleles. However, orangutan sequences group together in both kinds of C4d sequence dendrograms and seem to have arisen from an ancestor different from that of chimpanzee, gorilla and man C4d sequences. Finally, further data have been obtained that support trans-species conservation of A-ness and B-ness and the existence of trans-specifically conserved allelic motifs, both in intronic and exonic sequences.     

Polymorphism and signatures of selection in the multimammate rat DQB gene

In order to test if DQB is a good candidate marker to investigate the relationship between major histocompatibility complex genes and pathogens in natural populations of Mastomys natalensis, we assessed the polymorphism and evolutionary history of this gene. Twenty-four individuals were genotyped for exon 2 of DQB using capillary electrophoresis single-strand conformation polymorphism, cloning, and sequencing. We found 21 different alleles. Four individuals show three alleles implying a duplication event in the history of this gene. Each distinct sequence translates to give a distinct amino acid sequence and there are strong signals of positive selection on peptide binding sites. Signals of recombination were found in the sequences suggesting that recombination has played a role in generating allelic diversity. Although trans-taxon polymorphism is present at the interspecific level in DQB exon 2 sequences of Mus species, we did not find any evidence of allele sharing among Muridae genera. This indicates a temporal limit of DQB allele sharing in Muridae of less than 8 Mya. In conclusion, although DQB seems to be a good marker to investigate pathogen-driven selection, the polymorphism of gene copy number may restrict its utility in natural populations.     

Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis

We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis. A 163 by DNA segment from a 5′ portion of the gyrA gene was amplified from the DNA of each mutant strain. After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis). Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains. The patterns were classified into 13 types, and the DNA sequence of each type was determined. A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles. Eight were single base pair substitutions, four were substitutions of two consecutive base pairs, and one was a substitution of three consecutive base pairs. Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes. We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait.     

Linkage of C4 and C4 deficiency to BF and GPLA

The C4, Bf, and GPLA phenotypes of homo- and heterozygous C4-deficient guinea pigs were studied. The electrophoretic patterns suggest that the deficiency in circulating C4 results from an impaired structural gene, allelic to the C4^F, C4^S, and C4^S1 alleles at the C4 locus. In family studies, support for linkage of C4 and Bf to theGPLA system was obtained. The defective gene appears to be the fourth allele, which is rare, in the polymorphism of the fourth component of guinea pig complement.Abbreviations used in this paper are as follows Bf locus for properdin factor B - MHC major histocompatibility complex - GPLA major histocompatibility complex of the guinea pig     

Amphitrite ornata, a Marine Worm, Contains Two Dehaloperoxidase Genes

Amphitrite ornata, a terebellid polychaete, inhabits marine environments that are contaminated by biogenically produced halometabolites. These halogenated organic compounds are toxic and quite diverse. To survive in this environment, A. ornata produces a novel dehaloperoxidase (DHP I) that detoxifies haloaromatic compounds. In this study we identified and characterized two dehaloperoxidase genes, designated dhpA and dhpB, from an A. ornata complementary DNA library. The deduced amino acid sequences (DHP A and DHP B) of the two dhp genes both contain 137 amino acid residues, but they differ at 5 amino acid positions. Allelic variation was observed for both genes as well. Polymerase chain reaction–restriction fragment length polymorphism assays of genomic DNA from 19 in individuals showed that each individual contains both the dhpA and the dhpB genes. Therefore, the two types of DHP are encoded by separate genes and are not alleles of a single gene. Furthermore, DHP A and DHP B may have different substrate specificities since they have amino acid differences in the active site. Accepted January 16, 2001.     

Polymorphism and selection in the major histocompatibility complex <Emphasis Type="Italic">DRA</Emphasis> and <Emphasis Type="Italic">DQA</Emphasis> genes in the family Equidae

The major histocompatibility complex genes coding for antigen binding and presenting molecules are the most polymorphic genes in the vertebrate genome. We studied the DRA and DQA gene polymorphism of the family Equidae. In addition to 11 previously reported DRA and 24 DQA alleles, six new DRA sequences and 13 new DQA alleles were identified in the genus Equus. Phylogenetic analysis of both DRA and DQA sequences provided evidence for trans-species polymorphism in the family Equidae. The phylogenetic trees differed from species relationships defined by standard taxonomy of Equidae and from trees based on mitochondrial or neutral gene sequence data. Analysis of selection showed differences between the less variable DRA and more variable DQA genes. DRA alleles were more often shared by more species. The DQA sequences analysed showed strong amongst-species positive selection; the selected amino acid positions mostly corresponded to selected positions in rodent and human DQA genes.     

Positive selection and interallelic recombination at the merozoite surface antigen-1 (MSA-1) locus of Plasmodium falciparum.

DNA sequences of alleles at the merozoite surface antigen-1 (MSA-1) gene locus of the malaria parasite Plasmodium falciparum show evidence of repeated past recombination events between alleles. These include both (1) nonreciprocal recombination events that have homogenized certain gene regions among alleles and (2) reciprocal recombination events that have combined allelic segments with divergent evolutionary histories, thereby enhancing allelic diversity. In three different gene regions, the rate of nonsynonymous nucleotide substitution significantly exceeds that of synonymous nucleotide substitution, implying that positive Darwinian selection has acted to diversify alleles at the amino acid level. The MSA-1 polymorphism seems to be quite ancient; the two major allelic types have been maintained for approximately 35 Myr.     

Mhc class II B gene evolution in East African cichlid fishes

 A distinctive feature of essential major histocompatibility complex (Mhc) loci is their polymorphism characterized by large genetic distances between alleles and long persistence times of allelic lineages. Since the lineages often span several successive speciations, we investigated the behavior of the Mhc alleles during or close to the speciation phase. We sequenced exon 2 of the class II B locus 4 from 232 East African cichlid fishes representing 32 related species. The divergence times of the (sub)species ranged from 6000 to 8.4 million years. Two types of evolutionary analysis were used to elucidate the pattern of exon 2 sequence divergence. First, phylogenetic methods were applied to reconstruct the most likely evolutionary pathways leading from the last common ancestor of the set to the extant sequences, and to assess the probable mechanisms involved in allelic diversification. Second, pairwise comparisons of sequences were carried out to detect differences seemingly incompatible with origin by nonparallel point mutations. The analysis revealed point mutations to be the most important mechanism behind allelic divergences, with recombination playing only an auxiliary part. Comparison of sequences from related species revealed evidence of random allelic (lineage) losses apparently associated with speciation. Sharing of identical alleles could be demonstrated between species that diverged 2 million years ago. The phylogeny of the exon was incongruent with that of the flanking introns, indicating either a high degree of convergent evolution at the peptide-binding region-encoding sites, or intron homogenization. Received: 6 December 1999 / Revised: 15 February 2000     

Analysis of genetic diversity at the DQA loci in African cattle: evidence for a BoLA-DQA3 locus

 We describe the development of a polymerase chain reaction (PCR)-based approach for analysis of genetic diversity at the DQA loci in African Bos indicus and Bos taurus cattle. This approach, equally effective in European and Asian cattle breeds, detects the presence or absence of DQA1 and most duplicated DQA2 genes. Nucleotide and predicted amino acid sequence analysis of the highly polymorphic second exons, in addition to analysis of the locus-specific and relatively non-polymorphic transmembrane, cytoplasmic, and 3-prime untranslated regions, has provided evidence for considerable diversity between each of the duplicated DQA2 genes. Therefore, we propose the designation BoLA-DQA3 for the previously unpublished alleles at the second DQA2 locus. Fourteen distinct PCR restriction fragment length polymorphism (RFLP) patterns, each identifying families of alleles at three DQA loci, can be distinguished. Nucleotide sequence analysis of new PCR-RFLP patterns from 193 Kenyan Boran, Ethiopian Arsi (B. indicus), and Guinean N’Dama (B. taurus) cattle identified 13 DQA1 alleles within eight major allelic families, five DQA2 alleles within a single allelic family, and seven DQA3 alleles within three major allelic families. Received: 19 February 1997 / Revised: 28 February 1997     

Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum

Summary Allelic complexity is a key feature of self-incompatibility (S) loci in gametophytic plants. We describe in this report the allelic diversity and gene structure of the S locus in Solanum tuberosum revealed by the isolation and characterization of genomic and cDNA clones encoding S-associated major pistil proteins from three alleles (S ₁, S _r1, S ₂). Genomic clones encoding the S₁ and S₂ proteins provide evidence for a simple gene structure: Two exons are separated by a small intron of 113 (S ₁) and 117 by (S ₂). Protein sequences deduced from cDNA clones encoding S₁ and S_r1 proteins show 95% homology. 15 of the 25 residues that differ between these S ₁and S _r1alleles are clustered in a short hypervariable protein segment (amino acid positions 44–68), which corresponds in the genomic clones to DNA sequences flanking the single intron. In contrast, these alleles are only 66% homologous to the S ₂allele, with the residues that differ between the alleles being scattered throughout the sequence. DNA crosshybridization experiments identify a minimum of three classes of potato S alleles: one class contains the alleles S ₁, S _r1and S ₃, the second class S ₂and an allele of the cultivar Roxy, and the third class contains at present only S ₄. It is proposed that these classes reflect the origin of the S alleles from a few ancestral S sequence types.     

Population diversity and antibody selective pressure to Plasmodium falciparum MSP1 block2 locus in an African malaria-endemic setting

Background

Genetic evidence for diversifying selection identified the Merozoite Surface Protein1 block2 (PfMSP1 block2) as a putative target of protective immunity against Plasmodium falciparum. The locus displays three family types and one recombinant type, each with multiple allelic forms differing by single nucleotide polymorphism as well as sequence, copy number and arrangement variation of three amino acid repeats. The family-specific antibody responses observed in endemic settings support immune selection operating at the family level. However, the factors contributing to the large intra-family allelic diversity remain unclear. To address this question, population allelic polymorphism and sequence variant-specific antibody responses were studied in a single Senegalese rural community where malaria transmission is intense and perennial.

Results

Family distribution showed no significant temporal fluctuation over the 10 y period surveyed. Sequencing of 358 PCR fragments identified 126 distinct alleles, including numerous novel alleles in each family and multiple novel alleles of recombinant types. The parasite population consisted in a large number of low frequency alleles, alongside one high-frequency and three intermediate frequency alleles. Population diversity tests supported positive selection at the family level, but showed no significant departure from neutrality when considering intra-family allelic sequence diversity and all families combined. Seroprevalence, analysed using biotinylated peptides displaying numerous sequence variants, was moderate and increased with age. Reactivity profiles were individual-specific, mapped to the family-specific flanking regions and to repeat sequences shared by numerous allelic forms within a family type. Seroreactivity to K1-, Mad20- and R033 families correlated with the relative family genotype distribution within the village. Antibody specificity remained unchanged with cumulated exposure to an increasingly large number of alleles.

Conclusion

The Pfmsp1 block2 locus presents a very large population sequence diversity. The lack of stable acquisition of novel antibody specificities despite exposure to novel allelic forms is reminiscent of clonal imprinting. The locus appears under antibody-mediated diversifying selection in a variable environment that maintains a balance between the various family types without selecting for sequence variant allelic forms. There is no evidence of positive selection for intra-family sequence diversity, consistent with the observed characteristics of the antibody response.     

Molecular charge and electrophoretic mobility in cetacean myoglobins of known sequence

Fourteen myoglobins of known sequence were examined by polyacrylamide gel electrophoresis at five pH values. Gels at each pH divided the sequences into six to eight distinct classes, while the combination of the results of three gels at different pH levels distinguished 13 of 14, or 93%, of the sequences. The relative mobility of the myoglobins in the gels is significantly correlated with the charges of the proteins calculated from the pK values of the ionized groups. Major differences in mobility corresponded to expected differences in charge due to the amino acid substitutions between sequences. In addition to sequences differing in the total number of acidic and basic residues, those differing from each other in the total number of histidines were distinguished on low-pH gels. One pair of sequences differing by the exchange of lysine for arginine was separated on high-pH gels, as predicted from the differences in ionization of these two amino acids. On gels at pH 10.4, there was greater deviation of electrophoretic mobility from charge than on other gels, possibly due to the influence of amino acid substitutions in the neighborhood of lysine residues. Manipulation of the concentration and composition of the gels did not change the separation of the sequences from each other. Examination of myoglobins by gel electrophoresis at a wide range of pH values allows discrimination of nearly all amino acid substitutions and demonstrates the close relationship between titration and relative electrophoretic mobility.This work was supported by NIH Grants GM 24849 to R. C. Lewontin and CA 28854 to M. Skolnick.