Sequence analysis of a polymorphic Mhc class II gene in Pacific salmon

Polymorphism of the nucleotide sequences encoding 149 amino acids of linked major histocompatibility complex (Mhc) class II 131 and 132 peptides, and of the intervening intron (548–773 base pairs), was examined within and among seven Pacific salmon (Oncorhynchus) species. Levels of nucleotide diversity were higher for theB1 sequence than forB2 or the intron in comparisons both within and between species. For the codons of the peptide binding region of the BI sequence, the level of nonsynonymous nucleotide substitution (d_N) exceeded the level of synonymous substitution (d_S) by a factor of ten for within-species comparisons, and by a factor of four for between-species comparisons. The excess of d_N indicates that balancing selection maintains diversity at this salmonidMhc class II locus, as is common forMhc loci in other vertebrates. Levels of nucleotide diversity for both the exon and intron sequences were greater among than within species, and there were numerous species-specific nucleotides present in both the coding and noncoding regions. Thus, neighbor-joining analysis of both the intron and exon regions provided phylogenies in which the sequences clustered strongly by species. There was little evidence of shared ancestral (trans-species) polymorphism in the exon phylogeny, and the intron phylogeny depicted standard relationships among the Pacific salmon species. The lack of shared allelicB1 lineages in these closely related species may result from severe bottlenecks that occurred during speciation or during the ice ages that glaciated the rim of the north Pacific Ocean approximately every 100 000 years in the Pleistocene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U34692-U34720     

DNA sequence variation in a 3.7-kb noncoding sequence 5' of the CYP1A2 gene: implications for human population history and natural selection

CYP1A2 is a cytochrome P450 gene that is involved in human physiological responses to a variety of drugs and toxins. To investigate the role of population history and natural selection in shaping genetic diversity in CYP1A2, we sequenced a 3.7-kb region 5' from CYP1A2 in a diverse collection of 113 individuals from three major continental regions of the Old World (Africa, Asia, and Europe). We also examined sequences in the 90-member National Institutes of Health DNA Polymorphism Discovery Resource (PDR). Eighteen single-nucleotide polymorphisms (SNPs) were found. Most of the high-frequency SNPs found in the Old World sample were also found in the PDR sample. However, six SNPs were detected in the Old World sample but not in the PDR sample, and two SNPs found in the PDR sample were not found in the Old World sample. Most pairs of SNPs were in complete linkage disequilibrium with one another, and there was no indication of a decline of disequilibrium with physical distance in this region. The average +/- SD nucleotide diversity in the Old World sample was 0.00043+/-0.00026. The African population had the highest level of nucleotide diversity and the lowest level of linkage disequilibrium. Two distinct haplotype clusters with broadly overlapping geographical distributions were present. Of the 17 haplotypes found in the Old World sample, 12 were found in the African sample, 8 were found in Indians, 5 were found in non-Indian Asians, and 5 were found in Europeans. Haplotypes found outside Africa were mostly a subset of those found within Africa. These patterns are all consistent with an African origin of modern humans. Seven SNPs were singletons, and the site-frequency spectrum showed a significant departure from neutral expectations, suggesting population expansion and/or natural selection. Comparison with outgroup species showed that four derived SNPs have achieved high (>0.90) frequencies in human populations, a trend consistent with the action of positive natural selection. These patterns have a number of implications for disease-association studies in CYP1A2 and other genes.     

Organization of Mhc class II A and B genes in the tilapiine fish Oreochromis

Perch-like fishes of the family Cichlidae are models for the study of speciation. An important tool in these studies is the major histocompatibility complex (Mhc) and its organization. The present study takes the first step toward the elucidation of the Mhc class II gene organization in the tilapiine fish Oreochromis niloticus (Orni). Using class II A- and class II B-specific probes, Mhc-bearing clones were identified and isolated from a bacterial artificial chromosome (BAC) library. The analysis of these clones by a combination of molecular, genetic-mapping, and phylogenetic methods led to the identification of nine class II A and 15 class II B loci. Genes at these loci constitute two families, which we designate as class IIa and class IIb families. Each of the families contains A and B loci. Some genes in both families are expressed and functional. The two families differ in their chromosomal location (they are unlinked) and their mode of evolution. The class IIa family genes are conserved across different teleost taxonomical orders, whereas the class IIb family genes are apparently products of multiple, more recent, rounds of gene duplications. The rounds established at least five monophyletic groups of genes. The founding unit of each monophyletic group might have been a pair of class II A and B loci.     

Sequence,expression, and mapping of a rat Mhc class I b gene

The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence databased and have been assigned the accession number X70066.     

Songbird genomics: analysis of 45 kb upstream of a polymorphic Mhc class II gene in red-winged blackbirds (Agelaius phoeniceus)

Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative gamma-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.     

Nucleotide sequence of a type II DNA topoisomerase gene. Bacteriophage T4 gene 39.

T4 DNA topoisomerase is a type II enzyme and is thought to be required for normal T4 DNA replication T4 gene 39 codes for the largest of the three subunits of T4 DNA topoisomerase. I have determined the nucleotide sequence of a region of 2568 nucleotides of T4 DNA which includes gene 39. The location of the gene was established by the identification of the first fifteen amino acids in the large open reading frame in the DNA sequence as those found at the amino-terminus of the purified 39-protein. The coding region of gene 39 has 1560 bases, and it is followed by two in-frame stop codons. The gene is preceded by a typical Shine-Dalgarno sequence as well as possible promoter sequences for E. coli RNA polymerase. T4 39-protein consists of 520 amino acids, and it has a calculated molecular weight of 58,478. By comparing the amino acid sequences, T4 39-protein is found to share homology with the gyrB subunit of DNA gyrase. This suggests that these topoisomerase subunits may be equivalent functionally. Some of the characteristics of the 39-protein and its structural features predicted from the DNA sequence data are discussed.     

Partial nucleotide sequence of a bovine major histocompatibility class II DR beta-like gene

A genomic clone containing a bovine DR beta-like gene, BoDR beta II, was isolated from a bovine genomic library and characterized by restriction enzyme mapping and nucleotide sequencing of exon regions. Alignment of this sequence with the human DR beta cDNA sequence allowed identification of exon/intron boundaries. The clone contains a 13.3-kilobase (kb) insert, and includes 1.3 kb 5' of the beta 1 exon and 6.7 kb 3' of the transmembrane (TM) exon. Open reading frames were present in the BoDR beta exons sequenced. Nucleotide identities of the bovine beta 1, beta 2 and TM exons with the corresponding human DR beta exons were 73, 91 and 83%, respectively. Nucleotide identities of these exons with those of a previously described bovine DR beta-like pseudogene, BoDR beta I, were 69, 95 and 81%, respectively. Although a limited amount of sequence data was obtained for the intron regions, a 71% identity was found within a 514-nucleotide region immediately 3' to the beta 2 exons in BoDR beta I and BoDR beta II. A series of GT residues followed by a longer series of GA residues began about 35 nucleotides 3' of the beta 1 exon in both BoDR beta I and BoDR beta II.     

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed.     

Mapping and nucleotide sequence of a newHLA class II light chain gene,DQB3

A genomic clone specifying a new HLA class II antigen β chain,DQB3, was isolated from a human genomic phage library using aDQB1 cDNA probe under low stringency conditions. Southern hybridization and nucleotide sequence analyses identified the β2 domain exon (exon 3) with several deleterious mutations and the CP-TM-CY exon [connecting peptide, transmembrane, and cytoplasmic regions, (exon 4)], but the first, second, and fifth exons encoding the 5′ UT-leader, the β1 domain, and the 3′ UT domain of normal β chains, respectively, were entirely missing. The nucleotide sequences of these two exons were distinct from those of other class II β chain genes, but slightly more related to theDQB1 andDQB2 genes than to other class II genes. TheDQB3 sequence mapped betweenDQA2 andDQB1, 15 kb upstream fromDQA2, by analysis of overlapping cosmid clones. This mapping was supported by the fact thatTaq I,Msp I, andBam HIDQB3 polymorphisms were perfectly correlated with theDQA2 polymorphism and not with any polymorphisms in theDR orDQ subregion, suggesting the presence of a hot spot for recombination betweenDQB3 andDQB1. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M26577.     

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed. Received: 25 May 1998 / Accepted: 29 June 1998     

Dynamics of Mhc evolution in birds and crocodilians: amplification of class II genes with degenerate primers

Genes of the major histocompatibility complex (Mhc) are the most polymorphic functional loci in mammalian populations, but little is known of Mhc variability in natural populations of nonmammalian vertebrates. To help extend such studies to birds and relatives, we present a pair of degenerate primers that amplify polymorphic segments of one chain (the β chain) of the class II genes from the major histocompatibility complex (Mhc) of archosaurs (birds + crocodilians). The primers target two conserved regions lying within portions of the antigen-binding site (ABS) encoded by the second exon and amplify multiple genes from both genomic DNA and cDNA. The pattern of nucleotide substitution in ABS codons of 51 sequences amplified and cloned from five species of passerine birds and an alligator (Alligator mississippiensis) indicates that archosaurian class II β genes are subject to selective forces similar to those operating in mammalian populations. Hybridization of a genomic clone generated by the primers revealed highly polymorphic bands in a sample of Florida scrub jays (Aphelocoma coerulescens coerulescens). Because the primers amplify only part of the ABS from multiple class II genes, they will be useful primarily for generating species specific clones, thereby providing a critical inroad to more detailed structural and evolutionary studies.     

Complete genome sequence of Paracoccus sp. strain AK26: Insights into multipartite genome architecture and methylotropy

The present study reports the functional annotation of complete genome of methylotrophic bacterium Paracoccus sp. strain AK26. The 3.6 Mb genome with average GC content of 65.7% was distributed across five replicons; including chromosome (2.7 Mb) and four extrachromosomal replicons pAK1 (471Kb), pAK2 (189Kb), pAK3 (129Kb) and pAK4 (84 Kb). Interestingly, nearly 23% of the Cluster of Orthologous Group (COG) of proteins were annotated on extrachromosomal replicons and 185Kb genome content was attributed to segregated 19 genomic island regions. Among the four replicons, pAK4 was identified as essential and integral part of the genome, as supported by codon usage, GC content (66%) and synteny analysis. Comparative genome analysis for methylotrophy showed mechanistic variations in oxidation and assimilation of C1 compounds among closely related Paracoccus spp. Collectively, present study reports the functional characterization and genomic architecture of strain AK26 and provides genetic basis for quinone and isoprenoid based secondary metabolites synthesis using strain AK26.     

Partial nucleotide sequence of a bovine major histocompatibility class II DRβ-like gene

Summary. A genomic clone containing a bovine DRβ-like gene, BoDRβ II , was isolated from a bovine genomic library and characterized by restriction enzyme mapping and nucleotide sequencing of exon regions. Alignment of this sequence with the human DRβ cDNA sequence allowed identification of exon/intron boundaries. The clone contains a 13.3-kilobase (kb) insert, and includes 1.3kb 5' of the β₁ exon and 6.7kb 3' of the transmembrane (TM) exon. Open reading frames were present in the BoDRβ exons sequenced. Nucleotide identities of the bovine β₁, β₂ and TM exons with the corresponding human DRβ exons were 73, 91 and 83%, respectively. Nucleotide identities of these exons with those of a previously described bovine DRβ-like pseudogene, BoDRβ I , were 69, 95 and 81%, respectively. Although a limited amount of sequence data was obtained for the intron regions, a 71% identity was found within a 514-nucleotide region immediately 3' to the β₂ exons in BoDRβ I and BoDRβ II . A series of GT residues followed by a longer series of GA residues began about 35 nucleotides 3' of the β₁ exon in both BoDRβ I and BoDRβ II .     

Nucleotide sequence of a new class A beta-lactamase gene from the chromosome of Yersinia enterocolitica: implications for the evolution of class A beta-lactamases

Summary The nucleotide sequence has been determined of a 1400 by fragment from the chromosome of Yersinia enterocolitica containing the gene for -lactamase I. An ORF of 882 by was identified, which could code for a polypeptide of 294 amino acids, closely related to other -lactamases of molecular class A. Amino acids 1–30 could constitute a signal peptide. The mature protein would be 264 amino acids long with a calculated pI of 6.2. Alignment of the amino acid sequence of the class A -lactamases suggested the existence of two subgroups in the same class, and this is discussed in the context of the evolution of the enzymes.This sequence will appear in the EMBL Data Library under the accession number X57074     

The mitochondrial genome of Saprolegnia ferax: organization, gene content and nucleotide sequence

The mitochondrial genome of the peronosporomycete water mold Saprolegnia ferax has been characterized as a 46 930 bp circle containing an 8618 bp large inverted repeat (LIR). Eighteen reading frames encode identified subunits of respiratory complexes I, III, IV and V; 16 encode polypeptides of small and large mitoribosome subunits; and one encodes a subunit of the sec-independent protein translocation pathway. Of four additional putative reading frames three are homologues of those found in the related Phytophthora infestans genome. Protein encoding loci in the tightly compacted genome typically are arranged in operon-like clusters including three abutting and two overlapping pairs of reading frames. Translational RNAs include the mitochondrial small and large subunit rRNAs and 25 tRNA species. No tRNAs are encoded to enable translation of any threonine or the arginine CGR codons. The LIR separates the molecule into 19 274 bp large and 10 420 bp small single copy regions, and it encodes intact duplicate copies of four reading frames encoding known proteins, both rRNAs, and five tRNAs. Partial 3' sequences of three additional reading frames are duplicated at single copy sequence junctions. Active recombination between LIR elements generates two distinctive gene orders and uses the duplicated 3' sequences to maintain intact copies of the partially duplicated loci.     

Challenges in microarray class discovery: a comprehensive examination of normalization,gene selection and clustering

Background  

Cluster analysis, and in particular hierarchical clustering, is widely used to extract information from gene expression data. The aim is to discover new classes, or sub-classes, of either individuals or genes. Performing a cluster analysis commonly involve decisions on how to; handle missing values, standardize the data and select genes. In addition, pre-processing, involving various types of filtration and normalization procedures, can have an effect on the ability to discover biologically relevant classes. Here we consider cluster analysis in a broad sense and perform a comprehensive evaluation that covers several aspects of cluster analyses, including normalization.