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 Therefore, we propose the designation BoLA-DQA3 for the previously unpublished alleles at the second DQA2 locus.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     

The possible evolutionary significance of repeat elements near and within an early chorion gene in the late chorion locus of Bombyx mori

Summary Three -type early chorion gene copies (6F76.1, 6F76.2, and 6F76.3) are dispersed in the late region of chorion locus Chl-2. Detailed analysis of the 5-flanking region and the intron of 6176.1 shows that they contain sequences that are homologous to Bombyx mori Bm l repeat elements. Interestingly, the Bm l -type segment of the intron is interrupted by the insertion of a sequence that shows significant similarities with part of an intron of B. mori and Bombyx mandarina fibroin genes, and with part of the 3-flanking region of B. mori prothoracicotropic hormone and tRNA-Glu genes; this sequence may represent a new repetitive, possibly transposable, element of B. mori. Following the Bm1-homologous sequence of the 6176.1 5-flanking region and preceding the gene promoter region, a short DNA segment shows sequence motifs that are also present in the ErA.1 promoter region. The occurrence of these sequences near one end or within the Bm1 repeat element is suggestive of complex sequence transfer events. Comparative analysis of known B. mori chorion -gene promoters and of Bm1 repeat elements suggests, with marginal statistical significance, that these two sets of sequences contain common elements.Offprint requests to: G. Rodakis     

Sequence variation at theSec-1 locus of rye,Secale cereale (Poaceae)

This paper describes the structure of a 9.2-kb repeat unit of DNA, which represents one-secalin gene and spacer sequence located at theSec-1 locus on the short arm of chromosome 1 of rye. The gene units at theSec-1 locus comprise 1.1 kb representing the gene and 8.1 kb of spacer sequence separating the genes. A sequence comparison of nine genes and their promoter regions from theSec-1 locus, reveals that there is greater variation within the coding sequence than there is within the promoter regions. The gene sequence variation is discussed in terms of the size variation seen for the-secalin proteins in rye species. The results include a comparison of promoter sequences from members of the Triticeae to examine the degree of conservation between other seed storage protein genes.     

Highly Variable Polymorphism of the α-Amylase Gene Family in <Emphasis Type="Italic">Litopenaeus</Emphasis><Emphasis Type="Italic">vannamei</Emphasis> (Crustacea Decapoda)

-Amylase from the tropical shrimp Litopenaeus vannamei presents a high degree of polymorphism and at least eight different electromorphs are detected by electrophoresis. Based on nucleotide sequences, three cDNAs have been previously characterized. In this paper we report on the organization and the evolution of corresponding -amylase genes, determined after PCR amplification. Three AMY genes have been characterized, spanning over 3.3 kb and encoding mature proteins of 495 amino acids (aa), which are all expressed in the digestive gland. The existence of nine short introns, ranging from 86 to 454 bp, located at the same positions for each of the different genes, and presenting no similarity between them, is reported. Between 11 and 15% of changes are observed in the coding aa sequences of genes II and III compared to the gene I sequence respectively. One 5 putative promoter sequence has been sequenced and shows no classical TATA box upstream to the coding sequence. Based on the intron size difference, a single PCR (producing the S–R fragments) allows the separation of a partial gene I (750 bp), corresponding to cDNA 20, from the others (650–680 bp). Sequencing different S–R PCR fragments from one shrimp shows at least eight different haplotypes. A complex microsatellite repeat is present in intron 6 of gene II. Using size and sequence differences in this repeated portion, it is possible to characterize two gene subfamilies (IIa and IIb) encoding previously described cDNAs 28 and 37, respectively. For the gene II family, two to four alleles are present in one shrimp corresponding to these two genes. Within the Panama natural population, 35 different alleles are shown at this locus. Regarding -amylase gene structure in the shrimp, many recombinants are present from a set of individuals and constitute an important mechanism of evolution of -amylase function. Accession numbers: AJ132379, L. vannamei -amylase gene I; AJ133526, gene II; AJ133119, gene III     

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.     

Mapping and characterization of the DQ subregion of the ovine MHC

A map of the ovine MHC class II DQ subregion has been constructed from overlapping cosmid clones. This region consists of two loci linked on a linear tract of 130 kb DNA. Each locus consists of a DQA and a DQB gene in a tail-to-tail orientation. The genes in each locus are transcribed but only those designated DQ1 express class II molecules at the surface of mouse L cells following DNA-mediated gene transfection. The DQA1 and DQB1 genes are separated by 11kb while the DQA2 and B2 genes are 25 kb apart. The loci are separated by 22 kb.     

Organization of the genes encoding chalcone synthase in Pisum sativum

To analyze the regulation of defense-related genes by signal molecules produced by phytopathogens, we isolated genes that encode chalcone synthase (CHS) in Pisum sativum. We have obtained seven independent genomic clones that contain at least seven classes of CHS genes, identified by the hybridization analysis to CHS cDNA and by the restriction mapping analysis. Two of the genomic clones (clone 5 and 6) each contain two CHS genes in a tandem repeat. The nucleotide sequence analysis of CHS genomic clone 5 revealed that PsCHS1 and PsCHS2 were corresponding genes of the CHS cDNA clones, pCC6 and pCC2, respectively, as reported earlier. Both genes are interrupted by a single intron of 88 nucleotides with identical sequences, although exonic sequences and 5-flanking sequences are divergent. Nucleotide sequences of the introns in five other classes of CHS genes showed that three classes had an intron of 87 nt with a striking homology to each other, but that the intron of the other two classes of CHS genes showed heterogeneity both in size and nucleotide sequence. 5-upstream regions of PsCHS1 and PsCHS2 did not show sequence homology except the 31 bp identical sequence that contains the CCTACC motif resembling the box-1 sequence. Both PsCHS1 and PsCHS2 genes are shown to be induced by fungal elicitor by a primer extension analysis and a transient transformation analysis using pea protoplasts prepared from suspension cultured-cells.     

Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations

The highly polymorphic swine leucocyte antigen (SLA) genes are among the most important determinants of swine immune responses to disease and vaccines. Accurate and effective SLA genotyping methods are required to understand how SLA gene polymorphisms affect immunity, especially in outbred pigs with diverse genetic backgrounds. In this study, we present a simple and rapid molecular‐based typing system for characterizing SLA class II alleles of the DRB1, DQB1 and DQA loci. This system utilizes a set of 47 sequence‐specific PCR primers developed to differentiate alleles by groups that share similar sequence motifs. We applied this typing method to investigate the SLA class II diversity in four populations of outbred pigs (n = 206) and characterized a total of 19 SLA class II haplotypes, six of which were shared by at least three of the sampled pig populations. We found that Lr‐0.1 (DRB1*01XX–DQB1*01XX–DQA*01XX) was the most prevalent haplotype with a combined frequency of 16.0%, followed by Lr‐0.2 (DRB1*02XX–DQB1*02XX–DQA*02XX) with 14.6% and Lr‐0.15b (DRB1*04XX–DQB1*0202–DQA*02XX) with 14.1%. Over 70% of the pigs (n = 147) had at least one copy of one of these three haplotypes. The PCR‐based typing system described in this study demonstrates a reliable and unambiguous detection method for SLA class II alleles. It will be a valuable tool for studying the influence of SLA diversity on various immunological, pathological and physiological traits in outbred pigs.     

Expression of an α-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus

For expression of the -galactosidase gene from Cyamopsistetragonoloba in Kluyveromyces marxianus CBS 6556 we have used the promoter of the homologous inulinase-encoding gene (INU1). The INU1 gene has been cloned and sequenced and the coding region shows an identify of 59% with the Saccharomyces cerevisiae invertase gene (SUC2). In the 5'-flanking region of INU1 we found a sequence (TAAATCCGGGG) that perfectly matches to the MIG1 binding consensus sequence (WWWWTSYGGGG) of the S. cerevisiae GAL1, GAL4 and SUC2 genes. Using the K. marxianus INU1 promoter and prepro-signal sequence, we obtained a high -galactosidase production level (153 mg/l) and a secretion efficiency of 99%. Both the production level and the secretion efficiency were significantly reduced when the INU1 pro-peptide was deleted. With either the S. cerevisiae PGK or GAL7 promoter we could obtain only low -galactosidase production levels (2 mg/l). Correspondence to: R. J. Planta     

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.     

Structure of genes and an insertion element in the methane producing archaebacterium Methanobrevibacter smithii

Summary DNA fragments cloned from the methanogenic archaebacterium Methanobrevibacter smithii which complement mutations in the purE and proC genes of E. coli have been sequenced. Sequence analyses, transposon mutagenesis and expression in E. coli minicells indicate that purE and proC complementations result from the synthesis of M. smithii polypeptides with molecular weights of 36,697 and 27,836 respectively. The encoding genes appear to be located in operons. The M. smithii genome contains 69% A/T basepairs (bp) which is reflected in unusual codon usages and intergenic regions containing approximately 85% A/T bp. An insertion element, designated ISM1, was found within the cloned M. smithii DNA located adjacent to the proC complementing region. ISM1 is 1381 bp in length, has 29 bp terminal inverted repeat sequences and contains one major ORF encoded in 87% of the ISM1 sequence. ISM1 is mobile, present in approximately 10 copies per genome and integration duplicates 8 bp at the site of insertion. The duplicated sequences show homology with sequences within the 29bp terminal repeat sequence of ISM1. Comparison of our data with sequences from halophilic archaebacteria suggests that 5GAANTTTCA and 5TTTTAATATAAA may be consensus promoter sequences for archaebacteria. These sequences closely resemble the consensus sequences which precede Drosophila heatshock genes (Pelham 1982; Davidson et al. 1983). Methanogens appear to employ the eubacterial system of mRNA: 16SrRNA hybridization to ensure initiation of translation; the consensus ribosome binding sequence is 5AGGTGA.     

Co-regulation with genes of phospholipid biosynthesis of the CTR/HNM1-encoded choline/nitrogen mustard permease in Saccbaromyces cerevisiae

An 815 by region of the promoter of the Saccharomyces cerevisiae gene CTR/HNM1, encoding choline permease was sequenced and its regulatory function analysed by deletion studies in an in-frame promoter-lacZ construct. In addition to the TATA box, a 10 by motif (consensus 5-CATGTGAAAT-3) was found to be mandatory for CTR/HNM1 expression. This decamer motif is located between nucleotides –262 and –271 and is identical in 9 of 10 by with the regulatory motif found in the S. cerevisiae INO1 and CHO1 genes. Constructs with the 10 by sequence show high constitutive expression, while elimination or alterations at three nucleotide positions, of the decamer motif in the context of an otherwise unchanged promoter leads to total loss of -galactosidase production. Expression of the CTR/HNM1 gene in wild-type cells is regulated by the phospholipid precursors inositol and choline; no such influence is seen in cells bearing mutations in the phospholipid regulatory genes INO2, INO4, and OPI1. There is no regulation by INO2 and OPI1 in the absence of the decamer motif. However constructs not containing this sequence (promoter intact to positions –213 or –152) are still controlled by INO4. Other substrates of the choline permease, i.e. ethanolamine, nitrogen mustard and nitrogen half mustard do not regulate expression of CTR/HNM1.     

In vitro organogenesis from diploid tissues of Cycas revoluta Thunb.

Leaf base and mesocotyl explants derived from in vitro-grown seedlings of Echinochloa colona were cultured on Murashige and Skoog's (MS) medium containing various concentrations of benzyladenine (BA), -naphthaleneacetic acid (NAA) and kinetin. Leaf base and mesocotyl segments exhibited optimal morphogenetic response by using 6.66 M BA with 2.68 M NAA. Induction of rooting from regenerated shoots was readily achieved in half strength MS medium without organics and growth regulators. Histological studies revealed the sequence of shoot bud regeneration in the monocot system. The in vitro-raised plants were established in chromite minewaste.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - MS medium Murashige & Skoog's (1962) medium     

Polymorphism of the DQA1 promoter region (QAP) and DRB1, QAP,DQA1, DQB1 haplotypes in systemic lupus erythematosus

We have investigated the DNA polymorphism for the DQA1 promoter region (QAP) and HLA-class II DRB1, DQA1, and DQB1 genes in 178 central European patients with Systemic lupus erythematosus (SLE) using polymerase chain reaction and Dig-ddUTP labeled oligonucleotides. Increased frequencies of DRB1*02 and *03 are confirmed by DNA typing. In addition, the frequencies of DQA1*0501, *0102 and DQB1*0201, *0602 alleles are increased in the patients as compared to controls. The strongest association to SLE is found with DRB1*03 and DQB1*0201 alleles (p<10^–7, p corr. <10^–5 and p<10^–6, p corr. <10^–4, respectively). By investigating the DQA1 promoter region in the SLE patients we have detected nine different QAP variants. Increased frequencies of QAP1.2 and QAP4.1 are observed in patients as compared to controls (p <0.05, p corr. = n. s.). Analysis of linkage disquilibria demonstrates a very strong association between QAP variants and DQA1, DRB1 alleles. Certain QAP variants are completely associated with DQA1 and DRB1 alleles, whereas others can combine with different DQA1 and DRB1 alleles. All DRB1*02-positive patients and controls carry QAP1.2, and all DRB1*03-positive patients and controls carry QAP4.1. Conversely, the QAP1.2 variant appears only in DRB1*02 haplotypes, while the QAP4.1 variant can be observed in DRB1*03, *11, and *1303 haplotypes. Based on the strong linkage disequilibria between DRB1-DQA1-DQB1 genes and between DRB1-QAP-DQA1, we have deduced the four-point haplotypes for DRB1-QAP-DQA1-DQB1 in patients and controls. Two haplotypes DRB1*02-QAP1.2-DQA1*0102-DQB1*0602-and DRB1*03-QAP4.1-DQA1*0501-DQB1*0201 are significantly increased in patient as compared to controls (p<0.01, p corr. = n.s., RR = 1.8 and p <10^–7, p corr. <10^–5, RR = 3.1, respectively). The analysis of relative risks attributed to the various alleles of QAP, DQA1, and DQB1 as well as the investigation of the deduced DRB1-QAP-DQA1-DQB1 haplotypes leads to the conclusion that QAP4.1 and DQA1*0501 on the DR3 haplotypes are probably not involved in SLE susceptibility. There is no evidence for the involvement of DQ2 / dimers coded in transposition. Thus, susceptibility to SLE is on the DR3 haplotype most probably localized at DRB1 or telomeric of DRB1, while for the DR2 haplotype such orientation cannot be given. SLE study group members: M. Baur, A. Corvetta, H. Ehrfeld, J. Frey, J. R. Kalden, F. Krapf, B. Lang, G. G. Lange, K. Pirner, C. Rittner, E. Röther, P. Schneider, H. P. Seelig, S. Seuchter, W. Stangel, C. Specker, P. Späth, H. Deicher. Correspondence to: Z. Yao.     

Cloning and expression of a thermostable exo-α-1,4-glucosidase gene from Bacillus stearothermophilus ATCC12016 in Escherichia coli

The gene coding for a thermostable exo--1,4-glucosidase (-glucoside glucohydrolase: EC 3.2.1.20) of Bacillus stearothermophilus ATCC 12016 was cloned within a 2.8-kb AvaI fragment of DNA using the plasmid pUC19 as a vector and Escherichia coli JM109 as a host. E. coli with the hybrid plasmid accumulated exo--1,4-glucosidase mainly in the cytoplasm. The level of enzyme production was about sevenfold higher than that observed for B. stearothermophilus. The cloned enzyme coincided absolutely with the B. stearothermophilus enzyme in its relative molecular mass (62 000), isoelectric point (5.0), amino-terminal sequence of 15 residues (Met-Lys-Lys-Thr-Trp-Trp-Lys-Glu-Gly-Val-Ala-Tyr-Gln-Ile-Tyr-), the temperature dependency of its activity and stability, and its antigenic determinants.Correspondence to: Y. Suzuki