Allodeterminants and evolution of a novel HLA-B5 CREG antigen, HLA-B SNA.

A novel HLA-B5 CREG gene, HLA-B SNA was cloned and the primary structure was determined. The sequence data showed that HLA-B SNA was identical to HLA-B51 except the alpha 1 domain in which one amino acid substitution at residue 74 and 5 amino acid substitutions associated with the Bw4/Bw6 epitopes were observed between these Ag. The comparison with other HLA-B locus genes suggested that HLA-B SNA evolved from HLA-B51 by gene exchange or recombination at the exon 2 between HLA-B51 and B8. A total of 10 of 14 HLA-B51-specific CTL clones showed significantly weak or no recognition of HLA-B SNA Ag. They also gave the same degree of a lysis of Hmy2CIR cells expressing the HLA-B35/51 chimeric Ag composed of the alpha 1 domain of HLA-B35 and other domains of HLA-B51 as that of Hmy2CIR cells expressing the HLA-B SNA Ag. These results demonstrated that amino acid substitutions within positions 77-83 associated with the HLA-Bw4/Bw6 epitopes have an influence on recognition of the HLA-B SNA antigen by HLA-B51-specific CTL.     

HLA-B51 and HLA-Bw52 differ by only two amino acids which are in the helical region of the alpha 1 domain

Genes encoding the serologically cross-reactive HLA-B51 and HLA-Bw52 molecules were isolated and the exons sequenced. HLA-B51 genes obtained from Caucasian and Oriental individuals were identical. HLA-Bw52 differs from HLA-B51 by four nucleotide substitutions in exon 2 encoding the alpha 1 domain. These comprise one isolated silent substitution in codon 23 and a cluster of three coding substitutions in codons 63 and 67. Amino acid substitutions of N----E at position 63 and F----S at position 67 are the only differences between HLA-B51 and HLA-Bw52 and these residues are postulated to form HLA-B51 specific epitopes. HLA-B51 could have been formed from HLA-Bw52 by the combination of a genetic exchange with HLA-B8 and a point mutation. Similarity of HLA-B51 and HLA-Bw52 with HLA-Bw58 suggest they also share a common ancestor.     

Genetic and serological heterogeneity of the supertypic HLA-B locus specificities Bw4 and Bw6

Gene cloning and sequencing of theHLA-B locus split antigens B38 (B16.1) and B39 (B16.2) allowed localization of their subtypic as well as their public specificities HLA-Bw4 or-Bw6 to the α-helical region of the α 1 domain flanked by the amino acid positions 74–83. Comparison of their amino acid sequences with those of otherHLA-B-locus alleles established HLA-Bw6 to be distinguished by Ser at residue 77 and Asn at residue 80. In contrast, HLA-Bw4 is characterized by at least seven different patterns of amino acid exchanges at positions 77 and 80–83. Reactivity patterns of Bw4-or Bw6-specific monoclonal antibodies reveal two alloantigenic epitopes contributing to the HLA-Bw4 or-Bw6 specificity residing next to the region of highest diversity of the α 1 domain.     

Complotypes in individuals of African origin: frequencies and possible extended MHC haplotypes

We analyzed the frequency distribution of 106 complotypes [four allele sets of the major histocompatibility complex (MHC) genes for the complement proteins factor B, C2, C4A, and C4B] from 32 Black families residing in Boston and Washington, DC. Twenty-five different complotypes were identified, among which there were four complotypes that had not been previously observed in our large database of complotypes compiled from family studies of Boston Caucasians and that are, presumably, unique to individuals of African origin. These four African-derived complotypes areFC(1,90)0, FC63, S1C2,17, andSC(3,2,90)0. The frequencies of two of these four unique Black complotypes,FC(1,90)0 andFC63, were increased significantly when compared to Caucasians (p_corr <0.00042, p_corr=0.00294, respectively). The complotypeFC(1,90)0 was in positive linkage disequilibrium withHLA-DR3 haplotypes containing theB locus antigens Bw42, Bw52, Bw53, and Bw58, whileFC63 was associated withHLA-Bw70,-DR5. These findings demonstrate the extensive polymorphism of complotypes in Blacks, and also suggest that it may be possible to define unique extended haplotypes of African origin.     

Optimization of α-amylase and glucoamylase production by recombinant strains ofSaccharomyces cerevisiae

Summary Replacement of the regulatory sequence of theBacillus amyloliquefaciens α-amylase gene (AMY1) by the yeast alcohol dehydrogenase gene promoter (ADC1 _p) resulted in increased levels of extracellular α-amylase production inSaccharomyces cerevisiae. Negative regulation of glucoamylase synthesis by theSTA10-encoded repressor was alleviated by replacing the nativeSTA2 gene promoter fromS. cerevisiae var.diastaticus withADC1 _p. Enhanced degradation of starch was achieved when the modified versions of theAMY1 andSTA2 genes were introduced jointly intoS. cerevisiae.     

Inter- and intra-genomal chromosome pairing in an interspecific hybrid and its bearing on basic chromosome number inPennisetum

Meiosis in the interspecific hybrid betweenPennisetum typhoides (2n=14; genomeAA) andP. purpureum (2n=28; genomesA′A′BB) has been studied with particular reference to allosyndetic and autosyndetic pairing of chromosomes. Although up to nine bivalents occurred in the hybrid, never more than five were observed to be (heteromorphic)AA′ bivalents (range 1–5). It has been concluded thatA andA′ genomes are onlypartially homologous. It has further been inferred that the two genomes are evolutionarily related and could have arisen from a common progenitor withx=5 chromosomes or from related species withx=5 chromosomes. Autosyndetic pairing of chromosomes within thetyphoides complement (A genome) and within theA′ genome ofpurpureum have been reported here for the first time. Intra-haploid pairing to a probable maximum of two bivalents within each of the three genomes of the hybrid, viz.,A, A′ andB, further suggestsx=5 as the phyletically basic number in the genusPennisetum. It has been inferred thatx=7 is a secondarily basic number, having been derived fromx=5. The occurrence of a species withn=5 inPennisetum, viz.,P. ramosum, substantiates this view. Further support in favour of this conclusion comes from the secondary association of bivalents in dipoidP. typhoides. Thus, the apparently diploid species,Pennisetum typhoides with2n=14 chromosomes is considered to be a “secondary diploid” having a secondarily balanced number ofx=7. On the basis of the results obtained by the author is conjunction with the available evidence from the literature, it is suggested thatx=5 may be the original basic number for the entire grass family and seven, the most preponderant number in it, and other higher numbers derived from it subsequently during the course of evolution.     

Comparison of the structure of HLA-Bw47 to HLA-B13 and its relationship to 21-hydroxylase deficiency

Adrenal 21-hydroxylase deficiency is strongly associated with HLA-Bw47. This rare HLA allele and the HLA-B13 allele are both found in positive genetic linkage disequilibrium with HLA-A3, -Cw6, -DR7 and also display serological cross-reactivity. To investigate the relationship between these two alleles at the structural level, the nucleotide sequences of the HLA-B13 and HLA-Bw47 genes have been determined. They differ by 28 nucleotides, resulting in 14 amino acid substitutions: 5 in the 1 domain, 8 in the 2 domain, and 1 in the transmembrane region. Comparison of HLA-Bw47 nucleotide sequence with other HLA-B sequences shows a segment of 228 by identical with B44 in the a 1 domain and a segment of 218 by identical with B27 in the a2 domain, but only a 91 by segment of identity with B13 in the al domain. The complex pattern of substitutions and their degree of divergence indicate that HLA-B13 and HLA-Bw47 alleles are not related by a simple mutational event.     

HLA-D typing in 111 multiple sclerosis patients: Distribution of four HLA-D alleles

Multiple sclerosis patients-111 of them-were typed for theHLA-D allelesw1, w2, andw3 and a new determinant, EI. Statistically significant increase was obtained for thew2 andw3 frequencies as compared to healthy controls. The distribution of HLA-D phenotypes among MS patients revealed a good fit according to the Hardy-Weinberg law. By calculating linkage disequilibrium parameters, theHLA-B7,Dw2 allele combination was found to be more closely associated than in normal controls, whereas forHLA-Bw35, Dw1, no linkage disequilibrium could be detected in the patients' group. From these data we conclude that in multiple sclerosis, the disturbance affects the frequency ofDw3 and the gametic association between alleles of theHLA-B andD loci, as well as the already known increase ofHLA-B7 andDw2.     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

Presence ofenv-like sequences inQuercus suber retrotransposons

The main difference between LTR retrotransposons and retroviruses is the presence of theenvelope (env) gene in the latter, downstream of thepol gene. Theenv gene is involved in their infectious capacity. Here we report the presence ofenv-like sequences in the genome ofQuercus suber (cork oak), one of the most economically important Portuguese species. These gene sequences were isolated through DNA amplification betweenRNaseH conserved motifs and 3’ LTR, based on the structure ofcopia retrotransposons. Phylogenetic analysis revealed that almost all the clones isolated are clustered withCyclops-2, aTy3-gypsy element identified inPisum sativum, except one clustered withgypsy andcopia retroelements found in different species. This suggests the existence of a potential ancestral sequence of theenv gene, prior to the separation ofTy3-gypsy andTy1-copia retrotransposons. Additionally, the isolatedenv-like sequences showed 26–39% of homology withenv-like sequences characterized in viruses. The origin ofenv-like sequences in retrotransposons from host plant taxa is discussed.     

Beh?et’s disease in HLA-B*51 negative Germans and Turks shows association with HLA-Bw4-80I

Introduction

Behçet’s disease (BD) as systemic vasculitis of unknown etiology is associated with HLA-B*51 in European and Asian populations. HLA-A*26 was claimed as an additional BD susceptibility marker in Japanese and Greek patients. This study was performed to test for HLA associations in HLA-B*51 negative German and Turkish BD populations.

Methods

In total, 65 German and 46 Turkish patients lacking HLA-B*51 were analyzed in comparison to healthy HLA-B*51 negative Germans (n = 1500) and Turks (n = 130). HLA-A/B genotypes were determined by SSOP. P-values with correction for multiple testing (p_c), χ2-test and odds ratio (OR) were used for statistical evaluation.

Results

HLA-A*26 was significantly more frequent in HLA-B*51⁻ German patients [p_c = 0.0076, OR = 3.23, 95% CI 1.63 to 6.39] than in respective controls. HLA-A*26 was also elevated in a smaller group of Turkish patients versus the controls. Significant association of HLA-Bw4 with isoleucine at amino-acid position 80 (HLA-Bw4-80I) was found in the HLA-B*51⁻ German cohort of BD patients [p_c = 0.0042, OR = 2.35, 95% CI 1.41 to 3.93) and in the Turkish patients in comparison to the respective controls [p = 0.025, OR = 2.17, 95% CI 1.09 to 4.31]. On the contrary, HLA-Bw4-80 T was reduced in both HLA-B*51⁻ BD patient cohorts.

Conclusions

The study shows a significant association of HLA-Bw4-80I present on HLA-B*51 as well as on other B-locus molecules with BD. This indicates that distinctive Bw4 epitopes on HLA-B locus molecules could play a role in BD pathogenesis. The study also indicates an association with HLA-A*26 in German and Turkish BD patients as a genetic risk factor independent of HLA-B*51.     

Differential transport requirements of HLA and H-2 class I glycoproteins

Transport of human and mouse major histocompatibility complex class I glycoproteins has been examined in a transport deficient B-lymphoblastoid cell line × T-lymphoblastoid cell line (B-LCL × T-LCL) hybrid, 174 × CEM. T2 (T2). This cell line expresses no detectable endogenous HLA-B5 and reduced levels of HLA-A2 on its surface although these molecules are synthesized. In order to study this defect further, either HLA-Bw58 or HLA-B7 genomic clones were transfected into T2. Metabolic labeling and immune precipitation demonstrated biosynthesis of the Bw58 or 137 glycoprotein. However, like the endogenous HLA-B5 molecule, neither HLA-Bw58 nor HLA-B7 was expressed at the cell surface. The cloned genes were properly expressed on the surface of C1R, a control B-LCL. To determine if mouse class I alleles had the same transport requirements as the human class I glycoproteins, either mouse H-2D ^p or H-2K ^b class I genes were introduced into T2. Surprisingly, the H-2 class I glycoproteins were transported to the cell surface normally. These data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport.     

Interleukin-1α-induced changes in chromium-51 absorption, tissue retention, and urinary excretion in rats

The effects of interleukin-1 alpha (IL-1α) on chromium-51 absorption, tissue retention, and urinary excretion were studied in adult male Sprague-Dawley rats. Ten rats were deprived of food for 12 h, injected intraperitoneally with mouse recombinant IL-1α (1 Μg/kg of body weight in phosphate-buffered saline [PBS]) or control (0.1% bovine serum albumin [BSA] in PBS). Two hours after dosing with the IL-1α, rats were fed 50 ΜL (200 ΜCi, 0.36 Μg Cr) of⁵¹CrCl₃ by micropipet. Blood was collected from the tail at 0.25, 0.5, 1, 2, and 4 h. Six hours after dosing with⁵¹CrCl₃, rats were exsanguinated and blood and tissues were sampled. The IL-lα significantly decreased chromium-51 in blood, urine, and some tissues compared to the control. The decreased absorption, retention, and urinary excretion of chromium-51 from⁵¹CrCl₃ in this study may be due to IL-1α-mediated increases in the production of prostaglandins and/or decreased production of gastric acid.     

HLA-C “blank” alleles express class I gene products. Biochemical analysis of four different HLA-C “blank” polypeptides

Monoclonal antibodies (mAbs) W6/32, HC10, and 4E were used to precipitate class I antigens from 21 selected individuals with at least oneHLA-C “blank” allele. In 19 of these individuals, characteristicHLA-C banding patterns which could be precipitated by all three HLA class I mAbs were observed on one-dimensional isoelectric focusing gels-obviously the gene products ofHLA-C “blank”. At least four allelic HLA-C “blank” gene products with different isoelectric points could be discerned. All of them segregated withHLA-C “blank” haplotypes in informative families; two of them were associated withHLA-B51, one withHLA-B38, and one withHLA-B18. Reactivity of the HLA-C “blank” heavy chains with mAb W6/32 indicates that they are able to associated with beta-2 microglobulin, and hence are most probably expressed at the cell surface.     

Secretory expression in Escherichia coli and Bacillus subtilis of human interferon alpha genes directed by staphylokinase signals

Summary A DNA segment covering the signal sequence coding region, the ribosome binding site, and the promoter of the staphylokinase (sak) 42D gene (Behnke and Gerlach 1987) was cloned into pUC19 to form a portable expression-secretion unit (ESU). Fusion of human interferon α₁ (hIFNα₁) and hybrid hIFNα_1/2 genes to thissak ESU resulted in secretory expression of the two gene products in bothEscherichia coli andBacillus subtilis. While most of the IFNα was exported to the periplasmic space ofE. coli, about 99% was secreted to the culture medium by recombinantB. subtilis strains. The total yield inE. coli was 1.2×10⁵ IU/ml. This level of expression and export led to instability of the recombinant strains that was spontaneously relieved in vivo by inactivation of thesak ESU through insertion of an IS1 element. No such instability was observed withB. subtilis although expression and secretion levels reached even 3×10⁶ IU/ml. Proteolytic degradation of IFNα by extracellular proteases was avoided by a combination of constitutive expression and secretion during the logarithmic growth phase and the use of exoprotease-reduced host strains. The IFNα₁ protein purified fromB. subtilis culture supernatant was correctly processed, carried the expected 11 amino acid N-terminal elongation that resulted from DNA manipulations and proved to be homogenous in Western blotting experiments. The same recombinant plasmid that directed efficient secretion of hIFNα₁ inB. subtilis gave poor yields when introduced intoStreptococcus sanguis.     

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.     

Karyotypic evolution,morphological variability and phylogeny inMedicago sect.Intertextae

Karyotype and external morphological analyses were conducted onMedicago ciliaris, M. intertexta, M. muricoleptis andM. granadensis which comprise theIntertextae section of the genusMedicago. All species were found to have 2n = 16 chromosomes (= 2 ×), including one pair of satellite chromosomes in each respective complement. Karyotypic evolution in theIntertextae involves changes in absolute chromosome size and in centromeric and relative size symmetry. Numerical taxonomic analyses were conducted independently on 17 karyotypic features and on 16 features of external morphology. The results of the two sets of analyses proved comparable, withM. ciliaris andM. intertexta forming a fairly close pair, and the remaining two species appearing to have more distant relationships to each other and to the first pair. These observations are consistent with the infertility relationships and chorologies of the species. It is suggested thatM. muricoleptis andM. granadensis are derived from theM. ciliaris/intertexta species complex withM. granadensis arising fromM. muricoleptis, or these two species independently evolving from a common species complex. Chromosomal and numerical analyses suggest thatM. ciliaris is the most primitive andM. granadensis the most derived species of theIntertextae.     

An additional regulator,TsaQ, is involved with TsaR in regulation of transport during the degradation of<Emphasis Type="Italic"> p</Emphasis>-toluenesulfonate in<Emphasis Type="Italic"> Comamonas testosteroni</Emphasis> T-2

The degradation of p-toluenesulfonate (TSA) by Comamonas testosteroni T-2 is initiated by a transport system (TsaST) and enzymes (TsaMBCD) encoded on the tsa transposon, Tntsa, on the TSA plasmid (pTSA). Tntsa comprises an insert of 15 kb between two IS1071 elements. The left-hand 6 kb and the right-hand 6 kb are nearly mirror images. The regulator of the tsaMBCD₁ genes (right-hand side) is the centrally located LysR-type TsaR, which is encoded upstream of tsaMBCD₁ on the reverse strand. The other centrally located genes are tsaS and tsaT, encoded downstream of tsaR and on the same strand as both tsaR and tsaMBCD₂. The latter four genes are not expressed. Downstream of tsaD₁ (tsaD₂) is tsaQ₁ (tsaQ₂) and another open reading frame of unknown function. The tsaQ genes have identical sequences. Sequence analysis indicated that TsaQ could be an IclR-type regulator, whose expression during degradation of TSA was proven by data from RT-PCR. Both copies of tsaQ could be knocked-out by homologous recombination. Double mutants failed to grow with TSA but grew with p-toluenecarboxylate (TCA), which is also degraded via TsaMBCD. This showed TsaQ to be essential for the degradation of TSA but not TCA. We attributed this to regulation of the transport of TSA, especially to regulation of the expression of tsaT, which was expressed solely during growth with TSA. Seven independently isolated bacteria containing the tsa operon were available. Those six which contained tsaT on Tntsa also contained tsaQ. The promoter region of tsaT was found to be a target of the regulator TsaR. Band-shift data indicate that TsaR is required for the expression of tsaT, which suggests that tsaR and tsaQ_1,2, together with tsaMBCD₁, belong to a common regulatory unit.