Open reading frame sequencing and structure-based alignment of polypeptides encoded by RT1-Bb, RT1-Ba, RT1-Db, and RT1-Da alleles

MHC class II genes are major genetic components in rats developing autoimmunity. The majority of rat MHC class II sequencing has focused on exon 2, which forms the first external domain. Sequence of the complete open reading frame for rat MHC class II haplotypes and structure-based alignment is lacking. Herein, the complete open reading frame for RT1-B, RT1-B, RT1-D, and RT1-D was sequenced from ten different rat strains, covering eight serological haplotypes, namely a, b, c, d, k, l, n, and u. Each serological haplotype was unique at the nucleotide level of the sequenced RT1-B/D region. Within individual genes, the number of alleles identified was seven, seven, six, and three and the degree of amino-acid polymorphism between allotypes for each gene was 22%, 16%, 19%, and 0.4% for RT1-B, RT1-B, RT1-D, and RT1-D, respectively. The extent and distribution of amino-acid polymorphism was comparable with mouse and human MHC class II. Structure-based alignment identified the 65–66 deletion, the 84a insertion, the 9a insertion, and the 1a–1c insertion in RT1-B previously described for H2-A. Rat allele-specific deletions were found at RT1-B76 and RT1-D90–92. The mature RT1-D polypeptide was one amino acid longer than HLA-DRB1 due to the position of the predicted signal peptide cleavage site. These data are important to a comprehensive understanding of MHC class II structure-function and for mechanistic studies of rat models of autoimmunity.Nucleotide sequence data reported are available in the GenBank database under the accession numbers AY626180–AY626189 for all RT1-Bb sequences, AY626190–AY626199 for all RT1-Ba sequences, AY626200-AY626209 for all RT1-Db sequences and AY626210–AY626219 for all RT1-Da sequences.     

Identical RT1 class II molecules are expressed by rat RT1m and RT1c haplotypes

The RT1m haplotype of MNR rats has been suggested to be a recombinant RT1 haplotype inheriting RT1.A (class I) alleles from RT1a (DA) and RT1.B (class II) alleles from RT1c (AUG). Additional serologic and biochemical assays, however, have suggested that RT1m and RT1c share a single identical RT1.B molecule, although differing in the expression of the second RT1.B molecule. To resolve this contradiction, RT1.B class II molecules, comparable to I-A and I-E molecules in mice, expressed by the RT1c and RT1m haplotypes were immunoprecipitated by cross-reactive mouse anti-Ia antibodies and were compared by two-dimensional gel electrophoresis and by high pressure liquid chromatographic separation of tryptic peptides. Respective subunits expressed by the two haplotypes co-migrate on two-dimensional gels and have identical tryptic peptide maps. The results at the protein level were confirmed at the DNA level by Southern blot analysis of MNR and AUG genomic DNA. Identical restriction fragments associated with the RT1m and RT1c haplotypes hybridized with each of the DC1 beta, DR alpha, and DR beta cDNA probes. The results at both the protein and DNA levels suggest that the RT1m and RT1c haplotypes share identical expressed alleles at the RT1.Ba, RT1.Bb, RT1.Bc, and RT1.Bd loci.     

Polymorphism and mapping of the class II genes in the rat: RT1.B,RT1.D,and RT1.H,a new DP-like region

The major histocompatibility complex of the rat (RTI) encodes the class II molecules involved with antigen presentation and cell to cell communication. The organization of these class II genes has been studied by Southern blot hybridization using genomic DNA from inbred and recombinant rat strains digested with various restriction endonuclease and hybridized under stringent conditions with probes for mouse class II and human class II genes. Analysis of the restriction fragment length polymorphisms has mapped the class II genes relative to each other. We have confirmed the order of the - and -chain genes in the RT1.B region, mapped the RT1.D region relative to RT1.B and showed that it has - and -chain loci, and identified a new HLA-DP-like locus, RT1.H, to the RT1.A side of RT1. B. The RT1. H and RT1.H genes map to the region around the recombination point in R22, and there appears to be a hot spot of recombination in RT1.H. The H and D genes have high levels of polymorphism; B , B ,and H have intermediate levels of polymorphism, and D has a low level of polymorphism.     

The major histocompatibility complex of the rat,RT 1

Recombinational analysis has shown that the rat MHC,RT1 is divided into two regions:RT1.A, which codes for class I (transplantation) antigens, andRT1.B, which controls the humoral immune response and proliferative response to allogeneic cells as well as the expression of class II (Ia) antigens. Serological and sequence studies suggest that there might be more than one antigen-coding locus within theRT1.A region. Results obtained by sequential immunoprecipitation reveal that both regions code for at least two gene products. By implication, theRT1 complex must therefore harbor at least four loci;RT1.A andD coding for class I glycoproteins (45,000 daltons); andRT1.B andE coding for class II (Ia) glycoproteins (35,000 and 28,000 daltons).     

The major histocompatibility complex of the rat,RT1

The antigenic determinants expressed on RBC and lymphocytes and coded for by the MHC, RT1,of the MNR (RT1 ( m )) rat strain were compared to those of the BN.DA(RT1 ( a )), ALB (RT1 ( b )), and AUG (RT1 ( c )) strains by direct cytotoxicity and absorption analysis with RT1 typing sera, sera produced against MNR cells, and sera produced in MNR responders against cells carrying thea, b, andc haplotype determinants. The results indicate that MNR shares major class I (A) antigens with DA, and major class II (B) determinants with AUG, but that MNR differs from DA and AUG with respect to both classes of determinant. It appears, therefore, that the MNR haplotype does not represent a simple composite of the two other haplotypes,RT1 ( a ) andRT1 ( c ), as reported earlier.     

Pharmacophoric features of nucleosidic HIV-1RT inhibitors

A quantum pharmacological study on nucleosidic inhibitors of HIV-1RT has been performed. The main aim of this study is to discuss the pharmacophoric features (conformational and electrostatic) of nucleosidic inhibitors and compare them with normal substrate dNTP. Present study stresses on the need to refine nucleosidic drugs, as combination therapy to date is still one of the best remedies for AIDS. The results of ab initio HF calculations indicate very little effect of 3' substituent on ring puckering and suggest that potency regulation may be via very intricate phosphate-catalytic triad interactions. Our MESP maps also show charge complementarity between the drug and receptor.     

An immunochemical procedure for the purification of rat class I antigens (RT1.A/RT1.E) from detergent-solubilized erythrocytes

An immunochemical procedure using two immunoaffinity columns for the isolation of rat Class I antigens from detergent-solubilized DA erythrocytes is described. The protocol yields a pure Class I preparation as assessed by silver staining of sodium dodecyl sulfate-polyacrylamide gels and comparison of the amino acid composition with that previously determined for H-2K/D. The antigen preparation is capable of specifically blocking the hemagglutination of DA erythrocytes by Class I-specific alloantiserum.     

Evolutionary lineages of RT1.Ba in the Australian Rattus

In this study, the evolutionary history of the variable second exon of RT1.Ba and its adjoining intron b are compared across a number of species and subspecies of the Australian RATTUS: Three lineages are identified in the second intron across a range of Rattus species. Two of these lineages, separated by the insertion of a probable rodent short interspersed nucleotide element and by point mutations outside the indel region, are both found in each of the major clades of the endemic Australian RATTUS: This pattern of ancestral polymorphism is reflected in the adjoining exon 2 sequences, although phylogenetic constraints confirm that the clustering is not identical to that of the associated intron sequences. In addition, the coding sequences show evidence of the retention of ancestral polymorphism, with identical exon sequences found in two divergent species, and some indication of gene conversion detected for the exon sequences.     

The distribution of Tap2 alleles among laboratory rat RT1 haplotypes

We are reporting the cDNA sequences of Tap2 from two cim ^a and two cim ^b rat strains. Comparison of the cDNA sequences shows that these alleles fall into two groups, which we refer to as Tap2-A and Tap2-B. We found that alleles from the Tap2-B group are more closely related to the mouse homologue than are Tap2-A alleles, and among the 48 nucleotides which differ between the Tap2-A and Tap2-B cDNAs, three affect restriction sites. We defined pairs of oligonucleotides which allow amplification of the regions bearing these restriction sites from genomic DNA or cDNA, and this technique has been successful for the genotyping of all of the 56 laboratory strains of Rattus norvegicus tested and for five cell lines tested so far. All 14 known RT1 standard haplotypes were tested, and 7 found to belong to the Tap2-B group, and 7 to Tap2-A. We also found that intron sizes among the alleles of the Tap2-B group fall into two subgroups, providing further insight into the phylogency of these various haplotypes.