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.     

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.     

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.     

Characterization of and Ethanol Hyper-production by Clostridium thermocellum I-1-B

An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological proper­ ties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50%. decrease of growth yield are 27 and 16g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156 h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512mM) ethanol, 8.5 g/liter (94mM) lactate, 2.9 g/liter (48mM) acetate, and 0.9 g/liter (20mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.     

Characterization of multiple alleles of the T-cell differentiation marker ART2 (RT6) in inbred and wild rats

ART2 (RT6) belongs to the family of mono-ADP-ribosyltransferases (ARTs). ART2 is a T-cell differentiation marker expressed by the majority of mature peripheral T cells in the rat. The two known ART2 allotypes display approximately 95% amino acid identity. We sequenced the ART2 coding regions from 18 inbred rat strains and found two additional alleles, termed Art2 ^a2 and Art2 ^b2 . Monoclonal antibody Gy12/61 specifically reacted with Art2 ^a2 but not Art2 ^a1 lymph node cells. Expression of ART2 allotypes in Jurkat cells confirmed this specificity. A polymerase chain reaction (PCR) assay using restriction fragment length polymorphisms is described, which allows the easy discrimination of Art2 alleles. All four laboratory rat alleles, as well as an additional sequence variant, were found amongst 18 wild rat DNA samples. PCR analysis confirmed the selective presence of a rodent identifier (ID) element in the Art2 ^a but not the Art2 ^b alleles in all rats studied. Analysis of Art2 ^a1 and Art2 ^b2 genes showed greater divergence in coding than in non-coding regions. Together with the finding of a high number of non-synonymous mutations leading mostly to non-conservative amino acid substitutions clustered on the side facing away from the cell surface, this suggests that the Art2 polymorphism has been subject to selection.     

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.     

Characterization of cytochrome P450 2D6 alleles using the Invader system

The cytochrome p450 (CYP) superfamily comprises enzymes that play an essential role in the transformation of medically relevant compounds. Accurate genotyping of polymorphisms in members of this family is drawing increasing interest because certain allelic variants may result in either loss of efficacy or toxic accumulation of therapeutic agents. Debrisoquine 4-hydroxylase, or CYP2D6, is among the most widely studied of the CYPs. The complexity of the CYP2D6 genomic region, including pseudogenes, gene deletions, and gene duplications, has offered numerous challenges to developing a genotyping strategy. We describe a comprehensive CYP2D6 genotyping strategy that employs both a PCR/Invader genotyping assay system and an Invader genomic copy number assay The Invader system is a homogeneous, isothermal, highly specific, and robust signal amplification system. Resultsfrom II CYP2D6 assays in an alle frequency study compare well to published allele frequency values for Caucasians. Further, Invader assays provided unambiguous genotyping determinations for 100% of the 171 samples that yielded a visible PCR product on an agarose gel. A copy number assay yielded only one equivocal result in 205 samples. We identified 17 single-copy individuals and 17 three-copy (or more) individuals.     

Generation of the HLA-B35, -B5, -B16, and B15 groups of alleles studied by intron 1 and 2 sequence analysis

 HLA-B is the most polymorphic of the major histocompatibility complex classical class I loci. This polymorphism is mainly in exons 2 and 3, which code for the molecule’s α1 and α2 domains and include the antigenic peptide binding site. Recent studies have indicated that not only exons but also the intron 2 region may be involved in the generation of certain HLA-B alleles such as B ^* 3906 and B ^* 1522. To study the degree of intron 2 participation and the mechanisms that generate polymorphism at the HLA-B locus, intron 1 and 2 sequences from the HLA-B35, -B5, -B16 and -B15 groups of alleles were obtained. A group-specific intronic polymorphism was found: namely, B ^* 5301 shows intron 1 and 2 sequences identical to those found in all B35 alleles studied. On the other hand, B ^* 5101 and B ^* 52012 show the same intron 1 and 2 sequences and their intron 1 is the same as that found in the B35 group. This suggests that B5 and B35 groups of alleles may have arisen from a common ancestor. All known B16 alleles show the same introns 1 and 2, with the exception of B ^* 39061 and B ^* 39062, and all B15 alleles also bear the same introns 1 and 2, with the exception of B ^* 1522. Variability at intron 1 is more restricted than at intron 2, and the use of intron 1 for HLA-B allele phylogenetic analysis is better for grouping alleles of a postulated common origin. In conclusion, there is a remarkable conservation of intronic sequences within related HLA-B alleles, which probably reflects a common origin and perhaps a selective force avoiding DNA changes. Intronic sequences are also potentially useful to design DNA typing strategies. Received: 11 March 1997 / Revised: 29 May 1997     

Characterization and comparative analysis of HMW glutenin 1Ay alleles with differential expressions

Background  

High-molecular-weight glutenin subunits (HMW-GSs) have been considered as most important seed storage proteins for wheat flour quality. 1Ay subunits are of great interest because they are always silent in common wheat. The presence of expressed 1Ay subunits in diploid and tetraploid wheat genotypes makes it possible to investigate molecular information of active 1Ay genes.     

Characterization of ABC transporter ABCB1 expressed in human neural stem/progenitor cells

We investigated the localization and functional expression of the ABC transporter ABCB1 in human fetal neural stem/progenitor cells (hNSPCs). RT-PCR analysis revealed ABCB1 gene expression in hNSPCs. We found a single band in immunoblotted hNSPCs lysates probed with ABCB1 antibody, and detected ABCB1 at the hNSPCs cell membrane by immunocytochemistry and subcellular fractionation. ABCB1 inhibitors and substrate, and ATP-depleting agents enhanced hNSPCs' rhodamine 123 accumulation, and hNSPCs microsomes had vanadate-sensitive ATPase activity. ABCB1 and nestin expression decreased during hNSPCs differentiation, while the astroglial marker GFAP increased. ABCB1 may maintain hNSPCs in an undifferentiated state and could be a neural stem/progenitor marker.     

Characterization of grape Gibberellin Insensitive1 mutant alleles in transgenic Arabidopsis

We generated 12 different mutations in the grape Gibberellin Insensitive1 (VvGAI1) sequences, transformed them into Arabidopsis under the control of 35S, Arabidopsis GAI or grape GAI1 promoter, and evaluated the impact of these mutant alleles on plant growth and development. These VvGAI1 sequence variants included some mimics of the known GAI-like mutant alleles discovered in grape, wheat, barley, corn, Brassica, and Arabidopsis. In general, plant height and related traits such as length of internodes and inflorescences were significantly reduced for most of the mutant alleles studied, regardless of which promoter was used. Interestingly, the numbers of rosette leaves and lateral branches were generally reduced when a 35S promoter was used to express the mutant alleles, but increased when an Arabidopsis or grape GAI promoter was used. Furthermore, the 35S plants often displayed curly and small leaves. In contrast, the leaves of the plants carrying mutant alleles controlled by a GAI promoter were of variable size, dark green and rarely curly. In addition, when certain VvGAI1 mutant alleles were under the control of the grape GAI1 promoter, the number of pods on inflorescences was significantly increased, but some of the pods produced few seeds due to partial sterility. On the basis of the systematic evaluation of various VvGAI1 mutant alleles in Arabidopsis, we concluded that the VvGAI1 mutant alleles mimicking the GAI or GAI-like mutant variants discovered in wheat, barley and Brassica could potentially be useful for the improvement of grapevine plant architecture.     

HLA-A, -B, -C, -DRB1 and -DQB1 alleles associated with different hematological diseases in Chinese population

The purpose of this study was to explore the association between human leukocyte antigens (HLA)-A, -B, -C, -DRB1 and -DQB1 allele polymorphisms and different hematological diseases in Chinese groups. Retrospective analyses of HLA genotyping data in high-resolution for patients with acute myeloid leukemia (AML, 766 cases), chronic myeloid leukemia (CML, 330 cases), acute lymphoblastic leukemia (ALL, 605 cases), aplastic anemia (AA, 229 cases), myelodysplastic syndrome (MDS, 204 cases) were performed, and the susceptible or protective HLA alleles of the above-mentioned diseases were analyzed by Chi-square test and Fisher exact test with unrelated hematopoietic stem cell donors as control. The Results indicated that A*0201, B*4402, C*0701, DRB1*1201, DRB1*1401, and DQB1*0602 might be susceptible genes of AML, while A*1101, A*3303, B*5801, C*0302, DRB1*0301, DQB1*0201 and DQB1*0502 might be protective genes of AML. A*3303 might be a protective gene of CML, and DRB1*1401 might be a susceptible gene of CML. ALL's susceptible genes included A*0201, A*0210, B*5201, DRB1*1201, DRB1*1401 and DQB1*0602, but its protective genes included DQB1*0502. For AA, A*0201, A*0206, B*1511, DRB1*0901, DRB1*1401, DQB1*0303, DQB1*0602 might be susceptible genes, while A*3303, B*5801, C*0302, DRB1*1602 and DQB1*0502 might be protective genes. A*0201, A*0206, B*1511, DRB1*0901, DRB1*1401, DQB1*0303. A*0201, B*1558, B*4801, B*5201, DRB1*1401, DRB1*1501, and DQB1*0602 might be susceptible genes of MDS, and A*3303, B*4601, B*5801, C*0302, and DRB1*0901 might be protective genes of MDS. On the basis of HLA high-resolution genotyping for the first time, this study comprehensively analyzed HLA alleles associated with different hematological diseases in the Chinese population, which should provide clues for further study on the pathogenesis of these diseases.     

Characterization of variant alleles of cytochrome CYP2D6 in a Spanish population

The CYP2D6 gene codes for a P450 monooxygenase which is involved in the biotransformation of a large number of commonly prescribed drugs. Adverse drug effects and therapeutic failure can be related to abnormal CYP2D6 activity. We investigated the allele and genotype frequencies of cytochrome P4502D6 in a Spanish population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes in our population and to design a feasible CYP2D6 genotyping protocol. The study included 105 healthy unrelated Spanish Caucasian volunteers. CYP2D6 genotyping was performed by a combination of long-PCR, direct sequencing and allele-specific real-time PCR. The frequency of the wild-type CYP2D6*1 allele was 31%. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity showed frequencies of 40.47, 2.38 and 1.90%, respectively. Frequencies of defective alleles *3, *4, *5 and *6 were 0.95, 13.8, 3.33 and 0.95%, respectively. The defective CYP2D6 alleles *7, *8, *12, *14, *15 and *21 were not found. Duplicated CYP2D6 alleles were detected at a frequency of 4.27%. Our protocol allows the identification of the four inactive CYP2D6 alleles (*3, *4, *5 and *6) and the detection of alleles with CYP2D6 *1, CYP2D6 *2 and CYP2D6*4 gene duplications. Testing for this reduced CYP2D6 allele set would facilitate its use in clinical practice by assisting in the development of individualized pharmacotherapy.