Showing papers by "Richard A. Flavell published in 1985"

Duplicated gene pairs and alleles of class I genes in the Qa2 region of the murine major histocompatibility complex. a comparison

Abstract: DNA restriction maps of the major histocompatibility complex and hybridization with low copy probes have previously revealed strong homology between the Q6-Q7 and the Q8-Q9 class I gene pairs in the Qa2 region of the C57BL/10 mouse. After DNA sequence analysis of the Q7, Q8 and Q9 genes, we have compared the Q7 gene with its apparent allele, 27.1, from the BALB/c mouse; the 99% homology between Q7 and 27.1 indicates that this is a non-polymorphic gene. Comparison of Q7 with Q9, its homologue in the Q8-Q9 gene pair, revealed greater than 99% homology, thus supporting our proposal that the Qa2 region has evolved by the duplication of gene pairs. Q7 was also found to be homologous (93%) to Q8, the second member of the Q8-Q9 pair. However, the first exon (encoding the leader sequence) as well as the first intron of Q7 and Q8, which are presumably not subject to strong selective pressure, are essentially identical in nucleotide sequence (having only one mismatch), which suggests that greater than 200 bp of DNA may have been exchanged by gene conversion. Furthermore, transcripts of both Q7 and Q8 would have termination codons derived from the exon that normally encodes the transmembrane domain, thus these genes could encode either membrane-bound class I proteins that lack a cytoplasmic protein domain or class I proteins that are secreted.

The control of nucleolus volume in wheat, a genetic study at three developmental stages

Abstract: The major nucleoli in the wheat variety Chinese Spring are formed at the nucleolus organisers (NORs) on chromosomes 1B and 6B. Minor nucleoli are formed from the NORs on chromosomes 5D and 1A. Nucleolar volume is poorly correlated with the number of ribosomal RNA genes in the NOR region. The nucleolus on chromosome 1B is twice the volume of that on chromosome 6B even though only half as many ribosomal RNA genes reside at the 1B locus compared with the number at the 6B locus. Nucleolus size is correlated with the size of the NOR constriction seen in metaphase chromosomes. When major NORs are deleted, the volumes of the remaining nucleoli increase to compensate for the deletions. Varying the dosage of many of the chromosomes in the total complement influences the volume of the minor nucleoli. Chromosomes 1D and 6B have been shown to carry genes regulating total nucleolar volume. Although total nucleolar volume differs between cells at different stages of development, the genetic variation influencing volume appears to have similar effects in root tip, premeiotic and young pollen cells.

Tissue-specific, inducible and functional expression of the E alpha d MHC class II gene in transgenic mice.

Abstract: We have introduced the class II E alpha d gene into (C57BL/6 X SJL) F2 mice which do not express their endogenous E alpha gene. The mRNA expression of the E alpha d gene shows the same tissue distribution as the endogenous class II genes except in the case of one mouse, which carried 19 copies of the E alpha d gene. In this mouse expression of E alpha d mRNA was seen in all tissues tested. Expression of the transgene was induced by gamma-interferon in isolated macrophages from the transgenic mice. In addition, fluorescence activated cell sorter (FACS) analysis, mixed lymphocyte response and antigen-presentation experiments showed that the product of the transferred gene is expressed on the cell surface and functions as a major histocompatibility complex restriction element. Transmission of the gene occurred only with female transgenic mice, all males were infertile or did not transmit the gene, suggesting an effect of the transferred DNA sequence on male reproductive function.

Differences in methylation on the active and inactive human X chromosomes

Abstract: Summary Methylation of CCGG sites was examined in four regions of the X chromosome with four X-chromosome clones, three obtained by cloning random segments and one encoding a structural gene. In DNA from human peripheral blood cells unmethylated sites correlating with the inactive X chromosome were detected in the vicinity of two of the random clones and also in the vicinity of a cloned sequence of the X-linked phosphoglycerate kinase gene (PGK). The third random clone covered a region whose methylation pattern was unchanged between the active and inactive X chromosomes. Differential methylation at the sites detected appears to have no functional role in the maintenance of the inactive X chromosome since both active and inactive X chromosomes were found to be undermethylated in DNA from human lymphoblastoid cells.

Organization and expression of the MHC of the C57 black/10 mouse.

Abstract: Our laboratory has been studying the class I and II genes of the major histocompatibility complex (MHC) of the C57 BL/IO (BIO) mouse (Fig. 1). The class I genes, for example H-2K and H-2D, encode approximately 45,000 mw polymorphic glycoproteins that are expressed as a heterodimer, with jfi2-microglobulin on the surface of virtually all cells. The 45K polypeptide consists of 3 extracellular domains, called here al, a2, and a3; it is anchored in the membrane by a short transmembrane segment and has a cytoplasmic C terminal region of some 35 amino acids. The association with ^-microglobulin confers a symmetry on the molecule which is shown in Fig. Ib. The role of class I gene products is believed to be that of a recognition element for cytotoxic T cells (CTL). The antigenspecific receptor(s) of the CTL only recognize viral glycoproteins when they are associated with these class I proteins on the cell surface. A notable feature of the traditional class I MHC molecules (H-2K and H-2D) is the high frequency of polymorphic alleles found in the murine population (Klein 1975); this polymorphism is believed to play a role in the function of these molecules. In addition to the polymorphic class I molecules, several class I genes are found in the Tla complex (Steinmetz et al. 1982a, Evans et al. 1982, Flavell et al. 1983, Winoto et al. 1983, Weiss et al. 1984) (Fig. la) which appear to encode structurally similar but less polymorphic molecules such as Qa2,3 and TL which are structurally homologous to H-2K and H-2D. These molecules are apparently only expressed on certain populations of lymphocytes. The class II molecules of the MHC, I-A and 1-E, are also heterodimers consisting of a 35,000 mw a chain and a 29,000 mw p chain. The I-A region of the MHC encodes the A^, Aa, and Ep chains and the I-E region encodes the Ea chain (Fig. la). Class II molecules are expressed primarily on the surface of B lymphocytes and antigen-presenting cells such as macrophages and dendritic cells. These class II molecules, together with foreign antigen, activate the helper

The I region of the C57BL/10 mouse: characterization and physical linkage to H-2K of an SB beta-like class II pseudogene, psi A beta 3.

Abstract: In the C57BL/10 mouse, 140 kilobases (kb) of the I region (I-Ab, I-Eb) were isolated as recombinant cosmids. The class II genes A beta 2, A beta 1, A alpha,E beta 1, E beta 2, and E alpha are located from centromere to telomere in a region of approximately equal to 110 kb, which shows that the I region in the b haplotype has a similar overall organization to those described for the d, k, and wr7 haplotypes. In addition to these genes, we have also isolated a class II gene, psi A beta 3, which is physically linked to the class I H-2K region, 75 kb telomeric to the H-2Kb gene. This orients the H-2K region on the genetic map with the H-2Kb gene being located toward the I region. The sequence of the beta 2 domain of psi A beta 3 is similar to the immunoglobulin-like domain of other class II genes. Interestingly, it shows 83% nucleotide homology to the human SB beta gene, the same homology that was seen previously between the immunoglobulin-like exons of A beta 1 and DC beta and between E beta 1 and DR beta, respectively. It is likely, therefore, that psi A beta 3 represents a member of a third SB-like class II gene family present in addition to I-A and I-E genes and that the divergence of the SB family predates the speciation of rodents and primates. Comparison of the DNA sequence of the exon encoding the beta 2 domain of psi A beta 3 in the b or k haplotypes with functional class II genes shows that a deletion of eight nucleotides has occurred, such that the psi A beta 3 sequence cannot be translated into a functional class II protein. This suggests that psi A beta 3 is a pseudogene.

Secretion of a soluble class I molecule encoded by the Q10 gene of the C57BL/10 mouse.

Abstract: The DNA sequence of the Q10 genes appears to be highly conserved amongst strains of mice and has only been found to be transcribed in the liver An examination of the nucleotide sequence of the exon that normally encodes the transmembrane domain of class I molecules suggested that the Q10 gene encodes a secreted protein We have established this by showing that L cells transformed with an expression vector containing the Q10 gene secrete a class I molecule which was identified with an antiserum raised against a peptide predicted by the Q10 transmembrane exon Both the L cell-derived Q10 molecule and a class I protein immunoprecipitated from serum with this anti-peptide antiserum have mol wts of approximately 38 000; the Q10 molecule secreted by L cells is heterogeneous in mol wt This heterogeneity was drastically reduced after endoglycosidase F treatment, suggesting that Q10 molecules secreted into the serum by the liver may be glycosylated differently from those secreted by L cells Endoglycosidase F treatment of both the L cell and serum forms of the soluble molecule yielded two products with mol wts of approximately 32 000 and 35 000; this is consistent with the observation that the predicted Q10 protein sequence has two potential glycosylation sites In contrast to previous published results, the Q10 molecule reacted with rabbit anti-H-2 antisera which is consistent with its greater than 80% homology to the classical transplantation antigens

Allospecific cytolytic T lymphocytes recognize conformational determinants on hybrid mouse transplantation antigens.

Abstract: Alloreactive cytolytic T cell (CTL) lines and clones have been used to identify the sites of polymorphism of antigens of the major histocompatibility complex (MHC). Specific CTL were generated against wild-type H-2b products by cells from H-2b mutant mice that had one or a few amino acid changes in either the alpha 1 or alpha 2 domains of the Kb or Db class I molecules. These CTL populations, which might be expected to react with determinants expressed on single MHC domains, were examined for lytic activity on L cells expressing newly constructed hybrid class I molecules. Transformed cell lines expressing native class I molecules or hybrid class I molecules in which the alpha 1 and alpha 2 domains of H-2Kb had been substituted by those domains of H-2Db were lysed by H-2Db-specific CTL. Similarly, all H-2Kb-specific CTL recognized hybrid molecules in which the alpha 1 and alpha 2 domains of H-2Kb were inserted into the H-2Db molecule. In contrast, exchange of the alpha 1 domains of H-2Kb and H-2Db resulted in a total loss of recognition by Kb and Db-specific CTL. These results suggest that the allodeterminants recognized by H-2 mutant CTL are influenced by interactions between the alpha 1 and alpha 2 domains, findings similar to those seen using conventional alloreactive T cells (11). These results were compared to the binding of alloreactive mAbs, including 5 new mAbs specific for the Kb molecules. Finally, it was shown that primary and secondary CTL responses could be generated by direct sensitization against hybrid class I molecules, demonstrating that these molecules express neoantigenic determinants recognized by alloreactive CTL.

Recognition of interspecies hybrid class I histocompatibility antigens by antigen-specific cytolytic T lymphocytes.

Abstract: Two reciprocal interspecies hybrid class I histocompatibility genes have been constructed between genomic clones of human HLA-A2 and murine H-2Kb. The proteins encoded by these genes have been designated A21+2/Kb, where the polymorphic domains, alpha 1 and alpha 2, of HLA-A2 are linked to the carboxyl-terminal domains (alpha 3, transmembrane, and intracytoplasmic domains) of H-2Kb, and Kb1+2/A2, where the alpha 1 and alpha 2 domains of the H-2Kb antigen are linked to the carboxyl-terminal domains of HLA-A2. These genes have been transfected and expressed in recipient mouse L cells and human RD (rhabdomyosarcoma) cells. Both hybrid antigens were found to be serologically intact when tested with a panel of antigen-specific monoclonal antibodies. The monoclonal antibody W6/32, which recognizes a monomorphic determinant on all HLA-A, -B, and -C antigens, recognizes the alpha 1 and/or the alpha 2 domain, rather than the more conserved alpha 3 domain. Human cytolytic T lymphocytes (CTL) specific for the HLA-A2 antigen recognized the A2 and A21+2/Kb proteins only when expressed in human cells and not when expressed in mouse cells, even when surface antigen levels were 10-fold greater on the mouse cells than on the human cells. In contrast, a long-term, murine anti-H-2b CTL line not only lysed mouse L-cell lines that expressed the parental Kb and hybrid Kb1+2/A2 antigens but also lysed the Kb and Kb1+2/A2 human cell RD transformants as well. In both cases, the level of CTL recognition and lysis of the transformants that expressed the native antigen Kb was greater than of those transformants that expressed the hybrid antigen Kb1+2/A2. These data suggest that the carboxyl-terminal domains play some role in CTL allorecognition. The lack of human CTL recognition of HLA molecules expressed in mouse L cells, however, cannot be explained by the presence of a xenogeneic carboxyl terminus. Since murine CTL can recognize their target antigen when expressed on the surface of human cells, the possibility remains either that a ligand necessary for other molecular interactions of human CTL may be absent on mouse target cells or that murine and human CTL differ in affinity of binding to target antigens in the absence of accessory-molecule interactions.

Evolution and expression of the transplantation antigen gene family

Abstract: We have cloned 26 different class I genes that are located in the major histocompatibility complex of the C57BL/10 mouse. Two of the three class I genes found in the H-2 complex encode the H-2Kb and H-2Db antigens; the other 23 class I genes map to the adjacent Tla complex. We have grouped the cosmids containing these genes into three clusters: one cluster links the H-2K and I-A regions, one cluster links the H-2D and Qa-2 regions, and the final cluster maps to the TL region. The class I gene organizations in the Qa-2 and TL regions of the C57BL/10 and BALB/c mice are generally similar, but there are several polymorphic segments. The Qa-2 region of both mice seems to have evolved by the duplication of gene pairs; furthermore, the H-2K region may have been generated by the translocation of a gene pair from the Qa-2 region. We have evidence that several of the genes in the Qa-2 region are expressed.