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Biomedical subjects

S M Weissman

Publications and source records attributed to S M Weissman.

At least 91 records · Page 5Linked to original sources
PubMed1990

The amino acid sequence of the human RNA polymerase II 33-kDa subunit hRPB 33 is highly conserved among eukaryotes.

We have cloned and sequenced a cDNA of 1766 base pairs in length encoding the 275 amino acids of hRPB 33, the third largest subunit of human RNA polymerase II. The DNA was isolated by screening of a human lambda gt11 cDNA library with oligonucleotides designed on the basis of the amino acid residue analysis of the bovine material. The hRPB 33 amino acid sequence is highly conserved between Saccharomyces cerevisiae and human. Overall, 45% of the amino acid residues are identical with the yeast homologue RPB 3, and 65% of the amino acids are identical in the two major conserved regions at residues 0-103 and 151-197. hRPB 33 is also homologous to yeast RPC 5. The amino acid sequence of hRPB 33 showed no obvious homology with bacterial RNA polymerase or with any of its sigma factors.

Amino Acid Sequence↗
PubMed1990

Selective enrichment of a large size genomic DNA fragment by affinity capture: an approach for genome mapping.

A method to enrich large size DNA fragments obtained by digestion with rare cutting restriction endonucleases was developed and applied for the isolation of a 150 kb SfiI fragment containing the beta-globin gene cluster. The digested DNA is rendered single stranded at the ends by diffusing a strand specific exonuclease into an agarose plug containing DNA. The plug is melted and solution hybridization is then performed with a bridge RNA containing specific sequences from the end of a desired fragment linked to a common probe sequence. The common probe sequence is annealed to a biotinylated RNA and the resulting tripartite hybrid is retained onto a solid matrix containing avidin and specifically released by ribonuclease action. Enrichments of greater than 350 fold have been achieved consistently. Such directed purification of large DNA fragments without cloning can considerably expedite mapping and gene localization in a complex genome and facilitate the construction of sublibraries from defined regions of the genome.

Base Sequence↗
PubMed1990

Self-tolerance to HLA focuses the response of immunized HLA-transgenic mice on production of antibody to precise polymorphic HLA alloantigens.

HLA class I-transgenic mice express HLA antigen on their tissues and establish self-tolerance to the expressed monomorphic and polymorphic determinants. When challenged with skin grafts and lymphoid cells of a second HLA-transgenic mouse expressing a different HLA molecule, a specific immune response is elicited that is focused on the determinants specified by the allelic HLA differences between donor and recipient transgenic mice. In the studies described, this has led to the production of a number of monoclonal antibodies with specificity for the HLA-Cw3 and HLA-B7 crossreactive groups of class I antigens. These results indicate that immunization of appropriate transgenic strains of mice with murine cells expressing a different HLA allelic transgene should permit the generation of monoclonal antibodies of narrow specificity against virtually any polymorphic epitope on HLA antigens.

Animals↗
PubMed1990

Purification of two transcription factors required for initiation by mammalian RNA polymerase II.

We have purified two general transcription factors (FA and FE) necessary for specific transcription by mammalian RNA polymerase II to near homogeneity. Both activities are associated with peptides of approximately 33 kDa. FA and FE do not replace one another and show different kinetics of action in a sarkosyl block assay. In particular, FE participated in a rapid reaction after the formation of an initial complex with the other transcription factors. Furthermore, FE can associate with purified calf thymus RNA polymerase II.

Animals↗
PubMed1990

Expression of human globin genes in transgenic mice carrying the beta-globin gene cluster with a mutation causing G gamma beta + hereditary persistence of fetal hemoglobin.

We have introduced into the mouse germ line the 40-kilobase (kb) Kpn I fragment containing the beta-globin gene cluster from an individual with a non-deletion form of hereditary persistence of fetal hemoglobin (HPFH) believed to be due to a point mutation at position -202 of the G gamma-globin gene. The G gamma-globin gene, as well as the beta-globin gene, was expressed in adult erythroid tissues of the resulting transgenic mice. The level of expression of the G gamma-globin gene was about 20% of that of the beta-globin gene. Others have previously shown that cloned individual normal human beta- and gamma-globin genes containing a limited amount of 5'- and 3'-flanking DNA are expressed in a manner similar to that of their corresponding murine homologs during development in transgenic mice. In contrast, we have observed that the pattern of expression of the normal (non-mutated) A gamma- and beta-globin genes in the 40-kb insert was different from that of their corresponding murine homologs. The beta-globin gene remained inactive at the fetal stage, whereas the normal A gamma-globin gene was expressed beyond the embryonic (yolk sac) stage into the fetal stage of development and then became inactive in adult erythroid cells. The pattern of expression of the human globin transgenes during mouse development resembles that observed during human development. These results suggest that the gross organization of the human beta-like globin gene cluster is important for stage-specific expression of each human globin gene during development.

Animals↗
PubMed1990

Transcription analysis, physical mapping, and molecular characterization of a nonclassical human leukocyte antigen class I gene.

The human major histocompatibility complex contains approximately 20 class I genes, pseudogenes, and gene fragments. These include the genes for the three major transplantation antigens, HLA-A, HLA-B, and HLA-C, as well as a number of other genes or pseudogenes of unknown biological significance. Most of the latter have C + G-rich sequences in their 5' ends that are unmethylated in the B-lymphoblastoid cell line 3.1.0. We investigated one of these genes, HLA-H, in more detail. The gene is, overall, strongly homologous in sequence to HLA-A but differs in several potentially significant ways, including changes in conserved promoter sequences, a single-base deletion producing a translation termination codon in exon 4, and a region of sequence divergence downstream of the transcribed portion of the gene. Nevertheless, mouse L cells transfected with the gene accumulated small amounts of apparently full-length polyadenylated RNA. A portion of this RNA begins at the transcription site predicted by analogy to certain class I cDNA clones, while another portion appears to begin shortly upstream. L cells transfected with a hybrid gene containing the first three exons of HLA-H and the last five exons of HLA-B27 accumulated full-length HLA transcripts at the same level as cells transfected with an HLA-B27 gene; both levels are at least 15- to 20-fold higher than that directed by HLA-H alone. In addition, we isolated a cDNA clone for HLA-H that contains a portion of intron 3 attached to a normally spliced sequence comprising exons 4 through 8. These results suggest that low levels of translatable mRNA for the truncated class I heavy chain encoded by HLA-H are produced under physiologic circumstances and that sequences 3' of intron 3 decrease the levels of stable transcripts.

Amino Acid Sequence↗
PubMed1989

Isolation and molecular characterization of a cDNA encoding the 23-kDa subunit of human RNA polymerase II.

We have shown that antibodies against native calf thymus RNA polymerase II and antibodies against its 23-kDa subunit cross-reacted with the 23-kDa subunit of human RNA polymerase II. Immunoglobin G (IgG) against the 23-kDa subunit of calf thymus RNA polymerase II inhibited transcription in vitro from the adenovirus major late promoter. By immunoscreening of a human placenta lambda gt11 cDNA library with IgG against native CT RNA polymerase II and with IgG against its 23-kDa subunit, we isolated and characterized a full length 1.2-kilobase cDNA. We also generated oligonucleotide probes from a sequence of amino acid residues obtained by a modified peptide microsequencing procedure. The cDNAs isolated both from oligoscreening and immunoscreening were identical. The amino acid sequence deduced from the nucleotide sequence analysis indicates a polypeptide of 197 amino acid (23 kDa). The in vitro translation product of human cDNA HP-23 was precipitated by IgG against the 23-kDa subunit of CT RNA polymerase II. The amino acid sequence deduced from HP-23 showed no obvious homology with Escherichia coli RNA polymerase subunits or with any of its sigma factors.

Adenoviridae↗
PubMed1989

Multiple enhancer-like sequences in the HLA-B7 gene.

Enhancer-like sequences have previously been identified in the promoter region of the mouse major histocompatibility complex (MHC) class I genes. We have screened for such sequences in and around a human MHC class I gene, HLA-B7. Various restriction fragments of the B7 gene were assayed for their ability to enhance transcription of a bacterial chloramphenicol acetyltransferase gene from a simian virus 40 promoter in transiently transfected mouse LTA cells. Our results demonstrate that enhancer activity is located in introns 3 and 5 as well as 5' to the transcription initiation site. RNase protection experiments corroborate the results. Preliminary experiments indicate that B7 enhancers are active in various cell types. The role of these enhancers in B7 gene expression is not known at present. We speculate that the position of the enhancer elements may be related to the occurrence of Hpa II tiny fragment islands.

Animals↗
PubMed1989

Resolution and partial characterization of factors required for in vitro transcription by mammalian RNA polymerase II.

Multiple protein factors from HeLa cells are necessary for the accurate initiation of transcription on minimal promoters in vitro. We have partially purified these factors by chromatographic methods. In addition to RNA polymerase II, six factors A-F (FA, FB, FC, FD, FE, and FF) necessary for initiation at the beta-globin promoter start site in vitro have been identified. Certain of these (FA, FC, and FE) have been purified to near homogeneity. The present purification scheme yields sufficient amounts of purified material for the more detailed characterization and cloning of the genes for these activities. Among these factors, FD and FF were required with template DNA at an early step of formation of the initiation complex, whereas FB, FA together with FC, and FE were effective when added at successively later stages in the process of complex formation.

Cell Nucleus↗
PubMed1989

cDNA sequences of two inducible T-cell genes.

We have previously described a set of human T-lymphocyte-specific cDNA clones isolated by a modified differential screening procedure. Apparent full-length cDNAs containing the sequences of 14 of the 16 initial isolates were sequenced and were found to represent five different species of mRNA; three of the five species were identical to previously reported cDNA sequences of preproenkephalin, T-cell-replacing factor, and a serine esterase, respectively. The other two species, 4-1BB and L2G25B, were inducible sequences found in mRNA from both a cytolytic T-lymphocyte and a helper T-lymphocyte clone and were not previously described in T-cell mRNA; these mRNA sequences encode peptides of 256 and 92 amino acids, respectively. Both peptides contain putative leader sequences. The protein encoded by 4-1BB also has a potential membrane anchor segment and other features also seen in known receptor proteins.

Adjuvants, Immunologic↗
PubMed1988

Cell surface expression and alloantigenic function of a human class I MHC heavy chain gene (HLA-B7) in transgenic mice.

We have introduced the gene encoding the heavy chain of the human MHC class I Ag HLA-B7 into transgenic mice. The gene was shown to be expressed at both the RNA and protein level. Cell surface HLA-B7 was detected on whole spleen cells by immunoprecipitation and on purified T cells by flow cytometry (FACS). Normal mice immunized with H-2-syngeneic B7-transgenic spleen cells generated CTL capable of killing transgenic cells and B7-expressing human JY cells. Anti-HLA mAb blocked the killing of JY cells. These results indicate that the human class I Ag HLA-B7 can be expressed at the surface of transgenic spleen cells in the absence of human beta 2-microglobulin, and that a significant fraction exists in a form recognizable by nontransgenic CTL as a major histocompatibility Ag unrestricted by H-2.

Animals↗
PubMed1988

Tissue-specific and cell surface expression of human major histocompatibility complex class I heavy (HLA-B7) and light (beta 2-microglobulin) chain genes in transgenic mice.

We introduced the human genes HLA-B7 and B2M encoding the heavy (HLA-B7) and light [beta 2-microglobulin (beta 2m)] chains of a human major histocompatibility complex class I antigen into separate lines of transgenic mice. The tissue-specific pattern of HLA-B7 RNA expression was similar to that of endogenous class I H-2 genes, although the HLA-B7 gene was about 10-fold underexpressed in liver. Identical patterns of RNA expression were detected whether the HLA-B7 gene contained 12 or 0.66 kilobase(s) (kb) of 5' flanking sequence. The level of expression was copy number dependent and as efficient as that of H-2 genes; gamma interferon enhanced HLA-B7 RNA expression in parallel to that of H-2. In addition to the mechanism(s) responsible for gamma interferon-enhanced expression, there must be at least one other tissue-specific mechanism controlling the constitutive levels of class I RNA. Tissue-specific human beta 2m RNA expression was similar to that of mouse beta 2m, including high-level expression in liver. Cell surface HLA-B7 increased 10- to 17-fold on T cells and on a subset of thymocytes from HLA-B7/B2M doubly transgenic mice compared to HLA-B7 singly transgenic mice. The pattern of expression of HLA-B7 on thymocytes resembled that of H-2K as opposed to H-2D. These results confirm that coexpression of both human chains is required for efficient surface expression and that HLA-B7 may share a regulatory mechanism with H-2K, which distinguishes it from H-2D.

Animals↗
PubMed1987

Megabase-scale mapping of the HLA gene complex by pulsed field gel electrophoresis.

In the study of the genetic structure of mammalian chromosomes, there exists a "resolution gap" between molecular cloning experiments and meiotic linkage analyses. This gap has discouraged attempts to construct full-scale genetic maps of mammalian chromosomes. The organization of the human major histocompatibility complex was examined within this range by pulsed field gel electrophoresis. The data obtained indicate that the complex spans over 3000 kilobases and enable the construction of a megabase-scale molecular map. These results indicate that the techniques employed in DNA extraction, enzymatic digestion, electrophoresis, and hybridization are suitable for the efficient analysis of megabase regions of mammalian chromosomes and effectively bridge the resolution gap between molecular cloning and classical genetics.

Animals↗