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S M Weissman

Publications and source records attributed to S M Weissman.

At least 127 records · Page 7Linked to original sources
PubMed1985

Molecular organization of the class I genes of human major histocompatibility complex.

In this brief review, our main emphasis has been on the analysis of the sequence diversity among various class I genes and their functional implications. The availability of complete nucleotide sequences of 7 different genes representing different loci allowed us to derive a consensus sequence. One mouse MHC Class I gene was included in these comparisons as a representative of H2 genes Evolutionary patterns can be seen on the basis of divergence of various genes from the derived consensus sequence. At least 1 human gene which has a promoter similar to that of H2 genes and which contains a single initiation codon following this promoter (unlike all other human genes and like all the H2 genes) has been identified. Both variable and homology regions can be identified in the entire length of the gene. While exons show relatively strong conservation of sequences, the introns have many variable regions, introns 6 and 7 being the most heterogeneous. Stretches of conserved nucleotide sequences are noticed at the 3' regions of most introns. Estimation of total number of class I genes is presented on the basis of cloning experiments, and the abundance of 1 particular pseudogene is discussed.

Alleles↗
PubMed1985

Synthesis of predominantly unspliced cytoplasmic RNAs by chimeric herpes simplex virus type 1 thymidine kinase-human beta-globin genes.

The herpes simplex virus type 1 thymidine kinase (tk) gene lacks introns and produces stable mRNA in the absence of splicing. We have prepared a hybrid gene by placing the first exon, first intron (first intervening sequence, designated IVS1), and most of the second exon of the normal human beta-globin gene into the 3' untranslated region of the tk gene. Although this hybrid gene contains all globin sequences presumed necessary for the splicing of IVS1, predominantly, unspliced stable cytoplasmic RNA is produced in both long- and short-term expression assays. Moreover, stable unspliced cytoplasmic RNA is detected whether the intron is situated in a sense or an antisense orientation. Efficient splicing of IVS1 is obtained either by deleting the majority of tk coding sequences or by relocating the globin sequences from the 3' to the 5' untranslated region of the tk gene.

Animals↗
PubMed1984

Human fetal globin DNA sequences suggest novel conversion event.

DNA sequencing studies of two recently cloned human A gamma globin alleles has revealed a number of base differences which are clustered in the large intron (IVS-2). One allele has a previously undescribed IVS-2 sequence. Most of the allelic differences can be explained as resulting from a gene conversion event involving G gamma as a donor. A novel feature of this event is that three G gamma-like regions occur interspersed among unconverted areas of the A gamma gene. We propose that an alternating purine-pyrimidine run which is located between two of the converted sites is the initiation site of the conversion event. Consistent with models of gene conversion, this poly (purine-pyrimidine) tract has single-stranded characteristics in supercoiled plasmids as assayed by S1-nuclease.

Alleles↗
PubMed1984

G gamma beta+ hereditary persistence of fetal hemoglobin: cosmid cloning and identification of a specific mutation 5' to the G gamma gene.

Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition in which the normal shutoff of fetal hemoglobin (Hb F) production fails to occur. In the G gamma beta+ type of HPFH, erythrocytes of adult heterozygotes contain approximately equal to 20% Hb F, which is almost exclusively of the G gamma-globin variety, without increased levels of gamma-globin chains from the nearby A gamma-globin gene. Unlike some forms of HPFH, no major deletions in the globin gene cluster have been found by genomic blotting in the G gamma beta+ variety. We report here a family with this condition, from which cosmid clones of the beta-globin gene cluster from the G gamma beta+ HPFH allele have been obtained. Sequencing around the fetal genes has identified a point mutation 202 base pairs 5' to the G gamma-globin gene that is present in genomic DNA of 3/3 unrelated individuals with G gamma beta+ HPFH but in none of more than 100 non-HPFH individuals. Although the mutation could represent a tightly linked polymorphism, its location in a region suggested by recent data to be important in tissue-specific control of gene expression suggests the possibility that the -202 mutation accounts for the phenotype. The sequence created resembles elements of other eukaryotic promoters known to be important for efficient transcription.

Base Sequence↗
PubMed1984

Directional cloning of DNA fragments at a large distance from an initial probe: a circularization method.

The principle of a DNA cloning procedure that directionally generates genomic DNA fragments 50-2000 kilobases away from an initial probe is presented. The method depends on partial digestion of high molecular weight genomic DNA and subsequent ligation at very low concentration to generate covalent DNA circles. A library of the junction fragments from these circles can then be constructed. Biological or physical selection of the junction pieces can be achieved by incorporating a marker DNA fragment into the covalent circles. A 45-kilobase cosmid fragment has been successfully used to test the procedure. At appropriately low ligation concentrations (0.8 micrograms/ml), approximately equal to 90% of the ligated DNA is present as monomeric circles. Larger DNA fragments will require reducing the DNA concentration as the inverse square root of the DNA length. A suppressor tRNA gene has been tested as the selectable marker gene. Ligation of the digested circles into an amber-mutated lambda phage and propagation in a sup- host allows only the phage that contain junction fragments to produce plaques. Potential applications of this approach, such as mapping of complex genetic loci or moving from a linked gene toward a gene of interest, are presented and discussed.

Bacteriophage lambda↗
PubMed1984

Transcripts of human HLA gene fragments lacking the 5'-terminal region in transfected mouse cells.

Clones of mouse L cells transfected with a human HLA-B7 gene fragment lacking the 5' segment of exon 2 and all upstream sequences express HLA-specific transcripts of various lengths. These include species that correspond in size to full-length HLA-B7 mRNA. The level of these transcripts is increased in cells treated with interferon. It is probable that the full-length transcripts arise as a result of the linkage of the HLA-B7 gene fragments with DNA segments providing transcription initiation or polyadenylylation signals.

Animals↗
PubMed1984

Mouse mammary tumor virus-related sequences in mouse lymphocytes are inducible by 12-O-tetradecanoyl phorbol-13-acetate.

cDNA libraries from EL-4 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) were screened for TPA-inducible sequences by differential hybridization. The most abundant inducible species was a sequence similar to that of mouse mammary tumor virus (MMTV). Induction of the mRNA corresponding to the MMTV-related sequences was already evident 30 min after TPA treatment, whereas the maximum accumulation occurred after 20 h of exposure to TPA. TPA also increased levels of MMTV-related RNA in the normal spleen cells of BALB/c and C57BL/6 mice. The level of RNA expression corresponding to MMTV-related sequences, however, was markedly elevated in EL-4 cells as compared with normal spleen cells. Southern blots of EL-4 cell DNA showed that the MMTV-related sequences were inserted into multiple locations of the EL-4 genome. Sequence analysis revealed that the MMTV-related cDNA clones included a part of the env gene and the right long terminal repeat of MMTV. However, the cDNA sequences were substantially different from published MMTV proviral sequences, most notably because of a contiguous deletion of 491 base pairs in the open reading frame within the U3 region.

Amino Acid Sequence↗
PubMed1984

Concordance of a point mutation 5' to the G gamma globin gene with G gamma beta +. Hereditary persistence of fetal hemoglobin in the black population.

Hereditary persistence of fetal hemoglobin (HPFH) is a genetically heterogeneous and clinically benign condition characterized by persistent expression of fetal hemoglobin (Hb F) into adulthood. In the G gamma beta + type, no major deletions in the globin gene cluster occur; adult heterozygotes produce approximately 20% Hb F, which results from overproduction of G gamma chains, with no apparent increase in production from the adjacent A gamma gene. We have recently described a point mutation 202 base pairs 5' to the cap site of the G gamma gene in an individual with G gamma beta + HPFH. This mutation abolishes a normal ApaI restriction endonuclease site, and thus can be detected by blotting of genomic DNA. We present here further data on the ApaI mutation: (1) It occurs in six of seven families with G gamma beta + HPFH. (2) In three families, detailed haplotype analysis using 11 polymorphic restriction sites in the beta globin cluster has been done. The two that carry the missing ApaI site are identical but the third, which has a normal ApaI pattern, differs from the other two in at least two sites, one of which is a new polymorphic Nco I site between the delta and beta globin genes. This suggests the possibility of a different HPFH mutation in the third family. (3) The haplotype of the G gamma beta + HPFH chromosome carrying the ApaI mutation is different from that of 108 beta A chromosomes of black individuals that have been tested. (4) The G gamma ApaI site is normal in 61 beta A and 109 beta S alleles from non-HPFH black individuals, including 22 who share the same haplotype for the intragenic G gamma, A gamma HindIII polymorphisms. These data add support to the possibility that the -202 mutation is actually causative of the G gamma beta + HPFH phenotype.

Alleles↗
PubMed1983

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen.

Mouse LTK- cells (H-2k) were transfected with a series of recombinant plasmids consisting of the herpes simplex virus type 1 thymidine kinase (TK) gene linked to fragments of SV40 DNA coding for portions of SV40 T antigen in pBR322, and TK+ transformants (LTK+) were selected in HAT medium. The TK+ transformants were analyzed for SV40 transplantation rejection antigen (TrAg) at the cell surface by reacting them with cytotoxic lymphocytes (CTL) generated to SV40 TrAg in C3H/HeJ (H-2k) mice. The results indicated that the cells transformed by pVBETK-1 and synthesizing full size SV40 large T antigen were efficiently lysed by SV40 CTL. In addition, cells transformed by the plasmid pVBt1TK-1 and synthesizing a truncated 33 K T antigen were also found to be susceptible to lysis by the CTL. However, LTK+ cells that were transformed with the plasmid pVBt2TK-1 and which synthesized a truncated T antigen of 12.3 K did not provide a target for SV40 CTL nor did pVBETK-1-transformed cells that did not express any of the SV40 tumor antigens. Only the pVBETK-1-transformed cells that express 94 K T antigen were able to immunize mice against a challenge of syngeneic SV40-transformed cells. These results suggest that the TrAg expression at the cell membranes of transformed cells may be associated with the proximal half of SV40 T antigen.

Animals↗
PubMed1983

Structure and nucleotide sequence of the heavy chain gene of HLA-DR.

We have used a 175-nucleotide-long primer extension product corresponding to the 5' end of HLA-DR alpha-chain mRNA to isolate a genomic clone from a human DNA library. The entire HLA-DR alpha gene is contained in two contiguous EcoRI fragments spanning about 7.5 kilobases (kb); most of the sequence has been determined. The 5' end of the gene is contained in a 4.4-kb fragment, and the coding segments and the 3' untranslated region are contained in a 3.1-kb fragment. The gene is split into five exons. The 5' untranslated region, the leader peptide, and the first two NH2-terminal amino acids are fused into the first exon. Exons 2 and 3 represent two extracellular coding domains of mature p34. The transmembrane domain, cytoplasmic domain, and part of the 3' untranslated region are merged into a fourth exon. The rest of the 3' untranslated region is in exon 5. The predicted amino acid sequence of mature p34, as deduced from its gene structure, has 229 residues and reveals a single potential disulfide loop (between cysteine residues 107 and 163) as well as a 22-amino acid residue membrane integrated segment (residues 193-214). Fifteen amino acids (residues 215-229) reside on the cytoplasmic side of the plasma membrane. There is considerable amino acid sequence homology between the second external domains of p34 and p29, as well as the immunoglobulin-like third domain of HLA-B7, and beta 2-microglobulin and the homologous constant region domains of the light and heavy chains of immunoglobulins.

Amino Acid Sequence↗
PubMed1983

Recognition by xenogeneic cytotoxic T lymphocytes of cells expressing HLA-A2 or HLA-B7 after DNA-mediated gene transfer.

Murine L cells expressing HLA-A2 or -B7 antigens were isolated after cotransformation of thymidine kinase-negative cells with the herpes simplex virus thymidine kinase gene and the genomic clones containing either the HLA-A2 or -B7 genes. Monoclonal antibody binding analyses demonstrated the stable cell surface expression of HLA antigens by these cells at levels of up to 40% of the amount expressed by the human B lymphoblastoid cell line, JY. The HLA-A2 and -B7 antigens expressed by the L cells retained all of the antibody-defined, heavy-chain-associated antigenic determinants but lacked those determinants associated with human beta 2-microglobulin. These HLA transformants were capable of functioning as targets for monoclonal cytotoxic T lymphocytes (CTL) that specifically recognize the HLA-B7 or -A2 antigens expressed by JY cells. However, the efficiency of lysis, relative to the JY cell line, was 50-99% for individual CTL. In addition, not all of these CTL were capable of lysing the appropriate transformants. Because the antigens appear by serological criteria to be structurally intact and expressed at high levels, these results suggest that the complementation of the HLA heavy chains with mouse, rather than human, beta 2-microglobulin may alter the antigenic determinants that are important for CTL recognition.

Animals↗
PubMed1983

Different 3' end points of deletions causing delta beta-thalassemia and hereditary persistence of fetal hemoglobin: implications for the control of gamma-globin gene expression in man.

DNA at the end point of the gene deletion associated with one form of hereditary persistence of fetal hemoglobin (HPFH) was cloned and used as a probe in gene mapping experiments to analyze the extent and approximate 3' end points of various deletions associated with HPFH and delta beta-thalassemia. The deletions in the two known forms of deletion-type HPFH were shown to be considerably more extensive than in the two cases of delta beta-thalassemia studied. The overall extents of the deletions in the two types of HPFH were quite similar in both cases and the 3' end points were located at a minimum distance of approximately equal to 52 and 57 kilobases from the 3' extremity of the beta-globin gene. In contrast, the 3' end points of the deletions in the two forms of delta beta-thalassemia were located approximately equal to 5 and 10 kilobases to the 3' side of the beta-globin gene. The extent of these deletions and the nature of the DNA brought into the vicinity of the gamma-globin genes by the deletions may therefore be a more important influence on the phenotype of the deletions than the specific nature of the DNA sequences that are deleted within the non-alpha-globin gene cluster as a result of the mutations.

Base Sequence↗
PubMed1983

Use of synthetic oligonucleotide probes complementary to genes for human HLA-DR alpha and beta as extension primers for the isolation of 5'-specific genomic clones.

We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR alpha and beta by extending on poly(A)+ mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2 terminus of both polypeptides. The synthesis of the probe for the alpha-chain DNA was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted sequence. The synthesized 20-mer was then used to generate a 175-nucleotide cDNA probe which was shown to encode the appropriate amino acids for the alpha chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR alpha. For the beta polypeptide an 18-mer homologous to the NH2-terminal sequence of a cDNA clone from another B-cell line was used to extend on poly(A)+ mRNA isolated from a B-cell line. Preliminary sequence analysis of a 175-base-long extension product indicates a match of the cDNA sequence to the published sequence of a clone for HLA-DR beta. Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.

Amino Acid Sequence↗
PubMed1983

Sequences locating the 5' ends of the major simian virus 40 late mRNA forms.

The 5' sequences of late mRNA specified by several constructed or naturally occurring deletion or duplication mutants of simian virus 40 were examined. The mutants included viruses with various small deletions centered about 25 nucleotides upstream from the major transcription initiation site, as well as viruses containing tandem duplications of a sequence of 50 nucleotides or less embedding the major transcription initiation site. The results show that the sequences 25 to 30 nucleotides upstream from the major initiation site in the position of the TATA box of other polymerase II promoters are not essential for the precise localization of the initiation site of late mRNA. Rather, we deduce that the major late mRNA start site is determined primarily by sequences located very close to the initiation site, and that the relative abundance of the 5' ends with this initiation site is modulated by nearby downstream sequences. Modification of six nucleotides adjacent upstream to the initiation site almost completely prevents the utilization of this site. Various deletions and substitutions of sequences 21 nucleotides or more downstream from the major initiation site causes upstream shifts in the localization of the most abundantly utilized 5' ends. The sequences immediately downstream from the major simian virus 40 initiation sites contain inverted symmetries that could give rise to secondary structures in either single-stranded DNA or RNA; the possibility that these inverted symmetries function in transcription initiation at the level of DNA structure rather than in RNA stabilization is discussed. Finally, we present additional evidence that precursor species with certain 5' termini are selectively spliced to form 19S RNA, whereas other 5' termini are preferred for forming the 16S RNA splice. We discuss the possibility that this is a consequence of the influence of leader structure on downstream splicing events.

Base Sequence↗