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S M Weissman

Publications and source records attributed to S M Weissman.

At least 109 records · Page 6Linked to original sources
PubMed1987

Contingent replication assay (CRA) procedure for rapid isolation of enhancers.

A rapid procedure for the isolation of functional enhancer sequences consists of the construction of a shotgun DNA library in SV40-based plasmid shuttle vectors which depend on an enhancer for replication, the replication in monkey (CVI) cells of those vectors into which an enhancer sequence was inserted, the selective cleavage of unreplicated vectors by DpnI and the recovery of the replicated vectors by transfection into Escherichia coli. We describe conditions for the fusion of protoplasts to CVI cells, under which conditions the probability of only one type of plasmid entering a cell is increased and thus complementation and rescue of enhancer-less plasmids are decreased. The effectiveness of the procedure is demonstrated by the recovery of enhancers from bovine papillomavirus and Moloney murine sarcoma virus.

Bovine papillomavirus 1↗
PubMed1987

Structure, expression, and molecular mapping of a divergent member of the class I HLA gene family.

A class I gene distinct from HLA-A, -B, or -C was identified in a cosmid clone and transfected into mouse L cells. The gene, placed adjacent to the polyoma enhancer, produced a full-length class I mRNA and high levels of a 43-kDa protein in the cytoplasm. The surface expression of the gene product required its association with human beta 2-microglobulin. The protein was recognized by a xenoantiserum raised against a mixture of human B- and T-cell lines. The product was also serologically reactive with the HLA framework monoclonal antibodies. The complete nucleotide sequence of the gene was determined and a specific oligonucleotide probe was synthesized. This probe was used to identify a full-length mRNA transcript in a B-lymphoblastoid cell line (JY). The gene was mapped within a 190-kilobase Not I restriction fragment located in the telomeric portion of the human major histocompatibility complex. Distinct features of the gene include the structure of the promoter, the position of the translation initiation site, a frameshift mutation at the carboxyl terminus, the insertion of an Alu repeat element in the eighth exon, divergence in the derived amino acid sequence, and the lack of expression of the gene in some cells.

Amino Acid Sequence↗
PubMed1987

cDNA cloning and sequence of MAL, a hydrophobic protein associated with human T-cell differentiation.

We have isolated a human cDNA that is expressed in the intermediate and late stages of T-cell differentiation. The cDNA encodes a highly hydrophobic protein, termed MAL, that lacks a hydrophobic leader peptide sequence and contains four potential transmembrane domains separated by short hydrophilic segments. The predicted configuration of the MAL protein resembles the structure of integral proteins that form pores or channels in the plasma membrane and that are believed to act as transporters of water-soluble molecules and ions across the lipid bilayer. The presence of MAL mRNA in a panel of T-cell lines that express both the T-cell receptor and the T11 antigen suggests that MAL may be involved in membrane signaling in T cells activated via either T11 or T-cell receptor pathways.

Amino Acid Sequence↗
PubMed1987

Isolation and initial characterization of multiple species of T-lymphocyte subset cDNA clones.

A modified differential screening procedure was applied to analyze cDNA libraries of cloned helper T lymphocytes (Th) and cytolytic T lymphocytes (CTL). Negative and positive differential screening and RNA blot analysis were used to identify cDNA clones that were expressed preferentially in Th or CTL. Seven clones corresponded to previously described T-cell genes, and 16 additional types of cDNA clones were isolated, 9 from Th and 7 from CTL. Of these, 3 were expressed in both Th and CTL, 7 were expressed in only Th, and 6 only in CTL. These clones were analyzed for induction after stimulation by interleukin 2 or Con A or after stimulation of the T-cell antigen receptor (TCR). Three different patterns of expression were seen: induction only by Con A, induction by Con A and interleukin 2, and induction by Con A and TCR stimulation. The approach is potentially useful for analyzing paths of T-cell differentiation and detecting cDNA clones encoding unrecognized cytokines.

Animals↗
PubMed1987

L1 repeat elements in the human epsilon-G gamma-globin gene intergenic region: sequence analysis and concerted evolution within this family.

We have deduced the sequence of a composite long interspersed repeated DNA in primates and herein describe its relationship to a complex repeat element (L1Heg) located in the interval linking the human epsilon- and G gamma-globin genes. The main element of L1Heg is 3' truncated and interrupted by the insertion of the 3' end of a second L1 element. Transposition of L1Heg into this intergenic locus generated a 62-bp duplication of flanking sequences. In contrast, insertion of the second repeat may have been mediated by homology between donor and target sequences. The main repeat represents a novel class of abundant elements whose sequences have diverged from other rodent and primate LINES approximately 1.3 kb downstream from the 5' terminus of L1Heg. Comparison of L1Heg with the sequences of two other related L1 members revealed a complex set of rearrangements confined within a region that resembles the long terminal repeats of other types of retroposons. The boundaries of conversion-like events were defined on the basis of the clustering of nucleotide sequence variants common to two or more nonallelic 3' L1H elements. Several of these events are apparently initiated or resolved within a common 150-bp region that coincides with the 3' terminus of a pan-mammalian open reading frame. This analysis showed that concerted genetic interactions and random drift both contribute appreciably to sequence variation within this set of L1H members.

Animals↗
PubMed1987

Expression of the affected A gamma globin gene associated with Greek nondeletion hereditary persistence of fetal hemoglobin.

The overexpressed A gamma globin gene in the Greek type of nondeletion hereditary persistence of fetal hemoglobin has a unique single-base substitution located at position -117 relative to the site of transcription initiation. This gene and its normal counterpart were transferred into cultured cell lines by using a retroviral vector. The only difference in expression between the transferred normal and mutant gamma genes was observed in the human erythroleukemia cell line KMOE after exposure of the cells to cytosine arabinoside, a condition that resulted in an adult pattern of endogenous globin gene expression by the cells and was associated with increased expression of the mutant gene.

Animals↗
PubMed1986

Beta zero thalassemia caused by a base substitution that creates an alternative splice acceptor site in an intron.

A thalassemic beta-globin gene cloned from a haplotype I chromosome contains a T to G transversion at position 116 of IVS1 which results in the generation of an abnormal alternative acceptor splice site. Transient expression studies revealed a 4-fold decrease in the amount of RNA produced with greater than 99% of it being abnormally spliced despite preservation of the normal acceptor splice site at position 130. These results suggest that the mutation at IVS1 position 116 results in beta zero thalassemia. A closely related mutation at position 110 of IVS1 also generates a novel acceptor site and results in a similar decrease in total mRNA produced, but approximately 20% of the mRNA produced is normally spliced and thus the phenotype is that of beta + thalassemia. These observations suggest that short range position effects may play a dramatic role in the choice of potential splice acceptor sites. We demonstrate the presence of abnormally spliced mRNA in reticulocytes of affected individuals and show the mutation at IVS1 position 116 segregating from the mutation at IVS1 position 110 in a three generation pedigree. The mutation results in the creation of a MaeI restriction site, as do a number of other thalassemic mutations, and we demonstrate some difficulties that may arise in the differential diagnosis of these mutations.

Amino Acid Sequence↗
PubMed1986

HLA-JY328: mapping studies and expression of a polymorphic HLA class I gene.

The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk- cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed--a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus.

Animals↗
PubMed1986

Rapid plasmid library screening using RecA-coated biotinylated probes.

A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

Adenosine Triphosphate↗
PubMed1986

Expression of the late gene of simian virus 40 under the control of the simian virus 40 early-region promoter in monkey and mouse cells.

We constructed a recombinant plasmid (pVNR4) with the simian virus 40 (SV40) early promoter positioned 30 nucleotides upstream from the major SV40 late transcription initiation site at residue 325. After transfection of the recombinant plasmid DNA into COS and mouse L cells, the transcripts of the SV40 late region were analyzed by S1 nuclease and primer extension analysis. The following are the principal findings. (i) The 16S and 19S late RNAs used the characteristic wild-type splice; no detectable levels of 19S unspliced RNA were observed. (ii) The majority of the late RNAs were heterogeneous and initiated in the early region (upstream and downstream from the Hogness-Goldberg sequence), and a minor population initiated at residue 325, the principal 5' terminus of the wild-type late RNA. (iii) During SV40 lytic infection there was a shift in initiation sites used to transcribe the early region from sites that are downstream to sites which are upstream (up RNA) of the origin of DNA replication. We observed that unlike lytic infection, T antigen and viral DNA replication were not needed for the appearance of up RNA in mouse L cells. (iv) In mouse L cells late RNAs were made, and the residue 325 5' end was utilized in the absence of T antigen or DNA replication. (v) In COS cells we found down RNA and up RNA transcribed from the extrachromosomally replicating plasmid but only down RNA produced by the integrated SV40 genome.

Animals↗
PubMed1985

The genomic organisation and nucleotide sequence of the HLA-SB(DP) alpha gene.

We have isolated a unique fragment of the HLA-DR alpha gene and probed human genomic DNA at low stringency to search for homologous sequences. A minimum of six non-polymorphic cross-hybridizing high molecular weight fragments were found in all DNAs examined. In order to obtain molecular clones of these cross-hybridizing fragments, we constructed lambda and cosmid libraries of human DNA and screened them at low stringency with the HLA-DR alpha gene specific subclone. We have isolated clones corresponding to each of the six fragments and, in this paper, describe those which contain the gene encoding HLA-SB(DP) alpha.

Amino Acid Sequence↗
PubMed1985

Structure and polymorphism of class I MHC antigen mRNA.

We have used cDNA cloning and primer extension techniques to determine the complete nucleotide sequence of HLA-B7 mRNA. The 5'-untranslated sequence of the mRNA is rather short and the putative promoter has weak homology to the conventional "TATA" sequences. The 5' end and the polyadenylation site define the transcription unit of the B7 gene to be about 3.5 kb long. Comparison of the translated nucleotide sequence of this cDNA with the amino acid sequence of the heavy chain of the B7 antigen showed two amino acid differences. In addition, comparison with the sequences of the coding and untranslated regions of several HLA and H-2 genes showed that the class I histocompatibility molecules consist of four variable segments separated by three regions of homology. Analysis of the DNA polymorphisms revealed that in the variable segments the majority of the nucleotide substitutions are nonsilent, while in the homology regions the majority of substitutions are silent. Further analysis of the nature of the amino acid substitutions revealed the predominance of nonconservative replacements/changes in both the variable segments and the homology regions except the transmembrane part of the molecule. Selection at both the protein and the codon levels contributes to the pattern of mutations found in class I histocompatibility molecules.

Amino Acid Sequence↗
PubMed1985

HLA-B locus polymorphism: studies with a specific hybridization probe.

The large number of class I histocompatibility genes (HLA) and their extensive homology has made it difficult to assign bands on genomic Southern blots to known genes. Therefore, we have tried to obtain nucleic acid probes for class I genes that are locus specific or have restricted locus specificity. Computer sequence-homology analysis was used to compare the nucleic acid sequences of two genomic clones, one coding for the HLA-B7 antigen (JY150) and one containing a class I pseudogene (pHLA12.4). A sequence in the 3' untranslated region with very low homology was identified. This sequence from the HLA-B7 gene was subcloned into M13 phage. This fragment, JY150/C5, hybridized with two genomic bands in DNA from human HLA homozygotes--presumably the HLA-B locus gene and a closely related gene. The probe was used to assess restriction fragment polymorphism at the HLA-B locus in homozygous consanguineous cell lines. This analysis permitted the association of certain polymorphic restriction enzyme fragments with some alleles of this locus. However, many HLA-B alleles have identical restriction fragments produced by a number of restriction endonucleases.

Alleles↗