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Biomedical subjects

R Treisman

Publications and source records attributed to R Treisman.

70 records · Page 4Linked to original sources
PubMed1983

Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes.

Transcriptional analysis of five different cloned beta-thalassaemia genes introduced into cultured mammalian cells revealed specific defects in transcription and RNA splicing. A single base change 87 base pairs to the 5' side of the mRNA cap site significantly lowers the level of transcription and therefore appears to represent a promoter mutation. Three genes contain different single base changes in the first intervening sequence (IVS) 5' splice site. One mutation, at IVS1 position 1, inactivates the splice site completely; the other two, at IVS1 positions 5 and 6, reduce its activity. Each mutation activates the same three cryptic splice sites. The fifth gene contains a single base change within IVS2 at position 745, which results in the formation of abnormal beta-globin RNA that contains an extra exon.

Alleles↗
PubMed1983

Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products.

When the human beta-globin gene is transfected into Hela cells, no beta-globin RNA is detected unless the gene is linked to a viral transcription enhancer. In this paper we show that trans-acting adenovirus and herpesvirus (pseudorabies) transcriptional regulatory proteins can circumvent this enhancer requirement for detectable beta-globin transcription in transient expression assays. The viral gene products can be provided by constitutively expressed, integrated viral genes in established cell lines, by viral infection of permissive cells, or by transfection of cells with bacterial plasmids carrying the viral immediate-early genes. These results demonstrate the utility of transient expression assays for studying regulatory mechanisms involving trans-acting factors. Analysis of beta-globin promoter mutants indicates that between 75 and 128 bp of sequence 5' to the mRNA cap site is required for enhancer-dependent transcription in Hela cells. In contrast, beta-globin transcription in the presence of viral immediate-early gene products requires only 36 bp of 5'-flanking sequence, which includes the TATA box. Thus both cis and trans-acting viral factors activate beta-globin gene transcription in transient expression experiments, but the mechanisms by which they act appear to be fundamentally different.

Adenoviruses, Human↗
PubMed1983

cis and trans activation of globin gene transcription in transient assays.

We examined the effects of the simian virus 40 enhancer sequence on transcription of cloned human alpha- and beta-globin genes shortly after their introduction into cultured mammalian cells. We find that (i) detectable transcription of the beta-globin gene but not the alpha-globin gene requires linkage to the enhancer; (ii) the enhancer increases the amount of beta-globin RNA at least 100-fold but results in only a 5- to 10-fold increase in the amount of alpha-globin RNA; (iii) plasmid replication does not increase the level of beta-globin RNA, regardless of linkage to the enhancer, but does result in an approximately equal to 50-fold increase in the level of alpha-globin RNA; (iv) the enhancer is not required for and does not increase transcription of either gene in 293 cells, an adenovirus 5-transformed human kidney cell line. We also show that an enhancer sequence is not required for activity of the normally enhancer-dependent simian virus 40 early promoter in 293 cells, indicating that these cells contain a trans-acting factor(s) that circumvents the requirement for the enhancer sequence.

Alpha-Globulins↗
PubMed1983

Plasmid vectors for the rapid isolation and transcriptional analysis of human beta-globin gene alleles.

We describe the construction and characterization of miniplasmid vectors that can be used to isolate and express normal and mutant alleles of the human beta-globin gene. These vectors, designated pi SV beta plasmids, contain a bacterial origin of replication and selectable marker, a 5'-flanking beta-globin DNA fragment that can be used for recombination screening (Seed, 1983), and simian virus 40 (SV40) sequences that allow accurate and efficient expression of the beta-globin gene transfected into mammalian cells. We show that pi SV beta plasmids can be used to select cloned beta-globin genes from a bacteriophage lambda library of genomic DNA, and that plasmids containing the beta-globin gene linked to the SV40 enhancer sequence can be excised from the phage, circularized and recovered by transformation of Escherichia coli. Analysis of the beta-globin transcripts produced by the recovered pi SV beta recombinant plasmids after transfection into COS cells and replication to high copy number, indicates that the beta-globin gene is accurately transcribed, but a substantial fraction of the transcripts are the result of readthrough from sites within the vector. In contrast, when these plasmids are transferred into HeLa cells beta-globin RNA is accurately initiated and little readthrough transcription is observed. These results indicate that HeLa cells are more suitable than COS cells for studying mutant beta-globin genes, even though the copy number of the pi SV beta plasmids is much higher in COS cells.

Alleles↗
PubMed1982

A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing.

We have studied the structure and transcription of a cloned human beta-globin gene from a fetus diagnosed for beta 0 thalassemia. The sequence of the beta 0 gene differs from that of a normal beta-globin gene at positions 1 and 74 of the second intervening sequence (IVS2). The position 1 change alters the GT dinucleotide conserved at 5' splice sites, while the position 74 change is a common sequence polymorphism. When the cloned beta 0 gene is introduced into HeLa cells by use of an SV40-derived plasmid vector, two abnormally spliced cytoplasmic beta-globin RNAs are detected. The predominant RNA differs from normal beta-globin mRNA by the insertion of the first 47 nucleotides of IVS2 between exons 2 and 3. The less abundant RNA comprises the normal first exon spliced directly to the third. Analysis of nuclear RNA suggests that the beta 0 transcript is inefficiently spliced and that the removal of the two intervening sequences is coupled.

Base Sequence↗
PubMed1982

Bovine papillomavirus vector that propagates as a plasmid in both mouse and bacterial cells.

We report the construction of a bovine papillomavirus (BPV)-derived recombinant plasmid that propagates as an extrachromosomal element in both mouse and bacterial cells. Plasmids composed of a subgenomic transforming fragment of BPV DNA, a deletion derivative of pBR322, and a 7.6-kilobase fragment of DNA from the human beta-globin gene cluster efficiently induce focus formation on mouse C127 cells. BPV-beta-globin hybrids are maintained in the transformed cells as plasmids with a copy number of about 10-30 per cell. Plasmids indistinguishable from the input DNA have been recovered by transformation of bacteria with low molecular weight DNA from transformed mouse cells. The human beta-globin gene linked to BPV DNA is transcribed from its own promoter at a high level in these cells. The expression of BPV-linked cellular genes in conjunction with the ability to shuttle DNA between bacteria and mammalian cells may provide a rapid means of analyzing and recovering genes that confer an identifiable phenotype upon mammalian cells.

Animals↗
PubMed1981

Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein.

A modified polyoma virus genome has been constructed which can encode the middle-T protein, but not the large-T or small-T proteins. This was achieved, starting with the full length viral DNA inserted into a plasmid vector, by replacing a small genomic restriction fragment spanning the middle-T intervening sequence with the equivalent fragment from a cloned partial cDNA copy of the middle-T protein mRNA. Transfection of the modified viral DNA into cultured rat cells efficiently induced the formation of transformed cell foci which gave rise to cell lines that grew as tumours after injection into Fisher rats. The only viral early-region antigen synthesized by the cell lines was the middle-T protein. Expression of the middle-t protein is therefore sufficient to establish and maintain a transformed state. The viral mRNA produced by two of the transformed cell lines was structurally indistinguishable from the normal middle-T mRNA found in productively infected cells, suggesting that RNA splicing is not an essential step in the biogenesis of this messenger.

Animals↗
PubMed1981

The structures of the spliced mRNAs encoding polyoma virus early region proteins.

The polyoma virus early region mRNAs synthesized during productive infection of mouse cells have been characterized at the nucleotide level. One- and two-dimensional agarose gel fractionation of nuclease S1-resistant RNA-DNA hybrids was used to establish basic structures. The two splice donors and the two splice acceptors were positioned more precisely by high resolution S1-gel mapping with terminally labeled DNA probes and polyacrylamide gels. The nucleotide sequences across the three splice joints were established by cloning and sequencing partial cDNA copies of the mRNAs. In combination with data on the polyadenylated 3'-end previously published, and the detailed analysis of the capped 5'-ends presented elsewhere, the present data complete the description of a family of differentially spliced mRNAs able to encode the known early region gene products, small-T, middle-T, and large-T proteins.

Animals↗
PubMed1980

Characterisation of polyoma late mRNA leader sequences by molecular cloning and DNA sequence analysis.

The leader sequences of two of the three polyoma virus late mRNAs were characterised by molecular cloning and DNA sequence analysis. A short single-stranded DNA fragment complementary to the 5' end of the body of mVP1 was used to prime cDNA synthesis, and double-stranded cDNA was inserted into a derivative of pAT 153. Analysis of fourteen mVP1 cDNAs and two mVP3 cDNAs allowed the precise determination of the leader-body joints, and demonstrated that the majority of polyoma late leader sequences consist of exact tandem repeats of a 57 nucleotide sequence present only once in the genomic DNA at 66-67 m.u. The sequences in the genomic DNA borderline this unit are typical of those found at RNA splice points. Leader sequences contained on average three to four repeat units. Nuclease S1 mapping of total late mRNA demonstrated that most mRNA 5' ends map heterogeneously in the 50 nucleotides 5' to the repeated sequence unit. The structures of the leader sequences strongly suggest that they are generated by appropriate splicing events from a tandemly repeated transcript of the entire circular viral genome.

Base Sequence↗
PubMedDate not supplied

Simian virus 40 enhancer increases number of RNA polymerase II molecules on linked DNA.

Since the discovery of transcription enhancer sequences in the genomes of the DNA viruses simian virus 40(SV40) and polyoma virus, these elements have been shown to play an important part in the control of both viral and cellular gene expression. Enhancer elements act in cis to increase the amount of RNA produced from linked genes in a manner largely independent of distance and orientation. The mechanisms by which enhancers act are not understood; in particular, it is not known whether the enhancer-dependent increase in the level of stable RNA reflects an increase in the rate of transcription. To address this question, we have used an in vitro nuclear transcription assay to examine the effect of the SV40 enhancer on transcription of cloned human beta-globin genes transiently introduced into HeLa cells. We show here that the SV40 enhancer acts at least in part to increase the number of RNA polymerase II molecules transcribing the linked gene.

Cell Nucleus↗