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S M Kingsman

Publications and source records attributed to S M Kingsman.

At least 73 records · Page 4Linked to original sources
PubMed1989

Host-vector systems.

In 1980 it was only possible to express foreign genes in bacteria and a few easily cultured animal cells. During the subsequent eight years specialized vectors have been developed to allow the genetic manipulation of a wide range of both prokaryotes and eukaryotes. One of the major goals of biotechnology in 1980 was to use host cells as 'factories' for the production of proteins that were only available in minute quantities from natural sources. This has already lead to a new generation of pharmaceutical products. Advances in our understanding of host-vector systems have defined new goals. The basic concepts of expression vector design will be illustrated. Some of the new goals are discussed with particular reference to the exploitation of novel host-vector systems to develop vaccines and anti-viral agents against AIDS.

Cloning, Molecular↗
PubMed1989

HIV-1 TAT "activates" presynthesized RNA in the nucleus.

Replication of the human immunodeficiency virus (HIV-1) depends upon the viral TAT protein. TAT stimulates gene expression via a target response sequence (TAR) located within the HIV-1 LTR. As TAR is located in the transcribed region it could act as a signal in either the DNA, the RNA, or both. To test whether TAT acts on transcription and/or posttranscriptionally, we produced TAT in yeast and monitored its activity after microinjection into the nucleus or cytoplasm of Xenopus oocytes. The TAT protein stimulated TAR-dependent expression, but this activation was not inhibited by transcriptional inhibitors. Furthermore, TAR-containing RNA, produced in vitro, was "activated" by TAT after coinjection into oocytes. This activation only occurred, however, when the RNA was injected into the nucleus and not into the cytoplasm. Our data indicate, therefore, that in the Xenopus system TAT acts on presynthesized RNA and that the nucleus is involved in this action.

Animals↗
PubMed1989

Multiple cooperative interactions constrain BPV-1 E2 dependent activation of transcription.

Transcription directed by the BPV-1 long control region (LCR) is conditional upon activation by the virally encoded E2 protein. Within the 1.0 kb LCR there are five separate regions, A to E, that contain E2 responsive enhancers. The smallest functional region, A, is only 38 bp and contains two copies of the consensus sequence ACC(N)6GGT that is known to function as an E2 binding site in vitro. We show that a pair of these constitutes a minimal functional E2 responsive enhancer element but that the strength of enhancer activity is dramatically reduced both by increasing the spacing between them and by removing the dual elements from the proximity of other key promoter elements. Furthermore, pairs of dual elements activated transcription to varying levels depending upon their spatial arrangement and promoter proximity. We have also identified a low level constitutive enhancer in the D region which lacks an E2 consensus binding site but which can be activated by E2. We show that the activation potential of this constitutive enhancer is increased by association with a single E2 binding site suggesting some cooperation/interaction between viral and cellular enhancer proteins.

Base Sequence↗
PubMed1989

Efficient activation of transcription in yeast by the BPV1 E2 protein.

The full-length gene product encoded by the E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV1) is a transcriptional transactivator. It is believed to mediate its effect on the BPV1 long control region (LCR) by binding to motifs with the consensus sequence ACCN6GGT. The minimal functional cis active site, called the E2 response element (E2RE), in mammalian cells comprises two copies of this motif. Here we have shown that E2 can function in Saccharomyces cerevisiae by placing an E2RE upstream of a synthetic yeast assay promoter which consists of a TATA motif and an mRNA initiation site, spaced correctly. This E2RE-minimal promoter is only transcriptionally active in the presence of E2 protein and the resulting mRNA is initiated at the authentic start site. This is the first report of a mammalian viral transactivator functioning in yeast. The level of activation by E2 via the E2RE was the same as observed with the highly efficient authentic PGK promoter where the upstream activation sequence is composed of three distinct elements. Furthermore a single E2 motif which is insufficient in mammalian cells as an activation site was as efficiently utilized in yeast as the E2RE (2 motifs). Previous studies have shown that mammalian cellular activators can function in yeast and our data now extend this to viral-specific activators. Our data indicate however that while the mechanism of transactivation is broadly conserved there may be significant differences at the detailed level.

Base Sequence↗
PubMed1989

A novel method for the purification of HIV-1 p24 protein from hybrid Ty virus-like particles (Ty-VLPs).

The self-assembly properties of a protein encoded by the yeast retrotransposon Ty can be exploited to produce large amounts of recombinant, particulate fusion proteins as hybrid Ty virus-like particles (Ty-VLPs). This system has now been adapted to allow the release of the additional protein by incorporation of a protease cleavage site between the yeast carrier protein and the protein of interest. The purification of the additional protein is facilitated by exploiting the ease with which Ty-VLPs can be purified from other yeast cell components due to their particulate nature. We have used this modified system to produce hybrid particles containing the HIV-1 p24 protein downstream of the recognition sequence for the blood coagulation factor Xa. The p24 was released from the particles by proteolytic cleavage and rapidly separated from the residual particulate material using centrifugation and standard chromatography techniques. This procedure has been used to purify milligram quantities of HIV-1 p24 protein that reacts with anti-p24 sera and elicits the production of p24-specific antibodies in experimental animals.

Animals↗
PubMed1989

Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1.

The promoter of the yeast glycolytic gene encoding phosphoglycerate kinase (PGK) contains an upstream activation sequence between bases -538 and -402 upstream of the initiating ATG. The upstream activation sequence contains multiple functional elements, including an essential region called the activator core (AC) sequence and three copies of the pentamer 5'-CTTCC-3'. The AC sequence shows strong homology to the consensus binding sites for the yeast proteins RAP1 (GRF1) and TUF. We have demonstrated that the yeast protein which interacts with the AC sequence is the DNA-binding protein RAP1. Expression of the PGK gene is found to be regulated according to the carbon source in the growth medium. PGK mRNA levels are high in yeast cells grown in glucose medium but low in yeast cells grown in media containing carbon sources such as pyruvate and acetate. This carbon source regulation of transcription was found to be mediated, in part, via regulation of RAP1 binding to the AC sequence. The promoters of many other yeast glycolytic genes also contain consensus RAP1-binding sites and copies of the CTTCC pentamer. This suggests that RAP1 may be involved in transcriptional control of many other glycolytic genes in addition to the PGK gene.

Antibodies↗
PubMed1988

HIV expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems.

The pol gene of the human immunodeficiency virus (HIV-1) is expressed as a gag:pol fusion, arising from a ribosomal frameshift that brings the overlapping, out-of-phase gag and pol genes into translational phase. In this study, we show that HIV frameshifting is mediated by a very short sequence in the viral RNA. We demonstrate the importance of a homopolymeric run within this sequence and conclude that HIV frameshifting is not dependent on stem-loop structures downstream from the frameshift site. Our analysis also indicates that the sequence requirements are identical in mammalian and yeast systems.

Animals↗
PubMed1988

The UAS of the yeast PGK gene is composed of multiple functional elements.

The UAS (upstream activator sequence) of the yeast PGK gene contains a transcriptional activator domain located between bases -479 and -402 upstream from the initiating ATG. This region of the UAS contains three direct repeats of the sequence 5'CTTCC3'. The roles in transcriptional activation of these repeats and other sequences within the activator domain were investigated. When short regions containing the repeats were removed PGK expression was considerably reduced, the magnitude of the effect depended upon which CTTCC block was absent. Sequences between -473 and -458 which did not contain a CTTCC block were also shown to be necessary for high levels of PGK expression. A DNA fragment containing activator sequences up to -473 was shown to interact specifically in vitro with a yeast nuclear protein extract. DNase I footprinting identified a protected region between -473 and -458 and single base changes in DNase I sensitivity at the CTTCC repeats.

Chromosome Deletion↗
PubMed1988

Reconstruction of the yeast 2 micron plasmid partitioning mechanism.

The effect of the yeast 2 micron circle encoded REP1 and REP2 gene products on plasmid partitioning and copy number control was analyzed by removing the open reading frames from their normal sequence context and transcriptional control regions and directing their expression using heterologous promoters in [cir0] host strains. Both the REP1 and REP2 gene products are directly required at appropriate levels of expression to reconstitute the 2 microns circle partitioning system in conjunction with REP3 and the origin of replication. The level of expression of REP2 appears to be critical to re-establishing proper partitioning and may also play a role in monitoring and thereby regulating the plasmid copy number. Constitutive expression of the REP1 and REP2 open reading frames using heterologous expression signals can be used to reconstruct efficient plasmid partitioning even in the absence of FLP-mediated plasmid amplification and a functional D open reading frame.

DNA Replication↗
PubMed1988

Upstream and downstream transcriptional control signals in the yeast retrotransposon, TY.

The yeast retrotransposon, Ty, shares many structural and functional features with retroviral proviruses. These include production of a terminally redundant major transcript. There are also two less abundant transcripts of 5.0 kb and 2.2 kb. Ty transcription is regulated by cell-type, that is it is reduced 5-20 fold in a/alpha diploids as compared to haploids. However control of expression of Ty is not well understood. By deletion analysis we have identified regions of the element which are involved in the activation and regulation of transcription. These signals are found both upstream and downstream of the mRNA start site. The downstream signals are within the region encoding the major Ty proteins. This organisation of transcriptional control signals is discussed with reference to the organisation of control signals in other yeast genes and in retroviral proviruses and other retro-elements.

Base Sequence↗
PubMed1988

Synthesis of a gene for the HIV transactivator protein TAT by a novel single stranded approach involving in vivo gap repair.

The synthesis of a gene for the HIV TAT protein is described using a novel approach that capitalises on the ability to synthesise oligonucleotides of greater than 100 bp in length. It involves the synthesis of large oligomers covering one strand of the desired gene in its entirety and the use of small complementary bridging and adapter oligonucleotides to direct the assembly and cloning of the large oligomers. After ligation to the cloning vector the partially single stranded intermediate is transformed directly into the recipient bacterial host where the plasmid is repaired. The synthetic tat gene has been expressed in HeLa cells and is shown to trans-activate TAR+ but not TAR- HIV LTR-CAT constructs.

Amino Acid Sequence↗
PubMed1988

A heat shock element in the phosphoglycerate kinase gene promoter of yeast.

The phosphoglycerate kinase (PGK) promoter is often employed in yeast expression vectors due to its very high efficiency. Its activity in unstressed cells has been shown to be due to an upstream activator site (UASPGK) at -402 to -479. Since levels of PGK mRNA can sometimes be elevated by heat shock of yeast cultures this investigation determined how specific deletions of PGK promoter sequences effect levels of PGK mRNA both before and after heat shock. A series of PGK promoter deletions was inserted on a high copy plasmid into cells having a TRP1 gene disruption of the solitary chromosomal PGK locus. This enabled PGK transcripts of plasmid and chromosomal origin to be distinguished by virtue of their different sizes. Certain deletions lacking UASPGK displayed activities that were very low in unstressed cells, but which increased fifty to one-hundred fold after heat shock. With UASPGK present heat shock had only a relatively small or negligible effect on PGK mRNA levels. Heat shock activation was abolished when the -256 to -377 region with homology to the heat shock element consensus of eukaryotes was deleted in addition to UASPGK, but was unaffected by the deletion of regions further downstream containing TATA- and CAAT- sequence motifs. This is the first demonstration of a heat shock element, an activator site normally found upstream of eukaryotic heat shock protein genes, as a natural constituent of a high efficiency glycolytic promoter. It is proposed that PGK may be one member of a small subset of yeast genes that are highly expressed in unstressed cells yet possess a heat shock element to ensure their continued transcription after heat shock.

Base Sequence↗
PubMed1988

An AT rich region of dyad symmetry is a promoter element in the yeast TRP1 gene.

Transcription from the yeast TRP1 promoter results in two classes of transcript, I and II, that are influenced by different promoter elements. The 5' flanking region contains a region of dyad symmetry (RDS) which contains a 12 nucleotide AT rich inverted repeat, separated by a 21 bp spacer region. The RDS lies within a region of the promoter required for transcription of class II RNAs. A series of internal deletions and insertions have been constructed in vitro around the RDS and the effect of each mutation on transcription has been analysed. Deleting either of the repeats abolishes class II transcription and disruption of both repeats influenced the levels of the larger class I transcripts. Deletion of the spacer had no effect but increasing the length to 33 bp reduced transcription. These results show that the RDS is an important component of the TRP1 promoter, that both repeats must be preserved and that there is some constraint on the spacing of the repeats for maximal function.

Base Sequence↗
PubMed1988

Polyvalent recombinant antigens: a new vaccine strategy.

Recombinant DNA technology can be used to produce individual proteins from infectious organisms and these have the potential to be used as subunit vaccines. In many cases however these proteins display a low immunogenicity. One reason for this might be the poor presentation of the antigen to the cells of the immune system. A number of new recombinant DNA approaches are now being tried to improve the production and the presentation of antigens. One method is to use a carrier protein encoded by a retrotransposon of yeast called the Ty element. This protein can be linked to a wide range of viral antigens via the construction of hybrid genes. The fusion proteins assemble in yeast into 60 nm virus-like particles (VLPs) that display the additional antigens on the surface. These hybrid Ty-VLPs are good immunogens even in the absence of adjuvant. Similar approaches to the production of polyvalent particulate antigens involve using carrier proteins derived from hepatitis B and polio viruses.

Antigens, Viral↗
PubMed1988

Complete nucleotide sequence of a mouse VL30 retro-element.

The complete nucleotide sequence of a mouse retro-element is presented. The cloned element is composed of 4,834 base pairs (bp) with long terminal repeats of 568 bp separated by an internal region of 3,698 bp. The element did not appear to have any open reading frames that would be capable of encoding the functional proteins that are normally produced by retro-elements. However, some regions of the genome showed some homology to retroviral gag and pol open reading frames. There was no region in VL30 corresponding to a retroviral env gene. This implies that VL30 is related to retrotransposons rather than to retroviruses. The sequence also contained regions that were homologous to known reverse transcriptase priming sites and viral packaging sites. These observations, combined with the known transcriptional capacity of the VL30 promoter, suggest that VL30 relies on protein functions of other retro-elements, such as murine leukemia virus, while maintaining highly conserved cis-active promoter, packaging, and priming sites necessary for its replication and cell-to-cell transmission.

Amino Acid Sequence↗
PubMed1987

Multiple cis-active elements in the long control region of bovine papillomavirus type 1 (BPV-1).

A 1.0 kb region of the BPV-1 genome (the long control region, LCR), contains controls for transcription and the origin of replication. Transcription directed by the LCR is activated by the viral encoded E2 protein. To define the essential cis acting elements that are required to control transcription we have constructed a series of deletions throughout the LCR. We have identified three important domains in the LCR, two of which respond to E2. We have analysed the ability of small subcloned regions of the E2 responsive domains to act as enhancers in a heterologous assay system. This has led to the identification of five independent E2 responsive elements. We have shown that a fragment of only 38 base pairs is sufficient to respond to activation by E2. We also present evidence to suggest two types of E2 responsiveness that result in strong or weak activation. Strong response is correlated with the presence of the sequence 5'-ACCG/TNNNC/TCGGTGC-3' whereas weak response is correlated with the presence of a related sequence 5'-ACC(N)6GGT-3'. The contribution of these multiple elements to viral transcription is discussed.

Bovine papillomavirus 1↗