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M J Dobson

Publications and source records attributed to M J Dobson.

At least 55 records · Page 3Linked to original sources
PubMed1987

A transcriptional activator is located in the coding region of the yeast PGK gene.

Expression of heterologous genes from the PGK promoter on high copy number plasmids in yeast is relatively poor compared to the intact PGK gene because of low steady-state RNA levels. In this paper we show that low levels of heterologous RNA are not due to instability of mRNA but result from inefficient transcription due to a defect in RNA synthesis. A comparison of RNA levels from homologous and heterologous transcription units allowed the identification of a positive activator for transcription within the PGK coding region which is required for efficient expression of the PGK gene. Deletion of this region, the "downstream activator sequence", causes a six to ten fold reduction in transcriptional efficiency from the PGK 5' noncoding region.

Base Sequence↗
PubMed1985

The Ty transposon of Saccharomyces cerevisiae determines the synthesis of at least three proteins.

Two new Ty determined proteins have been identified by placing a Ty transcriptional unit under the control of a high efficiency yeast expression vector. One of these proteins is the product of a post-translational processing event and it binds nucleic acids. A previously identified protein, pl (Tyl-15), has also been shown to bind nucleic acids and to be modified by phosphorylation.

Amino Acid Sequence↗
PubMed1985

Variants within the yeast Ty sequence family encode a class of structurally conserved proteins.

The Ty transposable elements of Saccharomyces cerevisiae form a heterogeneous family within which two broad structural classes (I and II) exist. The two classes differ by two large substitutions and many restriction sites. We show that, like class I elements a class II element, Tyl-17, also appears to contain at least two major protein coding regions, designated TYA and TYB, and the organisational relationship of these regions has been conserved. The TYA genes of both classes encode proteins, designated p1 proteins, with an approximate molecular weight of 50 Kd and, despite considerable variation between the TYA regions at the DNA level, the structures of these proteins are remarkably similar. These observations strongly suggest that the p1 proteins of Ty elements are functionally significant and that they have been subject to selection.

Amino Acid Sequence↗
PubMed1985

Factors affecting heterologous gene expression in Saccharomyces cerevisiae.

The 'promoter' fragment from the yeast phosphoglycerate kinase (PGK) gene has been used to direct the expression of human interferon-alpha-2 (IFN alpha 2) on a high-copy-number plasmid in yeast. The yields of IFN alpha 2 are only 1-3% of yeast total protein, whereas the maximum yield of PGK produced by the PGK gene on a high-copy-number plasmid is at least 50%. IFN alpha 2 is turned over more rapidly than PGK but in addition a major reason for the relatively low level of IFN alpha 2 is that IFN-specific RNA levels are much lower. This does not reflect differences in plasmid copy number or integrity, or differences in the 5' and 3' untranslated regions of the transcripts or DNA flanking regions. It appears that the presence of heterologous coding sequences, or the absence of specific yeast sequences causes a reduction in heterologous RNA levels in yeast.

Cloning, Molecular↗
PubMed1984

Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence.

Base Sequence↗
PubMed1984

The identification and high level expression of a protein encoded by the yeast Ty element.

Transcription of the yeast Ty element, Ty1 -15, was placed under the control of the efficient PGK promoter on the yeast expression vector, pMA91 . In extracts of yeast transformants containing these constructions a new 52 K basic polypeptide was detected by one- and two-dimensional electrophoresis and was shown, by hybrid arrested translation, to be specifically encoded by the Ty element. The protein coding region was mapped to the first 1.45 kb of the transcribed region of Ty1 -15. These data show for the first time that Ty elements encode proteins and illustrate the general usefulness of high efficiency expression vectors for the detection of rare products.

Base Sequence↗
PubMed1983

Expression in Saccharomyces cerevisiae of human interferon-alpha directed by the TRP1 5' region.

The complete 5' flanking region of the yeast TRP1 gene encoding N-(5'-phosphoribosyl)- anthranilate isomerase, a nonabundant protein, has been cloned and the nucleotide sequence data has been extended from -102 to -440. The CT block--CAAG structure common to all efficiently expressed yeast genes is altered in the 5' region of TRP1 and a sequence postulated to be involved in general amino acid regulation is absent. There are two possible TATA boxes at -224 and -262. TRP1, in common with HIS3, HIS4 and TRP5 has a region of dyad symmetry upstream of the coding sequence which may play a role in initiation of transcription. The relative efficiency of gene expression directed by the complete 5' TRP1 region was assessed by comparison with that of PGK by inserting a cDNA for a human interferon-alpha downstream of their respective 5' regions. The respective interferon yields indicate that their in vivo expression capabilities are a function of their 5' regions.

Aldose-Ketose Isomerases↗
PubMed1983

The complete amino acid sequence of yeast phosphoglycerate kinase.

The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined. The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene. The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes. Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis. Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure. The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371). Both these regions contribute to the nucleoside phosphate-binding site. A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes. The yeast has strikingly fewer methionine, cysteine and tryptophan residues.

Amino Acid Sequence↗
PubMed1983

Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.

We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.

Animals↗
PubMed1982

Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae.

The position of the yeast phosphoglycerate kinase (PGK) gene has been mapped on a 2.95kb Hind III fragment. We have determined the nucleotide sequence of the 5' flanking region and compared this sequence with those from 16 other yeast genes. PGK, like all other yeast genes has an adenine residue at position -3. It has two possible TATA boxes at positions -114 and -152 and a CAAT box at -129. In addition we have defined a structure at position -63 to -39 that is common to all yeast genes that encode an abundant RNA. This structure is a CT-rich block followed, about 10 nucleotides later, by the sequence CAAG.

Amino Acid Sequence↗
PubMed1982

Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae.

The 5' control region of the yeast phosphoglycerate kinase gene (PGK) was fused to the coding sequence of a human interferon-alpha. This PGK-interferon fusion was then introduced into yeast on a high copy number 2mu-based plasmid vector. Strains containing this plasmid produced a PGK-interferon-alpha fusion protein as 1-2% of cell protein and the expression of interferon activity was regulated by the availability of a fermentable carbon source. The system is capable of making as much as 15 mg of human interferon-alpha per litre of batch culture.

Amino Acid Sequence↗
PubMed1982

Sequence and structure of yeast phosphoglycerate kinase.

The structure of yeast phosphoglycerate kinase has been determined with data obtained from amino acid sequence, nucleotide sequence, and X-ray crystallographic studies. The substrate binding sites, as deduced from electron density maps, are compatible with known substrate specificity and the stereochemical requirements for the enzymic reaction. A carboxyl-imidazole interaction appears to be involved in controlling the transition between the open and closed forms of the enzyme.

Amino Acid Sequence↗
PubMed1981

Sequence variation in the LEU2 region of the saccharomyces cerevisiae genome.

The LEU2 regions present on yeast plasmid vectors come from two sources, a series of strains derived from S288c and strain M127. The LEU2 region from the S288c series contains a Tyl-17 element with its associated delta sequences and a small repetitive RNA gene while the LEU2 region from M127 which is present on pJDB248, lacks the Tyl-17 element, but carries a delta sequence and a small RNA gene. The various LEU2 plasmids currently in use vary with respect to these sequences depending on which restriction fragment from the region is present on the recombinant molecule. In addition, strain M127 contains three LEU2 homologous sequences that are represented by different EcoRI fragments and which segregate independently at meiosis. Therefore, there are at least four forms of the centromere-distal EcoRI fragment of the LEU2 locus in the Saccharomyces cerevisiae gene pool; these are 7.1 kb, 1.9 kb, 1.48 kb and 1.15 kb long.

3-Isopropylmalate Dehydrogenase↗
PubMedDate not supplied

Purification and characterisation of an Aspergillus niger invertase and its DNA sequence.

A secreted invertase was purified 23-fold by ultrafiltration, ion-exchange, and gel filtration chromatography from the culture supernatant of 18 h sucrose-grown cultures of Aspergillus niger. The purified enzyme hydrolysed sucrose and raffinose but there was no detectable hydrolysis of inulin, melezitose or PNPG. Invertase activity was optimal at pH 5.5 and 50 degrees C. The molecular mass of reduced invertase was 115 kDa, as determined by SDS gel electrophoresis. The native molecular weight of between 225 kDa and 250 kDa, estimated by electrophoresis under non-denaturing conditions, suggests that the protein is a dimer of identical subunits. The suc1 gene encoding this protein was completely-sequenced. The translated sequence yields a protein of 566 amino acids with a calculated molecular mass of 61 kDa, suggesting that carbohydrates represent about 50% of the mass of the protein.

Amino Acid Sequence↗