Biomedical subjects

S S Ner

Publications and source records attributed to S S Ner.

9 recordsLinked to original sources

PubMed1989

A method for introducing random single point deletions in specific DNA target sequences using oligonucleotides.

We describe a method for the generation of random point deletions in any target DNA sequence using synthetic mixed oligonucleotides. A mixed pool of oligonucleotides, which contain single nucleotide deletions randomly distributed throughout the full length, was generated by a modification of the synthesis cycle of an automated DNA synthesiser that allowed the inefficient incorporation of nucleotide monomers during each cycle of synthesis. A family of oligonucleotides was used to prime in vitro synthesis of the complementary strand of a cloned DNA fragment in an M13 vector which had previously been passaged through a dut-, ung- Escherichia coli host. Strong selection for progeny from the newly synthesised strand is provided by transforming the heteroduplex into a dut+, ung+ host. This procedure introduced point deletions at 10-25% efficiency. It has been used to introduce point deletions into operator sequences which bind the yeast regulatory proteins encoded by MATa1 and MAT alpha 2.

Base Sequence↗

PubMed1989

Role of intron splicing in the function of the MATa1 gene of Saccharomyces cerevisiae.

The MATa1 gene of Saccharomyces cerevisiae is unique in yeast cells in that it contains two short intervening sequences (IVS1 and IVS2) 54 and 51 nucleotides long. The 3' intron is inefficiently spliced and results in the accumulation of transcript with only the first intron removed, leading to the speculation that the gene may produce different protein products by alternative splicing patterns. We have used in vitro mutagenic techniques to construct intronless MATa1 genes and have introduced point substitutions in the 5'-TACTAAC-3' internal conserved sequence of each intron to identify the protein product that is required for repression of haploid-specific genes. Analysis of these constructs for the ability to repress expression of an HO::lacZ fusion and for the ability to allow diploid cells to undergo sporulation during conditions of starvation revealed that the gene is functional with two, one, or no introns and that the only functional protein is the one produced when both introns are spliced from the mRNA.

Amino Acid Sequence↗

PubMed1988

Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate.

Asp-362, a potential key catalytic residue of Escherichia coli citrate synthase (citrate oxaloacetate-lyase [pro-3S)-CH2COO- ----acetyl-CoA), EC 4.1.3.7) has been converted to Gly-362 by oligonucleotide-directed mutagenesis. The mutant gene was completely sequenced, using a series of synthetic oligodeoxynucleotides spanning the structural gene to confirm that no additional mutations had occurred during genetic manipulation. The mutant gene was expressed in M13 bacteriophage and produced a protein which migrated in an identical manner to wild-type E. coli citrate synthase on SDS-polyacrylamide gels and which cross-reacted with E. coli citrate synthase antiserum. The mutant gene was subsequently recloned into pBR322 for large scale purification of the protein, and the resulting plasmid, pCS31, used to transform the citrate synthase deletion strain, W620. The mutant enzyme purified in an analogous manner to wild-type E. coli citrate synthase and expressed less than 2% of wild-type enzyme activity. The activity of the partial reactions catalysed by citrate synthase was similarly affected suggesting that this residual activity may be due to contaminating wild-type enzyme activity. The mutant citrate synthase retains a high-affinity NADH-binding site consistent with the protein preserving its overall structural integrity. Oxaloacetate binding to the protein is unaffected by the Asp-362 to Gly-362 mutation. Binding of the acetyl-CoA analogue, carboxymethyl-CoA, could not be detected in the mutant protein indicating that the lack of catalytic competence is due primarily to the inability of the protein to bind the second substrate, acetyl-CoA.

Acetyl Coenzyme A↗

PubMed1988

A simple and efficient procedure for generating random point mutations and for codon replacements using mixed oligodeoxynucleotides.

A very simple and highly efficient procedure for the generation of single and multiple substitutions in segments of DNA is described which has no requirements for conveniently placed restriction sites, and allows all DNA sequences to be equally accessible. A mixed pool of oligodeoxynucleotides is synthesized by contaminating the monomeric nucleotides with low levels of the other three nucleotides such that the full-length oligonucleotide contains on the average one to two changes per molecule. This pool is used in priming in vitro synthesis of the complementary strand of cloned DNA fragments in M13 or pEMBL vectors which have previously been passed through a dut-, ung- Escherichia coli host. Strong selection for the newly synthesized strand is provided by transforming the heteroduplex into a dut+, ung+ host. Single and multiple substitutions in the carboxy-terminal coding region of the MATa1 gene of Saccharomyces cerevisiae are introduced at high efficiency (25-55%) and the changes are identified by direct sequencing alone. The same principle can be used to generate multiple sets of changes at any specified codon.

Codon↗

PubMed1988

A rapid droplet method for Sanger dideoxy sequencing.

A method for performing the dideoxy sequence reaction on petri dishes is described. It allows rapid manipulation of clones and provides large amounts of sequence information quickly and without the need for elaborate laboratory equipment.

Base Sequence↗

PubMed1986

Sequence and interspecies transfer of an aminoglycoside phosphotransferase gene (APH) of Bacillus circulans. Self-defence mechanism in antibiotic-producing organisms.

The APH gene of a butirosin-producing Bacillus circulans was cloned and shown to be expressed in Escherichia coli and Streptomyces lividans. The gene was sequenced and a possible developmentally regulated promoter identified. When the deduced protein sequence was compared with those from transposon Tn5, transposon Tn903, Streptomyces fradiae, Staphylococcus aureus and Streptococcus faecalis, significant homology was found, indicating that the genes may have a common origin.

Bacillus↗

PubMed1986

The synthesis and use of oligodeoxynucleotides in plasmid DNA sequencing.

A convenient procedure for the synthesis and purification of oligonucleotides is described. 16-base long primers synthesised by this method were used to investigate DNA sequencing using plasmid DNA as a template. This allowed the further analysis of the E. coli glt A sequence coding for citrate synthase and enabled determination of the 5'-non-coding regulatory region of the aminoglycoside phosphotransferase gene.

Base Sequence↗

PubMed1983

The use of bacteriophage M13 carrying defined fragments of the Escherichia coli gltA gene to determine the location and structure of the citrate synthase promoter region.

The gltA gene from Escherichia coli, which encodes citrate synthase, has been located on a 3.24 Kb HindIII/EcoRl restriction fragment. This region contains one restriction site for BamHl and two for BglII. Defined restriction fragments from this region were cloned into suitably cleaved replicative form M13mp8 and M13mp9. The recombinants (M13gtlA1 leads to 10) were isolated as single stranded DNA and characterised on the basis of molecular weight and DNA sequence. The single stranded DNA was converted to the double stranded replicative form and used to transform E. coli strain JM103 from which bacteriophage were isolated. Infection of JM103 with different bacteriophage followed by measurement of expressed citrate synthase activity showed that the complete gltA gene must span the BamHl restriction site, that the control region was on the 5'-terminal side of this restriction site and that the coding region for citrate synthase protein commenced on the 3'-terminal side. Analysis of the DNA sequence of this region allowed us to confirm this model, to identify the start sequence for translation of the structural gene and a number of sequences controlling the initiation of transcription. Of special interest is the fact that there must be an extensive leader sequence (305 nucleotides) separating the predicted sites for initiation of transcription and translation.

Base Sequence↗

PubMed1983

Citrate synthase activity in Escherichia coli harbouring hybrid plasmids containing the gltA gene.

A hybrid plasmid, pDB2, was constructed by ligating a 3.24 kb EcoRI/HindIII fragment of the Escherichia coli chromosome into pBR322. This was used to transform a gltA mutant which was devoid of citrate synthase activity. The resultant strain expressed very high citrate synthase activity and this enabled a simplified purification of the homogeneous enzyme in high yield. The subunit Mr was estimated as 47000-49000 by SDS gel electrophoresis, which closely resembles the eukaryotic form of the enzyme. Evidence for some conservation of sequence between the two proteins was revealed in the acid cleavage pattern at aspartyl-prolyl residues. In addition to coding for the structural gene for citrate synthase, the 3.24 kb EcoRI/HindIII fragment also retained the genetic structure necessary for control of enzyme synthesis since the expression of enzyme activity in the strain harbouring pDB2 was still subject to glucose repression.

Citrate (si)-Synthase↗