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B Polisky

Publications and source records attributed to B Polisky.

49 records · Page 3Linked to original sources
PubMed1982

Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome.

We have isolated two mutant plasmid derivatives of ColE1 that exhibit temperature-sensitive replication properties. Both mutants have a normal copy number at 30 degrees C but increase their copy number 30- to 40-fold after a shift in temperature to 42 degrees C. A plasmid-encoded enzyme, beta-lactamase (penicillinase, EC 3.5.2.6), undergoes a 30- to 40-fold increase in specific activity concomitant with the increase in plasmid copy number. The copts phenotype of these mutants is not due to the synthesis of a temperature-sensitive polypeptide. Both mutations are located in an untranslated region of the plasmid genome encoding two overlapping transcripts involved in plasmid replication: a small transcript known as RNA I that acts as a negative control element in replication and a large transcript that has been characterized as the replication primer in vitro. The mutations alter the sequence encoding the primer but lie immediately 5' to the initiating nucleotide of RNA I, in the RNA I promoter region. The possibility that the temperature-dependent plasmid DNA amplification is a consequence of a temperature-sensitive RNA I promoter was tested by inserting the RNA I promoters from the wild-type and mutant plasmids into a plasmid in which galactokinase expression is dependent upon an exogenous promoter. These experiments demonstrate that the mutant promoters are not temperature-sensitive. Rather, the mutations may affect the secondary structure of the replication primer in a region important for RNA I interaction.

Bacterial Proteins↗
PubMed1982

Control of ColE1 DNA replication: the rop gene product negatively affects transcription from the replication primer promoter.

A 600-base-pair region essential for ColE1 and pMBl plasmid replication contains two promoters responsible for the synthesis of two RNA molecules central to copy number control. One promoter directs synthesis of the primer RNA precursor. The second promoter directs the synthesis of a small RNA molecule, RNAl, which acts in trans to inhibit processing of the RNA primer precursor. We have fused each promoter to the beta-galactosidase structural gene contained in a lambda phage. Expression of the RNAl promoter in lysogens is not influenced by the presence of wild-type pMBl or ColEl plasmids residing in the cell. Transcription from the RNA primer promoter, however, is repressed by the product of a trans-acting plasmid gene product, which we have designated rop (for repressor of primer). The rop gene maps downstream from the replication origin in a region that encodes a polypeptide of 63 amino acids whose sequence is completely conserved in pMBl and ColE1. We propose that this polypeptide is the rop gene product and that it regulates plasmid DNA replication by modulating the initiation of transcription of the primer RNA precursor.

Amino Acid Sequence↗
PubMed1981

A single base-pair alteration is responsible for the DNA overproduction phenotype of a plasmid copy-number mutant.

The Cop- plasmid pOP1 delta 6 is a recessive copy-number mutant derived from Col E1.pOP1 delta 6 exists at 200-300 copies per chromosome in E. coli, while Col E1 exists at 10-15 copies per chromosome. We have investigated the molecular basis for DNA overproduction by pOP1 delta 6 by mapping the mutation to a restriction fragment of the plasmid genome, which is about 400 bp from the origin of replication. The mutation is a single base-pair alteration-a GC leads to TA transversion. The alteration changes the nucleotide sequence of two RNA elements known to be synthesized from opposite DNA strands in the same region of the plasmid genome; a small, nontranslated RNA known as RNA1 and the primer RNA required for initiation of DNA replication in vitro. The mutation is located in a GC-rich region of dyad symmetry, which precedes the termination signal for RNA1 transcription. When pOP1 delta 6 DNA is transcribed in vitro, RNA1 is not observed. Rather, several new transcripts of a larger size are observed resulting from readthrough transcription of RNA1. In conjunction with previous genetic evidence, these results indicate that RNA 1 may be a negative modulator of Col E1 DNA replication and that its secondary structure is critical to its function.

Base Composition↗
PubMed1978

Isolation and characterization of ColE1-derived plasmid copy-number mutant.

The plasmid pBGP120 is a ColE1 derivative that contains elements of the Escherichia coli lac operon and the Tn3 transposon. We have selected and isolated a copy-number mutant of pBGP120. In exponentially growing cultures, the copy-number mutant, pOP1, represents approximately 30% of total intracellular DNA compared to about 5% for pBGP120. Plasmid-encoded beta-galactosidase monomer can represent 50% of newly synthesized protein in cells carrying pOP1. pOP1 is structurally unstable in certain genetic backgrounds and under certain growth conditions, breaking down to a smaller sized plasmid that retains the DNA overproducer phenotype and the Tn3 transposon. The smaller overproducer plasmid, pOP1delta6, is generated by a continuous deletion of sequences located between one end of the Tn3 transposon and a site about 630 nucleotides from the EcoRI site in the beta-galactosidase structural gene of pOP1. pOP1delta6 retains the ColE1 origin of replication but has lost the lac promotor and operator and most of the beta-galactosidase structural gene. pOP1delta6 exists at approximately 210 copies per chromosome in exponentially growing cells.

Bacteriocin Plasmids↗
PubMed1978

Regulated expression by readthrough translation from a plasmid-encoded beta-galactosidase.

We have characterized expression of beta-galactosidase from a plasmid cloning vehicle, pBGP120, which carries most of the lacZ gene and contains a single EcoRI site near the end of lacZ. In addition, we have examined expression of heterologous DNA inserted at the position of the EcoRI site. The EcoRI site was shown to be within the sequence coding for beta-galactosidase and its precise location and phase were deduced. Insertion of heterologous EcoRI-generated DNA fragments altered the molecular weight of the plasmid-encoded beta-galactosidase polypeptide. Those insertions that were in the correct phase were expressed at a high level as a fused protein. The different forms of beta-galactosidase polypeptides produced by various hybrid plasmids were all stable proteins. The level of expression of the plasmid-encoded beta-galactosidase was several times higher than maximal expression of chromosome-encoded beta-galactosidase, suggesting that expression is proportional to gene copy number. The expression of the plasmid lacZ gene was controlled by cyclic AMP. When grown in a cya strain (DG74), expression was dependent on exogenous cyclic AMP. Although in normal strains there was insufficient lac repressor to inactivate all copies of the plasmid, repressor regulation was restored when the plasmid was grown in a strain (M96) that overproduces the lac repressor.

Cyclic AMP↗
PubMed1976

A plasmid cloning vehicle allowing regulated expression of eukaryotic DNA in bacteria.

We have constructed a plasmid cloning vehicle in which transcription of inserted heterologous DNA fragments can be regulated by a defined bacterial operator and promoter. The lambda plac 5 EcoRIDNA fragment containing the operator, promoter, and beta-galactosidase gene of the lactose operon was linked to the ColE1 derivative plasmid pSF2124, creating a plasmid designated pBGP100, pBGP100 contains one EcoRI site at the lac DNA/pSF2124 DNA junction and another at the lambda DAN/pSF2124 DNA junction. We deleted the latter EcoRI site to generate a plasmid (pBGP120) retaining a single EcoRI site at the lac DNA/nSF2124 DNA junction. To determine whether DNA introduced at the EcoRI site of pBGP120 was expressed under lactose control, we inserted the EcoRI fragment containing 28S ribosomal DNA of Xenopus laevis, creating the hybrid plasmid pBGP123. RNA-DNA hybridization of pulse-labeled RNA from cells containing pBGP123 showed that induction of the lac operon increases the percentage of labeled RNA complementary to Xenopus 28S DNA about 9-fold. This vehicle may be of use for production of eukaryotic gene products in bacteria.

Animals↗
PubMed1975

Location of histones on simian virus 40 DNA.

The physical location of histone molecules in a simian virus 40 DNA-histone complex isolated from purified virions was examined using site-specific restriction endonucleases. The complex contains four host histone species but lacks histone F1. Histones prevent complete cleavage of SV40 DNA by two restriction enzymes, HindIII and EcoRI. From the pattern of DNA fragments resulting from cleavage of the histone-DNA complex by the HindIII endonuclease, which makes six breaks on purified SV 40 DNA, we have concluded that histones are randomly arranged on SV40 DNA relative to restriction enzyme cleavage sites. The EcoRI endonuclease, which makes one break in SV40 NDA, was used to determine the degree of physical coverage of the SV 40 DNA molecule by histones. We observed that 80% of the EcoRI sites in the complex are accessible to the enzyme while 20% are "closed." This degree of coverage is consistent with the mass ratio of DNA:histone in the complex as revealed by the buoyant density of the formaldehyde-fixed complex. We conclude that the histones in the complex are located randomly on the SV 40 genome and cover approximatley 20% of the DNA. These results suggest that the histone species F2b, F2al, F2a2, and F3 are bound without regard to nucleotide sequence of SV 40 DNA.

Binding Sites↗
PubMed1975

Specificity of substrate recognition by the EcoRI restriction endonuclease.

The substrate specificity of the EcoRI restriction endonuclease can be varied in vitro by changing the pH and the ionic environment of the reaction. Phosphodiester bond cleavage occurs at a DNA hexanucleotide sequence d(N-G-A-A-T-T-C-N)/d(N-C-T-T-A-A-G-N) when the ionic strength is high, 100 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, and the pH is approximately 7.3. Lowering the ionic strength to 25 mM Tris-HCl, 2 mM MgCl2, and adjusting the pH to 8.5 reduces the recognition specificity of the EcoRI endonuclease to the tetranucleotide sequence, d(N-A-A-T-T-N)/d(N-T-T-A-A-N). The enzymatic activity responsible for this substrate recognition is referred to as EcoRI. Cleavage of pVH51 plasmid DNA under EcoRI conditions results in a number of partial digest fragments, some of which disappear slowly over a prolonged digestion period. This suggests that different recognition sites are cleaved at different rates. Comparison of DNA fragment patterns of modified and unmodified pVH51 DNA indicates that the canonical EcoRI sequence is the most rapidly cleaved site under EcoRI conditions. DNA modified in vivo by the EcoRI methylase is not cleaved by the EcoRI endonuclease under standard conditions, but is cleaved under EcoRI conditions at sites other than the standard EcoRI substrate.

Base Sequence↗