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D Vapnek

Publications and source records attributed to D Vapnek.

At least 19 recordsLinked to original sources
PubMed1983

Chimeric plasmid that replicates autonomously in both Escherichia coli and Neurospora crassa.

A hybrid pBR322 plasmid (designated pDV1001) containing two functional Escherichia coli antibiotic resistance genes (kanr and camr) and a qa-2+ gene from Neurospora crassa transforms N. crassa qa-2- mutants to qa-2+ with a frequency of ca. 5 X 10(-5) per regenerated spheroplast (ca. 100 transformants per microgram of plasmid DNA). This plasmid can replicate autonomously without integrating into the N. crassa genome. The autonomously replicating hybrid plasmid was detected in N. crassa transformants by Southern gel hybridizations. DNA from these transformants can be recovered by retransformation back into E. coli aroD recipients and selection for chloramphenicol resistance. These E. coli transformants complement an aroD mutant. The hybrid plasmid DNA present in the E. coli transformants remains unchanged on the basis of DNA restriction enzyme analyses. The original, nonhomokaryotic N. crassa transformants can be maintained on a selective medium, but there is as yet no evidence that the self-replicating plasmid can be transmitted through meiosis. In addition, the self-replicating plasmid often integrates into the N. crassa genome and then is inherited in a generally stable fashion through meiosis. Our findings suggest that this plasmid, or some derivative of it, will prove useful as a routine shuttle vector for cloning genes in N. crassa.

Cloning, Molecular↗
PubMed1982

Cloning vectors derived from bacterial plasmids.

A wide variety of plasmid cloning vectors, most of which utilize the basic Co1E1I replicon have been constructed. Utilizing these vectors, in conjunction with the newly developed techniques of gene isolation and oligonucleotide synthesis, essentially any gene which can be identified can be cloned. We anticipate that future work in this area will be directed at improving techniques for the regulated expression of cloned genes and the further development of plasmid replicons in which the copy number can be readily controlled.

Bacteriophage lambda↗
PubMed1982

5'-Untranslated sequences of two structural genes in the qa gene cluster of Neurospora crassa.

The coding regions of two genes (qa-2 and qa-3) in the qa gene cluster of Neurospora crassa have been localized by nucleotide sequence analysis combined with data on previously determined NH2-terminal amino acid sequences for the proteins that these genes encode. The start point of transcription for each of these genes has been determined by nuclease S1 mapping experiments with poly(A)+RNA isolated from quinic acid-induced cultures of N. crassa. The sequences of approximately 200 nucleotides 5' to the start point of transcription have been compared with each other and with those of other eukaryotes. The results show that neither of these regions for the qa-2 nor the qa-3 genes share any significant homology with sequences apparently conserved in higher eukaryotic promoters (-25 and -70 regions). However, the qa-2 and qa-3 sequences do show homology with each other in these regions. Comparison of the 5'-flanking regions of these Neurospora genes with those of several Saccharomyces cerevisiae genes reveals a number of similarities in the region preceding the translation initiation codons.

Base Sequence↗
PubMed1982

Mutants of plasmid prophage P1 and elevated copy number: isolation and characterization.

A series of mutations of the P1 plasmid prophage that lead to increased copy number was isolated and analyzed. The copy number of the mutants was elevated at least five- to eightfold relative to wild-type P1, as determined by single-cell resistance to antibiotics, activity of enzymes, content of superhelical DNA, and reassociation kinetics. The copy number of two of the mutants was temperature dependent. Based on dominance tests, the mutants fell into two classes, cis specific and recessive. The latter class included a temperature-sensitive copy mutant. The existence of a class of recessive mutants suggests that the replication of the P1 plasmid is negatively regulated.

Ampicillin↗
PubMed1981

Versatile cloning vectors derived from the runaway-replication plasmid pKN402.

Two cloning vectors have been constructed employing runaway-replication mutants of plasmid R1. One of these, pMOB45, carries tetracycline and chloramphenicol resistance. The other, pMOB48, carries chloramphenicol resistance, lacOP, and an assayable part of the lacPOZ operon. Both of these plasmids can be amplified to high levels by heat induction, which condition does not lead to inhibition of protein synthesis; thus the plasmid can produce large amounts of DNA and protein. In pMOB48, a unique BamHI site is present near the amino-terminus of the beta-galactosidase gene. Chimeras formed by the insertion of restriction fragments at this site can be detected on X-gal plates, and can be used for the lacIq-controlled expression of proteins which are fused to the amino-terminus of beta-galactosidase. Induction with IPTG at 40 degrees C leads to the synthesis of extremely high levels of proteins whose gene have been cloned into this site.

Chloramphenicol↗
PubMed1979

In vivo transcription and translation of R-plasmid 538-1 DNA in Escherichia coli.

In vivo transcription and translation of R-plasmid 538-1 in E. coli was analyzed. Transcription of individual restriction fragments was determined qualitatively by utilizing the techniques developed by Southern (27), and quantitatively by carrying out DNA-RNA filter hybridization. The most active region of R-plasmid transcription in strains repressed for conjugal transfer was found to occur in the region of the R-plasmid carrying the antibiotic resistance genes. In plasmids derepressed for conjugal transfer, a high level of transcription from the transfer gene region was also observed. When strains carrying R538-1drd were induced with Hg++ a high level of transcription was observed from the region of the R-plasmid carrying the genes for Hgr. Hybrid ColE1 plasmids carrying restriction fragments from the antibiotic resistance region of R538-1 were segregated into minicells. Labeling of the minicells with 35S-methionine allowed identification of the proteins coded by the fragments. A limited number of proteins were detected, and several of these have been correlated with the antibiotic resistance genes carried by R538-1.

Bacterial Proteins↗
PubMed1979

Recombination between the plasmid prophages P1 and P7 and the E. coli chromosome.

The prophages of the related temperate phages P1 and P7, which normally exist as plasmid DNA, suppress E. coli dnaA(Ts) by integrating into the host chromosome. Integratively suppressed strains may either be capable of producing phage or may have prophage deletions. In strains containing non-defective prophages, the location of the site on the prophage used for integrative recombination was identified by use of restriction analysis and DNA-DNA hybridization techniques. At least seven different integration sites were found on the prophage; the site used most often may be at the 'end' of the genetic map generated by vegetative phage crosses. For suppression of P1 and P7, the sites on the host chromosome utilized for prophage integration are not distributed randomly.

Chromosomes, Bacterial↗
PubMed1978

Transcription and translation in E. coli of hybrid plasmids containing the catabolic dehydroquinase gene from Neurospora crassa.

Two hybrid plasmids which carry the gene for Neurospora crassa catabolic dehydroquinase (C-DHQase) and complement an aroD6 (dehydroquinase-deficient) auxotroph of Escherichia coli have been analyzed. One of these contains a 2.9 kilobase (kb) fragment cloned in the HindIII site of plasmid pBR322 (pVK57) and the other contains a 6.8 kb fragment cloned in the PstI site (pVK88). Restriction enzyme mapping of these plasmids has demonstrated that the 2.9 kb fragment is totally contained within the 6.8 kb fragment. When the polarity of either the HindIII fragment or PstI fragment was reversed with respect to pBR322 no effect was observed on either the ability of the hybrid to complement an aroD- auxotroph or on the level of C-DHQase activity. In vivo transcription of plasmid pVK88 in both orientations was analyzed by RNA-DNA hybridization and by the techniques developed by Southern (1975). Approx. 40% of the plasmid-directed transcription occurred from the cloned PstI fragment and 60--70% of these N. crassa transcripts were encoded by the 2.9 kb HindIII fragment. The Southern technique allowed a further localization of the region of most extensive transcription to a 1.8 kb HindIII-EcoRI fragment. Biochemical analysis revealed that the C-DHQase protein produced by strains harboring pVK57 and pVK88 in either orientation was identical to the N. crassa enzyme. Furthermore, when these plasmids were segregated into minicells and labeled with 14C amino acids, the C-DHQase protein was synthesized at a level comparable to other plasmid-encoded proteins. Taken together, these experiments demonstrate that transcription is efficiently initiated in E. coli from a site on the cloned N. crassa DNA and that the resulting C-DHQase mRNA is efficiently and accurately translated.

DNA Restriction Enzymes↗