PubMed Health⌕ Search

Biomedical subjects

A Shafferman

Publications and source records attributed to A Shafferman.

At least 91 records · Page 5Linked to original sources
PubMed1984

Mutations not altering the symmetrical sequences in the trp operator yield a constitutive phenotype.

An E. coli trp promoter operator mutant was constructed, having two base pair alterations at position -4 and -1 relative to the transcription initiation site (+1). Expression of chloramphenicol acetyltransferase gene under this trp promoter operator suggests that it is almost fully constitutive. This trp Oc in vitro derived mutant differs from previously isolated Oc mutants in that its twofold symmetry sequence is identical to that of the wild type trp operator. The base substitution in the operator does not affect the functionality of the trp promoter. The trp Oc promoter DNA fragment is engineered so that it can be manipulated conveniently for efficient expression of various genes in E. coli.

Acetyltransferases↗
PubMed1983

Structural properties of the beta origin of replication of plasmid R6K.

The beta origin of replication of plasmid R6K, one of three active R6K origins of replication, requires most or all of a 1962-base pair (bp) sequence for activity. The nucleotide sequence of a portion of this functional beta origin was determined in an earlier study (Stalker, D., Kolter, R., and Helinski, D. (1982) J. Mol. Biol. 161, 33-43). In this work, the sequence of the remaining portion of this 1964-bp segment was obtained. In addition to its activity as an origin of replication, this sequence also contains sufficient information for autonomous replication in Escherichia coli. A 277-bp region containing seven 22-bp direct repeats is present at one end of the beta origin segment (Stalker, D., Kolter, R., and Helinski, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1150-1154) while the other end contains a 140-bp sequence that includes a relaxation complex site. The 277-bp direct repeat region is required for activity of the beta origin. The start of the beta origin of replication as mapped by electron microscopy (Crosa, J. (1980) J. Biol. Chem. 255, 11075-11077) lies approximately 1000 bp away from the 277-bp region. The pi structural gene, which makes up most of the sequence between the direct repeats and the beta origin, is required in cis for beta origin activity. The pi protein also is required for beta origin activity but can be provided in trans. The nucleotide sequence just beyond the pi structural gene and within or near the start of beta origin of replication contains an open reading frame for a 151-amino acid protein. Deletions ranging from 94 bp to 1590 bp were obtained within the 1964-bp beta origin region. In every case, the deletion results in loss of origin activity even when the deleted sequence plus adjacent regions are provided in trans. These observations suggest a requirement for a specific secondary structure over an extensive region for beta origin activity.

Base Composition↗
PubMed1983

Genetic instability of an artificial palindrom DNA sequence.

A short DNA palindrom, produced by head to head ligation of a 29 bp DNA fragment, was inserted into a 27,000 bp plasmid DNA element composed of two functional replicons (R6K, ColE1). Several plasmid types containing a single copy of this palindrom in different locations of insertion on the R6K sequence were obtained. The palindrom was engineered to possess a unique EcoRI recognition sequence at its axis of symmetry. The presence of this restriction site allowed to monitor the genetic stability of the artificial palindrom at their different insertion loci. Out of 5 different insertion locations, one (in pAS807) was found to lead to a significant destabilization of the palindrom. This insertion site lies within the replication control region of R6K. We have shown that the inserted palindrom in pAS8O7 does not affect the functionality of the R6K replication origins. Excission of the palindrom sequences from pAS8O7 was not accompanied by loss of the adjacent R6K DNA sequences. Different deletion derivatives of pAS807 were generated in-vitro in order to determine the driving unit of DNA sequences around the palindrom that are involved in its excision. The results imply that large DNA structure(s) around the palindrom are involved in its excission. Complete deletion of R6K sequences from either the left or the right side of the palindrom resulted in new configurations which stabilized the palindrom. A configuration of R6K DNA sequences exceeding 270 bp long sequence from both sides of the palindrom are necessary for the transition from a palindrom stable to palindrom unstable state. In addition evidence is presented to show that the excision process of palindrom sequences requires a functional polymerase I but not the gene product of recA.

Base Sequence↗
PubMed1981

Conditional expression of the vesicular stomatitis virus glycoprotein gene in Escherichia coli.

Bacterial plasmids that directed expression of the vesicular stomatitis virus glycoprotein (G-protein) gene under control of the tryptophan operon regulatory region were constructed. A plasmid directing the synthesis of a G-protein-like protein (containing the NH2-terminal segment of seven amino acids encoded by the trpE gene fused to the complete G-protein sequence lacking only its NH2-terminal methionine) could be transformed into trpR+ (repressed) but not into trpR- (derepressed) cells. This result suggested initially that derepressed synthesis of the G-protein-like protein encoded by this plasmid was lethal in Escherichia coli. Deletion of the sequence encoding the large hydrophobic segment near the COOH terminus of G-protein did not overcome this lethality. Lethality of derepressed synthesis was overcome by deletion of the G-protein gene region encoding 10 amino acids in the hydrophobic NH2-terminal domain (signal peptide). Tryptic peptide mapping demonstrated that the G-protein-like protein and some truncated proteins encoded by the plasmid contained G-protein protein sequences. Antisera to vesicular stomatitis virus precipitated the G-protein-like protein, showing that it shares antigenic determinants with the authentic G-protein protein.

Base Sequence↗
PubMed1979

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c.

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH3CH(OH), CO2.-, O2.-, and e-aq' and the oxidation of the reduced cytochrome c derivatives by Fe(CN)3-6 were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.

Acetylation↗
PubMed1979

Intramolecular electron transfer and binding constants in iron hexacyanide-cytochrome c complexes as studied by pulse radiolysis.

Internal oxidation and reduction rates of horse cytochrome c in the complexes CII . Fe(III)(CN)6(3)- and CIII . Fe(II)(CN)6(4)-, are 4.6 . 10(4)s-1 and 3.3 . 10(2)s-1, respectively. The binding site of the iron hexacyanide ions on either CII or CIII are kinetically almost indistinguishable; binding constants range from 0.87 . 10(3) to 2 . 10(3)M-1. The present pulse radiolytic kinetic data is compared with that from NMR, T-jump and equilibrium dialysis studies.

Animals↗
PubMed1979

ColE1 DNA sequences interacting in cis, essential for mitomycin-C induced lethality.

Protein synthesis and survival of colicinogenic bacteria carrying different ColE1 deletion and insertion mutants, were followed in presence or absence of the inducing agent mitomycin-C. It is concluded that active colicin is not involved in the process leading to death of the induced colicinogenic cell. The region of ColE1, essential for cell induced lethality, is carried on the DNA between map positions 0.74 to 0.27. In this region the DNA sequences carried between 0.74 to 0.79 and 0.0 to 0.27 are essential, while those located between 0.79 to 0.0 are nonessential for commitment to death. A cis interaction of the ColE1 DNA sequences in the essential regions is probably necessary for cell induced lethality. Some possibilities for such a cis interaction are suggested and discussed.

Bacterial Proteins↗
PubMed1978

A DNA segment within the colicin E1 structural gene on ColE1 affecting immunity to colicin.

Insertion of DNA at the EcoRI site of ColE1 results in increase of immunity to colicin killing in E. coli harboring such recombinant ColE1 plasmid as compared to E. coli (ColE1). This effect is neither due to cis or trans interactions originating from the inserted foreign DNA fragment, nor to changes in plasmid copy number. This defect in the immunity mechanism is not trans complemented for by wild type ColE1. Increase in immunity can also be obtained by deleting a DNA segment from the ColE1 genome. This segment is approximately 120 bp left to the EcoRI site within the colicin structural gene. It is concluded that the structure of DNA per se, around the EcoRI site, within colcin structural gene, is the structure which affects immunity expression.

Colicins↗
PubMed1978

Cleavage maps of a tetracycline plasmid from Staphylococcus aureus.

The cleavage maps of a Staphylococcus aureus plasmid, pSN1 (2.75 megadaltons), conferring tetracycline resistance, were determined. Cleavage maps are given for HpaI and HindIII restriction endonucleases by using the single HpaII site as a reference point. Nucleases EcoRI, BamHI, SalI, and HaeIII have no sites on this plasmid.

DNA Restriction Enzymes↗