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D G Scraba

Publications and source records attributed to D G Scraba.

At least 55 records · Page 3Linked to original sources
PubMed1983

Studies of the interaction of RecA protein with DNA.

Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.

DNA Topoisomerases, Type I↗
PubMed1983

Bacteriophage phi W-14: the contribution of covalently bound putrescine to DNA packing in the phage head.

Bacteriophage phi W-14 is unusual because its DNA contains 12 mol% of the hypermodified pyrimidine, alpha-putrescinylthymine. The phi W-14 virion is similar in morphology to T4, except that the phi W-14 head is isometric rather than prolate, there is no collar-whisker structure associated with the neck, the tail fibers are short (approximately 15 nm), and the base plate terminates in small plates or knobs rather than spikes. The contractile tail sheath of phi W-14 appears to have a right-handed helical arrangement of subunits with a pitch in the extended form of approximately 20 nm. The "stacked disk" appearance of the tail sheath visible on negatively stained particles has a periodicity of 3-4 nm. The protein shell of the head has a similar thickness (2-3 nm) to that of T4. The phi W-14 virion contains at least 17 different polypeptide species. Based on measurements from electron micrographs of negatively stained phage particles on the same grid square, the volume of the phi W-14 head was estimated to be approximately 72% that of the T4 head. Surprisingly, however, the lengths of the DNA molecules released from phi W-14 and T4 heads by osmotic shock were 59.6 +/- 1.9 and 62.1 +/- 2.4 microns, respectively. am42 is an amber mutant of phi W-14 in which there is only 5 mol% putThy in the DNA made in the nonpermissive host. am42 virions are morphologically normal, but the length of the DNA released from these virions is only 53.1 +/- 3.1 microns. We conclude that phi W-14 DNA is packed much more compactly than T4 DNA into a virion of similar morphology and comparable complexity and that the tight packing is a consequence of, and dependent upon, the presence of putThy in phi W-14 DNA.

Bacteriophages↗
PubMed1983

Structure of fumarate reductase on the cytoplasmic membrane of Escherichia coli.

The terminal electron transfer enzyme fumarate reductase has been shown to be composed of a membrane-extrinsic catalytic dimer of 69- and 27-kilodalton (kd) subunits and a membrane-intrinsic anchor portion of 15- and 13-kd subunits. We prepared inverted membrane vesicles from a strain carrying the frd operon on a multicopy plasmid. When grown anaerobically on fumarate-containing medium, the membranes of this strain are highly enriched in fumarate reductase. When negatively stained preparations of these vesicles were examined with an electron microscope, they appeared to be covered with knob-like structures about 4 nm in diameter attached to the membrane by short stalks. Treatment of the membranes with chymotrypsin destroyed the 69-kd subunit, leaving the 27-, 15-, and 13-kd subunits bound to the membrane; these membranes appeared to retain remnants of the structure. Treatment of the membranes with 6 M urea removed the 69- and 27-kd subunits, leaving the anchor polypeptides intact. These vesicles appeared smooth and structureless. A functional four-subunit enzyme and the knob-like structure could be reconstituted by the addition of soluble catalytic subunits to the urea-stripped membranes. In addition to the vesicular structures, we observed unusual tubular structures which were covered with a helical array of fumarate reductase knobs.

Cell Membrane↗
PubMed1983

Formation and possible functions of alpha-putrescinylthymine in bacteriophage phi W-14 DNA: analysis of bacteriophage mutants with decreased levels of alpha-putrescinylthymine in their DNAs.

The DNA synthesized in the nonpermissive host by the noncomplementing mutants am36 and am42 of bacteriophage phi W-14 contains about half the wild-type level of alpha-putrescinylthymine (putThy) and a correspondingly greater level of thymine. The mechanisms whereby thymine nucleotides are excluded from replicating DNA are functional in both mutants because neither of them incorporates exogenous thymidine into DNA. It is proposed that (i) in wild-type phi W-14, the conversion of hydroxymethyluracil to putThy at the polynucleotide level is sequence specific, but that to thymine is nonspecific; and (ii) in the mutants, the sequence-specific recognition is impaired so that more thymine and less putThy are formed. The thymine-rich DNA can be packaged into phage particles. In the case of am42, the phage particles are morphologically indistinguishable from and have essentially the same polypeptide composition as wild-type particles. However, the DNA molecules they contain are about 11% shorter than those in wild-type phage, am42rev4, a revertant of am42, contains DNA with about 70% of the normal level of putThy; these molecules are about 3% shorter than wild-type DNA. The properties of am42 and am42rev4 are consistent with the suggestion that putThy facilitates the very tight packing of phi W-14 DNA (Scraba et al., Virology 124:152-160, 1983). It also appears that the putThy content of phi W-14 DNA can be reduced by no more than 30% without adversely affecting the production of viable progeny; for example, the burst size of am42rev4 is about 25% of that of the wild type.

Bacteriophages↗
PubMed1982

Formation of 9-nm filaments from pilin monomers obtained by octyl-glucoside dissociation of Pseudomonas aeruginosa pili.

Pili isolated from Pseudomonas aeruginosa PAK were solubilized in the detergent octyl-glucoside. Subsequent removal of the detergent by dialysis resulted in the formation of short rods of about 9 nm in diameter and various lengths. The aggregation process was followed by ultracentrifugation, viscometry, and electron microscopy to show that the aggregates produced in this way are distinct from pili produced by the bacteria, both in diameter, as measured by electron micrographs, and in their inability to compete in a biological assay for pili.

Adsorption↗
PubMed1981

Heteroduplex analysis of tRNAVal3b genes from the 90BC and 84D sites of Drosophila melanogaster.

Two recombinant DNA plasmids coding for Drosophila tRNAVal3b hybridize to two different loci on the drosophila genome. Plasmid pDt41R hydridizines at the 90BC site, and pDt78R hybridizes at the 84D site. Heteroduplexes of the two plasmids were examined by electron microscopy. The study shows that the Drosophila segments have significant homology only over the length of the tRNAVal3b genes.

Animals↗
PubMed1979

The isolation of Mengo virus stable non-capsid polypeptides from infected L cells and preliminary characterization of an RNA polymerase activity associated with polypeptide E.

Isolation of the Mengo virus stable non-capsid virus polypeptides E, F, G and I from infected L cells has been achieved. Unstable precursors were eliminated by incubation in the presence of pactamycin and capsid polypeptides were removed by ultracentrifugation and affinity chromatography. Subsequent sodium dodecyl sulphate (SDS)-hydroxylapatite chromatography resolved the non-capsid proteins into two major peaks which comprised F plus G and E plus I, respectively. The individual polypeptide species were separated by gel filtration on Sephadex G-100 in the presence of SDS. Polypeptide E was isolated in an undenatured form by gel filtration of infected cell extracts (from which precursor and capsid polypeptides had been removed) on Bio-Gel A-5m agarose beads. Purified polypeptide E was found to co-sediment with Mengo virion RNA during centrifugation in a sucrose density gradient and it was also capable of binding to poly(A)-Sepharose. Assay mixtures containing polypeptide E exhibited an RNA polymerase activity which was dependent upon exogenous virus RNA template and oligo(U) primer and which was not affected by the addition of virus capsid polypeptides or extracts from uninfected cells.

Chromatography↗
PubMed1979

Electron microscopic analysis of partially replicated bacteriophage T7 DNA.

Partially replicated bacteriophage T7 DNA was isolated from Escherichia coli infected with UV-irradiated T7 bacteriophage and was analyzed by electron microscopy. The analysis determined the distribution of eye forms and forks in the partially replicated molecules. Eye forms and forks in unit length molecules were aligned with respect to the left end of the T7 genome, and segments were scored for replication in each molecule. The resulting histogram showed that only the left 25 to 30% of the molecules was replicated. Several different origins of DNA replication were used to initiate replication in the UV-irradiated experiments in which 32P-labeled progeny DNA from UV-irradiated phage was annealed with ordered restriction fragments of T7 DNA (K. B. Burck and R. C. Miller, Jr., Proc. Natl. Acad. Sci. U.S.A. 75:6144--6148, 1978). Both analyses support partial-replica hypotheses (N. A. Barricelli and A. H. Doermann, Virology 13:460--476, 1961; Doermann et al., J. Cell. comp. Physiol. 45[Suppl.]:51--74, 1955) as an explanation for the distribution of marker rescue frequencies during cross-reactivation; i.e., replication proceeds in a bidirectional manner from an origin to a site of UV damage, and those regions of the genome which replicate most efficiently are rescued most efficiently by a coinfecting phage. In addition, photoreactivation studies support the hypothesis that thymine dimers are the major UV damage blocking cross-reactivation in the right end of the T7 genome.

DNA Replication↗