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L Mindich

Publications and source records attributed to L Mindich.

At least 55 records · Page 3Linked to original sources
PubMed1985

cDNA cloning of portions of the bacteriophage phi 6 genome.

Phage phi 6 has a genome consisting of three pieces of double-stranded RNA. Single-stranded RNA was prepared from phi 6 nucleocapsids by in vitro transcription with the phage RNA polymerase. These transcripts were polyadenylated and used as templates for the preparation of cDNA copies. The resulting DNA was cloned into the PstI restriction nuclease site of plasmid pBR322. Insert-bearing plasmids were annealed to phi 6 RNA to assign the inserts to their proper segments. In this way we identified inserts corresponding to the large, medium, and small segments. Two large overlapping inserts of the small segment constitute the complete complement of the segment as determined by the sequence analysis of the DNA. In vitro coupled transcription and translation showed that the small segment inserts were able to direct the synthesis of the four known genes in the small segment. Two overlapping inserts in the medium segment constitute the entire segment and were shown to direct the in vitro synthesis of two of the three known proteins of the medium segment. Several inserts bearing about one-third the complement of the large segment were also isolated, and one of these directed the synthesis of a peptide that resembles protein P1. Restriction endonuclease maps were prepared for the inserts, and by in vitro synthesis it was possible to refine the genetic map of phi 6. A chimeric plasmid was constructed that combines plasmids pUC8 and RSF1010. Inserts placed on this plasmid were transformed to Pseudomonas phaseolicola, the natural host of phage phi 6. It was possible to refine further the genetic map by complementation of nonsense mutants of phi 6 with the cDNA.

Acetyltransferases↗
PubMed1984

Characterization of the DNA-protein complex at the termini of the bacteriophage PRD1 genome.

DNA of bacteriophage PRD1 has protein P8 at its termini. Extracts of infected cells are able to derivatize P8 in vitro with labeled dGTP. Two early proteins, P1 and P8, products of genes I and VIII, respectively, are the only phage proteins necessary for the formation of the protein P8-dGMP complex. This was shown by complementation of extracts from cells infected with mutants and by use of extracts from cells carrying cloned genes I and VIII. With Escherichia coli mutants that are temperature sensitive for DNA synthesis, it was possible to show that the formation of the protein P8-dGMP complex was dependent upon the host replication apparatus. The analysis of the purified protein P8-dGMP complex by hydrolysis and enzymatic digestion showed that there is a covalent phosphodiester bond between tyrosine and 5'-dGMP.

Bacteria↗
PubMed1983

Molecular cloning of bacteriophage PRD1 genomic fragments.

DNA from bacteriophage PRD1 was extracted and partially digested with restriction endonuclease HaeII. The digest was cloned into the PstI site of plasmid pBR322 by homopolymer tailing with guanidylate tails on the plasmid and cytidylate tails on the phage DNA. Insert bearing plasmids were isolated by transforming E. coli strains for tetracycline resistance and screening for ampicillin sensitivity. These strains were then screened for the ability to accomplish marker rescue of nonsense mutants of bacteriophage PRD1. Additional clones were isolated by screening transformants with radioactively labeled probe PRD1 DNA fragments using colony hybridization. A genetic map was generated by the marker rescue capabilities of overlapping cloned inserts. This map allowed the ordering of fourteen of the known PRD1 complementation groups.

Bacteriophages↗
PubMed1983

Establishment of a physical and genetic map for bacteriophage PRD1.

DNA was isolated from the lipid-containing bacteriophage PRD1 and subjected to restriction endonuclease analysis. The total genome size is 14.7 kb. PRD1 DNA was resistant to cutting by fifteen restriction endonucleases with six base specificity. HaeII made thirty-seven cuts in the DNA, MboI made one cut, and MnlI made six cuts. DNA that was not treated with protease yielded two fewer fragments when treated with HaeII. Evidence is presented to indicate that the PRD1 DNA has protein at the ends of the DNA. The thirty-eight HaeII fragments were ordered using the ladder technique of Smith and Birnstiel (1976) on MboI and MnlI fragments of the genome. Clones of HaeII partial digests of PRD1 DNA in pBR322 were analyzed by HaeII digestion and were then assigned to specific regions of the genome by their HaeII fragment composition. A comparison of the marker rescue characteristics of the cloned DNA with the overall restriction fragment map generated a physical map of the genome. Some genes that have not been mapped because of a lack of mutants or leakiness at restrictive conditions were mapped by studying the in vitro protein synthesis of restriction endonuclease fragments.

Bacteriophages↗
PubMed1983

Characterization of phi 6 mutants that are temperature sensitive in the morphogenetic protein P12.

P12 is a morphogenetic protein necessary for the envelopment of the bacteriophage phi 6 nucleocapsid with the viral membrane. Gene 12 is located along with three other genes on the smallest chromosome of the virion. ts mutants in P12 were obtained by first characterizing the isoelectric focusing behavior of phi 6 proteins and then screening ts mutants of phi 6 that had previously been assigned to chromosome C for changes in the behavior of P12. In this manner, three independently isolated mutants were identified and were found to have morphogenetic consequences at restrictive temperatures similar to gene 12 nonsense mutants in nonsuppressor cells in that only unenveloped nucleocapsids were formed. When infected cells were labeled at restrictive temperature, 27 degrees, and then shifted to 21 degrees, normal phage particles were formed; however, the hydrophobic membrane proteins in the particles were not labeled, indicating that functional P12 must be present at the time of synthesis of the membrane proteins for them to assemble into virions or that the defective P12 leads the membrane proteins into a nonfunctional pathway.

Capsid↗
PubMed1983

Morphogenesis of bacteriophage phi 6: a presumptive viral membrane precursor.

Bacteriophage phi 6 has a lipid- and protein-containing membrane as its outer covering. Two phi 6-coded proteins are known to be required to produce enveloped phage particles: one is P9, the major phi 6 membrane structural protein, and the other is P12, a nonstructural protein without which membrane fails to assemble around phage nucleocapsids. A particle containing phospholipid, P9, and two minor phi 6 membrane proteins has been found in cells pulse labeled with protein precursors at late times after infection. The P9 particle can be chased into phage and is dependent on active P12 for its formation. Models are presented in which the role of the P9 particle in phi 6 membrane assembly is discussed.

Models, Biological↗
PubMed1983

Identification of a protein bound to the termini of bacteriophage PRD1 DNA.

Lipid-containing bacteriophage PRD1 has a double-stranded DNA genome of about 14,500 nucleotide base pairs. The phage can infect Escherichia coli and Salmonella typhimurium as well as other gram-negative bacteria harboring an appropriate plasmid. [35S]methionine label is incorporated into the DNA band early in infection. The label remains associated with DNA through phenol extraction and boiling with sodium dodecyl sulfate. Nuclease treatment of the genome released a protein which migrated as an early phage-specific protein (P8). This protein is also necessary for phage DNA replication. By restriction enzyme analysis it was shown that protein was associated with the terminal restriction fragments. Extracts of infected cells catalyzed the labeling of protein P8 with [alpha-32P]dGTP.

Bacteriophages↗
PubMed1982

Isolation of nonsense mutants of lipid-containing bacteriophage PRD1.

We isolated nonsense mutants of bacteriophage PRD1, a lipid-containing polyhedral virus capable of infecting many genera of gram-negative bacteria. These mutants were grouped into 19 classes on the basis of genetic complementation and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. PRD1 infection led to the synthesis of at least 25 viral proteins, 17 of which were components of mature virions. The synthesis of proteins fell into the following three classes: very early, middle early, and late. Two of the very early proteins, P1 and P8, had an effect on DNA synthesis, host protein synthesis shutoff, and the turning on of middle and late protein synthesis. Another very early protein, P12, was involved in the shutoff of early protein synthesis. Two genes were identified as affecting lysis of the host. One appeared to be a lysin, whereas the other was an accessory lytic factor.

Genetic Complementation Test↗
PubMed1982

Assembly of bacteriophage PRD1: particle formation with wild-type and mutant viruses.

Bacteriophage PRD1 contains DNA, 17 proteins, and lipid. The assembly pathway involves the formation of empty particles that contain lipid and all of the proteins of mature virions, with the possible exception of one. The major and minor capsid proteins, P3 and P5, occur as soluble multimers before they appear in the empty particles. Nonsense mutants of PRD1 that involve structural proteins of the virion other than P3 form particles that are missing only the defective protein. Those mutants that are unable to form P3 do not form particles. Mutations in two other genes that code for nonstructural proteins (P10, which is membrane bound, and P17, which is soluble) result in the absence of particles. Protein P2 is necessary for adsorption to host cells. Protein P9 is necessary for particle filling with DNA, whereas P20 and P22 are necessary for stable DNA packaging. Electron micrographs of infected cells confirmed the gradient analysis of particle formation. No free vesicles were observed in mutants that could not form complete empty particles, indicating that there are no free intermediate particles before the empty virions.

Bacteriophages↗
PubMed1982

Structure of the lipid-containing bacteriophage PRD1: disruption of wild-type and nonsense mutant phage particles with guanidine hydrochloride.

The lipid-containing bacteriophage PRD1 was disrupted, and the subviral particles were studied. Guanidine treatment released two phage proteins (P3 and P5). These proteins form the polyhedral capsid. The remaining phage proteins were associated with the phage membrane vesicle. The vesicle was capable of forming a tubular structure. The isolated phage membrane vesicles aggregated readily. We found that aggregation and tube formation were associated with specific phage proteins (P11 and P18, respectively) by using protease treatment and an analysis of nonsense mutant phage particles. In addition, the possibility that free vesicles might be precursors to empty virions was studied.

Bacteriophages↗
PubMed1979

Cell wall lysin as a component of the bacteriophage phi 6 virion.

Cell wall lytic activity was found in particles of the lipid-containing bacteriophage ø6. The activity can be extracted from the virion with Triton X-100 in the presence of salt. This treatment removes the membrane-like envelope of the virion which includes five proteins. The lysin requires detergent for in vitro activity. Virus particles formed in nonsuppressor cells by several classes of ø6 nonsense mutants contained the lysin activity; however, particles formed by a mutant (unable to make proteins P5 and P11) had very low activity; high activity was produced when particles were formed in a suppressor host. A study of the time course of the appearance of the lysin during infection showed that it appeared and increased in cells infected with wild-type virus and in suppressor cells infected with a mutant of class 511, but it did not increase in nonsuppressor cells infected with the class 511 mutant. It is concluded that protein P5 is a component of the lysin and that the role of its activity is in both early and late stages of infection. In particular, the lysin may be necessary for the passage of the infecting core of the virion through the cell wall of the bacterium, as well as in the final lysis necessary for the liberation of progeny phage. A mutant of the virus that produces a larger-than-normal protein P10 does not induce normal lysin activity in host Pseudomonas phaseolicola HB10Y, although it does in strain ERA Pseudomonas pseudoalcaligenes. This indicates that protein P5 is probably not sufficient for lysin activity, but the nature of the interaction between P5 and P10 is unknown.

Bacterial Proteins↗
PubMed1978

A mutation that increases the activity of nonsense suppressors in Escherichia coli.

We have isolated a new mutation, ups, that amplifies the suppressor activity of all the nonsense suppressors we have tested so far at low but not at high temperature. The properties of ups make it a very useful tool to improve the systems of temperature sensitive suppressors thus far described. ups maps between 25 to 27 min on the E. coli genetic map (Bachmann et al., 1976) and has no suppressor activity of its own. Its effects on translational fidelity are not influenced by mutations for ribosomal drug resistance. Thus, ups is different from ram which exhibits cooperative control of translation with other ribosomal proteins. The possible functions of ups in the cell are discussed.

Chromosome Mapping↗