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Biomedical subjects

P Model

Publications and source records attributed to P Model.

At least 73 records · Page 4Linked to original sources
PubMed1985

Direct cloning of the trxB gene that encodes thioredoxin reductase.

A strain was constructed which contains mutations in the genes encoding thioredoxin (trxA) and thioredoxin reductase (trxB) such that filamentous phage f1 cannot grow. The complementation of either mutation with its wild-type allele permits phage growth. We used this strain to select f1 phage which contain a cloned trxB gene. The location of the gene on the cloned fragment was determined, and its protein product was identified. Plasmid subclones that contain this gene overproduce thioredoxin reductase.

Cloning, Molecular↗
PubMed1984

Gene X of bacteriophage f1 is required for phage DNA synthesis. Mutagenesis of in-frame overlapping genes.

The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis. Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II. By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid. The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid. Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired. We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A* of phage phi X174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed.

Coliphages↗
PubMed1984

Specificity of translational regulation by two DNA-binding proteins.

The gene V protein of the filamentous bacteriophages fl, fd and M13, and the gene 32 protein of bacteriophage T4 share the property of binding strongly and co-operatively to single-stranded nucleic acids, especially DNA. Moreover, both are capable of repressing the translation of specific mRNAs (gene 32 protein its own, and gene V protein that of the filamentous phage gene II), both in vivo and in vitro. If the mechanism of repression by either of these proteins were based solely on its ability to bind single strands co-operatively, then the other would be expected to mimic or interfere with its effect in vitro. We have found no such mimicry or interference, even at protein concentrations high enough to have substantial non-specific effects on translation. This suggests that the sites of repression on the mRNAs must offer something other than simple "unstructuredness" for binding and repression to occur.

Bacteriophages↗
PubMed1984

A rho-dependent transcription termination signal in bacteriophage f1.

The bacteriophage f1 intergenic region distal to gene IV encodes a rho-dependent transcription termination signal. Terminator function in vivo and in vitro is dependent upon active Escherichia coli rho protein, although the RNA 3' ends detected in vivo differ from those seen in vitro. The minimal sequence required for terminator function in a heterologous plasmid system encompasses approximately 100 nucleotides distal to gene IV, which can be drawn as a large hairpin structure. The in vivo rho-dependent 3' end occurs within this sequence, while the in vitro rho-dependent 3' ends occur just distal to it. In vivo in a rho mutant host, f1 transcripts pass through the rho-dependent sites and stop within a sequence of high potential secondary structure near the f1 origin of DNA replication. This sequence alone causes transcription termination in the heterologous plasmid system in vivo. In vitro in the absence of rho protein, transcription does not terminate within this sequence. The RNA 3' ends detected in these studies do not occur within A + T-rich sequences.

Base Sequence↗
PubMed1984

Modification of DNA by reducing sugars: a possible mechanism for nucleic acid aging and age-related dysfunction in gene expression.

Reducing sugars react nonenzymatically with protein amino groups to initiate a process called nonenzymatic browning. Long-lived proteins, such as collagen and the lens crystallins, accumulate sufficient modification in vivo that they acquire many of the chemical properties characteristic of aged proteins. We have obtained evidence that nucleic acids also can undergo nonenzymatic modification by sugars. Incubation of DNA or nucleotides with glucose 6-phosphate (Glc-6-P) produces spectral changes similar to those described for nonenzymatic browning proteins. The occurrence of chemical modification was verified by measuring the transfection efficiency of viral DNA after incubation with glucose and Glc-6-P. A loss of transfection potential occurred that was first order with respect to time and sugar concentration. The rate of inactivation by Glc-6-P was 25 times that of glucose; 8 days of incubation with 150 mM Glc-6-P decreased transfection by 4 orders of magnitude. Glc-6-P also produced strand scission in a time- and concentration-dependent manner. We conclude that glucose, Glc-6-P, and possibly other sugars can react with DNA to produce significant structural and biological alterations. Since nucleic acids are long-lived molecules in the resting cell, the accumulation of these addition products might be a mechanism for the decreased genetic viability characteristic of the aged organism.

Aging↗
PubMed1984

Characterization of the cloned fip gene and its product.

A DNA fragment encoding the fip (filamentous phage production) gene from Escherichia coli, when cloned in a filamentous phage vector, restored to the phage ability to assemble progeny in fip mutant hosts. The fip gene was located just upstream of and transcribed in the same direction as the rho gene. Minicells containing fip+ phage or plasmids synthesized a 12,500-dalton protein that was missing or truncated when the Fip+ phenotype was inactivated by insertion of Tn5. The fip protein was cytoplasmic and was partially purified.

Bacterial Proteins↗
PubMed1984

Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid.

To determine whether the fip gene of Escherichia coli, which is required for filamentous phage assembly, is required for cell viability, we replaced the chromosomal copy of the gene with an inactive copy introduced on a plasmid. We found that the fip gene is dispensable. The method we devised, which should be generally useful, was also tested with an inactivated rho gene. As expected, the rho gene is essential.

Alleles↗
PubMed1983

Transcription in bacteriophage f1-infected Escherichia coli. Messenger populations in the infected cell.

Transcription of bacteriophage f1 DNA in vivo occurs in two independent regions. They are separated from one another by a strong terminator just downstream from gene VIII on one side, and by the filamentous phage intergenic space on the other. One of these regions contains genes II, V, VII, IX and VIII, and is actively transcribed. In this region there are a number of promoters but only one effective terminator. Thus, most of the RNAs that come from this region overlap and share sequences close to the termination site. The other region, which contains genes III, VI, I and IV, is transcribed much less actively. This region gives rise to a long (approximately 4 X 10(3) bases) RNA that covers the entire region, and several RNAs that overlap in the region closest to their 5' termini. Several other RNAs appear to overlap only with the 4 X 10(3) base transcript. Thus, not only the frequency but the organization of transcription differs in the two portions of the genome.

Coliphages↗
PubMed1983

A bacterial gene, fip, required for filamentous bacteriophage fl assembly.

An Escherichia coli mutant which does not support the growth of filamentous bacteriophage fl allows phage fl DNA synthesis and gene expression in mutant cells, but progeny particles are not assembled. The mutant cells have no other obvious phenotype. On the basis of experiments with phage containing nonlethal gene I mutations and with mutant fl selected for the ability to grow on mutant bacteria, we propose an interaction between the morphogenetic function encoded by gene I of the phage and the bacterial function altered in this mutant. The bacterial mutation defines a new gene, fip (for filamentous phage production), located near 84.2 min on the E coli chromosome.

Chromosome Mapping↗
PubMed1982

Effects of bacteriophage f1 gene III protein on the host cell membrane.

Plasmids which encode bacteriophage f1 coat protein genes VIII and III are responsible for a number of unusual properties suggesting that they have a drastic effect on the bacterial outer membrane. Analysis of several such recombinant plasmids and selection of mutant plasmids unable to cause this effect established that the properties were caused by gene III protein or its amino-terminal fragment.

Amino Acid Sequence↗
PubMed1982

The replication of bacteriophage f1: gene V protein regulates the synthesis of gene II protein.

Two filamentous phage gene products are required for the replication of phage DNA. One of these, the gene II protein, is a site-specific endonuclease required for all phage-specific DNA synthesis. The other, the gene V protein, is a single-stranded DNA-binding protein required only for single-strand synthesis. Purified gene V protein, when added to an in vitro protein synthesizing system programmed by f1 DNA, specifically inhibits the synthesis of gene II protein. Inhibition seems to be translational, since synthesis of gene II protein from an RNA template is also inhibited by gene V protein. Gene V protein control of gene II expression can account for the regulation of the level of expression of the filamentous phage genome.

Coliphages↗
PubMed1982

Filamentous phage pre-coat is an integral membrane protein: analysis by a new method of membrane preparation.

We show, using a simple, rapid fractionation method, that the precursor to the filamentous phage major coat protein is an integral membrane protein. The method, which consists of treatment of Escherichia coli with 0.1 N NaOH followed by centrifugation, leaves a subset of inner and outer membrane proteins in the NaOH pellet. Most proteins partition into the NaOH pellet (membrane) or supernatant (cytoplasm and periplasm) in a manner consistent with their subcellular location as determined by more conventional techniques. We find no evidence for cytoplasmic filamentous phage pre-coat protein in either wild-type of mutant-infected cells. Our evidence suggests that a protein identified as "soluble procoat" by K. Ito, G. Mandell and W. Wickner may be the amber fragment of a different phage protein.

Bacterial Proteins↗
PubMed1982

A prokaryotic membrane anchor sequence: carboxyl terminus of bacteriophage f1 gene III protein retains it in the membrane.

Gene III protein of bacteriophage f1 is inserted into the host cell membrane where it is assembled into phage particles. A truncated form of gene III protein, encoded by a recombinant plasmid and lacking the carboxyl terminus, does not remain in the membrane but instead appears to slip through it. Fusion of a hydrophobic "membrane anchor" from another membrane protein, the gene VIII protein, to the truncated gene III protein (by manipulation of the recombinant plasmid) restores membrane anchoring. A model for the relationship of gene III protein with the Escherichia coli membrane is discussed.

Cell Membrane↗
PubMed1981

An E. coli gene coding for a protamine-like protein.

Several open reading frames exist in the region of the tRNATyr/1 gene of the E. coli genome. One such sequence encodes a polypeptide 33 amino acids in length that, on the basis of size and amino acid sequence, bears a striking resemblance to the protamines found in trout sperm. DNA from the transducing phage phi 80 tRNATyr/1 su3+ directs in vitro synthesis of two small basic proteins that are not made when homologous DNA containing a deletion that overlaps both the su3+ gene and the gene for the putative protamine-like protein is used. DNA from phage that have regained the parental su3+ phenotype again direct in vitro synthesis of the basic proteins. Synthesis of basic protein is inhibited by the presence of ppGpp, as would be expected if the mRNA is part of a large RNA transcript that starts at the promoter for the tRNATyr/1 gene.

Escherichia coli↗
PubMed1981

A mutation downstream from the signal peptidase cleavage site affects cleavage but not membrane insertion of phage coat protein.

Morphogenesis of filamentous phage includes synthesis of the phage major coat protein in precursor form, its insertion into the host cell plasma membrane, its cleavage to the mature form of the protein, and its assembly there into virions. The M13 mutant am8H1R6 encodes a coat protein in which leucine replaces glutamic acid as residue 2 of the mature protein [Boeke, J. D., Russel, M. & Model, P. (1980) J. Mol. Biol. 144, 103-116]. The coat protein precursor produced by this variant is a poor substrate for the Escherichia coli signal peptidase both in vivo and in vitro. This pre-coat protein, which is eventually processed and assembled into viable phage particles, is associated with the membrane fraction of the infected cell. We conclude that the domain recognized by the signal peptidase extends beyond the signal peptide itself. Furthermore, membrane association and signal peptide cleavage can be separated temporally under conditions that permit membrane insertion, cleavage, and phage assembly.

Coliphages↗