Biomedical subjects
Publications and source records attributed to R E Webster.
Morphogenesis of the filamentous bacteriophage f1 occurred at adhesion zones between the inner and outer membranes of the host cell. Quantitation of adhesion zones in cells infected with mutant phage strains suggested that the phage gene I protein may be involved in the formation of adhesion zones for phage assembly.
We describe the identification of two mutations in bacterial genes, designated as fipB and fipC, which resulted in temperature-sensitive morphogenesis of bacteriophage f1. These mutations mapped at separate loci but had to be present simultaneously to block f1 production at 41.5 degrees C. One mutation defined the locus fipB at 85.3 min on the Escherichia coli linkage map; the other defined the locus fipC, which mapped very close to rpsL at 73 min. Since these mutations did not appear to affect phage DNA replication, gene expression, or protein localization, they probably interfered with the its life cycle at the level of assembly. fipB mutants were partially deficient in adsorption of bacteriophage lambda, and fipB and fipC mutants leaked beta-lactamase into the medium, suggesting that the mutations affect outer-membrane structure or function.
The gene V protein/phage single-stranded (SS) DNA complex is an intermediate in the assembly of the filamentous bacteriophage f1. The minor protein content of this complex isolated from wild-type and amber mutant phage-infected Escherichia coli bacteria has been analyzed. Other than the gene V protein, none of the proteins found in purified samples of the complex correspond to any known phage gene products. In particular, the minor coat proteins found in the mature phage particle do not appear to be components of the cytoplasmic gene V protein/f1 SS DNA complex. However, approximately 1-3 molecules of E. coli single-stranded DNA binding protein (SSB) copurify with the complex and may be stably associated with this structure in vivo.
The region of the bacteriophage f1 genome near the gene IV/intergenic region (IG) junction previously was shown to contain a sequence necessary for efficient packaging of single-stranded (SS) DNA into phage particles (the f1 "morphogenetic signal") [G. P. Dotto, V. Enea, and N. D. Zinder (1981) Virology 114, 463-473]. The DNA content of f1 phage/pBR322 chimeric plasmid-transformed, phage-infected bacteria has been investigated. The chimeric plasmids, constructed by Dotto et al. (1981), contained the f1 origins of DNA replication and, in some cases, also contained the "morphogenetic signal." Chimeric plasmid SS DNA was detected in phage-infected bacteria harboring any one of these chimeric plasmids, and the majority of this SS DNA was found complexed to the phage gene V protein. Therefore, the "morphogenetic signal" is not required for the formation of the gene V protein/f1 SS DNA complex but instead must function at a later stage of filamentous phage morphogenesis.
Crosslinking reagents were used to interrupt the process of filamentous phage morphogenesis and investigate the orientation in which nascent virions are extruded through the host cell membrane. Infected bacteria with emerging phage particles were crosslinked with glutaraldehyde. Immunoferritin-labeling studies on these emerging phage using anti-A protein IgG suggested that extrusion begins with the C protein end. To confirm this, phage extruding from infected bacteria were frozen using the reversible crosslinker dimethyl 3,3'-dithiobis-propionimidate and fragments of emerging phage were isolated by shearing. Protein analysis of these fragments showed them to be enriched in C protein relative to A protein, as predicted if phage extrusion begins with the C protein end. The production of multiple-length phage particles (polyphage) by nonpermissive bacterial hosts infected with amber mutant phage strains was also studied. Polyphage were produced upon infection with amber mutants in genes III, VI, VII, and IX which code for proteins found at the ends of the mature phage particle. No polyphage were produced by mutants in the other genes tested. Gene III amber mutants produce noninfective polyphage, but those produced by genes VII and IX are infective. Gene VI amber mutants appear to produce unstable, noninfective polyphage particles.
The gene II region of bacteriophage f1 DNA codes for two proteins, the 46 kd gene II protein and the 13 kd gene X protein, which results from an in-phase start at codon 300 of gene II. Using antigene II protein IgG, we show that the intracellular concentration of both proteins is controlled by the phage gene V protein. In wild-type f1-infected cells, the amount of gene II protein reaches a plateau of about 1500 molecules per cell at 20 min after infection, as measured by blot immunoassay. Similarly, the amount of gene X protein reaches a peak of about 500 molecules per cell around 10 min after infection. In contrast, when the gene V protein is inactive, both gene II and gene X proteins continue to accumulate at a high rate for at least 40 min after infection. This difference is caused by decreased synthesis of gene II and gene X proteins in the presence of gene V protein, which represses the translation of these two proteins.
The filamentous bacteriophage f1 can be transformed into a spherical particle (spheroid) or an intermediate shortened filament with a flared end (I-forms) by exposure to a chloroform-water interface at 22 or 4 degrees C, respectively. The protein composition of bacteriophage f1 spheroids and I-forms was examined by separating the proteins from the purified. [35S]cysteine-labeled particles by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Quantitation of the radioactivity on the gels showed that I-forms and spheroids contain the same complement of minor coat proteins as do untreated f1 phage. This composition is unchanged after removal of the DNA, either by digestion with micrococcal nuclease or by centrifugation of the particles through CsCl density gradients, indicating that none of the minor coat proteins is held in the particles solely through an interaction with the DNA. We also examined the location of the A protein in I-forms by decoration with ferritin-conjugated antibodies and examination under the electron microscope and found that the A protein is located specifically at the flared end of the I-form particle, through which the DNA is extruded and at which contraction into spheroids begins. The implications of these results with regard to the orientation of the DNA within the capsid and the process of infection are discussed.
We have isolated and characterized the 2 major proteins of a dense complex which accumulate in EScherichia coli cells infected with bacteriophage f1 under conditions where the phage gene V protein is inactive (Webster, R. E., and Rementer, M. (1980) J. Mol. Biol. 139, 393-405). The amino acid composition and NH2- and COOH-terminal sequences of the larger polypeptide (estimated molecular weight of 46,000) correspond to those predicted from the DNA sequence for the f1 gene II protein. The other polypeptide (estimated molecular weight of 14,000) has the amino acid composition and COOH-terminal sequence predicted for the f1 X protein, which previously had been found only as a product of an in vitro transcription-translation reaction. The X protein contains N-formylmethionine, cross-reacts with antibodies against gene II protein, and is present in wild type f1-infected bacteria. Thus, X protein is the product of f1 gene X (10), which is contained entirely in, and translated in phase with, gene II.
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The orientation of the major coat (B) protein of the bacteriophage f1, an integral membrane protein in the cytoplasmic membrane of infected Escherichia coli, was examined. Pyridoxal 5'-phosphate and [3H]NaBH4 were used to label the cytoplasmic membrane proteins in spheroplasts and membrane vesicles of E. coli infected with bacteriophage f1. Under the conditions described, tritium incorporation was almost completely dependent on the presence of pyridoxal 5'-phosphate and little if any of the cytoplasmic proteins were labeled when the reaction was applied to intact spheroplasts. The major coat protein was isolated from the cytoplasmic membranes labeled in this manner and the chymotryptic peptides were analyzed for the presence of tritium in the pyridoxamine 5'-phosphate conjugate. When the proteins were labeled in the intact spheroplast, only the NH2-terminal chymotryptic peptide of the coat protein was labeled. If the proteins were labeled during osmotic lysis of the spheroplasts or in isolated vesicles, the chymotryptic peptide containing the COOH terminus of the coat protein as well as the NH2-terminal peptide was labeled. The NH2-terminal peptide was labeled to approximately the same extent as occurred in the intact spheroplast. These results are consistent with the hypothesis that the mature f1 coat protein asymmetrically spans the cytoplasmic membrane of the infected host with its NH2 terminus exposed on the outside and COOH terminus exposed on the cytoplasmic surface.
The location within the virion of the A, C, and D minor coat proteins of the filamentous bacteriophage fl has been analyzed. The A protein is present in approximately 5 copies/particle and is located at the tip of normal length phage, miniphage, and fl/pBR322 chimeric phage, a longer than normal length phage. The mole ratios of the A, C, and D proteins are the same for each type of particle, consistent with a model of phage organization in which the minor coat proteins are clustered near or at the ends of the phage. Normal length phage were fragmented by passing them through a French press, and those fragments that contained the A protein were separated from those that did not by treating the mixture with anti-A protein antibody. Analysis of the protein compositions of the two populations of fragments showed that the A and D proteins were found together in one population of fragments and that most, it not all, of the C protein was found in the other. These results show that the D protein is located near or at the A protein end of the phage and that the C protein is located in a region near or at the opposite end. Treatment of the virion with proteases which lowered the infectivity of the phage resulted in particles in which only the A protein was cleaved to any detectable extent. These particles remained resistant to the action of micrococcal nuclease.
The minor coat proteins of the filamentous bacteriophage fl and fd have been isolated and characterized. The phage have approximately 5 copies each of the A protein (product of gene III) and the D protein (product of gene VI). The phage also contains about 10 copies of the C protein. Preliminary evidence suggests that the "C protein" may actually be a mixture of proteins, the major component of which is the product of gene IX. The D protein is located near or at the A protein end of the phage, and the C protein is located in a region near or at the other end. The DNA appears to be oriented iun the virion such that the DNA which specifies the intergenic region is located close to the C protein end of the phage and that which codes for the gene III protein is located near the A and D protein end.
The C and D proteins from bacteriophage fd and fl have been purified and characterized. Since the DNA sequence is known, the amino acid composition of these purified proteins indicated that they are coded for by the phage, the C protein being the product of Gene IX and the D protein specified by Gene VI. The molecular weights of the C and D proteins were calculated from the DNA sequences to be 3,650 and 12,350, respectively. These values are close to the molecular weights observed after polyacrylamide gel electrophoresis of the proteins in sodium dodecyl sulfate. Since the C and D proteins can be selectively labeled with radioactive cysteine and arginine, it was possible to estimate the number of the C and D protein molecules relative to the number of A protein molecules which had been accurately determined in previous studies. Based on these results, the average phage particle contains 5 A, 5 D, and 10 C protein molecules.
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We have analyzed eight major phage-specific mRNA species which are synthesized following infection of Escherichia coli with bacteriophage fl. The approximate half-lives of these RNAs appear to be inversely proportional to their lengths. Three species have the properties of primary transcripts. They are labeled very rapidly with [3H]uracil, and they co-migrate by sodium laruyl sulfate-urea polyacrylamide gel electrophoresis with the three major RNAs transcribed from replicative form DNA in vitro using [gamma-32P]GTP as the labeled ribonucleoside triphosphate. At least three of the remaining RNAs synthesized in vivo arise by some type of processing reaction. The processed species contain, as determined by oligonucleotide analysis, the information for the gene VIII protein and the 3'-terminal oligonucleotide expected for a transcription termination event at the rho-independent site following gene VIII. The two primary transcripts which could be analyzed also have this same 3'-terminal oligonucleotide. These results suggest that the processed mRNAs are the 3'-terminal cleavage products of primary transcripts which had terminated at the rho-independent site.
Examined the utility of the WAIS OA, BD, PIQ and FSIQ scores in combination with age, apparent degree of emotional severity and psychiatric disability in predicting the vocational success of 180 psychiatric outpatients. Although no significant statistical differences were obtained in a cros-validation procedure indicating that psychiatric disability affected the efficacy of the derived regression equations, substantial differences in predictable variance accounted for were found to be related to the nature of the psychiatric disability. This must be attended to in attempts to predict the vocational success of psychiatric outpatients.
We have isolated a conditional lethal mutant of bacteriophage 12 which makes plaques only on E. coli strains carrying a UGA suppressor. It grows normally in nonsuppressing hosts but does not lyse such strains. The mutation complements with amber mutations in each of the three known phage cistrons. These observations lead us to postulate the existence of a fourth gene in the RNA phage.
[3H]Uracil-pulse-labeled RNA from Escherichia coli infected with f1 bacteriophage was fractionated on polyacrylamide gels containing urea. Eight phage-specific RNA species were present with approximate lengths ranging from 2100 to 400 nucleotides. The amount of the seven largest species was increased when the infected bacteria were incubated at 41 degrees C. When the RNA was isolated and used as message in an in vitro protein-synthesizing system, most of the RNA species appeared to direct the synthesis of the phage gene VIII protein. The six largest species also directed the synthesis of the phage gene V protein. Some of the labeled smaller RNA species increased in amount after addition to rifampicin, suggesting that they may have resulted from cleavage of larger RNA species. These particular smaller RNA species also were present in infected bacteria containing a mutant RNase III. The data are discussed in terms of the regulation of synthesis of the phage-specific proteins.