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Lysozymes have proved useful for analyzing the relation between protein structure and function and evolution. In bacteriophage T4, the major soluble lysozyme is the product of the e gene, gpe (gene product = gp). This lysozyme destroys the wall of its host, Escherichia coli, at the end of infection to release progeny particles. Phage T4 contains two additional lysozymes that facilitate penetration of the baseplates into host cell walls during adsorption. At least one of these, a 44-kD protein, is encoded by gene 5. We show here that a segment of the gp5 lysozyme amino acid sequence, deduced from the DNA sequence of gene 5, is remarkably similar to that of the T4 gene e lysozyme. Both T4 lysozymes are somewhat similar to the lysozyme of the Salmonella phage P22, but there is little significant DNA sequence homology among the two T4 lysozyme genes and the P22 lysozyme gene. We speculate that these lysozymes are adapted to differences in the composition of the cell walls of E. coli and S. typhimurium. The cloned gene 5 of the phage T4 directs synthesis of a 63-kD precursor protein that is approximately 19 kD larger than the gene 5 protein isolated from baseplates. Gp5 first associates with gp26 to form the central hub of this structure.(ABSTRACT TRUNCATED AT 250 WORDS)
The S1 gene nucleotide sequences of 10 type 3 (T3) reovirus strains were determined and compared with the T3 prototype Dearing strain in order to study sequence diversity in strains of a single reovirus serotype and to learn more about structure-function relationships of the two S1 translation products, sigma 1 and sigma 1s. Analysis of phylogenetic trees constructed from variation in the sigma 1-encoding S1 nucleotide sequences indicated that there is no pattern of S1 gene relatedness in these strains based on host species, geographic site, or date of isolation. This suggests that reovirus strains are transmitted rapidly between host species and that T3 strains with markedly different S1 sequences circulate simultaneously. Comparison of the deduced sigma 1 amino acid sequences of the 11 T3 strains was notable for the identification of conserved and variable regions of sequence that correlate with the proposed domain organization of sigma 1 (M.L. Nibert, T.S. Dermody, and B. N. Fields, J. Virol. 64:2976-2989, 1990). Repeat patterns of apolar residues thought to be important for sigma 1 structure were conserved in all strains examined. The deduced sigma 1s amino acid sequences of the strains were more heterogeneous than the sigma 1 sequences; however, a cluster of basic residues near the amino terminus of sigma 1s was conserved. This analysis has allowed us to investigate molecular epidemiology of T3 reovirus strains and to identify conserved and variable sequence motifs in the S1 translation products, sigma 1 or sigma 1s.
This report describes a model for the structure of the reovirus cell-attachment protein sigma 1. S1 gene nucleotide sequences were determined for prototype strains of the three serotypes of mammalian reoviruses. Deduced amino acid sequences of the S1-encoded sigma 1 proteins were then compared in order to identify conserved features of these sequences. Discrete regions in the amino-terminal two-thirds of sigma 1 sequence share characteristics with the fibrous domains of other cellular and viral proteins. Most of the amino-terminal one-third of sigma 1 sequence is predicted to form an alpha-helical coiled coil like that of myosin. The middle one-third of sigma 1 sequence appears more heterogeneous; it is predicted to form a large region of beta-sheet that is followed by a region which contains two short alpha-helical coiled coils separated by a smaller region of beta-sheet. The two beta-sheet regions are each proposed to form a cross-beta sandwich like that suggested for the rod domain of the adenovirus fiber protein (N. M. Green, N. G. Wrigley, W. C. Russell, S. R. Martin, and A. D. McLachlan, EMBO J. 2:1357-1365, 1983). The remaining carboxy-terminal one-third of sigma 1 sequence is predicted to form a structurally complex globular domain. A model is suggested in which the discrete regions of sigma 1 sequence are ascribed to morphologic regions seen in computer-processed electron micrographic images of the protein (R. D. B. Fraser, D. B. Furlong, B. L. Trus, M. L. Nibert, B. N. Fields, and A. C. Steven, J. Virol. 64:2990-3000, 1990.
Adenovirus E1A dependent trans-activation of transcription involves the utilization of cellular promoter specific transcription factors. One such factor termed E2F is important for the transcription of the viral E2 gene and appears to be a rate limiting component targeted during the trans-activation event. Since E2F is of cellular origin and likely to be involved in cellular gene control, we have identified E2F binding sites in cellular genes. Examples include the c-myc, c-myb and N-myc protoncogenes, the DHFR gene and the EGF receptor gene. The transcription of these genes is regulated by cell proliferation signals and each falls into the so-called immediate early class: genes that are activated independent of new protein synthesis. Because of these common properties of regulation, we have addressed the possible role of E2F in growth factor dependent activation of transcription. Expression of a c-myc promoter driven CAT gene, transfected into quiescent 3T3 cells, is stimulated by serum addition whereas an identical gene containing mutations in the E2F binding sites is not responsive. The DNA binding activity of E2F is increased 4-fold upon serum stimulation and the kinetics of activation parallel activation of c-myc transcription. Furthermore, this increase in E2F activity is independent of new protein synthesis indicating that serum stimulation results in an activation of a pre-existing factor. These results thus provide strong evidence linking E2F and proliferation dependent control of transcription. We also believe that the E2F transcription factor is the first example of a regulator of the class of immediate early genes that is slowly activated by stimulation of cell proliferation.
The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.
The S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes were determined to gain insight into the structure and function of the S2 translation product, virion core protein sigma 2. The S2 sequences of the type 1 Lang, type 2 Jones, and type 3 Dearing strains are 1,331 nucleotides in length and contain a single large open reading frame that could encode a protein of 418 amino acids, corresponding to sigma 2. The deduced sigma 2 amino acid sequences of these strains are very conserved, being identical at 94% of the sequence positions. Predictions of sigma 2 secondary structure and hydrophobicity suggest that the protein has a two-domain structure. A larger domain is suggested to be formed from the amino-terminal three-fourths of sigma 2 sequence, which is separated from a smaller carboxy-terminal domain by a turn-rich hinge region. The carboxy-terminal domain includes sequences that are more hydrophilic than those in the rest of the protein and contains sequences which are predicted to form an alpha-helix. A region of striking similarity was found between amino acids 354 and 374 of sigma 2 and amino acids 1008 and 1031 of the beta subunit of the Escherichia coli DNA-dependent RNA polymerase. We suggest that the regions with similar sequence in sigma 2 and the beta subunit form amphipathic alpha-helices which may play a related role in the function of each protein. We have also performed experiments to further characterize the double-stranded RNA-binding activity of sigma 2 and found that the capacity to bind double-stranded RNA is a property of the sigma 2 protein of prototype strains and of the S2 mutant tsC447.
Evidence obtained with an improved in vivo assay of fimbrial phase variation in Escherichia coli supported a revised understanding of the roles of fimB and fimE in the site-specific DNA rearrangement with which they are associated. A previously proposed model argued that fimB and fimE play antagonistic, unidirectional roles in regulating the orientation of the invertible DNA element located immediately upstream of fimA, the gene encoding the major subunit of type 1 fimbriae. This conclusion, though, is based on an in vivo DNA inversion assay using recombinant plasmid substrates under conditions that, among other things, were incapable of detecting recombination of the fim invertible element from the on to the off orientation. Using a modified system that overcome this and several additional technical problems, we confirmed that fimB acts independently of fimE on the invertible element and that the additional presence of fimE results in the preferential rearrangement of the element to the off orientation. It is now demonstrated that fimE can act in the absence of fimB in this recombination to promote inversion primarily from on to off. In contrast to the previous studies, the effect of fimB on a substrate carrying the invertible element in the on orientation could be examined. It was found that fimB mediates DNA inversion from on to off, as well as from off to on, and that, contrary to prior interpretations, the fimB-associated inversion occurs with only minimal orientational preference to the on phase.
The major DNA adduct (greater than 95% total) resulting from the bioactivation of ethylene dibromide by conjugation with GSH is S-(2-(N7-guanyl)ethyl)GSH. The mutagenic potential of this adduct has been uncertain, however, because the observed mutagenicity might be caused by other adducts present at much lower levels, e.g. S-(2-N1-adenyl)ethyl)GSH. To assess the formation of other potential adducts, S-(2-(N3-deoxycytidyl)ethyl)GSH, S-(2-(O6-deoxyguanosyl)ethyl)GSH, and S-(2-(N2-deoxyguanosyl)ethyl)GSH were prepared and used as standards in the analysis of calf thymus DNA modified by treatment with [1,2-14C]ethylene dibromide and GSH in the presence of rat liver cytosol; only minor amounts (less than 0.2%) were found. A forward mutation assay in (repair-deficient) Salmonella typhimurium TA100 and sequence analysis were utilized to determine the type, site, and frequency of mutations in a portion of the lacZ gene resulting from in vitro modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)GSH, an analog of the ethylene dibromide-GSH conjugate. An adduct level of approximately 8 nmol (mg DNA)-1 resulted in a 10-fold increase in mutation frequency relative to the spontaneous level. The spectrum of spontaneous mutations was quite varied, but the spectrum of S-(2-chloroethyl)GSH-induced mutations consisted primarily of base substitutions of which G:C to A:T transitions accounted for 75% (70% of the total mutations). All available evidence implicates S-(2-(N7-guanyl)ethyl)GSH as the cause of these mutations inasmuch as the levels of the minor adducts are not consistent with the mutation frequency observed in this system. The sequence selectivity of alkylation was determined by treatment of end-labeled lac DNA fragments with S-(2-chloroethyl)GSH, cleavage of the DNA at adduct sites, and electrophoretic analysis. Comparison of the sequence selectivity with the mutation spectrum revealed no obligate relationship between the extent of adduct formation and the number of mutations which resulted at different sites. We suggest that the mechanism of mutagenesis involves DNA sequence-dependent alterations in the interaction of the polymerase with the (modified) template and incoming nucleotide.
A cDNA representing a 5.2-kb defective, endogenous murine leukemia proviral sequence (EPI-EPS) was isolated from a C57BL/6 mouse cDNA epididymal library. Northern blot analysis demonstrated that EPI-EPS was predominantly expressed in the C57BL/6 mouse epididymis and vas deferens with 10-fold lower expression in the seminal vesicle, kidney, and submandibular gland. Analysis of tissues from other inbred strains of mice as well as the wild mouse, Mus musculus musculus, showed a similar pattern of tissue expression. EPI-EPS expression was also highly androgen regulated in both the reproductive and nonreproductive tissues of the C57BL/6 strain. However, a differential response to testosterone replacement was observed between tissues. Expression of EPI-EPS mRNA in the epididymis and vas deferens exhibited only a partial recovery to precastration levels after testosterone replacement; in the kidney and submandibular gland there was a complete recovery of EPI-EPS expression. Finally, EPI-EPS expression was also highly restricted in the female tissues, with expression limited to the oviduct and uterus. EPI-EPS, however, was not estrogen regulated in the female. These results suggest that a proviral sequence, EPI-EPS, is expressed in M. m. musculus and several inbred strains of mice due to its integration near a highly tissue-specific and androgen-regulated genetic locus.