J Biol Chem, 1982 Aug 10, 257(15), 8972 - 9 Bacteriophage T4 gene 45 . Sequences of the structural gene and its protein product; Spicer EK et al.; Bacteriophage T4 gene 45 codes for a protein whose functions are required for both T4 DNA replication and T4 late gene transcription . To facilitate studies of the interactions of 45 protein with the T4 DNA replication complex and with RNA polymerase, we have determined the primary structure of 45 protein . The amino acid sequence of 45 protein has been determined by correlating nucleotide sequence analysis of gene 45 with protein chemistry studies of 45 protein . Our studies indicate that gene 45 codes for a polypeptide containing 227 amino acids, with a calculated Mr = 24,710 . The coding region of gene 46 is preceded by a putative promoter containing sequences which are homologous to Escherichia coli RNA polymerase recognition and binding regions . In addition, there are sequence similarities in the translation initiation regions of gene 45 and the rIIB gene, which may relate to their common regulation by regA protein. J Virol, 1982 Aug, 43(2), 714 - 20 Evidence for an internal component of the bacteriophage T4D tail core: a possible length-determining template; Duda RL et al.; The length of the T4 tail is precisely regulated in vivo at the time of polymerization of the tail core protein onto the baseplate . Since no mutations which alter tail length have been identified, a study of in vivo-assembled tail cores was begun to determine whether the structural properties of assembled cores would reveal the mechanism of length regulation . An assembly intermediate consisting of a core attached to a baseplate (core-baseplate) was purified from cells infected with a T4 mutant in gene 15 . When core-base plates were treated with guanidine hydrochloride, cores were released from baseplates . The released cores had the same mean length as cores attached to baseplates . Electron micrographs of these cores showed partial penetration of negative stain into one end, and, at the opposite end, a modified tip which often appeared as a short fiber projecting from the core . When cores were purified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two minor proteins and the major core protein were detected . One minor protein, the product of gene 48 (gp48), was present in at least 72% of the amount found in core-baseplates, relative to the amount of the major core protein . These findings suggest that cores contain a fibrous structure, possibly composed of gp48, which may form a "ruler" that specifies the length of the T4 tail. J Virol, 1982 Aug, 43(2), 697 - 704 Endo-N-acetylneuraminidase associated with bacteriophage particles; Kwiatkowski B et al.; A bacteriophage (phi 1.2) has been isolated for Escherichia coli K235 (O1:K1:H-) . phi 1.2 is specific for the host capsular polysaccharide (colominic acid) . The phage forms plaques with acapsular halos and thus carries a glycanase activity for colominic acid, a homopolymer of alpha (2 leads to 8)-linked N-acetylneuraminic acid (NeuNAc) residues . Upon incubation with purified phi 1.2 particles, a solution of K1 polysaccharide loses viscosity and consumes increasing amounts of periodate . Also, by gel filtration, the production of colominic oligosaccharides (down to a size of two to three NeuNAc residues) can be demonstrated . No NeuNAc monomers, however, are formed . The capsules of E . coli strains with the K92 antigen, which consists of NeuNAc residues linked by alternating alpha (2 leads to 8) and alpha (2 leads to 9) bonds, are also depolymerized by the phi 1.2 enzyme . Under the electron microscope, phage phi 1.2 is seen to belong to Bradley's morphology group C (D . E . Bradley, Bacteriol . Rev . 31:230-314, 1967); it has an isometric head, carrying a baseplate with six spikes . By analogy to other virus particles with host capsule depolymerase activity, it is probable that the phi 1.2 endo-N-acetylneuraminidase activity is associated with these spikes. J Virol, 1982 Aug, 43(2), 655 - 63 Genetic studies on capsid-length determination in bacteriophage T4 . II . Genetic evidence that specific protein-protein interactions are involved; Doherty DH; A bacteriophage T4 mutation (ptg19-80c) located in gene 23, which encodes the major structural protein of the T4 capsid, results in the production of capsids of abnormal length . Mutations outside gene 23 which partially suppress ptg19-80c have been described in the accompanying paper (D . H . Doherty, J . Virol . 43:641-654, 1982) . Characterization of these suppressors was extended . A complementation test suggested that the suppressors were in genes 22 and 24 . These genes coded for the major component of the morphogenetic core of the capsid precursor and the vertex protein of the capsid, respectively . The suppressor mutations were found to have no obvious phenotype in the absence of ptg19-80c . Suppression was shown to be allele specific: other ptg mutations at different sites in gene 23 were not suppressed by the suppressors of ptg19-80c . These results indicated that specific interactions among the three proteins gp22, gp23, and gp24 may play a role in the regulation of T4 capsid-length determination . Current models for capsid-length determination are considered in the light of these results. J Virol, 1982 Aug, 43(2), 641 - 54 Genetic studies on capsid-length determination in bacteriophage T4 . I . Isolation and partial characterization of second-site revertants of a gene 23 mutation affecting capsid length; Doherty DH; The T4 mutation ptg19-80 affects the mechanism of capsid-length determination . It is located in gene 23, which encodes the major structural protein of the capsid . The mutation results in the production of abnormal-length capsids in high frequencies . This paper describes the isolation and partial characterization of second-site revertants of ptg19-80 . In the course of their analysis, it was discovered that ptg19-80 is itself a double mutation consisting of a gene 23 mutation (ptg19-80c), which causes the morphogenetic defect, and a suppressor mutation located near the lysozyme gene . Phenotypic characterization of nine pseudo-wild-type revertants of this double-mutation revealed that these revertants all produced lower frequencies of abnormal capsids than did ptg19-80 . Seven of these revertants were shown to contain two suppressor mutations, one mapping in or near gene 22 and done mapping in or near gene 24 . Both mutations were required for suppression . These suppressors displayed no discernible phenotype in the absence of ptg19-80c. J Bacteriol, 1982 Aug, 151(2), 750 - 5 Specific mutator effects of ung (uracil-DNA glycosylase) mutations in Escherichia coli; Duncan BK et al.; Studies of trpA reversions revealed that G:C leads to A:T transitions were stimulated about 30-fold in E . coli ung mutants, whereas other base substitutions were not affected . A dUTPase (dut) mutation, which increases the incorporation of uracil into DNA in place of thymine, had no significant effect on the rate of G:C leads to A:T transitions . The results support the proposal that the glycosylase functions to reduce the mutation rate in wild-type cells by acting in the repair of DNA cytosine residues that have undergone spontaneous deamination to uracil . Further support was provided by the finding that when lambda bacteriophages were treated with bisulfite, an agent known to produce cytosine deamination, the frequency of clear-plaque mutants was increased an additional 20-fold by growth on an ung host . Bisulfite-induced mutations of the cellular chromosome, however, were about equal in ung+ and ung strains; it was found that during the treatment of ung+ cells with bisulfite, the glycosylase was inactivated. J Bacteriol, 1982 Aug, 151(2), 718 - 22 Roles of lipopolysaccharide and outer membrane protein OmpC of Escherichia coli K-12 in the receptor function for bacteriophage T4; Yu F et al.; The roles of lipopolysaccharide and OmpC, a major outer membrane protein, in the receptor function for bacteriophage T4 were studied by using Escherichia coli K-12 strains having mutations in the ompC gene or in genes controlling different stages of lipopolysaccharide synthesis . The receptor activity for T4 was monitored by (i) T4 sensitivity of intact cells, (ii) phage inactivation activity of cell envelopes, and (iii) phage inactivation activity of specimens reconstituted from purified OmpC and lipopolysaccharide . It was found that (i) in the presence of the OmpC protein, the essential region of the lipopolysaccharide for the receptor activity was the core-lipid A region that includes the heptose region, whereas the glucose region was not necessarily required for the receptor function; (ii) the OmpC protein was not required at all when the distal end of the lipopolysaccharide was removed to expose a glucose residue at the distal end; and (iii) when cells lacked both the OmpC protein and the glucose region, they became extremely resistant to T4 . Based on these findings, the roles of the OmpC protein and lipopolysaccharide in T4 infection are discussed. Biochimie, 1982 Aug-Sep, 64(8-9), 839 - 44 Are pyrimidine dimers tolerated during DNA replication of UV-irradiated parvovirus minute-virus-of-mice in mouse fibroblasts? Vos JM, Rommelaere J. The replication of the single-stranded DNA of parvovirus Minute-Virus-of-MIce (MVM) was depressed when virus was exposed to UV-light prior to infection of mouse fibroblasts . Most of the viral DNA containing pyrimidine dimers was permanently blocked in its conversion to double-stranded replicative forms (RF) . Yet dimers might be tolerated to a low extent, considering that a minor fraction of parental RF molecules was sensitive to the action of the UV-specific endonuclease V of bacteriophage T4, UV-irradiation of the cells prior to infection with UV-damaged MVM increased the levels of both parental RF and total viral DNA synthesized . The sensitivity of parental RF molecules to the UV-specific endonuclease was little enhanced by preirradiation of the cells and did not appear to be sufficient to account for the stimulation of RF formation in those cultures . Since parvoviral single-stranded DNA is not a substrate for nucleotidyl excision repair, one interpretation of these results would be that the process(es) activated in preirradiated cells overcome(s) barriers to viral DNA replication other than elongation blocks at pyrimidine dimers . Alternatively, pyrimidine dimers tolerated in pretreated cultures might become protected from attack by the UV-endonuclease. Biochimie, 1982 Aug-Sep, 64(8-9), 623 - 7 DNA replication and indirect induction of the SOS response in Escherichia coli; D'Ari R et al.; The SOS response can be induced indirectly in Escherichia coli by infection with UV irradiated bacteriophage P1, lambda or M13 . Induction, monitored quantitatively by means of a sfiA::lac operon fusion, was stronger with the plasmid phage P1 than with lambda, but the kinetics were similar, showing that plasmid and non-plasmid phages are not fundamentally different in their ability to produce indirect induction . In the absence of lambda DNA replication the level of induction was strongly reduced, indicating that the attempt to replicate damaged DNA results in induction of the SOS response . The slight residual induction observed in the absence of DNA replication suggests the existence of a second pathway leading from DNA lesions to induction of the SOS response. Biochimie, 1982 Aug-Sep, 64(8-9), 643 - 54 Pyrimidine dimer-DNA glycosylases: studies on bacteriophage T4-infected and on uninfected Escherichia coli; Bonura T et al.; Pyrimidine dimer (PD)-DNA glycosylase activity has been reported in both the M . luteus and phage T4 UV endonucleases . In the present studies the T4 PD-DNA glycosylase has been purified close to physical homogeneity using an assay that measures the release of free thymine from UV-irradiated poly ({H5} dT):poly (dA), after the photo-reversal of thymine-thymine dimers . The activity has also been demonstrated in vivo following infection of UV-irradiated E . coli uvr- cells with phage T4 . Under these conditions the T4 PD-DNA glycosylase accounts quantitatively for all thymine-containing PD excised from {3H} labeled E . coli DNA . In vitro the T4 PD-DNA glycosylase has an associated AP endonuclease activity that incises UV-irradiated DNA 3 to the apyrimidinic sites created by the glycosylase . However, the glycosylase/AP endonuclease reaction mechanism in vitro does not appear to be a concerted one . In addition, a T4 phage with a temperature-sensitive mutation in the denV gene shows wild-type levels of survival at the permissive temperature, despite the fact that in vitro, extracts of E . coli infected with this mutant show no detectable phage-coded AP endonuclease at 28 degrees C . Thus the exact role of the T4 AP endonuclease in the incision of UV-irradiated DNA dimer in vivo is not clear . The ratio of excised non-containing nucleotides to dimer-containing nucleotides following infection of UV-irradiated E . coli with phage T4 denV+ yields a calculated average repair patch size of approximately 7 nucleotides . In contrast, the calculated average patch size in uninfected E . coli is approximately 70 nucleotides . Thus the extent of excision/resynthesis of UV-irradiated DNA may be determined by the specific mode of incision of the DNA at PD . When uninfected E . coli (uvr+) is exposed to UV radiation, a fraction of the excised thymine-containing PD contain photolabile thymine, suggesting the presence of PD-DNA glycosylase in E . coli . The role of this putative activity in the metabolism of UV-irradiated DNA is under investigation. Zh Mikrobiol Epidemiol Immunobiol, 1982 Aug, (8), 66 - 9 {Characterisation of major biological properties of Bordetella bacteriophages}; Siniashina LN et al.; The main biological properties (morphology of negative colonies, parameters of adsorption and single development cycle) of B . pertussis and B . bronchiseptica phages, isolated spontaneously and by induction with mitomycin C, were studied . To compare these characteristics, one B . parapertussis indicator strain was used, and the experiments were carried out under identical conditions . Highly active sera were obtained with the use of complete Freund's adjuvant . B . pertussis phages isolated from the strains of different serovars were serologically related, but not identical, and differed in their constant characterizing their rate of neutralization with homologous antisera . The adsorption of the phages on homologous strains was more intensive than on the cells of B . parapertussis indicator strain . However, the authors failed to observe the further development of the phages in the host cells. Proc Natl Acad Sci U S A, 1982 Aug, 79(16), 4937 - 41 Nucleotide sequences involved in bacteriophage T4 gene 32 translational self-regulation; Krisch HM et al.; We have determined the nucleotide sequence of a cloned segment of the bacteriophage T4D chromosome, which contains the regulatory sequences and the structural gene (gene 32) for the single-stranded DNA binding protein (gp32) . The amino acid sequence predicted by translation of the structural gene agrees well with that published for gp32 {Williams, K . R., Lo-Presti, M . B., Setoguchi, M . & Konigsberg, W . H . (1980) Proc . Natl . Acad . Sci . USA 77, 4614-4617} . To localize the nucleotide sequence involved in translational self-regulation of gene 32, we have constructed a series of plasmids in which gene 32 is fused to an amino-terminal deletion mutant of the beta-galactosidase gene of Escherichia coli . Expression of a beta-galactosidase fusion protein that contains only the first seven amino acids of gp32 is still repressed by gp32 . The ribosomal binding site of gene 32 is flanked by a repetitive A+T-rich sequence . Preferential and cooperative binding of gp32 to this region of its mRNA could inhibit translation initiation and, thus, would account for the autoregulation. J Virol, 1982 Aug, 43(2), 566 - 73 New physical map of bacteriophage T5 DNA; Rhoades M; The locations of 103 cleavage sites, produced by 13 restriction endonucleases, were mapped on the DNA of bacteriophage T5 . Single- and double-digest fragment sizes were determined by agarose gel electrophoresis, using restriction fragments of phi X174 DNA and lambda DNA as molecular weight standards . Map coordinates were determined by a computer-based least-squares procedures (J . Schroeder and F . Blattner, Gene {Amst} 4:167-174, 1978) . The fragment sizes predicted by the final map are all within 2% of the measured values . Based on this analysis, T5st(+) DNA contains 121,300 base pairs (Mr, 80.3 X 10(6) and has a terminal repetition of 10,160 base pairs (Mr, 6.7 X 10(6)) . Restriction endonuclease analysis after treatment with exonuclease III and a single-strand-specific endonuclease allowed precise localization of five of the natural single-chain interruptions in T5 DNA . Revised locations for several T5 deletions were also determined. J Bacteriol, 1982 Aug, 151(2), 609 - 19 Mutations affecting regulation of methionine biosynthetic genes isolated by use of met-lac fusions; Mulligan JT et al.; Fusions of the lac genes to the promoters of four structural genes in the methionine biosynthetic pathway, metA, metB, metE, and metF, were obtained by the use of the Mu d(Ap lac) bacteriophage . The levels of beta-galactosidase in these strains could be derepressed by growth under methionine-limiting conditions . Furthermore, growth in the presence of vitamin B12 repressed the synthesis of beta-galactosidase in strains containing a fusion of lacZ to the metE promoter, phi(metE'-lacZ+) . Mutations affecting the regulation of met-lac fusions were generated by the insertion of Tn5 . Tn5 insertions were obtained at the known regulatory loci metJ and metK . Interestingly, a significant amount of methionine adenosyltransferase activity remained in the metK mutant despite the fact that the mutation was generated by an insertion . Several Tn5-induced regulatory mutations were isolated by screening for high-level beta-galactosidase expression in a phi(metE'-lacZ+) strain in the presence of vitamin B12 . Tn5 insertions mapping at the btuB (B12 uptake), metH (B12 dependent tetrahydropteroylglutamate methyltransferase), and metF (5,10-methylenetetrahydrofolate reductase) loci were obtained . The isolation of the metH mutant was consistent with previous suggestions that the metH gene product is required for the repression of metE by vitamin B12 . The metF::Tn5 insertion was of particular interest since it suggested that a functional metf gene product was also needed for repression of metE by vitamin B12. Eur J Biochem, 1982 Aug, 126(2), 227 - 33 The Fi-gene product of bacteriophage lambda . Purification and properties; Benchimol S et al.; The FI gene product of bacteriophage lambda has been purified extensively using a biochemical assay that measures assembly of lambda phage particles in vitro . The molecular weight of the native protein was estimated to be 21700 with an S20, w of 2.1 S and a Stokes radius of 2.5 nm . The molecular weight in dodecylsulfate was estimated to be 19000 . The protein is highly acidic with an isoelectric point less than 4.1. J Virol, 1982 Aug, 43(2), 753 - 5 Stability of Lambdoid bacteriophage heads: antagonism between polyamines and tryptamine; Deeb SS et al.; The biological activity of heads of bacteriophages phi 80 and lambda in in vitro assembly with tails was inhibited by dialysis, filtration on gels, and treatment with tryptamine . Inhibition by these three treatments could be prevented but not reversed by putrescine . Other diamines with shorter or longer carbon chain lengths were either less effective or not effective at all . It is suggested that tryptamine acts by loosening the tightly packed DNA in heads, whereas putrescine stabilizes it. Biochim Biophys Acta, 1982 Jul 30, 698(1), 29 - 34 Role of 3-methyladenine-DNA glycosylase in host-cell reactivation of methylated T7 bacteriophage; Mamet-Bratley MD et al.; Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway . For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1 . These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage . Quantitative analysis of the data, using the results of Verly et al . (Verly, W.G., Crine, P., Bannon, P . and Forget, A . (1974) Biochim . Biophys . Acta 349, 204-213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5-10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant . Thus, 3-methyladenine may be as toxic a lesion as an apurinic site. Nature, 1982 Jul 22, 298(5872), 393 - 6 Ligation of oligonucleotides by pyrimidine dimers--a missing 'link' in the origin of life? Lewis RJ, Hanawalt PC. One of the principal photochemical reactions of DNA on exposure to UV is the formation of intrastrand cyclobutane-type pyrimidine dimers . The efficiency of this reaction depends on both the wavelength of the UV2 and the specific nucleotide sequence in the DNA . The formation of the pyrimidine dimer and its repair in living cells have been studied extensively . We have examined the possibility that pyrimidines at the ends of DNA strands may be adequately juxtaposed for dimer formation by the presence of a complementary strand, even when no phosphodiester linkage joins their sugars . In these conditions the formation of a dimer will 'ligate' two DNA strands end-to-end . We report here that thymidine oligonucleotides annealed to polydeoxyadenylate can be ligated end-to-end by UV irradiation, via thymine dimerization of the terminal nucleotides in adjacent oligonucleotides . The linkages are susceptible to direct photoreversal by 254 nm UV, as expected for cyclobutane-type thymine dimers, but they are not cleaved by the bacteriophage T4 endonuclease V, a dimer-specific DNA repair enzyme . We demonstrate that the ligating dimers are also resistant to photolyase from Escherichia coli . Although the phosphodiester backbone is not required for dimer formation, it is required for recognition of dimers by these DNA repair enzymes . We discuss the possibility that high molecular weight polynucleotides in primordial seas might have been generated from oligonucleotides by pyrimidine dimerization under the intense solar UV flux unattenuated by an ozone layer. Nucleic Acids Res, 1982 Jul 10, 10(13), 3981 - 93 Insertion sites and the terminal nucleotide sequences of the Tn4 transposon; Hyde DR et al.; The nucleotide sequences at the ends of the Tn4 transposon (mercury spectinomycin and sulfonamide resistance) have been determined . They are inverted repeated sequences of 38 nucleotides with three mismatched base pairs . These sequences are strongly homologous with the terminal sequences of Tn501 (mercury resistance) but less so with those of Tn3 (ampicillin resistance) . The Tn4 transposon generates pentanucleotide members (Tn3, Tn1000, Tn501, Tn551, IS2) with the exception of Tn1721 and bacteriophage Mu . Among the three Tn4 insertion sites examined here, two of them occurred near a nonanucleotide sequence in perfect homology with part of the terminal inverted-repeat sequence of Tn4 and the third insertion occurred near a sequence of partial homology to one end of Tn4 . All three insertions were in the same orientation such that IRb is proximal to its homologous sequence on the recipient DNA. J Virol, 1982 Jul, 43(1), 67 - 72 Isolation and preliminary characterization of amber mutants of bacteriophage phi W-14 defective in DNA synthesis; Miller PB et al.; Of 42 amber mutants of bacteriophage phi W-14, 6 were defective in DNA synthesis . Three of the mutants synthesized DNA in the nonpermissive host, but were defective in post-replicational modification of the DNA . The DNA synthesized by two of these mutants, am36 and am42, contained more thymine and less alpha-putrescinylthymine than did wild-type DNA; that synthesized by the third mutant, am37, contained the normal amount of thymine, no alpha-putrescinylthymine, and hydroxymethyluracil . The properties of these mutants suggested that the presence of the normal amount of alpha-putrescinylthymine in phi W-14 DNA was essential for the production of viable progeny . Three of the mutants, am6, am35, and am45, failed to synthesize DNA in the nonpermissive host . These mutants were analogous to the DNA off mutants of T4 . Nonpermissive cells infected with DNA off mutants accumulated dATP, dGTP, dCTP, and hydroxymethyl dUTP, but not dTTP or alpha-putrescinyldeoxythymidine triphosphate, confirming that both thymine and alpha-putrescinylthymidine in phi W-14 DNA are formed from hydroxymethyluracil at the polynucleotide level . The synthesis of phi W-14 DNA is unusual because (i) thymine is formed from hydroxymethyluracil at the polynucleotide level, (ii) the hypermodification forming alpha-putrescinylthymine is essential, and (iii) thymine and alpha-putrescinylthymine must be made in the correct proportions . Complementation tests showed that the mutants defined three genes involved in DNA polymerization and two genes involved in post-replicational modification. J Virol, 1982 Jul, 43(1), 365 - 7 In vitro packaging of bacteriophage T7 DNA requires ATP; Masker WE; Removal of nucleoside triphosphates from extracts prepared from bacteriophage T7-infected Escherichia coli results in a stringent requirement for added ATP to form infective phage particles by in vitro packaging of bacteriophage T7 DNA . Optimal packaging efficiency was achieved at a concentration of about 1.25 mM . Other nucleoside triphosphates could be substituted for ATP, but none of the common nucleoside triphosphates was as effective as ATP in promoting in vitro encapsulation. J Invest Dermatol, 1982 Jul, 79 Suppl 1, 60s - 64s The collagen alpha-2 chain gene; Tolstoshev P et al.; A number of DNA sequences specific for collagen messenger RNAs and genes have been isolated, cloned in bacterial plasmids or bacteriophages, and studied in detail . Such sequences have been used to study regulatory mechanisms underlying the production of type I collagen in fibroblasts in culture, fibroblasts after viral transformation, and in tissues and organs during embryonic and fetal development . It is clear that a variety of mechanisms, transcriptional, translational and post-translational, are used by cells to regulate collagen production . The study of isolated collagen gene fragments coding for the alpha 2 collagen chain in sheep and chick have shown that many genes are very large, and are interrupted by as many as 50 intervening sequences . Additionally, the structure of the genes in the regions coding for the helical regions of the protein provides evidence that collagen genes may have arisen from the reduplication of a DNA segment containing a primordial collagen gene sequence . The availability of specific cloned collagen gene sequences will allow the precise chromosomal location of the collagen genes as well as the number and the linkage relationships between these genes . In addition, genetic disorders of connective tissue where alterations in collagen structure are implicated will now be amenable to analysis at the DNA level. J Bacteriol, 1982 Jul, 151(1), 487 - 90 Novel selection for tetracycline- or chloramphenicol-sensitive Escherichia coli; Craine BL; A method for selecting tetracycline- or chloramphenicol-sensitive Escherichia coli cells from a population of predominantly resistant cells is described . This method depends on the inability of drug-sensitive cells to induce lambda receptors in the presence of chloramphenicol or tetracycline, protein synthesis-inhibiting drugs . The addition of bacteriophage lambda vir to a mixture of drug-sensitive and drug-resistant cells, induced for lambda receptors in the presence of tetracycline or chloramphenicol, preferentially kills the drug-resistant cells (which are capable of inducing lambda receptors) . The result is a culture enriched for the sensitive cells . Several common strains used for transformation were compared for their ability to be selected . E . coli 294 was found to be superior. Proc Natl Acad Sci U S A, 1982 Jul, 79(14), 4298 - 302 "Nonrandom" DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method; Poncz M et al.; We describe a rapid "nonrandom" DNA sequence analysis procedure that facilitates the nucleotide sequence determination of large contiguous regions of DNA . The method consists of cloning a restriction endonuclease fragment of interest into bacteriophage M13 followed by construction of a series of nuclease BAL-31 deletion mutants originating from a single site in M13 that is close to the DNA insert . Determination of the size of the deletion mutant is accomplished by hybridization to a complementary single-stranded probe derived from M13 containing that total insert followed by nuclease S1 treatment . Single-stranded M13-insert DNAs of progressively smaller sizes are isolated and analyzed by using a site-specific M13 DNA primer and the dideoxy chain-termination method . In this way, analysis of the DNA sequence proceeds from one end of the total insert to the other in a nonrandom fashion due to generation of a controlled overlapping set of deletion mutants. Biochimie, 1982 Jul, 64(7), 495 - 502 Cloning of a nitrogen fixation (nif) gene cluster of Azospirillum brasilense; Quiviger B et al.; Homology was detected between the structural genes for the nitrogenase complex of K . pneumoniae (nifHDK genes) and the total DNA of several Azospirillum strains . Bacteriophage lambda gt 7-ara6 was used to construct a gene bank of A . brasilense strain 7000 DNA and a recombinant phage carrying a 6.7 kb Eco RI fragment, termed AbRI, was selected by hybridization with the K . pneumoniae nif probe . Using heteroduplex analysis the extent of the homology of the AbRI fragment and the K . pneumoniae nif genes was found to be approximately 5 kb . Proteins encoded by the AbRI fragment were examined after infection of E . coli minicells. Zh Mikrobiol Epidemiol Immunobiol, 1982 Jul, (7), 79 - 82 {Analysis of insertions of the determinant of drug resistance to kanamycin and chloramphenicol into bacteriophage lambda att80}; Shatalin KIu et al.; The insertion sites of elements Tn9 and Tn601 which determine chloramphenicol and kanamycin resistance have been detected restriction analysis . The functioning of transposons i.e . their stability or instability, has been found to influence the specificity of their insertions into the genome of lambda att80 bacteriophage . During transposition from stable integration sites both transposons are inserted into the regions of the lambda att80 bacteriophage genome, definite for each transposon . However, during transposition from the site of unstable integration both determinants of drug resistance are inserted into different regions of the phage genome. Mikrobiologiia, 1982 Jul-Aug, 51(4), 632 - 5 {Effect of freezing modes on the survival of Escherichia coli bacteriophages}; Tsutsaeva AA et al.; The object of this work was to study the effect of freezing down to--196 degrees C at different cooling and warming rates on the survival of T3, T4 and phiX174 phages . Phage particles survived when T3 phage was frozen at a rate of 20-400 degrees/min and phiX174 phage at a rate of 20-45 degrees/min . The survival rate of T4 phage was highest when it was frozen at a rate of 45 degrees/min . The survival of the phages depended also on the regime of warming . The susceptibility of the phages to freezing correlated with their sensitivity to osmotic shock in NaCl and sucrose solutions. Proc Natl Acad Sci U S A, 1982 Jul, 79(14), 4362 - 6 Conservative integration of bacteriophage Mu DNA into pBR322 plasmid; Liebart JC et al.; In order to clarify the first step in Mu integrative recombination, we have infected a bacterial strain harboring the plasmid pBR322 and isolated Mu DNA in a supercoiled form associated with this plasmid . These structures show an association of Mu with PBR322 without any preliminary replication. J Gen Microbiol, 1982 Jul, 128(7), 1631 - 4 Isolation and mapping of glutathione reductase-negative mutants of Escherichia coli K12; Davis NK et al.; Two independent mutants defective in glutathione reductase (EC 1.6.4.2) were isolated in an Escherichia coli K12 strain lysogenized with bacteriophage Mu . The prophage was lost (and the ability to reduce glutathione regained) by 32% of the xylose-positive transductants when T4GT7 was used as the vector, but the markers were not cotransduced by P1 . Similarly, the prophage site and malA were cotransduced by T4GT7 but not by P1 . The gor gene maps between min 77 and 78 on the E . coli genome, and the mutation causes no growth defect. J Bacteriol, 1982 Jul, 151(1), 62 - 7 Promoter mapping and selection of operator mutants by using insertion of bacteriophage Mu in the argECBH divergent operon of Escherichia coli K-12; Beny G et al.; The analysis of a large number of Arg mutants obtained by inserting phage Mu in the argECBH cluster of genes confirmed the "facing" arrangement proposed earlier for the promoters of argE (argEp) and argCBH (argCBHp) and clarified remaining ambiguities regarding the localization of argEp . Casadaban and Cohen's Mu d lac phages (M . Casadaban and S . N . Cohen, Proc . Natl . Acad . Sci . U.S.A . 76:4530-4533, 1979) were used to construct strains where either an intact or a truncated lacZ gene was fused to argC or argB . Several operator-constitutive mutations could be selected for in such strains; the mutations affected both arms of the cluster, thereby defining one common operator region for both directions of transcription. Gene, 1982 Jul-Aug, 19(1), 127 - 38 Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin; Moir D et al.; A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector . The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide . In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the Mr-value) is the major in vitro translation product of RNA from that tissue . Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene. Biokhimiia, 1982 Jul, 47(7), 1212 - 5 {Effect of the antitumor antibiotic bleomycin on DNA polymerase activity}; Naryzhnyi SN et al.; Treatment with bleomycin activates considerably a repair synthesis of DNA in rat liver chromatin in vitro and can cause loosening of the nucleoprotein complex, which facilitates the accessibility or repair enzymes for lesions in chromatin DNA . The bleomycin action on DNA-template increases severalfold the rate of synthesis catalyzed by DNA polymerase beta inhibits the activity of DNA polymerase I from Escherichia coli and suppresses severalfold the activity of DNA polymerase alpha and DNA polymerase of bacteriophage T4 . The effect of bleomycin consists in a prevailing increase of nicks and minimal gaps in DNA as compared to the rise of moderate gaps, thus suggesting that bleomycin is a gamma-mimetic. Biochim Biophys Acta, 1982 Jun 30, 697(3), 371 - 7 Heat- and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA; Wang RY et al.; 5-methylcytosine residues in DNA underwent deamination at high temperatures . Furthermore, their rate of deamination at neutral or alkaline pH was greater than that of cytosine residues in DNA . As sources of {14C}-5-methylcytosine-containing DNA, we used bacteriophage XP-12 DNA, in which 5-methylcytosine residues completely replace C residues, and calf thymus DNA experimentally substituted with {14C} 5-methylcytosine residues . Upon incubation at 95 degrees C in a physiological buffer or at 60 degrees C in 1 M NaOH, the respective rates of deamination of 5-methylcytosine residues were about 3- and 1.5-times those on cytosine residues . Under the same conditions, the free 5-methyldeoxycytidine was converted to thymidine more rapidly than deoxycytidine was converted to deoxyuridine . The reactions at physiological pH and elevated temperature suggest that deamination of 5-methylcytosine residues may yield a significant portion of spontaneous mutations in vivo, especially in view of the lack of thymine-specific mismatch repair systems with specificity and efficiency comparable to that of uracil excision repair systems. Eur J Biochem, 1982 Jun 15, 125(1), 63 - 8 DNA synthesis at a fork in the presence of DNA helicases; Kuhn B et al.; In a mixture of Escherichia coli DNA polymerase III holoenzyme, single-strand-binding protein, artificially forked lambda bacteriophage DNA with primer annealed to the leading side of the fork, dNTPs and ATP, DNA synthesis is enhanced by helicase II, less so by helicases, I, III or rep protein of E . coli or T4 phage helicase . The effect of helicase II depends on ATP, it is enhanced by helicase III, and it is not observed using DNA polymerase I or T4 DNA polymerase . In the absence of dNTPs helicase II is less active than helicase I or T4 helicase in unwinding the forked DNA . We believe that helicase II both shifts the forks and stimulates DNA polymerase III . The results support the conclusion derived from previous studies that helicase II is part of the DNA-synthesizing system of E . coli. J Biol Chem, 1982 Jun 10, 257(11), 5994 - 6 beta-Thalassemia in a Kurdish Jew . Single base changes in the T-A-T-A box; Poncz M et al.; We recently described a "non-random" sequencing procedure for DNA inserts in bacteriophage M13 using Bal 3 nuclease and the dideoxy chain termination method (Poncz, M., Solowiejczyk, D., Ballantine, M., Schwartz, E., and Surrey, S . (1982) Proc . Natl . Acad . Sci . U . S . A., in press) . Using this procedure, we have determined the nucleotide sequence of a cloned human beta-globin gene from a Kurdish Jew with beta +-thalassemia major . Comparison with the previously reported human beta-globin gene sequences (1-3) reveals a change in the "T-A-T-A" box . This region 5' to the capping site was previously demonstrated to be critical for the proper transcription in vitro of several different eukaryotic genes (4-7) . This is the first report of a T-A-T-A box modification found in association with a spontaneously occurring human genetic disorder . In addition to this mutation, other base changes, an insertion, and a deletion in the cloned gene were found in the 5' and 3' flanking regions. J Biol Chem, 1982 Jun 10, 257(11), 6529 - 36 The biosynthesis of membrane-bound M13 coat protein . Energetics and assembly intermediates; Zimmermann R et al.; The major coat protein of bacteriophage M13 spans the plasma membrane of infected cells prior to its assembly into extruding virus . It is initially made as a precursor, termed procoat, with a 23-residue leader sequence at its NH2 terminus . Procoat is found bound to the inner surface of the plasma membrane . The electrical potential of the cell membrane is required for procoat insertion and conversion to coat protein, although the order of these events has been unknown . We now report studies of the conversion of a mutant procoat (procoat-R6) from the virus M13am8H1R6 to mutant coat (coat R6) . The behavior of procoat-R6 differs from that of the wild type procoat in three respects . (i) Pulse-labeled procoat-R6 is largely found inserted across the cell membrane . This suggests that the active site of leader peptidase is on the periplasmic membrane face and that insertion normally precedes processing for wild type procoat as well . (ii) Despite the greater abundance of inserted procoat-R6 in M13am8H1R6-infected cells than inserted procoat in wild type infections, procoat-R6 is processed to coat-R6 more slowly than procoat is converted to coat . (iii) The membrane insertion and proteolytic processing of procoat-R6 are almost completely insensitive to uncouplers . We present a working model for the energetics and assembly intermediates of coat protein biosynthesis. J Biol Chem, 1982 Jun 10, 257(11), 6184 - 93 Nucleic binding affinity of bacteriophage T4 gene 32 protein in the cooperative binding mode; Bobst AM et al.; This study reports on various parameters which affect the binding stoichiometry for complexes of bacteriophage T4 gene 32 protein (P32) and single stranded polynucleotides (determined by UV absorbance and fluorescence quenching) and presents results of a quantitative electron spin resonance assay to determine physiologically effective binding affinity differences of nucleic acid binding proteins . The assay employs macromolecular spin probes (spin-labeled nucleic acids) which are used to determine the fraction of saturation in competition experiments with unlabeled nucleic acids . It was found that the fraction of complexed spin-labeled polynucleotides can be directly monitored by ESR with a two-component analysis approach when ligands such as poly(L-lysine), gene 5 protein (P5) of filamentous bacteriophage fd, and gene 32 protein (P32) of bacteriophage T4 are used . The ESR data unequivocally show that: 1) the binding stoichiometry for poly(L-lysine), P5 and P32 is nucleotide/lysine, 4 nucleotides/P5 monomer, and 10 nucleotides/P32 monomer, respectively; and 2) under physiologically relevant buffer conditions the relative affinity of P32 in the cooperative binding mode for polythymidylic acid is about 4 times greater than for polydeoxyinosinic acid and about 12 times greater than for polyinosinic acid, and the relative affinity of P32 for polydeoxyinosinic acid is about 3 times greater than for polyinosinic acid. J Biol Chem, 1982 Jun 10, 257(11), 6488 - 93 Intermediate stages in enzymatic replication of bacteriophage fd duplex DNA; Geider K et al.; Using purified enzymes, double strand replication of phage fd DNA has been dissected into several intermediate steps . (i) Phage fd gene 2 protein cleaves supercoiled phage fd replicative form at a specific site in the viral strand (Meyer, T . F., Geider, K., Kurz, C., and Schaller, H . (1979) Nature 278, 365-367) . (ii) Relaxed covalently closed circular replicative form DNA which is also formed by gene 2 protein as a side product in the initiation reaction preceding replication is converted into supercoils by DNA gyrase . (iii) The enzyme forms a noncovalent complex at the generated nick that is necessary for initiation of subsequent unwinding . (iv) The Escherichia coli rep helicase (rep protein) and E . coli DNA binding protein I unwind the double-stranded DNA . (v) Concomitant DNA replication by E . coli DNA polymerase III holoenzyme results in the formation of rolling circle intermediates . The double-stranded core of the rolling circle remains in an open form, thus allowing continued synthesis during several rounds of replication . (vi) Processing of replicated viral DNA can be subdivided into the cleavage and the circularization of viral single strands . Comparative studies of fd and phi X174 replication in vitro have revealed differences in the kinetics of individual steps besides an apparent contrast in the conformation of rolling circle intermediates in the electron microscopy where fd DNA features extended tails rather than looped-back structures observed for phi X174 DNA. J Virol, 1982 Jun, 42(3), 767 - 72 Identification of some bacteriophage T4 prereplicative proteins on two-dimensional gel proteins; Cook KS et al.; Profiles of bacteriophage T4 early proteins resolved by a two-dimensional nonequilibrium pH gradient electrophoresis system (P . Z . O'Farrell, H . M . Goodman, and P . H . O'Farrell, Cell 12:1133--1142, 1977) are presented . Over 65 phage-induced proteins were resolved . Amber or deletion mutants were used to identify 17 proteins in the gel patterns as the products of specific genes. J Virol, 1982 Jun, 42(3), 1123 - 6 Escherichia coli mutant which restricts T7 bacteriophage has an altered RNA polymerase; Shanblatt SH et al.; We have previously described an Escherichia coli K-12 mutant, Y49, which restricts the growth of bacteriophage T7 and causes the accumulation of short DNA molecules and head-related particles during infection . We now show that the basis for these effects is the inability of the T7 gene 2 product to inactivate the Y49 RNA polymerase during infection, similar to what has been shown by DeWyngaert and Hinkle (J . Biol . Chem . 254:11247--11253, 1979) for the BR3 and tsnB strains of E . coli. J Lab Clin Med, 1982 Jun, 99(6), 895 - 907 A new method of screening for inherited disorders of galactose metabolism; Paigen K et al.; A method has been developed for detecting elevated levels of galactose and galactose-1-phosphate in routine blood samples of newborns and has been successfully applied as a screening procedure for galactosemia in several laboratories . The procedure utilizes a strain of Escherichia coli that becomes resistant to bacteriophage C21 in the presence of galactose . The presence of galactose or galactose-1-phosphate is detected as a zone of bacterial growth around blood spots placed on a dish in which the bacteria are otherwise killed by phage . The diameter of the growth zone is proportional to the concentration of total blood galactose . The procedure has the potential of detecting all metabolic abnormalities that can lead to the accumulation of galactose or galactose-1-phosphate . Over a million newborn infants have now been tested by this procedure in three countries . In the New England Regional Screening Program, 12 galactosemic children were detected in 825,403 live births . One additional case, a sibling of a previously diagnosed galactosemic, was not allowed any milk feeding and was detected by an enzymatic test of cord blood . The combined frequency was 1:63,000 . No problems of interference by antibiotics were apparent . Use of the test in Switzerland and in Japan also allowed the discovery of infants with UDP galactose 4-epimerase deficiency . Our experience suggests that the test provides an efficient and reliable means of detecting congenital defects of galactose metabolism with a very low frequency of errors . It can also be used to monitor blood galactose levels in the management of galactosemic children. J Bacteriol, 1982 Jun, 150(3), 1400 - 4 Chromosomal location of att P7, the recA-independent p7 integration site used in the suppression of Escherichia coli dnaA mutations; Chesney RH et al.; Conjugational and transductional analyses were used to determine the chromosomal location of attP7, the recA-independent integration site used by bacteriophage P7 to suppress host dnaA mutations . The site of integration was found to be between tolC and dnaG . An increase in transduction frequencies was observed for markers surrounding attP7 when P7 was integrated . Under these conditions, all pairs of markers in this region, including those separated by attP7, were cotransduced at frequencies higher than normal, indicating the possible production of P7 specialized transducing particles. J Bacteriol, 1982 Jun, 150(3), 1130 - 7 Regulation of phenylalanine biosynthesis in Escherichia coli K-12: control of transcription of the pheA operon; Gowrishankar J et al.; Bacteriophage lambda ppheA-lac was used to obtain strains of Escherichia coli K-12 in which pheA and lacZ are each transcribed from a separate pheA promoter . Mutants in which both beta-galactosidase and chorismate mutase P-prephenate dehydratase (the pheA gene product) were derepressed were isolated, and a transacting gene (pheR) was identified . pheR was mapped at min 93 on the E . coli chromosome; pheR mutants acquired the wild-type phenotype when either F117 (which covers the 93-min region) or F116 (which covers min 59 to 65) was introduced into the cell . A rifampin resistance mutation, rpoB366, was found to derepress transcription of the pheA operon . pheR and rpoB366 affected two different systems for the phenylalanine-mediated control of pheA . A mutation in miaA (trpX), a gene known to be involved in attenuation in the tryptophan operon, was also shown to increase transcription of the pheA gene. Cell, 1982 Jun, 29(2), 329 - 35 The replication of bacteriophage f1: gene V protein regulates the synthesis of gene II protein; Model P et al.; 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. Proc Natl Acad Sci U S A, 1982 Jun, 79(11), 3403 - 7 Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22; Goldenberg D et al.; Newly synthesized tail spike polypeptide chains mature from trypsin- and NaDodSO4-sensitive unfolded chains to trypsin- and NaDodSO4-resistant native trimers with a t1/2 of 5 min at 30 degrees C . A metastable intermediate in subunit folding and assembly was trapped by chilling and isolated by electrophoresis through nondenaturing gels in the cold . A fraction of the intermediate could be matured into native trimers in vitro by incubating at physiological temperature . Mixing experiments with electrophoretically distinct mutant proteins showed that the precursor that matured in vitro represented three tail spike polypeptide chains already associated with each other but not fully folded . Identification of this intermediate reveals that the processes of polypeptide chain folding and subunit assembly are coupled in this large structural protein. Proc Natl Acad Sci U S A, 1982 Jun, 79(11), 3398 - 402 P1 site-specific recombination: nucleotide sequence of the recombining sites; Hoess RH et al.; Site-specific recombination between molecules of bacteriophage P1 DNA occurs at sites called loxP and requires the action of a protein that is the product of the P1 cre gene . Although recombination between two loxP sites is very efficient, recombination between loxP and a unique site in the bacterial chromosome (loxB) is inefficient and generates two hybrid lox sites called loxR and loxL . We present here the nucleotide sequences of all four lox sites . Analysis of these sequences indicates that (i) a region of extensive homology is not present at the loxP X loxB crossover point, in contrast to the 15-base pair common-core sequence in the bacteriophage lambda att sites, and (ii) the sites contain a region of dyad symmetry with 8- to 13-base pair inverted repeats separated by an 8- to 9-base pair sequence . The loxP X loxB crossover point falls in the sequence that separates the inverted repeats, and deletions that remove either the left or the right inverted repeat of loxP inactivate the site . These two observations are consistent with the conclusion that the region of dyad symmetry is important in los recombination . We have shown further that the loxP X loxP crossover point occurs in a 63-base pair sequence containing the loxP X loxB crossover point, suggesting that, despite the great difference in efficiencies of the two reactions, the crossover points may occur at the same place in both . Explanations for the different recombination properties of the various lox sites are discussed. J Virol, 1982 Jun, 42(3), 951 - 62 Transcriptional regulation of bacteriophage SPO1 protein synthesis in vivo and in vitro; Heintz N et al.; There are six classes of SPO1 transcripts which are, at least partially, regulated independently of each other . Analysis of proteins made in infections by phage mutants defective in DNA synthesis, or in genes which positively control transcription, permitted each protein to be assigned to one transcription class . Most of the late proteins belong to transcription class m2l . There seem to be few, if any, phage proteins in the l class whose mRNA synthesis depends absolutely on phage DNA synthesis, UV irradiation of host cells allowed the detection of many additional early proteins . The early proteins detected in vivo were compared with proteins synthesized in vitro, using bacterial or gp28 phage-modified RNA polymerase in an Escherichia coli cell-free system . Proteins characterized in vivo as belonging to the e transcription class could be made efficiently in vitro only when transcription was performed by bacterial RNA polymerase . em proteins could be elicited through the use of either bacterial or gp28 polymerase, indicating that their genes can be transcribed in either the early or the middle mode. Am Rev Respir Dis, 1982 Jun, 125(6), 640 - 3 Bacteriophage types of Mycobacterium tuberculosis in the United States; Jones WD Jr et al.; Strains of Mycobacterium tuberculosis from various geographic areas within the continental United States were typed according to their susceptibility to 4 mycobacteriophages . Of 462 wild isolates studied, 34% were phage type 1 (previously designated A0), 42% were type 2 (A1), 2.6% were type 5 (A4), 13% were type 7 (A6), and 20.1% were type 8 (B) . Distribution of types was essentially unaffected by geographic location, sex, age, or ethnic origin of the patient or by resistance of the isolate to antituberculosis drugs . Major types were further divided by susceptibility of the strains to lysis by 8 auxiliary phages, 2 of which were phages newly evaluated in this study . A new numbering system is proposed for designating major phage types of M . tuberculosis. Cell, 1982 Jun, 29(2), 337 - 45 Translational control of bacteriophage f1 gene II and gene X proteins by gene V protein; Yen TS et al.; 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. Gene, 1982 Jun, 18(3), 297 - 307 Bacteriophage T4 as a generalized DNA-cloning vehicle; Casna NJ et al.; We have designed a method for inserting foreign DNA segments into bacteriophage T4 . A plasmid containing T4 DNA is opened within the T4 sequence and the foreign DNA is inserted in vitro . Recombination in vivo, between T4 and the doubly chimeric plasmid, results in insertion of the foreign DNA into the genome of viable T4 phage . We have demonstrated the method by inserting a 203-bp DNA fragment from the lactose operon of Escherichia coli, into the dispensable region of the rIIB gene of T4 . With minor modifications, the method should make possible the cloning of very large DNAs into any one of a large number of sites on the T4 chromosome. Gene, 1982 Jun, 18(3), 231 - 8 M13 vectors for selective cloning of sequences specifying initiation of DNA synthesis on single-stranded templates; Ray DS et al.; M13 cloning vectors have been developed for the selection of DNA sequences capable of directing initiation of DNA synthesis on single-stranded templates . These vectors are derived from viable M13 mutants containing large deletions in the region of the complementary strand origin . The deletion mutants are defective in the conversion of viral single strands to the duplex replicative form (SS leads to RF) both in vivo and in vitro, give a reduced phage yield and form turbid plaques . A receptor site for foreign single strand initiation determinants has been introduced into the mutants by the insertion of EcoRI linker sequences at the deletion sites . Specific cloned sequences from bacteriophage G4 RF and from Co1E1 DNA restore a clear plaque type and normal phage growth . Selection of clear-plaque isolates obtained by transfection with RF from one of these vectors, M13 delta E101, carrying inserted Co1E1 HaeIII fragments resulted in the selective cloning of one specific fragment, the HaeIII-E fragment . Insertion of either the H or L strand of the HaeIII-E fragment into the M13 delta E101 viral strand gives a clear plaque phenotype, indicating the presence of initiation determinants on both the H- and L-strands of the Co1E1 HaeIII-E fragment . These cloning vectors provide a new means for the functional dissection of replication origins and for the identification of DNA sequences that determine the enzymatic mechanism of discontinuous synthesis along the length of the bacterial chromosome . The ability to assess initiation capability on the basis of plaque morphology also provides a means for rapid genetic analysis of initiation determinants. Cell, 1982 Jun, 29(2), 561 - 71 DNA intermediates in transposition of phage Mu; Harshey RM et al.; Transposable genetic elements can insert into DNA sites that have no homology to themselves . Evidence that there is a physical linkage between a transposable element and its target DNA sequence during transposition comes from studies on bacteriophage Mu DNA transposition in which plasmids containing Mu DNA have been shown to attach to host DNA . We report the isolation of key structures, seen after induction of Mu DNA replication, after cloning lac operator into Mu DNA and using the lac repressor-operator interaction to trap Mu DNA on nitrocellulose filters . We have localized Mu sequences within these structures in the electron microscope by visualizing the lac operator-repressor interaction after binding with ferritin-conjugated antibody . This analysis shows that key structures contain replicating Mu DNA linked to non-Mu DNA and that replication can begin at either end of Mu. Proc Natl Acad Sci U S A, 1982 Jun, 79(11), 3560 - 4 Estimation of phylogenetic relationships from DNA restriction patterns and selection of endonuclease cleavage sites; Adams J et al.; The distribution of cleavage sites and their related sequences have been analyzed for 54 restriction endonucleases in the genome of human mitochondrial DNA; in three papova viruses, BK, simian virus 40, and polyoma; and in three bacteriophages, phi X174, fd, and G4 . The results show that the cleavage sites and related sequences for most of the restriction enzymes tested are distributed nonrandomly . These results (i) constitute prima facie evidence for the action of natural selection, either direct or indirect on the restriction sites, and (ii) suggest that estimates of phylogenetic relationship, based on a phenetic approach using restriction enzyme data, will be biased. Proc Natl Acad Sci U S A, 1982 Jun, 79(11), 3498 - 502 Bacteriophage lambda DNA packaging: scanning for the terminal cohesive end site during packaging; Feiss M et al.; Bacteriophage lambda packages the DNA of the related phage 21 poorly {Hohn, B . (1975) J . Mol . Biol . 98, 93--106} . To understand the nature of the packaging defect, the interaction of the cohesive end site (cos) specific for phage 21 (cos phi 21) with phage lambda terminase has been investigated . The ability of lambda terminase to cleave cos phi 21 was studied in vitro; lambda terminase cleaved cos phi 21 only 1% as well as it cleaved the phage lambda cohesive end site (cos lambda) . In vitro packaging experiments showed that the lambda and 21 packaging specificities observed in vivo are also found in vitro . The cos cleavage reaction was modified so that competition experiments could be performed; these experiments showed that cos phi 21 was unable to bind lambda terminase, thus identifying the nature of the defect . Previous work {Feiss, M., Fisher, R . A., Siegele, D . A., Nichols, B . P . & Donelson, J . E . (1979) Virology 92, 56--67} has shown that the base pairs giving lambda or 21 packaging specificity are at the left end of the chromosome, outside the 22-base-pair symmetry region that includes the annealed cohesive ends . Therefore, terminase binding to cos requires interactions with base pairs to the Nu1 side of the cohesive end symmetry segment . The evidence supports the proposition that cos consists of adjacent sites for binding of terminase and for nicking by terminase . Because cos phi 21 can be cut by lambda terminase to terminate DNA packaging, it is proposed that the terminase that binds and nicks at the initial cos site is brought into contact with the terminal cos site by the packaging process . Terminase recognizes and nicks the cohesive end sequence of the terminal cos without requiring the binding site. J Bacteriol, 1982 Jun, 150(3), 1122 - 9 Construction from Mu d1 (lac Apr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of lambda ppheA-lac; Gowrishankar J et al.; Bacteriophage Mu d1 (lac Aprr) was used to obtain strains of Escherichia coli K-12 in which the lac genes are expressed from the promoter of pheA, the structural gene for the enzyme chorismate mutase P-prephenate-dehydratase . A derivative of bacteriophage lambda which carries the pheA-lac fusion was prepared; the method used is generally applicable for the construction, from Mu dl lysogens, of specialized transducing lambda phage carrying the promoter-lac fusions . A restriction enzyme cleavage map of lambda ppheA-lac for the enzymes HindIII and PstI is presented. Mol Biochem Parasitol, 1982 Jun, 5(6), 391 - 400 The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum and identification of individual clones by hybridisation; Goman M et al.; The DNA of Plasmodium falciparum has been purified and fragmented with the restriction endonucleases EcoRI and HindIII . The fragments have been incorporated in vitro into derivatives of bacteriophage lambda to make libraries in which most of the parasite DNA is represented . By Southern hybridisation we have been able to recover from these libraries specific clones containing (a) repetitive DNA sequences, (b) rRNA gene(s) and (c) sequences homologous to an actin gene probe . Parasite DNA from two independent sources differs markedly in the pattern of its repetitive DNA visualised by hybridisation to our repetitive clone . By contrast, the rRNA genes of the two isolates prove to be carried on identically sized fragments. Biochim Biophys Acta, 1982 May 31, 697(2), 235 - 42 Cloning of genes for bacteriophage T5 stable RNAs; Ksenzenko VN et al.; One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage lambda XIII and the plasmid pBR322 as vectors . Recombinant DNA molecules were studied by hybridization with in vivo 32P-labeled T5 4-5 S RNAs on nitrocellulose filters . Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments . For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed . It was shown that the EcoRI 440 fragment contains the gene for tRNA 10 (tRNAAsp), the EcoRI/HindIII 220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNAHis), and the EcoRI/HindIII 960 fragment contains only a part of the gene for tRNA 9 (tRNAGln) . The arrangement of these genes on the physical map of T5 phage was as follows: -tRNAGln-tRNAHis-RNA III-RNA I-...-tRNAAsp. Science, 1982 May 28, 216(4549), 946 - 51 Suppression of transcription termination by phage lambda; Ward DF et al.; The bacteriophage lambda N gene product positively controls development by preventing termination of transcription at terminator sites critical to the sequential expression of phage genes . Many host transcription factors, including RNA polymerase, are involved in N gene action . Recent findings have shown that ribosomal proteins are also involved . The current understanding of how the N protein affects transcription termination is reviewed, and a possible model and current problems are discussed. J Biol Chem, 1982 May 25, 257(10), 5711 - 5 Characterization of the free radical of mammalian ribonucleotide reductase; Graslund A et al.; Mouse fibroblast 3T6 cells, selected for resistance to hydroxyurea, were shown to overproduce protein M2, one of the two nonidentical subunits of mammalian ribonucleotide reductase . Packed resistant cells gave an EPR signal at 77 K very much resembling the signal given by the tyrosine-free radical of the B2 subunit of Escherichia coli ribonucleotide reductase . Also, the M2-specific free radical was shown to be located at a tyrosine residue . Of the known tyrosine-free radicals of ribonucleotide reductases from E . coli, bacteriophage T4 infected E . coli and pseudorabies virus infected mouse L cells, the M2-specific EPR signal is most closely similar to the signal of the T4 radical . The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures can be explained by slightly different angles of the beta-methylene group in relation to the plane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around the tyrosines . The pronounced difference in microwave saturation between the E . coli B2 tyrosine radical EPR signal and the M2 signal could be due to their different interactions with unspecific paramagnetic ions or with the antiferromagnetically coupled iron pair, shown to be present in the E . coli enzyme and postulated also for the mammalian enzyme . A difference in the iron-radical center between the bacterial and mammalian ribonucleotide reductase is also observed in the ability to regenerate the free radical structure . In contrast to the B2 radical, the M2 tyrosine free radical could be regenerated by merely adding dithiothreitol in the presence of O2 to a cell extract where the radical had previously been destroyed by hydroxyurea treatment. Eur J Biochem, 1982 May 17, 124(2), 245 - 52 The interaction of the A and A* proteins of bacteriophage phi X174 with single-stranded and double-stranded phi X DNA in vitro; van der Ende A et al.; The binding of the bacteriophage phi X 174-coded A and A* proteins to single-stranded (ssDNA) and double-stranded (dsDNA ) phi X DNA was studied by electron microscopy . The interaction of the A* protein with ssDNA and dsDNA was also studied by sedimentation velocity centrifugation . It was shown that the binding of the A and A* proteins to ssDNA occurs in a non-cooperative manner and requires no or very little sequence specificity under the conditions used here . Both protein-ssDNA complexes have the same compact structure caused by intrastrand cross-linking through the interaction of protein molecules with separate parts of the ssDNA molecule . The A protein does not bind to phi X dsDNA in the absence of divalent cations . The A* protein does bind to dsDNA, although it has a strong preference for binding to ssDNA . The structure of the A* protein-dsDNA complexes is different from that of the A* protein-ssDNA complexes, as the former have a rosette-like structure caused by protein-protein interactions . High ionic strengths favour the formation of large condensed aggregates. Biochemistry, 1982 May 11, 21(10), 2570 - 2 Stereochemical course of nucleotidyl transfer catalyzed by bacteriophage T7 induced DNA polymerase; Brody RS et al.; The bacteriophage T7 induced DNA polymerase, consisting of the phage specified gene 5 protein associated with Escherichia coli thioredoxin, catalyzes the copolymerization of SP-dATP alpha S with dTTP, producing the alternating of polymer poly{dTs-A)} by a mechanism involving inversion of configuration at P alpha . Degradation of poly{d(5s-A)} by the nucleolytic action of E . coli DNA polymerase produced the dinucleotide pdTps-dA, whose configuration at the phosphorothioate diester was assigned as R by comparison of the phosphorus-31 nuclear magnetic resonance chemical shift (55.0 ppm downfield from H3PO4) with that of an authentic sample . Further degradation by alkaline phosphatase to Rp-dTps-dA (55.6 ppm downfield from H3PO4) confirmed the configuration . The stereochemistry provides no evidence of a double displacement mechanism. J Biol Chem, 1982 May 10, 257(9), 5286 - 95 Studies of in vitro transcription by calf thymus RNA polymerase II using a novel duplex DNA template; Kadesch TR et al.; Addition of 10 to 100 oligodeoxycytidylate residues to the 3'-OH termini of T7 bacteriophage DNA produces a highly efficient template for transcription in vitro with purified calf thymus RNA polymerase II . Transcription initiates rapidly and selectively at the oligo (dC) ends of such a template and essentially all of the active RNA polymerase II molecules are then committed to a long period of RNA chain elongation . This allows the direct study of the RNA chain elongation and termination reactions and also permits determination of the concentration of active RNA polymerase II that is present . From 15 to 25% of the RNA polymerase molecules in our current preparations are active in these reactions . RNA chain elongation by calf thymus RNA polymerase II is relatively slow (7 nucleotides/s) even at saturating substrate concentrations . The in vitro elongation process appears to be discontinuous, with elongating polymerase molecules pausing for significant periods at certain sequences along the DNA . There is a low, but measurable frequency of RNA chain termination at some sites; however, the majority of elongating transcripts can grow to large sizes (over 6000 nucleotides) . Surprisingly, over 60% of the active calf thymus RNA polymerase II molecules form a long DNA-RNA hybrid during in vitro transcription and displace the nontranscribed DNA from the template to produce a characteristic split end structure . DNA-RNA hybrids are also formed during transcription by RNA polymerase II from duplex DNA templates lacking 3' oligo(dC) tails, which takes place predominantly at single strand breaks or ends . Thus the transcriptional elongation reaction carried out by calf thymus RNA polymerase II in vitro differs in several respects from that which must take place in vivo. J Biol Chem, 1982 May 10, 257(9), 5211 - 9 A novel endonuclease specified by bacteriophage lambda . Purification and properties of the enzyme; Benchimol S et al.; A DNA endonuclease whose expression is under the control of the b region of bacteriophage lambda has been partially purified from an induced lambda lysogen . In a reaction that requires single-stranded DNA, ATP, and Mg2+, the lambda-induced endonuclease makes one double strand break in pBR322 and other covalently closed circular DNA molecules, converting these substrates into unit-length linear forms . The double strand break in pBR322 DNA occurs at one of several preferred sites . Linear DNA appears not to be a good substrate for the enzyme. J Biol Chem, 1982 May 10, 257(9), 5201 - 10 Bacteriophage lambda DNA packaging in vitro . The involvement of the lambda FI gene product, single-strand DNA, and a novel lambda-directed protein in the packaging reaction; Benchimol S et al.; The FI gene product (gp) of bacteriophage lambda is required during phage head assembly in vivo . Mutations in this gene lead to an accumulation of immature concatemeric lambda DNA and of proheads that appear normal and are competent for DNA packaging in vitro . This phenotype can be taken as evidence of a failure to couple DNA and proheads for packaging/maturation . In contrast to the requirement for gpFI in vivo, the packaging of lambda DNA in vitro occurs efficiently in the complete absence of gpFI . However, if ssDNA is included at the outset of the in vitro packaging reaction, DNA packaging is blocked . This block to packaging is relieved by addition of gpFI . Thus packaging of lambda DNA in vitro can be made dependent of gpFI by the inclusion of ssDNA at the outset of the reaction . Inhibition of DNA packaging by ssDNA appears to be mediated by a lambda b region-directed protein (packaging inhibitor, ben protein) that is present in the crude extracts of cells used to support the early steps of the packaging reaction . Neither ssDNA nor the packaging inhibitor alone has significant inhibitory effect on packaging; both components are required together to effect the inhibition that is relieved by gpFI . The packaging inhibitor was extensively purified and shown to have endonucleolytic activity . Several lines of evidence are presented to support the idea that both the inhibitory and endonucleolytic activities are functions of the same protein . Although gpFI relieves the inhibition imposed by the ben protein in packaging, gpFI fails to block the DNA cleavage activity of the ben protein in the standard endonuclease assay. Appl Environ Microbiol, 1982 May, 43(5), 1098 - 103 Uptake of bacteriophage f2 through plant roots; Ward RL et al.; A model system was designed to measure viral uptake through the roots of plants and translocation to distal plant parts . For this study, uptake of bacteriophage f2 was measured in corn and bean plants growing in hydroponic solutions . Few phage were detected in plants with uncut roots . However, when roots of both plant types were cut just before exposure to very high concentrations of phage, the amount of phage uptake was several orders of magnitude greater than with uncut roots, but still was considerably less than that which was theoretically possible . Furthermore, cut roots were rapidly repaired, thus inhibiting uptake, and the amount of uptake in plants with cut roots was proportional to phage exposure levels . Finally, phage were transported to all plant parts examined, but their survival times within each portion of the plants appeared to be of limited duration . All of these factors tend to minimize the possible public health significance associated with viral uptake through the root systems of plants. Ann Microbiol (Paris), 1982 May-Jun, 133(3), 377 - 86 {Attempts of ultrastructural and biochemical characterisation of cell wall deficient "Brucella" (L forms) (author's transl)}; Schmitt-Slomska J et al.; In previous studies the in vivo conversion of Brucella suis to L-form state was put in evidence . The L forms isolated from mouse spleen had original structural aspects in common: the absence of the cell wall layer and the extracellular multilayer "membranous" structures . The biological characterization of these L forms and the preliminary identification of specific chemical markers of the bacterial envelope is reported in the present study, performed with the stable L forms well-growing in the liquid media . The electron microscopy confirmed the absence of cell wall and the presence of numerous dense multilayer membranous structures in the L forms cultivated for a long time on appropriate media . This aspect was changed in the L forms adapted to growth on the ordinary medium for brucella: numerous small dense bodies limited by unit membrane were observed . The chemical analysis of stable L forms showed the absence of diaminopimelic acid, confirming the lack of peptidoglycan . The result of chemical determination in L forms of the Na-2-keto-3-deoxyoctonate was negative . However, biological assays suggested that outer membrane components such as LPS and receptors for the bacteriophage Weybridge remained in the L forms, albeit in reduced amount as compared to parental brucella. J Gen Virol, 1982 May, 60(Pt 1), 135 - 46 Kinetics and characterization of the proteins synthesized during infection by bacteriophage PM2; Brewer GJ et al.; Using two-dimensional gel electrophoresis, we have examined the proteins whose synthesis is stimulated in Alteromonas espejiana by infection with the membrane-containing bacteriophage PM2 . In addition to four virus structural proteins, 11 non-structural proteins have been resolved and identified by their apparent isoelectric points and molecular weights . The relative rate of synthesis of each of the proteins was determined during the course of infection . Synthesis of the earliest proteins began around 10 min after infection . Synthesis of the virus structural proteins as a group did not begin until about 25 min after infection . In contrast to these structural proteins, the rate of synthesis of most of the non-structural virus proteins began to decline between 30 and 35 min after infection . This time preceded the onset of cell lysis marked by ion leakage (47 min); it corresponded to the beginning of packaging of virus DNA, removing that DNA from replication and transcription . Protein processing could not be demonstrated by pulse-chase labelling . These 15 proteins account for all of the coding capacity of the virus DNA . The virus origin of 14 of these proteins was established in an in vitro transcription-translation system programmed by PM2 DNA. J Virol, 1982 May, 42(2), 595 - 601 Function of an internal bacteriophage T7 core during assembly of a T7 procapsid; Serwer P et al.; A DNA-free, proteinaceous procapsid of bacteriophage T7 (capsid I) has been shown in previous studies to consist of an external, spherical shell (envelope) and an internal, cylindrical core with fibrous projections that connect the core to the envelope . To determine the role of the core in assembly of the envelope of capsid I, the kinetics of appearance of capsid I and possible intermediates in capsid I assembly (AG particles) were determined in the presence and absence of the core . For obtaining these data, agarose gel electrophoresis was used and appeared to be a technique more accurate and efficient than techniques used for obtaining similar data in the past . The results of these experiments were: (i) in the presence of the core, AG particles behaved kinetically as intermediates in the assembly of capsid I; (ii) in the absence of the core, assembly of capsid I terminated prematurely and AG particles accumulated . These and other data have been interpreted by assuming that: AG particles are breakdown products of precursors of capsid I; these precursors have uncorrected errors in the assembly of their envelope; and a function of the core is to correct these errors. J Virol, 1982 May, 42(2), 583 - 94 Detection and characterization of agarose-binding, capsid-like particles produced during assembly of a bacteriophage T7 procapsid; Serwer P et al.; It has previously been shown that: (i) during infection of its host, the DNA bacteriophage T7 assembles a DNA-free procapsid (capsid I), a capsid with an envelope differing physically and chemically from the capsid of the mature bacteriophage, and (ii) capsid I converts to a capsid (capsid II) with a bacteriophage-like envelope as it packages DNA . Lysates of phage T7-infected Escherichia coli contained a particle (AG particle) which copurified with capsid II during buoyant density sedimentation, velocity sedimentation, and solid support-free electrophoresis, but was distinguished from capsid II by its apparent diversity during electrophoresis in agarose gels . Treatment of AG particles with trypsin converted most of them to particles that comigrated with trypsin-treated capsid II during electrophoresis in agarose gels . Irreversible binding of AG particles to agarose gels was shown to contribute to the apparent diversity of AG particles during agarose gel electrophoresis . The results of quantitation of AG particles and of capsid I and capsid II in lysates of a nonpermissive host infected with T7 amber mutants suggested that, in site of their capsid II-like properties, most AG particles were produced during assembly of capsid I and not during DNA packaging . The presence of AG particles in T7 lysates explains contradictions in previous data concerning the pathway of T7 assembly. J Virol, 1982 May, 42(2), 422 - 31 Late events in T4 bacteriophage DNA replication . III . Specificity of DNA reinitiation as revealed by hybridization to cloned genetic fragments; Halpern M et al.; Through the use of the technique of hybridization to cloned genes, the site specificity of the reinitiation of T4 DNA replication was examined at late times after infection, when a large amount of DNA had accumulated in the infected cell . Replication was examined under two conditions; (i) when there was recombination but the repair of the recombinants was inhibited, and (ii) when recombination was followed by covalent joining . When no covalent repair of recombinant was allowed, reinitiation occurred in the areas known to be also involved in the initiation of replication of the parental molecule: thus late reinitiation, if covalent joining is prevented, is site specific . When there was covalent joining, reinitiation displayed no apparent site specificity . The results are discussed in light of the possibility that at late times after infection recombinant intersections act as primers . The similarity of the model proposed to the "break-and-copy" model for lambda phage and the fitness of the proposed model to the genetic phenomena described by others are emphasized. J Gen Microbiol, 1982 May, 128 (Pt 5), 973 - 9 Mode of action of colicin S8; Garcia ME et al.; The mode of action of colicin S8 has been studied and compared with that of other colicins . Two minutes after the addition of colicin S8 to bacteria a considerable proportion of the colicin is inaccessible to trypsin . Treatment of bacteria with colicin S8 renders them more sensitive to lysis by sodium dodecyl sulphate and also inhibits motility . Like colicins K and E1, colicin S8 provokes lysis of bacteria superinfected with bacteriophage T4 . Colicin S8, unlike colicin E2, prevents replication of bacteriophage T4 . The incorporation of isoleucine or uracil into bacteria is inhibited by colicin S8 but, unlike colicins K and E1, the effect is multiplicity-dependent . A rapid method of titration of colicin S8 is described . The results are discussed with emphasis on the possible rearrangements at the bacterial surface and the possibility that there is more than one type of specific receptor for colicin S8. Proc Natl Acad Sci U S A, 1982 May, 79(10), 3120 - 4 Trp aporepressor production is controlled by autogenous regulation and inefficient translation; Kelley RL et al.; We constructed a trpR-lacZ gene fusion that specifies a hybrid protein that has full beta-galactosidase activity . The gene fusion was associated with the unaltered trpR transcription and translation control region; thus, hybrid beta-galactosidase production was an indicator of expression of the trp aporepressor (trpR) operon . To facilitate in vivo expression studies, a DNA segment containing the trpR-lacZ gene fusion and the trpR controlling region was transferred to bacteriophage lambda and subsequently inserted into the bacterial chromosome . Analyses of hybrid beta-galactosidase production showed that the trpR operon is regulated autogenously but that the rate of synthesis of aporepressor varies only 4- to 5-fold in response to changes in the intracellular concentration of tryptophan . Under comparable conditions, the trp operon is regulated by trp repressor approximately 70-fold . Therefore, the operators of the trp operon and the trpR operon must have very different affinities for trp repressor in vivo . The promoter controlling trpR expression was found to be moderately active . Nevertheless, there are only about 50-300 molecules of trp aporepressor per cell . The low aporepressor level appears to be due to inefficient translation of trpR mRNA. J Bacteriol, 1982 May, 150(2), 916 - 24 Roles of cell surface components of Escherichia coli K-12 in bacteriophage T4 infection: interaction of tail core with phospholipids; Furukawa H et al.; The cell surface of Escherichia coli K-12, reconstituted from the OmpC protein, lipopolysaccharide, and the peptidoglycan layer, was active as a receptor for phage T4, resulting in the contraction of the tail sheath and the penetration of the core through the cell surface (Furukawa et al., J . Bacteriol . 140:1071--1080, 1979) . In the present work the process of DNA ejection from the contracted T4 phage particle was studied . Contracted phage particles were adsorbed to phospholipid liposomes by the core tip . This adsorption resulted in ejection of phage DNA . Either phosphatidylglycerol or cardiolipin was active for the DNA ejection . A proton motive force across the liposome membrane was not required for these processes . The process of DNA ejection, however, was temperature dependent, whereas the adsorption of the core tip to liposomes took place at 4 degrees C . Based on these observations together with those in the previous paper, the process of T4 infection of E . coli K-12 cells is discussed with special reference to the roles of cell surface components. Proc Natl Acad Sci U S A, 1982 May, 79(9), 2763 - 7 Isolation and characterization of rat skeletal muscle and cytoplasmic actin genes; Nudel U et al.; Southern blots of rat genomic DNA indicate the existence of at least 12 EcoRI DNA fragments containing actin gene sequences . By using specific probes and stringent conditions of hybridization, it was found that only one of these fragments contains sequences of the skeletal muscle alpha-actin gene . Recombinant bacteriophages originating from eight different actin genes were isolated from rat genomic DNA libraries . One of them, Act 15, contains the skeletal muscle actin gene . Another clone, Act I, contains a gene coding for a cytoplasmic actin, identified tentatively as the beta-actin gene . Both genes have a large intron very close to the 5' end of their transcribed region, followed by several small introns . DNA sequence analysis and comparison with the available data on actin genes in other organisms indicated an interesting relationship between the positions of introns and the evolutionary relatedness . Several intron sites are conserved from at least the echinoderms to the vertebrates; others appear to be present in some actin genes and not in others. Cell, 1982 May, 29(1), 219 - 25 Instability of transposase activity: evidence from bacteriophage mu DNA replication; Pato ML et al.; Transposition of genetic elements involves coupled replication and integration events catalyzed in part by a class of proteins called transposases . We have asked whether the transposase activity of bacteriophage Mu (the Mu A protein) is stable and capable of catalyzing multiple rounds of coupled replication/integration, or whether its continued synthesis is required to maintain Mu DNA replication . Inhibition of protein synthesis during the lytic cycle with chloramphenicol inhibited Mu DNA synthesis with a half-life of approximately 3 min, demonstrating a need for continued protein synthesis to maintain Mu DNA replication . Synthesis of specific Mu-encoded proteins was inhibited by infecting a host carrying a temperature-sensitive suppressor, at permissive temperature, with Mu amber phages, then shifting to nonpermissive temperature . When Aam phages were used, Mu DNA replication was inhibited with kinetics essentially identical to those with chloramphenicol addition; hence, it is likely that continued synthesis of the Mu A protein is required to maintain Mu DNA replication . The data suggest that the activity of the Mu A protein is unstable, and raise the possibility that the Mu A protein and other transposases may be used stoichiometrically rather than catalytically. Proc Natl Acad Sci U S A, 1982 May, 79(10), 3097 - 100 Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins; Steitz TA et al.; It is shown that there is a structural similarity between the presumed DNA-binding regions of the Escherichia coli catabolite gene activator protein ("CAP") and the cro repressor protein ("cro") from bacteriophage lambda . The correspondence between the two proteins is particularly striking for a structural unit consisting of two consecutive alpha-helices . The 24 alpha-carbon atoms that constitute the two-helical structural units in the two proteins can be superimposed with a root-mean-square disagreement of 1.1 A . It is shown that this agreement is very unlikely to be due to a chance correspondence . For both CAP activator and cro repressor proteins it is the second alpha-helix of the two-helical unit that has been proposed to bind within the major groove of left-handed or right-handed B DNA, respectively {McKay, D . B . & Steitz, T . A . (1981) Nature (London) 290, 744-749; Anderson, W . F., Ohlendorf, D . H., Takeda, Y . & Matthews, B . W . (1981) Nature (London) 290, 754-758} . The structural correspondence between CAP and cro seen here, together with other recent evidence of sequence homologies between cro, CAP, and other proteins that bind double-stranded DNA, suggests that the two-helical unit is likely to be a common feature of many DNA-binding proteins . The results also suggest that some principles of specific protein-double-stranded DNA interaction may be general and include recognition via alpha-helices fitting into the major groove of the DNA. Nature, 1982 Apr 29, 296(5860), 828 - 32 Enzymatic synthesis of bacteriophage fd viral DNA; Meyer TF et al.; An enzyme system with requirements similar to those for replication of phage fd replicative form (RF) DNA in bacteriophage fd-infected cells has been reconstituted with purified fd gene 2 protein, and DNA polymerase III holoenzyme, DNA binding protein I and rep-protein (rep-helicase) of Escherichia coli . The system generates viral circular single strands, which are infective for E . coli spheroplasts . Parental and newly synthesized DNA are covalently connected in early stages of replication, as expected for DNA replication using the rolling circle mechanism . Single-stranded tails of the rolling circle intermediates are cleaved after a full round of replication by gene 2 protein and circularized by the same enzyme molecule. Biochim Biophys Acta, 1982 Apr 26, 697(1), 25 - 30 Rapid inactivation of bacteriophage T7 by ascorbic acid is repairable; Richter HE et al.; Treatment of bacteriophage T7 with ascorbic acid resulted in the rapid accumulation of single-strand breaks in the DNA with double-strand breaks appearing only after incubation times of 20 min or longer . The single-strand breaks were responsible for a rapid inactivation of the phage as assayed by immediate plating of the phage-bacteria mixture on nutrient agar . Incubation of the phage-bacteria mixture in liquid medium prior to plating allowed a host cell reactivation process to repair the nicks and reactivate the phage . Non-reversible inactivation of the phage was a slower process which could be correlated with the appearance of double-strand breaks in the phage DNA . Host cell reactivation of the phage was also manifested in the phenomena of delayed lysis and delayed appearance of the concatemeric DNA replication intermediate. J Biol Chem, 1982 Apr 10, 257(7), 3896 - 904 Initiation, pausing, and termination of transcription in the threonine operon regulatory region of Escherichia coli; Gardner JF; The 6 S leader RNA transcript from the Escherichia coli threonine operon controlling region was synthesized in vitro using purified RNA polymerase and restriction fragment DNA templates . The terminated leader transcript was analyzed by RNase T1 digestion followed by electrophoresis on 20% polyacrylamide, 8 M urea gels . Oligonucleotides of 7, 8, 13, 15, and 35 bases in length were detected and correlated with the known DNA sequence . The kinetics of RNase T1 digestion indicated that the RNA forms extensive secondary structure, especially at the 3'-terminus of the transcript . The sites of transcription initiation were determined by labeling the 5'-end of the transcript with {gamma-32P}ATP or -GTP followed by direct RNA sequencing . The DNA sequence preceding the initiation site shows homology with the equivalent regions of other bacterial and bacteriophage promoters . The transcription termination sites were determined by mapping of the RNase T1 oligonucleotides arising from the 3'-terminus of the transcript . Comparison of the mobilities of the 3'-oligonucleotides with the mobilities of standards on 20% polyacrylamide, 8 M urea gels indicated that the RNA contains a heterogeneous 3'-terminus . The two predominant oligonucleotides were CU7 and CU8 . The 3'-terminus of the transcript also contains a region of dyad symmetry immediately preceding a stretch of uridine residues, characteristic of other rho-independent transcripts . In addition, kinetic studies indicated that RNA polymerase pauses approximately 50 base pairs upstream from the site of termination . The pause site appears to be immediately distal to another region of dyad symmetry. Eur J Biochem, 1982 Apr 1, 123(2), 415 - 20 Biological activity and a partial amino-acid sequence of Escherichia coli DNA-binding protein I isolated from overproducing cells; Beyreuther K et al.; DNA-binding protein I from Escherichia coli was purified from cells carrying the ssb gene on a multicopy plasmid . In comparison to the strain without the recombinant plasmid the DNA-binding protein was over-produced more than 20-fold . The amount of the protein was measured after the purification steps by gel electrophoresis and radial immunodiffusion . The protein was purified to homogeneity and was active in replication assays like the wild-type DNA-binding protein . The assays were enzymatic replication of single-stranded and double-stranded fd DNA . E . coli DNA-binding protein I was further subjected to amino acid sequence analysis . A monomer of the protein consists of 187 residues which correspond to a molecular weight of 19715 with 5% error in analysis . The sequence of the amino-terminal 40 residues was determined and includes several basic residues of the protein . Sequence comparison between the DNA-binding protein I from E . coli and that coded by bacteriophage fd reveals similarities suggesting that DNA-binding protein I may use amino-terminal residues for binding to DNA like the phage protein. J Bacteriol, 1982 Apr, 150(1), 16 - 26 Fusions of flagellar operons to lactose genes on a mu lac bacteriophage; Komeda Y; Previous studies have defined 29 genes necessary for synthesis of the Escherichia coli flagellar apparatus . This study analyzed the transcriptional control of flagellar genes, using Mu d (Apr lac) phage to generate flagellar mutants by insertion . These mutants contained operon fusions of flagellar genes to the lac genes of the Mu d phage and allowed the measurement of flagellar operon expression by detection of beta-galactosidase activity . These fusion mutants expressed the enzyme activity constitutively, and an autogenous regulation mechanism was not revealed . Lambda transducing phages carrying these chromosomal fla-lac fusions were also isolated and used to examine the effect of different fla mutations on expression of each flagellar operon . The results showed that flagellar operons are divided into six classes; (class 1) the flbB operon, which controls all of the other flagellar operons; (class 2) the flaU and flbC operons, which are controlled by the flbB operon gene products and are not required for the expression of other Fla operons; (class 3) the flbA, flaG, flaD, flaN, flaB, and flaA operons, which are under flbB operon control and are required for the expression of other fla operons; (class4) the flaZ operon, which is controlled by the gene products of the group 1 and 3 operons and is required for hag transcription; (class 5) the mocha and flaS operons, which are controlled by the gene products of the group 1 and 3 operons; and (class 6) the hag operon . These results are discussed with respect to the possible assembly sequence of the fla gene products. Proc Natl Acad Sci U S A, 1982 Apr, 79(8), 2442 - 6 Adenoviral protein-primed initiation of DNA chains in vitro; Ikeda JE et al.; The initiation of DNA chains by the 80-kilodalton form of the adenovirus terminal protein has been studied . This protein, which can be covalently linked to dCMP, is isolated complexed to a 140-kilodalton protein possessing DNA polymerase activity . In the presence of adenovirus DNA-protein, the formation of the 80-kilodalton protein-dCMP complex requires the addition of ATP and nuclear extract from uninfected cells in addition to Mg2+ and dCTP . When single-stranded DNA is used in place of the adenovirus DNA-protein, the formation of the 80-kilodalton protein-dCMP complex occurs in the absence of ATP and nuclear extract . In the presence of the four dNTPs, the complex yields DNA chains of various sizes between 100 and 300 nucleotides . The products formed with bacteriophage phi X174 single-stranded circular DNA as the template are site specific, predominantly derived from the sequences between nucleotides 2363 and 2977 and between nucleotides 3760 and 4206 . These small dNA chains are blocked at their 5' ends with the 80-kilodalton protein but possess free 3'-OH ends that are susceptible to degradation by exonuclease III and can be elongated to replicative form II products with DNA polymerase I of Escherichia coli or eukaryotic DNA polymerase beta preparations . A protein priming model explaining the different requirements for initiation with adenovirus DNA-protein and with phi X174 DNA is presented. J Virol, 1982 Apr, 42(1), 91 - 9 Nucleotide sequences at the phi X gene A protein cleavage site in replicative form I DNAs of bacteriophages U3, G14, and alpha 3; Heidekamp F et al.; Gene A protein, a bacteriophage phi X174-encoded endonuclease involved in phi X replicative form (RF) DNA replication, nicks not only phi X RFI DNA but also RFI DNAs of several other spherical single-stranded DNA bacteriophages . The position of the phi X gene A protein nick and the nucleotide sequence surrounding this site in RF DNAs of the bacteriophages U3, G14, and alpha 3 were determined . Comparison of the nucleotide sequences which surround the nick site of the gene A protein in RF DNAs of phi X174, G4, St-1, U3, G14, and alpha 3 revealed that a strongly conserved 30-nucleotide stretch occurred in RF DNAs of all six phages . However, perfect DNA sequence homology around this site was only 10 nucleotides, the decamer sequence CAACTTGATA . The present results support the hypothesis that, for nicking of double-stranded supercoiled DNA by the phi X gene A protein, the presence of the recognition sequence CAACTTGATA and a specific gene A protein binding sequence upstream from the recognition sequence are required . The sequence data obtained so far from phages U3, G14, St-1, and alpha 3 have been compared with the nucleotide sequences and amino acid sequences of both phi X and G4 . According to this comparison, the evolutionary relationship between phages G4, U3, and G14 is very close, which also holds for phages alpha 3 and St-1 . However, the two groups are only distantly related, both to each other and to phi X. J Virol, 1982 Apr, 42(1), 12 - 9 Mechanism of replication of bacteriophage phi X174 XX . Sensitivity of nascent DNA to single-strand-specific nucleases; Matthes M et al.; We reported earlier that dephosphorylated nascent phi X174 viral strand DNA molecules were less extensively degraded from the 5' end by spleen exonuclease than were non-nascent molecules . Experiments described here revealed that the insensitivity to the 5'-OH end-specific nuclease was more evident among the longer molecules in the population than among the shorter, all of the molecules being less than unit length in size . The smallest molecules in the population were about as sensitive to the enzyme as the control molecules and hence must possess unblocked 5'-terminal nucleotides . Degradation of the nascent DNA with the 3' end-specific snake venom phosphodiesterase revealed only a small enrichment for {3H}thymidine near the 3' end, seemingly insufficient to account completely for the apparent insensitivity of the longer molecules to spleen exonuclease . When the nascent molecules were isolated without the use of proteolytic enzymes, some pronase-sensitive material was found associated with the DNA, particularly the longer molecules . We suggest that the resistance of the longer nascent (pronase-treated) molecules to spleen exonuclease occurs because they have remnants of the viral gene A or A* protein covalently bound to the 5' end. J Bacteriol, 1982 Apr, 150(1), 429 - 32 Determining the phoM map location in Escherichia coli K-12 by using a nearby transposon Tn10 insertion; Wanner BL et al.; A phoR strain was constructed with transposon Tn10 inserted near the phoM+ locus . This was done without any prior knowledge of the phoM map location . Subsequently, we defined the phoM map position by screening tetracycline-sensitive (Tcs) derivatives for mutants which were both alkaline phosphatase negative (ther phoR phoM double mutant phenotype) and auxotrophic simultaneously . Some of these mutants were Thr- . Bacteriophage P1-mediated transductions were used to confirm that phoM and its nearby Tn10 insertion were closely linked to thr . Unexpectedly, 7 of 10 mutants analyzed also had mutations unlinked to the phoM-thr-Tn10 region . These may represent a new type of Tn10-promoted molecular event which is caused by transposition of a Tn10 end (IS10). J Virol, 1982 Apr, 42(1), 301 - 5 Mechanism of replication of bacteriophage phi X174 XIX . Initiation of phi X174 viral strand DNA synthesis at internal sites on the genome; Matthes M et al.; Bacteriophage phi X174 viral strand DNA molecules shorter than genome length found late in the infectious cycle in Escherichia coli were 5' end labeled with 32P . Hybridization of the 32P-labeled molecules to restriction enzyme fragments of phi X replicative form DNA revealed an excess of phi X molecules whose 5' ends mapped in HaeIII fragments Z3 and Z4 in comparison with fragments Z1 and Z2 . This suggests that initiation of phi X174 viral strand DNA synthesis may occur at internal sites on the complementary strand . There are several appropriately located sequences that might serve as n' (factor Y) recognition sequences and thereby facilitate discontinuous synthesis of the viral strand. J Virol, 1982 Apr, 42(1), 176 - 85 Transfection of Escherichia coli spheroplasts with a bacteriophage Mu DNA-protein complex; Chase CD et al.; We disrupted bacteriophage Mu particles by freeze-thaw treatment and recovered the DNA by CsCl density gradient centrifugation . This CsCl-purified DNA had a buoyant density which was indistinguishable from that of phenol-extracted Mu DNA . It was, however, 10(3) times more infective than phenol-extracted DNA for spheroplasts of exoV endI Escherichia coli . Infectivity was destroyed by proteinase K as well as by pancreatic DNase, indicating that the infective form was a DNA-protein complex . The infective properties of the complex demonstrated that the protein protects . Mu DNA against degradation by exonuclease V and that it serves at least one other function in bacteriophage Mu infection . The infectivity of the CsCl-purified DNA was due to a small class of highly infective molecules which sedimented 1.2 . times faster than phenol-extracted Mu DNA on neutral sucrose gradients . This change in sedimentation rate is best explained by the formation of protein-linked circular monomers or linear dimers of Mu DNA . In vitro labeling of the DNA-protein complex, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that the CsCl-purified DNA contained a noncovalently associated 65,000-dalton polypeptide . A 65,000-dalton protein was also found to be a minor component of the bacteriophage Mu particle . No protein was found in phenol-extracted Mu DNA . These results suggest that the 65,000-dalton protein is necessary for successful phage infection and is normally injected into the host cell with the Mu genome. J Virol, 1982 Apr, 42(1), 1 - 11 Cloned bacteriophage phi X174 DNA sequence interferes with synthesis of the complementary strand of infecting bacteriophage phi X174; van der Avoort HG et al.; The insertion of a particular phi X DNA sequence in the plasmid pACYC177 strongly decreased the capacity of Escherichia coli cells containing such a plasmid to propagate bacteriophage phi X174 . The smallest DNA sequence tested that showed the effect was the HindII fragment R4 . This fragment does not code for a complete protein . It contains the sequence specifying the C-terminal part of the gene H protein and the N-terminal part of the gene A protein, as well as the noncoding region between these genes . Analysis of cells that contain plasmids with the "reduction sequence" showed that (i) the adsorption of the phages to the host cells is normal, (ii) in a single infection cycle much less phage is formed, (iii) only 10% of the infecting viral single-stranded DNA is converted to double-stranded replicative-form DNA, and (iv) less progeny replicative form DNA is synthesized . The reduction process is phi X174 specific, since the growth of the related G4 and St-1 phages was not affected in these cells . The effect of the recombinant plasmids on infecting phage DNA shows similarity to the process of superinfection exclusion. Eur J Biochem, 1982 Apr 1, 123(2), 253 - 60 Solid-phase sequence analysis of polypeptides eluted from polyacrylamide gels . An aid to interpretation of DNA sequences exemplified by the Escherichia coli unc operon and bacteriophage lambda; Walker JE et al.; An approach to sequencing proteins by the solid-phase method combined with isolation of proteins and polypeptides by gel electrophoresis is described . Mixtures of proteins or polypeptides resulting from digests are fractionated in the presence of dodecylsulphate in polyacrylamide gels . They are detected with Coomassie blue, eluted, selectively reacted with p