Phage

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 porous glass derivatives and sequenced in their amino-terminal regions with the aid of a new microsequencer . Alternatively they can be analysed or digested with enzymes and fingerprinted . It is a relatively rapid method of purifying proteins for sequence analysis which we have used to provide partial protein sequence data to complement DNA sequences . Nine genes, four from the unc operon of Escherichia coli encoding the alpha, beta, gamma and epsilon subunits of ATP synthase and five for capsid proteins of bacteriophage lambda, have been identified by this method.

Nucleic Acids Res, 1982 Mar 11, 10(5), 1579 - 91
Digestion of highly modified bacteriophage DNA by restriction endonucleases; Huang LH et al.; The ability of thirty Type II restriction endonucleases to cleave five different types of highly modified DNA has been examined . The DNA substrates were derived from relatively large bacteriophage genomes which contain all or most of the cytosine or thymine residues substituted at the 5-position . These substituents were a proton (PBS1 DNA), a hydroxymethyl group (SP01 DNA), a methyl group (XP12 DNA), a glucosylated hydroxymethyl group (T4 DNA), or a phosphoglucuronated, glucosylated 4,5-dihydroxypentyl group (SP15 DNA) . Although PBS1 DNA and SP01 DNA were digested by most of the enzymes, they were cleaved much more slowly than was normal DNA by many of them . 5-Methylcytosine-rich XP12 DNA and the multiply modified T4 and SP15 DNAs were resistant to most of these endonucleases . The only enzyme that cleaved all five of these DNAs was TaqI, which fragmented them extensively.

J Biol Chem, 1982 Mar 10, 257(5), 2556 - 62
Purification and characterization of normal and mutant forms of T4 endonuclease V; Nakabeppu Y et al.; Endonuclease V of bacteriophage T4 has been purified to physical homogeneity from T4D-infected Escherichia coli 1100 . The enzyme, whose molecular weight is 16,000, possessed two distinct catalytic activities, a pyrimidine dimer-DNA glycosylase and an apurinic/apyrimidinic endonuclease . They acted on UV-irradiated poly(dA) . poly(dT) in a sequential manner; the glycosylase cleaved the N-glycosyl bond between the 5'-pyrimidine of a dimer and the corresponding sugar and then the endonuclease hydrolyzed a phosphodiester bond on the 3'-side of the apyrimidinic site . The 5'-termini thus generated were phosphorylated by T4 polynucleotide kinase only after they had been subjected to direct photoreversal and then treated with alkaline phosphatase . By using two phage mutants, uvs-5 and uvs-13, it was shown that occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities . Suppression of the mutation of uvs-5 rendered both activities partially active . When the mutation of uvs-13 was suppressed, a mutant form of enzyme that possessed only a glycosylase activity was produced . This suggests that there are two distinct domains in a single enzyme, each of which corresponds to one of the activities.

Biofizika, 1982 Mar-Apr, 27(2), 237 - 43
{Model of reversible phage interaction with cell receptors}; Ivanitskii GR et al.; A model of the bacteriophage behavior at the reversible adsorption stage was investigated . The ratio of concentration of long fibres bound with receptors on bacterial surface to the total concentration of the fibres was taken to be efficiency range of reversible adsorption . To determine this value the expressions for concentrations of all the intermediate forms of the long fibres binding were obtained . The relationship between reversible adsorption efficiency and concentrations of receptors with various values of corresponding constants was computed . The conditions providing different types of cooperativeness in the behavior of the baseplate elements in the process of reversible adsorption were analysed.

Can J Biochem, 1982 Mar, 60(3), 232 - 42
Injection defect in alkylated and depurinated T7 bacteriophage: analysis by DNA ejection; Mamet-Bratley MD et al.; Using DNA ejection in vitro as a model, we have studied the DNA injection defect caused by alkylation and depurination of T7 bacteriophage . Phage was alkylated with 0.02 M methyl methanesulfonate for 2 h at 37 degrees C; alkylated phage was then incubated 24 h at 30 degrees C to induce depurination . These samples were treated with formamide to cause DNA ejection without dissociation of the phage capsid . After ejection, the phage preparations were analyzed by electron microscopy . DNA lengths in capsid-DNA complexes were measured; relative numbers of full, empty, and partially empty phage heads were determined . To establish the direction of DNA ejection, E . coli RNA polymerase was bound to capsid-DNA complexes . The results showed that DNA was partially ejected from both alkylated and depurinated phages . In the alkylated sample, RNA polymerase was bound to the DNA end distal to the capsid; this showed that ejection started from the genetic left end . We interpret these results to show, in confirmation of earlier results obtained by marker rescue, that alkylation causes T7 phage to partially inject its DNA, starting from the genetic left end . For depurinated phage, our results suggest that partial DNA injection is responsible, in this case as well, for the already documented injection defect.

Appl Environ Microbiol, 1982 Mar, 43(3), 659 - 63
Chemical factors influencing adsorption of bacteriophage MS2 to membrane filters; Farrah SR; Antichaotropic salts, such as magnesium sulfate, and metal chelators, such as citrate ions, promoted adsorption of bacteriophage MS2 to membrane filters . In contrast, compounds that disrupt hydrophobic interactions, such as chaotropic salts, urea, Tween 80, and ethanol, did not promote adsorption of MS2 to membrane filters and counteracted the ability of magnesium sulfate to promote such adsorption . These results provide evidence that magnesium sulfate promotes the association of MS2 with membrane filters primarily by strengthening hydrophobic interactions between the virus and the filters.

J Bacteriol, 1982 Mar, 149(3), 889 - 96
Construction of a genetic map for Caulobacter crescentus; Barrett JT et al.; RP4-mediated conjugation has been used to transfer large fragments of chromosomal material in Caulobacter crescentus . In this system, conjugation proceeds from multiple origins, and haploid recombinants are recovered at frequencies of 10(-6) and 10(-7) per donor cell . The data from five-factor crosses were subjected to computer-assisted crossover analyses as a rapid method to determine marker order . Using this information and data from additional two- and three-factor crosses mediated by RP4 or the generalized transducing bacteriophage phi Cr30, we constructed the first genetic map for C . crescentus.

Gene, 1982 Mar, 17(3), 323 - 35
Nucleotide sequence of the major early region of bacteriophage phi 29; Yoshikawa H et al.; The nucleotide sequence of the left end of bacteriophage phi 29 DNA has been determined . Together with data reported earlier (Yoshikawa et al., 1981), this sequencing comprises the major early genetic region of this viral genome (5708 bp) . Computer analysis of the DNA sequences revealed that there are up to fifteen open reading frames which could encode polypeptides containing more than thirty amino acids . The DNA sequence also revealed a number of potential regulatory signals, promoters and ribosome binding sites . The initiation and the termination of transcription and probable early gene products are discussed.

Z Naturforsch {C}, 1982 Mar-Apr, 37(3-4), 321 - 5
Periodicities of dinucleotide self-information values in phi X174 DNA; Furlong NB et al.; The natural DNA sequence of bacteriophage, phi X174, when analyzed as a "text" of dinucleotides, is shown to display an easily detectable degree of non-randomness by the distribution of values of dinucleotide self-information along the sequence . Self-information corresponding to occurrences of dinucleotides separated by a single nucleotide is found to be somewhat higher than the values which precede or follow it for every third nucleotide position along the sequence . Consequently autocorrelation coefficients of these values display a strong periodicity and harmonic analysis of the values shows a spike at a value of 3 . Self-information autocorrelation periodicity is used as a test of the effect of randomizing portions of the sequence . Any one or two of the three nucleotides in each triplet of the sequence can be chosen at random without losing dinucleotide self-information periodicity except when both the 1st and 3rd nucleotide of all of the triplets in the major phi X174 protein reading frame are randomized . Periodicity is also lost when sequences are generated by randomizing triplets . Autocorrelation and harmonic analysis also indicate other less marked periodic features of dinucleotide self-information values of the nature sequence; non-random features are suggested at periods of 12, 20 and 24 nucleotides.

J Bacteriol, 1982 Mar, 149(3), 1166 - 70
Stimulation of groE synthesis in Escherichia coli by bacteriophage lambda infection; Kochan J et al.; We found that infection of Escherichia cell by lambda results in at least a twofold stimulation in the rate of synthesis of one of the products of groE . To determine what lambda-coded factors were responsible for this stimulation, numerous phage lambda mutants carrying bio substitutions were analyzed for their ability to stimulate groE synthesis . Our results revealed that the main factor(s) which is responsible for stimulating groE synthesis is located between the endpoints of the lambda bio69 and lambda bio252 substitutions, a region of DNA coding for bet, gam, kil, and cIII.

J Bacteriol, 1982 Mar, 149(3), 1050 - 63
Effect of bacteriophage lambda infection on synthesis of groE protein and other Escherichia coli proteins; Drahos DJ et al.; We used two-dimensional gel electrophoresis to quantitate the changes in rates of synthesis that follow phage lambda infection for 21 Escherichia coli proteins, including groE and dnaK proteins . Although total protein synthesis and the rates of synthesis of most individual E . coli proteins decreased after infection, some proteins, including groE protein, dnaK protein, and stringent starvation protein, showed increases to rates substantially above their preinfection rates . Infection by lambda Q- affected host synthesis in the same way as infection by gamma+, whereas infection by lambda N- showed no detectable effect on host synthesis . Deletion of the early genes between att and N abolished the effect, and shorter deletions in this region gave intermediate effects . By this sort of deletion mapping, we show that a large part, though not all, of the effect of lambda infection on host protein synthesis can be ascribed to the early region that contains phage genes Ea10 and ral . We compared the changes in protein synthesis after infection with the changes that occur in uninfected cells upon heat shock or amino acid starvation . The spectrum of changes that occurred on infection was very different from that seen after heat shock but quite similar to that seen during amino acid starvation . Despite this similarity of the effects of lambda infection and starvation, we did not detect any increase in the level of guanosine tetraphosphate during infection . We show that the groE protein is the same protein as B56.5 of Lemaux et al . (Cell 13:427-434, 1978) and A protein of Subramanian et al . (Eur . J . Biochem . 67:591-601, 1976).

Ann Neurol, 1982 Mar, 11(3), 285 - 91
Detection of herpes simplex virus mRNA in latently infected trigeminal ganglion neurons by in situ hybridization; Tenser RB et al.; Latent infection of the trigeminal ganglion with herpes simplex virus type 2 (HSV-2) was studied in guinea pigs by in situ DNA hybridization . Frozen ganglion sections from animals killed during the period of latent virus infection were studied under nondenaturing conditions . Some sections were treated with deoxyribonuclease (DNase) or ribonuclease (RNase) before incubation with HSV DNA probes . HSV probes consisted of viral DNA nick translated and labeled in vitro with tritiated nucleotides . Bacteriophage lambda DNA, similarly prepared, was used as a control probe . The lambda probe was negative in all situations, including HSV-2-infected monolayer cells in cell culture . HSV-2 probes produced heavy label and, therefore, evidence of hybridization with HSV-2-infected monolayer cells . When HSV-2 probes were incubated with latently infected ganglion sections, hybridization was detected in 71% of guinea pigs and 46% of ganglia . Label was seen only in neurons, and in positive ganglia 0.3 to 5% of neurons were labeled . The amount of label was markedly decreased by pretreatment of ganglion sections with RNase but not DNase, indicating that the DNA probes hybridized to HSV messenger RNA in the latently infected ganglia.

Cell, 1982 Mar, 28(3), 531 - 41
Partially deficient methylation of cytosine in DNA at CCATGG sites stimulates genetic recombination of bacteriophage lambda; Korba BE et al.; Lambda bacteriophages grown on arl mutants of Escherichia coli ("Arl-" phages) display intermediate levels of cytosine methylation: less 5-methylcytosine (m5C) than phages grown on wild-type bacteria ("Arl+" phages) but more than phages grown on dcm mutants, and thus lacking the methylated sequences (Cm5CATGG) characteristic of E . coli K-12 bacteria ("Dcm-" phages) . "Arl-" phages are one twelfth as resistant to Eco RII restriction (recognition site CCATGG) as "Arl+" phages, but 40-fold more resistant than "Dcm-" phages . Chromatographic analyses show the 5-methylcytosine content of "Arl-" DNA to be one third that of "Arl+" DNA . Altered cytosine methylation frequency correlates with two previously described properties of "Arl-" phages, increased genetic recombination and unusual sensitivity of phage DNA to endonuclease S1, which are absent in phages grown on dcm or dcm arl bacteria . Methylated/unmethylated heteroduplex DNA prepared in vitro (one strand from Eco RII-modified phages/one from "Dcm-" phages) is highly recombinogenic but not S1-sensitive . We hypothesize that hemimethylated CCATGG sites in "Arl-" DNA are necessary and sufficient for enhanced recombination, and necessary but not sufficient for S1 sensitivity.

J Bacteriol, 1982 Mar, 149(3), 1071 - 81
Physical and genetic localization of ilv regulatory sites in lambda ilv bacteriophages; Gray JE et al.; A set of nine lambda dilv phages were used to transduce bacterial recipients containing point mutations or deletions in the ilv genes located at 84 min on the Escherichia coli K-12 chromosome . This genetic analysis indicated that two phages carry the entire ilvGEDAC cluster; others carry the complete ilvC gene and, in addition, bacterial DNA that extends to a termination point between ilvA and ilvC, within ilvD, within ilvE, or within ilvG . DNA extracted from the lambda dilv phages was digested with EcoRI, HindIII, KpnI, PstI, SalI, and SmaI . The restriction maps revealed that these phages were generated after insertion at four distinct insertion sites downstream (clockwise) of ilvC . The physical relationships between the various phages were further examined by electron microscopic heteroduplex analysis . The physical maps of the phages thus generated were straightforward and in complete accord with the genetic data . No evidence for genetic rearrangements of ilv DNA in the phage was obtained, thus validating conclusions based on the use of these phages in previous and ongoing research projects . Bacterial cells with deletions of the ilv genes were made lysogenic with lambda dilv phage to examine the regulation of ilv genes present in the phage . The results confirm previous studies showing that one site for control by repression and derepression is upstream (counterclockwise) of ilvG . It was shown, in addition, that the activities of dihydroxy acid dehydrase and threonine deaminase were increased when the prototrophic lysogens were grown with 20 mM leucine . Since this increase was exhibited even when the ilvG-linked control region was not carried by the lambda dilv phage, additional control sites must be located within the ilvEDA region of the ilvGEDA transcription unit.

Gene, 1982 Mar, 17(3), 247 - 58
Transcription termination: sequence and function of the rho-independent tL3 terminator in the major leftward operon of bacteriophage lambda; Luk KC et al.; For cloning, assaying the function and sequencing terminators, we have constructed the pD12 plasmid, in which the late promotor p'R of phage lambda controls the expression of the galK gene of the pK03 plasmid of McKenney et al . (1981) . The lambda tL3 terminator region was cloned in this plasmid between the promoter and the galK gene, and found to be 90-94% effective in preventing galactokinase expression in both rho+ and rho- hosts . Is is also active in vitro, both in the presence or absence of the rho factor . The termination point is located at 4320 bp to the left of the SL startpoint of the PL-RNA, just downstream of gene exo . We have sequenced 356 bp of the hitherto uncharted lambda DNA to the right of the TaqI cut, which in turn is 110 bp to the right of the b522 deletion at 63.9% lambda . The tL3 terminator has several features common to other rho-independent termination sequences, including an 81% G+C-rich region of 2X8-bp symmetry ("stem") with a 5-bp intervening "loop", partially overlapping and followed by a sequence transcribed into the pyrimidine-rich CCUUUCU-OH 3' terminus of the RNA . The termination point that follows the last U was determined by the S1 mapping technique.

Ann Microbiol (Paris), 1982 Mar-Apr, 133(2), 225 - 33
Antitermination of transcription by the N-gene protein of bacteriophage lambda: recent progress and remaining problems; Greenblatt J; The N-gene protein of bacteriophage lambda recognizes sequences called nut in the lambda early operons and acts to prevent termination of the transcription of these operons . Part of the mechanism of action of N protein involves binding to the nusA protein of Escherichia coli, a transcription termination factor which associates directly with RNA polymerase . It is likely that N protein forms a ternary complex with the nusA protein and RNA polymerase at the nut site . Evidence is presented that translating ribosomes are not involved in N-protein action . It is still not known how RNA polymerase is modified to become termination-resistent as a result of N protein action.

Eur J Biochem, 1982 Mar 1, 122(3), 515 - 8
Competition of rifampicin with binding of substrate and RNA to RNA polymerase; Kessler C et al.; The rate of formation of dinucleoside tetraphosphate, pppApU, from ATP and UTP by RNA polymerase on the A1 promoter of the mutant D111 of bacteriophage T7 is distinctly and specifically reduced not only by the third template-directed nucleotide, CTP, but also by CMP . The inhibitory effect of CMP is not changed when the enzyme contains prebound rifampicin . The synthesis of pppApU is also strongly reduced after preincubation of the enzyme with RNA . This inhibitory effect of RNA is, however, distinctly diminished by rifampicin bound to the enzyme prior to the addition of RNA . On the other hand RNA can suppress the specific binding of the antibiotic to the RNA polymerase subassembly alpha 2 beta.

Nucleic Acids Res, 1982 Feb 11, 10(3), 1105 - 12
Nucleotide sequence of the bacteriophage T4 gene 57 and a deduced amino acid sequence; Herrmann R; A 693 basepair cloned fragment of bacteriophage T4 DNA, which supports specifically growth of T4 amber mutants in gene 57, has been sequenced . A polypeptide can be deduced from this sequence, that is either 54 or 60 amino acids long depending which of two AUG codons, 18 nucleotides apart, are used for initiation . The size of this deduced polypeptide is compatible with the size of a single polypeptide (based on polyacrylamide gel electrophoresis) synthesized in vivo in E . coli under the direction of the cloned T4 DNA fragment.

Nucleic Acids Res, 1982 Feb 11, 10(3), 821 - 32
Oligonucleotide-directed mutagenesis of gene IX of bacteriophage M13; Simons GF et al.; The synthetic oligodeoxyribonucleotide pCGAAAGACTACAC has been applied as a site-specific mutagen to introduce a T leads to G transversion mutation at nucleotide position 1223 of the M13 DNA sequence . The in vitro-induced conversion of a TAT codon into a TAG at this position resulted in gene IX mutants with an amber mutant character thereby confirming that this reading frame defines a gene of an essential phage protein . The gene IX amber mutants obtained grew well on SuI (Ser) and SuIII (Tyr) suppressing strains but could not be propagated on SuII (Gln) and SuVI (Leu) strains . Complementation studies show that amber mutants in genes V and VII exert a polar effect on gene IX expression suggesting that these three contiguous genes form an operon . In addition, we demonstrate the in vitro synthesis of gene IX-protein in a coupled transcription-translation system.

J Biol Chem, 1982 Feb 10, 257(3), 1462 - 7
Photochemical cross-linking of the gene 5 protein.fd DNA complex from fd-infected cells; Paradiso PR et al.; The gene 5 protein.fd DNA complex, which is an intermediate in the life cycle of fd bacteriophage, was isolated from fd-infected cells . After ultraviolet irradiation, approximately 10% of the gene 5 protein was covalently linked to fd DNA via the sulfhydryl group of the single cysteine residue at position 33 in the gene 5 protein . When intact, infected cells were first irradiated with ultraviolet light and the gene 5 protein.DNA complex subsequently isolated, less than 1% of the bound protein in the complex was found to be cross-linked . While these results agree with our previous findings based on irradiation of the gene 5 protein.fd DNA complex formed from purified gene 5 protein and fd DNA, they differ from the results of Lica and Ray ((1977) J . Mol . Biol . 115, 45-59) who found high yields of cross-linked gene 5 protein.fd DNA complex after ultraviolet irradiation of intact cells.

J Virol, 1982 Feb, 41(2), 501 - 7
Adsorption of the tailed mycoplasma virus L3 to cell membranes; Haberer K et al.; The adsorption properties of the tailed bacteriophage L3 to Acholeplasma laidlawii cells were studied . Adsorption followed a biphasic curve . Reversibility and virus heterogeneity were not sufficient to explain the break in the adsorption curve . Binding studies showed that each colony-forming unit could bind about 350 virions . The electrostatic nature of L3 adsorption was indicated by the effect of cations, pH, and temperature on the adsorption rate constant . L3 adsorption appeared to have a requirement for Ca2+, which could not be replaced by the mono- and divalent cations examined . Ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid inhibition of adsorption was totally reversed by added Ca2+ . The effects of EDTA, proteases, and lectins on absorption indicated that membrane proteins are the L3 receptors . The model for L3 adsorption is a multivalent one involving lateral diffusion of adsorbed virions and receptor proteins.

Proc Natl Acad Sci U S A, 1982 Feb, 79(4), 1101 - 5
Two alternative mechanisms for initiation of DNA replication forks in bacteriophage T4: priming by RNA polymerase and by recombination; Luder A et al.; We show that bacteriophage T4 has two alternative mechanisms to initiate DNA replication; one dependent on Escherichia coli RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6), and one dependent on general recombination . Continued DNA synthesis under recombination-defective conditions was sensitive to rifampin, an inhibitor of RNA polymerase . On the other hand, DNA synthesis accelerated in spite of the present of rifampin if recombination occurred.

Proc Natl Acad Sci U S A, 1982 Feb, 79(3), 865 - 9
Isolation and chromosomal localization of unique DNA sequences from a human genomic library; Kao FT et al.; Recombinant bacteriophage lambda from a human genomic library were screened to indentify human DNA inserts having only unique sequences . Unique human inserts were found in about 1% of the phage screened . One recombinant phage, P3-2, was studied in detail . It contains a human insert of 14.7 kilobases with four internal EcoRI cleavage sites . A restriction map was constructed for EcoRI and BamHI sites . Hybridization of the 32P-labeled P3-2 probe to a Southern blot of EcoRI-digested total human DNA yielded distinct bands at positions corresponding to the human insert fragments contained in P3-2 . By using a series of human-Chinese hamster somatic cell hybrids containing unique combinations of human chromosomes, the human DNA segment in phage P3-2 was assigned to human chromosome 22 by blot hybridization and synteny analysis . In addition, another human DNA segment, 11.4 kilobases, in phage P3-10 was assigned to human chromosome 10 by similar procedures . With this approach, more unique DNA sequences can be isolated, assigned to specific human chromosomes, and used as genetic markers for gene mapping and linkage, polymorphism, and other genetic studies in the human genome.

Eur J Biochem, 1982 Feb, 122(2), 319 - 26
1H NMR studies of the binding of bacteriophage-M13-encoded gene-5 protein to oligo(deoxyadenylic acid)s of varying length; Alma NC et al.; The binding of gene-5 protein to oligo(deoxyadenylic acid)s varying in length from 2 to 16 nucleotides has been studied by titrating the protein with the oligonucleotides and recording the 1H NMR spectra at 360 MHz . To obtain information about the mode of binding of the protein the aromatic parts of the spectra have been analysed by performing spectral simulations, starting from the assignments obtained from nuclear Overhausfer enhancements at 500 MHz {Alma, N . C . M., Harmsen, B . J . M., Hull, W . E., Van der Marel, G., Van Boom, J.H., and Hilbers, C . W . (1981) Biochemistry, 20, 4419-4428} . The 1H NMR spectra of the complexes of gene-5 protein with (dA)8, (dA)12 and (dA)16 appear to be identical except for differences in linewidth . The 1H NMR spectra of the complexes with the smaller oligonucleotides (dA)2, (dA)3 and (dA)4 differ from each other and from the spectra obtained from the complexes with longer oligonucleotides . However, binding of all oligonucleotides basically influences the same aromatic residues, namely two tyrosines and one phenylalanine . In the protein-oligonucleotide complexes, one protein monomer covers three nucleotide residues, in contrast to the stoichiometry of 1:4 found for protein-polynucleotide complexes . It was found that the binding to oligonucleotides is cooperative and ionic-strength-dependent but far less so than found for the binding to polynucleotides.

Proc Natl Acad Sci U S A, 1982 Feb, 79(4), 983 - 7
Template-directed pausing in in vitro DNA synthesis by DNA polymerase a from Drosophila melanogaster embryos; Kaguni LS et al.; The activity of Drosophila melanogaster DNA polymerase alpha on DNA-primed single-stranded DNA templates has been examined . The DNA templates contain a 1471-nucleotide sequence from the heavy-strand origin region of mouse mtDNA inserted into the single-stranded bacteriophage vector M13Gori1 . Preferred sites for pausing of in vitro DNA synthesis have been mapped within the cloned mtDNA insert and in the G4 cDNA strand origin which is contained within the vector DNA . Analysis of nascent DNA strands from replicative intermediates has revealed that pause sites are discrete and lie both at the positions of predicted stable dyads and in regions lacking the potential for formation of such structures . The patterns of kinetic pause sites observed for Escherichia coli DNA polymerase III holoenzyme is qualitatively similar to that found for DNA polymerase alpha . A subset of the observed kinetic pause signals are recognized by E . coli DNA polymerase I under similar conditions.

Proc Natl Acad Sci U S A, 1982 Feb, 79(4), 1129 - 33
Quantitative model for gene regulation by lambda phage repressor; Ackers GK et al.; A statistical thermodynamic model has been developed to account for the cooperative interactions of the bacteriophage lambda repressor with the lambda right operator . The model incorporates a general theory for quantitatively interpreting cooperative site-specific equilibrium binding data . Values for all interaction parameters of the model have been evaluated at 37 degrees C, 0.2 M KCl, from results of DNase protection experiments in vitro {A . D . Johnson, B . J . Meyer, & M . Ptashne, Proc . Natl . Acad . Sci . USA (1979) 76, 5061-5065} . With these values, the model predicts repression curves at the divergent promoters PR and PRM that control transcription of genes coding for the regulatory proteins cro and repressor, respectively . At physiological repressor concentrations, repression at PR is predicted to be nearly complete whereas PRM is predicted to remain highly active . The results demonstrate the importance of cooperative interactions between repressor dimers bound to the adjacent operator sites OR1 and OR2 in maintaining a stable lysogenic state and in allowing efficient switchover to the lytic state during induction.

Eur J Biochem, 1982 Feb, 122(2), 381 - 6
The receptor of bacteriophage lambda: evidence for a biosynthesis dependent on lipid synthesis; Pages C et al.; Inhibition of lipid synthesis in cerulenin-treated cells or in a mutant strain defective in sn-glycerol-3-phosphate acyltransferase after glycerol deprivation, results in a marked decrease of insertion of lamB protein into the outer membrane . No lambda receptor was found in any other cell compartment or in the medium under these conditions . The LamB protein synthesis was inhibited by about 70% in the absence of lipid synthesis . The residual 30% protein produced during inhibition of fatty-acid or phospholipid synthesis, was probably incorporated into the outer membrane since no further incorporation was observed after resumption of these syntheses . Besides OmpF and OmpC protein {Bocquet-Pages, C., Lazdunski, C., and Lazdunski, A . (1981) Eur . J . Biochem . 118, 105-111}, at least four other proteins of the outer membrane are also subject to alteration of levels in the absence of lipid synthesis . Under these conditions the uptake of maltose, like the uptake of 5'AMP {Bocquet-Pages, C., Lazdunski, C., and Lazdunski, A . (1981) Eur . J . Biochem . 118, 105-111}, was inhibited as much as 60% . These results are discussed with regard to the biosynthesis and assembly of the outer membrane proteins.

J Virol, 1982 Feb, 41(2), 657 - 73
Involvement of the bacterial groM gene product in bacteriophage T7 reproduction . I . Arrest at the level of DNA packaging; Kuhn AH et al.; The multiplication of bacteriophage T7 is blocked in Escherichia coli M . The genetic determinant of this ability (groM) to inhibit T7 growth was transferred to an E . coli K-12 recipient by means of conjugation . We determined at which precise step T7 maturation is blocked . Phage-directed protein and DNA synthesis as well as degradation of host DNA were not qualitatively affected . Instead of infective phages, only preheads were produced . These, however, were maturable in vitro . The newly synthesized phage DNA accumulated in a concatemeric form and matured from its tetrameric or longer forms (very fast sedimenting DNA) only into its dimeric form (fast-sedimenting DNA) or longer forms . The following step, i.e., the maturation of the dimeric to unit-length DNA, was not observed . Since the concatemeric form of T7 DNA accumulated in spite of the presence of maturable preheads, it is likely that the maturation process was blocked at the level of DNA packaging . As intermediates in the packaging process, we found some prehead-DNA complexes . We interpreted these as true assembly intermediates (or breakdown products thereof), since the attached DNA was still in its concatemeric form . This shows that the very first DNA packaging step, the binding of the progeny DNA to the preheads, was obviously not blocked . Rather, a later step, such as the filling of the preheads with T7 DNA or the stabilization of completely packaged particles (i.e., the final cutting of the concatemers into unit-size length), was inhibited.

Biosci Rep, 1982 Feb, 2(2), 123 - 34
Autoregulation of repressor synthesis in bacteriophage lambda imm434; Pastrana R; A 550-bp DNA fragment derived from the immunity region of phage lambda imm434 and carrying the wild-type prm promoter has been inserted upstream of the lacZ gene, in the beta 2 region of an 'in-vitro'-constructed transducing phage . In strains lysogenized with this phage, the rates of transcription from the 434-prm promoter can be assayed as units of beta-galactosidase in the absence or in the presence of increasing levels of 434 repressor . The results obtained indicate the existence of a mechanism of autoregulation for repressor maintenance in strains lysogenic for wild-type phage 434 . The rate of transcription from prm in a (434) lysogen is of the same magnitude as that in a (lambda ) lysogen, and, as in the case of lambda , high levels of repressor repress prm whereas low levels stimulate it . An estimation of the strength of the leftward promoters located in the right half of the lambda beta 2 region compared with 434 prm was also obtained.

Proc Natl Acad Sci U S A, 1982 Feb, 79(3), 724 - 8
Simple rapid method for the synthesis of radioactively labeled cDNA hybridization probes utilizing bacteriophage M13mp7; Ricca GA et al.; Double-stranded cDNA sequences for rat alpha 1-acid glycoprotein and rat glutathione S-transferase mRNAs were inserted into the Pst I site of bacteriophage M13mp7 and used to develop a new method for preparing specific cDNA hybridization probes directly from cloned template DNA . A palindrome sequence surrounding the Pst I site in the vector DNA permitted single-stranded DNA isolated from the recombinant phage to fold back, thus forming a stable hybrid bounded on the ends by a large loop of M13mp7 single-stranded DNA and a small loop of inserted foreign DNA . A primer corresponding to an internal sequence of the foreign DNA was hybridized, then Escherichia coli DNA polymerase I was used to synthesize a 32P-labeled complementary DNA copy of the cloned inserted DNA . The single-stranded cDNA reaction product was easily isolated by subsequent sedimentation through alkaline sucrose gradients . Gel electrophoresis of the labeled cDNA product, after denaturation with glyoxal, indicated a single discrete band with an electrophoretic mobility corresponding to the length of the inserted DNA sequence . About 95% of the cDNA product formed S1 nuclease-resistant hybrids in hybridization reactions with excess RNA in solution . DNA sequences complementary to the M13mp7 vector DNA were not detected in the cDNA product . Thus, these M13mp7-derived probes are the functional equivalent of cDNA copies to mRNAs and can be employed for quantitative measurements of mRNA concentration . This simple, rapid method probably can be used for most cloned DNA sequences to yield single-stranded radioactively labeled DNA, without contaminating vector DNA sequences, for virtually any hybridization requirement.

J Gen Virol, 1982 Feb, 58(Pt 2), 263 - 71
Inability to transmit scrapie by transfection of mouse embryo cells in vitro; Borras MT et al.; Infectivity of nucleic acid from highly infectious mouse scrapie brains was studied by transfecting the nucleic acid in vitro prior to inoculation into animals . Foetal mouse brain and whole mouse embryo cultures were chosen as the cells used for the transfection . As an internal control, infectious nucleic acid was recovered from bacteriophage phi X174 and whole virus particles were obtained when cultures were transfected with herpes simplex virus DNA . In contrast, none of the animals inoculated with nucleic acid preparations derived from scrapie-infected tissues developed scrapie disease either with or without transfection procedures . These findings (i) show transfection techniques fail to elicit evidence of a scrapie-specific infectious nucleic acid and (ii) confirm the observation that scrapie is not a viroid.

J Bacteriol, 1982 Feb, 149(2), 694 - 9
Physical mapping and cloning of bacteriophage T4 anti-restriction endonuclease gene; Dharmalingam K et al.; We have proposed that the ability of T4 to produce non-glucosylated progeny after a single cycle of growth on a galU rglA rglB+ mutant of Escherichia coli is due to the initiation of the rglB+ function by a phage-coded, anti-restriction endonuclease protein . Based on this hypothesis, we screened T4 deletion mutants for failure to give a burst in this host . The absence of an arn gene in phage mutants lacking the 55.5- to 58.4-kilobase region is verified by their inability to protect secondary infecting non-glucosylated phage from rglB-controlled cleavage . A functional arn gene was cloned on plasmid pBR325, and the 0.8-kilobase insert DNA was shown to be homologous to the DNA missing in the arn deletion phage.

J Bacteriol, 1982 Feb, 149(2), 529 - 33
P1 transduction mapping of the trg locus in rac+ and rac strains of Escherichia coli K-12; Bitner RM et al.; The trg locus, which had been located at min 31 in the cotransduction gap in the terminus region of the chromosome of Escherichia coli, has been mapped by transduction with bacteriophage P1 . This locus exhibited no cotransduction with fnr when rac+ strains were used . If rac strains were used, which removed approximately 27 kilobase pairs of DNA, trg and fnr exhibited 8.2% cotransduction . Although this mapping of trg at min 31.1 considerably reduces the size of the cotransduction gap, trg exhibited no cotransduction with a Tn10 insertion located on the other side of the gap at min 34.2.

J Biol Chem, 1982 Jan 10, 257(1), 362 - 5
Properties of the N gene transcription antitermination protein of bacteriophage lambda; Greenblatt J et al.; The product of the N gene of bacteriophage lambda prevents the termination of lambda early transcription . Here we describe the physical properties of pure lambda N protein . N protein is small and very basic . The apparent Mr of N protein during electrophoresis in the presence of sodium dodecyl sulfate is 12,500 . It contains 22 mol % (arginine plus lysine) and only one methionine . The methionine residue is at the blocked NH2 terminal since N protein is not detectably shortened by reaction with cyanogen bromide . When use is made of the DNA sequence of the N gene region of lambda DNA (Franklin, N . C., and Bennett, G . N . (1979) Gene 8, 107-119), the lack of an internal methionine residue, the size, and the amino acid composition of N protein can be used to predict that N protein contains 107 amino acids (calculated Mr = 12,241) and that its coding sequence begins at position 223 of the lambda pL operon mRNA . N protein can be assayed by its ability to stimulate endolysin synthesis in vitro in a reaction programmed with lambda N- DNA . N protein activity is heat-stable and trypsin-sensitive . Its sedimentation velocity in a sucrose gradient and its Stokes' radius indicate that N protein is an extremely asymmetric monomer (f/fmin = 1.6) . The relationship between this high degree of molecular asymmetry and the sequence which N protein must recognize in lambda nucleic acid is discussed.

J Biol Chem, 1982 Jan 10, 257(1), 366 - 9
Structure of the tyrosyl radical in bacteriophage T4-induced ribonucleotide reductase; Sahlin M et al.; Ribonucleotide reductase induced by bacteriophage T4 in Escherichia coli contains an organic free radical necessary for enzymatic activity . Its EPR spectrum at 77K is similar to but not identical with that of the corresponding radical in the enzyme from uninfected E . coli studied previously . Isotope substitutions now show that the radical in the T4-induced enzyme also is localized to a tyrosine residue with its spin density delocalized over the aromatic ring of tyrosine . The difference between the radicals of the T4-induced and the E . coli ribonucleotide reductases, as reflected in the hyperfine patterns of their EPR spectra, is suggested to be due to slightly different radical geometries, resulting from a twist of about 10 degrees around the bond between the aromatic ring and the methylene group in the tyrosine radical . Hydroxyurea destroys the free radicals of both ribonucleotide reductases and also their catalytic activities . Both enzymes are considerably more sensitive to hydroxyurea during catalysis than in the noncatalytic state . However, when compared to the bacterial ribonucleotide reductase, the T4-induced enzyme shows an overall approximately 10 times higher sensitivity to hydroxyurea, judging from the drug concentrations needed to destroy the radicals and inhibit the activities . This result may reflect a difference in accessibility for the drug to the active sites of the enzymes.

Z Allg Mikrobiol, 1982, 22(9), 629 - 37
Alteration of interaction with virulent bacteriophage Ta1 during differentiation of Thermoactinomyces vulgaris; Kretschmer S; During its life cycle Thermoactinomyces vulgaris 1227 and 1261 substrate mycelium showed three modes of interaction with the virulent phage Ta1, each expressed at a distinct morphological stage . Primary mycelium arising from spores 50 min after start of germination was the only stage which propagated phages upon infection . However, infection of growing secondary mycelium or of late sporulation stages resulted in a loss of phage . The lack of phage production upon infection of growing secondary mycelium was not related to sporogenesis, since it appeared already 3.5 hours before the beginning of sporulation . If phages were added to the secondary mycelium at the beginning of spore formation, the phage genome became integrated in the developing spores in a heat-stable state . Allowing outgrowth of these prophage-carrier spores, phages were produced similarly as in germ tubes arising from normal spores infected at the time of inoculation . The growing secondary substrate mycelium was characterized by competence for the uptake of exogenous DNA . Since at the stage of competence phages were neither produced nor was phage DNA trapped, the earlier reported lack of transfection in undisturbed differentiating T . vulgaris is now understandable.

Hemoglobin, 1982, 6(1), 27 - 36
Construction of human gene libraries from small amounts of peripheral blood: analysis of beta-like globin genes; Poncz M et al.; We describe a rapid procedure for constructing cloned human genomic libraries from small amounts of peripheral blood . High molecular weight DNA is isolated from 5-20 ml peripheral blood, partially cleaved with Eco R1, and 8-22 kb fragments are cloned using bacteriophage Charon 4A and suitable E . coli host . Using the approach we have isolated and characterized several non-alpha globin clones from a Kurdish Jew with homozygous beta thalassemia . The ability to isolate suitable amounts of high molecular weight DNA from peripheral blood provides a relatively simple means of constructing human gene libraries representing a variety of hemoglobin disorders.

Dev Comp Immunol, 1982 Winter, 6(1), 171 - 4
Specificity for tail fibers of a humoral crab factor neutralizing T2 bacteriophage; Donnelly C et al.; It is now becoming clear that invertebrates, animals lacking the ability to make humoral antibodies, are capable of recognizing self from non-self . In several instances this recognition is manifest in normal ("unimmunized") animals by a rapid clearance of certain foreign materials from the circulation One example of such a rapid innate reaction involves the clearance of T2 bacteriophage from the blue crab (1) . Other studies have indicated that blue crab plasma contained a factor capable of neutralizing in vitro T2 bacteriophage and hence, by inference, was responsible for the in vivo clearance reaction with this virus (2) . The purpose of the work undertaken here was to establish the nature of the specificity of this in vitro neutralizing factor . The strategy involved utilizing purified T2 bacteriophage tail fibers to inhibitthe neutralization of T2 bacteriophage by crab plasma.

Biophys J, 1982 Jan, 37(1), 253 - 62
Bilayer acyl chain dynamics and lipid-protein interaction: the effect of the M13 bacteriophage coat protein on the decay of the fluorescence anisotropy of parinaric acid; Wolber PK et al.; Nanosecond fluorescence polarization anisotropy decay is used to determine the effect of the bacteriophage M13 coat protein on lipid bilayer acyl chain dynamics and order . The fluorescent acyl chain analogues cis- and trans-parinaric acid were used to determine the rate and extent of the angular motion of acyl chains in liquid crystalline (39 degrees C) dimyristoylphosphatidylcholine bilayers free of coat protein or containing the coat protein at a protein:lipid ratio of 1:30 . Subnanosecond time resolution was obtained by using synchrotron radiation as the excitation source for single photon counting detection . Previous measurements of Forster energy transfer from coat protein tryptophan to cis- or trans-parinaric acid have shown that these probes are randomly distributed in the bilayer with respect to the protein . The anisotropy decay observed for pure bilayers has the form of a rapid drop, followed by a nonzero constant region extending from roughly 3 ns to at least 12 ns . The magnitude of the anisotropy in the plateau region is simply related to the acyl chain order parameter . The effect of the M13 coat protein is to increase the acyl chain order parameter significantly while having only a small effect on the rate of angular relaxation . This behavior is rationalized in terms of a simple microscopic model . The order parameters for pure lipid and coat protein containing bilayers are compared to 2H-NMR values.

Biophys J, 1982 Jan, 37(1), 243 - 51
19F nuclear magnetic resonance studies of the coat protein of bacteriophage M13 in synthetic phospholipid vesicles and deoxycholate micelles; Dettman HD et al.; The nonlytic, filamentous coliphage M13 offers an excellent model system for the study of membrane-protein interactions . We prepare derivatives of the protein containing fluorine-labeled amino acids and use 19F nuclear magnetic resonance (NMR) to study the protein in both deoxycholate micelles and phospholipid vesicles . We have previously described the in vivo preparation of an m-fluorotyrosyl derivative of M13 coat protein and also a method for incorporation of high levels of this protein into small, uniformly sized phospholipid vesicles of defined composition . Herein we describe the in vivo preparation and the characterization of an m-fluorophenylalanine derivative . We simultaneously compare the environment and mobility of the tyrosine and phenylalanine residues (the former in the hydrophobic region of the protein and the latter in the hydrophilic regions) as influenced by bile salt detergent or lipid interactions.

Infect Immun, 1982 Jan, 35(1), 343 - 9
Characterization of an inducible bacteriophage from a leukotoxic strain of Actinobacillus actinomycetemcomitans; Stevens RH et al.; A bacteriophage, designated phi Aa17, was isolated by mitomycin C induction from leukotoxic Actinobacillus actinomycetemcomitans strains 651 . Electron microscopy of the virus revealed particles with regular, nonelongated, polyhedral heads, and tails consisting of a contractile sheath and core . Spikes emanated from the base of the tail . The head had a diameter of 70 nm . The fully extended tail sheath had a length of 127 nm and a diameter of 22 nm . In its contracted form, the tail sheath measured 47 nm in length and 25 nm in diameter . The phage had a buoyant density of 1.370 in CsCl, and its genome was found to be double-stranded DNA . A single-cycle growth curve revealed that the phage had a latent period of 30 min and a burst size of 435 PFU per cell . The host range of the phage was examined, and A . actinomycetemcomitans strains ATCC 29523 and ATCC 29524 were found to be phage sensitive, whereas strains Y4, ATCC 29522, 2043, 652, 651, 627, 2097, N27, 2112, and 511 were resistant . The host range of this virus does not suggest any association between the phage and leukotoxin production.

Mol Gen Genet, 1982, 186(1), 44 - 9
Participation of the host protein(s) in the morphogenesis of bacteriophage P22; Joshi A et al.; Spontaneous mutants of S . typhimurium resistant to thiolutin are conditionally non-permissive for phage P22 development (Joshi and Chakravorty 1979) . At 40 degree C non-infective phage particles are produced . Phage development in two nonpermissive hosts (18/MC4 and 153/MC4) has been studied in detail . The steps at which the phage morphogenesis is interfered with differ in the two mutants . The electron micrograph of the particles produced in the mutant 18/MC4 reveals the presence of normal-looking particles; these particles contain phage DNA, adsorb to the permissive host but fail to inject their DNA . The particles produced in the mutant 153/MC4 which fail to adsorb to the host are found to be tail fibre-less . These observations indicate the involvement of host protein(s) in phage P22 morphogenesis.

Mol Gen Genet, 1982, 186(1), 135 - 9
Bacteriophage Mu DNA replication is stimulated by non-essential early functions; Goosen T et al.; The replication of a spontaneous Kil- mutant of bacteriophage Mu has been investigated . The Kil- mutation (Mucts62-13/4), which was introduced into a defective prophage, is pleiotrophic, leading to the loss of also the Gam, Cim and Sot functions . The mutation is caused by an insertion with the characteristics of IS1, located just outside the B gene . Mucts62-1 3/4 phages form extremely small plaques on wildtype indicator strains . The replication of the insertion mutant as compared to Mucts62 is strongly reduced . Normal replication could be restored by relieving the polarity of the insertion or by complementation with defective prophages which express all early functions . Apparently, early genes other than A and B are involved in normal Mu DNA replication.

Mol Gen Genet, 1982, 186(1), 122 - 6
Suppression of the lexC (ssbA) mutation of Escherichia coli by a mutant of bacteriophage P1; Johnson BF; A new mutant of bacteriophage P1 designated lxc that suppresses the phenotype of lexC and ssbA mutants of Escherichia coli was isolated and characterized . The properties of lexC mutants suppressed by the lxc mutation include temperature sensitive growth at 42 degrees C, sensitivity to ultraviolet light and alkylating agents, and a nonmutagenic response following exposure to ultraviolet irradiation . A bac mutant of bacteriophage P1 that suppresses the temperature sensitivity of dnaB mutants does not affect the phenotype of lexC or ssbA mutants . Neither the lxc or bac mutations affect the ultraviolet light sensitivity of strains with the mutations uvrA155, lexA102, or recA56.

Mol Gen Genet, 1982, 186(1), 101 - 5
Reversion of bacteriophage T4rII mutants by high levels of pyrimidine deoxyribonucleosides; DeVries JK et al.; High levels of pyrimidine deoxyribonucleosides, but not purine deoxyribonucleosides, increase the reversion rate of bacteriophage T4rII mutants to r+ . This increased reversion rate is not observed when a thymidine kinase mutation is introduced into the bacteriophage, but the high reversion rate persists when the host, E . coli, harbors a thymidine kinase mutation.

Mol Gen Genet, 1982, 185(3), 404 - 7
Mutability of bacteriophage M13 by ultraviolet light: role of pyrimidine dimers; Schaaper RM et al.; The role of pyrimidine dimers in mutagenesis by ultraviolet light was examined by measuring the UV-induced reversion of six different bacteriophage M13 amber mutants for which the neighboring DNA sequences are known . The mutational response at amber (TAG) codons preceded by a guanine or adenine (where no pyrimidine dimer can be formed) were compared with those preceded by thymine or cytosine (where dimer formation is possible) . Equivalent levels of UV-induced mutagenesis were observed at both kinds of sites . This observation demonstrates that there is no requirement for a pyrimidine dimer directly at the site of UV-induced mutation in this single-stranded DNA phage . UV irradiation of the phage was also performed in the presence of Ag+ ions, which specifically sensitize the DNA to dimer formation . The two methods of irradiation, when compared at equal survival levels (and presumably equal dimer frequencies), produced equivalent frequencies of reversion of the amber phage . We believe these results indicate that while the presence of pyrimidine dimers may be a prerequisite for UV mutagenesis, the actual mutagenic event can occur at a site some distance removed from a dimer.

J Virol, 1982 Jan, 41(1), 88 - 96
Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system; Radany EH et al.; An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented . Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity) . In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers . Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system . Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine . Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.

J Virol, 1982 Jan, 41(1), 222 - 7
Polymannose O-antigens of Escherichia coli, the binding sites for the reversible adsorption of bacteriophage T5+ via the L-shaped tail fibers; Heller K et al.; A study of the adsorption kinetics of T5+ and the tail fiber-less mutant hd-2 to lipopolysaccharides of various Escherichia coli strains demonstrated T5+ binding to the O-antigen of th O8 and O9 types . Incorporation of radioactive mannose into the phosphomannose isomerase-deficient strain E . coli F860 O9 pmi allowed the derivation of the number of O-antigens per cell required to increase T5 adsorption . With more than 500 O-antigen molecules, acceleration of T5+ adsorption was observed . The highest adsorption rate was obtained when nearly all lipopolysaccharide molecules were substituted with a polymannose O-antigen . Inhibition studies with purified components of an enzymatically degraded lipopolysaccharide of the O8 type showed that among the mannosides tested the smallest unit, the trimannoside, was the strongest inhibitor of T5+ binding . We conclude that the reversible preadsorption to the O8 and O9 polymannose antigens increases the rate of infection via the cellular receptor protein encoded by the fhuA (formerly tonA) gene.

Tokai J Exp Clin Med, 1982 Jan, 7(1), 85 - 93
Inhibitory effect of hydroquinone and related agents on multiplicity reactivation and genetic recombination of bacteriophage T4; Maezawa H et al.; The multiplicity reactivation of T4D phage irradiated with UV was inhibited by treatment of infected cells with 0.5 mM hydroquinone (HQ) . HQ also inhibited genetic recombination of T4B rII mutants . The recombination frequency was reduced by treatment of infected cells with HQ in a buffer when compared with an untreated control . In addition to HQ, the effects of quinone, an oxide form of HQ, mercaptoethanol and alpha-tocopherol, antioxidants, were also investigated . Quinone and mercaptoethanol inhibited recombinations but alpha-tocopherol did not.

J Cell Biochem, 1982, 18(3), 363 - 75
Structural characteristics of the short-tail fibers of T4 bacteriophage; Zorzopulos J et al.; The characteristics of pure preparations of short-tail fibers of bacteriophage T4 have been studied in the optical and electron microscope . Three main structures were observed: 1) spheres of 8.1 nm diameter; 2) fibers 43 nm long and 3.8 nm thick; and 3) fibers 54 nm long and 3.2 nm thick . Both types of fibers exhibited a regular beaded appearance . The 43-nm fibers were the most abundant structure . During the process of purification of the short-tail fibers, the formation of aggregates was observed each time the material containing the short-tail fibers was dialyzed against saline solutions . These aggregates became increasingly fibrous (as observed in the optical microscope) as the material used was increasingly enriched in short-tail fibers . Finally, most of the aggregates were of the fibrous type when they were formed from a purified preparation of short-tail fibers . In the electron microscope, it was found that the filamentous aggregates were organized in well-defined bundles . The amino acid composition of the highly purified short-tail fibers was also determined . Among the known fibrous proteins, the ones that most resemble the amino acid composition of the short-tail fibers are actin and fibrinogen . These observations are discussed in relation to the T4 short-tail fiber structure and their localization on the hexagonal baseplate of the T4 tail structure.

Eur J Respir Dis Suppl, 1982, 122, 36 - 47
Glucocorticoid-binding proteins in rat liver; Gustafsson JA et al.; The distribution, metabolism and protein-binding of {3H} corticosterone in the rat liver was studied with respect to time and sex . The maximum recovery from the cell nuclei occurred 5 min after injection of the steroid . At this time, ten times more radioactivity was recovered from the liver cell nuclei of male animals compared to females . The recovered radioactivity in the cell nuclei was identified as mainly unmetabolised corticosterone and 5 alpha-dihydrocorticosterone . The radioactivity bound to the cytosolic glucocorticoid receptor protein also appeared to consist of unmetabolised corticosterone and 5 alpha-dihydrocorticosterone . However, 5 alpha-dihydrocorticosterone was found to have very little biological activity . The glucocorticoid receptor was found to exist in two forms with Stokes radii of 6.1 and 3.6 nm, respectively, following in vivo labelling with {3H} dexamethasone . The 6.1 nm form could be converted into the 3.6 nm form by incubation with trypsin, papain, alpha-chymotrypsin or an extract of purified rat liver lysosomes . Further incubation resulted in an even smaller form with a Stokes radius of 1.9 nm . With the help of biospecific adsorption chromatography based on the differential affinity of the activated and nonactivated complexes for DNA, the 6.1 nm form of the glucocorticoid-receptor complex was purified to near homogeneity by a rapid and simple technique . An immunoglobulin fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor contained specific antibodies to the receptor . The antibodies cross-reacted with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus . Activated glucocorticoid receptor protein binds selectively in vitro to a cloned fragment of murine mammary tumor virus DNA . In contrast, the receptor fails to bind selectively to DNA restriction fragments from E coli plasmids pBR 322 and RSF 2124 or from bacteriophages alpha and T4.

Mol Gen Genet, 1982, 186(2), 185 - 92
Effects of bacteriophage f1 gene III protein on the host cell membrane; Boeke JD et al.; Plasmids which encode bacteriophage f1 coat protein genes VIII and III are responsible for a number of unusual properties suggesting that they have a drastic effect on the bacterial outer membrane . Analysis of several such recombinant plasmids and selection of mutant plasmids unable to cause this effect established that the properties were caused by gene III protein or its amino-terminal fragment.

Proc Natl Acad Sci U S A, 1982 Jan, 79(2), 248 - 52
The genome of frog virus 3, an animal DNA virus, is circularly permuted and terminally redundant; Goorha R et al.; We examined the structure of the frog virus 3 (FV 3) genome by using electron microscopic and biochemical techniques . The linear FV 3 DNA molecules (Mr approximately 100 x 10(6) formed circles when partially degraded with bacteriophage lambda 5'-exonuclease and annealed, but not when the annealing was done without prior exonuclease digestion . The results suggest that the DNA molecules contain direct terminal repeats . The repeated region composed about 4% of the genome . Complete denaturation of native FV 3 DNA molecules followed by renaturation produced duplex circles each bearing two single-stranded tails at different points along the circumference . The tails presumably represent the terminal repeats . The formation of duplex circles suggests that the FV 3 genome is circularly permuted . This is further borne out by (i) failure to identify a specific restriction endonuclease fragment containing the label when the molecular ends were radiolabeled by using the polynucleotide kinase procedure, and (ii) similarity in the restriction patterns of virion DNA and large concatemeric replicating viral DNA as revealed by endonucleolytic cleavage of both DNAs with HindIII . From the above data, we conclude that the FV3 genome is both circularly permuted and terminally redundant--unique features for an animal virus.

Proc Natl Acad Sci U S A, 1982 Jan, 79(1), 101 - 5
Protein dynamics by solid-state NMR: aromatic rings of the coat protein in fd bacteriophage; Gall CM et al.; The motions of the aromatic amino acids of the fd bacteriophage coat protein are described by solid-state 2H, 13C, and 15N NMR . Tryptophan-26 is immobile on time scales as slow as 10(3) HZ . The phenylalanine and tyrosine rings undergo 180 degree flips about the C beta--C gamma bond axis more often than 10(6) HZ as well as small-amplitude rapid motions in other directions.

Mol Gen Genet, 1982, 187(2), 347 - 53
Unusual suppression properties of lysogens containing derivatives of phi 80 psu3+; Feinstein SI et al.; Nonsense codon suppressing lysogens of E . coli have been made using phi 80 psu3+-A2 and phi 80 psuoc+-A2, heat-sensitive amber and ochre suppressing derivatives, respectively, of bacteriophage phi 80 . The various lysogens selected differ in strength of suppression as well as in heat sensitivity of suppressor function . Heat-resistant derivatives, some still carrying the A2 mutation, can be selected from the heat sensitive parents . Mapping experiments indicate that the phi 80 derivatives integrate at the tyrTV locus, which contains two copies of tRNA1Tyr . The origin of the various suppressor phenotypes appears to be related to the great variety of distinctive recombination events possible either between the incoming tRNA1Tyr gene and the host copies, or among the three copies of this gene in the lysogens.

Natl Cancer Inst Monogr, 1982, 60, 257 - 67
Mutagenesis and cellular responses to DNA damage; Walker GC et al.; Treatment of Escherichia coli with DNA-damaging agents results in the increased expression of a set of din (damage-inducible) genes . We have studied the regulation and function of these genes by using the Mud(Ap, lac) bacteriophage to obtain fusions of the beta-galactosidase structural gene to the promoters of various din genes . By this technique, we have shown that the uvrA, uvrB, and umuC genes are induced by UV and other DNA damaging agents . The products of the uvrA and uvrB genes are required for the excision repair of pyrimidine dimers and other bulky lesions; the umuC gene product is required for most chemical mutagenesis in E . coli . Genetic analyses of all of the din-lac fusions isolated to date indicate that lexA is the direct repressor of each of the din genes and that proteolytic cleavage of the lexA protein is required for their induction . We have also been studying the mechanism by which the clinically isolated plasmid pKM101 increases the susceptibility of cells to chemical mutagenesis . Inasmuch as the effects of pKM101 on mutagenesis are recA+ lexA+ -dependent and the plasmid can suppress the nonmutability of a umuC mutant, it seems likely that pKM101 may carry an analog of the chromosomal umuC gene . By insertion mutagenesis using Tn5, we identified an approximately 2,000-base pair region of pKM101 which is necessary for its effects on mutagenesis.

Ultramicroscopy, 1982, 7(3), 221 - 32
Discrimination of protein and nucleic acids by electron microscopy using contrast variation; Kuhlbrandt W; A contrast variation method is described by which protein and nucleic acid components of nucleoprotein assemblies can be distinguished in the electron microscope . Using contrasting media of variable electron scattering density, the contribution to image contrast of protein or nucleic acid can be matched out . The protein and DNA components of T4 bacteriophages were visualized separately in this way . The distribution of RNA and protein in ribosomes was studied by processing images of crystalline sheets of ribosomes embedded in a range of contrasting media . Projection maps of crystalline ribosomes suggest that the ribosomal RNA is located predominantly in the central region of the particle.

Mol Gen Genet, 1982, 188(2), 211 - 8
Genetic analysis of two bacterial RNA polymerase mutants that inhibit the growth of bacteriophage T7; Buchstein SR et al.; The Escherichia coli mutants 7009 and BR3 are defective in the growth of bacteriophage T7 . We have previously shown that both of these mutant hosts produce an altered RNA polymerase which is resistant to inhibition by the T7 gene 2 protein (De Wyngaert and Hinkle 1979) . In both strains, the mutation which prevents T7 growth is closely linked to rifA (rpoB) . Both mutants are complemented by transformation with a multicopy plasmid carrying rpoB and rpoC but not by a plasmid carrying only rpoB . This indicates that the mutations reside in rpoC, the structural gene for the beta' subunit of RNA polymerase . When a single copy of the wildtype rpoC allele is introduced into the mutant using the transducing phage lambda drifd18, the mutant allele is dominant over wildtype . The lambda drifd18 transductant also remains unable to support the growth of T7 in the presence of rifampin . This supports our conclusion that the mutation is in rpoC . We have measured the growth of T7 phage, the kinetics of phage DNA synthesis, and the structure of replicative DNA intermediates in several transductants, and compared these results with those obtained in the original mutant strains.

Mol Gen Genet, 1982, 188(1), 139 - 42
Studies on the expression of outer membrane protein 2 in escherichia coli; Fralick JA et al.; The relative level of protein 2 expressed in the outer membrane of strains of Escherichia coli K-12 lysogenized with bacteriophage PA-2 was found to be influenced by both the growth temperature and lc+ gene dosage . An increase in either of these parameters was accompanied by an increase in the level of protein 2 up to an apparent saturation level . Any increase in the amount of protein 2 was accompanied by a concomittant decrease in the amount of OmpF and OmpC porins . This inverse relationship led to the maintenance of an approximately constant protein mass per unit of peptidoglycan . Our results are discussed in light of recent genetic studies on the regulation of the OmpF and OmpC porins and can be explained through the competition of these three matrix proteins for a common export or insertion site.

Mol Gen Genet, 1982, 186(4), 540 - 7
Only one gene is required for the glpT-dependent transport of sn-glycerol-3-phosphate in Escherichia coli; Ludtke D et al.; Deletion and point mutants defective in the glpT-dependent sn-glycerol-3-phosphate transport system were isolated and located on the Escherichia coli chromosome . They mapped in glpT in the clockwise order gyrA, glpA, glpT at around 48 min on the Escherichia coli linkage map . The mutations within glpT were ordered by deletion mapping, three factor crosses, and by crosses involving lambda transducing bacteriophages carrying glpT-lac operon fusions . Results obtained using these fusion phages indicated that glpT is transcribed in the counterclockwise direction on the E . coli linkage map . Complementation analysis using these mutants revealed only one complementation group . Thus, one gene is necessary and sufficient for the proton motive force-dependent sn-glycerol-3-phosphate transport system.

Mol Gen Genet, 1982, 186(4), 497 - 500
Mechanism of pBR322 transduction mediated by cytosine-substituting T4 bacteriophage; Takahashi H et al.; A cytosine-substitution type mutant of bacteriophage T4 (T4dC phage) has been shown to mediate the transfer of plasmid pBR322 . The transduction frequency was around 10(-2) per singly infected cell at low multiplicity of infection . The transductants contained either a monomer or multimers of pBR322 . The transducing capacity of T4dC phage was resistant to methylmethanesulfonate treatment . The results of Southern blotting experiments have indicated that the pBR322 DNA exists as head-to-tail concatemers in the transducing particles . The mechanism of transfer of pBR322 mediated by T4dC phages is discussed.

Ann Microbiol (Paris), 1982 Jan, 133A(1), 43 - 7
Arrangement of bacteriophage lambda receptor protein (LamB) in the Escherichia coli cell surface; Yamada H et al.; The lambda receptor protein (LamB) was assembled into an ordered hexagonal lattice structure with a lattice constant of about 7.8 nm in the presence of lipopolysaccharide . Both the heptose-containing polysaccharide region and the fatty acid region are suggested to be involved in the interaction with LamB . The lattice structure was preferably formed on the peptidoglycan layer when the lipoprotein was covalently bound to this layer . In the presence of chloroform, the ordered hexagonal lattice structure was active in the receptor function for lambda, resulting in phage adsorption and DNA ejection.

Genetika, 1982, 18(1), 24 - 35
{W-reactivation and W-mutagenesis in lambda and T7 bacteriophages: a comparative study of the action of ultraviolet radiation (254 nm) and of the photosensitizing agents, 8-methoxypsoralen and angelicin}; Zavil'gel'skii GB et al.; Monoadducts and interstrand cross-links are formed in DNA after psoralen plus light treatment of bacteriophage lambda . Survival and clear plaque mutation frequency of lambda after photosensitization with 8-methoxypsoralen (8-MOP) are increased when the wild type host is slightly UV-irradiated (W-reactivation and W-mutagenesis) . The recA13, lexA1 and uvrA6 mutations block W-reactivation and W-mutagenesis of lambda treated with 8-MOP plus light . Using the technique of "repeated irradiation" we showed that the mutagenic effect of 8-MOP plus light treatment on phage is due mainly to formation of cross-links in DNA . The mutagenic activity of monoadducts had been studied by using angular furocoumarin, angelicin which forms mainly monoadducts in DNA . Upon W-mutagenesis of phage lambda treated with angelicin plus light a high mutagenic effect is observed . The results indicate that the mutagenic activity of monoadducts is 15-20 fold slower as compared to that of cross-links . W-reactivation and W-mutagenesis of UV-irradiated (254 nm) bacteriophage lambda are also observed after 8-MOP plus light treatment of Escherichia coli uvrA and wild type hosts . It is possible that the difference in mutagenic activity of psoralen adducts could depend on the repair mechanism of adducts: cross-links repair in bacterial and lambda DNA is controlled by lexA gene (error-prone SOS-repair mechanism), while monoadducts can be efficiently repaired by error-free excision and recombination.

EMBO J, 1982, 1(11), 1445 - 53
A site-specific, conservative recombination system carried by bacteriophage P1 . Mapping the recombinase gene cin and the cross-over sites cix for the inversion of the C segment; Iida S et al.; The bacteriophage P1 genome carries an invertible C segment consisting of 3-kb unique sequences flanked by 0.6-kb inverted repeats . With insertion and deletion mutants of P1 derivatives the site-specific recombinase gene cin for C inversion) has been mapped adjacent to the C segment and the cix sites (for C inversion cross-over) have been located at the outside ends of the inverted repeats . Inversion of the C segment functions as a biological switch and controls expression of the gene(s) responsible for phage infectivity carried on the C segment . The cin gene product can promote recombination between a 'quasi- cix ' site on plasmid pBR322 and a cix site on P1 DNA . The junctions formed on the resulting co-integrate can also serve as cix sites . This observation implies a potential evolutionary process to bring genes under the control of a biological switch acting by DNA inversion.

EMBO J, 1982, 1(10), 1287 - 93
Isolation and characterization of the rat tryptophan oxygenase gene; Schmid W et al.; Tryptophan oxygenase (TO, EC 1.13.1.12) from rat liver is subject to glucocorticoid and developmental control . To study the mechanism of regulation, TO mRNA sequences and the chromosomal TO gene were cloned . From a cDNA library prepared from rat liver poly(A)+ RNA enriched for TO mRNA, a recombinant plasmid containing TO cDNA sequences was identified by translation of hybrid-selected RNA and immunoprecipitation with antibodies directed against TO . This cDNA clone hybridizes to a mRNA 2000 bases long that is inducible by dexamethasone . With this clone as probe we isolated from a bacteriophage lambda rat DNA library genomic clones which together span a region of 32 kilobase pairs (kb) . Heteroduplex analysis revealed that the gene extends over 19 kb and is interrupted by at least 11 introns . To characterize the presumptive control region the DNA sequence around the 5' end of the TO gene was determined . S1 nuclease protection experiments revealed two separate start sites for TO mRNA transcription within this region.

EMBO J, 1982, 1(10), 1217 - 24
Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda; Zabeau M et al.; Hybrid plasmids carrying cro-lacZ gene fusions have been constructed by joining DNA segments carrying the PR promoter and the start of the cro gene of bacteriophage lambda to the lacZ gene fragment carried by plasmid pLG400 . Plasmids in which the translational reading frames of the cro and lacZ genes are joined in-register (type I) direct the synthesis of elevated levels of cro-beta-galactosidase fusion protein amounting to 30% of the total cellular protein, while plasmids in which the genes are fused out-of-register (type II) produce a low level of beta-galactosidase protein . Sequence rearrangements downstream of the cro initiator AUG were found to influence the efficiency of translation, and have been correlated with alterations in the RNA secondary structure of the ribosome-binding site . Plasmids which direct the synthesis of high levels of beta-galactosidase are conditionally lethal and can only be propagated when the PR promoter is repressed . Deletion of sequences downstream of the lacZ gene restored viability, indicating that this region of the plasmid encodes a function which inhibits the growth of the cells . The different applications of these plasmids for expression of cloned genes are discussed.

Mol Gen Genet, 1982, 188(1), 77 - 90
On the role of the single-stranded DNA binding protein of bacteriophage T4 in DNA metabolism . I . Isolation and genetic characterization of new mutations in gene 32 of bacteriophage T4; Doherty DH et al.; The product of gene 32 of bacteriophage T4 is a single-stranded DNA binding protein involved in T4 DNA replication, recombination and repair . Functionally differentiated regions of the gene 32 protein have been described by protein chemistry . As a preliminary step in a genetic dissection of these functional domains, we have isolated a large number of missense mutants of gene 32 . Mutant isolation was facilitated by directed mutagenesis and a mutant bacterial host which is unusually restrictive for missense mutations in gene 32 . We have isolated over 100 mutants and identified 22 mutational sites . A physical map of these sites has been constructed and has shown that mutations are clustered within gene 32 . The possible functional significance of this clustering is considered.

Mol Gen Genet, 1982, 187(2), 248 - 53
Isolation of a recombinant lambda phage carrying nusA and surrounding region of the Escherichia coli K-12 chromosome; Holowachuk EW et al.; A recombinant bacteriophage lambda, lambda argG-6, has been isolated which carries the argG gene and neighbouring loci on an EcoRI-generated 15.5 Kb DNA fragment from the Escherichia coli chromosome . The locations of the argG, nusA and pnp genes on the 15.5 Kb DNA fragment have been determined . In the case of nusA, a Tn5 insertion and sub-cloning of restriction fragments were used to locate the gene . The gene products of nusA and pnp have been identified on one- and two-dimensional polyacrylamide gels . The clockwise gene order was found to be argG-nusA-pnp.

J Gen Virol, 1982 Jan, 58 Pt 1, 181 - 90
Condensed DNA structures derived from bacteriophage heads; Virrankoski-Castrodeza V et al.; Bacteriophage lambda particles were rendered osmotically fragile by incubation, spread over hypophase and examined by electron microscopy . When water was used as hypophase, condensed structures were released from the phage heads and treatment of these with cytochrome c or several alternative proteins resulted in the release of free, relaxed DNA . Phage were pretreated with nitrogen mustard, a bifunctional alkylating agent; when the condensed structures from such phage particles were treated with protein, DNA was released in small supercoiled domains . This confirmed a previous finding that bacteriophage DNA has a supercoiled topology and suggests that the winding pattern of DNA in the phage might involve small domains of coiled DNA analogous to nucleosomes . Such a conformation could be consistent with other studies on the arrangement of DNA in phage heads if the domains have parallel axes.

Genetika, 1982, 18(8), 1221 - 30
{Specificity of Tn9 insertion into the genome of bacteriophage lambda att80 depending on its preceding location}; Zakharenko VI et al.; It was shown that the site of previous integration (the donor site) of Tn9 affects the specificity of its next integration into the target molecule--phage lambda att80 DNA . The transposon integration sites were mapped by restriction and heteroduplex analysis following Tn9 transposition from chromosomal sites of Escherichia coli K-12 differing in location and Tn9 stability . When transposed from chromosomal galT::IS1 gene, Tn9 inserted into the site with coordinates 44,5 +/- 2 kb of lambda att80; when transposed from chromosomal attTn9A site, the transposon inserted into the sites with coordinates 31 +/- 0,7 kb or 33,3 +/- 0,5 kb . In the course of transposition of Tn9 from chromosomal attTn9N site the transposon inserted into the lambda att80 site with coordinates 26,5 +/- 5 kb . In the latter case, the increase of Tn9 single-stranded loop and the appearance of two new HindIII cleavage sites were observed in heteroduplex experiments . The data were interpreted as indicating structural rearrangements of Tn9 or linked sequences in the course of transposition.

Adv Biophys, 1982, 15, 1 - 17
Molecular evolution in papova viruses and in bacteriophages; Soeda E et al.; By comparing the DNA sequences of three eukaryotic papova viral genomes, we attempt to show the very close relative phylogeny among the viral species and their host species, and that therefore the viral species appear to have evolved with their hosts . A comparison of the DNA data also reveals that the rate of nucleotide substitutions at the third positions of codons is much faster than at the first and second positions, though the rate varies depending upon the genes . The estimated rates of amino acid substitutions in homologous genes among the three virus species appear to be considerably faster than the rates known for various vertebrate genes . The comparison reveals further that the rate of silent substitutions is faster than that of replacement substitutions . The DNA sequence data on bacteriophages phi X174 and G4 enable us to examine the patterns of nucleotide substitutions in overlapping genes as well as in nonoverlapping genes . It then becomes evident that overlapping genes have a quite different substitutional pattern with respect to the position of nucleotides in codons than do nonoverlapping sequences . In nonoverlapping regions the third positions usually change the fastest among the three codon positions . This pattern does not apply to overlapping genes, which are coded in the same region but with different reading frames . It will be shown that the younger of the two overlapping genes appears to be very tolerant to nucleotide substitutions at any codon position . Further more, the rate of substitution at each nucleotide site appears to be determined by the rate of the corresponding site in the older of the two overlapping genes.

Mol Gen Genet, 1982, 185(3), 520 - 2
Cloning of bacteriophage T5 promoters; Ksenzenko VN et al.; Bacteriophage T5 was subjected to combined hydrolysis with the restriction endonucleases PstI and HindIII and the resulting fragments were inserted into the plasmid pBR322 . Selection of transformants for Aps-Tcr-phenotype made it possible to screen the hybrid plasmids that contained promoter sequences in the cloned fragments . Two PstI/HindIII fragments, 720 bp (51% of the T5 DNA length) and 1,200 bp (70%) were cloned in this study . Tcr levels for these plasmids were as high as 18 micrograms/ml and 75 micrograms/ml, respectively . The presence of Escherichia coli RNA polymerase binding sites on both fragments was shown using the nitrocellulose filter assay . These binding sites are situated between 35 bp and 95 bp from the HindIII cleavage site on the 1,200 bp fragment; and within 420 bp from the HindIII site on the 720 bp fragment.

Mol Gen Genet, 1982, 185(3), 462 - 7
Transcriptional termination sites in the b2 region of bacteriophage lambda that are unresponsive to antitermination; Burt DW et al.; A bacteriophage lambda cloning vector carrying the trp/lacW205 substitution is described . The vector facilitates the fusion in vitro of genetic control signals to the lacZ structural gene of Escherichia coli . This system was used to define transcriptional termination sites in the lambda b2 region . This region contains termination sites that are unresponsive to the lambda antiterminating proteins pQ and pN.

Mol Gen Genet, 1982, 185(3), 457 - 61
Influence of phage T3 and T7 gene functions on a type III(EcoP1) DNA restriction-modification system in vivo; Kruger DH et al.; The ocr+ gene function (gp 0.3) of bacteriophages T3 and T7 not only counteracts type I (EcoB, EcoK) but also type III restriction endonucleases (EcoP1) . Despite the presence of recognition sites, phage DNA as well as simultaneously introduced plasmid DNA are protected by ocr+ expression against both the endonucleolytic and the methylating activities of the EcoP1 enzyme . Nevertheless, the EcoP1 protein causes the exclusion of T3 and T7 in P1-lysogenic cells, apparently by exerting a repressor-like effect on phage gene expression . T3 which induces an S-adenosylmethionine hydrolase is less susceptible to the repressor effect of the SAM-stimulated EcoP1 enzyme . The abundance of EcoP1 recognition sites in the T7 genome is explained by their near identity with the T7 DNA primase recognition site.

Mol Gen Genet, 1982, 185(1), 120 - 8
Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG Gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis; Lupski JR et al.; A 24 kilobase pair region of the E . coli chromosome surrounding the dnaG gene has been cloned and characterized . A lambda phage library was constructed by ligating a Sau3A( decreases GATC) partial DNA digest of the entire E . coli chromosome into the lambda BamHI(G decreases GATCC) cloning vector charon 28 . Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome . This library was probed with a nick-translated plasmid (pRRBl) containing the rpoD gene, which maps adjacent to dnaG at 66 min . Four bacteriophages: lambda 3, lambda 4, lambda 5, lambda 6 that hybridized to the probe were isolated from the 2,500 plaques screened . One phage recombinant lambda 4, was shown to contain the dnaG gene . Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of lambda 4 using different restriction of fragments . Plasmids pGL444 and pBS10 5 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated . The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement of dnaGts chromosomal marker at nonpermissive 40 degrees C . In this manner a correlated physical and genetic map of dnaG was determined . A large number of Tn5 inserts map to a specific 900 b.p . region which we propose may be involved in the regulation of dnaG gene expression.

Proc Natl Acad Sci U S A, 1982 Jan, 79(2), 238 - 42
Posttranscriptional control of bacteriophage lambda gene expression from a site distal to the gene; Guarneros G et al.; The bacteriophage lambda int gene product, integrase, recombines the phage DNA with the host DNA at specific sites on each to accomplish lysogeny . The int gene is transcribed from two promoters, PL and PI, each regulated positively by lambda proteins . The expression of integrase is also controlled from a site, sib, in the b region of the phage genome . This is a unique regulatory site because it is located distal to the structural gene in relation to the promoters . The expression of int from the PL promoter is inhibited when sib is present . This effect appears to be specific for PL because sib does not cause inhibition of PI-dependent int synthesis . lambda mutants that contain alterations in the site have been isolated . Sequence analyses of the mutations reveal single base changes, spanning 37 base pairs (bp) in the b region, some 240 bp beyond the int gene . Another mutant, hef13, which has a phenotype similar to that of sib, introduces a nucleotide change within the same 37-bp region . The sib and hef mutations cluster within a region of dyad symmetry . Regulation of int synthesis by sib occurs after transcription of the int gene . There is no difference in the rate of PL-promoted int mRNA synthesis in either sib+ or sib- phage infections, yet int mRNA is less stable in the sib+ infection . Because RNase III host mutants are defective in sib regulation, processing of the PL mRNA at sib by this endoribonuclease may cause int mRNA decay and decrease int synthesis.

Gene, 1982 Jan, 17(1), 55 - 63
Systematic alteration of the nucleotide sequence preceding the translation initiation codon and the effects on bacterial expression of the cloned SV40 small-t antigen gene; Gheysen D et al.; In the preceding paper (Derom et al., 1981) we described the cloning in bacterial plasmids of the simian virus 40 (SV40) small-t antigen gene under transcriptional control of the bacteriophage lambda pL promoter . Systematic variation of the distance and/or nucleotide sequence between the Shine-Dalgarno ribosome interaction sequence and the small-t translation initiation codon leads to considerable differences in production of small-t by the different plasmids . Secondary structure models derived for the different mRNAs confirm our previous conclusions about the requirement first for an accessible start codon and second for an accessible ribosome interaction site for efficient translation initiation . Secondary structure models for mRNAs from plasmids containing the small-t gene under control of the lac promoter are in agreement with these conclusions.

Gene, 1982 Jan, 17(1), 45 - 54
High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter; Derom C et al.; Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter (pL) of bacteriophage lambda . Upon temperature induction the best of our constructions expressed a small-t-related 19 000-dalton polypeptide in an amount corresponding to approx . 2.5% of total de novo protein synthesis . This 19 000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis . In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent Mr of 14 500 . This 14 500-dalton polypeptide was shown to be related to authentic small-t . Presumably the secondary structure of the mRNA starting at pL is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon.

Gene, 1982 Jan, 17(1), 27 - 44
Phasmids: hybrids between ColE1 plasmids and E . coli bacteriophage lambda; Brenner S et al.; Plasmids carrying cloned lambda att sites may be integrated into the bacteriophage genome by the site-specific recombination mechanism of lambda . The cross, referred to as "lifting" the plasmid, requires mixed infection of an Escherichia coli strain carrying the plasmid with two appropriately constructed "lifting" lambda phages . One phage donates a short left arm and the other donates a short right arm . These two short arms are of insufficient length to produce a viable phage genome and yield no recombinants when crossed on standard bacteria . However, viable recombinants are obtained when the genome length is extended by integration of one or more plasmids . We call these recombinants phasmids . They contain multiple att sites introduced at the ends of the integrated plasmids, and in the presence of integrase, recombination between these att sites can be exploited to effect release of the plasmid components . These novel genetic elements can be used in a variety of ways as vectors in genetic manipulation experiments . Sequences cloned in phasmids may be studied as a component of either a plasmid and or of a phage, and easily interconverted between the two states.

Gene, 1982 Jan, 17(1), 19 - 26
Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs; Chumakov IM et al.; A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions . From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences . This mos-specific probe has been used for screening a human gene library cloned in bacteriophage lambda Charon 4A . Of these, 19 clones have been isolated containing mos-related sequences . By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus . Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene . The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed . By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen . The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.

Proc Natl Acad Sci U S A, 1982 Jan, 79(1), 31 - 5
Rat preprocarboxypeptidase A: cDNA sequence and preliminary characterization of the gene; Quinto C et al.; Rat carboxypeptidase A cDNA clones have been isolated from a cDNA library prepared from pancreatic mRNA . An almost complete mRNA sequence has been deduced that predicts a polypeptide having 78% amino acid sequence homology with bovine carboxypeptidase A . The amino acid sequence of the activation and signal peptides of the carboxypeptidase A precursor were inferred from the nucleotide sequence . The cDNA was used as a probe to identify DNA fragments containing carboxypeptidase A sequences in a bacteriophage lambda library of rat genomic DNA . Heteroduplexes revealed that the DNA coding sequence occupies 5.5 kilobases and is interrupted by nine intervening sequences . The nucleotide sequence of the 5' end of the gene and the adjacent flanking region provides information on the site of initiation of transcription and the putative control regions . There is no evident relationship between the localization of intervening sequences in the gene and functional/structural domains of the protein.

EMBO J, 1982, 1(12), 1559 - 64
The head protein D of bacterial virus lambda is related to eukaryotic chromosomal proteins; Witkiewicz H et al.; Bacteriophage lambda structural head protein D has physiochemical properties in common with eukaryotic chromosomal proteins . It has a low affinity for hydroxylapatite, it is heat stable and acid soluble . Moreover, it cross-reacts immunologically with histones H2A and H2B . The deduced primary structure of the D protein shows striking homology to calf chromosomal high mobility group HMG-14 protein . There are two clusters of four ( LSAK , ASDE ) and one of three (APA) identical amino acid residues . Additionally the cluster ETK of protein D occurs three times in HMG-14 and 14 single identical residues are present . A mechanism for an alternative to a nucleosomal mode of nuclear DNA condensation and a possible function of HMG proteins are discussed.

Mol Gen Genet, 1982, 187(1), 79 - 86
Bacteriophage lambda initiators: preparation from a strain that overproduces the O and P proteins; Tsurimoto T et al.; A recombinant plasmid was constructed which carries bacteriophage lambda initiator genes O and P under control of tandemly arranged PL and PR promoters . These promoters were repressed by a thermosensitive repressor, cI857, at low temperature, but became active when the culture was incubated at 42 degrees C . Upon elevation of the temperature, the O and P proteins were overproduced to the extent that they constituted several per cent of the total E . coli cellular proteins . Both the O and P proteins have been purified to apparent homogeneity, and were shown to consist of 298 and 233 amino acid residues, respectively . The amino acid composition and the terminal partial amino acid sequence of each protein were determined . Through these analyses, the locations of the O and P genes in the known lambda DNA sequence were determined . The termination codon for the O gene overlaps with the initiation codon for the P gene . The purified O protein binds specifically to the replication origin of lambda (lambda ori) in accordance with our previous observations . The purified P protein inhibits an ATPase activity of dnaB protein.

Mol Gen Genet, 1982, 187(1), 162 - 5
Effect of bacteriophage phi X174 infection on the conformation of Escherichia coli DNA; Pal SK et al.; The cistron A proteins of bacteriophage phi X174 inhibit the synthesis of beta-galactosidase of host Escherichia coli . A drastic reduction in the rate of transcription of the lac gene is observed in infected cells . This loss in the efficiency of transcription is due to conformational changes in the host DNA . Probably the host DNA is nicked at a few sites along its length and some of its negative superhelical twists are released.

Acta Biochim Pol, 1982, 29(3-4), 227 - 33
Expression of the replication region of phage lambda DNA cloned into pBR322 in E . coli minicells; Gorska I et al.; Replication region of bacteriophage lambda DNA was cloned into pBR322 plasmid by the use of two restriction enzymes--PstI and HindIII . The restriction analysis of four obtained plasmids revealed that lambda DNA was cloned in both orientations . Recombinant plasmids were transferred to the minicell-producing strain of E . coli and synthesis of the plasmid-mediated proteins was analysed by polyacrylamide-gel electrophoresis . All four recombinant plasmids produced lambda DNA replication proteins pO and pP as well as some proteins specific for pBR322 . The orientation of cloned fragment did not affect the synthesis of lambda DNA replication proteins.

Mol Gen Genet, 1982, 188(1), 143 - 8
2-aminopurine induced DNA repair in E . coli; Maenhaut-Michel G et al.; The survival of UV-irradiated lambda phages is increased when host bacteria are grown in the presence of the base analogue 2-aminopurine (2AP) before infection . This increase in survival, which we have called "2AP-reactivation" depends upon the concentration of 2AP and the time of exposure to 2AP . 2AP-reactivation can be distinguished from Weigle-reactivation in that it is not accompanied by an increase in mutagenesis, does not act on the single-stranded DNA bacteriophage phi X174, and occurs in recA and lexA bacteria . 2AP reactivation does not appear to involve known systems of recombinational repair, as it occurs in recB and recF bacteria, or excision repair, as it occurs in uvrA and uvrB bacteria . It is however dependent upon DNA polymerase I.

Mol Gen Genet, 1982, 187(2), 320 - 5
A rho-independent termination caused by the cloned inverted nut L site of phage lambda; Luk KC; For studying the termination activity of inverted nutL site of bacteriophage lambda, we have constructed a plasmid carrying the nutL fragment oriented reversely with respect to cloned lambda promoter p'R-directed transcription . The results of in vitro transcription on this plasmid template and S1 mapping assay reveal that the termination of p'R-promoted transcription at inverted nutL site is a rho-independent event . This nutL terminator shares several features with the other known sites of transcription termination, including (i) a uridine-rich 3' terminal RNA sequences,--UUAAUUUUU-OH, (ii) a GC-rich region in the DNA immediately preceding the site of termination, (iii) a region of dyad symmetry in the DNA which, in transcript, is capable of forming a stable hairpin containing four GC base pairs and one AU base pair in its stem.

Mol Gen Genet, 1982, 187(2), 231 - 5
Zygotic induction of the rac locus can cause cell death in E . coli; Feinstein SI et al.; Conjugational transfer of the rac locus of E . coli K-12 into a Rac- recipient strain (i.e . rac+ X rac-) results in the killing of a majority of the recipient cells . The efficiency of killing depends somewhat on the plating medium, and can be as high as 98% . The killing is not observed in the rac+ X rac+, rac- X rac- or rac- X rac+ configurations . The rac locus, which has the properties of a cryptic prophage, may carry a function analogous to the kil function of bacteriophage lambda, or may instead cause killing by some replication related process.

Mol Gen Genet, 1982, 186(4), 558 - 65
Physical evidence for the temporal transition of transcription in bacteriophage lambda; Erdile LF et al.; A high proportion of intracellular lambda DNA molecules are found to have D-loops, when isolated under four different conditions: (1) lambda Ots after 7 min at 31 degrees C in the presence of chloramphenicol; (2) lambda Ots after 7 min at 31 degrees C without chloramphenicol; (3) lambda Ots after 30 min at 42 degrees C; and (4) lambda cIIcIII after 50 min at 37 degrees C . The great majority of these D-loops contain RNA and are produced by E . coli RNA polymerase . In the presence of chloramphenicol, D-loops are mostly limited to the immediate early regions of the major leftward and rightward operons . At early times, with no chloramphenicol present, D-loops map primarily within the delayed early regions of the two major operons . At late times, D-loops are found mostly within the major late operon of the bacteriophage DNA . This physical evidence corroborates evidence of the temporal transition in lambda transcription obtained by other means . Chloramphenicol is shown to block the transition from immediate early to delayed early transcription.

Mol Gen Genet, 1982, 186(4), 548 - 57
Nucleotide sequence analysis of in vivo recombinants between bacteriophage lambda DNA and pBR322; King SR et al.; The nucleotide sequences involved in the illegitimate recombination of four recombinants between bacteriophage lambda DNA and pBR322 in E . coli (lambda TA6, lambda KA3, lambda TA1R, and lambda KA7) were determined . Each resulted from recombination between regions of homology of 10 to 13 base pairs . The presence of a recA+ allele was found to stimulate recombination between lambda DNA and pBR322 approximately 10-fold . Lambda TA6, lambda KA3, and lambda KA7 were isolated in the presence of a recA+ allele and therefore, may have been generated by the recA recombination system . However, lambda TA1R was isolated in a recA mutant, and was presumably generated by a different recombination system . The possibility that it was generated by DNA gyrase is discussed . Two recombination events were required to form lambda KA7, which may indicate that it also was generated by DNA gyrase.

Mol Gen Genet, 1982, 186(3), 419 - 26
Effect of bacterial host repair systems on the viability of hydroxylamine and methyl methanesulfonate treated T4 and lambda bacteriophages; Janion C; Survival of HA1 or MMS-treated T4 and lambda phages was estimated in bacterial cells differing in their ability to repair DNA . It has been found that the mismatch repair system of the bacterial host, which involves mutS mutR MutL uvrE and dam loci, does not excise, or does so to only a limited extent, the nonpaired bases from DNA of HA or MMS-treated phages . Mutation in polA, both in the polymerase as well as in the 5' leads to 3' exonuclease activity, have a small effect on survival of HA-treated phages, whereas mutation in the polymerase activity has a pronounced effect on survival of MMS-treated phages . There was a difference in the effect of polA mutations on survival of MMS-treated T4 and lambda phages; the survival of the former was less affected than the latter . Induction of SOS response has no effect on repair of HA and MMS-treated phages . Pretreatment of bacterial host (including the ada- mutant) with low doses of alkylating agents increases the survival of MMS (but not HA)-treated phages; pretreatment of bacteria with HA has no effect on survival of HA-treated phages . Three lines of evidence: the different inactivation rates of MMS-treated T4 and lambda phages, variation in the effect of polA mutations on survival of T4 and lambda phages, and a different level of adaptive response in ada- cells towards of MMS-treated T4 and lambda phages, suggest that the patterns of DNA methylation in T4 and lambda phages are different.

Mol Gen Genet, 1982, 186(3), 315 - 21
Nucleotide sequence of the immunity region of bacteriophage Mu; Priess H et al.; The leftmost 1590 bp of Mu DNA covering the immunity region have been sequenced . This region encodes the cI repressor, the cII or ner function and the beginning of gene A . An open reading frame extends from position 863 to 342 on the l-strand corresponding to cI protein with a molecular weight of 19212 . It is preceded by a sequence resembling a promoter . To the right of the HindIII site an open reading frame extends from position 1099 to 1323 corresponding to cII or ner protein (molecular weight of 8505) followed by the beginning of gene A at position 1328 . Between position 863 and 1099 promoters for leftward and rightward transcription and operator-like structures can be recognized in the sequence . The promoter for rightward transcription overlaps with the HindIII site and coincides with a RNA polymerase binding site as demonstrated by electron microscopy.

Mol Gen Genet, 1982, 185(3), 468 - 72
The cis-specificity of the Q-gene product of bacteriophage lambda; Burt DW et al.; A trp/lacW205 substitution, fused to the late region of bacteriophage lambda, provided a convenient assay for phage late gene expression in the presence or absence of lambda pQ . Comparison of lacZ expression from Q+ and Q- phages showed that late gene expression was markedly Q-dependent (263-fold difference) . A cis/trans comparison of lambda pQ action showed a 180-fold difference in lacZ expression . The results suggest that pQ in only significantly active when supplied in cis to its site of action.

Mol Gen Genet, 1982, 185(3), 424 - 9
Amplification of maize ribulose bisphosphate carboxylase large subunit synthesis in E . coli by transcriptional fusion with the lambda N operon; Gatenby AA et al.; The maize chloroplast gene coding for the large subunit of ribulose bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) has been placed under the transcriptional control of the bacteriophage lambda promoter PL, by fusion with the lambda N operon located on a multicopy plasmid . Transcription from PL was repressed at 32 degrees C by the presence in the E . coli chromosome of a cIts gene that specifies a temperature-sensitive repressor . After inactivation of the repressor at 45 degrees C unmoderated transcription of the chloroplast gene occurred from the PL promoter . Translation was probably initiated from a chloroplast Shine-Dalgarno sequence located five nucleotides from the N-terminal methionine initiation codon to yield a polypeptide the same size as that synthesised in maize . This direct translation results in a level of expression of the chloroplast gene corresponding to approximately 2% of the total E . coli cell protein as ribulose bisphosphate carboxylase large subunits . Transcriptional fusions with the lambda N operon should provide a generally applicable, simple method for the amplification and regulation of chloroplast gene expression in E . coli.

EMBO J, 1982, 1(1), 107 - 14
Bacteriophage T4 late promoters: mapping 5' ends of T4 gene 23 mRNAs; Kassavetis GA et al.; More than thirty 5' ends of RNA molecules which transverse the bacteriophage T4 late gene, gene 23, have been mapped by nuclease protection and reverse transcription, to a 1.7 kilobase pair (kbp) region upstream of gene 23 . Most of these 5' ends arise from RNA processing and degradation . Two sites at which RNA synthesis is initiated have been identified by guanylylation of diphosphate- or triphosphate-terminated RNA with vaccinia virion "capping" enzyme and are situated approximately 0.1 and 0.9 kbp upstream of gene 23 . At least one other promoter lies more than 1.7 kbp upstream of gene 23, that is, outside of the mapped region.

Mol Gen Genet, 1982, 186(2), 217 - 20
Thymine inhibits suppression by an Escherichia coli nonsense and frameshift suppressor; Cheung PK et al.; A mutant strain of Escherichia coli suppressed a frameshift and some UAG, UAA, and UGA mutants of bacteriophage T4 at 37 degrees C but not at 31 degrees C . This suppression was inhibited by the addition of thymine or thymidine to the medium used to test phage growth . Furthermore, the suppressor strain required thymine or thymidine for growth on minimal medium at 43 degrees C and if this auxotrophy was removed by reversion or recombination the strain no longer suppressed . These results suggest a link between thymidine nucleotide biosynthesis and suppression.

Ultramicroscopy, 1982, 8(1-2), 191 - 206
Three-dimensional structure of proteins determined by electron microscopy; Aebi U et al.; Recent developments in specimen preparation and image processing techniques have made it possible to determine the three-dimensional structure of proteins by electron microscopy . Periodic supramolecular aggregates of the protein under investigation are requiring to minimize radiation damage and to maximize the signal-to-noise ratio of structural detail . Useful information about the fine structure of the protein (e.g . binding sites for interacting molecules, antigenic determinants) can often be obtained by stoichiometric labeling of the ordered arrays with interacting molecules or antibody fragments, and computing difference maps from the reconstructions of the labeled and native structures . The use of this approach to molecular structure determination of proteins will be discussed in light of our work with bacteriophage and actin.

J Virol, 1982 Jan, 41(1), 330 - 3
Plasmid pR386 renders Escherichia coli cells restrictive to the growth of bacteriophage T4 unf mutants; Herman RE et al.; The introduction of the F1 incompatibility group plasmid pR386 Tc into several common laboratory strains of Escherichia coli rendered them restrictive to the growth of bacteriophage T4 unf mutants, which are defective in unfolding the host genome . The growth inhibition was temperature dependent . The single mutant unf39 x 5 exhibited an efficiency of plating of less than 10(-8) at 27 degrees C . However, at 37 degrees C, complete growth inhibition occurred only when host DNA degradation was also absent.

J Biol Chem, 1981 Dec 25, 256(24), 12636 - 9
Beta protein of bacteriophage lambda promotes renaturation of DNA; Kmiec E et al.; The protein encoded by the red beta gene of bacteriophage lambda was found to promote reannealing of complementary single strands of DNA . Reannealing activity was optimal at pH 6.0 and required a divalent cation . A threshold temperature of at least 15 degrees C was necessary in order to detect activity . The reaction was linear with time for about 20 min, but the extent of reaction was dependent upon the amount of beta protein added . Reannealing of complementary single strands was confirmed by measuring increased lability of DNA to lambda exonuclease . When protein preparations from red- lysogens were tested for ability to promote reannealing, high activity was observed in a mutant altered in the gene for exonuclease, but there was low activity in a mutant altered in the gene for beta protein.

Nucleic Acids Res, 1981 Dec 11, 9(23), 6591 - 9
Some characteristics of apurinic sites in single- and double-stranded biologically active phi X174 DNA; Lafleur MV et al.; With biologically active DNA of the bacteriophage phi X174, both single and double-stranded, some physico-chemical and biological parameters of the depurination reaction are studied . It is shown that in single-stranded DNA each apurinic site is lethal, while in double-stranded RFI-DNA only about 5% of these sites are lethal . Furthermore it is concluded that the apurinic sites are formed at different rates in single- and double-stranded DNA and also the conversion into breaks of the apurinic sites is different for both forms of DNA.

Biophys J, 1981 Dec, 36(3), 743 - 57
Assembly of bacteriophage T7 . Dimensions of the bacteriophage and its capsids; Stroud RM et al.; The dimensions of bacteriophage T7 and T7 capsids have been investigated by small-angle x-ray scattering . Phage T7 behaves like a sphere of uniform density with an outer radius of 301 +/- 2 A (excluding the phage tail) and a calculated volume for protein plus nucleic acid of 1.14 +/- 0.05 x 10(-16) ml . The outer radius determined for T7 phage in solution is approximately 30% greater than the radius measured from electron micrographs, which indicates that considerable shrinkage occurs during preparation for electron microscopy . Capsids that have a phagelike envelope and do not contain DNA were obtained from lysates of T7-infected Escherichia coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA by means of temperature shock (capsid IV) . In both cases the peak protein density is at a radius of 275 A; the outer radius is 286 +/- 4 A, approximately 5% smaller than the envelope of T7 phage . The thickness of the envelope of capsid II is 22 +/- 4 A, consistent with the thickness of protein estimated to be 23 +/- 5 A in whole T7 phage, as seen on electron micrographs in which the internal DNA is positively stained . The volume in T7 phage available to package DNA is estimated to be 9.2 +/- 0.4 x 10(-17) ml . The packaged DNA adopts a regular packing with 23.6 A interplanar spacing between, DNA strands . The angular width of the 23.6 A reflection shows that the mean DNA-DNA spacing throughout the phage head is 27.5 +/- less than 2.2 A . A T7 precursor capsid (capsid I) expands when pelleted for x-ray scattering in the ultracentrifuge to essentially the same outer dimensions as for capsids II and IV . This expansion of capsid I can be prevented by fixing with glutaraldehyde; fixed capsid I has peak density at a radius of 247 A, 10% less than capsid II or IV.

J Virol, 1981 Dec, 40(3), 890 - 900
Semiconservative DNA replication is initiated at a single site in recombination-deficient gene 32 mutants of bacteriophage T4; Dannenberg R et al.; We have investigated, by electron microscopy, replicative intermediate produced early after infection of Escherichia coli with two phage T4 gene 32 mutants (amA453 and tsG26) which replicate their parental DNA but are defective in secondary replications and in moderating the activities of recombination nucleases . Under conditions completely restrictive for progeny production, both of these mutant produced replicative intermediates, each containing a single internal loop . Both branches of these loops were double stranded; i.e., both leading and lagging strands were synthesized . The replicative intermediates of these mutants qualitatively and quantitatively resembled early replicating wild-type T4 chromosomes after solitary infection of E . coli . However, in contrast to intracellular wild-type T4 DNA isolated from multiple infection, the mutant DNAs showed neither multiple branches nor multiple tandem loops . These results demonstrate that a truncated gene 32 protein which consists of less than one-third of the wild-type T4 helix-destabilizing protein can facilitate the functions of T4 replication proteins, specifically those of T4 DNA polymerase and priming proteins . Our results also support the hypothesis that the generation of multiple tandem loops or branches in vegetative T4 DNA depends on recombination (Mosig et al., in B . Alberts, ed., Mechanistic Studies of DNA Replication and Genetic Recombination, p . 527-543, Academic Press, Inc., New York, 1980).

J Virol, 1981 Dec, 40(3), 822 - 9
Bacteriophage T4 alc gene product: general inhibitor of transcription from cytosine-containing DNA; Kutter EM et al.; The alc gene of bacteriophage T4 was originally defined on the basis of mutations which allow late protein synthesis directed by T4 DNA containing cytosine rather than hydroxymethylcytosine . The question remained whether the normal alc gene product (gpalc) also blocks the transcription of early genes from cytosine-containing DNA . Complementation experiments were performed between hydroxymethylcytosine-containing phage which direct gpalc synthesis but carry mutations in a given gene(s) and cytosine-containing phage carrying that gene(s) . The required protein would then have to be directed by the cytosine-containing DNA: it is looked for directly on polyacrylamide gels or through its physiological effects or both . For all early proteins examined in this way, no synthesis was observed when 95 to 100% of the hydroxymethylcytosine was substituted by cytosine in the infecting DNA, whereas there was significant synthesis with 75% substitution or less . The results indicate that gpalc is carried in with the infecting DNA or is made very early to block transcription of all cytosine-containing DNA.

J Virol, 1981 Dec, 40(3), 790 - 9
Monocistronic and polycistronic bacteriophage T4 gene 23 messages; Young ET et al.; We studied transcription of T4 late genes by in vitro translation of size-fractionated late RNA and by hybridization of T4 late RNA to plasmids containing identified T4 late genes . We identified mRNA species that coded for the late proteins gp10, gp18, gp21, gp22, gp23, and gp24 . Functional mRNA's that coded for the early proteins gp32 and IPIII were also detected after fractionation of late RNA . As in preparations of early RNA, gene 32 message activity was present in two species of RNA, which had molecular weights of approximately 0.5 x 10(6) and 0.8 x 10(6), and IPIII message activity was present in multiple species of RNA . Gene 24 and gene 10 message activities migrated as single species that had approximate molecular weights of 0.5 x 10(6) and 1.2 x 10(6), respectively . mRNA activity for gp18 migrated heterogeneously . We detected multiple transcripts from gene 23 by in vitro translation and by hybridization of late RNA to plasmids containing genes 21 through 23 . Both types of analysis indicated that the major gene 23 transcript had a molecular weight of 0.75 x 10(6) . In addition, two gene 23 transcripts having molecular weights of about 1.0 x 10(6) and 1.3 x 10(6) were present; these RNA species also coded for gp21 and gp22 . Physical linkage of transcripts from genes 21, 22, and 23 was demonstrated by hybridization.

J Gen Virol, 1981 Dec, 57(Pt 2), 365 - 73
Comparison of the lipid-containing bacteriophages PRD1, PR3, PR4, PR5 and L17; Bamford DH et al.; Five broad host range lipid-containing bacteriophages PRD1, PR3, PR4, PR5 and L17 isolated from different parts of the world were compared using biological and structural criteria . Virus morphology as well as genome sizes appeared to be identical . However, these viruses could be distinguished by restriction endonuclease mapping and by their structural protein patterns in SDS--gel electrophoresis . The viruses studied thus form a very close group of lipid-containing bacteriophages . We suggest PRD1 as a model organism for this group and that the group be called the PRD1 phage group.

Med Biol, 1981 Dec, 59(5-6), 389 - 93
Polyamine biosynthesis in Escherichia coli: construction of polyamine-deficient mutants; Tabor H; Previous work is summarized on the biosynthetic pathway for polyamines in Escherichia coli . Deletion mutants have been obtained in the various biosynthetic steps, resulting in cells with no polyamines . These mutants grow at one-third the rate of polyamine-supplemented cultures and can serve as suitable hosts for bacteriophages T4, T7, Q beta, and f2 . The major effects of polyamine deficiency in these polyamine-deficient strains are: (i) these cells do not serve as hosts for bacteriophage gamma and (ii) polyamine-deficient male strains have defects which are attributable to a decrease in the stability of the male pili, namely, decreased numbers of recombinants in Hfr crosses and poorer adsorption of the male-specific bacteriophages f1, f2, and Q beta . One polyamine-deficient strain has been developed which becomes absolutely dependent on polyamines for growth if it also contains a specific rpsL (strA) mutation.

J Virol, 1981 Dec, 40(3), 958 - 62
Modification of RNA polymerase from Escherichia coli by pre-early gene products of bacteriophage T5; McCorquodale DJ et al.; RNA polymerase from cells of Escherichia coli infected with T5 were recovered as a complex with two pre-early phage-coded polypeptides, the 60,000-dalton product of gene A1 and a previously reported 11,000-dalton polypeptide . This RNA polymerase complex had altered transcriptional specificity, in that it transcribed pre-early genes less efficiently than it did early genes.

J Virol, 1981 Dec, 40(3), 772 - 89
Sizes of bacteriophage T4 early mRNA's separated by preparative polyacrylamide gel electrophoresis and identified by in vitro translation and by hybridization to recombinant T4 plasmids; Young ET et al.; We determined the sized of specific T4 prereplicative nRNA's by preparative polyacrylamide gel electrophoresis, and we used the following two techniques to identify specific gene transcripts; cell-free protein synthesis accompanied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to distinguish T4 polypeptides and hybridization to recombinant plasmids containing T4 DNA of known genetic composition . In our first analysis, the use of nonsense and in-phase deletion mutants allowed unambiguous identification of the functional transcripts that encoded genes 32, rIIB, and rIIA . In addition, we identified the functional transcript that encoded genes 43, 45, 30, 39, and 52, the beta-glucosyl transferase gene, and the deletion 293 region . A single peak of mRNA activity that coded for gp43, gp39, gprIIA, beta-glucosyl transferase, and the polypeptide encoded in the deletion 293 region was present; the other polypeptides were encoded in multiple mRNA species, gp46 and gp32 were encoded by two mRNA's and gp52 and gprIIB were encoded by three nRNA's . By hybridizing fractionated, pulse-labeled early RNA to cloned restriction fragments of T4 DNA, we identified the same specific transcripts for genes 43, 52, and rIIB . In addition, a lower-molecular-weight RNA (presumably degraded nRNA) was present even in pulse-labeled RNA preparations . The distribution of pulse-labeled RNAs that hybridized to gene 39, gene 30, gene rIIA, gene 40 plus gene 41, and gene 42 plus the beta-glucosyl transferase gene indicated extensive degradation . We detected cotranscription of genes rIIA and rIIB by rehybridization of RNA first annealed to an rIIB plasmid and then eluted and annealed to an rIIA plasmid . The size distributions of normal and chloramphenicol-treated RNAs that hybridized to plasmids containing T4 immediate early gene 30, gene 39, gene 40 plus gene 41, and gene 42 plus the beta-glucosyl transferase gene were not significantly different.

J Bacteriol, 1981 Dec, 148(3), 853 - 60
General method for fine mapping of the Escherichia coli K-12 lamB gene: localization of missense mutations affecting bacteriophage lambda adsorption; Hofnung M et al.; lamB is the structural gene for the bacteriophage lambda receptor, a multifunctional protein located in the outer membrane of Escherichia coli K-12 . We present a method for deletion mapping of any lamB mutations with a recognizable pheno-type . This method involves a transducing phage constructed by in vitro recombination which can also be used for complementation, deoxyribonucleic acid sequence, and in vitro protein synthesis studies with the mutated lamB gene . Using this method, we mapped 18 lamB missense mutations which confer resistance to phage lambda h+ (wild-type host range) . The main results were the following . (i) None of the 18 mutations was located in the first 4 deletion intervals out of the 11 of the genetic map . (ii) These mutations were clustered according to their phenotype as follows . (a) Class I mutations, which allow growth of lambda h and lambda hh* (one-step and two-step host range mutants of lambda, respectively), were located in three regions--three in interval V, four in interval VIII-IX, and three in interval X-XI . Only the last three mutations still allowed growth of phage K10 which also uses the lambda receptor, and two of them still allowed reversible binding of lambda h+ . (b) All seven class II mutations allowed only growth of lambda hh* and mapped in interval V . These results are discussed in the frame of a genetic approach to the functional topology of the lambda receptor.

J Bacteriol, 1981 Dec, 148(3), 845 - 52
In vivo and in vitro functional alterations of the bacteriophage lambda receptor in lamB missense mutants of Escherichia coli K-12; Braun-Breton C et al.; lamB is the structural gene for the bacteriophage lambda receptor in Escherichia coli K-12 . In vivo and in vitro studies of the lambda receptor from lamB missence mutants selected as resistant to phage lambda h+ showed the following . (i) Resistance was not due to a change in the amount of lambda receptor protein present in the outer membrane but rather to a change in activity . All of the mutants were still sensitive to phage lambda hh*, a two-step host range mutant of phage lambda h+ . Some (10/16) were still sensitive to phage lambda h, a one-step host range mutant . (ii) Resistance occurred either by a loss of binding ability or by a block in a later irreversible step . Among the 16 mutations, 14 affected binding of lambda h+ . Two (lamB106 and lamB110) affected inactivation but not binding; they represented the first genetic evidence for a role of the lambda receptor in more than one step of phage inactivation . Similarly, among the six mutations yielding resistance to lambda h, five affected binding and one (lamB109) did not . (iii) The pattern of interactions between the mutated receptors and lambda h+ and its host range mutants were very similar, although not identical, in vivo and in vitro . Defects were usually more visible in vitro than in vivo, the only exception being lamB109 . (iv) The ability to use dextrins as a carbon source was not appreciably affected in the mutants . Possible working models and the relations between phage infection and dextrins transport were briefly discussed.

Gene, 1981 Dec, 16(1-3), 99 - 106
Cloning of bacteriophage T5 DNA fragments . II . Isolation of recombinants carrying T5 PstI fragments; Davison J et al.; The adjacent PstI-J, I and G fragments of the phage T5 DNA molecule (4.4, 4.6 and 7.2 kb, respectively) have been cloned in plasmid pBR322 and their locations verified by Southern blot analysis . The PstI I and G fragments overlap the previously cloned HindIII-P and G fragments and like those, contain no known genetic markers . In addition, one of the 12 newly isolated T5 mutants maps in this PstI-IG region . Thus, the size of the "empty" region between genes D15 and D17, which we have previously observed on the genetic map, extends to at least 11.8 kb . In contrast, the PstI-J fragment carried part of the D12 gene and the intact D14 and D15 genes . This clone is of particular interest since the D15 gene product is a nuclease and is responsible for the positive control of late gene transcription . The orientation of these genes relative to the T5 DNA molecule has been determined by a combination of restriction, deletion and complementation analyses.

Gene, 1981 Dec, 16(1-3), 89 - 96
Bacteriophage T5 growth in Escherichia coli containing PstI fragments of the colicin Ib plasmid; Pinkerton T et al.; PstI restriction fragments of the colicin Ib (ColIb) plasmid have been cloned into the Apr gene of the pBR322 vector . Colicin-producing clones (Col+) all contained two common PstI (L and U) fragments, 2000 and 800 bp long, respectively . All of these colicin producers were found to permit the normal growth of bacteriophage T5; the presence of the whole ColIb plasmid causes an abortive T5 or BF23 phage infection . None of the other clones selected for their inability to propagate T5 produced colicin . The clones (Abi+) that permitted only an abortive infection by T5 contained a single restriction fragment, O (1600 bp), which was not found in any of the colicin producers . Likewise, the specific fragments (L and U) found in the Col+ clones were not found in the Abi+ clones . These data are very hard to reconcile with the hypothesis of a colicin-induced cell deterioration after T5 phage infection.

Gene, 1981 Dec, 16(1-3), 35 - 58
Nucleotide sequence and genome organisation of filamentous bacteriophages fl and fd; Beck E et al.; The DNA sequence of the filamentous phage F1, consisting of 6407 nucleotides, has been determined . When compared with the DNA sequence of the related filamentous phage fd (Beck et al., 1978), the f1 sequence is one nucleotide shorter and differs in 180 positions from the fd DNA . Only ten of these base exchanges cause amino acid exchanges in the known gene products . Most of the exchanges in f1 are the same as in M13 (Van Wezenbeek et al., 1980), showing a near identity of these two phage (there are only 59 nucleotide differences) . Regulatory units for replication, transcription, and translation are in their essential parts identical in all three phage.

Gene, 1981 Dec, 16(1-3), 107 - 18
Cloning of bacteriophage T5 DNA fragments . III . Expression in Escherichia coli mini-cells; Brunel F et al.; Use has been made of the mini-cell system to study polypeptide synthesis from cloned EcoRI, HindIII and PstI fragments of T5 DNA . The correlation of certain gene products with known genes has been established, as well as the physical mapping of genes not yet identified genetically . In some cases, it has been possible to demonstrate the presence of T5 promoters on the cloned DNA fragments . The design of experiments to avoid certain artifacts inherent in the use of the mini-cell system is discussed.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7271 - 5
Incorporation into DNA of the base analog 2-aminopurine by the Epstein-Barr virus-induced DNA polymerase in vivo and in vitro; Grossberger D et al.; The Epstein-Barr virus (EBV)-induced intracellular DNA polymerase was assayed in vitro for the ability to utilize the mutagenic nucleotide analog 2-aminopurine deoxyribose triphosphate (d2apTP), incorporating it as the corresponding monophosphate into DNA or poly{d)(A-T)} template . Bacteriophage T4, lymphocyte alpha, and the EBV particle-associated DNA polymerases were assayed simultaneously for direct comparison . Unlike these three polymerases, which were capable of distinguishing between d2apTP and dATP with a strong preference for the latter, the EBV-induced DNA polymerase only weakly distinguished between dATP and d2apTP and incorporated substantial amounts of d2apTP into template . Detergent-treated lymphocyte nuclei undergoing a high level of EBV DNA synthesis were shown to incorporate the 2-aminopurine analog of dATP into viral DNA . The relative inability of the EBV-induced DNA polymerase to distinguish between the two purine nucleotides reported here is consistent with previous reports on the ready incorporation of other nucleotide analogs into DNA polymerases induced by other herpesviruses . Because most antiherpes agents currently in use or under study are nucleotide analogs, the viral mutagenic properties of these drugs should be examined.

Gene, 1981 Dec, 15(4), 365 - 78
Characterization of coliphage lambda hybrids carrying DNA fragments from Herpes simplex virus type 1 defective interfering particles; Denniston KJ et al.; We describe the characterization of 34 hybrid lambda bacteriophages carrying EcoRI fragments obtained from DNA of defective interfering particles of the Patton strain of Herpes simplex virus type 1 (HSV-1) . All cloned fragments contained S region terminal repeat sequences (TRs) fused to unique HSV-1 DNA . Several fragments contained deletions and rearrangements not described previously for DNA of HSV-1 defective interfering particles . A model describing the generation of defective interfering DNA based on recombination events involving the terminal "a" sequence as presented.

Eur J Biochem, 1981 Dec, 121(1), 27 - 31
The isolation, mapping and transcription in vitro of a beta 0-thalassaemia globin gene; Jackson IJ et al.; The red blood cell precursors of a patient with homozygous beta 0-thalassaemia have previously been shown to contain nuclear, but not cytoplasmic, beta-globin-specific transcripts . We describe the isolation of a beta-globin gene from this patient as a recombinant bacteriophage chromosome . Restriction-enzyme cleavage-site mapping experiments demonstrate no detectable deletions, insertions or major rearrangements in this thalassaemia gene . Two different techniques show that the gene isolated is transcribed as efficiently in vitro as the normal beta-globin gene.

J Virol, 1981 Dec, 40(3), 839 - 47
Genetic recombination of bacteriophage T7 in vivo studied by use of a simple physical assay; Lee D et al.; A new physical method was developed to assay genetic recombination of phage T7 in vivo . The assay utilized T7 mutants that carry unique restriction sites and was based on the detection of a new restriction fragment generated by recombination . Using this assay, we reexamined the genetic requirements for recombination of T7 DNA . Our results were in total agreement with previous findings in that recombination required the products of genes 3 (endonuclease), 4 (primase), 5 (DNA polymerase), and 6 (exonuclease) . Recombination was found to be independent of DNA ligase and DNA packaging and maturation functions.

Cell, 1981 Dec, 27(3 Pt 2), 533 - 42
Processing of mRNA by ribonuclease III regulates expression of gene 1.2 of bacteriophage T7; Saito H et al.; A bacteriophage T7 mutation, HS9, is phenotypically defective in gene 1.2, although it maps outside the gene . The single nucleotide change responsible for the HS9 mutation lies within the RNAase III recognition site immediately following gene 1.2 . This RNAase III recognition site, responsible for the processing of the mRNA encoding genes 1.1 and 1.2, contains two cleavage sites, separated by 29 bases . The HS9 mutation prevents cutting by RNAase III at one site in vitro, yielding a mRNA containing an additional 29 bases at its 3' end . The ten second-site reversion mutations of HS9 are all located in the RNAase III recognition site and either restore or eliminate cutting at both sites . RNAase III mutants of Escherichia coli phenotypically suppress the HS9 mutation . We propose that the extra 29 bases at the 3' end of the mRNA hybridize to the ribosome-binding site of gene 1.1; gene 1.1 immediately precedes gene 1.2 on the same mRNA molecule . Such hybridization prevents the initiation of translation of this mRNA containing gene 1.1 . A strong polar effect represses the translation of gene 1.2.

Biochim Biophys Acta, 1981 Nov 13, 662(1), 131 - 7
Lysozyme activity of bacteriophage T4 ghosts; Szewczyk B et al.; Bacteriophage T4 ghosts were found to possess lysozyme (mucopeptide N-acetylmuramoylhydrolase, EC 3.2.1.17) activity . This enzyme is probably responsible for the lysis from without, observed at high multiplicity of infection, a process independent of the presence of the e gene product which is also a lysozyme . The ghost lysozyme and e lysozyme differed with respect to their requirements for maximal catalytic activity and to some extent in substrate specificity . The ghost lysozyme was released from phage particle by the action of Triton X-100.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5797 - 809
Purification and characterization of a uracil-DNA glycosylase from the yeast . Saccharomyces cerevisiae; Crosby B et al.; An activity which releases free uracil from bacteriophage PBS1 DNA has been purified over 10,000 fold from extracts of Saccharomyces cerevisiae . The enzyme is active on both native and denatured PBS1 DNA and is active in the absence of divalent cation, and in the presence of 1 mM EDTA . The enzyme has a negative molecular weight of 27,800 as estimated by glycerol gradient centrifugation and gel filtration . Enzyme activity has been recovered after denaturation in SDS and electrophoresis in an SDS polyacrylamide gel . This analysis suggests that the enzyme consists of a single polypeptide chain of about 27,000 daltons . Normal levels of uracil-DNA glycosylase activity were found in partially purified extracts of the nitrous-acid sensitive rad18-2 mutant of yeast.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5587 - 99
Fine mapping of secondary structures of fd phage DNA in the region of the replication origin; Huang CC et al.; A synthetic heptaribonucleotide, GACCCCC, which is complementary to a unique site on fd bacteriophage DNA, primes DNA synthesis of fd by T4 bacteriophage DNA polymerase . The rate of the GACCCCC-primed DNA synthesis was not uniform as reflected by the appearance of discrete DNA fragments as replication intermediates on an alkaline agarose gel . After 10 minutes of synthesis a significant fraction of the DNA product ran as a single band with a length of about 1960 nucleotides . We have isolated this DNA fragment, hybridized back to unlabeled fd DNA template, and mapped the Taq I restriction fragments by urea polyacrylamide gel electrophoresis . This fine mapping procedure has located two major pause sites at fd nucleotide positions 5575 and 5674 . These sites reside in the stem of two very stable hairpin helices near the origin of DNA replication of fd . Models for the functional roles of these two hairpin helices are presented.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5679 - 88
Specific deletion of DNA sequences between preselected bases; Panayotatos N et al.; Blunt-end ligation of a "filled-in" HindIII, Sal I, Ava I or Bcl I restriction site with a DNA fragment having A, G, C, or T as the terminal 3' nucleotide regenerates the corresponding restriction site . A combination of this property with the action of BAL 31 nuclease which progressively removes base-pairs from the ends of linear DNA, can generate deletions extending to desired pre-selected nucleotides, and introduces unique restriction sites at those positions . Similarly other restriction sites can be used to select for the deletion of sequences between specific di-, tri-, tetra- and penta-nucleotides . Using this method, 10 base pairs were deleted from the end of a restriction fragment carrying the late promoter for bacteriophage T7 gene 1.1, to create a molecule with a unique restriction site at the initiation codon for translation.

J Biol Chem, 1981 Nov 10, 256(21), 11259 - 65
Bacteriophage f1 gene II and X proteins . Isolation and characterization of the products of two overlapping genes; Yen TS et al.; We have isolated and characterized the 2 major proteins of a dense complex which accumulate in EScherichia coli cells infected with bacteriophage f1 under conditions where the phage gene V protein is inactive (Webster, R . E., and Rementer, M . (1980) J . Mol . Biol . 139, 393-405) . The amino acid composition and NH2- and COOH-terminal sequences of the larger polypeptide (estimated molecular weight of 46,000) correspond to those predicted from the DNA sequence for the f1 gene II protein . The other polypeptide (estimated molecular weight of 14,000) has the amino acid composition and COOH-terminal sequence predicted for the f1 X protein, which previously had been found only as a product of an in vitro transcription-translation reaction . The X protein contains N-formylmethionine, cross-reacts with antibodies against gene II protein, and is present in wild type f1-infected bacteria . Thus, X protein is the product of f1 gene X (10), which is contained entirely in, and translated in phase with, gene II.

J Bacteriol, 1981 Nov, 148(2), 734 - 5
Improved generalized transducing bacteriophage for Caulobacter crescentus; Bender RA; Caulobacter phage phi Cr30T, a temperature-sensitive derivative of the lytic, generalized transducing phage phi Cr30, was isolated as a double temperature-sensitive recombinant in a cross between phi Cr30ts1 and phi Cr30ts2, phi Cr30T mediated generalized transduction of Caulobacter crescentus at frequencies comparable to those of phi Cr30 and eliminated the requirement for irradiation of transducing lysates to prevent killing of transductants on the plate.

J Bacteriol, 1981 Nov, 148(2), 712 - 5
Role of lipopolysaccharide in the receptor function for bacteriophage TuIb in Escherichia coli; Yu F et al.; Bacteriophage TuIb required lipopolysaccharide in addition to the OmpC trimer as a receptor component . Both the fatty acid and polysaccharide regions of lipopolysaccharide were shown to participate in the receptor function . The roles of lipopolysaccharide and outer membrane proteins in the receptor function for T-even type bacteriophages are discussed.

Mol Biol (Mosk), 1981 Nov-Dec, 15(6), 1385 - 96
{Interactions of gene 5 protein of bacteriophage f1 with oligonucleotides of a certain composition and sequence}; Veiko NN et al.; Interaction of gene 5 protein of bacteriophage f1 with a set of oligodeoxyribonucleotides (including those with 1,N6-ethenoadenine), which simulate a site of attachment of protein on DNA has been studied . The tyrosine and ethenoadenine fluorescence and CD of the complexes of the protein with the oligonucleotides have been measured . It has been revealed that the stoichiometry of the complexes depends on the protein--oligonucleotide ratio . It is demonstrated that protein binding changes the degree of hydration of the oligonucleotides and the geometry of stacking, without marked unstacking . Tyrosine residues partially intercalate in the oligonucleotide . It is possible that one of the tyrosines sits between the second and the third and one of the phenylalanines--between the third and the forth bases, of the pentanucleosidetetraphosphate from the 5'-end.

Gene, 1981 Nov, 15(2-3), 207 - 14
Nucleotide-sequence heterogeneity and sequence rearrangements in influenza virus cDNA; Fields S et al.; Double-stranded cDNA has been synthesized from influenza virus RNA and cloned into derivatives of the bacteriophage M13 for sequence analysis . The characterization of over 200 clones has permitted an analysis both of nucleotide sequence heterogeneity and of clones containing unusual rearrangements of sequence . Heterogeneity, due to genetic variability in the RNA population and to in vitro synthetic errors, was detected at the low level of one nucleotide difference per 3 700 nucleotides . By contrast, gross sequence rearrangements were identified in eight clones . Inversions of sequence within the same cDNA molecule were the predominant type of rearrangement, and three mechanisms for producing such inversions are discussed . In addition, we observed rarer clones containing sequence from one RNA molecule joined to that from another molecule.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 6754 - 8
SOS induction and autoregulation of the himA gene for site-specific recombination in Escherichia coli; Miller HI et al.; The himA gene of EScherichia coli controls the lysogenization of bacteriophage lambda at the level of catalysis of site-specific recombination and expression of the lambda int and cI genes required for lysogenic development . We have analyzed the regulation of himA by two methods: (i) beta-galactosidase synthesis from a lacZ gene inserted into the himA gene and (ii) detection of radioactive HimA protein after fractionation by two-dimensional gel electrophoresis . We find that himA- mutations produce enhanced expression of the himA gene, indicating that HimA protein controls its own synthesis . The himA gene is also induced by treatment of cells with UV or mitomycin C, suggesting control by the inducible DNA repair (SOS) system regulated by the LexA and RecA proteins . Regulation of himA follows the pattern expected for a typical SOS gene: constitutive high expression in mutants that have inactive LexA or the altered RecA conferred by the recA441 (tif1) mutation and low noninducible expression in a mutant that has a deleted recA gene . We conclude that the himA gene is a component of the inducible SoS response, repressed by LexA and induced by the capacity of activated RecA to cleave LexA . We suggest that HimA may be subject to SOS induction because it functions as an "acquisitionase" for new genetic material and thus is of special utility under conditions of impaired capacity for growth of the bacterial population.

J Bacteriol, 1981 Nov, 148(2), 399 - 405
Use of lipophilic cation-permeable mutants for measurement of transmembrane electrical potential in metabolizing cells of Escherichia coli; Hirota N et al.; Some lipopolysaccharide-defective mutants of Escherichia coli showed, without ethylenediaminetetraacetic acid treatment, a quick and high uptake of lipophilic cations such as triphenylmethylphosphonium and tetraphenylphosphonium . The rate and amount of uptake were comparable to those of an ethylenediaminetetraacetic acid-treated wild type . Transmembrane electrical potential, which was calculated from the distribution of these lipophilic cations between the inside and outside of the mutant cells, was about -150 mV at pH 7.5 and showed a strong dependency on the external pH . One of the E . coli mutants, the acrA mutant, was found to be also permeable to dicyclohexylcarbodiimide, an H+-adenosine triphosphatase inhibitor, and 1-anilino-8-naphthalene sulfonate, a fluorescent dye . The acrA mutant was vigorously motile and highly sensitive to many bacteriophages and colicins . Thus, the acrA mutant is quite useful for the quantitative measurement of transmembrane electrical potential by lipophilic cations in intact and metabolizing cells especially in relation to motility and actions of colicins and bacteriophages.

Biofizika, 1981 Nov-Dec, 26(6), 991 - 4
{Photochemical stability of aromatic amino acids under picosecond laser UV-irradiation}; Gurzadian GG et al.; Two-step photochemical decomposition of aromatic amino acids under picosecond laser UV-irradiation was investigated . These results were compared with the photochemical stability of nucleic acid bases . Using the known ratio between the nucleic acid bases and aromatic amino acids in native bacteriophages lambda and phi X174 it was shown that picosecond laser UV-inactivation of viruses occurred due to the photodegradation of nucleic acid.

J Bacteriol, 1981 Nov, 148(2), 739 - 43
Effects of recB21, recF143, and uvrD152 on recombination in lambda bacteriophage-prophage and Hfr by F- crosses; Howard-Flanders P et al.; The effects of the mutation pairs recB21 recF143 and recB21 uvrD152 on the frequency of genetic recombination were investigated in lambda phage-prophage crosses under homoimmune conditions . To prevent recombinants from being formed by the phage red system, these experiments were performed with phages and prophages carrying red and gam mutations . Both spontaneous and damage-induced recombination was measured, the phages being either undamaged or treated with trimethylpsoralen and 360-nm light to cross-link the phage DNA . Control and damaged phages were allowed to infect lysogenic host cells under conditions in which phage gene expression was repressed and phage DNA replication was blocked by lambda immunity . Although the double mutations recB21 recF143 and recB21 uvrD152 reduced recombination in Hfr by F- crosses to 0.3 to 0.02% of the wild-type controls, the presence of these pairs of mutations in the host lysogens had relatively little effect on the results of the phage-prophage crosses . In the latter system, recB21 recF143 reduced spontaneous and damaged-induced recombination by less than threefold whereas recB21 uvrD152 increased it to three times the wild-type level, the increase being attributable to the uvrD mutation . Evidently, the gene products of recB,C uvrD, and recF wee not needed for lambda phage-prophage recombination under repressed conditions.

J Virol Methods, 1981 Nov, 3(4), 241 - 9
A portable device for the rapid concentration of viruses from large volumes of natural freshwater; Logan KB et al.; A portable device for the rapid concentration of viruses from natural freshwaters described and its performance in field use is evaluated . The system handled up to 500 litres of water in less than 90 min at a cost of only 2 pounds per sample . Where the samples contained sufficient bacteriophages for detection by direct plating the apparent phage recoveries were greater than 75% . Plant and animal viruses were also concentrated from waters with this system.

J Virol, 1981 Nov, 40(2), 431 - 9
Structural organization and biological activity of molecular clones of the integrated genome of a BALB/c mouse sarcoma virus; Andersen PR et al.; BALB/c mouse sarcoma virus (BALB-MSV) is a spontaneously occurring transforming retrovirus of mouse origin . The integrated form of the viral genome was cloned from the DNA of a BALB-MSV-transformed nonproducer NRK cell line in the Charon 9 strain of bacteriophage lambda . In transfection assays, the 19-kilobase-pair (kbp) recombinant DNA clone transformed NIH/3T3 mouse cells with an efficiency of 3 X 10(4) focus-forming units per pmol . Such transformants possessed typical BALB-MSV morphology and released BALB-MSV after helper virus superinfection . A 6.8-kbp DNA segment within the 19-kbp DNA possessed restriction enzyme sites identical to those of the linear BALB-MSV genome . Long terminal repeats of approximately 0.6 kbp were localized at either end of the viral genome by the presence of a repeated constellation of restriction sites and by hybridization of segments containing these sites with nick-translated Moloney murine leukemia virus long terminal repeat DNA . A continuous segment of at least 0.6 and no more than 0.9 kbp of helper virus-unrelated sequences was localized toward the 3' end of the viral genome in relation to viral RNA . A probe composed of these sequences detected six EcoRI-generated DNA bands in normal mouse cell DNA as well as a smaller number of bands in rat and human DNAs . These studies demonstrate that BALB-MSV, like previously characterized avian and mammalian transforming retroviruses, arose by recombination of a type C helper virus with a well-conserved cellular gene.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 6643 - 6
The dicyclohexylcarbodiimide-binding protein c of ATP synthase from Escherichia coli is not sufficient to express an efficient H+ conduction; Friedl P et al.; Bacteriophage Mu was inserted into the unc genes of Escherichia coli . The resulting mutation AS12 had a polar effect on the unc operon: membranes of the mutant AS12 contained the dicyclohexylcarbodiimide-binding protein c and the protein a as sole subunits of the ATP synthase . It was shown by peptide mapping and amino acid analysis of the fragments that protein c from mutant AS12 was identical with the wild-type protein c . The absence of subunit b in mutant AS12 drastically lowered the H+ conduction dependent on the membrane-integrated moiety (F0) of the ATP synthase . This suggests that both subunits b and c are necessary for an efficient expression of H+ conduction.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 6628 - 32
Purified glucocorticoid receptors bind selectively in vitro to a cloned DNA fragment whose transcription is regulated by glucocorticoids in vivo; Payvar F et al.; Activated glucocorticoid receptor protein, purified to 40-60% homogeneity from rat liver extracts, binds selectively in vitro to a cloned fragment of murine mammary tumor virus (MTV) DNA . The DNA fragment tested contains about half of the sequences present in intact MTV DNA, and its rate of transcription, like that of the intact viral element, is strongly stimulated by glucocorticoids when it is introduced into the genome of a receptor-containing cell . In contrast, the receptor fails to bind selectively to DNA restriction fragments from E . coli plasmids pBR322 and RSF2124 or from bacteriophages lambda and T4 . Preliminary experiments to localize regions within MTV DNA responsible for selective binding have revealed thus far one subfragment that fails to bind the receptor and one selectively bound subfragment that maps far downstream from the 5' terminus of the normal RNA transcript . These studies are consistent with the notion that steroid receptors may modulate rates of transcription by recognizing specific DNA sequences within or near the regulated genes.

Gene, 1981 Nov, 15(2-3), 273 - 8
Cloned fragments of human adenovirus type-12 DNA; Vogel S et al.; The following restriction endonuclease fragments of human adenovirus type 12 (Ad12) DNA have been cloned in plasmid or bacteriophage lambda vectors using standard protocols: the EcoRI-A*, -B, -D, -E, and -F fragments, the BamHI-B, -C, -D, -F, -G, -H, and -I fragments, the HindIII-F and -I fragments, and the PstI-A, -D, -F, -G, and -H fragments . The EcoRI-A* fragment comprises the right terminal 5 kb of Ad12 DNA including the terminal 143 bp.

Gene, 1981 Nov, 15(2-3), 167 - 76
Modification of the bacteriophage vector M13mp2: introduction of new restriction sites for cloning; Rothstein R et al.; The construction of two new derivatives of the bacteriophage cloning vector M13mp2 is described . One derivative, mWJ22, contains a new HindIII site while the other, mWJ43, contains a new BamHI site . These new sites were both introduced at the EcoRI site at amino acid five of the 145 amino acid-long fragment of Escherichia coli beta-galactosidase within the phage . The new restriction sites do not disrupt the blue color detection system of M13mp2; therefore insertion of cloned fragments results in colorless plaques on indicator plates for the new derivatives.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 7019 - 23
RNA splicing mutation in an aberrantly rearranged immunoglobulin lambda I gene; Hozumi N et al.; The mouse cell line MOPC 315 is an IgA (lambda II)-producing myeloma . We have studied a derivative of MOPC 315 that secretes normal lambda II chains but no heavy chain . This derivative, MOPC 315-26, was found to contain a rearranged lambda I gene in addition to a rearranged lambda II gene . The rearranged lambda I gene was cloned into bacteriophage lambda DNA and its structure was studied . The lambda I gene was found to have arisen by an aberrant recombination event that resulted in a single base insertion at the site of V-J region joining . In addition, the gene contained numerous point mutations in the vicinity of the junction of the V and J regions . Two point mutations occurred in the donor splice sequence normally used for the removal of the intron between the J and C regions, suggesting that the RNA synthesized from the aberrantly rearranged lambda I gene would be unable to undergo proper RNA splicing.

Nucleic Acids Res, 1981 Oct 10, 9(19), 4863 - 78
Control of promoter utilization by bacteriophage T4-induced modification of RNA polymerase alpha subunit; Goldfarb A et al.; After infection of Escherichia coli cells, bacteriophage T4 induces several changes in the host DNA-dependent RNA polymerase . A well-characterized chemical change is a two-step ADP-ribosylation of the enzyme's alpha subunit (1) . In order to investigate the effect of this change on RNA polymerase transcriptional properties in an in vitro system, we have reconstituted the enzyme from separated individual subunits which were obtained from normal or T4-modified RNA polymerases . It is demonstrated that the enzymes containing T4-modified alpha differ from the enzymes with normal alpha in two respects: (i) their overall activity on T4 DNA is reduced and (ii) they fail to utilize certain T4 promotors while efficiently utilizing other promoters . Among the promoters which are switched off by alpha modification are the two promoters of the D region and one of the two promoters of the T4 tRNA gene cluster . The differential effect of alpha modification on the expression of the tRNA and the D regions in vitro correlates with the previously established pattern of their transcription in vivo . It is suggested that the T4-induced ADP-ribosylation of RNA polymerase alpha subunit is involved in the shutoff of the early bacteriophage genes at the late stage of phage development.

J Biol Chem, 1981 Oct 10, 256(19), 9951 - 8
The orientation of the major coat protein of bacteriophage f1 in the cytoplasmic membrane of Escherichia coli; Ohkawa I et al.; The orientation of the major coat (B) protein of the bacteriophage f1, an integral membrane protein in the cytoplasmic membrane of infected Escherichia coli, was examined . Pyridoxal 5'-phosphate and {3H}NaBH4 were used to label the cytoplasmic membrane proteins in spheroplasts and membrane vesicles of E . coli infected with bacteriophage f1 . Under the conditions described, tritium incorporation was almost completely dependent on the presence of pyridoxal 5'-phosphate and little if any of the cytoplasmic proteins were labeled when the reaction was applied to intact spheroplasts . The major coat protein was isolated from the cytoplasmic membranes labeled in this manner and the chymotryptic peptides were analyzed for the presence of tritium in the pyridoxamine 5'-phosphate conjugate . When the proteins were labeled in the intact spheroplast, only the NH2-terminal chymotryptic peptide of the coat protein was labeled . If the proteins were labeled during osmotic lysis of the spheroplasts or in isolated vesicles, the chymotryptic peptide containing the COOH terminus of the coat protein as well as the NH2-terminal peptide was labeled . The NH2-terminal peptide was labeled to approximately the same extent as occurred in the intact spheroplast . These results are consistent with the hypothesis that the mature f1 coat protein asymmetrically spans the cytoplasmic membrane of the infected host with its NH2 terminus exposed on the outside and COOH terminus exposed on the cytoplasmic surface.

Nucleic Acids Res, 1981 Oct 10, 9(19), 4833 - 45
ClaI . a new restriction endonuclease from Caryophanon latum L; Mayer H et al.; From Caryophanon latum L site specific restriction endonuclease (ClaI) has been purified, which recognises tha DNA hexanucleotide palindrome 5'-A-T-C-G-A-T-3' . Staggered cleavage generates DNA restriction fragments with 5'-terminal pCG extensions . A CLaI map of bacteriophage lambda has been determined, which indicates cleavage inhibition due to adenine methylation at over lapping ClaI-GATC recognition sequences . Plasmid pBR322 is cut only once, in the tetracycline promoter region, and can, therefore, be used as a vector system for cloning fragments derived from ClaI digestions, and in addition for fragments generated by TaqI, HpaII, and several other enzymes.

Eur J Immunol, 1981 Oct, 11(10), 757 - 64
Phagocytosis and degradation of DNA-anti-DNA complexes by human phagocytes . I . Assay conditions, quantitative aspects and differences between human blood monocytes and neutrophils; Lamers MC et al.; The uptake in vitro was studied of 3H-labeled DNA-anti-DNA complexes by neutrophils and monocytes from human blood . Complexes were prepared from 3H-labeled circular double-stranded (dS) DNA of bacteriophage PM2 and anti-dsDNA-containing sera from patients with systemic lupus erythematosus . After phagocytosis, cells and medium were separated . The cells were treated with DNase to remove adherent and noningested complexes before the cell-associated radioactivity was counted . Thus, only complexes inside the cells were measured . The medium was analyzed for acid-precipitable radioactivity . In this way, we found that neutrophils only phagocytose the complexes, whereas monocytes phagocytose the complexes and degrade the antigen . In contrast, both types of phagocyte degraded the antigen in tetanus-anti-tetanus complexes . The degradation took place after phagocytosis, inside the cells . The difference in DNA degradation between neutrophils and monocytes correlated with the difference in acid DNase activity of the lysosomal fractions: monocytes contained DNase activity, neutrophils did not . With complexes made from DNA with 131I-labeled anti-DNA, we found that both cell types degraded the antibody . Uptake of complexes and degradation of antigen increased with incubation time and cell concentration and was saturable with respect to complex concentration . The processes were inhibited by 5 mM mono-iodoacetic acid or by low temperatures.

J Cell Biol, 1981 Oct, 91(1), 26 - 44
Sperm morphogenesis in wild-type and fertilization-defective mutants of Caenorhabditis elegans; Ward S et al.; Taking advantage of conditions that allow spermatogenesis in vitro, the timing and sequence of morphological changes leading from the primary spermatocyte to the spermatozoon is described by light and electron microscopy . Together with previous studies, this allows a detailed description of the nuclear, cytoplasmic, and membrane changes occurring during spermatozoan morphogenesis . By comparison with wild type, abnormalities in spermatogenesis leading to aberrant infertile spermatozoa are found in six fertilization-defective (fer) mutants . In fer-1 mutant males, spermatids appear normal, but during spermiogenesis membranous organelles (MO) fail to fuse with the sperm plasma membrane and a short, though motile . pseudopod is formed . In fer-2, fer-3, and fer-4 mutants, spermatids accumulate 48-nm tubules around their nuclei where the centriole and an RNA containing perinuclear halo would normally be . In all three mutants, spermatids still activate to spermatozoa with normal fusion of their MOs, but the pseudopods formed are aberrant in most fer-2 and fer-4 spermatozoa and in some fer-3 spermatozoa . In fer-5 mutant males, spermatozoa do not form . Instead, defective spermatids with crystalline inclusions and abnormal internal laminar membranes accumulate . In fer-6 mutant males, only a few spermatozoa form and these have defective pseudopods . These spermatozoa retain their fibrous bodies, a structure which normally disassembles in the spermatid . The time of appearance of developmental abnormalities in all of these mutants correlates with the temperature-sensitive periods for development of infertility . The observation that each of these mutants has a different and discreet set of morphological defects, a structure which normally disassembles in the spermatid . The time of appearance of developmental abnormalities in all of these mutants correlates with the temperature-sensitive periods for development of infertility . The observation that each of these mutants has a different and discreet set of morphological defects, a structure which normally disassembles in the spermatid . The time of appearance of developmental abnormalities in all of these mutants correlates with the temperature-sensitive periods for development of infertility . The observation that each of these mutants has a different and discreet set of morphological defects shows that the strict sequence of morphogenetic events that occurs during wild-type spermatogenesis cannot arise because each event is dependent on previous events . Instead, spermatozoa, like bacteriophages, must be formed by multiple independent pathways of morphogenesis.

J Virol, 1981 Oct, 40(1), 309 - 13
Genetic and amber fragment maps of genes 46 and 47 of bacteriophage T4D; Wiberg JS et al.; We constructed genetic recombinational maps of genes 46 and 47 by using five amber mutants in gene 46, nine amber mutants in gene 47, and two-factor crosses . Two different amber fragments in gene 46 and three different amber fragments in gene 47 were detected on polyacrylamide slab gels in the presence of sodium dodecyl sulfate . The genetic maps agreed with the amber fragment maps; taken together, the data oriented all of the sites in both genes with respect to each other . Given the relative map positions of genes 46 and 47 determined genetically by Epstein et al . (Cold Spring Harbor Symp . Quant . Biol . 28:375-394, 1963), our results extend and reinforce the work of Hercules and Sauerbier (J . Virol . 12: 872-881, 1973) and that of Minner and Bernstein (J . Gen . Virol . 31:277-280, 1976), which indicated that the direction of transcription and translation of these genes if counterclockwise on the T4 genetic map (i.e., from gene 47 toward gene 46).

J Virol, 1981 Oct, 40(1), 248 - 57
Second-step transfer of bacteriophage T5 DNA: purification and characterization of the T5 gene A2 protein; Snyder CE Jr et al.; Second-step transfer of bacteriophage T5 DNA requires the function of the T5 pre-early proteins A1 and A2 . We have isolated and characterized the gene A2 protein as part of an effort to determine the mechanism of second-step transfer . The A2 protein was purified by DNA-cellulose column chromatography followed by gel filtration and ion-exchange column chromatography . The A2 protein's identity was confirmed by two-dimensional gel electrophoresis . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer gel filtration in 6 M guanidine hydrochloride demonstrated a molecular weight of 15,000 for the A2 polypeptide . Migration of the A2 protein through gel filtration columns under nondenaturing conditions, in combination with sedimentation behavior, indicated dimerization of the A2 polypeptide . The existence of the A2 dimer was confirmed by protein cross-linking with dimethyl suberimidate and analysis of the cross-linked proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The amino acid composition, degree of polymerization, DNA-binding ability, and physical characteristics of the T5 gene A2 protein are consistent with a function of the A2 protein in DNA transfer.

J Virol, 1981 Oct, 40(1), 197 - 203
Deletion analysis of the cloned replication origin region from bacteriophage M13; Cleary JM et al.; A cloned 270-nucleotide fragment from the origin region of the M13 duplex replicative form DNA confers an M13-dependent replication mechanism upon the plasmid vector pBR322 . This M13 insert permits M13 helper-dependent replication of the hybrid plasmid in polA cells which are unable to replicate the pBR322 replicon alone . Using in vitro techniques, we have constructed several plasmids containing deletions in the M13 DNa insert . The endpoints of these deletions have been determined by DNA sequence analysis and correlated with the transformation and replication properties of each plasmid . Characterization of these deletion plasmids allows the following conclusions . (i) The initiation site for M13 viral strand replication is required for helper-dependent propagation of the chimeric plasmid . (ii) A DNA sequence in the M13 insert, localized between 89 and 129 nucleotides from the viral strand initiation site, is necessary for efficient transformation of polA cells . A chimeric plasmid containing the viral strand initiation site, but lacking this additional 40 nucleotide M13 sequence, transforms helper-infected cells at a frequency approximately 10(4)-fold less than that of plasmids containing this additional DNA segment . (iii) The entire M13 complementary strand origin can be deleted without affecting M13-dependent transformation by the hybrid plasmids . We propose a model in which replication of one strand of duplex chimera initiates by nicking at the gene II protein nicking site in the viral strand of the M13 insert, followed by asymmetric single-strand synthesis . Initiation of the complementary strand possibly occurs within plasmid sequences.

Can J Biochem, 1981 Oct, 59(10), 799 - 801
Differential effects of ethanol and the rpsL1 (strA1) ribosomal mutation on the synthesis of an unusual protein coded by bacteriophages R17 and MS2 RNA; Laughrea M; During translation of R17 and MS2 RNA, ethanol stimulates the synthesis of a coat-related protein which has identical electrophoretic mobility to that of polypeptide 7 studied by Atkins and his colleagues . Streptomycin stimulates the synthesis of this polypeptide and broadens the protein band . In contrast the ribosomal protein S12 mutation rpsL1 (strA1) has no detectable effect on its synthesis.

Proc Natl Acad Sci U S A, 1981 Oct, 78(10), 5973 - 7
Reinitiation of translation from the triplet next to the amber termination codon in the absence of ribosome-releasing factor; Ryoji M et al.; Ribosome releasing factor (RR factor) releases ribosomes from mRNA at the termination codon in Escherichia coli . In the absence of this factor, polypeptides with molecular weights very close to the molecular weight of bacteriophage R17 coat protein were synthesized in vitro under the direction of a mutant R17 phage RNA having an amber mutation at codon 7 of the coat cistron . The major coat-protein-like product shared all the R17 coat protein sequence except that the seven NH2-terminal amino acids were missing . The minor product had the complete coat protein sequence starting from formylmethionine except for a probable amino acid substitution at codon 7 (UAG) . Addition of RR factor inhibited the synthesis of the major protein . These results indicate that, in the absence of RR factor, the ribosome that has released the NH2-terminal hexapeptide at the amber codon stays on the mRNA and subsequently reinitiates translation "in phase" immediately after the amber codon without formylmethionine.

J Virol, 1981 Oct, 40(1), 65 - 77
Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity associated with the T4 das mutation; Mickelson C et al.; Lethal, amber mutations in T4 genes 46 and 47 cause incomplete degradation of host DNA, premature arrest of phage DNA synthesis, accumulation of abnormal DNA replication intermediates, and defective recombination . These phenotypes can be explained by the hypothesis that genes 46 and 47 control a DNA exonuclease, but in vitro demonstration of such a nuclease has not yet been reported . Membrane and supernatant fractions from 46- and 47- mutant-infected and 46+ 47+ control-infected cells were assayed for the presence of the protein products of these genes (i.e., gp46 and gp47) and for the ability to degrade various DNA substrates to acid-soluble products in vitro . The two proteins were found only on membranes . The membrane fraction from 46- 47- mutant-infected cells digested native or heavily nicked Escherichia coli DNA to acid-soluble products three to four times slower that the membrane fraction from control-infected cells . No such effect was found in the cytoplasmic fractions . The effect on nuclease activity in membranes was the same whether 46- and 47- mutations were present singly or together . NaClO4, a chaotropic agent, released both gp46 and gp47 from 46+ 47+ membranes, as well as the DNase activity controlled by genes 46 and 47 . DNA cellulose chromatography of proteins released from membranes by NaClO4 showed that gp46 and gp47 bound to the native DNAs of both E . coli and T4 . Thus, the overall enrichment of gp46 and gp47 relative to total T4 protein was 600-fold (10-fold in membranes, 2-fold more upon release from membranes by NaClO4, and 30-fold more upon elution from DNA cellulose) . T4 das mutations, which partially suppress the defective phenotype of 46- and 47- mutants, caused a considerable increase in vitro DNase activity in both membrane and cytoplasmic fractions, We obtained evidence that the das+ gene does not function to inhibit E . coli exonuclease I or V, endonuclease I, or the UV endonuclease of gene uvrA or to decrease the activity of T4 exonuclease A or the T4 gene 43 exonuclease.

Eur J Biochem, 1981 Oct, 119(3), 663 - 8
Bacteriophage fd gene-2 protein . Processing of phage fd viral strands replicated by phage T7 enzymes; Harth G et al.; Bacteriophage T7 gene 4 protein and DNA polymerase of the phage were used to study the viral strand synthesis of bacteriophage fd in vitro . Cleavage of supercoiled phage fd replicative form (RF) by fd gene 2 protein produced a nick at a specific site in the viral strand . The cleaved double-stranded DNA was unwound by T7 gene 4 protein and T7 DNA polymerase and the 3' end of the nicked strand simultaneously extended according to the rolling circle mechanism . After a complete round of DNA synthesis fd gene 2 protein cleaved the viral strand presumably at the same site, where the endonuclease cuts fd RF I, and subsequently sealed the single-stranded linear DNA into a circle . The reaction products were analyzed by velocity sedimentation, gel electrophoresis and electron microscopy . Most of the single-stranded DNA synthesized was circular . No host proteins were required for the formation of the single-stranded circles . Strand switching of the T7 DNA polymerase indicated by double-stranded tails of the rolling circle structures reduced the yield of viral single-stranded circles in this enzyme system.

J Gen Microbiol, 1981 Oct, 126(Pt 2), 413 - 25
Is the IS1-flanked r-determinant of the R plasmid NR1 a transposon?
Iida S, Hanni C, Echarti C, Arber W.
The 23 kilobase multiple drug resistance r-determinant (r-det) of the R plasmid NR1 is an IS1-mediated transposon, Tn2671 . Drug-resistant Escherichia coli transductants isolated after infection with bacteriophage P1::Tn2671 derivatives carry the intact r-det in their chromosomes . Independently isolated transductants carry the r-det at different locations on the chromosome . From the E . coli chromosome, Tn2671 can transpose to various locations on the phage P7 genome . Throughout these processes, r-det is maintained as a stable unit . Various possible molecular mechanisms, which all might contribute with characteristic frequencies to the transposition of Tn2671, are discussed . The results presented are relevant to the understanding of mechanisms for a wide spreading of drug resistance genes.

Biochem J, 1981 Oct 1, 199(1), 273 - 6
The stereochemical course of phosphoryl transfer catalysed by polynucleotide kinase (bacteriophage-T4-infected Escherichia coli B); Jarvest RL et al.; Polynucleotide kinase (bacteriophage-T4-infected Escherichia coli B) catalyses the transfer of the {gamma-16O,17O,18O}phosphoryl group from 5'{gamma(S)-16O,17O,18O}ATP to 3'-AMP with inversion of configuration at the phosphorus atom . The simplest interpretation of this observation is that the {gamma-16O,17O,18O}phosphoryl group is transferred directly from ATP to the co-substrate by an 'in-line' mechanism.

J Gen Virol, 1981 Oct, 56(Pt 2), 267 - 74
Partial exclusion of bacteriophage T2 by bacteriophage T4: induction of early enzymes by excluded T2; Okker RJ; In crosses between bacteriophages T2 and T4 most early genes of T2 are partially excluded from the progeny . Six genes of T4 affect the exclusion of six corresponding exclusion-sensitive sites in T2, each gene being specific for the exclusion of one site . Mutants of T4 in these genes have been isolated (ex mutants) . The induction of the gene product (deoxycytidine triphosphate nucleotidohydrolase, dCTPase) of the strongly excluded T2 gene 56 was determined . The dCTPase was induced in the presence of the replication inhibitor oxolinic acid to prevent possible artefacts from unequal replicaton rates of T2 and T4 . The rate of T2 dCTPase induction was 30% of the control in T2 X T4 infections in which all six exclusion-sensitive sites were excluded and was 57% in infections in which the sites except 56 were excluded . The dCTPase induction was 67% of the control with exclusion of the 56 site only and equalled the control when no T2 site was excluded . Inhibition of dCTPase induction under conditions of exclusion is partly due to exclusion of neighboring sites and partly due to exclusion of the 56 site.

J Virol, 1981 Oct, 40(1), 211 - 23
den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities; McMillan S et al.; Recent studies have shown purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phage T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA . Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase . In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA . Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV+ phage . Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells . Furthermore, the addition of DNA containing apurinic or apyrimidinic sites to reactions containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA . On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity.

J Virol, 1981 Oct, 40(1), 204 - 10
Evidence that the UV endonuclease activity induced by bacteriophage T4 contains both pyrimidine dimer-DNA glycosylase and apyrimidinic/apurinic endonuclease activities in the enzyme molecule; Warner HR et al.; We performed experiments to determine whether the phage T4-induced UV endonuclease activity is a single protein containing both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities . The UV endonuclease activity is induced by the denV gene and codes for the glycosylase activity . We obtained several kinds of evidence that the protein containing the glycosylase activity also contains the apyrimidinic endonuclease activity . After chromatography on DEAE-cellulose, the two activities copurified during phosphocellulose chromatography and Sephadex G-100 chromatography, with a constant ratio of activities across the activity peaks . On Sephadex G-100 columns the molecular weights of the two activities agreed within 2,500 or less . When an extract of cells infected with the T4 V1 mutant was purified in exactly the same way as an extract of cells infected with T4 V1+, neither glycosylase nor apyrimidinic endonuclease activity was detected in the normal elution position of the T4 UV endonuclease activity . The glycosylase and apyrimidinic endonuclease activities were induced with similar kinetics, which were characteristic of immediate early rather than delayed early enzymes . This correlated well with the presumed major role of these activities in repairing thymine dimers in parental DNA before DNA replication begins . Finally, glycosylase and apyrimidinic endonuclease activities were lost in parallel during incubation of the enzyme at 46 degree C . Our results indicated that both of these enzyme activities are contained in the same enzyme molecule and, probably, in the same polypeptide.

Antimicrob Agents Chemother, 1981 Oct, 20(4), 425 - 32
Mechanism of action of cinodine, a glycocinnamoylspermidine antibiotic; Greenstein M et al.; The mechanism of action of cinodine, a glycocinnamoylspermidine antibiotic, was investigated . Upon addition of cinodine to growing cultures of Escherichia coli, a rapid decline in viable cell numbers was observed . Culture turbidity continued to increase for a short period before plateauing . Microscopic examination indicated that the antibiotic-treated cells continued to elongate with subsequent formation of serpentine-like structures . Radioisotopic-labeling studies of E . coli demonstrated that deoxyribonucleic acid (DNA) synthesis was immediately and irreversibly inhibited upon addition of cinodine . Ribonucleic acid synthesis was reduced after a significant delay, whereas protein synthesis remained unaffected . There was a minor degree of inhibition of incorporation of radiolabeled diaminopimelic acid into cell wall material . Cinodine likewise inhibited bacteriophage T7 DNA synthesis in infected E . coli cells . After inhibition of E . coli DNA synthesis by cinodine, intracellular DNA degradation was observed . Equilibrium dialysis studies demonstrated that the drug physically bound to DNA . These data indicate that cinodine functions as a potent irreversible inhibitor of bacterial and phage DNA synthesis.

J Biol Chem, 1981 Sep 25, 256(18), 9680 - 3
Structure and expression of human globin genes introduced into mouse fibroblasts; Chen MJ et al.; The human delta- and beta-globin genes, contained in a recombinant bacteriophage (lambda H beta G1), were introduced into mouse fibroblasts by cotransformation with a plasmid (chi 1) containing the herpes simplex thymidine kinase gene using the calcium phosphate precipitation technique . A molar ratio of lambda H eta G1 to chi 1 DNA of 3:1 was used . Four of the eleven stable transformants obtained contained intact delta- and beta-globin genes as determined by Southern blot analysis . To assess methylation in the segment of human DNA introduced into mouse cells, digestion with Hpa II or Msp I alone or with a second restriction enzyme was performed . The sites examined near the human delta- and beta-globin genes in transformed cells were not methylated . RNA extracted from the transformed cells was analyzed by RNA-cDNA hybridization; no more than 100 copies of human beta-globin mRNA/cell were found . Although hypomethylation of sites surrounding expressed globin genes in erythroid cells has been described, this property is not sufficient to ensure a high level of expression in fibroblasts.

J Biol Chem, 1981 Sep 25, 256(18), 9662 - 7
Cloning and structural characterization of an estrogen-dependent apolipoprotein gene; Wiskocil R et al.; ApoVLDLII is a major polypeptide component of avian very low density lipoprotein . Its production in the liver of the maturing female chick is developmentally synchronized with vitellogenesis . The apoVLDLII gene is normally dormant in males but can be activated by the administration of estrogen . In these studies, we describe the isolation of three recombinant bacteriophages that contain apoVLDLII genes . The genes appear to be identical from preliminary restriction analyses, although heterogeneity in flanking sequences is evident . The structure of the gene has been characterized and its organization correlated with the structure of the apoVLDLII polypeptide.

J Biol Chem, 1981 Sep 10, 256(17), 9246 - 53
Purification and properties of the Escherichia coli protein factor required for lambda integrative recombination; Nash HA et al.; A purified preparation of the Escherichia coli integration host factor (IHF) displays two polypeptides of apparent molecular weight 11,000 and 9,500 when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . Under nondenaturing conditions, IHF appears to exist as a 1:1 complex of these two polypeptides . Integrative recombination takes place in vitro when purified IHF and purified Int, a product of a bacteriophage lambda gene, are the only proteins added to reaction mixtures . No recombination is detected in the absence of either protein . The characteristics of the recombination reaction carried out by these two purified proteins are described . Purified IHF binds to DNA; in the presence of Int, a ternary complex is formed at one of the specific recombination sites . IHF hs no detectable endonuclease or topoisomerase activity . Several possibilities for the role of IHF in recombination are considered.

Rev Infect Dis, 1981 Sep-Oct, 3(5), 926 - 33
Agents of mycobacterial variations; Mankiewicz E; Mycobacterial variations are determined by physicochemical agents or by genetic transfers . The pioneer work done on the study of the effects of light, radiation, heat, nutritional factors, and drugs is reviewed . Technical and conceptual problems arise in the study of genetic transfer by means of conjugation and transformation in mycobacteria . Transduction and lysogenic conversion are shown to be responsible for important changes in bacterial and colony morphology, enzymatic activities, bacteriophage and drug resistance . Results of recent experiments with different strains of bacille Calmette-Guerin (BCG) suggest a relationship between phage conversion and the degree of immunogenic potential.

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