Genetika, 1983 Jun, 19(6), 940 - 4 {Lysogenic conversion induced by phage phi 80 . II . Mapping of the cor locus}; Kozyrev DP et al.; The cor locus determines lysogenic conversion of the lambdoid phi 80 phage . The cor locus was cloned from phi 80, phi 80pt1, phi 80pt0, phi 80pt4A and lambda hy51 DNAs in the pBR322 plasmid . According to the physical and genetic data, the cor locus was located on the genetic map of phi 80 bacteriophage at the 40.3 divided by 43.5% lambda . It is closely linked to the gene 13 of phi 80 phage and has a position similar to that of lom gene of lambda phage. J Virol, 1983 Jun, 46(3), 895 - 900 Physiological properties of a T7-T3 recombinant bacteriophage that productively infects strains of Escherichia coli that harbor the F plasmid; Spence JL et al.; The T7-T3 recombinant phage BO2, whose isolation and physical properties are described in the accompanying paper (Molineux et al., J . Virol . 46:881-894, 1983), has been characterized by physiological means after infection of male (F plasmid-containing) and female strains of Escherichia coli . Single-step growth analyses have shown that the hybrid phage gives a burst about two-thirds that of T7 or T3 on females and about one-half that of T3 on males . This reduced burst size can be correlated with altered kinetics of macromolecular synthesis, probably at the level of transcription . The T3 insertions of BO2, designated M1 and M2, that are essential for growth of the hybrid phage on male strains, have been shown to be trans acting and can rescue T7 from F-mediated restriction . The nature of the gene products encoded by the M1 and M2 regions and their role in overcoming the abortive infection of males by T7 are discussed. J Gen Virol, 1983 Jun, 64 (Pt 6), 1355 - 63 Correlation of genetic loci and polypeptides specified by bacteriophage T1; Ritchie DA et al.; The effects of amber mutations in the 24 known essential genes of phage T1 on phage-directed protein synthesis were examined by SDS-polyacrylamide gel electrophoresis of radiolabelled polypeptides from infected non-permissive cells . Mutations in nine genes (genes 1, 2, 3, 3.5, 5, 7-8, 10, 13.3 and 15) each resulted in the failure to synthesize a single polypeptide . Since the synthesis of each of these polypeptides was at least partially restored in permissive infections and wholly restored in am+ revertant infections we conclude that the affected polypeptide is the primary gene product . Mutations in genes 13.7, 16 and 17, which are required for head formation, are all defective in the synthesis of a 68000 mol . wt . non-structural polypeptide . Mutants in the head genes 13 and 14 fail to cleave P7p to P7, the major structural component of proheads . Gene 14 mutants also fail to make a 45000 mol . wt . non-structural polypeptide which appears to be involved with the gene 13 product in the P7p cleavage reaction . The protein products of the DNA genes 1, 2 and 3.5 are, as expected, synthesized predominantly early in infection whereas those of the remainder, which determine head and tail formation, are made in greater amounts late in infection . Gene 3, a tail gene which has been mapped within the 'early' DNA gene cluster, codes for a 75000 mol . wt . polypeptide synthesized predominantly late in infection . This observation suggests that the early genes 1, 2, 3.5 and 4 do not form an operon under the control of a single early promoter. J Bacteriol, 1983 Jun, 154(3), 1064 - 76 A bacterial gene, fip, required for filamentous bacteriophage fl assembly; Russel M et al.; An Escherichia coli mutant which does not support the growth of filamentous bacteriophage fl allows phage fl DNA synthesis and gene expression in mutant cells, but progeny particles are not assembled . The mutant cells have no other obvious phenotype . On the basis of experiments with phage containing nonlethal gene I mutations and with mutant fl selected for the ability to grow on mutant bacteria, we propose an interaction between the morphogenetic function encoded by gene I of the phage and the bacterial function altered in this mutant . The bacterial mutation defines a new gene, fip (for filamentous phage production), located near 84.2 min on the E coli chromosome. Cell, 1983 Jun, 33(2), 623 - 8 Bacteriophage Mu: a transposing replicon; Higgins NP et al.; Mu DNA replication has been carried out in vitro on cellophane discs in the presence of dBUTP . If the DNA is sheared to 80 kb pieces, the Mu replicas band anomalously in CsCl gradients between hybrid and light DNA density positions . The intermediate density DNA comprises semiconservatively replicated Mu sequences, flanked by unreplicated light DNA . This and previous data are consistent with replication occurring within Mu boundaries . Both the synthesis of Mu sequences and the intermediate density DNA are abolished by protein synthesis inhibition in vivo just prior to lysis on cellophane discs . These observations indicate that at least some steps in bona fide Mu transposition-replication are being observed in vitro. Proc Natl Acad Sci U S A, 1983 Jun, 80(12), 3787 - 91 Expression of Plasmodium falciparum blood-stage antigens in Escherichia coli: detection with antibodies from immune humans; Kemp DJ et al.; Many proteins produced by blood stages of the malaria parasite Plasmodium falciparum are natural immunogens in man . As an approach to determining which of these are relevant to protective immunity we have constructed an expression library of P . falciparum cDNA sequences, cloned in Escherichia coli . The cDNA sequences were inserted into the beta-galactosidase gene of an ampicillin-resistant derivative of the temperature-sensitive lysogenic bacteriophage lambda gt11 . About 5% of the resulting clones expressed P . falciparum sequences as polypeptides fused to beta-galactosidase . We have identified many clones that express P . falciparum antigens by immunological screening in situ with antibodies from immune human sera that inhibit P . falciparum growth in vitro . The antigen-positive clones contain P . falciparum cDNA sequences, as determined by hybridization . Some express polypeptides that are larger than beta-galactosidase and react both with antibodies to beta-galactosidase and with antibodies from humans immune to P . falciparum . The cloned P . falciparum antigens should facilitate new approaches to the identification of potential vaccine molecules. Proc Natl Acad Sci U S A, 1983 Jun, 80(12), 3632 - 6 Isolation of novel human genomic DNA clones related to human interferon-beta 1 cDNA; Sehgal PB et al.; Southern blot-hybridization analyses of human DNA (from Namalwa lymphoblastoid cells) digested with the restriction endonuclease EcoRI were carried out under optimal conditions with two human fibroblast interferon (IFN-beta 1) cDNA probes, pD19 and pD24, which contain IFN-beta 1 inserts 0.8 and 0.7 kilobase (kb) long, respectively . The analyses revealed the presence of several hybridizable DNA fragments, including two of lengths 6.8 and 5.5 kb, in addition to the classical IFN-beta 1 genomic DNA fragment of length approximately equal to 2.0 kb . We have screened a human DNA library in lambda bacteriophage Charon 4A by using a 32P-labeled IFN-beta 1 insert cDNA (pD24) and thereby isolated six strongly positive human genomic DNA clones . One of these (lambda B37) represents the classical human IFN-beta 1 gene; another (lambda B37) contains a 6.8-kb EcoRI DNA fragment(s) which cross-hybridizes with the IFN-beta 1 cDNA insert probes pD19 and pD24; and the remaining four (which are identical to each other and are exemplified by lambda B4) contain two EcoRI DNA fragments approximately 5.5 and 9 kb long which also cross-hybridize the IFN-beta 1 cDNA probes . A mRNA 0.9 kb long derived from the classical IFN-beta1 gene is expressed in poly(I) . poly(C)-induced human diploid fibroblasts (FS-4 strain) . Induced FS-4 cells also contain polyadenylylated RNA 1.8, 3, 5, and approximately equal to 8 kb long derived from the lambda B3 gene, all of which appear to code for biologically active human IFN-beta as tested by using the Xenopus laevis oocyte translation assay . These data strongly indicate that lambda B3 represents a novel functional IFN-beta gene . A 12-kb polyadenylylated RNA, derived from lambda B4, is expressed constitutively at a low level in FS-4 cells, but the amount of this RNA increases 5-7 hr after exposure of the cells to poly(I) . poly(C). Pediatrics, 1983 Jun, 71(6), 964 - 7 Transient immunodeficiency during asymptomatic Epstein-Barr virus infection; Bowen TJ et al.; In vivo and in vitro humoral and cellular immune responses were studied in a 2 1/2-year-old girl immediately before, during, and after an asymptomatic infection with Epstein-Barr virus . During the acute EBV infection, the patient's peripheral blood mononuclear cells were deficient in immunoglobulin synthesis and suppressed the in vitro immunoglobulin synthesis of normal allogeneic cells . In vitro mitogen transformation of lymphocytes was reduced . In vivo antibody responses to the T cell-dependent antigens bacteriophage phi X 174 and Keyhole limpet hemocyanin were markedly depressed . These studies suggest that suppressor cells induced during acute EBV infection not only suppress immunoglobulin synthesis in vitro, but also interfere with in vivo antibody synthesis. J Virol, 1983 Jun, 46(3), 829 - 40 Molecular cloning and characterization of endogenous feline leukemia virus sequences from a cat genomic library; Soe LH et al.; Recombinant bacteriophage lambda clones from a cat genomic library derived from placental DNA of a specific pathogen-free cat were screened to identify endogenous feline leukemia virus (FeLV) sequences . Restriction endonuclease mapping of four different clones indicates that there are a number of similarities among them, notably the presence of a 6.0- to 6.4-kilobase pair (kbp) EcoRI hybridizing fragment containing portions of sequences homologous to the gag, pol, env, and long terminal repeat-like elements of the infectious FeLV . The endogenous FeLV sequences isolated are approximately 4 kbp in length and are significantly shorter than the cloned infectious FeLV isolates, which are 8.5 to 8.7 kbp in length . The endogenous elements have 3.3- to 3.6-kbp deletions in the gag-pol region and approximately 0.7- to 1.0-kbp deletions in the env region . These deletions would render them incapable of encoding an infectious virus and may therefore be related to the non-inducibility of FeLV from uninfected cat cells and the subgenomic expression of these endogenous sequences in placental tissue . It appears that there is conservation in the ordering of restriction sites previously reported in the proviruses of the infectious FeLVs in sequences corresponding to the pol and env boundary as well as the region spanning the env gene of the endogenous clones, whereas a greater divergence occurs among restriction sites mapped to the gag and part of the pol regions of the infectious FeLV . Such deleted, FeLV-related subsets of DNA sequences could have originated either by germ-line integration of a complete ecotropic virus followed by deletion, or by integration of a preexisting, defective, deleted variant of the infectious virus. J Virol, 1983 Jun, 46(3), 778 - 87 Interaction of a DNA-binding protein, the product of gene D5 of bacteriophage T5, with double-stranded DNA: effects on T5 DNA polymerase functions in vitro; Fujimura RK et al.; The gene D5 product (gpD5) of bacteriophage T5 is a DNA-binding protein that binds preferentially to double-stranded DNA and is essential for T5 DNA replication, yet it inhibits DNA synthesis in vitro . Mechanisms of inhibition were studied by using nicked DNA and primed single-stranded DNA as a primer-template . Inhibition of T5 DNA polymerase activity by gpD5 occurred when double-stranded regions of DNA were saturated with gpD5 . The 3' leads to 5' exonuclease associated with T5 DNA polymerase was not very active with nicked DNA, but inhibition of hydrolysis of substituents at 3'-hydroxyl termini by gpD5 could be observed . T5 DNA polymerase appears to be capable of binding to the 3' termini even when double-stranded regions are saturated with gpD5 . The interaction of gpD5 with the polymerases at the primer terminus is apparently the primary cause of inhibition of polymerization. Biochimie, 1983 Jun, 65(6), 317 - 24 Vectors for high conditional expression of cloned genes; Leplatois P et al.; Several multicopy plasmids carrying the control region of bacteriophage lambda lysogeny, including the gene of a thermosensitive repressor CI857 have been constructed . The phages allow high expression of proteins under the transcription control of lambda promoter PR and translation control of Cro . The method has been assayed by measuring expression of either intact beta-galactosidase, truncated beta-galactosidase or beta-galactosidase fused to a mitochondrial gene product . It is shown that use of a strain with low endogeneous proteolytic activities strongly improves the conditional yield of foreign proteins, so that a high overproduction can be achieved (10-20 per cent of the total protein content of the cell). Chem Biol Interact, 1983 Jun, 44(3), 261 - 74 Phototoxicity of phenothiazine derivatives . I . Inactivating and mutagenic effects on bacteriophage øX174; Merville MP et al.; Inactivation of oX174 bacteriophages as a function of the irradiation time in the near-UV and in the presence of triflupromazine (TFPZ), promazine (PZ), chlorpromazine (CPZ) or methoxypromazine (MTPZ) proceeds according to single hit kinetics . Acepromazine (ACPZ) has no significant activity . At low concentrations (0.1 mM) TFPZ and PZ are the most active compounds . Higher concentrations (up to 5 mM) result in a protective effect by these two compounds but cause increased inactivation rates in the case of MTPZ or CPZ . Photoinactivation mediated by TFPZ or CPZ increases the reversion frequency of a oXamber mutant . Neither MTPZ nor PZ sensitization induces mutagenesis . The effect of NaN3 on the phage inactivation rate varies depending upon both the sensitizer and the concentration of the quencher . Phage inactivation in an N2 atmosphere is measurable only in the presence of high concentrations of CPZ and MTPZ . The drugs do not show any selectivity for calf thymus DNA or bovine serum albumin, at least as measured by dialysis equilibrium experiments. Mutat Res, 1983 Jun, 112(3), 129 - 37 Differential survival and chloramphenicol-insensitive error-prone repair of hydroxylamine-inactivated phi X174 bacteriophage mutants; Mukherjee S et al.; Features of inactivation, repair and concomitant mutagenesis of hydroxylamine-treated phi X174 bacteriophages are reported here . (1) For reasons unknown, the nonsense phage mutants tested here were far more sensitive to hydroxylamine than the wild-type phage . In contrast, the sensitivities of these same phi X174 mutants to UV-irradiation are indistinguishable . (2) Hydroxylamine-treated amber phages mutated to ochre but not to wild-type particles, i.e., G leads to A transition events were recovered . (3) The repair of phi X174 phages from hydroxylamine-induced damage was error-prone, but unlike UV damage, did not require protein synthesis de novo . Possible mechanisms of these novel features are discussed. Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3439 - 43 Clusters of point mutations are found exclusively around rearranged antibody variable genes; Gearhart PJ et al.; We have examined the nucleotide sequences of a series of murine antibody genes derived from one kappa light chain gene in order to gain insight into the mechanism that specifically mutates variable genes . Six rearranged VK167 genes from hybridoma and myeloma cells were cloned from bacteriophage lambda libraries . The sequences were compared to the germ-line sequence of the VK167 gene, the JK genes, and the CK gene to identify sites of mutation . Four of six rearranged genes had extensive mutation which occurred exclusively in a 1-kilobase region of DNA centered around the V-J gene . No mutations were found at more distant sites in the intervening sequence or in the constant gene . The frequency of mutation was approximately 0.5% (32 mutations per 6,749 base pairs) . Mutations were mostly due to nucleotide substitutions with no preference for transitions or transversions . The location of mutations around each gene indicates that they occur in clusters at random sites . The observation of mutations in the intervening sequence downstream from the JK5 gene rules out models for the mechanism of mutagenesis that rely solely on gene conversion or recombination . The distribution and high frequency of mutations are most easily explained by a mechanism of error-prone repair that occurs during several cycles of cell division. Cell, 1983 Jun, 33(2), 509 - 18 The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro; Mount SM et al.; The ability of purified U1 small nuclear RNA-protein complexes (U1 snRNPs) to bind in vitro to two RNAs transcribed from recombinant DNA clones by bacteriophage T7 RNA polymerase has been studied . A transcript which contains sequences corresponding to the small intron and flanking exons of the major mouse beta-globin gene is bound in marked preference to an RNA devoid of splice site sequences . The site of U1 snRNP binding to the globin RNA has been defined by T1 ribonuclease digestion of the RNA-U1 snRNP complex . A 15-17-nucleotide region, including the 5' splice site, remains undigested and complexed with the snRNP such that it can be co-precipitated by antibodies directed against the U1 snRNP . Partial proteinase K digestion of the U1 snRNP abolishes interaction with the globin RNA, indicating that the snRNP proteins contribute significantly to RNA binding . No RNA cleavage, splicing, or recognition of the 3' splice site by U1 snRNPs has been detected . Our results are discussed in terms of the probable role of U1 snRNPs in the messenger RNA splicing of eucaryotic cell nuclei. Nature, 1983 May 26, 303(5915), 349 - 52 Complete nucleotide sequence of an immunoglobulin VH gene homologue from Caiman, a phylogenetically ancient reptile; Litman GW et al.; Immunoglobulin variable (V) gene regions typify extensive multigenic families in terms of overall size, chromosomal arrangement and presence of large numbers of apparent pseudogenes . A unique mechanism of somatic reorganization involving recombination of VH, D and JH or VL and JL segments accompanies the differentiation of lymphoid cells and together with somatic mutation and other types of recombination accounts for V-region diversity . Although these processes have been well characterized in higher mammals, little is known concerning their origin and diversification during phylogenetic time . Previously, we described the blot-hybridization characteristics of murine VHIII probes with restriction enzyme-digested genomic DNA isolated from several phylogenetically critical species, including Caiman crocodylus, a modern representative of an ancient reptilian subclass . Here we have used a murine probe, S107V, to select homologous clones from a library of Caiman genomic DNA constructed in a lambda bacteriophage . The complete nucleotide sequence of a Caiman gene homologous to the murine VH gene and its adjacent 5' and 3' region is described . Comparison of the sequence with mammalian prototypes shows evidence of considerable organizational and structural homology extending outside the presumed VH-coding region and including elements believed to be involved in somatic recombination . Inferences about the evolution of this multigenic family can now be extended to the level of phylogenetic class. J Biol Chem, 1983 May 25, 258(10), 6250 - 4 Stimulation of calf thymus DNA alpha-polymerase by ATP; Wierowski JV et al.; ATP stimulates the activity of the A and C forms of calf thymus DNA alpha-polymerase on several natural and synthetic primer-templates . Stimulations ranging from 1.5- to 8-fold were observed on gapped bacteriophage fd replicative form DNA, poly(dA) x oligo(dT)10, and poly(dT) x oligo(A)10, at ATP concentrations of 1-5 mM . CTP, dATP, and dCTP can substitute for ATP but are less effective . The nonhydrolyzable ATP analogs, adenyl-5'-yl imidodiphosphate and adenosine 5'-O-(thiotriphosphate), and other deoxy- and ribonucleoside triphosphates are essentially inactive for stimulation . Stimulation does not result from polymerase-associated DNA primase activity . The size of products synthesized processively by each enzyme on the two homopolymer templates was determined by gel filtration of primers extended under conditions where the enzyme did not react with a given 3'-OH terminus more than once . The size of products synthesized by the A and D forms on poly(dA) x oligo(dT)10 increased by a factor of 3-6 in the presence of ATP . This suggests a direct effect of ATP on the primer elongation reaction . Finally, presynthesis incubation of enzyme plus template at 37 degrees C in the presence or absence of ATP demonstrates that ATP stabilizes the enzyme against breakdown. J Mol Biol, 1983 May 25, 166(3), 341 - 60 Gene 20 product of bacteriophage T4 . II . Its structural organization in prehead and bacteriophage; Driedonks RA et al.; The location of gene 20 product of bacteriophage T4 in phage and phage percursors has been determined by immunochemical analysis of polyacrylamide gels, immunoturbidimetry and immunoelectron microscopy . The protein is present at the membrane attachment site of the prehead, a head precursor, and is accessible to the antibodies in the solution . It is present at the tail attachment site of the capsid, partially buried in the structure . In complete phage particles it is totally buried in the structure . It is in contact with the major shell proteins, gp23 and gp23*, respectively, in preheads and capsids, as revealed by partial crosslinking experiments . It forms the upper collar of the neck in necked tails . The lower collar is constructed from other gene products . On the basis of these data a structural model of the neck region of the phage has been derived . This model is consistent with a number of events in phage assembly, such as the role of gp20 in head assembly and DNA packaging, prehead detachment from the bacterial membrane and head-tail attachment . The symmetry mismatch known to occur between head and tail has been localized at the gp20-gp23* contact area. J Biol Chem, 1983 May 25, 258(10), 6064 - 72 Properties of Bacteriophage T4 ribonucleoside diphosphate reductase subunits coded by nrdA and nrdB mutants; Cook KS et al.; As a part of the study of the bacteriophage T4-induced deoxyribonucleotide synthetase complex, an investigation has been made of the T4 ribonucleoside diphosphate reductases formed by a series of mutants of nrdA and B, the genes coding, respectively, for the alpha 2 and beta 2 subunits of the enzyme . dATP affinity columns were used to isolate the enzyme by a single-step procedure . The molecular weights of the alpha and beta chains have been found to be 84,000 and 43,500, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Since alpha 2 beta 2 is bound to dATP affinity columns through allosteric effector sites on alpha 2, it is possible to monitor the binding of beta 2 to alpha 2 . dTTP- and ATP-Sepharose columns did not bind T4 alpha 2 beta 2, although the corresponding nucleoside triphosphates are effectors of the enzyme and although the alpha 2 subunit of the host enzyme binds to these columns . Missense mutants of nrdA and B forming alpha 2 and beta 2 subunits that lacked catalytic activity but retained the ability to form the alpha 2 beta 2 complex have been described . The 50,000-dalton fragment formed by an amber mutant of nrdA did not bind to the dATP affinity column, providing evidence that a region of the carboxyl-terminal segment of the alpha chain is required for retention . The beta 2 subunit appears to protect the alpha 2 protein . On infection by nrdB mutants not forming beta 2, the alpha protein chain was cleaved specifically to form 3 protein chains of 61,000, 57,000, and 24,500 daltons, which retain the ability to bind to dATP-Sepharose . Some effects of mutation on the interaction of the alpha and beta chains of the enzyme with the deoxyribonucleotide synthetase complex have been examined. J Biol Chem, 1983 May 10, 258(9), 5798 - 803 Purification of phi X174 gene C protein; Aoyama A et al.; The product of gene C of bacteriophage phi X174 is required for the replication of phiX174 single-stranded DNA in Escherichia coli cells infected with phi X174 . The protein has been purified to homogeneity using an in vitro complementation system . The protein exhibits a molecular weight of 5800 under denaturing conditions . The NH2-terminal amino acid sequence of the protein is (NH2)-Met-Arg-Lys, which is consistent with the sequence predicted from the nucleotide sequence of gene C . The protein has an affinity for single-stranded DNA but less for double-stranded DNA. FEBS Lett, 1983 May 8, 155(2), 291 - 4 Studies of the structure of bacteriophage lambda cro protein in solution . Analysis of the circular dichroism data; Bolotina IA et al.; The secondary and tertiary structures of bacteriophage cro protein were studied by circular dichroism . The pH dependence of this structure was investigated: cro protein is stable over pH 4.5-10.5 . At these pH-values cro protein contains approximately 35% alpha-helix, approximately 20% antiparallel beta-structure and approximately 15% beta-turn, while the remaining part of the protein molecule is in the irregular state . The secondary and tertiary structures of the protein are modified abruptly at more acid and more alkaline pH-values . The curves characterizing the secondary and tertiary structures of the protein are symbatic . The effect of Gu-HCl on the secondary and tertiary structures of cro protein at 22 degrees C and pH 7.2 was studied also . The conformational transition occurs within 0.6-1.9 M Gu-HCl . The changes in the secondary and tertiary structures of the protein have a symbatic character . Thermal denaturation of cro protein was examined . A possible mechanism of the protein denaturation is discussed. Nature, 1983 May 5-11, 303(5912), 84 - 6 Evidence for a conservative pathway of transposition of bacteriophage Mu; Akroyd JE et al.; During its lytic cycle bacteriophage Mu uses repeated transposition as a mode of DNA synthesis . These transpositional events are undoubtedly replicative, and presumably semi-conservative . In a Mu lysogen this type of transposition can start immediately after prophage induction . However, in an infective cycle the Mu genome (which is injected into the host cell as a linear molecule flanked by short random sequences of bacterial DNA) must first become integrated into the host chromosome . Little is known about how this occurs apart from the fact that the bacterial sequences at either end of the Mu genome are lost in the process . The integration is thus similar to a transposition event . In an attempt to determine whether this type of Mu transposition (between a linear donor molecule and a circular recipient) is also semi-conservative we have analysed the progeny phage arising from an infective cycle in which the parental DNA was heterozygous for a known genetic marker . The expectation is that if integration of the infecting Mu genome occurs by a single semi-conservative transpositional event then pure phage bursts should be produced as the genetic information on only one strand would be preserved throughout the lytic cycle . The experiments reported here do not support this expectation in that the infected cells yield mixed bursts, suggesting that Mu integration is a conservative, rather than a semi-conservative event. Can J Microbiol, 1983 May, 29(5), 627 - 9 Bacteriophages of Halobacterium halobium: virion DNAs and proteins; Rohrmann GF et al.; The DNAs from bacteriophages Hh-1 and Hh-3 that infect Halobacterium halobium were characterized . Both phages contain linear double-stranded DNA and show no relatedness based on restriction endonuclease fragment patterns . Hh-1 DNA has 67.05% guanine plus cytosine (G+C) and a molecular weight of 24.6 X 10(6), whereas Hh-3 DNA has 62.15% G+C and a molecular weight of 19.4 X 10(6) . Polyacrylamide gel electrophoresis indicated that the major proteins of the two phages are of different molecular weights. J Antibiot (Tokyo), 1983 May, 36(5), 588 - 94 High frequency transduction of R-factor encoded gentamicin resistance by bacteriophage P1Cm; Piepersberg W et al.; No transposition of plasmid-coded gentamicin resistance from more than fifty different R-plasmids onto a deletion derivative of bacteriophage lambda was found . In contrast, 13 out of 17 R-plasmids gave rise to the formation of high frequency transducing (hft) hybrids of phage P1Cm . All the hft P1Cm derivatives transduced other antibiotic resistances in addition to gentamicin resistance . The DNA sequences found to be integrated in the prophage genomes of hft phages were generally longer than 15 kb, and ranged up to 60 kb . In most cases analyzed the points of insertions were close to the IS1 elements resident in P1Cm . In part of the hybrid phages the entire R-plasmids were cointegrated . One plasmid (pWP14a) cointegrated preferentially into a Bg1II fragment of P1Cm containing an invertible structure (C-loop) . Eleven out of 16 R-plasmids showed homology to IS1. Biophys J, 1983 May, 42(2), 171 - 80 DNA and protein lattice-lattice interactions in the filamentous bacteriophages; Marzec CJ et al.; The relations between the protein coats and DNAs of the four filamentous bacteriophages fd, Xf, Pf1, and Pf3 are considered . These viruses have similar morphologies, yet show a diversity of detailed structure, having different protein coat symmetries (helical and rotational), different coat protein sizes (44-50 amino acids per subunit) and sequences, different nucleotide axial translations (2.3-5.5 A), and different ratios of nucleotides per coat protein subunit (integers 1.0 and 2.0, and nonintegers approximately 2.4) . These divergences are all reconciled quantitatively by means of two theoretical concepts: the pitch connection and the restricted pitch connection . The pitch connection relates protein and DNA surface lattices with arbitrary, nonintegral nucleotide/subunit ratios in a nonrandom way . The restricted pitch connection selects a preferred set of n/s values . Both relations are derived formally in a mathematical appendix . The available structural data are explained, including the fd DNA pitch indicated by x-ray diffraction photos and the similar DNA morphologies of Xf and fd . Predictions are made for the existence of nonclassical inverted DNA structures (I-DNA) in Pf1 and Pf3. Virology, 1983 May, 127(1), 177 - 93 Morphogenesis of filamentous bacteriophage f1: orientation of extrusion and production of polyphage; Lopez J et al.; Crosslinking reagents were used to interrupt the process of filamentous phage morphogenesis and investigate the orientation in which nascent virions are extruded through the host cell membrane . Infected bacteria with emerging phage particles were crosslinked with glutaraldehyde . Immunoferritin-labeling studies on these emerging phage using anti-A protein IgG suggested that extrusion begins with the C protein end . To confirm this, phage extruding from infected bacteria were frozen using the reversible crosslinker dimethyl 3,3'-dithiobis-propionimidate and fragments of emerging phage were isolated by shearing . Protein analysis of these fragments showed them to be enriched in C protein relative to A protein, as predicted if phage extrusion begins with the C protein end . The production of multiple-length phage particles (polyphage) by nonpermissive bacterial hosts infected with amber mutant phage strains was also studied . Polyphage were produced upon infection with amber mutants in genes III, VI, VII, and IX which code for proteins found at the ends of the mature phage particle . No polyphage were produced by mutants in the other genes tested . Gene III amber mutants produce noninfective polyphage, but those produced by genes VII and IX are infective . Gene VI amber mutants appear to produce unstable, noninfective polyphage particles. J Virol, 1983 May, 46(2), 673 - 7 On the sequential packaging of bacteriophage P22 DNA; Adams MB et al.; Bacteriophage P22 is thought to package daughter chromosomes serially along concatemeric DNA . We present experiments which show that the average DNA packaging series length increases with time after infection, which supports this model . In addition, we have analyzed the effect on average series length of lowering the amount of the various individual proteins involved in DNA packaging . These results support the notion that the protein products of gene 2 and gene 3 are both more stringently required for initiation of sequential DNA packaging series than for their extension, and they are compatible with a model for the control of series length in which that length is determined, at least in part, by a competition between series initiation events and extension events. J Virol, 1983 May, 46(2), 629 - 31 Evidence that bacteriophage T4 eph1 is a missense hoc mutation; Childs JD et al.; An electrophoretic mutation of bacteriophage T4, eph1, appears to code for a missense hoc (highly antigenic outer capsid) protein . This is based on the observation that particles lacking hoc protein (hoc- particles), after incubation in a crude extract of Escherichia coli infected with phage carrying the eph1 mutation acquired the electrophoretic mobility of the eph1 strain (the electrophoretic mobility of the eph1 strain itself is slower than that of hoc- particles) . Thus, it is likely that during infection of E . coli with the eph1 strain, a hoc protein is made that has a lower negative charge than normal hoc protein but can nevertheless bind to particles lacking hoc protein . These results confirm that eph1 is a hoc mutation. Stain Technol, 1983 May, 58(3), 161 - 70 Characterization of five commercially available samples of acridine yellow; Houba-Herin N et al.; Commercial samples of acridine yellow, all labeled C.I . 46025, have been analyzed by thin layer chromatography, UV and visible light spectroscopy, mass spectrometry, and photodynamic efficiency in the inactivation of bacteriophage phi X174 . Three types of sample were clearly delineated: i) true acridine yellow (3,6-diamino-2,7-dimethylacridine) whose spectral and chromatographic properties are very close to those of proflavine (3,6-diaminoacridine); ii) a pure but different dye tentatively identified as euchrysine (3,6-diamino-2,7,9-trimethylacridine), since on the basis of mass spectral data, it contains an additional methyl group not fixed on the amino groups; and iii) a complex dye with its own special properties and whose main yellow component has a molecular weight and a mass spectrum compatible with an overall formula of C16H16N2S . The three types of dye could be distinguished on the basis of simple tests . Acridine yellow is photodynamically almost as efficient as proflavine, but the two other dyes are very poor sensitizers. Virology, 1983 May, 127(1), 124 - 33 Role of gene 8 product in morphogenesis of bacteriophage T3; Nakasu S et al.; The product of gene 8 (gp8) of T3 phage is one of the minor head proteins located at the phage head-tail junction . To determine the role of gp8, an amber (8-) and four temperature-sensitive mutants (ts8) were characterized by sedimentation analysis, polyacrylamide gel electrophoresis, and extract complementation . Neither DNA-containing particles nor empty particles were formed in cells infected with 8- . In addition, prohead assembly was greatly reduced . Prohead assembly was also blocked in cells infected with all ts8 mutants at 42 degrees and with some ts8 even at 37 degrees . Proheads containing gpts8 were converted to empty heads when cell lysates were treated with chloroform . The protein compositions of proheads showed that the minor head proteins, gp8, gp15, and gp16, were lost from proheads formed in cells infected with ts8, but these minor proteins were present in proheads formed in cells infected with double mutants of ts8 and 5- or 19-, which are defective in DNA synthesis or DNA maturation, respectively . In vitro complementation experiments suggested that a ts mutation in gene 8 affected not only DNA packaging but also subsequent assembly steps . From these results, it is concluded that gp8 plays multiple roles in T3 phage morphogenesis, including prohead assembly, prohead stabilization, DNA packaging, and subsequent events. Proc Natl Acad Sci U S A, 1983 May, 80(10), 2814 - 8 Relationship between promoter structure and template specificities exhibited by the bacteriophage T3 and T7 RNA polymerases; Bailey JN et al.; To explore the basis for the template specificities of the bacteriophage T3 and T7 RNA polymerases (EC 2.7.7.6), we determined the nucleotide sequences of six promoters recognized by the T3 RNA polymerase and compared them with the previously determined promoter sequences recognized by the bacteriophage T7 RNA polymerase . Recombinant plasmids containing random Hpa II and Taq I fragments of T3 DNA were screened for T3 promoter activity in vitro in a transcription assay using purified T3 RNA polymerase . Five promoters for the T3 RNA polymerase were identified in this manner and their sequences were determined; the sequence of an additional promoter was determined directly from a genomic DNA fragment . In five of the T3 promoters an identical 16-base-pair sequence (A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-A) extends from -12 to +4 (initiation occurring with GTP at +1); this sequence is preceded by a 6-base-pair A + T region . The remaining promoter contains an inserted C at position -1 and an A at the +1 position . The sequence of the 5' end of the RNA transcript from the latter promoter confirms that transcription is initiated with ATP at the +1 position . Previously, late T3 or T7 transcripts had not been found to initiate with ATP . The highly conserved T3 promoter sequence was compared to the T7 promoter consensus sequence . The fundamental difference between the two kinds of phage promoters is the occurrence of G-A at positions -11 and -10 in the T7 promoter, whereas there is a single C at position -10 in the T3 promoter. J Bacteriol, 1983 May, 154(2), 772 - 9 Genetic and physical characterization of lysogeny by bacteriophage MX8 in Myxococcus xanthus; Orndorff P et al.; Myxophage MX8 can initiate a lysogenic cycle in Myxococcus xanthus . The lysogenic phage was gentically stable in vegetative cells and persisted in the latent state through many cell generations in the absence of extracellular phage reinfection . The latent state also was stable during the host developmental cycle, since myxospores transmitted latent MX8 genetic information to future progeny cells . DNA hybridization experiments to probe the structure of the lysogenic phage provided physical evidence that MX8 formed a prophage . During lysogenization, MX8 DNA was cut at a specific site (attP) on phage DNA, and we have concluded that genetic recombination between attP and a bacterial DNA site (attB) leads to integration of MX8 DNA and formation of stable MX8 prophage . The genetic and physical properties of MX8 that we describe should make MX8 useful in the analysis of development of M . xanthus by genetic methods. Genetika, 1983 May, 19(5), 744 - 8 {General method for selecting mutations in bacterial and phage genes essential for the development of bacteriophage mu}; Piruzian ES et al.; A method has been developed for a relatively easy selection of bacterial mutations, either inhibiting or promoting Mu growth, and of various mutations in the genome of the Mu phage itself . Spontaneous or induced mutations inhibiting Mu growth are tested in a bacterial strain carrying a Mucts62 prophage in the chromosome and a defective Muc+ prophage within an unstable and frequently eliminated plasmid (the "operational" strain) . The cells of such a strain form colonies at 43 degrees C owing to the domination of the c+ allele, though a portion of the cells in a growing colony lose the plasmid with the Muc+ gene and die, dut to the induction of the Mucts prophage . In this case, a large number of phage particles are produced which cause cell lysis during the growth of a colony of the "operational" strain on a Mu-sensitive Escherichia coli lawn . The absence of a lysis zone around a colony of the "operational" strain would indicate a clone that is unable to produce viable Mu particles, as a result of a mutation in the bacterial or the phage genome . The situation is reverse when a strain incapable of maintaining Mu growth is used as the "operational" strain . Clones are tested for the ability to produce Mu on colour indicator media where the "operational" cells form coloured colonies and the tester cells form a colourless lawn . The method has been proved effective in a study conducted to test the use of Mu-lambda-Mu structures with defective lambda prophages flanked by identically oriented Mu genomes for selecting deletion mutants of Mu. Genetics, 1983 May, 104(1), 1 - 9 Involvement of gene 49 in recombination of bacteriophage T4; Miyazaki J et al.; The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants . When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype . However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor . Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvsX, but recombination between rII markers was not. Proc Natl Acad Sci U S A, 1983 May, 80(10), 2809 - 13 M13 procoat and a pre-immunoglobulin share processing specificity but use different membrane receptor mechanisms; Watts C et al.; Bacteriophage M13 procoat is accurately processed to transmembrane coat protein by salt-washed or N-ethylmaleimide-treated rough microsomes from dog pancreas . These treatments inhibit the processing of eukaryotic secreted protein precursors . M13 procoat can assemble into dog pancreas microsomes post-translationally . Thus, the microsomal proteins needed for assembly may be determined by the nature of the precursor protein itself . These results, and our finding that the mouse IgG kappa chain fragment precursor is processed by Escherichia coli leader peptidase, also suggest that the cleavage specificity of leader (signal) peptidases and the properties of preproteins that render them suitable for cleavage have been conserved during evolution. J Bacteriol, 1983 May, 154(2), 938 - 45 DNA injection during bacteriophage T4 infection of Escherichia coli; Furukawa H et al.; The process of phage T4 DNA injection into the host cell was studied under a fluorescent microscope, using 4',6-diamidino-2-phenylindole as a DNA-specific fluorochrome . The phage DNA injection was observed when spheroplasts were infected with the artificially contracted phage particles having a protruding core . The DNA injection was mediated by the interaction of the core tip with the cytoplasmic membrane of the spheroplast . A membrane potential was not required for the process of DNA injection . On the other hand, DNA injection upon infection by intact noncontracted phage of the intact host cell was inhibited by an energy poison . Based on these observations, together with results from previous work, a model for the T4 infection process is presented, and the role of the membrane potential in the infection process is discussed. Anal Biochem, 1983 May, 131(1), 205 - 10 Detection of A + T-rich DNA in gels by differential fluorescence; Hyman BC et al.; The fluorochrome Hoechst 33258 preferentially forms complexes with A + T-rich duplex DNA, whereas ethidium bromide binds nucleic acids independent of base composition . Both compounds can be conveniently used to visualize DNA fractionated by gel electrophoresis . Determination of fluorescence emission from Hoechst 33258-stained restriction fragments normalized to fluorescence derived from the same sample after ethidium bromide staining provides a measure of emission due to A + T content, and allows easy identification of A + T-rich restriction fragments . To demonstrate the utility of this procedure, an A + T map of bacteriophage lambda DNA was constructed and found to be comparable to similar maps derived by alternate techniques . Analysis of recombinant plasmid DNAs with established nucleotide sequences demonstrated that the A + T content of individual restriction fragments could be estimated to within an accuracy of 5%. Biosci Rep, 1983 May, 3(5), 469 - 74 The lysine residues implicated in the gene 5 protein association sites; Bayne S et al.; Gene 5 protein, a DNA unwinding protein encoded by the bacteriophage fd, is self-associative in presence of DNA or oligonucleotides . The lysine residues implicated in the protein-protein binding domains have been identified after modification with acetimidate by means of peptide and amino acid analyses . These residues are Lys-7 and Lys-69. Gene, 1983 May-Jun, 22(2-3), 167 - 74 The restriction mapping of c gene deletions in Streptomyces bacteriophage phi C31 and their use in cloning vector development; Harris JE et al.; In addition to 20 previously mapped restriction sites in the DNA of phi C31, we have determined eight sites for SphI, four for EcoRV, and two for SstII; there are none for BglII or SstI . Nine sites were in a 12-kb segment of DNA containing no previously mapped sites . Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule . One of the deletions (delta C3) was obtained in a previously described phi C31c+::vph (viomycin phosphotransferase) derivative containing two PstI sites separated by 3.9-kb of inessential DNA . After in vitro PstI treatment, plaque-forming phages lacking the 3.9-kb fragment were obtained from the c+ phage but not from its delta C3 derivative . Thus a 36.2-kb genome, but not one of 34.4 kb, was able to give infectious virions . PstI-generated DNA fragments of up to 8 kb can be inserted in vitro into the delta C3 derivative with retention of the vph selective marker . With the insertion of a 6.03-kb PstI fragment of plasmid SCP2, the latter phage became a potential vector (with loss of vph) for BamHI-generated DNA fragments of up to 9 kb . In the course of this work, several ClaI sites in phi C31::pBR322 bifunctional replicons were shown to be lost when the DNA was propagated in a dam+ Escherichia coli strain . This will allow the use of such replicons for the cloning of ClaI-generated DNA fragments of up to 6.7 kb. Genetika, 1983 May, 19(5), 720 - 6 {Nature of the plasmids determining the unstable inheritance of transposon Tn9 in Escherichia coli K-12}; Mirkin SM et al.; Unstable inheritance of transposon Tn9 in the Escherichia coli strain KS7201 had been connected with its integration into a certain bacterial chromosome site (attTn9A) . However, the present work shows that the transposon is situated within an unstable plasmid in this strain . A possibility of such plasmid's formation, as a result of a deletion of a part of bacteriophage lambda DNA, is shown. Mol Cell Biol, 1983 May, 3(5), 914 - 21 Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells; Curran T et al.; The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1) . Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA . The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus . Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome . This substituted region contains v-fos, the transforming gene of FBJ-MSV . Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA . Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region . However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome . The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human). Virology, 1983 May, 127(1), 24 - 36 Site-specific recombination by Gin of bacteriophage Mu: inversions and deletions; Plasterk RH et al.; A 3000-bp invertible segment in the DNA of bacteriophage Mu determines the host range of the phage . The inversion is catalyzed by the phage-coded protein Gin; the recombination sites are short inverted repeats . Gin protein is only made in low amounts by Mu . To further investigate the Gin-mediated recombination reaction a Gin overproducing strain was constructed . The gin gene was cloned on a plasmid behind the PL-promotor of phage lambda . This results in a 100-fold higher inversion frequency of a Mu gin phage as compared to the situation when Gin is expressed from its own promoter . A test system was developed suitable for the detection of Gin action in vivo and in vitro: the lacZ gene of E . coli was cloned within the invertible region in such a way that it is only expressed when the region is in one specific orientation . Thus inversions can be detected or selected as a switch from Lac- to Lac+ . This system was used to determine the inversion frequency under different experimental conditions . The ability of Gin to catalyze deletions was investigated by inverting in vitro one of the two recombination sites using restriction enzymes and genetically marking the DNA between those sites . Deletions do occur, although at a lower frequency than inversions. Proc Natl Acad Sci U S A, 1983 May, 80(10), 3000 - 4 Isolation of genes expressed preferentially during sporulation in the yeast Saccharomyces cerevisiae; Clancy MJ et al.; A library of Saccharomyces cerevisiae DNA in the vector lambda Charon 28 was probed for sequences complementary to cDNA made from poly(A)+ RNA isolated from the well-sporulating yeast strain AP1 a/alpha . The RNA was isolated from cells that had been incubated 7, 9, 11, and 13 hr in sporulation medium . DNA complementary to poly(A)+ RNA from alpha/alpha(nonsporulating) AP1 was used as a control, and 46 bacteriophage that gave a stronger response with a/alpha cDNA than with alpha/alpha cDNA were obtained in a screening of three yeast genomes worth of DNA . Two of the bacteriophage appeared to contain a/alpha-specific genes, in that they hybridized to cDNA from vegetative a/alpha RNA . The rest appeared to correspond to a/alpha genes expressed preferentially during sporulation . Restriction endonuclease analysis of four of the cloned sequences revealed a single major region of transcription in each; these regions ranged in size from 2.5 to 4.0 kilobases . RNA blot analysis showed that, in three of the four cases, transcripts of two different sizes were homologous to the cloned sequence . In all four cases, the homologous transcripts appeared at about 7 hr and were decreasing in amount by 13 hr . These results provide evidence for transcriptional control of genes expressed during sporulation and for at least one group of genes that is turned on at about the time of meiosis I in sporulation. Proc Natl Acad Sci U S A, 1983 May, 80(9), 2452 - 6 Role of short regions of homology in intermolecular illegitimate recombination events; Marvo SL et al.; The structures of three recombinants between bacteriophage lambda DNA and plasmid pBR322 that were generated in a recA derivative of Escherichia coli are described . Each resulted from two illegitimate recombination events that resulted in the substitution of part of the lambda genome by part of the plasmid genome . The nucleotide sequences at the six lambda-plasmid junctions were determined and compared with the sequences of the lambda and plasmid genomes before recombination . Each recombination occurred at a short region of homology in the two genomes, and other short regions of homology were found near some of the junctions . The structures of these junctions are similar to those resulting from illegitimate recombination in animal cells . A model to explain how these multiple illegitimate recombination events could result from a cascade of DNA gyrase-catalyzed recombinations is discussed. Proc Natl Acad Sci U S A, 1983 May, 80(9), 2442 - 6 Affinity purification of bacteriophage T4 proteins essential for DNA replication and genetic recombination; Formosa T et al.; The bacteriophage T4 helix-destabilizing protein, the product of gene 32, has been immobilized on an agarose matrix and used for affinity chromatography of lysates of T4-infected Escherichia coli cells . At least 10 T4-encoded early proteins and 3 or 4 host proteins are specifically retained by this gene 32 protein column . Nine of the T4 proteins have been identified as being involved in either DNA replication or genetic recombination . Notably, the T4 DNA polymerase (gene 43 protein) and two major proteins in the recombination pathway (the products of genes uvsX and uvsY) are specifically bound . On a preparative scale, the column is useful for purification of the bound proteins. Mutat Res, 1983 May, 109(2), 155 - 68 Study of the expression of UVRA and SSB proteins in vivo in lambda hybrid phages containing the uvrA and ssbA genes of Escherichia coli; Alazard RJ; A 9.3-kb Eco RI fragment obtained by partial digestion of the plasmid pDR2000 and containing the uvrA and ssbA genes was subcloned in the insertion vector lambda gt4 . Two hybrid bacteriophages carrying this fragment inserted in opposite orientations were isolated and used to lysogenize a uvrA and an ssbA mutant of Escherichia coli . Both phages conferred to these host bacteria the ultraviolet resistance of the wild-type parent indicating full complementation of the uvrA and of the ssbA defect . Two polypeptides corresponding to the molecular weights of the UVRA protein (115 000 dalton) and of the SSB protein (18 500 dalton) were synthesized and amplified after infection of a UV-irradiated lambda ind- lysogen with these 2 hybrid phages . The UVRA protein was not amplified after infection of a lex A3 host while SSB was still produced in large amount . These results establish that uvrA is repressed by lexA in vivo whereas ssbA is not. Bioorg Khim, 1983 May, 9(5), 711 - 3 {Structure of a recombination site in the transducing bacteriophage lambda plac5 DNA}; Shpakovskii GV et al.; A recombination site in the transducing bacteriophage lambda plac5 DNA has been structurally elucidated . Comparison of primary structures of E . coli lac-operon (distal end of lacZ gene, Z-Y spacer, and proximal end of lacY gene) described earlier with corresponding segments of bacteriophages lambda CI857 and lambda plac 5-2 DNAs sequenced in this paper showed that the bacterial DNA insert ends immediately after Z-Y spacer, just before the initiating triplet ATG of lacY gene . It thus follows that in contrast to the earlier conception, the insert does not seem to include any part of lacY gene . The recombination sites in both phage and bacterial DNA contain structurally homological segments about 20 b . p . long (crossover region), with two extra basepairs in the bacterial DNA (AT in the sense-strand) . We suppose that the very dinucleotide plays a substantial role in initiation of recombinational event: causing formation of a nonperfect heteroduplex structure, it determines the T-A internucleotide bond to be endonucleolytically cut (crossover point) followed by exonucleolytic elimination of the extra links (AT) and reciprocal strand exchange . The second recombination site in lambda plac5 DNA has been localized by us within lacI gene as being close to the HindII site (nucleotides 854 to 859 of the gene) . The structures of the two regions of site-specific recombination may shed light upon mechanisms of the phage abnormal excision leading to formation of transducing phages. Virology, 1983 Apr 30, 126(2), 636 - 50 Regulation of transcription and DNA replication of bacteriophage phi 80; Gilbert WR et al.; Transcriptional mapping and DNA replication measurements have been used to characterize a series of phi 80 suppressor-sensitive mutants which are defective in genes 15, 14, 16, and 17 . These genes are localized within the inner right arm of the vegetative phi 80 DNA genome . The sus326 mutation in gene 15 leads to a decrease in major leftward (pL-att80) RNA levels and to a marked pleiotropic reduction in major rightward RNA synthesis; however, phi 80 DNA synthesis is reduced only moderately (about two-fold) . These findings are consistent with the gene 15 product being a positive control regulator that is essential for normal transcriptional development, in particular, beyond a termination signal(s) (tR) located between genes 16 and 17 . The sus8 and sus258 mutations (in genes 14 and 16, respectively) lead to severe blockage of both major rightward RNA transcription and phi 80 DNA synthesis . The products of genes 14 and 16 appear to be required for both autonomous phi 80 DNA replication and the "late" transcriptional development . The sus121 mutation in gene 17 reduces the level of "late" major rightward transcription (gene 17-1-13-att'80 segment) by about 10-fold but does not have any apparent effect on the levels of phi 80 DNA synthesis . These profiles identify the product of gene 17 as a "Q-type" positive control regulator for the "late" major rightward RNA . These studies reveal the functional characterization of four genes, the products of which are necessary for the efficient expression of the "early" RNA transcribed segments, autonomous DNA replication, and the production of normal levels of "late" (17-1-13-att'80) RNA synthesis. Virology, 1983 Apr 30, 126(2), 600 - 13 Nonsense mutants of the lipid-containing bacteriophage PR4; Davis TN et al.; Thirty-three nonsense mutants of phage PR4 representing 12 complementation groups were isolated . One or two mutants of each group were grown on a suppressor-negative (Su-) host and characterized by the following criteria (i) proteins synthesized, (ii) level of phage DNA synthesis, and (iii) ability to assemble particles . We determined the protein and phospholipid compositions of the particles assembled in an Su- host, the presence of DNA in the particles, and the ability of the particles to adsorb to host cells . Finally each complementation group was tested for the ability to lyse an Su- host . We have identified one protein required for DNA synthesis, five proteins required for proper assembly of the protein coat and lipid membrane of the phage, two proteins required for stable insertion of DNA into the virion, a protein required for adsorption, a protein required for attachment of the adsorption protein to the virion, and a phage-encoded lytic enzyme. Virology, 1983 Apr 30, 126(2), 678 - 87 Bacteriophage N4-induced transcribing activities in E . coli . III . A third cistron required for N4 RNA polymerase II activity; Zehring WA et al.; The requirement of a third gene product of molecular weight 15,000 for coliphage N4 middle RNA synthesis is described . Therefore, three proteins of molecular weights 15,000, 30,000, and 40,000, which constitute the N4 RNA polymerase II activity, are required for N4 middle RNA synthesis . A complementation assay for N4 RNA polymerase II activity, which requires a DNA-membrane complex carrying the 15,000-MW protein and a supernatant providing the 30,000- and 40,000-MW proteins, has been developed . This assay, which reproduces the requirement for the cistron 5 gene product in vitro provides a means for the purification of the soluble components of the N4 RNA polymerase II activity (to be reported elsewhere) . The cistron 5 gene product is also required for N4 RNA polymerase II to synthesize RNAs utilizing middle promoters resident in recombinant plasmids . Mechanisms by which the cistron 5 gene product affects N4 RNA polymerase II transcription in vivo and in vitro are discussed. Virology, 1983 Apr 30, 126(2), 658 - 68 The integrase promoter and T'I terminator in bacteriophages lambda and 434; Benedik M et al.; The lambda integrase promoter PI lies immediately downstream from a terminator T'I . The PI promoter is activated by cII protein during lysogenization . The function of T'I is unknown . A deletion (trp-lambda 29), whose fusion point lies within one stem of T'1 retains cII-activable promoter function but shows little if any transcription termination in vivo . The nucleotide sequence of PI is identical in lambda and the related phage 434 . However, the T'I sequences of the two phages, though located in the same position, are not detectably homologous . When restriction fragments carrying the promoter-terminator segments from each of these phages were inserted into the trp operon, both responded identically to cII activation, and both caused termination. Virology, 1983 Apr 30, 126(2), 563 - 75 AvaII and BglI restriction maps of bacteriophage Mu; Marrs CF et al.; The AvaII and BglI restriction maps of bacteriophage Mu were derived by restriction analysis of a series of plasmid clones containing segments of Mu DNA which, in combination, covered the entire Mu genome . The plasmids analyzed included pKN36, pKN54, pKN62, pKN50, pKN35, pKN27, pKN48, pKN82, and pKN56 from the collection of W . Schumann and E . G . Bade, and pCM02, a newly constructed plasmid containing the rightmost internal EcoRI-PstI fragment of Mu DNA . BglI cuts Mu DNA at 23 sites, producing 24 fragments which range in size from 0.05 kb up to the approximately 7-kb fragment derived from the right end . AvaII cuts Mu DNA at 17 sites (including 2 within the G segment), producing fragments which range in size from 0.17 to 8.9 kb . The derived maps were confirmed by results of hybridization of 32P-labeled, nick-translated plasmid DNA to AvaII- and BglI-digested Mu DNAs . Evidence for modification of one of the AvaII sites in E . coli was obtained. Biochemistry, 1983 Apr 26, 22(9), 2104 - 15 A 500-MHz proton nuclear magnetic resonance study of the structure and structural alterations of gene-5 protein-oligo(deoxyadenylic acid) complexes; Alma NC et al.; The complex of the gene-5 protein of bacteriophage M13 with octadeoxyadenylic acid {d(A)8} has been shown earlier to differ in various respects from the complex with polynucleotides {Alma, N . C . M., Harmsen, B . J . M., van Boom, J . H., van der Marel, G., & Hilbers, C . W . (1982) Eur . J . Biochem . 122, 319-326} . In this paper the gene-5 protein-d(A)8 complex is compared with the complex formation between the gene-5 protein and a mixture of longer oligonucleotides, i.e., d(A)25-30 . Nuclear Overhauser experiments have been performed on both systems to obtain structural information regarding the oligonucleotide protein interactions . In the experiments also oligonucleotides deuterated at the adenyl C8 positions have been used in order to distinguish between the adenyl H2 and H8 resonances . Combination of the experiments shows that the nucleotides in the complexes are situated in such a way that the adenyl H8 and the sugar H1' protons are near the protein surface while the adenyl H2 protons are relatively far removed from all other protons, indicating that this side of the base is pointing away from the protein surface . It is concluded that structurally different complexes can be obtained for the d(A)25-30 system . The complex with d(A)25-30 undergoes a structural transition when going from excess oligonucleotide to excess gene-5 protein . This transition is identified with the transition between the "oligonucleotide" and the "polynucleotide" binding mode . The information derived from the present NMR experiments combined with known data from X-ray diffraction and electron microscopic studies is used to propose a model for a possible orientation of the adenyl bases in the complex. Nucleic Acids Res, 1983 Apr 25, 11(8), 2427 - 45 Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo; Seed B; Cloned genes have been purified from recombinant DNA bacteriophage libraries by a method exploiting homologous reciprocal recombination in vivo . In this method 'probe' sequences are inserted in a very small plasmid vector and introduced into recombination-proficient bacterial cells . Genomic bacteriophage libraries are propagated on the cells, and phage bearing sequences homologous to the probe acquire an integrated copy of the plasmid by reciprocal recombination . Phage bearing integrated plasmids can be purified from the larger pool of phage lacking plasmid integrates by growth under the appropriate selective conditions. J Biol Chem, 1983 Apr 25, 258(8), 4666 - 7 The rat fibrinogen genes . Linkage of the A alpha and gamma chain genes; Kant JA et al.; We have utilized cDNA probes for the alpha, beta, and gamma chains of rat fibrinogen to isolate the corresponding genes from two rat genomic libraries constructed in bacteriophage Charon 4A . There is a single copy of each gene . Mapping of greater than 92 kilobase pairs of rat genomic DNA has shown that the gamma and alpha chain genes are directly linked in a 5'-3' direction in vivo. J Mol Biol, 1983 Apr 25, 165(4), 633 - 54 Fidelity of replication of bacteriophage phi X174 DNA in vitro and in vivo; Fersht AR et al.; Seven different revertants of bacteriophage phi X174am16 (AB5276G leads to T) have been isolated and the nature of the reversions determined by sequencing their DNA . The revertants each differ from am16 by just a single base substitution . These may be distinguished with varying degrees of ease by characteristic temperature sensitivities of growth . This has facilitated the determination of the frequency at which DNA polymerase III catalyses different types of substitution mutations in copying phi X174 DNA in vitro and in vivo . During the replicative form (RF) leads to single-stranded (SS) stage of replication in vitro, four different revertants may be readily produced according to well-defined rate laws on biasing the concentrations of dNTPs . Transversion mutations are found to be formed predominantly by purine x purine mismatching, whilst transitions are formed predominantly by G x T mismatching . The substitutions via G x T and G x A mismatches are estimated to occur at similar frequencies in vivo . The two most common revertants isolated in vivo, however, are not those readily produced during the RF leads to SS stage in vitro but are those produced on purine x purine mismatching in the SS leads to RF stage . The accuracy of the DNA polymerase in vitro appears to be similar to that in this stage in vivo . However, the overall accuracy of the RF leads to SS replication in vivo is more accurate than predicted from the measurements of the accuracy in vitro. Anal Biochem, 1983 Apr 15, 130(2), 328 - 33 Protein N-terminal analysis using fast atom bombardment mass spectrometry; Beckner CF et al.; Fast atom bombardment (FAB) mass spectrometry was employed to identify and quantitate dansyl amino acids obtained in the N-terminal analysis of proteins . FAB mass spectra of dansyl amino acids are characterized by intense quasimolecular ions formed by cationization of the molecular species and fragment ions produced by cleavage of the bonds on either side of the sulfanyl group . Investigation of the dansyl amino acid responses using dansyl aminobutyric acid as an internal standard showed that dansyl amino acids can be determined quantitatively at a level of 0.1 nmol . N-terminal residue analysis was performed on a number of proteins to substantiate the technique including bovine serum albumin, pepsinogen, trypsinogen, bromelain, ribonuclease A, and bacteriophage P-22 tail protein. Virology, 1983 Apr 15, 126(1), 267 - 78 Circular genetic map of satellite bacteriophage P4; Deho G; A genetic map of satellite bacteriophage P4 has been constructed by means of standard multifactor crosses . The genetic map appears to be a circular permutation of the mature DNA physical map . In addition, a set of markers appear to be linked both to the left and to the right of the same gene alpha . These facts suggest that the P4 genetic map is circular . Since terminal redundancy and/or cyclic permutation are not known to be present in P4 mature DNA, the circularity of P4 genetic map may reflect the physical circularity of the molecules involved in the recombination process . The low frequency of recombination and the strong negative interference observed are in agreement with the above hypothesis. J Biol Chem, 1983 Apr 10, 258(7), 4293 - 7 Bacteriophage Mu DNA replication in vitro; Higgins NP et al.; An in vitro system for bacteriophage Mu DNA replication using lysates on cellophane discs is described . Mu replication was monitored by DNA hybridization . Using a thermoinducible Mu lysogen, 30-50% of all DNA synthesis in vitro was Mu-specific . Mu DNA synthesis is semidiscontinuous . In the presence of the DNA ligase inhibitor NMN, about one-half of the DNA was in Okazaki pieces and one-half in large DNA . The Mu Okazaki pieces hybridized mainly to the Mu light strand; the large DNA hybridized mainly to the Mu heavy strand . Okazaki pieces isolated from uninfected cells also hybridized to 2000-3000 bases of host DNA present in Mu-separated strands . However, the host Okazaki pieces hybridize to both Mu strands symmetrically . Most, if not all, host sequences were represented in mature Mu viral DNA . The in vitro data are most consistent with models in which Mu sequences, oriented randomly in both directions in the host chromosome, have recruited a bacterial replisome which traverses the Mu genome from left to right. J Mol Biol, 1983 Apr 5, 165(2), 321 - 56 Structure similarity, difference and variability in the filamentous viruses fd, If1, IKe, Pf1 and Xf . Investigation by laser Raman spectroscopy; Thomas GJ Jr et al.; The filamentous bacteriophages fd, If1, IKe, Pf1, Xf and Pf3 in aqueous solutions of low, moderate and high ionic strength have been investigated as a function of temperature by laser Raman difference spectroscopy . By analogy with Raman spectra of model compounds and viruses of known structure, the data reveal the following structural features: the predominant secondary structure of the coat protein subunit in each virus is the alpha-helix, but the amount of alpha-helix differs from one virus to another, ranging from an estimated high of 100% in Pf1 to a low of approximately 50% in Xf . The molecular environment and intermolecular interactions of tyrosine, tryptophan and phenylalanine residues differ among the different viruses, as do the conformations of aliphatic amino acid side-chains . The foregoing features of coat protein structure are highly sensitive to changes in Na+ concentration, temperature or both . The backbones of A-DNA and B-DNA structures do not occur in any of the viruses, and unusual DNA structures are indicated for all six viruses . The alpha-helical protein subunits of Pf1, like those of Pf3 and Xf, can undergo reversible transitions to beta-sheet structures while retaining their association with DNA; yet fd, IKe and If1 do not undergo such transitions . Raman intensity changes with ionic strength or temperature suggest that transgauche rotations of aliphatic amino acid side-chains and stacking of aromatic side-chains are important structural variables in each virus. J Mol Biol, 1983 Apr 5, 165(2), 229 - 48 Nucleotide sequence of the lysozyme gene of bacteriophage T4 . Analysis of mutations involving repeated sequences; Owen JE et al.; The nucleotide sequence of the lysozyme (e) gene of bacteriophage T4 and approximately 130 additional nucleotides on each side has been determined . The 5'-end of the gene for internal protein III appears to be located about 70 base-pairs from the 3'-end of the lysozyme gene . Nucleotide sequence analysis of mutant e genes confirmed that three identified hotspots of frameshift mutations are runs of five A nucleotides in the wild-type gene . The endpoints of two deletions are direct repeats of eight base-pairs in the wild-type gene . Two frameshift mutations with high reversion frequencies are duplications of five or seven base-pairs . The cloning and nucleotide sequence determination of the lysozyme gene will facilitate further study of the molecular biology of T4 lysozyme. J Virol, 1983 Apr, 46(1), 260 - 9 Bacteriophage SPO1 structure and morphogenesis . III . SPO1 proteins and synthesis; Parker ML et al.; The virion proteins of SPO1 have been determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods on purified phage components and on phage lysates . The phage head contains 16 proteins, and the connector or neck structure has an additional 3 proteins not found in the head . The proximal part of the tail, composed of sheath, tube and connecting components, contains six proteins . The distal baseplate is the most complex structure, with 28 proteins identifiable on sodium dodecyl sulfate gels . The maximum number of proteins found in phage subassemblies is 53, which would account for nearly half the coding capacity of the SPO1 genome. J Virol, 1983 Apr, 46(1), 196 - 203 Transcriptional regulation of three double-stranded RNA segments of bacteriophage phi 6 in vitro; Emori Y et al.; Three double-stranded RNA segments of bacteriophage phi 6 (L, M, and S) were transcribed in vitro by a virion-associated RNA polymerase . Regulation of L transcription was distinct from regulation of M and S transcription . Transcription of the L segment, which codes for early proteins, required manganous ion and high concentrations of all four ribonucleoside triphosphates and was inhibited by polyamines such as spermine . Transcription of the M and S segments, which code for late proteins, required manganous or magnesium ion and relatively low concentrations of all ribonucleoside triphosphates except GTP and was enhanced by polyamines . Optimal conditions for L transcription were more stringent than those for M and S transcription . These two apparently different patterns produced in in vitro transcription presumably reflect the two distinct in vivo transcription patterns; i.e., (i) similar amounts of three single-stranded RNA species were transcribed from the three corresponding segments of double-stranded RNA (early pattern) and (ii) a much larger amount of single-stranded RNA species was transcribed from M and S segments than from the L segment (late pattern) . The early transcription pattern may be changed into the late pattern by a change of environment, such as substrate concentration . This suggests that the different enzymatic properties under the different environmental conditions of the virion-associated transcriptase are responsible for the transcriptional regulation throughout the infection cycle of bacteriophage phi 6. J Virol, 1983 Apr, 46(1), 250 - 9 Bacteriophage SPO1 structure and morphogenesis . II . Head structure and DNA size; Parker ML et al.; The capsid of bacteriophage SPO1 is icosahedral, and the subunit arrangement on the 87-nm-diameter head suggests the triangulation number T = 16 . The major capsid protein (45,700 daltons) is cleaved from a 47,700-dalton precursor . Tubular heads (polyheads) are produced by mutations in genes 5 and 8 and contain cores as well as capped ends . The lattice constant of these structures is 13.4 nm; diameter is 109.5 nm . The size of the double-stranded SPO1 DNA (containing 5' hydroxymethyl uracil in place of thymine) was measured by sedimentation analysis and electron microscopy and has a molecular weight of 86 X 10(6) (about 140 kilobase pairs), which is smaller than several previously reported values. J Virol, 1983 Apr, 46(1), 239 - 49 Bacteriophage SPO1 structure and morphogenesis . I . Tail structure and length regulation; Parker ML et al.; Bacteriophage SPO1, a structually complex phage with hydroxymethyl uracil replacing thymine, has been studied by structural and chemical methods with the aim of defining the virion organization . The contractile tail of SPO1 consists of a complex baseplate, a tail tube, and a 140-nm-long sheath composed of stacked disks (4.1 nm repeat), each containing six subunits of molecular weight 60,300 . The subunits are arranged in six parallel helices, each with a helical screw angle (omega 0) of 22.5 degrees . The baseplate was shown to undergo a structural rearrangement during tail contraction into a hexameric pinwheel . A mutation in gene 8 which produced unattached heads and tails also produced tails of different lengths . The tail length distribution suggests that the smallest integral length increment is a single disk of subunits . The structural arrangement of subunits in long tails is identical to that of normal tails, and the tails can contract . Many of the long tails showed partial stain penetration within the tail tube to a point which coincides with the top of a unit-length tail . The implications of these findings with respect to tail length regulation are discussed. Proc Natl Acad Sci U S A, 1983 Apr, 80(7), 2059 - 62 Stabilization of proteins by a bacteriophage T4 gene cloned in Escherichia coli; Simon LD et al.; The cloned bacteriophage T4 pin gene functions to stabilize several different kinds of proteins in Escherichia coli bacteria . Incomplete proteins such as puromycyl polypeptides, abnormal but complete proteins such as the lambda phage tsO protein, and labile eukaryotic proteins encoded by genes cloned in E . coli such as mature human fibroblast interferon are stabilized in cells in which the T4 pin gene is expressed . The cloned T4 pin gene does not seem to affect the turnover of normal E . coli proteins. J Bacteriol, 1983 Apr, 154(1), 130 - 8 Integration of the overproduced bacteriophage T5 receptor protein in the outer membrane of Escherichia coli; Menichi B et al.; The tonA gene codes for an outer membrane protein, a receptor of phage T5, the TonA protein . Strains harboring pLG513, a multicopy plasmid in which the tonA gene has been cloned, overproduced TonA protein, which appeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell envelope proteins as a 78,000-molecular-weight protein . Identical results have been observed by Plastow et al . (FEBS Lett . 131:262-264, 1981) with plasmid pLC19-19, in which the tonA gene has also been cloned . The activity of the TonA protein, measured by its capacity to inactivate phage T5, increased by five- to sixfold in purified envelopes of cells harboring pLG513 compared with cells lacking the plasmid . Solubilization of the cytoplasmic membrane by Triton-Mg2+ treatment did not increase this activity . However, partial solubilization of outer membrane proteins by Triton-EDTA unmasked further T5 receptor activity, resulting in a final increase of around 50-fold, a value more consistent with the expected gene dosage effect . Treatment of whole cells by trypsin in conditions in which trypsin is allowed to enter the outer membrane revealed that part of the overproduced T5 receptors were embedded in the outer membrane and masked by a trypsin-sensitive protein . In addition, no T5 receptor activity was detected in either the periplasmic space or the cytoplasm . These results suggest that all of the overproduced TonA molecules were synthesized in an active form and integrated in the outer membrane, but only a small fraction could be reached or recognized by phage T5 in vivo. Gene, 1983 Apr, 22(1), 103 - 13 Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication; Remaut E et al.; Improved expression vectors have been constructed which are derived from runaway-replication mutants of plasmid R1 and carry the strong leftward promoter (pL) of bacteriophage lambda . The activity of this promoter is controlled by a temperature-sensitive repressor, product of the phage gene cI cloned on a compatible plasmid . Heat induction leads to amplification of the plasmid copy number and at the same time turns on the promoter . At a short distance downstream from the promoter, unique EcoRI, BamHI, XbaI and HindIII sites are present . This system was used for high level expression of the T4 DNA-ligase gene; 3 h after induction the ligase amounted to about 20% of total cellular protein. Proc Natl Acad Sci U S A, 1983 Apr, 80(8), 2314 - 7 Recombination involving transposable elements: role of target molecule replication in Tn1 delta Ap-mediated replicon fusion; Muster CJ et al.; Donor DNA molecules carrying Tn1 or Tn3 deletion mutants do not need to replicate in order to participate in replicon fusion recombination events during which the Tn1/Tn3 element is duplicated . We have assayed Tn1 delta Ap-mediated replicon fusion events involving plasmid R388 and the bacteriophage lambda-derived plasmid p lambda CM, and we find that the role of the recipient molecule is distinct . When p lambda CM carries Tn1 delta Ap, replicon fusion occurs in more than 1% of all cells assayed, whether or not p lambda CM::Tn1 delta Ap can replicate . In contrast, when R388 carries Tn1 delta Ap, replicon fusion occurs only when the p lambda CM target can replicate . Blocks to p lambda CM replication by prophage repressor or amber mutations of the O and P cistrons reduce replicon fusion so that it occurs in less than 1 out of 10(5) cells assayed. Proc Natl Acad Sci U S A, 1983 Apr, 80(7), 2012 - 6 Switch in the transposition products of Mu DNA mediated by proteins: Cointegrates versus simple insertions; Harshey RM; Bacteriophage Mu is a self-contained mobile unit encoding functions that mediate its movement . There appear to be two alternate pathways for Mu DNA transposition that differ with respect to the end products they generate . During the lytic cycle of phage Mu growth the end products of transposition are predominantly cointegrates in an experimental system in which the induced Mu prophage is located on pSC101, a low-copy-number plasmid . On the other hand, Mu insertions into the host genome during lysogenization contain Mu DNA as simple insertions . Two Mu functions, encoded by the A and B genes, are required for Mu DNA transposition during its lytic growth . However, during lysogeny the product of gene B is not required for integration of Mu DNA . Evidence is presented here which shows that in the absence of the B gene product the majority of transposition events are simple insertions . This is in striking contrast to the situation in which the majority of the products are cointegrates in the presence of both A and B gene products . Additional evidence also suggests that these simple insertions do not arise through the resolution of cointegrate structures. Cell, 1983 Apr, 32(4), 1301 - 11 Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination; Abremski K et al.; Bacteriophage P1 encodes its own site-specific recombination system consisting of a site at which recombination takes place called loxP and a recombinase called Cre . A number of lambda and plasmid substrates containing two loxP sites have been constructed . Using these substrates we have shown both in vivo and in vitro that a fully functional loxP site is composed of no more than 60 bp . In vitro, when an extract containing Cre is used, recombination between loxP sites on supercoiled, nicked-circle or linear DNA occurs efficiently . The most surprising result from the in vitro studies is that 50% of the products of recombination between loxP sites on a supercoiled DNA substrate are present as free supercoiled circles . The ability to produce free products starting with a supercoiled substrate suggests a rather unique property of Cre-mediated lox recombination, the implications of which are discussed in terms of possible effects of the protein on the topology of the DNA molecule. Mutat Res, 1983 Apr, 109(1), 1 - 11 Bypass of pyrimidine dimers in DNA of bacteriophage T4 via induction of primer RNA; Cupido M; Bacteriophage T4 has a third pathway for repair of damaged DNA besides excision repair and recombination repair . This pathway is a mechanism for the toleration of lesions rather than the repair of lesions . The substrate for this process is gapped DNA copied from a damaged template . Evidence indicates that these gaps are filled, giving rise to daughter strands that are sensitive to heat and to treatments with RNAase . These daughter strands subsequently serve as templates for DNA that is resistant to RNAase . This third pathway is dependent upon gene 41 (RNA-priming protein), gene uvsZ (function unknown) and gene 30 (polynucleotide ligase) and is presumed to consist of 4 steps: (1) induction of primer RNA opposite the lesion in the template; (2) elongation of primers by DNA polymerase; (3) ligation of daughter-strand fragments, without removal of primer RNA; (4) replication of DNA carrying RNA sequences, giving homogeneous DNA strands . We have called this process 'Re-initiation repair'. J Bacteriol, 1983 Apr, 154(1), 505 - 7 IncI2 plasmids specify sensitivity to filamentous bacteriophage IKe; Bradley DE et al.; Bacterial strains carrying the derepressed incompatibility group IncI2 plasmids TP114drp-l or R721pilc were lysed by the filamentous bacteriophages IKe, I2-2, and X . Phage I2-2 was serologically related to IKe, but phage X was not . Phage IKe adsorbed to the tips of thick pili determined by the IncI2 plasmids, but not to the well-known thin I2 pili. J Mol Biol, 1983 Mar 25, 165(1), 109 - 24 The lipid-containing bacteriophage PR4 . Effects of altered lipid composition on the virion; Muller ED et al.; Phage PR4 was grown on a variety of Escherichia coli mutants defective in fatty acid and phospholipid metabolism . The composition of the phage lipids was modified by changing the composition of the host membrane phospholipids . The compositions of both the polar and the acyl moieties of the phospholipids were altered . The proportion of saturated fatty acids in the phage phospholipids was increased in increments from 44% of the total fatty acids to 55%, 61% and 69% of the total fatty acids using a host mutant with a temperature-sensitive defect in unsaturated fatty acid biosynthesis . The increase in saturated fatty acids led to a pronounced loss of infectivity when the phage were incubated at temperatures between 2 degrees C and 30 degrees C (temperatures below those at which the phage were grown) . The greater the level of saturated fatty acids in the phage phospholipids, the higher the temperature below which the phage were inactivated . Our results strongly suggest that the phage membrane undergoes a lipid phase transition, which can disrupt and inactivate the virion . The phospholipid composition of PR4 was also altered by using host mutants defective in phosphatidylethanolamine and/or cardiolipin synthesis . Phage PR4 grown on wild-type host strains contains 56% phosphatidylethanolamine, 37% phosphatidylglycerol, 4.6% cardiolipin and no detectable phosphatidylserine . However, in response to changes in the host, PR4 preparations were obtained with phospholipid compositions varying from 28% to 60% in phosphatidylethanolamine, from 22% to 39% in phosphatidylglycerol, from 1% to 15% in cardiolipin and containing as much as 35% phosphatidylserine . These changes in phospholipid composition did not affect the infectivity of the phage . Moreover, the increased level of phosphatidylglycerol in the phage relative to the host was not altered by these manipulations . It is concluded that the net charge of the phage membrane phospholipids is not involved in the selection or function of the viral phospholipids . We also present evidence suggesting that the phage and host membranes do not fuse during the course of infection. FEBS Lett, 1983 Mar 21, 153(2), 339 - 44 Interactions of the 26-39 fragment of the cro protein from lambda bacteriophage with nucleic acids; Mayer R et al.; A tetradecapeptide with a sequence identical to residues 26-39 of the cro protein from bacteriophage lambda has been synthesized . This peptide has no secondary structure in an aqueous buffer but adopts an alpha-helical conformation in the presence of 20% hexafluoroisopropanol . The fluorescence of the single tyrosyl residue of the cro protein fragment is quenched upon binding to nucleic acids . Proton magnetic resonance has been used to investigate complex formation of the cro protein fragment with a self-complementary decadeoxynucleotide d(AATTGCAATT) . Changes in resonance positions and linewidths have been observed for both partners in the 4 complexes which are obtained when either the single-stranded or double-stranded oligonucleotide is mixed with either the random coil or the alpha-helical peptide . These studies are presently extended to the specific complex formed by the cro protein fragment with the OR3 operator sequence. J Mol Biol, 1983 Mar 15, 164(4), 573 - 87 Structure and inherent properties of the bacteriophage lambda head shell . III . Spectroscopic studies on the expansion of the prohead; Kawaguchi K et al.; The head shell of bacteriophage lambda expands by about 20% in diameter when it packages the DNA molecule in vivo . The expansion reaction is essentially a conformational change of the major head protein molecules to a state of lower free energy and can also be triggered in vitro by treatment with 4 M-urea . In order to investigate the conformational change, we have measured the circular dichroism, fluorescence and difference absorption spectra of the lambda head shell before and after the expansion by the treatment with urea . The far-ultraviolet circular dichroism spectra and the fluorescence spectra show that the expansion is not accompanied by a great change in the secondary structure (29% alpha-helix, 23% beta-structure) and the environment (non-polar) of the tryptophan residues of the major head protein molecule . On the other hand, by measurements of the circular dichroism and difference absorption spectra in the near-ultraviolet region as well as by chemical modification experiments with tetranitromethane, we have found that one or two tyrosine residues of the major head protein are transferred from a polar, solvent-exposed to a non-polar, solvent-unexposed environment during the expansion . Judging from these results, the conformational change seems to be mainly intermolecular or interdomainal rather than intradomainal. Biochim Biophys Acta, 1983 Mar 10, 739(2), 216 - 24 Dissociation of the Pf1 nucleoprotein assembly complex and characterisation of the DNA binding protein; Kneale GG; During replication of bacteriophage Pf1, progeny viral strands are complexed with a single-stranded DNA binding protein, analogous to the gene 5 protein of bacteriophage fd . Using fluorescence spectroscopy, ultracentrifugation and DNA-cellulose chromatography, conditions for dissociation of the nucleoprotein have been investigated . The Pf1 protein is unusual in that it is not released from the DNA by 2 M NaCl . Complete separation occurs in 0.6-1.0 M MgCl2, leading to a procedure for the purification of the protein . Two subfractions of the protein can be isolated of isoelectric points 5.9 and 6.4 . The molecular weight of the native DNA binding protein has been studied by gel filtration and sedimentation . The major species in solution has a sedimentation coefficient of 2.3 S and a diffusion coefficient of 7.8 X 10(-7) cm2 . s-1, corresponding to a protein dimer (Mr = 30 800) . Protein tetramers are induced in the presence of octanucleotides, but not tetranucleotides . Analysis of the ultraviolet spectra of the DNA binding protein and the native nucleoprotein complex indicates a stoichiometry of 3.9 +/- 0.4 nucleotides per protein subunit . The molar extinction coefficient of the DNA when bound to the protein (epsilon 260 = 8100) suggests that the binding protein maintains the DNA in an extended (unstacked) conformation similar to that found in the mature Pf1 virion. Nature, 1983 Mar 31-Apr 6, 302(5907), 389 - 93 Mechanism of ribosome frameshifting during translation of the genetic code; Weiss R et al.; Some frameshift mutations are strongly suppressed by limitation for particular aminoacyl-tRNA species . Here, we show that ribosome frameshifting at a specific tryptophan codon during Trp-tRNA limitation accounts for suppression of a group of downstream frameshift alleles in the rIIB gene of bacteriophage T4 . Genetic and physiological observations strongly suggest that ribosome frameshifting at this position depends on the binding of a noncognate (leucine) tRNA. J Mol Biol, 1983 Mar 5, 164(3), 377 - 93 Transcription in bacteriophage f1-infected Escherichia coli . Messenger populations in the infected cell; La Farina M et al.; Transcription of bacteriophage f1 DNA in vivo occurs in two independent regions . They are separated from one another by a strong terminator just downstream from gene VIII on one side, and by the filamentous phage intergenic space on the other . One of these regions contains genes II, V, VII, IX and VIII, and is actively transcribed . In this region there are a number of promoters but only one effective terminator . Thus, most of the RNAs that come from this region overlap and share sequences close to the termination site . The other region, which contains genes III, VI, I and IV, is transcribed much less actively . This region gives rise to a long (approximately 4 X 10(3) bases) RNA that covers the entire region, and several RNAs that overlap in the region closest to their 5' termini . Several other RNAs appear to overlap only with the 4 X 10(3) base transcript . Thus, not only the frequency but the organization of transcription differs in the two portions of the genome. Nature, 1983 Mar 3, 302(5903), 72 - 4 An immunologically active chimaeric protein containing herpes simplex virus type 1 glycoprotein D; Weis JH et al.; Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) cause both persistent and latent infections, including recurrent cutaneous disease, lethal neonatal disease, central nervous system disease and other clinical syndromes . Modified live vaccines or conventionally prepared subunit vaccines have generally been unsuccessful in the treatment of HSV-1 and HSV-2 infections from the standpoints of safety and efficacy . It has been established that HSV-1 and HSV-2 infectivity may be neutralized in vitro with antisera directed specifically against each of the four major glycoproteins of the virus (gA/gB, gC, gD and gE) and antisera against glycoprotein gD, of either HSV-1 or HSV-2, are capable of neutralizing both HSV-1 and HSV-2 infectivity in vitro and in vivo . We have previously reported on the identification, DNA sequence and expression at low level in Escherichia coli of the gD gene of HSV-1 strain Patton . Here we describe construction of a hybrid gene encoding a chimaeric protein containing HSV-1 gD, bacteriophage lambda Cro and E . coli beta-galactosidase (gD-beta-gal) protein, which is expressed at high level in E . coli . Moreover, the chimaeric protein elicits antibodies in rabbits that not only immunoprecipitate gD from cells infected with HSV-1 and HSV-2 but also neutralize HSV-1 and HSV-2 infectivity in vitro. Radiobiologiia, 1983 Mar-Apr, 23(2), 230 - 3 {Effect of alpha particles on bacteriophage T4}; Leont'eva GA et al.; Exponential survival curves were obtained for a dry film culture of bacteriophage T4 Br+ after exposure to both alpha-particles and gamma-quanta . Relative biological effectiveness of alpha-particles was 4.68 with respect to survival . The mutation spectrum after alpha-irradiation slightly differed from that produced by gamma-radiation. Genetics, 1983 Mar, 103(3), 353 - 66 A major role for bacteriophage T4 DNA polymerase in frameshift mutagenesis; Ripley LS et al.; T4 DNA polymerase strongly influences the frequency and specificity of frameshift mutagenesis . Fifteen of 19 temperature-sensitive alleles of the DNA polymerase gene substantially influenced the reversion frequencies of frameshift mutations measured in the T4 rII genes . Most polymerase mutants increased frameshift frequencies, but a few alleles (previously noted as antimutators for base substitution mutations) decreased the frequencies of certain frameshifts while increasing the frequencies of others . The various patterns of enhanced or decreased frameshift mutation frequencies suggest that T4 DNA polymerase is likely to play a variety of roles in the metabolic events leading to frameshift mutation . A detailed genetic study of the specificity of the mutator properties of three DNA polymerase alleles (tsL56, tsL98 and tsL88) demonstrated that each produces a distinctive frameshift spectrum . Differences in frameshift frequencies at similar DNA sequences within the rII genes, the influence of mutant polymerase alleles on these frequencies, and the presence or absence of the dinucleotide sequence associated with initiation of Okazaki pieces at the frameshift site has led us to suggest that the discontinuities associated with discontinuous DNA replication may contribute to spontaneous frameshift mutation frequencies in T4. Proc Natl Acad Sci U S A, 1983 Mar, 80(5), 1392 - 6 Unusual structure of the chicken embryonic alpha-globin gene, pi'; Engel JD et al.; We report the DNA sequence of the globin locus encoding the chicken embryonic alpha-globin, pi' . The structure differs significantly from that of the two chicken adult alpha-globin genes, alpha A and alpha D, as well as from that of previously studied adult alpha-globin genes in that the introns of the pi' gene are substantially larger than those in adult alpha-globin loci . In contrast, the pi' introns are structurally similar to the only other expressed embryonic alpha-globin gene reported to date, the human zeta gene . While completing the sequence of the pi' gene, we determined that only one chromosomal locus within the chicken genome hybridizes to a pi' central exon probe . These data lead to the conclusion that if the equimolar chicken embryonic alpha-globin polypeptides, called pi and pi', are indeed independently transcribed, then that transcription occurs from alleles of the same gene; however, we favor the possibility that the pi gene does not actually exist . This conclusion is drawn from the observation that the two chromosomal alleles of embryonic alpha-globins (represented by recombinant bacteriophage lambda CaG5 and lambda CaG7) both encode pi'. Cell, 1983 Mar, 32(3), 681 - 94 Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei; Green MR et al.; To study the mechanisms of RNA splicing we have synthesized beta-globin mRNA precursors by in vitro transcription using a plasmid in which a human beta-globin gene is fused to an efficient bacteriophage promoter . The structural requirements for accurate splicing of the in vitro synthesized pre-mRNAs were investigated by injection into Xenopus oocyte nuclei . We detect splicing only if the pre-mRNA is capped in vitro prior to injection; uncapped RNA is rapidly degraded . In addition, we find that in vitro synthesized pre-mRNAs that are not polyadenylated or lack a normal 3' end are spliced following injection into oocytes . The observation that a purified pre-mRNA can be spliced in oocytes indicates that transcription and splicing are not obligatorily coupled . When an in vitro synthesized beta-globin pre-mRNA containing a splice junction mutation is microinjected, the affected junction is not spliced, indicating that sequences necessary for accurate splicing in human cells are also necessary for splicing in oocytes. Radiat Res, 1983 Mar, 93(3), 461 - 78 Characterization of an Escherichia coli mutant (radB101) sensitive to gamma and uv radiation, and methyl methanesulfonate; Sargentini NJ et al.; After N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis of Escherichia coli K-12 (xthA14), and X-ray-sensitive mutant was isolated . This sensitivity is due to a mutation, radB101, which is located at 56.5 min on the E . coli K-12 linkage map . The radB101 mutation sensitized wildtype cells to gamma and uv radiation, and to methyl methanesulfonate . When known DNA repair-deficient mutants were ranked for their gamma-radiation sensitivity relative to their uv-radiation sensitivity, their order was (starting with the most selectively gamma-radiation-sensitive strain): recB21, radB101, wild type, polA1, recF143, lexA101, recA56, uvrD3, and uvrA6 . The radB mutant was normal for gamma- and uv-radiation mutagenesis, it showed only a slight enhancement of gamma- and uv-radiation-induced DNA degradation, and it was approximately 60% deficient in recombination ability . The radB gene is suggested to play a role in the recA gene-dependent (Type III) repair of DNA single-strand breaks after gamma irradiation and in postreplication repair after uv irradiation for the following reasons; the radB strain was normal for the host-cell reactivation of gamma- and uv-irradiated bacteriophage lambda; the radB mutation did not sensitize a recA strain, but did sensitize a polA strain to gamma and uv radiation; the radB mutation sensitized a uvrB strain to uv radiation. J Bacteriol, 1983 Mar, 153(3), 1308 - 14 dnaB analog function associated with plasmid R100drd-1; Potter AA et al.; Plasmid R100 and a number of its derivatives were able to suppress the temperature sensitivity of strains carrying different alleles of the dnaB gene of Escherichia coli K-12 . R100drd-l and pAR132 were able to rescue a strain carrying the dnaB266(Am) mutation in the absence of any known amber suppressors . This was taken as evidence for the existence of an R100drd-l dnaB analog function . The R100drd-l dnaB analog was different from those of bacteriophages P1 and P7 in that it was able to support the growth of bacteriophage lambda in a dnaB266(Am) background . The dnaB analog was also shown to be thermosensitive . The structural gene for this protein lies within the EcoRI fragment D of R100drd-l. Gene, 1983 Mar, 21(3), 175 - 91 A cluster of leftward, rho-dependent t'J terminators in the J gene of coliphage lambda; Luk KC et al.; While searching for the N-unresponsive terminator described by Honigman (Gene 13 (1981) 299-309), a 1680-bp DNA fragment from within gene J of bacteriophage lambda was cloned into plasmid pD12 between promoter p'R and the galK gene . In vitro transcription of this plasmid and S1 mapping assays, together with nucleotide sequencing, demonstrated that this DNA fragment contains a cluster of at least four in rho+ Escherichia coli hosts and only 30% in rho- hosts at at 30 degrees C . At the elevated temperature of 42 degrees C, the rho-dependent termination component of the t'J cluster becomes somewhat leaky, with the readthrough increasing by about twofold . The t'J terminators appear to be less responsive to nutR- and N-mediated antitermination (efficiency of 84-87%) than tL3, which responds to the same antitermination function with an efficiency of 97% . The relationship between the t'J cluster and the same antitermination function with an efficiency of 97% . The relationship between the t'J cluster and the highly N-unresponsive leftward termination signal tJ, which is also located within the J gene region (Gottesman et al., J . Mol . Biol . 140 (1980) 57-75; Honigman, Gene 13 (1981) 299-309), is unknown. Biofizika, 1983 Mar-Apr, 28(2), 217 - 20 {Packing of binding stained DNA strands in bacteriophage lambda}; Kishchenko GP et al.; Distribution of stainable DNA strands in phage lambda has been studied by polarized fluorescence . The effect of tight DNA-packing on fluorescence depolarization of complex dye-DNA was calculated . It is shown that stainable DNA in the phage is not concentrated in the central region . The arrangement of acridine orange molecules on the surface layers of the packed DNA is the most probable one. Virology, 1983 Mar, 125(2), 403 - 18 The tL2 cluster of transcription termination sites between genes bet and ral of coliphage lambda; Luk KC et al.; The major leftward transcription of bacteriophage lambda is controlled by several terminators (t), including tL1, tL2, tL3, and others . The tL2 termination site, which was placed by Salstrom and Szybalski (Virology 88, 252-260, 1978) between lambda genes bet and ral, was found to consist of a cluster of four leftward terminators . As not to change the numbering of other leftward terminators, these were designated as tL2a, tL2b, tL2c, and tL2d . As determined by S1 nuclease mapping of the tL2-terminated in vitro transcripts, the normally pL-initiated major leftward lambda transcription should encounter termination points at 1653 bp (tL2a; between genes Ea10 and ral), 2089 bp (tL2b; between genes cIII and Ea10), 2441-2442 bp, and 2483 bp (tL2c and tL2d; both within gene gam) from the sL startpoint (= +1) . All terminators were cloned in a pBR322-derived plasmid between the p'R promoter and the galK gene, and their in vivo termination efficiencies are 69% (tL2a), 53% (tL2b), and 38% (tL2c + tL2d), measured as reduction of galK expression in rho+galK- hosts at 30 degrees . The tL2a and tL2b), terminators depend little on the rho factor, whereas the efficiency of tL2c + tL2d decreases from 38 to only 14% in the rho- host . When shifted to 42 degrees, the termination efficiency of tL2b decreases from 53 to only 36%, while the other tL2 terminators are much less affected by increasing the temperature . The calculated joint efficiency of the entire tL2 cluster is 90%, which is in perfect agreement with the 90% termination efficiency reported by Salstrom and Szybalski (1978) for tL2 . However, the natural location of tL2c and tL2d within the actively translated gam gene may interfere with their termination function . Under in vitro conditions, tL2c and tL2d are active only in the presence of rho factor, whereas tL2a and tL2b do not require rho . The structure of the tL2 terminators resembles that of some other known termination sites: a perfect (8 bp for tL2a and tL2b) or imperfect (8-9 bp) dyad symmetry and a T6 (tL2a), T5 (tL2b), T4 (tL2c) or TTATT sequence (tL2d) toward the 3' end of the mRNA-like DNA strand. J Virol, 1983 Mar, 45(3), 971 - 6 Role of R loops in recA-independent homologous recombination of bacteriophage lambda; Matsumoto T et al.; We have previously shown that the DNA-dependent RNA polymerase of Escherichia coli can promote homologous recombination of ba