Mutat Res, 1984 Feb, 139(2), 45 - 9 Lack of induction of non-targeted mutations in intact bacteriophage by UVB (313 nm), UVA (334 nm, 365 nm) and visible (405 nm) irradiation of host cells; Tyrrell RM; Mutation to virulence has been measured in intact bacteriophage lambda 15 infected into host cells pre-treated with UVC (254 nm), UVB (313 nm), UVA (334 nm, 365 nm) or visible (405 nm) radiations . We have confirmed that UVC radiation leads to a large enhancement (maximum enhancement factor of 140 in wild-type) of the background spontaneous mutation frequency (non-targeted mutagenesis) and have further shown that this is at least partially dependent on excision repair (maximum enhancement factor of 14 in uvrA strain) . In contrast, UVB (313 nm) radiation enhances the mutation frequency by less than a factor of 2 . Longer wavelength UVA radiation (334 nm, 365 nm) actually reduces the mutation frequency to 25% of the background levels presumably by reducing the levels of viral replication occurring in the host cells . A visible wavelength (405 nm) has no effect on mutation frequency over the fluence range employed. Biochemistry, 1984 Jan 31, 23(3), 522 - 9 1H NMR (500 MHz) of gene 32 protein--oligonucleotide complexes; Prigodich RV et al.; In concentrated solutions, gene 32 single-stranded DNA binding protein from bacteriophage T4 (gene 32P) forms oligomers with long rotational correlation times, rendering 1H NMR signals from most of the protons too broad to be detected . Small flexible N- and C-terminal domains are present, however, the protons of which give rise to sharp resonances . If the C-terminal A domain (48 residues) and the N-terminal B domain (21 residues) are removed, the resultant core protein of 232 residues (gene 32P) retains high affinity for ssDNA and remains a monomer in concentrated solution, and most of the proton resonances of the core protein can now be observed . Proton NMR spectra (500 MHz) of gene 32P and its complexes with ApA, d(pA)n (n = 2, 4, 6, 8, and 10), and d(pT)8 show that the resonances of a group of aromatic protons shift upfield upon oligonucleotide binding . Proton difference spectra show that the 1H resonances of at least one Phe, one Trp, and five Tyr residues are involved in the chemical shift changes observed with nucleotide binding . The number of aromatic protons involved and the magnitude of the shifts change with the length of the oligonucleotide until the shifts are only slightly different between the complexes with d(pA)8 and d(pA)10, suggesting that the binding groove accommodates approximately eight nucleotide bases . Many of the aromatic proton NMR shifts observed on oligonucleotide complex formation are similar to those observed for oligonucleotide complex formation with gene 5P of bacteriophage fd, although more aromatic residues are involved in the case of gene 32P.(ABSTRACT TRUNCATED AT 250 WORDS) Virology, 1984 Jan 30, 132(2), 239 - 49 Phage head assembly in bacteriophage T1; Ramsay N et al.; Phage-related structures found in wild-type and mutant T1 infections were examined by sedimentation analysis, electron microscopy, and polyacrylamide gel electrophoresis . Phage-particle polypeptides P7 (molecular weight 33,000) and P11 (molecular weight 16,000) were identified as major head proteins and P10 (molecular weight 26,000) was shown to be the major tail protein . A DNA-free head-like structure containing P7 but not P11 was present in wild-type and a number of mutant infections . In view of its possible role as a precursor to mature heads, this structure was termed the prohead . Mutants in two genes, am10 (gene 13) and am45 (gene 14), synthesised only tails in nonpermissive infections . Mutants in eight of the head genes, am23 (gene 4), am283 (gene 13.3), am216 (gene 13.7), ts257 (gene 14.5), am11 (gene 15), am4 (gene 16), am7 (gene 17), and am30 (gene 18), synthesised proheads and tails but not other structures . am37 (gene 12) synthesised proheads, tails, and empty heads . Studies with a tail-defective mutant suggested that empty heads were produced from the breakdown of DNA-filled heads . P11, the T1 gene 13.3 product, is analogous to the phage lambda gene D protein: both are major capsid proteins which appear to stabilise the later stages of head filling . These data are reviewed according to the general principles established for phage-head assembly and a tentative pathway for T1-head assembly is proposed. J Mol Biol, 1984 Jan 25, 172(3), 301 - 23 Self cleavage of a precursor RNA from bacteriophage T4; Watson N et al.; We found that a precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Spl; precursor of species 1) has the capacity to cleave itself in a specific position . This cleavage is similar to a cleavage carried out by the aid of a protein, RNase F, that has been previously identified . This cleavage could lead to the maturation of an RNA (species 1) found in T4-infected E . coli cells . The reaction is time and temperature-dependent and is relatively slow as compared to the protein-dependent reaction . It requires at least a monovalent cation and is aided by non-ionic detergents . In the absence of detergent the cleavage can occur but at a reduced rate . The substrate does not contain hidden nicks and a variety of experiments suggest that it does not contain a protein . Moreover, we found no indication that the cleavage is due to contaminating nucleases in the substrate or in the reagents . The intact secondary and tertiary structures of the molecule are necessary for the cleavage to occur . The finding of a self cleaving RNA molecule has interesting evolutionary implications. Biochemistry, 1984 Jan 17, 23(2), 340 - 9 Mechanism of DNA binding to the gene 5 protein of bacteriophage fd; Brayer GD et al.; A model for the bimolecular complex arising from the interaction of single-stranded DNA with the gene 5 DNA binding protein (G5BP) of bacteriophage fd is proposed on the basis of difference Fourier analyses and the correlation between structural and physicochemical data . The essential DNA binding element is the G5BP dimer which provides two antiparallel DNA binding channels, each constructed from amino acid contributions of both monomers within the pair . These channels display identical bonding environments but opposite polarities as a consequence of the inherent dyad symmetry within each dimer unit . The two channels are separated by 30 A, and each is 10 A wide and 40 A long . We propose that DNA binding is a consequence of two general sets of interactions . Aromatic side chains of G5BP stack upon nucleic acid bases, and the DNA phosphate backbone is bound by a series of appropriately positioned lysyl and arginyl side chains . The DNA conformation is fully extended, and each binding channel can accommodate up to five nucleotides . Only minor conformational changes in the native G5BP structure are required to optimize the binding of DNA . The G5BP-DNA complexation model presented here serves to explain some of the mechanistic features associated with the role this protein plays in the formation of a nucleoprotein helix during the bacteriophage fd life cycle. Eur J Biochem, 1984 Jan 16, 138(2), 247 - 51 Functional aspects of Escherichia coli rep helicase in unwinding and replication of DNA; Baumel I et al.; The gene for Escherichia coli rep helicase (rep protein) was subcloned in a pBR plasmid and the protein overproduced in cells transformed with the hybrid DNA . The effect of purified enzyme on strand unwinding and DNA replication was investigated by electron microscopy . The templates used were partial duplexes of viral DNA from bacteriophage fd::Tn5 and reannealed DNA from bacteriophage Mu . The experiments with the two DNA species show DNA unwinding uncoupled from replication . The single-stranded phage fd::Tn5 DNA with the inverted repeat of transposon Tn5 could be completely replicated in the presence of the E . coli enzymes rep helicase, DNA binding protein I, RNA polymerase and DNA polymerase III holoenzyme . A block in the unwinding step increases secondary initiation events in single-stranded parts of the template, as DNA polymerase III holoenzyme cannot switch across the stem structure of the transposon. Nucleic Acids Res, 1984 Jan 11, 12(1 Pt 1), 227 - 36 PMAP, PMAPS: DNA physical map constructing programs; Polner G et al.; Computer programs are described, which facilitate the construction of the restriction site (physical) map of DNA molecules . By knowing the length of each fragment and its degree of error in the single and the double restriction enzyme digestions, the programs give all the possibilities for the physical map . This method is applicable to linear DNA molecules . Several examples are presented which indicate the high efficiency of the programs in constructing restriction site maps for the 62 Kb chromosome of bacteriophage 16-3 . We have constructed complex maps (i.e . EcoRI map with 16 and EcoRV with 11 fragments). Nucleic Acids Res, 1984 Jan 11, 12(1 Pt 1), 1 - 9 Knowledge-based simulation of genetic regulation in bacteriophage lambda; Meyers S et al.; We have developed a general-purpose computer program for the functional simulation of regulatory genetics . This simulator is knowledge-based and was developed using the Unit System, a software tool for the acquisition, representation, and manipulation of hierarchically organized knowledge . The advantages of a knowledge-based design are presented, and the simulator's architecture is described . Its performance on the decision between lytic and lysogenic growth in Bacteriophage Lambda is reported. J Biol Chem, 1984 Jan 10, 259(1), 20 - 2 Failure of translational repression in the phage f2 op3 mutant is not due to an altered coat protein-RNA interaction; Carey J et al.; A secondary phenotype of the op3 mutant of RNA bacteriophage f2 is the absence of translational repression of the phage replicase gene by the phage coat protein . We have synthesized RNA fragments corresponding to the site of translational repression for both the wild type and the op3 mutant . Using a quantitative assay, we show that the affinity of the closely related R17 coat protein for the mutant and wild type RNA fragments is the same . In addition, we find that the op3 and R17 coat proteins bind to the wild type RNA fragment with essentially identical dissociation constants . Thus, the altered regulation of replicase protein synthesis in the op3 mutant does not appear to be due simply to a reduced affinity of the translational repressor for its target site. FEBS Lett, 1984 Jan 9, 165(2), 238 - 42 Fluorescence polarization study of tertiary structure of DNA within bacteriophage lambda; Shurdov MA et al.; The fluorescence polarization of acridine orange-stained, oriented lambda phages was measured . The parameters of DNA packing within the phage head cos2 theta and cos4 theta were calculated (theta, angle between the direction of a small segment of DNA and the phage axis) . It is shown that simple models of lambda phage DNA tertiary structure are not consistent with calculated values . A new model is proposed. Science, 1984 Jan 6, 223(4631), 69 - 71 Activation of antitumor agent gilvocarcins by visible light; Elespuru RK et al.; Gilvocarcins that are antitumor agents are activated by low doses of visible light to induce bacteriophage lambda in Escherichia coli . This result is dependent on interaction with DNA . Gilvocarcin M, an analog without antitumor activity, failed to induce the prophage after light exposure, thus demonstrating a correlation between photosensitizing and antitumor activities . These results raise several possibilities regarding the mode of action of gilvocarcins as antitumor agents in vivo, involving light or enzymatic activating systems, which could be exploited in human cancer therapy. J Mol Biol, 1984 Jan 5, 172(1), 1 - 22 A rho-dependent transcription termination signal in bacteriophage f1; Moses PB et al.; The bacteriophage f1 intergenic region distal to gene IV encodes a rho-dependent transcription termination signal . Terminator function in vivo and in vitro is dependent upon active Escherichia coli rho protein, although the RNA 3' ends detected in vivo differ from those seen in vitro . The minimal sequence required for terminator function in a heterologous plasmid system encompasses approximately 100 nucleotides distal to gene IV, which can be drawn as a large hairpin structure . The in vivo rho-dependent 3' end occurs within this sequence, while the in vitro rho-dependent 3' ends occur just distal to it . In vivo in a rho mutant host, f1 transcripts pass through the rho-dependent sites and stop within a sequence of high potential secondary structure near the f1 origin of DNA replication . This sequence alone causes transcription termination in the heterologous plasmid system in vivo . In vitro in the absence of rho protein, transcription does not terminate within this sequence . The RNA 3' ends detected in these studies do not occur within A + T-rich sequences. FEBS Lett, 1984 Jan 2, 165(1), 121 - 7 The purification of 5-enolpyruvylshikimate 3-phosphate synthase from an overproducing strain of Escherichia coli; Duncan K et al.; The Escherichia coli aroA gene which codes for the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase) has been cloned from the lambda-transducing bacteriophage lambda pserC . The gene has been located on a 4.7 kilobase pair PstI DNA fragment which has been inserted into the multiple copy plasmid pAT153 . E . coli cells transformed with this recombinant plasmid overproduce EPSP synthase 100-fold . A simple method for the purification of homogeneous enzyme in milligram quantities has been devised . The resulting enzyme is indistinguishable from enzyme isolated from untransformed E . coli. Biophys Struct Mech, 1984, 10(4), 229 - 39 UV-induced small structural changes in the T7 bacteriophage studied by melting methods; Toth K et al.; UV optical absorption and circular dichroism (CD) properties (spectra and melting curves) of T7 bacteriophage were investigated to detect "in situ" structural damages which can be related to the biological inactivation due to UV irradiation . UV doses (0.2-1.2 kJ/m2 at 254 nm) near to the biologically effective minimal dose were applied where the initial genetic damage (approximately 10 events/phage) was observed . The decrease of the melting temperature of the helix-coil transition and the broadening of the transition range indicate the destabilization of the intraphage structure due to the presence of about 0.1-0.6% damaged base concentration. Genetics, 1984 Jan, 106(1), 17 - 27 Regulation of a bacteriophage T4 late gene, soc, which maps in an early region; Macdonald PM et al.; We have sequenced and analyzed the expression of an early region of the bacteriophage T4 genome that surprisingly contains a late gene, soc . soc is oriented in the same direction as early genes, like the T4 lysozyme gene . Northern hybridization of early and late T4 RNA, using cloned T4 restriction fragments as probes, identified two long early transcripts and a short late transcript, all containing the soc-coding sequence . Thus, soc is transcribed both early and late . It is, however, translated only late . The inhibition of soc translation from the long early transcripts can be explained by formation of a hairpin in the RNA that sequesters the soc ribosome-binding site . The transcript initiated at the late promoter cannot form this hairpin and is, therefore, translated. Cell, 1984 Jan, 36(1), 211 - 9 Defining a bacteriophage T4 late promoter: absence of a "-35" region; Elliott T et al.; We performed a deletion analysis to identify the minimal DNA sequence required for the function of the T4 late promoter, P23 . A minipromoter derivative of P23 was constructed, containing 35 bp of T4 DNA from -18 to +17 with respect to the transcriptional initiation site . This derivative retains the TATAAATA homology and is competent to serve as a T4 late promoter both in vitro and in vivo . Its transcriptional activity in vivo is regulated identically to a wild-type plasmid-borne P23, requiring the function of T4 genes essential for late transcription, but not requiring T4 DNA replication . Recombination with the T4 phage chromosome is not significant for mini-P23 activity in vivo. J Virol, 1984 Jan, 49(1), 293 - 6 Bacteriophage P22 capsids with a subgenome length of packaged DNA; Serwer P et al.; Bacteriophage P22 assembles a DNA-free procapsid that subsequently packages P22 DNA . To study the packaging of bacteriophage P22 DNA, attempts were made to isolate P22 capsids with a subgenome length of packaged DNA . With the use of cesium chloride buoyant density sedimentation and agarose gel electrophoresis, the following capsids with a subgenome length of packaged DNA were isolated and characterized: (i) a capsid with the solid-support-free electrophoretic mobility and radius of the DNA-free P22 procapsid; (ii) a capsid with the solid-support-free electrophoretic mobility and radius of the mature P22 bacteriophage; and (iii) a capsid with a solid-support-free electrophoretic mobility and possibly a radius intermediate to those of the procapsid and bacteriophage. Mol Gen Genet, 1984, 195(3), 459 - 65 Codon specificity of starvation induced misreading; Johnston TC et al.; Mistranslated derivatives of the coat protein of the bacteriophage MS2 were isolated from infected cells starved for asparagine . This protein contains a high level of lysine for asparagine substitutions . By peptide analysis and amino acid sequencing we show that there is a six-fold greater frequency of errors at AAU codons than at AAC codons . This ratio is the same as that found in unstarved cells where the overall error frequency is 100-fold less . We also demonstrate that, at least for AAC codons, context affects error frequency. Mol Gen Genet, 1984, 195(3), 411 - 7 Transcription in bacteriophage f1-infected Escherichia coli: RNA synthesized on DNA of deletion mutant PII shows the existence of a two-site terminator; La Farina M et al.; Two different transcripts are synthesized on the DNA of deletion mutant PII of bacteriophage f1 in E . coli cells infected with this miniphage . Both RNA species appear to be primary transcripts and differ by about 100 nucleotides at their 3'OH end . Mapping of these molecules on the miniphage genome suggests that a two-site terminator is active at the end of the I region of transcription of bacteriophage f1. Dev Comp Immunol, 1984 Summer, 8(3), 611 - 22 Structural and functional analysis of spontaneous anti-nitrophenyl antibodies in three cyprinid fish species: carp (Cyrinus carpio), goldfish (Carassius auratus) and tench (Tinca tinca); Vilain C et al.; High spontaneous anti-trinitrophenyl (TNP) activities were found in three Cyprinid fish species: Carp (Cyprinus carpio), Goldfish (Carassius auratus) and Tench (Tinca tinca) . The molecules involved, isolated by affinity chromatography on dinitrophenyl-lysine Sepharose (DNP-lysine-Sepharose), had the main characteristics of a high molecular weight immunoglobulin (IgM-like) . Affinity measurements were performed on natural anti-DNP/TNP antibodies isolated from nine individual tench sera, using the inhibition of DNP-T4 bacteriophage inactivation technique . The antibodies analysed were more specific for TNP than for DNP . No activity was found against paranitrophenyl hapten . Affinities were all very low, even for TNP . In the three species, natural anti-DNP/TNP antibodies constitute as much as 11 to 16% of the total immunoglobulin concentration . This high level of nitrophenyl-binding serum immunoglobulins either suggests the existence of a particular regulatory mechanism in fish or reflects a generally low antibody diversity in these species. J Mol Evol, 1984-85, 21(2), 97 - 111 Comparison of goose-type, chicken-type, and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution; Weaver LH et al.; The three-dimensional structure of goose-type lysozyme (GEWL), determined by x-ray crystallography and refined at high resolution, has similarities to the structures of hen (chicken) egg-white lysozyme (HEWL) and bacteriophage T4 lysozyme (T4L) . The nature of the structural correspondence suggests that all three classes of lysozyme diverged from a common evolutionary precursor, even though their amino acid sequences appear to be unrelated (Grutter et al . 1983) . In this paper we make detailed comparisons of goose-type, chicken-type, and phage-type lysozymes . The lysozymes have undergone conformational changes at both the global and the local level . As in the globins, there are corresponding alpha-helices that have rigid-body displacements relative to each other, but in some cases corresponding helices have increased or decreased in length, and in other cases there are helices in one structure that have no counterpart in another . Independent of the overall structural correspondence among the three lysozyme backbones is another, distinct correspondence between a set of three consecutive alpha-helices in GEWL and three consecutive alpha-helices in T4L . This structural correspondence could be due, in part, to a common energetically favorable contact between the first and the third helices . There are similarities in the active sites of the three lysozymes, but also one striking difference . Glu 73 (GEWL) spatially corresponds to Glu 35 (HEWL) and to Glu 11 (T4L) . On the other hand, there are two aspartates in the GEWL active site, Asp 86 and Asp 97, neither of which corresponds exactly to Asp 52 (HEWL) or Asp 20 (T4L) . (The discrepancy in the location of the carboxyl groups is about 10 A for Asp 86 and 4 A for Asp 97.) This lack of structural correspondence may reflect some differences in the mechanisms of action of the three lysozymes . When the amino acid sequences of the three lysozyme types are aligned according to their structural correspondence, there is still no apparent relationship between the sequences except for possible weak matching in the vicinity of the active sites. C R Acad Sci III, 1984, 299(8), 275 - 80 {Demonstration of a sudden change in the use of codons in the vicinity of transcription termination}; Limaiem J et al.; A characteristic profile of fluctuations in the use of codons is seen in bacteriophages, Mammal mitochondria and animal viruses . Following DNA in the direction of transcription, one goes slowly from an area rich in codons ending by C to an area rich in codons ending by T and then one falls abruptly in an area rich in C . The termination of transcription is located in the area where the use of codons changes suddenly . It seems that the choice of codons ending by T or C is directed by the necessity to have a variation in the stability of the DNA . We propose a dynamic model where large scale variations of the stability of the DNA regulates the speed of propagation of the RNA-polymerase. Adv Biophys, 1984, 17, 97 - 146 Molecular organization of the head of bacteriophage Teven: underlying design principles; Yanagida M et al.; Structure and assembly of the bacteriophage T4 head are described as revealed by results obtained in this laboratory . Subunit arrangement of the major coat protein, soc and hoc in the head shell has been determined (Figs . 2 and 21) . Two new approaches for studying the assembly pathway are presented: wild type infection at 19 degrees C and gene 23 cold sensitive mutants . We propose an assembly pathway in which the prehead is formed in one direction, starting from the neck and ending at the distal cap . The processes of proximal and distal capping of the head shell are distinctly different . Novel structural intermediates such as a naked core and cup-like particles are shown . A model on head length determination is presented, based on negative control by the core on distal capping . Interesting aspects in the maturation of the head are reviewed . An overall scheme of the head assembly is shown in Fig . 20 . Comparative studies among Teven and RB phages showed that the particle morphology is strictly conserved while certain so-called non-essential and essential proteins are significantly varied . A phylogenetic relation among these phages is constructed from calculation of distances (Fig . 25). Mol Gen Genet, 1984, 195(1-2), 77 - 82 Isolation and characterization of regulatory mutations affecting the expression of the guaBA operon of Escherichia coli K-12; Tiedeman AA et al.; We isolated strains of Escherichia coli K 12 in which the lac structural genes were fused to the structural genes of the guaBA operon . These strains were used to isolate regulatory mutations that increased the expression of the guaBA operon under normal repressing conditions as compared to the wild type parental fusion strain . Three classes of guaBA specific regulatory mutations were identified . Class I regulatory mutations were trans-acting and unlinked to the guaBA operon as shown by bacteriophage P1 transduction . Class II regulatory mutations were tightly linked to the guaBA operon, cis-dominant to the wild type allele in a cis-trans analysis and were regarded as control region mutations . Class III regulatory mutations were tightly linked to the guaBA operon and trans-recessive to the wild type allele in a cis-trans analysis . We have designated the locus responsible for the class III regulatory mutations as guaR . The guaR locus is tightly linked and was mapped to the counterclockwise side of the guaBA operon . The guaR locus is proposed to specify a trans acting regulatory element involved in the regulation of the guaBA operon. Mol Gen Genet, 1984, 196(1), 53 - 8 In vitro template activity of 0.3 mRNA from wild type and initiation mutants of bacteriophage T7; Ohsawa H et al.; Bacteriophage T7 0.3 mRNA synthesised and processed in vitro has been purified starting from the DNA of T7+ as well as from that of two initiation mutants of T7 (CR17 with a U----C transition in the initiation codon and CR35b whose potential Shine and Dalgarno (S-D) interaction is interrupted by a G----A transition) . These mRNAs were used as templates to direct the binding of fMet-tRNA and the synthesis of 0.3 protein in both E . coli and wheat germ cell-free systems . The initiation codon mutant displayed approximately 50% inhibition of fMet-tRNA binding and 0.3 protein synthesis in both systems . The S-D sequence mutant, on the other hand, was found to be less affected than the initiation triplet mutant (20%-40% inhibition) in both fMet-tRNA binding and template activity in the E . coli system . In the wheat germ system, which does not make use of the S-D interaction, however, this mutant displayed normal template activity suggesting that the inhibition obtained in the E . coli system, albeit slight, is due to the impairment of the S-D interaction and not to an alteration of the mRNA secondary or tertiary structure caused by the base substitution. J Virol, 1984 Jan, 49(1), 20 - 5 O antigen-dependent mutant of bacteriophage T5; Heller KJ et al.; A T5 mutant is described which showed normal infection of Escherichia coli F but virtually no infection of cells lacking the E . coli F O antigen . This was due to very poor adsorption to the O antigen-deficient cells . Inactivation kinetics with anti-T5 serum and adsorption and desorption kinetics to receptor-deficient E . coli F cells suggested that the mutation did not affect the L-shaped tail fibers which mediate binding to the O antigen . Proof was obtained from genetic data; the structural gene for the L-shaped tail fibers mapped at a different position on the T5 chromosome than did the mutated gene . Since binding of the T5 mutant to the FhuA receptor protein was strongly inhibited by ferrichrome and since the mutation could not be crossed into phage BF23, we conclude that the mutation affects the receptor binding protein of the T5 tail. J Bacteriol, 1984 Jan, 157(1), 126 - 9 Suppression by thymidine-requiring mutants of Escherichia coli K-12; Herrington MB et al.; Thymidine-requiring strains of Escherichia coli isolated by trimethoprim selection often simultaneously acquire the ability to suppress bacteriophage T4 nonsense mutations . Suppression is lost in Thy+ revertants and recombinants, but is sometimes retained in thyA plasmid-bearing transformants . Suppression is restricted in Strr derivatives of the Thy- mutants, indicating that suppression occurs at the level of translation. Adv Exp Med Biol, 1984, 179, 185 - 91 The origin of DNA replication of bacteriophage f1 and its interaction with the phage gene II protein; Dotto GP et al.; The origin of DNA replication of bacteriophage f1 consists of two functional domains: 1) a "core region", about 40 nucleotides long, that is absolutely required for viral (plus) strand replication and contains three distinct but partially overlapping signals, a) the recognition sequence for the viral gene II protein, which is necessary for both initiation and termination of viral strand synthesis, b) the termination signal, which extends for 8 more nucleotides on the 5' side of the gene II protein recognition sequence, c) the initiation signal that extends for about 10 more nucleotides on the 3' side of the gene II protein recognition sequence; 2) a "secondary region", 100 nucleotides long, required exclusively for plus strand initiation . Disruption of the "secondary region" does not completely abolish the functionality of the f1 origin but does drastically reduce it (1% residual biological activity) . This region, however, can be made entirely dispensable by mutations elsewhere in the phage genome. Mol Gen Genet, 1984, 194(1-2), 232 - 40 A physical map of bacteriophage T4 including the positions of strong promoters and terminators recognized in vitro; Gram H et al.; We present a linearized physical map of the genome of bacteriophage T4 . This map contains the cleavage sites for restriction enzymes SmaI, KpnI, SalI, BglII, XhoI, XbaI, ClaI , HaeII, EcoRI, and EcoRV . It also contains about 200 TaqI sites . The promoter sites recognized in vitro and a number of rho independent terminators have also been mapped. Mol Biol (Mosk), 1984 Jan-Feb, 18(1), 39 - 47 {C1 and cro repressors of lambda phages . Isolation of strains of bacteriophage lambda imm434 superproducing repressor cro}; Bespalova IN et al.; A collection of recombinant plasmids directing the increased synthesis of the cro-repressor of lambda imm434 bacteriophage under control of the lacZ promoter of Escherichia coli has been constructed . About 87 000 molecules of cro-repressor monomer per cell, e . g . 0,7% of total cell protein are synthesized by the overproducers . Strain bearing one of such plasmids--pBS71-1 . Several recombinant plasmids in which the cro-gene is placed under the control of early genes promoter of lambda phage (pl, pr, prm) have been also prepared . Plasmid pIL206, constructed on the basis of pIL203 vector, was used as a source of lambda-promoters. Mol Biol (Mosk), 1984 Jan-Feb, 18(1), 30 - 8 {C1 and cro repressors of lambda phages . I . Construction of vectors for expression of cro repressor of bacteriophage lambda imm434}; Bespalova IN et al.; Eight derivatives of recombinant plasmid pBRcro434, that consists of pBR322 and fragment of immunity region of phage lambda imm434 have been constructed and characterised . These derivatives contain the deletions in the region adjacent to OR3 operator and in the structural gene of cro-repressor of lambda imm434 . The deletions have been produced by the treatment of pBRcro434 with exonuclease III of Escherichia coli and S1 nuclease of Aspergillus orizae and precisely mapped . The unique EcoRI-restriction sites have been reconstructed with the aim of using this deletion plasmids as a vectors for cloning. Mol Biol (Mosk), 1984 Jan-Feb, 18(1), 227 - 33 {Kinetic characteristics of the ATP-pyrophosphate isotope exchange catalyzed by RNA ligase from T4 bacteriophage}; Zagrebel'nyi SN et al.; The dependence of initial rate v0 of ATP--PPi exchange reaction catalyzed by RNA-ligase of bacteriophage T4 on the concentration of ATP(s), pyrophosphate (z) and Mgcl2 has been determined . The dependence of v0 on s and z described by the equation v0 = k-1k2E0/(k-1 + K2) (1 + K1/s + k2/z) has been obtained for the reaction of E + S in equilibrium ES in equilibrium E1 + Z, where E--enzyme, E1--adenylylenzyme, S--ATP, Z--pyrophosphate, K1 and K2--constants of equilibrium, k-1, k2--velocity constants of transition of ES to E + S and E1 + Z, E0--complete concentration of enzyme . The low inhibition of the ATP--PPi exchange by the acceptor A(pA)2 and donors pAp, p(Ap)3, pCp has been shown . The dependence of v0 on the concentration of MgCl2 is consent with the incorporation of only dimagnesium salts of substrates in the isotope-exchange reaction. Prikl Biokhim Mikrobiol, 1984 Jan-Feb, 20(1), 24 - 30 {Isolation of highly purified RNA ligase from bacteriophage T4}; Zagrebel'nyi SN et al.; A technique for isolation of RNA-ligase of bacteriophage T4 was proposed . It is mainly based on the using of Soviet materials and sorbents and includes seven purification stages . The technique enables to isolate about 80 000 units of active enzyme from 100 g of E . coli B cells infected with the phage . T4am N82; that makes up 20% of the activity of the cell extract . The obtained preparations of RNA-ligase are homogeneous by the data of electrophoresis and practically, free of endo- and exonuclease admixtures. Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 535 - 9 In vivo formation of gene fusions encoding hybrid beta-galactosidase proteins in one step with a transposable Mu-lac transducing phage; Casadaban MJ et al.; A Mu-lac bacteriophage transposon, MudII301 (Ap, lac), was constructed to form hybrid protein gene fusions . When it integrates into structural genes in the appropriate direction and reading phase, transcription and translation from outside gene controlling regions can proceed across 116 nucleotides from the right end of Mu into lacZ codons to form hybrid proteins that are enzymatically active for beta-galactosidase . Integration can be obtained either by infection to form lysogens or by transposition during growth of a lysogen . The size of the hybrid protein product either corresponds to or, in the cases of translation restart or protein degradation, is a minimal estimate of the distance of the Mu insertion from the translation initiation site of the gene . Hybrid proteins formed by insertions in randomly selected genes and in the araB and A genes were examined by polyacrylamide gel electrophoresis. Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 414 - 8 Direct isolation of the functional human thymidine kinase gene with a cosmid shuttle vector; Lau YF et al.; We have developed a new recombinant DNA cloning system to isolate directly the functional unit of the human thymidine kinase (TK) gene . The system utilizes a cosmid vector that can shuttle cloned DNA sequences between bacteria and mammalian cells . A complete human cosmid library was constructed and DNA from the total library was transfected to mouse L cells deficient in TK (LTK-) by calcium phosphate precipitation . The transfected cells were then selected with hypoxanthine/aminopterin/thymidine (HAT) medium, and one HAT-resistant cell clone was isolated . This cell line became resistant to HAT selection by acquiring the TK gene derived from the human cosmid library . As the cosmid vector contains the cohesive ends of the bacteriophage, we could directly retrieve the human DNA sequences from the transformed mouse L cells . Total DNA from the transformed TK+ L cells was packaged in vitro with lysogenic bacterial extracts and used to infect Escherichia coli . One of the two recombinant cosmids isolated contained a 43.8-kilobase human DNA insert and was capable of converting TK- L cells to the TK+ phenotype in both acute and stable transformation assays . Thus, we have isolated the functional human TK gene in this recombinant cosmid . The gene was further localized on a 14.5-kilobase BamHI DNA fragment, and it transcribed a mature mRNA of about 1,500 nucleotides . This method of gene isolation has several special features: (i) an intact structural gene can be cloned directly based on its function without knowledge of its amino acid or nucleotide sequence; (ii) the functional gene sequences can be recovered faster and more efficiently than with the usual DNA transfection method; and (iii) in conjunction with cell-sorting techniques, this method can be used to clone genes encoding cell surface markers. Mol Gen Genet, 1984, 193(2), 205 - 9 Location of the rho gene and characterization of lambda ilv-gal derivatives of lambda ilv-rho bacteriophage; Calhoun DH et al.; The location of the rho gene and its position relative to the ilv genes of Escherichia coli K-12 was analyzed using genetic criteria, restriction enzyme cleavage, and maxicell analysis . Plasmids were constructed with deletions of the rho gene introduced in vitro, and lambda ilv-gal derivatives of lambda ilv-rho bacteriophage were isolated by recombination in vivo . A HindIII restriction fragment of 8 kilobases (kb) previously shown to contain at least part of the rho gene (Gray et al . 1981) was cloned into plasmid pMC81 . This vector has transcription stop sites that present read-through expression of cloned genes from either direction, and cloning sites upstream of the lacZ gene coding for beta-galactosidase . The position of the rho gene and flanking sequences required for its expression were further localized to a region of approximately 2 kb by introducing deletions using restriction enzyme treatment of these plasmids . A promoter in the rho region was found to direct beta-galactosidase synthesis in these plasmid derivatives . Derivatives of lambda ilv-rho phage were isolated in vivo by pyrophosphate chelation selection for phage with reduced genome size . Restriction enzyme analysis of twelve of these derivatives revealed an unexpected bias towards phage recombinants as opposed to simple internal deletions. Genetics, 1984 Jan, 106(1), 1 - 16 Cloning and physical mapping of an early region of the bacteriophage T4 genome; Macdonald PM et al.; We have cloned DNA restriction fragments from the largely nonessential region of bacteriophage T4 located between genes 39 and 56 . The cloned DNA fragments were used to construct a precise map of the sites in this region recognized by eight restriction endonucleases . This restriction map allowed us to compare the cytosine-containing T4 DNA used for cloning with the hydroxymethylcytosine-containing DNA of wild-type T4; there were no detectable rearrangements in the region tested . We were also able to determine the physical locations of several deletion end points and of several genes. Cell, 1984 Jan, 36(1), 197 - 202 Stoichiometric use of the transposase of bacteriophage Mu; Pato ML et al.; The transposase of bacteriophage Mu (gene A protein) mediates the coupled replication and integration processes that constitute transposition during the lytic cycle . Our previous results showed that the activity of the A protein is unstable, as its continued synthesis is required to maintain Mu DNA replication throughout the lytic cycle . We present here the results of experiments in which the A protein is used stoichiometrically and must be synthesized de novo for each round of Mu DNA replication . Induction of a Mu lysogen in the absence of DNA replication allows accumulation of potential for a single round of Mu DNA replication . Once achieved, this potential is stable even in the absence of further protein synthesis . Release of inhibition of DNA replication leads to a single semi-conservative replicative transposition event, followed by later rounds only if additional synthesis of the A protein is allowed. Mol Gen Genet, 1984, 193(1), 104 - 9 A hotspot for transition mutations in the rIIB gene of bacteriophage T4 . I . The extent of the hotspot; Singer BS; We have previously demonstrated that the sequence 5'TGGCAA 3' located at codons 32-33 of the rIIB gene of bacteriophage T4 is a hotspot for transition mutations (Nelson et al . 1981) . Here I report the properties of the same TGGCAA sequence introduced into the gene at codons 11-12 . The sequence is highly mutable in both locations, suggesting that its high mutability is due to features of the TGGCAA itself and is not dependent on the immediate juxtaposition of additional external sequences . Within this sequence, at either location, only the transition at the central G:C pair frequently arises spontaneously or by 2-aminopurine or ethylmethane sulfonate mutagenesis . However, the 3' G:C pair, in addition, is highly mutable after nitrous acid or hydroxylamine treatment . This suggests that, within the TGGCAA sequence, there are two hotspots which are targeted by different mutagens. IARC Sci Publ, 1984, (57), 731 - 9 Genetic and biochemical factors affecting the induction of bacteriophage lambda by N-nitroso compounds; Elespuru RK et al.; As part of our effort to validate a biochemical (prophage) induction assay (BIA) as a screening test for carcinogens, we have tested more than 100 N-nitroso compounds . An enzyme, beta-galactosidase, is induced as an indirect consequence of DNA damage to the host, as part of the 'SOS' response . Besides the obvious practical importance of detecting this class of carcinogen, there is the question of the mechanism by which these compounds work . Mutagenesis by one compound, N-methyl-N'-nitro-N-nitrosoguanidine, is known to proceed by both SOS (recA)-dependent and SOS-independent pathways . Mispairing due to O6 alkylation of guanine is thought to be responsible for the SOS-independent pathway; however, there has been little consideration of recA-dependent functions, of which phage induction is one . Although nitrosamides could be detected as phage inducers in our assay, N-nitrosamines in the presence of rat liver 9 000 X g supernatant usually gave no response . We found that the use of several mutant strains, particularly a lexA mutant, in combination with hamster liver 9 000 X g supernatant, allowed us to detect most N-nitrosamines reasonably well, in either a spot test or a quantitative tube assay . Induction in a lexA strain was most unexpected, since this mutation usually diminishes the expression of SOS functions induced by ultra-violet light . Because the genetic and biochemical conditions that favour phage induction are different from those that favour mutagenesis, it seems likely that the lesions in DNA leading to the two biological end-points are different. Cold Spring Harb Symp Quant Biol, 1984, 49, 699 - 705 DNA interactions during bacteriophage lambda site-specific recombination; Bauer CE et al.; Extensive research on site-specific recombination has provided many details, particularly with respect to the protein-DNA interactions . However, very little is known about the molecular mechanism of recombination during synapsis and strand exchange . Presumably, these steps of recombination involve various forms of DNA-DNA, DNA-protein, and protein-protein interactions . One stage at which DNA-DNA interactions may be occurring is at the level of synapsis where the recombining DNAs are juxtaposed . In this paper we have presented evidence that homology-dependent DNA interactions do occur within the overlap region before strand exchange . This interaction is presumably at the synaptic stage of recombination . Furthermore, we have demonstrated that the homology-dependent interactions require that only one strand of attB have homology to attP . Another stage in recombination at which DNA-DNA interactions could occur is during strand exchange where complementary strands from the recombining parents are paired and resealed . We have also presented evidence that homology-dependent DNA interactions occur during strand exchange prior to the resealing of the strands and that disruption of this interaction results in nonreciprocal recombination . Taken together, these results suggest that DNA-DNA interactions during reciprocal site-specific recombination occur during at least two stages in the reaction. Adv Exp Med Biol, 1984, 179, 77 - 89 Initiation of DNA synthesis on single-stranded DNA templates in vitro promoted by the bacteriophage lambda O and P replication proteins; LeBowitz JH et al.; The bacteriophage lambda O and P protein replication initiators, in conjunction with six purified Escherichia coli replication proteins, replicate the single-stranded chromosomes of phages M13 and phi X174 to a duplex form . Several discrete steps are involved in this DNA synthesis reaction . In an ATP-dependent step that precedes priming, the lambda O and P proteins interact with the Escherichia coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto DNA coated with single-stranded DNA binding protein . This creates a stable prepriming intermediate, isolable by gel filtration, that is rapidly primed and replicated upon the addition of primase and DNA polymerase III holoenzyme . Each of the eight proteins required for this nonspecific single strand replication reaction also have physiological roles in the replication of the bacteriophage lambda chromosome in vivo . We propose a scheme for the lambda O and P protein-dependent initiation of DNA synthesis that may be relevant to strand initiation events occurring during lambda DNA replication. Adv Exp Med Biol, 1984, 179, 231 - 40 Gene A protein interacting with recombinant plasmid DNAs containing 25-30 b.p . of the phi X174 replication origin; Fluit AC et al.; Synthetic oligodeoxyribonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p . of the 30 b.p . origin region of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence) . The double-stranded DNA fragments were cloned into the kanamycin resistance gene of pACYC177 (AmpR, KmR) . Transformants were picked up by antibiotic selection and filter-hybridization using one of the oligodeoxyribonucleotides as a probe . Approximate lengths of the inserts were determined by restriction enzyme analysis . Exact length and orientation of each insert was determined by DNA sequencing . Plasmid DNA with an insert homologous to the first 25 b.p . of the phi X174 origin is not nicked by the gene A protein . However, plasmid DNA containing the 27 b.p . fragment in either orientation is nicked by the gene A protein, as well as plasmid DNAs containing the first 28 b.p . or the complete 30 b.p . conserved origin region of the isometric phages. Mol Gen Genet, 1984, 197(1), 169 - 74 Transduction of multi-copy plasmid pBR322 by bacteriophage Mu; Teifel-Greding J; The temperate bacteriophage Mu transduces the 4363 bp multi-copy plasmid pBR322 at frequencies similar to those of chromosomal markers . Plasmid transducing particles contain DNA molecules of Mu DNA length . Plasmid DNA is transduced as a head-to-tail oligomer that becomes circularized in the recipient cell . The rec system of the donor strain participates in oligomer formation and the rec system of the recipient strain is required for oligomer circularization . Possible mechanisms that may explain the origin of plasmid transducing particles are discussed. Mol Gen Genet, 1984, 197(1), 104 - 8 Integration of bacteriophages lambda and phi 80 in wild-type Escherichia coli at secondary attachment sites . I . Formation of secondary lysogens; Kholodii GY et al.; The family of lambdoid phages displays a varying specificity of integration into the host chromosome . The lambda phage DNA failed to get inserted at the secondary site(s) of the gal operon (frequency less than 2.6 X 10(-8) in the presence of the primary (normal) att site . By contrast, phi 80 and the lambda att80 hybrid (lambda X phi 80) became integrated into wild-type Escherichia coli at at least two secondary att sites of the btuB locus, and the latter near purE and purC as well (frequency 2 X 10(-3)-10(-4) . The integration of phi 80 and lambda att80 into btuB occurred with about the same frequency as in cells in which the normal insertion site had been deleted (0.7-4.0 X 10(-6) . An analysis of the secondary lysogens with the prophage in btuB showed them to be polylysogens; the additional prophage(s) was found at the primary att site . We also failed to observe the integration into other loci of phi 80 and lambda att80 with the formation of secondary monolysogens (frequency less than 0.0035 at MOI = 10(-3) or 10) . It is presumed that these prophages become integrated at secondary att sites only if the primary site is occupied. Mol Gen Genet, 1984, 196(3), 401 - 6 Bacteriophage lambda replication proteins: formation of a mixed oligomer and binding to the origin of lambda DNA; Zylicz M et al.; The purified bacteriophage lambda replication proteins O and P sediment separately in metrizamide gradients of low ionic strength as dimers . Together they interact with each other forming an oligomer, composed of two molecules of lambda O and one molecule of lambda P . The lambda O-P oligomer is active in the in vitro replication of ori lambda-containing DNA . Equilibrium sedimentation in preformed metrizamide density gradients under conditions that separate DNA-protein complexes from free proteins was employed in order to study possible interactions among the lambda replication proteins and ori lambda DNA . It was found that the lambda P protein binds specifically to ori lambda-containing plasmid DNA only in the presence of lambda O protein . About 100 molecules of lambda O and 10 molecules of lambda P form a complex with the ori lambda DNA . The lambda DNA-lambda O-lambda P complex was shown to be active in an in vitro replication system . Since the physical interactions between ori lambda and lambda O and between lambda P and the Escherichia coli dnaB replication protein are well documented, the evidence for a lambda O-P interaction presented in this paper provides the missing link in the molecular mechanism that enables lambda to direct the host replication machinery to the replication of its own DNA. Mol Gen Genet, 1984, 195(1-2), 62 - 9 An integration-proficient int mutant of bacteriophage lambda; Enquist LW et al.; We have isolated and characterized a novel int mutant of phage lambda . This mutant promotes efficient recombination between the phage and bacterial attachment sites, but, unlike wild type, does not promote efficient recombination of any other pair of attachment sites tested in most conditions . In particular, recombination between two phage or two prophage attachment sites is poor relative to the wild type frequency . We attribute this unusual phenotype to differences in the distribution of int protein binding sites among different attachment sites (Ross and Landy 1982, 1983) . We suggest that int protein molecules bound to one of two recombining DNAs interact with empty sites or with bound proteins on the other, and that the mutant protein acts efficiently only if the distribution of protein binding sites within the two attachment sites is that of the attP-attB pair . Similar discrimination among attachment site pairs by wild type int protein may also modulate recombination frequencies. Mol Gen Genet, 1984, 196(1), 170 - 2 Hek: an Escherichia coli function involved in functional expression of the kil gene of bacteriophage Mu; Goosen N et al.; An Escherichia coli mutant has been isolated (Hek) in which the kil gene of bacteriophage Mu is not functionally expressed . The hek locus has been mapped between rpoD (66.2 min) and argR (69.5 min) on the E . coli chromosome . No influence of the hek mutation on phage or E . coli development could be detected. Int Arch Allergy Appl Immunol, 1984, 75(3), 219 - 26 A comparison of the antibody responses of badgers (Meles meles) and rabbits (Oryctolagus cuniculus) to some common antigens; Higgins DA et al.; The primary and secondary antibody responses of rabbits and badgers were compared after intravenous inoculation of inactivated influenza A virus, sheep erythrocytes (SRBCs), bovine serum albumin (BSA) or bacteriophage psi X174 . BSA was also given as a primary injection by the intramuscular route in solution or in Freund's incomplete or complete adjuvant, followed by an intravenous secondary inoculation without adjuvant . Antibody responses were monitored by: haemagglutination inhibition and neutralization tests for influenza virus; direct and antiglobulin haemagglutination tests for SRBCs; indirect haemagglutination test and the Farr method for antigen-binding capacity (ABC) for BSA; neutralization of psi X174 . Rabbits gave good responses to all antigens, but the response of badgers was generally poor . After intravenous administration, badgers gave a good response only to psi X174, but even then they produced less antibody than rabbits receiving 100 times less antigen; the immune elimination of phage was more rapid and antibody appeared about 48 h earlier in rabbits than in badgers . Intramuscular administration of BSA and the use of adjuvants improved the badgers' response, with greatest improvement in ABC . These results indicate that badgers display relatively poor immune responses to a variety of antigens. Mol Gen Genet, 1984, 194(3), 373 - 6 Structure and function of the repressor of bacteriophage lambda . II . Isolation and characterization of a lambda mutant which produces repressor having higher affinity for operators; Nag DK et al.; By mutagenizing a lambda cIts (lambda cI857) lysogen, a lambda mutant has been isolated with a wild-type phenotype . This mutant phage lysogenizes with low efficiency and produces a low burst . Though the initial rates of repressor synthesis in Escherichia coli after infection with wild-type and mutant lambda are the same, the maximum level of repressor that is synthesized in the latter case is only about 30% of that synthesized in the former . Virulent lambda plates on the lysogen of mutant lambda with slightly less efficiency producing very tiny plaques . Operator-binding studies made in vitro with purified mutant and wild-type repressors show that the binding curve of the former repressor is a rectangular hyperbola while that of the latter is sigmoid . The half-lives of the complexes of mutant and wild-type repressors with right operator are 133 and 27 min, respectively . All these results suggest that the mutant repressor possibly has a higher affinity for the operators . This mutant has been named lambda cIha (ha = high affinity). Gene, 1984 Jan, 27(1), 3 - 11 Genetics of bacteriophage phi 80--a review; Rybchin VN; The genetic maps of bacteriophage lambda and lambdoid phage phi 80 are compared . The gene organization of phi 80 is very similar to that of lambda, as shown by isolation and characterization of many am, ts and c (clear) mutants of the phage . In general, the essential genes located in the same position on the genetic map of the phages lambda and phi 80 fulfill the same functions . These include the gene clusters coding for the head and tail proteins, genes for DNA synthesis, and the genes controlling lysogeny and late gene expression . The specific regulatory features of phi 80 in relation to the N function of lambda are discussed, but they require further clarification . The two phages differ in immunity specificity, host range, conversion property and temperature sensitivity. Plasmid, 1984 Jan, 11(1), 65 - 73 Replication defective RP4 plasmids recovered after chromosomal integration; Grinter NJ; pHH6000 is a composite replicon made by the in vitro ligation of the IncP plasmid RP4 to a fragment of bacteriophage lambda capable of autonomous replication . Derivatives were selected in which it had integrated into the Escherichia coli chromosome by homologous recombination with the resident lambda prophage, and plasmids were subsequently regenerated from the integrated molecules . Although of the same molecular size as pHH6000, all had altered properties: those recovered from the chromosome of cells simultaneously carrying a distinguishable autonomous IncP plasmid showed a 100- to 1000-fold reduction in their ability to become established in a lambda lysogen; those regenerated from cells with no autonomous IncP plasmid were no longer RP4 replicons, now being dependent on replication functions encoded by the lambda DNA they carry and therefore unable to form a plasmid in a lambda lysogen . This second class of plasmids still exhibited normal RP4 incompatibility and stability even though neither property is encoded by the lambda replicator DNA . It was concluded that expression of RP4 incompatibility and partitioning control do not require an intact RP4 replicon . The data also suggest that the presence in the chromosome of a normal RP4 molecule may be deleterious to the host, although the manner in which the integrated molecules were obtained allows other explanations . The composite plasmids replicating from cloned lambda genes should be useful in analysis of the regulated distribution of RP4 molecules at cell division. Proc Natl Acad Sci U S A, 1984 Jan, 81(1), 130 - 4 Two-dimensional 1H NMR study of the lambda operator site OL1: a sequential assignment strategy and its application; Weiss MA et al.; The solution structure and dynamics of the 17-base-pair synthetic operator site OL1, which is recognized by the cI and Cro repressors of bacteriophage lambda, is studied by two-dimensional NMR methods . A sequential assignment strategy for nucleic acids is proposed and illustrated by the assignments of the base and sugar protons of OL1. Mol Gen Genet, 1984, 193(2), 322 - 6 Evidence that the cro repressor inhibits expression of the bacteriophage lambda P gene at high multiplicities of infection; Zambetti GP et al.; The activity of the lambda P gene product at various multiplicities of infection (m.o.i.) was examined in CI- conditions using an assay which measures the disappearance of the rapidly-sedimenting closed-circular (c.c.) form of phage DNA . When cells were infected with lambda CI857 at multiplicities of 5 phage/cell or less, between 65%-75% c.c . DNA was lost during incubation . If the multiplicity of infection was increased to 10 phage/cell or greater, a marked inhibition in the cleavage of c.c . DNA was observed . When bacteria were infected with either lambda CI857CII2002 or lambda CI857cro27 at low m.o.i., the usual 65%-75% decrease in the percentage of c.c . phage DNA occurred during incubation . In contrast, no losses in c.c . DNA were noted after infection with lambda CI857cro27susP3 . At high m.o.i., the cleavage of c.c . DNA was inhibited after infection with lambda CI857CII2002, but not after infection with lambda CI857cro27 . It is concluded that at high m.o.i . in CI- infections, the expression of gene P is unaffected by the CII gene product, but is inhibited by the increased intracellular levels of cro protein. J Bacteriol, 1984 Jan, 157(1), 165 - 70 Proteinase sensitivity of bacteriophage lambda tail proteins gpJ and pH in complexes with the lambda receptor; Roessner CA et al.; Previous studies have shown that bacteriophage lambda initially binds to liposomes bearing its receptor protein by the tip of the tail fiber (type 1 complex) . It then associates more directly so that the hollow tail tube is in direct contact with the membrane (type 2 complex) . DNA can be injected across the lipid bilayer into the liposome from type 2 complexes . We show here that gpJ, the tail fiber protein, becomes more sensitive to proteolytic degradation in type 2 complexes, indicating that the tail fiber does not pass into the liposome and that the tail fiber may undergo a conformational change in type 2 complexes . Another bacteriophage protein, pH, is sensitive to proteolytic degradation in free bacteriophage, type 1 complexes, or type 2 complexes formed with free receptor, but is resistant to proteinases in type 2 complexes formed with liposomes . This finding suggests that pH associates with the membrane . We suggest that this association is part of the mechanism by which a transmembrane hole for DNA entry is formed. Chromosoma, 1984, 89(3), 218 - 27 Molecular and genetic studies on the euchromatin-heterochromatin transition region of the X chromosome of Drosophila melanogaster . 1 . A cloned entry point near to the uncoordinated (unc) locus; Miklos GL et al.; A recombinant Charon 4 bacteriophage has been isolated on the basis of RNAs which are enriched in the head of the adult Drosophila melanogaster and hence are likely to be of neural origin . The cloned insert maps to the near vicinity of the uncoordinated locus in polytene chromosome band 19E8 . This band is within the transition zone between the euchromatic and heterochromatic regions of the X chromosome, a region which has been well characterized cytogenetically . The insert contains both repetitious and low copy number sequences, some of which vary extensively in both frequency and restriction fragment size between different laboratory strains . One particular family of moderately repeated sequences occurs predominantly in divisions 19 and 20 of the X chromosome and perhaps the distally located X heterochromatin . The molecular landscape surrounding the initial entry point contains many repeated sequences and is thus unlike those observed in most published chromosomal walks . The possible significance of the presence of repeated families in the distinct properties of this region are discussed. Mol Biol (Mosk), 1984 Jan-Feb, 18(1), 197 - 204 {EcoRV restrictase: physical and catalytic properties of homogenous enzyme}; Kuz'min NP et al.; With the use of the strain-overproducer restriction endonuclease R.EcoRV was isolated and purified to homogeneity . The molecular mass of the enzyme was determined by gel filtration and polyacrylamide gel electrophoresis to be 25 000 daltons . According to the data of immunological tests R.EcoRV differs in its antigenic characteristics from restriction endonucleases R.EcoRI and R.EcoRII . Dependence of enzyme activity on pH, ionic strength, temperature, presence of divalent cations (Mn2+, Mg2+, Co2+, Zn2+, Ni2+ and Cd2+) and organic solvents (glycerol, dimethylsulfoxide, ethanol) has been studied . It was shown that under conditions of replacement of Mg2+ for Mn2+ or after addition of organic solvents relaxation of R.EcoRV specificity takes place . It was shown also that R.EcoRV is able to digest T-even bacteriophage DNAs with different types and extents of modification . DNA modified by the action of MR.EcoRV system in vivo is susceptible to R.EcoRV in vitro . Under conditions of relaxed specificity noncanonical sites are susceptible to R.EcoRV attack . The fragments resulted may be cloned in canonical pBR322 EcoRV site. Mol Biol (Mosk), 1984 Jan-Feb, 18(1), 130 - 9 {Properties of cloned promoters}; Rechinskii VO et al.; Strong early bacteriophage T7 promoters A2 and A3 and also A2 and lac UV5 promoters with altered segments downstream the initiation of RNA start point were cloned using specially constructed plasmid vectors pBRS188 and PBRS240 . The relative signal strengths of these promoters in vivo and in vitro were evaluated and the kinetic parameters of their interaction with RNA polymerase were determined . It has been shown that the nucleotide sequence of the transcribed region plays a significant role in specific promoter-RNA polymerase interaction and that the rate-limiting step of RNA synthesis initiation is different for various promoters. J Mol Evol, 1984-85, 21(2), 150 - 60 Selection pressures on codon usage in the complete genome of bacteriophage T7; Sharp PM et al.; We searched the complete 39,936 base DNA sequence of bacteriophage T7 for nonrandomness that might be attributed to natural selection . Codon usage in the 50 genes of T7 is nonrandom, both over the whole code and among groups of synonymous codons . There is a great excess of purine- any base-pyrimidine (RNY) codons . Codon usage varies between genes, but from the pooled data for the whole genome (12,145 codons) certain putative selective constraints can be identified . Codon usage appears to be influenced by host tRNA abundance (particularly in highly expressed genes), tRNA-mRNA (one such interaction being perhaps responsible for maintaining the excess of RNY codons) and a lack of short palindromes . This last constraint is probably due to selection against host restriction enzyme recognition sites; this is the first report of an effect of this kind on codon usage . Selection against susceptibility to mutational damage does not appear to have been involved. Adv Exp Med Biol, 1984, 179, 221 - 30 Gene A protein of bacteriophage phi X174 is a highly specific single-strand nuclease and binds via a tyrosyl residue to DNA after cleavage; Van Mansfeld AD et al.; The sequence specificity of the endonuclease activity of gene A protein and A* protein was studied using synthetic oligonucleotides containing (part of) the sequence of the origin of phi X RF DNA replication and single-stranded (ss) DNA fragments of phi X and G4 . From a comparison of the sequences that are cleaved a consensus sequence for cleavage of ssDNA by gene A protein has been deduced . This consensus sequence occurs in ssDNA of both phi X and G4 at the origin and at one additional site . This is surprising since the rolling circle mechanism demands that gene A protein cleaves at the origin only . However, it could be shown that in the presence of SSB protein the ssDNAs of phi X and G4 are only cleaved at the origin, which is probably due to a strong gene A protein binding site, the key sequence, which forms part of the 30 b.p . origin region of phi X and related bacteriophages . Gene A protein and A* protein bind covalently to the DNA at the 5'-end of the cleavage site . Using a uniquely, internally 32p-labelled oligonucleotide as a substrate, it was shown that gene A protein and A* protein are bound via a tyrosyl residue to the 5'-phosphate of the phosphodiester bond which is cleaved. Mol Gen Genet, 1984, 197(2), 261 - 71 Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome; Albin R et al.; Mutations at the cpxA locus of Escherichia coli K-12 affect cellular processes that are not otherwise related . We have now determined the physical and genetic structure of the E . coli chromosome in the region of cpxA (87.5 min) . Our results indicate that cpxA is a single gene . Previous studies showed cpxA to be linked to tpiA . We therefore isolated two tpiA+ recombinant plasmids, pRA200 and pRA300, from EcoRI and BamHI digests of F'133, respectively . By genetic complementation or enzyme overproduction, the 9.5 kb EcoRI fragment in pRA200 was shown to include glpK, tpiA and cdh . The 13.6 kb BamHI fragment of pRA300 lacks glpK, but includes tpiA, pfkA and cpxA . Neither fragment complemented a deletion of the rha operon . These data indicate the chromosomal gene order: 87 min-rha-cpxA-pfkA-cdh-tpiA-glpK-88 min . The EcoRI and BamHI fragments overlap in an interval corresponding to about 8.2 kb of DNA . The total region of the E . coli K12 chromosome covered by the two fragments is about 15 kb . A terminal 2 kb EcoRI-BamHI fragment from pRA300 complemented the chromosomal cpxA2{Ts} allele with respect to isoleucine and valine synthesis, RNA bacteriophage sensitivity and surface exclusion in Hfr strains, and envelope protein composition . Complementation occurred when the fragment was subcloned in pBR325 but not when it was subcloned in pBR322, suggesting that the 2 kb fragment lacks expression sequences that are supplied by cat (chloramphenicol acetyltransferase gene) expression sequences of pBR325 . The cpxA locus on the E . coli chromosome was established with respect to two chromosomal Tn10 insertions by a combination of genetic and physical analyses . The locus established by those analyses was consistent with the location of the 2 kb EcoRI-BamHI fragment in the physical map of the region . Physical analyses of (rha-pfkA) and (rha-tpiA) deletion strains showed that they lack cpxA and surrounding genes . Since these strains were viable, cpxA is not essential under all growth conditions. Mol Gen Genet, 1984, 197(1), 129 - 36 Cloning and analysis of pif, replication and leading regions of the F plasmid; Jackson R et al.; We describe the molecular cloning of BglII fragments of the hybrid plasmid pRS5 (pSC101 and EcoRI fragments of F; f7, f5, f3 and f6) . The clones isolated were examined for the expression of F-specified replication, incompatibility, mobilization and inhibition of T7 bacteriophage multiplication . Proteins directed by the BglII clones were labelled in Escherichia coli K12 maxicells and analyzed by SDS-polyacrylamide gel electrophoresis . The sizes of previously reported proteins, encoded by the replication, incompatibility and leading regions encompassed by these plasmids have been confirmed in this study . In addition, the results demonstrate that a pif gene, which encodes an 80,000 dalton polypeptide essential for the inhibition T7 phage multiplication, is located on the BglII fragment that spans the junction of EcoRI fragments f7 and f5. Mol Gen Genet, 1984, 195(1-2), 5 - 9 Rho-dependence of the terminator active at the end of the I region of transcription of bacteriophage f1; La Farina M et al.; Infection of rho- Escherichia coli cells with deletion mutant PII of bacteriophage f1 results in a miniphage RNA population composed of transcripts longer than those synthesized in the infection of rho+ cells . This indicates a Rho dependence of the terminator active at the end of the I region of transcription of bacteriophage f1 . An estimate of the length of a transcript, which represents a good fraction of the RNA that passes beyond the terminator, indicates that the hairpin structure where synthesis of complementary strand DNA initiates also acts as a fairly efficient Rho-independent terminator. Mol Gen Genet, 1984, 195(1-2), 44 - 51 Cloning of the integration and attachment regions of bacteriophage P4; Pierson LS 3rd et al.; The integration and attachment regions of bacteriophage P4 have been cloned into a multicopy plasmid . This plasmid can integrate into the E . coli chromosome at the same location as the parent phage . Integration increases the stability of the plasmid and allows it to be retained even under conditions in which a non-integrated plasmid would be lost . None of the genes needed for P4 lytic growth is required for integration . The P4 integration and attachment regions have been cloned on separate plasmids . A plasmid that carries the attachment site can integrate into the chromosome only if another plasmid that carries the P4 integration functions is present . A plasmid that carries only this trans-acting integration function cannot integrate . Using deletion mutants of the plasmid, the maximum size of the region needed for integration has been determined to be 1.6 kb, of which no more than 1.2 kb codes for the integrase protein . A nonsense mutant defective in integration has been isolated by using a rapid screening procedure that identifies unstable plasmids. Mol Gen Genet, 1984, 196(1), 117 - 22 Transposon Tn1 intra-molecular transposition; Bishop R et al.; A system for the direct selection of intra- and inter-molecular transposition events has been used to show that intra-molecular transposition of Tn1 generates deletions and inversions and requires the tnpA but not the tnpR gene product, as predicted by current models of transposition . Intra-molecular Tn1 transposition is much less limited by 'transposition immunity' than inter-molecular transposition, and occurs at frequencies comparable to those for inter-molecular transposition . The selection system, which uses the bacteriophage lambda cI- PR region as a target can be used to select, quantify, and characterize any spontaneous or induced mutations. J Mol Appl Genet, 1984, 2(5), 436 - 46 Isolation and initial characterization of the alcohol dehydrogenase gene from Drosophila affinidisjuncta; Brennan MD et al.; Recombinant bacteriophages containing the alcohol dehydrogenase (ADH) gene from Drosophila affinidisjuncta have been isolated by virtue of their cross-hybridization to the previously cloned ADH gene from D . melanogaster . Within the 17 kilobases of cloned DNA represented in the phage genomes, the sequences hybridizing to the D . melanogaster ADH gene lie roughly in the center . The only region of detectable hybridization to cDNA made from templates of D . affinidisjuncta larval poly(A)-containing RNA maps to the same portion of the cloned DNA . Verification that the phages carry the ADH structural gene was obtained by hybrid-selecting ADH mRNA, translating it in vitro, and immunoprecipitating the resulting ADH polypeptide . Analysis of genomic DNA suggests that the ADH gene and most flanking sequences are present only once in the haploid genome . However, 3' to the ADH gene, two separable repetitive elements are found . Both repetitive elements are probably small and poorly conserved in the genome, and neither interferes with localization of the ADH gene, by in situ hybridization, to a position near the base of the third chromosome . Analysis of ADH transcripts demonstrates that there are at least four RNAs produced by the ADH gene . Two size classes of RNA are seen at each stage of development . In addition, ADH transcripts from larvae and adults differ from one another in a reproducible manner. Mol Gen Genet, 1984, 195(3), 541 - 3 Isolation and some properties of bacteriophage alpha3 gene J mutant; Kodaira K et al.; To elucidate the in vivo function of the J gene of microvirid (isometric) phages, we isolated several strains carrying a double mutation in J and H genes from phage alpha3 and then constructed single mutants each having an amber codon in the J gene . The J mutants could not multiply in suppressor-less hosts and were deficient in single-stranded progeny DNA synthesis . Nucleotide sequences of the wild-type and mutant alpha# J genes were analyzed to determine the mutation sites . The amino acid sequence of the J gene was also deduced from the nucleotide sequence and compared with those of phiX174 and G4. Biol Cell, 1984, 51(3), 389 - 94 Immunolabelling of bacteriophage lambda receptor protein (LamB) on thin sections of E . coli embedded in Lowicryl; Whitehouse RL et al.; LamB is one of the major cellular proteins when E . coli is grown in the presence of maltose and is localized in the outer membrane . Previous immunolabellings obtained with monoclonal antibodies showed that this protein is a transmembrane protein and led to the detection of 4 epitopes exposed on the cell surface and 2 located on the inner surface of the outer membrane (Scheckman et al., 1983) . In the present study, we have used this biological model in order to see whether these two classes of epitopes could be distinguished by immunocytochemical labelling performed on thin sections of E . coli embedded in Lowicryl K4M (Carleman et al., 1982) . The optimal conditions of fixation and embedding were first established for labelling with poly- or monoclonal antibodies detected by Protein A-gold complexes . The analysis of gold particle distribution on each side of the outer membrane after labelling with a polyclonal serum or after its adsorption on intact bacteria allowed us to conclude that the resolution of immunolabelling on thin sections was about 20 nm . The use monoclonal antibodies met with difficulties due mostly to the nonspecific labelling of the cytoplasm . Although this nonospecific labelling was decreased by fixing bacteria with paraformaldehyde alone, only one antibody gave a correct specific labelling after high dilution (1/3000) . The gold particle distribution obtained with this antibody confirmed the location on the cell surface of this epitope. J Mol Biol, 1983 Dec 25, 171(4), 577 - 80 Packaging of DNA into bacteriophage heads: a model; Harrison SC; A model is suggested for the geometry of DNA entry into a bacteriophage head . It accounts for recent observations indicating absence of a unique, ordered sequence of windings in the packaged DNA. J Mol Biol, 1983 Dec 25, 171(4), 401 - 18 Domain structure and quaternary organization of the bacteriophage P22 Erf protein; Poteete AR et al.; The structure and activities of the recombination-promoting P22 Erf protein were examined in vitro . Treatment of the protein with elastase produces a stable amino-terminal fragment, consisting of amino acid residues 1 to (approximately) 136 . We have purified this fragment, designated fragment B, to apparent homogeneity by gel filtration chromatography . Fragment B retains the oligomeric structure and single-stranded DNA binding specificity of intact Erf . It differs, however, in lacking the ability of intact Erf to bind single-stranded DNA into large aggregates following mild heat treatment of the protein . In addition, its binding to DNA may be weaker than that of intact Erf . Intact Erf sediments through a sucrose gradient as a discrete species with an apparent S20,w of approximately 11 X 7 S . Its sedimentation behavior is affected little, if at all, by concentration . Fragment B also sediments as a discrete species at approximately 10 X 4 S . In the electron microscope, intact Erf appears as rings, with 10 to 14 small projecting structures resembling the teeth of a gear . Fragment B is similar, except that it appears to lack the peripheral structures . From these observations, we conclude that Erf consists of at least two structurally and functionally distinct domains, and that it has a discrete ring-like oligomeric structure. J Mol Biol, 1983 Dec 25, 171(4), 383 - 99 Computer simulation of ribosome editing; Menninger JR; A stochastic model of protein synthesis was modified by including the process of dissociating peptidyl-tRNA from ribosomes . To simulate ribosome editing, the probability of dissociation was assumed to be high if the peptidyl-tRNA was erroneous; that is, if it resulted from transfer of a peptide to an aminoacyl-tRNA that was inappropriate relative to the mRNA codon . The effects of amino acid starvation on protein synthesis were simulated both by increasing the probability of such erring at and by reducing the conditional probability of elongation at "hungry" codons, those whose correct amino acid was in short supply . These probabilities were varied systematically to simulate tryptophan limitation during synthesis of coat protein from bacteriophage MS2 . Significant reduction, during starvation, in the synthesis of complete coat protein required large reductions in the probability of elongation at hungry codons but only small increases in the probability of erring . Enhanced dissociation of peptidyl-tRNA during starvation, followed rapidly by dissociation of ribosomes from mRNA, led to reductions in mean polysome size, a result that had been interpreted by others as due to some effect of starvation on the initiation of protein synthesis . Results from experiments by Goldman (1982) on the cell-free synthesis of MS2 coat protein during tryptophan starvation could be mimicked in detail by the computer simulations . A simple competition between correct and erroneous amino acids was sufficient to explain the tryptophan dependence of complete coat protein and internal peptide syntheses . Values for the Michaelis constants were derived from the computer simulations. J Biol Chem, 1983 Dec 25, 258(24), 15206 - 13 Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI; Bodnar JW et al.; The cleavage of specific DNA sequences by the restriction endonucleases AluI, DdeI, HinfI, RsaI, and TaqI has been studied by monitoring the effect of various nucleotide modifications on the rate of DNA digestion . Bacteriophage fd DNA was completely substituted in one strand with a single nucleotide analog, using an in vitro primed DNA synthesis reaction on a single-stranded viral DNA template . Twelve deoxynucleotide analogs were incorporated into these DNA substrates: 2-aminopurine, 2,6-diaminopurine, deoxytubercidin, deoxyuridine, 5-bromodeoxyuridine, 5-allylamine deoxyuridine, 5-biotinyl deoxyuridine, deoxypseudouridine, deoxyinosine, 8-azadeoxyguanosine, 5-iododeoxycytidine, and 5-bromodeoxycytidine . The restriction enzymes tested varied considerably in their ability to digest hemi-substituted DNAs containing these modified nucleotides . Structural alterations in the base pairs immediately adjacent to the phosphodiester bonds cleaved by the enzyme reduced the rate of enzyme activity most dramatically, and in most cases more than a single determinant on each base pair altered activity . Interactions with nucleotides outside the recognition site seem to have little importance in the binding or catalytic activity of these enzymes. J Theor Biol, 1983 Dec 21, 105(4), 631 - 45 A kinetic model for virus binding which involves release of cell-bound virus-receptor complexes; Incardona NL; A general kinetic mechanism is presented for reversible binding of viruses to cells followed by an irreversible step that initiates the delivery of the viral genome . A novel feature is additional pathways for the release of both virus-occupied and unoccupied receptors from cells . Due to one simplifying assumption, it does not apply at low receptor densities . However, it is sufficiently general to be applicable to ligand binding and internalization for those systems in which ligand diffusion is rate limiting . Three different versions of the model fit the usual kinetic data for the binding of an eclipse mutant of bacteriophage phi X174 to Escherichia coli . However, in each case binding to cell-bound receptors is irreversible . Therefore, this explains the apparent failure of this system to obey the Law of Mass Action . One version of the model also predicts that the release rate of lipopolysaccharide receptors from the outer membrane may be significantly lowered when virus is bound to these receptors. J Mol Biol, 1983 Dec 15, 171(3), 297 - 317 Structure and inherent properties of the bacteriophage lambda head shell . IV . Small-head mutants; Katsura I; Missense mutants of bacteriophage lambda that produce small proheads were found among prophage mutants defective in the major head protein gpE . Measurements of the sedimentation coefficient and molecular weight of the small proheads showed that they have the T = 4 structure composed of 240 molecules of gpE instead of the wild-type T = 7 structure composed of 420 molecules of gpE . When the phage mutants were grown in groE mutants of Escherichia coli, they produced small unprocessed proheads, which contained a smaller number (about 60) of the core protein (gpNu3) molecules than normal unprocessed proheads, which contain about 180 molecules of gpNu3 . This shows that the major head protein determines the size of not only the shell but also the core of unprocessed proheads . These mutants by themselves produce very few mature small-headed phage particles, partly because the lambda DNA molecule, whose cos sites are separated at a distance of 48,500 bases, is too long to be packaged into the small proheads . However, the small proheads can package shorter DNA in vivo and in vitro at somewhat reduced efficiency, if the length or a multiple of the length between the cos sites of the DNA is 13,000 to 19,000 bases. J Biol Chem, 1983 Dec 10, 258(23), 14619 - 25 The bacteriophage lambda terminase . Partial purification and preliminary characterization of properties; Gold M et al.; The maturation of bacteriophage lambda DNA and its packaging into preformed heads to produce infectious phage is under the control of the two leftmost genes on the lambda chromosome, i.e., Nu1 and A . Based on its ability to complement lambda A- phage-infected cell extracts for packaging of lambda DNA in vitro, a single protein, designated terminase (ter) has been extensively purified using adsorption, ion exchange, and affinity column chromatography . The final preparation represents an approximately 60,000-fold purification over the activity found in crude extracts and is about 30 to 80% homogeneous as judged by visualizing the protein after electrophoresis in sodium dodecyl sulfate-polyacrylamide gel . In addition to packaging, terminase can also catalyze the endonucleolytic cleavage of lambda cohesive-end site DNA; both of these reactions require ATP . In some preparations, certain terminase fractions of extreme purity require protein factors present in extracts of uninfected Escherichia coli in order to catalyze the cohesive-end site cleavage reaction . On ion exchange columns purified terminase co-chromatographs with a DNA-dependent ATPase activity, hydrolyzing ATP to ADP and Pi in the presence of any of several types of DNA tested including those of non-lambda origin . The molecular weight of the native enzyme is 117,000 and appears to be a hetero-oligomer composed of 2 nonidentical subunits . The most likely composition of terminase is one gpA (gene product of A), Mr = 74,000 and two gpNu1, Mr = 21,000. Biochemistry, 1983 Dec 6, 22(25), 5869 - 78 Mechanism and role of cooperative binding of bacteriophage fd gene 5 protein to single-stranded deoxyribonucleic acid; Shimamoto N et al.; The highly cooperative binding of fd gene 5 to single-stranded DNA was studied kinetically by rapid photo-cross-linking and stopped-flow UV absorption measurements . The observed change in absorbance was shown to be due to the binding by direct evidence of rapid photo-cross-linking of the bound proteins to fd DNA . The bimolecular rate constant obtained for the association was 1.6 X 10(10) M-1 s-1 (in terms of the molecular concentration of DNA), which was concluded to be diffusion controlled . The breakdown of cluster complexes on fd DNA was induced by the addition of large excess amounts of short single-stranded DNA . The breakdown took place in about 1 s . The kinetic process of redistribution of dissociated proteins was monitored by rapid photo-cross-linking and subsequent electrophoresis of the cross-linked complex . The dissociated proteins first formed isolated complexes, but later they were again converted into the cluster . The kinetic results showed that the cooperativity originated from the stabilization of the protein-DNA complex by the cluster formation, not from the accelerated association in the cluster formation . This kind of cooperative binding was shown to perform negative feedback control in the cluster formation . On the basis of the kinetic results obtained, we proposed a model for the regulatory role of the fd gene 5 protein in the synthesis of single-stranded fd DNA. J Mol Biol, 1983 Dec 5, 171(2), 229 - 32 Pf1 bacteriophage replication--assembly complex . X-ray fibre diffraction of the high humidity form; Kneale GG et al.; The helical intracellular nucleoprotein complex of Pf1 bacteriophage has been studied by X-ray fibre diffraction in various hydration states . The helix pitch changes from 44 A in dry fibres to 55 A in wet fibres, whereas the unit rise between subunits in the helix apparently does not change with humidity . This result indicates that the nucleoprotein assembly twists more readily than it stretches . This is consistent with its biological role of tightening the viral DNA into a more compact form for packaging in the virion. J Virol, 1983 Dec, 48(3), 770 - 3 Molecular cloning of the c-fms locus and its assignment to human chromosome 5; Roussel MF et al.; Molecular clones of the retroviral oncogene v-fms were used to isolate recombinant bacteriophages containing c-fms proto-oncogene sequences from a human placental DNA library . Viral and cellular fms sequences were used in Southern blotting experiments with a panel of 32 human X mouse somatic cell hybrids to assign the human c-fms proto-oncogene to human chromosome 5. J Biomol Struct Dyn, 1983 Dec, 1(3), 743 - 53 A proposal for a specific double-helical structure in which the polynucleotide strands intercalate instead of forming base-pairs; Viswamitra MA et al.; Double helices, since the discovery of the DNA structure by Watson and Crick, represent the single most important secondary structural form of nucleic acids . The secondary structures of a variety of polynucleotide helices have now been well characterised with hydrogen-bonded base-pairs as building blocks . We wish to propose here the possibility, in a specific case, of a double stranded helical structure without any base-pair, but having a repeat unit of two nucleotides with their bases stacked through intercalation . The proposal comes from the initial models we have built for poly(dC) using the stacking patterns found in the crystal structures of 5'-dCMPNa2 which crystallises in two forms depending on the degree of hydration . These structures have pairs of nucleotides with the cytosine rings partially overlapping and separated by 3.3A . Using these as repeat units one could generate a model for poly(dC) with parallel strands, having a turn angle of 30 degrees and a base separation of 6.6A along each strand . Both right and left handed models with these parameters can be built in a smooth fashion without any obviously unreasonable stereochemical contacts . The helix diameter is about 13.5A, much smaller than that of normal helices with base-pair repeats . The changes in the sugar-phosphate backbone conformation in the present models compared to normal duplexes only reflect the torsional flexibility available for extension of polynucleotide chains as manifested by the crystal structures of drug-inserted oligonucleotide complexes . Intercalation proposed here could have some structural relevance elsewhere, for instance to the base-mismatched regions on the double helix and the packing of noncomplementary single strands as found in the filamentous bacteriophage Pf1. J Biomol Struct Dyn, 1983 Dec, 1(3), 611 - 20 Natural occurrence of left-handed (Z) regions in PM2 DNA; Miller FD et al.; Bacteriophage PM2 DNA, a ccc genome of high apparent superhelical density, contains left-handed (Z) regions as detected by competitive radioimmunoassay, agarose gel electrophoresis of DNA: antibody complexes and immunoelectron microscopy . The latter technique, in conjunction with partial blockage of restriction endonuclease sites by bound antibody, was used to map the left-handed regions along the DNA molecule . A cluster of four to five antibody molecules (approximately 25% of bound antibody) was located within map units 0.05-0.18 of the single Hpa II restriction site . Sequence analysis of part of this region showed the presence of several areas of high alternating purine-pyrimidine content . A strong correlation is observed between alternating pyrimidine-purine tracts of significant length and antibody binding sites. Proc Natl Acad Sci U S A, 1983 Dec, 80(24), 7400 - 4 Purification and characterization of the unusual deoxynucleoside, alpha-N-(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)glycinamide, specified by the phage Mu modification function; Swinton D et al.; Bacteriophage Mu encodes a protein that modifies approximately equal to 15% of DNA adenine residues to a new and unusual form . Modified DNA was enzymatically digested to deoxynucleosides, and the products were fractionated by HPLC . A modified adenine nucleoside, designated dA'x, was purified and its molecular structure was established by mass spectrometry . We show that dA'x is alpha-N-(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)-glycinamide . The dA'x obtained from DNA was indistinguishable from the synthetic product with respect to its chromatographic behavior (HPLC and gas chromatography) and mass spectrum . Acid hydrolysis degrades dA'x to produce N6-carboxymethyladenine; this compound corresponds to the base Ax observed in earlier studies. J Virol, 1983 Dec, 48(3), 616 - 26 An Escherichia coli gene required for bacteriophage P2-lambda interference; Ghisotti D et al.; The gene old of bacteriophage P2 is known to (i) cause interference with phage lambda growth; (ii) kill recB- mutants of Escherichia coli after P2 infection; and (iii) determine increased sensitivity of P2 lysogenic cells to X-ray irradiation . In all of these phenomena, inhibition of protein synthesis occurs . We have isolated bacterial mutants, named pin (P2 interference), able to suppress all of the above-mentioned phenomena caused by the old+ gene product and the concurrent protein synthesis inhibition . Pin mutations are recessive, map at 12 min on the E . coli map, and identify a new gene . Satellite bacteriophage P4 does not plate on pin-3 mutant strains and causes cell lethality and protein synthesis inhibition in such mutants . P4 mutants able to grow on pin-3 strains have been isolated. Proc Natl Acad Sci U S A, 1983 Dec, 80(24), 7456 - 60 DNA sequences necessary for packaging of bacteriophage lambda DNA; Hohn B; The extent of DNA flanking the "cohered cohesive end" site of bacteriophage lambda DNA, which is required for packaging, was determined by using defined DNA fragments and a cosmid in vivo packaging assay . From the right end of lambda DNA a 20- to 36-base-pair stretch extending from the center of the cohered cohesive ends is shown to be required, whereas the packaging efficiency of cosmids extending to 70 base pairs into the left lambda arm is reduced to 10% (compared to a fragment extending until about 80 base pairs) . A 60-base-pair stretch of the left arm leaves an efficiency of only 1% . The segment thus delineated, by the nature of the assay, is both necessary and sufficient for the binding of packaging proteins to the DNA, the packaging of DNA itself, the DNA cleavage, and successful injection of the DNA into a bacterial host . By contrast, in vitro packaging of restriction fragments of mature lambda DNA directly demonstrated the selectivity of the packaging proteins for the fragment originating from the left end of the DNA . The results of the two complementary experiments are discussed in terms of the various steps before, during, and after packaging for which different sequences flanking and including the cohered cohesive ends might be required. Proc Natl Acad Sci U S A, 1983 Dec, 80(24), 7385 - 9 Structural organization and expression of human DNA sequences related to the transforming gene of avian myeloblastosis virus; Franchini G et al.; Bacteriophage libraries of human DNA were screened for sequences homologous to the transforming gene (v-myb) of avian myeloblastosis virus . The three overlapping clones isolated were shown to contain a total of 1.0 kilobase pair (kbp) of sequence related to v-myb distributed over 6.2 kbp . Restriction enzyme mapping and heteroduplex analysis revealed the presence of five myb-related domains interrupted by four stretches of non-homology . To study the extent of human DNA coding sequences that constitute the myb gene homologue, c-myb (human), probes spanning about 30 kbp were prepared from the clones and used to study transcription in a human hematopoietic cell line (MOLT-4) . Each of the probes hybridized a 4.5-kilobase transcript, which suggests that either the c-myb (human) gene encompasses 30 kbp or it contains two or more transcription units that each give rise to a mRNA of 4.5 kilobases. Gene, 1983 Dec, 26(2-3), 301 - 2 Location of the abi, col and imm genes on pHU011, a colicin Ib plasmid derivative; Gottlieb JH et al.; A cleavage site map of pHU011, a derivative of the colicin Ib plasmid containing the complete SalI-B fragment ligated to pBR322, has been determined . Sites of cleavage by PstI were determined using the Smith and Birnstiel {Nucl . Acids Res . 3 (1976) 2387-2398} method of mapping, whereas those for XbaI, XhoI, and HindIII were determined by double digestions or digestion of isolated fragments . In addition, the sites of the abi gene, which causes the abortive infection by T5 bacteriophage, and of the colicin (col) gene have been determined . The results indicate that these genes are not contiguous. Gene, 1983 Dec, 26(2-3), 159 - 63 Location of the Rz gene in bacteriophage lambda; Taylor A et al.; We report the nucleotide sequence of the Tn903 insertion in phage lambda dk23 identifying the Rz gene . The insertion is located after bp 46008 in the lambda nucleotide sequence . This is 43 bp after the start of the Rz gene, which is 459 bp long . Complementation experiments verify that the lysis defect in the lambda Rz::Tn903 mutant lies on a restriction fragment spanning the lysis region. Gene, 1983 Dec, 26(1), 91 - 9 New versatile cloning and sequencing vectors based on bacteriophage M13; Kieny MP et al.; A new pair of cloning and sequencing vectors based on bacteriophage M13mp7 has been developed . These vectors (M13tg130 and M13tg131) contain, in addition to the EcoRI, BamHI, HindIII, SmaI, SalI and PstI sites present in other vectors {cf., M13mp8 and M13mp9, Messing and Vieira, Gene 19 (1982) 269-276}, unique restriction recognition sequences for the enzymes EcoRV, KpnI, SphI, SstI and XbaI . A restriction site for the enzyme BglII has been incorporated into the polylinker region of one of the vector pair to permit rapid discrimination between the two vectors. Radiat Res, 1983 Dec, 96(3), 462 - 75 Contributions of various types of damage to inactivation of T4 bacteriophage by protons; Gialanella G et al.; Mean doses for damage induced by 3.7-MeV protons in T4 phage were measured for the following effects: inactivation, killing, adsorption, DNA injection, capsid rupture with DNA release, and single- and double-strand DNA breaks . These effects have been related to phage survival in the same experiment because of the variability inherent in such measurements . The experiments were carried out in nutrient broth, phosphate buffer, and phosphate buffer plus histidine as suspension media . The following conclusions can be drawn: (i) DNA double-strand breakage is the dominant cause of inactivation in nutrient broth; (ii) scavengers protect the DNA inside the capsid to only a small degree; (iii) indirect actions affect functions associated with proteins; (iv) DNA release, as measured by capsid rupture, accounts for only a small percentage of the loss of viability; (v) essentially all DNA from adsorbed phage is injected even though a large proportion of the DNA contains double-strand breaks. Eur J Biochem, 1983 Dec 1, 137(1-2), 119 - 24 On the cytotoxicity of vitamin C and metal ions . A site-specific Fenton mechanism; Samuni A et al.; The toxicity of ascorbate towards phage lambda and the phages T2-T7 has been investigated . At room temperature the T-odd and lambda bacteriophages are highly susceptible to ascorbate-induced damage, whereas the T-even phages are practically resistant . The toxicity of ascorbate is dependent on the presence of copper (or iron) and oxygen, although oxygen is not required in the presence of H2O2 . Hydrogen peroxide is essential for the ascorbate-induced phage inactivation and the damage is prevented by catalase . At the concentrations used, most of the copper ions are bound to the phage particles . Chelating agents such as EDTA or histidine fully protect the phages, whereas salicylate only reduces the rate of phage inactivation . OH scavengers such as sucrose, formate, mannitol, tert-butyl alcohol or poly(ethylene glycol) have no protective effect . Experiments with DNA labeled phages indicate that both phage adsorption and DNA injection are impaired as a result of the exposure to ascorbate and copper . The failure to express the viral genetic information as a result of single and double-strand breaks in the DNA, probably also contribute to the loss of the plaque-forming ability of the phages . The results are interpreted in terms of a 'site-specific' Fenton mechanism according to which the binding of the transition metal ions to the biological target is a prerequisite for the production of damage . The bound metal ion is reduced either by O(2), ascorbate or other reductants and is subsequently reoxidized by H2O2 yielding OH . radicals . This cyclic redox reaction of the metal generates OH . radicals which react with vital macromolecules with a high probability of causing 'multi-hit' damage . This 'site-specific' formation of OH . radicals, which takes place near the target molecules, accounts both for the high damaging efficiency and for the failure of OH . scavengers to protect against it. Chem Biol Interact, 1983 Dec, 47(3), 293 - 305 Antiinflammatory drugs: protection of a bacterial virus as an in vitro biological measure of free radical activity; Hiller KO et al.; The effects of the hydroxyl free radical (OH), the superoxide free radical (O2-) and the trichloromethyl peroxy free radical (CC13O2) on the survival of bacteriophage T2 have been studied in the absence and presence of several non-steroidal anti-inflammatory drugs (NSAID) . The trichloromethylperoxy radical derived from carbon tetrachloride is considerably more effective than the hydroxyl radical in inactivating the virus: the superoxide radical has only a minor inactivating effect . All the NSAID investigated (flurbiprofen, ibuprofen, sulindac, piroxicam, benoxaprofen, mefenamic acid, diflunisal, aspirin, D-penicillamine, indomethacin and metiazinic acid) inhibit inactivation by OH . This is in agreement with the high rate constants of reaction with this radical determined using the fast reaction technique of pulse radiolysis, i.e . (k greater than 10(9) M-1 S-1) . The sulphur-containing drugs, metiazinic acid, piroxicam, penicillamine and sulindac as well as the indole derivative indomethacin, protect the virus from inactivation by the model peroxy radical CC13O2 (the dose modifying factor, DMF greater than 20) . In contrast, acetylsalicylic acid related drugs, such as diflunisal, the anthranilic acid derivative, mefenamic acid, and some phenylpropionic acid derivatives, such as flurbiprofen, exhibit only a very small or no protective effect (DMF less than 2) . As with OH, the ability of the drugs to protect the virus from inactivation by the peroxy radical is in agreement with their corresponding rate constants of reaction determined by pulse radiolysis. Cell, 1983 Dec, 35(3 Pt 2), 795 - 803 The mechanism of phage lambda site-specific recombination: site-specific breakage of DNA by Int topoisomerase; Craig NL et al.; We demonstrate that the topoisomerase activity of bacteriophage lambda Int protein introduces single-strand breaks into duplex DNA at specific sites . Strand breakage is accompanied by the covalent linkage of Int to DNA . The linkage connects a residue in Int to the 3' phosphate of DNA at the site of breakage; the other breakage product has a 5' OH terminus . Int is the first procaryotic topoisomerase shown to break DNA in this manner . We find that in att sites, Int breaks DNA within the 15 bp homologous core . These sites of Int topoisomerase action result from the interaction of Int with "junction-type" recognition sequences (CAACTTNNT), and Int topoisomerase acts between the 7th and 8th bases of this sequence . The sites of breakage within the cores of attP and attB coincide exactly with positions where breakage and reunion occur during Int-dependent recombination . These results indicate that Int topoisomerase executes strand exchange during recombination. Cell, 1983 Dec, 35(3 Pt 2), 785 - 94 In vitro transposition of bacteriophage Mu: a biochemical approach to a novel replication reaction; Mizuuchi K; The transposition-replication reaction of phage Mu has been reproduced in a cell-free reaction system . Two assay methods were used for the detection of transposition products . The first method uses lambda DNA as the target of transposition and a plasmid containing the ends of Mu DNA and an ampicillin-resistance gene as the donor; after the reaction, in vitro lambda packaging allows the scoring of ampr transducing phages generated by transposition . In the second method, the products made in the presence of a radioactive precursor for DNA synthesis are directly analyzed by gel electrophoresis and unique product species are identified . The reaction requires a donor DNA carrying the two Mu ends in their proper relative orientation, extracts containing the A and B gene products of Mu, and host factor(s) . RNA synthesis by E . coli RNA polymerase is not required for the reaction . The products include both cointegrates and simple inserts . Both types of products show incorporation of radioactive DNA precursors; however, simple inserts do not seem to undergo a full round of DNA replication. Proc Natl Acad Sci U S A, 1983 Dec, 80(23), 7284 - 8 Site-specific recombination of yeast 2-micron DNA in vitro; Vetter D et al.; Most strains of the yeast Saccharomyces cerevisiae harbor several copies of a 2-micron plasmid circle DNA termed "2 micron." This circular plasmid contains two 599-base-pair precise inverted repeats across which a site-specific inversion event occurs in vivo . This inversion is promoted by a plasmid-encoded function called "FLP." We have cloned the FLP gene of 2-micron DNA under control of a strong yeast promoter and transformed yeast cells with a plasmid containing the cloned FLP gene . Cell-free extracts from such a transformant promote highly efficient inversion of 2-micron DNA in vitro . The reaction requires a cation and works efficiently on supercoiled, relaxed circular, or linear DNA . The FLP activity bears certain similarities to the cre protein, a site-specific recombinase encoded by bacteriophage P1. J Virol, 1983 Dec, 48(3), 647 - 53 In vitro recombination of bacteriophage T7 DNA detected by a direct physical assay; Lee D et al.; We developed a simple, direct, physical assay to detect genetic recombination of bacteriophage T7 DNA in vitro . In this assay two mature T7 DNA molecules, each having a unique restriction enzyme site, are incubated in the presence of a cell-free extract from T7-infected Escherichia coli cells . After extraction of the DNA, restriction enzyme digestion, and agarose gel electrophoresis, genetic recombination is detected by the appearance of a novel recombinant DNA band . Recombination frequencies as high as 13% have been observed . We used this assay to determine the genetic requirements for in vitro recombination . In agreement with results obtained previously with a biological assay, T7 recombination in vitro appears to proceed via two distinct pathways. J Virol, 1983 Dec, 48(3), 573 - 9 Molecular cloning of the Aleutian disease virus genome: expression of Aleutian disease virus antigens by a recombinant plasmid; Mayer LW et al.; Three nonoverlapping segments representing approximately 80% of the 4.8-kilobase pair Aleutian disease virus (ADV-G) duplex genome were molecularly cloned into either bacteriophage M13mp9 (M13bm2 = 0.07 to 0.15 map unit; M13bm1 = 0.15 to 0.54 map unit) or plasmid pUC8 (pBM1 = 0.54 to 0.88 map units) . In addition the 0.54- to 0.88-map unit segment of a Danish isolate of ADV (DK ADV) was also cloned into pUC8 (pBM2) . The recombinant plasmids pBM1 and pBM2 induced expression of several polypeptides in Escherichia coli JM103 that were specifically recognized by sera from mink infected with ADV . The same three proteins with approximate molecular weights of 55,000, 34,000, and 27,000 were detected both by immune blotting and by immunoprecipitation of {35S}methionine-labeled JM103 (pBM1) . None of these proteins were recognized in JM103 or JM103 (pUC8), nor were they detected by sera from normal mink . Purified pBM1 and pBM2 DNA appeared identical in size by gel analysis and contour length measurement, and electron microscopic heteroduplex mapping revealed no visible areas of heterology . However, restriction endonuclease mapping showed that pBM2 was different from pBM1, indicating that this segment of the ADV genome was similar but not identical for two strains of ADV (ADV-G and DK ADV) . Furthermore, when cloned DNA from ADV-G was labeled with {32P}dCTP by nick translation, DNA relatedness to several field strains of ADV (Utah I, Pullman, and DK), but not to mink enteritis virus or cellular DNA, was shown by Sou