Mol Gen Mikrobiol Virusol, 1988 Aug, (8), 12 - 7 {Transactivation of p'R promoter of phage lambda}; Troianovskaia IN et al.; The host-vector system for efficient expression of the cloned genes under the control of transactivated promoter p'R of bacteriophage lambda has been elaborated . The Q protein activating p'R promoter is coded by the defective prophage constructed in vitro by means of excision of the late phage genes between the distant sites of the restriction endonuclease MluI and change of the central SalI fragment carrying the kill gene for the kanamycin resistance gene . The general recombination system is impaired during the change, thus the bacteriophage DNA can be obtained from the induced RecA cells as a plasmid DNA . The induction of the prophage results in a sharp increase of beta-lactamase synthesis (30% of soluble cell protein) under the control of p'R promoter in a plasmid derived of pBR322. Mol Gen Genet, 1988 Aug, 213(2-3), 325 - 31 In vivo effect of DNA repair on the transition frequency produced from a single O6-methyl- or O6-n-butyl-guanine in a T:G base pair; Chambers RW et al.; We have previously reported some effects of DNA repair on the transition frequencies produced by an O6-methyl-guanine (MeG) or an O6-n-butyl-guanine (BuG) paired with C at the first position of the third codon in gene G of bacteriophage phi X174 form I' DNA (Chambers et al . 1985) . We now report experiments in which the transition is produced from T:MeG or T:BuG, instead of C:MeG or C:BuG, located at this site . The site-modified DNAs were transfected into cells with normal DNA repair as well as into cells with repair defects (uvrA, uvrB, uvrC, recA, uvrArecA) . The lysates were screened for phage carrying the expected transition using a characteristic change in phenotype . The data demonstrate that the transition frequency from T:BuG is low (0.3% of total phage progeny) in cells with normal repair (Escherichia coli AB1157) and increases 7-fold in uvrA cells (E . coli AB1886) . A similar increase is seen in uvrB and uvrC cells (AB1885, AB1884) . These data, like our previous data, indicate BuG is repaired primarily by excision . In contrast to this, the transition frequency from T:MeG is high (5 +/- 2%) in cells with normal repair . After induction of alkyl transfer repair in E . coli AB1157, the transition frequency goes up 5-fold . Compared with cells with normal repair, the transition frequency goes up 2-fold in uvrA, uvrB and uvrC cells; it goes up 1.5-fold in recA cells (E . coli AB2463) . The data reinforce our earlier conclusion that MeG is repaired primarily by alkyl transfer, but the ABC excinuclease as well as RecA protein inhibit this repair process.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1988 Aug, 85(15), 5439 - 43 Translation initiation controls the relative rates of expression of the bacteriophage lambda late genes; Sampson LL et al.; The late operon of bacteriophage lambda contains the genes encoding the morphogenetic proteins of the phage . These genes are transcribed equally from the single late promoter . Although the functional half-lives of the mRNA for the various genes of this operon vary less than 2-fold, their relative rates of expression have been shown to vary by nearly 1000-fold . This variation could result from differing rates of translation initiation, from overlapping upstream translation, or from differential elongation rates due to the presence of codons for which the corresponding tRNAs are rare . To distinguish between these possibilities, we have cloned sequences surrounding the initiator codons of several of these genes and measured their ability to drive synthesis of hybrid lambda-beta-galactosidase proteins . The rates of expression of the hybrid genes thus produced correlate very well with the natural rates of expression of the corresponding phage genes, suggesting that the rate of initiation of translation controls the relative expression rates of these genes. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Aug, 269(2), 147 - 55 Bacteriophages of Bordetella sp.: features of lysogeny and conversion; Holzmayer TA et al.; It has been the purpose of this paper to study molecular-biological features of the Bordetella bacteriophage interaction with the host cell during lysogeny and conversion as well as to determine the degree of homology between genomes of homologous and heterologous bacteriophages . Genomes of bacteriophages from B . pertussis 134, 41405 and B . bronchiseptica 214 were studied . Heteroduplex and restriction analyses revealed a heterogeneity of bacteriophage populations, and their DNAs were found to differ in size and position of inserts . As shown by blot hybridization, the bacteriophage genome is not inserted into the chromosome of the lysogenic cell but apparently exists as an autonomous plasmid replicon . It has been established that during conversion only a part of the phage genome is inserted into the chromosome of the recipient cell. J Biomol Struct Dyn, 1988 Aug, 6(1), 35 - 49 Resonance Raman spectroscopy of complexes of the helix destabilizing proteins GP32 and GP5 with poly(rA) and poly(dA); Otto C et al.; The bacteriophage T4 helix destabilizing protein (hdp) gp32 and its complexes with poly(rA) and poly(dA) were studied with ultra-violet resonant Raman spectroscopy . The UV-resonant Raman (UV-RR) spectrum of the complex of gp5, the coat protein of bacteriophage M13, with poly(dA) was also measured and is compared with the spectrum of the gp 32/poly(dA) complex . The excitation wavelength was 245.1 nm . This is on the far UV-side of the first absorption bands of adenine and near a "window" in the protein absorption spectrum . The overlap of fluorescence due to chromophores present in the protein and resonance Raman scattering was prevented by this choice of wavelength . The spectra of the protein/polynucleotide complexes are compared with the native nucleotide spectra measured at varying temperatures . The hyperchromicity which is expected when a nucleotide changes from a stacked to an unstacked conformation was not observed for poly(rA), neither upon temperature increase nor on protein binding . In both cases poly(dA) revealed a clear hyperchromicity . This different behavior of poly(rA) and poly(dA) is probably a consequence of their different conformations . The contributions of the proteins to the spectra is weak except for two bands, at 1550 and 1610 cm-1 due to tryptophan (in case of gp32) and one band near 1610 cm-1 due to tyrosine and phenylalanine. Can J Microbiol, 1988 Aug, 34(8), 1022 - 4 Phage f2 desorption from clay in estuarine water using nonionic detergents, beef extract, and chaotropic agents; Armon R et al.; Experimentally adsorbed bacteriophage f2 was eluted from clay particles in estuarine water using 1% Tween, 80.3% beef extract, and 0.3 M NaNO3 with 54% recovery . Replacing sodium nitrate with tetrasodium pyrophosphate (0.4 M) increased the recovery to 81% . Estuarine sediments treated with 1% Tween 80 revealed significantly higher male-specific phage elutions. Mol Gen Mikrobiol Virusol, 1988 Aug, (8), 17 - 23 {SOS-induction of the RP4 plasmid tet-determinant}; Skavronskaia AG et al.; Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested . Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light . The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4 . The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes . Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon . The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell . Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed. J Gen Virol, 1988 Aug, 69 ( Pt 8), 2033 - 42 Gene sequence and mapping data from Marek's disease virus and herpesvirus of turkeys: implications for herpesvirus classification; Buckmaster AE et al.; Purified DNAs from Marek's disease virus (MDV) and the herpesvirus of turkeys (HVT) were randomly sheared and cloned into the M13 bacteriophage . Two-hundred and ten MDV and 130 HVT clones were sequenced to give representative samples of the genome sequences . The predicted amino acid sequences from these gammaherpes-viruses were compared to known sequences from other herpesviruses using computer analysis . Thirty-five MDV and 24 HVT genes were identified by comparison with varicella-zoster virus (VZV), an alphaherpesvirus . However, only 14 MDV and seven HVT genes, giving generally lower homology scores, were found by comparison with Epstein-Barr virus (EBV), a gammaherpesvirus, indicating that MDV and HVT sequences bear greater similarity to VZV than to EBV sequences . A number of sequences were mapped by hybridizing labelled M13 clones to Southern blots of restriction fragments of MDV or HVT DNA . The results were consistent with the MDV and HVT genomes being collinear with VZV. Nucleic Acids Res, 1988 Jul 25, 16(14A), 6385 - 96 Influence of fd gene 2-protein and the viral replication origin on the compatibility of pfd-plasmids; Geider K et al.; Plasmids with the replication origin of bacteriophage fd, the pfd-plasmids, were investigated for compatibility in E . coli cells expressing fd gene 2-protein . This was measured by transformation of Ca-treated cells with and without a residing pfd-plasmid . When the two plasmids contained the complete intergenic region of bacteriophage fd, they were fully compatible in contrast to the situation in which at least one plasmid had a shortened origin for viral strand replication . This incompatibility effect was partially compensated for by a pfd-plasmid with a short origin and with the fd gene 2 . The fd replication origin on a colEl plasmid did not affect compatibility in polA+ cells indicating its idling in the presence of the colEl origin . It can be concluded that a short replication origin requires high amounts of gene 2-protein in contrast to the long origin . Accumulation of replication intermediates severely interferes with host cell metabolism. Nucleic Acids Res, 1988 Jul 25, 16(14A), 6531 - 46 RNA-primed initiation sites of DNA replication in the origin region of bacteriophage lambda genome; Yoda K et al.; Using DNA molecules synthesized in the early stage of lambda phage infection, deoxynucleotides at the transition sites from primer RNA to DNA synthesis have been mapped in the 1.5 kbase area of the lambda phage genome containing the genetically defined replication origin (ori lambda) . Sites in the 1-strand (the polarity of the 1-strand is 5' to 3' from the left to the right direction of the lambda phage genetic map) were distributed both inside and outside of the ori lambda, whereas the sites in the r-strand (the strand in the opposite polarity) were mainly distributed more than three hundred nucleotides apart from the ori lambda to the right . A CPuPu sequence was found at -12 to -10 region of transition sites of the r- and the 1-strands in the frequency of 80% and 70%, respectively, and over 60% of the CPuPu sequences were CAG . Properties of the transition sites are discussed in relation to the primer synthesis. Nucleic Acids Res, 1988 Jul 25, 16(14A), 6353 - 60 Detection of an epitope, not required for polymerization, that is conserved between E.coli DNA polymerases I and III and bacteriophage T4 DNA polymerase; Franden MA et al.; Monoclonal antibodies directed against the alpha subunit of the DNA polymerase III holoenzyme (1) of E . coli were tested for cross-reactivity with a variety of polymerases . We found that one monoclonal antibody bound to E . coli DNA polymerase I as well as to DNA polymerase III . A weaker, but specific, interaction was also detected with T4 DNA polymerase . We exploited the proteolysis procedure developed by Setlow, Brutlag and Kornberg (2) to determine which domain of DNA polymerase I contained the conserved epitope . Contrary to expectations, it was not found in the polymerase domain, but in the 5'----3' exonuclease domain . This reveals a sequence or structure, sufficiently important to be conserved among these polymerases, that is not directly involved in the polymerization reaction. J Mol Biol, 1988 Jul 20, 202(2), 233 - 43 Deletion formation in bacteriophage T4; Singer BS et al.; We have manipulated the dispensable region of the rIIB gene of bacteriophage T4 in order to study the generation of deletions involving direct repeats . We show that recombination between different parental chromosomes is one source of the deletions we have studied . We have also investigated the effects of structure, base composition and distance on deletion formation . We demonstrate that the potential to form structure in single-stranded DNA has variable effects on the frequency of deletion formation and conclude that, in some cases, slipped mispairing during DNA synthesis can make a substantial contribution to deletion frequencies . The G + C richness of the direct repeats involved in deletion formation is an important parameter of the frequency of deletion formation . We have confirmed that increasing the distance between direct repeats decreases deletion frequency. J Biol Chem, 1988 Jul 15, 263(20), 9831 - 9 The effect of the T7 and Escherichia coli DNA-binding proteins at the replication fork of bacteriophage T7; Nakai H et al.; In this paper we compare the effect of single-stranded DNA-binding proteins of bacteriophage T7 (gene 2.5 protein) and of Escherichia coli (SSB) at the T7 replication fork . The T7 gene 4 protein acts processively as helicase to promote leading strand synthesis and distributively as primase to initiate lagging strand synthesis by T7 DNA polymerase . On a nicked double-stranded template, the formation of a replication fork requires partial strand displacement so that gene 4 protein may bind to the displaced strand and unwind the helix catalytically . Both the T7 gene 2.5 protein and E . coli SSB act stoichiometrically to promote this initial strand displacement step . Once initiated, processive leading strand synthesis is not greatly stimulated by the single-stranded DNA-binding proteins . However, the T7 gene 2.5 protein, but not E . coli SSB, increases the frequency of initiation of lagging strand synthesis by greater than 10-fold . The results suggest a specific interaction of the T7 gene 2.5 protein with the T7 replication apparatus. J Biol Chem, 1988 Jul 15, 263(20), 9818 - 30 Leading and lagging strand synthesis at the replication fork of bacteriophage T7 . Distinct properties of T7 gene 4 protein as a helicase and primase; Nakai H et al.; Reactions at the replication fork of bacteriophage T7 have been reconstituted in vitro on a preformed replication fork . A minimum of three proteins is required to catalyze leading and lagging strand synthesis . The T7 gene 4 protein, which exists in two forms of molecular weight 56,000 and 63,000, provides helicase and primase activities . A tight complex of the T7 gene 5 protein and Escherichia coli thioredoxin provides DNA polymerase activity . Gene 4 protein and DNA polymerase catalyze processive leading strand synthesis . Gene 4 protein molecules serving as helicase remain bound to the template as leading strand synthesis proceeds greater than 40 kilobases . Primer synthesis for lagging strand synthesis is catalyzed by additional gene 4 protein molecules that undergo multiple association/dissociation steps to catalyze multiple rounds of primer synthesis . The smaller molecular weight form of gene 4 protein has been purified from an equimolar mixture of both forms . Removal of the large form results in the loss of primase activity but not of helicase activity . Submolar amounts of the large form present in a mixture of both forms are sufficient to restore high specific activity of primase characteristic of an equimolar mixture of both forms . These results suggest that the gene 4 primase is an oligomer which is composed of both molecular weight forms . The large form may be the distributive component of the primase which dissociates from the template after each round of primer synthesis. Biochemistry, 1988 Jul 12, 27(14), 5240 - 5 Thermal denaturation of T4 gene 32 protein: effects of zinc removal and substitution; Keating KM et al.; Gene 32 protein (g32P), the single-stranded (ss) DNA binding protein from bacteriophage T4, is a zinc metalloprotein . The intrinsic zinc is one of the factors required for the protein to bind cooperatively to a ssDNA lattice . We have used differential scanning calorimetry to determine how the thermodynamic parameters characterizing the denaturation of g32P are affected by removal or substitution of the intrinsic zinc . Over a wide concentration range (1-10 mg/mL), the native Zn(II) protein unfolds at a tm of 55 degrees C with an associated mean enthalpy change of 139 kcal mol-1 . Under the same conditions, the metal-free apoprotein denatures over a relatively broader temperature range centered at 49 degrees C, with a mean enthalpy change of 84 kcal mol-1 . Substitution of Zn(II) in g32P by either Cd(II) or Co(II) does not significantly change the enthalpy of denaturation but does affect the thermal stability of the protein . All metallo forms of g32P when bound to poly(dT) undergo highly cooperative denaturational transitions characterized by asymmetric differential scanning calorimetry peaks with increases in tm of 4-5 degrees C compared to the unliganded metalloprotein . Removal of the metal ion from g32P significantly reduces the cooperativity of binding to poly(dT) {Giedroc, D . P., Keating, K . M., Williams, K . R., & Coleman, J . E . (1987) Biochemistry 26, 5251-5259}, and presumably as a consequence of this, apo-g32P shows no change in either the shape or the midpoint of the thermal transition on binding to poly(dT).(ABSTRACT TRUNCATED AT 250 WORDS) Nucleic Acids Res, 1988 Jul 11, 16(13), 6205 - 21 Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP; Inokuchi Y et al.; We report the nucleotide sequence of the Group IV RNA bacteriophage SP . The entire sequence is 4276 nucleotides long . Four cistrons have been identified by comparison with the related Group III phage Q beta . The maturation protein contains 449 amino acids, the coat protein contains 131 amino acids, the read-through protein contains 330 amino acids and the replicase beta-subunit contains 575 amino acids . SP is 59 nucleotides longer than Q beta . We have analyzed both sequence and structural conservation between SP and Q beta and shown that the sequences for the coat and central region of the replicase are strongly conserved between the two genomes . We also show that the S and M replicase binding sites of Q beta are strongly conserved in SP . Interestingly, the base composition of SP and Q beta differ significantly from one another, and most of the differences can be accounted for by a strong preponderance of U in the third position of each codon of Q beta relative to SP . We also compare conserved hairpins associated with potential coat protein and replicase binding sites. Nucleic Acids Res, 1988 Jul 11, 16(13), 5895 - 914 Characterization of the origins of replication of bacteriophage phi 29 DNA; Gutierrez J et al.; The origins of replication of phi 29 DNA have been studied by analyzing the activity as templates in the phi 29 in vitro replication system of E . coli recombinant plasmids and M13 derivatives containing phi 29 DNA terminal sequences . Plasmid pITR, containing the 6 bp long inverted terminal repeat of phi 29 DNA, was shown to be essentially inactive . The analysis of a series of deletion derivatives of plasmid pID13, that contains the 73 and 269 bp from the left and right phi 29 DNA ends, respectively, indicated that the minimal origins of replication are comprised within the mutagenesis at these sequences was carried out . Changes of the second or third A into a C completely abolished the template activity . In the case of changes at position from 4 to 12, only 3 out of 14 mutations reduced the template activity; these 3 mutations were double changes and 2 of them affected the inverted terminal repeat . The results suggest that the sequence requirement at the end-proximal region of the origin of replication is more strict than that at the distal region. Nucleic Acids Res, 1988 Jul 11, 16(13), 5727 - 40 Site-directed mutagenesis in the DNA linking site of bacteriophage phi 29 terminal protein: isolation and characterization of a Ser232----Thr mutant; Garmendia C et al.; By site-directed mutagenesis we have changed the serine residue 232 of the phi 29 terminal protein, involved in the covalent linkage to dAMP for the initiation of replication, into a threonine residue . The mutant terminal protein has been purified to homogeneity and shown to be inactive in the formation of the initiation complex; nevertheless, the mutant protein retains its ability to interact with the phi 29 DNA polymerase and with the DNA . The results obtained indicate a high specificity in the linking site of the terminal protein. J Mol Biol, 1988 Jul 5, 202(1), 59 - 66 Properties of a mutant Cre protein that alters the topological linkage of recombination products; Abremski K et al.; The bacteriophage P1 Cre-loxP site-specific recombination system consists of two components: the Cre recombinase protein and the loxP DNA sequence where recombination takes place . We report here on the analysis of a mutation in the cre structural gene that produces a mutant protein with altered recombination properties . The mutant protein, Cre111, carries out recombination at a much slower rate than the wild-type Cre protein . To determine why the reaction is slow, we have examined a number of activities associated with Cre-mediated recombination . Our results indicate that the binding of Cre111 to the loxP site is comparable to wild-type Cre . Furthermore, the rate at which Cre111 resolves Holliday structures, an intermediate in this recombination reaction, is also comparable to wild-type Cre . Thus, DNA binding and resolution of the intermediate are not affected, suggesting that either synapsis, the process of bringing two lox sites together, or the first strand exchange event, could be affected in the mutant protein . The types of DNA products formed following recombination of a supercoiled substrate can reflect mechanisms of synapsis . Wild-type Cre generates mainly topologically unlinked and unknotted circular products . This suggests that the wild-type protein brings two lox sites together in a way that excludes the entanglement of supercoils present in the substrate DNA . In contrast, when Cre111 recombines a supercoiled molecule it generates many complicated catenanes and knotted DNA products . Presumably, the supercoils present in the DNA substrate are being trapped in the reaction products.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1988 Jul 5, 202(1), 11 - 6 Clone size distributions of mutations induced by ethyl methanesulfonate in bacteriophage T4; Drake JW; Size distributions of mutant clones can reveal important aspects of the mutation process . Previously published data on mutant clones induced by ethyl methanesulfonate (EMS) in bacteriophage T4 generated a distribution that was essentially flat, implying a mutagenic mechanism involving only rare mispairing by reacted bases . Here, methods for estimating the spontaneous component of such a distribution are used to generate a corrected distribution . The corrected distribution is strongly peaked, implying frequent (but not obligatory) mispairing . Frequent mispairing is in accord with current views of the fates of DNA lesions believed to mediate EMS-induced mutagenesis. J Mol Biol, 1988 Jul 5, 202(1), 17 - 34 Estimation of in-vivo miscoding rates; Ripley LS; The replication of premutagenic DNA lesions generates mutant progeny in patterns that distinguish lesions that rarely produce a mutation per DNA replication from those that frequently do so . The quantitative aspects of this distinction were tested in studies of heat-mutagenized bacteriophage T4 . Previous T4 studies had demonstrated that transition mutations produced at G.C base-pairs depended upon heat-induced DNA lesions distinct from those responsible for transversions at G.C pairs . In this study the transversion mutations are shown to arise in patterns predicted for mutations produced from lesions that miscode rarely (fewer than 10% per replication) . In contrast, the transition mutations arise in patterns predicted for mutations produced from lesions that miscode at about 20 to 60% per replication . The fact that the two classes of DNA lesions are distinguishable as predicted by the quantitative model suggests that such studies may in general be useful in quantifying the behavior of mutation-generating DNA lesions . The method employed also estimates the frequency of premutagenic lesions in DNA. J Biol Chem, 1988 Jul 5, 263(19), 9427 - 36 The mechanism of homologous DNA strand exchange catalyzed by the bacteriophage T4 uvsX and gene 32 proteins; Kodadek T et al.; A strand exchange reaction between a single-stranded DNA circle and a homologous linear double-stranded DNA molecule is catalyzed by a mixture of two T4 bacteriophage proteins, the uvsX protein (a DNA-dependent ATPase that resembles the recA protein) and the gene 32 protein (a helix-destabilizing protein) . The products are different from those formed in the corresponding recA protein-catalyzed reaction; rather than producing a linear single strand plus a nicked circular double-stranded (form II) DNA molecule as the final products, interlinked DNA networks are rapidly generated . Electron microscopy reveals that these networks form from multiple pairing reactions that involve the recombination intermediates . Since the uvsX protein is present in substoichiometric quantities, it presumably recycles to catalyze these successive pairing events . Recycling of the uvsX protein has been more directly examined in an assay that monitors the rate of uvsX protein-catalyzed branch migration . The branch migration reaction is rapidly inhibited by dilution of the uvsX protein or by the addition of a heterologous competitor DNA, showing that the uvsX protein-DNA filaments that catalyze strand exchange are dynamic structures . The evidence suggests that individual uvsX protein monomers are continuously entering and leaving the cooperatively formed filament in a cycle that is strongly affected by their ATP hydrolysis. Blood, 1988 Jul, 72(1), 314 - 21 Molecular cloning, expression, and chromosomal localization of a human gene encoding the CD33 myeloid differentiation antigen; Peiper SC et al.; Monoclonal antibodies of the CD33 cluster group recognize a 67-kilodalton (Kd) protein, designated p67, expressed on the surface of normal human myeloid progenitors and leukemic cells from most patients with acute myelogenous leukemia . The human gene encoding p67 was isolated in a mouse genetic background after DNA-mediated gene transfer and fluorescence-activated cell sorting (FACS) for transformants that bound the monoclonal antibody MY9 . After three serial rounds of gene transfer and cell sorting, multiple independently derived tertiary mouse cell transformants were obtained that expressed p67 . Southern blot analysis revealed that these transformants shared restriction fragments containing highly reiterated human DNA sequences . Two shared EcoRI fragments of 3.3-kilobase (kb) and 9.5-kb pairs were molecularly cloned into bacteriophage vectors . A subsegment of the 3.3-kb fragment lacking repeated sequences was then used as a unique sequence probe to isolate two independent cosmid clones . Cells transfected with DNA from both cosmid clones bound MY9, and the human p67 protein was demonstrated by immunoprecipitation . NFS mice inoculated with a mouse cell transformant coexpressing p67 and the v-fms oncogene product produced antisera that specifically immunoprecipitated p67 from human leukemic cell lines, mouse cell transformants, and mouse cells transfected with the biologically active cosmid clones . The human p67 locus was previously assigned to chromosome 19 by screening a panel of rodent X human somatic cell hybrids with the unique sequence probe . The gene was sublocalized to the q13.3 region of chromosome 19 by in situ hybridization . RNA transcripts of approximately 1.6 kb and 1.4 kb were identified in polyadenylated RNA from human myeloid leukemia cell lines using a probe from the genomic locus . Manipulation of the cloned p67 gene may provide insight into the function of its product and mechanisms regulating its expression. Virology, 1988 Jul, 165(1), 317 - 20 Monoclonal antibodies to the major structural proteins of bacteriophage phi 6; Olkkonen VM et al.; A panel of 38 monoclonal antibodies to the five major structural proteins of phi 6 was generated and characterized . The panel includes antibodies recognizing the receptor recognition protein P3, the major hydrophobic envelope protein P9, the nucleocapsid surface protein P8, and the nucleocapsid proteins P1 and P4, which are involved in the viral RNA polymerase activity and form the internal protein skeleton of the nucleocapsid . Six out of the fourteen antibodies to the receptor recognition protein, P3, showed neutralizing activity, interfering with the adsorption of phi 6 to host cells. Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4633 - 7 DNA twisting and the affinity of bacteriophage 434 operator for bacteriophage 434 repressor; Koudelka GB et al.; The affinity of the Escherichia coli phage 434 operator for phage 434 repressor is affected by changes in the sequence of the noncontacted base pairs near the operator's center . The results presented here show that base composition near the center of the operator affects the operator's affinity for repressor by altering the ease with which the operator can be overtwisted into the proper configuration for complex formation . We show that both DNA flexibility and repressor flexibility influence the strength of the repressor-operator interaction: an operator with a single-strand nick at its center has a higher affinity for repressor than does the intact operator: and a repressor bearing a mutation that results in a relaxed dimer interaction is less sensitive than is wild type to changes in the flexibility of the operator . We show that the effect of noncontacted base pairs on operator affinity is independent of the slight overall bend of the operator seen in the repressor-operator complex . Central sequence effects on affinity for repressor are independent of the identity of adjacent base pairs, suggesting that the structure of the individual base pairs, not interactions between them, are responsible for the different torsional rigidities of different operators. Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4610 - 4 One-dimensional diffusion of Escherichia coli DNA-dependent RNA polymerase: a mechanism to facilitate promoter location; Ricchetti M et al.; The mechanism of promoter location by DNA-dependent RNA polymerase of Escherichia coli was investigated . The occupancies of DNA fragments carrying the A1 promoter of bacteriophage T7 were analyzed as a function of the length of flanking sequences adjacent to the promoter . Competition between the promoters on different fragments showed qualitatively that DNA sequences downstream of the promoter enhanced promoter occupancy, whereas upstream flanking sequences had little or no influence on occupancy . This was studied quantitatively by using a set of DNA fragments with four identical A1 promoters (I-IV) equidistant from each other, but with different lengths of flanking sequences upstream from promoter I and downstream from promoter IV . The relative occupancies of these promoters showed that downstream DNA sequences of up to 250 base pairs increased the occupancy of the adjacent promoter, whereas upstream sequences longer than 70 base pairs had little or no effect on occupancy . Promoter occupancies measured as a function of the length of the downstream flanking DNA sequences were fit by a published theory that takes into account an enhancement of signal-sequence location by linear diffusion. J Gen Microbiol, 1988 Jul, 134 ( Pt 7), 1773 - 8 Characterization of SE-3, a virulent bacteriophage of Saccharopolyspora erythraea; Smorawinska M et al.; SE-3 is a virulent bacteriophage isolated from a large-scale culture of Saccharopolyspora erythraea, an erythromycin producer . The host range of the phage is narrow, limited to some strains of this species . Another strain of Sac . erythraea, and a strain of Sac . hirsuta, are able to adsorb phage particles but do not sustain their complete multiplication . SE-3 is closely related to the phage SE-5 as shown by DNA restriction mapping . The differences between SE-3 and SE-5 genomes are apparently limited to two DNA segments flanked by short inverted repeats, visualized by electron microscopy. Mol Gen Mikrobiol Virusol, 1988 Jul, (7), 42 - 7 {Complementary addressed modification of single- and double-stranded DNA by alkylating derivatives of oligonucleotides isolated by partial DNA fragmentation}; Gaidamakov SA et al.; Reagents for complementary addressed modification of nucleic acids are proposed to be synthesized on the base of oligonucleotides obtained by partial chemical fragmentation of DNA . The alkylating 4-(N-2 chlorethyl-N-methylamino) benzyl-5'-phosphamide derivatives of 5'-{32P}-labelled oligonucleotides obtained from single and double-stranded DNA cloned in bacteriophage M13 mp9 have been synthesized . The alkylated derivatives of oligonucleotides selectively modify the complementary tracts of single-stranded DNA-target . They are also able to modify the complementary regions in double-stranded supercoiled plasmid DNA. J Gen Microbiol, 1988 Jul, 134 ( Pt 7), 1765 - 71 Characterization of bacteriophage phi C69 of Saccharopolyspora erythraea and demonstration of heterologous actinophage propagation by transfection of Streptomyces and Saccharopolyspora; Katz L et al.; A bacteriophage, designated phi C69, isolated from a culture of Saccharopolyspora erythraea was characterized . The phage propagates on Sac . erythraea NRRL 2338 but does not infect 10 Streptomyces or 3 Micromonospora species tested . It infects Sac . erythraea NRRL 2359 but does not produce infectious phage particles in this host . phi C69 is approximately 40 kb in length and contains cohesive ends . A cos fragment containing ligated phage DNA ends was cloned in Escherichia coli . Restriction maps of the phage DNA and the cos fragment for several enzymes are shown . Transfection of both Sac . erythraea and Streptomyces lividans with phi C69 resulted in approximately equal titres of infectious phage particles produced from approximately the same number of regenerating cells . Transfection of Sac . erythraea with DNA from Streptomyces phages SH10 and KC404 also resulted in the production of infectious phage particles . The basis for differences among hosts in susceptibility to infection by various actinophages is discussed. Mol Gen Genet, 1988 Jul, 213(1), 30 - 5 Nucleotide sequence of the rci gene encoding shufflon-specific DNA recombinase in the IncI1 plasmid R64: homology to the site-specific recombinases of integrase family; Kubo A et al.; Shufflon is a novel type of DNA rearrangement in which four DNA segments are flanked by seven 19-bp repeat sequences . The site-specific recombination between any inverted repeats results in an inversion of the DNA segment(s) either independently or in groups . The recombination is mediated by a gene designated rci . We have determined the nucleotide sequence of the rci gene and found that it encodes a basic protein with 384 amino acid residues . The rci gene was fused with lacZ and its gene product was identified by Western blot analysis . The Rci protein shows regional homologies to the site-specific recombinases encoded by the bacteriophage genomes, including those of lambda, phi 80, P22, P2, 186, P4 and P1. Mikrobiologiia, 1988 Jul-Aug, 57(4), 623 - 8 {Attachment of long fibrils at various stages of assembly of the virus particle of a bacteriophage}; Abuladze NK et al.; Bacteriophage T4 fibrillar structural elements were obtained when the basal plates and their complexes with the core were complemented in vitro with long tail fibrils . The organisation and functioning of the complexes were studied using PAAG electrophoresis, electron microscopy and sedimentation analysis . About 80% of the particles attached fibrils and were biologically active structures, i.e . could participate in the process of infection . Apparently, the organisation of virus particles is not a strictly regulated and consistent process at all the steps, but only at certain stages of their formation. Genes Dev, 1988 Jul, 2(7), 801 - 6 In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage; Vinson CR et al.; We have used a recombinant bacteriophage that expresses the DNA-binding domain of C/EBP to optimize conditions for a screening technique that may facilitate the cloning of genes that encode sequence-specific DNA-binding proteins . The method relies on the expression of cDNA inserts in bacteriophage lambda gt11 . Fusion protein adsorbed onto nitrocellulose filters is probed with radioactive, double-stranded DNA as a ligand . Two procedures greatly increase the level of binding between ligand and recombinant fusion protein . First, nitrocellulose filters are processed through a denaturation/renaturation regimen using 6 M guanidine hydrochloride . Second, synthetic DNA corresponding to the specific binding site is catenated extensively using DNA ligase . The combination of these procedures leads to remarkably strong detection signals . Specific DNA-binding signals can be detected on duplicate filters, and filters can be washed and reused by repeating the cycle of denaturation/renaturation. Appl Environ Microbiol, 1988 Jul, 54(7), 1731 - 7 Transduction of Escherichia coli by bacteriophage P1 in soil; Zeph LR et al.; Transduction of Escherichia coli W3110(R702) and J53(RP4) (10(4) to 10(5) CFU/g of soil) by lysates of temperature-sensitive specialized transducing derivatives of bacteriophage P1 (10(4) to 10(5) PFU/g of soil) (P1 Cm cts, containing the resistance gene for chloramphenicol, or P1 Cm cts::Tn501, containing the resistance genes for chloramphenicol and mercury {Hg}) occurred in soil amended with montmorillonite or kaolinite and adjusted to a -33-kPa water tension . In nonsterile soil, survival of introduced E . coli and the numbers of E . coli transductants resistant to chloramphenicol or Hg were independent of the clay amendment . The numbers of added E . coli increased more when bacteria were added in Luria broth amended with Ca and Mg (LCB) than when they were added in saline, and E . coli transductants were approximately 1 order of magnitude higher in LCB; however, the same proportion of E . coli was transduced with both types of inoculum . In sterile soil, total and transduced E . coli and P1 increased by 3 to 4 logs, which was followed by a plateau when they were inoculated in LCB and a gradual decrease when they were inoculated in saline . Transduction appeared to occur primarily in the first few days after addition of P1 to soil . The transfer of Hg or chloramphenicol resistance from lysogenic to nonlysogenic E . coli by phage P1 occurred in both sterile and nonsterile soils . On the basis of heat-induced lysis and phenotype, as well as hybridization with a DNA probe in some studies, the transductants appeared to be the E . coli that was added . Transduction of indigenous soil bacteria was not unequivocally demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS) Gene Anal Tech, 1988 Jul-Aug, 5(4), 80 - 2 Small-scale purification of bacteriophage lambda DNA by an airfuge centrifugation step in cesium chloride gradients; Mirande M et al.; A rapid and efficient procedure for purifying bacteriophage lambda DNA is described . This small-scale purification involves isolation of bacteriophage particles on cesium chloride gradients . Using an Airfuge ultracentrifuge, the centrifugation step can be readily achieved in 90 minutes . The method allows a 1-day purification of up to 12 independent lambda DNA (20-40 micrograms each) . The recovered DNA, essentially devoid of RNA and DNA contaminants, is efficiently cut by restriction endonucleases and can serve as starting material for the ligation of DNA fragments in other cloning vehicles. Genetics, 1988 Jul, 119(3), 477 - 84 Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage lambda terminase; Wu WF et al.; The large subunit of phage lambda terminase, gpA, the gene product of the phage A gene, interacts with the small subunit, gpNul, to form functional terminase . Terminase binds to lambda DNA at cosB to form a binary complex . The terminase:DNA complex binds a prohead to form a ternary complex . Ternary complex formation involves an interaction of the prohead with gpA . The amino terminus of gpA contains a functional domain for interaction with gpNul, and the carboxy-terminal 38 amino acids of gpA contain a functional domain for prohead binding . This information about the structure of gpA was obtained through the use of hybrid phages resulting from recombination between lambda and the related phage 21 . lambda and 21 encode terminases that are analogous in structural organization and have ca . 60% sequence identity . In spite of these similarities, lambda and 21 terminases differ in specificity for DNA binding, subunit assembly, and prohead binding . A lambda-21 hybrid phage produces a terminase in which one of the subunits is chimeric and had recombinant specificities . In the work reported here; a new hybrid, lambda-21 hybrid 67, is characterized . lambda-21 hybrid 67 is the result of a crossover between lambda and 21 in the large subunit genes, such that the DNA from the left chromosome end is from 21, including cosB phi 21, the 1 gene, and the first 48 codons for the 2 gene . The rest of the hybrid 67 chromosome is lambda DNA, including 593 codons of the A gene . The chimeric gp2/A of hybrid 67 binds gp1 to form functional terminase.(ABSTRACT TRUNCATED AT 250 WORDS) Virology, 1988 Jul, 165(1), 103 - 14 Late stages in bacteriophage lambda head morphogenesis: in vitro studies on the action of the bacteriophage lambda D-gene and W-gene products; Perucchetti R et al.; The in vitro maturation of bacteriophage lambda can be divided into discrete steps . Concatemers of lambda DNA bind terminase to form complex I . This DNA-terminase complex then binds a prohead to form a ternary complex (II) . Complex II in turn can be converted to infectious phage by the addition of extracts containing the products of the phage genes D, W, FII, as well as phage tails . By using in vitro complementation assays gpD and gpW have been partially purified and their interactions with complex II studied . gpD can bind to complex II in vitro to form a new complex (III) which can be isolated by sedimentation on neutral sucrose gradients . This complex requires only the addition of gpW, gpFII, and phage tails to form mature phage particles . The sedimentation of complex III is virtually identical to that of complex II; however, the resistance of the former to inactivation by DNase is higher, likely due to the partial packaging of the DNA . In similar experiments it was shown that gpW cannot bind to complex II but can effectively interact with complex III . This latter reaction converts complex III to a DNase-resistant form which sediments in a manner identical to that of full phage heads (complex IV) . After isolation of the complex IV only gpFII and tails are required for mature phage formation in vitro . gpW is a heat-stable protein of molecular weight approximately 10,000. J Bacteriol, 1988 Jul, 170(7), 3089 - 93 Replication forks of Escherichia coli are not the preferred sites for lysogenic integration of bacteriophage Mu; Sivan S et al.; The question of whether bacteriophage Mu prefers replication forks for lysogenic integration into Escherichia coli chromosomes was tested by using two different systems . In the first, inactivation of genes was scored in synchronized cultures infected by Mu at various times . No increase in the mutation frequency of a gene was found after infection at the time of its replication . In the second, the composition of colonies formed by bacteria lysogenized by Mu was determined; the newly formed lysogens should give rise to mixed colonies (containing lysogenized as well as nonlysogenized bacteria), uniform colonies, or both, depending on the mode of integration . Both types of colonies were found, and the fraction of uniform colonies was proportional to the relative length of the unreplicated segment of an average chromosome in the culture . The results in both systems clearly preclude the possibility that a lysogenizing Mu integrates with high preference at the chromosome replication forks. Mol Gen Genet, 1988 Jul, 213(1), 134 - 9 DNA sequence analysis of point mutations in traA, the F pilin gene, reveal two domains involved in F-specific bacteriophage attachment; Frost LS et al.; Six missense point mutations in traA (WPFL43,44,45,46,47 and 51), the gene encoding F pilin in the transfer region of the F plasmid, have been characterized for their effect on the transfer ability, bacteriophage (R17, QB and fl) sensitivity and levels of piliation expressed by the plasmid . The sequence analysis of the first five of these mutations revealed two domains in the F pilin subunit exposed on the surface of the F pilus which mediate phage attachment . These two domains include residues 14-17 (approximately) and the last few residues at the carboxy-terminus of the pilin protein . One of these mutants had a pleiotropic affect on pilus function and was thought to have affected pilus assembly . The sixth point mutant (WPFL51), previously thought to be in traA, was complemented by chimeric plasmids carrying the traG gene of the F transfer region, which may be involved in the acetylation of the pilin subunit . A traA nonsense mutant (JCFL1) carried an amber mutation near the amino-terminus which is well suppressed in SuI+ (supD) and SuIII+ (supF) strains . Neither the antigenicity of the pilin nor the efficiency of plating of F-specific bacteriophages were affected when this plasmid was harbored by either suppressor strain . A second amber mutant (JCFL25) which is not suppressible, carried its mutation in the codon for the single tryptophan in F pilin, suggesting that this residue is important in subunit interactions during pilus assembly . Two other point mutants (JCFL32 and 44) carried missense mutations in the leader sequence (positions 9 and 13) which affected the number of pili per cell presumably by altering the processing of propilin to pilin. J Gen Virol, 1988 Jul, 69 ( Pt 7), 1683 - 94 Molecular cloning and restriction enzyme mapping of an African swine fever virus isolate from Malawi; Dixon LK; DNA prepared from a field isolate of African swine fever virus, which causes high mortality and severe disease in domestic pigs, was cloned in bacteriophage lambda and plasmid vectors . Clones containing DNA inserts overlapping with each other and together covering the complete genome, apart from short fragments close to the cross-linked termini of the genome, were obtained . A complete restriction enzyme site map of the genome for three enzymes was deduced. J Bacteriol, 1988 Jul, 170(7), 3016 - 24 A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease; Skorupski K et al.; A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease has been identified . This pin (proteolysis inhibition) gene was selected for its ability to support plaque formation by a lambda Ots vector at 40 degrees C . Southern blot experiments indicated that this T4 gene is included within the 4.9-kilobase XbaI fragment which contains gene 49 . Subcloning experiments showed that T4 gene 49.1 (designated pinA) is responsible for the ability of the Ots vector to form plaques at 40 degrees C . Deficiencies in Lon protease activity are the only changes known in E . coli that permit lambda Ots phage to form plaques efficiently at 40 degrees C . lon+ lysogens of the lambda Ots vector containing pinA permitted a lambda Ots phage to form plaques efficiently at 40 degrees C . Furthermore, these lysogens, upon comparison with similar lysogens lacking any T4 DNA, showed reduced levels of degradation of puromycyl polypeptides and of canavanyl proteins . The lon+ lysogens that contained pinA exhibited other phenotypic characteristics common to lon strains, such as filamentation and production of mucoid colonies . Levels of degradation of canavanyl proteins were essentially the same, however, in null lon lysogens which either contained or lacked pinA . We infer from these data that the T4 pinA gene functions to block Lon protease activity; pinA does not, however, appear to block the activity of proteases other than Lon that are involved in the degradation of abnormal proteins. J Biol Chem, 1988 Jun 15, 263(17), 8413 - 9 The Nu1 subunit of bacteriophage lambda terminase; Parris W et al.; The maturation and packaging of bacteriophage lambda DNA are catalyzed by the phage terminase enzyme . Terminase is composed of two protein subunits, gpNu1 and gpA . The holoenzyme is multifunctional in vitro; it binds to and cleaves lambda DNA at the cos site (where cos represents cohesive-end site), packages DNA into lambda proheads, and is also a DNA-dependent ATPase . The genes of the two subunits have been cloned separately into powerful expression vectors which allow for very high levels of protein overproduction . The gpNu1 protein has been purified to homogeneity and has a monomeric molecular weight of 21,200, in close agreement with the Mr of 20,444 expected from its amino acid sequence . Both gel filtration and sedimentation velocity centrifugation indicate that the native gpNu1 protein exists as a Mr greater than 500,000 aggregate . The sequence of the first 20 amino acids and the overall composition both match those predicted by the nucleotide sequence of the Nu1 gene . Purified gpNu1 is able to complement gpA-containing extracts in both lambda DNA packaging and cos cleavage assays . The Nu1 gene amino acid sequence predicts DNA binding by the protein, and gpNu1 does show specific binding to lambda DNA by filter binding assays . Also, as predicted from its sequence, gpNu1 exhibits ATPase activity; but in contrast to the holoenzyme, this activity is DNA-independent. Gene, 1988 Jun 15, 66(1), 121 - 34 Expression vectors permitting cDNA cloning and enrichment for specific sequences by hybridization/selection; Pruitt SC; A set of vectors is described which allow the efficient cloning of full-length cDNAs, using a modification of the method of Okayama and Berg {Mol . Cell Biol . 2 (1982) 161-170}, and enrichment of specific sequences directly from cDNA libraries by hybridization/selection . The vectors pcDpolyB+ and pcDpolyB- are derived from an expression vector described previously {Okayama and Berg, Mol . Cell Biol . 3 (1983) 280-289} and allow expression of cloned cDNAs in eukaryotic cells from the simian virus 40 early region promoter . The vectors BSB+ and BSB- contain convenient priming sites for sequence analysis and the T3 and T7 RNA polymerase promoters, allowing synthesis of transcripts homologous to either strand of the cDNA . Each of these vectors also contains the intergenic region from the bacteriophage f1 permitting synthesis of single-stranded (ss) copies of the cDNA libraries . Enrichment for cDNAs containing sequences homologous to the hypoxanthine phosphoribosyl transferase gene from an ss copy of a cDNA library by hybridization/selection is demonstrated . Levels of enrichment sufficient for the direct cloning of specific sequences without requiring colony or plaque hybridizations were obtained . Libraries constructed from different cell types can be screened against each other to create sublibraries highly enriched in sequences specific to a single cell type . The availability of cDNA expression libraries enriched for cell-type-specific cDNAs should greatly enhance the efficiency with which cDNAs can be identified on the basis of functional assays. Biochemistry, 1988 Jun 14, 27(12), 4357 - 67 Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-{Pt(NH3)2(d(GpG}}, built into a specific site in a viral genome; Naser LJ et al.; A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-{Pt(NH3)2(d(GpG}} intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II) . The duplex dodecamer d(AGAAGGCCTAGA).d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site . A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant . The dodecamer d(TCTAGGCCTTCT) was synthesized by the solid-phase phosphotriester method and platinated by reaction with cis-{Pt(NH3)2(H2O)2}2+ (yield 39%) . Characterization by pH-dependent 1H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines . The platinated oligonucleotide was phosphorylated in the presence of {gamma-32P}ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome . Approximately 80% of the gapped duplexes incorporated a dodecanucleotide in the ligation reaction . Of these, approximately half did so with the dodecanucleotide covalently joined to the genome at both 5' and 3' termini . The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII . The cis-{Pt(NH3)2(d(GpG}} cross-link completely inhibited StuI cleavage, which was fully restored following incubation of the platinated genome with cyanide to remove platinum as {Pt(CN)4}2- . Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-{Pt(NH3)2(d(GpG}} cross-link induces localized weakening of the DNA double helix . In addition, double- and single-stranded genomes, in which the cis-{Pt(NH3)2(d(GpG}} cross-link resides specifically in the plus strand, were constructed . Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes . In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-{Pt(NH3)2(d(GpG}} cross-link is lethal to the single-stranded bacteriophage. Biochemistry, 1988 Jun 14, 27(12), 4350 - 7 Sugar pucker and phosphodiester conformations in viral genomes of filamentous bacteriophages: fd, If1, IKe, Pf1, Xf, and Pf3; Thomas GJ Jr et al.; The laser Raman spectra of filamentous viruses contain discrete bands which are assignable to molecular vibrations of the encapsidated, single-stranded DNA genomes and which are informative of their molecular conformations . Discrimination between Raman bands of the DNA and those of the coat proteins is facilitated by analysis of viruses containing deuterium-labeled amino acids . Specific DNA vibrational assignments are based upon previous studies of A-, B-, and Z-DNA oligonucleotide crystals of known structure {Thomas, G.J., Jr., & Wang, A.H.-J . (1988) in Nucleic Acids and Molecular Biology (Eckstein, F., & Lilley, D.M.J., Eds.) Vol . 2, Springer-Verlag, Berlin} . The present results show that canonical DNA structures are absent from six filamentous viruses: fd, If1, IKe, Pfl, Xf, and Pf3 . The DNAs in three viruses of symmetry class I (fd, If1, IKe) contain very similar nucleoside sugar puckers and glycosyl torsions, deduced to be C3'-endo/anti . However, nucleoside conformations are not the same among the three class II viruses examined: Pf1 and Xf DNAs contain similar conformers, deduced to be C2'-endo/anti, whereas Pf3 DNA exhibits bands usually associated with C3'-endo/anti conformers . Conformation-sensitive Raman bands of the DNA 3'-C-O-P-O-C-5' groups show that in all class I viruses and in Pf1 the ssDNA backbones do not contain regularly ordered phosphodiester group geometries, like those found in ordered single- and double-stranded nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS) Nucleic Acids Res, 1988 Jun 10, 16(11), 5055 - 66 A comparison of two phage coat protein-RNA interactions; Wu HN et al.; The interaction between the coat protein of the group I bacteriophage fr with its translational operator site is compared with the previously studied R17 interaction . The sequence of the two RNA binding sites differ by 2 of 20 nucleotides and two coat proteins by 17 of 129 amino acids . An analysis of the binding of fr coat protein to 24 operator variants revealed that the two proteins recognize operator sequences in virtually the same way . However, fr coat protein binds to nearly every RNA 6 to 14-fold tighter than R17 coat protein . Since the fr operator is a weaker binding variant and the fr coat protein shows a different temperature dependence of binding, it is unlikely that the two systems have different Kas in vivo . RNA fragments containing the operator sequences can initiate the capsid assembly with both fr and R17 coat protein . Surprisingly, the two coat proteins can form a mixed capsid in vitro. Nucleic Acids Res, 1988 Jun 10, 16(11), 4789 - 800 Expression of the E.coli hemolysin secretion gene hlyB involves transcript anti-termination within the hly operon; Koronakis V et al.; The genes hly A and hly B dictating synthesis and secretion of hemolytic toxin by Escherichia coli are transcribed as part of an operon hly C, hly A, hly B but are separated by an inverted repeat and poly-T sequence characteristic of a rho-independent terminator . The hly A-hly B intergenic sequence caused a reduction of in vivo transcriptional read-through from the gal promotor into gal K when inserted in the pKG1900 terminator-probe vector and also in vitro 3' to the bacteriophage T7 luminal diameter 10 promotor . Hybridisation of mRNA generated by the hly determinant of recombinant DNA pBR202-312 to an antisense RNA probe spanning the hly intergenic sequence revealed specific termination of 80% of in vivo hly transcripts within the poly-T sequence, immediately preceding the hly B translation start . Extension of the hly determinant with 3.5kbp of hly promotor-proximal DNA sequence raised intracellular and cell-free hemolytic activity 3-fold and 20-fold respectively and increased markedly the secretion of the 107Kd hemolysin protein (HlyA) . Parallel hybridisation of in vivo mRNA to hly A and hly B antisense probes revealed that levels of hly A and hly B mRNA were increased by approximately 3-fold and 90-fold . The dramatically enhanced level of hly B mRNA resulted primarily from specific suppression of transcription termination at the intergenic rho-independent terminator from 80% to 1%, thus allowing virtually all hly A transcripts to elongate into hly B. J Biol Chem, 1988 Jun 5, 263(16), 7604 - 9 Isolation and characterization of the mouse ornithine decarboxylase gene; Katz A et al.; Mouse ornithine decarboxylase (ODC) genomic clones were isolated from a bacteriophage lambda genomic library representing mouse myeloma 653-1 cells which over-produce ODC due to amplification of an active ODC gene . Sequence analysis of the amplified ODC gene revealed that ODC mRNA is encoded by 12 exons, 10 of which (exons 3 to 12) code for the ODC protein . Exon 12 also corresponds to the 3' noncoding region of the two species of ODC mRNA which are formed by alternative utilization of two polyadenylation signals separated from each other by 422 nucleotides . The transcription initiation site was mapped by S1 nuclease protection and by primer extension analysis . The 5' flanking region is extremely rich in G + C and contains typical promoter motifs such as the TATA box and SP1 transcription factor binding sites . Joining the 5' flanking region to the Escherichia coli chloramphenicol acetyltransferase structural gene and its introduction into mouse cells resulted in the expression of a high level of chloramphenicol acetyltransferase activity . Comparing the sequence of the ODC gene to our previously published sequence of ODC cDNA revealed a disagreement between the sequences located 5' to the AvaI site and demonstrated that this region of our previously reported cDNA represents a cloning artifact . The portion of the correct 5' noncoding region encoded by exon 1 is extremely rich in G + C and includes potential secondary structures which may be involved in translational regulation of ODC mRNA. J Biol Chem, 1988 Jun 5, 263(16), 7478 - 86 Primary structure of T4 DNA polymerase . Evolutionary relatedness to eucaryotic and other procaryotic DNA polymerases; Spicer EK et al.; Bacteriophage T4 gene 43 codes for the viral DNA polymerase . We report here the sequence of gene 43 and about 70 nucleotides of 5'- and 3'-flanking sequences, determined by both DNA and RNA sequencing . We have also purified T4 DNA polymerase from T4 infected Escherichia coli and from E . coli containing a gene 43 overexpression vector . A major portion of the deduced amino acid sequence has been verified by peptide mapping and sequencing of the purified DNA polymerase . All these results are consistent with T4 DNA polymerase having 898 amino acids with a calculated Mr = 103,572 . Comparison of the primary structure of T4 DNA polymerase with the sequence of other procaryotic and eucaryotic DNA polymerases indicates that T4 DNA polymerase has regions of striking similarity with animal virus DNA polymerases and human DNA polymerase alpha . Surprisingly, T4 DNA polymerase shares only limited similarity with E . coli polymerase I and no detectable similarity with T7 DNA polymerase . Based on the location of specific mutations in T4 DNA polymerase and the conservation of particular sequences in T4 and eucaryotic DNA polymerases, we propose that the NH2-terminal half of T4 DNA polymerase forms a domain that carries out the 3'----5' exonuclease activity whereas the COOH-terminal half of the polypeptide contains the dNTP-binding site and is necessary for DNA synthesis. J Mol Biol, 1988 Jun 5, 201(3), 517 - 35 Autogenous regulatory site on the bacteriophage T4 gene 32 messenger RNA; McPheeters DS et al.; We have identified the binding site on the bacteriophage T4 gene 32 mRNA responsible for autogenous translational regulation . We demonstrate that this site is largely unstructured and overlaps the initiation codon of gene 32 as previously predicted . Co-operative binding of gene 32 protein to this site specifically blocks the formation of 30 S-tRNA(fMet)-gene 32 mRNA ternary complexes and initiation of translation . The translational operator is bound co-operatively by gene 32 protein and this binding is facilitated by a nucleation site far upstream from the initiation codon . A similar unstructured mRNA lacking this nucleation site is also bound co-operatively, but only at concentrations of gene 32 protein higher than those needed to repress binding of ribosomes to the gene 32 mRNA . Some sequence-specific interactions may also influence this binding . Comparison of the bacteriophage T2, T4 and T6 gene 32 operator sequences leads us to propose that the nucleation site is a pseudoknot. J Mol Biol, 1988 Jun 5, 201(3), 487 - 96 Maltose transport and starch binding in phage-resistant point mutants of maltoporin . Functional and topological implications; Charbit A et al.; The relationships between the bacteriophage lambda binding site, the starch binding site and the pore formed by maltoporin (LamB protein, lambda receptor protein) were investigated . Bacteria with single amino acid substitutions in the maltoporin sequence, which were previously shown to be strongly reduced in phage lambda sensitivity, were assayed for maltose- (and maltodextrin) selective pore functions . Maltose transport assays was performed at low substrate concentrations, under conditions where LamB is limiting for transport . It revealed three classes of mutants . Class A is composed of mutants with no effect on transport (substitutions at amino acid residues 154, 155, 259, 382 and 401); class B corresponds to mutants with a significant but variable reduction in transport (sites 148, 151, 152, 163, 164, 245, 247 and 250); class C is represented by a single mutant for which transport is almost completely abolished (site 18) . Starch binding was assayed by two different methods that gave compatible results . In class A mutants, binding was normal, while no binding was observed in the class C mutant . Binding was impaired to various extents in category B mutants . There was a correlation between the level of impairment of starch binding and impairment of maltose transport, consistent with the notion that the residues influencing starch binding are inside, or in close proximity to, the pore . These results, together with previous data on starch-binding mutants that were not affected in phage binding (substitutions at residues 8, 74, 82, 118 and 121), suggest that the binding sites for starch and phage lambda overlap but are distinct . Mutations affecting transport and starch binding are located in the first third of the protein and in the region of residues 245 to 250 . Mutations affecting phage adsorption are located mainly in the last two-thirds of the protein . The topological constraints suggested by the results with the available mutants altered in the lamB gene were used to propose a revised model of maltoporin folding across the outer membrane as well as to define the outlines of footprints of macromolecular binding sites (phage, starch and monoclonal antibodies) on the surface of the protein. Appl Environ Microbiol, 1988 Jun, 54(6), 1637 - 41 Morphological diversity of ruminal bacteriophages from sheep and cattle; Klieve AV et al.; Large numbers of bacteriophages (2 x 10(7) to 1 x 10(8)/ml) were present in ruminal fluid from sheep and cattle . Twenty-six distinct types were identified and placed in three morphological groups; several phages possessed unusual structural features . The large numbers and diversity of phages observed indicates a possible role in bacterial lysis and hence in the population dynamics of the ruminal bacteria. J Bacteriol, 1988 Jun, 170(6), 2866 - 9 Enrichment of the bacteriophage PR4 membrane in phosphatidylglycerol is not essential for phage assembly and infectivity; Vanden Boom T et al.; The membrane phospholipids of bacteriophage PR4 grown on wild-type Escherichia coli are markedly enriched in phosphatidylglycerol (PG) relative to host phospholipids . To investigate the role of PG in phage assembly and infectivity, we propagated PR4 on an E . coli mutant defective in PG synthesis . The PG content of PR4 grown on the mutant host accounted for 0.4% of the total viral phospholipids, representing a 90-fold decrease in PG relative to the PG content of phage grown on a wild-type host . Phosphatidylethanolamine and phosphatidic acid, the two major phospholipid species present in these phage preparations, accounted for 88.4 and 9.4% of the total viral phospholipids, respectively . This drastic alteration of the phage phospholipid composition had little or no adverse effect on either the stability or infectivity of the phage . We conclude that the enrichment of the PR4 virion in PG does not reflect an absolute structural requirement of the phage and is not essential for phage infectivity. Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4162 - 5 Mutant 16S ribosomal RNA: a codon-specific translational suppressor; Murgola EJ et al.; We have isolated an unusual codon-specific translational suppressor in Escherichia coli . The suppressor resulted from a spontaneous mutation in a chromosomal gene during a selection for suppressors of the auxotrophic nonsense mutation trpA(UGA211) . The suppressor allows readthrough of UGA mutations at two positions in trpA and at two sites in bacteriophage T4 . It does not, however, suppress amber (UAG) or ochre (UAA) mutations that were tested in both genomes, some of which were at the same positions as the suppressible UGA mutations . The suppressor also does not allow mistranslation of the UGA-related trpA missense mutations UGG at positions 211 and 234, AGA at 211 and 234, CGA at 211, or UGU and UGC at 234 . The suppressor mutation was mapped by genetic procedures to position 89 on the E . coli genetic map . Localization of the suppressor mutation to rrnB was achieved by cloning it in the low-copy-number plasmid pEJM007 by in vivo recombination from the chromosome . Recloning in bacteriophage M13 and subsequent DNA sequence analysis allowed the identification of the suppressor mutation as a deletion of the cytidylic acid residue at nucleotide position 1054 of the 16S ribosomal RNA . The mutant EcoRI-Xba I fragment from the suppressor gene was recloned, from M13, in an otherwise wild-type rrnB in the plasmid pEJM007, and UGA suppression was examined . The UGA-suppressing activity of the reconstructed suppressor-containing pEJM007 was indistinguishable from that of the original recombinant suppressor-containing plasmid . This result demonstrates that the C1054 deletion in 16S rRNA is both necessary and sufficient for UGA suppression . The existence of this mutant suggests an important role for rRNA in codon recognition, at least for accurate polypeptide chain termination. J Bacteriol, 1988 Jun, 170(6), 2850 - 4 The fadL gene product of Escherichia coli is an outer membrane protein required for uptake of long-chain fatty acids and involved in sensitivity to bacteriophage T2; Black PN; The fadL+ gene of Escherichia coli encodes an outer membrane protein (FadL) essential for the uptake of long-chain fatty acids (C12 to C18) . The present study shows that in addition to being required for uptake of and growth on the long-chain fatty acid oleate (C18:1), FadL acts as a receptor of bacteriophage T2 . Bacteriophage T2-resistant (T2r) strains lacked FadL and were unable to take up and grow on long-chain fatty acids . Upon transformation with the fadL+ clone pN103, T2r strains became sensitive to bacteriophage T2 (T2s), became able to take up long-chain fatty acids at wild-type levels, and contained FadL in the outer membrane. J Ultrastruct Mol Struct Res, 1988 Jun, 99(3), 189 - 202 Head structure of bacteriophages T2 and T4; Baschong W et al.; The length-to-width ratios of bacteriophage T2 and T4 heads and stereometric angles specifying the prolate icosahedral T2 capsid were evaluated on electron micrographs recorded from samples prepared by a variety of methods . The copy numbers of the major capsid protein, gp23*, of T2 and T4 phages were compared by quantitative gel electrophoresis . Taken together, the resulting values are most compatible with triangulation numbers T = 13 and Q = 21 for both T2 and T4, thus confirming the previously proposed capsid architecture of T4 revealed by indirect measurements and thereby eliminating the repeatedly reported discrepancy between T2 and T4 in favor of a common Q number of 21 corresponding to 960 copies of gp23*. J Bacteriol, 1988 Jun, 170(6), 2493 - 500 Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli; Moreau PL; Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes . (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected . These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein . However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair . Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA. J Virol, 1988 Jun, 62(6), 2124 - 33 Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro; Polo JM et al.; Genetic loci affecting Sindbis virus pathogenesis in neonatal mice have been examined by using a full-length cDNA clone of the virus (Toto1101) . The full-length cDNA is linked to a bacteriophage SP6 promoter to facilitate the synthesis of infectious RNA transcripts in vitro . Virus derived from Toto1101 showed reduced virulence (attenuation) in neonatal mice . Replacement of the E1 glycoprotein and 6K genes of Toto1101 with cloned E1 and 6K genes derived from a virulent Sindbis virus strain, AR339 (SB), resulted in a new construct, TR2000, that gave rise to virulent virus . Sequence determinations for the entire substituted regions of TR2000, Toto1101, and related virulent and attenuated strains identified three coding differences in E1 between Toto1101 and TR2000 . These differences, individually or in combination, may be responsible for the attenuated phenotype . Previous studies in this laboratory identified another attenuating mutation at amino acid position 114 of the E2 glycoprotein (N.L . Davis, F.J . Fuller, W.G . Dougherty, R.A . Olmsted, and R.E . Johnston, Proc . Natl . Acad . Sci . USA 83:6771-6775, 1986) . Substitution of Arg-114 in the mutant SB-RL for Ser-114 of SB appears to confer three distinguishing phenotypes: attenuation in neonatal mice, increased sensitivity to specific E2 monoclonal antibodies, and accelerated penetration of BHK cells . Replacement of TR2000 sequences containing the codon for amino acid 114 of E2 with corresponding fragments from cDNA clones of SB or SB-RL produced two strains of Sindbis virus (TR2100 and TR2200) which were isogenic except for the E2 114 codon (Ser and Arg, respectively) . The three diagnostic phenotypes cosegregated according to the origin of the codon for amino acid 114 of E2, confirming the dramatic effect of this single amino acid substitution on these three phenotypes. Gene, 1988 May 30, 65(2), 259 - 68 Purification and characterization of the DNA-binding protein Ner of bacteriophage Mu; Allet B et al.; The construction is described of a plasmid (pL-ner) which directs the high-level production of the bacteriophage Mu Ner protein in Escherichia coli . The protein, recovered in the soluble cellular fraction, was susceptible to in vivo proteolytic processing, in many host strains, but not in E . coli B, a natural lon- prototroph . A simple purification method is described which takes advantage of the basic nature of the protein . The purified protein was shown to be physically and chemically homogeneous and to have an amino acid sequence identical to that predicted for the authentic protein . The protein was also shown to have in vitro biological activity, as measured by specific binding to a DNA fragment containing the consensus Ner-binding sequence, and in vivo biological activity as the protein produced by the pL-ner plasmid allowed lysogenic-like maintenance of a Mu prophage c mutant unable to synthesise a functional Mu repressor. Nucleic Acids Res, 1988 May 25, 16(10), 4595 - 605 Integration host factor of Escherichia coli regulates early- and repressor transcription of bacteriophage Mu by two different mechanisms; van Rijn PA et al.; Integration host factor (IHF) of E . coli positively regulates both early and repressor transcription of bacteriophage Mu . In this paper we show that although binding of IHF to the same binding site is responsible for both types of transcription regulation, the mechanisms by which these regulations occur are different: Activation of transcription from the early promoter (Pe) requires a helix-dependent orientation of IHF- and RNA polymerase binding sites on the DNA helix with a limited distance between both sites . Activation of repressor transcription shows no helix dependency between promoter and IHF binding site and the distance between both sites can be enlarged at least by 100 base pairs without affecting the positive control . A possible mechanism for both types of transcription stimulation will be discussed. J Biol Chem, 1988 May 25, 263(15), 6960 - 3 Inhibition of the RNA polymerase-catalyzed synthesis of RNA by marcellomycin . Preferential interference of the inhibitor with the stabilization of the ternary promoter-RNA polymerase-nascent RNA complex; Kriebardis T et al.; Marcellomycin is a strong inhibitor of the Escherichia coli RNA polymerase-catalyzed synthesis of RNA from the strong A promoters of bacteriophage T7 DNA . Marcellomycin inhibits preferentially the last phase of transcription initiation . During this phase a stabilized ternary complex is formed consisting of RNA polymerase, DNA template, and a nascent RNA oligonucleotide about 11 nucleotides long, resulting from the extension of the RNA dinucleotide component of the corresponding early ternary complex . Marcellomycin is also responsible for minor inhibition of the formation of the open binary RNA polymerase-template complex, which serves as the precursor of the ternary complex . These findings suggest that marcellomycin may be a potentially useful tool in the study of the late stages of transcription initiation . The present findings may also contribute to a better overall understanding of the mode of drug action at the level of individual genes. Nucleic Acids Res, 1988 May 25, 16(10), 4483 - 98 In vitro transcription and translational efficiency of chimeric SP6 messenger RNAs devoid of 5' vector nucleotides; Jobling SA et al.; A plasmid containing the bacteriophage SP6 promoter, designated pHSTO, permits in vitro transcription of RNAs devoid of vector-derived nucleotides . This vector has been characterized for relative transcriptional activity using constructs which alter the conserved nucleotides extending beyond the SP6 transcriptional initiation site . SP6 polymerase efficiently transcribes cDNA inserts which contain a guanosine (G) nucleotide at position +1 relative to the SP6 promoter; however, inserts with an adenosine (A) or pyrimidine at position +1 are not transcribed . Several cellular and viral cDNAs have been transcribed into translatable messenger RNA using this vector; however, SP6 polymerase will not transcribe the A-T rich untranslated leader from alfalfa mosaic virus RNA 4 efficiently unless the viral mRNA cap site is separated from the transcriptional initiation site by twelve base pairs of vector DNA . Chimeric messenger RNAs were created by linking the untranslated leader sequence of several viral mRNAs to the coding region of barley alpha-amylase, and the resultant mRNAs were translated in a wheat germ extract to determine relative translational efficiencies . The untranslated leader sequences of turnip yellow mosaic virus coat protein mRNA and black beetle virus RNA 2 did not increase translational efficiency, while the tobacco mosaic virus leader stimulated translation significantly . The results indicate that substitution of a cognate untranslated leader sequence with a leader derived from a highly efficient mRNA does not necessarily predict enhanced translational efficiency of the chimeric mRNA. FEBS Lett, 1988 May 23, 232(2), 308 - 12 Structural requirements of choline derivatives for 'conversion' of pneumococcal amidase . A new single-step procedure for purification of this autolysin; Sanz JM et al.; Tertiary amines appear to be the minimal structure needed to convert in vitro the inactive form (E-form) of pneumococcal amidase to the catalytic active form (C-form) . Diethylethanolamine was one of the compounds that converted the E-form, a finding that has been used successfully to develop an affinity chromatography system in DEAE-cellulose for the rapid and efficient purification of lytic enzymes of pneumococcus and its bacteriophages. J Mol Biol, 1988 May 20, 201(2), 239 - 46 Oxidative damage in DNA . Lack of mutagenicity by thymine glycol lesions; Hayes RC et al.; Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is a base damage common to oxidative mutagens and the major stable radiolysis product of thymine in DNA . We assessed the mutagenic potential of thymine glycols in single-stranded bacteriophage DNA during transfection of Escherichia coli wild-type and umuC strains . cis-Thymine glycols were induced in DNA by reaction with the chemical oxidant, osmium tetroxide (OsO4); modification of thymines was quantitated by using anti-thymine glycol antibody . Inactivation of transfecting molecules showed that one lethal hit corresponded to 1.5 to 2.1 thymine glycols per phage DNA in normal cells, whereas conditions of W-reactivation (SOS induction) reversed 60 to 80% of inactivating events . Forward mutations in the lacI and lacZ' (alpha) genes of f1 and M13 hybrid phage DNAs were induced in OsO4-treated DNA in a dose-dependent manner, in both wild-type and umuC cells . Sequence analysis of hybrid phage mutants revealed that mutations occurred preferentially at cytosine sites rather than thymine sites, indicating that thymine glycols were not the principal pre-mutagenic lesions in the single-stranded DNA . A mutagenic specificity for C----T transitions was confirmed by OsO4-induced reversion of mutant lac phage . Pathways for mutagenesis at derivatives of oxidized cytosine are discussed. J Mol Biol, 1988 May 20, 201(2), 327 - 38 Control of cell division by sex factor F in Escherichia coli . III . Participation of the groES (mopB) gene of the host bacteria; Miki T et al.; Cell division of F+ bacteria is coupled to DNA replication of the F plasmid . Two plasmid coded genes, letA (ccdA) and letD (ccdB) are indispensable for this coupling . To investigate bacterial genes that participate in this coupling, we attempted to identify the target of the division inhibitor (the letD gene product) of the F plasmid . Two temperature-sensitive growth defective mutants were screened from bacterial mutants that escaped the letD product growth inhibition that occurs in hosts carrying an FletA mutant . Phage P1-mediated transduction and complementation analysis indicated that the temperature-sensitive mutations are located in the groES (mopB) gene, which is essential for the morphogenesis of several bacteriophages and also for growth of the bacteria . The nucleotide sequence of the promoter region of the gene in which the temperature-sensitive mutations had occurred was virtually identical with that of the groES gene of Escherichia coli; furthermore the sequence of the first five amino acid residues and the overall amino acid composition predicted from the nucleotide sequence of the gene match those of the purified GroES protein . The temperature-sensitive mutants did not allow the propagation of phage lambda at 28 degrees C and formed long filamentous structures without septa at 41 degrees C, as is observed in the case of groES mutants . Growth of the two groES mutants tested was not inhibited by the F plasmid with the letA mutation . These observations suggest to us that the morphogenesis gene groES plays a key role in coupling between replication of the F plasmid and cell division of the host cells. Anal Biochem, 1988 May 15, 171(1), 192 - 6 Purification by ammonium sulfate precipitation of bacteriophage lambda gt11 DNA for restriction analysis of cloned cDNA inserts; Ziai MR et al.; A rapid and efficient method to purify lambda gt11 DNA is described . This technique involves precipitation of intact bacteriophage particles with ammonium sulfate, followed by phage lysis with sodium dodecyl sulfate, proteinase K, and alkaline treatment . The quality of DNA for subsequent restriction analysis, infectivity, subcloning, and radiolabeling is comparable to that isolated by cesium chloride banding or ion exchange chromatography . The yield of the phage DNA is, however, two to eight times higher than that obtained by other conventional methods of lambda gt11 purification . Furthermore the time required to process the bacteriophage lysate is approximately 2 h and therefore more rapid than other currently used methods. J Immunol, 1988 May 15, 140(10), 3640 - 5 Characterization of lipopolysaccharide-induced macrophage gene expression; Tannenbaum CS et al.; A cDNA library from LPS-treated murine peritoneal macrophages has been screened by differential hybridization with radiolabeled cDNA from untreated and LPS-treated macrophages . Six clones hybridizing with mRNA sequences present in LPS-treated cells but not in controls were selected for further characterization . When the recombinant bacteriophage DNA from each clone was used as a probe in Northern analysis of total RNA from LPS-treated macrophages, inducible mRNA ranging from 1.45 to 6.4 kb were seen . In five of six cases, the mRNA expression was undetectable in untreated macrophage cultures . All but one clone identified mRNA that were inducible even in the presence of cycloheximide, indicating the independence of such gene expression from protein synthesis; none of the genes were superinduced by this treatment . The time course of expression differed among the individual genes . Four were induced transiently, whereas two showed stable increasing accumulation through an 8-h period after stimulation . In addition, four of the genes were seen within 30 min of stimulation, whereas two were seen only after 2 to 4 h . Two genes were induced only by treatment with LPS, whereas four were also induced in response to other agents, including IFN-gamma, macrophage CSF, and PMA . The insert sequences from these recombinant clones did not hybridize with a set of cDNA encoding other inducible gene products, including TNF, IL-1, ornithine decarboxylase, c-myc, c-fos, JE, or KC . Thus, these six cDNA appear to encode inducible macrophage genes that are distinct from one another as well as from a selection of previously described early genes . Although their functional identity remains indeterminate, they may encode previously described early proteins induced in macrophages treated with LPS. Nucleic Acids Res, 1988 May 11, 16(9), 3907 - 18 DNA unwinding and inhibition of T4 DNA ligase by anthracyclines; Montecucco A et al.; The ability to alter DNA tertiary structure of ten anthracycline derivatives whose antitumor potency is known was studied by an assay that makes use of nicked circular DNA and bacteriophage T4 DNA ligase . This assay allows the detection of tertiary structure alterations caused by DNA binding of both intercalating and non-intercalating drugs . The determination of these events can be obtained at different temperatures in the range of activity of DNA ligase . The results indicate that anthracyclines alter the DNA tertiary structure but this property does not correlate with their cytotoxic or antitumor activities . An additional interesting finding was that several anthracyclines inhibit T4 DNA ligase . The inhibition can be complete and is a cubic function of drug concentration . The inhibition of DNA ligase does not correlate with the ability of anthracyclines to alter the tertiary structure of DNA but is dependent from the presence of an amino group on the sugar ring. Nucleic Acids Res, 1988 May 11, 16(9), 4053 - 67 Analysis of the entire nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar L1 . Evidence for involvement in DNA replication; Hatt C et al.; Chlamydia trachomatis serovar L1/440/LN possesses a 7498bp plasmid which was designated pLGV440 . The plasmid was cloned at the BamH1 site of pAT153 into Escherichia coli and the recombinant plasmid was designated pCTL1 . A detailed restriction endonuclease map of pCTL1 was constructed . A fragment of the chlamydial plasmid was shown to function as a promoter in E . coli when placed upstream of the lacZ gene . The entire plasmid was sequenced by the chain termination method . Open reading frames were identified from the resulting consensus sequence together with a candidate for the plasmid origin of replication consisting of four perfect tandem repeats of a 22bp sequence, an A:T rich sequence and an open reading frame which could generate a 34.8kdal product . The predicted polypeptide products of the open reading frames were compared by computer with all reported protein sequences . Homology of the predicted polypeptide product of an open reading frame to the E . coli dnaB protein and the analogous product of gene 12 of bacteriophage P22 is described. Biochim Biophys Acta, 1988 May 6, 950(1), 75 - 80 Mapping and regulation of the pifC promoter of the F plasmid; Kennedy M et al.; The pif region of the F plasmid, which causes abortive infection of Escherichia coli by T7 bacteriophage, is autogenously controlled by the product of the pifC gene . Here we describe the identification of the pif operon promoter by S1-nuclease mapping, and show that it is autoregulated at the transcriptional level and that its activity is modulated by integration host factor. J Mol Biol, 1988 May 5, 201(1), 19 - 30 Conformational isomerization of the Holliday junction associated with a cruciform during branch migration in supercoiled plasmid DNA; Dickie P et al.; The variable positions of a branch-migrating cruciform junction in supercoiled plasmid DNA were mapped following cleavage of the DNA with bacteriophage T7 endonuclease I . T7 endonuclease I specifically cleaved, and thereby resolved, the Holliday junction existing at the base of the cruciform in the circular bacterial plasmid pSA1B.56A . Cruciform extrusion of cloned sequences in pSA1B.56A (containing a 322 base-pair inverted repeat insert composed of poxvirus telomeric sequences) topologically relaxed the plasmid substrate in vitro . Thus, numerous crossover positions were identified within the region of cloned sequences, reflecting the range of superhelical densities in the native plasmid preparation . Endonuclease I-sensitive crossover positions, mapped to both strands of the viral insert following the T7 endonuclease I digestion of either plasmid preparations or individual topoisomers, were regularly separated by approximately ten nucleotides . The appearance of sensitive crossovers every ten nucleotides corresponds to a change in linking difference (delta Lk) of +/- 2 in the circular core domain of the plasmid during branch point migration . In contrast, individual topoisomers of a plasmid preparation differ in linking number in increments of +/- 1 . Thus, the observed linearization of each individual topoisomer following enzyme treatment, as a result of resolution of the crossovers associated with each topoisomer, showed that branch point migration to sensitive crossover positions must have occurred facilely . T7 endonuclease I randomly resolved across either axis of the cruciform, though some discrimination (related to the sequence specificity of the enzyme) was observed . The ten-nucleotide spacing between sensitive crossover positions is accounted for by an isomerization of the cruciform junction on branch point migration . An hypothesis is that this isomerization was imposed upon the cruciform junction by the change in helix twist (delta Tw) in the two branches that compose the topologically closed, circular domain of the plasmid . T7 endonuclease I may discriminate between the various isomeric forms and cleave a sensitive conformation that appears with every turn of branch migration which leads to the extrusion, or absorption, of two turns of helix from the circular core. J Mol Biol, 1988 May 5, 201(1), 91 - 100 Bacteriophage T3 connector: three-dimensional structure and comparison with other viral head-tail connecting regions; Donate LE et al.; The bacteriophage T3 connector, which consists of 12 copies of protein gp8, has been studied by image processing of electron micrographs from negatively stained ordered aggregates . A three-dimensional reconstruction of T3 connectors was obtained by collection of tilted views and using the direct Fourier method, up to 2.3 nm resolution . The reconstructed unit cell contains two connectors whose main structural features are essentially identical, but facing in opposite directions . The T3 connector has a height of about 10.9 nm, with two clearly defined domains: a wider one 14.4 nm in diameter, with 12 morphological units in the periphery, and a narrower one, 9.7 nm in diameter . There is a channel clearly defined in the narrower domain that almost closes along the wider domain . Comparison of the three-dimensional structure obtained for the connector of phages T3 and phi 29, and that of the neck extracted from phage phi 29 particles, reveals striking similarities and significant differences . A model for a general connector to account for the common functions carried out by these viral assemblies is discussed together with the possible role of the channel for DNA translocation. J Mol Biol, 1988 May 5, 201(1), 115 - 25 Spatial arrangement of DNA-dependent RNA polymerase of Escherichia coli and DNA in the specific complex . A neutron small angle scattering study; Heumann H et al.; In this paper we demonstrate that neutron small angle scattering is a suitable method to study the spatial arrangement of large specific protein-DNA complexes . We studied the complex of DNA-dependent RNA polymerase of Escherichia coli and a 130 base-pair DNA fragment containing the strong promoter A1 of bacteriophage T7 . Contrast variation of the complex with deuterium allowed us to "visualize" either RNA polymerase, or DNA, or both components in situ . From the corresponding scattering curves information was derived about: (1) Conformational changes of RNA polymerase and DNA by complex formation: comparison of the scattering profiles of the isolated and complexed components showed that by specific complex formation the cross-section of RNA polymerase decreases, while the DNA fragment does not undergo a gross conformational change . (2) The spatial arrangement of RNA polymerase and DNA in the specific complex from the cross-sectional radii of gyration of the complex the normal distance dn between the centre of gravity of the RNA polymerase and the axis of the DNA fragment was derived as 5.0 (+/- 0.3) nm . On the basis of these and footprinting data a low resolution model of the RNA polymerase-promoter complex is proposed . The main feature of this model is the positioning of RNA polymerase to only one side of the DNA. J Biol Chem, 1988 May 5, 263(13), 6202 - 8 Effect of bacteriophage T4 DNA topoisomerase gene 39 on level of beta chain of ribonucleoside diphosphate reductase in a T4 nrdB mutant; Cook KS et al.; Bacteriophage T4 ribonucleoside diphosphate reductase consists of alpha 2 and beta 2 subunits encoded by genes nrdA and nrdB, respectively, and plays a central role in the T4-induced deoxyribonucleotide synthetase complex . The accompanying paper describes the decreased rate of synthesis of deoxyribonucleotides after infection by the T4 mutant, nrdB93, and the suppression of this defect by a second mutation in gene 39, coding for one of the three protein chains of T4 DNA topoisomerase . In this study we examined these effects at the protein level . On infection by nrdB93 not only was the beta 93 protein chain altered, as shown by its migration relative to the wild type protein in electrophoretic gels and by its temperature sensitivity, but the infected cells showed very low levels of the protein . However, on infection with the double mutant of nrdB93 and 39-01 (gene 39) the concentration of beta 93 chain returned to the values of beta protein found with wild type phage . A double mutant bearing nrdB93 and an amber mutation of gene 39 also suppressed the nrdB93 defect . By contrast, a temperature-sensitive mutant of gene 39, A41, did not show suppression at either 30 or 41 degrees C . Amber mutations in the two other genes coding for T4 DNA topoisomerase, 52 and 60, did not suppress the defect . We propose that the deficiency in the quantity of beta 93 chain and the suppression of this defect occur at the transcriptional or translational expression of the nrdB93 gene and that a specific domain of the gene 39 protein, not acting in the capacity of T4 DNA topoisomerase, inhibits the expression. J Biol Chem, 1988 May 5, 263(13), 6193 - 201 Defect in synthesis of deoxyribonucleotides by a bacteriophage T4 nrdB mutant is suppressed on mutation of T4 DNA topoisomerase gene; Wirak DO et al.; Bacteriophage T4 infection is known to induce the formation of a complex of enzymes effecting the de novo synthesis of deoxyribonucleoside triphosphates, which in turn are channeled into T4 DNA replication . The first step in this pathway is catalyzed by a ribonucleoside diphosphate reductase, comprised of subunits coded by T4 genes nrdA and nrdB . Maximum rates of synthesis of the pyrimidine deoxyribonucleotides and of DNA replication in vivo also require a type II DNA topoisomerase encoded by T4 genes 39, 52, and 60 . We report the identification of a unique mutant, nrdB93, and the suppression of its defective deoxyribonucleotide synthesis by a gene 39 mutation, 39-01 . After infection by 39-01, DNA synthesis and plaque formation were temperature-sensitive, but nearly wild type rates of deoxyribonucleotide synthesis were retained at all temperatures . The nrdB93 mutation had a profound effect on deoxyribonucleotide synthesis at 41 degrees C; even at the permissive temperature of 30 degrees C, synthesis was reduced to 30% of that of wild type or 39-01 . However, on infection at 30 degrees C by the double mutant, 39-01 nrdB93, the level of deoxyribonucleotide synthesis again reached that of wild type phage infections; involvement of the comparable host enzyme in the suppression process has been excluded . Suppression of the effect of nrdB93 by 39-01 implicates the gene 39 product in the regulation of nrdB expression . The accompanying paper (Cook, K . S., Wirak, D . O., Seasholtz, A . F., and Greenberg, G . R . (1988) J . Biol . Chem . 263, 6202-6208) examines the nature of the suppression process at the molecular level. Biochemistry, 1988 May 3, 27(9), 3210 - 5 Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication; Kodadek T et al.; We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA . The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme . The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA . When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion . In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site . We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand. Genetika, 1988 May, 24(5), 791 - 802 {The casein genes of Bos taurus . II . Isolation and characteristics of the beta-casein gene}; Gorodetskii SI et al.; The expression of the casein genes in mammary gland cells is regulated by peptide and steroid hormones . To study underlying regulatory mechanisms, the bovine beta-casein gene was isolated and characterized from lambda bacteriophage bovine DNA library . The beta-casein gene is 8.6 kb long and is 7.8 times longer than the mature casein mRNA coded for by 9 exons . The genomic clones incorporate additional 8.5 and 4.5 kb of the 5'- and 3'-flanking regions . The nucleotide sequences of 5' and 3' ends of the beta-casein gene are determined . Conserved sequences identical or homologous to potential sites of binding with the nuclear factor CTF/NF-1, glucocorticoid and progesterone receptors were identified . The regulatory region of the casein gene contains two different TATA signals flanking the duplication site in the promoter region. Virology, 1988 May, 164(1), 81 - 90 The structure of three bacteriophage T4 genes required for tail-tube assembly; Ishimoto LK et al.; Three different protein molecules copurify with T4 tail tubes after the tubes are released from the baseplate by guanidine hydrochloride treatment . These tube-associated proteins (TAPs) are the products of genes 29, 48, and 54 . To further investigate the structural roles that these proteins may play in T4 tail assembly we have cloned and sequenced the genes coding for these proteins and have deduced their predicted amino acid sequences . The sequence data reveal a region of amino acid sequence similarity between gp54 and the T4 tail-tube structural protein, gp19 . We believe that this region of similarity is significant and consistent with the role gp54 may play in initiating T4 tail-tube polymerization. Mutat Res, 1988 May, 199(1), 183 - 90 Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an 'uncloneable' phenotype; Mita S et al.; Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors . For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome . All of 50 recombinant M13 clones containing this 'uncloneable' fragment had one or more changes in nucleotide sequence . Each clone contained at least one alteration in two nucleotide positions within the tRNAThr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNAThr . These results imply that the HeLa mitochondrial tRNAThr gene is responsible for the 'uncloneable' phenotype of this region of human mitochondrial (mt) DNA . A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNAThr gene . 56 mutations were single-base substitutions; 5 were deletions . Approximately 80% of the base substitution mutations were A:T----G:C transitions . A preference for A:T----G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals . This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin. J Bacteriol, 1988 May, 170(5), 2276 - 82 Transduction of plasmid DNA in Streptomyces spp . and related genera by bacteriophage FP43; McHenney MA et al.; A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus . The transducing particles contained linear concatemers of plasmid DNA . Lysates of FP43 prepared on S . griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA . Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared . FP43 lysates prepared on S . griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora. Appl Environ Microbiol, 1988 May, 54(5), 1297 - 9 Selective stabilization by the bacteriophage 434 repressor of the plasmid expressing bovine growth hormone in Escherichia coli; Giladi H et al.; The maintenance of a plasmid vector-host system that selects for bacteria carrying the plasmid without the need for antibiotics is described . In this system, the bacteriophage 434 repressor gene cloned on the plasmid protects the host from lysis by a lambda imm434 cI- prophage . Cells that occasionally lose the plasmid are killed by prophage induction and therefore do not accumulate in the growing culture . The presence of the phage 434 repressor in the cells does not interfere with the process of lambda repressor inactivation and the high-level production of bovine growth hormone. Radiat Res, 1988 May, 114(2), 319 - 30 Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22; Hartman PE et al.; Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C) . T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+ . Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H2O2 or some product derived therefrom was predominant in causing inactivation of plaque formation . Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected . A similar effect was found for the polyamine, spermidine . In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection . L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man . L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals. J Virol, 1988 May, 62(5), 1723 - 9 Heat cleavage of bacteriophage T4 gene 23 product produces two peptides previously identified as head proteins; Robinson DR et al.; During studies on the intracellular protein pools of bacteriophage T4, we found that amber mutants in gene 23 blocked the synthesis of a 20-kilodalton (kDa) protein . Radiolabeled amino acid pulses showed that the protein appears at 8 min postinfection with kinetics similar to those of other major late species . Pulse-chase experiments demonstrated that the 20-kDa protein behaves like a primary product and also revealed a 29-kDa protein which, like other proteins cleaved during head assembly, appeared only after a long chase . Both species have been identified as constituents of the T4 head and have resisted previous efforts to identify their genetic origin . The dependence of the 20- and 29-kDa head proteins on the presence of gene 23 protein (gp23) and the observation that the sum of their masses equalled that of mature cleaved gp23 suggested that these two proteins were derived from this major capsid species . Evidence is presented demonstrating that heating samples before electrophoresis causes peptide bond cleavages in gp23, leading to the formation of the two peptides . As predicted by the results of Rittenhouse and Marcus (Anal . Biochem . 138:442-448, 1984), the cleavage occurs at Asp-336-Pro-337 and at two other Asp-Pro sites . Limited heat-induced proteolysis followed by two-dimensional gel analysis provided a peptide map of gp23 useful in the characterization of its assembly-related cleavages. J Gen Microbiol, 1988 May, 134 ( Pt 5), 1333 - 8 Charac