FEBS Lett, 1986 Nov 10, 208(1), 7 - 10 The phage Mu repressor c and IS30 transposase proteins are significantly related; Dalrymple B; The IS30 transposase exhibits significant amino acid sequence homology to the phage Mu repressor c in the amino- and carboxy-terminal regions of the proteins . The conserved sequences include the proposed Mu repressor DNA binding site, which is also related to the proposed Mu and D108 transposase DNA binding sites . The carboxy-terminal homologies are characterised by two almost complete, and one partial, somewhat diverged amino acid sequence repeats . Only weak homologies to this domain are present in the Mu transposase (Mu A) . Nevertheless, a clear link between an insertion sequence and a bacteriophage has been established. Biochemistry, 1986 Nov 4, 25(22), 6768 - 78 N-terminal domain of the bacteriophage lambda repressor: investigation of secondary structure and tyrosine hydrogen bonding in wild-type and mutant sequences by Raman spectroscopy; Thomas GJ Jr et al.; Laser Raman spectroscopy has been employed to investigate structures of the lambda repressor N-terminal fragment, which recognizes operator DNA . Examination of repressor fragments containing deuterated amide groups and specifically labeled deuteriotyrosines has enabled the assignment of many of the conformation-sensitive Raman bands . By use of Fourier deconvolution and signal averaging techniques, the spectra of both wild-type and mutant sequences have been obtained as a function of the total protein concentration in aqueous solution over the range 5-100 mg/mL . This analysis has permitted monitoring of the monomer-dimer association of the repressor fragment and determination of the effects of dimerization upon individual side-chain interactions and main-chain secondary structure . The spectra are interpreted to reveal the hydrogen-bonding environments of four tyrosines of the N-terminal fragment (Y22, Y60, Y85, and Y88) . The fifth tyrosine (Y101) is known from NMR experiments to be exposed to solvent molecules . The results show that in the dimer Y22 and Y85 are each acceptors of a strong hydrogen bond from a positive donor group, while Y88 is the donor of a strong hydrogen bond to a negative acceptor and Y60, like Y101, is involved in both a donor role and an acceptor role . Y60, Y85, and Y88, which are all near the dimer interface, undergo a collective change in hydrogen-bonding environment with dissociation of the dimer . The net effect of this change is the conversion of one acceptor tyrosine, deduced to be Y88, to a combined donor and acceptor role . The Raman results also indicate a predominantly alpha-helical structure for the N-terminal fragment in aqueous solution, with 70 +/- 4% of the residues incorporated into helical domains . The amount of alpha-helix determined from the Raman spectrum is consistent with X-ray and prediction results and is altered neither by the mutations C85----Y85 and C88----Y88 nor by dissociation of the dimer. Eur J Biochem, 1986 Nov 3, 160(3), 571 - 8 DNA synthesis in vitro with an endoplasmic-reticulum-DNA-polymerase complex from unfertilized sea urchin eggs; Shioda M; An endoplasmic-reticulum-DNA-polymerase complex was prepared from unfertilized sea urchin eggs and its DNA-synthesizing activity was examined using single-stranded DNA of bacteriophage fd as a template . The complex catalyzed the ribonucleotide-dependent DNA synthesis which required dNTPs, NTPs, Mg2+ and single-stranded DNA . The DNA synthesis was sensitive to aphidicolin and N-ethylmaleimide but was resistant to 2',3'-dideoxyribosylthymine 5'-triphosphate (ddTTP) and alpha-amanitin, suggesting the involvement of DNA polymerase alpha . In parallel with the DNA synthesis, a small amount of RNA was synthesized in the presence of 100 micrograms/ml alpha-amanitin . The Km value of ribonucleotides for the RNA synthesis coincided with that for the DNA synthesis, suggesting a correlation between the DNA and RNA syntheses . Labelling of the products with {gamma-32P}ATP followed by DNA digestion with pancreatic DNase I revealed the attachment of an oligoribonucleotide (7-11 bases in length) at the 5' ends of the DNA products . These observations suggest that in DNA synthesis, primer RNA synthesis occurs first, followed by DNA chain elongation . During 1-90-min incubation, the amount of the DNA synthesized increased but the length was not significantly increased . Over 80% of the number of synthesized DNA molecules comprised a single population of short DNA fragments (60-200 bases, on average 120 bases in length) and the number of fragments increased, depending on the incubation time . However, DNA fragments of various sizes (about 100-6000 bases) were synthesized with DNA polymerase alpha solubilized from the endoplasmic-reticulum-DNA-polymerase complex . All this evidence suggests that in vitro, the complex preferentially synthesizes a particular size of short DNA fragments . The significance of the fragments is discussed. J Bacteriol, 1986 Nov, 168(2), 833 - 8 Molecular proof that bacteriophage T4 alc and unf genes are the same gene; Snyder L et al.; The DNA of bacteriophage T4 normally has a substituted base, hydroxymethylcytosine, instead of the usual cytosine . The bacteriophage shuts off host transcription after infection presumably by specifically blocking transcription of cytosine DNA . If T4 incorporates cytosine into its own DNA, this shutoff mechanism is directed back at itself and blocks its own transcription . Mutations which overcome this transcriptional block are in the T4 alc gene, and alc mutations allow the propagation of T4 with cytosine in their DNA (L . Snyder, L . Gold, and E . Kutter, Proc . Natl . Acad . Sci . USA 73:3098-3102, 1976) . By genetic criteria, alc is the same as another gene, unf, whose product is required for the unfolding of the bacterial nucleoid after infection (K . Sirotkin, J . Wei, and L . Snyder, Nature {London} 265:28-32, 1977; D . P . Snustad, M . A . Tigges, K . A . Parson, C . J . H . Bursch, F . M . Caron, J . F . Koerner, and D . J . Tutas, J . Virol . 17:622-641, 1976; M . Tigges, C . J . H . Bursch, and D . P . Snustad, J . Virol . 24:775-785, 1977) . The product of the alc gene has been identified as a 19-kilodalton protein (R . E . Herman, N . Haas, and D . P . Snustad, Genetics 108:305-317, 1984; E . Kutter, R . Drivdahl, and K . Rand, Genetics 108:291-304, 1984), and an open reading frame has been proposed to be the alc gene based on its size and map position (E . Kutter, R . Drivdahl, and K . Rand, Genetics 108:291-304, 1984) . We used marker rescue techniques and DNA sequencing to confirm that this open reading frame is the alc gene . We also present a molecular proof that alc and unf are the same gene . While these results do not rigorously exclude the possibility that Unf and Alc are different activities of the same protein, they strongly support the conclusion that the unfolding of the bacterial nucleoid the blockage of transcription are but different manifestations of the same activity. J Nat Prod, 1986 Nov-Dec, 49(6), 971 - 80 The use of recombinant DNA techniques to study tylosin biosynthesis and resistance in Streptomyces fradiae; Cox KL et al.; A substantial amount of information on the biosynthesis of tylosin has been obtained over the past ten years . Physiological studies and experiments with tylosin-blocked (tyl) mutants have suggested the probable pathway by which tylactone is converted to tylosin . The development of recombinant DNA methodology for streptomycetes in general, and for Streptomyces fradiae in particular, has allowed us to apply gene cloning techniques in further studies of tylosin biosynthesis in S . fradiae . The macrocin O-methyltransferase (MOMT), which catalyzes the last step in tylosin biosynthesis, was purified, and the sequence of the 35 amino acids at its amino-terminus was determined . A synthetic 44 base oligonucleotide probe was constructed on the basis of the amino acid sequence . The probe was used to identify sequences containing the MOMT structural gene in bacteriophage and cosmid libraries of S . fradiae DNA . Complementation of tyl mutants with the cloned DNA sequences identified nine tyl biosynthetic genes (tylC, D, E, F, H, J, K, L, and M) in a 42 kb stretch of DNA . Genes complementing four mutant classes, tylA, B, G, and I were not found . A tylosin-resistance gene, tlrB, was located just left of the tyl gene cluster . Tylosin-sensitive mutants of S . fradiae, which were isolated from regenerated protoplasts and which have pleiotropic deficiencies in tylosin biosynthesis, contained deletions which included at least some of the identified tyl loci and one or both of two tylosin-resistance genes, tlrB and tlrC . Possible schemes for the functional organization of the tyl region of the S . fradiae genome are discussed. Genetics, 1986 Nov, 114(3), 705 - 16 Frameshift suppression by thyA mutants of Escherichia coli K-12; Herrington MB et al.; We have extended our previous study on the suppression of frameshift mutants by Escherichia coli thyA mutants by assaying suppression of 15 rIIB frameshift mutants of bacteriophage T4 on one of our suppressing thyA mutant strains . The majority of insertion mutants were suppressible, whereas none of the deletion mutants tested was suppressible . Frameshift suppression could be inhibited by adding thymidine to the assay medium, but was not affected by the presence of a restrictive rpsL mutation in the host strain . We suggest that the frameshift suppression event occurs at a nonsense codon generated by the frameshift mutation. J Bacteriol, 1986 Nov, 168(2), 534 - 40 New locus (ttr) in Escherichia coli K-12 affecting sensitivity to bacteriophage T2 and growth on oleate as the sole carbon source; Morona R et al.; The nature of resistance to phage T2 in Escherichia coli K-12 was investigated by analyzing a known phage T2-resistant mutant and by isolating new T2-resistant mutants . It was found that mutational alterations at two loci, ompF (encoding the outer membrane protein OmpF) and ttr (T-two resistance), are needed to give full resistance to phage T2 . A ttr::Tn10 mutation was isolated and was mapped between aroC and dsdA, where the fadL gene (required for long-chain fatty acid transport) is located . The receptor affected by ttr was the major receptor used by phage T2 and was located in the outer membrane . Phage T2 was thus able to use two outer membrane proteins as receptors . All strains having a ttr::Tn10 allele and most of the independently isolated phage T2-resistant mutants were unable to grow on oleate as the sole carbon and energy source, i.e., they had the phenotype of fadL mutants . The gene fadL is known to encode an inner membrane protein . The most likely explanation is that fadL and ttr are in an operon and that ttr encodes an outer membrane protein which functions in translocating long-chain fatty acids across the outer membrane and also as a receptor for phage T2. J Bacteriol, 1986 Nov, 168(2), 1014 - 8 Expression of the bacteriophage T4 denV structural gene in Escherichia coli; Recinos A 3rd et al.; The expression of the T4 denV gene, which previously had been cloned in plasmid constructs downstream of the bacteriophage lambda hybrid promoter-operator oLpR, was analyzed under a variety of growth parameters . Expression of the denV gene product, endonuclease V, was confirmed in DNA repair-deficient Escherichia coli (uvrA recA) by Western blot analyses and by enhancements of resistance to UV irradiation. Proc Natl Acad Sci U S A, 1986 Nov, 83(22), 8452 - 6 Gene 32 protein, the single-stranded DNA binding protein from bacteriophage T4, is a zinc metalloprotein; Giedroc DP et al.; Gene 32 protein (g32P) isolated from bacteriophage T4-infected Escherichia coli and from an overproduction vector derived from the plasmid pKC30 contains 1 mol of tightly incorporated Zn(II) per mol of protein . A linear incorporation of three molar equivalents of p-hydroxymercuriphenylsulfonate (PMPS) results in a linear release of 1.1 mol of Zn(II) from the protein . Reversal of formation of the g32P-PMPS complex with thiol in the presence of EDTA results in a zinc-free apo-g32P . Cd(II) and Co(II) can be exchanged with the intrinsic Zn(II) ion . The Cd(II) protein shows a charge-transfer band at approximately 250 nm . The Co(II) protein shows a set of absorption bands typical of a tetrahedral Co(II) complex (epsilon max = 660 M-1 X cm-1 at 645 nm), and two intense charge-transfer bands are present at 355 nm (epsilon = 2,250 M-1 X cm-1) and 320 nm (epsilon = 3,175 M-1 X cm-1) . These observations are consistent with three cysteines as ligands to the Zn(II) ion in g32P . Zn(II) g32P undergoes precise limited proteolysis by trypsin to produce the small fragments A and B and the core, g32P-(A + B) . Under identical conditions, apo-g32P is hydrolyzed rapidly beyond the g32P-(A + B) stage to produce many proteolyzed fragments . Fluorescence quenching experiments show that at low protein concentration apo-g32P has markedly altered binding affinity for poly(dT) relative to native g32P . Three of the four cysteines of g32P are found in a tyrosine-rich sequence corresponding to residues 72-116 and implicated in DNA binding by 1H NMR investigations . Zn(II) appears to provide a conformational element contributing to DNA binding by coordinating the cysteine and possibly histidine side chains in the sequence -Cys-X3-His-X5-Cys-X2-Cys-, residues 77-90, located in the DNA binding domain of g32P. Proc Natl Acad Sci U S A, 1986 Nov, 83(22), 8501 - 5 Mutagenic potential of O4-methylthymine in vivo determined by an enzymatic approach to site-specific mutagenesis; Preston BD et al.; O4-Alkylthymine-DNA adducts have been implicated as causative lesions in chemical mutagenesis and carcinogenesis . To directly assess the mutagenic potential of these adducts in vivo, we have designed an enzymatic technique for introducing nucleotide analogues at predetermined sites of biologically active DNA . Escherichia coli DNA polymerase I was used in vitro to incorporate a single O4-methylthymine residue at the 3' terminus of an oligonucleotide primer opposite the adenine residue of the amber codon in bacteriophage phi X174 am3 DNA . After further extension of the primer with unmodified nucleotides, the partial-duplex product was transfected into E . coli spheroplasts . Replication of the site-specifically methylated DNA in E . coli deficient in O4-methylthymine-DNA methyltransferase (ada-) yielded 10-fold more mutant progeny phage than replication of nonmethylated DNA; no increase in mutation frequency was observed after replication in repair-proficient (ada+) E . coli . The DNA from 20 independently isolated mutant plaques all contained A.T----G.C transitions at the original site of O4-methylthymine incorporation . These data demonstrate that O4-methylthymine induces base-substitution mutations in E . coli and suggest that this adduct may be involved in mutagenesis by N-nitroso methylating agents . This enzymatic technique for site-specific mutagenesis provides an alternative to the chemical synthesis of oligonucleotides containing altered bases. Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8064 - 8 Isolation and sequence determination of a cDNA clone related to human cytochrome P-450 nifedipine oxidase; Beaune PH et al.; Human liver cytochrome P-450NF is the form of cytochrome P-450 responsible for the oxidation of the calcium-channel blocker nifedipine, which has been reported to show polymorphism in clinical studies . By screening a bacteriophage lambda gt11 expression cDNA library, we isolated two clones: NF95 with an insert length of 0.8 kilobases which gave a stable fusion protein and NF25 with an insert length of 2.2 kilobases . The two clones were both sequenced and shown to be identical in their overlapping section . The sequence of NF25 is 77% similar to that reported for a rat cytochrome "P-450PCN" cDNA (PCN = pregnenolone-16 alpha-carbonitrile) . The similarity decreases to 45-53% when the sequence is compared to human cytochromes P-450 belonging to other families {i.e., "pH P-450(1)," "P1-450," "P3-450," and "P-450MP." The deduced amino acid sequence is 73% similar to that of rat cytochrome P-450PCN, and the first 21 amino acids are identical to those reported for human liver cytochrome "P-450p." Sections of these clones were nick-translated and used as probes for analyses of human mRNA and genomic DNA . The number and size of bands indicate that P-450NF belongs to a multigene family, the so-called pregnenolone-16 alpha-carbonitrile-inducible family. Virology, 1986 Nov, 155(1), 289 - 92 Interaction of the bacteriophage phi 29 connector protein with the viral DNA; Herranz L et al.; The protein that forms the connector of phage phi 29, p10, binds to DNA . Apparently, p10 binding is not sequence specific . Nevertheless, the presence of the terminal protein (p3) covalently attached to the ends of phi 29 DNA produces a significant increase of p10 molecules bound to the DNA ends, thus suggesting a terminal protein-mediated recognition of DNA ends by the phage connector . As the p3-DNA complex is the substrate for phage phi 29 DNA packaging, these results may reflect a direct implication of the phage connector in the packaging process. Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8122 - 6 Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase; Fuerst TR et al.; DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome . The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells . Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions . When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels . Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region . The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system. Mol Gen Mikrobiol Virusol, 1986 Nov, (11), 14 - 8 {Irreversible changes in phage DNA after its protonation in solution and inside the virion detected by the transfection method}; Sukhorukov BI et al.; The dependence of irreversible structural changes in phage lambda DNA on the degree of its protonation in a solution and inside the virion has been found by measuring the transfection activity of bacteriophage . The different effect of ionic strength on pH-dependence of the irreversible changes in the structure of DNA upon its protonation in a solution or in situ has been registered and explained . The insignificant shift of pH from neutral region value in 0.1 M NaCl has resulted in a damaging effect of H+ ions on compact DNA in situ as compared to the DNA in a solution . The effect of H+ ions on compact DNA in situ is mainly based on the formation of noncovalent intermolecular DNA-protein and DNA-DNA linkages. Mol Biol (Mosk), 1986 Nov-Dec, 20(6), 1638 - 44 {The effect of the phage lambda ral gene on the level of synthesis of the EcoK restriction endonuclease beta-subunit}; Zinkevich VE et al.; E . coli hsd genes were subcloned from lambda 642 (ral+) into lambda L47.1 vector (ral-after replacement) . The influence of bacteriophage lambda ral gene on the expression efficiency of hsdS kappa, hsdM kappa genes was investigated . It was shown, that its presence in vitro enhanced the synthesis of beta-subunit, hsdM gene product, and the increase of modification in vivo was observed . It is proposed that the increase of modification rate of lambda phage fully unmodified DNA is connected with the appearance of E . coli DNA methylase consisting of beta- and gamma-subunits but lacking alpha-subunit. Genetika, 1986 Nov, 22(11), 2572 - 82 {Bacterial transposons and the mechanisms of their transposition}; Il'ina TS; The published data on molecular mechanisms of transposons movement inside and between genomes are reviewed . The replicative mechanism of transposition of the family of Tn3-like elements is discussed, as well as the modes of bacteriophage Mu, Tn9, Tn10, Tn903 transposition . The factors affecting the choice of transposition pathways are analysed. J Virol, 1986 Nov, 60(2), 787 - 92 Cloning the complete rIIB gene of bacteriophage T4 and some observations concerning its middle promoters; Shinedling S et al.; We cloned the intact T4 rIIB gene by joining plasmids carrying gene fragments . rIIB was expressed at a low level under control of the lac promoter, and the clone complemented rIIB mutants . We suspect that earlier attempts to clone the intact gene were unsuccessful because of transcription from T4 middle-mode promoters . These promoters are silent early in infection but are recognized when resident on a plasmid in an uninfected cell. J Virol, 1986 Nov, 60(2), 436 - 49 Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment; Rosel JL et al.; The sequence of the 8,600-base-pair HindIII H fragment, located at the center of the vaccinia virus genome, was determined to analyze several late genes . Seven major complete open reading frames (ORFs) and two that started from or continued into adjacent DNA segments were identified . ORFs were closely spaced and present on both DNA strands . Some adjacent ORFs had oppositely oriented overlapping termination codons or contiguous stop and start codons . Nucleotide compositional analysis indicated that the A-T frequency was consistently lowest in the first codon position . The sizes of the polypeptides predicted from the DNA sequence were compared with those determined by polyacrylamide gel electrophoresis of cell-free translation products of mRNAs selected by hybridization to cloned single-stranded DNA segments or synthesized in vitro by bacteriophage T7 RNA polymerase . Six transcripts that initiated within the HindIII H DNA fragment were detected, and of these, four were synthesized only at late times, one was synthesized only early, and one was synthesized early and late . The sites on the genome corresponding to the 5' ends of the transcripts were located by high-resolution nuclease S1 analysis . For late genes, the transcriptional and translational initiation sites mapped within a few nucleotides of each other, and in each case the sequence TAAATGG occurred at the start of the ORF . The extremely short leader and the absence of A or G in the -3 position, relative to the first nucleotide of the initiation codon, distinguishes the majority of vaccinia virus late genes from eucaryotic and vaccinia virus early genes. Mol Biol (Mosk), 1986 Nov-Dec, 20(6), 1562 - 9 {1H-NMR study of the OR1 operon in bacteriophage lambda . I . Assignment of signals from imino protons and adenine C2 protons}; Kirpichnikov MP et al.; An oligodeoxyribonucleotide composed of 17 residues, d(TATCACCGCCAGAGGTA), and a complementary chain were synthesized . Their duplex was identical with the operator OR1, the binding site for bacteriophage lambda cro and c1 repressors . The 1H NMR spectra (500 MHz) of the duplex imino and aromatic protons were studied at 10, 20 and 25 degrees C . Signals from the imino protons of complementary base pairs and from the C2 protons of adenine (with the exception of the duplex terminal nucleotides) were assigned using the NOE technique and the known characteristics of short DNA fragment melting . No signals from the imino protons of the terminal base pairs were detected even at 10 degrees C due to fraying which increased as the temperature was raised . The assignment of signals can be used to identify centers of interaction between the operator OR1 and repressors, as well as to study possible local changes in DNA geometry. J Virol, 1986 Nov, 60(2), 564 - 8 Ion etching of bacteriophage lambda: evidence that the right end of the DNA is located at the outside of the phage DNA mass; Brown JC et al.; Bacteriophage lambda was etched in an Ar+ plasma under conditions in which the capsid and some of the DNA were eroded (by sputtering) from the particle surface . Analysis of the DNA remaining in etched phage demonstrated an enrichment in sequences derived from the left end and middle of the genome; sequences from the right end were selectively lost . The results suggest that the DNA in the mature phage is arranged with its left end toward the center and its right end toward the exterior of the overall DNA mass . Since the left end is the first to enter the phage prohead, the results are most compatible with the view that prohead filling also proceeds from the center to the exterior of the cavity . The suggested arrangement of lambda DNA is comparable to that observed in phage T4 and is consistent with the spiral-fold model of packaged DNA. J Bacteriol, 1986 Nov, 168(2), 1048 - 50 Secondary attachment site for bacteriophage lambda in the guaB gene of Escherichia coli; Thomas MS et al.; lambda gua transducing bacteriophages were used to identify and sequence the secondary attachment site for lambda in the guaB gene of Escherichia coli . The sequence matched the primary core sequence at nine positions, and a putative integrase binding-site overlapped the left core-arm junction . Recombinational crossover occurred between nucleotides -3 and +2 of the core region. Genetics, 1986 Nov, 114(3), 669 - 85 Bacteriophage T4 endonucleases II and IV, oppositely affected by dCMP hydroxymethylase activity, have different roles in the degradation and in the RNA polymerase-dependent replication of T4 cytosine-containing DNA; Carlson K et al.; Bacteriophage T4 mutants defective in gene 56 (dCTPase) synthesize DNA where cytosine (Cyt) partially or completely replaces hydroxymethylcytosine (HmCyt) . This Cyt-DNA is degraded in vivo by T4 endonucleases II and IV, and by the exonuclease coded or controlled by genes 46 and 47.-Our results demonstrate that T4 endonuclease II is the principal enzyme initiating degradation of T4 Cyt-DNA . The activity of endonuclease IV, but not that of endonuclease II, was stimulated in the presence of a wild-type dCMP hydroxymethylase, also when no HmCyt was incorporated into phage DNA, suggesting the possibility of direct endonuclease IV-dCMP hydroxymethylase interactions . Endonuclease II activity, on the other hand, was almost completely inhibited in the presence of very small amounts of HmCyt (3-9% of total Cyt + HmCyt) in the DNA . Possible mechanisms for this inhibition are discussed.-The E . coli RNA polymerase modified by the products of T4 genes 33 and 55 was capable of initiating DNA synthesis on a Cyt-DNA template, although it probably cannot do so on an HmCyt template . In the presence of an active endonuclease IV, Cyt-DNA synthesis was arrested 10-30 min after infection, probably due to damage to the template . Cyt-DNA synthesis dependent on the unmodified (33-55-) RNA polymerase was less sensitive to endonuclease IV action. EMBO J, 1986 Nov, 5(11), 3029 - 37 Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface; Charbit A et al.; The LamB protein is a trimeric integral outer membrane protein from Escherichia coli K12 which functions as a pore for maltodextrins and a receptor for bacteriophages . When inserted into two selected sites of LamB, a foreign antigen, the C3 epitope from poliovirus, was exposed at the cell surface with its normal antigenic properties . Since these genetic insertions did not affect in any essential way the routing, activity and folding of the LamB protein, we conclude that the two corresponding LamB sites are at the cell surface as predicted by our recent model . We discuss the implications of our results for the study of protein topology with a single epitope and the direct cloning and cell surface expression of epitopes of interest as well as the development of live vaccines or diagnostic tests. Virology, 1986 Oct 30, 154(2), 357 - 68 Termination and reinitiation signals of bacteriophage phi X174 rolling circle DNA replication; Fluit AC et al.; The nucleotide sequence requirements for termination and reinitiation of rolling circle DNA replication within the 30-bp phi X174 origin region were studied . Plasmids were constructed which contained a complete and a partial phi X174 origin region in the same orientation . The partial origin consisted of the first 16, 24, 25, 26, 27, or 28 bp of the origin region . Plasmids harboring a complete origin region are subject to rolling circle DNA replication and packaging of single-stranded plasmid DNA into phage coats in phi X174 or G4 phage infected cells . The plasmids with a complete and partial origin region were tested in these in vivo transduction systems . The results lead to the following conclusions: The phi X174 and G4 in vivo transduction systems are useful in studying termination and reinitiation of rolling circle DNA replication . The first 24 bp of the origin region are sufficient for termination of a round of rolling circle DNA replication coupled to DNA packaging . The first 16 bp, however, are not recognized as a termination signal . Reinitiation of rolling circle DNA replication coupled to DNA packaging on a partial origin region occurs with low frequency. J Biol Chem, 1986 Oct 25, 261(30), 14256 - 65 Transcription at bacteriophage T4 variant late promoters . An application of a newly devised promoter-mapping method involving RNA chain retraction; Kassavetis GA et al.; Bacteriophage T4 late promoters display a conserved sequence that extends over about 18 base pairs, in which the central 8-base pair sequence (TATAAATA in the nontranscribed strand) is absolutely conserved . Transcription by T4-modified RNA polymerase in vitro nevertheless initiates within a cluster of 6 overlapping variant T4 late promoters that deviate from the absolutely conserved sequence at one or two positions . One of these variant promoters is dominant, and its sequence implies that two of the absolutely conserved nucleotides (underlined above) are not essential . Three other variant promoters that contain only one of the deviations found in the dominant variant promoter are, at best, utilized weakly, suggesting that sequences outside the absolutely conserved segment are important for promoter function . A newly devised method, based on arrest of RNA chain elongation with ribonucleotide analogs and its reversal, has been used to precisely map initiation within the overlapping promoter cluster . Multiple cycles of RNA chain arrest, pyrophosphorolysis, and resynthesis can be executed . This process permits a precise stepwise control of the advance of a transcription complex through a gene. J Mol Biol, 1986 Oct 20, 191(4), 601 - 13 Spectrum of spontaneous frameshift mutations . Sequences of bacteriophage T4 rII gene frameshifts; Ripley LS et al.; The DNA sequences of 185 independent spontaneous frameshift mutations in the rIIB gene of bacteriophage T4 are described . Approximately half of the frameshifts, including those at hot spot sites, are fully consistent with classical proposals that frameshift mutations are produced by a mechanism involving the misaligned pairing of repeated DNA sequences . However, the remaining frameshifts are inconsistent with this model . Correlations between the positions of two base-pair frameshifts and the bases of DNA hairpins suggest that local DNA topology might influence frameshift mutation . Warm spots for larger deletions share the property of having endpoints adjacent to DNA sequences whose complementarity to sequences a few base-pairs away suggest that non-classical DNA misalignments may participate in deletion mutation . A model for duplication mutation as a consequence of strand displacement synthesis is discussed . In all, 15 frameshifts were complex combinations of frameshifts and base substitutions . Three of these were identical, and have extended homology to a sequence 256 base-pairs away that is likely to participate in the mutational event; the remainder are unique combinations of frameshifts and transversions . The frequency and diversity of complex mutants suggest a challenge to the assumption that the molecular evolution of DNA must depend primarily upon the accumulation of single nucleotide changes. J Mol Biol, 1986 Oct 20, 191(4), 589 - 99 Mitochondrial gene URFN of Neurospora crassa codes for a long polypeptide with highly repetitive structure; Burger G et al.; The mitochondrial DNA of Neurospora crassa contains a long potential gene, designated URFN, which is located immediately downstream from the CO1 gene . These two genes are encoded in different reading frames and overlap by 13 codons . URFN is 633 triplets long and terminates at a UAG stop codon . Its codon usage is atypical for N . crassa mitochondrial exons and introns, and resembles that of the long open reading frame (ORF) of the mitochondrial plasmid present in N . crassa strain Mauriceville . Multiple sequence repetitions occur in the presumptive URFN polypeptide, most notably a seven-times reiterated motif of 16 to 18 amino acid residues length . The hydropathy pattern shows that the N-terminal third of the URFN polypeptide is predominantly apolar and includes several potentially membrane-spanning stretches; the remaining part is hydrophilic . Calculation of the secondary structure predicts a high proportion (47%) of alpha-helix conformation . The longest alpha-helix contains 40 residues . No similarities to other mitochondrial genes or reading frames have been found, except a significant homology over a stretch of 16 amino acid residues between the N-terminal part of URFN and a well-conserved sequence in the C-terminal region of CO1 . The repetitive region in URFN resembles a similarly repetitive stretch in an unassigned reading frame from bacteriophage lambda . Three arguments support the view that URFN is translated . The open reading frame has a considerable length; URFN is transcribed into a mRNA including the overlapping CO1 gene; URFN is most probably conserved among all the various Neurospora species examined thus far, strongly suggesting that it codes for an essential protein. Virology, 1986 Oct 15, 154(1), 168 - 79 Physical characterization of the genome of feline herpesvirus-1; Rota PA et al.; The physical structure of the genome of feline herpesvirus-1, a major upper respiratory tract pathogen of cats, was studied . Purified FHV-1 DNA was analyzed by restriction endonuclease and gamma 5' exonuclease digestion, blot hybridization, and electron microscopy . To facilitate further studies, nine bacteriophage clones were isolated which contained 85% of the viral genome as SalI inserts, and DNA from these clones was used in blot hybridization experiments and as substrates for restriction digest analysis . Data from these studies permitted construction of a SalI and partial HindII and EcoRI restriction maps of the viral genome . FHV-1 DNA is approximately 134 kb in size and is composed of a long (L) and a short (S) segment . The long segment (U1) is 104 kb in size and is composed of unique DNA . The adjacent S segment is approximately 30 kb in size and contains a central portion of unique DNA (Us) which is approximately 8 kb in size . The Us region is bounded by inverted repeat sequences which are 11 kb in size . Therefore, the physical structure of the FHV-1 genome is similar to the genomes of other alpha-herpesviruses. Eur J Biochem, 1986 Oct 15, 160(2), 363 - 70 Multiple crosslinks of proteins S7 and S9 to domains 3 and 4 of 16S ribosomal RNA in the Escherichia coli 30S particle; Chiaruttini C et al.; RNA-protein cross-links were introduced into Escherichia coli 30S subunits by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide . 16S rRNA, cross-linked to 30S ribosomal proteins, was isolated and hybridized with seven single-stranded bacteriophage M13-DNA probes . These probes, each carrying an inserted rDNA fragment, were used to select contiguous RNA sections covering domains 3 and 4 (starting at nucleotide 868 and ending at the 3'OH terminus) of the 16S rRNA . The proteins covalently linked to each selected RNA section were identified by two-dimensional polyacrylamide gel electrophoresis . Proteins S7 and S9 were shown to be efficiently cross-linked to multiple sites belonging to both domains. Cell, 1986 Oct 10, 47(1), 81 - 7 Multiple self-splicing introns in bacteriophage T4: evidence from autocatalytic GTP labeling of RNA in vitro; Gott JM et al.; RNA from T4-infected cells yielded multiple end-labeled species when incubated with alpha-32P-GTP under self-splicing conditions . One of these corresponds to the previously identified intron from the td gene of T4, while others appear to represent additional group I introns in T4 . Two loci distinct from the td gene were found to hybridize to a mixed alpha-32P-GTP-labeled T4 RNA probe . These mapped in or near the unlinked genes nrdB and nrdC . A fragment from the nrdB region that contains the intron has been cloned and shown to generate characteristic group I splice products with RNA synthesized in vivo and in vitro . Multiple introns, and the prospect that these occur within several genes in the same metabolic pathway, suggest a possible regulatory role for splicing in T4. Nucleic Acids Res, 1986 Oct 10, 14(19), 7751 - 65 Nucleotide sequence of a type II DNA topoisomerase gene . Bacteriophage T4 gene 39; Huang WM; T4 DNA topoisomerase is a type II enzyme and is thought to be required for normal T4 DNA replication T4 gene 39 codes for the largest of the three subunits of T4 DNA topoisomerase . I have determined the nucleotide sequence of a region of 2568 nucleotides of T4 DNA which includes gene 39 . The location of the gene was established by the identification of the first fifteen amino acids in the large open reading frame in the DNA sequence as those found at the amino-terminus of the purified 39-protein . The coding region of gene 39 has 1560 bases, and it is followed by two in-frame stop codons . The gene is preceded by a typical Shine-Dalgarno sequence as well as possible promoter sequences for E . coli RNA polymerase . T4 39-protein consists of 520 amino acids, and it has a calculated molecular weight of 58,478 . By comparing the amino acid sequences, T4 39-protein is found to share homology with the gyrB subunit of DNA gyrase . This suggests that these topoisomerase subunits may be equivalent functionally . Some of the characteristics of the 39-protein and its structural features predicted from the DNA sequence data are discussed. Cell, 1986 Oct 10, 47(1), 99 - 106 Synthesis of bacteriophage phi X174 in vitro: mechanism of switch from DNA replication to DNA packaging; Aoyama A et al.; Replication of a replicative form DNA of bacteriophage phi X174 initiates by rolling-circle synthesis of the viral DNA followed by discontinuous synthesis of the complementary DNA . Gene C protein of phi X174, which is involved in DNA packaging, inhibits the rolling-circle DNA synthesis by binding to the initiation complex in vitro . The gene C protein-associated initiation complex can synthesize and package the viral DNA to produce infectious phage when supplemented with phi X174 gene J protein and the prohead . Multiple rounds of phage synthesis occur without dissociation of the gene C protein from the complex . These results indicate that gene C protein is central in the switch from replication of a replicative form DNA to synthesis and concomitant packaging of viral DNA into phage capsid, which occurs in the late stage of infection. Biochemistry, 1986 Oct 7, 25(20), 5865 - 72 Investigation of complexes formed between gene 32 protein from bacteriophage T4 and heavy-atom-modified single-stranded polynucleotides using optical detection of magnetic resonance; Khamis MI et al.; Optical detection of triplet-state magnetic resonance (ODMR) is employed to study the complexes formed between gene 32 protein (GP32), a single-stranded DNA-binding protein from bacteriophage T4, and the heavy-atom-derivatized polynucleotides poly(5-HgU) and poly(5-BrU) . The triplet-state properties of some of the tryptophan (Trp) residues in the complexes are dramatically different from those in the free protein, in that they are subject to an external heavy-atom effect . Direct evidence for the presence of a heavy-atom effect, and hence a close-range interaction between mercurated or brominated nucleotide bases and Trp residues in the complex, is provided by the observation of the zero-field (D) + (E) ODMR transition of Trp, which is not normally observed in the absence of a heavy-atom perturbation . The amplitude-modulated phosphorescence-microwave double-resonance (AM-PMDR) technique is employed to selectively capture the phosphorescence spectrum originating from the heavy-atom-perturbed Trp residue(s) in the GP32-poly(5-HgU) complex . Arguments based on our experimental results lead to the conclusion that the heavy-atom perturbation arises from aromatic stacking interactions between Trp and mercurated bases . Wavelength-selected ODMR measurements reveal the existence of two environmentally distinct and spectrally different types of Trp in GP32 . One of these types is perturbed selectively by the heavy atom and hence undergoes stacking interactions with the heavy-atom-derivatized bases of the polynucleotide while the second type of Trp residue is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS) EMBO J, 1986 Oct, 5(10), 2737 - 44 A computer aided thermodynamic approach for predicting the formation of Z-DNA in naturally occurring sequences; Ho PS et al.; The ease with which a particular DNA segment adopts the left-handed Z-conformation depends largely on the sequence and on the degree of negative supercoiling to which it is subjected . We describe a computer program (Z-hunt) that is designed to search long sequences of naturally occurring DNA and retrieve those nucleotide combinations of up to 24 bp in length which show a strong propensity for Z-DNA formation . Incorporated into Z-hunt is a statistical mechanical model based on empirically determined energetic parameters for the B to Z transition accumulated to date . The Z-forming potential of a sequence is assessed by ranking its behavior as a function of negative superhelicity relative to the behavior of similar sized randomly generated nucleotide sequences assembled from over 80,000 combinations . The program makes it possible to compare directly the Z-forming potential of sequences with different base compositions and different sequence lengths . Using Z-hunt, we have analyzed the DNA sequences of the bacteriophage phi X174, plasmid pBR322, the animal virus SV40 and the replicative form of the eukaryotic adenovirus-2 . The results are compared with those previously obtained by others from experiments designed to locate Z-DNA forming regions in these sequences using probes which show specificity for the left-handed DNA conformation. Cancer Biochem Biophys, 1986 Oct, 8(4), 327 - 35 Adriamycin-mediated introduction of a limited number of single-strand breaks into supercoiled DNA; Haidle CW et al.; It was reported previously that Adriamycin converts form I covalently closed circular, supercoiled bacteriophage PM2 DNA to the relaxed circular form II DNA; no form III linear DNA was produced as a result of the extracellular action of Adriamycin in the presence of NADH-dehydrogenase . When form II DNA, produced by the action of Adriamycin, was treated with the BAL 31 nuclease, a single sharp DNA band after agarose gel electrophoresis indicated the presence of only full-length linear form III DNA . As one of its activities, the BAL 31 nuclease introduces a single-strand break in the complementary strand opposite a preexisting single-strand break . When form II DNA, produced by the action of gamma irradiation, was reacted with the BAL enzyme, the resulting linear DNA molecules exhibited a broad range of molecular weights, indicating the presence of many single-strand breaks in the substrate form II DNA . When the Adriamycin-produced form II DNA was treated with restriction endonucleases that cleave PM2 DNA at a single site, either with or without pretreatment with the BAL enzyme, the formation of only full-length linear DNA was observed . Thus, the drug is capable of introducing one or only a very limited number of single-strand breaks into supercoiled DNA; furthermore, these breaks are introduced at random sites along the DNA molecules. J Inorg Biochem, 1986 Oct-Nov, 28(2-3), 155 - 69 Zinc metalloproteins involved in replication and transcription; Giedroc DP et al.; RNA polymerase (RPase) from E . coli contains two tightly incorporated Zn(II) ions, while the monomeric RPase from bacteriophage T7 does not contain zinc and does not require Zn(II) in the assay . One of the two Zn(II) ions can be differentially removed from E . coli RPase with p-hydroxymercuriphenylsulfonate (PMPS) combined with EDTA and thiol . The resultant Znl or ZnA RPase shows no alteration in transcription initiation and elongation rate from sigma-specific promoters . Biosynthesis of a Co2 RPase and formation of CoA RPase by similar treatment shows the tetrahedral-type Co(II) d-d absorption bands to be associated only with the Co(II) at the A site with maxima at 760 (epsilon = 800), 710 (epsilon = 900), 602 (epsilon = 1500), and 484 (epsilon = 4000) nm . Sulfur to Co(II) charge transfer bands are present at 350 (epsilon = 9600) and 370 (epsilon = 9500) nm . The absorption characteristics strongly suggest that the A site is a tetrathiolate site . While DNA polymerases do not in general appear to contain zinc, gene 32 protein (g32P) from bacteriophage T4, an accessory protein essential for DNA replication and recombination and translational control in the T4 life cycle, is a Zn(II) metalloprotein and contains 1 gram atom of tightly incorporated Zn(II) . PMPS displaces the zinc by reacting with three SH groups . Apo-g32P shows markedly altered DNA binding properties . Co(II) substitution gives a protein with intense d-d transitions typical of a tetrahedral Co(II) complex with absorption maxima at 680 (epsilon = 480), 645 (epsilon = 660), 605 (epsilon = 430), 355 (epsilon = 2250), and 320 (epsilon = 3175) nm . The data support a 3 Cys, 1 His coordination site located in the middle of the DNA binding domain of g32P . Data thus far suggest that the Zn(II) binding sites in multisubunit RNA polymerases and in accessory proteins involved in polynucleotide biosynthesis are more likely to play structural or allosteric (regulatory) roles rather than directly participating in catalysis. Mol Cell Biol, 1986 Oct, 6(10), 3559 - 62 Expression of the denV gene of coliphage T4 in UV-sensitive rad mutants of Saccharomyces cerevisiae; Valerie K et al.; A plasmid containing the denV gene from bacteriophage T4, under the control of the yeast alcohol dehydrogenase I (ADC1) promoter, conferred a substantial increase in UV resistance in the UV-sensitive Saccharomyces cerevisiae mutants rad1-2 and rad3-2 . The UV resistance of the denV+ yeast cells was cell cycle dependent and correlated well with the level of the denV gene product as measured by immunoblotting and by a photoreversal assay for pyrimidine dimer-DNA glycosylase activity. EMBO J, 1986 Oct, 5(10), 2719 - 28 The mom gene of bacteriophage Mu: the mechanism of methylation-dependent expression; Seiler A et al.; Transcription of the DNA modification gene (mom) of bacteriophage Mu requires methylation of three GATC sites upstream of the mom promoter by the Escherichia coli deoxyadenosine methylation function (Dam) . The three sites map within a 40-bp segment termed region I . Small deletions, inversions, duplications and specific point mutations have been introduced in region I . Their effect on mom expression has been studied in dam+ and dam strains . Dam-dependent expression of the mom gene requires a specific arrangement of the three GATC sites and the presence of the methylated base in at least two of the three sites . We show that mom specific modification is regulated by a host protein . The Mom function is expressed in dam strains if they are defective in one component of the methylation-instructed mismatch correction system, mutH . We suggest that the product of mutH functions as a transcriptional repressor by binding to region I. Proc Natl Acad Sci U S A, 1986 Oct, 83(20), 7668 - 72 Cloning and expression of Bradyrhizobium japonicum uptake hydrogenase structural genes in Escherichia coli; Zuber M et al.; To identify the structural genes for the components of Bradyrhizobium japonicum uptake hydrogenase (Mr 60,000 and 30,000), we have expressed these genes in Escherichia coli and shown that the products cross-react with antibodies to the respective hydrogenase subunits . We constructed subclones of overlapping DNA fragments from an uptake hydrogenase-complementing cosmid, pHU52 {Lambert, G . R., Cantrell, M . A., Hanus, F . J., Russell, S . A., Haddad, K . R . & Evans, H . J . (1985) Proc . Natl . Acad . Sci . USA 82, 3232-3236}, in pMZ 545, a plasmid expression vector . DNA fragments inserted into one or more of the four cloning sites downstream from the E . coli lac operon promoter (Plac) on pMZ 545 generate transcriptional, but not translational, fusions . Two subclones that directed the synthesis of Mr 60,000 and 30,000 proteins in E . coli "maxicells" were identified . The DNA inserts from these subclones were then inserted down-stream of the bacteriophage lambda PL promoter on a transcriptional fusion vector . When the PL promoter was activated in vivo by heat inactivation of the temperature sensitive cI repressor of lambda in an appropriate E . coli strain, the respective fragments expressed higher levels of Mr 60,000 and 30,000 proteins that could be detected in immunoblots . These data provide direct evidence for the presence of uptake hydrogenase structural genes on the uptake hydrogenase-complementing cosmid pHU52. J Exp Med, 1986 Oct 1, 164(4), 1160 - 70 Isolation of a Treponema pallidum gene encoding immunodominant outer envelope protein P6, which reacts with sera from patients at different stages of syphilis; Peterson KM et al.; A phage directing the synthesis of an abundant 45-kD Treponema pallidum surface protein was isolated from an EMBL-4 bacteriophage lambda library of T . pallidum DNA . The recombinant phage was identified using an mAb that was directed toward an immunodominant, outer envelope T . pallidum protein designated P6 . The recombinant P6 protein possessed the same mol mass as the native treponemal antigen detected from total T . pallidum protein preparations, confirming the cloning of the structural gene for this molecule . Furthermore, E . coli was transformed by a 4.5-kb Eco RI lambda insert fragment subcloned into the plasmid vector pUC19 . These transformed cells expressed and translocated the 45-kD protein to their outer membranes . Finally, all sera from patients with different stages of syphilis (primary, secondary, and latent) contained antibody reactive to this protein. Protein Eng, 1986 Oct-Nov, 1(1), 67 - 74 Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering; Mead DA et al.; Chimeric phage-plasmid expression vectors were constructed from pUC18/19 plasmids by cloning a single-stranded DNA (ssDNA) origin of replication from bacteriophage f1 and inserting a bacteriophage T7 promoter within the beta-galactosidase gene . A T7 promoter permits in vivo or in vitro expression of single proteins by the translation of T7 RNA polymerase transcripts . Insertional inactivation of the T7 promoter-containing beta-galactosidase gene permits a simple blue-to-white color cloning assay . Compared with several helper phages that were examined, superinfection with M13K07 resulted in the highest yields of the pTZ plasmids as ssDNA viral particles . These ssDNA promoter plasmids are uniquely suited for protein engineering because they simplify cloning, oligonucleotide directed mutagenesis, verification by enzymatic sequence analysis, and expression of mutant proteins from a single vector . These vectors were utilized to eliminate an efficient transcriptional terminator of T7 RNA polymerase in the cDNA of bovine preproparathyroid hormone by oligonucleotide directed mutagenesis . The mutation changed the codon for phenylalanine-19 in the signal peptide to alanine . In a cell-free system the mutant cDNA transcripts were translated into preproparathyroid hormone, which was converted to proparathyroid hormone in the presence of microsomal membranes. Proteins, 1986 Oct, 1(2), 116 - 24 Translational repression in vitro by the bacteriophage T4 regA protein; Adari HY et al.; The bacteriophage T4 translational repressor regA protein has been purified from an overproducing strain, and its activity has been studied in simple in vitro protein synthesis reactions . RegA protein was found to inhibit the translation of T4 genes 44, 45, and ORF45-1 in a concentration-dependent fashion . Expression of two other T4 genes which are insensitive to regA protein in vivo, genes 32 and 43, was unaffected by the presence of regA protein . Specific inhibition of synthesis of genes 44, 45, and ORF 45-1 proteins was achieved with 5-20 microM concentrations of regA protein, without the addition of any other T4 encoded proteins or cofactors . When in vitro protein synthesis was performed in two steps, uncoupling translation from transcription, regA protein had an inhibitory effect regardless of whether it was added at the initiation of transcription or only at the translation step . This indicates that regA protein functions during the translation step of protein synthesis in vitro in agreement with previous in vivo studies of regA protein. Proc Natl Acad Sci U S A, 1986 Oct, 83(20), 7703 - 7 Human delta-aminolevulinate dehydratase: nucleotide sequence of a full-length cDNA clone; Wetmur JG et al.; Two cDNAs encoding human delta-aminolevulinate dehydratase (ALA-D; porphobilinogen synthase; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, were identified, recloned into bacteriophage M13, and sequenced by primer extension . The first clone with an 827-base-pair (bp) pEX-ALA-D cDNA insert, shown to contain DNA sequences that were colinear with four bovine ALA-D peptide sequences, was used to screen a pKT218 human liver library . A second clone containing a 1200-bp insert was identified that contained an open reading frame of 990 bp as well as 5' (66 bp)- and 3' (94 bp)-untranslated regions, the latter terminating in poly(dA) . The predicted N-terminal amino acid sequence was colinear with the first 13 residues of microsequenced ALA-D purified from human erythrocytes . The ATG initiation codon was preceded by ACGCC, a functional initiation sequence, while an upstream (position -32), in-phase AACTG ATG sequence was entirely nonhomologous with the initiation consensus sequence and, therefore, presumed to be nonfunctional . The unusual polyadenylylation signal, AGTAAA, has been reported only in the human HRAS1 gene . The nucleotide sequences of the two cDNA clones differed at position 730 or 733 and encoded two differently charged amino acids . This nucleotide difference may be the basis for the polymorphic charge isozymes of human ALA-D . The sequence encoding this zinc metalloenzyme contained a cysteine- and histidine-rich binding site for zinc and an unusual region of charge complementarity surrounding the active lysine residue in the catalytic site. Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7221 - 5 Putative diazepam binding inhibitor peptide: cDNA clones from rat; Mocchetti I et al.; cDNA clones corresponding to the polypeptide that has been shown to be an endogenous diazepam binding inhibitor and may act as a physiological ligand for the benzodiazepine/beta-carboline receptor have been isolated from bacteriophage lambda recombinant libraries from rat hypothalamus, total brain, and liver . The clones contain an open reading frame corresponding to 87 amino acids . A signal sequence is not present . In addition to high levels of mRNA in various brain regions, RNA blot analysis reveals an abundance of diazepam binding inhibitor mRNA in many peripheral organs (e.g., testes, kidney, liver, and heart) that are known to be rich in peripheral benzodiazepine recognition sites . The size of the mRNA in all tissue examined is approximately 0.7 kilobase . Southern blot analysis of genomic DNA suggests the presence of about six genes in the rat, some of which may be pseudogenes. Proteins, 1986 Oct, 1(2), 176 - 87 The restoration of electron micrographs blurred by drift and rotation; Carragher B et al.; We have investigated the restoration of electron micrographs exhibiting blurring due to drift and rotation . Blurring due to drift arises in micrographs taken of a specimen which is moving relative to the image plane . A related problem is that of rotational blurring which arises in micrographs of thin sections of helical particles viewed in cross section . The twist of the particle within the finite thickness of the section causes the image to appear rotationally blurred about the helical axis . Restoration algorithms were evaluated by applying them to the restoration of blurred model images degraded by additive Gaussian noise . Model images were also used to investigate how an incorrect estimate of the point spread function describing the blur would effect the restoration . Images were, if necessary, geometrically transformed to a space in which the point spread function of the blur can be considered as linear and space invariant as, under these conditions, the restoration algorithms are greatly simplified . In the case of the rotationally blurred images this procedure was accomplished by transforming the image to polar coordinates . The restoration techniques were successfully applied to blurred micrographs of bacteriophage T4 and crystals of catalase . The quality of the restoration was judged by comparisons of the restored images to undegraded images . Application to micrographs of rotationally blurred cross sections of helical macrofibers of sickle hemoglobin resulted in a reduction in the amount of rotational blurring. Appl Environ Microbiol, 1986 Oct, 52(4), 737 - 43 Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli; O'Neill GP et al.; A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein . To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E . Remaut, H . Tsao, and W . Fiers, Gene 22:103-113, 1983) . With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase . The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride . Since the codon usage of the exoglucanase gene is so markedly different from that of E . coli genes, the overproduction of the exoglucanase in E . coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism. Mol Gen Mikrobiol Virusol, 1986 Oct, (10), 33 - 9 {Deletion of the phage lambda att80 Tn9 genome; the type of intramolecular recombination}; Pokrovskaia MS et al.; The intramolecular deletion-generating recombination which transforms lambda bacteriophage genomes into the plasmids (named pLS) proved to be site-specific to a certain extent . Using electron microscopy heteroduplex analysis three preferential sites for this recombination were found in seven independent pLS isolates studied . Att-sites were not registered to be involved in the formation of deletions in isolates studied . It was shown that recombination operating in our system was independent of the phage int and bacterial recA genes. Proc Natl Acad Sci U S A, 1986 Oct, 83(20), 7638 - 42 Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction; Dodson M et al.; The O protein of bacteriophage lambda localizes the initiation of DNA replication to a unique site on the lambda genome, ori lambda . By means of electron microscopy, we infer that the binding of O to ori lambda initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication . We can define three stages of this prepriming reaction, the first two of which we have characterized previously . First, dimeric O protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some . Second, lambda P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A + T-rich region adjacent to the O binding sites . Third, the addition of the DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB . The unwinding reaction is unidirectional, proceeding "rightward" from the origin . The minimal DNA sequence competent for unwinding consists of two O binding sites and the adjacent A + T-rich region to the right of the binding sites . We conclude that the lambda O protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication . Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control. J Bacteriol, 1986 Oct, 168(1), 357 - 64 Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing; Groisman EA et al.; New mini-Mu transposons with plasmid replicons were constructed with additional features for in vivo DNA cloning and lac gene fusing in Escherichia coli . These mini-Mu replicons can be used to clone DNA by growing them with a complementing Mu bacteriophage and by using the resulting lysate to transduce Mu-lysogenic cells . These mini-Mu phage have selectable genes for resistance to kanamycin, chloramphenicol, and spectinomycin-streptomycin, and replicons from the high-copy-number plasmids pMB1 and P15A and the low-copy, broad-host-range plasmid pSa . The most efficient of these elements can be used to clone genes 100 times more frequently than with the previously described mini-Mu replicon Mu dII4042, such that complete gene banks can be made with as little as 1 microliter of a lysate containing 10(6) helper phage . The 39-kilobase-pair Mu headful DNA packaging mechanism limits the size of the clones formed . The smallest of the mini-Mu elements is only 7.9 kilobase pairs long, allowing the cloning of DNA fragments of up to 31.1 kilobase pairs, and the largest of them is 21.7 kilobase pairs, requiring that clones carry insertions of less than 17.3 kilobase pairs . Elements have been constructed to form both transcriptional and translational types of lac gene fusions to promoters present in the cloned fragment . Two of these elements also contain the origin-of-transfer sequence oriT from the plasmid RK2, so that clones obtained with these mini-Mu bacteriophage can be efficiently mobilized by conjugation. Dev Biol, 1986 Oct, 117(2), 574 - 80 A transient expression assay for tissue-specific gene expression of alcohol dehydrogenase in Drosophila; Martin P et al.; The regulation of expression of the alcohol dehydrogenase gene of Drosophila was examined by injecting plasmids containing the gene directly into preblastoderm embryos and subsequently staining for alcohol dehydrogenase activity in somatic cells of larvae and adults . The alcohol dehydrogenase genes introduced in this manner were expressed normally in both adults and larvae; i.e., alcohol dehydrogenase activity was found exclusively in tissues where it would normally be expressed . Activity was found in some cells in more than 90% of all surviving third instar larvae, but not all cells which would normally express the enzyme were positive, presumably due to the random distribution of the injected DNA to the cells of the embryo . Regulated expression was not dependent on the vector used: tissue-specific expression was obtained from alcohol dehydrogenase genes inserted in the P-element vector, Carnegie-4; in pBR322; in pUC18; or in bacteriophage lambda . The bulk of the injected DNA was not integrated into the chromosome and appeared to persist throughout development as supercoiled and nicked circles . Using the procedure and in vitro mutagenesis, we were able to show that the alcohol dehydrogenase gene was expressed in a normal tissue-specific manner in larvae if there were 777 nucleotides of upstream information present. J Virol, 1986 Oct, 60(1), 97 - 104 Topoisomerase II and other DNA-delay and DNA-arrest mutations impair bacteriophage T4 DNA packaging in vivo and in vitro; Zachary A et al.; A survey of DNA packaging in vivo and in vitro during infections caused by T4 DNA-delay and DNA-arrest amber mutants revealed a common DNA packaging-deficient phenotype . Electron microscopy revealed high proportions of proheads partially filled with DNA in vivo, indicating normal initiation but incomplete encapsidation . In contrast, exogenous mature T4 DNA was packaged in vitro by several early-gene mutant extracts . Detailed analysis of gene ts39 mutants (subunit of topoisomerase II) showed that in vivo packaging is defective, yet expression of late proteins appeared normal and the concatemeric DNA was not abnormally short or nicked . Although g39 amber mutant extracts packaged DNA in vitro, two of three ts39 mutant extracts prevented encapsidation of the exogenous DNA . The temperature-sensitive (ts) gp39 in a mutant topoisomerase II complex may have interfered with packaging in vivo and in vitro by interacting with DNA in an anomalous fashion, rendering it unfit for encapsidation . These results support the hypothesis that T4 DNA packaging is sensitive to DNA structure and discriminates against encapsidation of some types of defective DNA. J Virol, 1986 Oct, 60(1), 185 - 93 Use of lambda gt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins; Petrovskis EA et al.; A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambda gt11 . The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins . By the nature of the lambda gt11 vector, the cloned proteins were expressed in Escherichia coli as beta-galactosidase fusion proteins . The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera . The antisera were screened by several different assays, including immunoprecipitation of {14C}glucosamine-labeled PRV proteins . This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63 . The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome . The DNA sequence was determined for the region of the genome encoding gp63 and gI . It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV . The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI . It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved. Eur J Immunol, 1986 Oct, 16(10), 1231 - 5 Impaired humoral immune response in complement C3-deficient guinea pigs: absence of secondary antibody response; Bottger EC et al.; A recently described genetically controlled C3 deficiency (C3D) in guinea pigs (GP) provided a unique model for studying the role of C3 in the afferent limb of the humoral immune response in a direct manner . These C3D animals, which have only 5-7% of normal serum C3 level, were immunized with the bacteriophage phi chi 174, a T cell-dependent antigen, followed by a booster injection after 4 weeks (1.5 X 10(9) plaque-forming units/kg) . The formation of IgM and IgG antibody in the course of the primary and secondary response was determined and compared with a control group of inbred strain 2 GP . The C3D animals showed a markedly diminished antibody response to this antigen . Amplification of the antibody titer as well as regular isotype switching from IgM to IgG was absent in the secondary response . Increasing the amount of antigen to a high dose (1 X 10(10) plaque-forming units/kg) led to a normalization of the antibody response . The impairment in antibody formation resembles closely the impaired antibody response in C4-deficient or C2-deficient GP, which both have a block in activation of C3 via the classical pathway . However, in contrast to C4D GP or C2D GP the C3D GP do not exhibit serological characteristics of immune complex disease . They have normal levels of total serum IgM, of IgM anti-2,4-dinitrophenyl antibodies and of IgM rheumatoid factors. Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7348 - 52 The in vivo mutagenic frequency and specificity of O6-methylguanine in phi X174 replicative form DNA; Bhanot OS et al.; A bacteriophage phi X174-based site-specific mutagenesis system for the study of the in vivo mutagenic frequency and specificity of carcinogen-induced modification in DNA is presented . A (-)-strand primer containing O6-methylguanine in a specific site was hybridized to a single-stranded region in gene G of phi X gapped duplex DNA . The hybrid was enzymatically converted to replicative form DNA and was used to transform Escherichia coli cells . All gene G mutants generated by the modification were rescued by genetic complementation . An amber mutation in lysis gene E of the (+) strand of the replicative form DNA prevented lytic growth of wild-type phage derived from this strand . In each mutant-containing infective center produced from the transformed cells, gene G mutant phage were present in a 3:1 ratio compared to wild type . Thus, in vivo, O6-methylguanine in replicating phi X DNA has a mutagenic frequency of 75% . When repair of O6 methylguanine occurred, it was prereplicative . The mutations were due exclusively to the misincorporation of thymine. J Gen Microbiol, 1986 Oct, 132 ( Pt 10), 2907 - 17 Bacteriophages F0lac h, SR, SF: phages which adsorb to pili encoded by plasmids of the S-complex; Coetzee JN et al.; Phage F0lac is an RNA-containing phage which plates only on strains carrying the plasmid EDP208, a pilus derepressed derivative of the unique incompatibility plasmid F0lac . A host range mutant, phage F0lac h, was selected which plated on strains carrying the ungrouped plasmid pPLS::Tn5 and lysed strains carrying another ungrouped plasmid TP224::Tn10 or the Com9 plasmid R71 . An RNA-containing phage, SR, was isolated from sewage on bacteria harbouring plasmid pPLS::Tn5 . It was antigenically distinct from the above two phages but had the same host range as phage F0lac h . Phages F0lac h and SR adsorbed unevenly to the shafts of the conjugative pili . Another phage, SF, was filamentous and plated or propagated on strains carrying any of the above plasmids as well as on strains harbouring IncD or F-complex plasmids . Plasmids TP224::Tn10 and pPLS::Tn5 were compatible with representative plasmids of all Inc groups also encoding thick flexible pili . The four plasmids EDP208, R71, TP224::Tn10 and pPLS::Tn5 were compatible with one another except for the reaction of TP224::Tn10 in the presence of pPLS::Tn5 which was slightly ambiguous . The host ranges of the bacteriophages, together with the serological relatedness of the thick flexible pili determined by these four compatible plasmids, suggested that they constitute a new complex, here designated S. J Clin Invest, 1986 Oct, 78(4), 914 - 21 Molecular cloning, expression, and chromosomal localization of the gene encoding a human myeloid membrane antigen (gp150); Look AT et al.; DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gp150, was used to construct a bacteriophage lambda library . A single recombinant phage containing 12 kilobases (kb) of human DNA was isolated, and molecular subclones were then used to isolate the complete gp150 gene from a human placental genomic DNA library . The intact gp150 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells . Molecular probes from the gp150 locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants . The gp150 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization . The chromosomal location of the gp150 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells. J Bacteriol, 1986 Oct, 168(1), 449 - 51 Shielding of Escherichia coli outer membrane proteins as receptors for bacteriophages and colicins by O-antigenic chains of lipopolysaccharide; van der Ley P et al.; The accessibility of several outer membrane proteins for bacteriophages and colicins in isogenic smooth and rough Escherichia coli strains was investigated . The results show that O antigen carrying lipopolysaccharide is able to prevent access of all phages and colicins tested to their outer membrane protein receptors. J Biol Chem, 1986 Sep 25, 261(27), 12820 - 7 DNA binding by the bacteriophage SPO1-encoded type II DNA-binding protein, transcription factor 1 . Formation of nested complexes at a selective binding site; Greene JR et al.; The interactions of the phage SPO1-encoded Type II DNA-binding protein, transcription factor 1 (TF1), with one of its preferred binding sites in SPO1 DNA have been analyzed in detail . The results suggest that TF1 recognizes a high-affinity "core" binding site and that additional protein moieties can accrue to either side of the occupied core site to form higher order complexes . Close contacts between TF1 and the core binding site as well as some of the steric requirements for recognition of the core site were determined . Comparison of the nucleotide sequences of several preferred binding sites for TF1 reveals a striking lack of precise homology but does show common features. J Biol Chem, 1986 Sep 25, 261(27), 12723 - 32 Structure of the lc and nmpC outer membrane porin protein genes of lambdoid bacteriophage; Blasband AJ et al.; The lc gene of the lambdoid bacteriophage PA-2 and the nmpC gene located on a defective lambdoid prophage in the 12-min region of the Escherichia coli K12 chromosome have been sequenced . The porin proteins encoded by these two genes were almost identical, with only 4 of the 365 residues of the precursor forms of the proteins being different . The Lc and NmpC proteins were strongly homologous to the OmpC, ompF, and PhoE proteins, with greater than 56% of the residues identical in each case . Sequencing of the region flanking the lc gene allowed precise positioning of this gene with respect to the rightward cos site of the phage and to sequences which are homologous between PA-2 and lambda . In wild-type strains of E . coli K12, the nmpC gene is not expressed and contains an IS5 insertion near the 3' end of the coding region . This insertion deletes 18 residues from the COOH terminus of NmpC protein and adds 8 residues from an open reading frame extending into IS5 sequence . Expression of this form of the gene in an expression vector plasmid demonstrated that this altered protein is still capable of being translocated to the outer membrane . Plasmid expression experiments using lc-nmpC hybrid genes show that it is the presence of the IS5 insertion which prevents expression of the porin in wild-type E . coli K12 . In the nmpC mutant which expresses the protein, there has been a precise excision of the IS5 which regenerates a COOH terminus of NmpC protein which is identical to that of the Lc protein . Blot hybridization detected no mRNA transcripts from the wild-type nmpC gene, although transcripts were readily detected from the lc gene in PA-2 lysogens and from the nmpC mutant which has excised the IS5 . This indicates that IS5 affects the production or stability of transcripts from the adjacent nmpC gene. Biochemistry, 1986 Sep 23, 25(19), 5751 - 5 Processivity of T4 endonuclease V is sensitive to NaCl concentration; Ganesan AK et al.; We previously reported that endonuclease V of bacteriophage T4 reacts processively with pyrimidine dimers in UV-irradiated DNA, tending to react with all of the dimers on one DNA molecule before reacting with any dimers on another DNA molecule {Lloyd, R . S., Hanawalt, P . C., & Dodson, M . L . (1980) Nucleic Acids Res . 8, 5113-5127} . In this paper we show that this processivity depends upon salt concentration: it can be detected in 10 mM NaCl but not, by our methods, in 100 mM NaCl . In addition, we show that endonuclease V binds to unirradiated DNA in 10 mM NaCl but not in 100 mM NaCl . We conclude that T4 endonuclease V binds to pyrimidine dimers in a two-step process in 10 mM NaCl . It first binds electrostatically to undamaged sections of DNA, and it remains bound during the second step in which it "searches" for pyrimidine dimers . Our conclusion is analogous to the expanded target theory developed for Lac repressor {Berg, O . G., Winter, R . B., & von Hippel, P . H . (1981) Biochemistry 20, 6929-6948}. Biochemistry, 1986 Sep 23, 25(19), 5378 - 87 An Escherichia coli RNA polymerase tight-binding site on T7 DNA is a weak promoter subject to substrate inhibition; Prosen DE et al.; A specific Escherichia coli RNA polymerase tight-binding (TB) site on bacteriophage T7 has been located at 32,988 base pairs from the left end of T7 . This site is referred to as the T7 F promoter since it is fully active in vitro . Kinetics of association and dissociation have been measured by use of the abortive initiation assay and runoff transcription . The association constant, ka approximately 9 X 10(5) M-1 s-1, is of the same magnitude as ka for the T7 minor promoters . In competitive titration assays, the F promoter was found to be slightly weaker than the minor T7 E promoter at low RNA polymerase concentrations and, as expected, much weaker than the major T7 A3 promoter . An unusual RNA polymerase mediated inhibition of both the association rate and the transcriptional activity was observed at moderately high concentrations of polymerase . A mechanistic model analogous to enzyme substrate inhibition is presented. J Mol Biol, 1986 Sep 20, 191(2), 255 - 66 DNA sequence of the tail fiber genes 37, encoding the receptor recognizing part of the fiber, of bacteriophages T2 and K3; Riede I et al.; The DNA sequences of genes 37 of bacteriophages T2 and K3 are presented and compared with that of phage T4 . The corresponding proteins constitute, as dimers, the part of the long tail fibers that recognizes the bacterial receptor . The CO2H termini of the polypeptides are located at the free ends of the fibers . Morphologically, the three phages are essentially identical, but they use different receptors . The genes from phages T4, T2 and K3 encode proteins consisting of 1026, 1341 and 1243 amino acid residues, respectively . DNA-DNA hybridizations had shown earlier that genes 37, in contrast to the gene for the major capsid protein, of a number of T-even type phages are highly polymorphic . The deduced amino acid sequences now show that this polymorphism extends to the protein primary structures . About 50 NH2-terminal residues are conserved and are probably required for binding to the adjacent protein 36 . This area is followed by more or less irregularly spaced regions of non-homology, partial homology or complete homology . The heterogeneity is most prominent in a region encompassing about 600 CO2H-terminal residues of the T2 or K3 proteins . Nevertheless, the amino acid compositions of the three proteins are very similar and all are rich in glycine . It has been found that the receptor specificities of phages K3 and T2 are determined by protein 38, a polypeptide required for the efficient dimerization of protein 37 of phage T4 . Proteins 38 of phages K3 and T2 are functionally interchangeable, those of T4 and T2 or K3 are not . Proteins 37 of phages K3 and T2 possess a conserved sequence of 160 CO2H-terminal residues . This area is missing in the T4 protein . This region may serve as a binding site for polypeptides 38 of phages K3 and T2 . The overall picture of the protein primary structures of the three phages strongly suggests that the evolution of genes 37, which was most likely driven by selection for variations in receptor recognition specificities, has not been a steady process but has involved loss and gain of segments of DNA. J Mol Biol, 1986 Sep 20, 191(2), 267 - 72 Morphogenesis of the long tail fibers of bacteriophage T2 involves proteolytic processing of the polypeptide (gene product 37) constituting the distal part of the fiber; Drexler K et al.; Gene 37 of phage T2 codes for a protein that, as a dimer, constitutes the most distal, receptor-recognizing part of its long tail fibers . It was found that, from a plasmid carrying a fragment of gene 37 that lacked a region of the gene encoding 87 CO2H-terminal amino acid residues, a protein was expressed that was slightly larger than that present in the phage . This size difference could not be accounted for . The missing region of gene 37 and also gene 38 (which codes for the auxiliary protein required for dimerization of protein 37) were cloned . Plasmids were constructed with gene 37, or gene 37 together with gene 38, under inducible control . Independent of the presence of the latter gene, a protein was produced that had the same size as protein 37 in the phage . A pulse-chase experiment revealed that a precursor of protein 37 is synthesized and processed such that approximately 120 amino acid residues, most likely CO2H-terminal, are removed . Therefore, the protein produced from the truncated gene was larger because it cannot be processed . This fact also solved an old puzzle: an amber fragment of protein 37 of phage T2 had been found to be larger than the mature protein . The amber codon could be located 24 codons away from the normal stop codon . Obviously, the fragment cannot be processed . The existence of this fragment demonstrates that processing occurs during phage maturation. J Mol Biol, 1986 Sep 20, 191(2), 299 - 300 Structure of F-pili: reassessment of the symmetry; Marvin DA et al.; Reassessment of the X-ray fibre diffraction patterns of F-pili using a more accurate subunit molecular weight suggests that subunits in F-pili are related by a fivefold rotation axis around the pilus axis . The identity of this fivefold symmetry with the fivefold rotation axis that relates the subunits in fd bacteriophage supports a simple model for tip-to-tip adsorption of bacteriophage to pili. J Biol Chem, 1986 Sep 15, 261(26), 12414 - 9 Purification and properties of the groES morphogenetic protein of Escherichia coli; Chandrasekhar GN et al.; The morphogenesis of lambda proheads is governed by the products of at least four bacteriophage-coded genes (B, C, E and Nu3) and two host-coded genes (groES (mopB) and groEL (mopA)) . Earlier genetic experiments indicated that the phenotypes of some of the groES- mutations could be suppressed by mutations in the groEL gene, suggesting an interaction between the two groE proteins in vivo (Tilly, K., and Georgopoulos, C . P . (1982) J . Bacteriol . 149, 1082-1088) . The Mr 15,000 groES protein was overproduced and purified to homogeneity by monitoring its presence after polyacrylamide gel electrophoresis . Both gel filtration on an AcA34 sizing column and glycerol gradient centrifugation indicate that the groES protein possesses an oligomeric structure of Mr 80,000 . In agreement, electron microscopic pictures of the purified groES protein show that it possesses a symmetrical ring-like structure . The sequence of the first five amino acids and the overall composition of the purified protein match those predicted by the nucleotide sequence of the groES gene . The following results implicate a physical association between the groES and groEL proteins in vitro . The groES protein inhibits the weak ATPase activity of the groEL protein, with a maximal effect seen at a 1:1 molar ratio; the two proteins cosediment during glycerol gradient centrifugation in the presence of ATP and Mg2+; and the groES protein binds specifically to a groEL-affinity column . These results help explain why mutations in either of the groE genes exhibit similar phenotypes with respect to both lambda and bacterial growth. J Biol Chem, 1986 Sep 15, 261(26), 12352 - 61 Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells; Jahnsen T et al.; One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells . To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells . A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product . Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size . The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end . The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides) . An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA . Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively . Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA . Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene. Nucleic Acids Res, 1986 Sep 11, 14(17), 6901 - 14 Purification of the gam gene-product of bacteriophage Mu and determination of the nucleotide sequence of the gam gene; Akroyd JE et al.; The gam gene of bacteriophage Mu encodes a protein which protects linear double stranded DNA from exonuclease degradation in vitro and in vivo . We purified the Mu gam gene product to apparent homogeneity from cells in which it is over-produced from a plasmid clone . The purified protein is a dimer of identical subunits of 18.9 kd . It can aggregate DNA into large, rapidly sedimenting complexes and is a potent exonuclease inhibitor when bound to DNA . The N-terminal amino acid sequence of the purified protein was determined by automated degradation and the nucleotide sequence of the Mu gam gene is presented to accurately map its position in the Mu genome. Biochemistry, 1986 Sep 9, 25(18), 5098 - 102 Gene for human factor X: a blood coagulation factor whose gene organization is essentially identical with that of factor IX and protein C; Leytus SP et al.; Factor X is one of six vitamin K dependent proteins known to be involved in blood coagulation, the others being factor VII, factor IX, prothrombin, protein S, and protein C . In the present studies, recombinant bacteriophage containing overlapping DNA inserts coding for the gene for human factor X have been isolated and characterized . These DNA inserts code for almost the entire gene for factor X, extending from the prepro leader peptide through the 3' noncoding region of the transcription product . The organization of the gene for factor X was established by DNA sequencing to identify the location of the introns and exons in the gene . Seven introns and eight exons were identified and their intron/exon boundaries established . The seven introns interrupt the coding sequence at essentially identical locations in the amino acid sequence as the introns in the genes for human factor IX and protein C . In addition, the introns in the gene for factor X divide the coding sequence into discrete exons that code for potential structural and functional domains of the protein . This information provides strong evidence to support the suggestion that the vitamin K dependent proteins present in plasma have evolved from a single, common gene and that this ancestral gene arose through a process that involved the assembly of small protein coding units of DNA into a single gene. J Mol Biol, 1986 Sep 5, 191(1), 29 - 37 Mutations of bacteriophage lambda that define independent but overlapping RNA processing and transcription termination sites; Montanez C et al.; Bacteriophage lambda int gene expression is regulated differentially from transcripts originated at the pL and pI promoters . Transcripts initiated at pI terminate at the site tI and express int gene product efficiently . Polymerases starting at pL do not terminate at tI, due to the antiterminating activity of lambda N protein . The pL transcripts are unable to express Int protein efficiently because sib, a control site overlapping tI in the unterminated RNA, is processed by host RNase III . We have isolated lambda sib- mutants by their inability to inhibit int expression from pL transcripts . sib mutations were genetically mapped to the left of the lambda attachment site, and do not structurally alter this site for recombination . Several sib mutations do alter the nucleotide sequence of the overlapping sib and tI sites . The lambda sib- mutants tested prevent RNA processing but do not affect transcription termination in vivo. Science, 1986 Sep 5, 233(4768), 1050 - 6 Multiple DNA-protein interactions governing high-precision DNA transactions; Echols H; The precise association of DNA-binding proteins with localized regions of DNA is crucial for regulated replication and expression of the genome . For certain DNA transactions, the requirement for precision in localization and control is extremely high . High-precision events amenable to detailed biochemical analysis are the initiation of DNA replication and site-specific recombination by bacteriophage lambda and Escherichia coli . Recent experiments indicate that site-localization and control in these reactions involves the association of DNA-bound proteins to generate organized nucleoprotein structures in which the DNA is folded or wound . These specialized nucleoprotein structures are likely to provide the requisite accuracy for site localization and the necessary regulated reactivity to direct the DNA transaction . Multiple DNA-protein interactions are also required for controlled transcription of the eukaryotic genome . Distant upstream regulator and enhancer sequences may define protein-binding sites that form part of a reactive nucleoprotein structure capable of initiating transcription. J Virol Methods, 1986 Sep, 14(2), 189 - 91 A comparison of Selas and membrane filters for the sterilization of bacteriophage preparations; Schwan WR et al.; Lysates of three different coliphage were sterilized by filtration through Selas, Millipore GVWP, and Millipore GS filters . Phage titers were comparable when either the Selas or Millipore GVWP (hydrophilic) filters were used; however, the GVWP filters were faster and could accommodate more lysate before the filters clogged . The Millipore GS (hydrophobic) filters were unsatisfactory. Mutat Res, 1986 Sep, 162(2), 137 - 44 Survival and mutagenesis of bacteriophage T7 damaged by methyl methanesulfonate and ethyl methanesulfonate; Dodson LA et al.; We have examined survival and mutagenesis of bacteriophage T7 after exposure to the alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) . It was found that although both alkylating agents caused increased reversion of specific T7 mutations, EMS caused a higher frequency of reversion than did MMS . Exposure of the host cells to ultraviolet light so as to induce the SOS system resulted in increased survival (Weigle reactivation) of T7 phage damaged with either EMS or MMS . However, after SOS induction of the host we did not detect an accompanying increase in mutation frequency measured as either reversion of specific T7 mutants or by generation of mutations in the T7 gene that codes for phage ligase . Neither mutation frequency nor survival of alkylated phage was affected by the umuD,C mutation in the Escherichia coli host nor by the presence of plasmid pKM101 . This may mean that the mode of Weigle reactivation that is detected in T7 is not mutagenic in nature. J Virol, 1986 Sep, 59(3), 759 - 60 Nucleotide sequence and protein overproduction of bacteriophage T4 thioredoxin; LeMaster DM; The bacteriophage T4 thioredoxin gene was cloned and physically mapped to 47.6 kilobases from the reference BamHI site . The DNA sequence is consistent with that reported from earlier protein sequence studies . The gene was subcloned into a lambda pL overexpression vector which allowed for the isolation of approximately 5 mg/liter. Carcinogenesis, 1986 Sep, 7(9), 1569 - 76 Utilization of 1,N6-etheno-2'-deoxyadenosine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli; Revich GG et al.; To test whether vinyl chloride-induced mutagenesis might involve ambiguous base pairing of 1,N6-etheno-adenine (epsilon A) during DNA synthesis, we examined the base pairing potential of epsilon dATP during DNA synthesis catalyzed by Escherichia coli DNA polymerase I (Klenow fragment) . An electrophoretic assay of chain elongation was used to assess the degree to which epsilon dATP could substitute for each of the normal dNTPs during elongation of a primer annealed to a bacteriophage template . Despite the fact that the etheno bridge completely blocks normal Watson-Crick pairing of epsilon A with T, we observed that epsilon dATP could substitute for dATP during primer elongation (although inefficiently) . In addition, detectable substitution of epsilon dATP for dGTP and dCTP occurred, indicating that epsilon A exhibits ambiguous base pairing properties . The relative ease of epsilon dAMP incorporation (opposite template T, C and G) appeared to vary considerably at different positions along the template . The major form of epsilon A incorporation (replacement of A) was confirmed by measurements of epsilon dATP----epsilon dAMP turnover (a commonly used method for detecting misincorporation), and also by the demonstration that epsilon A was present in enzymatic hydrolysates prepared from DNA that was synthesized with epsilon dATP replacing dATP . A model for ambiguous base pairing of epsilon dATP is proposed, in which incorporation occurs via the protonated, syn form of epsilon dATP. Proc Natl Acad Sci U S A, 1986 Sep, 83(18), 6954 - 8 Frameshift mutations produced by proflavin in bacteriophage T4: specificity within a hotspot; Ripley LS et al.; Frameshift mutations were induced by proflavin in the rIIB gene of bacteriophage T4 . rIIB DNA from each of 48 independent frameshifts was inserted into M13mp8 and sequenced . Two-thirds of the frameshifts (33/48) lie contiguous to one another in 10 base pairs of the rIIB sequence . This hotspot differs markedly from previously characterized mutagen-induced frameshift hotspots . Distinctive features of the hotspot include the absence of locally repetitive sequences, particularly G X C runs, and the fact that many different sequence changes are induced within the hotspot sequence at appreciable frequencies . Among the 33 mutants at the hotspot, 8 distinguishable DNA sequence changes were seen . All of the mutations were deletions of a single base or duplications of one or more bases . Duplications were more frequent than deletions . The patterns of the base sequence changes suggest that two specific phosphodiester bonds within the hotspot sequence are sites at which proflavin-induced mutation is initiated. Proc Natl Acad Sci U S A, 1986 Sep, 83(17), 6543 - 7 Molecular cloning of cDNAs encoding rat and human medium-chain acyl-CoA dehydrogenase and assignment of the gene to human chromosome 1; Matsubara Y et al.; Rat liver mRNA encoding the precursor of medium-chain acyl-CoA dehydrogenase was purified to near homogeneity by polysome immunoadsorption using a polyclonal, monospecific antibody . A single-stranded, 32P-labeled cDNA probe was synthesized using the enriched mRNA as template and was used to screen directly 15,000 colonies from a total rat liver cDNA library constructed in pBR322 . One clone {600 base pairs (bp)} was positively identified by hybrid-selected translation combined with mitochondrial processing of translated products . Using the isolated rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened . Three overlapping clones (1100 bp, 500 bp, and 400 bp) were isolated and positively identified by hybrid-selected translation . The largest human cDNA clone was subcloned into the transcription vector pGEM-2, which contains a bacteriophage T7 RNA polymerase promoter . In vitro transcription of this recombinant, followed by in vitro translation, showed that the cDNA clone coded for approximately 80% of the medium-chain acyl-CoA dehydrogenase protein . The sizes of rat and human mRNAs encoding the precursor of medium-chain acyl-CoA dehydrogenase were 2.2 and 2.4 kilobases long, respectively, as determined by blot hybridization analysis of electrophoretically fractionated poly(A)+ RNA . Southern blot analysis of DNAs from human-rodent somatic cell hybrids with an isolated human cDNA assigned the gene coding for this enzyme to the short arm of chromosome 1, band p31 . The chromosomal assignment was confirmed by in situ hybridization of the probe to human metaphase cells . Direct screening of cDNA libraries using a highly enriched mRNA to generate a probe, as demonstrated in this study, may provide the most rapid and convenient approach to cDNA cloning of low-abundance mRNAs. Proteins, 1986 Sep, 1(1), 100 - 7 A novel method for selective isolation of C-terminal peptides from tryptic digests of proteins by immobilized anhydrotrypsin: application to structural analyses of the tail sheath and tube proteins from bacteriophage T4; Kumazaki T et al.; A novel method useful for selective isolation of the C-terminal peptide from a tryptic digestion mixture of a protein has been developed by taking advantage of a unique property of anhydrotrypsin, which has a strong specific affinity for the peptides containing arginine or lysine at their C-termini . Briefly, peptides produced by tryptic digestion of a protein are fractionated by affinity chromatography on a column of immobilized anhydrotrypsin . The C-terminal peptide is recovered in a breakthrough fraction, while the remainders are adsorbed on the column (unless the protein ends in arginine or lysine) . The breakthrough fraction is then subjected to reversed-phase high-performance liquid chromatography in order to purify the C-terminal peptide . Using this method, we have successfully isolated the C-terminal peptides from tryptic digests of the sheath protein (gp 18) and the tube protein (gp 19) of bacteriophage T4 . The analytical results on these peptides, together with the information on the N-terminal structures of the original proteins and on the nucleotide sequences of genes 18 and 19, allowed us to establish the complete primary structures of the two proteins. Eur J Epidemiol, 1986 Sep, 2(3), 178 - 81 Preliminary studies on bacteriophage typing of Mycobacterium tuberculosis strains isolated in Salamanca (Spain); Garcia-Rodriguez JA et al.; A phage typing of 202 strains of Mycobacterium tuberculosis isolated in Central and Northwest Spain was carried out . The commonest phage type was A (64%) and within this type A (45%) . This was followed in frequency by phage type B (26%) and in last place type I (10%) . No relationship was observed between the phage type and the geographical or anatomical origin of the strains. Genetika, 1986 Sep, 22(9), 2252 - 8 {Genetic analysis of Escherichia coli min81 mutation blocking the development of bacteriophage Mu}; Piruzian ES et al.; Data characterizing mim81 mutation obtained by the method for direct selection of transposition mutations are presented . The development of Mu is shown to be dramatically suppressed in the mutant strain both upon infection and after induction from the lysogenic state . Frequencies of lysogenization and mini-Mu-dependent formation of cointegrates in the mutant strain are comparable with those in the wild-type strain . Mu development prohibition is removed if expression of early Mu gene is provided from the modified Pe promoter . The results obtained make us believe that the mechanism of mim81 mutation action involves reduction of early gene expression to the level that is sufficient for Mu DNA integration into the chromosome during infection and for single replicative events, but insufficient for vegetative development of bacteriophage Mu. Mol Gen Genet, 1986 Sep, 204(3), 540 - 2 Structure and function of the repressor of bacteriophage lambda . III . Molecular cloning of the high-affinity mutant cI gene of lambda and studies of the properties of the clones; Das T et al.; The high-affinity mutant cI gene of lambda cIha (Nag et al . 1984) was cloned in the multicopy plasmid pBR322 . In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205 . Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of lambda cIha . Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of lambda. Mol Gen Genet, 1986 Sep, 204(3), 532 - 9 Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae; Birmingham VA et al.; A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae . The tylosin-resistance (Tylr) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment . The KpnI fragment contained all of the information required for the expression of the Tylr phenotype in S . lividans and S . griseofuscus . Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S . fradiae and several tylosin-sensitive (Tyls) mutants . The cloned tlrA gene failed to restore tylosin resistance in two Tyls mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyls mutant obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis . The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S . griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene . Twenty-eight kb of S . fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage lambda Charon 4 . Introduction of these DNA sequence into S . fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tylr gene is not closely linked to tylosin biosynthetic genes. Mol Gen Genet, 1986 Sep, 204(3), 477 - 84 Molecular cloning of the phosphate (inorganic) transport (pit) gene of Escherichia coli K12 . Identification of the pit+ gene product and physical mapping of the pit-gor region of the chromosome; Elvin CM et al.; The pit+ gene, encoding the phosphate (inorganic) transport system of Escherichia coli, was isolated from a library of E . coli genes inserted in the cosmid vector pHC79 . A 25.5-kb chromosomal DNA fragment was shown also to carry the gor locus encoding glutathione oxidoreductase . Physical mapping placed the two genes about 10 kb apart, confirming bacteriophage P1 mapping of the 77-min region . Subcloning and deletion analysis indicated that the entire pit+ gene was located within a 2.2-kb Sal1-Ava1 fragment . The pit+ gene product was identified by SDS-polyacrylamide gel electrophoresis as a 39-kdal inner membrane protein by two methods: 35S-methionine-labelling of minicells carrying pit+ plasmids or plasmids from which all or part of the pit+ gene was deleted . Overproduction of the Pit protein using a thermoinducible "runaway" replication plasmid . Complementation of the pit-1 mutant allele using a unit-copy-number pit+ plasmid indicated that the pit-1 mutation is recessive . Strains carrying a multicopy pit+ plasmid show a 10-fold increase in the initial rate of phosphate uptake; however there is no change in the steady-state level of 32Pi accumulation. Proc Natl Acad Sci U S A, 1986 Sep, 83(17), 6387 - 91 Isolation and characterization of a complementary DNA specific for human aromatase-system cytochrome P-450 mRNA; Evans CT et al.; A cloned complementary DNA sequence has been isolated from a human placental cDNA library in the bacteriophage expression vector lambda gt11 after screening with polyclonal antibodies against human placental aromatase-system cytochrome P-450 (P-450Arom) . A single recombinant clone, lambda hAROM1, was characterized by its ability to generate a beta-galactosidase fusion protein that reacted independently with polyclonal antibodies raised against beta-galactosidase and cytochrome P-450Arom and with monoclonal antibodies specific for cytochrome P-450Arom . The cDNA insert, which was found to be 1.8 kilobases in length, was radiolabeled and used to analyze poly(A)+ RNA isolated from human placenta and total RNA isolated from human adipose stromal cells cultured in the absence or presence of regulatory factors . The radiolabeled cDNA hybridized to several size species of mRNA in both placental and adipose stromal cell RNA fractions . Changes in the levels of adipose stromal cell RNA that hybridized to the cDNA insert were associated with comparable changes in the levels of translatable cytochrome P-450Arom mRNA and aromatase system activity . These findings are indicative that lambda hAROM1 contains DNA sequences complementary to human cytochrome P-450Arom mRNA and are suggestive that regulatory factors affect aromatase activity by altering the transcriptional activity of the cytochrome P-450Arom gene. J Bacteriol, 1986 Sep, 167(3), 905 - 19 Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu; Ross W et al.; We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition . All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivat