Phage

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 derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh-201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively . For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively . Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase . Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele . In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling . The extent of growth of Mu cts differed in the various gyrase mutants tested . Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants . In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested.

Clin Immunol Immunopathol, 1986 Sep, 40(3), 436 - 46
Transient immune deficiency in patients with acute Epstein-Barr virus infection; Junker AK et al.; To study the effect of primary Epstein-Barr virus (EBV) infection on antigen-specific antibody production, we immunized 17 college students who had developed acute infectious mononucleosis with the T-cell dependent neoantigen bacteriophage phi X174 . During the early phase of infectious mononucleosis, the proportion of peripheral blood lymphocytes displaying Ia and T8 (CD8) phenotypes was increased and the T helper/suppressor (T4/T8) ratio was decreased (less than 1) . These abnormalities disappeared during the convalescent phase . Correlating with EBV-induced changes in T lymphocytes, we demonstrated depressed humoral immune responses to bacteriophage phi X174 both in vivo and in vitro . In vitro coculture experiments indicated that the Ia+ suppressor T cells could inhibit antibody production and isotype switch . Removal of T8+ lymphocytes from patient T cells normalized in vitro antibody synthesis . In addition, impaired B-cell function was shown to be in part responsible for deficient antibody production . These studies demonstrate that infection with EBV affects both B and T lymphocytes and causes a broad-based transient immune deficiency in patients with uncomplicated infectious mononucleosis.

Proc Natl Acad Sci U S A, 1986 Sep, 83(18), 6716 - 20
Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation; Lundwall A et al.; Protein S is a cofactor of activated protein C; together they function as a regulator of blood coagulation . A human liver cDNA library constructed in bacteriophage lambda gt11 was screened with DNA fragments from a full-length bovine cDNA clone encoding protein S . Several cDNA clones were isolated and sequenced . The combined cDNA sequences encoded the mature protein and 15 residues of the leader sequence when compared to bovine protein S . Human protein S is a single-chain protein consisting of 635 amino acids with 82% homology to bovine protein S . After an NH2-terminal gamma-carboxyglutamic acid-containing region, there is a short region with thrombin-sensitive bond(s), followed by a region with four repeat sequences that are homologous to the precursor of mouse epidermal growth factor . In contrast to the other vitamin K-dependent plasma proteins, the COOH-terminal portion of human protein S does not show any resemblance to serine proteases.

J Bacteriol, 1986 Sep, 167(3), 1071 - 3
Cloning and expression of the ltf gene of bacteriophage T5; Heller KJ et al.; The unique 5-kilobase BamHI fragment of bacteriophage T5 was cloned into plasmid pBR322 . Location of the intact ltf gene on the cloned fragment was demonstrated by complementation of the ltf mutation of phage T5hd-2, identification of a plasmid-coded polypeptide of the same molecular weight as the polypeptide forming the L-shaped tail fibers, which binds to anti-T5 antibodies; and analyses of transposon Tn1000 insertions.

J Bacteriol, 1986 Sep, 167(3), 1035 - 42
Mutational analysis of bacteriophage lambda lysis gene S; Raab R et al.; A plasmid carrying the bacteriophage lambda lysis genes under lac control was subjected to hydroxylamine mutagenesis, and mutations eliminating the host lethality of the S gene were selected . DNA sequence analysis revealed 48 single-base mutations which resulted in alterations within the coding sequence of the S gene . Thirty-three different missense alleles were generated . Most of the missense changes clustered in the first two-thirds of the molecule from the N terminus . A simple model for the disposition of the S protein within the inner membrane can be derived from inspection of the primary sequence . In the first 60 residues, there are two distinct stretches of predominantly hydrophobic amino acids, each region having a net neutral charge and extending for at least 20 residues . These regions resemble canonical membrane-spanning domains . In the model, the two domains span the bilayer as a pair of net neutral charge helices, and the N-terminal 10 to 12 residues extend into the periplasm . The mutational pattern is largely consistent with the model . Charge changes within the putative imbedded regions render the protein nonfunctional . Loss of glycine residues at crucial reverse-turn domains which would be required to reorient the molecule to reenter the membrane also inactivate the molecule . Finally, a number of neutral and rather subtle mutations such as Ala to Val and Met to Ile are found, mostly within the putative spanning regions . Although no obvious explanation exists for this subtle and heterogeneous class of mutations, it is noted that all of the changes result in a loss of alpha-helical character as predicted by Chou-Fasman theoretical analysis . Alternative explanations for some of these changes are also possible, including a reduction in net translation rate due to substitution of a rare codon for a common one . The model and the pattern of mutations have implications for the probable oligomerization of the S protein at the time of endolysin release at the end of the vegetative growth period.

Mutat Res, 1986 Sep, 166(2), 187 - 98
A sensitive, enzymatic assay for the detection of closely opposed cyclobutyl pyrimidine dimers induced in human diploid fibroblasts; Lam LH et al.; A sensitive, enzymatic assay has been developed for the detection of closely opposed cyclobutyl pyrimidine dimers induced in UV-irradiated human diploid fibroblasts . In this assay closely opposed dimers are detected as bifilar enzyme-sensitive sites . Single-strand incisions are made at the positions of individual pyrimidine dimers through the action of M . luteus pyrimidine dimer-DNA glycosylase . Incisions at closely opposed dimers, effectively expressed as double-strand breaks, are quantified from the resulting reduction in DNA double-strand molecular weight as determined by velocity sedimentation through neutral sucrose density gradients . The stability of the bacteriophage lambda cos site under our reaction conditions indicates that opposed incisions must be relatively close to be expressed as a double-strand break . The dose response for the induction of bifilar enzyme-sensitive sites in mammalian cells was found to be complex but can be approximated by a function that increases as the 1.2-1.4 power of UV dose . The frequency of bifilar enzyme-sensitive sites observed decreased during postirradiation incubation of excision-repair-proficient human diploid fibroblasts with less than 20% still detectable at 24 h after irradiation with 5 J/m2 (254 nm) . By comparison, over 80% of the bifilar enzyme-sensitive sites induced in fibroblasts assigned to xeroderma pigmentosum complementation group A remained detectable 24 h after irradiation . The implications of these results for models addressing the induction and repair of closely opposed pyrimidine dimers are discussed.

J Mol Biol, 1986 Aug 20, 190(4), 587 - 91
Purification, crystallization and preliminary X-ray data of the bacteriophage MS2; Valegard K et al.; Single crystals of the bacteriophage MS2 have been produced by the vapour diffusion technique in the presence of 1.5% polyethylene glycol 6000 and 0.2 M-sodium phosphate buffer (pH 7.4) . These are the first bacteriovirus crystals diffracting to high resolution . The crystal space group is C2 with the unit cell parameters a = 467.9 A, b = 289.5 A, c = 275.6 A and beta = 121.8 degrees . The asymmetric unit contains one half of the virion . The maximum resolution limit of the X-ray diffraction data obtained from these crystals was 2.9 A . The purification of the virus material was done by mild procedures exclusively and involved precipitation with polyethylene glycol 6000 and size exclusion chromatography on Sepharose CL-4B.

J Mol Biol, 1986 Aug 20, 190(4), 577 - 86
Structure and inherent properties of the bacteriophage lambda head shell . V . Amber mutants in gene E; Katsura I; A total of 940 amber mutants in gene E of bacteriophage lambda was isolated to study the structure-function relationship of the gene product, the major capsid protein . The mutants were mapped to 43 mutation sites, most of which have been located, albeit tentatively, at exact points in the known base sequence, by deletion mapping and by the specificity of mutagenesis and the patterns of suppression . The patterns of suppression were interpreted in terms of both the efficiency of insertion of amino acid residues by suppressors and the exchangeability of amino acid residues . The exchangeability seems to be related to the hydrophilicity of the residues themselves and their environment, as well as to the functional similarity between the replaced and the inserted amino acid residues . Suppression of two of the mutations resulted in the production of characteristic aberrant head-related structures, each showing a defect in a different functional site in the protein . This, together with the approximate positions of some specific missense mutations as determined in this study, revealed the distribution of the functional sites along the polypeptide chain of the gene E product.

J Biol Chem, 1986 Aug 15, 261(23), 10736 - 43
Bacteriophage N4-coded 5'----3' exonuclease . Purification and characterization; Guinta D et al.; Bacteriophage N4 DNA replication requires the activity of a phage-induced exonuclease . We show here that the activity is phage coded . We have purified this enzyme to apparent homogeneity . It has a denatured molecular weight of 45,000 and exists in solution as a dimer . Duplex DNA is the preferred substrate which it degrades in a 5'----3' direction to 5' mononucleotides by a distributive mechanism . The enzyme does not act at a nick or a gap; indeed, it requires an end for activity . A possible role for this exonuclease in N4 replication is discussed.

J Biol Chem, 1986 Aug 15, 261(23), 10506 - 10
Development of an in vitro bacteriophage N4 DNA replication system; Rist JK et al.; An in vitro DNA replication system from bacteriophage N4-infected Escherichia coli has been developed . It requires MgCl2, all four deoxyribonucleoside triphosphates, and exogenously added N4 phage DNA; other DNAs are used inefficiently or not at all . Ribonucleoside triphosphates are not required, although they stimulate DNA synthesis . In vitro replication starts at the ends of the N4 genome and moves progressively inward . Initiation occurs through hairpin priming at the 3' ends of the genome, but shows a strong preference for the right end . Three N4 gene products (dnp, dbp, and exo) required in vivo for N4 DNA synthesis are absolutely required in the in vitro system . These findings are discussed with respect to the mode of N4 DNA replication.

J Immunol, 1986 Aug 15, 137(4), 1280 - 5
The role and mechanism of cobra venom factor-induced suppression of the humoral immune response in guinea pigs; Bottger EC et al.; The cobra venom factor (CVF)-mediated suppression of the humoral immune response to bacteriophage phi X 174, a T cell-dependent antigen, was investigated in guinea pigs . The suppression was markedly dependent on the amount of antigen used: decomplementation of animals diminished the humoral immune response when immunizing with low doses of antigen, whereas increasing the antigenic dose resulted in little or no diminution by CVF . In contrast to the results with normal animals, CVF had no influence on the humoral immune response of genetically C2-deficient guinea pigs; i.e., no suppression occurred above that due to the inherited complement deficiency state alone . We therefore postulate that CVF suppresses the humoral immune response by the depletion of C3, with no measurable role for complement split products under these experimental conditions.

J Mol Biol, 1986 Aug 5, 190(3), 509 - 12
Arrangement of double-stranded DNA packaged in bacteriophage capsids . An alternative model; Serwer P; Toroidal winding of double-stranded DNA in the protein capsids of bacteriophages has been proposed previously . An alternative model for the packaging and arrangement of DNA in bacteriophage capsids is presented here . By introducing sharp folds, the alternative model avoids toroidal winding and its accompanying difficulties . This alternative model is in agreement with the current data obtained with several different bacteriophages.

J Mol Biol, 1986 Aug 5, 190(3), 329 - 41
Topoisomerization of plasmid DNA in Escherichia coli infected with bacteriophage T4; Albright LM et al.; The degradation of host DNA, and the block to transcription of cytosine-containing DNA, which are a part of the normal course of infection by bacteriophage T4, can be eliminated in an appropriate T4 genetic background (designated as our reference type, or r.t.), so that T4 late promoters carried on plasmid DNA can function . The changes of topoisomer distribution that ensue when phage T4 r.t . infect Escherichia coli carrying a plasmid containing a T4 late promoter were analyzed . The linking number of the covalently closed circular plasmid DNA increased (implying relaxation) at the same time as the distribution of topoisomers became much broader . The relaxation of plasmid DNA was primarily, but not exclusively, due to T4 DNA topoisomerase II . The bacterial DNA topoisomerase II (gyrase) continued to function after phage infection to maintain some degree of superhelicity in plasmid DNA . When the DNA gyrase was inhibited by coumermycin or oxolinic acid, the topoisomer distribution became distinctly bimodal, part of the DNA remaining highly negatively supercoiled . It is argued that the observed post-infection topological changes involve relaxation of torsional stress and changes of binding by proteins that topologically constrain the plasmid DNA.

J Mol Biol, 1986 Aug 5, 190(3), 425 - 37
Specific binding of monomeric bacteriophage T3 and T7 RNA polymerases to their respective cognate promoters requires the initiating ribonucleoside triphosphate (GTP); Basu S et al.; Bacteriophage T3 and T7 RNA polymerases are monomeric proteins of Mr of about 100,000 . Each polymerase has stringent specificity for its own promoters that is present only on the homologous phage DNA template . Neither enzyme recognizes the heterologous phage promoters or Escherichia coli RNA polymerase promoters . In the present study, the interaction of T3 and T7 RNA polymerases with their respective cognate promoters was studied by DNase I footprinting techniques . These studies revealed an absolute requirement for the initiating nucleotide (GTP) for each phage RNA polymerase to bind specifically to and protect its cognate promoter from DNase I digestion . In the absence of the initiating nucleotide, both enzymes randomly bind DNA with lower affinity . No other nucleotide can substitute for GTP; however, the addition of GTP + ATP, which causes the synthesis of a hexamer RNA (pppGpGpGpApGpA), makes the DNA-RNA-protein complex highly stable . Nitrocellulose filter binding studies confirmed these observations . On the basis of these results we propose that the binding of the initiating nucleotide (in this case, GTP) drives the phage RNA polymerase into an "initiation conformation" in which the random DNA-binding property of the enzyme is converted to a promoter-specific recognition, and the polymerase is primed to initiate transcription.

J Biol Chem, 1986 Aug 5, 261(22), 10348 - 51
Isolation of the nuclear gene encoding a subunit of the yeast mitochondrial RNA polymerase; Kelly JL et al.; Antisera directed against the purified 145,000-dalton subunit of the Saccharomyces cerevisiae mitochondrial RNA polymerase have been used to immuno-screen a library of yeast genomic inserts constructed in the fusion protein expression vector, lambda gt11 . A 4-kilobase pair yeast DNA fragment inserted into one of the recombinant bacteriophages appears to contain most or all of the gene that encodes the 145,000-dalton subunit.

Bioorg Khim, 1986 Aug, 12(8), 1070 - 2
{Tritium labeling of RNA and protein of bacteriophage MS2}; Neiman LA et al.; Thermal activation of tritium gas is used for labeling of the nucleoprotein, phage MS 2 . The obtained preparation of tritiated phage has a specific radioactivity of 20-50 Ci/mmole, is considerably infectious and appears suitable for a wide range of studies . The radioactivity is distributed between intraphage RNA and phage outer protein (approximately 1:3 ratio) . Consequently, phage capsid is porous and sufficiently permeable for activated tritium atoms.

Anal Biochem, 1986 Aug 1, 156(2), 406 - 12
Rapid preparation of bacteriophage DNA for sequence analysis in sets of 96 clones, using filtration; Eperon IC; A method is described for the preparation of single-stranded DNA from clones in bacteriophage M13 vectors . This procedure allows multiples of 96 clones to be processed at once, utilizing filtration to remove host cells and simplifying the treatment of bacteriophage pellets . The DNA produced can be used for sequencing of mutagenesis.

Mol Cell Biol, 1986 Aug, 6(8), 2932 - 43
mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence; Adam SA et al.; We identified and produced antibodies to the major proteins that interact with poly(A)+ RNAs in the yeast Saccharomyces cerevisiae . The major proteins which were cross-linked by UV light to poly(A)+ RNA in intact yeast cells had apparent molecular weights of 72,000, 60,000, and 50,000 . The poly(A) segment of the RNA was selectively cross-linked to the 72,000-molecular-weight protein (72K protein) . Mice immunized with purified UV-cross-linked RNA-protein (RNP) complexes produced antibodies to the three major RNP proteins . A yeast genomic DNA library constructed in the lambda gt11 expression vector was screened with the anti-RNP serum, and recombinant bacteriophage clones were isolated . One recombinant phage, lambda YPA72.1, bearing a 2.5-kilobase insert, produced a large beta-galactosidase-RNP fusion protein . Affinity-selected antibodies from the anti-RNP serum on this fusion protein recognized a single 72K protein which was cross-linked to the poly(A) segment of RNA in the intact cell . Furthermore, the fusion protein of lambda YPA72.1 had specific poly(A)-binding activity . Therefore, lambda YPA72.1 encodes the 72K poly(A)-binding protein . Immunofluorescence microscopy showed that this protein was localized in the cytoplasm . Hybrid-selected mRNA translated in vitro produced the 72K poly(A)-binding protein, and mRNA blot analysis detected a single 2.1-kilobase mRNA . DNA blot analysis suggested a single gene for the poly(A)-binding protein . DNA sequence analysis of genomic clones spanning the entire gene revealed a long open reading frame encoding a 64,272-molecular-weight protein with several distinct domains and repeating structural elements . A sequence of 11 to 13 amino acids is repeated three times in this protein . Strikingly, this repeated sequence (RNP consensus sequence) is highly homologous to a sequence that is repeated twice in a major mammalian heterogeneous nuclear RNP protein, A1 . The conservation of the repetitive RNP consensus sequence suggests an important function and a common evolutionary origin for messenger RNP and heterogeneous nuclear RNP proteins.

J Gen Virol, 1986 Aug, 67 ( Pt 8), 1663 - 72
The characterization of a set of amber mutants of bacteriophage T5; Kay D et al.; A collection of amber mutants of bacteriophage T5 was analysed using an in vivo complementation test and assigned to 21 complementation groups . The incomplete phage structures produced by T5 amber mutant infection of the non-permissive host were examined . The mutants were allocated to four phenotypic types, as defined by an in vitro complementation test: those which produced functional heads, H; those which produced functional tails, T; those which produced inactive heads and functional tails, HI + T; and those which did not synthesize either heads or tails, 0 . Functional heads and inactive heads were indistinguishable in shape and size in the electron microscope . Four different patterns of DNA metabolism were observed when different amber mutants were grown under non-permissive conditions . They were the wild-type pattern (D+) in which host DNA degradation was followed by synthesis of phage DNA, host DNA degradation without phage DNA synthesis (D0), neither host DNA degradation nor phage DNA synthesis (DD0) and host DNA degradation with slight DNA synthesis (DS) . Upon infection of the non-permissive host with representatives of different complementation groups of mutants either normal lysis occurred or bacterial growth ceased without subsequent lysis . The phenotypic characteristics of the amber mutants were used for partial elucidation of the functions of the affected genes.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5382 - 6
5-Azido-2'-deoxyuridine 5'-triphosphate: a photoaffinity-labeling reagent and tool for the enzymatic synthesis of photoactive DNA; Evans RK et al.; We have synthesized the photoactive deoxyuridine nucleotide 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP) and used it to synthesize light-sensitive DNA by enzymatic incorporation . In the absence of ultraviolet light, 5-N3dUTP is a substrate for Escherichia coli DNA polymerase I . In in vitro DNA synthesis reactions using bacteriophage M13 single-stranded DNA as the template and 5-N3dUTP in place of dTTP, a photoactive complementary strand was synthesized by DNA polymerase I . The complementary strand was not synthesized when the 5-N3dUTP was substituted for dCTP or when it was exposed to ultraviolet light prior to the addition of DNA polymerase I . Using a synthetic lac operator template of 26 bases and a 15-base primer, we generated a photoactive 26-base-pair lac operator by enzymatically incorporating 5-N3dUMP with DNA polymerase I . Crosslinking of this photoactive DNA fragment to lac repressor was totally dependent on the presence of UV light and was reduced 78% by 150 microM isopropyl beta-D-thiogalactoside . Under the same conditions no crosslinking to lac repressor was observed using a nonphotoactive 26-base-pair lac operator . Photoactivatable deoxyuridine analogs have potential application as reagents to crosslink DNA binding proteins to 5-azidouracil-containing DNA and as active-site-directed photoaffinity labelling reagents.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5469 - 73
Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein; Tullius TD et al.; A method has been developed for making "footprints" of proteins bound to DNA . The hydroxyl radical, generated by reduction of hydrogen peroxide by iron(II), is the reagent used to cut the DNA . Hydroxyl radical breaks the backbone of DNA with almost no sequence dependence, so all backbone positions may be monitored for contact with protein . In addition to defining the DNA sequence in contact with the protein, hydroxyl radical footprints embody structural information about the DNA-protein complex . For example, hydroxyl radical footprints of the bacteriophage lambda repressor and Cro protein show directly that these proteins are bound to only one side of the DNA helix . Additional contacts of lambda repressor and Cro protein with DNA, not observed by other chemical footprinting methods, are revealed by hydroxyl radical footprinting.

J Bacteriol, 1986 Aug, 167(2), 744 - 8
Genetic analysis of Myxococcus xanthus and isolation of gene replacements after transduction under conditions of limited homology; O'Connor KA et al.; Genetic analysis of Myxococcus xanthus is greatly facilitated by the ability to introduce cloned DNA into M . xanthus to generate gene replacement and merodiploid strains . However, gene replacement strains are difficult to obtain when the region(s) of homology between the cloned DNA and the M . xanthus chromosome is limited (less than 1 kilobase) . We found that gene replacements can be obtained at an increased frequency by a two-step procedure involving the use of bacteriophage P1 to isolate merodiploid strains followed by generalized transduction to another M . xanthus strain by using phage Mx4.

Mol Gen Genet, 1986 Aug, 204(2), 359 - 61
sub, a host mutation that specifically allows growth of replication-deficient gene B mutants of coliphage P2; Sunshine M et al.; Bacteriophage P2 normally requires the products of its early genes A and B for lytic growth in its host, Escherichia coli C . A host mutation, sub-1, which allows P2 to grow without a functional B gene product is described . The sub-1 mutation is recessive and maps at approximately 10 min on the E . coli genetic map.

Genetics, 1986 Aug, 113(4), 797 - 810
Recombination between IS5 elements: requirement for homology and recombination functions; Timmons MS et al.; Intermolecular recombination between two IS5 elements was measured, using bacteriophage lambda recombination vectors, and was compared to recombination between two copies of an SV40 segment cloned into the same vectors . Experiments were conducted in the presence and in the absence of RecA and Red functions, and with the recombining inserts in the same or in reversed orientation . Under all conditions, IS5 elements recombined in a manner similar to the SV40 inserts, indicating that IS-encoded functions did not confer measurable additional intermolecular recombination ability to IS5 in E . coli K-12 . Bacteriophages containing reversed IS5 inserts, for which the 16 base pair (bp) termini are identical in 15 positions and which display 12 bp of uninterrupted homology, recombined at approximately the same low frequency under Rec+ and Rec- conditions, indicating that these short homologies were not good substrates for the Rec system . Bacteriophages having reversed inserts recombined better under Red+ than under Red- conditions, but the crossovers were located in nonhomologous regions flanking the element termini . This suggests that 12-bp homologies are not good substrates for the Red system.

DNA, 1986 Aug, 5(4), 299 - 304
Analysis of the organization and nucleotide sequence of the chromosomal gene for the beta-subunit of rat thyrotropin; Croyle ML et al.; The gene for the beta-subunit of rat thyrotropin has been isolated from a library of rat DNA fragments cloned in bacteriophage lambda . The complete nucleotide sequence of the gene has been determined including a portion of 5'- and 3'-flanking regions . The rat TSH-beta gene contains approximately 4879 nucleotides which ultimately lead to the production of a mRNA of about 554 nucleotides, exclusive of the 3' poly(A) tract . The first exon which represents only 5' untranslated sequences of the mRNA, is separated from the second exon by a very large, 3.9-kbp intervening sequence . The second and third exons are separated by a small, 377-bp intervening sequence . Southern blot analysis of total genomic DNA demonstrated that the rat genome contains sequences similar to the cloned gene, suggesting that no rearrangements occurred during the cloning process.

Virology, 1986 Aug, 153(1), 70 - 9
Transposition and replication of maxi-Mu derivatives of bacteriophage Mu; Faelen M et al.; The insertion of DNA fragments within the lac sequence of a MudI(Ap,lac) prophage resulted in the formation of a set of maxi-Mu genomes which were 39.8, 59, 85.6, and 88.2 kb long, respectively . The comparison of these maxi-Mu's with the 37.2-kb-long parental MudI(Ap,lac) indicated that the transposition frequency decreased as the length of the prophage increased . No replication of the two longest maxi-Mu's could be detected . The 59- and the 39.8-kb-long chimeric genomes were noted to replicate at approximately 1-2 and 30%, respectively, of the rate found with the MudI(Ap,lac) prophage . The length dependence of the transposition and replication could be explained by the impairment of an early step of the transposition/replication mechanism.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 6012 - 6
Isolation of point mutations in bacteriophage Mu attachment regions cloned in a lambda::mini-Mu phage; Burlingame RP et al.; Twenty-one derivatives of a lambda::mini-Mu phage containing point mutations in the Mu attachment regions were isolated after mutD mutagenesis and selection for relief from Mu-specific replicative interference of lambda growth . DNA sequence analysis revealed that the single left-end mutant had suffered a T----C transition at position 1 of the Mu sequence, while the remaining 20 right-end mutants contained single base-pair insertions or deletions within the terminal 19 base pairs . A genetic assay showed that the right-end mutations revealed by sequencing were necessary for relief of the replicative inhibition of lambda growth . The properties of these mutants suggest that the terminal 2-base-pair and subterminal 8-base-pair inverted repeats are important for Mu-specific replicative transposition.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5784 - 8
Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein; Birnbaum MJ et al.; Antibody raised against the human erythrocyte glucose transporter identified a recombinant lambda gt11 bacteriophage in a cDNA library prepared from immunoselected polysomal RNA from adult rat brain . The cDNA predicts a 492-amino acid protein that demonstrates 97.6% identity to the human hepatoma hexose carrier . The tissue distribution of the transporter mRNA is identical to that of immunologically identifiable protein and transport activity, except in liver in which high levels of transport are associated with little or no transporter mRNA or protein . As assayed by blot-hybridization analysis, mRNA from insulin-responsive and nonresponsive tissues are indistinguishable . These data suggest that a genetically unrelated protein is responsible for hexose transport in normal liver.

J Virol, 1986 Aug, 59(2), 203 - 9
Interruption-deficient mutants of bacteriophage T5: analysis of single-site mutants; Rhoades M; The properties of viable mutants of bacteriophage T5 that lack, singly, each of the four major sites at which single-chain interruptions normally occur in T5 DNA are described . The mutations responsible for loss of each interruption were mapped by analysis with HhaI, a restriction endonuclease with a cleavage site (pGCGC) that occurs at the 5' termini of the major interruptions (B . P . Nichols and J . E . Donelson, J . Virol . 22:520-526, 1977) . For each mutant tested, loss of a specific interruption resulted in loss of a specific HhaI cleavage site . Multiple single-site mutants were constructed to determine the effect of loss of more than one interruption on phage viability . These recombinants, including a phage that lacks the four major interruptible sites, were fully viable and did not exhibit a compensating increase in the frequency of minor interruptions . The effect of loss of a specific interruption on genetic recombination was tested in two-factor crosses with markers that occur close to, but on opposite sites of, the interruption . Loss of the interruptible site did not affect recombination frequency.

J Bacteriol, 1986 Aug, 167(2), 503 - 7
Role of ner protein in bacteriophage Mu transposition; Goosen N et al.; The Ner protein of bacteriophage Mu negatively regulates transcription initiated at the early promoter and at the major repressor promoter . The construction and isolation of a Ner- mutant of Mu is described . Ner is an essential function for Mu, because the mutant phage only forms plaques when complemented for Ner . Mutations in the repressor protein did not abolish the need for Ner . However, when transcription of the repressor gene c was blocked by deleting the major repressor promoter, Ner was no longer essential for normal Mu development.

Mol Gen Genet, 1986 Aug, 204(2), 322 - 7
In vivo loss of supercoiled DNA carrying a palindromic sequence; Leach D et al.; Interest in the fate of long palindromic DNA sequences in E . coli has been kindled by the observation that their inviability is overcome in recBC sbcB strains and that these hosts permit the construction of DNA libraries containing long palindromic sequences present in the human genome . In this paper we show that a reduction in the level of intracellular supercoiled DNA occurs as the result of the presence of a 530 bp palindrome in bacteriophage lambda . This reduction occurs in Rec+ and recA strains but not in strains lacking exonucleases V and I (recBC sbcB) . However, the DNA must be active (not repressed) for this reduction to be observed, since it is not seen in a Rec+ host lysogenic for phage lambda . These results argue against two hypotheses: firstly, that the palindrome causes inviability solely by interfering with packaging, and secondly, that it does so solely by interfering with recombination . Conversely, these results suggest that a feature of active monomeric DNA (probably its replication) is involved in inviability.

Virology, 1986 Aug, 153(1), 80 - 6
Subunit arrangement of the tail fiber of bacteriophage T3; Kato H et al.; A tail fiber of phage T3 is a trimer of the product of gene 17 (gp17) . Treatment of T3 phage particles with chymotrypsin resulted in cleavage of only the tail fiber protein, at a site near the distal end of the fiber, causing a decrease of about 10% in the size of gp17 in the treated virion . The N-terminal amino acid sequences of intact and cleaved tail fiber proteins were identical and corresponded to that deduced from the nucleotide sequence of gene 17 except for the absence of the initiation Met residue . These results indicate that cleavage of the tail fiber occurred near the C terminus and suggest that gp17 polypeptides are oriented parallel to each other in the tail fiber . Association of tail fibers with the tail involves the N-terminal region of gp17 . Under mild conditions of SDS-polyacrylamide gel electrophoresis, intact tail fibers dissociated from virions but cleaved ones did not . The nucleotide sequences indicate that T3 and T7 gp17 contain many sites that are potentially sensitive to chymotrypsin . In fact, free tail fibers, purified from T3-infected cells, were cleaved to many smaller fragments by chymotrypsin . These results suggest that the attachment of the tail fibers to the tail may induce a change(s) in the configuration and/or arrangement of gp17 to mask the sensitive sites from cleavage by chymotrypsin.

Dev Biol, 1986 Aug, 116(2), 539 - 42
Formation of nucleus-like structure in the cytoplasm of lambda-DNA-injected fertilized eggs and its partition into blastomeres during early embryogenesis in Xenopus laevis; Shiokawa K et al.; The fate of bacteriophage lambda-DNA was examined after injection into the fertilized eggs of Xenopus laevis . Injection of a large amount of lambda-DNA (ca . 24 ng) into a fertilized Xenopus egg induced the formation around the injected DNA of a giant nucleus-like structure which was surrounded by an apparently normal bilayered nuclear membrane with nuclear pore complexes . Southern blot analysis revealed the persistence of injected lambda-DNA until the blastula stage . The nucleus-like structure was partitioned into blastomeres during cleavage through a process of nuclear fission, and was maintained in a group of extraordinarily large blastomeres until the blastula stage.

Anal Biochem, 1986 Aug 1, 156(2), 354 - 6
Electrophoretic identification of fusion proteins expressed in single recombinant lambda-bacteriophage plaques; Saul A et al.; Sufficient fusion protein is present in single plaques produced by lytic, recombinant lambda bacteriophage to be detected by Coomassie blue staining following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels . Agar plugs containing single plaques can be loaded directly onto the stacking gel thus avoiding the need for extensive sample preparation.

Biochem Biophys Res Commun, 1986 Jul 31, 138(2), 945 - 52
Efficient screening of recombinant DNA junctions afforded by probing with synthetic "bridge" oligonucleotides; Recinos A 3rd et al.; Procedures have been developed which simplify and expedite the screening of recombinant DNA constructions for those which only exhibit the desired DNA-DNA junctions . A synthetic DNA oligonucleotide designed to span (or "bridge") sequences around correct restriction enzyme junctions was used as a hybridization probe for the rapid identification of those sequences in several molecular cloning methodologies . It facilitated analyses of the products of random ligation reactions, as well as constructions harbored in bacteria and bacteriophage . "Bridge" probes, {32P}-end-labeled to very high specific activity, remained useful after several hybridizations and often circumvented lengthy restriction analyses.

Nucleic Acids Res, 1986 Jul 25, 14(14), 5813 - 26
Translational regulation of expression of the bacteriophage T4 lysozyme gene; McPheeters DS et al.; The bacteriophage T4 lysozyme gene is transcribed at early and late times after infection of E . coli, but the early mRNA is not translated . DNA sequence analysis and mapping of the 5' ends of the lysozyme transcripts produced at different times after T4 infection show that the early mRNA is initiated some distance upstream from the gene . The early mRNA is not translated because of a stable secondary structure which blocks the translational initiation site . The stable RNA structure has been demonstrated by nuclease protection in vivo . After DNA replication begins, two late promoters are activated; the late transcripts are initiated at sites such that the secondary structure can not form, and translation of the late messages occurs.

J Biol Chem, 1986 Jul 25, 261(21), 9879 - 85
O6-methylguanine mutation and repair is nonuniform . Selection for DNA most interactive with O6-methylguanine; Topal MD et al.; Mutations were induced in the ampicillinase gene of a bacteriophage f1/pBR322 chimera both by incorporation of O6-methyl-dGTP opposite T during DNA replication in vitro and by site-directed mutagenesis using O6-methylguanine-containing oligonucleotides . After passage of the DNA through Escherichia coli, analysis of 151 O6-methyl-dGTP-induced mutations indicated a significantly greater number of unmutated mutation sites than expected, whereas the mutated sites generally fit a Poisson distribution . The unmutated sites are assumed to be caused by the inability of some sequences to tolerate the presence of a tetrahedral methyl group within the confines of a Watson-Crick helix (Toorchen, D., and Topal, M.D . (1983) Carcinogenesis 4, 1591-1597) . A consensus of the DNA sequences surrounding unmutated mutation sites was derived . The consensus sequence had significant similarity to the region of the rat Harvey ras oncogene containing the N-methyl-N-nitrosourea activated site for transformation (Zarbl, H., Sukumar, S., Arthur, A . V., Dionisio, M.-Z., and Barbacid, M . (1985) Nature 315, 382-385) . We propose that direct alkylation at O6 of a guanine present within the consensus sequence may produce a DNA conformation less subject to repair . Mutation by O6-methylguanine-containing oligonucleotides demonstrated that repair of the O6-methylguanine lesions varied at least 3-4-fold with position of the lesion.

Nucleic Acids Res, 1986 Jul 11, 14(13), 5481 - 97
Secondary structure as primary determinant of the efficiency of ribosomal binding sites in Escherichia coli; Looman AC et al.; Using a previously described vector (pKL203) we fused several heterologous ribosomal binding sites (RBSs) to the lacZ gene of E . coli and then studied the variation in expression of the fusions . The RBSs originated from bacteriophage Q beta and MS2 genes and the E . coli genes for elongation factor EF-Tu A and B and ribosomal protein L11 (rplK) . The synthesis of the lacZ fusion proteins was measured by an immuno precipitation method and found to vary at least 100-fold . Lac-specific mRNA synthesis follows the variation in protein production . It appears that there is a correlation between the efficiency of an RBS to function in the expression of the fused gene and the lack of secondary structure, involving the Shine and Dalgarno nucleotides (SDnts) and/or the initiation codon . This efficiency is context dependent . The sequence of the SD nts and the length and sequence of the spacer region up to the initiation codon alone are not able to explain our results . Deletion mutations, created in the phage Q beta replicase RBS, reveal a complex pattern of control of expression, probably involving the use of a "false" initiation site.

Nucleic Acids Res, 1986 Jul 11, 14(13), 5309 - 20
Structural organization and nucleotide sequence of mouse c-myb oncogene: activation in ABPL tumors is due to viral integration in an intron which results in the deletion of the 5' coding sequences; Lavu S et al.; Bacteriophage libraries of mouse DNA were screened for sequences homologous to the v-myb oncogene and two overlapping clones containing the v-myb related region were isolated . Restriction enzyme mapping, heteroduplex analysis and nucleotide sequence analysis revealed the presence of nine exons . Six of these exons are homologous to the v-myb region while the other three exons are derived from the 5' region which is deleted in the viral oncogene . The sequences downstream to the sixth v-myb exon are not included in the 17 kbp of DNA sequences analyzed in this study . Comparison of the structure of the normal c-myb clone with its rearranged couterpart present in plasmacytoid lymphosarcomas revealed that the rearrangements occur in this locus as a result of viral integration . Present studies demonstrate that such a viral insertion interrupts the c-myb coding region at a region identical to that observed in the generation of the v-myb gene of avian myeloblastosis virus and results in the synthesis of mRNAs that lack the same 5' coding region.

J Mol Biol, 1986 Jul 5, 190(1), 83 - 95
Amber mutants in gene 67 of phage T4 . Effects on formation and shape determination of the head; Keller B et al.; Two amber mutations in gene 67 of bacteriophage T4 were constructed by oligonucleotide-directed mutagenesis and the resulting mutated genes were recombined back into the phage genome and their phenotype was studied . The 67amK1 mutation is close to the amino terminus of the gene, and phage carrying this mutation are unable to form plaques on suppressor-negative hosts . A second mutation, 67amK2, which lies in the middle of the gene, three codons N-terminal to a proteolytic cleavage site, produces a small number of viable phage particles . In suppressor-negative hosts, both mutants produce polyheads and proheads . 67amK1 assembles only few proheads that have a disorganized core structure, as judged from thin sections of infected cells . The proheads and the mature phages of both mutants are mainly isometric rather than having the usual prolate shape . Depending on the 67 mutant and the host, between 20% and 73% of the particles that are produced are isometric, and 1 to 10% are two-tailed biprolate particles . 67amK2 phages grown on a supD suppressor strain that inserts serine in place of the wild-type leucine do not contain gp67* derived from gene product 67 (gp67) by proteolytic cleavage . This demonstrates the importance of the correct amino acid at this position in the protein . Other abnormalities in these 67amK2 phages are the presence of uncleaved scaffolding core proteins (IPIII and gp68), indicating a structural alteration in the prohead scaffold, resulting in only partial cleavage . In wild-type phages these proteins are found in the head only in the cleaved form . With double-mutants of 67 with mutations in the major shell protein gp23 no naked scaffolding cores were found, confirming the necessity of gp67 for the assembly or persistence of a "normal" core.

J Mol Biol, 1986 Jul 5, 190(1), 11 - 22
Modification enhancement by the restriction alleviation protein (Ral) of bacteriophage lambda; Loenen WA et al.; The product of the lambda ral gene alleviates restriction and enhances modification by the Escherichia coli K-12 restriction and modification system . An open reading frame (orf) located between genes N and Ea10 has been assigned to the ral gene . We have cloned this orf in a plasmid where its transcription is controlled by a thermolabile lambda repressor . Inactivation of the lambda repressor caused a 1000-fold reduction in K-specific restriction of unmodified lambda phage and a 100-fold increase in modification . In minicells transformed with ral+ plasmids, derepression resulted in the appearance of a polypeptide with a lower mobility than that predicted for a protein encoded by the orf attributed to ral; in a transcription and translation system in vitro DNA from a ral+ plasmid encoded a polypeptide with the same mobility . This polypeptide was absent when the plasmid DNA carried a mutant ral gene . The nucleotide sequence of this mutant gene defined two base changes, one of which inactivates the initiation codon of the orf . The K restriction endonuclease, which is also a K-specific methylase, is encoded by three genes designated hsdR, hsdM and hsdS, although the hsdR polypeptide is not essential for the methylase activity . We show that Ral enhances modification in a host strain lacking the entire hsdR gene, and lambda phages carrying the hsdM and S genes modify their own DNA inefficiently in the absence of Ral, despite the fact that derivatives of these phages provide efficient amplification of the K-specific methylase . Our data support a model in which, as a consequence of the interaction of Ral with either the hsdM or the hsdS polypeptide, the conformation of the enzyme is changed and the efficiency of methylation of unmodified target sites is enhanced . It has been postulated that Ral counteracts Rho, but in our experiments Ral did not relieve transcriptional polarity.

Mol Biol (Mosk), 1986 Jul-Aug, 20(4), 1048 - 52
{Measuring the bleomycin-induced paired damage of two DNA chains by a method of psoralen photocross-linking}; Gonikberg EM et al.; DNA of bacteriophage PM2 was allowed to react with bleomycin in the presence of Fe(II) and oxygen and the "paired" DNA lesions of two types were measured: (1) double-strand breaks, (2) lesions converted to double-strand breaks after introducing into the DNA a large number of psoralen cross-links (about 10(-2) per base pair) and alkali treatment . The mean numbers of each lesion type per DNA molecule are found to be proportional to the square of bleomycin concentration over the range of 3 X 10(-7) to 3 X 10(-6) M . These findings indicate that paired lesions are formed as a result of action of two bleomycin molecules at the same DNA site.

Mol Gen Genet, 1986 Jul, 204(1), 192 - 4
Mu-induced rifamycin-resistant mutations not located in the rpoB gene of Escherichia coli; Kollenda MC et al.; A new class of rifamycin-resistant mutants of Escherichia coli was obtained by lysogenic insertions of bacteriophage Mu Amp DNA . Rifamycin resistance is closely linked to the ampicillin resistance conferred by the prophage . Mapping by conjugation with auxotrophic markers revealed that the rifamycin-resistant mutations are located between 28 and 37 min on the E . coli chromosome standard map, some distance from the rpoB gene at 89.5 min . The DNA-dependent RNA polymerase of these mutants is highly sensitive to rifampicin.

Transplantation, 1986 Jul, 42(1), 14 - 9
Correction of equine severe combined immunodeficiency by bone marrow transplantation; Bue CM et al.; A 32-day-old horse with severe combined immunodeficiency was transplanted with equine bone marrow cells in an attempt to establish immunologic responsiveness . A histocompatible, mixed-leukocyte-culture-nonreactive, sex-matched, full sibling was used as the donor . Recipient total lymphocyte count, T and B lymphocyte numbers, and response of peripheral blood mononuclear cells to phytolectin stimulation increased by 14 days following transplantation . Circulating lymphocytes exceeded 1000 cells/microliter blood by 40 days posttransplantation, and by 170 days following transplantation, T and B lymphocyte numbers had reached normal values . The foal demonstrated significant primary and secondary antibody responses when immunized with bacteriophage phi X 174 at 100 and 142 days posttransplantation . Concentrations of IgG and IgM remained within the normal range following cessation of i.v . plasma therapy 156 days after transplantation . More than 300 days following transplantation, the foal remains healthy and is growing normally . At no time during the posttransplant period was there detectable evidence of graft-versus-host disease.

Mutat Res, 1986 Jul, 166(1), 1 - 8
Mechanism of inhibition of bacteriophage T7 DNA synthesis in Escherichia coli B cells infected by alkylated bacteriophage T7; Czaika G et al.; Quantitative analysis of DNA replication, in E . coli B cells infected by methyl methanesulfonate-treated bacteriophage T7, showed that production of phage DNA was delayed and decreased . The cause of the delay appeared to be a delay in host-DNA breakdown, the process which provides nucleotides for phage-DNA synthesis . In addition, reutilisation of host-derived nucleotides was impaired . These observations can be accounted for by a model in which methyl groups on phage DNA slow down DNA injection and also reduce the replicational template activity of the DNA once it has entered the cell . Repair of alkylated phage DNA may be required not only for replication but also for normal injection of DNA.

Mutat Res, 1986 Jul, 161(2), 113 - 8
Mutagenesis by normal metabolites in Escherichia coli: phenylalanine mutagenesis is dependent on error-prone DNA repair; Sargentini NJ et al.; In search of a model for the production of 'spontaneous' mutations induced by DNA damage produced during normal metabolism, 19 amino acids were tested for mutagenicity in Escherichia coli K-12 uvrB . Cystine, and, to a lesser extent, arginine and threonine were found to be antimutagenic; only phenylalanine was found to be mutagenic . At 2 mM, phenylalanine induced mutants at 1.5-2-fold above background {lacZ53(amber)----Lac+, rifampicin resistance (missense), and bacteriophage T6 resistance} . Tyrosine and, to a lesser extent, tryptophan (each at 2 mM) inhibited the mutagenicity of phenylalanine . Phenylalanine mutagenesis was detected in the uvrB strain, but not in the wild-type, uvrB umuC or uvrB lexA strains . Thus, phenylalanine seems to cause the production of excisable lesions ('UV-like'?) in DNA, which, if not excised, can induce mutations via error-prone DNA repair.

Clin Exp Immunol, 1986 Jul, 65(1), 90 - 9
Variability in B cell maturation and differentiation in X-linked agammaglobulinemia; Leickley FE et al.; Among seven males with X-linked agammaglobulinemia in an extended pedigree, serum immunoglobulins and antibodies were extremely low in all but one who had a normal IgA (78 mg/dl) and tetanus antibodies (1:19,683) . Following bacteriophage phi X 174 immunizations, the oldest failed to clear phage and had no primary or secondary antibody responses . The youngest had normal phage clearance, low primary and secondary antibody responses, and no amplification or switching to IgG . The other four affected had normal or slightly delayed phage clearance, low primary and secondary responses, but some amplification and switching from IgM to IgG which increased with age . Normal percentages of surface immunoglobulin positive cells were present in the two youngest patients, but all seven affected had very low percentages of cells reacting with monoclonal antibodies to B cell surface antigens . Immunoglobulin production by cultured blood B cells was very low and not increased by pokeweed mitogen . However, a majority of Epstein-Barr virus (EBV)-transformed lymphoblastoid cells derived from the blood of four of the patients bore IgD and IgM and reacted with all of the monoclonal antibodies to B cell antigens . Culture supernatants from those lines contained significant quantities of IgM and lesser amounts of IgG and IgA . The studies presented here provide further support for the hypothesis that the primary abnormality in X-linked agammaglobulinemia affects B cells at more than one stage of development rather than just at the level of the pre-B cell.

J Ultrastruct Mol Struct Res, 1986 Jul-Sep, 96(1-3), 189 - 93
Visualization of RNA polymerase bound to R-loop molecules improves electron microscopic analysis of in vitro transcription; Meyer J et al.; An electron microscope method is described which allows improved analysis of in vitro transcription . Transcription complexes are fixed with glutaraldehyde, subjected to R-loop conditions which allow the nascent RNA chains to hybridize to the DNA templates, and mounted for electron microscopy by a protein-free preparation method . An RNA polymerase molecule (or parts of it) associated with only one end of the R-loop identifies the polarity of the transcript, thus determining the origin and direction of transcription . The method was evaluated using known in vitro promoters on the bacteriophage P1 genome and was used for mapping of additional promoters in their vicinity.

J Lipid Res, 1986 Jul, 27(7), 706 - 12
Amino acid sequence of rat apolipoprotein A-II deduced from the nucleotide sequence of cloned cDNA; Nagashima M et al.; A rat apolipoprotein A-II cDNA clone was isolated from a rat liver cDNA library by in situ hybridization of bacteriophage plaques using a 32P-labeled human apoA-II cDNA as a probe . The cDNA insert from this clone was characterized by DNA sequencing . The amino acid composition derived from the DNA sequence data matched well with that of rat apoA-II reported earlier (Herbert et al . 1974 . J . Biol Chem . 249: 5718-5724), indicating that the cDNA insert coded for rat apoA-II . Further evidence was provided by a comparison of the amino acid sequence of rat apoA-II obtained here with that of human apoA-II (Brewer et al . 1972 . Proc . Natl . Acad . Sci . USA . 69: 1304-1308) . While the rat apoA-II cDNA insert did not code for the entire presegment, it had the same COOH-terminal residues of the presegment as well as the same prosegment (Ala-Leu-Val-Arg-Arg) as in human preproapoA-II, suggesting that rat apoA-II was also synthesized initially as preproapoA-II . Mature rat apoA-II contains 79 amino acids . Residue 6 of mature rat apoA-II is Asp, while it is Cys in human apoA-II, and this would account for the absence of dimeric forms of rat apoA-II in plasma . While the overall amino acid sequence homology between rat and human apoA-II is about 50%, the amphipathic alpha-helical structures, which are responsible for lipid-binding, seem to be conserved in the two proteins . The size of rat apoA-II mRNA was estimated to be about 600 nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Infect Immun, 1986 Jul, 53(1), 129 - 34
Expression of Mycoplasma pneumoniae antigens in Escherichia coli; Trevino LB et al.; A genomic library of Mycoplasma pneumoniae was generated by using bacteriophage lambda EMBL3 as the vector . Screening of the library for the expression of M . pneumoniae protein antigens with adsorbed anti-M . pneumoniae serum revealed strong reactivity from a third of those clones which contained mycoplasma DNA inserts . Three of the most highly reactive clones were analyzed in detail and found to synthesize discrete mycoplasma proteins . Two carried overlapping fragments of mycoplasma DNA which encoded a protein that was readily detected in Escherichia coli after infection with recombinant bacteriophage . The third clone contained a novel mycoplasma DNA fragment which directed the synthesis of two additional mycoplasma proteins . Further screening of the library with antiserum raised against the major M . pneumoniae adhesin protein P1 (165 kilodaltons {kDa}) yielded one clone which produced an immunologically reactive protein of 140 kDa . Adsorption of anti-P1 serum by this clone selected a population of antibodies that were reactive with M . pneumoniae adhesin P1 (165 kDa) . These results demonstrate that immunologically active M . pneumoniae proteins are synthesized in E . coli.

Mol Gen Genet, 1986 Jul, 204(1), 24 - 8
Conditionally lethal nusAts mutation of Escherichia coli reduces transcription termination but does not affect antitermination of bacteriophage lambda; Nakamura Y et al.; Termination of transcription at bacteriophage lambda terminators as well as at the Escherichia coli trp a attenuator was examined in the conditionally lethal mutant (nusAts11) defective in the NusA protein of E . coli . Experiments using terminator-assay lambda vectors revealed that the efficiency of termination at both rho-dependent (lambda tL1) and rho-independent (lambda tL2 and trp a) terminators decreases in the mutant . The mutation does not block lambda phage growth at either permissive or nonpermissive temperatures, nor does it affect the lambda Q protein antitermination activity at the t6s terminator . These results indicate that NusA is required for transcription termination, and that lambda N and Q-mediated antitermination may not require the NusA protein function in the nusAts11 mutant.

J Virol, 1986 Jul, 59(1), 31 - 6
In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein; Ruyechan WT et al.; The major herpes simplex virus DNA-binding protein, ICP8, was purified from cells infected with the herpes simplex virus type 1 temperature-sensitive strain tsHA1 . tsHA1 ICP8 bound single-stranded DNA in filter binding assays carried out at room temperature and exhibited nonrandom binding to single-stranded bacteriophage fd DNA circles as determined by electron microscopy . The filter binding assay results and the apparent nucleotide spacing of the DNA complexed with protein were identical, within experimental error, to those observed with wild-type ICP8 . Thermal inactivation assays, however, showed that the DNA-binding activity of tsHA1 ICP8 was 50% inactivated at approximately 39 degrees C as compared with 45 degrees C for the wild-type protein . Both wild-type and tsHA1 ICP8 were capable of stimulating viral DNA polymerase activity at permissive temperatures . The stimulatory effect of both proteins was lost at 39 degrees C.

Mol Biol (Mosk), 1986 Jul-Aug, 20(4), 974 - 84
{Spatial structure of the cro-repressor in a solution . I . Identification of interaction in a hydrophobic cluster using the nuclear Overhauser effect}; Kurochkin AV et al.; The structure of a bacteriophage lambda cro repressor hydrophobic globule was studied by the technique of 1H NMR spectroscopy at 500 MHz . The analysis of NOE difference spectra and building of the molecular models for the most probable fragments of the secondary structure allowed us to assign many signals in the protein spectrum and to identify the intramolecular interactions which stabilized the hydrophobic globule and the tertiary structure of the molecule . The results suggest that the structure of the cro repressor hydrophobic globule in solution coincides with the crystal one, although there are some differences in the mutual arrangement of the alpha 1 helix and the three-fold beta-sheet . Resonance assignment has made it possible to study conformational changes and specific interactions of the cro repressor by using suitable reporter groups.

Mol Gen Genet, 1986 Jul, 204(1), 141 - 7
Role of homology and pathway specificity for recombination between plasmids and bacteriophage lambda; King SR et al.; To determine the minimum amount of homology required for efficient recombination in Escherichia coli, we measured recombination frequencies between bacteriophage lambda and pBR322 derivatives containing lambda DNA fragments of various sizes by assaying for phages that could transduce the bla and ori genes of pBR322 . Efficient recombination required about 40 bp of homology; increases in homology above 40 bp resulted in proportionate increases in recombination, while decreases below 40 bp resulted in precipitous decreases in recombination . The recA+ gene stimulated recombination over the entire range of homologies tested . Restriction enzyme digests of several recombinant DNA molecules indicated that they contained the complete plasmid DNA inserted in the lambda genome as expected for a reciprocal crossover . Analysis of recombination frequencies in different recombination-deficient mutant strains indicated that the formation of lambda-plasmid cointegrates by homologous recombination proceeded predominantly by the RecBC pathway and very inefficiently, if at all, by the RecE and RecF pathways.

J Bacteriol, 1986 Jul, 167(1), 415 - 9
Translational regulatory signals within the coding region of the bacteriophage lambda cIII gene; Altuvia S et al.; Six independent mutations which enhance the lysogenic response were analyzed . The mutations cause single-base substitutions at three sites within the cIII coding sequence, one of which does not change the amino acid code . The mutations allow for elevated translation of the cIII gene, possibly via changes in the mRNA secondary structure.

J Bacteriol, 1986 Jul, 167(1), 191 - 200
Identification and characterization of mutations in Escherichia coli that selectively influence the growth of hybrid lambda bacteriophages carrying the immunity region of bacteriophage P22; Strauch MA et al.; Mutations in two Escherichia coli genes, sipA and sipB, result in a specific inhibition of the growth of certain hybrid lambdoid bacteriophages, lambda immP22, that have the early regulatory regions and adjacent genes from bacteriophage P22 . The sipB391 mutation maps near minute 56 and exerts the strongest inhibitory effect on the growth of the hybrid phages . The sipA1 mutation maps near minute 72 and plays an auxiliary role: enhancing the action of sipB391 . Such a role is not limited to sipA1, since there is a similar enhancement by the nusA1 and nusE71 mutations . The Sip-imposed restriction on the growth of lambda immP22 phages is not observed if the phage carries a mutation in the c1 gene . Perhaps this reflects the fact that the c1 product regulates phage DNA replication and is a major determinant in the decision governing whether the phage takes the lytic or lysogenic pathway . Consistent with this idea is the observation that lambda immP22 DNA replication is severely inhibited in bacteria carrying the sipB391 mutation . It is suggested that sip mutations exaggerate the normal role of c1 in limiting lytic growth . This causes a failure in the expression of sufficient amounts of some or all of the lytic gene products required for phage growth.

Clin Immunol Immunopathol, 1986 Jul, 40(1), 94 - 104
Complement, membrane glycoproteins, and complement receptors: their role in regulation of the immune response; Ochs HD et al.; To determine the effect of complement on the normal antibody response we studied seven patients with genetically determined complement component deficiencies, guinea pigs deficient of C4 and C2, respectively, and two patients whose neutrophils and monocytes lack the C3bi receptor . Patients deficient of early complement components (C4, C2, C3) have abnormal antibody responses to the T-cell-dependent antigen, bacteriophage phi X 174 . Complement-deficient guinea pigs (C4, C2) produce less antibody than normal guinea pigs and are unable to maintain measurable antibody levels; during secondary immunization they fail to develop amplification and to switch from IgM to IgG . This defect can be overcome by increasing the antigen dose or by injecting normal guinea pig serum at the time of the primary (but not the secondary) immunization . Patients with deficiency of the C3bi receptor were shown to have a significantly depressed antibody response to T-dependent antigens . We postulate that the contribution of complement to the mature humoral immune response is related to activation of C3 . The initial production of IgM following antigen injection leads to antigen-antibody complexes which interact with complement, to be nonspecifically trapped by C3b and C3bi receptors on B cells or macrophages . Thus antigen is selectively accumulated within the lymphoid organs and in turn may entrap antigen-specific B cells by interaction of the trapped antigen with surface immunoglobulin . As a result, close approximation between antigen, antibody, and a network of specific and nonspecific lymphoid cells is initiated, allowing generation of specific memory cells and initiation of a prompt mature antibody response on subsequent exposure to antigen.

Plasmid, 1986 Jul, 16(1), 63 - 71
The unique conjugation system of IncHI3 plasmid MIP233; Bradley DE; The conjugation system of the IncHI3 plasmid MIP233 was studied using a transfer-derepressed Tn5-insertion mutant . The conjugative pili of this plasmid were short pointed rods resembling rigid pili, with a well-defined modal length . Unlike plasmids with rigid pili, the MIP233 mutant mediated a surface + liquid conjugation system . The pili were serologically different from all known pilus types including H pili, and did not act as receptors for any known pilus-specific bacteriophage . They converted the surface conjugation system of RP4 to a surface + liquid one . Antiserum to pili of the mutant plasmid inhibited transfer of the wild-type plasmid MIP233, demonstrating that it contained only one transfer system.

J Infect Dis, 1986 Jul, 154(1), 110 - 20
Molecular characteristics of prion rods purified from scrapie-infected hamster brains; McKinley MP et al.; Purification of scrapie prions from hamster brains has demonstrated that the infectious particles contain one major protein, PrP 27-30 . This protein, which is required for and inseparable from scrapie infectivity, polymerizes into heterogeneous rod-shaped particles measuring 10-20 nm in diameter and 100-200 nm in length . We attempted to identify the minimal infectious unit by disrupting aggregates of the rods . Prolonged sonication resulted in progressive fragmentation of the rods into spherical particles with a mean diameter of 19 nm and short rods with a mean length of 60 nm . No change in scrapie infectivity accompanied this profound alteration in rod morphology . In contrast, brief sonication disrupted the ultrastructure of the filamentous bacteriophage M13 and led to a marked loss in infectivity . No consistent correlation could be made between scrapie prion infectivity and disruption of the rods by a variety of treatments . Proteases, acid, base, chaotropic agents, detergents, and heat were examined for their ability to alter the morphology of the rods . The lack of correlation between ultrastructural morphology of the rods and titers of prions is consistent with the hypothesis that the rods are aggregates of prions and are not fundamental particles themselves.

Nucleic Acids Res, 1986 Jun 25, 14(12), 4731 - 41
In vitro transcription of bacteriophage phi 29 DNA . Correlation between in vitro and in vivo promoters; Mellado RP et al.; The phi 29 DNA in vitro transcription initiation sites have been accurately mapped by S1 protection experiments . The results obtained indicated that the B . subtilis RNA polymerase containing the sigma 43 subunit basically recognized the same set of phi 29 promoters in vitro as those used in vivo . In addition, the sequence of the phi 29 early A2a promoter used both in vitro and in vivo has been determined as well as the precise nucleotide where initiation of transcription from the C2 promoter occurs in vitro.

Nucleic Acids Res, 1986 Jun 25, 14(12), 4881 - 97
Localization and DNA sequence analysis of the C gene of bacteriophage Mu, the positive regulator of Mu late transcription; Margolin W et al.; The C gene of bacteriophage Mu, required for transcription of the phage late genes, was localized by construction and analysis of a series of deleted derivatives of pKN50, a plasmid containing a 9.4 kb Mu DNA fragment which complements Mu C amber mutant phages for growth . One such deleted derivative, pWM10, containing only 0.5 kb of Mu DNA, complements C amber phages and transactivates the mom gene, one of the Mu late genes dependent on C for activation . The DNA sequence of the 0.5 kb fragment predicts a single long open reading frame coding for a 140 amino acid protein . Sequence analysis of DNA containing a C amber mutation located the base change to the second codon of this reading frame . Generation of a frameshift mutation by filling in a BglII site spanning codon 114 of this reading frame resulted in the loss of C complementation and transactivation activity . These results indicate that this open reading frame encodes the Mu C gene product . Comparison of the predicted amino acid sequence of the C protein with those of other transcriptional regulatory proteins revealed some similarity to a region highly conserved among bacterial sigma factors.

J Mol Biol, 1986 Jun 20, 189(4), 603 - 16
Structural and regulatory divergence among site-specific recombination genes of lambdoid phage; Leong JM et al.; The lambdoid bacteriophage phi 80 and P22 have site-specific recombination systems similar to that of lambda . Each of the three phage has a different insertion specificity, but structural analysis of their attachment sites suggests that the three recombination pathways share similar features . In this study, we have identified and sequenced the int and xis genes of phi 80 and P22 . phi 80 int and xis were identified using a plasmid recombination assay in vivo, and the P22 genes were mapped using Tn1 insertion mutations . In all three phage, the site-specific recombination genes are located directly adjacent to the phage attachment site . Interestingly, the transcriptional orientation of the phi 80 int gene is opposite to that of lambda and P22 int, resulting in convergent transcription of phi 80 int and xis . Because of its transcriptional orientation, phi 80 int cannot be expressed by the major leftward promoter, PL, and the regulatory strategy of phi 80 integration and excision must differ significantly from that of lambda . The deduced amino acid sequences of the recombination proteins of the three systems show surprisingly little homology . Sequences homologous to the lambda PI promoter are more conserved than the protein-coding sequences . Nevertheless, the Int proteins are locally related in the C-terminal sequences, particularly for a stretch of some 25 amino acid residues that lie approximately 50 residues from the C terminus . The Xis proteins can be aligned at their N termini.

J Mol Biol, 1986 Jun 20, 189(4), 597 - 602
Analysis of the ends of bacteriophage Mu using site-directed mutagenesis; Groenen MA et al.; We showed previously that two regions at the left end (L1 and L3) and one at the right end (R2) of bacteriophage Mu are essential for transposition . These regions all contain a 22 base-pair sequence with the consensus YGtTTCAYtNNAARYRCGAAAR, where Y and R represent any pyrimidine and purine, respectively . The Mu A protein binds to these regions in vitro, and weakly to sequences between nucleotides 1 and 30 of the right end (R1) and between nucleotides 110 and 135 of the left end (L2) . These weak A binding sites contain the sequence AARYRCGAAAR . Here we show, using site-directed mutagenesis, that the weak A binding sites are essential for transposition . Mutations in these weak A binding sites have a greater effect on transposition than mutations of corresponding base-pairs in the stronger A binding sites, located adjacent to these weak A binding sites . We confirm the importance of several of the conserved base-pairs in the consensus sequence YGtTTCAYtNNAARYRCGAAAR . The base-pairs in the A binding sites that are shown to be essential for transposition are all conserved in the ends of the related bacteriophage D108 . Furthermore, it is shown that the distance of 90 base-pairs between the two regions at the left end (L1 and L2L3) is essential.

Biochem Biophys Res Commun, 1986 Jun 13, 137(2), 869 - 75
Multiply branched DNA molecules from bacteriophage lambda: putative post-replicational repair DNA intermediates; Valenzuela MS et al.; Previous studies have shown that thymidine deprivation causes the formation of multiply branched molecules among bacteriophage lambda DNA replicative intermediates . In the present report, we present supporting evidence indicating that the induction of the SOS response is involved in this process . Moreover, close inspection of the DNA replicatives intermediates present in a recA deficient strain, shows an accumulation of gapped replicative intermediates . From these observations we postulate a model by which multiply branched DNA molecules may be intermediates or derived intermediates of a post-replicational repair pathway.

Nucleic Acids Res, 1986 Jun 11, 14(11), 4407 - 20
Hydrolysis by restriction endonucleases at their DNA recognition sequences substituted with mismatched base pairs; Shenoy S et al.; Restriction endonucleases were tested for their ability to catalyze the cleavage of mismatch-containing recognition sites in DNA . These mismatched base pairs were T.G, U.G, or A.C in covalently closed, circular heteroduplexes prepared by in vitro extension of chemically synthesized oligonucleotide primers annealed to a bacteriophage M13-derived viral DNA . None of the restriction enzymes was able to completely cleave the mismatch-containing recognition sites under standard conditions . However, three of them, SmaI, SalI, and SstI, catalyzed partial digestion leading to an accumulation of DNA singly nicked at the mismatched recognition site . The ability of SmaI and SstI to partially cleave at a mismatch was shown to depend on the nature and position of the mismatch within the corresponding recognition site . In contrast, little or no digestion was obtained with AccI, HincII, HindIII, and KpnI at mismatch-containing sites . Therefore, in some cases a transition-type substitution in only one strand of a recognition site inhibits restriction endonuclease-catalyzed digestion at that site although in others partial digestion occurs.

J Biol Chem, 1986 Jun 5, 261(16), 7537 - 43
Characterization of the DNA binding domain of bacteriophage lambda O protein; Wickner SH et al.; The lambda O and P gene products are required for the initiation of lambda DNA replication . In order to study the biochemistry of this process, we have constructed plasmids that carry the lambda O gene, P gene, and half of the O gene coding for the amino-terminal half of the O protein . Each is under the control of the inducible lambda promoter, PL . We have purified these three proteins from induced cells carrying the plasmids . Our results show that the amino-terminal portion of the O protein binds to the lambda origin of replication in a manner similar to the intact lambda O protein, demonstrating that the amino-terminal portion of O protein contains the DNA binding domain . Using chromatographic procedures, we have isolated a complex of lambda O and P proteins with lambda dv DNA . The amino-terminal portion of the O protein does not complex with P protein under the same conditions . This suggests that the specificity of the lambda O protein for P protein resides in the carboxyl-terminal half of the lambda O protein . Our results also show that, while the intact O protein is active in in vitro replication of lambda dv plasmid DNA, the amino-terminal portion of the O protein is inactive and is a competitive inhibitor of the lambda O protein in this reaction . These results confirm previous genetic observations that were interpreted as indicating a bifunctional structure for the lambda O protein with the amino-terminal domain recognizing the lambda origin of replication and the carboxyl-terminal domain interacting with the lambda P protein.

J Biol Chem, 1986 Jun 5, 261(16), 7178 - 83
Isolation of the gene for the testis-specific H1 histone variant H1t; Cole KD et al.; H1t is a testis-specific H1 variant found in pachytene spermatocytes and round spermatids of mammals . The H1t gene was isolated from the Sargent-Bonner library of recombinant lambda bacteriophage containing EcoRI fragments of rat liver DNA using a hybridization probe derived from a chicken H1 variant . The rat H1t gene encodes a 207-amino acid protein (ignoring the initiating methionine) that matches perfectly what is known of the sequence and composition of H1t isolated from rat testes . The gene lacks introns and has good matches to all the consensus sequences known to lie upstream from a variety of H1 genes from diverse organisms . It also has the standard downstream palindromic sequence that specifies the 3'-end of most histone messages . Accordingly, the features of the gene or its environs that restrict its expression to a particular phase of spermatogenesis are not yet evident.

Eur J Biochem, 1986 Jun 2, 157(2), 329 - 34
Different DNA-binding modes and cooperativities for bacteriophage M13 gene-5 protein revealed by means of fluorescence depolarisation studies; Bulsink H et al.; The binding of the bacteriophage-M13-encoded gene-5 protein to oligo(deoxythymidylic acid)s and M13 DNA was studied by means of tyrosyl fluorescence decay and fluorescence anisotropy measurements . The observed fluorescence decays could be described with two exponentials, characterised by the lifetimes tau 1 = 2.2 ns and tau 2 = 0.8 ns respectively . Only the amplitude of the longer-lifetime component is influenced by binding of the protein to DNA . This indicates that a part of the tyrosyl residues is involved in the binding . By means of fluorescence depolarisation measurements the rotational correlation time of the protein dimer is found to be 12.9 ns . In contrast to earlier measurements, carried out on the DNA-binding protein of phage Pf1 {Kneale, G . G . and Wijnaendts van Resandt, R . W . (1985) Eur . J . Biochem . 149, 85-93}, the observed rotational correlation times of the gene-5 protein pass through a maximum when the protein is titrated with oligo(deoxythymidylic acid)s . This is not observed upon titration with M13 DNA . Our measurements showed that for the oligo(deoxythymidylic acid)s there clearly is a decrease in the number of clustered proteins on the lattice in the case of excess nucleotide . This is a direct consequence of the much lower cooperativity of the binding to the oligonucleotides compared to the cooperativity characteristic of binding to polynucleotides . The number of nucleotides covered by a protein monomer is found to be less than or equal to 3 for the oligonucleotides and approximately equal to 4 for M13 DNA . Model calculations show that the 'time-window' through which the fluorescence depolarisation can be observed (i.e . the fluorescence lifetime) in this case significantly affects the 'measured' effective rotational correlation times.

Ultrasound Med Biol, 1986 Jun, 12(6), 511 - 7
The detection of temperature induced structural changes in T4B and T7 bacteriophages by means of high precision acoustic velocity measurements; Shilnikov GV et al.; High precision measurements of the velocity of 7-7.5 MHz ultrasonic waves in suspensions of both T4B and T7 bacteriophages as a function of temperature revealed the presence of a distinct transition in the physiological range of 35-45 degrees C . Data from acoustic measurements, sedimentation analysis and electron microscopy enabled us to identify this transition as being caused by the protein component of the phage and not the DNA . This transition does not depend on the position of the long tail fibers and may be part of some normal physiological process within the bacteriophage which presumably enhances its recognition and attachment to its host cell.

J Virol, 1986 Jun, 58(3), 951 - 4
End structure and mechanism of packaging of bacteriophage T4 DNA; Kalinski A et al.; We analyzed by restriction enzyme digestion the end structure of T4 phage DNA by comparing mature, concatemeric, first-packaged, and incompletely packaged DNAs . The structure of mature DNA was also studied using 3' end labeling with terminal transferase . Our data support the hypothesis that T4 DNA packaging is not initiated at specific packaging initiation sequences on the concatemeric precursor (cos or pac site mechanisms) but by a different packaging mechanism.

J Gen Microbiol, 1986 Jun, 132 ( Pt 6), 1685 - 93
Identification, mapping, cloning and characterization of a gene (sbmA) required for microcin B17 action on Escherichia coli K12; Lavina M et al.; We have identified mutations in three different chromosomal genes of Escherichia coli K12 which reduce sensitivity to microcin B17 . Mutations in ompF and ompR genes affected production of an outer membrane porin protein, OmpF, and resulted in reduced sensitivity to a number of other agents (colicins, bacteriophages) besides microcin B17 . The third class of mutants were specifically and highly resistant to microcin B17 . The mutations in these strains were mapped to a gene (sbmA), located at 8.7 min on the E . coli K12 chromosome, which is closely linked to phoA . The wild-type sbmA allele was cloned into multiple copy number plasmids, and its location within the cloned DNA fragment was further defined by mutagenesis with MiniMudII1681 . These insertion mutations resulted in in-frame fusions between the sbmA and lacZ genes, thereby allowing us to determine the direction of sbmA gene transcription . Plasmids carrying these gene fusions produced low levels of beta-galactosidase, indicating that the sbmA gene is poorly expressed . We have been unable to identify the sbmA gene product, but indirect evidence indicates that it might be an envelope protein involved in microcin uptake.

J UOEH, 1986 Jun 1, 8(2), 251 - 6
Origins of molecular biology in Japan; Obayashi M; The purpose of this paper is to discuss the origins of molecular biology in Japan . Japanese molecular biology does not have a long history since it started only after World War II . Especially, molecular genetics which uses "bacteriophage" had hardly been studied before the war and only a few scientists were interested in it immediately after the war . This is one of the origins of molecular biology in Japan . But there are other origins, one of which is the group formed by biologists, biochemists and physicists interested in nucleic acids . This group also started just after the war . Still another origin is the group of enzymologists . Enzymology was one of the main subjects of biochemistry from before the war . In Japan, biochemistry developed in conjunction with the medical and agricultural sciences from the pre-war era . These played an important role in introducing molecular biology from Europe and the United States after the war . A historical study of the development of molecular biology in Japan, comparing it with the history of molecular biology in Europe and the United States, should contribute to the elucidation of the features of the history of molecular biology in Japan.

Mol Gen Genet, 1986 Jun, 203(3), 445 - 50
Regulation of the plasmid state of the genetic element P4; Alano P et al.; After infection of sensitive cells in the absence of a helper phage, the satellite bacteriophage P4 enters a temporary phase of uncommitted replication followed by commitment to either the repressed-integrated condition or the derepressed-high copy number mode of replication . The transient phase and the stable plasmid condition differ from each other in the pattern of protein synthesis, in the rate of P4 DNA replication and in the expression of some gene functions . The regulatory condition characteristic of the P4 plasmid state affects a superinfecting genome, preventing the establishment of the P4 immune condition.

Biochem Int, 1986 Jun, 12(6), 921 - 31
Purification and properties of RNA polymerase of phage KB1; Verma M; Bacteriophage KB1 belongs to group C of Bradely's classification After infection a bacteriophage specific RNA polymerase is induced in infected cells . KB1 RNA polymerase is a stable enzyme and is easily purified to homogeneity in good overall yield . The activity resides in a single polypeptide chain of molecular weight about 90,000 . Synthesis of RNA by KB1 RNA polymerase requires a DNA template and Mg++ and like SP6 RNA polymerase, is strongly stimulated by either bovine serum albumin or spermidine . Thiol reactive reagents inhibit the enzyme, suggesting the presence of essential sulfhydryl residues . The enzyme possess a stringent promoter specificity . The KB1 RNA polymerase is also highly active in synthesis of poly(rG) with poly(dI).(dC) as template . My experiments suggest that the catalytic portion of the polymerase can be separated from the RNA polymerase holoenzyme.

J Inorg Biochem, 1986 Jun, 27(2), 113 - 21
Effects of exposure of DNA to methyl mercury on its activity as a template-primer for DNA polymerases; Frenkel GD et al.; A previous publication {Frenkel, Cain, and Chao, Biochem . Biophys . Res . Commun . 127, 849-856 (1985)} described the observation that double-stranded DNA which was briefly exposed to methyl mercury (MeHg) and purified to remove free methyl mercury was transcribed at a higher rate by RNA polymerase II from wheat germ . The specificity of this phenomenon has now been investigated by examining the activity of this MeHg-exposed DNA as a template-primer for DNA polymerases . DNA synthesis by the bacteriophage T4-induced DNA polymerase was higher with the MeHg-exposed DNA as a template-primer than with control DNA . In contrast, the rate of DNA synthesis by E . coli DNA polymerase I was lower with the MeHg-exposed DNA as template-primer . With both enzymes (as well as with RNA polymerase II), after denaturation of the MeHg-exposed and control DNAs the differences in template activity were either eliminated or markedly reduced . The enzymes are thus able to detect a MeHg-induced alteration in DNA . In contrast, circular dichroism, a physical method that is sensitive to conformational changes in DNA, did not detect any difference between the MeHg-exposed and control DNAs.

J Bacteriol, 1986 Jun, 166(3), 1137 - 40
Peptidoglycan association of bacteriophage T5 receptor in Escherichia coli K-12; Menichi B et al.; A total of 50% of the FhuA proteins (also called TonA proteins) present in Escherichia coli cells were associated with the peptidoglycan and 50% were free, whether or not this protein was overproduced . This FhuA-peptidoglycan association was made via the lipoprotein.

Proc Natl Acad Sci U S A, 1986 Jun, 83(11), 3614 - 8
Interactions of the RNA polymerase of bacteriophage T7 with its promoter during binding and initiation of transcription; Ikeda RA et al.; Promoters for T7 RNA polymerase have a highly conserved sequence of 23 continuous base pairs located at position -17 to +6 relative to the initiation site for the RNA . The complex of T7 RNA polymerase with the phage phi 10 promoter has been visualized indirectly by exploiting the ability of the polymerase to protect DNA sequences from cleavage by methidiumpropyl-EDTA X Fe(II) . The DNA contacts made by T7 RNA polymerase have been mapped during binding and during the subsequent initiation of transcription . The RNA polymerase alone protects 19 bases in a region from -21 to -3 . Synthesis of the trinucleotide r(GGG) expands the length of the sequence protected by the RNA polymerase and stabilizes the complex; 29 bases (-21 to +8) are protected, and the observed equilibrium association constant of the r(GGG) complex is 5 X 10(5) M-1 . The formation of a hexanucleotide mRNA, r(GGGAGA), further extends the protected region; 32 bases (-21 to +11) are protected . Finally, the synthesis of a pentadecanucleotide mRNA leads to a translocation of the region protected by the protein; the sequence now protected is reduced to 24 bases (-4 to +20).

J Biomol Struct Dyn, 1986 Jun, 3(6), 1055 - 66
Prediction of 3-D structure of the Cro protein from phage 434; Cygler M et al.; A comparative model building process has been utilized to predict the three-dimensional structure of the bacteriophage 434 Cro protein . Amino acid sequence similarities between the 434 Cro protein and other bacteriophage repressor and Cro proteins have been used, in conjunction with secondary structure prediction and the known structures of other base sequence specific DNA binding proteins, to derive the model . From this model the interactions between the 434 Cro protein and its operator DNA have been deduced . These proposed interactions are consistent with the known properties of the bacteriophage 434 Cro protein.

Immunol Invest, 1986 Jun, 15(4), 365 - 78
Nucleotide sequence of cDNA and derived amino acid sequence of rabbit complement component C3 alpha-chain; Kusano M et al.; The nucleotide sequence coding for 726 amino acid residues of the alpha-chain of rabbit C3 was determined from a cDNA clone . Subfragments of the cDNA produced by restriction endonucleases were inserted into the bacteriophage M13 and sequenced using the dideoxynucleotide technique . The derived amino acid sequence was compared with those of human and mouse C3, which have been previously reported {by De Brujn, M.H.L . and Fey, G.H . (1985) Proc . Natl . Acad . sci . USA 82, 708, and Westel, R.A . et al . (1984) J . Biol . chem . 259, 13857, respectively} . There was 79% or 78% homology in amino acid sequence between rabbit and human or mouse C3, respectively . All of the cysteinyl residues were conserved among the three molecules, and the sequence around the thioester site was also highly conserved . Several regions having low homology were found: one of them was the small fragment released by factor I cleavage.

Mol Gen Genet, 1986 Jun, 203(3), 505 - 10
Controlled gene expression utilising lambda phage regulatory signals in a cyanobacterium host; Friedberg D et al.; This study presents plasmid systems that utilize regulatory signals of bacteriophage Lambda to accomplish regulated expression of cloned genes in an A . nidulans R2 derivative strain . An operator-promoter region and the temperature-sensitive repressor gene cI857 of bacteriophage Lambda were employed . Linked to a cyanobacterial replicon, the plasmid vectors efficiently transformed Anacystis and were stably maintained within this host . The cat structural gene, encoding chloramphenicol acetyltransferase, was used to demonstrate that expression can be regulated by temperature shift . We have identified in extracts from the vector bearing Anacystis, a protein similar in size and immunology to the Lambda repressor . The systems described should allow controlled expression of adventitious genes in the cyanobacterial host.

Genetics, 1986 Jun, 113(2), 215 - 27
Direction of travel of RecBC recombinase through bacteriophage lambda DNA; Stahl FW et al.; We examined linkage relationships for RecBC-mediated recombination in lytic cycle crosses of lambda phages bearing two cohesive end sites (cos) oriented in the same direction . The relationships obtained imply that a given recombinant tends to be packaged from the cos site that is the nearer one to the right of the exchange . In view of the previously established coupling of entry of a recombinase at a cos cut and initiation of DNA packaging by that cos cut, these results imply that the recombinase (presumably the RecBC gene product) enters lambda's chromosome at the right end.

J Gen Virol, 1986 Jun, 67 ( Pt 6), 1123 - 33
DNA inversion in bacteriophage Mu: characterization of the inversion site; Schmucker R et al.; Gin-mediated site-specific recombination promotes inversion of the G segment of phage Mu . The crossover takes place between two 34 bp-long inverted repeat sequences flanking the G segment . We have characterized the inversion site, the target for the site-specific recombination mechanism . An artificial invertible segment was constructed which consists of parts of the invertible segments of Mu and phage P1, which in this respect are largely homologous . Upon inversion of this hybrid segment the crossover site could be located, by DNA sequencing, in the ACCT sequence of the centre of symmetry in the inverted repeat in Mu . The hybrid Mu-P1 segment inverts at a lower frequency than its parental invertible segments probably because of the mismatches between the inverted repeats of Mu and P1 . This suggests that base pairing between the inverted repeats is an intermediate step in recombination . Plasmids with subcloned G segments lacking the adjacent beta region of Mu or the corresponding region in P7, a relative of P1, are deficient in inversion . By analysis through site-specific mutagenesis of Mu DNA, an enhancer element with multiple recognition sites was identified which is necessary for efficient inversion . This component of the inversion site was located in a 170 bp segment within the Mu beta region, 30 bp to the right of the inverted repeat sequence, but can be separated from the crossover site by a 1200 bp insertion without losing its effect.

J Invest Dermatol, 1986 Jun, 86(6), 653 - 8
DNA alterations photosensitized by tetracycline and some of its derivatives; Piette J et al.; Bacteriophage M13 mp10 DNA were irradiated with near-UV light in the presence of tetracycline derivatives and primed with synthetic oligonucleotide to be used for DNA synthesis using Escherichia coli DNA polymerase . Chain terminations were observed by denaturing polyacrylamide gel electrophoresis and mapped precisely . All the synthesis stops occurred before or at the level of guanine residues, showing that the photoreaction mediated by tetracycline derivatives led to a preferential alteration of guanine residues . These lesions were demonstrated to be induced in DNA through a pathway involving singlet oxygen . Tetracycline derivatives also photoinduced the breakage of the DNA sugar-phosphate backbone monitored by the conversion of supercoiled phi X174 DNA to a relaxed form . This lesion was shown to be initiated by hydroxyl radicals . The production of this free radical has been confirmed by electron paramagnetic resonance (EPR) spin trapping experiments using 5,5-dimethyl-1-pyrroline-N-oxide as spin trap . In addition to the EPR signal due to OH radicals trapping another unassigned signal has been detected.

Virology, 1986 Jun, 151(2), 350 - 61
Cloning and sequencing of the genetic right end of bacteriophage T3 DNA; Yamada M et al.; The genetic right end of phage T3 DNA, from the beginning of gene 17, was cloned and sequenced . Genes 17, 18, and 19 were identified by comparing the sequence with the genetic map and by comparing the calculated and observed molecular weights of gene products . N-terminal amino acid sequence of the gene 17 product (gp17) predicted from the nucleotide sequence was consistent with the data from the analysis of purified gp17 . Gene 17.5 was identified as the lysis gene on the basis of the presence of a nonsense codon within an open reading frame in the sequence of DNA from an amber mutant of lysis gene . In addition, five potential genes have been identified . Sequences corresponding to a promoter for phage T7 RNA polymerase (Rosa and Andrews, 1981) and to a class-III promoter for phage T3 RNA polymerase (Sarker et al., 1985) were found . The genomic organization and the nucleotide and deduced amino acid sequences of T3 were compared with those of T7 . The genomic organizations of T3 and T7 were identical in this region . The sequence comparisons of T3 and T7 DNA point out the highly conserved sequences in all genes but also heavily varied regions in some genes . From these comparisons, possible implications with regard to structural and functional domains within several genes are discussed.

J Biomol Struct Dyn, 1986 Jun, 3(6), 1133 - 44
Bacteriophage lambda int protein may recognize structural features of the attachment sites; Nussinov R et al.; The bacteriophage lambda int protein binds to and promotes polynucleotide strand exchange within specific DNA segments called attachment sites . Previous work strongly suggests that the specificity of int protein action is based, at least in part, on its ability to recognize nucleotide sequences in the attachment sites . We suggest that int protein also recognizes structural features of the attachment sites such as the twist and roll angles between adjacent base pairs . This proposal is based on statistical analysis of the predicted twist and roll angles of a large collection of secondary attachment sites . The analysis shows that the oscillation patterns of these parameters are conserved in regions where int proteins binds.

Mol Cell Biol, 1986 Jun, 6(6), 2053 - 61
Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes; Carroll D et al.; When DNA molecules are injected into Xenopus oocyte nuclei, they can recombine with each other . With bacteriophage lambda DNAs, it was shown that this recombination is stimulated greatly by introduction of double-strand breaks into the substrates and is dependent on homologous overlaps in the recombination interval . With plasmid DNAs it was shown that little or no recombination occurs between circular molecules but both intra- and intermolecular events take place very efficiently with linear molecules . As with the lambda substrates, homology was required to support recombination; no simple joining of ends was observed . Blockage of DNA ends with nonhomologous sequences interfered with recombination, indicating that ends are used directly to initiate homologous interactions . These observations are combined to evaluate possible models of recombination in the oocytes . Because each oocyte is capable of recombining nanogram quantities of linear DNA, this system offers exceptional opportunities for detailed molecular analysis of the recombination process in a higher organism.

Proc Natl Acad Sci U S A, 1986 Jun, 83(11), 3904 - 8
Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene; Brotcorne-Lannoye A et al.; Untargeted UV mutagenesis of bacteriophage lambda--i.e., the increased recovery of lambda mutants when unirradiated lambda infects UV-irradiated Escherichia coli--is thought to be mediated by a transient decrease in DNA replication fidelity, generating mutations in the newly synthesized strands . Using the bacteriophage lambda cI857----lambda c mutation system, we provide evidence that the RecA protein, shown previously to be required for this mutagenic pathway, is no longer needed when the LexA protein is inactivated by mutation . We suggest that the error-prone DNA replication responsible for UV-induced untargeted mutagenesis is turned on by the presence of replication-blocking lesions in the host cell DNA and that the RecA protein is required only to derepress the relevant din gene(s) . This is in contrast to mutagenesis of irradiated bacteria or irradiated phage lambda, in which activated RecA protein has a second role in mutagenesis in addition to the cleavage of the LexA protein . Among the tested din genes, the dinB gene product (in addition to the uvrA and uvrB gene products) was found to be required for untargeted mutagenesis of bacteriophage lambda . To our knowledge, a phenotype associated with the dinB gene has not been reported previously.

Virology, 1986 Jun, 151(2), 296 - 314
Potential length determiner and DNA injection protein is extruded from bacteriophage T4 tail tubes in vitro; Duda RL et al.; Bacteriophage T4 tails contain a set of extended protein molecules in the central channel of the tail tube through which the DNA must exit during infection . Treatment of tails with guanidine hydrochloride separates the baseplates, leaving the tail tube and several specific tube-associated proteins . Methods were developed to purify these structures . Using specific antisera, immunoblotting, and electrophoretic analysis, these structures were shown to contain proteins gp19, 29, and 48 . Electron microscopy showed specifically defined stain penetration into the tail tube, a bulge at one end, and a short fiber extruded from the tube . These structures could be removed by proteases but the gp19 tube itself was resistant . Structural studies of tails and intact phage show that the bulge and fiber are at the end of the tube that interacts with the cell membrane during infection . Since the fiber did not protrude from baseplates or from incomplete (short) tube-baseplates, we propose that it is first assembled as a compact structure formed of six copies of a tube-associated protein, which elongates during tail tube formation to fill the central channel, span the length of the tube, and regulate its length . We suggest that the exit of this fiber during infection signals DNA ejection.

J Virol, 1986 Jun, 58(3), 835 - 42
Spontaneous lambda OR mutations suppress inhibition of bacteriophage growth by nonimmune exclusion phenotype of defective lambda prophage; Hayes S et al.; Survivor clones with defects in gene functions that participate in the replicative killing of thermally induced Escherichia coli constructs with integrated lambda N through P or cIII through P gene fragments were selected at a frequency of about 10(-6) . Among the population of survivors, clones were identified that exhibited normal lambda immunity at 30 degrees C, as shown by their ability to prevent the plating of lambda wild type and to support the plating of a nearly identical heteroimmune bacteriophage lambda imm434 . However, when placed at 42 degrees C to inactivate the cIts857 repressor, these survivor isolates excluded the plating of both lambda wild-type and lambda imm434 phages, a phenotype designated nonimmune exclusion (Nie) . Spontaneous mutants of lambda wild type were isolated that overcame the Nie phenotype and would plaque at 42 degrees C on cell lawns of these isolates . The acquired lambda se mutations suppressed nonimmune exclusion, prevented lysogenization by interrupting repressor expression from PRM, and made the phage insensitive to replicative inhibition . The se mutations were genetically mapped and sequenced within the rightward lambda operator site.

J Cell Biol, 1986 Jun, 102(6), 2106 - 14
A cloned cDNA encoding MAP1 detects a single copy gene in mouse and a brain-abundant RNA whose level decreases during development; Lewis SA et al.; Screening of a bacteriophage lambda gt11 cDNA expression library with a polyclonal anti-microtubule associated protein (MAP) antiserum resulted in the isolation of two non-cross-hybridizing sets of cDNA clones . One set was shown to encode MAP2 (Lewis, S . A., A . Villasante, P . Sherline, and N . J . Cowan, 1986, J . Cell Biol., 102:2098-2105) . To determine the specificity of the second set, three non-overlapping fragments cloned from the same mRNA molecule via a series of "walking" experiments were separately subcloned into inducible plasmid expression vectors in the appropriate orientation and reading frame . Upon induction and analysis by immunoblotting, two of the fusion proteins synthesized were shown to be immunoreactive with an anti-MAP1-specific antibody, but not with an anti-MAP2-specific antibody . Since these MAP1-specific epitopes are encoded in non-overlapping cDNAs cloned from a single contiguous mRNA, these clones cannot encode polypeptides that contain adventitiously cross-reactive epitopes . Furthermore, these cDNA clones detected an abundant mRNA species of greater than 10 kb in mouse brain, consistent with the coding requirement of a 350,000-D polypeptide and the known abundance of MAP1 in that tissue . The MAP1-specific cDNA probes were used in blot transfer experiments with RNA prepared from brain, liver, kidney, stomach, spleen, and thymus . While detectable quantities of MAP1-specific mRNA were observed in these tissues, the level of MAP1 expression was approximately 500-fold lower than in brain . The levels of both MAP1-specific and MAP2-specific mRNAs decline in the postnatal developing brain; the level of MAP1-specific mRNA also increases slightly in rat PC12 cells upon exposure to nerve growth factor . These surprising results contrast sharply with reported dramatic developmental increases in the amount of MAP1 in brain and in nerve growth factor-induced PC12 cells . The cDNA clones encoding MAP1 detect a single copy sequence in mouse DNA, even under conditions of low stringency that would allow the detection of related but mismatched sequences . The cDNAs cross-hybridize with genomic sequences in rat, human, and chicken DNA, but not with DNA from frog, Drosophila, or sea urchin . These data are discussed in terms of the evolution and possible biological role of MAP1.

J Cell Biol, 1986 Jun, 102(6), 2098 - 105
Brain-specific expression of MAP2 detected using a cloned cDNA probe; Lewis SA et al.; We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11 . The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule . Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum . Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein . The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues . Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus . We conclude that the expression of MAP2 is brain-specific . Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse . The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both . Under conditions of low stringency, the mouse MAP2 cDNA probe cross-hybridizes with genomic sequences from rat, human, and (weakly) chicken, but not with sequences in frog, Drosophila, or sea urchin DNA . Thus, there is significant interspecies divergence of MAP2 sequences . The implications of the above observations are discussed in relationship to the potential biological function of MAP2.

Science, 1986 May 30, 232(4754), 1113 - 5
Computer graphic display method for visualizing three-dimensional biological structures; Jimenez J et al.; A computer graphic display method that produces two-dimensional perspective views of three-dimensional objects is presented . The method is applied to the reconstruction at a resolution of 2.2 nanometers of the neck of bacteriophage phi 29, obtained from transmission electron micrographs processed by the direct Fourier method . The combined use of directed illumination, reflectance models, color, and different levels of transparency provides a powerful tool for a better interpretation of the three-dimensional structure, allowing improved correlation with genetic, structural, and biochemical data.

Biochim Biophys Acta, 1986 May 27, 867(1-2), 57 - 66
Identification of sites of cysteine misincorporation during in vivo synthesis of bacteriophage T7 0.3 protein; Rice JB et al.; The 0.3 protein encoded by coliphage T7 does not normally contain cysteine residues . Incorporation of {35S}cysteine can therefore be used to assay mistranslation . We have purified 0.3 protein, synthesized in the presence of {35S}cysteine, from T7 infected cells of E . coli and determined the locations of misincorporated cysteine residues . Analysis of the molecular weights (Mr) of {35S}cysteine-labeled tryptic peptides of 0.3 protein demonstrated that cysteine (encoded by UGU or UGC) is not extensively misincorporated, as might be predicted by substitution for arginine residues (encoded by CGU or CGC) . Edman degradation of the amino-terminal 50 residues of {35S}cysteine-labeled 0.3 protein determined that cysteine was most frequently misincorporated at position 15, which is correctly occupied by a tyrosine residue (encoded by UAC) . There are four other tyrosine codons (1 UAU; 3 UAC) in the region of the 0.3 protein studied, but these were not mistranslated . The context in which a codon is located must therefore be more important in causing mistranslation than the sequence of the codon itself . Misincorporation of {35S}cysteine was also found at positions 9 (ACC, asparagine), 16 (GAA, glutamic acid), 41 (GCC, alanine) and 42 (GAU, aspartic acid) . One mistranslation event appears to increase the likelihood that the following codon will also be mistranslated . This clustering of misincorporated {35S}cysteine residues was accentuated in 0.3 protein synthesized in the presence of streptomycin.

Nucleic Acids Res, 1986 May 27, 14(10), 4197 - 205
Identification and location of nine T5 bacteriophage tRNA genes by DNA sequence analysis; Desai SM et al.; Sequence analysis of two DNA fragments generated from bacteriophage T5 DNA by restriction with Hpa I and Hae III has resulted in the detection and localization of nine tRNA genes (His, two Ser genes, Leu, Val, Lys, fMet, Pro, and Ile) . The genes which code for tRNAs His and Leu are partials, whereas the remaining genes are complete . A majority of the tRNA genes are located in close proximity to one another . A unique feature of the Pro and Ile genes is that their DNA sequence overlap.

Nucleic Acids Res, 1986 May 27, 14(10), 4229 - 38
Two juxtaposed tyrosyl-OH groups participate in phi X174 gene A protein catalysed cleavage and ligation of DNA; van Mansfeld AD et al.; Bacteriophage phi X174 encoded gene A protein is an enzyme required for initiation and termination of successive rounds of rolling circle phi X DNA replication . This enzyme catalyses cleavage and ligation of a phosphodiester bond between nucleotide residues G and A at the phi X origin . The cleavage reaction which occurs during initiation involves formation of a free GOH residue at one end and a covalent bond between tyrosine-OH of the gene A protein and 5' phosphate of the A residue, at the other end of the cleavage site . During termination the covalently bound gene A protein cleaves the phosphodiester bond between G and A at the regenerated origin and ligates the 3' and 5' ends of the displaced genome-length viral DNA to form a circle . Since tyrosyl-OH mediated rearrangements of phosphodiester bonds in DNA may also apply to other enzymes involved in replication or recombination such as topoisomerases we have studied this interesting mechanism in greater detail . Analysis of 32P-labelled gene A protein-DNA complex by tryptic digestion followed by sequencing of 32P-containing peptides showed that two tyrosyl residues in the repeating sequence tyr-val-ala-lys-tyr-val-asn-lys participate in phosphodiester bond cleavage . Either one of these tyrosyl residues can function as the acceptor of the DNA chain . In an alpha-helix the side chains of these tyrosyl residues are in juxtaposition . An enzymatic mechanism is proposed in which these two tyrosyl-OH groups participate in an alternating manner in successive cleavage and ligations which occur during phosphodiester bond rearrangements of DNA.

J Biol Chem, 1986 May 25, 261(15), 7001 - 10
Studies of the DNA helicase-RNA primase unit from bacteriophage T4 . A trinucleotide sequence on the DNA template starts RNA primer synthesis; Cha TA et al.; The purified DNA replication proteins encoded by genes 41 and 61 of bacteriophage T4 catalyze efficient RNA primer synthesis on a single-stranded DNA template . In the presence of additional T4 replication proteins, we demonstrate that the template sequences 5'-GTT-3' and 5'-GCT-3' serve as necessary and sufficient signals for RNA primer-dependent initiation of new DNA chains . These chains start with primers that have the sequences pppApCpNpNpN and pppGpCpNpNpN, where N can be any one of the four ribonucleotides . Each primer is initiated from the T (A-start primers) or C (G-start primers) in the center of the recognized template sequence . A subset of the DNA chain starts is observed when one of the four ribonucleoside triphosphates used as the substrates for primer synthesis is omitted; the starts observed reveal that both pentaribonucleotide and tetraribonucleotide primers can be used for efficient initiation of new DNA chains, whereas primers that are only 3 nucleotides long are inactive . It was known previously that, when 61 protein is present in catalytic amounts, the 41 and 61 proteins are both required for observing RNA primer synthesis . However, by raising the concentration of the 61 protein to a much higher level, a substantial amount of RNA-primed DNA synthesis is obtained in the absence of 41 protein . The DNA chains made are initiated by primers that seem to be identical to those made when both 41 and 61 proteins are present; however, only those template sites containing the 5'-GCT-3' sequence are utilized . The 61 protein is, therefore, the RNA primase, whereas the 41 protein should be viewed as a DNA helicase that is required (presumably via a 41/61 complex) for efficient primase recognition of both the 5'-GCT-3' and 5'-GTT-3' DNA template sequences.

J Theor Biol, 1986 May 21, 120(2), 215 - 22
A unique four-stranded model of a homologous recombination intermediate; Hopkins RC; This paper proposes a model of four-stranded DNA synapsis during recombination between homologous segments of two DNA duplexes . The proposed intermediate is one of only two known models having relative chain orientations about the synaptic junction that are consistent with recent topological results on the integrative recombination of bacteriophage lambda . This model has the advantage of providing a mechanism for recognition of sequence homology between duplexes through specific hydrogen-bond formation; other models are discussed in comparison . The new model is based on an alternative family of DNA structures having chain directions opposite to those of the Watson-Crick family of structures . Idealized coordinates for generating both right- and left-handed forms of these alternative structures are presented for further study.

J Mol Biol, 1986 May 20, 189(2), 261 - 72
Isolation of bacteriophage T4 DNA polymerase mutator mutants; Reha-Krantz LJ et al.; More than 20 new bacteriophage T4 DNA polymerase mutants have been isolated by a procedure designed to select mutants with high spontaneous mutation rates . Some of the mutants produce the highest mutation frequencies that have been observed in T4 thus far . The design of the selection procedure allows for the isolation of mutator mutants that preferentially induce certain types of replication errors, and some of the mutator mutants have mutational specificities different from wild-type . The new mutants are clustered at just two sites in the DNA polymerase gene, and this result confirms an earlier observation.

J Mol Biol, 1986 May 20, 189(2), 285 - 92
Insertions of palindromic DNA sequences into the J-F intercistronic region of bacteriophage phi X174 interfere with normal phage growth; Muller UR et al.; The effect of hairpin (cruciform) size on the regulation of gene expression was investigated by cloning a series of palindromic sequences into the non-essential J-F intercistronic region of the bacteriophage phi X174 ins6 genome . Genetic stability of the insert sequence and its effect on the growth efficiency of the phage was used as an initial measure of the biological consequence of hairpin insertions . Multimers of increasing size of the BamHI linker sequence C-C-G-G-A-T-C-C-G-G were inserted into the PvuII site of the parental strain ins6 . The largest hairpin that could be constructed and maintained in the phi X174 genome had a stem length of 22 base-pairs and a loop size of four nucleotides (linker tetramer) . However, this structure proved to be disadvantageous to the phage and was rapidly deleted from its genome . Trimer inserts were more stable, but were eventually deleted also . Monomer and dimer inserts, though genetically stable, decreased the growth efficiency of the phage as judged by competitive growth experiments and measurements of burst size . The physical formation of these hairpins was shown by restriction digests of single-stranded DNA with BamHI and HpaII . We argue that these secondary structures form in vivo, at least in the single-stranded genome and the polycistronic mRNAs, and were responsible for the observed growth defects.

J Mol Biol, 1986 May 5, 189(1), 113 - 30
Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes; Studier FW et al.; A gene expression system based on bacteriophage T7 RNA polymerase has been developed . T7 RNA polymerase is highly selective for its own promoters, which do not occur naturally in Escherichia coli . A relatively small amount of T7 RNA polymerase provided from a cloned copy of T7 gene 1 is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid . Such transcription can proceed several times around the plasmid without terminating, and can be so active that transcription by E . coli RNA polymerase is greatly decreased . When a cleavage site for RNase III is introduced, discrete RNAs of plasmid length can accumulate . The natural transcription terminator from T7 DNA also works effectively in the plasmid . Both the rate of synthesis and the accumulation of RNA directed by T7 RNA polymerase can reach levels comparable with those for ribosomal RNAs in a normal cell . These high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation . It seems likely that a specific mRNA produced by T7 RNA polymerase can rapidly saturate the translational machinery of E . coli, so that the rate of protein synthesis from such an mRNA will depend primarily on the efficiency of its translation . When the mRNA is efficiently translated, a target protein can accumulate to greater than 50% of the total cell protein in three hours or less . We have used two ways to deliver active T7 RNA polymerase to the cell; infection by a lambda derivative that carries gene 1, or induction of a chromosomal copy of gene 1 under control of the lacUV5 promoter . When gene 1 is delivered by infection, very toxic target genes can be maintained silent in the cell until T7 RNA polymerase is introduced, when they rapidly become expressed at high levels . When gene 1 is resident in the chromosome, even the very low basal levels of T7 RNA polymerase present in the uninduced cell can prevent the establishment of plasmids carrying toxic target genes, or make the plasmid unstable.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Biol, 1986 May 5, 189(1), 243 - 8
A new membrane-associated DNA replication protein, the gene 69 product of bacteriophage T4, shares a patch of homology with the Escherichia coli dnaA protein; Mosig G et al.; A new phage T4 DNA replication protein, gp69, is found to be associated with membrane fractions, as predicted by the translated base sequence of gene 69 . In addition, gp69 shares a patch of homology with a segment of the Escherichia coli dnaA initiation protein . The patchy homology of dnaA protein and gp69 suggests that they may serve some similar functions, such as interactions with the same E . coli components in bacterial and viral DNA replication . We have shown before that gene 69 spans an origin of T4 DNA replication, and that this origin is preferentially associated with membrane fractions . We suggest the possibility that gp69 is involved in the attachment of this origin to the bacterial envelope.

J Mol Biol, 1986 May 5, 189(1), 131 - 41
Transcription termination and processing sites in the bacteriophage lambda pL operon; Hyman HC et al.; S1 nuclease mapping was performed on transcripts from the major leftward operon of the bacteriophage lambda in order to locate the 3' ends of stable RNA species produced in vivo . The analysis was carried out on RNA purified from either an induced lambda prophage or bacteria carrying a plasmid containing a large segment of lambda including the intact PL operon through the bet gene . The S1 nuclease mapping was performed on transcripts produced in the presence and the absence of the N antitermination function, and in the presence and the absence of either the RNase III processing enzyme or the Rho factor . The results of this work indicate that the intercistronic region between the N and ral genes of lambda contains three sites at which transcripts end under N-Rho+ conditions (positions on the lambda sequence: 34,826, 34,558 and 34,393) . The distal two correspond to the two sites previously described in this region as tL1 (on both sides of the BamHI site) . In the region between ral and Ea10, we mapped the 3' ends of three species of RNA . The 3' end of one species was found to be located 90 nucleotides proximal to tL2a, at 34,000 in the lambda sequence . The terminator at this site may be partially N-resistant . In an RNase III deficient host, an additional RNA species is formed . The 3' end of this RNA species is located at tL2a (33,910 on the lambda sequence) . In the presence of the antitermination N gene product, the readthrough transcripts are processed to form a 3' end at position 33,980 on the lambda sequence . These results suggest that elongation of transcription of the lambda PL operon is reduced gradually by clusters of termination located between genes and that the expression of the terminated products is further controlled by processing of the mRNA.

J Biol Chem, 1986 May 5, 261(13), 6107 - 18
Purification and characterization of the T4 bacteriophage uvsX protein; Formosa T et al.; Gene uvsX of bacteriophage T4 encodes a 40,000-dalton protein that plays a key role in the major pathway for genetic recombination in T4-infected cells . Mutations at the uvsX locus lead to increased sensitivity to various DNA-damaging agents, reduced phage bursts, decreased genetic recombination, and early arrest of DNA synthesis . Like the Escherichia coli recA protein, the purified uvsX protein is a DNA-dependent ATPase that catalyzes pairing between homologous single- and double-stranded DNA molecules in vitro (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H., (1985) Eur . J . Biochem . 148, 127-134) . At physiological salt concentrations, the uvsX protein binds tightly and cooperatively to single-stranded DNA, covering about five nucleotides per protein monomer; at lower salt concentrations, a similar type of binding to double-stranded DNA is detected (Griffith, J., and Formosa, T., (1985) J . Biol . Chem . 260, 4484-4491) . We show here that the ATPase activity of this protein is unusual in producing both ADP plus Pi and AMP plus PPi as products . Generating the fully active form of the ATPase is a cooperative process, apparently requiring that a protein monomer be bound to single-stranded DNA while surrounded by other ATP-bound monomers . The catalysis of homologous pairing by the uvsX protein is shown to be greatly stimulated by the presence of the T4 gene 32 protein, a helix-destablizing protein previously studied in this laboratory, and it requires continued ATP hydrolysis . We present a method that allows the purification of the uvsX protein to essential homogeneity . We also describe the complete purification of two proteins that bind to the uvsX protein: the T4 uvsY protein (16,000 daltons) and an E . coli host protein of 32,000 daltons whose gene is unknown . The host protein is likely to play a role in DNA metabolism, because it also binds to the T4 gene 32 protein and to DNA; the sequence of its amino-terminal 29 amino acids has been determined.

J Mol Biol, 1986 May 5, 189(1), 143 - 65
New reactions of the ribosomal RNA precursor of Tetrahymena and the mechanism of self-splicing; Inoue T et al.; The availability of Tetrahymena pre-rRNA of discrete size, produced by transcription of recombinant plasmids with bacteriophage SP6 RNA polymerase, has permitted a more detailed investigation of the self-splicing reaction . The predicted splicing intermediate, the product of cleavage by guanosine at the 5' splice site, was identified . This intermediate was tested in the intermolecular exon ligation reaction and found to be competent to undergo the second step of splicing . These results and others that evaluated the reactivity of the 5' and 3' splice sites independently show that splicing occurs in two separable steps . The 3' splice site was found to be susceptible to site-specific hydrolysis leaving a hydroxyl terminus . This is interpreted as an indication that the 3' splice site is activated for nucleophilic attack in general and for exon ligation in particular . Preliminary evidence for specific hydrolysis at the 5' splice site was also obtained . All of the newly characterized intervening sequence RNA-mediated reactions as well as those found previously are divided into three categories: transesterification by guanosine at sites following two or three pyrimidine nucleotides (and, as a minor reaction, at sites following other guanosine residues); transesterification by oligopyrimidines or by the 5' exon (which terminates with C-U-C-U-C-UOH) at the site following the 3'-terminal guanosine residue of the intervening sequence; and specific hydrolysis at the splice sites . One of the products of the reactions at the 3' splice site is a molecule that contains the 5' exon still attached to the intervening sequence . It has a 3'-terminal GOH and undergoes cyclization both at the normal cyclization site within the intervening sequence and at the 5' splice site . The finding that the splice site can act as a cyclization site, combined with the earlier observation that the normal cyclization site is subject to attack by guanosine mononucleotide, leads us to propose that all these reactions may be occurring in the same active site . Translocation (a conformational change) would then bring different oligopyrimidine sequences into the active site for attack by guanosine . On the basis of the experimental results, a model for the local structure at the active site is described . A key feature of the model is the interaction between the U at the end of the oligopyrimidine sequence, a G residue within the internal guide sequence in the intervening sequence, and another G residue that can be either the attacking group for transesterification or the 3'-terminal G of the intervening sequence.

Virology, 1986 May, 151(1), 110 - 8
Overproduction and purification of the products of bacteriophage T3 genes 18 and 19, two genes involved in DNA packaging; Hamada K et al.; The products of gene 18 (gp18) and gene 19 (gp19) of bacteriophage T3 are noncapsid proteins involved in DNA packaging . A restriction fragment containing gene 18 or 19 was cloned into the plasmid vector pNT45 under the control of the inducible leftward promoter (PL) of phage lambda . Induction of transcription of gene 18 or 19 by derepression of the PL promoter led to the synthesis of a high level of gp18 or gp19 . By using complementation of T3 DNA packaging in vitro as an assay, gp18 and gp19 were purified to near homogeneity . The overall yields of gp18 and gp19 were 1.4 mg and 0.35 mg, respectively, from 1 g wet wt cells . Addition of gp18 to the in vitro DNA packaging system resulted in increased phage production with increasing amounts of gp18 until 10% of the DNA was packaged into infectious phage particles . In contrast, addition of gp19 to the packaging system initially caused an increase in phage production, but increasing amounts of gp19 inhibited DNA packaging.

J Virol, 1986 May, 58(2), 450 - 8
DNA requirements at the bacteriophage G4 origin of complementary-strand DNA synthesis; Lambert PF et al.; An in vivo assay was used to define the DNA requirements at the bacteriophage G4 origin of complementary-strand DNA synthesis (G4 origin) . This assay made use of an origin-cloning vector, mRZ1000, a defective M13 recombinant phage deleted for its natural origin of complementary-strand DNA synthesis . The minimal DNA sequence of the G4 genome sufficient for the restoration of normal M13 growth parameters was determined to be 139 bases long, located between positions 3868 and 4007 . This G4-M13 construct was also found to give rise to proper initiation of complementary-strand synthesis in vitro . The cloned DNA sequence contains all the regions of potential secondary structure which have been implicated in primase-dependent replication initiation as well as additional sequence information . To address the role of one region which potentially forms a DNA secondary structure, the DNA sequence internal to the G4 origin was altered by site-directed mutagenesis . A 3-base insertion at the AvaII site as well as a 17-base deletion between the AvaI and AvaII sites both resulted in loss of origin function . The 17-base deletion was also generated within the G4 genome and found to dramatically reduce the infectious growth rate of the resulting phage . These results are discussed with respect to the role of the G4 origin as the recognition site for primase-dependent replication initiation and its possible role in stage II replication.

Virology, 1986 May, 151(1), 119 - 23
A defined in vitro system for packaging of bacteriophage T3 DNA; Hamada K et al.; Using purified components, we have constructed an in vitro system for packaging of mature phage T3 DNA . In addition to mature T3 DNA, the system contained T3 proheads and the products of gene 18 (gp18) and gene 19 (gp19) . The reaction required Mg2+, ATP, and polyvinyl alcohol . Spermidine was stimulatory but not absolutely required for the packaging reaction . Polyvinyl alcohol could be replaced by polyethylene glycol . The packaging efficiency decreased with decreasing molecular weight of the polymer, and low molecular weight polyols such as sucrose, sorbitol, and glycerol were inactive . The packaging reaction exhibited a sigmoidal relationship with respect to the concentration of ATP with the concentration for half maximal activity about 15 microM . A nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate), inhibited the packaging reaction.

Proc Natl Acad Sci U S A, 1986 May, 83(10), 3391 - 4
Yeast RPO41 gene product is required for transcription and maintenance of the mitochondrial genome; Greenleaf AL et al.; A 4-kilobase DNA fragment carried by a recombinant lambda gt11 bacteriophage appears to contain most of the coding information for the 145-kDa subunit of the Saccharomyces cerevisiae mitochondrial RNA polymerase . The RPO41 gene is located on chromosome VI, as determined by hybridization to electrophoretically separated yeast chromosomes . Hybridization and gene disruption/replacement experiments show that the RPO41 gene exists in a single copy and that its product is required for transcription and maintenance of the mitochondrial genome.

J Gen Virol, 1986 May, 67 ( Pt 5), 831 - 8
Two groups of capsule-specific coliphages coding for RNA polymerases with new promoter specificities; Dietz A et al.; Four bacteriophages (A16, CK235, phi 1.2 and K31) which specifically attack different encapsulated strains of Escherichia coli have been shown to be related to bacteriophage T7 (which is unable to grow on encapsulated hosts) . The conclusion that phages A16 and CK235 are related to T7 is based on similarities in the pattern of expression of intracellular phage proteins, early appearance, in infected host cells, of a phage DNA-specific RNA polymerase and hybridization (albeit to a low extent) of A16 DNA and of CK235 DNA to T7 DNA . The first two criteria also apply to phages phi 1.2 and K31 but hybridization of their DNAs with T7 DNA could not be detected . The RNA polymerases of CK235 and A16 have similar template specificities and the same applies to the RNA polymerases of phi 1.2 and K31 . None of the new RNA polymerases can use T7 DNA as template.

Z Gesamte Inn Med, 1986 May 1, 41(9), 249 - 55
{New knowledge and aspects in the use of biotechnology}; Kolb E; The clarification of the structure and the function of deoxyribonucleic acids with hereditary properties (genes) has led to the development of methods for their biochemical and chemical synthesis, respectively . Such synthetic genes may be introduced in bacterial cells with the help of carrier-DNA (vectors, e.g . plasmid- or bacteriophage-DNA) and may be used for the production of certain hormones (proinsulin, growth hormone), medicaments (interferons) and vaccines (e.g . covering proteins of viruses and of peptides from such ones respectively . Genes may also be introduced into the pronuclei of the zygote of mammals . Then in the germs, however, appear considerable losses . The creation of descendants with the same hereditary factors is possible in mammals up to now only by the dismemberment of the germ up to the eight-cell-stage . It is referred to ethical aspects of the use of gene-technical methods . Several problems in the application of biotechnical techniques in the reproduction are described.

Proc Natl Acad Sci U S A, 1986 May, 83(10), 3238 - 42
Regulation of bacteriophage P2 late-gene expression: the ogr gene; Christie GE et al.; The ogr gene product of bacteriophage P2 is a positive regulatory factor required for P2 late-gene transcription . We have determined the nucleotide sequence of the ogr gene, which encodes a basic polypeptide of 72 amino acids . P2 growth is blocked by a host mutation, rpoA109, in the alpha subunit of DNA-dependent RNA polymerase . The ogr52 mutation, which allows P2 to grow in an rpoA109 strain, was shown to be a single nucleotide change, in the codon for residue 42, that changes tyrosine to cysteine . The predicted amino acid sequence of the Ogr protein does not show similarity to DNA-binding proteins that are known to affect promoter recognition, to sigma factors, or to other characterized transcriptional regulatory proteins . We have inserted the ogr gene into a plasmid under control of the leftward promoter and operator of bacteriophage lambda . Thermal induction of ogr gene expression in this plasmid results in overproduction of a small protein that has been shown by complementation to possess Ogr function.

Cancer Res, 1986 May, 46(5), 2374 - 6
Inhibition of enzymic incision of thymine dimers by covalently bound guanine adducts of 4-nitroquinoline-1-oxide in DNA; Duker NJ et al.; The effects of the presence in DNA of covalently bound guanine adducts of the carcinogen 4-nitroquinoline-1-oxide on the pyrimidine dimer-DNA glycosylase, purified from bacteriophage T4-infected Escherichia coli, were investigated . E . coli DNA, labeled in thymine, photosensitized by silver nitrate, and irradiated by 254 nm monochromatic light, was the substrate . 4-Nitroquinoline-1-oxide was reduced to 4-hydroxyaminoquinoline-1-oxide and then reacted with irradiated DNA in the presence of seryl-AMP, yielding covalently bound adducts in DNA . These were assayed by high performance liquid chromatography . Enzyme activity was assayed by measuring release of labeled free thymine from directly photoreversed DNA after the reaction . Glycosylase activity was reduced against carcinogen-modified DNA, with the Vmax 38% of that against the control DNA; the Km was unaffected . Therefore, as with other modified purines, 4-nitroquinoline-1-oxide guanine modifications can reduce enzymic incision at thymine dimers . Left unrepaired, pyrimidine dimers are both mutagenic and carcinogenic . This is consistent with the possibility that interference with enzymic initiation of DNA excision repair of UV damage may be an indirect mechanism of mutagenesis by stable carcinogen-DNA adducts.

Mol Gen Mikrobiol Virusol, 1986 May, (5), 20 - 3
{Synthesis of aminoglycoside phosphotransferase is under control the PL-promoter of phage lambda}; Kiselev VI et al.; The recombinant plasmid pKP145 PL has been constructed containing the gene for aminoglycosidephosphotransferase (APT) . Expression of the APT gene is under the control of lambda bacteriophage PL promoter . Escherichia coli cells harbouring this plasmid synthesize APT in quantity up to 13-15% of the total cellular protein . The technique for isolation of APT from superproducing cells has been elaborated . Preparations of the enzyme devoid of contaminating bacterial proteins have been obtained.

Mol Cell Biol, 1986 May, 6(5), 1711 - 21
Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae; McIntosh EM et al.; The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids . Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells . The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert . Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene . Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site . Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene . Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript . The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini . Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids) . A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes . Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

Bioorg Khim, 1986 May, 12(5), 695 - 8
{Complete primary structure of DNA from the transducing bacteriophage lambda plac5}; Shpakovskii GV et al.; In studying molecular mechanisms of specialized transduction, primary structure of the junction between the E . coli gene lacI and the lambda phage locus Ea47 in transducing bacteriophage lambda plac5 has been established . Along with the lambda DNA and E . coli lac operon structures as well as with our earlier data on another phage-bacterial junction in lambda plac5, it lead to the complete sequence of lambda plac5 DNA, including the lac5 substitution, a wellknown segment of lambdoid cloning vehicles . The lambda plac5 DNA is shown to consist of 48645 b.p . distributed as follows: 19368 (lambda left arm) + 3924 (lac5 substitution) + 25353 (lambda right arm) . The presence of the phage pbL promoter near to the right end of the lac5 insert is shown . The lacI gene distal end in lambda plac5 proved to be considerably more long-stretched than it used to be believed, coding for 224 C-terminal amino-acid residues of lac repressor . The recombination studied in this paper, similarly to the abnormal prophage excision, occurred near to a Chi-like structure, which is partly homologous to the chi+lacZ site present in lambda plac5 . On the basis of the data obtained, a key role of the E . coli RecBC system and Chi sites in the formation of long-stretched deletions in the bacterial cell has been suggested.

Plasmid, 1986 May, 15(3), 182 - 90
pHG276: a multiple cloning site pBR322 copy number vector expressing a functional lac alpha peptide from the bacteriophage lambda PR promoter; Stewart GS et al.; The bacteriophage lambda early promoter PR has been used to direct the synthesis of lac alpha in a plasmid containing the multiple cloning site of pUC8 . The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI857 gene for temperature regulation of lac alpha expression . Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lac alpha and consequently, a Lac- phenotype in a host carrying the M15 deletion of lac . The potential of pHG276 as a fully regulated expression vector is examined.

Proc Natl Acad Sci U S A, 1986 May, 83(10), 3386 - 90
Illegitimate recombination at the replication origin of bacteriophage M13; Michel B et al.; Hybrids composed of phage M13 and plasmid pHV33 were used to study the formation of deletions in Escherichia coli . Eighty to ninety percent of the deletion endpoints were at the position of the nick introduced into the M13 replication origin by the phage gene II protein . This suggests the existence of a novel mechanism of illegitimate recombination.

J Bacteriol, 1986 May, 166(2), 609 - 17
IS1-dependent generation of high-copy-number replicons from bacteriophage P1 Ap Cm as a mechanism of gene amplification; Froehlich BJ et al.; Mutant P1 Ap Cm lysogens were isolated in which the drug resistance genes resident on the plasmid prophage P1 Ap Cm are amplified by a novel mechanism . The first step required for amplification is IS1-mediated rearrangement of the P1 Ap Cm prophage . The drug resistance genes are amplified from the rearranged P1 Ap Cm prophage by the formation of a plasmid (P1dR) which contains the two resistance genes . The P1dR plasmid is an independent replicon about one-half the size of P1 Ap Cm that can be maintained at a copy number eightfold higher than that at which P1 Ap Cm can be maintained . It contains no previously identified replication origin and is dependent on the Rec+ function of the host.

Plasmid, 1986 May, 15(3), 242 - 4
Plasmid expression vector using the lambda late promoter; Edlind T et al.; A plasmid expression vector called pQTE1 based on the late promoter, pR', and positive control gene Q of bacteriophage lambda has been constructed . This vector has unique cloning sites for placing exogenous DNA under control of pR' . Induction of expression of genes cloned into the pQTE1 plasmid leads to massive overproduction of the gene products . Also, transcription from the pR' promoter on pQTE1 appears to be insensitive to polarity effects.

Proc Natl Acad Sci U S A, 1986 May, 83(10), 3395 - 7
Methyl-directed repair of frameshift mutations in heteroduplex DNA; Dohet C et al.; DNA heteroduplexes with single unpaired bases of the four different kinds were prepared by annealing separated strands of bacteriophage lambda DNA and used to transfect Escherichia coli . Genetic analysis of the progeny phages obtained from transfected bacteria indicates that the E . coli mismatch repair system can recognize and repair heteroduplexes with single unpaired bases--i.e., frameshift/wild-type heteroduplexes . The repair of a particular strand of the heteroduplex is inhibited by full methylation of the adenines in the GATC sequences of that strand . Thus, it appears that the E . coli mismatch repair system can act on newly synthesized DNA strands to remove replication errors involving the insertion or deletion of a single base.

J Bacteriol, 1986 May, 166(2), 683 - 5
Differential induction of Escherichia coli autolysis by penicillin and the bacteriophage phi X174 gene E product; Halfmann G et al.; The behavior of the temperature-sensitive, penicillin-tolerant Escherichia coli mutant VC44 to endogenously induced autolysis by the bacteriophage phi X174 gene E product (gpE) was investigated . Expression of the cloned phi X174 lysis gene showed that cultures of strain VC44 grown at the restricted temperature were fully sensitive to endogenously induced autolysis . The results revealed that the modes of E . coli lysis induction by gpE and by penicillin differ and that the trigger mechanisms for autolysis depend greatly on the specific inducer used.

Mol Cell Biol, 1986 May, 6(5), 1751 - 9
A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells; Choi OR et al.; A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated . A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro . The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus . When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type . The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide . It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.

J Virol, 1986 May, 58(2), 459 - 67
Mechanism of in vitro synthesis of covalently linked dimeric RNA molecules by the poliovirus replicase; Lubinski JM et al.; Four RNA fragments of approximately 1,000 to 1,200 nucleotides, representing both the 5' and 3' termini of poliovirus plus- and minus-strand RNAs, were generated by transcription of poliovirus cDNA by using bacteriophage SP6 RNA polymerase . The copying of these templates by the poliovirus replicase invariably produced RNA products approximately twice the size of the templates . In experiments with templates uniformly labeled with 32P it was shown that some of the apparently double-length products were generated by extension from an internal site of the template . Filter hybridization of the labeled in vitro-synthesized products with various unlabeled templates suggested a second mechanism by which double-length molecules could be synthesized; the results can be best explained by de novo synthesis of the first strand by copying of the template RNA, followed by snap-back of the newly synthesized RNA, generating a template-primer structure for the synthesis of the second strand . Highly purified poliovirus replicase was able to support the synthesis of double-length RNA products in response to these templates . These reactions did not require host factor . In contrast, synthesis of genome-length copies of poliovirion RNA by the same replicase was absolutely dependent on added host factor . The synthesis of double-length RNA products did not require either the 3'-terminal poly(A) of plus RNA or sequences within the 3' termini of both plus- and minus-strand RNAs.

Nucleic Acids Res, 1986 Apr 25, 14(8), 3521 - 6
Sequences of three promoters for the bacteriophage SP6 RNA polymerase; Brown JE et al.; Fragments of SP6 DNA generated by cleavage with Hpa II or Taq I were cloned into the Cla I site of pBR322 and the recombinant plasmids were screened for the presence of SP6 promoter activity by transcription in vitro with purified SP6 RNA polymerase . Three plasmids having promoter activity and small inserts of SP6 DNA were characterized . Hybridization studies showed that all three cloned promoters arose from different regions of the SP6 genome . Comparison of the consensus promoter sequence (5' ATTTAGGtgGACACTATAGAAGgaG 3') with the consensus sequences of promoters recognized by the T3 and T7 RNA polymerases reveals a common core sequence (5'-CACTA-3') extending from -7 to -3, as well as other features that may be important in selective promoter recognition by the phage RNA polymerases.

J Biol Chem, 1986 Apr 25, 261(12), 5663 - 73
Cloning of the bacteriophage T4 uvsX gene and purification and characterization of the T4 uvsX recombination protein; Hinton DM et al.; The bacteriophage T4 uvsX gene is a nonessential gene required for normal levels of DNA repair, recombination, and replication . We demonstrate that plasmids containing the T4 DNA approximately 300-2900 base pairs upstream of T4 gene 41 express a biologically active uvsX protein . This uvsX protein imparts increased survival to UV-irradiated T4 uvsX- phage and decreases the T4 uvsX- mutant suppression of a conditionally lethal T4 mutant in the gene 49 recombination nuclease . The uvsX protein purified from cells with a uvsX+ plasmid catalyzes ATP hydrolysis to ADP and AMP and, in the presence of the T4 gene 32 helix-destablizing protein, ATP-dependent strand exchange between homologous circular single-stranded and linear duplex DNA . These results agree with the recent characterization of uvsX protein from T4-infected cells by Yonesaki et al . (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H . (1985) Eur . J . Biochem . 148, 127-134) and by Formosa and Alberts (Formosa, T., and Alberts, B.M . (1984) Cold Spring Harbor Symp . Quant . Biol . 49, 363-370) . In addition, we find that under some reaction conditions strand exchange is catalyzed by uvsX protein in the absence of 32 protein . The level of the uvsX protein expressed by the uvsX+ plasmids is high and independent of the orientation of the T4 DNA within the vector . This suggests that transcription promoter(s) lie upstream of the uvsX gene on the cloned T4 DNA . In vitro transcription of T4 DNA restriction fragments reveals two tandem promoters whose transcripts initiate approximately 500 and 600 nucleotides upstream of the uvsX gene and extend through the gene.

Eur J Biochem, 1986 Apr 15, 156(2), 285 - 9
Nucleotide sequence of the bacteriophage T5 DNA fragment which contains the gene for tRNAAsp; Shlyapnikov MG et al.; The nucleotide sequence of bacteriophage T5 tRNAAsp has been determined by conventional methods using thin-layer chromatography on cellulose for oligonucleotide fractionation . It exhibits several unusual features, such as (a) the displacement of the constant residues U-8, A-14 and R-15; (b) the presence of three G X U out of four base pairs in the D-stem . The gene for T5 tRNAAsp has been cloned in pBR 322 and sequenced . The analysis of the flanking regions shows the presence of two open reading frames on both sides of this gene . It has also been shown that the cloned gene is expressed in Escherichia coli, and RNase P is involved in the T5 tRNAAsp processing.

Virology, 1986 Apr 15, 150(1), 33 - 44
Host and phage-coded functions required for coliphage N4 DNA replication; Guinta D et al.; Escherichia coli strains containing mutations in various deoxyribonucleic acid synthesis cistrons have been tested for their ability to support bacteriophage N4 growth and, specifically, N4 DNA synthesis . N4 DNA synthesis is independent of the activity of the products of the E . coli dnaA, dnaB, dnaC, dnaE, dnaG, and rep genes . In contrast, N4 DNA replication requires the products of the dnaF, (ribonucleotide reductase) and lig (DNA ligase) genes of E . coli . N4 DNA replication, specifically processing of short DNA fragments requires the 5'-3' exonuclease activity of the polA gene product . However, its DNA polymerizing activity is not required . In addition, the sensitivity of N4 DNA synthesis to inhibitors or temperature-sensitive mutants of E . coli DNA gyrase suggests that this activity is required for N4 DNA synthesis . To date, we have found five N4 gene products required for N4 DNA replication: dbp (a single-stranded DNA binding protein), dnp (a DNA polymerase), dns (unknown function), vRNAp (the N4 virion-associated, DNA-dependent RNA polymerase) and exo (a 5'-3' exonuclease).

Biochem Biophys Res Commun, 1986 Apr 14, 136(1), 329 - 35
A bacteriophage-associated glycanase cleaving beta-pyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO); Altmann F et al.; A bacteriophage growing on Escherichia coli K13, K20, and K23 strains carries a glycanase that catalyzes the hydrolytic cleavage of the beta-ketopyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO) in the respective capsular polysaccharides . The main cleavage product of the K23 polysaccharide has been identified by 1H- and 13C-n.m.r . spectroscopy as beta beta Ribfl----7 beta KDOp2----3-beta Ribfl----7KDO . Cleavage of polysaccharides containing alpha-pyranosidic, or 5-substituted beta-pyranosidic KDO is not catalyzed by the enzyme.

Cell, 1986 Apr 11, 45(1), 71 - 9
Localized conversion at the crossover sequences in the site-specific DNA inversion system of bacteriophage P1; Iida S et al.; The crossover sites for site-specific C inversion consist of imperfect 12 bp inverted repeats with the dinucleotide TT at the center of symmetry . The phage P1 Cin recombinase acts not only at these cix sites but also less efficiently at cix-related sequences called quasi-cix sites, cixQ . When cixQ contains a central dinucleotide TT, crossover occurs in vivo at the 2 bp sequence TT in the normal and the quasi-cix sites . If cixQ carries only one T residue, inversion-associated localized conversion can occur at the mismatched position within the 2 bp sequence . The results indicate that Cin generates 2 bp staggered cuts in vivo and that reciprocal strand exchanges occur at these 2 bp crossover sequences.

J Mol Biol, 1986 Apr 5, 188(3), 487 - 90
Coliphage P2 late control gene ogr . DNA sequence and product identification; Birkeland NK et al.; The bacteriophage P2 late control gene ogr was cloned and precisely localized by deletion analysis in vitro . The DNA sequence of the ogr gene containing the ogr1 mutation was determined . The sequence translates into a basic protein of a molecular weight of 8300 . Plasmids overproducing the ogr gene product were constructed, and the ogr gene product was identified by polyacrylamide gel electrophoresis.

J Mol Biol, 1986 Apr 5, 188(3), 415 - 31
Information content of binding sites on nucleotide sequences; Schneider TD et al.; Repressors, polymerases, ribosomes and other macromolecules bind to specific nucleic acid sequences . They can find a binding site only if the sequence has a recognizable pattern . We define a measure of the information (R sequence) in the sequence patterns at binding sites . It allows one to investigate how information is distributed across the sites and to compare one site to another . One can also calculate the amount of information (R frequency) that would be required to locate the sites, given that they occur with some frequency in the genome . Several Escherichia coli binding sites were analyzed using these two independent empirical measurements . The two amounts of information are similar for most of the sites we analyzed . In contrast, bacteriophage T7 RNA polymerase binding sites contain about twice as much information as is necessary for recognition by the T7 polymerase, suggesting that a second protein may bind at T7 promoters . The extra information can be accounted for by a strong symmetry element found at the T7 promoters . This element may be an operator . If this model is correct, these promoters and operators do not share much information . The comparisons between R sequence and R frequency suggest that the information at binding sites is just sufficient for the sites to be distinguished from the rest of the genome.

J Mol Biol, 1986 Apr 5, 188(3), 403 - 13
Morphogenesis of f1 filamentous bacteriophage . Increased expression of gene I inhibits bacterial growth; Horabin JI et al.; We have cloned the gene I sequence of the filamentous bacteriophage f1 downstream from the lambda leftward promoter on a plasmid that also contains the temperature-sensitive lambda repressor, cI857 . Temperature induction of gene I protein (pI) resulted in rapid cessation of growth . This inhibition appears to involve a rapid decrease in synthesis of host protein and RNA . The ability of pI to cause this inhibition is not dependent on thioredoxin, a host factor that is necessary for phage morphogenesis and has been shown by genetic data to interact with pI . The inhibition does not appear to be mediated by the amino half of the protein, as induction of an identical plasmid construction of an amber mutant positioned two-thirds along gene I, does not affect cell growth . Analysis of the transcription products from the cloned gene I confirmed previous suggestions that a transcription terminator exists in the amino-terminal portion of the gene . In addition, there is no detectable promoter activity in the 152 bases immediately upstream from the gene . These data and the inability to overproduce pI argue for down-regulation of pI production . Radioactive labeling of proteins in maxi-cells and normal Escherichia coli cells identifies pI as a protein of about 39,000 Mr that partitions with the cell envelope . Pulse-chase experiments suggest that pI is not processed to any appreciable extent.

J Bacteriol, 1986 Apr, 166(1), 34 - 7
Analysis of forward mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine in the bacteriophage P22 mnt repressor gene; Lucchesi P et al.; We describe the isolation and genetic characterization of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutations in the phage P22 mnt repressor gene cloned in plasmid pBR322 . Mutations in the mnt repressor gene or its operator on this plasmid, pPY98, confer a tetracycline resistance phenotype, whereas the wild-type plasmid confers tetracycline sensitivity . Cells carrying pPY98 were briefly exposed to MNNG to give 20 to 40% survival and a 50- to 100-fold increase in tetracycline-resistant cells . DNA sequence analysis showed that 29 of 30 MNNG-induced mutations were GC-to-AT transitions and one was an AT-to-GC transition . About 80% of the mutations are in three hotspots . This mutation spectrum is consistent with the proposed mechanism of mutagenic action of MNNG, which involves mispairing of an alkylated base, O6-methylguanine . The mnt gene may be a useful target for determining mutagenic specificity at the nucleotide level because forward mutations are easily isolated, the target size is small, and the DNA sequence changes of mutations can be determined rapidly.

J Virol, 1986 Apr, 58(1), 142 - 51
Nucleotide sequence of the small double-stranded RNA segment of bacteriophage phi 6: novel mechanism of natural translational control; McGraw T et al.; The lipid-containing bacteriophage phi 6 has a genome composed of three segments of double-stranded RNA . We determined the nucleotide sequence of a cDNA copy of the smallest RNA segment . The coding sequences of the four proteins on this segment were identified . These sequences were clustered . Three of the genes had overlapping initiation-termination codons . All noncoding sequences were at the ends of the molecule . The genes of the small double-stranded RNA segment comprised two translational polarity groups . We propose that the translational coupling is the result of an inability of ribosomes to bind independently to two of the four genes . Translation of these genes occurred when ribosomes were delivered to them by translation of an upstream gene.

Genetics, 1986 Apr, 112(4), 727 - 39
Frameshift suppression in aminoacyl-tRNA limited cells; Weiss RB et al.; Under certain conditions aminoacyl-tRNA limitation can phenotypically suppress frameshift alleles . The observed suppression is due to an increase in abnormal translocation of ribosomes translating codons that have a short supply of aminoacyl-tRNA . The rIIB frameshift alleles of bacteriophage T4 are used here to pinpoint the sites of ribosome frameshifting caused by these hypothetical decoding errors . The data indicate that not all hungry codons are associated with abnormal translocation, only a relatively small subset . Analysis of the hungry codons which are associated with ribosome frameshifting points to the existence of severe context effects determining the shiftiness of these codons.

Lab Anim Sci, 1986 Apr, 36(2), 119 - 25
Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: clinical and immunologic studies; Tsai CC et al.; A simian acquired immunodeficiency syndrome (SAIDS) associated with retroperitoneal fibromatosis (RF) has been observed in several species of macaque at the Washington Regional Primate Research Center . Clinical signs were recurrent diarrhea, weight loss, mesenteric lymphadenopathy, and opportunistic infections . Most affected macaques in the later stages of illness showed marked immunodeficiency . Response of peripheral blood mononuclear cells to mitogens was impaired significantly . There was sharply depressed primary and secondary antibody response to the T-cell dependent antigen, bacteriophage phi X174 . Affected monkeys did not switch from IgM to IgG antibody following a secondary immunization, as did normal macaques . Twenty-four (67%) of 36 affected animals with progressive RF or deteriorated stages of illness had hypoproteinemia and hypoalbuminemia . Quantitative serum immunoglobulins of 23 cases showed that eight (35%) had hypogammaglobulinemia, six (26%) had hypergammaglobulinemia, and the remainder (39%) were within the normal range . Opportunistic infections were predominantly bacterial pathogens . Type D retrovirus appeared to be closely associated with RF-affected macaques (12/12 or 100%) . The case fatality rate (including animals sacrificed after prolonged illness) was 98% . The leading cause of death was due directly to RF lesions in 43%, to enterocolitis in 36%, septicemia in 12%, amyloidosis in 5%, and malignant lymphoma (2%) . Clinical, immunologic and pathologic changes reveal an acquired immunodeficiency syndrome that has many similarities to human AIDS . SAIDS and RF may be a useful model for studying human AIDS.

J Gen Microbiol, 1986 Apr, 132 ( Pt 4), 997 - 1007
No correlation exists between the conjugative transfer of the autotrophic character and that of plasmids in Nocardia opaca strains; Sensfuss C et al.; A new isolate of Nocardia opaca was obtained by enrichment culture for aerobic lithoautotrophic growth on CO2 and H2 . This strain, MR22, is very similar to N . opaca MR11 (formerly 1b) in functioning as a donor for genetic information determining the ability to grow lithoautotrophically (Aut character) in matings with Aut- strains of N . opaca or closely related heterotrophic species . The strain contains a plasmid, pHG33 of about 110 kb . A mutant was isolated from strain MR22 which was plasmid-free, and had lost the Aut character, resistance to 50 microM-thallium salt and susceptibility to the nocardia-specific bacteriophage phi B1 . As a recipient of the Aut character, this plasmid-free mutant was as well suited as plasmid-bearing Aut- strains of N . opaca . In matings with the mutant as recipient the frequency of Aut+ transconjugants per donor was 3 X 10(-4) with N . opaca MR11 (pHG31-a, Aut+, Tlr, Strs, phi B1s) and 2 X 10(-3) with N . opaca MR22 (pHG33, Aut+, Tlr, Strs, phi B1r) as donor . Phenotypic characterization of the transconjugants, which had been selected for the Aut marker, revealed that in many cases the Aut marker had been transferred without plasmid transfer . Furthermore, plasmid-free, Aut+ transconjugants functioned as donors for the Aut marker . Both plasmid-free and plasmid-bearing transconjugants transferred the Aut marker to the Aut- strains of N . opaca with a frequency which was one or two orders of magnitude higher than that of the wild-type strains . The plasmids pHG31-a and pHG33 code for thallium resistance (50 microM-thallium acetate) . The frequency of thallium-resistant transconjugants was 10(-1) to 10(-2) per donor; all thallium-resistant transconjugants contained the donor plasmid . We conclude that the plasmids pHG31-a of strain MR11 and pHG33 of strain MR22 of N . opaca carry the genetic information for thallium resistance but not the Aut character . As plasmid-free Aut+ strains can function as donors the Aut character is assumed to reside on the chromosome and to function as an independent self-transmissible genetic element.

J Biomol Struct Dyn, 1986 Apr, 3(5), 887 - 98
Binding stoichiometry of the gene 32 protein of phage T4 in the complex with single stranded DNA deduced from boundary sedimentation; Scheerhagen MA et al.; Short 145 base DNA fragments in complex with the helix destabilizing protein of bacteriophage T4, GP32, have been studied with boundary sedimentation . The sedimentation coefficient was determined as a function of concentration, protein-nucleic acid ratio, temperature and salt concentration . It can be concluded that the measured values reflect the properties of the saturated DNA-GP32 complex . A combination of the earlier obtained translational diffusion coefficient of the complex with the sedimentation coefficient yields its anhydrous molecular weight (Mw = 5.4.10(5) D), which corresponds to a size of the binding site of 10 nucleotides per protein . This procedure is not sensitive to the presence of non-binding protein molecules and to the assumed protein concentration, and therefore, it seems more reliable than a determination from titration experiments . Similar sedimentation measurements were performed with tRNA-complexes containing 76 nucleotides . The translational diffusion coefficient can be calculated from the measured rotational diffusion coefficient and assuming the same hydrodynamic diameter for this complex as obtained for the 145 b DNA complex . The molecular weight derived from the data then also leads to a binding site size of about 10 nucleotides . This suggests that also the short tRNA-complex forms an open, strongly solvated structure, as was proposed for the 145 b DNA-GP32 complex.

Mol Gen Mikrobiol Virusol, 1986 Apr, (4), 16 - 9
{Isolation of a restriction endonuclease with HindIII-specificity from Bordetella bronchiseptica}; Kalinin VN et al.; Site-specific restriction endonuclease BbrI has been found in bacteriophage resistant strain B . bronchioseptica 4994 . The technique was elaborated for purification of BbrI to the stage free of nuclease and phosphatase contamination . The yield of purified enzyme is 6000-20 000 units per 10 g of biomass . BbrI recognises and cleaves the same DNA sequence as HindIII with the formation of four-nucleotide cohesive ends . The simplicity of cultivation, security for human, presence of the single restriction endonuclease and the high level of its production make B . bronchioseptica 4994 a promising producer of BbrI restriction endonuclease, isoshizomeric to HindIII, for use in experimental practice in industry.

Anal Biochem, 1986 Apr, 154(1), 353 - 60
Specific-primer-directed DNA sequencing; Strauss EC et al.; A simple and rapid strategy for DNA sequence analysis based on the Sanger chain-termination method is described . This procedure utilizes full-sized inserts of 1 to 4 kb of DNA cloned into M13 bacteriophage vectors . After the sequence of the first 600-650 bp of the insert DNA has been determined with the commercially available universal vector primer, a specific oligonucleotide is synthesized utilizing the sequence data obtained from the 3' end of the sequence and used as a primer to extend the sequence analysis for another 600-650 nucleotides . Additional primers are synthesized in a similar manner until the nucleotide sequence of the entire insert DNA has been determined . General guidelines for the selection of oligonucleotide length and composition and the use of unpurified primers are discussed . The use of the specific-primer-directed approach to dideoxynucleotide sequence analysis, in association with highly purified single-stranded template DNA, reduces considerably the time required for the analysis of large segments of DNA.

Mol Gen Mikrobiol Virusol, 1986 Apr, (4), 9 - 16
{Expression of the chloramphenicol acetyltransferase gene is under control of various promoters of E . coli and phage lambda}; Mashko SV et al.; Plasmids have been constructed with the structural region of the cat gene being under the control of the lactose (lacUV5), tryptophane (trpOP), operons of Escherichia coli, the hybrid trp-lac (tac) promoter and early bacteriophage lambda promoters (PL, PR and PLIT) . The expression of chloramphenicolacetyltransferase gene in Escherichia coli cells harbouring such recombinant plasmids and pBR325 as well has been examined by determining the chloramphenicol resistance and studying the enzyme activity of Cm-acetylase . A high level of enzyme synthesis is connected with transcription from PL, PR and tac promoters.

Mol Biochem Parasitol, 1986 Apr, 19(1), 35 - 43
Cloning of Leishmania donovani genes encoding antigens recognized during human visceral leishmaniasis; Sheppard HW et al.; In this report we describe the construction and analysis of a genomic library of Leishmania donovani gene segments in the bacteriophage vector lambda gt11 . This cloning vector permits the expression of parasite polypeptides as fusion products with Escherichia coli beta-galactosidase . A group of 90 clones which express L . donovani antigens have been isolated from this library using various polyvalent antisera . Many of these clones appear to encode parasite membrane antigens some of which are recognized by sera from patients with visceral leishmaniasis (kala azar) . Some clones reacted with specific subsets of kala azar sera while others reacted with all kala azar sera tested . In addition, some of the clones appear to encode conserved antigens that are recognized by sera from mice immunized with L . major . Preliminary characterization of five clones indicated that each one contains a distinct genetic element and that at least four encode different fusion peptides.

Proc Natl Acad Sci U S A, 1986 Apr, 83(8), 2576 - 8
Mismatch-stimulated killing; Doutriaux MP et al.; DNA duplexes with or without mismatches and with or without adenine-methylated GATC sequences were prepared from separated strands of bacteriophage lambda DNA and used to transfect Escherichia coli . Unmethylated heteroduplexes containing one or more repairable mismatches transfect cells with a functioning mismatch repair system less efficiently than they transfect cells deficient in mismatch repair . No difference is observed when the duplexes contain no mismatch or a poorly repaired mismatch or when the heteroduplexes are fully or hemimethylated . These results and the phenotypes of E . coli dam mutants suggest that the E . coli mismatch repair system may introduce double-strand breaks in unmethylated DNA at or near repairable mismatches.

Nucleic Acids Res, 1986 Mar 25, 14(6), 2811 - 27
Promoter and nonspecific DNA binding by the T7 RNA polymerase; Smeekens SP et al.; T7 RNA polymerase plays an important role in both the transcription and replication of bacteriophage T7 . In this study we have used a nitrocellulose filter binding assay to examine the binding properties of the T7 RNA polymerase with T7 promoters cloned into plasmid DNAs . Promoter-specific binding was shown to be relatively insensitive to variations in the ionic strength of the incubation solution but dependent on the helical structure of the DNA . On the other hand, nonpromoter interior-site binding was independent of the superhelicity of the DNA but extremely sensitive to changes in the ionic strength . These results suggest that nonspecific binding results from ionic interactions between positively charged residues of the polymerase and the polyanionic backbone of the DNA, whereas promoter-specific binding is dependent upon base-specific contacts within the promoter sequence . A comparison between the transcriptional activity and binding strengths of the RNA polymerase to specific promoters indicates little correlation between these two properties . This suggests that differential promoter binding does not represent a major mechanism for regulating transcription in bacteriophage T7 . Instead, factors which influence the efficiency or rate of formation of the polymerase-promoter open complex are found to have the major role in determining transcriptional levels in this system.

Biochemistry, 1986 Mar 25, 25(6), 1216 - 21
Photosensitive DNA cleavage and phage inactivation by copper(II)-camptothecin; Kuwahara J et al.; Upon irradiation with 365-nm light, copper(II)-camptothecin significantly produced single- and double-strand breaks of DNA and also induced a marked inactivation of bacteriophage . The nucleotide sequence analysis exhibited considerably random DNA cleavage . The DNA strand scission by the camptothecin-Cu(II)-UV light system, as well as the phage inactivation, was strongly suppressed by bathocuproine and catalase, indicating participation of cuprous species and hydrogen peroxide in the reaction . The present results suggest that (1) Cu(II) ion may play an important role as a cofactor in antitumor action of camptothecin and (2) the combination of copper-camptothecin plus long-wave ultraviolet light is useful against certain cancer treatment as a new photochemotherapy.

Nucleic Acids Res, 1986 Mar 25, 14(6), 2611 - 20
An Epstein-Barr virus transcription unit is at least 84 kilobases long; Bodescot M et al.; We have studied the structure of the Epstein-Barr virus mRNAs expressed in B95-8, a productively-infected Marmoset cell line established from in vitro-infected B-lymphocytes . We constructed a cDNA library from the cytoplasmic polyadenylated RNAs of B95-8 in the lambda gt10 bacteriophage . We present here the analysis of a 3.5 kbp cDNA containing exons transcribed from the US, IR and UL regions of the viral genome . The corresponding transcription unit is at least 84 kbp long . Two exons are transcribed from the US region, five from the IR region and two from the UL region . The exons from the IR region consist of two tandem repeats of a unit containing two exons, 66 and 132 nucleotides, and of a third copy of the 66 nucleotide exon . The exons from the UL region contain an open reading frame coding for a 944 amino acid polypeptide . The C-terminal end of this polypeptide harbors three types of repeated sequences . The corresponding mRNA is the second described of a family of mRNAs produced by alternative splicing of exons transcribed from the US, IR and UL regions.

J Mol Biol, 1986 Mar 20, 188(2), 215 - 23
Interaction between gene II protein and the DNA replication origin of bacteriophage f1; Horiuchi K; The origin of DNA replication of the filamentous bacteriophage f1 binds its initiator protein (gene II protein) in vitro to form a complex that can be trapped on nitrocellulose filters . The binding occurs with both superhelical form DNA and linear DNA fragments . A number of defective mutants of the origin were tested for the ability to bind gene II protein . The region of DNA required for the binding is around a second palindrome downstream from the palindrome that contains the DNA replication initiation site . It overlaps, but is not identical to, the region required for the nicking reaction by the protein . The nicking site itself was dispensable for the binding . In vivo, a number of defective deletion mutants of the origin, when in a plasmid, inhibited growth of superinfecting phage if the intracellular level of gene II protein was low . In addition, these defective origins inhibited the activity of the functional phage origin located on the same replicon . The domain of the DNA sequence required for inhibition in vivo was consistent with that for the binding in vitro.

J Mol Biol, 1986 Mar 20, 188(2), 185 - 98
Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins; Kreuzer KN et al.; We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322 . During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles) . In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection . We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection, which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage . Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome . When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected . The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids; these have been shown to contain a special type of T4 replication origin.

J Biol Chem, 1986 Mar 15, 261(8), 3744 - 52
Positive and negative regulation of the Mu operator by Mu repressor and Escherichia coli integration host factor; Krause HM et al.; Bacteriophage Mu utilizes two converging promoters to regulate the lytic and lysogenic pathways . Messenger RNA encoding the repressor gene is synthesized leftward from a promoter (PcM) located 1066 base pairs from the Mu left end . This transcript overlaps and is complementary to RNA synthesized rightward from the early promoter (PE) for transposase and replication proteins which initiates transcription at base pair 1028 . Purified Mu repressor binds to three distinct operator sites (O1, O2, and O3); repressor binding at O2 blocks RNA polymerase binding at PE and repressor binding at O3 blocks RNA polymerase binding at PcM . O1 and O2 have higher affinity for repressor than O3, and transcription from PE is blocked at repressor concentrations that do not affect PcM . Thus, maintenance of the lysogenic state and autoregulation of the repressor gene is achieved by RNA polymerase transcription through DNA-repressor ensembles at O1 and O2 . Integration host factor (IHF) encoded by the Escherichia coli him A and him D genes binds to Mu operator DNA between sites O1 and O2 . IHF enhances transcription from PE 3-5-fold on supercoiled plasmid substrates in vitro and in Mu monolysogens in vivo . In vitro, IHF simultaneously decreases transcription from PcM 5-10-fold which results in a 25-fold change in lytic transcription relative to repressor transcription . A model for regulating the Mu lysis-lysogeny decision is presented.

J Biol Chem, 1986 Mar 15, 261(8), 3717 - 24
Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster . II . Isolation of the gene and characterization of the 5' flanking region; Gil G et al.; Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and microsomal HMG-CoA reductase are sequential enzymes in the cholesterol biosynthetic pathway; both are negatively regulated by cholesterol . In this paper, we report the isolation of overlapping bacteriophage lambda clones that encompass the gene for hamster cytoplasmic HMG-CoA synthase . The gene spans 20 kilobases and contains 11 exons and 10 introns . Under conditions of high-level expression in cultured hamster cells and hamster liver, approximately 50% of the mRNAs contain two introns in the region of the gene corresponding to the 5' untranslated region . The remaining 50% contain only one intron as a result of the direct splicing of exon 1 to exon 3 . The optional exon (exon 2) in the 5' untranslated region contains a 26-nucleotide sequence that is homologous to the 5' untranslated region of the mRNA for HMG-CoA reductase . Approximately 350 base pairs upstream of the transcription initiation site, the HMG-CoA synthase gene contains a sequence that is strongly homologous to the 72-base pair enhancer region of the SV40 virus . Approximately 125 nucleotides downstream of this region, the sequence CCGCCC, or its inverse complement GGGCGG, is repeated four times . Multiple copies of this sequence are present in the SV40 promoter and in the 5' flanking region of HMG-CoA reductase and several other cellular housekeeping genes . The availability of cloned genes for two consecutive negatively regulated enzymes of the cholesterol synthetic pathway should allow elucidation of the mechanism for this coordinate expression.

Science, 1986 Mar 14, 231(4743), 1273 - 6
X-ray diffraction from magnetically oriented solutions of macromolecular assemblies; Glucksman MJ et al.; A simple system was developed for obtaining x-ray diffraction patterns from magnetically oriented solutions of macromolecular assemblies . A small permanent magnet was designed that produces a magnetic field of 16 kilogauss in a volume of 1 cubic millimeter and is mountable on most x-ray cameras . Many subcellular structures have sufficient diamagnetic anisotropy that they exhibit orientation in dilute solution when placed between the poles of the magnet . Diffraction from solutions oriented in this magnet can provide substantially more structural information than small-angle scattering from isotropic solutions . In favorable cases, such as dilute solutions of filamentous bacteriophages, it is possible to produce oriented fiber diffraction patterns from which intensities along layer lines can be measured to 7-angstrom resolution . The magnetically induced birefringence observed in solutions of other macromolecular assemblies suggests that this technique may have broad applicability to subcellular structures.

Nucleic Acids Res, 1986 Mar 11, 14(5), 2365 - 80
Mutations induced by DNA polymerase alpha upon in vitro replication of M13mp8(+) DNA; Reckmann B et al.; The forward mutation of the lacZ part of the bacteriophage M13mp8 has been used to study the fidelity of the 9S DNA polymerase alpha from calf thymus during in vitro replication of single-stranded DNA . Errors leading to a loss of alpha-complementation were identified by DNA sequencing . The overall mutation rate of the lacZ target sequence was in the range of 1:300-1:1000 which is more than one order of magnitude higher than the spontaneous mutation rate . In a mutL host the mutation rate was nearly threefold higher as compared to the wildtype host . Base substitutions comprise 86% of the errors whereas base deletions amount to 12% . The addition of a base was detected only in one mutant out of 71 sequenced ones . The frameshift mutations occurred predominantly in runs of the same base . The frequencies of individual base substitution are in the order of 2 X 10(-4)-4 X 10(-4) for most of the mismatches . Mutations involving dCTP:T and dGTP:T mismatches are observed with a lower frequency, those involving dTTP:C mismatches with a higher frequency.

Nucleic Acids Res, 1986 Mar 11, 14(5), 2201 - 13
The isolation and nucleotide sequence of the complex AROM locus of Aspergillus nidulans; Charles IG et al.; The AROM locus of A . nidulans, which governs five consecutive steps in pre-chorismate aromatic amino acid biosynthesis, has been cloned in a bacteriophage vector . The nucleotide sequence of the locus reveals a single, open reading-frame of 4,812 base-pairs, apparently without introns . An internal segment of the A . nidulans AROM sequence has extensive homology with the E . coli aroA gene that encodes the 5-enolpyruvylshikimate 3-phosphate synthase.

Nucleic Acids Res, 1986 Mar 11, 14(5), 2287 - 300
The role of the loxP spacer region in P1 site-specific recombination; Hoess RH et al.; The lox-Cre site-specific recombination system of bacteriophage P1 is comprised of a site on the DNA where recombination occurs called loxP, and a protein, Cre, which mediates the reaction . The loxP site is 34 base pairs (bp) in length and consists of two 13 bp inverted repeats separated by an 8 bp spacer region . Previously it has been shown that the cleavage and strand exchange of recombining loxP sites occurs within this spacer region . We report here an analysis of various base substitution mutations within the spacer region of loxP, and conclude the following: Homology is a requirement for efficient recombination between recombining loxP sites . There is at least one position within the spacer where a base change drastically reduces recombination even when there is homology between the two recombining loxP sites . When two loxP sites containing symmetric spacer regions undergo Cre-mediated recombination in vitro, the DNA between the sites undergoes both excision and inversion with equal frequency.

Nucleic Acids Res, 1986 Mar 11, 14(5), 1985 - 9
A new restriction endonuclease from Spirulina platensis; Kawamura M et al.; Three restriction endonucleases, Sp1I, Sp1II and Sp1III have been purified partially from Spirulina platensis subspecies siamese and named . Sp1I cleaves bacteriophage lambda DNA at one site, phi X 174 RF DNA at two sites, but does not cleave pBR322 DNA . This enzyme recognizes the sequence 5'CGTACG3' 3'GCATCG5' and cuts the site indicated by the arrows . Sp1II is an isoschizomer of Tth111I and Sp1III is an isoschizomer of HaeIII.

J Mol Biol, 1986 Mar 5, 188(1), 39 - 48
Assembly and disassembly of bacteriophage T4 polyheads; Caldentey J et al.; The assembly of the product of bacteriophage T4 gene 23 (gp23), the uncleaved form of the main shell protein, has been studied . Assembly and disassembly follow the predictions for entropy-driven processes; assembly is strongly favored by conditions of high salt concentrations and high temperatures, whereas low salt and low temperatures promote disassembly . In the absence of the scaffolding core proteins in vitro, only polyheads, the tubular variant of the prohead, are produced . Kinetic studies show that the rate of polyhead dissociation depends on the concentration of associated protein, not on the number and length of the particles . Comparable to crystal formation, assembly of gp23 occurs above a critical concentration, which is dependent on salt concentration, pH and temperature . These characteristics are common to most self-assembling systems . The oligomeric states of gp23 have been investigated by analytical ultracentrifugation, which indicated the existence, at very low salt concentration and low temperature, of an equilibrium between monomers and higher oligomers, culminating in the hexamer . At pH 9.0 polyheads are completely dissociated into their monomeric gp23 subunits . Our data suggest that the hexamer is a true intermediate of polyhead assembly.

J Mol Biol, 1986 Mar 5, 188(1), 15 - 22
Phage and host genetic determinants of the specific anticodon loop cleavages in bacteriophage T4-infected Escherichia coli CTr5X; Kaufmann G et al.; Anticodon loop cleavages of two host tRNA species occur in bacteriophage T4-infected Escherichia coli CTr5X, a host strain restricting phage mutants deficient in polynucleotide kinase (pnk) or RNA ligase (rli) . The cleavage products accumulate with the mutants but are further processed in wt infection through polynucleotide kinase and RNA ligase reactions . Inactivating mutations in stp suppress pnk- or rli- mutations in E . coli CTr5X and, as shown here, also abolish the anticodon nuclease, implicating the stp product with this activity . We show also that there exist other suppressing mutations of a pnk- (pseT2) mutation that appear not to affect the anticodon nuclease and are not in stp . It has been shown that a single locus in E . coli CTr5X, termed prr, determines the restriction of pnk- or rli- mutants . A transductant carrying prr featured upon infection the anticodon nuclease reaction products, suggesting that prr determines the specific manifestation of this activity . However, prr does not encode the tRNA species that are vulnerable to the anticodon nuclease.

J Biol Chem, 1986 Mar 5, 261(7), 3112 - 5
Molecular cloning and sequence analysis of human placental alkaline phosphatase; Millan JL; The complete amino acid sequence of the precursor and mature forms of human placental alkaline phosphatase have been inferred from analysis of a cDNA . A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage lambda gt11 placental cDNA library with antibodies against CNBr fragments of the enzyme . The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental alkaline phosphatase . The mature protein is 513 amino acids long . The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn122 and Asn249 and a highly hydrophobic membrane anchoring domain at the carboxyl terminus of the protein . Significant homology exists between placental alkaline phosphatase and Escherichia coli alkaline phosphatase . Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced.

J Biol Chem, 1986 Mar 5, 261(7), 3441 - 50
Rat vitamin D binding protein . Determination of the full-length primary structure from cloned cDNA; Cooke NE; Vitamin D binding protein (DBP) is an abundant serum glycoprotein secreted by the liver which transports vitamin D sterols, binds to actin, and is found on the surface of B-lymphocytes and subpopulations of T-lymphocytes . In the current study, a cDNA to rat DBP mRNA was cloned from a bacteriophage lambda gt 11 rat liver expression library . This DBP cDNA clone was identified by immunoblotting and its identity was confirmed by immunoprecipitation of a 54-kDa protein after hybrid-assisted translation . Northern analysis and primer extension mapping of rat liver mRNA indicated that the full-length DBP mRNA contains 1700 bases . By DNA sequence analysis this 1655-base pair clone contains a single open reading frame encoding the 476-amino acid containing full-length DBP and includes its 16-amino acid signal sequence . Analysis of the sequence reveals about 40% nucleotide and 23% amino acid homology to both albumin and alpha-fetoprotein . The encoded DBP contains a characteristic placement of cysteine residues, identical to that in albumin, suggesting a similar secondary folding structure . Albumin and alpha-fetoprotein are composed of three internally homologous domains . DBP mRNA terminates 122 amino acids before the larger albumin mRNA in the third internal domain, but retains the characteristic homology among the first two domains and the truncated portion of the third domain . These data support the conclusion that DBP is a member of a multigene family which includes albumin and alpha-fetoprotein.

Eur J Biochem, 1986 Mar 3, 155(2), 383 - 90
Effect of polyamines on translation fidelity in vivo; McMurry LM et al.; Polyamines, when added to cell-free protein-synthesizing systems, have been shown either to reduce mistranslation or to increase it depending upon the composition of the reaction mixture . To study this question under conditions as natural as possible we investigated the effects of polyamines on the fidelity of protein synthesis in intact Escherichia coli bacterial cells, using strains which were auxotrophic for polyamine synthesis . Error was measured in two ways: the incorporation of {3H}histidine into coat protein of bacteriophage MS2, the gene of which does not code for histidine, and the synthesis of a basic variant of MS2 coat protein in which a lysine erroneously replaces an asparagine, causing a change in isoelectric point . We found that when cell cultures were supplemented with polyamines there was no effect on the first type of error and the second type decreased twofold . The measured errors occurred at the level of translation because their frequency increased in the presence of streptomycin and decreased in cells bearing a streptomycin-resistance mutation known to lower the occurrence of translational misreading . The average erroneous incorporation per mol coat protein in the presence of polyamines was 1.43 +/- 0.59 mmol histidine and 25-34 mmol lysine/asparagine substitution . The reason for the different effect of polyamines on the two types of error is not known but could be related to the difference between their corresponding frequencies or to codon-specific effects.

J Virol, 1986 Mar, 57(3), 960 - 7
Infection by choleraphage phi 138: bacteriophage DNA and replicative intermediates; Chowdhury R et al.; Choleraphage phi 138 contains a linear, double-stranded, circularly permuted DNA molecule of 30 X 10(6) daltons or 45 kilobase pairs . Upon infection, the host DNA is degraded, and synthesis of phage-specific DNA is detectable 20 min after infection . The phage utilizes primarily the host DNA degradation products for its own DNA synthesis . A physical map of phi 138 DNA was constructed with the restriction endonucleases Bg/II, HindIII, and PstI . A concatemeric replicative DNA intermediate equivalent to eight mature genome lengths was identified . The concatemer was shown to be the precursor for the synthesis of mature bacteriophage DNA which is subsequently packaged by a headful mechanism.

J Virol, 1986 Mar, 57(3), 759 - 64
Cell-free transcription and translation of isolated restriction fragments localize bacteriophage T5 pre-early genes; Blaisdell PW et al.; In vitro transcription and translation of isolated restriction fragments containing all or part of the terminal redundancies of bacteriophage T5 localized virtually every pre-early gene to a small 6.3-kilobase BglI fragment . Among these genes were those encoding the A1 and A2 proteins, which are responsible for complete entry of the viral genome into its host, and the deoxynucleoside 5'-monophosphatase . A 3.9-kilobase BglI fragment containing the remainder of the pre-early region induced no proteins under these same conditions . Proteins induced by fragments including the right and left terminal redundancies were also compared and found to be identical . DNA immediately flanking the pre-early regions induced few proteins in vitro . Thus, this technique has allowed the overall gene organization of the pre-early region of T5 to be described.

EMBO J, 1986 Mar, 5(3), 501 - 10
Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation; Newmeyer DD et al.; DNA (from bacteriophage lambda or Xenopus) is assembled into nucleus-like structures when mixed with an extract from Xenopus eggs . Electron microscopy shows that these in vitro-reconstituted nuclei possess complete double membranes; some, but not all, nuclei have pore complexes . Extracts depleted of their endogenous ATP (by addition of ATPases) cannot assemble nuclear envelopes visible by phase-contrast microscopy . Once synthetic nuclei are assembled, however, they are stable when ATP is subsequently depleted, although their chromatin becomes condensed . About one-fourth of the nuclei assembled in vitro from lambda DNA accumulate nuclear proteins such as nucleoplasmin . ATP depletion blocks nucleoplasmin accumulation both in vitro, in pre-assembled synthetic nuclei, and in vivo, in the nucleus of microinjected oocytes . However, nucleoplasmin previously accumulated by reconstituted nuclei or by the germinal vesicle in microinjected oocytes is retained after ATP depletion.

Mol Gen Genet, 1986 Mar, 202(3), 363 - 7
Identification of T4 gene 25 product, a component of the tail baseplate, as a 15K lysozyme; Szewczyk B et al.; The proteins synthesized in Escherichia coli B cells after infection with various T4 bacteriophage tail baseplate mutants were analysed by the immunoblotting method for the presence of the 15 Kilodalton lysozyme found in phage T4 particles . Using three different antisera: anti-phage, anti-baseplate and anti-15K lysozyme, it has been found that the 15K lysozyme is not present in lysates of bacteria infected with T4 gene 25 amber mutants . The 15K lysozyme was also found to be expressed in E . coli B cells transformed with a plasmid containing only a small portion of the T4 genome but which included T4 gene 25 . These observations indicate that the 15K lysozyme is the gene 25 product.

Mol Biol (Mosk), 1986 Mar-Apr, 20(2), 415 - 22
{Bacteriophages T3 and T7: transcription-dependent mechanism of phage DNA transport into the cell during infection}; Zavriev SK et al.; The mechanism by which bacteriophage T3 DNA is transported into the E . coli cell during infection was studied . The data obtained testify that bacteriophage T3, similarly to what we have earlier found for bacteriophage T7, introduces its DNA into the infected cell is a transcription-dependent way . A detailed discussion is presented on the occurrence of the transcription-coupled transport of viral DNA into the infected cell and on a number of general issues concerning the transport functions of template-directed enzymes.

Mol Biochem Parasitol, 1986 Mar, 18(3), 333 - 42
Expression of Schistosoma japonicum antigens in Escherichia coli; Saint RB et al.; A cloned library of DNA complementary to the mRNA of adult Schistosoma japonicum has been prepared and expressed as fusion proteins with Escherichia coli beta-galactosidase . Colonies expressing the S . japonicum cDNA clones were screened both with antibodies from individuals with a history of schistosomiasis and with antibodies obtained from a rabbit immunized with whole adult worms . In both cases colonies were detected which bound antibody, although the frequency of antigen-positive clones was much higher with the rabbit antiserum than with human sera . In both cases the proportion of colonies reacting with antibodies was markedly lower than that published for equivalent screens of Plasmodium falciparum cDNA with sera from individuals with a history of falciparum malaria . Several major S . japonicum antigens were identified by the affinity purification of antibodies using immobilised fusion proteins produced during lytic growth of the recombinant bacteriophage.

J Virol, 1986 Mar, 57(3), 754 - 8
Conservative replication and transcription of Saccharomyces cerevisiae viral double-stranded RNA in vitro; Nemeroff ME et al.; All double-stranded RNA viruses have capsid-associated RNA polymerase activities . In the reoviruses, the transcriptase synthesizes the viral plus strand in a conservative mode and the replicase synthesizes the viral minus strand, again conservatively . In bacteriophage phi 6 and in some fungal viruses, the transcriptase activity is semiconservative, acting by displacement synthesis . In this work we demonstrate Saccharomyces cerevisiae viral RNA replication in vitro for the first time and, using more sensitive techniques than those previously used, show that both the transcriptase and the replicase appear to act conservatively, like those of reovirus . There is therefore clearly no universal life cycle for the double-stranded RNA viruses.

Mutat Res, 1986 Mar, 165(2), 89 - 100
Cloning and expression of the 3' portion of the T4 denV gene as a lacZ fusion gene; Lloyd RS et al.; Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4 . This rabbit serum, from which endemic E . coli antibodies have been removed, reacts with a single protein from T4-infected E . coli with a molecular weight of 16078 dalton . It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay . A phage lambda gt11 T4 dC DNA library was screened for phage which produced a beta-galactosidase-endonuclease V fusion protein . Immunopositive clones were detected at a frequency of 0.25% of the plaques in the library . Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within lambda gt11 in a single orientation . Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native beta-galactosidase.

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