J Clin Lab Immunol, 1981 Sep, 6(2), 147 - 55 Effect of glucocorticosteroid therapy on the immune system of patients with nonimmunologically mediated dermatoses; Niwa Y et al.; Humoral and cellular immunologic studies were carried out to define the effect of steroid treatment on the immune system of patients with non-immunologically-mediated dermatoses . The cellular immune response as measured by PHA and Con A induced lymphoblastogenesis was highly impaired, being reduced in 79% and 66%, respectively, of the cases . The peripheral and bone marrow lymphocyte subpopulation and PPD skin reaction were only slightly affected by steroid administration . Histologic studies of lymph node biopsies showed atrophy, fibrosis and fat degeneration . The immunoglobulin and complement levels and humoral antibody responses to typhoid vaccine, diphtheria toxoid and bacteriophage phi--x 174 were mildly affected . The impairment of immune systems induced by steroids was largely reversible, while the PHA induced blastogenesis and pathological changes in the lymph nodes were irreversible . In comparison with experimental animal studies using massive doses of steroids, the immune system in man was not significantly affected by administration of small doses on a long term basis in clinical practice. J Biochem (Tokyo), 1981 Sep, 90(3), 779 - 83 Involvement of host cell gene products in conversion of bacteriophage S13 single-stranded DNa to duplex replicative form DNA in vitro; Watabe K et al.; The single-stranded circular DNA of bacteriophage S13 was converted to the duplex replicative form DNA by soluble extracts from Escherichia coli strain H560 (polA, endA) in vitro . The maximal conversion required four deoxyribonucleoside triphosphates, Mg2+, exogenous S13 DNA and ATP, but not CTP, UTP, or GTP . The conversion was blocked by N-ethylmaleinimide but not by rifampicin . The product was identified as a gapped duplex replicative form DNA . Using extracts from some thermosensitive mutants of E . coli defective in DNA replication, we found that dnaB and dnaC gene products are involved in the conversion stage of single-stranded DNA to duplex DNA in vitro. Proc Natl Acad Sci U S A, 1981 Sep, 78(9), 5749 - 53 Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli; Bagg A et al.; The product of the umuC gene is required for UV and chemical mutagenesis in Escherichia coli . By the use of the Mud(Ap, lac) bacteriophage, we have obtained an operon fusion of the lac structural genes to the promoter/regulatory region of the umuC gene . The strain containing the umuC::Mud(Ap, lac) fusion was identified on the basis of its UV nonmutability . Strains containing this putative null allele of umuC were (i) nonmutable by UV and other agents, (ii) slightly UV sensitive, and (iii) deficient in their ability to carry out Weigle reactivation of UV-irradiation bacteriophage lambda . The UV nonmutability of the strain could be suppressed by a derivative of the mutagenesis-enhancing plasmid pKM101 . beta-Galactosidase synthesis in umuC::Mud(Ap, lac) fusion strains was inducible by UV and other DNA-damaging agents . Genetic analysis of the regulation of beta-galactosidase in umuC::Mud(Ap, lac) strains suggests that the lexA protein is the direct repressor of the umuC gene and that a function of the recA protein, probably its protease activity, is required for the removal of the lexA repressor at the time of umuC induction. Proc Natl Acad Sci U S A, 1981 Sep, 78(9), 5618 - 22 Fast responses of bacterial membranes to virus adsorption: a fluorescence study; Bayer ME et al.; After collision with their host cells, virus particles may remain mobile on cell surfaces until they become attached at firm binding sites . We propose that a virion will arrive within a typical median time at such a site, generating a membrane signal such as an increased membrane fluorescence in cells labeled with the voltage-sensitive dyes 8-anilino-1-naphthalene-sulfonate (Mg-salt) (ANS), N-phenylnaphthylamine (NPA), or 3,3'-dipentyl-2,2'-oxacarbocyanine (di-O-C5{3}) . We found that the time span between virus adsorption and fluorescence response varies widely among phages and also depends on bacterial strain, metabolic state, and type of dye . di-O-C5{3}-labeled cells react within 1 sec to uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) . Cells labeled with ANS and NPA react to CCCP in 4-6 sec . Bacteriophages T4, T5, chi, and BF23, added to ANS-labeled cells, change the fluorescence in 9-15 sec . T-even ghosts cause a response at identical times . Baseplate-defective phage mutant T412- and isolated adsorption organelles of smaller viruses fail to cause an effect . di-O-C5{3}-labeled cells respond to T4 at a multiplicity of infection greater than or equal to 40 within 1 sec . A longer time (8 sec) is required at lower multiplicities . The receptor-degrading phages epsilon 15, epsilon 34, c 341, and K29 need the longest time (1 min for ANS) to cause a fluorescence increase . We suggest that the delayed fluorescence response is concomitant with the surface "walk" of the virion, which is terminated at an injection site . T4 tail sheath contraction coincides with the onset of the membrane fluorescence response. Cell, 1981 Sep, 25(3), 729 - 36 A novel role for site-specific recombination in maintenance of bacterial replicons; Austin S et al.; If daughter copies of unit-copy replicons recombine with each other, a replicon dimer results that cannot be partitioned equally to daughter cells at cell division . We present evidence that dimer formation interferes with plasmid equipartition in the case of a miniplasmid derived from the unit-copy plasmid prophage of bacteriophage P1 . Asymmetric partition occurs, leading to a relatively high rate of loss of the plasmid from the growing population . In contrast, the wild-type P1 plasmid is maintained very efficiently in host cells . We show that this efficient maintenance is due to the presence of the loxP-cre site-specific recombination system present on the intact P1 plasmid . This system promotes rapid recombination between two loxP sites on dimer molecules, resolving them into monomeric substrates for proper partition . We suggest that bacterial replicons that are maintained with great accuracy in recombination-proficient cells might also encode high-efficiency recombination systems. J Bacteriol, 1981 Sep, 147(3), 920 - 4 High-affinity arabinose transport mutants of Escherichia coli: isolation and gene location; Clark AF et al.; The gene araF, the product of which is the L-arabinose-binding protein--a component of the high-affinity L-arabinose transport system, was located on the Escherichia coli linkage map at 45 min . We established this location using bacteriophage P2 eductates and bacteriophage P1 cotransduction frequencies with the adjacent genetic loci, his (histidine biosynthesis) and mgl (methylgalactoside transport) . In addition, we isolated a number of mutants that phenotypically exhibited altered high-affinity L-arabinose transport capacities . At least two of these mutations were located in the araF gene, as binding protein purified from these strains exhibited altered in vitro arabinose-binding properties. J Bacteriol, 1981 Sep, 147(3), 720 - 7 In vitro host cell reactivation of alkylated bacteriophage T7 deoxyribonucleic acid by repair-deficient strains of Escherichia coli; Dodson LA et al.; An in vitro system capable of packaging bacteriophage T7 deoxyribonucleic acid (DNA) into phage heads to form viable phage particles has been used to monitor the biological consequences of DNA dam aged by alkylating agents, and an in vitro DNA replication system has been used to examine the ability of alkylated T7 DNA to serve as template for DNA synthesis . The survival of phage resulting from in vitro packaging of DNA preexposed to various concentrations of methyl methane sulfonate or ethyl methane sulfonate closely paralleled the in vivo situation, in which intact phage were exposed to the alkylating agents . Host factors responsible for survival of alkylated T7 have been examined by using wild-type strains of EScherichia coli and mutants deficient in DNA polymerase I (polA) or 3-methyladenine-DNA glycosylase (tag) . For both in vivo and in vitro situations, a deficiency in 3-methyladenine-DNA glycosylase dramatically reduced phage survival relative to that in the wild type, whereas a deficiency in DNA polymerase I had an intermediate effect . Furthermore, when the tag mutant was used as an indicator strain, phage survival was enhanced when alkylated DNA was packaged with extracts prepared from a wild-type strain in place of the tag mutant or by complementing a tag extract with an uninfected tag+ extract, indicating in vitro repair during packaging. Proc Natl Acad Sci U S A, 1981 Sep, 78(9), 5498 - 502 Knotted DNA from bacteriophage capsids; Liu LF et al.; The majority of the DNA prepared from tailless capsids of bacteriophage P2 by the phenol extraction procedure consists of monomeric rings that have their cohesive ends joined . Electron microscopic and ultracentrifugal studies indicate that these molecules have a complex structure that is topologically knotted; they have a more compact appearance and a higher sedimentation coefficient when compared with regular nicked P2 DNA rings . Linearization of these rings by thermal dissociation or repair of the cohesive ends by DNA polymerase I in the presence of all four deoxynucleoside triphosphates gives molecules that are indistinguishable from normal P2 DNA that has been similarly treated . The knotted nature of the majority of P2 head DNA is further supported by analyzing the products when these molecules are treated with ligase and the ligase-treated molecules are subsequently nicked randomly with DNase I . The data are consistent with the notion that, if such a molecule is first converted to a form that contains only one single-chain scission per molecule, strand separation gives a linear strand and a highly knotted single-stranded ring . The results suggest that the DNA packaged in tailless P2 capsids is arranged in a way that leads to the formation of a complex knot when the ends join . In an intact phage particle, the anchoring of one terminus of the DNA to the head-proximal end of the tail {Chattoraj, D . K . & Inman, R . B . (1974) J . Mol . Biol . 87, 11-22} presumably diminishes or prevents this kind of joining . The novel knotted DNA can be used to assay type II DNA topoisomerases that break and rejoin DNA in a double-stranded fashion. Proc Natl Acad Sci U S A, 1981 Sep, 78(9), 5421 - 24 Gene II of phage f1: its functions and its products; Dotto GP et al.; Plasmids harboring the amino-terminal part of bacteriophage f1 gene II confer to bacterial cells partial resistance to infection with the male-specific bacteriophages f1 and f2 . This effect (IP-2 phenotype) is due to the production of large amounts of an approximately 20,000-dalton polypeptide corresponding to the amino-terminal part of gene II protein . These results have allowed the isolation of clones producing functional gene II protein in large amounts . An in vitro assay has been developed to test the enzymatic activity of the gene II protein produced. Proc Natl Acad Sci U S A, 1981 Sep, 78(9), 5416 - 20 Cloning of bacteriophage fd gene 2 and construction of a plasmid dependent on fd gene 2 protein; Meyer TF et al.; Bacteriophage fd gene 2 was cloned in plasmid pBR325 . Cells carrying the hybrid plasmid produce about 200 times more enzymatically active fd gene 2 protein than did cells infected with phage fd wild type, as measured by replication of phage fd replicative form I in vitro . Cloned gene 2 supports replication of an artificial phage fd miniplasmid consisting of the origin of bacteriophage fd replication and a gene coding for kanamycin resistance . This plasmid occurs in high copy numbers and is viable only in cells carrying the cloned fd gene 2 or in cells infected with phage fd . Because the miniplasmid is not propagated in natural hosts, it can be considered a safe cloning vector . Its fusion with the gene 2 hybrid plasmid provides an autonomous replicon independent of the polA function of the host cell . fd gene 2 is the only phage-encoded trans-acting function required for replication of double-stranded fd DNA in vivo. Gene, 1981 Sep, 14(4), 251 - 62 Two-dimensional display of lambda and Escherichia coli restriction fragments separated by Hg-Cs2SO4 centrifugation and gel electrophoresis; Yamagishi H et al.; EcoRI restriction fragments derived from the DNA of bacteriophage lambda and Escherichia coli were fractionated by density gradient centrifugation of their mercury complexes in Cs2SO4 and subsequent electrophoresis on a horizontal agarose-gel slab . In this two-dimensional display, lambda fragments were resolved into six components and E coli fragments into more than 108 components . Bacterial chromosome regions contiguous to lambda prophage integrated at different sites were amplified by induction, and the EcoRI fragments were subjected to the two-dimensional analysis . As expected, the sets of amplified fragments were clearly different among the various lysogens . The approximate genome region affected by induction was estimated as one-tenth of the whole chromosome. Biokhimiia, 1981 Sep, 46(9), 1596 - 602 {Biosynthesis of early enzymes induced by bacteriophage T4}; Vaitkiavichene RS et al.; The biosynthesis of early enzymes of DNA-ligase, RNA-ligase, DNA-polymerase, polynucleotide kinase, exonuclease A induced by bacteriophage T4amN82 was studied . The maximal activity of DNA-ligase was observed at the 60th min after the infection, while that of the other enzymes was revealed at the 90th min and reached 4, 45, 529, 120 and 78 units per mg of protein for DNA-ligase, RNA-ligase, polynucleotide kinase, DNA-polymerase and exonuclease A, respectively . Bacteriophage T4amN82 induced the maximal biosynthesis of the tested enzymes, when E . coli B-23 cells were grown in medium I containing trypton bacto ("Merck", West Germany) and a yeast extract ("Difco", USA) . Similar events were observed when E . coli B-23 cells infected with phage T4amN82 were grown in a medium (II) with casein hydrolysate and yeast extract . Ultrasonication used for the disruption of E . coli B-23 cells infected with bacteriophage T4 had no effect on the enzyme activities. Cancer Res, 1981 Sep, 41(9 Pt 1), 3597 - 603 Detection of RNA complementary to herpes simplex virus DNA in human cervical squamous cell neoplasms; Eglin RP et al.; Nonneoplastic and neoplastic cervical biopsy specimens were examined by in situ hybridization to 125I-labeled DNA of herpes simplex virus (HSV), adenovirus, and bacteriophage lambda DNA's, and quantitative hybridization data were obtained using a Video Image Analyser . HSV-specific RNA was detected in 72% of cervical intraepithelial neoplasia, 60% of squamous cervical carcinomas, 2% of nonneoplastic cervices, and 9% of primary adenocarcinomas of the cervix . None of the tissues gave positive hybridization with adenovirus or lambda DNA probes . In paired biopsies of cervical intraepithelial neoplasia and nonneoplastic epithelium from 29 individuals, HSV-specific RNA was detected only in the epithelium of the neoplastic sample and not in the nonneoplastic control . Infectious HSV-2 was isolated from a low proportion (2%) of both ectocervical swabs and cell-free tissue extracts of patients examined, suggesting that the HSV-specific RNA detected in squamous cell neoplasms was not due to overt infections. Biochim Biophys Acta, 1981 Aug 27, 655(1), 96 - 101 Inhibition of the action of Escherichia coli transcription termination protein rho by poly(C) and heparin; Bektesh SL et al.; Poly(C) and heparin at low concentrations (1 microgram/ml) prevent the RNA synthesis termination protein rho from functioning during the biosynthesis of RNA from bacteriophage T7 DNA catalyzed by Escherichia coli RNA polymerase . Both of these polyanions inhibit the binding of rho to isolated T7 RNA . Heparin also inhibits rho ATPase when isolated RNA transcripts are used as cofactors . It is concluded that the polyanions inhibit termination by binding to the site on rho that is normally used for the initial interaction with a nascent RNA transcript in the rho-mediated release of RNA . Since one of the inhibitors, poly(C), is itself a potent activator for rho ATPase, it is also concluded that the ATP hydrolysis step that is required for rho termination has to be coupled to an action of rho on the RNA molecule to be released from the transcription complex. Nucleic Acids Res, 1981 Aug 25, 9(16), 3889 - 906 Transcription of sea urchin histone genes in Escherichia coli; Mellado RP et al.; DNA fragments comprising units of the repeated histone genes form the sea urchins Psammechinus miliaris and Echinus esculentus were placed under the control of bacteriophage Lambda promoters by cloning into lambda replacement vectors . Although promoter-like regions exist within the cloned fragments, transcription of the histone genes is controlled mainly, but not exclusively, by lambda PL promoter . A transcription map of the cloned P . miliaris histone DNA fragment was obtained . The order of histone genes in E . esculentus was deduced from electron microscopic analyses of heteroduplexes with P . miliaris histone genes, and is similar to that in P . miliaris . Translation products of the transcripts have not been found in E . coli. Nucleic Acids Res, 1981 Aug 25, 9(16), 3979 - 89 Novel topologically knotted DNA from bacteriophage P4 capsids: studies with DNA topoisomerases; Liu LF et al.; DNA molecules isolated from bacteriophage P4 are mostly linear with cohesive ends capable of forming circular and concatemeric structures . In contrast, almost all DNA molecules isolated form P4 tailless capsids (heads) are monomeric DNA circles with their cohesive ends hydrogen-bonded . Different form simple DNA circles, such P4 head DNA circles contain topological knots . Gel electrophoretic and electronmicroscopic analyses of P4 head DNA indicate that the topological knots are highly complex and heterogeneous . Resolution of such complex knots has been studied with various DNA topoisomerases . The conversion of highly knotted P4 DNA to its simple circular form is demonstrated by type II DNA topoisomerases which catalyze the topological passing of two crossing double-stranded DNA segments {Liu, L . F., Liu, C . C . & Alberts, B . M . (1980) Cell, 19, 697-707} . The knotted P4 head DNA can be used in a sensitive assay for the detection of a type II DNA topoisomerase even in the presence of excess type I DNA topoisomerases. J Biol Chem, 1981 Aug 25, 256(16), 8400 - 6 Primary structure of the low molecular weight nucleic acid-binding proteins of murine leukemia viruses; Henderson LE et al.; Murine leukemia viruses contain a low molecular weight basic protein, designated p10, which binds to single-stranded nucleic acids . The complete amino acid sequence of p10 from the Rauscher strain of virus has been determined . The partial amino acid sequences of p10s from Moloney, Friend, AKR, Gross, radiation leukemia, and BALB/2 viral strains have also been determined using microsequencing techniques . Rauscher p10 is composed of 56 amino acid residues; the other p10s are similar in size but differ from Rauscher by a few conservative amino acid substitutions . The structure of Rauscher p10 was compared to the structure of a functionally homologous protein from Rous avian sarcoma virus . The comparison revealed regions of amino acid sequence homologies which indicate a phylogenetic relationship between the murine and avian viral strains . The analyses revealed a periodic placement of three Cys residues and a Gly-His sequence . A structure involving these residues is found once in the murine protein and twice in the avian protein . A similar structure is seen in the single stranded nucleic acid binding protein of bacteriophage T4 . However, in the latter case, the order of amino acid residues is inverted. Nucleic Acids Res, 1981 Aug 11, 9(15), 3731 - 46 Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda; Villeponteau B et al.; A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe . Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA . Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes . The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized . The overall features of the map were confirmed by genomic Southern analysis . Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation . The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself . Probably such deletions explain the failure to recover this gene in previous attempts. In Vitro, 1981 Aug, 17(8), 695 - 700 Incorporation of bacteriophage DNA into the genome of cultured human lymphocytes; Wenger SL et al.; After exposure of phytohemagglutinin-stimulated human lymphocytes to high doses of tritiated-thymidine labelled phi X-174 or T2 bacteriophage, label from the phage genome became incorporated into lymphocyte DNA . Exposure to bacteriophage DNA, whether biologically active, inactive, or fragmented, had a depressive effect on lymphocyte DNA replication . Incorporation of label from phage DNA into the lymphocyte DNA, however, was maximum for biologically active phage. Eur J Biochem, 1981 Aug, 118(2), 371 - 7 Identification of transcription initiation sites for bacterial RNA polymerase and eukaryotic RNA polymerase B on the 5' end of the mouse beta-Globin gene; Crepin M et al.; Using a recombinant phage containing the mouse beta-Globin gene with lambda gtWES bacteriophage DNA, transcription initiation sites for Escherichia coli RNA polymerase and calf thymus RNA polymerase B were mapped at the 5' and 3' ends of the mouse beta-Globin gene . The bacterial enzyme was capable of initiating RNA synthesis at the 3' end site located at about 700 residues from the 3' end of the beta-Globin restriction enzyme map . Initiation at this site was more efficient than initiation at the known early lambda promotors (PL, PR) . Calf thymus RNA polymerase B initiated transcription at the same sites as the bacterial enzyme but in this case maximum efficiency was at the 5' end site as compared to the 3' end site . Initiation of transcription occurs in the region of the d(T-A-T-A-A) sequence . Initiation efficiency at the 5' end site, as probed by the maximum rate of transcription, was shown to depend partly upon the presence of the adjacent sequences upstream and downstream of the 5' initiation site. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4669 - 73 Translational regulation: identification of the site on bacteriophage T4 rIIB mRNA recognized by the regA gene function; Karam J et al.; The bacteriophage T4 gene regA encodes a protein that diminishes the expression of many unlinked early T4 genes . Previous work demonstrated that regA-mediated repression occurs after transcription . We report here on the identification of the target site on one regA-sensitive mRNA, the message encoding the phage T4 rIIB protein . The target for regA-mediated action overlaps the translational initiation domain of the rIIB messenger . The regA protein may be a repressor that operates translationally on a significant and interesting set of early phage T4 mRNAs. Mutat Res, 1981 Aug, 83(1), 1 - 14 Mutagenic specificity of nitrosomethylurea in bacteriophage T4; Ripley LS; In contrast to alkylating agents such as ethyl methanesulphonate which can induce mutation after treatment of free phage particles, nitrosamides induce mutations only when phage are treated intracellularly during infection of the host . The basis of this intracellular dependence is not currently understood . In this study the mutational specificity of nitrosomethylurea (NMU) in bacteriophage T4 was investigated by measuring the reversion of well-characterized mutants in the rII genes . While no mutation was produced after in vitro treatments of free phage, in vivo treatments strongly induced G:C leads to A:T transitions and substantially induced A:T leads to G:C transitions . Transversions and frameshift mutations were rarely induced . Although methyl methanesulfonate mutagenesis of T4 depends upon a phage-encoded error-prone repair system, NMU-induced mutagenesis is independent of this repair system . Similarities in mutagen specificity of nitrosamides and differences in DNA metabolism in T4 when compared to its host, Escherichia coli, suggest that T4 is well-suited for the study of mechanisms of nitrosamide mutagenesis. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 5041 - 5 Mutation in an intervening sequence splice junction in man; Orkin SH et al.; The alpha 2-globin gene of an individual with alpha-thalassemia associated with the absence of alpha 2 mRNA was cloned in bacteriophage . This mutant globin gene was normally active in transcription in vitro . The DNA sequence of the gene, however, revealed a pentanucleotide deletion within the 5' splice junction of the first intervening sequence . Following the G of the invariant G-T dinucleotide normally located within such junctions, a deletion of T-G-A-G-G was found . No other sequence abnormalities within the mutant gene were present . We speculate therefore that this deletion within the splice junction is the primary genetic defect in this individual with thalassemia and that loss of a functional splice junction results in failure of stable mRNA formation. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4936 - 40 Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA termination signal; Gentz R et al.; Downstream placement of a strong transcriptional termination signal has made possible the cloning of bacteriophage T5 promoters known to exhibit high signal strength . The cloning system constructed contains two easily assayable indicator functions whose expression is controlled by the integration of promoters and terminators, respectively . By assessing transcription within the indicator regions, the efficiency of promoters as well as termination signals can be determined in vitro and in vivo. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4877 - 81 Structural variation among human beta-tubulin genes; Cowan NJ et al.; A chicken beta-tubulin cDNA probe has been used to screen two independently generated human genomic libraries . Of 13 EcoRI fragments detectable in a human genomic Southern blot experiment, 7 correspond in size to EcoRI fragments isolated from recombinant bacteriophage . The location of beta-tubulin-specific regions and the direction of transcription were determined within each cloned fragment . One clone (5 beta) contained a beta-tubulin-specific region of 6.8 kilobase pairs (kbp) that included three intervening sequences as well as a number of inverted repeat structures . The remaining clones contained beta-tubulin-specific sequences that were close to or, in two cases, substantially less than 1.9 kbp long . Because mature human beta-tubulin mRNA is approximately 1.9 kbp long, these short DNA regions cannot on their own encode a functional beta-tubulin mRNA . Analysis using 3'- and 5'-specific probes derived from the chicken cDNA clone showed the presence of both of these end regions within one truncated tubulin-like sequence . A second short tubulin-specific region failed to hybridize with a 3'-specific probe . These short sequences are therefore likely to be examples of pseudogenes that have arisen by loss of a portion of DNA essential to the production of functional human beta-tubulin mRNA. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4867 - 71 Primary structure and evolution of rat growth hormone gene; Barta A et al.; The rat growth hormone gene was isolated on a cloned 11.4-kilobase EcoRI-generated DNA fragment from a bacteriophage "library" of chromosomal DNA . The structural gene sequence, approximately 2.1 kilobases long, was identified by hybridization to the corresponding cloned rat growth hormone cDNA and shown to contain four intervening sequences . The complete primary structure of the gene and the 5' and 3'-flanking regions was determined . The mosaic structure of exons and introns can be related to the different biological activities of growth hormone and to the evolution from ancestral sequences of a gene that was the precursor to the growth hormone and the related prolactin and placental lactogen (chorionic somatomammotropin) genes . The largest intron was found to contain a dispersed repetitive DNA sequence flanked by perfect 18-base pair direct repeats . The mobility of sequences of this kind could play a role in the observed variation of intron sizes and in rearrangements of mammalian genes. J Virol, 1981 Aug, 39(2), 510 - 8 Application of Arrhenius kinetic theory to viral eclipse: selection of bacteriophage phi X174 mutants; Incardona NL; Analysis of the bacteriophage phi X174 eclipse period in terms of Arrhenius kinetic theory suggests the following hypothesis: mutants should exist with two concomitant physiological characteristics as their phenotype . These are an eclipse rate lower than that of the wild type at permissive temperatures for plaque formation and an eclipse rate too low at lower temperatures to permit plaque development . Thus, enrichment of a mutagenized virus population for mutants that fail to eclipse during a short period at permissive temperatures should yield eclipse mutants with the cold-sensitive (cs; nonpermissive temperature, 25 degrees C), and not the temperature-sensitive (ts; nonpermissive temperature, 42 degrees C), plaque phenotype . In several trials, the frequency of the cs phenotype in the population increased from less than 0.2% to between 2 and 4% after the enrichment step, whereas the frequency of the ts phenotype remained unchanged (less than 0.2%) . Moreover, 80% of these cs mutants have eclipse rates that are 3- to 40-fold lower than that of the wild type at both 37 degrees C and 25 degrees C . The successful application of the Arrhenius theory to phi X eclipse may provide insights into the molecular mechanism whereby the phi X174 genome is delivered into the host cell . Since the eclipse kinetics of other nonenveloped viruses are similar to those of phi X174, kinetic theory may be broadly applicable in the selection and characterization of viral eclipse mutants. J Bacteriol, 1981 Aug, 147(2), 660 - 9 Arrangement of bacteriophage lambda receptor protein (LamB) in the cell surface of Escherichia coli: a reconstitution study; Yamada H et al.; The LamB protein purified in a solution of sodium dodecyl sulfate was assembled into an ordered hexagonal lattice structure with a lattice constant of about 7.8 nm in the presence of lipopolysaccharide . The LamB alone formed aggregates with some lattice structure . However, the regularity of the lattice was only maintained within a very small area . An ordered hexagonal lattice was also formed when the wild-type lipopolysaccharide was replaced by heptoseless lipopolysaccharide, lipid A, and even fatty acid . However, the lattice constants were appreciably smaller than that with the wild-type lipopolysaccharide . The results suggest that the heptose-containing polysaccharide region, as well as the fatty acid region, are involved in the interaction with the LamB protein . The LamB-lipopolysaccharide lattice was preferably formed on the peptidoglycan layer when the lipoprotein was covalently bound to this layer . These results indicate that the molecular arrangement of the LamB protein in the outer membrane is similar to that of matrix proteins, OmpC and OmpF, which exist as trimers . The ordered hexagonal lattice was active in the receptor function for lambda, resulting in phage adsorption and deoxyribonucleic acid ejection . Thus, this reconstitution system should provide a useful means of studying the mechanism of lambda infection. Blood, 1981 Aug, 58(2), 360 - 8 Recovery of antibody production in human allogeneic marrow graft recipients: influence of time posttransplantation, the presence or absence of chronic graft-versus-host disease, and antithymocyte globulin treatment; Witherspoon RP et al.; One-hundred fifty-three recipients of HLA-identical sibling marrow transplants for aplastic anemia or hematologic malignancy were injected with bacteriophage phi X174 (phage), pneumococcal polysaccharide antigen (PPA), or keyhole limpet hemocyanin (KLH) . Antibody levels were determined several times in the 6 wk after injection . Multiple regression techniques were used to determine what factors played significant roles in the antibody response . The most significant factors were the time elapsed from transplantation, chronic graft-versus-host disease (GVHD), and antithymocyte globulin (ATG) treatment . All patients had low antibody responses to all antigens in the first 180 days from transplant . Beyond 180 days patients without chronic GVHD showed antibody responses indistinguishable from those of normal donors . However, patients with chronic GVHD had the following impairments: (1) primary response to phage, (2) conversion from IgM to IgG in secondary response to phage, (3) secondary response to KLH, and (4) response to PPA . ATG treatment given to patients either prophylactically or therapeutically for acute GVHD was followed by lower primary responses to phage in the first 180 days and poor ability to switch from IgM to IgG antibody in the secondary response beyond 180 days postgrafting . Other factors did not yield additional significant information about ability to predict antibody responses including diagnosis, conditioning regimen, treatment in or out of laminar air flow rooms, transplantation, pretransplant refractoriness of the recipient to platelet transfusions from random donors, donor age or donor sex, and steroid administration for treatment for prevention of GVHD . The data indicate that, given enough time after transplantation, the ability to produce normal antibody function recovers except in those patients experiencing chronic GVHD. Gene, 1981 Aug, 14(3), 155 - 63 The nucleotide sequence and protein-coding capability of the transposable element IS5; Engler JA et al.; The nucleotide sequence of IS5, a bacterial insertion sequence, has been determined . It is 1195 bp long and contains an inverted terminal repetition of 16 bp with one mismatch . One open reading frame, spanning nearly the entire length of the element, could encode a polypeptide of 338 amino acids . Upon insertion into a DNA segment, IS5 causes a duplication of 4 bp . Based on seven examples, this site of insertion appears to be nonrandom, and the consensus target site sequence is C . T/A . A . G/A (or C/T . T . A/T . G on the opposite strand) . The nucleotide sequences of IS5 insertions into the B and cim genes of bacteriophage Mu have allowed tentative identification of the protein-coding frames of B and cim. J Virol, 1981 Aug, 39(2), 548 - 58 Partial purification and properties of an exonuclease inhibitor induced by bacteriophage Mu-1; Williams JG et al.; From an induced lysogen of bacteriophage Mu-1, we partially purified a substance of high molecular weight that blocks the action of several exonucleases on double-stranded DNA . The presence of the inhibitor in cell-free extracts is dependent on induction of a Mu prophage . The Mu-related inhibitor acts by binding to double-stranded DNA rather than by interacting with the DNase . The inhibitor protects linear duplex DNA of Mu, P22, and phi X174am3 from exonucleolytic degradation by recBC DNase and lambda exonuclease . Single-stranded DNA, however, is not protected by the inhibitor from degradation by either recBC DNase or exonuclease I . The inhibitor preparation contains a protein that binds to linear duplex DNA, but not to circular duplex DNA; ends are required for binding to occur . Single-stranded DNA is not a substrate for the binding protein . These and other results suggest that the binding protein and the inhibitor are the same activity. J Bacteriol, 1981 Aug, 147(2), 612 - 21 Structural studies of lambda transducing bacteriophage carrying bacterial deoxyribonucleic acid from the metBJLF region of the Escherichia coli chromosome; Krueger JH et al.; The structures of several lambda dmet and related lambda darg transducing phage were studied by restriction fragment mapping and electron microscopic measurements of homoduplexes and heteroduplexes . A new transducing phage (lambda dmet141), in which metF is the only functional gene of the cluster, was isolated . In contrast, lambda dmet117, which expresses the entire metBJLF cluster, has only 3 kilobases more bacterial deoxyribonucleic acid (DNA) than lambda dmet141 . An EcoRI restriction fragment of lambda dmet117, which carries the leftmost 6 kilobases of the bacterial DNA insert, was isolated and shown to contain a functional copy of metB . Small structural differences at the attachment sites of some of the phage were shown to result from different sites of lambda integration in the two parent insertion lysogens. J Histochem Cytochem, 1981 Aug, 29(8), 959 - 68 Mithramycin- and 4'-6-diamidino-2-phenylindole (DAPI)-DNA staining for fluorescence microspectrophotometric measurement of DNA in nuclei, plastids, and virus particles; Coleman AW et al.; The properties of two DNA-specific fluorochromes, 4'-6-diamidino-2-phenylindole (DAPI) and mithramycin, have been analyzed as reagents to quantitate cellular DNA by fluorescence microspectrophotometry . Optimal staining conditions and concentrations, and the effects of other cellular materials to which the dyes bind, have been evaluated in measurements of the DNA of rat, chick, and yeast nuclei, Gonyostomum chloroplasts, and T4 particles . Use of either fluorochrome permits a high degree of resolution of different DNA quantities in nuclei and in cell organelles, and the DAPI-DNA complex is sufficiently fluorescent to permit quantitation of the DNA content in genomes as small as those of individual T4 bacteriophage particles . Fluorescence of mithramycin- or DAPI-stained DNA is proportional to DNA quantity when DNA of the same has composition is compared . Quantitation does not appear to be affected discernably by the state of the DNA, whether in different stages of the cell cycle, in condensed chromosomes, or in noncycling, differentiated nuclei . The use of chicken red blood cells is recommended as an internal monitor for variations in staining conditions. J Biol Chem, 1981 Jul 25, 256(14), 7097 - 100 Evidence for the absence of DNA proofreading in HeLa cell nuclei; Wang ML et al.; {3H}2-Aminopurine deoxyribonucleoside triphosphate and {32P}dATP were added exogenously at equimolar concentrations to washed HeLa cell nuclei both in the presence and absence of cell cytoplasm . The observed ratio of 2-aminopurine/adenine deoxyribonucleotide incorporation into DNA was about 12%, which is consistent with 2-aminopurine misinsertion frequencies measured in cell-free assays, for various DNA polymerases including alpha-polymerase from calf thymus, Escherichia coli polymerase I, and several mutant and wild type bacteriophage T4 polymerases . Based on the 12% 2-aminopurine/adenine misincorporation ratio, we propose that proofreading of replicating DNA is not occurring in HeLa nuclei, and that discrimination against 2-aminopurine incorporation is governed primarily by a 1.1 kcal/mol difference in free energy between 2-aminopurine.thymine and adenine.thymine base pairs rather than by properties attributable to either the mammalian DNA polymerase or HeLa cell nuclear replication apparatus. Nucleic Acids Res, 1981 Jul 24, 9(14), 3335 - 54 Construction and characterization of recombinant plasmid DNAs containing sequences of the origin of bacteriophage phi X174 DNA replication; Heidekamp F et al.; The synthetic DNA fragment (formula, see text) (corresponding to nucleotides 4299-4314 of the phi X DNA sequence) was cloned into either the AmpR gene or the KmR gene of plasmid pACYC 177 . The DNA sequence of the KmR gene around the insertion site was determined by nucleotide sequence analysis of the pACYC 177 FnudII restriction DNA fragment N6 (345 b.p.) . Of five selected plasmid DNAs, which contained inserted DNA sequences in the antibiotic resistance genes, the nucleotide sequences at and around these insertions were determined . Two recombinant plasmids (pFH 704 and pFH 614) contain the hexadecamer sequence in tandem (tail-to-tail and tail-to-head) . In the recombinant plasmids pFH 812, pFH 903 and pFH 807 the DNA sequence homology with the phi X origin region was 14 (No . 4300-4313), 16 (No . 4299-4314) and 20 nucleotides (No . 4299-4318), respectively . None of the supercoiled recombinant plasmid DNAs is nicked upon incubation with phi X gene A protein . Moreover, the recombinant plasmid RFI DNAs cannot act as substitutes for phi X RFI DNA in the in vitro (+) strand synthesizing system . It has been shown earlier that single-stranded DNA, which contains the decamer sequence CAACTTGATA is efficiently nicked by the phi X gene A protein . The present results indicate that for nicking of double-stranded supercoiled DNA nucleotide sequence homology with the phi X origin region of more than 20 nucleotides is required . These results suggest a model for initiation of phi X RF DNA replication, which involves the presence of the recognition sequence CAACTTGATA of the phi X gene A protein as well as a second specific nucleotide sequence which is required for the binding of the phi X gene A protein . This binding causes local unwinding of the DNA double helix and exposure of the recognition sequence in a single-stranded form, which then can be nicked by phi X gene A protein. Nature, 1981 Jul 16, 292(5820), 215 - 20 Termination of transcription by nusA gene protein of Escherichia coli; Greenblatt J et al.; The nusA gene protein of Escherichia coli and N gene protein of bacteriophage lambda interact in vitro and cooperate in vivo to prevent transcription termination . In vitro the nusA gene protein causes RNA polymerase to pause in the tR2 terminator region of lambda DNA . A completed termination event at tR2 requires both the nusA gene protein and the previously described E . coli termination factor rho . The nusA gene protein is therefore both a transcription termination factor and a protein which couples antitermination factors to the elongating transcription complex. Nature, 1981 Jul 16, 292(5820), 212 - 5 The nus mutations affect transcription termination in Escherichia coli; Ward DF et al.; The nusA1 and nusB5 mutations in a partial suppression of polarity and thus transcription termination in Escherichia coli . As these mutations block the transcription antitermination activity of bacteriophage lambda N gene product, they paradoxically seem to enhance transcription termination at phage termination sites . The rho mutation HDF026 displays almost identical properties . The observations suggest that the nusA and nusB gene products may act as termination factors analogous to rho protein. Nature, 1981 Jul 9, 292(5819), 128 - 32 Purified lambda regulatory protein cII positively activates promoters for lysogenic development; Simatake H et al.; The bacteriophage lambda regulatory protein, cII, has been purified and shown to activate positively RNA transcription from the two phage promoters which coordinately regulate phage lysogenic development . To obtain this protein, the cII gene was cloned into a plasmid vector carrying the strong, regulatable lambda phage promoter PL such that it was overproduced to levels approaching 5% of cellular protein. Hoppe Seylers Z Physiol Chem, 1981 Jul, 362(7), 969 - 81 Relationship between the purity and molecular weight of calf thymus DNA; Welsh RS et al.; The molecular weight and purity of calf thymus DNA, prepared by different procedures of isolation, were determined . The highest molecular weight was obtained by the method of Blin and Stafford (12 x 10(7)) . This DNA contained 17% protein . The further purification of this DNA led both to increase of purity and to reduction of the molecular weight . The relationship between molecular weight and achieved purity was found to be a monotonic sigmoid-shape function ranging between the molecular weights of 12 x 10(7) and 12 x 10(6) and between the purities of 17 and 0.7% protein . After achieving this highest level of purity (0.7% protein content), despite repetition of the purification cycles, no further increase of purity or reduction of the molecular weight was achieved . The use of {14C}T4 bacteriophage DNA as internal standard demonstrated that the reduction of the molecular weight does not result from an artifact, such as shear or enzymatic degradation, but that the high-molecular-weight calf thymus DNA preparation must be considered to consist of either aggregates of small subunits stabilized by laterally attached proteins or of tandemly joined units connected by protein or peptide linkers. Farmaco {Sci}, 1981 Jul, 36(7), 629 - 47 Carbomethoxy-derivatives of psoralen: interactions with DNA and photobiological properties; Marciani Magno S et al.; The dark and photochemical interactions with DNA in vitro as well as the photobiological properties of two psoralen derivatives having a carbomethoxy-group inserted in 3 or 5' position of the furocoumarin nucleus were studied . 3-Carbomethoxy-4',8-dimethylpsoralen photoreacts with DNA in vitro to a very small extent and, as a consequence, it appears to be photobiologically ineffective . On the contrary, 5'-carbomethoxy-4,8-dimethylpsoralen appears very interesting, showing a photobinding and cross-linking capacity with DNA in vitro higher than that of 8-MOP . A similarly higher photobiological activity was also demonstrated, with respect to this reference compound, in experiments on inhibition of DNA and RNA synthesis in Ehrlich ascites tumor cells, and on the killing of bacteria and of T2 bacteriophage . Finally, this compound inhibited the tumor transmitting capacity of Ehrlich ascites tumor cells. J Invest Dermatol, 1981 Jul, 77(1), 39 - 44 Psoralen photochemistry and nucleic acid structure; Hearst JE; Many new psoralen derivatives have been synthesized in an effort to enhance their water solubility and their binding to nucleic acids . Availability of the very soluble strongly binding compounds has improved our abilities to follow the optical changes associated with the photochemistry of psoralens with DNA . Changes in both absorbance and fluorescence are presented in this review . A kinetic model for the photochemistry concludes that the detailed kinetics is dominated by the equilibrium constant for intercalation of the psoralen in the DNA, the quantum yield for photoaddition to DNA once intercalated and the quantum yield for photodestruction of the drug in water . With these 3 parameters the kinetics of photochemistry is predictable . The values of these parameters for numerous derivatives of 8-methoxypsoralen and 4,5',8 trimethylpsoralen are presented . Application of this photochemistry to a study of nucleic acid secondary structure in chromatin, fd bacteriophage, and in ribosomes is reviewed. Clin Chem, 1981 Jul, 27(7), 1157 - 64 Alternatives to radioimmunoassay: labels and methods; Schall RF Jr et al.; We review the following labels used as substitutes for radioisotopes in immunoassay systems: bacteriophages, chemiluminescence precursors, fluorochromes, fluorogens, fluorescence quenchers, enzymes, coenzymes, inhibitors, substrates, various particulates, metal atoms, and stable free radicals . New methods for performing immunoassays with these labels are described where appropriate . Methods that require no separation steps and offer special promise for easy automation are noted. Proc Natl Acad Sci U S A, 1981 Jul, 78(7), 4107 - 11 Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins: requirement for T7 RNA polymerase; Romano LJ et al.; The primary origin of bacteriophage T7 DNA replication is located 15% of the distance from the left end of the T7 DNA molecule . This intergenic segment is A + T-rich, contains a single gene 4 protein recognition site, and is preceded by two tandem promoters for T7 RNA polymerase {RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6} . Analysis by electron microscopy shows that T7 DNA polymerase {DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7} and gene 4 protein initiate DNA synthesis at randomly located nicks on duplex DNA to produce branched molecules . However, upon the addition of T7 RNA polymerase and ribonucleoside triphosphates 14% of the product molecules have replication bubbles, all of which are located near the primary origin observed in vivo; no such initiation occurs on T7 deletion mutant LG37 DNA, which lacks the primary origin . We have also studied initiation by using plasmids into which fragments of T7 DNA have been inserted . DNA synthesis on these templates is also dependent on the presence of T7 RNA polymerase and ribonucleoside triphosphates . DNA synthesis is specific for plasmids containing the primary origin, provided they are first converted to linear forms. J Virol, 1981 Jul, 39(1), 60 - 6 Leakage induced in Escherichia coli cells by A protein-RNA complexes from bacteriophage f2; Cody JD et al.; Complexes of f2 phage RNA and its A protein, or maturation protein, transfect Escherichia coli cells much better than does protein-free RNA . We used these complexes to introduce the bacteriophage f2 lysis gene into cells . The A protein-RNA complex was found to kill cells, probably by causing them to leak large macromolecules . Previously induced beta-galactosidase leaked from cells treated either with the A protein-RNA complex or with lethal but noninfectious complexes that had been treated with formaldehyde . This observation was consistent with an earlier finding that formaldehyde-treated f2 RNA stimulates the in vitro synthesis of a lysis protein . The complexes did not stimulate the rate of leakage of beta-galactosidase from a streptomycin-resistant mutant known to be lysis defective . On the other hand, the rate of leakage was increased in a double mutant resistant to both streptomycin and rifampin and which is lysed normally by f2 bacteriophage. Proc Natl Acad Sci U S A, 1981 Jul, 78(7), 4475 - 9 Retroregulation of the int gene of bacteriophage lambda: control of translation completion; Schindler D et al.; Bacteriophage lambda regulates the integration--excision reaction as a crucial aspect of the choice of pathway during lysogenic or lytic viral development . This control involves differential expression of the tightly linked, partially overlapping int and xis genes from two promoter sites: pI, positively regulated by cII/cIII proteins, and pL, positively regulated by N protein . After lambda infection, Int is synthesized from the pI transcript under cII regulation; however, very little Int is produced from the pL RNA because of the existence of a cis-acting regulatory element, sib, on the opposite side of the int gene from the pL promoter . Presumably sib serves to prevent unwanted synthesis of Int protein during the lytic response; the Int protein necessary for excisive recombination from a prophage can be supplied by pL transcription because sib is separated from int by prophage insertion . We have studied the effect of sib on nearby lambda genes by means of gel electrophoresis of labeled proteins from infected cells . Deletion of the sib region greatly enhances production of Int protein without substantial effect on Xis production; thus, sib regulation normally is highly specific for Int . When the sib region is moved past int and xis by deletion, regulation of the adjacent gene for the protein Ea22 occurs, suggesting that sib regulation can work on other genes . Although synthesis of wild-type Int is severely inhibited by sib, shorter Int protein fragments generated by nonsense mutations escape sib regulation, indicating that the regulation is translational and occurs near the completion stage of protein synthesis . Regulation by sib thus exhibits novel regulatory features: distal location, recombinational control, and regulation of the completion of protein synthesis . Because Int and Ea22 control is lost in a RNase III- host, we suggest that sib regulation might involve RNase III cleavage of a RNA duplex region that includes sib and the regulatory target (normally the int gene) . We note such a potential site within int. Proc Natl Acad Sci U S A, 1981 Jul, 78(7), 4251 - 5 DNA polymerase accuracy and spontaneous mutation rates: frequencies of purine.purine, purine.pyrimidine, and pyrimidine.pyrimidine mismatches during DNA replication; Fersht AR et al.; DNA from the am16 mutant of bacteriophage phi X174 may be replicated in vitro and expressed in vivo to give five classes of revertants . Each class may be specifically induced by the appropriate biasing of the concentrations of deoxynucleoside triphosphates in a predictable manner . The frequency of each reversion follows a kinetic rate equation relating it to the concentrations of the triphosphates involved in the substitution . The reversions corresponding to TAG leads to GAG, AAG, CAG, TGG, and TCG are calculated to occur with frequencies of 5 X 10(-7), 4 X 10(-7), 4 X 10(-7), approximately 2 X 10(-7), and approximately 5 X 10(-9), respectively, at the concentration of triphosphates found in vivo . The frequencies are in the range found for the reversion of the phage in vivo and so are consistent with errors in nucleotide selection by DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) III being largely responsible for the rate of spontaneous mutation in vivo . The relative frequency of mispairing leading to misincorporation is: purine.purine approximately purine.pyrimidine much greater than pyrimidine.pyrimidine, confirming predictions from model-building studies that transversions arise through purine.purine mismatches. J Virol, 1981 Jul, 39(1), 31 - 45 Genetic complementation by cloned bacteriophage T4 late genes; Jacobs KA et al.; Bacteriophage T4 containing nonsense mutations in late genes was found to be genetically complemented by four conjugate T4 genes (7, 11, 23, or 24) located on plasmid or phage vectors . Complementation was at a very low level unless the infecting phage carried a denB mutation (which abolishes T4 DNA endonuclease IV activity) . In most experiments, the infecting phage also had a denA mutation, which abolishes T4 DNA endonuclease II activity . Mutations in the alc/unf gene (which allow dCMP-containing T4 late genes to be expressed) further increased complementation efficiency . Most of the alc/unf mutant phage strains used for these experiments were constructed to incorporate a gene 56 mutation, which blocks dCTP breakdown and allows replication to generate dCMP-containing T4 DNA . Effects of the alc/unf:56 mutant combination on complementation efficiency varied among the different T4 late genes . Despite regions of homology, ranging from 2 to 14 kilobase pairs, between cloned T4 genes and infecting genomes, the rate of formation of recombinants after T4 den:alc phage infection was generally low (higher for two mutants in gene 23, lower for mutants in gene 7 and 11) . More significantly, when gene 23 complementation had to be preceded by recombination, the complementation efficiency was drastically reduced . We conclude that high complementation efficiency of cloned T4 late genes need not depend on prior complete breakage-reunion events which transpose those genes from the resident plasmid to a late promoter on the infecting T4 genome . The presence of the intact gene 23 on plasmids reduced the yield of T4 phage . The magnitude of this negative complementation effect varied in different plasmids; in the extreme case (plasmid pLA3), an almost 10-fold reduction of yield was observed . The cells can thus be said to have been made partly nonpermissive for this lytic virus by incorporating a part of the viral genome. Cell, 1981 Jul, 25(1), 269 - 76 Multilevel regulation of bacteriophage lambda lysogeny by the E . coli himA gene; Miller HI; Previous experiments have shown that the himA gene of E . coli specifies a protein that is required for bacteriophage lambda integration . lambda Forms clear plaques on himA mutants indicating a possible additional defect in the establishment of repression . We have tested the effects of a himA mutation on the establishment and maintenance of lambda repressor (cl) synthesis and on the synthesis of Int protein . The rate of synthesis of cl and Int after infection by lambda is severely reduced in a strain carrying a himA gene deletion . Synthesis of Int or repressor can occur in the himA- strain if the phage carry constitutive promoters for either the int gene or the cl gene . Maintenance of repression is unaffected by himA mutations as judged by repressor-stimulated transcription of PM fused to the lacZ gene . These results indicate that the himA gene participates in the regulation of the promoter sites specific of the establishment of lysogeny: PE for cl synthesis and PI, for Int production . Since the himA gene product is required also for lambda site-specific recombination, it appears that the himA gene regulates lambda lysogeny at several levels . I discuss the significance of this multilevel regulation to lambda development. Proc Natl Acad Sci U S A, 1981 Jul, 78(7), 4274 - 8 Sequences of the ssb gene and protein; Sancar A et al.; We have determined the sequences of the ssb gene and protein of Escherichia coli . The coding region of ssb is 534 base pairs and is preceeded and followed by dyad symmetries of 39 base pairs and 27 base pairs, respectively . The promoter for ssb is close to that for uvrA and these two genes are transcribed in opposite directions: ssb clockwise and uvrA counterclockwise on the standard E . coli genetic map . The DNA helix-destabilizing protein encoded by the ssb gene (single-strand binding protein) contains 177 amino acids and has a calculated molecular weight of 18,873 . Although there is no extensive sequence homology among the three helix-destabilizing proteins whose sequences are now known, both the E . coli and bacteriophage T4 DNA helix-destabilizing proteins do contain an acidic, alpha-helical region at their carboxy termini that may be functionally homologous . The remainder of the E . coli helix-destabilizing protein can be divided into two apparent domains on the basis of its amino acid sequence . The amino-terminal region (residues 1-105) contains 79% of the charged residues (27 out of 34 total) in the protein and is predicted to have a high degree of secondary structure (alpha helix and beta pleated sheet) . In contrast, the region including residues 106-165 contains only two charged amino acids and is devoid of alpha helix or beta pleated sheet. Proc Natl Acad Sci U S A, 1981 Jul, 78(7), 4036 - 40 Recombinant bacteriophages containing the integrated transforming provirus of Gardner--Arnstein feline sarcoma virus; Fedele LA et al.; The integrated DNA provirus of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) was molecularly cloned in a bacteriophage lambda vector . The cloned DNA fragment is 14.4 kilobase pairs long and contains a 6.7-kilobase provirus flanked by cellular sequences derived from nonproductively transformed mink cells . Transfection of mouse NIH/3T3 cells with the cloned DNA fragment induced foci of transformation at efficiencies of 10(4) focus-forming units/pmol of sarcoma virus DNA . Restriction endonuclease mapping and heteroduplex analyses were used to compare the GA-FeSV provirus with that of Snyder-Theilen (ST)-FeSV, a second strain that contains homologous transformation-specific sequences (v-fes) . Both viruses have the general structure 5'-gag-fes-env-c region-3', each having retained portions of the feline leukemia virus (FeLV) gag and env genes . In addition to segments shared by the two sarcoma viruses, GA-FeSV contains 1.7 kilobases of extra sequences not found in ST-FeSV . Of these, at least 400-500 base pairs located near the 5' end of v-fes encode a portion of the GA-FeSV polyprotein; the remaining 1.2 kilobases are derived from the FeLV env gene but do not appear to encode any detectable product related to the FeLV envelope glycoprotein . The close homology of the v-fes sequences shows that GA- and ST-FeSV were formed by recombination of FeLV with similar portions of a cat cellular gene (c-fes). Mol Biol (Mosk), 1981 Jul-Aug, 15(4), 883 - 93 {Restriction mapping of T4 bacteriophage late gene region which contains the origins of DNA replication}; Krutilina AI et al.; DNAs of lambda T4 recombinants 596-27 (genes 50-5), 596-30 (genes 50-8), 596-29 (genes 50-12), 591-16 (genes 6-8), 591-1 (genes 9-12), 596-13 (genes 13-16), 596-17 (genes 18-20) and 596-11 (genes 25-29) were mapped with the use of EcoRI, HindIII, SmaI, SalI and BamHI restriction enzymes . T4 dcDNA was digested with HindIII restriction endonuclease and resulting fragments were cloned into HindIII lambda vector 761 . The recombinants 761-7, 761-17, 761-19, 761-24, 761-44, 761-50, 761-55 contained the region of genes 25-48 and 761-42, 761-26 and 761-16 contained a single HindIII-fragment with genes 6-12 in both orientations . Data obtained with the DNA of the latter recombinants allowed to show the correctness of the map established earlier which did not contain a full set of overlapping sequences . As a result of the experiments reported, the position of EcoRI and HindIII recognition sites in the region of genes 50-20 and 25-48 was determined and in the region of genes 25-48 BglII and XhoI restriction sites were mapped . The location of a single BamHI restriction site in the region of gene 8 was also established. Nucleic Acids Res, 1981 Jun 25, 9(12), 2791 - 800 Mapping of in vitro transcription units and identification of primary transcripts of the D region of bacteriophage T4; Goldfarb A et al.; The D region of bacteriophage T4 is comprised of six closely linked genes which are situated between 161 kb and 165 kb on the T4 chromosome . We studied the transcription of these genes in vitro by using DNA templates derived from a series of deletion mutants in this region . The mixture of primary products made by Escherichia coli RNA polymerase were fractionated by gel electrophoresis into discrete RNA species . The results obtained together with the known map positions of the deletions allowed to identify four wild-type and several deletion-specific transcripts of the D region . The end points of these transcripts were approximately mapped . The results demonstrate that the D region has two promoters and two terminators, an organisation which is similar to the previously established organisation of the T4 tRNA gene cluster. Nucleic Acids Res, 1981 Jun 11, 9(11), 2509 - 15 Restriction enzyme digestion of hemimethylated DNA; Gruenbaum Y et al.; Hemimethylated duplex DNA of the bacteriophage phi X 174 was synthesized using primed repair synthesis is in vitro with E . coli DNA polymerase I followed by ligation to produce the covalently closed circular duplex (RFI) . Single-stranded phi X DNA was used as a template, a synthetic oligonucleotide as primer and 5-methyldeoxycytidine-5'-triphosphate (5mdCTP) was used in place of dCTP . The hemimethylated product was used as substrate for cleavage by various restriction enzymes . Out of the 17 enzymes tested, only 5 (BstN I, Taq I, Hinc II, Hinf I and Hpa I) cleaved the hemimethylated DNA . Two enzymes (Msp I and Hae III) were able to produce nicks on the unmethylated strand of the cleavage site . Msp I, which is known to cleave at CCGG when the internal cytosine residue is methylated, does not cleave when both cytosines are methylated . Another enzyme, Apy I, cleaves at the sequence CCTAGG when the internal cytosine is methylated, but is inactive on hemimethylated DNA in which both cytosines are methylated . Hemimethylated molecules should be useful for studying DNA methylation both in vivo and in vitro. J Biol Chem, 1981 Jun 10, 256(11), 5840 - 4 Transfer RNA cross-linked to the elongation factor Tu subunit of Q beta replicase does not inhibit Q beta RNA replication; Guerrier-Takada C et al.; One of the four subunits of bacteriophage Q beta RNA replicase is elongation factor Tu (EF-Tu), the host aminoacyl-tRNA (AA-tRNA) binding protein . To determine whether the RNA polymerase activity requires the tRNA binding site of EF-Tu, we reconstituted replicase with EF-Tu . GTP covalently bound to AA-tRNA . This cross-linked ternary complex (XLTC) was formed by the reaction of N epsilon-bromoacetyl-Lys-tRNA with EF-Tu-GTP . In an EF-Tu-dependent system for the reconstitution of replicase, XLTC restored polymerase activity at least as well as an equivalent amount of EF-Tu . Replicase reconstituted with XLTC was resolved from replicase containing EF-Tu by chromatography on phosphocellulose, a result which confirmed that the tRNA moiety was incorporated into the enzyme . Chromatographic analysis of reconstitution mixtures revealed that XLTC was incorporated into replicase as extensively as EF-Tu . From these results, it appears that the AA-tRNA binding site on EF-Tu is not required for the assembly or activity of Q beta RNA replicase . Furthermore, because the tRNA macromolecule is cross-linked to His-66 of the EF-Tu, the region surrounding His-66 must normally be exposed on the surface of the replicase. J Biol Chem, 1981 Jun 10, 256(11), 5810 - 3 Replication of phase fd RF with fd gene 2 protein and phage T4 enzymes; Meyer TF et al.; Bacteriophage fd replicative form DNA with a nick in the viral strand serves as a template for DNa replication with purified bacteriophage T4 enzymes . As anticipated from previous in vitro studies carried out with this system (Morris, C . F., Sinha, N . K., and Alberts, B . M . (1975) Proc . Natl . Acad . Sci . U.S.A . 72, 4800-4804), DNA is synthesized by a rolling circle mechanism . We show here that the DNA strands synthesized are processed by the phage fd gene 2 protein into unit length products, providing that the gene 2 protein is present at the moment when this DNA is made . The products are mostly unit length linear single strands, indicating that the circularization step normally catalyzed by gene 2 protein subsequent to its site-specific cleavage of an fd DNA strand occurs only inefficiently in this system . The gene 2 protein reduces the level of DNA synthesis by 2-fold at low concentrations, even though it only cleaves the DNA products efficiently at higher levels of the enzyme . This indicates that there are at least two different effects of the fd gene 2 protein in processing of viral fd DNA. Biochemistry, 1981 Jun 9, 20(12), 3586 - 91 5-{(Hydroxymethyl)-O-pyrophosphoryl}uracil, an intermediate in the biosynthesis of alpha-putrescinylthymine in deoxyribonucleic acid of bacteriophage phi W-14; Maltman KL et al.; In a nonpermissive host, an amber mutant, am 37, of bacteriophage phi W-14 synthesizes deoxyribonucleic acid (DNA) of considerably greater buoyant density than the DNA synthesized by wild-type phage . The am 37 DNA lacks the hypermodified pyrimidine, alpha-putrescinylthymine (putThy) . Instead, it contains a new modified base, 5-{(hydroxymethyl)-O-pyrophosphoryl}uracil (hmPPUra) . Extracts of cells infected with wild-type phi W-14 convert the hmPPUra in am 37 DNA to putThy when incubated with putrescine. Biochemistry, 1981 Jun 9, 20(12), 3579 - 85 Effects of (hydroxymethyl)trimethylpsoralen on structure and function of bacteriophage MS2 ribonucleic acid; Karathanasis SK et al.; Treatment of bacteriophage MS2 with 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen and 360-nm light caused a dose-dependent decline in the infectivity of the virus . Covalent photobinding of a single psoralen molecule on the phage genome was a lethal event . Ribonucleic acid (RNA) extracted from psoralen and light-treated virus had a dose-dependent 385-nm fluorescence emission but was unaltered in its physical properties compared to control RNA samples . Phage adsorption and penetration in Escherichia coli host cells were unaffected, but in vivo replication of the treated virus was affected to the same extent as infectivity . The cell-free translational activity of the MS2 RNA was also severely reduced after psoralen and light treatment of the phage . Examination of the in vitro translation products revealed that the synthesis of the viral replicase protein was most substantially affected . Psoralen treatment of purified, protein-free MS2 RNA promoted an even greater reduction in cell-free synthesis of all viral proteins . This difference in translational function was consistent with the observation that virion-free RNA bound approximately 4 times as much psoralen as did RNA treated within the phage capsid . It was concluded that the replicase gene is the most sensitive region of the viral RNA molecule for psoralen binding. Science, 1981 Jun 5, 212(4499), 1159 - 62 DNA sequence of two closely linked human leukocyte interferon genes; Lawn RM et al.; A single recombinant lambda bacteriophage isolated from a human genome library contains two closely related human interferon genes of the leukocyte or alpha type . The two genes are separated by 12 kilobase pairs and are oriented in the same direction with respect to transcription . Comparisons of the DNA sequences of these two genes and interferon complementary DNA clones indicate that the two interferon genes lack intervening sequences. Cell, 1981 Jun, 24(3), 669 - 77 Chromosomal arrangement of the chicken beta-type globin genes; Dolan M et al.; We have isolated the chicken beta-type globin genes from a library of chicken DNA--lambda Charon 4A recombinant bacteriophage . There are four beta-type genes within this segment of the genome; we believe this represents all of the beta-type genes of the chicken . The recombinant lambda C beta G1 contains the embryonic epsilon- and adult beta-globin genes . The hatching beta H- and embryonic rho-globin genes are found in the recombinant lambda C beta G2 . Although lambda C beta G1 and lambda C beta G2 do not physically overlap, we present evidence that all four genes are closely linked and transcribed from the same DNA strand . These experiments demonstrate that the chromosomal regions represented by lambda C beta G1 and lambda C beta G2 lie approximately 1.6 kb apart in the chicken genome . A third recombinant lambda C beta G3 extends the genomic locus studied in the vicinity of the beta-type globin genes to approximately 39 kb . The physical order of the chicken beta-type globin genes within this segment of the chromosome is 5' .. . rho--beta H--beta--epsilon .. . 3' . This arrangement is unique among the vertebrate beta-type globin gene clusters thus far examined, in that embryonic genes are located at the 5' and 3' ends of the cluster while the hatching and adult genes occupy central positions. Proc Natl Acad Sci U S A, 1981 Jun, 78(6), 3454 - 8 Changes in the promoter range of RNA polymerase resulting from bacteriophage T4-induced modification of core enzyme; Goldfarb A; Primary transcripts made in vitro on bacteriophage T4 DNA by RNA polymerase isolated from normal or T4-infected Escherichia coli were compared by gel electrophoresis . Bacteriophage-modified RNA polymerase fails to initiate transcription at certain promoters recognized by unmodified enzyme . In the T4tRNA gene region, only one of the two promoters is active with the modified RNA polymerase . Reconstitution of separated RNA polymerase components demonstrates that this change in promoter site selection results from the modification of core enzyme and not sigma factor. Proc Natl Acad Sci U S A, 1981 Jun, 78(6), 3433 - 7 recA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange; Cox MM et al.; The recA protein of Escherichia coli promotes the complete exchange of strands between full-length linear duplex and single-stranded circular DNA molecules of bacteriophage phi X-174, converting more than 50% of the single-stranded DNA into heteroduplex replicative form II-like structures . Kinetically, the reaction can be divided into two phases, formation of short heteroduplex regions (D loops) and extension of the D loops via branch migration . recA protein participates directly in both phases . D loops are formed efficiently in the presence of ATP or the nonhydrolyzable ATP analog adenosine 5'-{gamma-thio}triphosphate, whereas D-loop extension requires continuous ATP hydrolysis . Complete strand exchange requires a stoichiometric amount of recA protein and is strongly stimulated by the single-stranded-DNA-binding protein of E . coli. Eur J Biochem, 1981 Jun, 117(1), 1 - 6 Molecular properties of two mutant species of the elongation factor Tu; Van der Meide PH et al.; The molecular properties of two mutant species of the elongation factor Tu (EF-Tu), derived from either tuf A or tuf B, have been studied . One, designated EF-TuAR, is the product of a kirromycin-resistant tufA gene . The other designated EF-TuBO is a tuf B product and is present in a kirromycin-resistant mutant of Escherichia coli (LBE 2012) also harbouring the EF-TuAR species . EF-TuAR has been isolated in homogeneous form as a single gene product from the mutant strain LBE 2045, in which the tuf B gene has been inactivated by an insertion of the bacteriophage Mu . EF-TuBO has been isolated from LBE 2012 together with EF-TuAR in a 1:1 mixture . Fractionation of this mixture of DEAE-Sephadex A-50 resulted in an enrichment of EF-TuBO of about 80% . The properties of EF-TuAR and EF-TuBO have been compared to those of a kirromycin-sensitive species designated EF-TuAS, which was isolated from LBE 2045 by transduction of wild-type tuf A . We show here that all three EF-Tu species are fully competent to sustain polypeptide synthesis . All also appear to interact normally with guanine nucleotides and EF-Ts . Only in the presence of the antibiotic do the following differences appear . (a) Kirromycin causes EF-TuAS (wild-type tuf A gene product) to be retained on, and thus block, the ribosome . (b) EF-TuAR fails to bind the antibiotic and thus is capable of protein synthesis in its presence . (c) EF-TuBO fails to sustain polypeptide synthesis upon binding of kirromycin . It does not, however, block the ribosome, so the strain harbouring both this protein and EF-TuAR (LBE 2012) is kirromycin resistant. J Virol, 1981 Jun, 38(3), 833 - 9 Escherichia coli traD(Ts) mutant temperature sensitive for assembly of RNA bacteriophage MS2; Schoulaker-Schwarz R et al.; We report here a study on the temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39, carrying a traD(Ts) mutation, which is also temperature sensitive for group I RNA phages (MS2, f2, and R17) . It is shown that, when the mutant was infected with MS2 at 42 degrees C, phage RNA replicated; a 27S MS2 RNA and phage proteins were synthesized . However, neither PFU nor physical MS2 particles were formed, showing that phage assembly was inhibited . In addition, the high temperature affected the membranes of the host mutant: the mutant was hypersensitive to chemicals, and the electrophoretic pattern of the membranal proteins was modified . We suggest that the pleiotropic effects of the traD mutation on MS2 assembly and DNA transfer during conjugation were a result of the changes in the membrane of the mutant. Proc Natl Acad Sci U S A, 1981 Jun, 78(6), 3428 - 32 Resolution of cointegrates between transposons gamma delta and Tn3 defines the recombination site; Reed RR; Transposition of the genetically related insertion elements gamma delta and Tn3 is thought to involve two steps . In the case of transposition from one replicon to another, the first step is fusion of the parent and target replicons with the element appearing in direct orientation at the two junctions . In a subsequent reaction, the cointegrate structure is resolved via a site-specific recombination event . I have constructed two plasmids, each carrying segments of gamma delta and Tn3, that contain the internal resolution site . The tnpR gene product encoded by either Tn3 or gamma delta mediates intramolecular recombination between these two sites . The product of this recombination is a hybrid region that contains gamma delta and Tn3 sequences fused at the point of crossover . DNA sequence analysis of such recombinants indicates that the recombination occurs within a 19-base-pair (bp) region of exact homology between gamma delta and Tn3 . The site lies in the 160-bp center intercistronic region, 50 bp before the beginning of the tnpA gene . My results therefore suggest a model for the coupled regulation of the repressor (tnpR) and the transposase (tnpA) genes and site-specific recombination of transposition intermediates . The Tn3/gamma delta recombination system and bacteriophage lambda integration are compared. Proc Natl Acad Sci U S A, 1981 Jun, 78(6), 3328 - 32 Analysis of two divergent rat genomic clones homologous to the transforming gene of Harvey murine sarcoma virus; DeFeo D et al.; Harvey murine sarcoma virus (Ha-MuSV) is a mouse-rat recombinant retrovirus that encodes a protein designated p21, required for virally induced transformation . Using a radiolabeled DNA fragment from the p21 coding region, we have detected homologous DNA sequences in the normal DNA of rats and of several other vertebrate species . Moreover, many tested cells from these species contain low levels of a p21 protein that is highly related to viral 21 . Now we report two independent fragments from normal rat DNA containing sequences (sarc) homologous to the Ha-MuSV transforming region that were cloned in the bacteriophage vector Charon 4A . Sarc sequences in the one fragment are completely colinear with the viral sequences and share apparently all restriction endonuclease sites . Sarc sequences in the second fragment have several sets of intervening sequences and lack some restriction endonuclease sites found in the viral transforming region . Despite the presence of these intervening sequences in the second sarc fragment, we have been able to ligate this sarc fragment to the long terminal repeat sequence of HaMuSV and to induce cellular transformation and high levels of p21 expression upon transfection of this DNA to NIH 3T3 mouse cells . These results suggest that elevated levels of p21, normally expressed at low levels in a variety of cells, can induce cellular transformation. J Bacteriol, 1981 Jun, 146(3), 1170 - 3 dnaB125, a dnaB nonsense mutation; Sclafani RA et al.; A temperature-sensitive dnaB mutation, dnaB125, was shown to be a suppressed amber mutation . The effects of inserting different amino acids at the mutated site via amber suppressors were examined for both Escherichia coli and bacteriophage gamma growth . In addition, the dnaB125 amber allele was shown to be different from the previously described dnaB amber allele, dnaB266 . The extent of residual deoxyribonucleic acid synthesis observed in a supF(Ts) dnaB125 strain at high temperature revealed that the dnaB protein was present in excess and that deoxyribonucleic acid synthesis could continue for several generation equivalents without further production of dnaB protein. J Microsc, 1981 Jun, 122(Pt 3), 275 - 86 Comparative mass measurement of biological macromolecules by scanning transmission electron microscopy; Freeman R et al.; A method is described for measuring the mass/length or mass of molecular assemblies by comparative electron scattering in the STEM . Standard particles whose mass is well established (e.g . TMV or fd bacteriophage) are deposited on the electron microscope grid together with the sample to be measured . Images containing at least one sample and standard and with a clean, contamination-free background are chosen and stored on computer disc and then directly integrated . Use of a comparative technique does not require accurate determination of scattering parameters or instrumental geometry and requires only that the limits of linearity be established . The results of the mass/length measurements on phage pf 1, pili, muscle thick filaments and actin are in good agreement with existing molecular weight data and generally have a standard deviation of about 10% . The results for the total mass measurement of the multisubunit enzymes glutamate dehydrogenase and glutamine synthetase are also close to the literature values for their molecular weights . The results for the spherical, Semliki forest and tomato bushy stunt viruses are lower than expected, possibly reflecting some dissociation during preparation. Nucleic Acids Res, 1981 May 25, 9(10), 2281 - 95 Characterization of a cloned histone gene cluster of the newt Notophthalamus viridescens; Stephenson EC et al.; We report the cloning and characterization of a histone gene cluster of the newt Notophthalamus viridescens . Fragments containing newt histone genes were identified in whole genome Southern blots; these fragments were cloned into a bacteriophage lambda cloning vector constructed for this purpose . The positions of most of the histone genes were determined by hybridizing subcloned sea urchin histone genes to digests of the cloned newt gene cluster . The position of each gene was verified, and its polarity determined by sequencing a portion of each . The order of the genes in the cloned segment is H1-H3-H2B-H2A-H4, with each of the genes but H2B being transcribed in the same direction . Subcloned segments of the histone gene repeat were used to determine the size of each newt oocyte histone mRNA. Nucleic Acids Res, 1981 May 11, 9(9), 2187 - 94 The atp operon: nucleotide sequence of the region encoding the alpha-subunit of Escherichia coli ATP-synthase; Gay NJ et al.; Part of the atp (or unc) operon encoding the alpha, beta, gamma, delta, and epsilon subunits of Escherichia coli ATP-synthase has been cloned into the plasmid pACYC 184 . The DNA coding for the largest of these proteins, the alphas subunit, has been sequenced by cloning into the bacteriophage M13 and sequencing with dideoxy nucleotide chain terminators . It comprises 1539 nucleotides corresponding to a protein of 513 amino acids. Nucleic Acids Res, 1981 May 11, 9(9), 2173 - 85 Structure of the baboon endogenous virus genome: cloning of circular virus DNA in bacteriophage lambda; Noda M et al.; Linear, small and large circular forms of unintegrated viral DNAs were detected in Hirt supernatant fraction of human cultured cells infected with baboon endogenous virus M7 . The circular M7 DNAs were cloned in bacteriophage lambda, Charon 28 . Seventeen independent clones were isolated and analyzed by restriction endonuclease mapping . Nine clones were carrying a viral sequence of 8.6 kilobase pairs (kb) with two tandem repeats of 0.6 kb, which correspond to the large circular form of the unintegrated M7 DNA . Eight other clones had the viral insert of 8.0 kb, i . e., the small circular form, and were deleted one of the repeated sequences . The repeated sequences correspond to the long terminal repeats of 0.6 kb, located at both ends of the linear M7 DNA of 8.6 kb . One of the recombinants of the large circular M7 DNA had an inversion of 2.5 kb . One end of the inverted sequence was near the terminus of the long terminal repeats and the other in the gag gene region . The inversion seems to be occurred by integration of a viral DNA within itself during early periods of infection . The mechanism of the processes leading to integration is discussed from the structure of these unintegrated M7 DNAs as the precursors. Nucleic Acids Res, 1981 May 11, 9(9), 2037 - 53 Enzymatic properties of the bacteriophage phi X174 A protein on superhelical phi X174 DNA: a model for the termination of the rolling circle DNA replication; van der Ende A et al.; Incubation of phi X174 replication form I DNA with the A* protein of phi X174 in the presence of MN2+ results in the formation of three different types of DNA molecules: open circular form DNA (RFII), linear form DNA (RFIII) and the relaxed covalently closed form DNA (RFIV) . The RFII and RFIII DNAs are shown to be A* protein-DNA complexes by electron microscopy using the protein labeling technique of Wu and Davidson (1) . The linear double-stranded RFIII DNA molecule carries at one end a covalently attached A* protein whereas at the other end of the molecule the single-stranded termini are covalently linked to each other . The structure of the RFIII DNA shows its way of formation . The described properties of the A* protein indicate the way the larger A protein functions in the termination step of the rolling-circle type of phi X174 DNA replication. Cell, 1981 May, 24(2), 437 - 41 Membrane assembly: posttranslational insertion of M13 procoat protein into E . coli membranes and its proteolytic conversion to coat protein in vitro; Goodman JM et al.; The major coat protein (gene 8 product) of bacteriophage M13 is an integral membrane protein during infection of host cells . It is synthesized as a larger precursor (procoat) with a leader sequence of 23 amino acids at its amino terminus . In vivo studies have shown that procoat only inserts into the host-cell plasma membrane after its synthesis is completed . We now demonstrate that procoat can post-translationally insert into inverted cytoplasmic membrane vesicles from E . coli and can be processed proteolytically to yield coat protein . Procoat changes from an assembly-competent substrate to an incompetent (denatured) form within minutes after its synthesis; much of the procoat that accumulates during an hour of in vitro synthesis is therefore denatured . These studies emphasize the importance of stringent criteria for the demonstration of obligate cotranslational assembly. Mol Biol (Mosk), 1981 May-Jun, 15(3), 583 - 8 {Serological characterization of bacteriophage T3 carrying different "non-classical" modifications}; Gachechiladze KK et al.; The antigenic properties of bacteriophage T3 and a mutant T3/R7 which undergoes "non-classical" modification and restriction are compared . The "non-classical" modification of T3/R7 consists of a host-dependent, reversible change in the adsorption capacity of phage on different host strains . We have shown that this modification is connected with changes in the antigenic properties of phage components involved in phage absorption to the host cell . This means that, in contrast to the "classical" host-control-led modification and restriction of DNA, the "non-classical" modification and restriction of phage is based on protein modification. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2864 - 8 On the molecular basis of transition mutations: frequencies of forming 2-aminopurine.cytosine and adenine.cytosine base mispairs in vitro; Watanabe SM et al.; We address the question of whether substituting 2-aminopurine (APur) in place of adenine (Ade) in DNA can increase the frequency of base mispairing with cytosine . Using DNA polymerase alpha to measure the rates of inserting deoxycytidine and thymidine nucleotides in direct competition with each other for APur or Ade sites on synthetic copolymer DNA templates, we observe that the ratio of dCMP to dTMP insertion is increased by a factor of at least 230 when APur replaces Ade on a poly(dA) template and by a factor of 35 when APur replaces Ade on a poly(dC,dA) template . These data support the idea that APur.C base mispairs are directly involved in APur induction of A.T leads to G.C transition mutations . The observed misinsertion frequency of cytosine substituting for thymine opposite template APur sites is about 5% . This value is in excellent agreement with earlier predictions and measurements for APur.C heteroduplex-heterozygote frequencies in T4 bacteriophage in vivo. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2962 - 6 Conformational transitions in Pf3 and their implications for the structure and assembly of filamentous bacterial viruses; Thomas GJ Jr et al.; Laser Raman and circular dichroism spectra of filamentous bacteriophage Pf3 show that its coat protein is predominantly alpha-helical, similar to the subunits of bacteriophages Pf1 and fd . Unlike Pf1 and fd, however, the subunits of Pf3 are converted to beta-sheet structures by raising the temperature, the transition temperature depending upon phage and NaCl concentrations . On cooling, the beta structure reverts to an alpha structure the same as or similar to the native structure . On further heating it converts irreversibly to a second alpha-helical form different from the original one . The spectra also show that aromatic amino acid residues of Pf3 undergo dramatic changes in molecular environment during the alpha leads to beta transition . Similar transitions are observed to take place in the filamentous bacteriophage Xf. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2717 - 21 Alcohol dehydrogenase gene of Drosophila melanogaster: relationship of intervening sequences to functional domains in the protein; Benyajati C et al.; The gene that codes for Drosophila alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase EC 1.1.1.1) was identified in a bacteriophage lambda library of genomic Drosophila DNA by using ADH cDNA cloned DNA as a probe . The DNA sequence of the protein encoding region was shown to be in agreement with the amino acid sequence of the ADH . Two intervening DNA sequences (introns) were identified within the protein encoding region: one was 65 nucleotides and located between the codons for amino acid residues 32 and 33, and one was 70 nucleotides and located between the codons for amino acid residues 167 and 168 . Both contained the 5' G-T and 3' A-G dinucleotides characteristic of intron boundaries of eukaryotic genes . On the basis of secondary structure predictions, the first 140 amino acid residues of Drosophila ADH are in an alternating beta-sheet/alpha-helix arrangement which is characteristic of the coenzyme binding domain of dehydrogenases . The smaller of the two introns interrupts the domain predicted to bind the adenine portion of the coenzyme. Genetics, 1981 May, 98(1), 1 - 24 DNA rearrangements associated with reversion of bacteriophage Mu-induced mutations; Khatoon H et al.; Excision of transposable genetic elements from host DNA is different from the classical prophage lambda type of excision in that it occurs at low frequency and is mostly imprecise; only a minority of excision events restores the wild-type host sequences . In bacteriophage Mu, a highly efficient transposon, imprecise excision is 10-100 times more frequent than precise excision . We have examined a large number of these excision events by starting with mucts X mutants located in the Z gene of the lac operon of Escherichia coli . Mucts X mutants are defective prophages whose excision occurs at a measurable frequency . Imprecise excision was monitored by selecting for melibiose+ (Mel+) phenotype, which requires only a functioning lacY gene . Mel+ revertants exhibit an array of DNA rearrangements and fall in four main classes, the predominant one being comprised of revertants that have no detectable Mu DNA . Most of these revertants can further revert to Lac+ . Perhaps 5 base-pair duplications, originally present at prophage-host junctions, are left in these lacZ-Y+ revertants, and they can be further repaired to lacZ+ . Another class has, in addition to the loss of Mu DNA, deletions that extend generally, but not always, to only one side of the prophage . The other two classes of revertants, surprisingly, still have Mu DNA in the lacZ gene . One class has deletions in the Z gene, whereas, no deletions can be detected in the other . Many of the revertants in the last class can further revert to lacZ+, indicating that the lacY gene must have been turned on by a rearrangement within Mu DNA . Apparently, all of the detectable precise and most of the imprecise excision events require functioning of the Mu A gene . We suggest that a block in large-scale Mu replication allows the excision process to proceed. Cell, 1981 May, 24(2), 429 - 36 Structure of chi hotspots of generalized recombination; Smith GR et al.; Chi recombinational hotspots are sites around which the rate of Rec-promoted recombination in bacteriophage lambda is elevated . Examination of a derivative of lambda into which the plasmid pBR322 was inserted reveals that pBR322 lacks Chi sites . Using this lambda-pBR322 hybrid, we obtained mutations creating Chi sites at three widely separated loci within pBR322 . Nucleotide sequence analysis reveals that the mutations are single base-pair changes creating the octamer 5' GCTGGTGG 3' . This sequence is present at three previously analyzed Chi sites in lambda, and all analyzed mutations creating or inactivating these Chi sites occur within this octamer . We conclude that Chi is 5' GCTGGTGG 3', or its complement, or both. J Bacteriol, 1981 May, 146(2), 713 - 7 Identification of wild-type or mutant alleles of bacterial genes cloned on a bacteriophage lambda vector: isolation of uvrC(am) and other mutants; Auerbach JI et al.; We have identified lambda transducing bacteriophages carrying deoxyribonucleic acid repair or recombination genes of Escherichia coli K-12 by their ability to infect and express their bacterial genes in mutant cells in an agar overlay . This technique has been used to recognize transducing phages carrying uvrC+, ssb+, and other genes and to isolate phages carrying mutant alleles unable to complement ssb or uvrC cells . Several uvrC mutations were obtained which were suppressor sensitive. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2918 - 22 Molecular cloning of integrated simian sarcoma virus: genome organization of infectious DNA clones; Robbins KC et al.; The integrated form of simian sarcoma virus (SSV) was molecularly cloned in the Charon 16A strain of bacteriophage lambda . In transfection analysis, the recombinant viral DNAs demonstrated the ability to transform cells in tissue culture at high efficiency . Such transformants possessed typical SSV morphology, expressed simian sarcoma associated virus (SSAV) gag gene products in the absence of virus release, and released SSV after superinfection with a type C helper virus . A physical map of the 5.8-kilobase-pair (kbp) recombinant viral DNA clone, deduced from restriction endonuclease analysis, revealed a 5.1-kbp SSV genome containing 0.55-kbp-long terminal repeats flanked by 0.45 and 0.25 kbp of contiguous host cell sequences . By R-loop analysis, the viral DNA molecule contained two regions of homology to SSAV, separated by a 1.0-kbp nonhomologous region . This SSV-specific sequence was shown to be uniquely represented within the normal cellular DNA of diverse mammalian species, including human . Our results demonstrate that this primate transforming retrovirus arose in nature by recombination of a type C helper virus and a host cellular gene. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2883 - 7 DNA breakage and closure by rat liver type 1 topoisomerase: separation of the half-reactions by using a single-stranded DNA substrate; Been MD et al.; Circular single strands of bacteriophage phi X174 DNA are broken by rat liver DNA nicking-closing enzyme (type 1 topoisomerase) in low salt (50 mM KCl) at 37 degrees C, generating linear strands containing covalently bound enzyme {Been, M . D . & Champoux, J . J . (1980) Nucleic Acids Res . 8, 6129-6142} . The linear strands can be recircularized in the presence of 10 mM MgCl2 at 24 degrees C and 37 degrees C or 250 mM KCl at 24 degrees C . Recircularization is blocked when the hydroxyl group at the 5' terminus is phosphorylated . The linears generated by the nicking-closing enzyme can also be joined to other DNA fragments containing 5' hydroxyls, but not 5' phosphates . The linkage formed in both the intrastrand and interstrand reactions is stable to alkali . Reclosure of broken single strands is presumed to be analogous to the closure step that occurs durng nicking and closing cycles on duplex DNA. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2796 - 800 Expression of the denV gene of bacteriophage T4 cloned in Escherichia coli; Lloyd RS et al.; The denV gene of bacteriophage T4 has been cloned into Escherichia coli K-12 by inserting appropriate fragments of cytosine-containing T4 DNA into the Sal I site of the plasmid pBR322 . The denV gene codes for an enzyme that initiates the excision repair of pyrimidine dimers produced in DNA by UV . In uvrA recA mutants, deficient in an early step in excision repair, the cloned DNA results in enhanced UV resistance that is more pronounced in stationary- than in exponential-phase cultures . The expression of the cloned DNA also results in the enhanced survival of UV-irradiated phage lambda or of a denV mutant of phage T4 and in removal of dimers from the DNA of UV-irradiated cells. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2742 - 6 Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA; Nakabeppu Y et al.; T4 endonuclease V {endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1)}, which is involved in repair of UV-damaged DNA, has been purified to apparent physical homogeneity . Incubation of UV-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer . The activity to produce alkali-labile sites was optimal in a relatively broad pH range (pH 6.0-8.5), whereas the activity to form nicks had a narrow optimum near pH 6.5 . By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks . The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity {endodeoxyribonuclease (apurinic or apyrimidinic, EC 2.1.25.2} . The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide . An enzyme fraction from cells infected with bacteriophage T4v1, a mutant that is sensitive to UV radiation, was defective in both glycosylase and endonuclease activities . Moreover, occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities, and suppression of the mutation rendered both activities partially active . These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4 . Because the two activities exhibited different thermosensitivity, it was further suggested that conformation of the active sites for these activities may be different. Mol Biol (Mosk), 1981 May-Jun, 15(3), 538 - 46 {Cloning fragments of bacteriophage T5 DNA, determining expression of phage-dependent ligase}; Mel'nikov AA et al.; Cloning vector lambda gt-p MB9 has been used for cloning of DNA fragments of bacteriophage T5 produced by EcoR*I activity . One clone contains a DNA fragment of 2.2 Md which has been mapped at 67-71% on the physical map of the genome . Functional studies have shown that bacteriophage lambda gt-T5 can grow on E . coli lights 7 . Infection of this E.coli strain with phage lambda gt-T5 induces DNA-ligase activity which has been previously observed in E . Coli infected with bacteriophage T5. J Virol, 1981 May, 38(2), 621 - 31 Restriction alleviation by bacteriophages lambda and lambda reverse; Toothman P; Deletion analysis indicated that the phage lambda restriction alleviation gene(s) ral resides between the cIII and N genes . The Ral+ phenotype was expressed only when lambda ral+ carried a modification such that it was resistant to restriction by the host specificity system . Under these conditions, Ral function protected superinfecting unmodified phages from restriction by EcoK or EcoB but not from restriction by EcoP1 . Ral-protected phage DNA was not concomitantly K and B modified, but rather received only the modification specified by the system of the restricting host . Possible mechanisms for Ral action are discussed . Of the other lambdoid phages tested, the hybrid phage lambda rev had Ral activity, whereas phi 80vir and one lambda-P22 hybrid did not . The restriction alleviation activity of lambda rev called Lar, may be the same as the activity expressed in sbcA- strains of Escherichia coli, but it was functionally separable from exonuclease VIII activity (the product of the recE gene), which is also expressed in sbcA- strains. Cell, 1981 May, 24(2), 421 - 8 Interaction of the sigma factor and the nusA gene protein of E . coli with RNA polymerase in the initiation-termination cycle of transcription; Greenblatt J et al.; The nusA gene protein of E . coli is involved in regulating termination of transcription in vivo . In vitro it causes termination of transcription in the tR2 region of the PR operon of bacteriophage lambda . We have now used a nusA-Sepharose affinity column and gradient sedimentation experiments to show that the nusA protein binds directly, with great specificity, and with equimolar stoichiometry to the E . coli RNA polymerase core enzyme beta beta' alpha 2 . The RNA polymerase sigma subunit is able to displace the nusA protein from a beta beta' alpha 2-nusA complex, regenerating RNA polymerase holoenzyme beta beta alpha 2-sigma able selectively to initiate transcription at promoter sites . It is proposed that beta beta' alpha 2-nusA and beta beta' alpha 2-sigma are complementary forms of RNA polymerase that interchange with one another in the initiation-termination cycle of transcription. J Bacteriol, 1981 May, 146(2), 668 - 75 Cloning and restriction mapping of the alkaline phosphatase structural gene (phoA) of Escherichia coli and generation of deletion mutants in vitro; Inouye H et al.; The structural gene for alkaline phosphatase (phoA) of Escherichia coli was cloned into the PstI site of pBR322, from a transducing bacteriophage, lambda p(phoA-proC) . The restriction map of the plasmid was established . Based upon this information, several phoA deletion plasmids as well as a smaller phoA+ plasmid were constructed . The genetic map and restriction map were correlated by recombination analysis . Cells carrying one of the phoA+ plasmids overproduce alkaline phosphatase 10-fold upon phosphate limitation . However, both regulation and processing of the enzyme were found to be normal. Nature, 1981 Apr 30, 290(5809), 754 - 8 Structure of the cro repressor from bacteriophage lambda and its interaction with DNA; Anderson WF et al.; The three-dimensional structure of the 66-amino acid cro repressor protein of bacteriophage lambda suggests how it binds to its operator DNA . We propose that a dimer of cro protein is bound to the B-form of DNA with the 2-fold axis of the dimer coincident with the 2-fold axis of DNA . A pair of 2-fold-related alpha-helices of the repressor, lying within successive major grooves of the DNA, seem to be a major determinant in recognition and binding . In addition, the C-terminal residues of the protein, some of which are disordered in the absence of DNA, appear to contribute to the binding. J Biol Chem, 1981 Apr 25, 256(8), 4087 - 94 Two types of replication proteins increase the rate at which T4 DNA polymerase traverses the helical regions in a single-stranded DNA template; Huang CC et al.; We have recently developed an in vitro DNA synthesis system in which a synthetic heptaribonucleotide pairs with a unique site on a single-stranded fd DNA molecule and thereby primes the growth of new DNA strands from this single point (Huang, C.-C., and Hearst, J . E . (1980) Anal . Biochem . 103, 127-139) . In this report, we use this system to investigate the mechanism by which various bacteriophage T4 DNA replication proteins stimulate the T4 DNA polymerase . We find that with the "polymerase accessory proteins" present (the T4 gene 44/62 and 45 proteins), the DNA polymerase proceeds rather rapidly through the occasional hairpin helices which otherwise interrupt the progress of this enzyme along single-stranded DNA templates . By using a potent inhibitor of the 44/62 ATPase, ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)), we have obtained data which suggest that ATP hydrolysis is required for the formation of a polymerase accessory protein-DNA template complex, and that this complex then persists, serving as a sliding clamp which greatly increases the strength of binding between a T4 DNA polymerase molecule and its 3'OH primer template end . The progress of the T4 DNA polymerase though hairpin helices in the DNA template is also stimulated by addition of the T4 helix-destabilizing protein (gene 32 protein) . The effect of the 44/62 and 45 proteins is independent of the effect of the 32 protein in this assay, and the rate of polymerase travel over the strongest hairpin helices is increased more than 40-fold in the presence of these four additional proteins. Nucleic Acids Res, 1981 Apr 24, 9(8), 1941 - 7 Sequence of DNA complementary to a small RNA segment of influenza virus A/NT/60/68; Moss BA et al.; A small RNA segment from the influenza virus strain A/NT/60/68 (H3N2) was converted to cDNA and then to double-stranded DNA using synthetic oligodeoxynucleotide primers . The double-stranded form was cloned into the bacteriophage M1 3mp7 . Clones yielding single-strand recombinant templates in opposite orientation were sequenced by the Sanger dideoxynucleotide chain termination technique . The small viral RNA was 422 nucleotides long and the evidence indicated that it was formed by internal deletion of segment 3 . It also contained sequences homologous to segment 1. Nucleic Acids Res, 1981 Apr 24, 9(8), 1789 - 99 Purified bacteriophage lambda O protein binds to four repeating sequences at the lambda replication origin; Tsurimoto T et al.; The bacteriophage lambda O protein is needed for initiation of lambda DNA replication . Several lines of evidence suggest that initiation requires that this protein interacts with a specific sequence called ori (for origin) in lambda DNA . We have purified this protein to near homogeneity and studied the protection against nuclease cleavage of the origin DNA sequences . Our data demonstrate that the O protein binds within an interval of about 95 base pairs (bp), which contains four tandemly arranged 19bp repeating sequences, ATCCCTCAAAACGA (G)GG GAT(A) . At a low concentration of O protein, the inner two repeats are primarily covered, while binding to the outer two repeats requires a high concentration of O protein . From the molecular size of O protein (32,000 daltons), and the internal symmetry in each 19bp repeat, we inferred that the O protein may bind in dimeric form, and that the 95bp region may be filled only when four such dimers have bound . This interaction is discussed in connection with the "activation" of the ori by O protein leading to initiation of DNA synthesis. Experientia, 1981 Apr 15, 37(4), 339 - 40 Lack of distant relationship between lysozyme Ch and hen egg white lysozyme: computer comparison studies; Chang JJ et al.; A computer search, made for distant relationships between lysozyme Ch, hen egg white lysozyme, and bacteriophage T4 lysozyme, revealed no unusual similarities in their amino acid sequences . Also, antibodies generated against lysozyme Ch failed to cross react with hen egg white lysozyme and vice versa . These lysozymes most likely represent examples of convergent evolution. J Biol Chem, 1981 Apr 10, 256(7), 3593 - 7 Leader peptidase is found in both the inner and outer membranes of Escherichia coli; Zwizinski C et al.; Many membrane proteins are synthesized as transient precursors with an NH2-terminal leader (or signal) peptide . During insertion of these proteins into the membrane, leader peptides are removed by leader peptidase . One such enzyme has been detected in detergent extracts of Escherichia coli membranes and extensively purified us