Plasmid, 1986 Mar, 15(2), 159 - 62 Overproduction of proteins encoded by lambda replicon-integrated plasmid: effect of partial inactivation of cI repressor; Ohta A et al.; Composite plasmids containing the 7.2-kb fragment of bacteriophage lambda cI857cro27, essential for lambda DNA replication and its control, have been constructed . The plasmids contained, in addition, pBR322, a part of Co1E1, and the Escherichia coli pss gene, coding for phosphatidylserine synthase . When the plasmid-harboring cells were induced at different temperatures, the phosphatidylserine synthase production was maximum at 37 degrees C, and the specific activities at 37 degrees C continuously increased until the culture reached saturation . The results suggest that, when the cro gene is defective, only a transient temperature for inactivation of cI repressor provides both the increase in copy number of the plasmids and cellular activities that allow overproduction of the plasmid-encoded protein. Mol Gen Genet, 1986 Mar, 202(3), 467 - 70 The alpha-glucosyltransferases of bacteriophages T2, T4 and T6 . A comparison of their primary structures; Gram H et al.; With the aim of comparing the primary structures of gene products coded for by T-even bacteriophages we constructed clone libraries of the DNAs of bacteriophages T2 and T6 . Using hybrid M13 phages carrying the gene for the T4-coded alpha-glucosyl transferase (alpha gt) we isolated corresponding T2 and T6 clones . The nucleotide sequences of the three alpha gt genes and the amino acid sequences derived were compared . The differences between the genes and their products are discussed in terms of structure, function and evolutionary aspects. Genetics, 1986 Mar, 112(3), 409 - 20 A genetic analysis of primary products of bacteriophage lambda recombination; Huisman O et al.; Primary products of bacteriophage lambda recombination that display heterozygosity as a consequence of the presence of regions of heteroduplex DNA are rare in standard lambda crosses . Phage manifesting heterozygosity at a given allele are evident when recombinants, emerging from a cross, are selected for an exchange in a neighboring interval . We show that the abundance of such heterozygotes can be increased 10- to 20-fold by selection on an E . coli indicator that is defective in methyl-directed mismatch repair (mutL) . Thus, the activity of the methyl-directed mismatch repair system is, at least in part, responsible for the low frequency of detectably heterozygous phage emerging from a standard cross . In a mutL indicator, many primary products of recombination are replicated without the intervention of mismatch repair.--The products of a six-factor phage cross have been plated on a mutL indicator allowing visual detection of those phage products heterozygous for one of the allelic pairs, cI . By genetic analysis, we show that the heteroduplex regions of these primary products of recombination are on the average about 4 kb in length and can include as much as half of the lambda genome. J Virol, 1986 Mar, 57(3), 875 - 82 Involvement of host DNA gyrase in growth of bacteriophage T5; Constantinou A et al.; Bacteriophage T5 did not grow at the nonpermissive temperature of 42 degrees C in Escherichia coli carrying a temperature-sensitive mutation in gyrB {gyrB(Ts)}, but it did grow in gyrA(Ts) mutants at 42 degrees C . These findings indicate that the A subunit of host DNA gyrase is unnecessary, whereas the B subunit is necessary for growth of T5 . The necessity for the B subunit was confirmed by a strong inhibition of T5 growth by novobiocin and coumermycin A1, which interfere specifically with the function of the B subunit of host DNA gyrase . However, T5 growth was also strongly inhibited by nalidixic acid, which interferes specifically with the function of the A subunit . This inhibition was due to the interaction of nalidixic acid with the A subunit and not just to its binding to DNA, because appropriate mutations in the gyrA gene of the host conferred nalidixic acid resistance to the host and resistance to T5 growth in such a host . The inhibition by nalidixic acid was also not due to a cell poison formed between nalidixic acid and the A subunit (K . N . Kreuzer and N . R . Cozzarelli, J . Bacteriol . 140:424-435, 1979) because nalidixic acid inhibited growth of T5 in a gyrA(Ts) mutant (KNK453) at 42 degrees C . We suggest that T5 grows in KNK453 at 42 degrees C because its gyrA(Ts) mutation is leaky for T5 . Inhibition of T5 growth due to inactivation of host DNA gyrase was caused mainly by inhibition of T5 DNA replication . In addition, however, late T5 genes were barely expressed when host DNA gyrase was inactivated. Nucleic Acids Res, 1986 Feb 25, 14(4), 1845 - 61 Effect of SSB protein on cleavage of single-stranded DNA by phi X gene A protein and A* protein; van Mansfeld AD et al.; Gene A protein of bacteriophage phi X174 plays a role as a site-specific endonuclease in the initiation and termination of phi X rolling circle DNA replication . To clarify the sequence requirements of this protein we have studied the cleavage of single-stranded restriction fragments from phi X and G4 viral DNAs using purified gene A protein . The results show that in both viral DNAs cleavage occurs at the origin and at one additional site which shows striking sequence homology with the origin region . During rolling circle replication the single-stranded viral DNA tail is covered with single-stranded DNA binding (SSB) protein . Therefore, we have also studied the effect of SSB on phi X gene A protein cleavage . In these conditions only single-stranded fragments containing the complete or almost complete origin region of 30 bases are cleaved, whereas cleavage at the additional sites of phi X or G4 viral DNAs does not occur . A model for termination of rolling circle replication which is based on these findings is presented . Finally, we present evidence that the second product of gene A, the A* protein, cleaves phi X viral DNA at the additional cleavage site in the presence of SSB, not only in vitro but also in vivo . The functional significance of this cleavage in vivo is discussed. J Immunol, 1986 Feb 15, 136(4), 1482 - 9 The mouse Thy-1.2 glycoprotein gene: complete sequence and identification of an unusual promoter; Ingraham HA et al.; Recombinant bacteriophage and cosmid clones containing the gene for the mouse Thy-1.2 glycoprotein were isolated and characterized . The complete sequence of the gene was determined, including a previously unidentified exon located 2.1 kb upstream of the portion of the gene encoding the Thy-1.2 glycoprotein . The transcriptional initiation site was located by S1 nuclease protection mapping in both T lymphocytes and neural cells and was found to be located immediately upstream of this exon . S1 nuclease protection mapping was also used to define the 3' end of the Thy-1.2 transcription unit, and no evidence for alternate mRNA processing was found . Thus, the mouse Thy-1.2 gene is 5447 base pairs in length, including promoter sequences, rather than 2094 as previously described . The mouse and rat Thy-1 genes are highly homologous in both introns and exons . However, the mouse Thy-1 cDNA and rat Thy-1 cDNA differ significantly in sequence in the 5' untranslated region . This suggests that the transcriptional initiation site of the mouse and rat genes may be located at different positions within the genomic sequence and may be related to the differing tissue distribution of Thy-1 in the two species. J Biol Chem, 1986 Feb 5, 261(4), 1653 - 5 The ionic properties of the filamentous bacteriophages Pf1 and fd; Zimmermann K et al.; The surface charge of the filamentous bacteriophages Pf1 and fd was determined by polyelectrolyte titration covering a range from pH 3 to 9 . The isoelectric point was measured at pH 4.0 for Pf1 and at pH 4.2 for fd . Close agreement of experimental and calculated data was found, confirming the hypothesis that charged residues in the DNA are neutralized by oppositely charged residues of the coat proteins . Almost perfect consistence is reached when structural details of the viruses are included. J Biol Chem, 1986 Feb 5, 261(4), 1499 - 502 Sequences complementary to the brain-specific "identifier" sequences exist in L-type pyruvate kinase mRNA (a liver-specific messenger) and in transcripts especially abundant in muscle; Lone YC et al.; A sequence complementary to the brain-specific identifier sequence has been found in the 3' untranslated extension of the heavy 3.2-kilobase (kb) long liver L-type pyruvate kinase mRNA while it is absent in the other two 2- and 2.2-kb long pyruvate kinase mRNA species . A 53-base fragment corresponding to this identifier sequence was subcloned in both orientations in the single-stranded bacteriophage M13, both strands being used as probes to detect homologous sequence in different tissues . Both strands are transcribed in various tissues and are detected in heterogeneous high molecular weight RNA species which are especially abundant in the adult muscle . In addition, the probe identical to the identifier sequence recognized a discrete 0.6-kb RNA species in the muscle and the probe complementary to the identifier-sequence recognized the expected two small brain-specific identifier BC-1 and BC-2 RNAs described by Sutcliffe et al . (Sutcliffe, J . G., Milner, R . J., Bloom, F . E., and Lerner, R . A . (1982) Proc . Natl . Acad . Sci . U . S . A . 79, 4942-4946; Sutcliffe, J . G., Milner, R . J., Gottesfeld, J . M., and Lerner, R . A . (1984) Nature 308, 237-241; Milner, R . J., Bloom, F . E., Lai, C., Lerner, R . A., and Sutcliffe, J . G . (1984) Proc . Natl . Acad . Sci . U . S . A . 81, 713-717; Sutcliffe, J . G., Milner, R . J., Gottesfeld, J . M., and Reynolds, W . (1984) Science 225, 1308-1314). J Biol Chem, 1986 Feb 5, 261(4), 1782 - 5 The nucleotide sequence of the Escherichia coli K12 dnaJ+ gene . A gene that encodes a heat shock protein; Bardwell JC et al.; The Escherichia coli dnaJ gene product is required for bacteriophage lambda DNA replication at all temperatures . It is also essential for bacterial viability in at least some conditions, since mutations in it result in temperature-sensitive bacterial growth . We have previously cloned the dnaJ gene and shown that its product migrates as a Mr 37,000 polypeptide under denaturing conditions . Here we present the primary DNA sequence of the dnaJ gene . It codes for a processed basic protein (63 basic and 51 acidic amino acids) composed of 375 amino acids totaling Mr 40,973 . The predicted NH2-terminal amino acid sequence, overall amino acid composition, and isoelectric point agree well with those of the purified protein . We present evidence that the rate of expression of the dnaJ protein is increased by heat shock under the control of the htpR (rpoH) gene product. Anal Biochem, 1986 Feb 1, 152(2), 339 - 45 Determination of a particle's radius by two-dimensional agarose gel electrophoresis; Serwer P et al.; Electrophoresis in an agarose gel dilute enough to be almost nonretarding, followed by electrophoresis in an orthogonal direction into a more concentrated agarose gel, has been developed as a procedure to determine the radius of spherical particles . Unlike procedures of unidirectional electrophoresis in a single gel, the above procedure can be used to compare the radii of particles that differ in solid-support-free electrophoretic mobility . Accuracy of 0.3 nm has been achieved with particles 30 nm in radius . It was found that the apparent radius of the spherical capsid of bacteriophage P22 decreased by 3% during elevated temperature-induced ejection of DNA from the capsid . Though originally designed for use with multimolecular particles, the procedure described here should also be useful with monomolecular particles. J Ultrastruct Mol Struct Res, 1986 Feb, 94(2), 105 - 13 Bacteriophage T3 gene 8 product oligomer structure; Carazo JM et al.; The structure of the connector of bacteriophage T3 (built up by the product of gene 8) has been studied in two dimensions by combined use of translational and rotational image filtering procedures applied to tetragonal ordered aggregates of the former oligomers . This analysis, performed up to 1/1.6 nm-1 resolution, has revealed the existence of a 12-fold symmetry in the outermost region of the specimen (mainly between radii 5.2 and 6.7 nm), a 6-fold one in the inner region (between radii 1.7 and 3.2 nm), and a hole in its center . These features are very similar to the ones described for the connectors of other phages, such as T4, lambda, and phi 29, thus suggesting a common mechanism for the functions carried out by this viral region. Dev Biol, 1986 Feb, 113(2), 501 - 11 Calmodulin gene expression during sea urchin development: persistence of a prevalent maternal protein; Floyd EE et al.; Calmodulin gene expression during embryogenesis of the sea urchin Strongylocentrotus purpuratus was investigated . Several identical bacteriophages containing a cDNA insert encoding sea urchin calmodulin (CM-1) were identified by screening a lambda gt10 library of S . purpuratus gastrula-stage cDNAs with a chicken calmodulin cDNA sequence . A 1.2-kb cDNA fragment from CM-1 was subcloned into pUC-8 to give plasmid pCAL-8 . pCAL-8 contains a single open reading frame encoding 79 amino acids, a termination codon, and 0.9 kb of 3'-untranslated message . This sea urchin amino acid sequence shows 95% homology to amino acid residues 69-148 of the predicted sequence of chicken calmodulin . Northern analysis showed that pCAL-8 hybridizes to a single size (3.2 kb) of mRNA in both embryonic and adult somatic tissues . Genome blots suggested that there is a single calmodulin gene in the S . purpuratus genome . We used pCAL-8 to study calmodulin mRNA accumulation in S . purpuratus embryos . Calmodulin mRNA is present in the unfertilized egg at the level of a typical rare-class mRNA (1000-2000 transcripts) and accumulates approximately 100-fold to levels representing about 1/10th of 1% of the total mRNA in pluteus-stage cells . Synthesis of calmodulin, identified by two-dimensional gel electrophoresis, shows a similar developmental pattern . However, in spite of the very active synthesis of calmodulin during embryogenesis, most of the calmodulin in the pluteus is apparently provided for by an enormous store of calmodulin in the egg, corresponding to about 2% of the mass of total protein. Proc Natl Acad Sci U S A, 1986 Feb, 83(4), 1084 - 8 Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis; Harvey RP et al.; Cloned cDNAs have been isolated that encode a variant of hirudin, a potent thrombin inhibitor that is secreted by the salivary glands of the medicinal leech, Hirudo medicinalis . This variant probably corresponds to a form that has been purified from leech heads but differs in amino acid sequence from the hirudin purified from whole leeches . There are at least three hirudin transcripts detectable in leech RNAs that are different in size, site of synthesis, inducibility by starvation, and relationship to hirudin activity . The new hirudin variant predicted by the cDNA and the heterodisperse transcription products suggest a hirudin protein family . The hirudin cDNA was expressed in Escherichia coli under the control of the bacteriophage lambda PL promoter . The recombinant product is biologically active, inhibiting the cleavage by thrombin of fibrinogen and a synthetic tripeptide substrate. J Bacteriol, 1986 Feb, 165(2), 363 - 6 Cloning, overproduction, and purification of the B2 subunit of ribonucleoside-diphosphate reductase; Salowe SP et al.; The nrdB gene, which encodes the B2 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1), was cloned into multicopy plasmid pSPS2 . This vector, which contains the pL promoter of bacteriophage lambda and the tetracycline resistance gene of pBR322, was transformed into a lysogenic host with a thermolabile repressor . In the newly constructed strain, subunit B2 constituted approximately 25% of the soluble protein after heat induction, an overproduction of several hundredfold relative to the wild-type strain . Purification to homogeneity of the overproduced protein was accomplished by using DEAE and quaternary aminoethyl ion-exchange resins. Mol Cell Biol, 1986 Feb, 6(2), 502 - 10 Characterization and chromosome assignment of the human homolog of int-2, a potential proto-oncogene; Casey G et al.; int-2 is one of two cellular genes (int-1 and int-2) currently implicated in the genesis of mammary carcinomas by mouse mammary tumor virus and may constitute a novel cellular proto-oncogene . Using low-stringency hybridization with mouse int-2 probes, we established that homologous genes exist in a variety of mammalian species, including humans, but failed to detect related sequences in other classes and phyla . Recombinant bacteriophage clones and a single cosmid encompassing the human int-2 gene were isolated and characterized by restriction enzyme mapping . A survey of nine primary human breast tumors, three breast tumor cell lines, and three normal individuals revealed no evidence for gross amplification or rearrangement of the int-2 locus . Three distinct restriction fragment length polymorphisms were observed which could prove useful in future linkage studies . By a combination of in situ hybridization of metaphase chromosomes and somatic cell genetics, the human int-2 gene was mapped to chromosome 11, band q13. EMBO J, 1986 Feb, 5(2), 433 - 40 The integrase family of site-specific recombinases: regional similarities and global diversity; Argos P et al.; A combination of two methods for detecting distant relationships in protein primary sequences was used to compare the site-specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1 . This group of proteins exhibits an unexpectedly large diversity of sequences . Despite this diversity, all of the recombinases can be aligned in their C-terminal halves . A 40-residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein . This family of recombinases does not appear to be related to any other site-specific recombinases . Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well-conserved C-terminal region . We speculate that these residues contribute to the active site of this family of recombinases, and suggest that tyrosine-433 forms a transient covalent linkage to DNA during strand cleavage and rejoining. Nippon Sanka Fujinka Gakkai Zasshi, 1986 Feb, 38(2), 253 - 60 Measurement of human prolactin messenger RNA in decidual tissues using complementary DNA probe cloned in M13mp9 bacteriophage; Suganuma N et al.; A human prolactin (PRL) cDNA clone was digested with restriction enzyme Pst I and the resultant fragments were cloned into bacteriophage M13mp9 . Single stranded recombinant DNAs having a coding strand of the PRL cDNA were selected by hybridization with 125I-labeled PRL mRNA obtained from human prolactinoma tissue . One of the single stranded recombinant DNAs was purified by agarose gel electrophoresis and labeled with 125I to a specific activity of 1.4 X 10(8) cpm per microgram of DNA . The probe could be successfully used in RNA dot hybridization . Analysis of poly (A) RNAs from prolactinoma, liver and placental tissues revealed that this probe was specific to PRL mRNA sequence . Hybridization of poly (A) RNA from decidual tissue to this probe revealed the presence of PRL mRNA sequence . However, PRL mRNA in decidua was at least 20,000 times less than that in pituitary prolactinoma. Proc Natl Acad Sci U S A, 1986 Feb, 83(4), 922 - 6 Bacteriophage T4 DNA topoisomerase mediates illegitimate recombination in vitro; Ikeda H; We have found that purified T4 DNA topoisomerase promotes recombination between two phage lambda DNA molecules in an in vitro system . In this cross, T4 DNA topoisomerase alone is able to catalyze the recombination and produce a linear monomer recombinant DNA that can be packaged in vitro . ATP is not required for this recombination, while oxolinic acid stimulates it . The recombinant DNA molecules contain duplications or deletions, and the crossovers take place between nonhomologous and nonspecific sequences of lambda DNA . Therefore, the recombination mediated by the T4 DNA topoisomerase is an illegitimate recombination that is similar to that mediated by Escherichia coli DNA gyrase . A model was proposed previously in which DNA gyrase molecules that are bound to DNA associate with each other and lead to the exchange of DNA strands through the exchange of DNA gyrase subunits . This model is also applicable to the recombination mediated by T4 DNA topoisomerase. J Gen Virol, 1986 Feb, 67 ( Pt 2), 333 - 43 In vitro packaging of foreign DNA into heads of bacteriophage T1; Hug H et al.; The isolation of a collection of 44 morphologically T1-like phages is described . It is shown that these phages share some similarity with T1 in terms of cross-inactivation with anti-T1 serum, particle proteins and DNA packaging in vitro by the headful process . Virion DNA extracted from these phages was treated with T1 in vitro packaging extracts and the reaction mixtures were tested for the formation of infectious phage particles . The packaging efficiencies observed varied from about 1 to 100% of that of virion T1 DNA . Phage lambda virion DNA was packaged with an efficiency of between 0.01 and 2% (5 X 10(1) to 3 X 10(3) p.f.u./micrograms DNA), the shorter deleted derivative lambda L47 being packaged more efficiently than normal length lambda C1857 DNA . Virion DNA from phages T3 and T7 was also packaged at an efficiency similar to that for lambda . The in vitro packaging of T1 DNA requires the presence of the pac sequence which initiates headful packaging from a concatemeric precursor . The high efficiency of packaging DNA from some of the T1-like phages may indicate the presence of similar packaging sequences . However, in the case of lambda L47, which is known not to contain such a sequence, the in vitro DNA packaging reaction must occur by a secondary pathway unrelated to the headful mechanism. Virology, 1986 Feb, 149(1), 128 - 31 Mutations in bacteriophage lambda that alter phage dependence on the htpR gene product of Escherichia coli; Fuerst CR; Six mutants of lambda having reduced dependence on the htpR function of Escherichia coli were isolated from lambda cIts857 . Burst sizes in htpRts cells at 40.5 degrees were in the range of 10 to 20 particles per cell . Mapping and complementation analysis of one of the mutants suggested that the mutation in this isolate is in gene J . Additional evidence that the mutations in most of the isolates are in J was provided by the finding that all but one of the mutants differ from the parental phage in properties pertaining to extended host range. Virology, 1986 Jan 30, 148(2), 298 - 311 The overproduction and characterization of the bacteriophage Mu regulatory DNA-binding protein ner; Tolias PP et al.; The bacteriophage Mu ner gene has been cloned under the control of the lacUV5 promoter in the expression vector pOP95-15 . The gene products of the recombinant plasmid, pUD88, visualized by in vitro coupled transcription-translation, are the bacteriophage Mu ner protein (8 kDa) and a 23 KDa protein consisting of the amino terminus of gpA (Mu transposase) fused to the carboxy terminus of beta-lactamase . DNA-binding activity was measured by the retardation of migration of a 32P-labeled DNA restriction fragment (containing the presumed ner-binding sites) in polyacrylamide gels . We have demonstrated specific association of ner to its binding sites to occur within 30 sec after the addition of impure extracts of ner overproducing cells . Much of this binding was dissociated within 30 sec by competition with a 20-fold molar excess of specific unlabeled DNA restriction fragment, but was resistant to dissociation when competed with unlabeled heterologous DNA for as long as 45 min at 37 degrees . By adapting a method for DNA-footprinting using impure extracts of ner overproducing cells, we were able to determine that the ner-binding sites are located between nucleotides 1026 and 1058 from the Mu left end . These results support the hypothesis that ner is similar to the cro regulatory protein from bacteriophage lambda and acts to regulate Mu early gene expression and the choice between lytic and lysogenic development. J Biol Chem, 1986 Jan 25, 261(3), 1286 - 92 Signal recognition particle mediates the insertion of a transmembrane protein which has a cytoplasmic NH2 terminus; Holland EC et al.; The mechanism by which rat liver asialoglycoprotein receptor (rat hepatic lectin, RHL) is inserted into membranes has been investigated . RHL is a prototype for transmembrane proteins which are oriented with their NH2 termini in the cytoplasm and their COOH termini outside the cell . Such transmembrane proteins are synthesized without cleavable NH2-terminal signal sequences . An in vitro translation system has been developed in which RHL is translated from RNA produced in a bacteriophage SP6 in vitro transcription system . RHL produced in this way can be inserted cotranslationally in the correct orientation into dog pancreas microsomes . This insertion process has been shown to be dependent on the signal recognition particle and its receptor (the docking protein) in the microsomal membranes . A detailed mechanism for the insertion of this type of transmembrane protein into the lipid bilayer is proposed. Nucleic Acids Res, 1986 Jan 24, 14(2), 737 - 49 Thymine glycol lesions terminate chain elongation by DNA polymerase I in vitro; Clark JM et al.; Single-strand circular DNA from bacteriophage M13mp9 was chemically modified with osmium tetroxide to introduce specifically cis-thymine glycol lesions, a major type of DNA damage produced by ionizing radiation . An oligonucleotide primer was extended on damaged and undamaged templates using either the large fragment of E . coli pol I or T4 DNA polymerase . The reaction products were analysed by electrophoresis alongside a DNA sequence ladder . Synthesis on the damaged templates terminated at positions opposite thymine bases in the template . These results indicate that cis-thymine glycol lesions in single-strand DNA constitute blocks to synthesis by DNA polymerases in vitro . Surprisingly, replication halts after the correct nucleotide, dAMP, is inserted opposite the lesion . These results imply that the primary effect of the thymine glycol lesion is suppression of DNA synthesis and that the lesion is not a potent mutagen. Nucleic Acids Res, 1986 Jan 24, 14(2), 779 - 95 The full length coding sequence of rat liver androsterone UDP-glucuronyltransferase cDNA and comparison with other members of this gene family; Jackson MR et al.; Cloned cDNAs coding for hepatic UDP-glucuronyltransferase (UDPGT) have been isolated from a rat liver cDNA library in the expression vector bacteriophage lambda gt11 using anti-UDPGT antibodies . Four different mRNAs have been identified by sequencing of 15 UDPGT cDNA clones . The sequences of the four classes of cDNA were determined to be 85-95% homologous . Restriction fragments were isolated from the cDNA in each class and used as class specific probes . Hybridisation of these probes to northern blots of total RNA prepared from the livers of normal and genetically deficient Wistar rats identified the cDNA in class 4 with androsterone UDPGT . Translation of the cDNA sequence of clone rlug 23, the longest member of class 4, allowed determination of the complete amino acid sequence of androsterone UDPGT. J Mol Biol, 1986 Jan 20, 187(2), 197 - 212 Bacteriophage P1 cre gene and its regulatory region . Evidence for multiple promoters and for regulation by DNA methylation; Sternberg N et al.; The bacteriophage P1 site-specific recombination system consists of two components, a site, loxP, at which recombination occurs, and a recombinase protein, Cre . In this paper, we present the DNA sequence of the cre structural gene and its upstream regulatory region . Analysis of the sequence indicates: (1) that cre encodes a protein of 343 amino acids; (2) that cre and loxP are separated by a 434 base-pair region that contains a 73 amino acid open reading frame, orf1; and (3) that cre and orf1 are oriented with their amino-terminal ends proximal to loxP . We have identified three promoters that are located upstream of the cre structural gene . Their activities range from 7 to 10% of the activity of the galactose operon promoter . The promoter furthest from cre, pR1, contains two Dam methylation sites (5'-G-A-T-C-3') in its -35 region, and is sensitive to Dam methylation . Its transcription is three- to fourfold higher in a dam- host than it is in a dam+ host . The promoter closest to cre, pR3, signals the production of an RNA transcript that functions inefficiently for Cre protein synthesis because it lacks a ribosome recognition site . None of the three cre promoters is sensitive to proteins expressed by the P1 prophage, including the c1 repressor protein . To assess the role of cre in the P1 life-cycle, we isolated cre mutants and studied their behavior in recA+ and recA- hosts . Those studies indicate that Cre is dispensable for viral vegetative growth and lysogeny in a recA+ host, but is required for both processes in a recA- host . The cre requirement for lysogeny suggests that the protein is essential for the cyclization of newly injected terminally redundant virion DNA . The requirement for vegetative growth suggests that Cre also has a role to play in the viral lytic cycle after the viral DNA has been cyclized. J Mol Biol, 1986 Jan 20, 187(2), 225 - 39 Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K; Filutowicz M et al.; The three replication origins of the antibiotic resistance plasmid R6K require for their activity in Escherichia coli a DNA segment containing seven 22 base-pair direct repeats and a plasmid-encoded initiation protein (pi) . The pi protein functions in the negative control of R6K replication, in addition to its requirement for the initiation of replication . Construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pR promoter of bacteriophage lambda provided high levels of production of pi protein in E . coli . The pi protein was purified and shown to possess general DNA binding properties with a preference for DNA fragments containing the gamma origin of replication, the operator region of the pir gene and the R6K beta-origin region . Velocity sedimentation analysis indicates that the pi protein exists as a dimer in its native form . Agarose gel electrophoresis analysis of pi-gamma-origin complexes suggests that one pi dimer binds to each copy of the 22 base-pair direct repeats in the gamma origin region . Purified mutant pi protein obtained from a temperature-sensitive initiation mutant (pir 105-ts) exhibited temperature-sensitive binding activity to the gamma-origin region, whereas two mutant proteins exhibiting a high copy number phenotype were unaltered (pir104-cop) or slightly reduced (pir1-cop) in binding activity . The patterns of DNase I protection and enhancement were similar for the wild-type and mutant proteins examined. FEBS Lett, 1986 Jan 20, 195(1-2), 61 - 4 Cloning and DNA sequence of the 5'-exonuclease gene of bacteriophage T5; Kaliman AV et al.; The nucleotide sequence of the BalI-PstI fragment of T5 DNA, 1347 bp in length, coding for 5'-exonuclease (D15 gene), has been determined . A coding region of the gene contains 873 bp and is preceded by a typical Shine-Dalgarno sequence . The D15 gene belongs to a cluster, consisting of at least 3 genes, in which a termination codon of a preceding gene overlaps an initiation codon of the following one . The sequence contains an open reading frame for 291 amino acid residues . The molecular mass of the 5'-exonuclease calculated from the predicted amino acid sequence is 33 400 Da. J Mol Biol, 1986 Jan 20, 187(2), 213 - 24 hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein; Banuett F et al.; The level of the viral cII protein has been proposed to be the crucial determinant in the lysis-lysogeny decision of bacteriophage lambda . A new Escherichia coli locus (hflB) has been identified in which a mutation (hflB29) leads to high frequency of lysogeny by lambda . A double mutant defective in both hflB and the previously identified hflA gene displays a more severe Hfl- phenotype than either single mutant . The hflB locus is at 69 minutes on the E . coli map, 85% co-transducible with argG . The hflB29 mutation results in increased stability of the phage cII protein (increasing its half-life twofold) and is recessive to hflB+ . We conclude that the hflB+ locus is a negative regulator of cII, perhaps coding for or regulating a protease that acts on cII . In addition, we observe that the can1 mutation, an alteration of the cII gene that results in enhanced lysogenization, leads to increased stability of cII protein . These observations reinforce the view that the level of cII is a key factor in the lysis-lysogeny decision of lambda. Cell, 1986 Jan 17, 44(1), 137 - 45 Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging; Levis R et al.; Defective-interfering (DI) genomes of a virus contain sequence information essential for their replication and packaging . They need not contain any coding information and therefore are a valuable tool for identifying cis-acting, regulatory sequences in a viral genome . To identify these sequences in a DI genome of Sindbis virus, we cloned a cDNA copy of a complete DI genome directly downstream of the promoter for the SP6 bacteriophage DNA dependent RNA polymerase . The cDNA was transcribed into RNA, which was transfected into chicken embryo fibroblasts in the presence of helper Sindbis virus . After one to two passages the DI RNA became the major viral RNA species in infected cells . Data from a series of deletions covering the entire DI genome show that only sequences in the 162 nucleotide region at the 5' terminus and in the 19 nucleotide region at the 3' terminus are specifically required for replication and packaging of these genomes. Virology, 1986 Jan 15, 148(1), 198 - 209 Novel dimeric configurations from bacteriophage G4 replicative form DNA; Fishel RA et al.; The oligomeric fraction of the replicative form of phage G4 was prepared by sedimentation on three successive CsCl velocity gradients followed by resolution on CsCl-propidium diiodide equilibrium gradients and subfractions through the equilibrium gradients were examined by electron microscopy . The most frequent dimer species were the circular dimer, the singly linked catenane and the figure 8; these occurred in a ratio of 10:3:1 . The high enrichment for dimers and other oligomers made possible the observation and the determination of the frequency of occurrence of a number of minor species, some of them of novel configuration . These are (a) dimers similar to figure 8s except containing long, apparently four-stranded junctions common to the two halves (theta forms); (2) dimers similar to those in (1) except that the long junctions separate the two halves (dumbbell forms); (3) multiply catenated dimers with apparent right-handed intertwines; and (4) dimers containing a knot . Theta forms cleaved by EcoRI were shown to be stable under conditions in which EcoRI-treated figure 8s were resolved by branch migration. Biochemistry, 1986 Jan 14, 25(1), 251 - 6 Bacteriophage P22 Cro protein: sequence, purification, and properties; Poteete AR et al.; The DNA sequence of part of the bacteriophage P22 early regulatory region, including genes cro and c1, was determined . The protein product of the cro gene consists of 61 amino acid residues, and that of c1, 92 amino acid residues . Both genes were placed separately in plasmids from which they are expressed from a controllable promoter in vivo . Induced cells bearing the cro-expressing plasmid were used as a source for purifying and characterizing the Cro protein . The amino-terminal sequence of this protein was found to be as predicted by the DNA sequence; close agreement was also observed between its predicted and experimentally determined amino acid composition and molar extinction coefficient at 280 nm . In gel filtration experiments, Cro protein at concentrations around 10(-5) M appears to have a molecular weight of 8600, which is more consistent with monomers (6800) than with dimers (13 600) . Cro protein binds specifically to the three repressor binding sites in the P22 right operator; in order of decreasing affinity, these are OR3, OR1, and OR2. Biochemistry, 1986 Jan 14, 25(1), 21 - 5 Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product . Expression of the ssb gene under lambda PL control; Lohman TM et al.; We report a rapid procedure for the large-scale purification of the Escherichia coli encoded single-strand binding (SSB) protein, helix-destabilizing protein which is essential for replication, recombination, and repair processes in E . coli . To facilitate the isolation of large quantities of the ssb gene product, we have subcloned the ssb gene into a temperature-inducible expression vector, pPLc28 {Remaut, E., Stanssens, P., & Fiers, W . (1981) Gene 15, 81-93}, carrying the bacteriophage lambda PL promoter . A large overproduction of the ssb gene product results upon shifting the temperature of E . coli strains which carry the plasmid and also produce the thermolabile lambda cI857 repressor . After 5 h of induction, the ssb gene product represents approximately 10% of the total cell protein . The overexpression of the ssb gene and the purification protocol reported here enable one to isolate SSB protein (greater than 99% pure) with final yields of approximately 3 mg of SSB protein/g of cell paste . In fact, very pure (greater than 99%) SSB protein can be obtained after approximately 8 h, starting from frozen cells in the absence of any columns, although inclusion of a single-stranded DNA-cellulose column is generally recommended to ensure that the purified SSB protein possesses DNA binding activity . The ability to easily purify 1 g of SSB protein from 300-350 g of induced cells will facilitate physical studies requiring large quantities of this important protein. Nucleic Acids Res, 1986 Jan 10, 14(1), 141 - 58 Sequence landscapes; Clift B et al.; We describe a method for representing the structure of repeating sequences in nucleic-acids, proteins and other texts . A portion of the sequence is presented at the bottom of a CRT screen . Above the sequence is its landscape, which looks like a mountain range . Each mountain corresponds to a subsequence of the sequence . At the peak of every mountain is written the number of times that the subsequence appears . A data structure called a DAWG, which can be built in time proportional to the length of the sequence, is used to construct the landscape . For the 40 thousand bases of bacteriophage T7, the DAWG can be built in 30 seconds . The time to display any portion of the landscape is less than a second . Using sequence landscapes, one can quickly locate significant repeats. Nucleic Acids Res, 1986 Jan 10, 14(1), 109 - 26 Analysis of the occurrence of promoter-sites in DNA; Mulligan ME et al.; We show that the occurrence and homology score (1) of promoter-sites in DNA depends upon the base composition of the DNA . We used simple probability theory to calculate the mean homology score expected for all promoter-sites that had a specific match in the canonical hexamers . By using the square root of this mean score as a measure of significance, we objectively classify all promoter-sites which are reported . We tested the theoretical approach in two ways . First, we used the program (PROMSEARCH) to analyze approximately 150,000 base pairs of random sequence DNA with different base compositions and we found excellent agreement with the theoretical predictions . Our second test was the analysis of a number of sequences drawn from the GENBANK DNA sequence database . We have analyzed 20 bacterial and bacteriophage sequences, which consisted of at least one operon, for promoter-sites . We found no absolute preference for promoter-sites within noncoding regions . We show the results of analyzing the phages lambda, T7 and fd, and the E . coli lac operon . The major known promoters in these sequences were all found correctly . We discuss the question of the location of a number of minor promoter-sites and show how PROMSEARCH can be used to help identify the correct location of the promoter . This approach can be applied to the search for any DNA site and should allow greater objectivity when comparing DNA sequences for meaningful subsequences. FEBS Lett, 1986 Jan 6, 194(2), 245 - 8 T7 and E . coli share homology for replication-related gene products; Toh H; Recently, the complete nucleotide sequence of the bacteriophage T7 genome was determined and 50 genes were identified on the genome . We compared amino acid sequences of all the gene products of T7 and replication-related gene products of E . coli . As a result, we found that T7 and E . coli share homology for each pair of exonuclease, DNA primase and helix-destabilizing protein . For E . coli, these gene products are known to be involved in the process of discontinuous DNA replication . These observations suggest that T7 and E . coli have a common origin for a part of their replication systems. J Biol Chem, 1986 Jan 5, 261(1), 391 - 6 Bacteriophage P1 Cre-loxP site-specific recombination . Site-specific DNA topoisomerase activity of the Cre recombination protein; Abremski K et al.; Site-specific recombination in bacteriophage P1 occurs between two loxP sites in the presence of the Cre recombination protein . The structure of the 34-base pair loxP site consists of two 13-base pair inverted repeats separated by an 8-base pair spacer region . A mutation in the loxP site has been constructed which deletes one of the internal bases of the spacer region at the axis of dyad symmetry . This mutant loxP site shows a 10-fold reduction in recombination activity with a wild-type site both in vivo and in vitro . This low level of intramolecular recombination between a wild-type loxP site and the mutant loxP501 site is observed in vitro only when the DNA substrate is supercoiled . The majority of the supercoiled substrate is relaxed by the Cre protein, and on longer incubations, single-stranded nicks accumulate in the DNA . We have determined that these nicks occur in both the wild-type and the mutant sites . The positions of these nicks correspond to the positions of cleavage found during recombination of two wild-type sites, suggesting that the Cre protein is attempting to carry out recombination with the mutant site but most of the time this reaction is abortive . We have determined that the Cre protein relaxes a supercoiled topoisomer of a DNA substrate containing one wild-type site and one mutant site to yield a distribution of topoisomers whose linking numbers differ by steps of one, indicating that Cre can act as a type I topoisomerase. J Biol Chem, 1986 Jan 5, 261(1), 386 - 90 Formation of transmembrane channels in liposomes during injection of lambda DNA; Roessner CA et al.; Bacteriophage lambda binds to unilamellar liposomes bearing its receptor protein, LamB, and the lambda DNA can be injected into the internal aqueous space . During this process, transmembrane channels are formed in the liposomes which permit the entry and escape of small molecules, but not proteins . The channels are stable and persist after DNA injection. Gene, 1986, 43(1-2), 123 - 30 Molecular cloning of a cDNA for human delta-aminolevulinate dehydratase; Wetmur JG et al.; A cDNA encoding human delta-aminolevulinic acid dehydratase (ALA-D; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, was isolated from a human liver cDNA expression library . Of the original 17 clones selected with anti-ALA-D antibody, only four expressed anti-ALA-D epitopes as assessed by rescreening with antibody preabsorbed with purified antigen . Subsequent screening of the antibody-positive clones with mixed oligodeoxynucleotide (oligo) probes, synthesized to correspond to human N-terminal and bovine active-site peptide sequences, identified three clones which hybridized only with the oligo probes for the bovine amino acid (aa) sequences . Restriction endonucleases analysis revealed that these three clones contained the same 800-bp cDNA insert . This insert was recloned into bacteriophage M13mp18 and mp19 and sequenced by primer extension . The aa sequence predicted from the partial nucleotide sequence was found to be essentially colinear with the sequences of four bovine ALA-D peptides, totaling 35 non-overlapping aa residues. J Neurosci Res, 1986, 16(1), 37 - 49 Isolation and sequence of cDNA clones coding for the precursor to the gamma subunit of mouse muscle nicotinic acetylcholine receptor; Boulter J et al.; cDNA libraries have been constructed in plasmid (pBR322) and bacteriophage lambda gammagt10) vectors with poly (A+) RNA isolated from the nonfusing mouse muscle cell line BC3H-1 . The libraries were screened with a restriction fragment derived from a genomic clone coding for a human acetylcholine receptor gamma subunit . Several clones were obtained whose cDNA inserts possessed nucleotide and deduced amino acid sequence homology with acetylcholine receptor gamma subunits from Torpedo californica, chick, calf, and human . One isolate, lambda BMG419, has 88 nucleotides of 5'-untranslated sequence, an open reading frame of 1,557 nucleotides coding for the precursor to the mouse acetylcholine receptor gamma subunit, and 144 nucleotides of 3'-untranslated sequence . Alignment of the lambda BMG419-deduced amino acid sequence with homologs from other species predicts a precursor peptide of 519 amino acids and a mature protein of 497 amino acids, with nonglycosylated molecular weights of 58,744 and 56,424 daltons, respectively . Comparison of the deduced amino acid sequence of the mouse gamma subunit with Torpedo, chick, calf, and human sequences showed overall homologies of 54%, 67%, 90%, and 90%, respectively; however, significantly higher homologies were found in several putative functional domains . Radiolabeled lambda BMG419 has been used to identify homologous RNA species, one of approximately 2 kb and one of about 3.5 kb, in poly (A+) RNA prepared from BC3H-1 cells and denervated mouse limb muscle . gamma Subunit-coding RNA species are considerably more abundant in denervated than in innervated muscle, suggesting that neural regulation of the abundance of the gamma subunit is exerted through regulation of the amount of its mRNA. Gene, 1986, 43(3), 205 - 11 Nucleotide sequence identity of mitochondrial DNA from different human tissues; Monnat RJ Jr et al.; Recombinant DNA techniques have been used to search for mitochondrial (mt) nucleotide (nt) sequence differences between human tissues within an individual . mtDNA isolated from brain, heart, liver, kidney, and skeletal muscle of two different individuals was cleaved with SacI and XbaI, and then cloned in bacteriophage M13 . Partial nt sequence determination of 121 independently isolated recombinant M13 clones containing either the cytochrome oxidase subunit III gene or the D-loop region of human mtDNA revealed base substitution differences between individuals, and between each individual and the published human mtDNA sequence . A majority of these base substitutions were transitions . No systematic nt sequence differences were identified between tissues within an individual, however . These results suggest that mtDNA sequence alterations do not accompany organogenesis, and that somatic mutations do not accumulate in the mtDNA of different human tissues to a level of greater than one nt substitution per molecule. Gene, 1986, 42(1), 113 - 7 Synthesis of a fixed-length single-stranded DNA probe by blocking primer extension in bacteriophage M13; Liu JZ et al.; A simple and efficient technique has been developed for preparing radiolabeled single-stranded (ss) probes of determined length and high specific radioactivity . The human beta-globin gene intervening segment II (IVSII) fragment (0.9-kb) was inserted between the EcoRI and BamHI sites of M13mp11 and used as a template for ss probe synthesis . The M13 hybridization probe primer (M13 Hpp) was annealed to the recombinant M13mp11-beta IVSII template DNA . This M13 Hpp was next blocked by the enzymatic addition of a dideoxy adenosine monophosphate (ddAMP) residue to the 3' OH group of the primer . The M13 universal sequencing primer was then annealed and used to prepare an ss copy of the beta-IVSII fragment . Synthesis of the ss fragment was terminated by the presence of the dd-blocked M13 Hpp yielding a specific 0.9-kb ss beta-IVSII probe. Physiol Chem Phys Med NMR, 1986, 18(4), 275 - 85 Phage T7-inactivation test . A possibility of quantitative mutagenicity screening; Ronto G et al.; A new method is described for the quantitative characterization of the genotoxic effect of chemicals . The method is based on the determination of the inactivation of bacteriophage T7 and on the application of a simple mathematical model valid for the processes during, or at least in the initial stage of the interaction of chemicals and phages . A value characteristic for the chemical is defined and it is determined from the inactivation kinetics . Typical inactivation kinetic curves and some problems of the application of the model as well as the mutagenicity index values determined for about 30 substances are presented . The substances examined have mutagenicity index values covering a range of six orders of magnitude . The obtained values are compared with the results of different mutagenicity/carcinogenicity tests and discussed on the basis of data in the literature . The presented method is proposed to be applied for quantitative mutagenicity screening of chemicals. Gene, 1986, 49(2), 207 - 13 Construction of recombinant vaccinia virus strains using single-stranded DNA insertion vectors; Wilson EM et al.; The ability of single-stranded (ss) DNA, isolated from recombinant M13 bacteriophage, to direct the insertion of foreign genetic elements into the vaccinia virus (VV) genome was examined . An identical chimeric transcriptional unit {VV promoter/chloramphenicol acetyl transferase (CAT) gene embedded in DNA sequences encoding vaccinia virus thymidine kinase (TK)} was inserted into either the previously characterized plasmid insertion vector, pGS20, or into M13mp18 . It was found that the ss vector (M13mp18:TK/CAT) was four times more efficient than the plasmid vector (pGS20:CAT) in catalyzing homologous recombination of the cat gene by marker transfer into the VV genome . Furthermore, Southern blot analyses and CAT enzymatic activity assays confirmed that the structure of the M13-derived recombinant genomes were as expected and that the chimeric genes were fully active . Although the precise mechanism responsible for the ss DNA-catalyzed insertion event is not known, these results are discussed with respect to the advantages of using M13-based vectors with which to manipulate and insert genetic information into infectious VV recombinants. Gene, 1986, 49(2), 199 - 205 Linear DNA replication: inverted terminal repeats of five closely related Escherichia coli bacteriophages; Savilahti H et al.; The closely related lipid-containing bacteriophages PRD1, PR4, PR5, PR722 and L17 isolated from different parts of the world have double-stranded DNA genomes which replicate in a linear form . The nucleotide (nt) sequences of the genome termini of these viruses reveal 110-111-bp-long inverted terminal repeats (ITRs) . Both ends of the viral DNA are identical . The first 18 bp and the last 35 bp of the ITRs are totally conserved in all viruses . Between these conserved nt sequences there is a variable sequence, which enables us to divide the phages into two groups . Comparison of the virus ITRs led also to the identification of a 10-bp-long A + T stretch, where the only changes observed were transversions between A and T . The termini of the PRD1 virus family genomes exhibit sequence similarities to those of phi 29 and Cp-1 families. Gene, 1986, 48(1), 165 - 74 Nucleotide sequence and transcription of a human tRNA gene cluster with four genes; Chang YN et al.; A bacteriophage lambda clone containing a 20-kb human DNA segment was isolated and found to harbor a cluster of four tRNA genes . An 8.2-kb HindIII subfragment encompassing the genes was cloned into pBR322 for restriction mapping and DNA sequence analysis . The genes were found to be arranged as two tandem pairs, separated by 3 kb . A proline tRNAAGG gene is separated from a leucine tRNAAAG gene by a 724-bp intergenic region in the first pair, and a second proline tRNAAGG gene is 316 bp from a threonine tRNAUGU gene in the second pair, with the leucine tRNA gene being of opposite polarity to the other three genes . A putative Alu-like element was found to occur within a 2.0-kb DNA fragment, at least 0.7 kb from the tRNA gene cluster . The coding sequences of the two proline tRNAAGG genes are identical . The coding regions of all four tRNA genes contain consensus internal split promoter sequences and do not have intervening sequences nor the CCA trinucleotide found in mature tRNAs . The 3'-flanking regions of these four tRNA genes have normal RNA polymerase III termination sites of at least four consecutive T nucleotides . No apparent homologies occur between the 5'-flanking regions of these genes . All four tRNA genes are accurately transcribed in an in vitro HeLa cell-free system, and the RNase T1 fingerprints of the mature-sized tRNA transcripts were found to be consistent with the DNA sequences of the genes. Gene, 1986, 49(2), 245 - 51 The nucleotide sequence of gene 21 of bacteriophage T4 coding for the prohead protease; Keller B et al.; We have sequenced gene 21 coding for the bacteriophage T4 prohead protease . The sequence codes for a protein of 212 amino acids (aa) with an Mr of 23,251 . A second possible in-frame initiation site was also found which would code for an Mr 18,440 protein . Evidence is presented that this second site is used in vivo . The only striking homology of gp21 to other proteins is with the serine proteases . The protein is homologous to a short aa sequence around the active site, but has a His where the active site Ser is normally found . However, mutation of this His to Ser gave a functional protein that could not be inhibited by serine protease inhibitors . We have located three sites in the gene that give rise to temperature-sensitive mutations . One of these is towards the N-terminus of the gene, the other two flank the region that shows homology with serine proteases . Attempts to overproduce the protein in Escherichia coli failed due to the extreme lability of the enzyme . A frame-shift mutation in the gene was therefore constructed which allowed the synthesis of large amounts of a stable N-terminal fragment of the protein. Gene, 1986, 48(2-3), 251 - 6 Screening recombinant clones containing sequences homologous to Escherichia coli genes using single-stranded bacteriophage vector; Wei YG et al.; Detection and isolation of Escherichia coli clones carrying vectors with foreign DNA sequences partially homologous to specific E . coli genes is difficult because denatured DNA in the host genome can hybridize with the probe . In this paper we present a procedure which simplifies this task by using bacteriophage M13 as the cloning vector . The procedure takes advantage of the secretory properties of the phage, as well as the property of nitrocellulose membrane to bind protein and single-stranded DNA but not double-stranded DNA . This procedure is shown to be effective in identifying E . coli clones containing sequences of Chlamydomonas reinhardtii chloroplast DNA that are homologous to the rpoC gene of E . coli . We suggest that this procedure can be used generally for rapid isolation of DNA sequences that are homologous to E . coli genes. Appl Environ Microbiol, 1986 Jan, 51(1), 126 - 31 Transformation and transfection of anthracycline-producing streptomycetes; Lampel JS et al.; Streptomyces peucetius and Streptomyces strain C5, producers or anthracycline antibiotics, were converted to protoplasts from vegetatively growing mycelia . Conditions are described for maximal protoplast formation (greater than 99%) and for regeneration frequencies of up to 13% . Streptomycete plasmids pIJ61, pIJ702, and pIJ922, from the replicons SLP1, pIJ101, and SCP2, respectively, were isolated from Streptomyces lividans 66 and successfully introduced into S . peucetius and Streptomyces strain C5 by polyethylene glycol-mediated protoplast transformation . Frequencies of up to 10(6) transformations X microgram of plasmid DNA-1 were achieved by these procedures . Analyses showed that the two anthracycline-producing strains can stably harbor the plasmids without deletion of plasmid sequences or loss of the plasmids for several transfers through selective media . Fragments of DNA from S . peucetius ligated into pIJ702 and introduced into Streptomyces strain C5 were stable after several transfers through selective media . Both anthracycline producers also were sensitive to infection and transfection by actinophages KC401 and KC515, clear plaque derivatives of bacteriophage phi C31 . Optimal conditions were determined for the transfection of S . peucetius and Streptomyces strain C5 protoplasts with phi C31 KC401 and KC515 DNA with liposome-assisted, polyethylene glycol-mediated protoplast transfection. Gene, 1986, 48(2-3), 257 - 66 Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum; Carr LG et al.; The isopenicillin N synthetase (IPS) gene from Penicillium chrysogenum was isolated from a recombinant bacteriophage lambda library using the Cephalosporium acremonium IPS (cIPS) gene as a heterologous hybridization probe . The protein coding region of the P . chrysogenum IPS (pIPS) gene was about 74% homologous to the cIPS gene, and the predicted amino acid sequences of the encoded proteins were about 73% homologous . Escherichia coli cells with the pIPS gene contained IPS activity whereas untransformed cells were completely devoid of this enzymatic activity . The transformed cells were also shown to contain an abundant protein accounting for about 10% of total cell protein which reacted strongly with anti-cIPS antiserum. J Bacteriol, 1986 Jan, 165(1), 181 - 92 Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12; Coulton JW et al.; The fusion-generating phage lambda plac Mu1 was used to produce fusions of lacZ to fhuA, the gene encoding the ferrichrome-iron receptor (FhuA protein) in the outer membrane of Escherichia coli K-12 . Fusions to the fhuA gene in a delta (lac) strain were selected by their resistance to bacteriophage phi 80 vir . Ten independent (fhuA'-'lacZ) fusions were all Lac+ and were resistant to the lethal agents which require the FhuA protein as receptor, i.e., phi 80 vir, T5, T1, UC-1, and colicin M; none could utilize ferrichrome as the sole iron source . Specialized transducing phages were obtained by illegitimate excision from the chromosome of each of the fusion-bearing strains, and EcoRI fragments which encoded the fusions were subcloned into the high-copy plasmid pMLB524 . Physical mapping of the fusion-containing plasmids confirmed the presence of three restriction sites which were also located on the chromosomal DNA of sequences near the fhuA gene . The direction of transcription of the fhuA gene was deduced from the direction of transcription of the (fhuA'-'lacZ) gene fusion . Identification of the chimeric proteins was made by both radiolabeling cells and immunoprecipitating the LacZ-containing proteins with antibody to beta-galactosidase and by preparing whole cell extracts from Lac+ cells containing the cloned gene fusions . Two sizes of (FhuA'-'LacZ) proteins were detected, 121 kDa and 124 kDa . The DNA sequences at the unique fusion joints were determined . The sequence information allowed us to identify three distinct fusion joints which were grouped as follows, type I fusions, 5'-ACT GCT CAG CCA A-3'; type IIa fusions, 5'-GCG GTT GAA CCG A-3'; and type IIb fusions: 5'-ACC GCT GCA CCT G-3' . To orient these fhuA fusion joints, the complete nucleotide sequence of the fhuA gene was determined from a 2,902-base-pair fragment of DNA . A single open reading frame was found which translated into a 747-amino acid polypeptide . The signal sequence of 33 amino acids was followed by a mature protein with a molecular weight of 78,992 . Alignment of the amino acid sequence of the FhuA protein with the amino acid sequences presented for two other tonB-dependent receptor proteins in the outer membrane of E . coli showed an area of local homology at the amino terminus of all three proteins. Gene, 1986, 50(1-3), 69 - 85 The production of generalized transducing phage by bacteriophage lambda; Sternberg N; Generalized transduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes . However, throughout that time little progress has been made in understanding how generalized transducing particles are produced . The experiments presented in this paper use phage lambda to assess some of the factors that affect that process . The results of those experiments indicate: the production of generalized transducing particles by bacteriophage lambda is inhibited by the phage lambda exonuclease (Exo) . Also inhibited by lambda Exo is the production of lambda docR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos . In contrast, the production of lambda docL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by lambda Exo; lambda-generalized transducing particles are not detected in induced lysis-defective (S-) lambda lysogens until about 60-90 min after prophage induction . Since wild-type lambda would normally lyse cells by 60 min, the production of lambda-generalized transducing particles depends on the phage being lysis-defective; if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10-20-fold range . In contrast, if transducing lysates are prepared by the induction of a lambda lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers; if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the amount of prophage-proximal bacterial DNA in the cell . Measurements of DNA in transducing particles indicate that the rest of the increase is probably due to the preferential packaging of the prophage-proximal bacterial DNA . These results are most easily interpreted in terms of a model for the initiation of bacterial DNA packaging by lambda, in which the proteins involved (Ter) do not recognize any particular sequence in bacterial DNA but rather recognize some feature of the DNA tht is sensitive to lambda exonuclease, such as a nick or a double-stranded cut.(ABSTRACT TRUNCATED AT 400 WORDS) Gene, 1986, 50(1-3), 101 - 9 Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu; Patterson TA et al.; We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition . Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage lambda pL promoter . Derepression of pL leads to a high frequency of mini-Mu transposition (5.6 X 10(-2) which is dependent on the presence of the Mu ends and the Mu A and B proteins . Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described. Basic Life Sci, 1986, 40, 265 - 78 Extrachromosomal elements of extrachromosomal elements of Paramecium and their extrachromosomal elements; Quackenbush RL et al.; Expression of killer traits in Paramecium is due to a complex interaction between the lower eukaryote host and two or three elements that can be viewed either as extrachromosomal elements or as endosymbionts . In all cases, the determinants of the killer trait are carried by obligate bacterial endosymbionts belonging to the genus Caedibacter . However, the actual genetic determinants for expression of these traits are not an integral part of the symbiont genome . They are located on extrachromosomal genetic elements (plasmids or bacteriophages) which essentially are molecular endosymbionts of Caedibacter . In the case of the plasmids, they are associated with yet another set of extrachromosomal genetic elements, which are transposons . These transposons have been observed to move into new sites in the plasmids and even to disrupt expression of R body production and the killer trait . Thus, the transposons can be considered either as extrachromosomal elements of extrachromosomal elements (plasmids) of extrachromosomal elements (C . taeniospiralis) of paramecia, or as molecular parasites of molecular endosymbionts (plasmids) of bacterial endosymbionts of paramecia. Gene, 1986, 48(1), 101 - 8 Electron microscopic analysis of in vitro transposition intermediates of bacteriophage Mu DNA; Miller JL et al.; Bacteriophage Mu is a highly efficient transposon and the only moveable element for which an in vitro transposition system has been reported . Recently, this system has been used by Craigie and Mizuuchi {Cell 41 (1985) 867-876} to identify and biochemically characterize intermediates in the transposition process . We have utilized the in vitro transposition system to generate intermediates in the transposition process and have analyzed these intermediates by electron-microscopic methods . Partial denaturation mapping has shown the intermediates to be theta-shaped structures in which the phi X174 target DNA is joined to the mini-Mu plasmid at the ends of the Mu genome . Our results are in agreement with the previous biochemical studies and the type of intermediate we observe is exactly what is predicted by the Shapiro model of transposition {Proc . Natl . Acad . Sci . USA 76 (1979) 1933-1937}. Gene, 1986, 45(3), 311 - 6 Direct expression of urogastrone gene in Escherichia coli; Kishimoto F et al.; Human epidermal growth factor (urogastrone; UG) is a 53-amino acid polypeptide hormone . A 192-bp DNA fragment containing the coding sequence for methionyl UG (Met-UG) and the ribosome-binding site (RBS) was chemically synthesized and placed downstream from the promotor for the Escherichia coli outer-membrane lipoprotein gene (lpp) on a plasmid . E . coli cells harboring the plasmid directed the synthesis of Met-UG at 10(2)-10(3) molecules per cell . Next, the coding sequence for Met-UG was inserted in a runaway-replication plasmid and expressed under the control of the lpp promoter and the RBS derived from bacteriophage Mu cII gene . Upon heat induction, the cells harboring the recombinant plasmid synthesized 10(5) molecules of Met-UG per cell. Gene, 1986, 45(2), 193 - 201 A plasmid expression vector that permits stabilization of both mRNAs and proteins encoded by the cloned genes; Duvoisin RM et al.; Two new expression vectors have been constructed to take advantage of several useful properties of bacteriophage T4-infected Escherichia coli . These plasmids, pRDB8 and pRDB9, contain the promoter region and start codon of T4 gene 32, a contiguous multiple cloning site (MCS), and translation and transcription termination signals . DNA fragments inserted into the MCS are transcribed and translated at a high level in both uninfected and phage T4-infected cells . Furthermore, the extreme stability of the hybrid mRNA after infection permits the specific biosynthetic labeling of the protein encoded by the cloned gene . In addition, the cloned gene product is stabilized, since the host-mediated degradation of foreign proteins is inhibited by phage infection . The properties of this expression system were demonstrated with the constant region of a rabbit immunoglobulin lambda light chain (C lambda) gene . Although proteolytic degradation of the C lambda fusion protein was rapid in uninfected cells, degradation was blocked in phage-infected cells and the protein accumulated in greater amounts. J Inherit Metab Dis, 1986, 9 Suppl 1, 3 - 16 Introduction to recombinant DNA; Scott J; This paper describes the current state of knowledge of methods for analysing gene structure and localization . Illustrations are given of the preparation of complementary DNA libraries and their screening by positive-negative selection, the use of synthetic oligodeoxynucleotides and the use of antibodies . Analysis of the EGF precursor is used as an example to show its close relationship to plasma membrane receptor and its homology with the LDL receptor . Analysis of cloned genome DNA by use of bacteriophage lambda or cosmids gives useful information about gene regulation and evolution . Mutations by frame shift, point or missence mutations are discussed with reference to the LDL receptor and the apolipoproteins . The techniques of gene mapping by rat-human cell hybridization and hybridization in situ are illustrated, again with reference to genes coding for enzymes of cholesterol metabolism, the apolipoproteins and insulin-like growth factors . Finally the potential of in vitro mutagenesis and the injection of cloned DNA into the fertilized mouse ovum are discussed. Gene, 1986, 45(1), 77 - 86 Unusual occurrence of EcoP1 and EcoP15 recognition sites and counterselection of type II methylation and restriction sequences in bacteriophage T7 DNA; Schroeder C et al.; Selected and counterselected oligodeoxynucleotide sequences were identified in the total sequence of bacteriophage T7 DNA using a statistical criterion derived for a probability model of the Markov chain type . All extremely rare tetra- and pentadeoxynucleotides are (or contain) recognition sequences for the Escherichia coli DNA methylases dam or dcm . Most of the 37 hexadeoxynucleotides absent from T7 DNA are recognition sequences for type II modification/restriction enzymes of E . coli or related species . In contrast to most restriction sites counterselected during evolution, the EcoP1 site GGTCT occurs 126 times in the T7 genome, and phage T7 replication is severely repressed in P1-lysogenic host cells . We demonstrate that the frequency of the EcoP1 site is determined by that of the overlapping recognition sites for T7 primase, an essential phage enzyme . The recognition site of a type III enzyme, EcoP15, is also not counterselected . In T7 DNA all 36 EcoP15 sites are arranged in such a manner that the sequence CAGCAG is confined to the H strand, the complementary sequence CTGCTG to the L strand . This "strand bias" is highly significant and, therefore, very probably selected . A functional relation between this strand bias and the refractive behaviour of phage T7 to EcoP15 restriction is suspected. Gene, 1986, 44(1), 133 - 8 Cloning and expression of a chloramphenicol acetyltransferase gene in cytosine-substituted T4 bacteriophage; Noguchi T et al.; We have developed an efficient method for transferring foreign genes into the T4 phage genome . Any foreign genes inserted into the T4 uvsY gene cloned on plasmids can be transferred into a cytosine-substituted T4dC(delta NB5060) phage genome by a replacement type of recombination . To achieve this, we constructed chimeric plasmids which had a chloramphenicol acetyltransferase gene (cat) derived from transposon Tn9 inserted into the Bg/II site within the T4 uvsY gene on pBR322 . The cat gene was then transferred by in vivo recombination into the T4dC(delta NB5060) phage genome . Moreover, it was demonstrated that the cat gene in the hybrid T4dC phage was expressed upon phage infection and development. Gene, 1986, 43(1-2), 59 - 67 The sequence and mom-transactivation function of the C gene of bacteriophage Mu; Heisig P et al.; The mom gene of bacteriophage Mu encodes a DNA modification function . The gene is regulated on the transcriptional level by Dam-specific methylation and a trans-acting Mu function, and on a post-transcriptional level by the product of gene com . The gene encoding the transactivator has been cloned and mapped . By complementation analysis the activation function (also designated Dad) was shown to be the product of gene C . Transactivation of the mom promoter was shown in the following assay: the mom promoter and N-terminal part of com were fused in frame to lacZ . Cells containing such fusion plasmids were infected with M13 clones expressing C in the presence of IPTG and XGal . Successful transactivation results in the formation of blue plaques . Moreover, we have determined the sequence of gene C and found that it has a coding capacity of 140 amino acids . The promoter for C (pc) is likely to be located at least 0.5 kb upstream from the gene . A transcription terminator is found directly downstream from the C-coding region. Gene, 1986, 43(1-2), 155 - 67 Nucleotide sequence and transcription of a human glycine tRNAGCC gene and nearby pseudogene; Pirtle IL et al.; A bacteriophage lambda clone containing a 15.4-kb human DNA fragment was isolated and found to contain a glycine tRNA gene and, 758 bp away, a pseudogene, both with an anticodon of GCC . The nucleotide (nt) sequence of a 1362-bp segment of this clone, encompassing the gene, pseudogene, and their flanking regions, was determined . The gene and pseudogene have an identical sequence of eight nt (5'-CAGCTGGA-3') in their 5'-flanking regions immediately preceding the coding regions, as well as characteristic transcription termination sites of five consecutive T nt in the 3'-flanking regions . Neither of these genes has intervening sequences . Only one of the two genes was efficiently transcribed in vitro by RNA polymerase III in a HeLa cell-free system . During the course of transcription, primary transcripts of one gene were processed to yield mature-sized products . In contrast, the level of transcription of the second gene was significantly less than that of the first, and no mature-sized products could be detected . The nt sequence of the inefficiently transcribed gene has two base substitutions compared to the sequence of the efficiently transcribed gene, and the DNA sequence predicted from the human placental tRNAGlyGCC sequence . One of these nt substitutions is a C to T transition in the TTCG sequence within the B block of the characteristic internal split promoter sequence . The precursor-product relationships of the tRNA transcripts were established by comparing the RNase T1 and RNase A fingerprints of the precursors and products. Gene, 1986, 41(2-3), 315 - 9 Orientation and sequence analysis of right ends and target sites of bacteriophage mu and D108 insertions in the plasmid pSC101; Szatmari GB et al.; We have isolated four independent insertions of the entire 37-kb D108cts 10 genome in the low-copy-number plasmid pSC101 in vivo . They were all formed by replicative transposition during the D108 lytic cycle . The orientation of these four insertions was found to be the same, with the left ends facing towards pSC101 replication, and the right end facing in the direction of all pSC101 transcription, as was previously found for a Mucts62 insertion in pSC101, pMC321 . The exact sites of insertion of two of the D108 prophages, as well as the Mu prophage, have been determined by sequence analysis . All three insertions caused a 5-bp duplication of pSC101 sequences at the target site, as has been found for insertions formed by conservative integration upon lysogeny . Moreover, we have determined the nucleotide sequence of the first 75 bp of the right end of D108 and, though this end is interchangeable with the right end of Mu as a substrate for either phage's transposition functions, there are a number of nucleotide differences between them. Gene, 1986, 41(2-3), 193 - 200 Cloning and expression of the bacteriophage T3 RNA polymerase gene; Morris CE et al.; The gene that encodes the RNA polymerase of bacteriophage T3 (gene 1) has been cloned into a pBR322 derivative under the control of an inducible lacUV5 promoter . Large quantities of the protein are synthesized after induction of cells that carry this plasmid . RNA polymerase purified from these overproducing cells selectively initiates transcription from T3 promoter sequences as demonstrated by transcription of a dual promoter plasmid that carries both T3 and T7 promoters . Cells that carry the T3 RNA polymerase gene can complement amber mutants of T3 that are defective in gene 1 but not gene 1 amber mutants of T7, and vice versa; this experiment demonstrates the specificity of these enzymes in vivo. Gene, 1986, 41(1), 39 - 46 Plasmid pKUN9, a versatile vector for the selective packaging of both DNA strands into single-stranded DNA-containing phage-like particles; Peeters BP et al.; A versatile vector plasmid, pKUN9, has been constructed which, simply by infecting cells harboring this plasmid with either bacteriophage IKe or Ff (M13, fd, and fl), permits the selective packaging of both of its DNA strands into, single-stranded (ss) DNA-containing, phage-like particles . The plasmid, which is a derivative of plasmid pUC9 {Vieira and Messing, Gene 19 (1982) 269-276}, contains in opposite orientations the replication origins and contiguous packaging signals of the distantly related filamentous phages IKe and Ff . As a result of the selective packaging, both strands of a DNA fragment cloned in pKUN9 can be obtained in a single-stranded form and can be sequenced by the dideoxy method using commercially available (+) and (-) sequencing primers . In addition, plasmid pKUN9 possesses all unique properties incorporated in the M13mp phages and the pUC plasmids. Gene, 1986, 41(1), 129 - 34 Ultraviolet irradiation of DNA: a way of generating partial digests for rapid restriction site mapping; Whittaker PA et al.; UV-irradiation of DNA can inhibit the activity of certain restriction endonucleases because of thymine dimer formation within the enzyme recognition sequence . The number of sites affected depends upon the dose of UV, thus making it easier to control the extent of enzyme digestion than by either limiting the digestion time, or the amount of enzyme . Restriction-site maps of bacteriophage lambda recombinants are readily produced by labelling DNA using a radioactive oligonucleotide that is complementary to either the left or right cohesive end of lambda, irradiating the DNA with UV light, limit digesting with the appropriate enzyme, and calculating the size of the fragments detected after gel electrophoresis and autoradiography. Gene, 1986, 41(1), 103 - 11 Expression of immunogenically reactive diphtheria toxin fusion proteins under the control of the pR promoter of bacteriophage lambda; Zettlmeissl G et al.; The tox228 gene encoding the non-toxic, immunologically cross-reactive CRM228 mutant diphtheria toxin (DT) has been cloned downstream of the PR promoter and the cro translational initiation region of bacteriophage lambda carried by plasmid pCQV2 (Queen, 1983) . Efficient transcription but no appreciable amount of a translational product corresponding to complete DT could be detected in Escherichia coli hosts . Deletion of 320 bp from the C-terminal region of the B-fragment of DT, and fusion of the truncated tox228 gene to lacZ yielded several hybrid beta-galactosidases (beta Gal) in an E . coli lon- strain in addition to beta Gal . The various DT fragments fused to beta Gal were immunologically reactive and were identified with antibodies specifically directed against the A- or the B-fragment of DT . Antibodies raised against the DT-beta Gal fusion proteins in guinea pigs cross-reacted with wild-type DT and its B-fragment and protected Vero cells in tissue culture against the lethal action of DT . Immunized guinea pigs survived upon injection of a five-fold lethal dose of wild-type DT. Appl Environ Microbiol, 1986 Jan, 51(1), 211 - 3 Concentration of viruses in beef extract by flocculation with ammonium sulfate; Shields PA et al.; Bacteriophages and enteroviruses in water were adsorbed to positively charged filters (Virosorb 1MDS {AMF Cuno, Inc., Meriden, Conn.} or Seitz S {Republic Filters, Milldaler, Conn.}) . Adsorbed viruses were eluted by treating the filters with 10% beef extract, pH 9 . Organic flocculation of the beef extract at pH 3.5 permitted recovery of more than 40% of the enteroviruses tested but less than 15% of the bacteriophages present . A method was developed that uses salts at pH 7 to flocculate beef extract . Two volumes of saturated ammonium sulfate were added to beef extract, and both enteroviruses and bacteriophages were adsorbed to the flocs that formed . Greater than 70% of the enteroviruses and bacteriophages were recovered by centrifuging the sample and suspending the flocs in a small volume of distilled water. J Cell Biol, 1986 Jan, 102(1), 189 - 93 Alternatively spliced mRNAs code for different polypeptide chains of the chicken neural cell adhesion molecule (N-CAM); Murray BA et al.; Rabbit polyclonal antibodies directed against the chicken neural cell adhesion molecule (N-CAM) were used to isolate four overlapping cDNA clones from a chicken cDNA expression library in bacteriophage gamma gt11 . These clones collectively accounted for 3.8 kilobases of N-CAM mRNA sequence and hybridized specifically to two 6-7-kilobase brain polyadenylated RNA species that co-migrated with previously identified N-CAM mRNAs . DNA fragments derived from an internal region of the cloned cDNA sequences hybridized to the larger but not to the smaller N-CAM mRNA species, while fragments on either side of this region hybridized to both mRNAs . A cDNA fragment that recognized only the larger mRNA was subcloned into gamma gt11, and the expressed fusion protein was used to affinity-purify rabbit polyclonal antibodies; the antibodies recognized only the larger of the two structurally related N-CAM polypeptides . In contrast, when several cDNA clones that recognized both mRNAs were used to purify antibodies, the antibodies recognized both polypeptides . The results, in conjunction with other data indicating that there is one gene specifying N-CAM, suggest that different N-CAM polypeptides are synthesized from multiple N-CAM messages generated by alternative splicing of transcripts from a single N-CAM gene. J Bacteriol, 1986 Jan, 165(1), 219 - 25 Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine; Sladek TL et al.; Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JA1 and K2 cells . The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JA1 cells . This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable . DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JA1 . Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JA1 restricts DNA containing the sequence GAT 5-methylcytosine . We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence . In contrast, JA1 cells (which contain no methylated DNA bases) have a newly discovered type of restriction-modification system . From results of studies of the restriction of specifically methylated DNAs, we conclude that JA1 cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine . This is the first report of a DNA restriction activity specific for a single (methylated) base . Modification in this system is the absence of cytosine methylating activity . A restriction-deficient variant of strain JA1, which retains the JA1 modification phenotype, was isolated, indicating that JA1 cells have a gene product with restriction specificity for DNA containing 5-methylcytosine. J Bacteriol, 1986 Jan, 165(1), 107 - 15 fii, a bacterial locus required for filamentous phage infection and its relation to colicin-tolerant tolA and tolB; Sun TP et al.; We describe mutations in a new bacterial locus, designated fii, which do not allow the filamentous bacteriophage f1 to infect bacteria harboring the F plasmid . Mutations at this locus do not affect the ability of F plasmid-containing bacteria to undergo conjugation or be infected by the F plasmid-specific RNA phage f2 . The filamentous phage can still adsorb to the F sex pilus, but the DNA is unable to enter the bacteria . All fii mutants become tolerant to colicins E1, E2, and E3 . Strains with amber mutations in fii also are unable to plaque P1, even though they can be infected with this phage . Mutations in fii also prevent infection of bacteria harboring the N plasmid by the filamentous bacteriophage IKe . The fii locus maps adjacent to tolA, mutants of which demonstrate tolerance to high levels of the E and K colicins . The three genes tolA, tolB, and fii are shown to reside on a 4.3-kilobase fragment of the Escherichia coli chromosome . Each gene has been cloned into a chimeric plasmid and shown to complement, in trans, mutations at the corresponding chromosomal locus . Studies in maxicells show that the product of fii appears to be a 24-kilodalton protein which copurifies with the cell envelope . The product of tolA has been identified tentatively as a 51-kilodalton protein . Data from cloning, Tn5 mutagenesis, and P1 transduction studies are consistent with the gene order sucA-fii-tolA-tolB-aroG near 17 min on the E . coli map. Gene, 1986, 49(2), 273 - 82 Localization of the gam gene of bacteriophage mu and characterisation of the gene product; Akroyd J et al.; Using cloning techniques in conjunction with an in vitro assay for activity of the gam-coded protein (pgam), the gam gene has been located on a 930-bp fragment immediately to the right of an AccI site situated 5.75 kb from the left-hand end of the phage Mu genome . An analysis of the properties of pgam obtained from an overproducing clone indicates that it is a non-specific DNA-binding protein which interacts with linear duplex plasmid DNA having a variety of different termini and confers protection against exonuclease action (Gam function) . It also stimulates the frequency with which linear plasmid DNA transforms Escherichia coli to antibiotic resistance (Sot function) . The preliminary results reported here suggest that pgam is potentially a useful 'tool' in molecular biology, although the molecular details of pgam activity require further clarification. Gene, 1986, 49(2), 263 - 71 Factors which equalize the representation of genome segments in recombinant libraries; Wyman AR et al.; Genomic segments which contain inverted repetitions longer than 300 bp are frequently lost from recombinant libraries grown on rec+ hosts . We have found that 9% of phage lambda clones that contain 15-20-kb insertions of human or Drosophila DNA are inhibited on rec+ hosts and as a result will become under-represented in amplified genomic libraries . We have therefore examined several factors of both host and vector origin which affect the fidelity of representation of genomic sequences in recombinant DNA libraries constructed in bacteriophage lambda vectors . This loss may be diminished if the vector carries either a chi element or a functional gam gene . The most successful approach, however, involves using a host with mutations in recB, recC, and sbcB, or in recD . We have shown that recombinant clones which require such mutant hosts for growth are somewhat more likely to contain DNA derived from loci in the genome which are polymorphic than are clones recovered on conventional hosts. Gene, 1986, 47(2-3), 221 - 30 Gene fusion in vivo using transductional cointegrates; Struck DK et al.; A method is described which allows the efficient construction of hybrids between homologous genes . The technique is based on the phenomenon of cointegrate transduction in which the homology between cloned sequences present on a bacteriophage lambda vector and a plasmid vector is exploited to allow the packaging of a plasmid-phage recombinant . The size of the cointegrate molecule can be far beyond the normal packaging limit of lambda and still allow the transduction of plasmid-borne drug-resistance markers . This method allows the exchange of the 5' and 3' ends of the participating genes as well as the exchange of sequences residing between the end-points of homology between the two genes . Hybrids of either type were constructed between a sea urchin and a Drosophila actin gene using the transductional cointegrate method in vivo . This approach does not require the use of specialized phage or plasmid vectors and can also be used to screen plasmid libraries with a bacteriophage lambda probe. Gene, 1986, 46(2-3), 171 - 80 Repression of a mutant derivative of the pRE promoter of bacteriophage lambda by its activator, CII; Gussin GN et al.; A 2-bp insertion between the -10 and -35 regions of the pRE promoter of bacteriophage lambda reverses the effect of the activator protein, CII, on transcription from pRE in vitro . The mutant promoter is weakly constitutive in the absence of cII protein and repressed in its presence . This is in sharp contrast to wild-type pRE which is inactive in the absence of cII protein and stimulated at least 1000-fold in its presence (Shih and Gussin, 1984a; McClure and Hoopes, 1985) . These effects are explained by the creation of a new -35 region with weak homology to the -35 consensus sequence for Escherichia coli promoters, and by the altered spatial relationship between the -35 region and the CII-binding site . This interpretation was confirmed by analysis of double mutants containing known cy (pRE) mutations together with the 2-bp insertion . Insertion of 4 bp or deletion of 2 bp completely inactivates pRE in the presence or absence of cII protein, again indicating that activation is dependent upon proper spacing between the -35 region and the transcription start point. Gene, 1986, 43(3), 255 - 63 Selective inhibition of Escherichia coli recBC activities by plasmid-encoded GamS function of phage lambda; Friedman SA et al.; The gam locus of bacteriophage lambda encompasses two coding sequences with the same reading frame and translational stop, one corresponding to an Mr 11646 polypeptide (gamS gene), the other to an Mr 16349 polypeptide (gamL gene) . A DNA segment encoding gamS but not gamL was placed under lambda pR promoter control (regulated by the cIts857-coded repressor) on a multicopy plasmid, and an insertion mutation (gamS201) was constructed . Expression of gamS+, but not gamS201, inhibited Escherichia coli RecBC nuclease in vivo; the criteria were inhibition of chromosomal DNA degradation after UV irradiation and plating of T4 gene 2- phages . The recB+ C+ bacteria expressing gamS+ were completely or partially similar to recC- mutants with respect to certain phenotypes: defective plating of phages P1 and P2, ability to plate (in a recA- background) lambda red- gam- phages, reduced resistance to UV irradiation, defective SOS induction, decreased colony-forming ability. Gene, 1986, 42(3), 345 - 9 Use of the lysis gene of bacteriophage phi X174 for the construction of a positive selection vector; Henrich B et al.; DNA fragments generated by a variety of restriction enzymes can easily be cloned in the small (3.2-kb) positive-selection vector pUH84, which contains the modified lysis gene of bacteriophage phi X174 under transcriptional control of the lac promoter . Plasmid pUH84 does not yield transformants after introduction into Escherichia coli unless the lysis gene is inactivated by insertion of foreign DNA into one of the unique PstI, SalI, AccI, HincII, BamHI, or EcoRI sites . This highly efficient positive selection of recombinants requires neither the use of a distinct host strain nor a special induction of the lysis function . Transcription of fragments cloned into pUH84 may be effectively regulated by the lac promoter provided the host cells are cotransformed with the newly constructed plasmid pUH7 which carries the IQ allele of the lac repressor gene. Biosci Rep, 1986 Jan, 6(1), 103 - 12 The FI gene product of bacterial virus Lambda is related to the E . coli chromosomal protein NS2 and is involved in intracellular DNA organization; Witkiewicz H et al.; A likely function of the Lambda FI gene product (gpFI) is condensation of developmental forms of the bacteriophage DNA in the host cell . Several characteristics of gpFI support this hypothesis: it is similar in its structure and properties to E . coli NS proteins whose involvement in the bacterial DNA condensation has been established and it comigrates with DNA during fractionation of host cell lysate through a sucrose gradient. C R Acad Sci III, 1986, 302(1), 1 - 6 {Constraints acting on the base sequence in a polynucleotide . II . Distribution of complementary tetranucleotides in genes of Escherichia coli and bacteriophages lambda and T7}; Vigier P et al.; The complementary tetranucleotides tend to be equally favoured or avoid in E . coli, lambda and T7 genes . Constraints exist that act equally on nucleotide sequence of each one of both strings of DNA molecule, without any relation with the lecture phase. Proc Natl Acad Sci U S A, 1986 Jan, 83(1), 135 - 9 Cloning an expressed gene shared by the human sex chromosomes; Darling SM et al.; The existence of genes shared by mammalian sex chromosomes has been predicted on both evolutionary and functional grounds . However, the only experimental evidence for such genes in humans is the cell-surface antigen encoded by loci on the X and Y chromosomes (MIC2X and MIC2Y, respectively), which is recognized by the monoclonal antibody 12E7 . Using the bacteriophage lambda gt11 expression system in Escherichia coli and immunoscreening techniques, we have isolated a cDNA clone whose primary product is recognized by 12E7 . Southern blot analysis using somatic cell hybrids containing only the human X or Y chromosomes shows that the sequences reacting with the cDNA clone are localized to the sex chromosomes . In addition, the clone hybridizes to DNAs isolated from mouse cells that have been transfected with human DNA and selected for 12E7 expression on the fluorescence-activated cell sorter . We conclude that the cDNA clone encodes the 12E7 antigen, which is the primary product of the MIC2 loci . The clone was used to explore sequence homology between MIC2X and MIC2Y; these loci are closely related, if not identical. J Bacteriol, 1986 Jan, 165(1), 167 - 74 Translation initiation of bacteriophage lambda gene cII requires integration host factor; Mahajna J et al.; Escherichia coli integration host factor (IHF), a DNA-binding protein, positively regulates expression of the lambda cII gene . Purified IHF stimulates cII protein synthesis in vitro, suggesting a direct role for host factor in cII expression . Further evidence for a direct role for IHF was obtained with operon and gene fusions between cII and lacZ or cII and galE . Analysis of these fusions in vivo demonstrated that IHF is essential for the initiation of cII translation . Replacement of the entire cII coding sequence with lacZ yielded a gene fusion which was still IHF dependent . However, a cII-galE fusion carrying a hybrid ribosome binding region expressed galE in IHF mutants . These results indicate that sequences which make cII translation IHF dependent lie between the ribosome binding region and the initiating codon of cII . Failure to translate cII activates a transcription terminator located within cII and results in polar effects on downstream transcription . This polarity is suppressed by the lambda N antitermination function . When cloned into another context, the terminator is active in both wild-type and IHF mutant strains . The amino terminus of cII is located near an IHF binding site in a region with considerable dyad symmetry . The role of IHF in cII translation may be to prevent formation of an RNA-RNA duplex that sequesters the ribosome binding site of cII . The binding of IHF might influence RNA structure by altering the rate of the dissociation of RNA from the DNA template. Gene, 1986, 48(2-3), 183 - 93 A plasmid vector for cloning and expression of gene segments: expression of an HTLV-I envelope gene segment; Sisk WP et al.; We describe a cloning-expression vector system for selecting DNA fragments containing open reading frames (ORFs) and expressing them as beta-galactosidase (beta Gal) hybrid fusion proteins . The plasmid vector, pWS50, utilizes the very strong and easily regulated bacteriophage lambda promoter pL, and the efficient translation initiation signals of the N-terminal segment of the lambda cII gene . Fused distally to and out of translational phase with cII is the E . coli lacZ gene, lacking its own transcriptional and translational initiating signals . A unique restriction enzyme site (NruI) is located between the upstream regulatory sequences and the lacZ gene, which provides a cloning site for the insertion of blunt ended DNA fragments . In addition, there are two other unique restriction sites (NheI and BamHI) located in this region which can also be used as closing sites . If a DNA fragment does not contain any translation termination codons (i.e., an ORF), and is inserted correctly into the vector, the translational reading frame between cII and lacZ can be restored . Colonies containing these recombinants can be easily screened as LacZ+ on lactose indicator media . The beta-galactosidase fusion proteins produced from the LacZ+ recombinants are identified on sodium dodecyl sulfate polyacrylamide gels by their large size and high level of production . To test the ORF cloning-expression system, a segment of the human T-cell lymphotrophic virus type I envelope gene was cloned and expressed at high levels . The envelope-beta Gal fusion protein was recognized by antibodies in serum from a patient with adult T-cell leukemia. Gene, 1986, 41(2-3), 241 - 7 Synthesis of maize chloroplast ATP-synthase beta-subunit fusion proteins in Escherichia coli and binding to the inner membrane; Gatenby AA et al.; The maize chloroplast gene for the beta subunit (atpB) of the chloroplast CF1 component of ATPase from maize, when fused to either the lacZ or ral genes in the vectors pMC1403 or pHUB4, is expressed in Escherichia coli as a fusion protein with beta-galactosidase or with bacteriophage lambda Ral sequences . Some of the fusion proteins are converted to a membrane-bound form, as determined by differential and sucrose-gradient centrifugation . The specificity of membrane binding has been examined using E . coli unc mutants that are defective in binding of the F1 ATPase component to the F0 receptor site on the membrane, and by the use of two different length maize atpB::lacZ gene fusions . We show that the first 365 N-terminal amino acids (aa) of the maize beta subunit are involved in binding to the E . coli inner membrane, and that this binding is probably mediated by the bacterial F0 receptor. J Virol, 1986 Jan, 57(1), 258 - 66 Effects of bacteriophage fd infection on Escherichia coli HB11 envelope: a morphological and biochemical study; Bayer ME et al.; Phage fd-infected host bacteria revealed three characteristic changes in their envelope . (i) The preferred cleavage plane during freeze-fracturing shifted from the inner to the outer membrane (OM) . (ii) The total lipids of the OM of the infected cells increased by 25% without major alterations in the relative concentration of phospholipids . We propose that such an increase would to some extent contribute to the change in the freeze-fracture behavior of the OM; however, additional factors will have to play a role in the apparent fracture resistance of the inner membrane . (iii) Ultrathin sectioning and immunolabeling methods revealed that extrusion of fd phages takes place at membrane adhesion sites of the infected cells. Basic Life Sci, 1986, 38, 281 - 6 The repair of pyrimidine dimers via a DNA-glycosylase mechanism; Grafstrom RH; The "UV endonuclease" isolated either from M . luteus or bacteriophage T4 infected E . coli (the denV gene product) consists of two enzymatic activities on a single polypeptide chain: a pyrimidine dimer-DNA glycosylase and an AP endonuclease . The repair of pyrimidine dimers by this enzyme is initiated by the cleavage of the N-glycosylic bond of the 5' pyrimidine of the dimer that leaves the cyclobutane dimer still attached to the DNA through the N-glycosylic bond of the 3' pyrimidine of the dimer . This reaction results in the formation of an apyrimidinic site in the DNA . The second step in this repair pathway is the endonucleolytic cleavage of the DNA 3' to the AP site by the associated AP endonuclease . As a result, the nicked DNA contains DNA damage on both sides of the incision site: an apyrimidinic moiety on the 3' end and a thymine-thymidylate dimer on the 5' end . The enzymes prefer double stranded DNA over single stranded DNA, and thymine over cytosine at the 5' position of the dimer . The AP endonuclease activity prefers the AP site created by the pyrimidine dimer-DNA glycosylase on UV irradiated DNA over either apurinic or apyrimidinic DNA . This repair mechanism appears to be operative in vivo since DNA intermediates containing thymine-thymidylate dimer sites have been detected in UV irradiated T4 infected E . coli and in UV irradiated M . luteus . The cloned denV gene partially complements the UV repair deficient uvr A, B, C strains of E . coli. Gene, 1986, 41(1), 93 - 102 RNA splicing and in vivo expression of the intron-containing td gene of bacteriophage T4; Belfort M et al.; The splice junction sequence of td mRNA from T4-infected cells has been determined (5'....GGU-CUA....3') and shown to be identical to that of the RNA ligation product encoded by the cloned gene {Belfort et al . Cell 41 (1985) 375-382} . The RNA processing functions, T4 RNA ligase, T4 polynucleotide kinase, and the host prr gene product appear not to be essential for exon ligation; neither are the host endoribonucleases RNase III, RNase P and RNase E required for intron excision . While these results are consistent with the autocatalytic splicing mechanism demonstrated in vitro {Chu et al . J . Biol . Chem . 260 (1985) 10680-10688}, they leave unanswered the question of which protein(s), if any, might stimulate the in vivo reaction . Analysis of the products of the cloned td gene has led to identification of two td-encoded polypeptides, namely a polypeptide corresponding to the exon-I-coding sequence (NH2-TS), and the catalytically active thymidylate synthase (TS) . Kinetic and nucleotide sequence data provide evidence that NH2-TS is the product of the primary transcript and that TS is encoded by spliced mRNA . These results suggest that splicing may provide a switch controlling the relative expression of NH2-TS and TS, two proteins with markedly different temporal appearances despite their identical transcriptional and translational start sites. J Mol Biol, 1985 Dec 20, 186(4), 707 - 13 Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli . I . Primary structure of a pyrI gene encoding a modified regulatory subunit; Cunin R et al.; In a previous article, we have identified a lambda bacteriophage directing the synthesis of a modified aspartate carbamoyltransferase lacking substrate-co-operative interactions and insensitive to the feedback inhibitor CTP . These abnormal properties were ascribed to a mutation in the gene pyrI encoding the regulatory polypeptide chain of the enzyme . We now report the sequence of the mutated pyrI and show that, during the generation of this pyrBI-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene . A model is presented for the formation of this modified pyrI gene during the integrative recombination of the parental lambda phage with the Escherichia coli chromosome . An accompanying paper emphasizes the importance of the carboxy-terminal end of the regulatory chain for the homotropic and heterotropic interactions of aspartate carbamoyltransferase. J Mol Biol, 1985 Dec 20, 186(4), 759 - 71 Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes; Feiss M et al.; Bacteriophage lambda chromosomes are packaged in a polarized, sequential fashion from a multimeric DNA substrate . Mature chromosomes are generated when terminase introduces staggered nicks in the cohesive end sites (cos sites) bounding a chromosome . Packaging is polarized, to the initial and terminal cos sites for packaging a chromosome can be defined . To initiate packaging, terminase binds to cos at cosB, and subsequently cuts at cosN . To terminate packaging of a chromosome, a functional cosB is not required at the terminal cos . To explain this finding, it was proposed earlier that terminase scans for the terminal cosN, rather than any subsequent cosB, during packaging . In the work described here we performed helper packaging experiments to see whether processive action of terminase occurs during sequential packaging of lambda chromosomes . The helper packaging experiments involve trilysogens; strains carrying three prophages in tandem . Infection by a hetero-immune helper phage results in packaging of the repressed prophage chromosomes, since the prophage structure is analogous to the normal DNA substrate . Two chromosomes can be packaged from between the three cos sites of the prophages of a trilysogen . Both chromosomes are packaged even when the central cos is cosB- . Our interpretation of these data is that terminase is brought to the central cos by packaging; following cleavage of the central cos, the terminase remains bound to the distal chromosome; and terminase acts to begin packaging of the distal chromosome . The frequency at which terminase reads across the central cos to initiate packaging of the distal chromosome is in the range from 0.3 to 0.5 in our experiments . Reading across cos was found not to be greatly dependent on the state of cosB, indicating that cosB binding is only needed for packaging the first chromosome in a packaging series . A multilysogen was constructed in which the initial cos was cos+ and the distal cos sites were all cosB- . The initial and downstream chromosomes were found to be packaged . This result indicates that terminase that is brought to the central cos by packaging is not only able to initiate packaging of a downstream chromosome, but can also scan and terminate packaging of the downstream chromosome . A model is presented in which processive action of terminase is the basis for sequential packaging of lambda chromosomes. J Biol Chem, 1985 Dec 15, 260(29), 15907 - 13 Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage; Kuhn A et al.; The major coat protein (gene 8 protein) of bacteriophage M13 has been studied intensively as a model of membrane assembly, protein packing, and protein-DNA interactions . Because this protein is essential for assembly of the phage, very few mutants have been isolated . We have therefore cloned the gene 8 into a plasmid under control of the araB promoter . In the presence of arabinose, the cloned gene is expressed at a rate comparable to that in an M13-infected cell . Plasmid-derived procoat is inserted across the plasma membrane and processed to coat at a normal rate . The coat can support plaque formation by a defective M13 virus (M13am8) with an amber mutation in its procoat gene . This complementation assay was used to screen the mutageni