Mol Gen Genet, 1987 Jun, 208(1-2), 52 - 6 DNA sequences of bacteriophage P2 early genes cox and B and their regulatory sites; Haggard-Ljungquist E et al.; Part of the early operon of the temperate phage P2 of Escherichia coli, including genes cox (involved in prophage excision) and B (required for phage specific DNA synthesis), was sequenced . The results are consistent with an early promoter spanning the repressor binding sites, a leader sequence of about 80 bases which overlaps the leader sequence of the repressor gene for about 30 bases, and coordinate transcription of genes cox and B with a termination signal after the B gene . In addition, the data provide amino acid sequences for the Cox and B proteins of 91 and 166 residues, respectively and reveal a hitherto undetected coding sequence between genes cox and B that has the potential to produce a very basic polypeptide of 56 residues . Slight structural similarities between the P2 Cox protein and the analogous Xis protein of phage lambda were noted and the P2 B gene product was compared with proteins that interact with the DnaB protein of E . coli. Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 3997 - 4001 Ubiquitous upstream repression sequences control activation of the inducible arginase gene in yeast; Sumrada RA et al.; Expression of the yeast arginase gene (CAR1) responds to both induction and nitrogen catabolite repression . Regulation is mediated through sequences that both positively and negatively modulate CAR1 transcription . A short sequence, 5'-TAGCCGCCGAGGG-3', possessing characteristics of a repressor binding site, plays a central role in the induction process . A fragment containing this upstream repression sequence (URS1) repressed gene expression when placed either 5' or 3' to the upstream activation sequences of the heterologous gene CYC1 . Action of the URS and its cognate repressor was overcome by CAR1 induction when the URS was situated cis to the CAR1 flanking sequences . This was not observed, however, when it was situated downstream of a heterologous CYC1 upstream activation sequence indicating that URS function is specifically neutralized by cis-acting elements associated with CAR1 induction . Searches of sequences in various gene banks revealed that URS1-like sequences occur ubiquitously in genetic regulatory regions including those of bacteriophage lambda, yeast, mammalian, and viral genes . In a significant number of cases the sequence is contained in a region associated with negative control of yeast gene regulation . These data suggest the URS identified in this work is a generic repressor target site that apparently has been conserved during the evolution of transcriptional regulatory systems. Exp Parasitol, 1987 Jun, 63(3), 272 - 8 Toxoplasma gondii and Hammondia hammondi: DNA comparison using cloned rRNA gene probes; Johnson AM et al.; A mung bean nuclease genomic library of purified DNA from tachyzoites of the RH strain of Toxoplasma gondii was prepared in the bacteriophage lambda gtll and recombinants containing rRNA gene fragments were detected by hybridization with radiolabeled total RNA from the closely related coccidian Eimeria acervulina . Ten recombinants were chosen at random, and five of these were investigated further using probes for the genes of the large and small rRNA of Plasmodium berghei . An insert (called TG4) that hybridized only to the 3' end of the large rRNA coding region of P . berghei and an insert (called TG18) that hybridized only to the small rRNA coding region of P . berghei were purified by electrophoresis in low melting point agarose . Radiolabeled E . acervulina total RNA, TG4, and TG18, were then used to compare the sizes of the large and small rRNA gene fragments after DNA extracted from three strains of T . gondii, and the type strain of the closely related coccidian Hammondia hammondi were cut by one of a series of 10 restriction endonucleases . The patterns obtained for the three T . gondii isolates were identical to those obtained for H . hammondi, for each enzyme tested . In addition, the guanine plus cytosine (G + C) content of H . hammondi DNA was found to be almost identical to that obtained previously for T . gondii DNA. Virology, 1987 Jun, 158(2), 414 - 26 The functional boundaries of the Q-utilization site required for antitermination of late transcription in bacteriophage lambda; Somasekhar G et al.; Expression of the late genes of bacteriophage lambda requires, in addition to the host functions, the lambda p'R promoter, the antiterminator sequence qut, and the product of gene Q which interacts with the Q utilization (qut) site . In the absence of the Q function or qut site, the p'R-initiated transcription is blocked by the t'R terminator at the 194th nucleotide downstream of the start point, s'R, producing a short 6 S mRNA . In this study the position and boundaries of the qut site were deduced by constructing plasmids containing various portions of the p'R-qut region, the t'R1 terminator, and the reporter gene galK . We measured galK gene expression in response to the gamma Q gene product supplied in trans by a prophage or Q-expression plasmid . We show that among the lambda proteins, the Q gene product alone is necessary and sufficient for complete qut-mediated transcription antitermination in vivo . These antitermination experiments, employing plasmids that contain different lengths of lambda p'R-qut sequence, identified the right boundary of the qut site, which is located between +4 and +18 (for s'R = +1) . The functional left boundary of qut does not extend upstream from the -26th nucleotide of the p'R promoter, as based on the following experiments . The promoter function of the truncated (-26)p'R-s'R-(+18) sequence can be restored by fusion to the complete but qut-less p'R, pp, or PLac promoter; however, no antitermination was observed for such a p-(-26)p'R-s'R-(+18)-t'R-galK plasmid . Thus we conclude that the qut site partially overlaps with the p'R promoter sequence . However, promoters that contain the -10 region of p'R, s'R, and the +1 to +18 qut sequence did mediate Q-dependent antitermination when properly fused to the homologous or heterologous -35 promoter regions . Only those transcripts that start at s'R (+1 or very near to it) and also contain at least the first 18 nucleotides (actually greater than 4 and less than or equal to 18) of 6 S RNA appear to be a target for the Q-qut-mediated transcription antitermination, which acts not only at t'R but also at other Rho-independent or Rho-dependent terminators. Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4049 - 53 Role of homology in site-specific recombination of bacteriophage lambda: evidence against joining of cohesive ends; Nash HA et al.; Bacteriophage lambda integration and excision take place at specific loci called attachment sites . Earlier work has shown that efficient recombination requires the identical sequence to be present in both attachment sites throughout the seven-base-pair region between the points of strand exchange . A plausible model for the role of homology postulates that Int, the site-specific recombinase, makes double-strand breaks at attachment sites such that each broken end has a short single-strand protrusion . Recombination would then depend upon the capacity of these protrusions to form Watson-Crick helices--i.e., to anneal--a process that might require perfect complementarity between the cohesive ends . To test this model, we have studied Int-promoted crosses in which one attachment site is a heteroduplex . Specifically, we constructed sites in which the seven-base-pair region between the points of strand exchange contains one or more noncomplementary pairs . The double-strand break and annealing mechanism predicts that crosses with these heteroduplex sites should yield one completed recombinant and one broken site . We find that such nonreciprocal recombination is uncommon and that the typical outcome of crosses involving a heteroduplex site is a reciprocal recombinant in which both products are resealed . Moreover, the occasional appearance of nonreciprocal products can be explained by our finding that Int can cleave heteroduplex attachment sites after recombination is completed . Taken together, our data strongly indicate that bacteriophage lambda recombination does not proceed by the homology-dependent annealing of cohesive ends; acceptable alternatives for the role of homology are discussed. J Gen Microbiol, 1987 Jun, 133 ( Pt 6), 1631 - 9 glnA mutations conferring resistance to methylammonium in Escherichia coli K12; Servin-Gonzalez L et al.; Cells of Escherichia coli K12 were sensitive to 100 mM-methylammonium when cultured under nitrogen limitation, and resistant when grown with an excess of either NH4Cl or glutamine . Glutamine synthetase activity was required for expression of the methylammonium-sensitive phenotype . Mutants were isolated which were resistant to 100 mM-methylammonium, even when grown under nitrogen limitation . P1 bacteriophage transduction and F' complementation analysis revealed that the resistance-conferring mutations mapped either inside the glnA structural gene and/or elsewhere in the E . coli chromosome . Glutamine synthetase was purified from the wild-type and from some of the mutant strains . Strains carrying glnA-linked mutations that were solely responsible for the methylammonium-resistant phenotype yielded an altered enzyme, which was less active biosynthetically with either ammonium or methylammonium as substrate . Sensitivity to methylammonium appeared to be due to synthesis of gamma-glutamylmethylamide by glutamine synthetase, which was synthesized poorly, if at all, by mutants carrying an altered glutamine synthetase enzyme. Mol Gen Mikrobiol Virusol, 1987 Jun, (6), 38 - 42 {Host-dependent modifications of bacteriophage T3 expressing changes in adsorption properties (serological study)}; Gacheniladze KK et al.; The ability of the bacteriophage T3 to adsorb on the host cells of Escherichia coli W1655 changes depending on the host strain in which the phage was propagated before . This phenomenon is termed "non-classical" host-controlled modification in contrast to "classical" DNA modification . We demonstrate here that T3 phages with various non-classical modifications as well as the host range mutant T3hW differ from each other in the antigenic determinants of the phage adsorption protein. Virology, 1987 Jun, 158(2), 361 - 72 Expression of Japanese encephalitis virus antigens in Escherichia coli; Mason PW et al.; The expression of Japanese encephalitis virus (JE) cDNA in Escherichia coli has been used to study the functional organization of the viral genome . JE protein coding sequences were expressed in E . coli by subcloning random fragments of cloned cDNA (P.C . McAda, P.W . Mason, C.S . Schmaljohn, J.M . Dalrymple, T.L . Mason, and M.J . Fournier, 1987, Virology 158, 348-360) into the bacteriophage lambda gt11 expression vector . Over 120 lambda gt11 recombinants expressing viral protein sequences as beta-galactosidase fusion proteins were identified immunologically with monoclonal antibodies (MAbs) and polyclonal hyperimmune mouse ascites fluid (HMAF) . This expression and immunological detection strategy has been used to (1) map viral protein coding sequences to the JE genome; (2) demonstrate that contiguous viral protein coding regions can be expressed as single polypeptides in E . coli, providing functional confirmation for a long viral open reading frame; (3) localize important antigenic domains within the envelope protein E; and (4) identify in JE-infected cells a form of the glycosylated nonstructural protein NS1 that contains a hydrophobic C-terminal extension encoded by portions of the "ns2a" region of the JE genome. Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4171 - 5 Bacteriophage lambda cloning system for the construction of directional cDNA libraries; Meissner PS et al.; We have developed a bacteriophage lambda cloning vector, lambda ORF8, that can be used for the construction of cDNA libraries . The wild-type lambda genome contains five BamHI, five EcoRI, and seven HindIII restriction sites that have all been removed from the genome of lambda ORF8 . Sites for these endonucleases are present within the multiple cloning site of lambda ORF8 . We report a method for preparing cDNAs that can be cloned in a single orientation in our phage vector . The method utilizes the synthesis of double-stranded cDNA, including priming of first-strand synthesis by oligo(dT) . After completion of second-strand synthesis, a bifunctional oligodeoxynucleotide linker is ligated to the cDNA fragments . This linker, which contains a BamHI restriction site, will create a HindIII restriction site when ligated to the 3' end of cDNA fragments . Subsequent treatment of methylated cDNA with HindIII and BamHI endonucleases allows these fragments to be cloned directionally into lambda ORF8 . To demonstrate the utility of this cloning system, we prepared a library from 5 micrograms of mRNA isolated from phytohemagglutinin-stimulated human peripheral blood lymphocytes . The primary library contained 2 X 10(8) plaque-forming phage, at least 80% of which contain inserts . A portion of the library was examined for the presence of gamma-interferon-related clones to verify the method had generated a library that was representative of phytohemagglutinin-stimulated peripheral blood lymphocytes . This simple and efficient cDNA cloning system significantly reduces the amount of RNA and effort required for the preparation of large directionally cloned libraries. Biochem Biophys Res Commun, 1987 May 29, 145(1), 330 - 5 Pseudogene in the genome of bacteriophage lambda? Kypr J, Mrazek J. We find a region in the non-coding part of bacteriophage lambda genome that codes for the conserved fold which repressors and other proteins use for specific DNA binding . The region is involved in a long open reading frame exceeding one kilobase and is read in the same frame as gene A in the opposite strand . The putative translation product of this open reading frame has a highly ordered secondary structure with a predominance of alpha helices, which is typical of repressors . In addition, codon usage in this frame suggests a protein-coding region . However, there is a TGA stop codon located between the putative gene start point and the region coding for the DNA binding fold . It thus appears that bacteriophage lambda had one more DNA binding protein, perhaps repressor, in the past that was inactivated by a mutation. Nature, 1987 May 21-27, 327(6119), 252 - 4 Interactions between DNA and coat protein in the structure and assembly of filamentous bacteriophage fd; Hunter GJ et al.; Bacteriophage fd is a class I filamentous virus (others are M13 and f1) that comprises a circular, single-stranded DNA molecule enclosed in a cylindrical protein sheath to form a flexible particle approximately 890 nm long and 7 nm in diameter . The viral DNA contains 6,408 nucleotides incorporating 10 genes, and the protein sheath is composed of about 2,700 major coat protein subunits in a shingled helical array, the symmetry of which is defined by a fivefold rotational axis combined with a twofold screw axis of pitch 3.2 nm . The DNA extends throughout the length of the particle but is not base-paired and has a symmetry different from that of the protein helix . How the DNA is packed remains unclear but the number (2.4) of nucleotides packaged per major coat protein subunit is certainly not integral, in contrast with, say, the packaging of RNA in tobacco mosaic virus . The coat protein subunit is 50 amino-acid residues in length and, in the virus particle, adopts a largely alpha-helical conformation, with the long axis of the helix aligned close to the long axis of the filament . This protein is arranged with its negatively charged N-terminal region on the outside of the filament and its positively charged C-terminal region on the inside abutting the DNA . We report here that positive charge on one of the four lysine side chains in the latter region has a direct effect on DNA packaging, because when this charge is absent, elongated particles are produced with lengths that can be correlated with the residual positive charge in the C-terminal region of the coat protein subunit. J Mol Biol, 1987 May 20, 195(2), 323 - 31 Interaction of the bacteriophage P22 Arc repressor with operator DNA; Vershon AK et al.; Are repressor binds to a single, partially symmetric, 21 base-pair operator site that is centered between the -10 and -35 regions of the Pant promoter . Protection and interference experiments show that Arc makes contacts with the operator on one side of the DNA helix . Although Arc is a small protein (53 residues/subunit), it makes contacts that are farther from the center of the operator than those made by many larger repressors . These extended contacts include the phosphate groups at the ends of the 21 base-pair site . Under standard conditions (pH 7.5, 100 mM-KCl, 3 mM-MgCl2, 22 degrees C) half-maximal operator binding is observed at an Arc concentration of 2.5 X 10(-9) M and the protein-DNA complex is very stable (t1/2 approximately equal to 80 min). J Mol Biol, 1987 May 20, 195(2), 311 - 22 Bacteriophage P22 Mnt repressor . DNA binding and effects on transcription in vitro; Vershon AK et al.; We have examined the binding of Mnt repressor to operator DNA in vitro and have determined how this binding affects the level of transcription from two nearby promoters, Pant and Pmnt . Mnt binds to a region of DNA that overlaps the startpoint of transcription of Pant and the -35 region of Pmnt . Mnt represses transcription in vitro from Pant and enhances transcription from Pmnt . Protection and interference experiments show that Mnt binds to a single, 17 base-pair operator site . The operator sequence and the protein-DNA contacts are symmetric . Mnt makes major groove contacts on both faces of the operator DNA . At pH 7.5, 200 mM-KCl, 22 degrees C, the Mnt tetramer binds operator with high affinity (Kd = 2.2 X 10(-11M) and the protein-DNA complex is quite stable (t1/2 = 48 min) . Operator binding shows large dependencies on pH, salt concentration, and temperature. J Mol Biol, 1987 May 20, 195(2), 439 - 45 Bacteriophage P4 DNA replication . Nucleotide sequence of the P4 replication gene and the cis replication region; Flensburg J et al.; A 3100 base piece of DNA from the 11,500 base genome of bacteriophage P4 was analyzed for its nucleotide sequence . This segment of DNA contains two open reading frames of 106 and 777 amino acid residues; the latter of which is the coding sequence for the Mr 84,841 alpha protein, which is necessary for P4 DNA replication and is thought to act as a P4-specific DNA primase . A region of about 300 base-pairs localized just beyond the alpha gene and about 4500 bases from the origin of replication (ori), was defined as the locus for P4's cis replication region (crr) . This region is required for replication both in vivo and in vitro, and consists of two directly repeated sequences of 120 base-pairs that match one another at 98 positions . These directly repeated sequences are separated by 60 base-pairs, which are not necessary for replication . Each repeat in crr contains three copies of the octamer TGTTCACC that is found six times in ori . Either of the 120 base-pair repeat sequences in crr is sufficient for replication, and the entire crr can function in an inverted orientation . crr is also active at a distance of 1800 bases from the P4 origin of replication. Cell, 1987 May 8, 49(3), 347 - 56 Correct integration of retroviral DNA in vitro; Brown PO et al.; We have developed a cell-free system for studying the integration of retroviral DNA . In our assay, amber mutations in a bacteriophage lambda genome that serves as the target for integration are suppressed by integration of an MLV derivative that carries the E . coli supF gene . The structure of the reaction products is that expected from an authentic MLV integration reaction . Linear viral DNA from the cytoplasm of infected cells serves as a precursor, though not necessarily the immediate precursor, to the provirus integrated in vitro . The viral DNA in the infected cell appears to be tightly associated with the enzymatic machinery required for its integration . Supercoiling, chromatin structure, transcription, and replication are not required of the target DNA . Since no high-energy cofactor is necessary, the DNA breakage and joining steps in the integration reaction are probably coupled. Nature, 1987 May 7-13, 327(6117), 73 - 5 Determination of bacteriophage lambda tail length by a protein ruler; Katsura I; How the size and shape of living structures are determined by genetic information is one of the fundamental problems in biology . Here I describe a study in which the size of a biological supramolecular structure was changed in a predictable way by in vitro genetics, with the size both before and after manipulation being exactly determined . I have studied the tail of bacteriophage lambda, whose length is determined by the length of the 'ruler protein', the product of gene H . The length of the tail can be decreased or increased by deleting the middle part of gene H or by forming a small duplication there, and the length of the tail is proportional to the size of the protein . These results can be regarded as a special case of protein engineering, namely supramolecular protein engineering. Biochemistry, 1987 May 5, 26(9), 2465 - 71 A study of side reactions occurring during synthesis of oligodeoxynucleotides containing O6-alkyldeoxyguanosine residues at preselected sites; Borowy-Borowski H et al.; As part of our studies on the molecular mechanisms of mutation by carcinogens we have synthesized 12 oligonucleotides (15-mers) containing an O6-alkylguanine residue at a preselected position for use as primers in the enzymatic synthesis of biologically active DNA . Ten of these oligonucleotides are derived from a minus strand sequence carrying the modified nucleotide in the third codon of gene G of bacteriophage phi X174 DNA . Two others are derived from plus strand sequences carrying the modification in the 12th codon of the human Ha-ras protooncogene . During this work several potentially serious side reactions, which could complicate interpretation of mutagenesis data, were observed . This paper describes a detailed study of these reactions . Since we were unable to avoid undesirable side products, we developed simple chromatographic methods for detecting and removing them. Biokhimiia, 1987 May, 52(5), 707 - 14 {Cloning of DNA complementary to mRNA for proopiomelanocortin from the bovine, rat and human hypophysis . Hormonal regulation of proopiomelanocortin mRNA in the rat hypophysis}; Mertvetsov NP et al.; Cloning of DNA and complementary mRNA of bovine, rat and human proopiomelanocortin (POMC) was carried out . A structural analysis of the cloned cDNA of POMC was performed . Using restriction fragments of bovine, rat and human POMC cloned cDNA, probes for molecular hybridization based on one-chain bacteriophage M13 were made . Using the dot-hybridization technique with labeled {32P} POMC cDNA, the effect of 17 beta-estradiol and adrenalectomy on the POMC mRNA level in rat hypophysis was studied . The hormone was shown to significantly decrease the POMC mRNA content at a concentration of 4 and 8 micrograms per 100 g of body weight 4 hours after injection . Adrenalectomy caused a 2-3-fold increase in the POMC mRNA level in rat hypophysis after a period of 5-7 days . The possibility of polyhormonal control of POMC genome transcription is discussed. Microb Pathog, 1987 May, 2(5), 319 - 26 Serotyping and genotyping of encapsulated Escherichia coli K1 sepsis isolates with a monoclonal IgG anti K1 antibody and K1 gene probes; Frosch M et al.; Among infectious diseases caused by E . coli the capsular type K1 plays a predominant role . E . coli K1 isolates account for 80% of cases of E . coli neonatal meningitis and 30% of E . coli sepsis strains . Serotyping of K1 strains has conventionally relied upon the use of K1-specific bacteriophages or serum agar methods with polyvalent anti K1 serum . In the study present here, 187 E . coli sepsis isolates have been analysed for production of the K1 antigen using K1 phages, K1 serum agar plates and Latex agglutination and ELISA using an IgG2a anti K1 monoclonal antibody . In total, 33 sepsis isolates (about 18%) were identified as K1 positive, with three of these strains proving negative in all tests except those exploiting the monoclonal antibody . That these three strains elaborate the K1 antigen was confirmed by Southern blot experiments using cloned K1 antigen production genes as probes . The failure of the three strains in all the tests except those that use monoclonal antibody could be explained by apparent disruption of K1 gene sequences that encode functions essential for the export of capsular material to the cell surface . The superiority of tests based on monoclonal antibodies above the conventional methods for detection of K1 antigen is evident. J Gen Microbiol, 1987 May, 133 ( Pt 5), 1127 - 35 Factors influencing the survival and multiplication of bacteriophages in calves and in their environment; Smith HW et al.; Seven phages were fairly susceptible in vitro to the lethal effect of acidified whey, more so than the enteropathogenic Escherichia coli strains on which they were active . The low acidity that prevailed in the abomasum contents of calves shortly after a milk feed had little harmful effect on orally administered organisms of these phages; they flooded into the small intestine . The high acidity that prevailed later was lethal to orally administered phage organisms; few entered the small intestine . The lethal effect could be counteracted by giving CaCO3 in the feed . Low concentrations of phage-neutralizing antibodies were found in some serum samples from human beings, cattle and pigs . Antibodies to one of the seven phages were common in the human samples and antibodies to another, phage B44/1, were common in the cattle and pig samples and in bovine colostrum . Phage B44/1 antibodies in a sample of colostral whey were destroyed at pH 3.25 or less . Giving colostrum containing phage B44/1 antibodies with CaCO3 to a calf greatly reduced the numbers of orally administered phage B44/1 organisms in its alimentary tract . Antibodies to another phage were induced in the serum of a calf suffering from E . coli diarrhoea by treating it with that phage . The phages were as susceptible as the E . coli strains to the lethal action of formaldehyde and sodium hypochlorite . In contrast to the E . coli strains, they were almost completely resistant to phenol and chloroxylenol . The in vitro virulence of 21 phages varied according to the temperature at which tests were performed.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1987 May, 207(2-3), 517 - 8 Nonsense suppression context effects in Escherichia coli bacteriophage T4; Edelmann P et al.; Nonsense suppression by supE44 has been examined in a collection of 14 T4 gene 22 and gene 23 UAG mutants, for which the precise gene location is known . In concordance with previous studies, UAG followed by a pyrimidine was inefficiently suppressed . However, among positions with similar 3' nucleotides, there was considerable variation in suppression efficiency . The competition between supE44 and Release Factor 1 (RF 1) was also investigated following the introduction of a multicopy RF 1 plasmid . An inverse relationship between the efficiency of suppression and RF 1 competition was observed. Mol Gen Genet, 1987 May, 207(2-3), 224 - 32 Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli; Shinedling S et al.; Mutant ribosome binding sites of the bacteriophage T4 rIIB gene, resident on an 873 bp DNA fragment, were cloned into a plasmid vector as in-frame fusions to a reporter gene, beta-galactosidase . The collection of mutations included changes in the region 5' to the Shine/Dalgarno sequence, a mutation of the Shine/Dalgarno sequence, the alternate initiation codons GUG, AUA and ACG, and mutants in which several closely spaced initiation codons compete with each other on the same mRNA . The results show that the secondary structure variations we have installed 5' to the Shine/Dalgarno sequence have little effect on translation . GUG is essentially as good an initiator of translation as AUG when they are assayed on separate messages, but is outcompeted at least 50-fold in the sequence AUGUG . AUA and ACG are poor start codons, and are temperature sensitive . The initiation codon pair AUGAUA, in which the AUG is only two nucleotides from the Shine/Dalgarno sequence, displays a novel cold-sensitive phenotype. J Bacteriol, 1987 May, 169(5), 2158 - 64 Regulation of the aroH operon of Escherichia coli by the tryptophan repressor; Grove CL et al.; Regulation of expression of aroH, the structural gene for the tryptophan-sensitive 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase, by the tryptophan repressor and its corepressor, L-tryptophan, was studied in vivo by using aroH-lacZ fusions . Protein and operon fusions were constructed on multicopy plasmids and subsequently crossed in single copy to the bacterial chromosome via the specialized transducing bacteriophage lambda RZ5 . Analysis of the resulting lysogens demonstrated that aroH-lacZ expression in a trpR mutant strain varied four- to fivefold relative to an isogenic trpR+ strain under fully repressing conditions . In trpR+ strains containing either fusion, a modest (ca . 50%) change in activity was seen in response to the addition of L-tryptophan to the culture medium . These data demonstrate that aroH gene expression is only moderately regulated by the tryptophan repressor and that this regulation is at the level of transcription . Addition of L-phenylalanine, L-tyrosine, or Casamino Acids (Difco Laboratories, Detroit, Mich.) to the cell culture medium resulted in a tryptophan repressor-dependent derepression of aroH expression . We believe that this effect is caused by L-tryptophan limitation as a result of repression and feedback inhibition of the tyrosine- and phenylalanine-specific 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase isoenzymes . Derepression of aroH expression by the L-tryptophan analogs, 3-beta-indoleacrylic acid and indole-3-propionic acid, is also documented. Genetika, 1987 May, 23(5), 757 - 65 {Transfer of "artificial transposons" constructed on the basis of insertion element IS1}; Mirkin SM et al.; Terminal inverted repeats of the insertion element IS1 were synthesized chemically and plasmids containing these sequences flanking kanamycin-resistance gene in different combinations were constructed . Further incorporation of a whole-sized copy of the IS1 into such plasmids caused in some cases the autonomous transfer of Km-resistance from plasmid to bacteriophage lambda DNA . The transposition of the Km-resistance gene was only observed in those cases when the gene was enclosed between IS1 copy and one of the terminal repeats . The data obtained are discussed with regard to the evolution of bacterial transposons. Mol Gen Genet, 1987 May, 207(2-3), 395 - 401 Specificity of bacteriophage Mu excision; Nag DK et al.; To study the excision of bacteriophage Mu at the DNA sequence level, the Mu-derived phage lambda placMu3 was transposed to the transcribed but non-translated leader region of a plasmid-borne tetracycline (tet) resistance gene . Revertants (excision products) were then selected by Tet+ restoration of Tet+ and characterized . Of 21 independent Tet+ revertants, 17 contained simple deletions of most or all of lambda placMu3, while the other four contained more complex rearrangements in which one end of lambda placMu3 had been transposed, and most of the prophage had been deleted . The deletion endpoints were found in short direct repeats in each of the complex rearrangements and in 11 of the 17 simple deletion excisants . The results suggest models of slipped mispairing of template and nascent DNA strands facilitated by proteins of the Mu transposition machinery. J Virol, 1987 May, 61(5), 1751 - 5 In vivo transcription of bacteriophage phi 29 DNA: transcription termination; Barthelemy I et al.; The main early and late transcription termination sites in vivo in bacteriophage phi 29 DNA were determined by nuclease S1 mapping . Transcription of the phi 29 early genes located at the left end of the viral genome terminated at the very end of the DNA molecule and within the HindIII G fragment of the viral DNA . Transcription termination of the early genes located at the right end of the genome and that of the late viral genes overlapped in a specific region of the phi 29 DNA within the EcoRI D fragment . Stem-loop structures followed by uridine-rich tails could be derived close to the 3' ends of early and late mRNAs, suggesting Rho-independent transcription termination in phi 29 DNA. J Virol, 1987 May, 61(5), 1358 - 67 Map position and nucleotide sequence of the gene for the large structural phosphoprotein of human cytomegalovirus; Jahn G et al.; Human cytomegalovirus particles contain a phosphoprotein of 150,000 (pp150) apparent molecular weight in their matrix; the protein appears particularly reactive in Western blot analyses with human antisera . The gene for pp150 was mapped by screening a bacteriophage lambda gt11 cDNA expression library with monospecific rabbit antisera . Subsequent hybridization of cDNA with cosmid and plasmid clones containing the human cytomegalovirus strain AD169 genome mapped the gene to HindIII fragments J and N . The gene is transcribed into a late 6.2-kilobase RNA . The nucleotide sequence of this region was determined, and a transcription initiation site and two polyadenylation sites of an abundant transcript were located by primer extension and nuclease protection experiments . The reading frame for pp150, deduced from computer analyses, gives rise to a polypeptide of 1,048 amino acids in length; protein secondary structure analysis revealed multiple beta-pleated sheets in hydrophilic clusters, providing a possible explanation for the immunogenic properties of the polypeptide. J Bacteriol, 1987 May, 169(5), 2171 - 6 Regulation in Escherichia coli of the porin protein gene encoded by lambdoid bacteriophages; Blasband AJ et al.; Specialized lambda transducing phages carrying the cloned lc porin gene from the lambdoid bacteriophage PA-2, including various amounts of a sequence 5' to the start of transcription, were used to study the regulation of the porin gene . It was found that a cyclic AMP receptor protein consensus binding site 65 base pairs 5' to the start of transcription was required for catabolite repression of lc but was not sufficient for maximum expression under derepressing conditions . A sequence located more than 209 base pairs 5' to the start of transcription was necessary for maximum expression . By manipulating the copy number of the lc gene and the temperature and by measuring both the rate of synthesis of mRNA and the amount of Lc protein in the outer membrane, it was determined that the expression of lc is regulated primarily at the level of transcription and that expression is not autoregulated . Evidence is also presented that the silent phage porin gene nmpC of Escherichia coli K-12 is transcribed to the same extent as lc even though it does not give rise to a stable pool of mRNA . The structure of the 5' end of lc and nmpC is similar to that of ompF, and a model for transcriptional regulation is presented which may apply to all of these porin genes. J Bacteriol, 1987 May, 169(5), 2107 - 12 Identification of the cydC locus required for expression of the functional form of the cytochrome d terminal oxidase complex in Escherichia coli; Georgiou CD et al.; The aerobic respiratory chain of Escherichia coli contains two terminal oxidases which are differentially regulated . The cytochrome o complex predominates under growth conditions of high aeration, whereas the cytochrome d complex predominates when the oxygen tension is low . Either terminal oxidase will support aerobic growth . The goal of the work presented in this paper was to identify genes required for the expression of the functional form of the cytochrome d complex, other than the genes encoding the polypeptide components of the oxidase complex (cyd locus) . A strain lacking the cytochrome o complex (cyo mutant strain) was mutagenized by using a lambda-Mu hybrid hopper bacteriophage, lambda placMu53, which inserts randomly into the chromosome and carries a kanamycin resistance marker . Strains were isolated and examined which were unable to grow aerobically, i.e., which lacked functional cytochrome d complex, and which could not be complemented by introduction of the cyd gene on F-prime episomes . One strain was selected for characterization . The phage insert was mapped to min 18.9 on the genetic linkage map, defining a new genetic locus, cydC . Evidence described in the text suggests that the gene product is probably required for the synthesis of the unique heme d component of the cytochrome d complex. J Bacteriol, 1987 May, 169(5), 1812 - 7 Mutations in the lac P2 promoter; Donnelly CE et al.; We used site-directed mutagenesis to generate mutations in the -10 region of the lac P2 promoter . The mutations were crossed onto lambda bacteriophage carrying the lac regulatory elements and an intact lacZ gene, and the effects of the various mutations were determined in vivo and in vitro . Two of four mutations had effects on the start point of the P2-directed transcript and had very little effect on lac expression . Another mutation, which abolishes P2 promoter activity in vitro, also had very little effect on lac expression in vivo . We suggest that the P2 promoter plays little or no role in the activation of the P1 promoter by catabolite activator protein in complex with cyclic AMP. Sci Sin {B}, 1987 May, 30(5), 495 - 502 Inhibition of streptomycin-dependent mutation in Escherichia coli on the lytic growth of bacteriophage lambda; Tong KZ et al.; Spontaneous streptomycin-dependent mutants (StrDA) were isolated from Escherichia coli C600 . On C600 StrD, the lytic growth of phage lambda Nam and lambda cI857 was inhibited . After E . coli lysogenic strain 1.1485 (lambda cI857) mutated to StrDA, induction of lambda was decreased greatly . On StrDA of E . coli strains C600 and 1.1485 (lambda cI857), the plating efficiencies and burst sizes of phage T4 and T7 remained normal . Since StrDA is a mutation in the structural gene for ribosomal protein S12, the results obtained in the present study suggest that ribosomes of the StrDA mutants inhibit the lytic growth of lambda phage . The possibility that StrDA ribosomes inhibit the expression of lambda N gene is discussed based on the comparison of the genetic background of lambda cI857 and lambda Nam. EMBO J, 1987 May, 6(5), 1507 - 12 Organization of double-stranded DNA in bacteriophages: a study by cryo-electron microscopy of vitrified samples; Lepault J et al.; In this paper it is shown that conformation and packing of double-stranded DNA within the head of bacteriophages lambda and T4 can be assessed by cryo-electron microscopy of vitrified specimens . Electron diffraction patterns show that DNA within vitrified bacteriophages has a B conformation . Electron micrographs of vitrified bacteriophages show domains within the head formed by a approximately 2.5-nm striation and arising from the DNA packing . The number of differently oriented domains seen within a vitrified bacteriophage depends upon the geometry of the DNA container: the bacteriophage capsid . The packing of DNA within bacteriophages seems then to be governed by at least two phenomena . The first is the tendency of DNA to form local alignments (nematic liquid crystals) . The second is the orientation of these liquid crystals by the bacteriophage capsid . From these observations we propose a possible packaging mechanism: constrained nematic crystallization. J Bacteriol, 1987 May, 169(5), 2103 - 6 Bacteriophage lambda receptor site on the Escherichia coli K-12 LamB protein; Gehring K et al.; We have analyzed eight new phage-resistant missense mutations in lamB . These mutations identify five new amino acid residues essential for phage lambda adsorption . Two mutations at positions 245 and 382 affect residues which were previously identified, but lead to different amino acid changes . Three mutations at residues 163, 164, and 250 enlarge and confirm previously proposed phage receptor sites . Two different mutations at residue 259 and one at 18 alter residues previously suggested as facing the periplasmic face . The mutation at residue 18 implicates for the first time the amino-terminal region of the LamB protein in phage adsorption . The results are discussed in terms of the topology of the LamB protein. J Virol, 1987 May, 61(5), 1738 - 42 Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase; Broyles SS et al.; Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I) . A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I . Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene . The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate . A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized . Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites. J Biol Chem, 1987 Apr 25, 262(12), 5918 - 23 Characterization of a second gene product related to rabbit cytochrome P-450 1; Johnson EF et al.; A cDNA, p1-88, was cloned from a library constructed using rabbit liver mRNA . Sequence analysis indicates that p1-88 is highly similar (congruent to 95%) to the cDNA, p1-8, that encodes rabbit liver cytochrome P-450 1 and that had been isolated from the same library . The predicted amino acid sequence of the protein encoded by p1-88, P-450 IIC4, differs at 25 of 487 amino acids from that encoded by p1-8 . P-450 IIC4 was synthesized in vitro using rabbit reticulocyte lysate primed with RNA transcribed from the coding sequence of p1-88 using a bacteriophage T7 RNA polymerase/promoter system . P-450 IIC4 reacts with two monoclonal antibodies that recognize P-450 1 and exhibits the same relative electrophoretic mobility as P-450 1 . In contrast, the reactivity of a third monoclonal antibody recognizing P-450 1, 1F11, toward P-450 IIC4 synthesized in vitro is greatly diminished . The latter antibody extensively inhibits hepatic progesterone 21-hydroxylase activity and recognizes phenotypic differences among rabbits in the microsomal concentration of P-450 1 . This difference in the immunoreactivity of P-450 IIC4 and P-450 1 with the 1F11 antibody suggests that P-450 IIC4 does not contribute significantly to hepatic progesterone 21-hydroxylase activity . S1 nuclease mapping demonstrates that the expression of mRNAs corresponding to p1-88 are expressed to equivalent extents in rabbits exhibiting high and low expression of mRNAs corresponding to p1-8 . Thus, P-450 1 differs from the protein encoded by p1-88, in its regulation, immunoreactivity, and by inference its catalytic properties although the amino acid sequences of P-450 1 and P-450 IIC4 are highly similar (congruent to 95%). J Biol Chem, 1987 Apr 25, 262(12), 5677 - 81 Organization of the gene encoding the human beta-hexosaminidase alpha-chain; Proia RL et al.; The lysosomal enzyme, beta-hexosaminidase, is composed of two chains, alpha and beta . In Tay-Sachs disease, mutations in the gene encoding the alpha-chain produce a beta-hexosaminidase deficiency that results in the storage of its natural substrate, GM2 ganglioside . To obtain the background information for the eventual identification of the mutational errors in Tay-Sachs disease and to determine possible relationships between protein and gene structure, we have characterized the intron-exon organization of the human beta-hexosaminidase alpha-chain gene . Several overlapping clones were isolated from human genomic libraries constructed in cosmid and bacteriophage vectors . The cloned genomic DNA was analyzed by restriction endonuclease mapping, Southern blotting, and DNA sequencing . It was determined that the alpha-chain gene is approximately 35 kilobases long and is split into 14 exons . Sequences which resemble the "TATA" and "CAAT" transcriptional regulatory motifs are present at the 5' end of the gene . Differential transcription or processing of the most 3' exon of the gene results in two alpha-chain mRNAs with different 3'-untranslated regions . The first exon of the gene encodes the amino-terminal portion of the alpha-chain which is removed during the proteolytic maturation of the enzyme, raising the possibility that this segment may exist as a functional domain. Nucleic Acids Res, 1987 Apr 24, 15(8), 3257 - 73 The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo; Gallie DR et al.; A 67-nucleotide portion of the non-coding, 5'-leader sequence of tobacco mosaic virus RNA {defined as omega' (Gr . omega prime)} has been shown to enhance the translation of contiguous foreign gene transcripts both in vitro and in vivo . Chemically-synthesized omega', containing convenient linker sequences, was inserted into derivatives of an in vitro transcription plasmid (pSP64) between the bacteriophage-SP6 promoter and sequences coding for either chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) . Run-off in vitro transcripts, with or without a 5'-cap structure (G(5')ppp(5')G) and/or the omega' sequence, were tested in mRNA-dependent cell-free translation systems derived from rabbit reticulocyte lysate, wheat germ extract or Escherichia coli (MRE 600) . In all cases, the presence of omega' increased the translational expression of both reporter genes, typically between 2- to 10-fold . Electroporation of isolated mesophyll protoplasts from Nicotiana tabacum cv . Xanthi, or microinjection of oocytes from Xenopus laevis, with SP6-transcripts containing the CAT-coding region confirmed and extended the value of omega' as a potential translational enhancer of gene expression in vivo. Cell, 1987 Apr 24, 49(2), 221 - 7 T7 lysozyme inhibits transcription by T7 RNA polymerase; Moffatt BA et al.; The selectivity of T7 RNA polymerase for its own promoters is used to direct all transcription and replication to bacteriophage T7 DNA during infection . We now find that T7 lysozyme, which is known to cut a bond in the peptidoglycan layer of the cell wall, forms a specific complex with T7 RNA polymerase and inhibits transcription . Mutations that weaken this interaction have been found in the coding sequence for T7 RNA polymerase; an affinity column containing wildtype polymerase selectively binds T7 lysozyme, but a similar column containing mutant polymerase does not . The lysozyme-polymerase interaction ensures a controlled burst of late transcription during infection, and could possibly have some direct role in replication and/or control of lysis. Cell, 1987 Apr 24, 49(2), 253 - 62 Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA; Surette MG et al.; We report that two types of stable protein-DNA complexes, or transpososomes, are generated in vitro during the Mu DNA strand transfer reaction . The Type 1 complex is an intermediate in the reaction . Its formation requires a supercoiled mini-Mu donor plasmid, Mu A and HU protein, and Mg2+ . In the Type 1 complex the two ends of Mu are held together, creating a figure eight-shaped molecule with two independent topological domains; the Mu sequences remain supercoiled while the vector DNA is relaxed because of nicking . In the presence of Mu B protein, ATP, target DNA, and Mg2+, the Type 1 complex is converted into the protein-associated product of the strand transfer reaction . In this Type 2 complex, the target DNA has been joined to the Mu DNA ends held in the synaptic complex at the center of the figure eight . Supercoils are not required for the latter reaction. Science, 1987 Apr 24, 236(4800), 416 - 22 Direct evidence for DNA bending at the lambda replication origin; Zahn K et al.; Replication initiation in bacteriophage lambda appears to require wrapping of origin DNA on an approximately 50 angstrom radius in or around the complex with the initiator protein O . Since short lengths of DNA are not that flexible, it may be that runs of coherently spaced deoxyadenylate residues constitute bend sites in the ori sequence that facilitate the process . Earlier data showed that ori DNA has electrophoretic anomalies characteristic of bend sites and that these are augmented by initiator protein binding . Here origin bending is examined by direct measurement of the ability of polymerized ori sequences to form small circles . The smallest circles observed (84 residues) are compatible with the required radius of curvature . Bend sites within the O protein binding sites, bend sites in the spacers between them, plus the inherent flexibility of non-bent DNA in the origin may all contribute to origin bending . The data also show that a bend site is required for O protein binding to DNA. Biochem J, 1987 Apr 15, 243(2), 597 - 601 Cloning and sequencing of the medium-chain S-acyl fatty acid synthetase thioester hydrolase cDNA from rat mammary gland; Naggert J et al.; cDNA clones coding for the medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II) from rat mammary gland were identified in a bacteriophage lambda gt11 library and their nucleotide sequences were determined . The predicted coding region spans 263 amino acid residues and includes a sequence identical with that of a peptide derived from the enzyme active site . The rat thioesterase II cDNA sequence exhibits homology with that of a thioesterase found in duck uropygial glands. J Biol Chem, 1987 Apr 15, 262(11), 5288 - 92 Gene 1.2 protein of bacteriophage T7 . Effect on deoxyribonucleotide pools; Myers JA et al.; The gene 1.2 protein of bacteriophage T7, a protein required for phage T7 growth on Escherichia coli optA1 strains, has been purified to apparent homogeneity and shown to restore DNA packaging activity of extracts prepared from E . coli optA1 cells infected with T7 gene 1.2 mutants (Myers, J . A., Beauchamp, B . B., White, J . H., and Richardson, C . C . (1987) J . Biol . Chem . 262, 5280-5287) . After infection of E . coli optA1 by T7 gene 1.2 mutant phage, under conditions where phage DNA synthesis is blocked, the intracellular pools of dATP, dTTP, and dCTP increase 10-40-fold, similar to the increase observed in an infection with wild-type T7 . However, the pool of dGTP remains unchanged in the mutant-infected cells as opposed to a 200-fold increase in the wild-type phage-infected cells . Uninfected E . coli optA+ strains contain severalfold higher levels of dGTP compared to E . coli optA1 cells . In agreement with this observation, dGTP can fully substitute for purified gene 1.2 protein in restoring DNA packaging activity to extracts prepared from E . coli optA1 cells infected with T7 gene 1.2 mutants . dGMP or polymers containing deoxyguanosine can also restore packaging activity while dGDP is considerably less effective . dATP, dTTP, dCTP, and ribonucleotides have no significant effect . The addition of dGTP or dGMP to packaging extracts restores DNA synthesis . Gene 1.2 protein elevates the level of dGTP in these packaging extracts and restores DNA synthesis, thus suggesting that depletion of a guanine deoxynucleotide pool in E . coli optA1 cells infected with T7 gene 1.2 mutants may account for the observed defects. J Biol Chem, 1987 Apr 15, 262(11), 5280 - 7 Purification and characterization of the gene 1.2 protein of bacteriophage T7; Myers JA et al.; Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C . C . (1981) J . Virol . 37, 343-351) . In the absence of the T7 1.2 and E . coli optA gene products, the degradation of E . coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin . However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released . The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed . Purification of the {35S} methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis . The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence . We have found that extracts of E . coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection . Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein . No specific enzymatic activity has been found associated with the purified gene 1.2 protein. J Biol Chem, 1987 Apr 15, 262(11), 5238 - 47 The mannose permease of Escherichia coli consists of three different proteins . Amino acid sequence and function in sugar transport, sugar phosphorylation, and penetration of phage lambda DNA; Erni B et al.; The mannose permease of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation . It also functions as a receptor for bacterial chemotaxis and is required for infection of the cell by bacteriophage lambda where it most likely functions as a pore for penetration of lambda DNA . The permease consists of three different subunits, IIIMan, II-PMan, and II-MMan, which are encoded in a single transcriptional unit ptsLPM . The complete amino acid sequence of the subunits is deduced from the nucleotide sequence . IIIMan (35 kDa) is a hydrophilic protein which is transiently phosphorylated and most likely contains the active site for sugar phosphorylation . II-PMan (28 kDa) is very hydrophobic; II-MMan (31 kDa) is moderately hydrophobic . Both are integral membrane proteins and most likely form the transmembrane channel . All three subunits are required for sugar transport and phosphorylation; II-PMan and II-MMan alone are sufficient for penetration of lambda DNA . Truncated forms of II-MMan and II-PMan are described that mediate lambda DNA penetration but have no apparent sugar transport activity . Residual sugar phosphorylation activity is found with the truncated form of II-PMan . No obvious homologies at the level of amino acid sequence could be detected with other bacterial transport proteins. Nucleic Acids Res, 1987 Apr 10, 15(7), 2787 - 801 Purification and characterization of 3-methyladenine DNA glycosylase I from Escherichia coli; Bjelland S et al.; We have purified 3-methyladenine DNA glycosylase I from Escherichia coli to apparent physical homogeneity . The enzyme preparation produced a single band of Mr 22,500 upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis in good agreement with the molecular weight deduced from the nucleotide sequence of the tag gene (Steinum, A.-L . and Seeberg, E . (1986) Nucl . Acids Res . 14, 3763-3772) . HPLC confirmed that the only detectable alkylation product released from (3H)dimethyl sulphate treated DNA was 3-methyladenine . The DNA glycosylase activity showed a broad pH optimum between 6 and 8.5, and no activity below pH 5 and above pH 10 . MgSO4, CaCl2 and MnCl2 stimulated enzyme activity, whereas ZnSO4 and FeCl3 inhibited the enzyme at 2 mM concentration . The enzyme was stimulated by caffeine, adenine and 3-methylguanine, and inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and 3-methyladenine . The enzyme showed no detectable endonuclease activity on native, depurinated or alkylated plasmid DNA . However, apurinic sites were introduced in alkylated DNA as judged from the strand breaks formed by mixtures of the tag enzyme and the bacteriophage T4 denV enzyme which has apurinic/apyrimidinic endonuclease activity . It was calculated that wild-type E . coli contains approximately 200 molecules per cell of 3-methyladenine DNA glycosylase I. Nature, 1987 Apr 30-May 6, 326(6116), 888 - 91 A new-specificity mutant of 434 repressor that defines an amino acid-base pair contact; Wharton RP et al.; The repressor encoded by bacteriophage 434 binds to its operators by inserting a 'recognition' alpha-helix into the major groove of the DNA . We have identified an amino acid-base pair contact that determines (in part) the DNA-binding specificity of 434 repressor . The identification is based on the properties of a 'new-specificity' mutant, named Repressor {Ala 28}, which bears the substitution of Ala for Gln at the first residue of its recognition alpha-helix . Repressor {Ala 28} binds with high affinity to a particular doubly mutant operator bearing the same substitution at position 1 in each half-site, but does not bind to either the wild-type operator or to other mutant operators . We describe molecular models of residue 28-base pair 1 interactions that account for the binding specificities of both the mutant and wild-type proteins. Nature, 1987 Apr 30-May 6, 326(6116), 846 - 52 Structure of the repressor-operator complex of bacteriophage 434; Anderson JE et al.; The crystal structure of a specific complex between the DNA-binding domain of phage 434 repressor and a synthetic 434 operator DNA shows interactions that determine sequence-dependent affinity . The repressor recognizes its operators by its complementarity to a particular DNA conformation as well as by direct interaction with base pairs in the major groove. J Mol Biol, 1987 Apr 5, 194(3), 469 - 79 Recognition and cleavage of the bacteriophage P1 packaging site (pac) . II . Functional limits of pac and location of pac cleavage termini; Sternberg N et al.; Bacteriophage P1 initiates the processive packaging of its DNA at a unique site called pac . We show that a functional pac site is contained within a 161 base-pair segment of P1 EcoRI fragment 20 . It extends from a position 71 base-pairs to a position 232 base-pairs from the EcoRI-22 proximal side of that fragment . The 3' and 5' pac termini are located centrally within that 161 base-pair region and are distributed over about a turn of the DNA helix . The DNA sequence of the terminus region is shown below, with the large arrows indicating the positions of termini that are frequently represented in the PI population and the small arrows indicating the positions of termini that are rarely represented in the P1 population . (Sequence: in text) . Digestion of P1 virus DNA with EcoRI generates two major EcoRI-pac fragments, which differ in size by about five or six base-pairs . While the structure and position of the double-stranded pac ends of these fragments have not been determined precisely, the 5' termini at those ends probably correspond to the two major pac cleavage sites in the upper strand of the sequences shown above . The 161 base-pair pac site contains the hexanucleotide sequence 5'-TGATCAG-3' repeated four times at one end and three times at the other . Removal of just one of those elements from either the right or left ends of pac reduces pac cleavage by about tenfold . Moreover, the elements appear to be additive in their effect on pac cleavage, as removal of one and a half elements or all three elements from the right side of pac reduces pac cleavage 100-fold, and greater than 1000-fold, respectively. J Mol Biol, 1987 Apr 5, 194(3), 397 - 410 Translational repression: biological activity of plasmid-encoded bacteriophage T4 RegA protein; Miller ES et al.; The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs . We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda . Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties . It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control . Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets . The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli . The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites . Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ . The results show that no additional T4 products are required for RegA-mediated translational repression . Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor . Surprisingly, plasmid-generated RegA protein represses the synthesis of some E . coli proteins and appears to enhance selectively the synthesis of others . The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs. J Mol Biol, 1987 Apr 5, 194(3), 453 - 68 Recognition and cleavage of the bacteriophage P1 packaging site (pac) . I . Differential processing of the cleaved ends in vivo; Sternberg N et al.; The packaging of bacteriophage P1 DNA into viral capsids is initiated at a specific DNA site called pac . During packaging, that site is cleaved and at least one of the resulting ends is encapsidated into a P1 virion . We show here that pac is located on a 620 base-pair fragment of P1 DNA (EcoRI-20) . When that fragment is inserted into the chromosome of cells that are then infected with P1, packaging of host DNA into phage particles is initiated at pac and proceeds down the chromosome, unidirectionally, for about five to ten P1 "headfuls" (about 5 X 10(5) to 10 X 10(5) bases of DNA) . Using an assay for pac cleavage that does not depend on DNA packaging, we have identified a set of five amber mutations that are mapped adjacent to pac, and that define a gene (gene 9) essential for pac cleavage . Amber mutations that are located in genes necessary for viral capsid formation (genes 4, 8 and 23), or in a gene necessary for "late" protein synthesis (gene 10), do not affect pac cleavage . The latter result suggests that the synthesis of the pac cleavage protein is not regulated co-ordinately with other phage morphogenesis proteins . The products of pac cleavage were analyzed using two different DNA substrates . In one case, a single copy of pac was placed in the chromosome of P1-sensitive cells . When those cells were infected with P1, we could detect the cleavage of as much as 70% of the pac-containing DNA . The pac end destined to be packaged in the virion was detected five to 20 times more efficiently than was the other end . Since this result is obtained whether or not the infecting P1 phage can encapsidate the cut pac site, the differential detection of pac ends is not simply a consequence of one end being packaged and the other not . In a second case, pac was located in cells on a small (5 X 10(3) bases) multicopy plasmid . When those cells were infected with P1, neither pac end was detected efficiently after P1 infection, unless the cells carried a recBCD- mutation . In recBCD- cells, the results with plasmid-pac substrates were similar to those obtained with chromosomally integrated pac substrates . We interpret these results to mean that, following pac cleavage, the end destined to be packaged is protected from cellular nucleases while the other end is degraded by the action of at least two nucleases, one of which is the product of the host recBCD gene.(ABSTRACT TRUNCATED AT 400 WORDS) J Mol Biol, 1987 Apr 5, 194(3), 411 - 22 Initiation of bacteriophage P22 DNA packaging series . Analysis of a mutant that alters the DNA target specificity of the packaging apparatus; Casjens S et al.; Bacteriophage P22 is thought to package its double-stranded DNA chromosome from concatemeric replicating DNA in a "processive" sequential fashion . According to this model, during the initial packaging event in such a series the packaging apparatus recognizes a nucleotide sequence, called pac, on the DNA, and then condenses DNA within the coat protein shell unidirectionally from that point . DNA ends are generated near the pac site before or during the condensation reaction . The opposite end of the mature chromosome is created by a cut made in the DNA after a complete chromosome is condensed within the phage head . Subsequent packaging events on that concatemeric DNA begin at the end generated by the headful cut of the previous event and proceed in the same direction as the previous event . We report here the identification of a consensus nucleotide sequence for the pac site, and present evidence that supports the idea that the gene 3 protein is a central participant in this recognition event . In addition, we tentatively locate the portion of the gene 3 protein that contacts the pac site during the initiation of packaging. Bioorg Khim, 1987 Apr, 13(4), 559 - 61 {Primary structure of proline tRNA of bacteriophage T5}; Shliapnikov MG et al.; The uniformly 32P-labeled bacteriophage T5 proline tRNA has been isolated from phage-infected E . coli cells by two-dimensional PAGE . Its nucleotide sequence has been determined by conventional techniques (using TLC on cellulose for oligonucleotide fractionation) as follows: (Formula: see text) . The tRNA has the anticodon sequence UGG, which can presumably recognize the four proline-specific codons (CCN) . It has 70% homology with phage T4 tRNA(Pro). Bioorg Khim, 1987 Apr, 13(4), 533 - 8 {Primary structure of tRNAAsn of bacteriophage T5}; Shliapnikov MG et al.; Unformly 32P-labelled phage-specific tRNAAsn was isolated from bacteriophage T5-infected E . coli cells its oligonucleotide fragments were fractionated by thin-layer chromatography on cellulose and the tRNA's primary structure was determined as follows: (formula; see text) . Main features of the structure are: displacement of the constant residue A14 by U; absence of G27 X A43 and U30 X psi40 pairing in the anticodon stem; possibility of additional pairing in D-loop . Comparison of T5 tRNAAsn with other asparagine tRNAs is presented. Bioorg Khim, 1987 Apr, 13(4), 568 - 70 {Highly selective affinity labeling of DNA-dependent RNA-polymerase of bacteriophage T7}; Grachev MA et al.; T7 phage RNA polymerase was affinity labelled in the presence of its promoter by treatment with an ATP gamma-derivative (a phosphoamide obtained from 4-(N-chloroethyl, N-methyl)aminobenzylamine, or one of esters obtained from 2-methoxy-4-formylphenol, 4-formylphenol, and 2{N-(4-formylphenyl), N-methyl}-aminoethanol) followed by addition of {alpha-32P}GTP . The most efficient labelling took place with the alkylating phosphoamide reagent. Mol Biochem Parasitol, 1987 Apr, 23(3), 211 - 22 Cloning of a gene encoding the immunodominant surface antigen of Leishmania donovani promastigotes; Heath S et al.; This study describes the characterisation of externally oriented surface peptides of both morphological forms of Leishmania donovani, the causative agent of visceral leishmaniasis (kala-azar) . Using 125I surface labelling techniques and peptide extraction in the detergents Triton X-100 and Triton X-114, a major iodinable promastigote peptide at 63 kDa or 65 kDa (depending on detergent used) was identified . This peptide was demonstrated to be the immunodominant membrane peptide of L . donovani and was strongly recognised by human sera from parasitologically confirmed cases of kala-azar . This peptide was not demonstrated on the surface of tissue amastigotes, although in vitro translations of poly(A+) RNA from both promastigotes and amastigotes demonstrated that both forms possessed mRNA that directs the synthesis of a 63 kDa peptide . It is suggested therefore that in amastigotes this peptide may be a processed antigen . We also report the isolation of a recombinant cDNA clone in the bacteriophage vector lambda gt10 which encodes a 63 kDa polypeptide that is recognised by human kala-azar sera . It is proposed that this surface peptide could be used in a specific immunodiagnostic test for leishmaniasis. DNA, 1987 Apr, 6(2), 129 - 37 Spontaneous deletion mutants of bacteriophage Pf3: mapping of signals involved in replication and assembly; Luiten RG et al.; Defective deletion mutants (miniphages) arise spontaneously during serial propagation of the filamentous bacteriophage Pf3 . They contain a circular single-stranded (ss) DNA molecule which is up to 80% smaller than the wild-type single-stranded genome . Analysis of the genomic structure of three of these miniphages revealed that they consist of sequences that in the wild-type genome are flanked by direct repeats 5-8 nucleotides long; only one copy of these repeats was found again in the miniphage genome . One miniphage genome contained a tandemly duplicated sequence, and from its structure we concluded that the duplication had occurred after a primary deletion event . Also, short direct repeats must have been involved in the duplication process . We conclude that the deletion and duplication events have occurred by an identical recombination process, probably the "slipped mispairing" mechanism . In the presence of helper functions, the three miniphage genomes are replicated and assembled into ssDNA containing virus-like particles . Hence, the assembly signal and the replication origins for viral and complementary strand synthesis are located within the region shared by all three miniphage genomes, i.e., nucleotides 4092-4678 of the wild-type genome (Luiten et al., 1985). J Gen Virol, 1987 Apr, 68 ( Pt 4), 957 - 63 The head-tail linker protein of bacteriophage T5: genetic and immunological studies; Kay D et al.; We investigated the properties of amber mutants of coliphage T5 defective in a late stage of phage assembly . The non-functional heads synthesized by mutants am25 or am158 were unable to combine with functional tails to produce viable phage particles . These mutants were shown to lack a single protein, designated the head-tail linker protein (HTLp), which was identified by polyacrylamide gel electrophoresis and Western blot analysis and had an Mr of 18,000 . An extract containing the HTLp, when added to the HTLp-deficient heads, restored their ability to combine with functional tails. Virology, 1987 Apr, 157(2), 285 - 97 Characterization of morphogenetic intermediates and progeny of normal and alkylated bacteriophage T7; Karska-Wysocki B et al.; Analysis of thin sections of Escherichia coli B cells infected by normal (nonalkylated) or alkylated bacteriophage T7 showed that alkylation altered phage morphogenesis . To understand these morphogenetic alterations, we have isolated phage-related particles from infected-cell lysates by differential and sucrose gradient centrifugation . Cells infected by normal and by alkylated phage produced mature phage particles, empty heads, and proheads; however, production of proheads and mature phage particles was less in the case of alkylated phage . These lysates also contained sedimentable material which migrated more slowly than empty heads on sucrose gradients . In the case of alkylated phage, this peak contained radioactive material in amounts nearly equal to that in either proheads or empty heads; for normal phage, this peak represented a smaller fraction of the total radioactivity . Examination of the gradient fractions by electron microscopy revealed appreciable quantities of phage tails and tail-related particles . The same gradient fractions contained phage tail proteins: gene products (gps) 11, 12, and 17 as well as smaller amounts of gp 8, the head-tail connector . In addition, these fractions contained two other proteins which we believe to be of bacterial origin . These proteins may be related to tail formation or function as part of the phage receptor . On the basis of our data, we propose an alternative morphogenetic pathway for T7 tail formation, a pathway which would involve formation of a complex of tail proteins prior to association with the phage head. J Clin Periodontol, 1987 Apr, 14(4), 245 - 7 Association between bacteriophage-infected Actinobacillus actinomycetemcomitans and rapid periodontal destruction; Preus HR et al.; Actinobacillus actinomycetemcomitans was isolated from periodontal pockets in a patient suffering from prepubertal periodontitis . Electron microscopy revealed 3 different groups of bacteriophages in filtrates of subgingival plaque from all the active periodontal lesions . Phage infected A . actinomycetemcomitans in this patient was restricted to periodontal pockets which, according to standardized roentgenograms, had shown bone destruction during the past 12 months . A follow-up study of 7 months revealed that a "burned out" site which harbored noninfected A . actinomycetemcomitans, turned into an active site at the same time as the A . actinomycetemcomitans of that site became infected with the phages . These findings indicate a relationship between rapid prepubertal periodontal destruction and phage-infected A . actinomycetemcomitans. Jpn J Exp Med, 1987 Apr, 57(2), 117 - 24 Genetic recombination between closely linked makers of bacteriophage T4 . IV . Mutations which interfere with mismatch repair; Honda M; T4 phage mutations MCO1 and MCO3 reduced recombination between multiple closely linked markers preferentially and were located between gene 24 and gene 25 . The MCO1 and the MCO3 mutants complemented each other . The results of UV-cross reactivation experiments indicated that the MCO1 and the MCO3 mutants could rescue the markets of UV-damaged phage, but could not segregate them from flanking mutations . Therefore, it is concluded that MCO1 mutation and MCO3 mutation interfere with mismatch repair. J Biomol Struct Dyn, 1987 Apr, 4(5), 859 - 68 A preliminary structure for the DNA binding protein from bacteriophage IKe; Brayer GD; A modelling procedure has been utilized to obtain a preliminary three-dimensional structural model for the bacteriophage IKe DNA binding protein (IKe-DBP) based on the known high resolution X-ray diffraction structure of a functionally related protein (G5BP) from bacteriophage fd . The degree of structural homology observed is much higher than the 44% primary sequence identity between these proteins would indicate . These studies suggest IKe-DBP, like G5BP, is composed of a central three-stranded beta sheet from which protrude three extended beta loops . Furthermore, the IKe-DBP structural model can easily form, without conformational rearrangements, the compact dimer unit that is the functionally active species of G5BP . Structural comparisons show residues conserved in the primary sequence of both proteins tend to cluster in two regions . The first being essential for the maintenance of dimer association . The second about the two DNA binding channels which cross the face of each dimer . Based upon an earlier characterized G5BP-DNA complex, a model for DNA complexation to IKe-DBP is also presented. J Clin Microbiol, 1987 Apr, 25(4), 698 - 701 Atypical isolates of Brucella abortus from Canada and the United States characterized as dye sensitive with M antigen dominant; Ewalt DR et al.; A total of 41 Brucella isolates, examined by standard biotyping procedures, were found to be similar to Brucella abortus biovar 2 in dye sensitivity but had a dominant M antigen . Oxidative metabolic tests performed on 39 of the isolates confirmed them as B . abortus . Additional biochemical and bacteriophage susceptibility studies were performed on 35 of the isolates . The isolates had identical reactions in the various tests, except for one isolate which was resistant to lysis by all phage strains used . Two isolates were injected into guinea pigs and shown to be virulent . The isolates described in this study appear similar to atypical Brucella isolates previously reported in the United Kingdom and the United States and may form the basis of a new biovar, B . abortus biovar 10. Virus Res, 1987 Apr, 7(2), 169 - 83 The complete nucleotide sequence of bluetongue virus serotype 1 RNA3 and a comparison with other geographic serotypes from Australia, South Africa and the United States of America, and with other orbivirus isolates; Gould AR; The sequence of the RNA segment 3 of bluetongue virus (BTV) serotype 1 from Australia is presented along with its deduced amino acid sequence . DNA copies of this genome segment were inserted either into the E . coli plasmid pBR322 by homopolymeric tailing or by direct insertion of double-stranded DNA fragments generated by restriction endonuclease cleavage into the appropriate M13 bacteriophage vectors (Vieira, J . and Messing, J., 1982, Gene 19, 259-268) . Direct comparisons were made to the nucleotide sequence data of Purdy, M . et al., 1984 (J . Virol . 51, 754-759) and Ghiasi, H . et al., 1985 (Virus Res . 3, 181-190) for the United States of America (US) isolates of BTV, serotypes 10 and 17, respectively . A method for the rapid cloning, sequencing and alignment of orbivirus RNA 3 segments was utilised to compare other geographical isolates of BTV, as well as those of other orbivirus serotypes, in particular, epizootic haemorrhagic disease of deer virus (EHDV) and Warrego . The comparison of this sequence data reveals that BTV isolates can be separated into distinct geographical types which in turn are distinct from the other orbivirus isolates studied . The sequence conservation at the amino acid level for the gene product of RNA3 (VP3) does not enable distinctions to be made amongst the BTV isolates at a geographical level, but does afford easy distinction into the different orbivirus groups . A possible evolutionary schematic is presented for the orbiviruses studied. Proc Natl Acad Sci U S A, 1987 Apr, 84(8), 2160 - 4 Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer; Wahl GM et al.; We have designed cosmid vectors for rapid genomic "walking" and restriction mapping . These vectors contain the transcription promoters from either bacteriophage SP6, T7, or T3 flanking a unique BamHI cloning site . Mammalian expression modules encoding the dominant marker neomycin phosphotransferase or the amplifiable dihydrofolate reductase gene expressed from SV40 promoters were inserted for use in gene transfer studies . Restriction sites for the enzymes Not I and Sfi I, which cut mammalian DNA very infrequently, have been engineered near the transcriptional promoters to enable the excision of most inserts as single, full-length fragments . Genomic libraries representative of mouse, human, and hamster genomes were constructed by inserting 33- to 44-kilobase-pair (kbp) DNA fragments, generated by partial cleavage of genomic DNA with Mbo I or Sau3A, into the unique BamHI site . Digestion of recombinant cosmids with restriction enzymes that cleave frequently but do not disrupt the transcriptional promoters generates two small DNA templates for the synthesis of end-specific RNA probes to facilitate directional "walking." Cosmid restriction maps can be determined rapidly by one of several methods . The cosmids and methods we describe should have wide utility in determining the functional and structural organization of complex eukaryotic genomes and for physically linking distant genetic loci. Eur J Biochem, 1987 Apr 1, 164(1), 45 - 51 Overproduction and large-scale preparation of deoxyuridine triphosphate nucleotidohydrolase from Escherichia coli; Hoffmann I et al.; A recombinant plasmid, pHW1, directing the overproduction of the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) from Escherichia coli has been constructed . A 1900-base DNA fragment carrying the structural gene for the enzyme (dut) has been recloned into a runaway replication vector that also carries the strong leftward promoter (pL) of bacteriophage lambda . Upon temperature shift, an E . coli strain carrying the new plasmid gives an increase in dUTPase activity of about 600-fold in rich medium compared to wild-type bacteria . The 64-kDa protein corresponding to the mature form of the enzyme reaches 20% of the total protein content of the bacterial cell . Using this strain, a simplified procedure has been developed for the purification of dUTPase . The purification steps consist of extraction of the cytoplasmic proteins, ammonium sulfate precipitation, anion-exchange chromatography and gel filtration on FPLC . The new overproducing plasmid and the simplified purification procedure developed will make it possible to purify dUTPase in sufficient amounts for detailed characterization studies. EMBO J, 1987 Apr, 6(4), 1121 - 7 GATC sequences, DNA nicks and the MutH function in Escherichia coli mismatch repair; Langle-Rouault F et al.; Circular heteroduplex DNAs of bacteriophage phi X174 have been constructed carrying either a G:T (Eam+/Eam3) or a G:A (Bam+/Bam16) mismatch and containing either two, one or no GATC sequences . Mismatches were efficiently repaired in wild-type Escherichia coli transfected with phi X174 heteroduplexes only when two unmethylated GATC sequences were present in phi X174 DNA . The requirements for GATC sequences in substrate DNA and for the E . coli MutH function in E . coli mismatch repair can be alleviated by the presence of a persistent nick (transfection with nicked heteroduplex DNA in ligase temperature-sensitive mutant at 40 degrees C) . A persistent nick in the GATC sequence is as effective in stimulating mutL- and mutS-dependent mismatch repair as a nick distant from the GATC sequence and from the mismatch . These observations suggest that the MutH protein participates in methyl-directed mismatch repair by recognizing unmethylated DNA GATC sequences and/or stimulating the nicking of unmethylated strands. Genetika, 1987 Apr, 23(4), 622 - 9 {Transmission of amber mutants of bacteriophage T4 . III . Thermostability of the replication of amber mutants in cells of a non-permissive host is typical for the majority of phage tail genes}; Shalnene VIu et al.; The article deals with determination of the spreading of the earlier discovered phenomenon of the temperature sensitivity of multiplication of T4 phage amber mutants . On the basis of the study of the dependence of multiplication of 50 amber mutants in 22 genes of T4 phage tail in the cells of non-permissive host on the incubation temperature in the range of 15-41 degrees C, the following conclusion is drawn: temperature sensitivity of multiplication of amber mutants appears to be gene-specific and is widely spread among T4 phage genes, i.e . in the case of amber mutants the burst size decreases, even for 14 tail genes, by several orders with the increase in incubation temperature . Temperature sensitivity of multiplication is typical of amber mutants in the genes whose proteins are either of small number in a phage particle (several molecules) or play the role of catalytic factors . Moreover, genes, amber mutants of which possess temperature sensitivity of multiplication, map in defined clusters. Genetics, 1987 Apr, 115(4), 605 - 10 Repair of a mismatch is influenced by the base composition of the surrounding nucleotide sequence; Jones M et al.; Heteroduplexes with single base pair mismatches of known sequence were prepared by annealing separated strands of bacteriophage lambda DNA and used to transfect Escherichia coli . A series of transition (G:T and A:C) and transversion (G:A and C:T) mismatches located throughout most of the bacteriophage lambda cI gene has been examined . The results suggest that the transition mismatches are generally better repaired than the transversion mismatches and that, at least for the transversion mismatches studied, repair efficiency increases with increasing G:C content in the neighboring nucleotide sequence . This specificity of the E . coli mismatch repair system can account, in part, for the similar frequencies of base substitution mutations throughout the E . coli genome. Genetics, 1987 Apr, 115(4), 597 - 604 Cross-specificities between cII-like proteins and pRE-like promoters of lambdoid bacteriophages; Wulff DL et al.; We have investigated the activation of transcription from the pRE promoters of phages lambda, 21 and P22 by the lambda and 21 cII proteins and the P22 c1 (cII-like) protein, using an in vivo system in which cII protein from a derepressed prophage activates transcription from a pRE DNA fragment on a multicopy plasmid . We find that each protein is highly specific for its own cognate pRE promoter, although measureable cross-reactions are observed . The primary recognition sequence for cII protein on lambda pRE is a pair of TTGC repeat sequences in the sequence 5'-TTGCN6TTGC-3' at the -35 region of the promoter . This same sequence is found in 21 pRE, while P22 pRE has the sequence 5'-TTGCN6TTGT-3', which is the same as that of lambda ctr1, a pRE+ variant of lambda . lambda ctr1 pRE is half as active as lambda + pRE when assayed with either the lambda cII or the P22 c1 proteins . Therefore, the single base change in the P22 repeat sequence cannot explain why the P22 c1 protein is much more active with P22 pRE than lambda pRE . The dya5 mutation, a G----A change at position -43 of pRE, makes pRE a stronger promoter when assayed with either the lambda or 21 cII proteins or the P22 c1 protein . We conclude that efficient activation of a cII-dependent promoter by a cII protein requires sequence information in addition to the TTGC repeat sequences . We do not know the characteristics of the proteins which are responsible for the specificity of each protein for its own cognate promoter.(ABSTRACT TRUNCATED AT 250 WORDS) Cell Differ, 1987 Apr, 20(4), 253 - 61 Electron microscopic studies of giant nucleus-like structure formed by lambda DNA introduced into the cytoplasm of Xenopus laevis fertilized eggs and embryos; Shiokawa K et al.; When bacteriophage lambda DNA was injected into the cytoplasm of the fertilized egg of Xenopus laevis, giant nucleus-like structures were assembled around the injected DNA . These nucleus-like structures survived during cleavage and were partitioned into blastomeres at the blastula stage . The nucleus-like structures formed in the uncleaved fertilized eggs and the blastula cells were both surrounded by a bilayer nuclear membrane with nuclear pore complexes . The ultrastructural features of the lambda DNA-induced nucleus-like structure were considerably different from those of the normal blastula nucleus: although the nuclear pore complexes appeared to be normal, the 'nucleoplasm' was much too homogeneous as compared with that of the normal nucleus. Mutat Res, 1987 Apr, 177(2), 179 - 88 Metal-induced lethality and mutagenesis: possible role of apurinic intermediates; Schaaper RM et al.; The possible mechanisms by which various metals exert their mutagenic effects were investigated using both chemical and biochemical techniques . Ions of Cu, Ni and Cr enhanced the release of either adenine {Cu(II) and Ni(II)} or guanine {Cr(VI)} from DNA as measured in a chromatography assay, suggesting the possible importance of depurination in metal-induced mutagenesis . Transfection experiments with single-stranded bacteriophage phi X174 DNA indicated that micromolar levels of Cu(II), Cr(III), Cr(VI) and Pt(IV) are capable of causing extensive lethal damage to the phage DNA . In case of Cu(II) and Pt(IV) this damage proved mutagenic for phi X174 am3 after transfection of DNA into SOS-induced spheroplasts . For Cu(II) mutagenesis is likely due to the release of adenine residues from the phage DNA based on the abolishment of mutagenesis by alkali and the observed specificity of the phage revertants . The enhancement of the adenine depurination rate by Cu(II) was estimated to be as high as 10,000-fold. J Bacteriol, 1987 Apr, 169(4), 1750 - 2 Proton-motive-force-dependent step in the pathway to lysis of Escherichia coli induced by bacteriophage phi X174 gene E product; Witte A et al.; We examined the cellular effects after the expression of the cloned lysis gene E of bacteriophage phi X174 . Chloramphenicol prevented lysis only when added within the first minute of derepression of E synthesis, indicating that a time lag of several minutes exists between the synthesis of the E protein and the onset of cell lysis . Experiments with protonophores showed the existence of a subsequent step dependent on proton motive force at about 3 to 5 min before lysis. J Bacteriol, 1987 Apr, 169(4), 1585 - 92 Isolation and analysis of Escherichia coli mutants that allow increased replication of bacteriophage lambda; Keller JA et al.; Escherichia coli mutants were isolated that supported the growth of a lambda Ots and, in at least one case, a lambda Bts phage at the normally nonpermissive temperature of 39 degrees C . In one such strain, Ots and Bts suppression ability appeared to be a function of the guaB gene . Ots suppression by the mutant guaB strain was prevented if high levels of guanine or xanthine were present in the medium . No other base had any effect on Ots suppression in this strain . Other strains carrying spontaneous mutations resulting in guanine or xanthine auxotrophy (guaA or guaB lesions, respectively) all allowed lambda Ots replication at 39 degrees C; Ots suppression in these strains was also abolished by addition of guanine to the medium . Thus, reduced intracellular guanine levels resulting from guaA or guaB mutations appeared to suppress the inability of lambda Ots and, at least in some cases, Bts bacteriophage to form plaques at 39 degrees C . In burst size experiments, a guaB mutant produced a larger phage yield per infected cell of both lambda Ots and lambda O+ phage at 39 degrees C than did a similar guaB+ strain . It appeared that a lower-than-normal level of guanine (or a guanine derivative) in these cells may permit unusually efficient lambda replication . The fact that O+ and lambda Ots bursts in the guaB mutant were reduced significantly by addition of exogenous guanine to the medium is consistent with this suggestion . Another strain that suppresses the Ots allele has no known auxotrophic requirements, and suppression in this strain was unaffected by addition of guanine to the medium; however, addition of cytidine to the medium specifically eliminated Ots suppression in this strain . The mutation responsible for allowing Ots replication in this strain is unknown. Genes Dev, 1987 Apr, 1(2), 204 - 12 Control of gene expression in bacteriophage P22 by a small antisense RNA . II . Characterization of mutants defective in repression; Wu TH et al.; Phage P22 produces antirepressor protein early after infection from a transcript initiated at the Pant promoter . After the first few minutes of infection, transcription from Pant is repressed by a protein encoded by the arc gene . Antirepressor is not produced late in infection, even though the antirepressor gene, ant, is transcribed from the late operon promoter Plate . We describe the isolation of P22 mutants that synthesize antirepressor from the Plate transcript . The mutations inactivate a promoter Psar, which lies within the ant coding sequence and directs the synthesis of sar RNA, a small antisense regulatory RNA complementary to the ant ribosome binding site . Characterization of the Psar down-mutants shows that transcription from Psar interferes with synthesis of antirepressor from both the Plate and Pant transcripts . Since sar RNA represses synthesis of antirepressor in trans, we propose that sar RNA base-pairs with ant mRNA to inhibit antirepressor synthesis at a post-transcriptional level . The role and importance of sar RNA in P22 biology are discussed. Genes Dev, 1987 Apr, 1(2), 197 - 203 Control of gene expression in bacteriophage P22 by a small antisense RNA . I . Characterization in vitro of the Psar promoter and the sar RNA transcript; Liao SM et al.; The characterization in vitro of a newly discovered promoter (Psar) in the bacteriophage P22 immI region is described . Psar is located within the ant gene and is directed toward the major immI promoter, Pant . The entire intercistronic region between the P22 arc and ant genes (69 bp) is transcribed . The initiation and termination of sar (small antisense regulatory) RNA transcription are unusual . Frequent abortive initiation occurs in the presence of all four NTPs; RNA products 3-13 nucleotides in length are produced in about 15- to 25-fold larger numbers than full-length transcripts . Termination of sar RNA synthesis occurs after transcription of the first and second Ts of a TTTA sequence following a region of hyphenated dyad symmetry . The effects of convergent transcription between Pant and Psar were investigated on linear and supercoiled templates . Active transcription from Pant interferes with full-length transcription from Psar; several factors that interfere with Pant initiation (e.g., Pant down-mutation, Mnt repressor protein, Arc repressor protein) result in indirect activation of sar RNA synthesis . The sar RNA pairs rapidly with ant mRNA to form a stable stoichiometric complex . The location and properties of Psar suggest an important regulatory function for sar RNA as a negative effector of ant expression . The results of Wu et al . (this issue) support this suggestion. Biochemistry, 1987 Mar 24, 26(6), 1688 - 96 Effect of single amino acid changes in the region of the adenylylation site of T4 RNA ligase; Heaphy S et al.; Preparation and analysis of a series of mutants of bacteriophage T4 RNA ligase that carry single amino acid changes at or near the site of covalent reaction with ATP (adenylylation) are described . The mutant proteins were constructed by site-directed mutagenesis of the gene for T4 RNA ligase (g63) cloned in M13 vectors, transfer of the mutant genes into a lambda pL-containing expression plasmid, and subsequent expression in Escherichia coli . The results give further evidence that Lys-99 is the adenylylation site and that the residue is also important to step 3 in the RNA ligase mechanism (ligation between acceptor and adenylylated donor) . Mutations at Glu-100 or Asp-101 have no effect on adenylylation, but Asp-101 is shown to be crucial to both step 2 (transfer of adenylyl to donor) and step 3. Biochemistry, 1987 Mar 24, 26(6), 1532 - 8 Phage phi X174 probed by laser Raman spectroscopy: evidence for capsid-imposed constraint on DNA secondary structure; Incardona NL et al.; The Raman spectrum of the isometric bacteriophage phi X174 contains a number of well-resolved bands which have been assigned unambiguously to proteins of the capsid or to the single-stranded DNA (ssDNA) genome . Additional Raman bands of protein and DNA, which are partially overlapped in the spectrum of virus, have been resolution enhanced by Fourier deconvolution to permit improved semiquantitative measurement of spectral intensities and frequencies for structural conclusions . Raman conformation markers indicate that the ssDNA molecule within the capsid contains nucleosides of C2'-endo sugar pucker and anti-glycoside bond orientation, but the nucleic acid backbone lacks the geometry characteristic of B-form DNA . The Raman profile of encapsidated phi X DNA indicates a backbone more similar to heat-denatured DNA than to DNA containing hairpinlike secondary structure . This finding suggests limited interbase interactions in the packaged genome, which is presumably the result of constraints imposed by the viral capsid . Thus, the extensive pairing and stacking of bases indicated by Raman profiles from ssRNA viruses are not evident for the phi X174 chromosome . Overall, the proteins of the virion contain extensive beta-sheet and irregular secondary structures . Fourier deconvolution of the Raman amide I band provides an estimate of the percentage of total beta-sheet structure (approximately 60%) in all proteins of the virion . The amide III region of the spectrum confirms that beta-sheet and irregular domains are the predominant protein secondary structures . Samples of phi X174 concentrated for Raman spectroscopy by either ultracentrifugation or ultrafiltration exhibit nearly identical Raman spectra, indicating that either method can be employed to prepare intact virus without significant loss of DNA or protein components. J Mol Biol, 1987 Mar 20, 194(2), 231 - 43 Sense and antisense transcription of bacteriophage T4 gene 32 . Processing and stability of the mRNAs; Belin D et al.; Analysis of bacteriophage T4 gene 32 transcription has revealed a multiplicity of mRNAs . In plasmids, gene 32 is expressed primarily from a strong promoter that is shut off after phage infection . In a wild-type infection, gene 32 is initially transcribed from prereplicative polycistronic and monocistronic promoters; subsequently, a monocistronic late mRNA predominates . This transcript, as well as a post-transcriptionally processed product of the earlier mRNA, can be stable . The eventual degradation of the stable mRNAs is temporally regulated by the phage . Finally, the transcription termination region of gene 32 can function as an antisense promoter both in vitro and in vivo. J Mol Biol, 1987 Mar 20, 194(2), 349 - 52 Abortive infection of Escherichia coli F+ cells by bacteriophage T7 requires ribosomal misreading; Kruger DH et al.; The use of different precisely mapped T3/T7 recombinants strengthens the conclusion that abortive infection by T7 of F plasmid-carrying cells is due to the nucleotide sequence at the end of the T7 gene 1 . Furthermore, we demonstrate that the exclusion requires suppression of ochre stop codons, a phenomenon that occurs with low frequency in wild-type cells due to ribosomal misreading . The introduction of rspL mutations in which ribosomal misreading is reduced alleviates the exclusion and the presence of ochre tRNA suppressors increases its severity. J Mol Biol, 1987 Mar 20, 194(2), 205 - 18 Use of site-specific recombination as a probe of DNA structure and metabolism in vivo; Bliska JB et al.; We used site-specific recombination catalyzed by the bacteriophage lambda Int system to probe DNA structure and metabolism in vivo . In vitro, the complexity of catenated products was linearly proportional to substrate supercoil density . A system was developed that gave efficient, controlled Int recombination in Escherichia coli cells . From a comparison of the data obtained in vitro and in vivo, we conclude that Int recombination does have the same mechanism in vivo as it has in vitro, but that only 40% of the plasmid DNA linking deficit in E . coli cells may be in the interwound supercoil form demonstrated in vitro . We suggest that this is the effective level of supercoiling in vivo, because the remaining DNA is constrained in alternative forms by protein binding . The study of Int recombination in vivo also provides an assay for enzymes that decatenate circular molecules, such as those formed during DNA replication . We find that DNA gyrase is the principal decatenase in E . coli and that it acts spontaneously and rapidly. Nature, 1987 Mar 19-25, 326(6110), 312 - 4 Stimulation of protein-directed strand exchange by a DNA helicase; Kodadek T et al.; The protein-mediated exchange of strands between a DNA double helix and a homologous DNA single strand involves both synapsis and branch migration, which are two important aspects of any general recombination reaction . Purified DNA-dependent ATPases from Escherichia coli (recA protein), Ustilago (rec 1 protein) and phage T4 (uvsX protein) have been shown to drive both synapsis and branch migration in vitro . The T4 gene 32 protein is a helix-destabilizing protein that greatly stimulates uvsX-protein-catalysed synapsis, and the E . coli SSB (single-strand binding) protein stimulates the analogous recA-protein-mediated reaction to a lesser degree . One suspects that several other proteins also play a role in the strand exchange process . For example, a DNA helicase could in principle accelerate branch migration rates by helping to melt the helix at the branch point . The T4 dda protein is a DNA helicase that is required to move the T4 replication fork past DNA template-bound proteins in vitro . Previously, we have shown that the dda protein binds to a column that contains immobilized T4 uvsX protein . We show here that this helicase specifically stimulates the branch migration reaction that the uvsX protein catalyses as a central part of the genetic recombination process in a T4 bacteriophage-infected cell. J Biol Chem, 1987 Mar 15, 262(8), 3800 - 8 Interactions of a proteolytically nicked RNA polymerase of bacteriophage T7 with its promoter; Ikeda RA et al.; The association of nicked RNA polymerase of bacteriophage T7 (Ikeda, R . A., and Richardson, C . C . (1987) J . Biol . Chem . 262, 3790-3799) with the T7 phi 10 promoter has been examined by DNA cleavage protection . The phi 10 promoter consists of a 23-base pair consensus sequence that extends from -17 to +6 with respect to the site of the initiation of transcription (+1) . Nicked T7 RNA polymerase alone protects 20 bases from -21 to -2 (+/- 1) base at each border . Initiation and synthesis of the trinucleotide r(GGG) expands and shifts the sequence protected by nicked T7 RNA polymerase . Twenty-five bases are protected from -17 to +8 (+/- 1) . The polymerization of three additional ribonucleotides, synthesis of the hexamer r(GGGAGA), further expands the protected sequence . Twenty-seven bases are protected from -17-+10 (+/- 1) . Finally, the synthesis of a pentadecaribonucleotide transcript, r(GGGAGACCACGG), leads to the formation of a transcription complex that protects 22 bases from -2-+20 (+/- 1) . In comparison to the sequences protected by T7 RNA polymerase the sequences protected by the nicked enzyme are shortened at the 5' end and are translocated downstream much earlier during the initiation of transcription . It appears that a portion of the DNA contacts made at the amino terminus of T7 RNA polymerase are disrupted in the small fragment of nicked T7 RNA polymerase . The changes that are observed in the sequences protected by nicked T7 RNA polymerase are reflected in the physical characteristics of the DNA X enzyme complexes . The number of ion pairs formed by the r(GGG)-initiated complex of the nicked enzyme is reduced, and the association constant for the formation of the r(GGG)-initiated complex is decreased as compared to the intact T7 RNA polymerase. J Biol Chem, 1987 Mar 15, 262(8), 3790 - 9 Enzymatic properties of a proteolytically nicked RNA polymerase of bacteriophage T7; Ikeda RA et al.; The RNA polymerase of bacteriophage T7 is sensitive to cleavage by a protease associated with the membrane fraction of many strains of Escherichia coli . A major degradation product is a T7 RNA polymerase that is proteolytically cleaved between amino acids 172 (lysine) and 173 (arginine) (Tabor, S., and Richardson, C.C . (1985) Proc . Natl . Acad . Sci . U.S.A . 82, 1074-1078) . The cleavage splits the enzyme into a large fragment (Mr approximately 75,000) and a small fragment (Mr approximately 23,000) which remain tightly associated during the purification of nicked RNA polymerase . The protein retains RNA polymerase activity, but specific activity is reduced 3.5-fold . The proteolytic cleavage also reduces the Mg2+ requirements, increases the apparent Michaelis-Menten constants for the utilization of the ribonucleoside 5'-triphosphates, increases the temperature sensitivity, increases the sensitivity to inhibition by heparin, and increases the probability that a transcript will not be removed from the template . The reduced activity of nicked T7 RNA polymerase is apparently a consequence of inefficient initiation and premature termination . Nicked T7 RNA polymerase successfully initiates at the phi 10 promoter at half the efficiency of native T7 RNA polymerase . Transcripts synthesized by the nicked enzyme are also significantly shorter than transcripts synthesized by the native enzyme . In contrast, nicked T7 RNA polymerase and T7 RNA polymerase exhibit equivalent poly(dI).poly(dC)-dependent activity and equivalent polymerization velocities (60 bases/s at 25 degrees C). J Biol Chem, 1987 Mar 15, 262(8), 3553 - 61 Purification and properties of a bacteriophage-induced endo-N-acetylneuraminidase specific for poly-alpha-2,8-sialosyl carbohydrate units; Hallenbeck PC et al.; The soluble form of a bacteriophage-induced endo-N-acetylneuraminidase (Endo-N) specific for hydrolyzing oligo- or poly-alpha-2,8-linked sialosyl units in sources as disparate as bacterial and neural membrane glycoconjugates was purified approximately 10,000-fold and characterized . The enzyme appears homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a subunit Mr 105,000 . This corresponds to one of the higher Mr phage proteins which comprises 7.5% (by weight) of the total phage protein . The holoenzyme is active at neutral pH and has a Mr by gel filtration of 328,000, suggesting that the active enzyme is a trimer . Endo-N requires a minimum of 5 sialyl residues (DP5, where DP represents degree of polymerization) for activity . The limit digest products from the alpha-2,8-linked polysialic acid capsule of Escherichia coli K1 are DP4 with some DP3 and DP1,2 . DP2-4 do not appear to inhibit depolymerization of polysialic acid . Endo-N digestion of the polysialosyl moiety on neural cell adhesion molecules yields sialyl oligomers with DP3 and DP4 . The presence of a terminal sialitol changes both the distribution of limit digestion products and the apparent minimum substrate size . Higher Mr alpha-2,8-linked sialyl polymers (approximately DP200) are better substrates (Km 50-70 microM) than sialyl oligomers of approximately DP10-20 (Km 1.2 mM) . Endo-N activity is inhibited by DNA and several other poly-anions tested . An examination of the distribution of intermediate products shows that Endo-N binds and cleaves at random sites on the polysialosyl chains, in contrast to initiating cleavage at one end and depolymerizing processively . Endo-N can serve as a specific molecular probe to detect and selectively modify poly-alpha-2,8-sialosyl carbohydrate units which have been implicated in bacterial meningitis and neural cell adhesion. Biochem J, 1987 Mar 15, 242(3), 735 - 42 Organization and sequence of the human alpha-lactalbumin gene; Hall L et al.; A recombinant bacteriophage containing the entire alpha-lactalbumin gene was isolated from a human genomic library constructed in bacteriophage lambda L47 . Within this recombinant the 2.5 kb alpha-lactalbumin gene is flanked by about 5 kb of sequence on either side . The complete nucleotide sequence of the gene and its immediate flanking sequences were determined and compared with those of the rat alpha-lactalbumin gene . These studies showed that the size, organization and sequence of the exons have been highly conserved, whereas the introns have diverged considerably . In particular, the first intron of the human gene was found to contain an Alu repetitive sequence not present in the rat . A high degree of homology (67%) was also observed in the 5' flanking regions, extending as far as 655 nucleotide residues upstream of the transcriptional initiation site . Comparison of the 5' flanking sequences of these two alpha-lactalbumin genes with t