Phage

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 those of five casein genes has revealed the presence of a highly conserved region {consensus sequence: RGAAGRAAA(N)TGGACAGAAATCAA(CG)TTTCTA}, extending from position -140 to -110 in all seven sequences examined, suggesting a possible regulatory role in the hormonal control or tissue-specific expression of milk protein genes in the mammary gland.

Biochem Biophys Res Commun, 1987 Mar 13, 143(2), 593 - 9
A bacteriophage transcription terminator permits the cloning of a mammalian expression vector carrying the human preprorenin gene in E . coli; Weighous TF et al.; We constructed a plasmid-based expression vector carrying the murine metallothionein gene promoter, the human preprorenin gene, the Tn5 neomycin phosphotransferase II gene, and a complete bovine papilloma virus genome . We were unsuccessful in cloning the preprorenin gene in E . coli when it was inserted into a plasmid vector 3' to the metallothionein gene promoter . This result was consistent with our previous data suggesting that expression of the preprorenin gene is toxic to E . coli (Weighous, T.F . et al; (1986) Gene 45: 121-129) . We then inserted a DNA fragment from bacteriophage lambda carrying the t1 transcriptional terminator between the promoter and the preprorenin gene; this vector was stably maintained in E . coli . Prior to introducing the vector DNA into mouse cells for expression studies the terminator was removed . Mouse cells carrying the vector DNA secreted high levels of human prorenin for greater than six weeks.

Nucleic Acids Res, 1987 Mar 11, 15(5), 2069 - 88
In vitro molecular genetics as a tool for determining the differential cleavage specificities of the poliovirus 3C proteinase; Ypma-Wong MF et al.; We describe a completely in vitro system for generating defined poliovirus proteinase mutations and subsequently assaying the phenotypic expression of such mutations . A complete cDNA copy of the entire poliovirus genome has been inserted into a bacteriophage T7 transcription vector . We have introduced proteinase and/or cleavage site mutations into this cDNA . Mutant RNA is transcribed from the altered cDNA template and is subsequently translated in vitro . Employing such a system, we provide direct evidence for the bimolecular cleavage events carried out by the 3C proteinase . We show that specific genetically-altered precursor polypeptides containing authentic Q-G cleavage sites will not act as substrates for 3C either in cis or in trans . We also provide evidence that almost the entire P3 region is required to generate 3C proteinase activity capable of cleaving the P1 precursor to capsid proteins . However, only the 3C portion of P3 is required to generate 3C proteinase activity capable of cleaving P2 and its processing products.

Biochemistry, 1987 Mar 10, 26(5), 1373 - 81
Structure and dynamics of the Pf1 filamentous bacteriophage coat protein in micelles; Schiksnis RA et al.; The major coat protein of filamentous bacteriophage adopts its membrane-bound conformation in detergent micelles . High-resolution 1H and 15N NMR experiments are used to characterize the structure and dynamics of residues 30-40 in the hydrophobic midsection of Pf1 coat protein in sodium dodecyl sulfate micelles . Uniform and specific-site 15N labels enable the immobile backbone sites to be identified by their 1H/15N heteronuclear nuclear Overhauser effect and allow the assignment of 1H and 15N resonances . About one-third of the amide N-H protons in the protein undergo very slow exchange with solvent deuterons, which is indicative of sites in highly structured environments . The combination of results from 1H/15N heteronuclear correlation, 1H homonuclear correlation, and 1H homonuclear Overhauser effect experiments assigns the resonances to specific residues and demonstrates that residues 30-40 of the coat protein have a helical secondary structure.

J Mol Biol, 1987 Mar 5, 194(1), 105 - 17
Localization of a DNA-binding determinant in the bacteriophage P22 Erf protein; Murphy KC et al.; Four amber fragments of the recombination-promoting P22 Erf protein were characterized . The intact Erf monomer contains 204 amino acids . The amber mutations produce fragments of 190, 149, 130 and 95 amino acid residues, all of which are inactive in vivo . The 190 residue fragment is more susceptible to proteolysis in cell extracts than is intact Erf . It breaks down to a stable remnant that is slightly larger than the 149 residue fragment . The 149 and 130 residue fragments are stable; electron microscopy of the purified fragments reveals that they have similar morphologies, retaining the ring-like oligomeric structure, but lacking the tooth-like protruding portions of intact Erf . Intact Erf and the 149 residue fragment have similar affinities for single-stranded DNA; the affinity of the 130 residue fragment is 40-fold lower in low salt at pH 6.0 . The 95 residue fragment is unstable in vivo . These observations, combined with previous observations, are interpreted as suggesting that the boundary of the amino-terminal domain of the protein lies between residues 96 and 130, that certain residues between 131 and 149 form part of an interdomain DNA-binding segment of the protein, that the boundary of the carboxy-terminal domain lies to the C-terminal side of residue 149, and that the carboxy-terminal domain is not necessary for assembly of the ring oligomer, although it is essential for Erf activity in vivo.

J Mol Biol, 1987 Mar 5, 194(1), 31 - 9
DNA sequence of genes 38 encoding a receptor-recognizing protein of bacteriophages T2, K3 and of K3 host range mutants; Riede I et al.; Genes 38, which code for a receptor-recognizing protein present at the tip of the long tail fibers, have been sequenced from phages T2, the T-even-type phage K3 and its host range mutants K3hx, K3h1 and K3h1h . The genes from phages T2 and K3 code for proteins consisting of 262 and 260 amino acid residues, respectively . Fifty amino-terminal and 25 carboxy-terminal residues are highly conserved . The amino-terminal amino acids are most likely involved in binding to the neighboring protein 37 . Between residues 116 and 226 of the T2 protein and residues 116 and 223 of the K3 protein, sequences exist that are similar to sequences present in Escherichia coli outer membrane proteins and which serve as phage receptors . Most likely, all of these regions in the latter proteins are exposed on the cell surface and are part of their phage receptor areas . In the phage proteins, these sequences are flanked by stretches rich in glycine, perhaps providing an increased flexibility for the polypeptide at these sites; some "wobble" may be required during the protein 38-receptor interaction . The mutational alterations in the host range mutants were found in gene 38 . In the K3hx protein, a duplication of six base-pairs caused the wild-type sequence -Gly163-Lys-Leu-Ile- to be changed to -Gly163-Lys-Leu-Lys-Leu-Ile- . In the K3h1 protein, a glutamic acid residue at position 203 was substituted by a lysine . Both alterations occurred within areas similar to outer membrane proteins . Mutant K3h1h, derived from K3h1, exhibits an extended host range as compared to K3h1 . No mutational alteration, in addition to that found in K3h1, was found in g38 nor was the part of gene 37 that encodes the carboxy-terminal moiety of the protein altered . K3h1h may represent a "trigger-happy" phage . The results of this and other work show that the phage-phage receptor systems under study represent a primitive immune system.

J Mol Biol, 1987 Mar 5, 194(1), 23 - 30
T-even-type bacteriophages use an adhesin for recognition of cellular receptors; Riede I et al.; Protein 38 of the Escherichia coli phage T4 is thought to be required catalytically for the assembly of the long tail fibers of this phage . It is shown that this protein of phage T2 and the T-even-type phage K3 and Ox2 act differently . It was found that NH2-terminal fragments of the protein, expressed from cloned fragments of gene 38 of phage K3, bind to gene 38 amber mutants of phage T2 . Such phage or T2 gene 38 amber mutants, grown on a non-permissive host, possess a complete set of six tail fibers but are non-infectious . Both types of non-infectious phage could be repaired by incubation with an extract of cells harboring a cloned gene 38 of a host range mutant of phage K3, K3hx . The repaired phages had the host range of K3hx and not of T2 . Immuno-electron microscopy showed that protein 38 is located at the free ends of the long tail fibers of phages T2, K3 and Ox2 . The protein serves the recognition of the cellular receptor, i.e . it acts as an adhesin.

J Mol Biol, 1987 Mar 5, 194(1), 155 - 9
Mismatch repair of deaminated 5-methyl-cytosine; Jones M et al.; Deamination of 5-methyl-cytosine in double-stranded DNA produces a G-T mismatch . Heteroduplexes of bacteriophage lambda DNA containing a G-T mismatch at the site of a G-5-meC base-pair in one of the parental phages were constructed and used to transfect Escherichia coli cells . Genetic analysis of the progeny phages derived from such heteroduplexes suggests that, in E . coli, mismatches resulting from the deamination of 5-methyl-cytosine are repaired by a system requiring the E . coli dcm methylase and some, but not all, of the functions of the E . coli methyl-directed mismatch repair system . The repair appears to act only on the G-T mismatch and acts specifically to restore the cytosine methylation sequence.

Biofizika, 1987 Mar-Apr, 32(2), 342 - 3
{Study of local changes in the structure of phage DNA by electron microscopy}; Vecher AA et al.; DNA of defective bacteriophage PBSX was studied by electron microscopy . The presence and distribution of sites containing local structure changes were revealed using antibodies specific for the DNA molecules modified in situ . These structural changes are related to the capsid geometry, but not to the DNA primary sequence.

Biophys J, 1987 Mar, 51(3), 503 - 7
Dynamic light-scattering study on changes in flexibility of filamentous bacteriophage Pf1 with temperature; Sasaki S et al.; The temperature dependence of the flexibility of bacteriophage Pf1 was investigated by dynamic light scattering, and the following results were obtained: The gamma/K2 values measured at 1 degree-25 degrees C and at various K values were T/eta-scaled to 20 degrees C, where gamma is the first cumulant of the field correlation function of scattered light, K is the length of the scattering vector, T is the absolute temperature, and eta is the solvent viscosity at T . And it was found that the scaled gamma/K2 values at low K values were independent of temperature, whereas those at high K values increased sigmoidally and reversibly against temperature . This suggests that the virion is more flexible at temperatures above the transition temperature Tt . This characteristic temperature Tt depended on the pH of the suspension: Tt = 11 degrees C at pH 6.9 and Tt = 8 degrees C at pH 8.2.

Genetics, 1987 Mar, 115(3), 405 - 17
A new epistasis group for the repair of DNA damage in bacteriophage T4: replication repair; Wachsman JT et al.; The gene 32 mutation amA453 sensitizes bacteriophage T4 to the lethal effects of ultraviolet (UV) irradiation, methyl methanesulfonate and angelicin-mediated photodynamic irradiation when treated particles are plated on amber-suppressing host cells . The increased UV sensitivity caused by amA453 is additive to that caused by mutations in both the T4 excision repair (denV) and recombination repair (uvsWXY) systems, suggesting the operation of a third kind of repair system . The mutation uvs79, with many similarities to amA453 but mapping in gene 41, is largely epistatic to amA453 . The mutation mms1, also with many similarities to amA453, maps close to amA453 within gene 32 and is largely epistatic to uvs79 . Neither amA453 nor uvs79 affect the ratio of UV-induced mutational to lethal hits, nor does amA453 affect spontaneous or UV-enhanced recombination frequencies . Gene 32 encodes the major T4 ssDNA-binding protein (the scaffolding of DNA replication) and gene 41 encodes a DNA helicase, both being required for T4 DNA replication . We conclude that a third repair process operates in phage T4 and suggest that it acts during rather than before or after DNA replication.

Genetics, 1987 Mar, 115(3), 393 - 403
Genetic mapping of the amino-terminal domain of bacteriophage T4 DNA polymerase; Hughes MB et al.; The DNA polymerase of bacteriophage T4 is a multifunctional enzyme that harbors DNA-binding, DNA-synthesizing and exonucleolytic activities . We have cloned in bacterial plasmids about 99% of the structural gene for this enzyme (T4 gene 43) . The gene was cloned in six contiguous 5'-terminal DNA fragments that defined seven intragenic mapping regions . Escherichia coli hosts harboring recombinant plasmids carrying the gene 43 subsegments were used in marker-rescue experiments that assigned a large number of ts and nonsense polymerase mutations to different physical domains of the structural gene . Conspicuously, only one missense mutation in a large collection of mutants mapped in the 5'-terminal 450 base-pair segment of the approximately 2700 base-pair gene . To test if this indicated a DNA polymerase domain that is relatively noncritical for biological activity, we mutagenized a recombinant plasmid carrying this 5'-terminal region and generated new conditional-lethal mutations that mapped therein . We identified five new ts sites, some having mutated at high frequency (nitrosoguanidine hot spots) . New ts mutations were also isolated in phage genes 62 and 44, which map upstream of gene 43 on the T4 chromosome . A preliminary examination of physiological consequences of the ts gene 43 mutations showed that they exhibit effects similar to those of ts lesions that map in other gene 43 segments: some were mutators, some derepressed gene 43 protein synthesis and they varied in the severity of their effects on T4-induced DNA synthesis at nonpermissive temperatures . The availability of the gene 43 clones should make it possible to isolate a variety of lesions that affect different activities of the T4 DNA polymerase and help to define the different domains of this multifunctional protein.

J Bacteriol, 1987 Mar, 169(3), 1232 - 8
Cloning and characterization of the Escherichia coli lit gene, which blocks bacteriophage T4 late gene expression; Kao C et al.; Escherichia coli lit mutations inhibit gene expression late in infection by bacteriophage T4 . We cloned the lit gene from wild-type E . coli and three independent lit mutants . We present evidence that lit mutations {renamed lit(Con) mutations} cause overproduction of the lit gene product and that overproduction of this product causes the inhibition of gene expression . We also present evidence that the lit gene product is nonessential for E . coli growth, although the gene is common to most E . coli K-12 strains.

Mol Biol (Mosk), 1987 Mar-Apr, 21(2), 396 - 402
{Mechanism of reaction catalyzed by RNA-ligase from bacteriophage T4}; Zagrebel'nyi SN et al.; The dissociation constants of the complexes of RNA-ligase with acceptors, donors and the adenylylated donor A(5')ppAp have been determined on the basis of the inhibition of ATP-pyrophosphate exchange reaction . The dissociation constants of the complexes of the enzyme with "poor" acceptors (oligouridilates) have been shown to be slightly different from those with "good" acceptors (oligoadenylates) . The dependence of the reaction velocity of the formation of ligation products on the concentration of acceptors (pA)4, (pU)4 and the adenylylated donor A(5)ppAp has been studied . On the basis of the data obtained the conclusion about the random addition mechanism has been drawn . The reaction takes place in the steady-state conditions in the case of (pA)4 and in the equilibrium conditions--in the case of (pU)4.

EMBO J, 1987 Mar, 6(3), 809 - 14
Autoregulation of bacteriophage P2 repressor; Saha S et al.; The immunity repressor of bacteriophage P2 regulates the two convergent promoters, Pe and Pc, located in the early control region . Pe is the early promoter which is negatively regulated by the repressor . It was found, by DNase I protection studies, that the P2 repressor enhances the binding of RNA polymerase to Pc . Furthermore, under in vivo conditions the transcription initiated at Pc, measured as chloramphenicol acetyl transferase gene expression, is low in the absence of repressor but is stimulated by low repressor levels . With increasing repressor concentrations transcription from the Pc promoter decreases . Thus, the P2 repressor both negatively and positively regulates its own promoter.

Biokhimiia, 1987 Mar, 52(3), 512 - 8
{Purification and various properties of exonuclease from bacteriophage T4}; Kanopka AE et al.; Exonuclease A was isolated from bacteriophage T4-infected cells of E . coli . The molecular mass of the enzyme is approximately 42,000 Da, pH optimum is 7-8.5, pI is 4.05 . The enzyme activity depends on Mg2+, the optimal concentration of Mg2+ being 1-5 mM . The enzyme splits one- and two-helical DNA in the direction of 3'----5' and is a deoxyribonuclease splitting 5'-deoxynucleotides . The enzyme shows a practically equal affinity for one and two-helical DNA . The Km value for one- and two-helical DNA is 10 +/- 1 and 11 +/- 1 pmole of chain DNA, respectively . The Vmax value for one- and two-helical DNA is 61 +/- 5 and 45 +/- 5 pmole of nucleotides per min . Exonuclease A may be used for preparing substrates for DNA-polymerase T4 and Klenow fragment, i.e., during labeling of DNA at 3'-ends.

Genetika, 1987 Mar, 23(3), 440 - 8
{Effect of mutations of Escherichia coli K-12 chromosome on the integration of bacteriophage Mu introduced into cells by infection or conjugation}; Upmal MR et al.; We present the detailed research on the previously described Escherichia coli K-12 Mud- mutants with impaired development of bacteriophage Mu . The ability of Mu phage DNA to penetrate into mutant cells on infection was shown . If introduced into the cells or combined with mud mutation by recombination, the prophage may be induced, which results in phage Mu lythic development and phage burst from mutant cells . In the course of conjugative transfer into the mutant cells, within a DNA fragment of the lysogenic donor chromosome, MupAp1 prophage is not inherited by recombinants . At the same time, Mu prophage deficient in genes A and B, whose products are required for transposition, is inherited by the mutant with the usual frequency . These data enable us to conclude that the mud mutations disturb the stage of conservative transposition which is connected with the insertion of the Mu prophage into the chromosome, after excision from the linear DNA introduced into the cells via infection or conjugation.

Genetika, 1987 Mar, 23(3), 389 - 96
{Physical mapping of the immunity region of phage phi 80}; Vasinova NA et al.; Mapping of the sites of cleavage of five restriction endonucleases (BglI, BglII, EcoRV, KpnI and PvuII) in the immunity region of bacteriophage phi 80 DNA is described . The order of the 27 restriction sites was established . This helped to localize the phi 80 cI gene within the 640 bp fragment of the immunity region . Recombinant plasmids carrying phi 80 "kil" function were constructed . The function is suppressed by the cI-coded repressor . The deletion AB43 which is present in the phi 80 vir mutant is located within the phi 80 DNA fragment carrying the cI gene.

Virology, 1987 Mar, 157(1), 167 - 71
Expression and proteolytic processing of the darA antirestriction gene product of bacteriophage P1; Streiff MB et al.; The darA gene coding for one of the two bacteriophage P1 antirestriction functions is expressed late after infection or induction . The protein is made as a high-molecular-weight soluble precursor . This is proteolytically cleaved to the mature form, which is a structural component of the phage head . Defective mutants of the phage have been found in which the synthesis of gpdarA is normal but processing does not take place . These mutations all map to the same region of the P1 genome and we propose that they lie in the structural gene for the processing protease.

Virology, 1987 Mar, 157(1), 156 - 66
Two DNA antirestriction systems of bacteriophage P1, darA, and darB: characterization of darA- phages; Iida S et al.; Bacteriophage P1 is only weakly restricted when it infects cells carrying type I restriction and modification systems even though DNA purified from P1 phage particles is a good substrate for type I restriction enzymes in vitro . Here we show that this protection against restriction is due to the products of two phage genes which we call darA and darB (dar for defense against restriction) . Each of the dar gene products provides protection against a different subset of type I restriction systems . The darA and darB gene products are found in the phage head and protect any DNA packaged into a phage head, including transduced chromosomal markers, from restriction . The proteins must, therefore, be injected into recipient cells along with the DNA . The proteins act strictly in cis . For example, upon double infection of restricting cells with dar+ and dar- P1 phages, the dar+ genomes are protected from restriction while the dar- genomes are efficiently restricted.

Virology, 1987 Mar, 157(1), 117 - 26
The amino terminus of the bacteriophage D108 transposase protein contains a two-component sequence-specific DNA-binding domain; Tolias PP et al.; We have cloned various amino-terminal domains of the transposable bacteriophage D108 A protein (transposase) into a high-copy-number expression vector and visualized the D108 polypeptides and fusion proteins expressed by the recombinant plasmids . By using crude protein extracts made from strains harboring these recombinant plasmids, we have performed band competition assays and DNasel footprinting on a 32P-end-labeled DNA restriction fragment which contained the Mu right end (to which the Mu A protein binds) and have shown that these fusion proteins in crude extracts can specifically bind to this DNA substrate . Furthermore, we have divided the amino-terminal 13 kDa of the D108 A protein (which may contain two bi-alpha-helical protein structures) in half and have shown that each half is capable of independent binding to the Mu attR site . These results suggest that the D108 transposase protein contains multiple DNA-binding domains which may be required for the complex protein-DNA interactions of D108 transposition.

Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1244 - 8
Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins; McIntosh LP et al.; A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented . This approach is based on 15N (or 13C) labeling of selected residues in a protein . The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-15N (or 13C) spectrum . The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements . Using this approach, we have observed approximately 160 resonances from 15N-bonded protons in the backbone and sidechains of uniformly 15N-labeled T4 lysozyme (molecular mass = 18.7 kDa) . Partial proton-deuterium exchange can be used to simplify the 1H-15N spectrum of this protein . These resonances are identified by amino acid class using selective incorporation of 15N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple 15N and 13C labeling, and isotope-directed nuclear Overhauser effect measurements . For example, using a phenyl{15N}alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an alpha-helical region, we observe an isotope-directed nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.

Mikrobiologiia, 1987 Mar-Apr, 56(2), 227 - 31
{Decreased synthetic activity as a possible cause of the death of Escherichia coli bacteria during amino acid starvation}; Rybkin AI et al.; The work is concerned with studying the breakdown of proteins and RNA when a polyauxotrophic Escherichia coli strain is incubated in a salt solution without amino acids, phosphorus, nitrogen and glucose at 43 degrees C as well as the ability of starving bacterial cells to recommence protein and RNA synthesis (also in the course of phage T4 infection) and to reproduce bacteriophages T4, lambda and MS2 . Within the first two hours of the incubation, 12% of proteins and 40% of RNA break down to acid-soluble fragments . Then protein degradation stops while RNA decomposition goes on, but at a lower rate . Within 4-6 h of starvation, the rate of protein and RNA synthesis drops down 4-5 times and the survival rate equals 40-60% when the cells are transferred onto a complete medium . The quantitative characteristics of phages T4, lambda and MS2 reproduction fall down in prestarved cells . The authors speculate that E . coli cells die off in the course of starvation not because some unique structure is destroyed, but owing to the fact that the activity of enzymes and ribosomes gradually declines . As a result, the synthetic activity of the cell drops down abruptly and irreversibly because the enzymes are inactivated and RNA breaks down, which eventually causes cell death.

J Biol Chem, 1987 Feb 25, 262(6), 2696 - 703
Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli; Maurizi MR; Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0 . Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J . E., and Pearson, M . L . (1981) Cell 24, 225-233) . Under conditions used for N protein degradation, several lambda and E . coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease . Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP . This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro . The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP . By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions . Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease . N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule . In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine) . However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.

Nucleic Acids Res, 1987 Feb 25, 15(4), 1445 - 58
Isolation of polymorphic DNA fragments from human chromosome 4; Gilliam TC et al.; We have identified and characterized 40 DNA probes detecting restriction fragment length polymorphism (RFLP) on human chromosome 4 . Single copy human clones were isolated from a bacteriophage library enriched for chromosome 4 sequences . Each clone was hybridized to somatic cell hybrid DNAs for verification of its species and chromosomal origin and for regional localization . Sequences specific for chromosome 4 were tested for their ability to detect RFLPs in human DNA and their potential utility as genetic markers was assessed . Approximately 263,000 base pairs or 0.13% of the chromosome was screened for sequence variation . The estimate of heterozygosity calculated from this large body of data, H = 0.0021, indicates that the degree of sequence variation on chromosome 4 is comparable to other autosomes . The characterization of these 40 markers has tripled the number of polymorphic loci available for linkage studies on chromosome 4, making it feasible to begin construction of a detailed linkage map that will span the entire chromosome.

Biochemistry, 1987 Feb 24, 26(4), 1094 - 9
Cloning and sequence determination of a complementary DNA related to human liver microsomal cytochrome P-450 S-mephenytoin 4-hydroxylase; Umbenhauer DR et al.; A cDNA sequence related to the human cytochrome P-450 responsible for S-mephenytoin 4-hydroxylation (P-450MP) has been isolated from a human liver bacteriophage lambda gt11 library with antibodies specific for P-450MP . The total length of the cDNA is 2.5 kilobases (kb), of which there is a 1.6-kb EcoRI fragment coding for all but five amino acids corresponding to the N-terminus of the protein and including a small noncoding region at the 3' end . This 1.6-kb fragment has been sequenced and used as a probe to analyze human genomic DNA and liver RNA . The sequence shows extensive sequence similarity with that of rabbit liver cytochrome P-450 progesterone 21-hydroxylase {Tukey, R . H., Okino, S., Barnes, H., Griffin, K . J., & Johnson, E . F . (1985) J . Biol . Chem . 260, 13347-13354}, and this cDNA, like the rabbit clone, appears to be part of a multigene family . At least two liver mRNA species, 2.2 kb and 3.5 kb, hybridize to the cDNA sequence . The cloning of this gene should aid in analyzing the molecular basis for the genetic polymorphism of S-mephenytoin 4-hydroxylation reported in humans.

Science, 1987 Feb 20, 235(4791), 865 - 70
Immunological self, nonself discrimination; Guillet JG et al.; The ability of immunodominant peptides derived from several antigen systems to compete with each other for T cell activation was studied . Only peptides restricted by a given transplantation antigen are mutually competitive . There is a correlation between haplotype restriction, ability to bind to the appropriate transplantation antigen, and ability to inhibit activation of other T cells restricted by the same transplantation antigen . An exception was noted in that a peptide derived from an antigen, bacteriophage lambda cI repressor, binds to the I-Ed molecule in a specific way, yet is not I-Ed-restricted . Comparison of the sequence of the repressor peptide with that of other peptides able to bind to (and be restricted by) I-Ed and a polymorphic region of the I-Ed molecule itself revealed a significant degree of homology . Thus, peptides restricted by a given class II molecule appear to be homologous to a portion of the class II molecule itself . The repressor-derived peptide is identical at several polymorphic residues at this site, and this may account for the failure of I-Ed to act as a restriction element . Comparison of antigenic peptide sequences with transplantation antigen sequences suggests a model that provides a basis for explaining self, nonself discrimination as well as alloreactivity.

J Biol Chem, 1987 Feb 15, 262(5), 2066 - 76
DNA helicase II of Escherichia coli . Characterization of the single-stranded DNA-dependent NTPase and helicase activities; Matson SW et al.; Escherichia coli helicase II has been purified to near homogeneity from cells harboring a multicopy plasmid containing the structural gene for helicase II, uvrD . In this paper a detailed description of the single-stranded DNA-dependent nucleoside 5'-triphosphatase and helicase reactions catalyzed by helicase II is presented . The results of this study suggest that nucleoside 5'-triphosphate hydrolysis provides the energy required for translocation of the enzyme along single-stranded DNA . Measurements of the rate of ATP hydrolysis using a variety of single-stranded DNAs of known structure and length suggest a processive translocation mechanism for helicase II . Single-stranded DNA coated with either Escherichia coli single-stranded DNA binding protein (SSB) or bacteriophage T4 gene 32 protein fails to support helicase II ATPase activity . Moreover, helicase II is apparently unable to displace a molecule of bound SSB protein from single-stranded DNA when it is encountered in the process of translocation along a single-stranded DNA effector . The helicase reaction has been characterized using an in vitro strand displacement helicase assay . The helicase reaction requires concomitant nucleoside 5'-triphosphatase hydrolysis that is satisfied by the hydrolysis of either rATP or dATP . As the length of duplex DNA present in the partial duplex helicase substrate is increased from 71 base pairs to 343 base pairs, the fraction of duplex DNA molecules that are unwound by helicase II decreases in the absence of any accessory proteins . However, the total number of base pairs of duplex DNA unwound depends primarily on the amount of enzyme added to the helicase reaction and not on the length of the duplex DNA present in the partial duplex DNA substrate . These data suggest the number of base pairs of duplex DNA unwound is directly proportional with the concentration of helicase II in the reaction mixture . In addition, the rate of the unwinding reaction is independent of the length of the duplex DNA available for unwinding . Helicase II has been shown to dissociate from single-stranded DNA molecules infrequently acting as an ATPase . However, the enzyme dissociates from partial duplex helicase substrates more frequently . This suggests a more distributive reaction mechanism on duplex DNA than was observed on single-stranded DNA substrates . The fraction of 343-base pair partial duplex DNA molecules unwound by helicase II can be increased by the addition of appropriate concentrations of E . coli SSB to the reaction . This suggests that helicase II and SSB may act in a concerted reaction to unwind duplex DNA.

Anal Biochem, 1987 Feb 15, 161(1), 85 - 8
Preserving primary cDNA libraries; Klinman DM et al.; A technique for the long term storage of primary cDNA libraries in a form such that relevant DNA sequences can be readily identified and retrieved is described . cDNA libraries produced using the lambda gt 10 cloning vector were plated out on host bacteria in 0.7% top agarose supplemented with 30% glycerol . Nitrocellulose lifts of these libraries were made and stored . These lifts could be screened at a later time to permit identification of bacteriophage plaques containing specific cDNA inserts . The plated libraries were then transferred to a -70 degrees C freezer . The combination of freezing and glycerol treatment allowed the bacteriophage in these primary cDNA libraries to remain viable for significantly longer than 1 year.

J Biol Chem, 1987 Feb 15, 262(5), 2326 - 31
Purification and properties of Gal repressor:pL-galR fusion in pKC31 plasmid vector; Majumdar A et al.; The galR gene, which encodes the Gal repressor protein in Escherichia coli, has been fused to the strong pL promoter of bacteriophage lambda in plasmid pKC31 . The pL promoter is kept repressed by a thermolabilie lambda repressor, CIts857, to prevent cell killing . Heat induction of the pL-galR fusion plasmid synthesizes large amounts of active Gal repressor . The protein has been purified to homogeneity in three steps . The purification is greatly aided by the reversible insolubility of active repressor in crude extract at salt concentrations of less than 200 mM . The amino-terminal amino acid sequence determined by automated Edman degradation is: N-Ala-Thr-Ile-Lys-Asp-Val-Ala-Arg-Leu-Ala-Gly-Val-Ser-Val-Ala-Thr-Val- . Comparison of this sequence with that deduced from the DNA sequence of the galR gene showed that the formyl methionine residue preceding alanine at position 1 is cleaved off . The repressor is present in solution as a dimer of a 37-kDa subunit . The protein binds to gal DNA containing wild type and not mutant operator sequences . As predicted, this sequence-specific binding is inhibited by the presence of D-galactose or D-fucose, both of which are in vivo inducers of the gal operon . Gal repressor inhibits the expresison of gal operon by binding to two spatially separated operators which flank, but do not overlap, the gal promoter segment . Experiments to study the mechanism of repressor action are discussed.

Nucleic Acids Res, 1987 Feb 11, 15(3), 1227 - 43
Utilization of DNA photolyase, pyrimidine dimer endonucleases, and alkali hydrolysis in the analysis of aberrant ABC excinuclease incisions adjacent to UV-induced DNA photoproducts; Myles GM et al.; ABC excinuclease of Escherichia coli removes 6-4 photoproducts and pyrimidine dimers from DNA by making two single strand incisions, one 8 phosphodiester bonds 5' and another 4 or 5 phosphodiester bonds 3' to the lesion . We describe in this communication a method, which utilizes DNA photolyase from E . coli, pyrimidine dimer endonucleases from M . luteus and bacteriophage T4, and alkali hydrolysis, for analyzing the ABC excinuclease incision pattern corresponding to each of these photoproducts in a DNA fragment . On occasion, ABC excinuclease does not incise DNA exclusively 8 phosphodiester bonds 5' or 4 or 5 phosphodiester bonds 3' to the photoproduct . Both the nature of the adduct (6-4 photoproduct or pyrimidine dimer) and the sequence of neighboring nucleotides influence the incision pattern of ABC excinuclease . We show directly that photolyase stimulates the removal of pyrimidine dimers (but not 6-4 photoproducts) by the excinuclease . Also, photolyase does not repair CC pyrimidine dimers efficiently while it does repair TT or TC pyrimidine dimers.

Nucleic Acids Res, 1987 Feb 11, 15(3), 933 - 45
Differential gene expression during the amoebal-plasmodial transition in Physarum; Sweeney GE et al.; We have prepared cDNA libraries for amoebae and plasmodia of the acellular slime mould, Physarum polycephalum . Differential screening was used to isolate cell-type-specific cDNA clones (in bacteriophage M13) and both libraries yielded approximately 5% of such sequences . The amoebal- and plasmodial-specific clones were used to assay changes in transcription during the amoebal-plasmodial transition . The results obtained substantiate the view that the switch from amoebal to plasmodial characteristics occurs over several nuclear division cycles . With one exception, the specific cDNAs came from single-gene families . Southern blotting experiments also showed that they hybridised to identical restriction fragments from amoebal and plasmodial DNAs indicating that genomic rearrangements are unlikely to be involved in the regulation of these genes.

Biochemistry, 1987 Feb 10, 26(3), 847 - 54
Dynamics of fd coat protein in the bacteriophage; Colnago LA et al.; The dynamics of the coat protein in fd bacteriophage are described with solid-state 15N and 2H NMR experiments . The virus particles and the coat protein subunits are immobile on the time scales of the 15N chemical shift anisotropy (10(3) Hz) and 2H quadrupole (10(6) Hz) interactions . Previously we have shown that the Trp-26 side chain is immobile, that the two Tyr and three Phe side chains undergo only rapid twofold jump motions about their C beta-C gamma bond axis {Gall, C . M., Cross, T . A., DiVerdi, J . A., & Opella, S . J . (1982) Proc . Natl . Acad . Sci . U.S.A . 79, 101-105}, and that most of the backbone peptide linkages are highly constrained but do undergo rapid small amplitude motions {Cross, T . A., & Opella, S . J . (1982) J . Mol . Biol . 159, 543-549} in the coat protein subunits in the virus particles . In this paper, we demonstrate that the four N-terminal residues of the coat protein subunits are highly mobile, since both backbone and side-chain sites of these residues undergo large amplitude motions that are rapid on the time scales of the solid-state NMR experiments . In addition, the dynamics of the methyl-containing aliphatic residues Ala, Leu, Val, Thr, and Met are analyzed . Large amplitude jump motions are observed in nearly all of these side chains even though, with the exception of the N-terminal residue Ala-1, their backbone peptide linkages are highly constrained . The established information about the dynamics of the structural form of fd coat protein in the virus particle is summarized qualitatively.(ABSTRACT TRUNCATED AT 250 WORDS)

Science, 1987 Feb 6, 235(4789), 683 - 4
A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA; Vassart G et al.; The term "DNA fingerprint" has been used to describe the extensive restriction fragment length polymorphism associated with hypervariable minisatellites present in the human genome . Until now, it was necessary to hybridize Southern blots to specific probes cloned from human genomic DNA in order to obtain individual-specific restriction patterns . The present study describes the surprising finding that the insert-free, wild-type M13 bacteriophage detects hypervariable minisatellites in human and in animal DNA, provided no competitor DNA is used during hybridization . The effective sequence in M13 was traced to two clusters of 15-base pair repeats within the protein III gene of the bacteriophage . This unexpected use of M13 renders the DNA fingerprinting technology more readily available to molecular biology laboratories.

J Mol Biol, 1987 Feb 5, 193(3), 479 - 95
Sequence of a conditionally essential region of bacteriophage T3, including the primary origin of DNA replication; Schmitt MP et al.; The 3526 base-pair nucleotide sequence from near the end of bacteriophage T3 gene 1 to within the coding sequence of gene 2.5 is given . It includes the complete coding sequences for nine known or presumptive proteins, most of which are only conditionally essential for phage growth . The sequence includes five promoters for the phage RNA polymerase, the terminator for early (host enzyme-catalyzed) transcription, and two recognition sites for RNAase III . The primary origin of T3 DNA replication that is utilized by the phage in vivo has been localized to a 142 base-pair region . It has several features in common with the phage T7 origin of DNA replication, and exhibits considerable homology to recognition sites for the mRNA processing enzyme RNAase III . It is proposed that the primary origin of T3 DNA replication may have evolved directly from an RNAase III recognition site . The deletions present in a number of T3 mutant strains and the location of the nucleotide changes in several T3 strains that are defective in their ability to grow on F+-containing strains or on optA mutant hosts have been determined . We discuss how T3 may have become genetically isolated from its relatives in the T7-T3 group and simultaneously acquired novel biological and biochemical properties.

J Virol, 1987 Feb, 61(2), 597 - 9
Identification of the gene encoding an RNA polymerase-binding protein of bacteriophage T4; Williams KP et al.; One of five bacteriophage T4-specified proteins that bind to host RNA polymerase core has been purified and partially sequenced . A mixed oligonucleotide, based on the amino acid sequence, was used to probe genomic restriction fragments . The gene for this protein, previously designated the 15K protein, has been located between T4 genes 45 and 46 and designated rpbA.

J Microsc, 1987 Feb, 145 ( Pt 2), 159 - 77
Restoration of direct Fourier three-dimensional reconstructions of crystalline specimens by the method of convex projections; Carazo JM et al.; We consider the problem of the three-dimensional (3-D) reconstruction of objects by the direct Fourier method (DFM) and their restoration by the method of projections on to convex sets (POCS) . The main discussion is centered on the case of specimens arranged in a two-dimensional (2-D) crystal and imaged by transmission electron microscopy, although the conclusions could be extended to more general cases . We present results of the restoration of the 3-D reconstruction of a computer generated 2-D crystal under different conditions of data collection limitation . A preliminary application with a real biological specimen (the connector of bacteriophage phi 29) is also presented . These results indicate that POCS can be used practically, in certain cases, to restore 3-D reconstructions obtained by the DFM, giving grounds for the proposal of the study of a combined DFM + POCS (reconstruction + restoration) method for the determination of biological structures by electron microscopy and 3-D image processing.

Genetics, 1987 Feb, 115(2), 219 - 27
Studies on the recombination genes of bacteriophage T4: suppression of uvsX and uvsY mutations by uvsW mutations; Yonesaki T et al.; Genes uvsW, uvsX and uvsY are dispensable for T4 growth but are implicated in recombination and in the repair of damaged DNA . We found that large-plaque mutants arose efficiently from small-plaque uvsX and uvsY mutants at 42 degrees and were pseudorevertants containing a new mutation in uvsW . Using reconstructed double mutants, we confirmed that a mutation in uvsW partially increases the burst size and UV resistance of uvsX and uvsY mutants . At 41 degrees the uvsW mutation completely restores the arrest in DNA synthesis caused by mutations in genes uvsX, uvsY and 46, but at 30 degrees it only partially restores DNA synthesis in a gene 46 mutant and does not restore DNA synthesis in uvsX and uvsY mutants . Restored DNA synthesis at 41 degrees was paralleled by the overproduction of single-stranded DNA and gene 32 protein . Based on these findings, we propose that the uvsW gene regulates the production of single-stranded DNA and we discuss the phenotype of uvsW mutants and their suppression of some uvsX and uvsY phenotypes . Infection of restrictive cells with am uvsW mutants revealed a defect in the synthesis of a protein of molecular weight 53,000 daltons, suggesting that this protein is the uvsW gene product.

Am J Physiol, 1987 Feb, 252(2 Pt 1), C205 - 14
Stimulation of albumin gene transcription by insulin in primary cultures of rat hepatocytes; Lloyd CE et al.; The first goal of the work reported here was to prepare single-stranded DNA sequences for use in studies on the regulation of albumin gene expression . A double-stranded rat albumin cDNA clone was subcloned into the bacteriophage vector M13mp7 . Single-stranded recombinant clones were screened for albumin sequences containing either the mRNA strand or the complementary strand . Two clones were selected that contained the 1,200 nucleotide long 3' end of the albumin sequence . DNA from the clone containing the mRNA strand was used as a template for DNA polymerase I to prepare a radiolabeled, single-stranded cDNA to albumin mRNA . This radiolabeled cDNA probe was used to quantitate the relative abundance of albumin mRNA in samples of total cellular RNA . DNA from the clone containing the complementary strand was used to measure relative rates of albumin gene transcription in isolated nuclei . The second goal was to use the single-stranded DNA probes to investigate the mechanism of the insulin-mediated stimulation of albumin synthesis in primary cultures of rat hepatocytes . Addition of insulin to hepatocytes maintained in hepatocytes . Addition of insulin to hepatocytes maintained in a chemically defined, serum-free medium for 40 h in the absence of any hormones resulted in a specific 1.5- to 2.5-fold stimulation of albumin gene transcription that was maximal at 3 h and was maintained above control values for at least 24 h . The relative abundance of albumin mRNA and albumin secretion increased correspondingly within 24 to 30 h . These parameters remained above control levels for at least 60 h after addition of insulin . Maximal responses were attained at an insulin concentration of 100 nM and there was a close correspondence between albumin gene transcription and albumin secretion at each concentration tested . The rate of albumin gene transcription in nuclei isolated from livers of diabetic rats was reduced to 50% of the value recorded in control nuclei . Taken together, these findings demonstrate that insulin regulates synthesis of albumin at the level of gene transcription.

J Gen Virol, 1987 Feb, 68 ( Pt 2), 263 - 72
Abortive infection by bacteriophage Me1 of Escherichia coli K12 strains bearing the plasmid ColV, I-K94; Reakes CF et al.; Bacteriophage Me1 is unable to grow on Escherichia coli strains harbouring the ColV,I-K94 plasmid . The nature of this inhibition was investigated, and it was found not to be due to restriction, superinfection exclusion or receptor-mediated resistance, but to be a new example of plasmid-mediated abortive infection . Investigation of events occurring during abortive Me1 infection revealed some differences from previously described cases, especially with regard to late protein synthesis, which did occur, albeit showing abnormal amounts of some proteins . No major differences were observed in membrane permeability of productively and abortively infected cells . Phage-directed DNA synthesis was reduced in abortively infected cells . Comparative studies of Me1 and T4 revealed a striking similarity despite some minor differences.

Mol Pharmacol, 1987 Feb, 31(2), 152 - 8
Characterization of a rat liver cytochrome P-450UT-H cDNA clone and comparison of mRNA levels with catalytic activity; Churchill PF et al.; A rat liver cDNA library was prepared using the expression vector bacteriophage lambda gt11 and plaques were screened using polyclonal antibodies raised to purified rat liver cytochrome P-450UT-H, the major enzyme involved in debrisoquine 4-hydroxylation, bufuralol 1'-hydroxylation, and sparteine delta 5-oxidation . A clone was selected which contained a 1.3-kb insert . The Escherichia coli beta-galactosidase fusion protein had a molecular weight greater than that of native beta-galactosidase (and reacted with anti-P-450UT-H after electrophoresis) and was also shown to compete with microsomal P-450UT-H for anti-P-450UT-H, partially relieving catalytic inhibition by anti-P-450UT-H in rat liver microsomes . Hybrid selection experiments with the cloned cDNA also support the view that the insert is related to P-450UT-H . mRNA electrophoresis/hybridization experiments indicated that the 1.3-kb cDNA probe recognized primarily only a single size class of mRNA (2.0 kb) in rat liver . mRNA blotting and in vitro translation/immunoprecipitation experiments both indicated that levels of P-450UT-H mRNA are similar in male and female Sprague-Dawley rats . Dark Agouti strain rats of both sexes contained significantly less P-450UT-H mRNA than did Sprague-Dawley rats and the females had approximately one-half the level of the males . These results are consonant with sex and strain differences in measured levels of P-450UT-H and bufuralol 1'-hydroxylase and sparteine delta 5-oxidase activities . Analysis of genomic DNA indicated that several DNA restriction fragments hybridized to this partial length cDNA; no differences were found between the rat strains and sexes . The results suggest that the basis for the variation in P-450UT-H and its activities among rat strains and sexes is at the level of mRNA concentrations.

J Virol, 1987 Feb, 61(2), 366 - 74
Identification of two new bacteriophage T4 genes that may have roles in transcription and DNA replication; Hsu T et al.; We have identified two bacteriophage T4 genes, 45.1 and 45.2, that map in the intergenic space between phage replication genes 46 (which encodes a recombination initiation protein) and 45 (which encodes a bifunctional protein required in replication and transcription) . The existence of genes 45.1 and 45.2 had not been previously recognized by mutation analysis of the T4 genome . We cloned the T4 gene 45.1/45.2 segment, determined its nucleotide sequence, and expressed its two reading frames at high levels in bacterial plasmids . The results predicted molecular weights of 11,400 (100 amino acids) for gp45.1 and 7,500 (62 amino acids) for gp45.2 . We also determined that in T4-infected Escherichia coli, genes 45.1 and 45.2 are cotranscribed with their distal neighbor, gene 45, by at least one mode of transcription . In an accompanying report (K . P . Williams, G . A . Kassavetis, F . S . Esch, and E . P . Geiduschek, J . Virol . 61:600-603, 1987), it is shown that the product of gene 45.1 is the so-called T4-induced 15K protein, an RNA polymerase-binding protein of unknown role in phage development . Possibly, T4 genes 45.2, 45.1, and 45 constitute an operon for host RNA polymerase-binding phage proteins . Jointly with Williams et al., we propose the term rpb (RNA polymerase-binding) to refer to T4 genes whose products bind to the host RNA polymerase and have adopted the name rpbA for T4 gene 45.1.

Protein Eng, 1987 Feb-Mar, 1(2), 115 - 23
Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme; Sun DP et al.; Five different cysteine-containing mutants of the lysozyme from bacteriophage T4 were used to explore the feasibility of using site-directed mutagenesis to generate isomorphous heavy-atom derivatives for protein crystallography . Cysteines 54 and 97, present in wild-type lysozyme, can be readily reacted with mercuric ion to produce an excellent isomorphous heavy-atom derivative . Mutants with an additional cysteine at position 86, 146, 153 or 157, or with Cys 97 replaced by Val, were engineered by site-directed mutagenesis . The mutant lysozyme Thr 157----Cys reacts with mercuric chloride to give an excellent new derivative although Cys 157 is only approximately 60% substituted with the heavy atom . The cysteine at position 146 is largely buried but reacts readily with mercuric chloride . In this case the isomorphism is poor and the resultant derivative is of marginal quality . Cys 153 reacts rapidly with mercuric ion but the derivative crystals do not diffract . The mutant Pro 86----Cys does not yield a particularly good heavy-atom derivative . This is due in part to a loss of isomorphism associated with the mutation . In addition, Cys 86 shows very little reactivity towards mercurials even though it is fully exposed to solvent . The mutation Cys 97----Val was used to explore the possibility of creating an independent derivative by deleting a heavy-atom site already present in wild-type lysozyme . In all cases that were tested, the quality of the heavy-atom derivative was improved by using as an isomorphous pair mercury-substituted mutant versus non-substituted mutant rather than mercury-substituted mutant versus (non-substituted) wild-type lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Odontol Scand, 1987 Feb, 45(1), 49 - 54
Bacteriophage infection--a possible mechanism for increased virulence of bacteria associated with rapidly destructive periodontitis; Preus HR et al.; We have recently isolated several groups of bacteriophages infecting Actinobacillus actinomycetemcomitans from periodontal lesions in patients with rapidly destructive periodontitis . Bacteriophage infection of these bacteria in these patients was restricted to periodontal pockets showing radiographic evidence of recent bone loss and suggests an association between phage-infected A . actinomycetemcomitans and active periodontal disease . On the basis of the biological activity of bacteriophages we propose a working hypothesis to explain the mechanism by which a phage may increase bacterial virulence in periodontal disease.

Microb Pathog, 1987 Feb, 2(2), 147 - 53
Nucleotide sequence analysis of the structural genes for Shiga-like toxin I encoded by bacteriophage 933J from Escherichia coli; Jackson MP et al.; A nucleotide sequence analysis was performed on the Shiga-like toxin I genes previously cloned from the Escherichia coli bacteriophage 933J . Two structural genes designated slt-I A and slt-I B were found to be oriented on a single transcriptional unit with slt-I A preceding slt-I B . A 12 base pair gap separated the in-phase open reading frames of slt-I A and slt-I B . Putative ribosome binding sites were identified 5' to both the slt-I A and slt-I B genes . Translation of the SLT-I nucleotide sequence revealed that both the A and B subunits were synthesized with signal peptides . The molecular weight calculated for the mature A subunit was 32,211 while the molecular weight of the mature B subunit was 7690.

Mol Gen Genet, 1987 Feb, 206(2), 344 - 51
A cloned DNA fragment from bacteriophage P1 enhances IS2 insertion; Sengstag C et al.; A 1.75 kb DNA segment of the bacteriophage P1 genome is known to serve as a preferred target for IS2 insertions . The presence of this fragment in a plasmid expressing the galK gene dramatically increases the proportion of IS2 insertions among spontaneous galK- mutants . Subfragments from two different parts of the 1.75 kb segment independently stimulate IS2 insertion, while another subfragment does not . In the plasmids studied IS2 elements not only insert into the cloned P1 fragment but also into parts of the galK gene with similar probability and mostly in one orientation . Many insertion sites are unique but several specific sites within the preferred target are repeatedly used for IS2 integration . The experimental data are compatible with a proposed cooperative mechanism, according to which more than one attracting sequence on the same plasmid might significantly enhance the probability of a particular target region to attract IS2.

Genetika, 1987 Feb, 23(2), 228 - 38
{Molecular causes of thalassemia . IV . Cloning of the beta-globin gene in a patient with beta-thalassemia from Azerbaijan and determination of the point mutation in a minor intron}; Limborskaia SA et al.; On the basis of DNA from a beta-thalassemic patient, human gene library has been obtained using bacteriophage lambda EMBL4 as a vector . The recombinants contain human DNA insertions of 15 to 20 kb . The lambda A1 beta 1 clone has been isolated by the method of hybridization of phage plaque replicas to the HhaI fragment of JW102 plasmid containing human beta-globin cDNA . Restriction mapping revealed the presence of a 22 kb human DNA fragment comprising a portion of the beta-globin gene system . Subcloned fragments of beta-globin gene (within the pUC19 plasmid or phage MI3mp10) were sequenced using the Maxam and Gilbert method as well as that of Sanger . 2150 nucleotides in total were analysed . We have detected the point substitution G----A in the 110 nucleotide of minor intron, leading to the formation of an additional splicing site.

DNA, 1987 Feb, 6(1), 59 - 70
The human growth hormone gene locus: structure, evolution, and allelic variations; Hirt H et al.; Genomic clones containing the closely related genes for human growth hormone (hGH) and chorionic somatomammotropin (hCS) were obtained from genomic bacteriophage lambda and cosmid libraries . The entire GH/CS chromosomal locus was reconstructed utilizing overlapping restriction fragments characterized from the isolated clones . The hGH/hCS locus contains two GH genes and three CS genes spanning 48 kb of DNA in the order: 5'-(hGH-1/hCS-5/hCS-1/hGH-2/hCS-2)-3', confirming analysis of cosmid clones obtained from a different human library (Barsh et al., 1983) . To complete the characterization of the hCS genes, the nucleotide sequence of the hCS-5 gene was determined . Sequence analysis revealed a mutation of the 5' splice site at the exon II-intron B boundary, suggesting that the hCS-5 gene is a pseudogene . The nucleotide sequence of an allelic variant of the hCS-2 gene was determined and found to contain a single amino acid substitution and the deletion of a single codon . The hGH/hCS gene locus was further characterized by the localization of at least 27 Alu-type repetitive sequences and identification of three unique sequences in the vicinity of several hGH and hCS genes which define the probable breakpoints of the evolutionary duplication units . These data, combined with the nucleotide sequences of all five GH and CS genes, indicate that the hGH/hCS gene locus has evolved by duplication mechanisms . Evidence for the occurrence of at least one gene conversion event involving the hCS-1 gene precursor and the hCS-2 gene was found, indicating that the hGH/hCS gene locus has evolved by concerted mechanisms . The structure of the hCS genes is discussed in light of recent studies of CS genes from other mammalian species.

J Gen Virol, 1987 Feb, 68 ( Pt 2), 451 - 62
Human papillomavirus type 16 DNA from a vulvar carcinoma in situ is present as head-to-tail dimeric episomes with a deletion in the non-coding region; Kennedy IM et al.; A number of genital cancer biopsy samples were screened for the presence of human papillomavirus type 16 (HPV-16) DNA sequences . One of these samples (a vulvar carcinoma in situ) was found to contain more than 100 copies of HPV-16 DNA sequences per cell . Using this tumour DNA, a genomic library was constructed in bacteriophage lambda and the library was screened for recombinant phage containing HPV-16 sequences . Five recombinant phage clones were isolated and their DNA was analysed by restriction endonuclease digestion and blot hybridization . All five recombinants contained two copies of the HPV-16 genome present in a head-to-tail arrangement . The data are consistent with the presence of HPV-16 sequences in the tumour DNA arranged as genomic dimers in a circular episomal configuration . The HPV-16 genomes contained a deletion within the non-coding region, a region which includes the viral origin of DNA replication and transcriptional control sequences . Possible consequences of this deletion for viral replication and transcription are discussed.

Proc Natl Acad Sci U S A, 1987 Feb, 84(3), 774 - 8
Nucleotide sequence and characterization of the 5' flanking region of the rat Ha-ras protooncogene; Damante G et al.; Thyrotropin and cAMP stimulate growth of FRTL5 rat thyroid cells and increase c-Ha-ras mRNA levels . To study the mechanism by which thyrotropin enhances c-Ha-ras expression in the thyroid, we constructed a genomic library of FRTL5 DNA in the bacteriophage vector EMBL3 . Using a v-Ha-ras probe (0.7-kilobase Sst I-Pst I fragment), we isolated eight clones containing portions of the FRTL5 c-Ha-ras gene . Restriction mapping of one of these clones revealed a structure very similar to that previously reported for the rat c-Ha-ras gene . We determined the nucleotide sequence of exon 1 as well as 1.15 kilobases upstream from exon 1 . Blot-hybridization analysis of FRTL5 thyroid cell mRNA was performed with three DNA fragments upstream of exon 1 . Two of these probes were Pst I-Pst I fragments 0.4 and 0.55 kilobases long, 1.15 and 0.6 kilobases upstream of exon 1, respectively . The third probe, a 0.6-kilobase Pst I-HindIII fragment, was immediately upstream of exon 1 and included 54 base pairs of the 5' end of exon 1 . The data revealed an upstream ("-1") exon, consistent with the homology between the nucleotide sequences of our clone and the human c-Ha-ras gene in this region . Primer extension of a synthetic 30-mer oligonucleotide probe complementary to exon +1 on a FRTL5 mRNA template revealed three transcription start (cap) sites, 182, 169, and 153 bases upstream of the ATG codon . "CCAAT boxes" are present 65 and 100 bases upstream from the initial cap site . Three "GC boxes" and two C + G-rich inverted repeats characteristic of the binding site for the transcription regulation factor Sp1 are present in the 177 base pairs upstream of the initial cap site . Comparison of the rat and human c-Ha-ras -1 exons showed an area of poor homology immediately upstream of the human cap sites, followed further upstream by a region of close homology (approximately equal to 60 base pairs) . The nucleotide sequence of the rat -1 exon is more similar to the v-ras sequence than is the human . The v-ras gene is truncated relative to both human and rat -1 exons.

J Gen Virol, 1987 Feb, 68 ( Pt 2), 253 - 62
Controlling mechanisms for expression of the bacteriophage T4 beta-glucosyltransferase gene; Thylen C; The plasmid pTHF3047 carries a 2200 bp fragment containing the phage T4 beta-glucosyltransferase (beta gt) gene and part of the upstream gene 42 cloned in pBR313 under the control of the amp promoter P1 . In T4-infected cells the beta gt gene may be expressed by a mechanism antagonizing Rho action . The plasmid pTHF3047 expressed about threefold higher beta-glucosyltransferase activity in a strain carrying the polarity-suppressing rho-102 mutation than in an otherwise isogenic rho+ strain, and production of T4 alpha gt- beta gt- phage was strongly stimulated . The plasmid copy number and the total T4-specific transcription was the same in the two strains . Two T4-specific transcripts from the plasmid, 600 bases and 1850 bases, were identified by Northern hybridization . Comparison with the T4 and plasmid maps suggested that both transcripts were initiated at P1, the 600 base transcript ending at the Rho-dependent terminator t42 between gene 42 and beta gt, and the 1850 base transcript reading through this terminator to the end of the beta gt gene . This analysis places t42 at position 25.1 on the T4 map, and a Rho-independent beta gt terminator at position 23.8 . The three-fold higher beta gt expression in the rho- strain may be partially accounted for by Rho control of transcription . In the rho+ strain about half of the transcripts stopped at t42, while in the rho- strain readthrough appeared slightly higher . Thus, the t42 terminator was observed also on the plasmid, but appeared considerably less effective there than in the phage DNA in infected cells.

Proc Natl Acad Sci U S A, 1987 Feb, 84(3), 699 - 703
B protein of bacteriophage mu is an ATPase that preferentially stimulates intermolecular DNA strand transfer; Maxwell A et al.; A DNA strand-transfer reaction is an early step in the transposition of phage Mu . It has been shown that an efficient reaction in vitro requires, in addition to buffer and salt, only the Mu A protein, Mu B protein, host protein HU, ATP, and Mg2+ . We have determined that, of the three protein factors involved, only the Mu B protein has an ATPase activity . The Mu B ATPase is stimulated by Mu A protein and DNA but not by either of these factors alone . Double-stranded DNA is a much better cofactor than single-stranded DNA, but there is no apparent sequence specificity . In the absence of the Mu B protein and/or ATP, the intermolecular Mu DNA strand-transfer reaction is extremely inefficient, and the strand-transfer products are predominantly the result of an intramolecular reaction . This contrasts with the efficient intermolecular reaction that occurs if Mu B protein and ATP are provided . The Mu B protein, in the presence of Mu A protein and protein HU, therefore, seems to facilitate interactions between potential DNA target sites and pairs of Mu DNA ends.

Cell, 1987 Jan 30, 48(2), 205 - 17
Nuclear reconstitution in vitro: stages of assembly around protein-free DNA; Newport J; We have developed a cell-free system derived from Xenopus eggs that reconstitutes nuclear structure around an added protein-free substrate (bacteriophage lambda DNA) . Assembled nuclei are morphologically indistinguishable from normal eukaryotic nuclei: they are surrounded by a double membrane containing nuclear pores and are lined with a peripheral nuclear lamina . Nuclear assembly involves discrete intermediate steps, including nucleosome assembly, scaffold assembly, and nuclear membrane and lamina assembly, indicating that during reconstitution nuclear organization is assembled one level at a time . Topoisomerase II inhibitors block nuclear assembly . Lamin proteins and membrane vesicles bind to chromatin late in assembly, suggesting that these components do not interact with chromatin that is formed early in assembly . Reconstituted nuclei replicate their DNA; replication begins only after envelope formation has initiated, indicating that envelope attachment may be important for regulating replication.

Nucleic Acids Res, 1987 Jan 26, 15(2), 431 - 42
Structure of secondary cleavage sites of E . coli RNAaseIII in A3t RNA from bacteriophage T7; Gross G et al.; Five 'secondary' cleavage sites of E . coli RNAaseIII within the initiator RNA A3t (141 nucleotides) from bacteriophage T7 are described . Cleavage takes place apparEntly within or at the bottom of a short double-stranded stretch of RNA . Three secondary sites are efficiently cleaved in the presence of magnesium 2+ ions, two additional sites are cleaved in the presence of manganese 2+ ions at low monovalent salt concentrations (less than 0.05 M).

J Mol Biol, 1987 Jan 20, 193(2), 315 - 43
Pf1 Inovirus . Electron density distribution calculated by a maximum entropy algorithm from native fibre diffraction data to 3 A resolution and single isomorphous replacement data to 5 A resolution; Marvin DA et al.; We have calculated the electron density distribution of the Pf1 strain of filamentous bacteriophage by a maximum entropy method . In the calculation we included native X-ray fibre diffraction data extending to 3 A resolution in the meridional direction on 60 layerlines that are resolved to 4 A in the equatorial direction, and lower resolution data from a single isomorphous derivative iodinated on the Tyr25 residue . The electron density map indicates that the 46-residue protein subunit is a single, gently curved stretch of alpha-helix with its axis at an angle of about 20 degrees to the axis of the virion . The alpha-helix subunit curves around the virion axis by about 1/6 turn, and decreases from about 27 A radius to about 13 A radius in the virion as the amino acid sequence of the subunit runs from the N terminus to the C terminus . Nearest-neighbour alpha-helical subunits are about 10 A apart along their length, and the axis of each subunit makes an unexpected negative angle with its nearest neighbours in the virion . To confirm the validity of the maximum entropy calculation, we have varied the constraints on the calculation . All variations result in either a map that is close to the original map or a map that cannot be interpreted in terms of secondary structure: we find only one map that makes structural sense.

J Mol Biol, 1987 Jan 20, 193(2), 359 - 76
Gene 3 endonuclease of bacteriophage T7 resolves conformationally branched structures in double-stranded DNA; de Massy B et al.; Gene 3 endonuclease of bacteriophage T7 has been expressed from the cloned gene, purified, and characterized as to its activity on different DNA substrates . Besides its known strong preference for cutting single-stranded DNA rather than double-stranded DNA, the enzyme has a strong preference for cutting conformationally branched structures in double-stranded DNA, either X or Y-shaped branches . Three types of branched DNA substrates were used: relaxed circular DNAs containing large cruciform structures (a model for Holliday structures, presumed intermediates in genetic recombination); X-shaped molecules having a limited potential for branch migration, made from the cloned phage and bacterial arms of the lambda attachment site; and Y-shaped molecules, made by hybridizing molecules homologous except for a 2 X 21 base-pair palindrome in one of them . Gene 3 endonuclease cuts two opposing strands at or near the branchpoint to resolve these substrates into linear molecules, and does not cut the potentially single-stranded tips of the stem-and-loop structure generated from the palindrome . The position of the cleavage points on the equivalent arm of two X-shaped molecules, constructed from wild-type and mutant lambda attachment sites, show that the enzyme can cut at several different sites within or slightly 5' of the limited region of branch migration . The various activities of gene 3 endonuclease are consistent with the known role of this enzyme in genetic recombination, in maturation and packaging of T7 DNA, and in degradation of host DNA, and suggest that the enzyme recognizes a specific structural feature in DNA . Its cleavage specificity, ready availability, and ability to act at physiological pH and ionic conditions may make gene 3 endonuclease useful as a probe for specific DNA structures or for binding of proteins that alter DNA structure.

Mol Biochem Parasitol, 1987 Jan 15, 22(2-3), 195 - 202
cDNA clone encoding a high molecular weight antigen of Babesia bovis; Gill A et al.; An expression library was constructed by inserting cDNA copied from mRNA of the blood stages of Babesia bovis isolate KA into bacteriophage lambda gt11-amp3 . An antigen-positive cDNA clone detected by screening the library with antibodies from cattle vaccinated with the KA isolate was shown to encode part of a high-molecular weight polypeptide antigen of B . bovis . This molecule was a dominant immunogen and was found by immunofluorescence to be within the parasite in infected erythrocytes.

Nucleic Acids Res, 1987 Jan 12, 15(1), 119 - 40
The control of lambda DNA terminase synthesis; Murialdo H et al.; Nu1 and A, the genes coding for bacteriophage lambda DNA terminase, rank among the most poorly translated genes expressed in E . coli . To understand the reason for this low level of translation the genes were cloned into plasmids and their expression measured . In addition, the wild type DNA sequences immediately preceding the genes were reduced and modified . It was found that the elements that control translation are contained in the 100 base pairs upstream from the initiation codon . Interchanging these upstream sequences with those of an efficiently translated gene dramatically increased the translation of terminase subunits . It seems unlikely that the rare codons present in the genes, and any feature of their mRNA secondary structure play a role in the control of their translation . The elimination of cos from plasmids containing Nu1 and A also resulted in an increase in terminase production . This result suggests a role for cos in the control of late gene expression . The terminase subunit overproducer strains are potentially very useful for the design of improved DNA packaging and cosmid mapping techniques.

J Mol Biol, 1987 Jan 5, 193(1), 189 - 99
Structure of bacteriophage T4 lysozyme refined at 1.7 A resolution; Weaver LH et al.; The structure of the lysozyme from bacteriophage T4 has been refined at 1.7 A resolution to a crystallographic residual of 19.3% . The final model has bond lengths and bond angles that differ from "ideal" values by 0.019 A and 2.7 degrees, respectively . The crystals are grown from electron-dense phosphate solutions and the use of an appropriate solvent continuum substantially improved the agreement between the observed and calculated structure factors at low resolution . Apart from changes in the conformations of some side-chains, the refinement confirms the structure of the molecule as initially derived from a 2.4 A resolution electron density map . There are 118 well-ordered solvent molecules that are associated with the T4 lysozyme molecule in the crystal . Four of these are more-or-less buried . There is a clustering of water molecules within the active site cleft but, other than this, the solvent molecules are dispersed around the surface of the molecule and do not aggregate into ice-like structures or pentagonal or hexagonal clusters . The apparent motion of T4 lysozyme in the crystal can be interpreted in terms of significant interdomain motion corresponding to an opening and closing of the active site cleft . For the amino-terminal domain the motion can be described equally well (correlation coefficients approx . 0.87) as quasi-rigid-body motion either about a point or about an axis of rotation . The motion in the crystals of the carboxy-terminal domain is best described as rotation about an axis (correlation coefficient 0.80) although in this case the apparent motion seems to be influenced in part by crystal contacts and may be of questionable relevance to dynamics in solution.

J Mol Biol, 1987 Jan 5, 193(1), 97 - 113
Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein . Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA; Kowalczykowski SC et al.; The effect that Escherichia coli single-stranded DNA binding (SSB) protein has on the single-stranded DNA-dependent ATPase activity of RecA protein is shown to depend upon a number of variables such as order of addition, magnesium concentration, temperature and the type of single-stranded DNA substrate used . When SSB protein is added to the DNA solution prior to the addition of RecA protein, a significant inhibition of ATPase activity is observed . Also, when SSB protein is added after the formation of a RecA protein-single-stranded DNA complex using either etheno M13 DNA, poly(dA) or poly(dT), or using single-stranded phage M13 DNA at lower temperature (25 degrees C) and magnesium chloride concentrations of 1 mM or 4 mM, a time-dependent inhibition of activity is observed . These results are consistent with the conclusion that SSB protein displaces the RecA protein from these DNA substrates, as described in the accompanying paper . However, if SSB protein is added last to complexes of RecA protein and single-stranded M13 DNA at elevated temperature (37 degrees C) and magnesium chloride concentrations of 4 mM or 10 mM, or to poly(dA) and poly(dT) that was renatured in the presence of RecA protein, no inhibition of ATPase activity is observed; in fact, a marked stimulation is observed for single-stranded M13 DNA . A similar effect is observed if the bacteriophage T4-coded gene 32 protein is substituted for SSB protein . The apparent stoichiometry of DNA (nucleotides) to RecA protein at the optimal ATPase activity for etheno M13 DNA, poly(dA) and poly(dT) is 6(+/- 1) nucleotides per RecA protein monomer at 4 mM-MgCl2 and 37 degrees C . Under the same conditions, the apparent stoichiometry obtained using single-stranded M13 DNA is 12 nucleotides per RecA protein monomer; however, the stoichiometry changes to 4.5 nucleotides per RecA protein monomer when SSB protein is added last . In addition, a stoichiometry of four nucleotides per RecA protein can be obtained with single-stranded M13 DNA in the absence of SSB protein if the reactions are carried out in 1 mM-MgCl2 . These data are consistent with the interpretation that secondary structure within the natural DNA substrate limits the accessibility of RecA protein to these regions . The role of SSB protein is to eliminate this secondary structure and allow RecA protein to bind to these previously inaccessible regions of the DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Biol, 1987 Jan 5, 193(1), 223 - 6
Suppressor and novel mutants of bacteriophage T4 tRNA(Gly); McClain WH et al.; We have isolated a weak UGA suppressor of phage T4 tRNA(Gly) in which the anticodon is changed from UCC to UCA . Two secondary mutants lacking suppressor activity are atypical in accumulating tRNA(Gly) . Both mutations change the T stem of the cloverleaf model . One involved a G to A change at the 5' base position of the middle base-pair; the second involves a C to U change at a constant base position next to the T loop . The precursor RNAs of the mutants were cleaved in vitro with the catalytic RNA subunit of RNase P . Relative to normal precursor RNA, the precursor mutated at the middle base-pair position of the T stem was cleaved more rapidly, whereas the precursor mutated at the base-pair position next to the T loop was cleaved more slowly.

Mol Gen Genet, 1987 Jan, 206(1), 24 - 34
The bacteriophage T4 dexA gene: sequence and analysis of a gene conditionally required for DNA replication; Gauss P et al.; We have cloned and sequenced a bacteriophage T4 EcoRI fragment that complements T4 del (39-56) infections of an optA defective Escherichia coli strain . Bacteria containing this recombinant plasmid synthesize two new proteins with molecular weights of 9 and 26 kilodaltons . We have identified the gene encoding the 26 kilodalton protein as essential for T4 infections of optA defective E . coli . Genetic and biochemical results are consistent with the identification of this protein as the product of the dexA gene, which encodes a 3' to 5' exonuclease.

Mutat Res, 1987 Jan, 190(1), 1 - 6
Nitrous acid induced damage in T7 DNA and phage; Scearce LM et al.; T7 phage was exposed to 56 mM nitrous acid at pH 4.6 causing a 90% decrease in survival for each 10 min duration of exposure . The survival of phage made by encapsulating nitrous acid treated DNA into empty phage heads was nearly the same as the survival of phage exposed to nitrous acid in vivo . In contrast to previous reports, growth of SOS-induced wild-type E.coli showed no increase in survival . The survival of nitrous acid treated phage was not lowered when grown on E.coli strains deficient in DNA polymerase I, exonuclease III, and the uvrA component of the nucleotide excision-repair endonuclease . Therefore, these enzymes are not vital for repair of nitrous acid induced damage in bacteriophage T7.

J Virol, 1987 Jan, 61(1), 113 - 8
Prohead core of bacteriophage T4 can act as an intermediate in the T4 head assembly pathway; Kuhn A et al.; Bacteriophage T4 assembly was impaired in Escherichia coli hdB3-1 at an incubation temperature below 30 degrees C . Naked prohead cores (head scaffold) bound to the inner surface of the plasma membrane accumulated, and the major shell protein (gp23) precipitated into visible intracellular aggregates in the cytoplasm . Shifting the temperature to 42 degrees C allowed newly synthesized gp23 to assemble around the accumulated cores . We conclude that synchronous assembly of the scaffold and shell is not obligatory and that naked cores can serve as intermediates in the T4 assembly pathway.

J Cell Sci Suppl, 1987, 7, 277 - 85
Tobacco mosaic virus replicase and replicative structures; Young N et al.; The RNA-dependent RNA polymerase (replicase) mediating the replication of tobacco mosaic virus (TMV) has been investigated in a number of laboratories over a period of 20 years . Cell-free enzyme preparations have been prepared which can continue the synthesis of nascent complementary RNA, initiated in vivo; however, the enzyme does not require, nor does it respond to, exogenous viral RNA as a template . The presence in plants of a virus-stimulated, host-encoded RNA-dependent RNA polymerase (RdRp) has added confusion to this field; it is now generally conceded, however, that this enzyme is not the TMV replicase . Our recent studies have emphasized several aspects of TMV RNA replication . We have examined the nature of TMV replicative structures synthesized in vitro by utilizing a partially purified enzyme preparation isolated from TMV-infected tobacco tissue . Radiolabelled products of the reaction were analysed on agarose gels and fractions with the predicted electrophoretic migration and nuclease sensitivities of replicative form (RF) and replicative intermediate (RI) were isolated . These fractions were hybridized to a collection of bacteriophage M13 clones containing portions of the TMV genome of both plus and minus polarity . The nascent synthesis in the RI-like molecules was restricted to the plus viral strand, while the new synthesis in the RF-like molecules was of both plus and minus polarity . Solubilization of the membrane-bound replicase with the non-ionic detergent CHAPS has yielded complexes which remain in solution after high-speed centrifugation . The solubilized replication complexes have been utilized as starting material for enzyme purification by Sepharose 4B gel filtration chromatography . The intracellular site of synthesis of TMV RNA has been reinvestigated in the light of reports suggesting a nuclear site of replication . The conclusion for nuclear synthesis has been based on fractionation of subcellular homogenates of virus-infected leaves or mesophyll protoplasts and identification of virus-related proteins associated with these fractions . In our studies, however, we conclude that these procedures can be misleading in that the 126,000 Mr TMV protein (and replicase activity) were found in all fractions of the homogenate analysed . Double-stranded TMV RNA, on the other hand, was barely detectable in preparations of purified nuclei; instead it was concentrated in the post-nuclear supernatant, suggesting that the nucleus is not the site of TMV RNA synthesis.

Mol Gen Genet, 1987 Jan, 206(1), 181 - 4
Large non-homology in heteroduplex DNA is processed differently than single base pair mismatches; Dohet C et al.; Unmethylated DNA heteroduplexes with a large single stranded loop in one strand have been prepared from separated strands of DNA from two different strains of bacteriophage lambda, one of which has a approximately 800 base pair IS1 insertion in the cI gene . The results of transfections with these heteroduplexes into wild-type and mismatch repair deficient bacteria indicate that such large non-homologies are not repaired by the Escherichia coli mismatch repair system . However, the results do suggest that some process can act to repair such large non-homologies in heteroduplex DNA . Transfections of a series of recombination and excision repair deficient mutants suggest that known excision or recombination repair systems of E . coli are not responsible for the repair . Repair of large non-homologies may play a role in gene conversion involving large insertion or deletion mutations.

J Clin Invest, 1987 Jan, 79(1), 275 - 81
Molecular mechanisms of McArdle's disease (muscle glycogen phosphorylase deficiency) . RNA and DNA analysis; Gautron S et al.; Lack of muscle glycogen phosphorylase activity leads to McArdle's disease, a rare metabolic myopathy . To investigate its molecular basis at the nucleic acid level, we isolated muscle phosphorylase cDNA clones from a human cDNA library in Escherichia coli plasmid pBR 322 . Subcloning of one insertion of M13 bacteriophage permitted its definite identification by sequencing . Northern blot experiments revealed one specific messenger RNA of 3.4 kilobases found uniquely in tissues expressing muscle phosphorylase . We show that McArdle's disease exhibits a molecular heterogeneity at the messenger RNA level . In eight unrelated cases of McArdle's disease in which no inactive proteins had been detected, we assayed muscle biopsies for phosphorylase mRNA by Northern blotting . In five cases, no muscle phosphorylase mRNA could be detected, while in three other cases, normal length mRNA was present in lower amounts . Moreover, Southern blot analysis of DNA isolated from white blood cells in four McArdle patients revealed no major deletion or rearrangements of the phosphorylase gene as compared with controls.

Arch Immunol Ther Exp (Warsz), 1987, 35(5), 569 - 83
Results of bacteriophage treatment of suppurative bacterial infections in the years 1981-1986; Slopek S et al.; In the years 1981-1986 bacteriophage therapy was applied in 550 cases (100 treated in 1986) of suppurative bacterial infections . Positive results were obtained in 508 cases (92.4%) . In 38 cases (6.9%) a transient improvement was observed and in 4 cases (0.7%) phage treatment proved ineffective . Considering that majority of patients (518 cases, 94.2%) were resistant to antibiotic treatment, the results of phage therapy may be regarded as favorable.

Protein Seq Data Anal, 1987, 1(2), 99 - 102
The amino acid sequence of crystalline sheets: a proteolytic fragment of the major head protein (gP23) of bacteriophage T4; Tsugita A et al.; A peptide, obtained from T4 phage major capsid protein gP23 by chymotryptic digestion, has been shown to form two types of crystalline sheets, hexagonal and rectangular . The sheet peptide was subjected to conventional sequencing work . Together with the DNA sequence of gene 23, the results showed that the peptide consisted of 155 amino acids, located from 295 to 449 in the nascent gP23 and from 230 to 384 in the matured gP23.

Gene, 1987, 61(3), 329 - 38
Human cysteine-proteinase inhibitors: nucleotide sequence analysis of three members of the cystatin gene family; Saitoh E et al.; Three genes from the human cystatin gene family of cysteine-proteinase inhibitors have been isolated from a bacteriophage lambda library containing HindIII digests of human genomic DNA . Two of the genes code for salivary cystatin SN and SA, the third is a pseudogene . The cloned genes were identified with a probe made from a salivary cystatin cDNA . The complete nucleotide sequence of the gene that codes for the precursor form of the neutral salivary protein, cystatin SN, was determined . The gene, which we name CST1, contains three exons and two intervening sequences . The expected CAT and ATA boxes are present in the 5'-flanking region of the gene . Partial nucleotide sequence determination of a second gene revealed that it codes for the precursor form of the acidic salivary protein, cystatin SA . This gene, which we name CST2, has the same gene organization as CST1 . The complete nucleotide sequence of a third gene was determined . It does not contain a typical ATA box, and in addition, a premature stop codon and a frameshift deletion mutation occur within the gene . These inactivation mutations show that this gene, which we name CSTP1, is a cystatin pseudogene . These data combined with our genomic Southern-blot analyses show that the cystatin genes form a multigene family with at least seven members.

Biochem Soc Symp, 1987, 53, 115 - 21
Schistosome elastases: biological importance, structure, function and stage-specific expression; Newport G et al.; Larval schistosomes (Digenea: Trematoda) invade their definitive host by directly penetrating the skin . During the process they secrete a number of macromolecules, ostensibly to facilitate their entry . Among these we have identified and characterized a dominant proteolytic species: a serine protease capable of fragmenting keratin, types IV and VIII collagen, proteoglycan, fibronectin, laminin, and elastin . The enzyme exhibits the specificity characteristic of elastases, has a molecular mass of 30,000 Da and pI of 7.8, and is potently immunogenic in its native form . Specificity of the active site has been analysed, tetrapeptides having large hydrophobic or aromatic amino acids at size P1 serving as best substrates . The amino terminal 20 amino acids of the mature enzyme have been sequenced and the information derived has been used to construct an oligonucleotide (22-mer) complement of its corresponding mRNA . The latter has been used to establish, by Northern analysis, that expression of the enzyme is stage specific (differing in this respect from most schistosome immunogens), and under transcriptional control . Transcripts are encoded by a multigene family . Several cDNAs hybridizing to the oligonucleotide have been isolated, subcloned into bacteriophage M-13, and sequenced by the di-deoxy method . It is our expectation that this line of investigation will lead towards: (i) an anti-infection vaccine; (ii) a means for chemically preventing infection (using enzyme inhibitors), and/or (iii) a rapid diagnostic assay of prepatent infection.

Gene, 1987, 58(1), 67 - 76
A set of expression plasmids for the synthesis of fused and unfused polypeptides in Escherichia coli; Zaballos A et al.; A set of plasmid expression vectors for cloning of DNA fragments containing open reading frames has been obtained . The plasmids carry the strong leftward promoter of bacteriophage lambda and the translation initiation signals from either the gene ner of bacteriophage Mu or the gene 4 of bacteriophage phi 29 . The vectors could overexpress the cloned sequences as fusion peptides at the N terminus with the N-terminal segment of the phi 29 protein p4 or at the C terminus with the Escherichia coli beta-galactosidase from its 8th residue, or both . Alternatively, the cloned sequences could be directed to overproduce proteins in an unfused form . DNA fragments of the hemagglutinin gene from human influenza A virus, have been cloned in one of the plasmid vectors and some potential antigenic determinants have been characterized using monoclonal antibodies.

Gene, 1987, 58(1), 13 - 8
Overexpression and purification of a biologically active rifampicin-resistant beta subunit of Escherichia coli RNA polymerase; McKinney JD et al.; The gene rpoB (rifD 18), which encodes rifampicin-resistant beta subunit of Escherichia coli RNA polymerase, has been placed on an overexpression plasmid under the control of bacteriophage T7 promoter . Induction of the T7 RNA polymerase gene in the host cells resulted in extensive overproduction of the beta polypeptide . Most of the overproduced material was recovered from cell lysates in insoluble form and was solubilized by extraction with 6 M urea . Purified overproduced beta subunit was added, in molar excess, to urea-denatured rifampicin-sensitive RNA polymerase . Upon removal of urea by dialysis, the reconstituted enzyme became rifampicin-resistant, indicating that overproduced beta subunit can be efficiently assembled into functional holoenzyme.

Gene, 1987, 56(1), 61 - 70
Chemical synthesis of a gene coding for human angiogenin, its expression in Escherichia coli and conversion of the product into its active form; Denefle P et al.; A synthetic gene coding for human angiogenin was synthesized by solid support phosphoramidite chemistry as eight long oligodeoxynucleotides which were subsequently assembled and cloned in Escherichia coli . The gene was designed to use codons found in highly expressed E . coli proteins . A pBR322-derived expression vector was constructed containing the E . coli trp promoter, the ribosome-binding site of the bacteriophage lambda cII gene, the angiogenin coding sequence, and the transcription terminator region of the E . coli rrnB operon . Under tryptophan deprivation, angiogenin was strongly expressed in E . coli cells at a yield of 5-10% of total protein . The eukaryotic protein was found to be insoluble but could be easily renatured and purified . The purified angiogenin was demonstrated to be active as an angiogenic factor and exhibited a characteristic RNase activity.

Rheumatol Int, 1987, 7(4), 161 - 8
Structural requirements of DNA used in the Farr assay to detect antibodies directed against double-stranded DNA; Pollard KM et al.; The measurement of antibodies to DNA in SLE requires the use of double-stranded DNA (dsDNA), demonstrably free of single-stranded regions . Such dsDNA preparations can, however, contain other structural components . In this study DNA preparations with defined structure, both secondary (single- and double-stranded and random base-paired) and tertiary (superhelical and open circular), were used in the Farr assay to measure the DNA binding of sera from patients with SLE and related connective-tissue diseases . The presence of true single-stranded DNA regions in denatured DNA, native DNA, and dsDNA containing single-stranded regions increased the DNA binding measured in all sera . DsDNA, whether intact or containing small regions of random base-pairing, was bound by sera from the majority of patients with SLE but not by non-SLE sera . Superhelical dsDNA from bacteriophage PM2 was bound by SLE sera to a greater extent than linear dsDNA was . Inhibition experiments suggested that this difference in binding to DNA according to tertiary, as opposed to secondary, structure is because there are fewer available binding sites on superhelical dsDNA . DNA binding, as measured by the Farr assay, can thus be influenced by both secondary and tertiary DNA structure . Using superhelical DNA, advantage can be taken of the dsDNA form plus tertiary structure to enhance DNA binding of SLE sera beyond the levels achieved using linear dsDNA.

Physiol Chem Phys Med NMR, 1987, 19(1), 67 - 74
Salt effects on the bacteriophage T7-II structure and activity changes; Toth K et al.; Optically detected thermal stability and biological activity of phage T7 has been compared as the function of the ionic composition and strength of the buffers . The ionic strength range was studied between 20-140 mmol/1 . In Tris buffer containing only monovalent ions the biological activity of the phages decreases abruptly below 50 mmol/1 ionic strength . Structural studies show a logarithmic dependence between the ionic strength and the intraphage DNA stability and no significant change in the thermal stability of the whole phage . Mg2+ and Ca2+ ions at low concentration (1 mmol/1) given into a Tris buffer of 20 mmol/1 original ionic strength highly stabilize the biological activity, which stabilization is also to be seen in the intraphage DNA and also in the whole phage thermal denaturation process.

Physiol Chem Phys Med NMR, 1987, 19(1), 59 - 66
Salt effects on bacteriophage T7-I; Toth K et al.; The thermal denaturation as a measure of the structural stability of the nucleoprotein in bacteriophage T7 has been studied in dependence of the ionic environment . Optical density and circular dichroism melting curves measured at wavelengths characterizing either the DNA or protein conformational changes were compared to identify different steps of the denaturation and to follow the effect of the ions . Monovalent salts strengthen the helical structure of intraphage DNA logarithmically in the way as they do in the case of isolated double-stranded DNA . Mg2+ and Ca2+ at very low concentrations stabilize the DNA helicity . Higher divalent ion concentrations decrease the stability of the double helix because of the repulsive ionic interactions . The high structural sensitivity of DNA in the presence of Mg2+ and Ca2+ in this "in situ" environment can be related to the biological role of these ions.

Gene, 1987, 53(2-3), 257 - 64
Secondary structure at the bacteriophage G4 origin of complementary-strand DNA synthesis: in vivo requirements; Lambert PF et al.; The bacteriophage G4 origin of complementary strand DNA synthesis, G4 ori, contains several regions of potential secondary structure . In this study, we ask whether DNA secondary structure is important for G4 ori function in vivo . Point mutations were generated within a region of potential secondary structure so as to disrupt intrastrand base pairing . These mutations led to a strong temperature-dependent reduction in ori function in vivo . A double point mutation which introduces the same base substitutions without destabilizing intrastrand base pairing did not cause a temperature-dependent disruption in ori function . The double mutant did display a slight temperature-independent reduction in ori function compared to the wild-type G4 ori . Based on these findings, we conclude that DNA secondary structure, as well as recognition of specific sequences, is required for G4 ori activity in vivo.

Gene, 1987, 53(2-3), 181 - 90
The mapping of chromosomes in Saccharomyces cerevisiae . I . A cosmid vector designed to establish, by cloning into cdc-mutants, numerous start loci for chromosome walking in the yeast genome; Breter HJ et al.; A series of vectors for cosmid cloning in yeast has been derived from cosmid pHC79 . Vectors pMT4 through pMT6 contain two tandemly arranged cohesive end sites (cos) from the genome of bacteriophage lambda . Their design allows the rapid and simple preparation of cosmid arms by linearizing a vector at the unique PvuII-restriction site located between the two cos-sequences and then cutting the linearized molecule at one of its unique cloning sites for BamHI, ClaI, PvuI, SalI or ScaI . Cosmids generated with arms from the most advanced vector, pMT6, carry the origin of replication (ori) and the ApR gene from pBR322 and the TRP1/ARS1 and URA1 genes from Saccharomyces cerevisiae . A yeast genomic DNA library was established by packaging in vitro, into bacteriophage lambda preheads, of partially restricted yeast DNA fragments ligated to cosmid arms of vector pMT6 . About 80% of the clones thus obtained comprise inserts of contiguous genomic DNA over 30 kb in length . Unique DNA probes for the yeast genes CDC10, CDC39, HIS4, LEU2, and PGK1 have successfully been applied when testing for completeness of this library by isolating a series of overlapping cosmid clones that carry the respective genes . The library will thus be useful for the selection of cosmid clones which carry CDC genes from yeast by complementing first, with the vectorial yeast gene URA1, the pyrimidine auxotrophy of most cdc-strains and then, with the respective CDC wild-type genes, of the temperature-sensitive mutant alleles . Most CDC clones thus obtained will provide unique DNA probes which serve as randomly distributed start sequences within the yeast genome for overlap hybridization screening in chromosome mapping studies.

Microbios, 1987, 49(200-201), 161 - 9
Induction, purification and some properties of phage tail-like particles from Myxococcus coralloides D; Munoz J et al.; Myxococcus coralloides D was lysogenic for a defective prophage . The particles of the defective bacteriophage could be induced by ultraviolet light and mitomycin C, but the particles did not appear in the supernatants, unless the cells were lysed with chloroform . The phage tails were purified by using a two-phase separation method, ultracentrifugation, chromatography through Sepharose 4B, treatment with chloroform, dialysis and centrifugation on a sucrose gradient . The chemical analysis of the purified samples revealed that the phage tails contained only proteins, neither DNA nor RNA . The different parts of the phage tails (sheath, core and baseplate) did not have the same sensitivity to the chemical and physical agents which were assayed.

C R Acad Sci III, 1987, 304(3), 67 - 9
{A family of hypervariable minisatellites detected by means of a sequence derived from phage M 13}; Brocas H et al.; A new family of "hypervariable minisatellites" has been identified in the genome of man and of a variety of animal species using as a probe a DNA segment isolated from the M 13 bacteriophage . This finding provides a new series of hyperpolymorphic genetic markers and renders the "DNA-fingerprinting" methodology available to every molecular biology laboratory.

J Virol, 1987 Jan, 61(1), 159 - 66
Purified scrapie prions resist inactivation by UV irradiation; Bellinger-Kawahara C et al.; The development of effective purification protocols has permitted evaluation of the resistance of isolated scrapie prions to inactivation by UV irradiation at 254 nm . Prions were irradiated on ice with doses of UV light ranging up to 120,000 J/m2 . UV dosimetry experiments, performed with Saccharomyces cerevisiae plasmid DNA or eucaryotic cells, indicated that under these experimental conditions an incident UV dose of 10 J/m2 formed 2 thymine dimers per 5.1 X 10(6) daltons of eucaryotic cell DNA . The D37 values for scrapie prions ranged from 17,000 to 22,000 J/m2; D37 values were also determined for virus, viroid, and enzyme controls . The number of pyrimidine dimers formed was correlated with the D37 values obtained for irradiated prions and target nucleic acids . The D37 value for bacteriophage M13, 6.5 J/m2, occurred at a dose that would form 0.56 dimers per target genome; the D37 for potato spindle tuber viroid, 4,800 J/m2, occurred at a dose that would form about 24 dimers per target viroid . The D37 value for an EcoRI restriction site, a target of 12 bases, occurred at a dose that would correspond to the formation of 0.89 thymine dimers per target site . The D37 value for prions occurred at a dose that would form 1 dimer in every 4 bases of single-stranded target nucleic acid . If the putative scrapie nucleic acid were double-stranded and readily repairable after UV damage, then the prion D37 value could reflect a nucleic acid molecule of 30 to 45 base pairs . While the D37 value for prions fell within the range of pure protein targets, our experiments cannot eliminate the possibility that a prion contains a small, highly protected nucleic acid molecule.

Gene, 1987, 55(1), 9 - 18
Expression of a major bovine rotavirus neutralisation antigen (VP7c) in Escherichia coli; McCrae MA et al.; Sequences from genomic RNA segment 8 of the United Kingdom tissue-culture (t.c.)-adapted bovine rotavirus encoding a major viral neutralisation antigen VP7c have been expressed in Escherichia coli . Expression under the regulated control of the bacteriophage lambda pR promoter was as a C-terminal extension to E . coli beta-galactosidase (beta Gal) . Following temperature induction, high levels of the fusion protein were synthesised and accumulated in induced cells, making up 5%-15% of total bacterial cell protein after 2 h of induction . Immunisation of sero-negative rabbits and mice with gel-purified fusion-protein raised antibodies, which gave specific immunofluorescence with virus-infected cells and were able to immunoprecipitate proteins of the VP7 complex from such cells . Hyperimmune sera also gave a virus-type-specific reaction in a solid-phase enzyme-linked immunoabsorbant assay and neutralised virus infectivity in standard plaque-reduction assays.

Gene, 1987, 52(2-3), 279 - 83
Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region; Schauder B et al.; New expression vectors were constructed for use in strains of Escherichia coli . Their most important feature is a polylinker system that facilitates the insertion of a gene in an optimal relationship to the highly efficient E . coli atpE translational initiation region (from nucleotide -50 to the start codon) . Three ATG-containing restriction endonuclease sites can be used for the insertion of the 5' end of a gene at, or near to, its translational initiation codon . These sites may alternatively be used for the creation of a suitable translational start codon . Transcription is started by the bacteriophage lambda major promoters pR and pL in tandem and terminated by the bacteriophage fd terminator . Transcriptional initiation is very effectively repressed at 28-30 degrees C by the product of the bacteriophage lambda cIts857 gene, which is also present on the vectors . Full induction is achieved by shifting the incubation temperature to 42 degrees C . The combination of highly efficient transcriptional and translational signals on these vectors allowed high-level expression of sequences encoding human interferon beta and interleukin 2 and of the E . coli atpA, sucC and sucD genes.

Gene, 1987, 52(2-3), 155 - 64
The first-step transfer-DNA injection-stop signal of bacteriophage T5; Heusterspreute M et al.; Bacteriophage T5 is different from most phages in that its DNA is injected in two steps during infection . The region containing the injection stop signal (iss) has been cloned and sequenced and found to contain numerous large repeats and inverted repeats which may be part of the iss . The most impressive of these are the 31-bp repeat units (rb) which are present three times in 99 bp . The rb repeats, themselves, contain inverted repeats so that mutually exclusive stem-and-loop structures may potentially form, not only within the repeats, but also between them . Another pair of repeats (21 bp each) contains two sequences resembling DnaA protein-binding sites . The region sequenced also contains one of the T5 site-specific strand interruptions and this was found to lie at the base of a perfect 9-bp palindrome.

Mol Gen Mikrobiol Virusol, 1987 Jan, (1), 14 - 9
{Various characteristics of the anti-restriction mechanism in bacteriophage T5}; Chernov AP et al.; Bacteriophage T5 is not confined by the restriction systems of the second type EcoRII and EcoRV . Bacteriophage T5 DNA is not modified by EcoRII and EcoRV methylases in vivo . The sites of recognition for restriction endonuclease EcoRV are mapped at 24.4; 57.6; 68.5; 70.2% of T5 DNA, while the sites at 5.1; 7.6% are recognized by EcoRII, the sites at 5.75; 6.0 and 6.5% are recognized by HpaI in FST . A high activity of restriction endonucleases EcoRI and EcoRV is demonstrated in crude extracts of E . coli B834 (RI) and E . coli B834 (RV) cells infected by bacteriophage T5 . The simultaneous infection of E . coli B834 (RI) or E . coli B834 (RV) cells by the amber mutants of bacteriophage T5 and the suppressing phage lambda NM761 does not result in the protection of lambda DNA by the T5 anti-restriction mechanism . The presented data support the hypothesis that the anti-restriction mechanism of bacteriophage T5 is based on prevention of T5 DNA contacts with restriction enzymes by a specific phage protein.

Immunogenetics, 1987, 25(2), 116 - 22
Genetic organization of the ovine MHC class II region; Scott PC et al.; To study the class II genes of the major histocompatibility region of the sheep genome, human HLA class II genes corresponding to the known subregions in man (DR, DQ, DP, DO, and DZ) were used for Southern hybridization analysis of sheep DNA and to probe a sheep genomic library . Hybridizing bands were noted for all probes except DP alpha . DQ alpha and beta and DR beta appear to be present as multicopy genes, while DR alpha-, DZ alpha-, and DO beta- like genes appear to be single copy . All bands detected with the DP beta probe were also detectable with other beta chain probes . From eight lambda-bacteriophage clones of a sheep genomic library nine distinct class II genes were identified . These genes were characterized by differential hybridization analysis and restriction mapping . Two genes were DR beta-like, three DQ alpha-like and four DQ beta-like . The extensive cross-hybridization observed with beta chain probes was not seen with alpha chain probes . The results of this study suggest that the major histocompatibility complex class II region of the sheep has a similar genetic organization to that of man, with the provisional exception of the DP subregion.

Virology, 1987 Jan, 156(1), 74 - 83
Characterization of the integration site of the CMV mtr in a tumor cell line; Buonaguro FM et al.; Previous studies have shown that a 558-bp fragment of human cytomegalovirus (CMV) DNA contained within pCM4127 and designated CMV mtr can morphologically transform rodents cells in vitro . By cotransformation with pCM4127 and a plasmid conferring G418 resistance, pSV2neo, morphologically transformed NIH3T3 cell lines were isolated . Dot blot hybridization indicated that approximately 30% of the transformants retained CMV sequences . Two cell lines which retained viral DNA were chosen for further study . They were capable of anchorage independent growth and formed tumors in nude mice . Integrated viral sequences in the transformants and tumor cell lines were analyzed by Southern blotting . A bacteriophage lambda library was constructed using a tumor cell line which retained a single copy of the viral sequences, and a phage was isolated which contained the integrated plasmid and the flanking cellular sequences . A complex rearrangement between pCM4127 and pSV2neo had occurred . DNA sequence analysis showed integration of the plasmid sequences into repetitive mouse DNA and identified an adjacent mouse sequence.

J Gen Virol, 1987 Jan, 68 ( Pt 1), 39 - 46
Analysis of the role of the cysteine 171 residue in the activity of herpes simplex virus type 1 thymidine kinase by oligonucleotide-directed mutagenesis; Inglis MM et al.; The thymidine kinase (TK) gene from herpes simplex virus type 1 strain SC16 was cloned into bacteriophage M13 mp8 so that functional HSV-1 TK was expressed in bacteria infected with the recombinant bacteriophage, M13/TK . Oligonucleotide site-directed mutagenesis was then employed to introduce single nucleotide changes into the TK gene in M13/TK in order to alter the codon for cysteine 171 in the wild-type enzyme to a codon specifying either serine or glycine . Analysis of the mutant enzymes in bacterial extracts showed that these substitutions had little effect on the activity of the enzyme, indicating that the side chain of this residue is not involved in nucleoside binding and is not essential for the catalytic activity of the enzyme.

J Bacteriol, 1987 Jan, 169(1), 14 - 8
Characterization of the ftsB gene as an allele of the nrdB gene in Escherichia coli; Kren B et al.; A temperature-sensitive, salt-rescuable ftsB cell division mutant, MFT84, was found to be hydroxyurea sensitive on low-salt medium . Complementation studies with plasmids and a marker rescue study with bacteriophage M13 nrd indicated that ftsB is an allele of nrdB and that the mutation occurs in the region corresponding to nucleotides 6729 to 7032 of the nrdB gene . Enzymatic characterization demonstrated that the B2 subunit of ribonucleoside-diphosphate reductase encoded by ftsB was responsible for the decreased activity and the thermolability of the enzyme . The ftsB-encoded B2 subunit was activated by the addition of 0.1 M NaCl to an in vitro assay, corroborating the in vivo temperature-dependent salt requirement was a result of a defective B2 subunit.

J Virol, 1987 Jan, 61(1), 60 - 5
Integration of the DNA of filamentous bacteriophage Cflt into the chromosomal DNA of its host; Kuo TT et al.; It was demonstrated for the first time that filamentous bacteriophage Cflt, which contains single-stranded DNA, can incorporate its genome into that of its host . Evidence in support of the incorporation was obtained from a Southern blot hybridization analysis of DNA isolated from Cflt-lysogenized cells . DNAs from different Cflt-lysogenized cells were purified, and the integration patterns were compared . Because all integration patterns were identical and only one fragment in Cflt replicative-form DNA was missing, it appears that the integration was site specific . Only one complement of viral DNA was integrated per host chromosome . To determine the attachment site on the viral DNA, the physical map of EcoRI, XhoI, SstII, and BglII on Cflt DNA was constructed . Based on this physical map and a Southern blot hybridization analysis of lysogen DNA with these restriction endonucleases, we demonstrated that DNA sequences from all regions of the Cflt genome were represented in the integrated viral sequences . The attachment site on the viral genome was located at 69.2 to 73.8 min on the Cflt DNA.

J Cell Sci Suppl, 1987, 7, 33 - 40
Genome interactions which influence DNA palindrome mediated instability and inviability in Escherichia coli; Leach D et al.; The interaction of three factors determine the detrimental effect of a palindromic DNA sequence in Escherichia coli cells . The first is the nature of the palindrome (its length, extent of central asymmetry and perhaps its base sequence), the second is the genotype of the host cell and the third is the replicon within which it is located . In this paper we extend the genetic and physical characterization of lambda bacteriophages carrying a palindrome of approximately 560 base pairs . We also show that a palindrome of approximately 110 base pairs which can be cloned in a plasmid cannot be cloned in a phage M13 derivative . These observations are relevant to the choice of vectors used in the cloning of eukaryotic DNA containing palindromic sequences.

Gene, 1987, 61(2), 135 - 44
Functional elements of DNA upstream from the integrase operon that are conserved in bacteriophages 434 and lambda; Limberger RJ et al.; A 1488-bp restriction fragment of bacteriophage 434 DNA contains the integrase promoter and an adjacent nucleotide sequence (t'I) resembling a Rho-independent terminator . To identify and quantitate transcription termination, DNA segments were cloned into a plasmid between the galactose promoter and assayable galactokinase gene and tested for termination . Whereas the entire fragment effectively terminated transcription, a 331-bp restriction fragment containing the t'I terminator had only weak terminator activity . Random sequential deletions of the 434 DNA segment defined a strong terminator 650-bp upstream from t'I . This proposed Rho-independent terminator called tL4 consists of a 7-bp stem and 6-nt loop followed by a uridine-rich region in the RNA . Phage lambda contains an even stronger tL4 terminator that differs in 4 nt from 434 tL4 . Thus, despite some sequence divergence, terminator activity has been conserved in these phages . The 434 DNA segment was also tested for promoter activity . Rightward promoter activity (opposite to pL in the phage) was located about 200 bp to the right of tL4 and was followed by an open reading frame (ORF) capable of encoding a 91 amino acid protein . Promoter activity in the same approximate location was also found in phage lambda . Thus the rightward promoter, the tL4 and t'I terminators, and ORF-55 all are elements in this segment of the genome that are conserved for function despite sequence divergence.

Gene, 1987, 60(1), 129 - 35
Construction of an ordered overlapping library of bacteriophage P1 DNA in phage vector lambda D69; O'Regan GT et al.; A library of bacteriophage P1 DNA was constructed in the phage vector lambda D69 . The DNA of some 150 randomly chosen lambda-P1 hybrid phages containing P1 DNA fragments 5-10 kb in size was analyzed by restriction endonuclease digestion using enzymes EcoRI, BglII, and BamHI that cleave P1 DNA at known positions on the physical map of P1 . Approximately one third of the phages contained P1 DNA inserts having two or more restriction sites for any one of these enzymes, thus allowing the location of the insert to be determined with respect to the physical map . Genetic tests allowed detection of lambda-P1 hybrid phages possessing inserts with functional P1 ban and CmR genes . A subset of 18 phages was analyzed in more detail; their P1 DNA inserts comprise an ordered collection of overlapping P1 DNA fragments that cover almost 98% of the P1 genome.

Gene, 1987, 55(2-3), 345 - 51
The bacteriophage Mu com gene appears to specify a translation factor required for mom gene expression; Hattman S et al.; Expression of the bacteriophage Mu mom gene is subject to a variety of regulatory controls . Both the host Dam DNA-adenine methylase and the phage Mu C protein are required for mom gene transcription . In addition, the Mu com gene product is required for production of the mom protein . Because the com and mom genes overlap on the same mRNA transcript (with com being located proximal to the 5' end), it is likely that Com function is exerted after transcription initiation . To study the role of Com, two segments of Mu were cloned in both orientations (+ and -) into the HindIII site of the galactokinase expression vector, pKG1800; the HindIII site is located between the galK structural gene and its promoter . In (+) plasmids, the Mu DNA inserts were transcribed from the gal promoter in the same orientation as in the phage genome; (-) plasmids had the Mu DNA inserted in the reverse orientation . Each Mu insert contained the same segment of the mom gene from the 3' terminus, but differed in the extent of com gene included at the 5' terminus; one contained a truncated com gene and the other a complete com gene, as well as upstream Mu regulatory sequences . The results are summarized as follows: (1) both (-) plasmids produced only about 10% as much galactokinase activity following fucose induction as the parental vector, pKG1800; (2) plasmid pGTVH(+), with an intact com gene produced about 30% as much galactokinase as pKG1800; (3) plasmid pMTVH(+), with a truncated com gene, produced only about 10% as much enzyme as pKG1800.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1987, 54(2-3), 291 - 7
New mutations in the pRM promoter of bacteriophage lambda; Gussin GN et al.; A pRM-cI-lacZ fusion inserted into the b2 region of bacteriophage lambda imm21 was used to isolate mutations in the lambda pRM promoter . Among the mutations causing defects in synthesis of both repressor (cI gene product) and beta-galactosidase, new promoter mutations were identified at positions -11 and -32 relative to the cI transcription start point . Both mutations are changes in conserved (consensus) nucleotides in pRM, but the mutation at -11, which alters a more highly conserved nucleotide, has a somewhat greater effect on promoter function in vitro than does the mutation at -32 . We also isolated a mutation at -69 in the repressor-binding site OR1, which presumably prevents activation of pRM by repressor.

Gene, 1987, 54(2-3), 261 - 5
Location of genes D16, D17, and N4 encoding tail proteins on the physical map of bacteriophage T5; Krauel V et al.; Bacteriophage T5 DNA fragments were cloned into plasmid pBR322 . Recombinant plasmids complementing T5 amber mutants were isolated, and used as hybridization probes with T5 DNA in Southern blots . Employing this approach the three T5 genes D16, D17, and N4 were mapped with respect to the physical map of T5, and shown to be located at 74%, 72%, and 82% of the genome, respectively.

Gene, 1987, 55(1), 47 - 53
Detection of heterologous fusion proteins in Escherichia coli with a monoclonal antibody; Zweig M et al.; Several laboratories have constructed expression vectors for the production of heterologous fusion proteins containing the N-terminal 13 amino acids of the bacteriophage lambda cII-coded protein in Escherichia coli . We have prepared a monoclonal antibody to a synthetic peptide having this CII amino acid sequence and have found that this antibody reacts with authentic CII protein in Western blot tests and with most CII peptide-containing fusion proteins in both radioimmunoprecipitation and Western blot assays . However, there are some CII-hybrid protein species with which the antibody does not react . Our findings indicate that this antibody is a valuable tool for detecting and purifying expressed proteins and in studying their structure and function.

Acta Biochim Pol, 1987, 34(1), 29 - 34
Construction of a DNA-polymerase I overproducing plasmid and isolation of the enzyme; Bielawski K et al.; The polA gene of Escherichia coli coding for DNA polymerase I was cloned under the control of bacteriophage lambda promoter pL and gene N in a high copy number plasmid vector . The chromosomally located lambda cIts repressor gene kept the synthesis of the polA gene product at 28 degrees C at a low level . Raising the temperature to 43 degrees C resulted in inactivation of the repressor and overproduction of DNA polymerase I, which could easily be purified to homogeneity.

Gene, 1987, 51(2-3), 139 - 47
Post-transcriptional regulation of the bacteriophage Mu mom gene by the com gene product; Wulczyn FG et al.; The mom gene of bacteriophage Mu encodes a DNA modification function, the expression of which is detrimental to the host cell . This may be reflected by the tight regulation of the mom gene at the level of transcription initiation by the Mu C gene product and the host Dam function . In addition, mom expression requires the positive regulatory function Com . The com and mom genes comprise the mom operon with the com coding region partially overlapping that of mom . The degree of overlap is defined by experiments reported here . We have tested Com for activity as an antiterminator of mom transcription . We show that in the absence of Com, premature termination affects at most 33% of the transcription across the mom operon . Although no premature termination is observed in the presence of Com, these results are inconsistent with a role for Com as an antiterminator . Northern blot analysis of Com+ and Com- Mu phage mRNA confirms this conclusion . Two models for the post-transcriptional regulation of mom gene expression by Com are presented.

Gene, 1987, 51(1), 77 - 84
In vivo DNA cloning with a mini-Mu replicon cosmid and a helper lambda phage; Groisman EA et al.; A mini-Mu bacteriophage, containing the cohesive-end packaging site (cos) from a lambda-phi 80 hybrid phage, a high-copy-number plasmid replicon, and a kanamycin-resistance gene for independent selection, was constructed to clone genes in vivo . This mini-Mu element can be derepressed to transpose at a high frequency . DNA segments that become flanked by copies of this mini-Mu element in the same orientation can be packaged by a helper lambda phage . The resulting lambda lysate can be used to infect recipient cells where the injected DNA can circularize by annealing at the cos termini . Drug-resistant transductants obtained carry the mini-Mu-replicon cosmid element with inserts of different nucleotide sequences . These are analogous to recombinant DNA clones generated in vitro with restriction endonuclease cutting and ligase joining reactions replaced by the Mu transposition process . Clones of particular genes were isolated by their ability to complement specific mutations . Both recA+ and recA- recipient cells can be used with equal efficiency . Clones obtained with a helper lambda phage require the presence of the cos site in the mini-Mu replicon . They carry larger inserts than those isolated with the same mini-Mu element and Mu as a helper phage . The mini-Mu replicon-cosmid bacteriophage contains a lac-gene fusing segment for isolating fusions of lac operon DNA to gene control regions in the cloned sequences . Independent clones of a particular gene can be used to prepare a restriction map of the gene and its flanking regions.

Genetics, 1987 Jan, 115(1), 3 - 10
Synthesis of a trans-acting inhibitor of DNA maturation by prohead mutants of phage lambda; Murialdo H et al.; Bacteriophage lambda with mutations in genes that control prohead assembly and other head precursors cannot mature their DNA . In this paper we present evidence that the failure of these phage mutants to mature DNA is a reflection of a mechanism that modulates terminase nicking activity during normal phage development . We have constructed plasmids that contain the lambda-cohesive end site (cos) and the genes that code for DNA terminase, the enzyme that matures DNA by cutting at cos . The DNA terminase genes are under control of a thermosensitive cI repressor . These plasmids lack most of the genes involved in prohead morphogenesis and other head precursors . However, when repression is lifted by destruction of the thermosensitive repressor, the terminase synthesized is able to cut almost 100% of the plasmids . Therefore, these plasmids can mature in the absence of proheads and other head gene products . The plasmids are also able to complement mutants of lambda deficient in terminase and DNA maturation . However, in these complementation experiments, if the phage carry mutations in prohead genes E or B, not only is phage DNA maturation blocked, but the plasmid also fails to mature . These experiments show that, in the absence of proheads, phage lambda produces a trans-acting inhibitor of maturation . The genetic determinant of this inhibitor maps in a region extending from the middle of gene B to the end of gene C . A model is proposed in which the nicking activity of DNA-bound terminase is inhibited by the trans-acting inhibitor . Prohead (and other factors) binding to this complex would release the block to allow DNA cleavage and packaging.

J Bacteriol, 1987 Jan, 169(1), 430 - 3
Nonrandom minichromosome replication in Escherichia coli K-12; Koppes LJ et al.; The intervals between rounds of chromosome and minichromosome replication were measured by density shift experiments and found to be similar . Thus the minichromosome, a lambda asnA oriC bacteriophage, mostly replicates once each division cycle rather than randomly, despite its high copy number . Slight differences between the chromosome and the oriC plasmid are explained.

J Bacteriol, 1987 Jan, 169(1), 403 - 9
Rec dependence of mu transposition from P22-transduced fragments; Hughes KT et al.; Derivatives of bacteriophage Mu carrying a lac operon and a selectable drug resistance element (Mu d phages) are frequently used tools of bacterial genetics . Mu d prophages used in this way can be treated as transposons, in that the inserted material can be transduced from one strain to another by general transducing phages, such as P1 and P22 . When a Mu d prophage is transduced into a new recipient by P1 or P22, the Mu d element can transpose from the transduced fragment into the bacterial chromosome . Transposition of the Mu d element from a P22-transduced fragment shows several striking differences from transposition of a Mu d genome injected by a Mu virion . First, the frequency of transposition from a transduced fragment is greatly enhanced by a P22 helper genome . Second, transposition requires the host recA, B, and C functions . Transposition of Mu following injection by a Mu virion is rec independent . While the basis of these observations is not understood, we suggest that the Mu X protein, a 65-kilodalton protein injected by a Mu virion and required for Mu transposition, may not be packaged by P22 . We suggest that the effects seen reflect the behavior of a Mu genome in the absence of the X protein.

J Basic Microbiol, 1987, 27(4), 225 - 8
Temperature dependence of M pilus formation as demonstrated by electron microscopy; Karste G et al.; The formation of IncM plasmid encoded pili is dependent on the incubation temperature of the corresponding host strains . By labelling the short, rigid M pili with the donor specific bacteriophage luminal diameter M, the presence of pili at 30 degrees C but not at 37 degrees C incubation temperature could be demonstrated for E . coli K12 substrains carrying different IncM group plasmids . In contrast, such a temperature dependence of M pilus formation is not observed in S . typhimurium substrains.

Free Radic Res Commun, 1987, 2(4-6), 343 - 50
DNA damage by chemically generated singlet oxygen; Lafleur MV et al.; A naphthalenic endoperoxide was used as a non-photochemical source of singlet oxygen (1O2) to examine some interactions between this reactive oxygen species and DNA . High molecular weight DNA (ca . 10(8) daltons) was exposed to 120 mol m-3 1O2 (cumulative concentration) and analyzed for interstrand crosslinkage by hydroxyl apatite chromatography following formamide denaturation . No evidence for 1O2-induced interstrand crosslinking was obtained . The capacity of 1O2 to generate strand breaks in single-stranded (ss) and double-stranded (ds) DNA was investigated by sucrose gradient centrifugation analysis of bacteriophage phi X174 DNA . No direct strand breaks could be detected at neutral pH, whereas extensive strand breakage was observed after treatment with alkali . Possible biological consequences of 1O2-exposure were assessed by examining the plaque-forming capacity of ss and ds phi X174 DNA molecules using wildtype Escherichia coli spheroplasts as recipients . Without any further treatment with heat or alkali, exposure to the endoperoxide resulted in a time- and dose-dependent inactivation, ss DNA being considerably more sensitive than ds DNA . From the present results and those reported earlier (Nieuwint et al.,) we infer that 1O2-induced inactivation of phi X174 DNA is not due to DNA backbone breakage nor to interstrand crosslinking, but rather to some form of damage to the base or sugar moiety of the DNA, the exact nature of which remains to be elucidated.

J Cell Sci Suppl, 1987, 7, 41 - 50
The requirements for a high level of transposition of bacteriophage Mu; Groenen MA et al.; To analyse the functional and structural requirements of Mu transposition a special mini-Mu transposon was constructed . This mini-Mu, situated on a derivative of pBR322, has an easily selectable marker gene conferring chloramphenicol resistance (Cam) cloned between the ends of the mini-Mu . The genes required for transposition are cloned outside the ends and are under control of the PL promoter of bacteriophage lambda . To obtain a high frequency of transposition the A and B products are required . Under B- conditions both cointegrate formation and simple insertions are strongly reduced . The role of B in the transposition process is unknown and is discussed . Deletion analysis of the ends of the mini-Mu has revealed the existence of multiple sites required for transposition . The same sites were found to be strong A-binding sites . In addition two weak A-binding sites were found . Point mutations introduced into these last sites show that they are at least as important for transposition as the strong binding sites . The organization of the binding sites is asymmetric in Mu . At the left end two binding sites are situated more than 100 bp away from the end . These affect specifically the frequency of transposition . The distance between these two sites and the site close to the left end is extremely critical . At the right end also three A-binding sites are found, but only two seem to be important for transposition . Transposition with low frequency can be obtained with a mini-Mu with only one end . Secondary att sites are selected which all show some homology with an A-binding site . An absolute requirement for a 5'T at the artificial end is observed . Based on these results a simple scheme for the evolution of a transposon is discussed.

Dev Genet, 1987, 8(4), 233 - 47
Restriction enzyme evidence for Alu sequence-mediated dispersion of microinjected genes in transgenic mice; Rubinstein WS et al.; A human bacteriophage clone containing adult beta-globin genes with four Alu sequences was microinjected to produce transgenic mice . Southern blot analysis on the spleen of a transgenic mouse revealed an unusual hybridization pattern that suggested extensive dispersion of human DNA throughout the mouse genome . This pattern was reproducible using several restriction enzymes, including a noncutting enzyme . The hybridization pattern was not observed in other tissues, and sequences were not detected in progeny using the bacteriophage probe . However, hybridization of spleen DNA of offspring against a human Alu probe revealed genetic transmission of human Alu sequences . The results suggest dispersion of microinjected Alu sequences throughout the genome.

Gene, 1987, 58(2-3), 297 - 8
Convenient vectors for cloning and sequencing EcoRI and HindIII fragments; Edenberg HJ et al.; The polylinker regions of plasmid pUC and bacteriophage M13mp vectors have been specifically modified to provide alternative positions for cloning and reexcising EcoRI and HindIII fragments; the EcoRI and HindIII sites have been moved internal to BamHI and Bg/II sites . The location of EcoRI and HindIII sites in these HinEco vectors allows either selective linearization or excision of the cloned fragments at unique flanking sites.

Gene, 1987, 57(2-3), 193 - 201
Plasmid and bacteriophage vectors for excision of intact inserts; Lathe R et al.; Plasmid (pPolyIII) and bacteriophage lambda (EMBL301) vectors are described in which sites for the rare-cutting enzymes SfiI and NotI (8-bp, recognition sequences) flank the polylinker cloning region . Intact DNA inserts for introduction into cultured cells or into the early embryo are readily excised from the vectors . General-purpose miniplasmid cloning vectors pPolyI and pPolyII are also described, and the utility of the bacteriophage lambda vector is demonstrated in the construction of a bovine genomic library.

Chem Biol Interact, 1987, 64(1-2), 139 - 49
Phototoxic potential of afloqualone, a quinazolinone derivative, as determined by photosensitized inactivation of bacteriophage; Fujita H et al.; Effect of UV-A irradiation on bacteriophage lambda in the presence of afloqualone (AQ) was examined to obtain in vitro evidence for phototoxic potential of AQ, a centrally acting muscle relaxant . Neither AQ itself nor the long-lived photoproducts affected viability of the phage, but the phage was inactivated when it was irradiated in the presence of the drug . Photosensitized inactivation was efficiently repressed by the presence of radical scavengers such as hydroquinone, cysteamine and cystein but not by D-mannitol, benzoate, formate and dimethyl sulfoxide (.OH scavengers) . Methionine also inhibited inactivation as well . Sodium azide and tryptophan followed them, but 1,4-diazabicyclo{2.2.2}octaine (DABCO) did not reduce the inactivation rate . Deuterium effect was not observed . AQ-sensitized photoinactivation occurred even under anoxic conditions although the rate was lower than under aerobic conditions . In view of these results, Type I process is more suitable for explanation of AQ-sensitized photoinactivation than Type II process.

J Mol Evol, 1987, 25(4), 361 - 70
A highly conserved sequence in H1 histone genes as an oligonucleotide hybridization probe: isolation and sequence of a duck H1 gene; Tonjes R et al.; A 3.5-kb HindIII fragment of a histone gene cluster was isolated from a recombinant phage out of a duck genomic library . This DNA contains a duck H1 gene and its flanking sequences . The hybridization probe, which was used to screen for the H1 gene, had been designed on the basis of a comparative analysis of available H1 gene and protein data . Most H1 histones contain repeated motifs in their C-terminal domain, and these form part of an octapeptide (ser pro lys lys ala lys lys pro) that is highly conserved in many H1 histone proteins . A comparison of the duck H1 described here with two different published chicken H1 histone sequences reveals conservative amino acid exchanges at 22 (of 217 and 218, respectively) positions . The homology is maintained at the flanking sequences, and includes the putative H1 histone gene-specific signal structures and the established 3' stem and loop structures and the CAAGA box . The duck H1 gene and its flanking sequence have been found in identical arrangements in two recombinant bacteriophages, but minor sequence variations and genomic Southern blotting after HindIII digestion suggest that we have either isolated alleles of this genome segment or that the gene described may occur twice per haploid duck genome.

J Mol Evol, 1987, 25(4), 308 - 17
Conservation of delta-crystallin gene structure between ducks and chickens; Piatigorsky J et al.; A cloned chicken delta-crystallin cDNA was used to identify two putative delta-crystallin genes in the duck by Southern blot hybridization . A DNA fragment containing most of one of these genes was isolated from a library made in bacteriophage lambda Charon 28A containing genomic DNA from 14-day-old embryonic ducks . Electron microscopy, partial gene sequencing, primer extension analysis using duck mRNA, and comparison with the well-characterized chicken delta-crystallin genes suggest that our cloned duck delta-crystallin gene, like the chicken delta-crystallin genes, is 8-10 kb long and contains 17 exons . Hybridization and sequencing data show great similarity between the homologous 5' untranslated and coding exons of the duck and chicken delta-crystallin genes . Overall, the homologous introns also appear to have approximately 30% sequence similarity, and have been subject to deletion/insertion events . Our partial characterization of duck delta-crystallin gene sequences suggests that this avian and reptilian crystallin family has been conserved during evolution, as have the other crystallin gene families that are expressed in the eye lens.

Arch Virol, 1987, 96(3-4), 169 - 83
Characterization of a murine cytomegalovirus-induced immunosuppressive factor; Whyte PF et al.; Murine cytomegalovirus infection in spleen cultures resulted in the production of a soluble factor, VISF (virus-induced suppressive factor), which inhibited concanavalin A mitogenesis in fresh spleen cells . Its production was specific for MCMV, since infection of spleen cultures by Sindbis virus, or bacteriophages PM 2 and T 4, and the phagocytosis of latex beads, all failed to elicit VISF . Maximum appearance of the factor occurred within 24 hours p.i . in spleen cultures, and its source was identified as the population of spleen cells which adhered to a plastic culture dish within two hours at 37 degrees C . Non-adherent cells did not produce the factor . Its production was not inhibited by indomethacin . VISF could be concentrated by ultrafiltration on a YM 2 membrane filter, and it was readily fractionated by chromatography on sephadex G-25 . In relation to peptides of known molecular weight it appeared to be smaller than 1,400 daltons . Its ability to suppress concanavalin A mitogenesis was largely removed by digestion with proteinase K . Thus VISF appears to be a relatively small peptide or peptide-containing substance . It was purified further by HPLC.

Intervirology, 1987, 27(3), 172 - 6
RNA complementary to alfalfa mosaic virus RNA 4 is not translated in vitro; Nelson SE et al.; cDNA of the subgenomic messenger RNA (RNA 4) of alfalfa mosaic virus was ligated in both orientations into plasmids containing the bacteriophage SP 6 promoter . Upon transcription in vitro these plasmids yielded synthetic full-length complementary RNA and messenger-sense RNA . The complementary RNA contained an open reading frame of 417 nucleotides; the messenger-sense RNA encodes the virus coat protein . No translation products from the complementary RNA were detected in a wheat germ translation system, a reticulocyte lysate translation system, or Xenopus oocytes, whereas the messenger sense RNA was very active in these translation systems.

Intervirology, 1987, 27(2), 102 - 11
Analysis of Aleutian disease of mink parvovirus infection using strand-specific hybridization probes; Bloom ME et al.; A 7- to 95-map unit segment of DNA from Aleutian disease of mink parvovirus (ADV) was subcloned into a bacteriophage SP6 based transcription vector and used to produce radiolabeled viral RNA transcripts corresponding to either 'plus' or 'minus' sense . The radiolabeled transcripts were reacted against Southern blots of whole cell DNA from ADV infected cell cultures as hybridization probes . The 'plus' sense RNA probe hybridized both to duplex replicative forms (RFs) as well as to single-stranded virion DNA (SS DNA), which is 'minus' in sense . In contrast, the 'minus' sense RNA probe reacted preferentially with the duplex RFs . When these probes were tested against DNA extracted from mink infected with the virulent ADV-Utah I strain, RFs were detected at 10 days after infection in mesenteric lymph node, liver, spleen and gut, but only in gut and mesenteric lymph node at 43 days . SS DNA was noted in these tissues at 10, 43 and 60 days, and was more abundant than RFs . Only SS DNA at very low levels was observed in bone marrow cells . Serum contained large amounts of SS DNA (probably in virions) at 10 days, less at 43 days, and no detectable DNA at 60 days . These findings suggest that ADV replication may have occurred in the gut as well as lymphoreticular tissues, and that bone marrow was not a major site of ADV replication.

Gene, 1987, 55(2-3), 255 - 63
Genetically engineered diphtheria toxin fusion proteins carrying the hepatitis B surface antigen; Phalipon A et al.; Tripartite fusion proteins comprising the nontoxic mutant protein CRM228 of diphtheria toxin (DT), the hepatitis B virus surface antigen (HBsAg), and beta-galactosidase were obtained by expression of hybrid genes from the pR promoter of bacteriophage lambda and purification by affinity chromatography . The antigenicity and immunogenicity of the individual protein constituents were analyzed . A major neutralizing epitope of DT was inactivated by the HBsAg insertion into the DT B fragment . The fusion proteins elicited antibodies reactive with 22 nm HBsAg particles . This suggests a novel approach towards the use of DT mutants as immunogenic carriers of heterologous antigens.

Proc Natl Acad Sci U S A, 1987 Jan, 84(2), 339 - 43
Rat androgen-binding protein: evidence for identical subunits and amino acid sequence homology with human sex hormone-binding globulin; Joseph DR et al.; The cDNA for rat androgen-binding protein (ABP) was previously isolated from a bacteriophage lambda gt11 rat testis cDNA library and its identity was confirmed by epitope selection . Hybrid-arrested translation studies have now demonstrated the identity of the isolates . The nucleotide sequence of a near full-length cDNA encodes a 403-amino acid precursor (Mr = 44,539), which agrees in size with the cell-free translation product (Mr = 45,000) of ABP mRNA . Putative sites of N-glycosylation and signal peptide cleavage were identified . Comparison of the predicted amino acid sequence of rat ABP with the amino-terminal amino acid sequence of human sex hormone-binding globulin revealed that 17 of 25 residues are identical . On the basis of the predicted amino acid sequence the molecular weight of the primary translation product, lacking the signal peptide, was 41,183 . Hybridization analyses indicated that the two subunits of ABP are coded for by a single gene and a single mRNA species . Our results suggest that ABP consists of two subunits with identical primary sequences and that differences in post-translational processing result in the production of 47,000 and 41,000 molecular weight monomers.

Nucleic Acids Res, 1986 Dec 22, 14(24), 9797 - 809
A bacterial protein requirement for the bacteriophage lambda terminase reaction; Gold M et al.; The bacteriophage lambda terminase enzyme cleaves the cohesive-end sites of lambda DNA to yield the protruding 5'-termini of the mature molecule . In vitro, this endonucleolytic event requires a protein factor which has been isolated and purified from extracts of uninfected E . coli . The terminase host factor (THF) is a heat stable basic protein of M.W . approximately 22,000 . The integration host factor (IHF) protein of E . coli can efficiently substitute for THF in the terminase reaction; however, THF can be demonstrated to be physically present in, and isolated with full biological activity from extracts of cells defective or deficient in IHF.

J Mol Biol, 1986 Dec 20, 192(4), 853 - 67
Three-dimensional reconstruction of the connector of bacteriophage phi 29 at 1.8 nm resolution; Carazo JM et al.; The three-dimensional reconstruction of the connector of bacteriophage phi 29 has been obtained from tilt series of negatively stained tetragonal ordered aggregates under low-dose conditions and up to a resolution of (1/1.8) nm-1 . These connectors are built up as dodecamers of only one structural polypeptide (p10) . Two connectors form the crystal unit cell, each one facing in the opposite direction with respect to the plane of the crystal and partially overlapping . The main features of the two connectors that build the unit cell were essentially the same, although they were negatively stained in slightly different ways, probably due to their situations with respect to the carbon-coated support grid . The main features of the phi 29 connector structure revealed by this three-dimensional reconstruction are: the existence of two clearly defined domains, one with a diameter of around 14 nm and the other narrower (diameter approximately equal to 7.5 nm); an inner hole running all along the structure (around 7 to 8 nm in height) with a cylindrical profile and an average diameter of 4 nm; a general 6-fold symmetry along the whole structure and a 12-fold one in the wider domain; a clockwise twist of the more contrasted regions of both domains from the narrower towards the wider domain (the direction of DNA encapsidation) . These features are compatible with an active role for the connector in the process of DNA packaging.

J Mol Biol, 1986 Dec 20, 192(4), 793 - 803
Organization and expression of the satellite bacteriophage P4 late gene cluster; Dale EC et al.; The satellite bacteriophage P4 genes for capsid size determination (sid), transactivation (delta), and polarity suppression (psu) are cotranscribed at late times after infection from a single P4 late promoter (Psid) that lies to the left of the sid gene . While the -10 region of this promoter is similar to the consensus sequence for Escherichia coli RNA polymerase, the -35 region shares no homology with known classes of E . coli promoters . The -10 and -35 regions of Psid share no homology with the late gene promoters of helper phage P2 . Nonetheless, P4 late transcription is stimulated by coinfecting P2, as well as by P2 prophage . This stimulation depends on the P2 encoded transcription factor ogr; transcription from Psid is stimulated following the induction of the P2 ogr gene carried on a plasmid . P4 late transcription in the absence of P2 requires the P4 delta product, which is partially homologous to the P2 ogr gene product . DNA sequence analysis shows that the psu gene codes for a protein of Mr = 21,314 that is unrelated to the antitermination gene products of the lambdoid phages.

EMBO J, 1986 Dec 20, 5(13), 3691 - 6
Illegitimate recombination occurs between the replication origin of the plasmid pC194 and a progressing replication fork; Michel B et al.; Hybrids between plasmids pC194, pBR322 and the bacteriophage f1 undergo deletions in Escherichia coli . The deletions end most often between nucleotides 1445 and 1446 of pC194 . That site probably corresponds to a nick in the replication origin of this plasmid . The localization of the other deletion end appears to be determined by the position of the f1 replication fork . Two models accounting for these data are discussed.

EMBO J, 1986 Dec 20, 5(13), 3687 - 90
Transposition of mini-Mu containing only one of the ends of bacteriophage Mu; Groenen MA et al.; Transposition of mini-Mu containing only one of the ends of bacteriophage Mu was studied . Transposition was observed when plasmids containing this mini-Mu were used as the donor in a mating-out assay with the F factor POX38, which lacks all known transposable elements, as the target . Analysis of the plasmids isolated from the recipient strain showed that in most cases the mini-Mu containing plasmid and POX38 were fused and that a part of the plasmid had been duplicated, indicating the formation of co-integrates . To obtain the DNA sequences of the junctions between donor and recipient DNA, an F factor was constructed that made it possible to analyse these junctions directly . The results showed that several sequences can be used as an alternative end in transposition and that these alternative ends show homology with the consensus for a strong A binding site . Moreover, the first base pair at the junction was always a (TA) base pair . This base pair is situated at exactly the same position with respect to the sequence which has homology with the consensus for a strong A binding site as in the ends of Mu.

J Mol Biol, 1986 Dec 20, 192(4), 767 - 80
Escherichia coli protein synthesis initiation factor IF3 controls its own gene expression at the translational level in vivo; Butler JS et al.; Measurements of the relative synthesis rates of mRNAs transcribed from the gene (thrS) for threonyl-tRNA synthetase and the adjacent gene (infC) for initiation factor IF3 show four- to fivefold more infC mRNA than thrS mRNA in vivo, suggesting that infC expression can be controlled independently of thrS expression . S1 mapping experiments reveal the existence of two transcription initiation sites for infC mRNAs internal to the thrS structural gene . Both the mRNA measurements and the S1 mapping experiments indicate that the majority of infC transcription initiates at the infC proximal promoter . In agreement with these results, the deletion of the infC distal promoter from infC-lacZ gene fusions does not affect the expression of these gene fusions in vivo . Measurements of the relative synthesis rate of infC mRNA in vivo in infC- strains overproducing IF3 shows that infC mRNA levels are normal in these strains, thus suggesting that IF3 regulates the translation of infC mRNAs in vivo . Extension of these experiments using infC-lacZ gene fusions carried on lambda bacteriophage and integrated at the lambda att site on the Escherichia coli chromosome shows that the expression of infC-lacZ protein fusions, but not infC-lacZ operon fusions, is derepressed in two infC- strains . A cellular excess of IF3 represses the expression of an infC-lacZ protein fusion but not an infC-lacZ operon fusion . Measurements of the relative mRNA synthesis rates of hybrid infC-lacZ mRNA synthesized from an infC-lacZ protein fusion under conditions of a fourfold derepression or a threefold repression of hybrid IF3-beta-galactosidase expression shows that the hybrid infC-lacZ mRNA levels remain unchanged . These results indicate that the cellular levels of IF3 negatively regulate the expression of its own gene, infC, at the translational level in vivo.

Eur J Biochem, 1986 Dec 15, 161(3), 727 - 31
Cooperative DNA binding by lambda integration protein--a key component of specificity; Minter SJ et al.; Quantitative analysis of nitrocellulose filter binding data by the method of Clore, Gronenborn and Davies {(1982) J . Mol . Biol . 155, 447-466} has been used to show that lambda integration protein (Int) exhibits cooperativity in binding to specific recognition sites within the attachment site region (lambda attP) of bacteriophage lambda DNA . Optimal values of the equilibrium constant obtained were 3.0(+/- 1.0) X 10(10) M-1 for the P' site using a model of three sites with equal affinity and 1.9(+/- 0.4) X 10(10) M-1 for the P1 site on a two-site model . The value of the cooperativity parameter alpha is 172(+106)(-66) in all cases . The occurrence of a consensus recognition sequence is necessary but not sufficient for strong binding; cooperative interaction between Int molecules binding to adjacent members of an array of binding sites is also essential . The occurrence of binding site arrays distinguishes lambda attP very clearly from other DNA sequences containing single recognition sites by chance.

J Biol Chem, 1986 Dec 15, 261(35), 16682 - 8
The bacteriophage Mu N gene encodes the 64-kDa virion protein which is injected with, and circularizes, infecting Mu DNA; Gloor G et al.; Upon infection of Escherichia coli with bacteriophage Mu, a 64-kDa protein is injected into the host cell along with the phage DNA . This protein is involved in circularizing the infecting Mu DNA (Harshey, R . M., and Bukhari, A . I . (1983) J . Mol . Biol . 167, 427-441; Puspurs, A . H., Trun, N . J., and Reeve, J . N . (1983) EMBO J . 2, 345-352) . Its possible role in the integration of infecting Mu DNA and in the infection process remains to be established . To identify the source of this protein we have prepared antiserum to the protein purified from viral particles . We have shown that the antiserum is specific for the Mu N gene product . The antiserum has been used to immunologically screen a Mu DNA library cloned into an expression vector . Four clones have been shown to produce a protein of 64 kDa that is specifically bound by the antiserum . The only Mu gene common to all four clones is the N gene, as demonstrated by physical and genetic mapping . We have also demonstrated by peptide mapping that the cloned N gene product is identical to the 64-kDa protein found complexed with the injected Mu DNA.

Nucleic Acids Res, 1986 Dec 9, 14(23), 9311 - 27
The region of phage T4 genes 34, 33 and 59: primary structures and organization on the genome; Hahn S et al.; The product of gene 33 is essential for the regulation of late transcription and gene product 59 is required in recombination, DNA repair and replication . The exact functions of both proteins are not known . Restriction fragments spanning the genomic area of genes 33 and 59 have been cloned into phage M13 and a 4.9 kb nucleotide sequence has been determined . Translation of the DNA sequence predicted that gp33 contains 112 amino acids with a mol.wt . of 12.816 kd while gp59 is composed of 217 amino acids adding up to a mol.wt . of 25.967 kd . The genomic area studied here also contains 3 open reading frames of genes not identified to date and it is thought to include the NH2-terminal part of g34 . One of the open reading frames seems to code for the 10 kd protein, probably involved in the regulation of transcription of bacteriophage T4 . This protein is predicted to consist of 89 amino acid residues with a mol.wt . of 10.376 kd . Gene 33 and the gene for the 10 kd protein were cloned separately on high expression vectors resulting in over-production of the two proteins.

J Mol Biol, 1986 Dec 5, 192(3), 677 - 80
Directional control of site-specific recombination by bacteriophage lambda . Evidence that a binding site for Int protein far from the crossover point is required for integrative but not excisive recombination; Winoto A et al.; Phage lambda controls its integration and excision by differential catalysis of the forward and reverse reactions . The lambda Int protein is required for both directions, but Xis for excision only . To investigate the substrate requirements for directional control, we have characterized two mutations of the phage attachment site that are defective in integrative but not excisive recombination . Both of these mutations produce the same base change in the P'3 binding site for Int protein 79 base-pairs from the center of the crossover region for site-specific recombination . We infer that differential utilization of this distant binding site is crucial for directional control of recombination.

Cell, 1986 Dec 5, 47(5), 793 - 806
DNA synthesis dependent on genetic recombination: characterization of a reaction catalyzed by purified bacteriophage T4 proteins; Formosa T et al.; To simulate a reaction that occurs in T4-infected cells, we have developed an in vitro DNA synthesis system that requires seven highly purified proteins encoded by this bacteriophage: the DNA polymerase "holoenzyme" (four proteins), gene 32 protein, dda DNA helicase, and uvsX protein - an enzyme that catalyzes homologous DNA pairing and is functionally homologous to the recA protein . In the reaction observed, the 3'OH end of one single-stranded DNA molecule primes DNA synthesis using a double-stranded DNA molecule of homologous sequence as the template . The uvsX protein continuously removes the new DNA chain from its template, so that DNA is synthesized by a conservative mechanism . This type of reaction, which requires the cooperation of recombination and replication enzymes, seems likely to be a general feature of DNA metabolism.

J Mol Biol, 1986 Dec 5, 192(3), 513 - 27
Mutational analysis of integrase arm-type binding sites of bacteriophage lambda . Integration and excision involve distinct interactions of integrase with arm-type sites; Bauer CE et al.; Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome (attP) and the Escherichia coli genome (attB) results in a prophage flanked by the hybrid recombinant sites attL and attR . Each att site contains sequences to which proteins involved in recombination bind . Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int "arm-type" binding sites located within attP, attL and attR . Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int . Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm-type sites within attP are required for efficient integrative recombination . Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm-type sites in attL and attR . These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm-type sites.

Mutat Res, 1986 Dec, 175(4), 217 - 22
SCP1-dependent Weigle activation of the VP5 phage in Streptomyces coelicolor A3(2); Misuraca F et al.; The kinetics of induction of the UV-irradiated bacteriophage VP5 (Weigle reactivation) in Streptomyces coelicolor A3(2) strains with and without plasmid was investigated . Chloramphenicol (CAF) inhibits Weigle reactivation (WR) in UF strains (SCP1 absent) but not in SCP1+ strains of IF fertility (free plasmid) . CAF, moreover, inhibits protein synthesis in non-irradiated UF and IF strains . In UV-irradiated IF strains, on the other hand, protein synthesis takes place irrespective of CAF . Weigle reactivation appears to require protein synthesis: the SCP1 plasmid, by protecting protein synthesis from CAF inhibition in UV-irradiated strains, allows WR . The proteins synthesized after UV induction during the pre-incubation period were investigated and the results suggest that a new UV-induced protein, coded by a gene localized on the plasmid, interacts with the cellular SOS system.

J Virol, 1986 Dec, 60(3), 1145 - 7
The bacteriophage T4 regulatory protein gpunf/alc binds to DNA in the absence of RNA polymerase; Snustad DP et al.; DNA-cellulose chromatography and two-dimensional gel electrophoresis have been used to demonstrate the DNA-binding capacity of bacteriophage T4 gpunf/alc . The unf/alc protein does not bind to DNA via an association with RNA polymerase; gpunf/alc was shown to bind to DNA after separation from RNA polymerase and other large proteins by Sephadex chromatography.

J Biochem (Tokyo), 1986 Dec, 100(6), 1403 - 23
New strategies for the determination of macromolecular structure in solution; Altman RB et al.; Non-crystallographic approaches to the determination of protein structure must solve the problem of insufficient and low information content experimental data . Most successful methods augment experimentation with theoretical constraints (for example, potential energy functions or optimization error metrics) . We believe it is important to separate the contributions of experimentation and theory in the construction of protein structure . The PROTEAN system defines protein topology on the basis of experimental data alone . Its performance on three data sets, derived from the lac-repressor headpiece of E . coli, sperm whale myoglobin, and domain 1 of bacteriophage T4 lysozyme, indicates that there may be families of related conformations that are consistent with the experimental data . These conformations provide insight into the strengths and weaknesses in the data sets . They also provide a set of structures with which to begin theoretical refinements . We outline here a strategy which maintains a clear distinction between refinements based on theory and those based on experiment, and thus allows a careful analysis of the properties of such refinement methods.

Mol Gen Genet, 1986 Dec, 205(3), 523 - 9
Identification and purification of the N gene product of bacteriophage phi 80; Kanemoto K et al.; To confirm the in vivo observation that the N gene product of phi 80, phi 80-pN, prevents termination of transcription at the tL1 region and is therefore a transcription antitermination factor (Tanaka and Matsushiro 1985), we demonstrated that phi 80-tL1 is a rho-dependent terminator, similar to lambda-tL1, and that phi 80-pN has a transcription antitermination function at this site in an in vitro transcription system using a nucleic acid-free S-100 extract . In the presence of rho-protein, transcription termination at tL1 was suppressed completely with an S-100 extract prepared from Escherichia coli strain NT525 containing the pBN1-N+ plasmid . Starting from this pN-overproducing cell extract, we purified phi 80-pN to homogeneity by chromatography on DEAE-Sephacel, Sephadex G-150 and CM-Sephadex C-50 . The molecular weight of purified pN was about 12,000 and the NH2-terminal sequence was NH2-Met-Ile-Asp-Asp-Ile-Lys, which was consistent with the sequence deduced from the DNA sequence.

DNA, 1986 Dec, 5(6), 511 - 7
Cloning and expression in Escherichia coli of a synthetic DNA for hirudin, the blood coagulation inhibitor in the leech; Fortkamp E et al.; A 235-bp DNA coding for the leech blood coagulation inhibitor, hirudin, was chemically synthesized . The synthesis involved preparation of seven long oligodeoxyribonucleotide pairs which were assembled and cloned using a rapid and simple procedure . More than half of the transformed Escherichia coli cells expressed a biosynthetic polypeptide having biological properties which were very similar to authentic hirudin from the leech Hirudo medicinalis . To achieve efficient expression, we fused the hirudin DNA to a truncated C1 repressor gene of bacteriophage lambda to create a hybrid protein . An additional methionine at the fusion point allowed the active hirudin to be cleaved off by cyanogen bromide.

Jpn J Exp Med, 1986 Dec, 56(6), 315 - 9
Genetic recombination between multiple markers of bacteriophage T4 . III . A mutation which blocks a structural change of DNA required for strand exchange; Honda M; T4 phage mutation MCO5 is a non-lethal recessive mutation which blocks recombination between multiple closely linked markers and links to genes 24 and 25 . The three-factor cross, which required double exchange flanking the central marker for the formation of wild type recombinant, was blocked by MCO5 mutation . The blockage was restored by the UV irradiation of the parental phage carrying single mutation but not by the UV irradiation of the parental phage carrying double mutation . The MCO5 mutant reduced the rescue of the markers of UV-irradiated phage, although it had normal levels of sensitivity to UV in both single and multiple infections . From these results, it is concluded that MCO5 mutation blocks a structural change of DNA which is necessary for receiving DNA segment.

Proc Natl Acad Sci U S A, 1986 Dec, 83(24), 9353 - 7
Activation of transcription by the bacteriophage 434 repressor; Bushman FD et al.; Bacteriophage 434 encodes a repressor that, like bacteriophage lambda repressor, both activates and represses transcription . As in the lambda chromosome, a region of the 434 chromosome, called the right operator, contains three repressor binding sites (OR1, OR2, and OR3) that mediate these effects on two adjacent promoters . We now show that a part of the 434 repressor, the amino-terminal domain, activates leftward transcription when bound to OR2 . We show that 434 repressor bound to OR2 closely approaches (touches) RNA polymerase bound to the leftward promoter . Model building based on ethylation interference and other experiments suggests that in three cases, those involving lambda repressor, 434 repressor, and bacteriophage P22 repressor, and in spite of differences in detailed arrangements, transcription is activated by a contact between the repressor and the same part of RNA polymerase.

Proc Natl Acad Sci U S A, 1986 Dec, 83(23), 8878 - 82
Preferential DNA repair of an active gene in human cells; Mellon I et al.; Removal of pyrimidine dimers was measured in defined sequences in human cells amplified for the dihydrofolate reductase (DHFR) gene . We quantitated repair in specific restriction fragments by using the dimer-specific bacteriophage T4 endonuclease V and analysis by Southern blotting . Within 4 hr after 5- or 10-J/m2 UV irradiation, more than 60% of the dimers had been removed from a 20-kilobase fragment that lies entirely within the transcription unit of the DHFR gene and from a 25-kilobase fragment located in the 5' flanking region of the gene . Repair in the overall genome was measured by analyzing cellular DNA treated with T4 endonuclease V in alkaline sucrose gradients . Sixty-nine percent of the dimers were removed from the genome overall within 24 hr after irradiation, but only 25% were removed within 4 hr and 38% were removed within 8 hr . These results demonstrate a strong preferential rate of removal of dimers from the 50-kilobase region that includes the transcriptionally active DHFR gene compared to that in total cellular DNA . We confirmed that DHFR-containing DNA is repaired more rapidly than bulk DNA by using an approach that provides a direct comparison between repair in specific sequences and repair in total cellular DNA . We also show that the DHFR-containing sequences are repaired more rapidly than the nontranscribed repetitive alpha DNA sequences . Our finding of preferential early repair in a transcriptionally active region in overall repair-proficient cells suggests that selective dimer removal from active sequences may be a general characteristic of mammalian DNA repair.

Proteins, 1986 Dec, 1(4), 302 - 11
Isolation and analysis of arc repressor mutants: evidence for an unusual mechanism of DNA binding; Vershon AK et al.; We have isolated 64 different missense mutations at 36 out of 53 residue positions in the Arc repressor of bacteriophage P22 . Many of the mutant proteins with substitutions in the C-terminal 40 residues of Arc have reduced intracellular levels and probably have altered structures or stabilities . Mutations in the N-terminal ten residues of Arc cause large decreases in operator DNA binding affinity without affecting the ability of Arc to fold into a stable three-dimensional structure . We argue that these N-terminal residues are important for operator recognition but that they are not part of a conventional helix-turn-helix DNA binding structure . These results suggest that Arc may use a new mechanism for sequence specific DNA binding.

Jpn J Exp Med, 1986 Dec, 56(6), 309 - 14
Genetic recombination between closely linked markers of bacteriophage T4 . II . A mutation which reduces the formation of heteroduplex heterozygotes; Honda M; A T4 phage mutant MCO4 was isolated as a mutant that reduced the frequencies of recombinants between multiple closely-linked markers but not those between two markers . MCO4 mutation was a non-lethal, recessive mutation and was located nearby gene 24 and gene 25 . MCO4 mutation reduced the frequency of heteroduplex heterozygotes but not the frequency of terminally redundant heterozygotes . Therefore, MCO4 mutation appears to block the formation and/or the extension of heteroduplex regions or to shorten their life-time.

Virology, 1986 Dec, 155(2), 392 - 401
Modulation of in vivo and in vitro transcription of bacteriophage phi 29 early genes; Whiteley HR et al.; The majority of early transcripts of the phi 29 bacteriophage are produced throughout the lytic cycle but the levels of a class of transcripts from the right end of the phi 29 genome are significantly reduced late in the infection . We have isolated a phage early protein which selectively interferes with the initiation in vitro of transcription from promoters at the right end of the phi 29 genome . The amino acid sequence of the purified inhibitory protein correlates to the sequence predicted from the phi 29 gene 6 reading frame . In addition the inhibitory protein was not detectable in cells infected with phage mutated in gene 6 and the decrease in transcription did not occur in vivo when nonpermissive cells were infected with phi 29(sus6) . The results indicate that the gene 6 protein modulates transcription from the right side of the phi 29 genome.

J Cell Biol, 1986 Dec, 103(6 Pt 1), 2091 - 102
In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins; Newmeyer DD et al.; An in vitro system was developed that provides a quick microscopic assay for nuclear transport . The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin . This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold) . Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA . This transport requires the signal domain of nucleoplasmin . Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin . An active transport model would predict that nuclear transport be temperature- and energy-dependent and that inhibition of transport by either low temperature or energy depletion would be reversible . Both predictions were confirmed in our system . Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C . The effects of low temperature are reversible . As found for 125I-labeled nucleoplasmin (Newmeyer, D . D., J . M . Lucocq, T . R . Burglin, and E . M . De Robertis, 1986, EMBO (Eur . Mol . Biol . Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion . This effect is reversed by later ATP addition . Under ATP-depleted conditions non-nuclear proteins continue to be excluded . These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity . In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles . Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope.

J Bacteriol, 1986 Dec, 168(3), 1258 - 64
Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli; Lim CJ et al.; The gene encoding thioredoxin in Anabaena sp . strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity . DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E . coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E . coli strain lacking functional thioredoxin . Transformants that complemented the trxA mutation in E . coli were identified by increased colony size and confirmed by enzyme assay . Expression of the cloned Anabaena thioredoxin gene in E . coli was substantiated by subsequent purification and characterization of the algal protein from E . coli . The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin . The E . coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.

Mol Gen Genet, 1986 Dec, 205(3), 572 - 4
DNA inversion mediated by the r-determinant of plasmid NR1: evidence for the intramolecular replicative transposition of a 23 kb IS1-flanked transposon?
Iida S, Hanni C, Meyer J, Arber W.
The r-determinant (r-det) of the R plasmid NR1-Basel is a 23 kb, IS1-flanked transposon, called Tn2671, which has been shown to transpose to the genome of bacteriophage P7 . Among the derivatives of phage P7::r-det we found one which carried two copies of the r-det as inverted repeats and which also contained the P7 genome segment between them in inverted orientation . Its generation is best explained by assuming that the entire 23 kb Tn2671 transposon has undergone intramolecular replicative transposition.

Genetics, 1986 Dec, 114(4), 1061 - 79
Sequence and transcripts of the bacteriophage T4 DNA repair gene uvsY; Gruidl ME et al.; We have cloned, sequenced and analyzed transcription of the phage T4 uvsY gene . This gene is transcribed from a single gp MotA-dependent middle promoter to give a major transcript of approximately 930 nucleotides and a minor transcript of approximately 620 nucleotides . All in vivo and in vitro uvsY transcripts show anomalous migration in agarose gels . The uvsY transcript contains an open reading frame coding for an 137 amino acid {15.8 kilodaltons (kD)} UvsY protein and two unidentified open reading frames, ORF UvsY.-1 (9.0 kD) and ORF UvsY.-2 (6.0 kD) . Our DNA sequence differs in only three places from that published by TAKAHASHI et al . However, one of these changes alters the predicted carboxy terminus of the UvsY protein . Marker rescue experiments map gene 25 to the region upstream of uvsY . Gene 25 is likely, although not certain, to correspond to an ORF that is found upstream from uvsY and is translated in the same direction.

Virology, 1986 Dec, 155(2), 402 - 17
Generation of cDNA clones of the bacteriophage phi 6 segmented dsRNA genome: characterization and expression of L segment clones; Revel HR et al.; Bacteriophage phi 6 has three dsRNA genome segments of about 3.0, 4.0, and 6.4 kbp . More than 90% of the segmented phi 6 dsRNA genome has been cloned as subchromosomal cDNA fragments, generated by reverse transcription of denatured polyadenylated dsRNA, RNA removal, annealing, filling, size fractionation, tailing, and insertion at the PstI site of pBR322 . All of the large (L) segment is represented by five overlapping fragments, 98% of the small (S) segment is present in three fragments, and 67% of the medium (M) segment is contained in two fragments . Fragments have been aligned in linear arrays by Southern blot hybridization and restriction enzyme analysis . The orientation of the ordered fragments with respect to genomic RNA and phi 6 transcriptional direction was determined by comparison of terminal DNA sequences with RNA sequences at the genomic ends of phi 6 RNA . Expression of L segment clones using both Escherichia coli minicells and T7 polymerase/promoter vectors indicate that the order of known phi 6 genes on the large chromosome is: 5'--gene 7, gene 2, gene 4, gene 1--3' . cDNA complementation of a ts mutant, ts411, has located this mutation in gene 4.

Proc Natl Acad Sci U S A, 1986 Dec, 83(23), 8962 - 6
Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta; Chung BC et al.; Conversion of cholesterol to pregnenolone is mediated by P450scc {cholesterol, reduced-adrenal-ferrodoxin: oxygen oxidoreductase (side-chain-cleaving), EC 1.14.15.67} . RNA from several human adrenal samples was translated in vitro and immunoprecipitated with anti-bovine P450scc, indicating that P450scc mRNA represents about 0.5% of human adrenal mRNA in normal, hypertrophied, and malignant adrenals . A 1626-base-pair human adrenal P450scc cDNA was cloned in bacteriophage lambda gt10 . Primer extension data indicated P450scc mRNA is about 1850 bases long and that all adrenal P450scc mRNA has the same 5' end . A full-length clone containing 1821 bases was obtained from a human testis cDNA library to yield the complete sequence . The encoded human preP450scc contains 521 amino acids with a molecular weight of 60189.65 . The testis and adrenal sequences were identical; the human cDNA and amino acid sequences are 82% and 72% homologous, respectively, with the bovine sequences . P450scc cDNA was used to probe DNA from a panel of mouse-human somatic cell hybrids, showing that the single human P450scc gene lies on chromosome 15 . The human P450scc gene is expressed in the placenta in early and midgestation; primary cultures of placental tissue indicate P450scc mRNA accumulates in response to cyclic AMP.

Proc Natl Acad Sci U S A, 1986 Dec, 83(23), 8873 - 7
Identification of a domain within the phosphoprotein of vesicular stomatitis virus that is essential for transcription in vitro; Gill DS et al.; A full-length cDNA copy of the phosphoprotein (NS) mRNA of vesicular stomatitis virus (New Jersey serotype) was inserted into pGEM4 vector downstream of the promoter for bacteriophage SP6 RNA polymerase . Transcription of the cDNA in vitro resulted in the synthesis of NS mRNA, which was subsequently translated into NS protein in a cell-free rabbit reticulocyte system . The biological activity of the expressed NS protein was demonstrated by in vitro synthesis of mRNA by transcription-reconstitution with purified viral L protein and N-RNA template . Deletion mapping of the NS gene defined a specific domain between amino acid residues 213 and 247, which was essential for in vitro transcription . Removal of the COOH-terminal 21 amino acids, on the other hand, did not have a significant effect on transcription . This domain appears to be involved in efficient binding of NS protein to the N protein-RNA template.

J Virol, 1986 Dec, 60(3), 1002 - 11
A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived from viral cDNA clones; Huang MT et al.; The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8 . Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses . Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA . The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively . Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72 . The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone . The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis . By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA . The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.

Genetics, 1986 Dec, 114(4), 1041 - 60
Very short patch mismatch repair in phage lambda: repair sites and length of repair tracts; Lieb M et al.; Five amber mutations in the repressor (cI) gene of bacteriophage lambda recombine anomalously with nearby cI mutations . When any of these markers is used in four-factor crosses, cI+ recombinants that are expected to require three cross-overs occur at high frequencies . These recombinants are attributable to very-short-patch (VSP) repair of specific mismatches in DNA heteroduplexes formed during recombination between the markers flanking cI . The sites of the repair-prone mutations and the lengths of repair tracts have now been determined . Amber mutations subject to VSP repair are C to T transitions in 5'CCATGG, the sequence methylated by the product of gene dcm, and also in the related 5'CAGG or 5'CCAG sequences . Ambers arising in CAG sequences found in other contexts, or in codons other than CAG, were not subject to VSP repair . Repair tracts rarely, if ever, exceed ten nucleotides in length, and can be as short as two nucleotides . A repair-prone mutation does not stimulate recombination between flanking cI markers.

Proc Natl Acad Sci U S A, 1986 Dec, 83(23), 8844 - 8
Cloning of T4 gene 32 and expression of the wild-type protein under lambda promoter PL regulation in Escherichia coli; Shamoo Y et al.; Bacteriophage T4 gene 32 encodes a single-stranded DNA binding protein required for T4 DNA replication, recombination, and repair . Previous attempts at cloning gene 32 have failed due to a presumably deleterious effect on host cell viability . In addition, overexpression of gene 32 would be expected to be limited by the autoregulatory ability of the gene 32 product g32P . A repetitive A + T-rich sequence flanking the ribosome binding site of gene 32 has been implicated in this translational regulation . To circumvent these problems, the wild-type gene for g32P has been reconstructed in M13 using restriction fragments from T4 g32am453 and synthetic oligodeoxynucleotides so that it no longer includes its native promoter and putative autoregulatory region . The g32am453 codon TAG was changed back to TGG as in wild-type gene 32 using site-directed oligodeoxynucleotide mutagenesis . In vectors containing the lambda leftward promoter PL, gene 32 is overexpressed with the resulting transcripts being depressed at g32P concentrations that repress the wild-type gene 32 transcripts.

Proc Natl Acad Sci U S A, 1986 Dec, 83(23), 8829 - 33
Bacteriophage lambda cro mutations: effects on activity and intracellular degradation; Pakula AA et al.; Following random mutagenesis of the bacteriophage lambda cro gene, we have isolated missense mutations that affect approximately half of the 66 residue positions of Cro . About two-thirds of the mutations change residues involved in the maintenance of Cro structure and stability . The corresponding mutant proteins are severely degraded in the cell but often have specific activities near that of wild-type Cro . The remaining mutations affect residues involved in DNA binding . These mutant proteins are present at moderately reduced intracellular levels, but their specific activities are much lower than that of wild type.

J Bacteriol, 1986 Dec, 168(3), 1159 - 64
Roles of RecA protease and recombinase activities of Escherichia coli in spontaneous and UV-induced mutagenesis and in Weigle repair; Tessman ES et al.; The RecA protein has a second, direct role in the mutagenesis of Escherichia coli and bacteriophage lambda in addition to its first, indirect role of inducing the SOS system by enhancing the proteolytic cleavage of the LexA repressor protein . The need for RecA protease and recombinase functions in the direct role was examined in cells containing split-phenotype RecA mutations, in the absence of LexA protein . Spontaneous mutation of E . coli (his----his+) required both the protease and recombinase activities . The mutation frequency increased with increasing RecA protease strength . In contrast, UV-induced mutation of E . coli required only the RecA protease activity . Weigle repair and mutation of UV-irradiated phage S13 required only RecA protease activity, and even weak activity was highly effective; RecA recombinase activity was not required . RecA+ protein inhibited RecA (Prtc {protease constitutive} Rec+) protein in effecting spontaneous mutation of E . coli . We discuss the nature of the direct role of the RecA protein in spontaneous mutation and in repair and mutagenesis of UV-damaged DNA and also the implications of our results for the theory that SOS-mutable cryptic lesions might be responsible for the enhanced spontaneous mutation in Prtc Rec+ strains.

AIDS Res, 1986 Dec, 2 Suppl 1, S177 - 81
Prevention and treatment of Epstein-Barr virus (EBV)-associated lymphoproliferative diseases in immune deficient patients; Purtilo DT; Prevention of EBV-associated lymphoproliferative diseases in immune deficient individuals is preferred; however, standard therapy for the B cell lymphomas has been successful . Chemotherapy must be given cautiously lest further immune compromise result in opportunistic infections . Recently, Acyclovir has decreased morbidity of patients with acute infectious mononucleosis in immune competent persons . In contrast, immunodeficient patients with X-linked lymphoproliferative (XLP) syndrome do not seem to respond favorably . Hence, a prospective study is underway using prophylactic immunoglobulin containing (EBV)-specific antibodies . The mortality rate is 85% following EBV infection in XLP due to fatal infectious mononucleosis associated with fulminant hepatitis and virus-associated hemophagocytic syndrome, acquired hypogammaglobulinemia or malignant B cell lymphoma . We can detect XLP by noting failure of switching from IgM to IgG antibody production on secondary challenge with bacteriophage phi X174 . Also, linkage studies with the XLP locus using restriction fragment length polymorphisms are being done to detect affected males pre-EBV infection . Our rationale for prevention of phenotypes of XLP is based on observations that infants in tropical Africa and males with XLP do not develop EBV-induced diseases while neutralizing maternal antibodies are present . An EBV vaccine will be used, when available, in seronegative males with XLP . Prevention of acquired immune deficiency by screening blood for human immune deficiency virus, encouraging prudent life styles, development of specific immunosuppressive agents, development of new antiviral agents (i.e., DHPG), and identification of high risk seronegative patients offer possibilities for preventing life-threatening EBV-induced diseases.

J Biol Chem, 1986 Nov 25, 261(33), 15668 - 72
Purification and properties of the DNA invertase gin encoded by bacteriophage Mu; Mertens G et al.; The host range of bacteriophage Mu is regulated through an invertible segment . Inversion requires the presence of two properly oriented recombination sites and a recombinational enhancer sis . The reaction is catalyzed by the Mu-encoded DNA invertase Gin and a host factor termed factors for inversion stimulation (FISs) . We present a novel purification scheme for Gin . Purified Gin alone catalyzes the inversion reaction at very low efficiency recombining less than 0.8% of substrate molecules . When supplemented with FIS substrates containing the recombinational enhancer are recombined efficiently . Stoichiometric amounts of Gin are required for recombination.

J Biol Chem, 1986 Nov 25, 261(33), 15673 - 8
Purification and properties of the Escherichia coli host factor required for inversion of the G segment in bacteriophage Mu; Koch C et al.; G inversion in bacteriophage Mu requires the product of the DNA invertase gene gin and an Escherichia coli host factor termed FIS (factor for inversion stimulation) . A recombination substrate must contain two recombination sites, arranged as inverted repeats, and a recombinational enhancer sequence termed sis . FIS has been purified to homogeneity . The purified protein has a relative molecular weight of 12,000 when analyzed under denaturing conditions . The intact protein behaves as a dimer of relative molecular weight 25,000 in gel filtration analysis . The purified protein does not possess any recombinogenic activity when assayed in the absence of the DNA-invertase Gin . In the presence of purified Gin FIS is the only additional protein required for efficient inversion . By performing gel retention assays, we show that FIS is a DNA-binding protein, which specifically binds to DNA fragments containing the recombinational enhancer sis.

Nucleic Acids Res, 1986 Nov 25, 14(22), 9063 - 80
Site directed mutagenesis experiments suggest that Glu 111, Glu 144 and Arg 145 are essential for endonucleolytic activity of EcoRI; Wolfes H et al.; We have constructed a plasmid (pRIF 309+) carrying the EcoRI restriction endonuclease gene and the f1 origin of replication . Upon transformation of this plasmid into E . coli and infection with bacteriophage f1 single stranded plasmids are produced which can be used for sequencing and site directed mutagenesis . Using this single stranded DNA and synthetic oligodeoxynucleotides we have introduced point mutations at defined positions of the EcoRI gene . Since in pRIF309+ the EcoRI gene is under the control of the pL-promoter, high level expression of the mutated EcoRI gene could be obtained upon induction . Mutant EcoRI enzymes were purified to homogeneity and characterized in structural and functional terms . Our results demonstrate that the Glu 111----Gln, Glu 144----Gln and Arg 145----Lys -mutants adopt a very similar conformation as the wild type enzyme, but have by two orders of magnitude smaller specific activities than the wild type enzyme, mainly due to a reduction of the Vmax-value.

Nature, 1986 Nov 20-26, 324(6094), 260 - 2
Interaction of peptide antigens and class II major histocompatibility complex antigens; Guillet JG et al.; T lymphocytes require a foreign antigen to be presented on a cell surface in association with a self-transplantation antigen before they can recognize it effectively . This phenomenon is known as major histocompatibility complex (MHC) restriction . It is not clear how an incalculably large number of foreign proteins form unique complexes with a very limited number of MHC molecules . We studied the recognition properties of T cells specific for a peptide derived from bacteriophage lambda cI protein . Analogues of this peptide, as well as peptides derived from other unrelated antigens which can be presented in the context of the same MHC molecule, can competitively inhibit activation of these T cells by the cI peptide . Furthermore, these unrelated antigens can stimulate cI-specific T cells if certain specific amino-acid residues are replaced . Here we suggest a model in which all antigens give rise to peptides that can bind to the same site on the MHC molecule . T-cell recognition of this site (which is presumed to be polymorphic) with or without antigen bound can explain self-selection in the thymus and MHC restriction.

J Biol Chem, 1986 Nov 15, 261(32), 15217 - 24
Dissection of RNA-primed DNA synthesis catalyzed by gene 4 protein and DNA polymerase of bacteriophage T7 . Coupling of RNA primer and DNA synthesis; Nakai H et al.; Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates . T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H . and Richardson, C . C . (1986) J . Biol . Chem . 261, 15208-15216) . Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates . The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates . RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis . Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template . The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates . Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis . DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.

J Biol Chem, 1986 Nov 15, 261(32), 15208 - 16
Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7 . Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis; Nakai H et al.; Three proteins catalyze RNA-primed DNA synthesis on the lagging strand side of the replication fork of bacteriophage T7 . Oligoribonucleotides are synthesized by T7 gene 4 protein, which also provides helicase activity . DNA synthesis is catalyzed by gene 5 protein of the phage, and processivity of DNA synthesis is conferred by Escherichia coli thioredoxin, a protein that is tightly associated with gene 5 protein . T7 DNA polymerase and gene 4 protein associate to form a complex that can be isolated by filtration through a molecular sieve . The complex is stable in 50 mM NaCl but is dissociated by 100 mM NaCl, a salt concentration that does not inhibit RNA-primed DNA synthesis . T7 DNA polymerase forms a stable complex with single-stranded M13 DNA at 50 mM NaCl as measured by gel filtration, and this complex requires 200 mM NaCl for dissociation, a salt concentration that inhibits RNA-primed DNA synthesis . Gene 4 protein alone does not bind to single-stranded DNA . In the presence of MgCl2 and dTTP or beta, gamma-methylene dTTP, a gene 4 protein-M13 DNA complex that is stable at 200 mM NaCl is formed . The affinity of DNA polymerase for both gene 4 protein and single-stranded DNA leads to the formation of a gene 4 protein-DNA polymerase-M13 DNA complex even in the absence of nucleoside triphosphates . However, the binding of each protein to DNA plays an important role in mediating the interaction of the proteins with each other . High concentrations of single-stranded DNA inhibit RNA-primed DNA synthesis by diluting the amount of proteins bound to each template and reducing the frequency of protein-protein interactions . Preincubation of gene 4 protein, DNA polymerase, and M13 DNA in the presence of dTTP forms protein-DNA complexes that most efficiently catalyze RNA-primed DNA synthesis in the presence of excess single-stranded competitor DNA.

J Biol Chem, 1986 Nov 15, 261(32), 15006 - 12
Interaction of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage T7 . The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity; Huber HE et al.; DNA polymerase activity in Escherichia coli cells infected with bacteriophage T7 resides in a protein complex consisting of the T7 gene 5 protein and E . coli thioredoxin in a 1 to 1 stoichiometry . We have analyzed nine mutant thioredoxins, both in vivo and in vitro, for their ability to interact with the T7 gene 5 protein and stimulate the DNA polymerase and exonuclease activities inherent in gene 5 protein . The efficiency of plating of T7 on E . coli thioredoxin mutants depends strongly on the copy number of the respective mutant thioredoxin allele . Plating efficiencies at a constant copy number correlate well with the affinity of the purified mutant proteins for T7 gene 5 protein . The observed dissociation constant, Kobs, is increased between 5 and several hundredfold at 42 degrees C compared to wild-type thioredoxin . The maximum polymerase activity of the reconstituted gene 5 protein-thioredoxin complex at saturating concentrations of mutant thioredoxins, however, is reduced by less than 20% . Consequently, none of the mutant thioredoxins acts as a competitive inhibitor of wild-type thioredoxin . The active-site disulfide of thioredoxin is not essential for the activities of the gene 5 protein-thioredoxin complex . Both cysteines can be replaced without significantly affecting the maximum polymerase or exonuclease activities . Substitution or alkylation of either cysteine, however, reduces the affinity for gene 5 protein drastically, indicating that the active site is part of the thioredoxin surface involved in the protein-protein interaction.

J Biol Chem, 1986 Nov 15, 261(32), 15030 - 8
Nucleotide sequence of the structural genes for an anion pump . The plasmid-encoded arsenical resistance operon; Chen CM et al.; The structural genes for the arsenical pump of the conjugative R-factor R773 contained on a HindIII fragment of 4.3 kilobase pairs were cloned into bacteriophage M13 . A series of ordered deletions was created using Bal31 digestion, and the nucleotide sequence of the operon determined . Three open reading frames for genes arsA, arsB, and arsC were found . The arsA gene encodes a hydrophilic protein of 63,169 Da with two potential adenylate-binding sites . The arsB gene encodes a potentially membrane protein of 45,577 Da . The arsC gene encodes a 15,811-Da hydrophilic protein . The arsA and arsC gene products correspond to cytosolic proteins previously identified from minicell experiments . Isolated ArsA protein was shown to bind to dye-agarose columns which act as affinity resins for nucleotide-binding proteins . A model is proposed in which these gene products form an anion translocating ATPase for extrusion of arsenite and arsenate from resistant cells.

Biochim Biophys Acta, 1986 Nov 13, 868(2-3), 145 - 52
Nucleoside 5'-triphosphates with modified sugars as substrates for DNA polymerases; Chidgeavadze ZG et al.; A number of nucleoside 5'-triphosphate analogs were tested with Escherichia coli DNA polymerase I and Klenow fragment of the enzyme, bacteriophage T4 DNA polymerase and calf thymus DNA polymerase alpha . It was shown that 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates as well as a number of 3'-derivatives of dTTP(3'NH2) are able to terminate DNA synthesis catalyzed by each enzyme if the reaction is performed in the absence of natural substrates . ddNTP and dNTP(3'F) were found to be inactive with DNA polymerase alpha only, but araNTP(3'NH2) was inactive with E . coli DNA polymerase I . dTTP(3'N3), dGTP(3'N'3), dCTP(3'N3), araNTP(3'N3) and (alpha-thio)dTTP(3'F) were unable to inhibit any of the above-mentioned DNA polymerases, in contrast to reverse transcriptase, accessible to the most nucleotide analogs tested.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8637 - 54
Nucleotide sequence and analysis of the 58.3 to 65.5-kb early region of bacteriophage T4; Valerie K et al.; The complete 7.2-kb nucleotide sequence from the 58.3 to 65.5-kb early region of bacteriophage T4 has been determined by Maxam and Gilbert sequencing . Computer analysis revealed at least 20 open reading frames (ORFs) within this sequence . All major ORFs are transcribed from the left strand, suggesting that they are expressed early during infection . Among the ORFs, we have identified the ipIII, ipII, denV and tk genes . The ORFs are very tightly spaced, even overlapping in some instances, and when ORF interspacing occurs, promoter-like sequences can be implicated . Several of the sequences preceding the ORFs, in particular those at ipIII, ipII, denV, and orf61.9, can potentially form stable stem-loop structures.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8535 - 56
The purification of the Escherichia coli UvrABC incision system; Yeung AT et al.; The UvrA, UvrB and UvrC proteins of Escherichia coli have been purified in good yields to homogeneity with rapid three- or four-step purification procedures . The cloned uvrA and uvrB genes were placed under control of the E . coli bacteriophage lambda PL promoter for amplification of expression . Expression of the uvrC gene could not be amplified by this strategy, however, subcloning of this gene into the replication-defective plasmid pRLM24 led to significant overproduction of the UvrC protein . The purified UvrA protein, with its associated ATPase activity, has a molecular weight of 114,000, the purified UvrB is an 84,000 molecular weight protein and the UvrC protein has a molecular weight of 67,000.

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