J Virol, 1990 Dec, 64(12), 5988 - 96 Use of a cell-free system to identify the vaccinia virus L1R gene product as the major late myristylated virion protein M25; Franke CA et al.; A 25-kDa vaccinia virus (VV) virion protein, designated M25, is modified in vivo by covalent addition of myristic acid . The predicted amino acid sequences of all VV open reading frames which have been reported were searched for the sequence M-G-X-X-X-(S/T/A), which has been proposed to be the consensus recognition signal for cotranslational modification of proteins by N-myristyltransferase . This conserved signal was found at the amino terminus of a single locus, which corresponded to the leftmost rightward-reading open reading frame (L1R) initiating within the VV HindIII L DNA fragment . By using synthetic oligonucleotides in concert with polymerase chain reaction techniques, a chimeric gene consisting of open reading fram L1R fused to a bacteriophage T7 promoter was constructed and cloned into a plasmid vector . Transcripts derived from the wild-type expression plasmid (designated pL1G1) were translated in vitro in a wheat germ extract to yield a polypeptide with an apparent molecular mass of 25 kDa . This polypeptide was labeled with either {35S}methionine or {3H}myristic acid and comigrated with in vivo-labeled protein M25 on sodium dodecyl sulfate-polyacrylamide gels . Polyclonal antiserum generated in rabbits against a trpE:L1R fusion protein immunoprecipitated a 25-kDa protein labeled either in vitro (the L1R gene product, designated protein L1) or in vivo (from purified VV, protein M25), identifying the M25 protein as the gene product of open reading frame L1R . Chromatographic analysis of the protein L1-bound fatty acid moieties liberated after acid methanolysis resulted in recovery of greater than 99% of the fatty acid as myristate-associated label . Cell-free translation of proteins derived from a set of deletions from the carboxy terminus of the open reading frame L1R suggested that the site of myristylation maps near the amino terminus of protein L1 . This hypothesis was supported by cell-free translation of mutant L1R transcripts in which the penultimate glycine codon had been altered by site-directed mutagenesis to encode either an aspartic acid (pL1D1) or alanine (pL1A1) residue . In both cases, the mutant transcripts were translated into a 25-kDa protein which could be labeled in vitro with {35S}methionine but not with {3H}myristic acid . These data demonstrate that VV open reading frame L1R encodes a myristylated protein and provide evidence that the site of modification of protein L1 is the amino-terminal glycine residue. Virology, 1990 Dec, 179(2), 728 - 37 Expression of plasmid-encoded structural proteins permits engineering of bacteriophage T4 assembly; Duda RL et al.; A complementation system for studying bacteriophage T4 tail assembly has been developed and used to test the effects of nonviable mutations on the function of a specific T4 tail protein, gp48 . The complementation system assays the assembly function of gp48 without requiring that viable phage be produced, circumventing the operational problems of maintaining nonviable mutants of this lytic bacteriophage . The protein to be tested was preexpressed from cloned genes in a host cell prior to infection with the challenge phage . Assembly activity was assayed by monitoring the conversion of one tail assembly intermediate, the baseplate lacking gp48, into baseplates containing gp48 or into tube baseplates (or sheathed tails) assembled from such baseplates . Specific incorporation of gp48 into these structures was confirmed using gp48-specific antiserum, and the same serum was used in direct immunoelectron microscopy experiments to localize gp48 to the baseplate-proximal end of the T4 tail tube, at the site where the tube and sheath bind to the baseplate . The protein gp48 has been previously shown to be a baseplate protein, as well as a tail-tube-associated protein, and was tested for a possible role as a tail-length tape-measure protein . Tests with a deleted variant of gp48 were inconclusive because the protein was inactive . A variant of gp48, 20% longer than wild-type protein due to an internal duplication, was found to be partly functional in our assembly complementation system . This abnormally elongated protein allows several assembly steps to proceed, including the assembly of normal length T4 tails, implying that it does not specify tail length . The insertion-duplication variant of gp48 appears to have a defect in its interaction with the tail sheath protein, leading to abnormal sheath contraction. J Virol, 1990 Dec, 64(12), 5883 - 90 Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition; Marcy AI et al.; Eucaryotic, viral, and bacteriophage DNA polymerases of the alpha-like family share blocks of sequence similarity, the most conserved of which has been designated region I . Region I includes a YGDTDS motif that is almost invariant within the alpha-like family and that is similar to a motif conserved among RNA-directed polymerases and also includes adjacent amino acids that are more moderately conserved . To study the function of these conserved amino acids in vivo, site-specific mutagenesis was used to generate herpes simplex virus region I mutants . A recombinant virus constructed to contain a mutation within the nearly invariant YGDTDS motif was severely impaired for growth on Vero cells which do not contain a viral polymerase gene . However, three recombinants constructed to contain mutations altering more moderately conserved residues grew on Vero cells and exhibited altered sensitivities to nucleoside and PPi analogs and to aphidicolin . Marker rescue and DNA sequencing of one such recombinant demonstrated that the region I alteration confers the altered drug sensitivity phenotype . These results indicate that this region has an essential role in polymerase function in vivo and is involved directly or indirectly in drug and substrate recognition. Virology, 1990 Dec, 179(2), 694 - 700 The bacteriophage Mu gem gene: a positive regulator of the C operon required for normal levels of late transcription; Giusti M et al.; The gem product of bacteriophage Mu modulates synthesis of various host proteins and alters the host chromosome topology . To elucidate the role of the gem gene in Mu development, we analyzed the behavior of several mutants in this gene . The results, obtained with two Mu gem- phages, show that (1) phage growth is significantly delayed and inhibited, (2) early transcription is normal but late transcription is delayed and reduced, (3) DNA replication appears normal, and (4) the Mu C gene, whose product positively regulates Mu late genes, is one of the gem target sites . Transcription of a C promoter-lacZ fusion, carried by the pPH91 plasmid, is stimulated both after infection with Mu gem+ or Mu gem3 and is strains lysogenic for the same phages in the presence of viral immunity . These data suggest that the primary role of the gem product is modulation of gene expression . This control could be carried out by direct interaction with transcription factors or by changing DNA supercoiling. Mol Gen Mikrobiol Virusol, 1990 Dec, (12), 12 - 5 {Genetic control of resistance of Escherichia coli K12 to phage C1}; Likhacheva NA et al.; The bacteriophage C1 isolated in production cycle lysis belongs to the most common group of bacteriophages wide spread in nature and having the long noncontractile motile tail . Bacterial cells sensitivity to bacteriophage C1 is determined by functioning of the three different loci mapped in different regions of Escherichia coli map at 89, 75 and 61 min . The possibility of existence of a complex receptor for bacteriophage C1 is discussed. Genes Dev, 1990 Dec, 4(12A), 2223 - 33 RNase III-dependent hydrolysis of lambda cII-O gene mRNA mediated by lambda OOP antisense RNA; Krinke L et al.; The 77-nucleotide OOP antisense RNA of bacteriophage lambda complements lambda cII-O mRNA in a region that includes 55 nucleotides at the 3' end of the cII gene and 22 nucleotides in the intercistronic region between the cII and O genes . OOP RNA, produced from multicopy plasmids, inhibits lambda cII gene expression by approximately 100-fold through an RNase III-dependent mechanism . Using primer extension analysis of cellular RNA isolated from an induced lambda lysogen that contains an OOP DNA plasmid, we have identified a cleavage site in cII-O mRNA within the region of complementarity with OOP RNA, at 13 nucleotides from the 3' end of that region . Ribonuclease protection experiments demonstrate that almost all cII-O mRNA in this overlap region is cleaved when OOP RNA is overproduced in RNase III+ cells but not in RNase III- cells . RNA fragments are detected that extend into the O gene from the cleavage sites, while the sister fragments that extend into the cII gene cannot be detected and must be eliminated by additional hydrolytic events . Differences in levels of uncleaved mRNA between RNase III+ and RNase III- cells are much less at several hundred nucleotides to either side of the target region . An alternate OOP RNA-dependent hydrolytic process occurs in RNase III- cells that results in cleavages in one of two regions, one close to the cleavage site observed in RNase III+ cells, and the second several nucleotides beyond the end of the complementary region between OOP RNA and cII-O mRNA . In this latter case, the fragments that extend into the cII gene are stable, while the sister O gene fragments are destroyed, in direct contrast to the RNase III-dependent process. J Bacteriol, 1990 Dec, 172(12), 6641 - 50 Identification of a positive regulator of the Mu middle operon; Mathee K et al.; Transcription of bacteriophage Mu occurs in a regulatory cascade consisting of three phases: early, middle, and late . The 1.2-kb middle transcript is initiated at Pm and encodes the C protein, the activator of late transcription . A plasmid containing a Pm-lacZ operon fusion was constructed . beta-Galactosidase expression from the plasmid increased 23-fold after Mu prophage induction . Infection of plasmid-containing cells with lambda phages carrying different segment of the Mu early region localized the Pm-lacZ transactivation function to the region containing open reading frames E16 and E17 . Deletion and linker insertion analyses of plasmids containing this region identified E17 as the transactivator; therefore we call this gene mor, for middle operon regulator . Expression of mor under the control of a T7 promoter and T7 RNA polymerase resulted in the production of a single polypeptide of 17 kDa as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Insertion of a linker into mor substantially reduced the ability of Mu to form plaques . When growth of the mor mutant was assayed in liquid, lysis was delayed by about 50 min and the burst size was approximately one-fifth that of wild-type Mu . The mor requirement for plaque formation and normal growth kinetics was abolished when C protein was provided in trans, indicating that the primary function of Mor is to provide sufficient C for late gene expression . Comparison of the predicted amino acid sequence of Mor with other proteins revealed that Mor and C share substantial amino acid sequence homology. Virology, 1990 Dec, 179(2), 936 - 40 Production of lambda-phi 29 phage chimeras; Donate LE et al.; Proheads of bacteriophage lambda which carry the connector of phage phi 29 instead of that of lambda have been produced in vitro . These hybrid proheads have a structure similar to that of normal lambda proheads . Furthermore, the chimeric proheads can package both lambda and phi 29 DNA . These data show that the connector domains involved in both head assembly and DNA packaging are functionally similar . The DNA-containing lambda-phi 29 proheads can be complemented in vitro with phi 29 tails to yield infective particles capable of DNA transfer. J Radiat Res (Tokyo), 1990 Dec, 31(4), 340 - 53 Neutron and gamma-irradiation of bacteriophage M13 DNA: use of standard neutron irradiation facility (SNIF); Singh SP et al.; We describe here the use of the Van de Graaff accelerator as a source of high energy neutrons for biological irradiation . Single-stranded bacteriophage M13 DNA was chosen as the system to determine the relative biological effectiveness of monoenergetic neutrons . A Standard Neutron Irradiation Facility (SNIF) was established using a 3 MV Van de Graaff accelerator . The 2D (d,n)3He nuclear reaction was used to produce neutron fluxes of 3 x 10(8) cm 2 sec-1 yielding dose rates as high as 50 Gy h-1 . A detailed description of the neutron source, neutron fluence measurement, dose calculation and calibration are included . Exposure of single-stranded bacteriophage M13 DNA to 90 Gy of neutrons reduced survival to 0.18% of the unirradiated value . 500 Gy of gamma-rays were required for the same level of killing, and RBE was estimated at 6 based on Do values . Determination of the extent of DNA damage after exposure to cleavage using gel electrophoresis, gave RBE values of 6-8 which was very similar to that observed for bacteriophage survival . The facility described here provides a reproducible source of high energy monoenergetic neutrons and dose levels suitable for experiments designed to measure DNA damage and effects on DNA synthesis. Microbiologia, 1990 Dec, 6(2), 94 - 9 Structure of the DNA of five bacteriophages infecting Micromonospora; Caso JL et al.; The physical maps of the DNA of five bacteriophages (Mm1, OM2, OM3, Mm4 and Mm5) which infect Micromonospora are presented . The restriction analyses showed that all of them had linear, double-stranded DNA, but only four (Mm1, OM2, Mm4 and Mm5) presented cohesive ends . The phages showed no relationship in terms of their restriction maps or of DNA-DNA hybridization, with the exception of Mm4 and Mm5, which resulted to be very similar . Phage Mm5 presented a high level of resistance to chelating agents, although deletion mutants, all of them showing a single detection of 1.4 kb, were obtained by using extremely selective conditions. Curr Eye Res, 1990 Dec, 9(12), 1185 - 93 Ultraviolet light induced DNA damage and repair in bovine lens epithelial cells; Kleiman NJ et al.; DNA damage caused by UV-B and UV-A irradiation and the rate of repair of such damage was quantitated in bovine lens epithelial cell cultures using a modified alkaline elution methodology . Two enzymes, bacteriophage T4 endonuclease V, which cleaves at the site of pyrimidine dimers, and E . coli endonuclease III, which cleaves at the site of thymine glycols, were utilized . Pyrimidine dimers were not detected after UV-A irradiation of lens cultures with up to 400 J/m2 . In contrast, after exposure to as little as 2 J/m2 of UV-B irradiation, large numbers of pyrimidine dimers were observed . At higher fluences, thymine glycols were also found . Significant levels of DNA-DNA crosslinking were suggested by reduced rates of elution of DNA from cells treated with both UV-B irradiation and H2O2 in comparison to treatment with H2O2 alone . Protein-DNA crosslinks, in contrast, were not observed . The rate of repair of UV-B induced DNA damage was quantitated by harvesting cells at various times after the UV-B exposure . Single-strand breaks were never observed immediately after UV-B exposure but appeared later during the repair phase . In contrast to the repair of H2O2 induced DNA damage, which is largely completed within 30 min of exposure, more than 50% of the UV-B light induced DNA damage remained unrepaired five hours after exposure . This difference between the rate of repair of H2O2 and UV-B induced DNA damage could provide valuable insights into the nature of DNA damaging agents in the lens environment and may reflect underlying differences in the potential for epithelial cell DNA mutation in response to various DNA damaging insults. Virus Res, 1990 Dec, 18(1), 21 - 8 Expression of bluetongue virus serotype 17 NS1 protein from a cloned gene; Grubman MJ; A full-length copy of the coding region of segment 6 from bluetongue virus (BTV) serotype 17 was constructed from five overlapping cDNA clones . The gene coding for the NS1 protein was cloned into an expression plasmid under the control of a bacteriophage T7 promoter and expressed both in vitro and in Escherichia coli BL21(DE3) cells which contain a T7 RNA polymerase gene in their chromosome . Expression in both systems resulted in the synthesis of a protein comigrating with NS1 and a minor polypeptide comigrating with another viral-induced protein, NS1a, sometimes seen in BTV-infected cells . The proteins induced in E . coli were synthesized to high levels as insoluble products. J Gen Microbiol, 1990 Dec, 136 ( Pt 12), 2395 - 404 Characterization of FP22, a large streptomycete bacteriophage with DNA insensitive to cleavage by many restriction enzymes; Hahn DR et al.; Bacteriophage FP22 has a very broad host range within streptomycetes and appeared to form lysogens of Streptomyces ambofaciens ATCC 15154 . FP22 shared strong cross-immunity and antibody cross-reactivity with bacteriophage P23, but not with seven other streptomycete bacteriophages . FP22 particles had a head diameter of 71 nm and a tail length of 307 nm . The FP22 genome was 131 kb, which is the largest bacteriophage genome reported for streptomycetes . The G + C content of the genome was 46 mol% and restriction mapping indicated that FP22 DNA had discrete ends . NaCl- and pyrophosphate-resistant deletion mutants were readily isolated and the extent of the deletions defined at least 23 kb of dispensable DNA in two regions of the genome . The DNA was not cleaved by most restriction endonucleases (or isoschizomers) which have been identified in the streptomycetes, including the tetranucleotide cutter MboI (GATC). Carcinogenesis, 1990 Dec, 11(12), 2103 - 9 Role of DNA polymerase 3'----5' exonuclease activity in the bypass of aminofluorene lesions in DNA; Strauss BS et al.; N-(Deoxyguanosin-8-yl)-2-(acetylamino)fluorene (AAF-G) adducts in the DNA of bacteriophage M13 can be converted to N-(deoxyguanosin-8-yl)-2-aminofluorene (AF-G) adducts in situ by treatment with 1.0 M NaOH for 45 min at room temperature . The conversion is accompanied by a dramatic increase in the transfection activity of the samples which is correlated with the measured deacetylation of the acetylaminofluorene adduct . The pair of substrates (AAF-G/AF-G) with adducts at identical places in the DNA has been used to study bypass synthesis catalyzed by T7 DNA polymerase, an altered T7 DNA polymerase from which the 3'----5' exonuclease has been genetically removed by an 84 nucleotide deletion (Sequenase 2), T4 DNA polymerase and Escherichia coli DNA polymerase I . All polymerases appear blocked at acetylaminofluorene lesions . Sequenase 2 is apparently able to add nucleotides opposite the acetylaminofluorene lesion but is unable to catalyze further elongation . T7 DNA polymerase, including thioredoxin and with an active 3'----5' exonuclease, is unable to bypass aminofluorene adducts, whereas Sequenase 2 bypasses the lesions readily . The data support the view that the elongation step is rate limiting in synthesis past lesions and that low 3'----5' exonuclease activity allows the priming nucleotide opposite the altered template site to remain in position long enough for elongation past particular adducts. Gene, 1990 Nov 30, 96(1), 9 - 15 Characterization of the cos sites of bacteriophages P2 and P4; Ziermann R et al.; A 641-bp cos-containing P2 DNA fragment was sequenced and compared to the P4 cos region . Alignment of the P2 and P4 cos regions shows a homologous region of 55 bp that has only three mismatches and contains a completely conserved region of dyad symmetry . A number of P4- and P2-derived cosmids were tested in an in vivo transduction assay in order to determine the minimal cos region required for packaging . These experiments show that the common region of 55 bp is sufficient for transduction with low frequency, but that a 125-bp cos-containing fragment contains all the information for transduction with optimal frequency. Gene, 1990 Nov 30, 96(1), 59 - 65 Analysis of the tsx gene, which encodes a nucleoside-specific channel-forming protein (Tsx) in the outer membrane of Escherichia coli; Bremer E et al.; The tsx gene of Escherichia coli encodes an outer membrane protein, Tsx, which constitutes the receptor for colicin K and bacteriophage T6, and functions as a substrate-specific channel for nucleosides and deoxynucleosides . The mini-Mu element pEG5005 was used to prepare a gene bank in vivo, and this bank was used to identify T6-sensitive strains carrying the cloned tsx gene . Subcloning of the tsx gene into the multicopy plasmid, pBR322, resulted in a strong overproduction of Tsx . The sequence of a 1477-bp DNA segment containing tsx and its flanking regions was determined . An open reading frame (ORF) was found which was followed by a pair of repetitive extragenic palindromic sequences . This ORF translated into a protein of 294 amino acids (aa), the first 22 aa of which showed the characteristic features of a bacterial signal sequence peptide . The putative mature form of Tsx is composed of 272 aa with a calculated Mr of 31418 . The aa sequence of Tsx shows an even distribution of charged residues (52 aa) and lacks extensive hydrophobic stretches . No significant homologies of Tsx to the channel-forming proteins OmpC, OmpF, PhoE and LamB from the E . coli outer membrane were detected . Using nuclease S1, we identified two transcription start points for the tsx mRNA which were separated by approx . 150 bp . Genetic data suggest that the synthesis of the larger mRNA species is directed by a weak promoter (P1) that is controlled by the DeoR repressor, whereas the smaller mRNA species is directed by the main promoter P2, which is negatively controlled by the CytR repressor and positively affected by the cyclic AMP/catabolite activator protein complex. Gene, 1990 Nov 30, 96(1), 133 - 6 Temperature-mediated regulation and downstream inducible selection for controlling gene expression from the bacteriophage lambda pL promoter; Lowman HB et al.; We have examined in detail the effects of various induction temperatures on the expression of a heterologous fusion gene controlled by the bacteriophage lambda PL promoter in a heat-inducible Escherichia coli expression system which utilizes the CIts857 repressor . Experiments performed over a temperature range spanning 29-42 degrees C indicate that, under our conditions, temperatures as low as 29 degrees C may be required to fully repress the CI857-controlled transcription from PL, and that the highest protein yields are obtained after induction at 36 degrees C for 6 h . We cloned the cat reporter gene downstream from a heterologous gene controlled by PL and found that cat expression at a low induction temperature permits the monitoring of productive transcription through the heterologous gene and thus aids in selecting transformants that are capable of producing the heterologous protein in E . coli. Nature, 1990 Nov 29, 348(6300), 454 - 5 Fitness of RNA virus decreased by Muller's ratchet; Chao L; Why sex exists remains an unsolved problem in biology . If mutations are on the average deleterious, a high mutation rate can account for the evolution of sex . One form of this mutational hypothesis is Muller's ratchet . If the mutation rate is high, mutation-free individuals become rare and they can be lost by genetic drift in small populations . In asexual populations, as Muller noted, the loss is irreversible and the load of deleterious mutations increases in a ratchet-like manner with the successive loss of the least-mutated individuals . Sex can be advantageous because it increases the fitness of sexual populations by re-creating mutation-free individuals from mutated individuals and stops (or slows) Muller's ratchet . Although Muller's ratchet is an appealing hypothesis, it has been investigated and documented experimentally in only one group of organisms--ciliated protozoa . I initiated a study to examine the role of Muller's ratchet on the evolution of sex in RNA viruses and report here a significant decrease in fitness due to Muller's ratchet in 20 lineages of the RNA bacteriophage phi 6 . These results show that deleterious mutations are generated at a sufficiently high rate to advance Muller's ratchet in an RNA virus and that beneficial, backward and compensatory mutations cannot stop the ratchet in the observed range of fitness decrease. Biochemistry, 1990 Nov 27, 29(47), 10702 - 9 Heterogeneous initiation due to slippage at the bacteriophage 82 late gene promoter in vitro; Guo HC et al.; RNAs synthesized in vitro by purified Escherichia coli RNA polymerase from a bacteriophage 82 promoter are heterogeneous at the 5' end . We show that this heterogeneity results from variable addition of extra adenine residues, allowed by slippage of the initial oligonucleotide pppAAA-OH against its DNA template sequence TTT . Slippage backward by one base allows another A to be added, giving pppAAAA-OH, and this cycle can continue more than 20 times before it is ended by incorporation of UMP encoded by the fourth template base A . Slippage is abolished by mutation of the TTT template sequence to TGT and is sensitive to the concentrations of UTP and ATP in the reaction mixture . Analysis of deletions, substitutions, and point mutants implies that the slippage reaction requires only the existence of TTT at the initiation site of the template strand, although changes in neighboring nucleotides slightly affect its efficiency. Nucleic Acids Res, 1990 Nov 25, 18(22), 6553 - 7 Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication; Pakula TM et al.; Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E . coli cells harbouring a gene XII-containing plasmid . Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments . Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy . Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific . Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA . Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication. Nucleic Acids Res, 1990 Nov 25, 18(22), 6491 - 5 Solid-phase synthesis of H-Phe-Tyr-(pATAT)-NH2: a nucleopeptide fragment from the nucleoprotein of bacteriophage phi X174; Dreef-Tromp CM et al.; The preparation of the nucleopeptide H-Phe-Tyr-(pATAT)-NH2 could be realized via a solid phase phosphitetriester approach and by using the protected protecting group 2-(tert-butyldiphenylsilyoxymethyl)-benzoyl for the masking of the N6-amino function of deoxyadenosine . The latter protecting group can be removed under mild conditions with fluoride ion. Nucleic Acids Res, 1990 Nov 25, 18(22), 6587 - 94 Purification of RNA and RNA-protein complexes by an R17 coat protein affinity method; Bardwell VJ et al.; We describe an affinity chromatography method to isolate specific RNAs and RNA-protein complexes formed in vivo or in vitro . It exploits the highly selective binding of the coat protein of bacteriophage R17 to a short hairpin in its genomic RNA . RNA containing that hairpin binds to coat protein that has been covalently bound to a solid support . Bound RNA-protein complexes can be eluted with excess R17 recognition sites . Using purified RNA, we demonstrate that binding to immobilized coat protein is highly specific and enables one to separate an RNA of interest from a large excess of other RNAs in a single step . Surprisingly, binding of an RNA containing non-R17 sequences to the support requires two recognition sites in tandem; a single site is insufficient . We determine optimal conditions for purification of specific RNAs by comparing specific binding (retention of RNAs with recognition sites) to non-specific binding (retention of RNAs without recognition sites) over a range of experimental conditions . These results suggest that binding of immobilized coat protein to RNAs containing two sites is cooperative . We illustrate the potential utility of the approach in purifying RNA-protein complexes by demonstrating that a U1 snRNP formed in vivo on an RNA containing tandem recognition sites is selectively retained by the coat protein support. J Mol Biol, 1990 Nov 20, 216(2), 315 - 25 Localization of the proteins gp7, gp8 and gp10 in the bacteriophage T4 baseplate with colloidal gold: F(ab)2 and undecagold: Fab' conjugates; Watts NR et al.; We report the localization of the proteins gp7, gp8 and gp10 in the bacteriophage T4 baseplate . Proceeding on the assumption that these proteins occupy discrete locations, we have decorated baseplates and tails with immunological probes . Using 5 nm diameter colloidal gold: F(ab')2 conjugates, we show that proteins gp7 and gp10 are located directly at the vertex, with gp10 positioned in the pin directly below gp7 . gp8 is located beside gp7 towards the centre of the baseplate . Using a novel undecagold: Fab' conjugate we have also determined the radial positions of gp7 and gp8 in baseplates that have transformed to stars . A mechanism for the nature of the hexagon-to-star transformation is proposed. J Mol Biol, 1990 Nov 20, 216(2), 327 - 34 Receptor-recognizing proteins of T-even type bacteriophages . The receptor-recognizing area of proteins 37 of phages T4 TuIa and TuIb; Montag D et al.; Escherichia coli phages of the T4 family (T4, TuIa, TuIb) recognize their cellular receptors by means of a C-terminal region of protein 37; a dimer of this polypeptide (1026 residues in T4) is located at the distal part of the long tail fibers . Virions of the T2 family use protein 38 (which is attached to the free end of protein 37) for this purpose . The corresponding areas of genes 37 belonging to TuIa and TuIb were cloned and sequenced . Comparison of the deduced protein primary structures, including those of T4 and lambda Stf (Stf most likely representing a subunit of the side tail fibers of phage lambda) showed that an area of 70 to 100 residues is characterized by very variable sequences, while the sequences of the adjacent 43 to 44 C-terminal residues as well as those upstream from the variable region are highly homologous . The variable regions are flanked and interrupted seven or eight times by the motif His-x-His-y, with x and y most often being Ser or Thr; furthermore, the locations of these repeated tetrapeptides are conserved . Using hybrid phages obtained by recombination of one phage with cloned fragments of gene 37 of another, it could be shown that the area of this gene encoding receptor specificity includes the variable area . The situation is analogous to the known receptor-recognizing region of proteins 38 belonging to the T2-type family, except that the repeating sequence is of a different nature . In T4, receptor specificity is coded for by 382 base-pairs of the 3'-end of the gene, starting exactly at the variable area . It was found that T4 can use the outer membrane protein OmpC or lipopolysaccharide as receptors with the same efficiency, and it is proposed that the 70 residues of the variable part of the protein serve to bind to both ligands. J Mol Biol, 1990 Nov 20, 216(2), 243 - 50 A short DNA sequence from lambda phage inhibits protein synthesis in Escherichia coli rap; Perez-Morga D et al.; The Escherichia coli rap mutant inhibits vegetative growth of bacteriophage lambda . Phage mutations termed bar, which overcome the rap defect, have been mapped to three genetic loci in the pL operon . Plasmids with a lambda wild-type bar DNA segment cloned downstream from an active promoter cannot be maintained in rap mutant bacteria . The viability of a rap mutant strain decreases rapidly after induction of transcription through bar regions present on plasmids . Under these (restrictive) conditions the expression of plasmid-encoded beta-lactamase and plasmid DNA replication are arrested, but plasmid RNA synthesis continues for several hours . Analysis of protein extracts from E . coli rap cells containing bar plasmids revealed that both plasmid and bacterial protein synthesis are inhibited under restrictive conditions . In addition, unlike other RNAs tested, the chemical half-life of bar RNA increases 3.5-fold relative to the half-life of bar RNA under permissive conditions . We propose that transcription through the bar region, or the accumulation of bar RNA, results in an irreversible defect in cellular mRNA translation . This defect eventually kills the rap cells, and thus prevents bar plasmid maintenance. J Biol Chem, 1990 Nov 15, 265(32), 19961 - 5 Characterization of the 5'-flanking region of the rat protein kinase C gamma gene; Chen KH et al.; The 5'-flanking region of protein kinase C (PKC) gamma gene was identified from a rat liver genomic library in a bacteriophage lambda Charon 4A . A 3.6-kilobase (kb) genomic fragment containing the 5'-flanking region, first exon, and first intron was isolated and sequenced . The transcriptional initiation site, identified by S1 mapping and primer extension, was located 243 base pairs upstream from the translational initiation site . Promoter activity of a DNA segment spanning the 5'-flanking region was demonstrated by both in vitro transcription using HeLa cell nuclear extracts and chloramphenicol acetyltransferase assay by transfection of 293 cells with a PKC gamma-CAT fusion construct . Chloramphenicol acetyltransferase assay revealed that a fragment of about 0.16 kb from the transcriptional initiation site was sufficient for promoter activity in these cells, and the construct containing up to 1.6 kb from the cap site was expressed at a similar level . This promoter-active fragment contains several regions similar to defined transcriptional elements in other mammalian promoters, such as those for stimulatory protein 1 (Sp1), activator proteins 1 and 2 (AP1, AP2), c-myc, cAMP regulatory element-binding protein (CREB), and enhancer core (EnhC) . Investigation of the genomic structure of PKC gamma gene may lead to the identification of cis-elements controlling tissue-specific and developmental stage-specific expression of PKC gamma. J Biol Chem, 1990 Nov 15, 265(32), 19800 - 6 Cloning and sequencing of a cDNA for the hemolymph juvenile hormone binding protein of larval Manduca sexta; Lerro KA et al.; A cDNA for the hemolymph juvenile hormone binding protein (JHBP) of larval Manduca sexta has been cloned and sequenced . The JHBP was purified to homogeneity from fifth instar larval hemolymph using gel filtration chromatography, ion exchange chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Polyclonal rabbit antibodies, generated in response to this protein, were used to identify and isolate JHBP cDNAs from a fat body expression library in bacteriophage lambda ZAPII . Eleven putative JHBP cDNA clones were isolated and subcloned into Bluescript plasmid; cDNA inserts were approximately 750 base pairs in length . A 36-kDa immunoreactive protein was expressed from these plasmids; this beta-galactosidase fusion protein, like the authentic 32-kDa JHBP, was specifically photoaffinity labeled with {3H} epoxyhomofarnesyl diazoacetate (EHDA) . Single-stranded DNA from one clone was sequenced by the Sanger dideoxynucleotide method, using deletion and custom primer techniques . A mature translation product was identified which had 226 amino acid residues, a molecular mass of 25,111 daltons, and a predicted isoelectric point (pI) of 5.40 . The cDNA correctly predicts the N-terminal amino acid sequence and the amino acid composition of an authentic M . sexta hemolymph JHBP . A computer search of protein and nucleic acid data bases failed to reveal any related sequences . Thus, M . sexta hemolymph JHBP appears to be the first member of a new superfamily of insect hormone binding proteins. Biochemistry, 1990 Nov 13, 29(45), 10317 - 22 Three-dimensional model and molecular dynamics simulation of the active site of the self-splicing intervening sequence of the bacteriophage T4 nrdB messenger RNA; Nilsson L et al.; The secondary and 3D structure of the active site of the self-splicing T4 nrdB RNA has been modeled on a graphics workstation by use of the suggested 3D arrangement of the active site of the Tetrahymena IVS {Kim, S.H., & Cech, T.R . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 8788-8792} as a guideline . The initially obtained crude structure was then subjected to molecular mechanics energy minimization and molecular dynamics simulation to relax tensions . In this process the energy decreased considerably and gave a final structure that deviated by 3 A {root mean square (rms)} from the initial structure . The cofactor guanosine (and the competitive inhibitor arginine) was docked to a proposed {Michel, F., Hanna, M., Green, R., Bartel, D.P., & Szostak, J.W . (1989) Nature 342, 391-395} binding site, where it was found to fit rather well . A minor modification of the binding mode easily brought the O3' end of the guanosine within 2 A of the phosphodiester bond where the primary cleavage occurs. Biochemistry, 1990 Nov 13, 29(45), 10357 - 64 Incorporation of dA opposite N3-ethylthymidine terminates in vitro DNA synthesis; Bhanot OS et al.; N3-Ethylthymidine (N3-Et-dT) was site specifically incorporated into a 17-nucleotide oligomer to investigate the significance of DNA ethylation at the central hydrogen-bonding site (N3) of thymine . The 5'-(dimethoxytrityl)-protected N3-Et-dT was converted to the corresponding 3'-phosphoramidite and used to incorporate N3-Et-dT at a single site in the oligonucleotide during synthesis by the phosphite triester method . The purified N3-Et-dT-containing oligomer was ligated to a second 17-mer to yield a 34-nucleotide template with N3-Et-dT present at position 26 from the 3'-end . The template DNA, which corresponds to a specific sequence at gene G of bacteriophage phi X174, was used to study the specificity of nucleotide incorporation opposite N3-Et-dT . At 10 microM dNTP and 5 mM Mg2+, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 96% immediately 3' to N3-Et-dT and 4% after incorporation of a nucleotide opposite N3-Et-dT (incorporation-dependent blocked product) . DNA replication past the lesion (postlesion synthesis) was negligible . Incorporation opposite N3-Et-dT increased with increased dNTP concentrations, reaching 35% at 200 microM . Postlesion synthesis remained negligible . DNA sequencing of the incorporation-dependent blocked product revealed that dA is incorporated opposite N3-Et-dT consistent with the "A" rule in mutagenesis . Formation of the N3-Et-dT.dA base pair at the 3'-end of the growing chain terminated DNA synthesis . These results implicate N3-Et-dT as a potentially cytotoxic lesion produced by ethylating agents. J Biol Chem, 1990 Nov 5, 265(31), 19007 - 14 Characterization of bacteriophage T4 regA protein-nucleic acid interactions; Webster KR et al.; The bacteriophage T4 regA protein is a translational repressor of a group of T4 early mRNAs . We have characterized the binding of regA protein to polynucleotides and to specific RNAs . Binding to nucleic acids was monitored by the quenching of the intrinsic tryptophan fluorescence of regA protein . regA protein exhibited differential affinities for the polynucleotides examined, with the order of affinity being poly(rU) greater than poly(dT) greater than poly(dU) = poly(rG) greater than poly(rC) = poly(rA) . The binding site size calculated for regA protein binding to poly(rU) was n = 9 +/- 1 nucleotides . Cooperativity was observed in binding to multiple-site oligonucleotides, with a cooperativity parameter (omega) value of 10-22 . To study the specific interaction between regA protein and T4 gene 44 mRNA, the affinity of regA protein for synthetic gene 44 RNA fragments was measured . The association constant (Ka) for regA protein binding to gene 44 RNA fragments was 100-fold higher than for binding to nontarget RNA . Study of variant gene 44 RNA fragments indicated that the nucleotides required for specific binding are contained within a 12-nucleotide sequence spanning -12 to -1, relative to the AUG codon . The bases of five nucleotides (indicated in upper case type) are critical for specific regA protein interaction with the gene 44 recognition element, 5'-aaUGAGgAaauu-3' . These studies further showed that formation of a regA protein-RNA complex involves a maximum of 2-3 ionic interactions and is primarily an enthalpy-driven process. Cell, 1990 Nov 2, 63(3), 609 - 18 Structure of RNAs replicated by the DNA-dependent T7 RNA polymerase; Konarska MM et al.; The DNA-dependent RNA polymerase of bacteriophage T7 efficiently and specifically replicates two structurally related RNAs, termed X and Y RNAs . Replication of both RNAs involves synthesis of complementary strands initiated with pppC and pppG . RNAs transcribed from DNA template containing the established sequences of X and Y RNAs were efficiently replicated by T7 RNA polymerase . Both RNAs possess palindromic sequences with a dual axis of symmetry, permitting formation of hairpin-, dumbbell-, or cloverleaf-type structures . The template must consist of RNA and not DNA sequence, and the terminal unpaired dinucleotides of the RNA are necessary for replication . Nucleotidyl transferase activity of E . coli adenylates the unpaired CCOH dinucleotide at the 3' end of a C strand of X RNA . This feature, as well as the length (64 nucleotides) and compact structure of X and Y RNAs, suggests that they may resemble tRNA molecules and tRNA-like structures at the 3' termini of many plant viral RNA genomes. J Clin Microbiol, 1990 Nov, 28(11), 2383 - 8 High-level production of Escherichia coli STb heat-stable enterotoxin and quantification by a direct enzyme-linked immunosorbent assay; Urban RG et al.; A convenient and sensitive enzyme-linked immunosorbent assay (ELISA) for the STb heat-stable enterotoxin of Escherichia coli was developed and used to quantify STb production by strains with a high level of expression . Based on an antigenic profile of the secreted form of STb, a synthetic peptide (STb3-27) spanning the major predicted epitope was synthesized, coupled to keyhole limpet hemocyanin, and used to immunize rabbits . Anti-STb3-27 antibodies were affinity purified on a synthetic peptide-Sepharose 4B column and used in a direct-binding STb ELISA . Based on a highly purified form of toxin as a standard, the ELISA detected as little as 1 to 2 ng of STb from crude culture filtrates . ELISA data revealed that natural STb-producing strains elaborate little STb in defined-medium cultures relative to that elaborated by a recombinant strain harboring a cloned copy of the estB gene . Replacement of the endogenous STb promoter with any of several highly active promoters, including a bacteriophage T7 promoter, a beta-galactosidase promoter, and a tryptophan-beta-galactosidase hybrid (tac) promoter, increased the yield of STb 10- to 20-fold over levels obtained by an E . coli strain harboring the recombinant estB gene . The high level of STb antigen detected by the ELISA correlated with intestinal secretory activity . The combination of a convenient assay and effective hyperproduction of STb will serve as a basis for a large-scale toxin purification strategy. Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8687 - 91 Tat-responsive region RNA of human immunodeficiency virus 1 can prevent activation of the double-stranded-RNA-activated protein kinase; Gunnery S et al.; Transcription from the human immunodeficiency virus type 1 promoter gives rise to short cytoplasmic transcripts of approximately 60 nucleotides as well as to longer mRNAs . These RNAs contain the Tat-responsive region sequence, which is capable of assuming a stem-loop structure and has been implicated in the regulation of both transcription and translation . It has been reported that Tat-responsive region RNA inhibits translation in vitro through activation of an interferon-induced protein kinase, the double-stranded-RNA-activated inhibitor, which phosphorylates eukaryotic initiation factor 2 . We show that the activation property is due to double-stranded RNA that often contaminates RNA synthesized in vitro using bacteriophage RNA polymerases . After purification, high concentrations of Tat-responsive region RNA inhibit the activation of double-stranded RNA-activated inhibitor, suggesting that it may serve to protect human immunodeficiency virus type 1 infection from a cellular defense mechanism. J Electron Microsc Tech, 1990 Nov, 16(3), 235 - 9 Modification of the Polaron sputter-coater unit for glow-discharge activation of carbon support films; Benada O et al.; We describe a modification of the Polaron sputter-coater unit series 11 HD enabling activation of carbon support films for electron microscopy of macromolecules and macromolecular assemblies . The modification is simple and the device can be used in two modes, for sputter-coating of SEM samples and for glow-discharge activation of carbon support films . Examples of protein-free spreading of DNA and negative staining of bacteriophage particles on activated carbon support films are presented. Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8665 - 9 An essential arginine residue for initiation of protein-primed DNA replication; Hsieh JC et al.; A group of proteins that act as primers for initiation of linear DNA replication are called DNA-terminal proteins (terminal proteins) . We have found a short stretch of conserved amino acid sequence among the terminal proteins from six different sources . The location of this sequence motif is also similar among the different terminal-proteins . To determine the functional role of this terminal-protein domain in DNA replication, we have studied the bacteriophage PRD1 system . The PRD1 terminal protein and DNA polymerase genes were cloned into expression vectors, and the recombinant plasmids were used for constructing PRD1 terminal protein mutants . Site-directed mutagenesis and functional analysis showed that one of the two arginines (Arg-174) in the conserved sequence is critical for the initiation complex-forming activity of the PRD1 terminal protein . Replacement of Arg-174 by noncharged amino acids resulted in nonfunctional terminal protein . Phenylglyoxal, an alpha-dicarbonyl compound that reacts with the guanidino group of arginine, inhibits initiation complex formation between PRD1 terminal protein and dGMP . On the basis of these results, we propose that Arg-174 represents, at least in part, the binding site for phosphate groups of dGTP. J Bacteriol, 1990 Nov, 172(11), 6469 - 75 Enterohemolysin production is associated with a temperate bacteriophage in Escherichia coli serogroup O26 strains; Beutin L et al.; A temperate bacteriophage that determines the expression of enterohemolysin was isolated from Escherichia coli O26 strain C3888 . The genetic determinant associated with enterohemolysin production (E-Hly determinant) was cloned from EcoRI-digested bacteriophage DNA in vector plasmid pUC8 . pUC8 recombinant plasmid pEO19 carries a 3.7-kb EcoRI insert of phage DNA, and enterohemolysin was expressed in E . coli K-12 after transformation . Hemolysin-negative derivatives of pEO19 were generated by transposon mutagenesis with Tn1725 . By subcloning, the phage E-Hly determinant was assigned to a 2,150-bp piece of DNA which is flanked by EcoRI and AccI restriction sites . The enterohemolysin-producing recombinant strains and wild-type strain C3888 express a 60-kDa protein which was detected in the bacterial outer membrane by Western immunoblotting . Biologically active enterohemolysin was detected only in bacteria grown to the stationary phase, and the hemolysin was not released into the culture medium . Lysis of erythrocytes was inhibited by 30 mM dextran 4, which functions as an osmotic protectant without destroying the enterohemolysin itself. J Bacteriol, 1990 Nov, 172(11), 6323 - 32 Bacteriophage T4 nrdA and nrdB genes, encoding ribonucleotide reductase, are expressed both separately and coordinately: characterization of the nrdB promoter; Tseng MJ et al.; We examined the expression of the bacteriophage T4 nrdA and nrdB genes, which encode the alpha 2 and beta 2 subunits, respectively, of ribonucleoside diphosphate reductase, the first committed enzyme in the pathway of synthesis of the deoxyribonucleoside triphosphates . T4 nrdA, located 700 bp upstream from nrdB, has been shown previously to be transcribed by two major transcripts: a prereplicative, polycistronic message, TU, orginating at an immediate-early promoter, PE, that is 3.5 kb upstream from nrdA, and a postreplicative message commencing from a late promoter in its 5' flank . We have found a third promoter initiating a transcript at 159 nucleotides upstream from the reading frame of nrdB . PnrdB functions only in the presence of the T4 motA gene product, which is required for middle (time) promoters, and therefore the onset of nrdB transcription is delayed more than 2 min after infection . Because of the distance of nrdA from PE, the inception of nrdA transcription (delayed early) coincides closely with that of nrdB . An apparent termination site, tA, occurs about 80 bp downstream from nrdA . Some of the polycistronic mRNA reading through the site after 5 min contributes to nrdB transcription . nrdA and nrdB genes in an uninfected host have been reported to be transcribed only coordinately . In contrast, T4 nrdA and nrdB are initially transcribed separately onto the PE and PnrdB transcripts, respectively, but at about 5 min after infection are transcribed both coordinately and on separate transcripts . Evidence is presented that TU coordinately transcribes a deoxyribonucleotide operon in the order: frd, td, gene 'Y,' nrdA, nrdB . Since the beta 2 subunit is known to be formed after the alpha 2 subunit, the expression of the nrdB gene determines the onset of deoxyribonucleoside triphosphate synthesis and thus of T4 DNA replication. J Bacteriol, 1990 Nov, 172(11), 6189 - 93 The level of a transcript required for production of a Streptomyces coelicolor antibiotic is conditionally dependent on a tRNA gene; Guthrie EP et al.; In Streptomyces coelicolor A3(2), bldA mutants are conditionally defective in aerial mycelium formation and fail to synthesize all four antibiotics produced by bldA+ strains . Previous studies showed that bldA specifies the tRNA for the rarely used leucine codon UUA . Here we describe experiments examining the abundance in a bldA mutant of a transcript involved in antibiotic production . With use of a bacteriophage-based integrative vector, a promotorless xylE reporter gene was inserted into a previously undescribed gene for an early step in biosynthesis of the red antibiotic undecylprodigiosin, located in the red gene cluster . With this transcriptional fusion present at unit copy number in the chromosome, xylE expression in a bldA+ strain was maximal late in growth in a liquid production medium and was virtually absent in a bldA mutant . On plates of a different medium, the bldA mutant was able to produce undecylprodigiosin and to express the red::xylE fusion, but both abilities were repressed by increasing the concentration of phosphate in the medium . These experiments showed that the undecylprodigiosin deficiency of bldA mutants cannot be accounted for by the presence of TTA codons in the red structural genes, but rather that bldA influences red gene mRNA abundance . In low-phosphate conditions, an alternative regulatory pathway can lead to red gene expression. Carcinogenesis, 1990 Nov, 11(11), 2009 - 14 Characterization and genotoxicity of DNA adducts caused by 2-naphthyl isocyanate; Tamura N et al.; Calf thymus DNA and M13mp9 RF DNA were modified with {ring-3H}2-naphthyl isocyanate (NIC) and analyzed by reverse-phase HPLC following enzymatic hydrolysis . In each case, essentially, a single radioactive component, which co-chromatographed with authentic N4-2-naphthyl-carbamoyl-2'-deoxycytidine (NCdC), was detected . In order to explore the biological potential of this adduct, mp9 RF DNA modified with NIC was introduced into Escherichia coli strains using a calcium chloride technique . The plaque-forming efficiencies of DNA decreased with increasing adduct level, and the decreases were more pronounced in Uvr endonuclease-deficient strains (i.e . AB1886, uvrA; AB1885, uvrB; AB1884, uvrC) as compared to JM103 (Uvr endonuclease proficient) and JM101 RH03 (recA) . These results suggest that these lesions, NCdC adducts, can be removed by the Uvr endonuclease repair system . Mutations were detected as the loss of ability of the bacteriophage to complement the defective beta-galactosidase of the host cells . Induction of SOS functions in the host cells enhanced the mutation frequency to 0.089%, i.e . greater than or equal to 4-fold greater than in non-SOS-induced cells, in transfections with RF DNA that contained 100 adducts/molecule . The mutagenic potency of this cytidine lesion is lower than that of the guanine-C8 adducts of 2-aminofluorene and 2-acetylaminofluorene as reported previously for this mutagenesis system. Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8716 - 20 A DNA gyrase-binding site at the center of the bacteriophage Mu genome is required for efficient replicative transposition; Pato ML et al.; We have discovered a centrally located site that is required for efficient replication of bacteriophage Mu DNA and identified it as a strong DNA gyrase-binding site . Incubation of Mu DNA with gyrase and enoxacin revealed a cleavage site 18.1 kilobases from the left end of the 37.2-kilobase genome . Two observations indicate a role for the site in Mu replication: mutants of Mu, able to grow on an Escherichia coli gyrB host that does not allow growth of wild-type Mu, were found to possess single-base changes resulting in more efficient gyrase binding and cleavage at the site . Introduction of a 147-base-pair deletion that eliminated the site from a prophage inhibited the onset of Mu replication for greater than 1 hr after induction. Carcinogenesis, 1990 Nov, 11(11), 2005 - 8 Strand scission in DNA by quercetin and Cu(II): identification of free radical intermediates and biological consequences of scission; Fazal F et al.; Quercetin was shown to reduce oxygen to superoxide . In the presence of Cu(II), the hydroxyl radical was formed . The strand scission of DNA was shown to occur under conditions in which Cu(II), quercetin and either hydrogen peroxide or oxygen were present and superoxide was not a necessary intermediate . Strand scission involved the hydroxyl radical and a radical DNA intermediate . The strand scission reaction was shown to account for the biological activity of quercetin as assayed by bacteriophage inactivation. Biotechniques, 1990 Nov, 9(5), 578 - 80, 582-3 Detection of large cDNA inserts within crude lambda gt11 lysates: a rapid and sensitive method; Asundi V et al.; A method is presented for the isolation of bacteriophage lambda DNA and the rapid identification of large cDNA inserts within crude phage lysates . The primary screening of a lambda gt11 cDNA library with a 32P-radiolabeled cDNA probe yielded 21 putative positive clones . A phage "spot-blot" analysis was employed to quickly screen these potential recombinants . This eliminated 9 of the 21 clones as the result of false positive signals . The remaining 12 recombinant phage were amplified on agarose-based media, and phage DNA was isolated using a modified plate lysate procedure . The DNA thus obtained from these crude lysates could be easily digested with EcoRI and examined by Southern blot analysis . The resulting blot was hybridized with the same cDNA probe used in the initial screening of the library . Thus, two clones harboring the longest cDNA insert were identified from a mixed phage population and were subsequently plaque purified . The procedure is rapid, sensitive, reproducible, inexpensive and allows the processing of several clones at once without sacrificing the quality or yields of the DNA preparation . Furthermore, the method obviates the need for plaque purifying all the positives obtained from the initial screening of a cDNA library. J Bacteriol, 1990 Nov, 172(11), 6540 - 50 Specificity determinants in the attachment sites of bacteriophages HK022 and lambda; Nagaraja R et al.; The Int proteins of bacteriophages HK022 and lambda promote recombination between phage and bacterial attachment sites . Although the proteins and attachment sites of the two phages are similar, neither protein promotes efficient recombination between the pair of attachment sites used by the other phage . To analyze this difference in specificity, we constructed and characterized chimeric attachment sites in which segments of one site were replaced with corresponding segments of the other . Most such chimeras recombined with appropriate partner sites in vivo and in vitro, and their differential responses to the Int proteins of the two phages allowed us to locate determinants of the specificity difference in the bacterial attachment sites and a central segment of the phage attachment sites . The location of these determinants encompasses three of the four core-type binding sites for lambda Int: C, B, and most importantly, B' . The regions corresponding to the C' core binding site and the arm-type binding sites of lambda Int play no role in the specificity difference and, indeed, are well conserved in the two phages . We found, unexpectedly, that the effect of replacement of an Int-binding region on the recombinational potency of one chimeric site was reversed by a change of partner . This novel context effect suggests that postsynaptic interactions affect the specificity of recognition of attachment sites by Int. Protein Expr Purif, 1990 Nov, 1(2), 159 - 68 Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi: high level expression and purification of a soluble recombinant form of OspA; Dunn JJ et al.; The ospA gene of Borrelia burgdorferi encodes an outer membrane protein which is a major antigen of the Lyme disease agent . Two sequence-specific sets of oligonucleotide primers were used to specify the amplification of the ospA coding sequence by the polymerase chain reaction . One set allowed the entire ospA sequence to be amplified, while the other primed amplification of a truncated form of ospA lacking the first 17 codons specified by the wild-type ospA structural gene, residues believed to constitute a signal sequence which normally would direct localization of the ospA protein to the Borrelia cell's outer membrane . Each set of primers also contained sequences near their 5' ends which facilitated cloning of the amplified DNA directly into a high level expression system based on bacteriophage T7 genetic elements . We showed that the full-length OspA protein is synthesized poorly in Escherichia coli and it is associated with the insoluble membrane fraction . In contrast, the truncated form can be expressed to very high levels and it is soluble . The truncated protein was purified to homogeneity and partially characterized . Its N-terminal sequence and molecular weight derived from sodium dodecyl sulfate-polyacrylamide gel electrophoresis agree with those deduced from the DNA sequence . It is a monomer with a native molecular weight of 28,000 and it is very resistant to digestion by trypsin even though it is rather rich in lysine residues (16 mol%) . Recombinant OspA protein synthesized in E . coli is recognized by antibodies in sera of Lyme patients, which suggests that the protein may be useful in immunoassays and as a possible immunogen to protect against Lyme borreliosis. Virology, 1990 Nov, 179(1), 217 - 27 The ImmC region of phage P1 codes for a gene whose product promotes lytic growth; Baumstark BR et al.; The ImmC region of the temperate bacteriophage P1 contains c1, a gene that codes for a repressor of lytic growth . Located in the region upstream of c1 are four open reading frames capable of coding for low-molecular-weight proteins . The efficiency of lysogeny by P1+Cm was found to be reduced by almost 10(5)-fold when the host cells carry this region of ImmC on a multicopy plasmid . The sequences responsible for interfering with lysogen formation were localized to one of the small open reading frames (orf4) within ImmC . Insertions and deletions within orf4 suppress the virulent phenotype of P1virC mutants when introduced into the phage by recombination . These virC-suppressed mutant phage were found to be incapable of lytic growth unless the product of orf4 is provided in trans . The presence of orf4 was observed to interfere with repression by the c1 protein of ImmC-encoded promoters fused to lacZ . For this reason, we suggest that orf4 corresponds to coi, a gene previously proposed to code for an inactivator of c1-mediated repression. Mol Microbiol, 1990 Nov, 4(11), 1839 - 46 The par region of pSC101 affects plasmid copy number as well as stability; Manen D et al.; The par locus is a segment of pSC101 that has been identified as a cis-acting determinant of plasmid stability . We show that par also determines copy number and must, therefore, play a role in plasmid replication . The segregation defect, but not the copy-number reduction, of par- replication origins is completely suppressed by a short sequence from the bacteriophage lambda gene O which is present in plasmid pKO-4 . Thus, replication and segregation functions are separable from each other. Mol Gen Mikrobiol Virusol, 1990 Nov, (11), 25 - 8 {Loss of adsorptive ability--the reason for inactivation of the RM2 bacteriophage during storage}; Gonikberg EM et al.; The kinetics of bacteriophage PM2 inactivation at storage was compared with the kinetics of bacteriophage adsorption on Alteromonas espejiana BAL-31 host cells . Adsorption ability and infectivity are lost with the same rate at temperatures 4-28 degrees C suggesting the loss of adsorption ability to result in bacteriophage inactivation . At higher temperatures infectivity is lost more rapidly than the ability of adsorption . The single hit kinetics of adsorption ability loss suggests the simple model of independent inactivation of 12 antireceptors located at the tops of icosaedric capsid to be erroneous . At bacteriophage inactivation the major port of protein I, a fragment of antireceptors, is preserved in the capsid composition. Mol Microbiol, 1990 Nov, 4(11), 1891 - 7 Instability of bacteriophage Mu transposase and the role of host Hfl protein; Gama MJ et al.; The activity of the transposase of bacteriophage Mu is unstable, requiring the protein to be synthesized throughout the lytic cycle (Pato and Reich, 1982) . Using Western blot analysis, we analysed the stability of the transposase protein during the lytic cycle and found that it, too, is unstable . The instability of the protein is observed both in the presence and the absence of Mu DNA replication, and is independent of other Mu-encoded proteins and the transposase binding sites at the Mu genome ends . Stability of the protein is enhanced in host strains mutated at the hfl locus; however, stability of the transposase activity is not enhanced in these strains, suggesting that functional inactivation of the protein is not simply a result of its proteolysis. J Gen Virol, 1990 Nov, 71 ( Pt 11), 2709 - 17 Identification of seroreactive regions of the human papillomavirus type 16 protein E4, E6, E7 and L1; Muller M et al.; Small fragments of the DNA of human papillomavirus type 16 (HPV-16) were randomly cloned into the bacteriophage fd which expresses the resulting peptides as part of its capsid . Antisera raised against different HPV-16 fusion proteins were used for screening of the phage clones and the reacting peptides were determined by sequencing the inserted HPV-16 DNA fragments of the positive recombinants . Seroreactive regions of the proteins derived from the E4, E6, E7 (two regions) and L1 (three regions) open reading frames could be found by this approach . Of these seven regions, four were defined by at least two overlapping inserts, thus limiting the domains to between 10 and 15 amino acids . In the case of the E4 open reading frame, the same region identified by immunoscreening was also found when synthetic overlapping octapeptides were tested by ELISA with the anti-E4 antiserum . Using an approach to predict 'receptor-like' regions within the respective proteins, five of the seven regions were also identified . From the data on these regions, synthetic peptides were produced and used for the detection of antibodies against HPV-16 proteins in human sera by ELISA. Gene, 1990 Oct 30, 95(1), 9 - 15 Deletion analysis of a bacteriophage P2 late promoter; Grambow NJ et al.; We have fused the promoter (PF) for the P2 late FETUD operon to the gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a plasmid vector . Synthesis of CAT in Escherichia coli strains carrying this plasmid requires the product of the P2 ogr gene or the satellite phage P4 transactivation gene, delta . Our results demonstrate that these phage-encoded transcriptional regulatory proteins are necessary and sufficient for activation of P2 late transcription in this reporter plasmid . Positive regulation of cloned PF is severely impaired in a host strain carrying the rpoA109 mutation . Expression from the cloned promoter thus approximates those features of P2 late transcription that have been shown to occur during normal P2 infection . To define sequences required for promoter function, sequential upstream deletions of PF were generated using BAL 31 nuclease, and the mutant promoters were assayed for cat expression . A sequence between nucleotides -69 and -64 from the transcription start point was found to be essential for promoter activity . This coincides with a region of homology conserved among all four P2 late gene promoters and the two P4 late promoters, and includes an element of dyad symmetry. Gene, 1990 Oct 30, 95(1), 25 - 30 Deletions at the N terminus of bacteriophage phi 29 protein p6: DNA binding and activity in phi 29 DNA replication; Otero MJ et al.; Deletions corresponding to the first 5 or 13 amino acids (aa), not counting the initial Met, have been introduced into the N terminus of the phage phi 29 protein p6 . The activity of such proteins in the in vitro phi 29 DNA replication system, their capacity to interact with the phi 29 DNA ends, and their interference with the wild type (wt) protein p6 activity have been studied . The initiation activity of protein p6 decreased considerably when 5 as were deleted and was undetectable when 13 aa were removed . The mutant proteins were unable to specifically interact with the phi 29 DNA ends . These results indicate the need of an intact N terminus for the activity of protein p6 . However, such N-truncated proteins inhibited both the specific binding of the wt protein p6 to the phi 29 DNA ends and its activity in phi 29 DNA replication. J Biol Chem, 1990 Oct 25, 265(30), 18511 - 7 A bacteriophage P1-encoded modulator protein affects the P1 c1 repression system; Velleman M et al.; Bacteriophage P1 encodes a tripartite immunity system composed of the immC, immI, and immT region . Their basic genetic elements are the c1 repressor of lytic functions, the c4 repressor which negatively regulates antirepressor synthesis, and the bof gene, respectively . The function of the latter will be described here . We have cloned and sequenced the bof gene from P1 wild type and a P1 bof amber mutant . Based on the position of a TAG codon of the bof amber mutant the bof wild type gene was localized . It starts with a TTG codon, comprises 82 codons, and is preceded by a promoter structure . The bof protein (Mr = 7500) was overproduced in Escherichia coli from a bof recombinant plasmid and was purified to near homogeneity . The N-terminal amino acids predicted from the DNA sequence of the bof gene were confirmed by sequence analysis of the bof protein . Using a DNA mobility shift assay, we show that bof protein enhances the binding of c1 repressor to the operator of the c1 gene . In accordance with this result, in transformants of Escherichia coli, containing both a bof- and a c1-encoding plasmid, c1 expression is down-regulated . We conclude that bof acts as a modulator protein in the repression of a multitude of c1-controlled operators in the P1 genome. J Biol Chem, 1990 Oct 25, 265(30), 18311 - 7 Properties of overexpressed phage T5 D15 exonuclease . Similarities with Escherichia coli DNA polymerase I 5'-3' exonuclease; Sayers JR et al.; The D15 gene of the bacteriophage T5, thought to encode an exonuclease, was cloned into an M13 phage on a 1344-base pair fragment . The deduced amino acid sequence of 291 residues (Kaliman, A . V., Krutilina, A . I., Kryukov, V . M., and Bayev, A . A . (1986) FEBS Lett . 195, 61-64) shows a high degree of homology with the first 320 amino acid residues of Escherichia coli DNA polymerase I, the region containing the enzyme's 5'-3' exonuclease activity . Recombinant M13 phage DNA was manipulated by oligonucleotide-directed mutagenesis to enable subcloning into a high efficiency expression vector, allowing the production of large amounts of enzyme for physical characterization and crystallization trials . The enzyme was purified to homogeneity . The purified enzyme is active on both native and heat-denatured DNA and shows no endonuclease activity on either double-stranded closed-circular or nicked DNA . The enzyme is also able to degrade some oligonucleotides in a manner which depends not only on the nucleotide sequence but also on the state of hybridization of the potential substrate . The mode of action of this enzyme is similar to, although not identical to that of the 5'-3' exonuclease activity of E . coli DNA polymerase I. Nucleic Acids Res, 1990 Oct 25, 18(20), 5961 - 7 Selection for mutations in the PR promoter of bacteriophage lambda; Brown S et al.; Insertion of DNA containing PR, the early rightward promoter of bacteriophage lambda, is lethal to M13-derived vectors when the promoter directs transcription (using the '+' strand as template) toward the M13 origin of replication (ori) . Lethality can be relieved by mutation of PR, repression of the promoter by the lambda cl repressor, or by insertion of a strong transcription terminator between PR and ori . We have used selection for plaque formation in the absence of repressor to isolate 14 different mutations at 8 sites in PR . This method of isolating promoter mutants in vivo is applicable generally to strong promoters whose activity is regulated either positively or negatively. J Biol Chem, 1990 Oct 25, 265(30), 18682 - 9 Synthesis of DNA by human immunodeficiency virus reverse transcriptase is preferentially blocked at template oligo(deoxyadenosine) tracts; Williams KJ et al.; The genome of human immunodeficiency virus (HIV) and especially the envelope gene are mutated with unusually high frequency during in vivo replication . Recent studies indicate that HIV reverse transcriptase (RT) is unusually error prone and that the number of generated mutations is disproportionately high within repetitive base sequences . To study the ability of recombinant and wild-type HIV RT to traverse specific homo-oligomeric stretches, we used bacteriophage M13 DNA templates that contain different oligo(purine) and oligo(pyrimidine) inserted tracts . The progress of HIV RT along these templates was potently inhibited from further progression only at a (dA)16 insert . Comparison with other polymerases indicates that the almost complete blockage of polymerization beyond an oligo(dA) insert is unique to HIV RT and Moloney murine leukemia virus RT, which has high sequence homology with HIV RT . The extent of termination of HIV RT at the oligo(dA) run is not affected by alterations in the concentration of KCl, Mg2+, dNTP, or by a decrease in pH . Obstruction of HIV RT opposite the oligo(dA) insert is not alleviated by moving the primer position further upstream from the oligo(dA) insert . Lastly, HIV RT purified directly from virions is also specifically arrested at an oligo(dA) tract . Competition experiments indicate that the concentration of active HIV RT in the presence of M13(dA)16 DNA is similar to that observed in the presence of M13(dG)16 DNA . In addition, preincubation of M13(dA)16 DNA with HIV RT does not subsequently inhibit avian myeloblastosis virus RT from successfully traversing the (dA)16 insert . Therefore, it appears that the blockage of chain elongation of HIV RT at the (dA)16 insert is not the result of trapping the enzyme at this site. J Biol Chem, 1990 Oct 25, 265(30), 18561 - 7 Pore formation associated with the tail-tip protein pb2 of bacteriophage T5; Feucht A et al.; Upon binding of bacteriophage T5 tails to purified FhuA receptor protein the tail-tip protein pb2 became extremely sensitive to trypsin and other proteases . However, when T5 tails were bound to FhuA integrated into liposomes, pb2 was found to retain some resistance to trypsin . Electron microscopic examination of tail-liposome complexes supported the idea that trypsin resistance of pb2 in such complexes was caused by insertion of the tail-tip into the liposomes . pb2 was isolated from tails by treatment with sodium dodecyl sulfate and was further purified by gel filtration using a fast protein liquid chromatography system . pb2 obtained with this procedure was most likely monomeric . It was extremely sensitive to trypsin . When reconstituted into black lipid bilayer membranes, it formed pores with an average single-channel conductance of 4.6 nanosiemens in 1 M KCl . Zero-current potential measurements showed only a very slight preference, if any, for cations over anions . The data are compatible with pb2 forming a large water-filled transmembrane channel . The functioning during infection of pb2 in cytoplasmic membrane depolarization and phage DNA uptake into the cell is discussed. Biochim Biophys Acta, 1990 Oct 23, 1087(2), 199 - 204 Unique hairpin structures occurring at the replication origin of phage G4 DNA; Hirao I et al.; Recently we reported that a DNA fragment, GCCAAAGC, forms an extraordinarily stable hairpin structure with two G-C pairs at the terminus and A-A-A stacked structure . The sequence is present at the replication origin of bacteriophage G4 ssDNA, and so on . Several kinds of possible hairpin structures, corresponding to the replication origin of phage G4, were synthesized and their secondary structures were examined . It was found that the fragments are able to form interconvertible hairpin structures depending on the length of the base-paired regions . The hairpin structure consisting of GCGAAAGC was not digested by the exonuclease activity of T4 DNA polymerase and it was stable enough to be only minimally bound by a single-stranded DNA binding protein. Biochemistry, 1990 Oct 23, 29(42), 9765 - 71 Reduced tendency to form a beta turn in peptides from the P22 tailspike protein correlates with a temperature-sensitive folding defect; Stroup AN et al.; A family of mutants of the P22 bacteriophage tailspike protein has been characterized as temperature sensitive for folding (tsf) by King and co-workers {King, J . (1986) Bio/Technology 4, 297-303} . There is substantial evidence that the tsf mutations alter the folding pathway but not the stability of the final folded protein . Several point mutations are known to cause the tsf phenotype; most of these occur in regions of the tailspike sequence likely to take up reverse turns . Hence, it has been hypothesized that the correct folding of the P22 tailspike protein requires formation of turns and that the mutations causing tsf phenotypes interfere at this critical stage . We have tested this hypothesis by study of isolated peptides corresponding to a region of the P22 tailspike harboring a tsf mutation . Comparison of the tendencies of wild-type and tsf sequences to adopt turn conformations was achieved by the synthesis of peptides with flanking cysteine residues and the use of a thiol-disulfide exchange assay . We find that the wild-type sequence, either as a decapeptide (Ac-CVKFPGIETC-CONH2) or as a dodecapeptide (Ac-CYVKFPGIETLC-CONH2), has a 3-5-fold greater tendency for its termini to approach closely enough to form the intramolecular disulfide than do the peptide sequences corresponding to the tsf mutant sequences, which have a Gly----Arg substitution (Ac-CVKFPRIETC-CONH2 or Ac-CYVKFPRIETLC-CONH2) . A peptide with a D-Arg substituted for the Gly has a slightly higher turn propensity than does the wild type . Together with data from nuclear magnetic resonance analysis of the oxidized peptides, this suggests that a type II beta turn is favored by the wild-type sequence . Our results on isolated peptides from the P22 tailspike protein support the model for its folding that includes reverse turn formation as a critical step. J Theor Biol, 1990 Oct 21, 146(4), 501 - 11 Selection for lysis inhibition in bacteriophage; Abedon ST; For Escherichia coli cells that have been infected by T-even bacteriophages (phages T2, T4, and T6), the adsorption of a second T-even phage results in an increase in the length of the original phage infection and an associated increase in the number of phages produced by the same infected cell . This is a phage encoded response called lysis inhibition . In this study the ecological significance of lysis inhibition is explored . In particular it is argued that lysis inhibition is an adaptive response to environments containing high concentrations of infected cells and low concentrations of uninfected cells. J Biol Chem, 1990 Oct 15, 265(29), 17393 - 6 A novel functional domain of an alpha-like DNA polymerase . The binding site on the herpes simplex virus polymerase for the viral UL42 protein; Digard P et al.; Most DNA-dependent DNA polymerases exist as a complex with one or more noncovalently bound accessory proteins, whose presence is necessary for the correct functioning of the holoenzyme . Using the herpes simplex virus DNA polymerase as a representative member of the alpha-polymerase family, we have recreated the association between the polymerase and its accessory protein UL42 in vitro through the translation in rabbit reticulocyte lysate of bacteriophage RNA polymerase-generated transcripts encoding the two polypeptides . Study of the ability of deleted versions of the polymerase protein to bind UL42, as detected by coimmunoprecipitation of the two polypeptides, defined a carboxyl-terminal region of the DNA polymerase that was both necessary and sufficient for the association . This domain is distinct from regions of the protein previously characterized as involved in catalysis . The results suggest a strategy for the design of novel targeted antiviral drugs, which would disrupt the DNA polymerase-UL42 complex. Biochemistry, 1990 Oct 2, 29(39), 9241 - 9 Role of the Cro repressor carboxy-terminal domain and flexible dimer linkage in operator and nonspecific DNA binding; Hubbard AJ et al.; A series of mutations comprising single and multiple substitutions, deletions, and extensions within the carboxy-terminal domain of the bacteriophage lambda Cro repressor have been constructed . These mutations generally affect the affinity of repressor for specific and nonspecific DNA . Additionally, substitution of the carboxy-terminal alanine with several amino acids capable of hydrogen-bonding interactions leads to improved specific binding affinities . A mutation is also described whereby cysteine links the two Cro monomers by a disulfide bond . As a consequence, a significant improvement in nonspecific binding and a concomitant reduction in specific binding are observed with this mutant . These results provide evidence that the carboxy terminus of Cro repressor is an important DNA binding domain and that a flexible connection between the two repressor monomers is a critical factor in modulating the affinity of wild-type repressor for DNA. Radiat Res, 1990 Oct, 124(1 Suppl), S16 - 22 Microdosimetry and Katz's track structure theory . I . One-hit detectors; Zaider M; A microdosimetric treatment of the response of one-hit detectors to radiation is formulated and compared with the model proposed by R . Katz, S . C . Sharma, and M . Homayoonfar (in Topics in Radiation Dosimetry, Suppl . I (F . H . Attix, Ed.), pp . 317-383, Academic Press, New York, 1972) within the framework of their track-structure theory . It is shown that radial dose distributions (on which the track structure theory is based) are generally poor substitutes for the exact microdosimetric distributions except when (a) the target is much larger than the radial extent of the track or (b) the "effective" specific energy in the target (alpha z, see text) is negligibly small . Since neither one of these conditions is generally satisfied, it is suggested that a meaningful search for one-hit detectors be based on a microdosimetric description of the stochastics of energy deposition . An analysis of the phi x-174 bacteriophage inactivation data is presented. Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 8070 - 4 Cleavage of the bacteriophage P1 packaging site (pac) is regulated by adenine methylation; Sternberg N et al.; The packaging of bacteriophage PI DNA is initiated when the phage packaging site (pac) is recognized and cleaved and continues until the phage head is full . We have previously shown that pac is a 162-base-pair segment of P1 DNA that contains seven DNA adenine methyltransferase methylation sites (5'-GATC) . We show here that cleavage of pac is methylation sensitive . Both in vivo and in vitro experiments indicate that methylated pac is cleavable, whereas unmethylated pac is not . Moreover, DNA isolated from P1 phage and containing an uncut pac site was a poor substrate for in vitro cleavage until it was methylated by the Escherichia coli DNA adenine methyltransferase . Comparison of that uncut pac DNA with other viral DNA fragments by digestion with methylation-sensitive restriction enzymes indicated that the uncut pac DNA was preferentially undermethylated . In contrast, virion DNA containing a cut pac site was not undermethylated . We believe these results indicate that pac cleavage is regulated by adenine methylation during the phage lytic cycle. J Gen Virol, 1990 Oct, 71 ( Pt 10), 2257 - 64 Ribozymes that cleave potato leafroll virus RNA within the coat protein and polymerase genes; Lamb JW et al.; Two ribozymes were synthesized which were designed to cleave potato leafroll virus (PLRV) positive strand RNA within the regions known to encode the viral coat protein and the predicted RNA polymerase gene . DNA sequences encoding the ribozymes were inserted into the Escherichia coli plasmids pTz18R and pTz19R under the control of the bacteriophage T7 promoter and enzymically active RNA molecules generated by transcription by T7 RNA polymerase in vitro . Each ribozyme cleaved its cognate site in RNA derived from either cDNA or PLRV particles . Ribozyme cleavage sites within the polymerase gene and coat protein gene were determined and shown to be at the predicted sequence immediately downstream from a GUC motif . An altered version of the ribozyme which recognized the sequence in the coat protein gene was isolated in which a single adenosine residue in the enzymic loop of the ribozyme was deleted . This mutated ribozyme was unable to cleave RNA molecules containing the coat protein ribozyme target site. Biochem J, 1990 Oct 1, 271(1), 265 - 8 Use of ATP, dATP and their alpha-thio derivatives to study DNA ligase adenylation; Montecucco A et al.; Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-{alpha-35S}thio-ATP . However, the joining reaction does not take place in the presence of the deoxyribotriphosphates . Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives . Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction . In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix. J Bacteriol, 1990 Oct, 172(10), 6135 - 8 groE genes affect SOS repair in Escherichia coli; Liu SK et al.; Repair of UV-irradiated bacteriophage in Escherichia coli by Weigle reactivation requires functional recA+ and umuD+C+ genes . When the cells were UV irradiated, the groE heat shock gene products, GroES and GroEL, were needed for at least 50% of the Weigle reactivation of the single-stranded DNA phage S13 . Because of repression of the umuDC and recA genes, Weigle reactivation is normally blocked by the lexA3(Ind-) mutation (which creates a noncleavable LexA protein), but it was restored by a combination of a high-copy-number umuD+C+ plasmid and a UV dose that increases groE expression . Maximal reactivation was achieved by elevated amounts of the Umu proteins, which was accomplished in part by UV-induced expression of the groE genes . By increasing the number of copies of the umuD+C+ genes, up to 50% of the normal amount of reactivation of S13 was achieved in an unirradiated recA+ host. J Virol, 1990 Oct, 64(10), 4851 - 7 Regulated expression of nuclear genes by T3 RNA polymerase and lac repressor, using recombinant vaccinia virus vectors; Rodriguez D et al.; Recombinant vaccinia viruses that express the bacteriophage T3 RNA polymerase (VV-T3pol) or the Escherichia coli lac repressor (VV-lacI) under control of the early-late vaccinia promoter P7.5 were constructed . To determine whether phage polymerase and lac repressor can function in the nucleus of mammalian cells, the bacterial chloramphenicol acetyltransferase (CAT) gene was cloned downstream of a T3 promoter (PT3-CAT) or downstream of a T3 promoter-lac operator fusion element (PT3Olac-CAT), and these reporter gene cassettes were introduced stably into NIH 3T3 or Ltk- cells . Infection of 3T3/PT3-CAT or Ltk-/PT3-CAT cells by VV-T3pol led to rapid expression of CAT (greater than 20 ng of CAT protein per 10(6) cells) . The presence of hydroxyurea (which blocks virus DNA replication) did not prevent CAT production . When 3T3/PT3Olac-CAT cells were infected with both VV-T3pol and VV-lacI (multiplicities of infection of 2.5 and 10, respectively), greater than 30-fold repression of CAT gene activity by lac repressor was observed . This could be reversed to unrepressed levels by the presence of 10 mM o-nitrophenyl-beta-D-galactoside (IPTG) in the medium . Regulated expression of the target gene was observed with cell lines that had been maintained for over 1 year (greater than 50 passages in culture), and Southern blot analysis revealed the presence of the CAT gene only in the nuclear fraction in these cells, demonstrating the stability of the target gene . These results indicate that vaccinia virus-encoded proteins can function in the mammalian nucleus and provide the basis for a genetic system in which essential vaccinia virus genes, placed in the chromosome of a cell, can be used to complement defective virus particles . This approach may prove useful for other virus systems. Virology, 1990 Oct, 178(2), 373 - 83 Complementation of a vesicular stomatitis virus glycoprotein G mutant with wild-type protein expressed from either a bovine papilloma virus or a vaccinia virus vector system; Lefkowitz EJ et al.; Using a complementation assay, we have evaluated the potential of two eukaryotic expression systems to produce functional virus proteins . The first expression system was based on a bovine papilloma virus (BPV) eukaryotic expression vector which contained a copy of the gene for the membrane glycoprotein G of vesicular stomatitis virus (VSV) . This vector was transfected into a mouse cell line, and transformed cell clones constitutively expressing VSV G protein were selected . These cell clones were then screened for their ability to support the replication of a temperature-sensitive G mutant of VSV (tsO45) at the permissive and nonpermissive temperatures . A 100-fold increase in tsO45 titer was observed in some of the G protein-producing cell lines in comparison with nonproducing cells . These results were compared with complementation by VSV G protein expressed from a second expression system utilizing a vaccinia virus (VV) recombinant which produced bacteriophage T7 RNA polymerase . T7 RNA polymerase expressed in cells infected with the vaccinia recombinant produced VSV G transcripts from a plasmid which had been transfected into these cells . This plasmid contained the VSV G gene cloned between T7 RNA polymerase initiation and termination signals . VSV G protein expressed by this system was able to complement tsO45 replication at the nonpermissive temperature, and yielded much greater levels of complemented virus than the BPV system . When calcium phosphate-mediated transfection was used to introduce the VSV G plasmid vector into cells infected with the VV recombinant, a complementation efficiency as high as 1500-fold was obtained . Using lipofectin-mediated transfection, a 15,000-fold increase in virus titer could be obtained in G protein-producing cells in contrast to nonproducing cells . At the nonpermissive temperature, yields of temperature-sensitive virus were within 10-fold of the yields obtained at the permissive temperature . Virus produced in this system was shown to be a pseudotype which contained wild-type G protein in the viral envelope but still maintained the temperature-sensitive genotype . This expression system will be used to study the extent to which the integrity of the G coding sequence of wild-type VSV might be altered in the absence of selection pressure for functional G protein during VSV replication. J Bacteriol, 1990 Oct, 172(10), 6106 - 11 Differential translation of cell division proteins; Mukherjee A et al.; Cloned division genes (ftsQ and ftsA) and the gene for beta-lactamase (bla) were transcribed in vivo from a bacteriophage T7 promoter under conditions which blocked the use of other promoters . The different coding regions of single mRNAs were translated with widely different efficiencies, such that the ratio of beta-lactamase production to FtsQ production was about 75:1 . The relative rates of translation of the division proteins reflected their relative rates of production from normal chromosomal promoters (FtsA greater than FtsQ) . We show that the low rates of production of FtsQ and FtsA proteins are due to their ribosome-binding sequences and that there is no obligatory translational coupling between them, despite the close proximity of the genes . Levels of translation of FtsA are shown to be proportional to levels of transcription, and therefore there is no evidence of variable regulation of translation. Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 8165 - 9 Protein-DNA conformational changes in the crystal structure of a lambda Cro-operator complex; Brennan RG et al.; The structure of a complex of bacteriophage lambda Cro protein with a 17-base-pair operator has been determined at 3.9-A resolution . Isomorphous derivatives obtained by the synthesis of site-specific iodinated DNA oligomers were of critical importance in solving the structure . The crystal structure contains three independent Cro-operator complexes that have very similar, although not necessarily identical, conformations . In the complex, the protein dimer undergoes a large conformational change relative to the crystal structure of the free protein . One monomer rotates by about 40 degrees relative to the other, this being accomplished primarily by a twisting of the two beta-sheet strands that connect one monomer with the other . In the complex, the DNA is bent by about 40 degrees into the shape of a boomerang but maintains essentially Watson-Crick B-form . In contrast to other known protein-DNA complexes, the DNA is not stacked end-to-end . The structure confirms the general features of the model previously proposed for the interaction of Cro with DNA. Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 8095 - 9 Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library; Mullinax RL et al.; We have applied a molecular biology approach to the identification of human monoclonal antibodies . Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction . These products, containing coding sequences for numerous immunoglobulin heavy- and kappa light-chain variable and constant region domains, were inserted into modified bacteriophage lambda expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library . Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library . These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested . We estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen. J Bacteriol, 1990 Oct, 172(10), 5617 - 23 Translational efficiency of phi X174 lysis gene E is unaffected by upstream translation of the overlapping gene D reading frame; Blasi U et al.; The lysis gene E of bacteriophage phi X174 is entirely embedded in gene D . Expression studies of genes D and E in Escherichia coli minicells and lysis times obtained in the presence or absence of D translation showed that the simultaneous expression of gene D does not affect protein E production . Thus, unlike other overlapping gene pairs, gene E expression is independent from the upstream translation of gene D . lacZ fusion studies and primer extension inhibition analysis (toeprinting) revealed an intrinsically weak E ribosome-binding site, which seems to be the major factor determining the low expression rate of the gene and thus proper scheduling of cell lysis. EMBO J, 1990 Oct, 9(10), 3253 - 60 The p53 tumour suppressor protein is phosphorylated at serine 389 by casein kinase II; Meek DW et al.; The entire coding sequence of wild-type mouse p53 was expressed in Escherichia coli under control of the PL promoter of bacteriophage lambda . The bacterial p53 protein had identical mobility to p53 from SV3T3 cells on SDS polyacrylamide gels and was recognized in bacterial lysates by three p53-specific monoclonal antibodies, including PAb246 which is specific for wild-type mouse p53 . Immunoprecipitates of the bacterial p53 were phosphorylated by a highly purified preparation of rat casein kinase II; the stoichiometry of incorporation was approximately 1 mol of phosphate per mol of p53 . The phosphorylated residue was identified by phosphopeptide mapping as serine 389, which is a major site of p53 phosphorylation in vivo . p53 (serine 389) kinase activity was detected on lysates of SV3T3 cells; this activity co-purified with casein kinase II on phosphocellulose and Mono Q columns and was inhibited by heparin . Immunoprecipitates of the p53-T antigen complex from SV3T3 cells also had associated serine 389 kinase activity . Phosphorylation of serine 389 by this kinase was potently inhibited by heparin and quenched by excess unlabelled GTP . The data indicate that p53 is a physiological substrate of casein kinase II, which is stimulated in response to mitogens, phosphorylates nuclear oncoproteins, and may play a role in the transduction of extracellular signals to the nucleus. Indian J Biochem Biophys, 1990 Oct, 27(5), 275 - 9 Molecular cloning of human satellite DNA sequences and their use in detecting DNA polymorphism; Ehtesham NZ et al.; A approximately 400 bp HaeIII human genomic satellite DNA band was cloned into pUC18 to construct a partial library . A fragment of bacteriophage M13 containing a sequence homologous to the human minisatellite core was cloned in pUC18 and was used as a probe to isolate a approximately 350 bp human satellite clone (pTRF5.6) from the partial library . Other clones from this library showed a wide variation in terms of size and hybridization to the pTRF5.6 clone . Human DNA from different individuals was digested with restriction enzymes, Southern transferred and probed with TRF5.6 . Individual-specific complex pattern of DNA bands was produced . TRF5.6, therefore, could be useful as a probe for detecting genetic polymorphism. Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 7963 - 7 A monoclonal antibody that neutralizes Epstein-Barr virus, human cytomegalovirus, human herpesvirus 6, and bacteriophage T4 DNA polymerases; Tsai CH et al.; A monoclonal antibody (mAb) designated 55H3 was produced by using chemically induced Epstein-Barr virus genome-positive B95-8 cells . mAb 55H3, which reacted with an 85- to 80-kDa polypeptide, neutralized Epstein-Barr virus-encoded DNA polymerase activity in crude extracts of chemically induced M-ABA, HR-1, and B95-8 cells, as well as the partially purified Epstein-Barr virus DNA polymerase in a dose-dependent manner . The mAb also neutralized the virus-encoded DNA polymerase activity from cells infected with human cytomegalovirus, human herpesvirus 6, and the purified bacteriophage T4 DNA polymerases . However, mAb 55H3 did not neutralize the DNA polymerase activities encoded for by herpes simplex virus types 1 and 2, the reverse transcriptase of avian myeloblastosis virus, or Escherichia coli DNA polymerase 1 (Klenow fragment) . These results suggest that mAb 55H3 recognizes an epitope common to some herpesviruses and T4 DNA polymerases and further supports the hypothesis that these organisms are evolutionarily related. Biochem Biophys Res Commun, 1990 Sep 28, 171(3), 988 - 95 Isolation and sequencing of mouse angiogenin DNA; Bond MD et al.; The mouse genomic DNA for angiogenin, a potent blood vessel inducing protein, has been isolated from a bacteriophage library using the human angiogenin gene as a probe . The 1129 bp fragment contains 499 bp in the 5' flanking region, 192 bp in the 3' flanking region, and 438 bp coding for the mature protein (121 amino acids) and signal peptide (24 amino acids) . Potential TATA box and AATAAA polyadenylation sequences are present, and a consensus sequence for an intron 3' boundary occurs 16 bp upstream of the Met-(24) codon, suggesting the presence of an intron in the 5' region . The protein sequence inferred from the DNA is 76% identical to that of human angiogenin, and matches the sequences obtained previously from tryptic peptides of a serum-derived mouse angiogenin . The critical catalytic residues of human angiogenin are conserved in the mouse protein, as are the six cysteines necessary for disulfide bond formation. Nucleic Acids Res, 1990 Sep 25, 18(18), 5401 - 6 Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector; Mellits KH et al.; Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro . Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation . We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI . Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays . We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI . This method appears to be generally applicable. J Mol Biol, 1990 Sep 20, 215(2), 287 - 99 Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA; Serwer P et al.; When bacteriophage T7 gene 6 exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed . By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made . (1) Most degradation of intracellular DNA requires the presence of T7 gene 3 endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene 3 and gene 6 products are absent . (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene 6 exonuclease . (3) Fragments produced in the absence of gene 6 exonuclease are linear and do not further degrade; precursors of the fragments are non-linear . (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication . On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene 6 exonuclease is caused by cleavage at branches . The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene 6 exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene 6 exonuclease during a wild-type T7 infection . Observation of joining of genomes in the absence of gene 6 exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication . The observed selective shortening of the left end indicates that gene 6 exonuclease is required for formation of most, possibly all, mature left ends. J Mol Biol, 1990 Sep 20, 215(2), 237 - 44 Characterization of the interaction between topoisomerase II and DNA by transcriptional footprinting; Thomsen B et al.; The interaction between calf thymus topoisomerase II and DNA has been characterized using a transcription assay . A highly preferred recognition sequence for topoisomerase II was inserted in either direction downstream from a promoter specific for a bacteriophage RNA polymerase . The presence of topoisomerase II-DNA complexes on the template provoked blockage of transcription, yielding RNA transcripts terminated 5' to the topoisomerase II binding site . A footprint of topoisomerase II, derived from transcription towards the complex from either side, revealed that eukaryotic topoisomerase II binds a region of 28 base-pairs with a highly protected central core of 22 base-pairs . The binding region was located symmetrically around the topoisomerase II-mediated cleavage site . In agreement with this result, optimal topoisomerase II-mediated cleavage was observed with a DNA substrate consisting of a 28-mer oligonucleotide homologous to the protected region . Stepwise removal of base-pairs from the ends of the 28-mer gradually reduced the level of enzyme-mediated cleavage. Science, 1990 Sep 14, 249(4974), 1261 - 5 Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase; Ostrander EA et al.; Fusion of the DNA-binding domain of yeast GAL4 protein to the amino terminus of bacteriophage T7 RNA polymerase yields a chimera that retains the characteristics of its components . The presence of the GAL4 peptide allows the chimeric enzyme to anchor itself on the DNA template, and this anchoring in turn drives the formation of a supercoiled DNA loop, in linear or circular templates, when RNA synthesis at the polymerase site forces a translocation of the DNA relative to the site . Nonspecific interaction between the chimeric enzyme and DNA appears to be sufficient to effect supercoiling during transcription . Transcription by the chimeric polymerase is strictly dependent on the presence of a T7 promoter; thus it provides a tool in vitro and in vivo for specifically supercoiling DNA segments containing T7 promoter sequences. Biochem Biophys Res Commun, 1990 Sep 14, 171(2), 581 - 8 Structure and fluctuations of bacteriophage T4 glutaredoxin modelled by molecular dynamics; Nilsson O et al.; A 120 ps molecular dynamics (MD) trajectory for bacteriophage T4 glutaredoxin was calculated including non-inertial solvent effects . The potential energy attains an equilibrated regime after the first 20 ps . The r.m.s . difference of all non-hydrogen atoms between X-ray and average MD structures for the regular secondary structure is 0.99A which shows that the MD simulation reproduces the essentials of the structure with high accuracy . Loop displacements are detected, shown by the larger full structure all non-hydrogen atom r.m.s . difference of 1.2A . The fluctuation pattern derived from MD agrees fairly well with that derived from X-ray isotropic temperature factors . The active site is a stable structural region in this MD modellization . Structural changes are put in context with the protein's function. Biochemistry, 1990 Sep 11, 29(36), 8207 - 10 Folding of the reduced form of the thioredoxin from bacteriophage T4; Borden KL et al.; The folding pattern for bacteriophage T4 thioredoxin is similar to that of the oxidized form {Borden, K . L . B., & Richards, F . M . (1990) Biochemistry 29, 3071-3077} . Equilibrium and kinetic studies were carried out by fluorescence and circular dichroism techniques . The same box model proposed for the oxidized form, with four identifiable states, can accommodate most of the data: N----Uc----Ut----It----N, where N is the native state, Uc is the unfolded species with Pro 66 in the cis form, Ut is the unfolded species with Pro 66 in the trans form, and It is a trans-Pro 66 intermediate with a volume comparable to that of N . However, the relative importance of the different components is shifted between the oxidized and reduced proteins . In spite of the small size of the disulfide loop, the Cys 14-Cys 17 bond appears to be important in stabilizing It . The tertiary structure as monitored by near-UV CD and fluorescence indicates that the reduced form is significantly less stable than its oxidized counterpart; however, the two secondary structures, as seen by far-UV CD, are very similar . The intermediate It behaves as though it is cold denaturated at 4 degrees C. Nucleic Acids Res, 1990 Sep 11, 18(17), 5031 - 6 Frameshift suppression at tandem AGA and AGG codons by cloned tRNA genes: assigning a codon to argU tRNA and T4 tRNA(Arg); Spanjaard RA et al.; Arginine is coded for by CGN (N = G, A, U, C), AGA and AGG . In Escherichia coli there is little tRNA for AGA and AGG and the use of these codons is strongly avoided in virtually all genes . Recently, we demonstrated that the presence of tandem AGA or AGG codons in mRNA causes frameshifts with high frequency . Here, we show that phaseshifts can be suppressed when cells are transformed with the gene for tRNA(T4Arg) or E . coli tRNA(argU,Arg) demonstrating that such errors are the result of tRNA depletion . Bacteriophage T4 encoded tRNA(Arg) (anticodon UCU) corrects shifts at AGA-AGA but not at AGG-AGG, suggesting that this tRNA can only read AGA . Similarly, comparison of the translational efficiencies in an argU (Ts) mutant and in its isogenic wild type parent indicates that argU tRNA (anticodon UCU) reads AGA but not AGG . An argU (Ts) mutant barely reads through AGA-AGA at 42 degrees C but translation of AGG-AGG is hardly, if at all, affected . Overexpression of argU+ relaxes the codon specificity . The thermosensitive mutant in argU, previously called dnaY because it is defective in DNA replication, can be complemented for growth by the gene for tRNA(T4Arg) . This implies that the sole function of the argU gene product is to sustain protein synthesis and that its role in replication is probably indirect. J Mol Biol, 1990 Sep 5, 215(1), 31 - 9 Identification of a region of the bacteriophage T3 and T7 RNA polymerases that determines promoter specificity; Joho KE et al.; Bacteriophages T7 and T3 encode DNA-dependent RNA polymerases that are 82% homologous, yet exhibit a high degree of specificity for their own promoters . A region of the RNA polymerase gene (gene 1) that is responsible for this specificity has been localized using two approaches . First, the RNA polymerase genes of recombinant T7 x T3 phage that had been generated in other laboratories in studies of phage polymerase specificity were characterized by restriction enzyme mapping . This approach localized the region that determines promoter specificity to the 3' end of the polymerase gene, corresponding to the carboxyl end of the polymerase protein distal to amino acid 623 . To define more closely the region of promoter specificity, a series of hybrid T7/T3 RNA polymerase genes was constru