Proc Natl Acad Sci U S A, 1992 Jan 15, 89(2), 763 - 7 A DNA polymerase alpha pause site is a hot spot for nucleotide misinsertion; Fry M et al.; In this study we examined whether the arrest of DNA polymerase alpha (pol alpha)-catalyzed DNA synthesis at template pause sites entails terminal nucleotide misincorporation . An approach was developed to identify the 3'-terminal nucleotide in nascent DNA chains that accumulate at pause sites . A radioactive 5'-end-labeled primer was annealed to a bacteriophage M13mp2 single-stranded DNA template and elongated by pol alpha . Individual DNA chains that were accumulated at pause sites were resolved by sequencing gel electrophoresis, isolated, and purified . These DNA chains were elongated by pol alpha by using four annealed synthetic DNA templates, each of which contained a different nucleotide at the position opposite the 3' terminus of the arrested chain . Owing to the high preference of pol alpha for matched-over-mismatched primer termini, only those templates that contain a nucleotide that is complementary to the 3' terminus of the isolated pause-site chain are copied . Electrophoresis of product DNA showed the extent of copying of each template and thus identified the 3'-terminal nucleotide of the pause-site chains . We found that product DNA chains terminate with a noncomplementary 3'-terminal nucleotide opposite pause sites within the sequence 3'-d(AAAA)-5' at positions 6272-6269 of the M13mp2 genome . pol alpha catalyzed misincorporation of dG or dA into the 3' terminus of nascent chains opposite two of the M13mp2 template dA residues . A similar analysis of a different pause site did not reveal significant misincorporation opposite template dC . These results suggest that some but not all sites at which pol alpha pauses may constitute loci of mutagenesis. J Biol Chem, 1992 Jan 15, 267(2), 1225 - 30 DNA-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 DNA polymerase; Blanco L et al.; In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5'-dAMP . Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer . The nature of the TP-dAMP linkage was shown to be identical (a O-5'-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication . Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases . However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield . This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site . Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction . According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a "non-DNA-instructed" protein-primed deoxynucleotidylation. J Biol Chem, 1992 Jan 15, 267(2), 1190 - 7 Saccharomyces cerevisiae elongation factor 2 . Genetic cloning, characterization of expression, and G-domain modeling; Perentesis JP et al.; The elongation factor 2 (EF-2) genes of the yeast Saccharomyces cerevisiae have been cloned and characterized with the ultimate goal of gaining a better understanding of the mechanism and control of protein synthesis . Two genes (EFT1 and EFT2) were isolated by screening a bacteriophage lambda yeast genomic DNA library with an oligonucleotide probe complementary to the domain of EF-2 that contains diphthamide, the unique posttranslationally modified histidine that is specifically ADP-ribosylated by diphtheria toxin . Although EFT1 and EFT2 are located on separate chromosomes, the DNA sequences of the two genes differ at only four positions out of 2526 base pairs, and the predicted protein sequences are identical . Genetic deletion of each gene revealed that at least one functional copy of either EFT gene is required for cell viability . Messenger RNA levels of yeast EF-2 parallel cellular growth and peak in mid-log phase cultures . The EF-2 protein sequence is strikingly conserved through evolution . Yeast EF-2 is 66% identical to, and shares over 85% homology with, human EF-2 . In addition, yeast and mammalian EF-2 share identical sequences at two critical functional sites: (i) the domain containing the histidine residue that is modified to diphthamide and (ii) the threonine residue that is specifically phosphorylated in vivo in mammalian cells by calmodulin-dependent protein kinase III, also known as EF-2 kinase . Furthermore, yeast EF-2 also contains the Glu-X-X-Arg-X-Ile-Thr-Ile "effector" sequence motif that is conserved among all known elongation factors, and its GTP-binding domain exhibits strong homology to the G-domain of Escherichia coli elongation factor Tu (EF-Tu) and other G-protein family members . Based upon these observations, we have modeled the G-domain of the deduced EF-2 protein sequence to the solved crystallographic structure for EF-Tu. Eur J Biochem, 1992 Jan 15, 203(1-2), 53 - 8 Rat liver DNA ligases . Catalytic properties of a novel form of DNA ligase; Elder RH et al.; A novel form of rat liver DNA ligase (molecular mass 100 kDa) can be differentiated from DNA ligase I by several biochemical parameters . It is a more heat-labile enzyme and unable to join blunt-ended DNA, even in the presence of poly(ethylene glycol) concentrations which stimulate such joining by DNA ligase I and T4 DNA ligase . It also lacks the AMP-dependent nicking/closing reaction, which is a property of all other DNA ligases tested so far, including DNA ligase I from rat liver . Both rat liver DNA ligases were inhibited by deoxyadenosinetriphosphate, however this inhibition was competitive with respect to ATP, for DNA ligase I (Ki 22 microM) and non-competitive for the 100-kDa DNA ligase (Ki 170 microM) . These results support the idea that, when compared with other DNA ligases, the novel form of DNA ligase has a unique AMP-binding site, may have an absolute requirement for single-strand breaks and, furthermore, may have an altered reaction mechanism to that which is conserved from bacteriophage to mammalian DNA ligase I. Gene, 1992 Jan 15, 110(2), 251 - 6 Synthesis of the biologically active beta-subunit of human nerve growth factor in Escherichia coli; Negro A et al.; The gene (NGFB) encoding the beta subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters PR and PL, and expressed in Escherichia coli . The recombinant protein represented approximately 3% of the total cellular protein . Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule . Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons . These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E . coli. J Biol Chem, 1992 Jan 15, 267(2), 938 - 43 Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion; Lee JI et al.; Leader peptidase of Escherichia coli spans the plasma membrane twice with its amino terminus on the periplasmic surface of the membrane and its large carboxyl-terminal domain protruding into the periplasm . To monitor the transfer of the amino terminus of leader peptidase to the periplasm, we have constructed a fusion protein between the 18-residue amino-terminal periplasmic domain of Pf3 bacteriophage coat protein and the beginning of leader peptidase . We find that neither the SecA or SecY proteins nor a transmembrane electrochemical potential is required for insertion of the amino terminus, while the transfer of the carboxyl-terminal domain of leader peptidase has these requirements . The first 35 residues of leader peptidase, which include the first hydrophobic domain and the carboxyl-terminal positively charged cluster, are sufficient to insert the amino terminus . When positively charged residues are introduced before the first transmembrane segment, translocation of the amino terminus is abolished . These studies in protein membrane topogenesis, showing that there are different requirements for amino and carboxyl termini insertion, indicate that multiple mechanisms exist even within the same protein. Biochemistry, 1992 Jan 14, 31(1), 57 - 65 Site-specific enthalpic regulation of DNA transcription at bacteriophage lambda OR; Koblan KS et al.; Binding of cI repressor to DNA fragments containing the three specific binding sites of the right operator (OR) of bacteriophage lambda was studied in vitro over the temperature range 5-37 degrees C by quantitative footprint titration . The individual-site isotherms, obtained for binding repressor dimers to each site of wild-type OR and to appropriate mutant operator templates, were analyzed for the Gibbs energies of intrinsic binding and pairwise cooperative interactions . It is found that dimer affinity for each of the three sites varies inversely with temperature, i.e., the binding reactions are enthalpy driven, unlike many protein-DNA reactions . By contrast, the magnitude of the pairwise cooperativity terms describing interaction between adjacently site-bound repressor dimers is quite small . This result in combination with the recent finding that repressor monomer-dimer assembly is highly enthalpy driven (with delta H degrees = -16 kcal mol-1) {Koblan, K . S., & Ackers, G . K . (1991) Biochemistry 30, 7817-7821} indicates that the associative contacts between site-bound repressors that mediate cooperativity are unlikely to be the same as those responsible for dimerization . The intrinsic binding enthalpies for all three sites are negative (exothermic) and nearly temperature-invariant, indicating no heat capacity changes on the scale of those inferred in other protein-DNA systems . However, the three operator sites are affected differentially by temperature: the intrinsic binding free energies for sites OR1 and OR3 change in parallel over the entire range, delta H0OR1 = -23.3 +/- 4.0 kcal mol-1 and delta H0OR3 = -22.7 +/- 1.2 kcal mol-1.(ABSTRACT TRUNCATED AT 250 WORDS) Dev Genet, 1992, 13(2), 97 - 102 Bacteriophage lambda DNA fragments replicate in the Paramecium macronucleus: absence of active copy number control; Kim CS et al.; We show that bacteriophage lambda DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules . These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase . The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes . We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur . Instead, the results suggest that injected DNA is replicated once per cell division. Nature, 1992 Jan 9, 355(6356), 137 - 43 Atomic structure of single-stranded DNA bacteriophage phi X174 and its functional implications; McKenna R et al.; The mechanism of DNA ejection, viral assembly and evolution are related to the structure of bacteriophage phi X174 . The F protein forms a T = 1 capsid whose major folding motif is the eight-stranded antiparallel beta barrel found in many other icosahedral viruses . Groups of 5 G proteins form 12 dominating spikes that enclose a hydrophilic channel containing some diffuse electron density . Each G protein is a tight beta barrel with its strands running radially outwards and with a topology similar to that of the F protein . The 12 'pilot' H proteins per virion may be partially located in the putative ion channel . The small, basic J protein is associated with the DNA and is situated in an interior cleft of the F protein . Tentatively, there are three regions of partially ordered DNA structure, J Mol Biol, 1992 Jan 5, 223(1), 67 - 78 Nucleosome arrays inhibit both initiation and elongation of transcripts by bacteriophage T7 RNA polymerase; O'Neill TE et al.; We have examined the effects of nucleosome cores on the initiation and elongation of RNA transcripts by phage T7 RNA polymerase in vitro . A transcription template, pT207-18, was constructed containing tandemly repeated 207 base-pair (bp) nucleosome positioning sequences from a sea urchin (Lytechinus variegatus) 5 S RNA gene inserted between the T7 and SP6 transcription promoters of pGEM-3Z . Nucleosome cores were reconstituted onto supercoiled, closed circular pT207-18 DNA and double label transcription experiments were performed to determine the effects of nucleosome cores on the initiation and elongation of transcripts by T7 RNA polymerase . Both transcript initiation and elongation were inhibited, the extent of the inhibition being directly proportional to the number of nucleosome cores reconstituted onto the pT207-18 DNA templates . Time course transcription experiments indicated that nucleosome cores caused a reduction in the equilibrium length of transcripts and not mere retardation of elongation rates . Continuous regularly spaced linear arrays of nucleosomes were obtained by digesting reconstituted nucleosomel pT207-18 templates with DraI, for which a unique restriction site lies within the nucleosome positioning region of the 207 bp 5 S rDNA repeat sequence . After in vitro transcription with T7 RNA polymerase an RNA ladder with 207 nucleotide spacing was obtained, indicating that transcription can occur through continuous arrays of positioned nucleosome cores . It is demonstrated that nucleosome cores partially inhibit the elongation of transcripts by T7 RNA polymerase, while allowing passage of the transcribing polymerase through each nucleosome core at an upper limit efficiency of 85% . Hence, complete transcripts are produced with high efficiency from short nucleosomal templates, while the production of full-length transcripts from long nucleosomal arrays is relatively inefficient . The results indicate that nucleosome cores have significant inhibitory effects in vitro not only on transcription initiation but on transcription elongation as well, and that special mechanisms may exist to overcome these inhibitory effects in vivo. J Mol Biol, 1992 Jan 5, 223(1), 55 - 66 Direct interaction between two Escherichia coli transcription antitermination factors, NusB and ribosomal protein S10; Mason SW et al.; The Escherichia coli proteins NusB and ribosomal protein S10 are important for transcription antitermination by the bacteriophage lambda N protein . We have used sucrose gradient co-sedimentation and affinity chromatography with immobilized ribosomal protein S10, a glutathione S-transferase-S10 fusion protein, and NusB to show that NusB binds directly and very selectively to S10 . The interaction is non-ionic and has an estimated Kd value of 10(-7) M . We hypothesize that NusB binds to N-modified transcription complexes primarily by interacting with S10. J Mol Biol, 1992 Jan 5, 223(1), 23 - 5 GTPase activity of bacteriophage T4 sheath protein; Serysheva II et al.; We show by nuclear magnetic resonance studies that, following GTP hydrolysis during phage T4 sheath contraction, GDP remains bound to the sheath protein (gp18), whereas orthophosphate is released . gp18 in the contracted state has GTPase activity and can hydrolyse exogenous GTP; the reaction is calcium-dependent and displays high substrate specificity . The process comprises two steps: (1) displacement of GDP from gp18 by exogenous GTP, and (2) GTP hydrolysis proper . The first step appears to be rate-limiting and to be accelerated when the nucleotide-protein interaction is mechanically disrupted by sonication. J Biol Chem, 1992 Jan 5, 267(1), 166 - 72 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions; Cheng KC et al.; Mutations caused by oxidative DNA damage may contribute to human disease . A major product of that damage is 8-hydroxyguanine (oh8Gua) . Because of differences in experimental design, the base pairing specificity of oh8G in vivo is not completely resolved . Here, oh8dGTP and DNA polymerase were used in two complementary bacteriophage plaque color assays to examine the mutagenic specificity of oh8Gua in vivo . The first is a reversion assay that detects all three single-base substitutions caused by misreading of guanine analogues inserted at a specific site . oh8Gua at that site gave a mutation frequency of 0.7% . Twenty-two of the 23 mutations were G----T substitutions . The second assay, a forward mutation assay, tests the mispairing potential of any altered nucleotide 1) during incorporation as substrate nucleotide, and 2) after multiple incorporations into a single-stranded DNA gap region of M13mp2 . Substituting oh8dGTP for dGTP during polymerization produced 16% mutants; two classes of mutations were observed, both caused by pairing of oh8Gua with A . Seventy-six of 78 mutations were A----C substitutions, and two were G----T substitutions . These assays thus illustrate mutagenic replication of oh8Gua as template causing G----T substitutions and misincorporation of oh8Gua as substrate causing A----C substitutions, both caused by oh8Gua.A mispairs. J Biol Chem, 1992 Jan 5, 267(1), 455 - 61 The purification and properties of the scaffolding protein of bacteriophage lambda; Ziegelhoffer T et al.; The Nu3 gene of bacteriophage lambda resides within a cluster of genes that specify structural components of the bacteriophage head . Previous experiments indicate that the Nu3 gene product (gpNu3) is associated with immature proheads but is not detectable in mature proheads or bacteriophage particles, hence its classification as a scaffolding protein . The Nu3 gene has been cloned and overexpressed, and its protein product has been purified . The purified protein is biologically active, as demonstrated by its ability to complement a gpNu3-deficient extract in an in vitro assembly reaction . The sequence of the amino terminus of the protein indicates that translation of Nu3 starts at nucleotide position 5,342 on the standard lambda DNA sequence, yielding a protein with a calculated Mr of 13,396 . A combination of gel exclusion chromatography and velocity sedimentation gradient data indicates that gpNu3 possesses an unusually elongated shape. Nature, 1992 Jan 2, 355(6355), 89 - 91 DNA twisting and the effects of non-contacted bases on affinity of 434 operator for 434 repressor; Koudelka GB et al.; The bacteriophage 434 repressor regulates gene expression by binding with differing affinities to the six operator sites on the phage chromosome . The symmetrically arrayed outer eight base pairs (four in each half-site) of these 14-base-pair operators are highly conserved but the middle four bases are divergent . Although these four base pairs are not in contact with repressor, operators with A.T or T.A base pairs at these positions bind repressor more strongly than those bearing C.G or G.C, suggesting that these bases are important for the repressor's ability to discriminate between operators . There is evidence that the central base pairs influence operator function by constraining the twisting and/or bending of DNA . Here we show that there is a relationship between the intrinsic twist of an operator, as determined by the sequence of its central bases, and its affinity for repressor; an operator with a lower affinity is undertwisted relative to an operator with higher affinity . In complex with repressor, the twist of both high- and low-affinity operators is the same . These results indicate that the intrinsic twist of DNA and its twisting flexibility both affect the affinity of 434 operator for repressor. Intervirology, 1992, 33(1), 61 - 4 Absence of a gene dosage effect during bacteriophage T3- and T7-coded RNA polymerase synthesis; Horn D et al.; The rates of synthesis of phage-coded RNA polymerase upon infection of Escherichia coli by bacteriophages T3 or T7 were measured at different MOIs under permissive and non-permissive conditions . At MOIs from 1 to 15, these rates did not vary appreciably, at MOIs of about 20 there was a slight depression in the synthesis rate . The reason for this absence of a positive gene dosage effect is unknown. Intervirology, 1992, 33(1), 6 - 16 Isolation and characterization of a portal protein-DNA complex from dsDNA bacteriophage; Zachary A et al.; A portal protein-DNA complex was isolated from bacteriophage disrupted in 70% acetic acid . This treatment dissociated most of the phage structural proteins, while the portal protein ring remained attached to the highly condensed naked DNA . In the absence of a headful of DNA, the portal ring was not protected . Purification of the portal ring-DNA complex by density gradient centrifugation showed that a dsDNA fragment of about 40 bp was protected from DNase digestion by this association, suggesting it was internalized within the 140 A long, 20-40 A in diameter T4 apical channel of the ring dodecamer . A portal ring-DNA complex could be isolated from a number of phages (T4, T7, Phi29), and the end(s) of phage T7 DNA was (were) found to be preferentially associated with the ring complex. Biochem J, 1992 Jan 1, 281 ( Pt 1), 203 - 10 Structure and expression of the rat epididymal secretory protein I gene . An androgen-regulated member of the lipocalin superfamily with a rare splice donor site; Girotti M et al.; The complete rat epididymal secretory protein I (ESP I) gene was isolated from a genomic library constructed in bacteriophage lambda Charon 4A . The complete nucleotide sequence of the gene and its immediate 5' and 3' flanking sequences were determined . Interesting features include the presence of a rare, but functional, splice donor site (...GC) and the presence of a putative androgen-receptor-binding element . A detailed analysis of ESP I regulation was carried out after castration and subsequent testosterone treatment, demonstrating the requirement for androgens . Efferent-duct ligation and cryptorchism, on the other hand, had no effect on the steady-state concentrations of ESP I transcripts . Comparison of the exon/intron organization of the ESP I gene with those of members of the lipocalin superfamily provides strong support for a common ancestral origin. Mol Immunol, 1992 Jan, 29(1), 21 - 30 Cloning and expression of the variable regions of mouse myeloma protein MOPC315 in E . coli: recovery of active FV fragments; Cheadle C et al.; Recombinant DNA techniques were used to clone and express the FV portion of MOPC315, a mouse myeloma protein with a high affinity for 2,4-dinitrophenyl (DNP) . The FV fragment consists of a heterodimer of heavy and light chain variable domains (VH and VL) . Two separate bacterial plasmid constructs, containing either a variable region cDNA for the light chain or a variable region synthetic gene for the heavy chain demonstrated high levels of expression (150-200 mg/L) under control of the bacteriophage T7 promoter . Recombinant chains were initially recovered as inclusion bodies and then dissolved separately in 8 M urea, combined together, and refolded by subsequent chaotrope removal . Biologically active FV was affinity purified from the chain mixture by specific binding to DNP-lysine-Sepharose . Yields of active material as high as 20% were obtained with activity confirmed by fluorescence quench analysis . The purified FV displayed a binding affinity of 4.8 +/- 0.3 x 10(-7) M which was identical to the native FV . Chimeric FVs composed of recombinant and native chain mixtures yielded similar results . Recombinant MOPC315 FV activity was also obtained using a single chain construct (sFV), in which recombinant VH and VL were linked via a (Gly4Ser)3 spacer region . Binding affinity of the sFV was shown to be the same as the recombinant and native FVs . The ease of purification and characterization of active MOPC315 as the recombinant and native FVs . The ease of purification and characterization of active MOPC315 FV makes this system useful in the study of the optimization of antibody production in bacteria. Genes Dev, 1992 Jan, 6(1), 149 - 59 Specificity of Escherichia coli endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site; Ehretsmann CP et al.; Endoribonuclease RNase E has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs . We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32 . A series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo . The same mutations were transferred into T4 by homologous recombination . In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations . Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro . The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro . These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W = A or U) at the cleavage site . Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing . Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure. Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 11 - 5 Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins; Murray MT et al.; Storage of maternal mRNAs as nontranslated ribonucleoprotein (RNP) complexes is an adaptive strategy in various vertebrate and invertebrate oocytes, for rapid translational recruitment during embryonic development . Previously, we showed that Xenopus laevis oocytes have a soluble cytoplasmic pool of mRNA-binding proteins and particles competent for messenger RNP assembly in vitro . Here we report the isolation of cDNAs for the most abundant messenger RNPs, the 54- and 56-kDa polypeptide (p54/p56) components of the approximately 6S mRNA-binding particle, from an ovarian expression library . The nucleotide sequence of p56 cDNA is almost identical to that recently reported for the putative Xenopus transcription factor FRG Y2 . p54 and p56 are highly homologous and are smaller than expected by SDS/PAGE (36 kDa and 37 kDa) due to anomalous electrophoretic mobility . They lack the "RNP consensus motif" but contain four arginine-rich "basic/aromatic islands" that are similar to the RNA-binding domain of bacteriophage mRNA antiterminator proteins and of tat protein of human immunodeficiency virus . The basic/aromatic regions and a second conspicuous 100-amino acid "domain C" of p54 and p56 are conserved in the following DNA-binding proteins: human proteins dpbA, dpbB, and YB-1, rat protein EFIA, and Xenopus protein FRG Y1, all reported to bind to DNA; domain C is homologous to the major Escherichia coli cold-stress-response protein reportedly involved in translational control . Antibodies raised against a peptide of domain C have identified similar proteins in Xenopus somatic cells and in some mammalian cells and tissues . We conclude that p54 and p56 define a family of RNA-binding proteins, at least some of which may be involved in translational regulation. J Bacteriol, 1992 Jan, 174(2), 619 - 22 Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo; Iost I et al.; We show that in Escherichia coli at 32 degrees C, the T7 RNA polymerase travels over the lacZ gene about eightfold faster than ribosomes travel over the corresponding mRNA . We discuss how the T7 phage might exploit this high rate in its growth optimization strategy and how it obviates the possible drawbacks of uncoupling transcription from translation. J Bacteriol, 1992 Jan, 174(2), 595 - 600 Gene V protein-mediated translational regulation of the synthesis of gene II protein of the filamentous bacteriophage M13: a dispensable function of the filamentous-phage genome; Zaman GJ et al.; Introduction of a deletion in the genome of wild-type M13 bacteriophage that eliminates translational repression of M13 gene II by its cognate gene V protein had no effect on phage viability . Furthermore, it was noted that gene V protein of phage IKe, a distant relative of M13, does not function as a translational repressor of its cognate gene II protein . The data strongly indicate that the gene V protein-mediated control of gene II expression in bacteriophage M13 is an evolutionary relic of the ancestral filamentous-phage genome and thus dispensable for proper filamentous-phage replication. Virology, 1992 Jan, 186(1), 192 - 200 Synthesis of full-length transcripts of beet western yellows virus RNA: messenger properties and biological activity in protoplasts; Veidt I et al.; Full-length cDNA of beet western yellows virus genomic RNA has been cloned behind the bacteriophage T7 RNA polymerase promoter of the transcription vector BS(-) . The in vitro run-off transcription product obtained in the presence of T7 RNA polymerase and m7GpppG cap has the same messenger properties as natural viral RNA in in vitro translation systems . The full-length transcript was also able to infect Chenopodium quinoa protoplasts inoculated by electroporation . Infection could be followed by the appearance of viral coat protein in the inoculated protoplasts and the de novo synthesis of viral RNA . Site-directed mutagenesis experiments revealed that expression of beet western yellows virus open reading frame 1 and the C-terminal portion of open reading frame 6 were not required for infection of protoplasts . Additional experiments with these mutants and mutants in the other viral open reading frames should provide information concerning the requirements for beet western yellows virus replication and, ultimately, the role of virus genes in other important steps in the virus infection cycle, such as aphid transmission. Blood Cells, 1992, 18(1), 57 - 73; discussion 74 Use of 8-methoxypsoralen and long wavelength ultraviolet radiation for decontamination of platelet concentrates; Corash L et al.; Transmission of viral diseases through blood products remains a problem in transfusion medicine . We have developed a photochemical decontamination system (PCD) for platelet concentrates (PC) utilizing treatment with long wavelength ultraviolet radiation (UVA, 320-400 nm) and 8-methoxypsorlan (8-MOP) . This system is capable of inactivating 25-30 logs/hour of bacteria E . coli or S . aureus, 6 logs/hour of bacteriophage fd, 0.9 log/hour of bacteriophage R17, and 1.1 logs/hour of feline leukemia virus (FeLV) in PC . Immediately following 6 hours of PCD treatment, platelet integrity and function of PCD-treated and control PC were equivalent . After overnight storage, PCD-treated and control PC platelet properties were equal, but there was a slight reduction in TXB-2 production of PCD-treated PC compared to controls . Following PCD treatment, PC were stored for 48 to 96 hours . Platelet counts, morphology scores, extracellular LDH levels, aggregation response, dense body (db) content, and alpha granule (alpha g) content of PCD-treated and control PC were comparable . We assessed the ability of the PCD technique to inactivate intracellular and extracellular virus, quantified the degree of DNA adduct formation in contaminating lymphocytes, and measured the inhibition of polymerase chain reaction (PCR) mediated amplification of intracellular DNA . High titers of cell-free murine cytomegalovirus added to human platelet concentrates (final concentration 10(6)) were inactivated by PCD within 30 minutes . Cat renal fibroblasts infected at high levels with feline rhinotracheitis virus (FeRTV) were seeded into PC followed by PCD treatment with inactivation of 4.8 logs of FeRTV within 10 minutes . Purified human lymphocytes were seeded into PC and treated with PCD in the presence of 3H 8-MOP . Six hours of PCD treatment resulted in the formation of 9.3 to 12.8 8-MOP adducts per 1000 base pairs (bp) of DNA . PCR amplification of a 242 bp segment at the HLA-DQ alpha locus was examined . Inhibition of PCR DNA amplification was dependent on the numbers of 8-MOP adducts formed, and no amplification was present when greater than 12 adducts per 1000 bp were formed . These studies indicate that PCD can effectively inactivate high titers of cell-associated and cell-free virus seeded into standard human PC . The efficiency of DNA adduct formation can be quantitated, and the level of 8-MOP adduct formation in lymphocytes contaminating PC is comparable to the level of adduct formation in cellular DNA reported in the absence of platelets. Blood Cells, 1992, 18(1), 27 - 41; Discussion 41-2 Photochemical inactivation of viruses and bacteriophage in plasma and plasma fractions; Morel P et al.; Transfusion-associated transmission of viral diseases remains a problem . A number of methods have been developed to inactivate viral pathogens in plasma and plasma fractions, including: dry heating, wet heating, solvent-detergent treatment, and immunoaffinity purification . While some of these methods successfully inactivate pathogenic viruses, inactivation may be incomplete or result in damage to labile plasma proteins . We have developed a method of photochemical decontamination (PCD) using psoralens and long wavelength ultraviolet light to inactivate pathogenic viruses . In the present study, a spectrum of model viruses have been added to plasma and plasma fractions to examine the efficiency of photochemical decontamination and the effects on labile plasma coagulation factors . Both RNA and DNA viruses have been inactivated under conditions which permit preservation of coagulation protein function . PCD technology appears to offer a promising solution to decontamination of blood products. Arch Virol, 1992, 124(3-4), 201 - 9 Frequency of morphological phage descriptions; Ackermann HW; Bacteriophages are listed by morphotypes and host genera . At least 4.007 phages, belonging to 13 virus families, have been described since 1960 . About 3,850 phages (96%) are tailed and 154 phages (4%) are cubic, filamentous, or pleomorphic . Siphoviridae or phages with long noncontractile tails constitute 60% of tailed phages . Phages are found in over 100 bacterial genera including archaebacteria and rickettsiae . Their distribution is very uneven and probably reflects the evolutionary history of bacteria. Annu Rev Immunol, 1992, 10, 239 - 65 In vitro antibodies: strategies for production and application; Morrison SL; The approaches to the production of antibodies (Ab) using the techniques of genetic engineering and expression are reviewed . Genetic engineering facilitates the production of proteins tailormade for an intended use . Bacterial and mammalian expression systems are commonly used for the production of Ab and Ab-like molecules . While genomic or cDNA cloning can be used to obtain the relevant variable regions, PCR-based cloning approaches facilitate the acquisition of additional binding specificities . Large numbers of different chimeric Abs with murine variable regions joined to constant regions from human and other species have been expressed and found to exhibit the expected binding specificities and effector functions . These molecules have been used to study the structural basis of effector functions such as complement activation and Fc receptor binding, and potentially they may be used as therapeutic agents . Carbohydrate has been shown to influence both variable and constant region function . Single-chain Abs and fusion proteins with Ab binding specificities joined to nonimmunoglobulin sequences provide a source of Ab-like molecules with novel properties, and genetically engineered Ab-like molecules provide a source of useful antigens . Combinatorial libraries produced in bacteriophage present an alternative to hybridomas for the production of Abs with desired combining specificities . Issues of the immunogenicity of the recombinant molecules are addressed. Electrophoresis, 1992 Jan-Feb, 13(1-2), 87 - 92 The use of radioactive bacteriophage proteins as X-Y markers for silver stained two-dimensional electrophoresis gels and quantification of the patterns; Gersten DM et al.; We have demonstrated the feasibility of using bacteriophage ghost proteins, tritiated by metabolic labeling, as a set of standard markers for two-dimensional gels in which the proteins are to be detected by silver staining . The results indicate that a 2.5 microgram load of phage proteins yields a reproducible silver pattern of 48 spots . The spots can also be readily identified by radioautography and radiofluorography, establishing their value as a standard constellation of markers . Quantification of these patterns by computerized densitometry indicates a general agreement between detection by silver staining and detection by radiofluorography. Electron Microsc Rev, 1992, 5(2), 283 - 309 Bacteriophage structure; Wurtz M; The purpose of this review is to provide information of the role played by electron microscopy in respect of bacteriophage structure . This 40 years' "love story" between phages and microscopy was a valuable contribution to the progress of scientific knowledge in molecular biology . In spite of the rather drastic treatment required for electron microscopical analysis, it was possible to reveal the molecular organization and morphogenic pathway of many of the bacteriophages cited in this paper. Arch Virol, 1992, 124(1-2), 69 - 82 The species concept and its application to tailed phages; Ackermann HW et al.; A recently proposed polythetic definition of virus species appears easily applicable to bacteriophages . Criteria for classification of tailed phages are evaluated . Morphology, DNA homology, and serology are the most important criteria for delineation of species, but no single criterion is satisfactory . Dot-blot hybridization and seroneutralization may suggest false relationships by detecting common sequences in the DNA of otherwise unrelated phages . Species of tailed phages can be defined by a combination of morphology and DNA homology or serology . A procedure for identification of novel phages is outlined . Phage names should include elements of host names. J Cell Sci, 1992 Jan, 101 ( Pt 1), 183 - 9 Analysis of p53 expression in human tumours: an antibody raised against human p53 expressed in Escherichia coli; Midgley CA et al.; A cDNA encoding the complete normal human p53 protein was expressed in Escherichia coli using an expression system based on the bacteriophage T7 promoter . The cDNA was adapted so that the full-length protein was produced without fusion to any other sequence . Large amounts of the protein were isolated and the purified protein used to produce very high titre polyclonal antibodies to p53 . These new antibodies permit the sensitive detection of p53 and p53 complexes in ELISA and immunoblotting assays . Most importantly, they also permit the detection of p53 in archival tumour material that has been conventionally fixed in formalin and embedded in paraffin wax . Using this reagent we have found that aberrant expression of p53 is a frequent feature of human breast cancer . We are able to recognise six different classes of p53 expression pattern that may be of help in the subclassification of breast tumours. Zentralbl Bakteriol, 1992 Jan, 276(2), 213 - 20 Isolation and characterization of bacteriophages specific for capsular antigens K3, K7, K12, and K13 of Escherichia coli; Nimmich W et al.; Four bacteriophages recognizing the Escherichia coli capsular antigens K3, K7, K12, and K13, respectively, were isolated from pooled sewage samples . The nucleic acid of these phages was identified as double-stranded DNA of different size (phi K3, 71.3; phi K7, 32.8; phi K12, 42.9; phi K13, 43.4 kbp) . Three of these phages belonged to Bradley's morphology group C and were specific for K3, K7, and K12 antigens, respectively . The phage phi K13 (Bradley's group A) attacked not only E . coli K13 strains but also E . coli producing the closely related K20 and K23 antigens . It is suggested that the common basic repeating units occurring in these capsular polysaccharides are the primary receptor of phi K13 . It could be demonstrated that the four phages were able to depolymerize enzymatically the capsular polysaccharides isolated from the respective host strains. Arch Microbiol, 1992, 157(4), 381 - 8 Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli; Witte A et al.; Expression of cloned gene E of bacteriophage PhiX174 induces lysis by formation of a transmembrane tunnel structure in the cell envelope of Escherichia coli . Ultrastructural studies of the location of the lysis tunnel indicate that it is preferentially located at the septum or at polar regions of the cell . Furthermore, the diameter and shape of individual tunnel structures vary greatly indicating that its structure is not rigid . Apparently, the contours of individual lysis tunnels are determined by enlarged meshes in the peptidoglycan net and the force produced at its orifice, by the outflow of cytoplasmic content . Once the tunnel is formed the driving force for the lysis process is the osmotic pressure difference between cytoplasm and medium . During the lysis process areas of the cytoplasmic membrane which are not tightly attached to the envelope are extended inward by the negative pressure produced during lysis . After cell lysis external medium can diffuse through the lysis tunnel filling the inner cell space of the still rigid bacterial ghosts. EMBO J, 1992 Jan, 11(1), 305 - 13 The isolation and characterization of mutants of the integration host factor (IHF) of Escherichia coli with altered, expanded DNA-binding specificities; Lee EC et al.; The integration host factor (IHF) of Escherichia coli is a small, basic protein that is required for lambda site-specific recombination and a variety of cellular processes . It is composed of two subunits, alpha and beta, that are encoded by the himA and hip (himD) genes, respectively . IHF is a sequence-specific DNA-binding protein and bends the DNA when it binds . We have used the bacteriophage P22-based challenge phage selection to isolate suppressor mutants with altered, expanded DNA binding specificities . The suppressors were isolated by selecting mutants that recognize variants of the phage lambda H'IHF recognition site . Two of the mutants recognize both the wild-type and a single variant site and contain amino acid substitutions at positions 64 (Pro to Leu) or 65 (Lys to Ser) of the alpha subunit . These substitutions are in a region of the protein that is predicted to contain a flexible arm that interacts with DNA . Three other mutants, which recognize the wild-type and a different variant site, contain amino acid substitutions at position 44 (Glu to Lys, Val or Gly) of the beta subunit . These substitutions are in the middle of a predicted beta-strand of the subunit . We discuss the possible mechanisms of suppression by the mutants in terms of a model of the IHF-DNA complex proposed by Yang and Nash {Cell, 57, 869-880 (1989)}. Protein Seq Data Anal, 1992, 5(1), 43 - 5 The superfamily of UvrA-related ATPases includes three more subunits of putative ATP-dependent nucleases; Koonin EV et al.; It is demonstrated that the amino acid sequences of the products of E . coli genes sbcC and prrC, and bacteriophage P2 gene old encompass the four conserved motifs typical of the superfamily of UvrA-related ATPases . A more pronounced statistically significant similarity was revealed between SbcC protein, bacteriophage T4 endonuclease component gp46 and bacteriophage T5 protein D13 . It is suggested that the newly identified members of the superfamily might all be ATPase components of the respective nucleases, and that the reactions catalyzed by these enzymes are probably ATP dependent. Genetica, 1992, 87(2), 95 - 100 The SbcCD protein of Escherichia coli is related to two putative nucleases in the UvrA superfamily of nucleotide-binding proteins; Leach DR et al.; The derived amino-acid sequences of the proteins encoded by E . coli genes sbcC and sbcD have been compared with other protein sequences using computer assisted methods . This work has shown that SbcC and D, which inhibit the propagation of replicons containing long palindromic DNA sequences, are distantly related to two putative bacteriophage nucleases . These nucleases both comprise two polypeptide chains which are the products of genes 46 and 47 of bacteriophage T4 (gp 46 and gp 47) and genes D13 and D12 of bacteriophage T5 (gp D13 and gp D12) . The comparisons reveal that SbcC, gp 46 and gp D13 are more closely related to each other than are SbcD, gp 47 and gp D12 . SbcC appears to have undergone a partial duplication of an ancestral sequence . These proteins all contain motifs common to the superfamily of nucleotide-binding proteins that includes UvrA and the cystic fibrosis transmembrane regulator CFTR. Crit Rev Immunol, 1992, 12(3-4), 125 - 68 Genetically engineered antibodies: progress and prospects; Wright A et al.; Techniques of genetic engineering and expression have been applied to the production of antibodies in a variety of expression systems . Novel antibodies have been produced with a variety of modifications: as chimeric antibodies, as "humanized" antibodies, with catalytic groups, as bifunctional or fusion proteins, and as functional fragments such as Fabs or Fvs . The domain structure of the antibody is favorable to such manipulation; the novel proteins often retain their antibody-derived activity and acquire new properties as well . Chimeric and complementarity-determining region (CDR)-grafted antibodies have been effective in immunotherapy, but problems of immunogenicity remain . Combinatorial libraries produced in bacteriophage may present an alternative to animal immunization as a source of antigen-binding specificities . Structural and mutational analysis of variable regions is providing useful information about the requirements of the variable region for antigen binding . Careful analysis and comparison of effector functions among immunoglobulin isotypes may be applied to the design of effective therapeutic antibodies. Genetica, 1992, 86(1-3), 259 - 67 Lambdoid phages as elements of bacterial genomes (integrase/phage21/Escherichia coli K-12/icd gene); Campbell A et al.; The lambdoid phages are a group of related temperate bacteriophages that lysogenize by site-specific recombination with the bacterial chromosome . Various members of the group have different specific chromosomal insertion sites, despite the fact that the enzymes catalyzing the insertion (integrases) appear to be all descended from a common ancestor . Insertion sites are not located randomly on the E . coli chromosome but are restricted to one segment of the map; also, most prophages are oriented in the same direction along the chromosome . Lambdoid phage 21 inserts within the isocitrate dehydrogenase gene and introduces an alternative 165 bp 3' end for that gene . A defective element (e14) inserts at the same position . We suggest that this mode of insertion arose from insertion of an ancestral phage to the right of icd which then picked up part of the icd gene by abnormal excision and speculate that, at an earlier time, phages may have arrived at their present locations by a process of chromosomal walking. DNA Seq, 1992, 2(6), 411 - 3 Nucleotide sequence of bovine interleukin-6 cDNA; Droogmans L et al.; We report the cloning of bovine interleukin-6 (IL-6) cDNA . The clone was isolated from a bovine-leukemia virus (BLV)-induced B cell-lymphosarcoma cDNA library cloned in the bacteriophage lambda gt11 . The cDNA encodes a full length IL-6 protein made of 208 amino acids with 65, 53, 42 and 42% homology to published sequences of porcine, human, mouse and rat IL-6, respectively . The significance of IL-6 expression in a BLV-induced tumor is briefly discussed. DNA Seq, 1992, 2(6), 405 - 9 The nucleotide sequence between genes 31 and 30 of bacteriophage T4; Nivinskas R et al.; The nucleotide sequence of the 2994 bp T4 phage DNA fragment between genes 31 and 30 is presented . The fragment contains 7 complete open reading frames in the direction of early transcription and two early promoters, PE128.6 and PE128.2, which we show to cause difficulties in cloning DNA from this genomic region . Our data complete the nucleotide sequence and the organization of genes in the genomic region between T4 genes 31 and 30. Crit Rev Biotechnol, 1992, 12(5-6), 437 - 62 Protein engineering of antibodies; Sandhu JS; This article reviews the technical advances in antibody engineering and the clinical applications of these molecules . Recombinant DNA technology facilitates the construction and expression of engineered antibodies . These novel molecules are designed to meet specific applications . Although genomic and cDNA cloning have been used widely in the past to isolate the relevant antibody V domains, at present, the PCR-based cloning is the preferred system . Bacterial and mammalian expression systems are used commonly for the production of antibodies, antibody fragments, and antibody fusion proteins . A range of chimeric antibodies with murine V domains joined to C regions from human and other species have been produced and found to exhibit the expected binding characteristics and effector functions . Humanized antibodies have been developed to minimize the HAMA response, and bifunctional immunoglobulins are being used in tumor therapy and diagnosis . Single chain antibodies and fusion proteins with antibody specificities jointed to nonimmunoglobulin sequences provide a source of antibody-like molecules with novel properties . The potential applications of minimal recognition units and antigenized antibodies are described . Combinatorial libraries produced in bacteriophage present an alternative to hybridomas for the production of antibodies with the desired antigen binding specificities . Future developments in this field are discussed also. Mamm Genome, 1992, 3(10), 550 - 8 A mouse genomic library in the bacteriophage P1 cloning system: organization and characterization; Pierce JC et al.; Using the bacteriophage P1 cloning system, we have constructed a two to three times coverage, high-molecular-weight (HMW) genomic library from mouse C127 fibroblast cells . The library consists of about 127,500 clones with an average insert size of about 70 kb that are organized into 300 primary pools containing approximately 425 clones per pool . For screening purposes the primary pools are combined into secondary pools (approximately 4250 clones each) and tertiary pools (approximately 21,250 clones each) . Screening is performed by the polymerase chain reaction (PCR) with DNA isolated from the secondary and tertiary pools . We have screened the library for 13 different mouse sequences and have detected 11 . Clones generated from two of the eleven positive screens were isolated from the library (those containing the c-fos and G alpha i2 genes) and were further characterized . Direct double-stranded sequencing of DNA from P1 clones with primers bordering the insert provided sequence information from each end of the cloned DNA. Microbiol Immunol, 1992, 36(7), 731 - 6 A phage in Bartonella bacilliformis; Umemori E et al.; Bacteriophage-like particles were found in Bartonella bacilliformis culture . The particles consisted of head (icosahedral), 40 nm in diameter, and tail, 16 nm in length. DNA Seq, 1992, 2(5), 329 - 33 DNA sequence characterization of the G gene region of bacteriophage Mu; Baxa CA et al.; The nucleotide sequence of a 1.2 kb region of bacteriophage Mu DNA was determined . This region contains the 3' end of the F gene, the complete G gene, and the 5' end of the I gene, all late genes involved in Mu virion morphogenesis . Identity of the G gene open reading frame was confirmed by sequencing four Gam mutations . The G open reading frame is predicted to encode proteins of 16.7 or 17.2 kDa, depending on which of two possible start codons are used to initiate translation . Four new nuB mutations in the DNA gyrase-binding site between the G and I genes were also sequenced and found to be identical to the nuB103 mutation sequenced previously. Genes Chromosomes Cancer, 1992 Jan, 4(1), 69 - 74 Specific metaphase and interphase detection of the breakpoint region in 8q24 of Burkitt lymphoma cells by triple-color fluorescence in situ hybridization; Ried T et al.; Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the breakpoint in 8q24 of the Burkitt lymphoma cell line J1 was used for the specific delineation of this breakpoint in individual tumor cells . With this approach, tumor-specific breakpoints in translocation chromosomes can be detected at all stages of the cell cycle with high specificity. Microbiol Immunol, 1992, 36(2), 161 - 7 Organization of the ribosomal RNA genes in Treponema phagedenis and Treponema pallidum; Fukunaga M et al.; The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3 . A restriction map of the fragment was constructed and the organization of the rRNA genes was determined . The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S . Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T . phagedenis strain Reiter and T . pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome . These results suggest that both pathogenic and non-pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes. Trends Genet, 1992 Jan, 8(1), 11 - 6 Cloning high molecular weight DNA fragments by the bacteriophage P1 system; Sternberg NL; The cloning of high molecular weight genomic DNA promises to provide the means of mapping chromosomes, isolating genes, and understanding long-range effects on gene expression . This review describes the background and some recent advances in cloning of high molecular weight DNA using the bacteriophage P1 system. Biotechnology (N Y), 1992 Jan, 10(1), 66 - 73 Production and functional characterization of a recombinant fragment of von Willebrand factor (vWF): an antagonist to platelet receptor GP Ib; Prior C et al.; We expressed a recombinant peptide fragment (Ser445-Val733) of human von Willebrand factor (vWF), containing the binding domain for the platelet receptor of GP Ib, in E . coli . This 33 kD peptide blocks binding of the intact vWF molecule to GP Ib in the presence of modulators . Thus, it offers potential as an antithrombotic agent . High level expression was achieved in a plasmid construct driven by the bacteriophage T7 promoter . The peptide was solubilized from inclusion bodies in strong chaotrope, then reduced and alkylated . Following purification, formulation at pH 3.5, and lyophilization, the reconstituted experimental product (RG 12986) exists as an equilibrium of monomer and dimer species . When formulated above pH 5.0, soluble aggregates are formed; these solutions have less bioactivity than RG 12986 . Interestingly, the non-aggregated state of RG 12986 remains conserved following dilution and incubation with platelet-poor plasma . The overall purification/low pH formulation strategies may be applicable to other E . coli recombinant proteins having a tendency to aggregate following removal of chaotrope near physiologic pH when in a concentrated format. Immunogenetics, 1992, 36(4), 208 - 12 Genomic organization of a polymorphic duplicated region centromeric of HLA-B; Leelayuwat C et al.; The region between tumor necrosis factor (TNF) and HLA-B in the central major histocompatibility complex (MHC) is polymorphic and associated with several autoimmune diseases . The polymorphisms are haplospecific or haplotypic and retained within the same MHC ancestral haplotype (AH) . We have cloned this region from four AHs into lambda bacteriophage and found that a highly polymorphic region in the TNF-HLA-B interval is duplicated . Clones from this region isolated from three MHC AHs show two populations . The regions, designated CL1 and CL2, have different sizes of Bam HI fragments carrying the duplicated sequences . These fragments correspond to those seen after Bam HI restriction fragment length polymorphism (RFLP) analysis of genomic DNA from the same cell lines . Pulsed field gel electrophoresis analysis shows that both CL1 and CL2 are in the central MHC and are about 16 kilobases apart . DNA cloning and RFLP analysis demonstrate that the CL region is highly polymorphic but retained within an MHC AH . Polymorphism and duplication are common characteristics of the genes found in the MHC and therefore the CL sequences have the potential to be interesting in this respect. Rev Latinoam Microbiol, 1992 Jan-Mar, 34(1), 53 - 60 Genetic characterization of the mechanism by which certain strains of Escherichia coli survive in high kanamycin concentrations; Cisneros Benavides ME; By genetic studies, it was tried to find the mechanism by which a bacterial fraction from different isolated clinical cultures resistant to 25 micrograms/ml of kanamycin can grow in media containing 500 micrograms/ml of kanamycin (at a frequency of about 10(-5)) . This study was done in six clinical isolates of Escherichia coli resistant to more than three antibiotics . The results from the bacterial fraction (subpopulation) resistant to high concentrations of kanamycin in the level of resistance to aminoglycoside and non-aminoglycoside antibiotics, in the conjugation experiments, and in the percentage of resistant bacteria to 500 micrograms/ml of kanamycin when the subpopulations were subsequently cultivated in the absence of antibiotics suggest that genetic amplification occurred when one of the strains was growing in the presence of 500 micrograms/ml of kanamycin . Moreover, this strain increased its frequency of survival in high kanamycin concentrations when it was transduced by bacteriophage P1, propagated in cultures resistant to 500 micrograms/ml of kanamycin. J Bacteriol, 1992 Jan, 174(1), 102 - 7 Cloning, nucleotide sequence, overexpression, and inactivation of the Escherichia coli 2-keto-4-hydroxyglutarate aldolase gene; Patil RV et al.; Having previously determined the complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli (C . J . Vlahos and E . E . Dekker, J . Biol . Chem . 263:11683-11691, 1988), we amplified the gene that codes for this enzyme by the polymerase chain reaction using synthetic degenerate deoxyoligonucleotide primers . The amplified DNA was sequenced by subcloning the polymerase chain reaction products into bacteriophage M13; the nucleotide sequence of the gene was found to be in exact agreement with the amino acid sequence of the gene product . Overexpression of the gene was accomplished by cloning it into the pKK223.3 expression vector so that it was under control of the tac promoter and then using the resultant plasmid, pDP6, to transform E . coli DH5 alpha F'IQ . When this strain was grown in the presence of isopropyl beta-D-thiogalactopyranoside, aldolase specific activity in crude extracts was 80-fold higher than that in wild-type cells and the enzyme constituted approximately 30% of the total cellular protein . All properties of the purified, cloned gene product, including cross-reactivity with antibodies elicited against the wild-type enzyme, were identical with the aldolase previously isolated and characterized . A strain of E . coli in which this gene is inactivated was prepared for the first time by insertion of the kanamycin resistance gene cartridge into the aldolase chromosomal gene. Plant Mol Biol, 1992 Jan, 18(2), 353 - 61 Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system; Bayley CC et al.; The Cre-lox site-specific recombination system of bacteriophage P1 was used to excise a firefly luciferase (luc) gene which had previously been incorporated into the tobacco genome . The excision event was due to site-specific DNA recombination between two lox sequences flanking the luc gene and was catalyzed by the Cre recombinase introduced by cross-fertilization . Recombination resulted in the fusion of a promoter with a distally located hygromycin phosphotransferase (hpt) coding sequence and the excision event was monitored as a phenotypic change from expression of luc to expression of hpt . The efficiency of recombination was estimated from the exchange of gene activity and confirmed by molecular analysis . The relevance to potential applications of site-specific deletion-fusion events for chromosome engineering are discussed. Neuron, 1992 Jan, 8(1), 59 - 70 Efficient expression of rat brain type IIA Na+ channel alpha subunits in a somatic cell line; West JW et al.; Type IIA rat brain Na+ channel alpha subunits were expressed in CHO cells by nuclear microinjection or by transfection using a vector containing both metallothionein and bacteriophage SP6 promoters . Stable cell lines expressing Na+ channels were isolated, and whole-cell Na+ currents of 0.9-14 nA were recorded . The mean level of whole-cell Na+ current (4.5 nA) corresponds to a cell surface density of approximately 2 channels active at the peak of the Na+ current per microns 2, a density comparable to that observed in the cell bodies of central neurons . The expressed Na+ channels had the voltage dependence, rapid activation and inactivation, and rapid recovery from inactivation characteristic of Na+ channels in brain neurons, bound toxins at neurotoxin receptor sites 1 and 3 with normal properties, and were posttranslationally processed to a normal mature size of 260 kd . Expression of Na+ channel cDNA in CHO cells driven by the metallothionein promoter accurately and efficiently reproduces native Na+ channel properties and provides a method for combined biochemical and physiological analysis of Na+ channel structure and function. J Neurosci, 1992 Jan, 12(1), 268 - 77 Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells; Yang XC et al.; The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells . Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter . Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells . However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors . Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps . The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes . These differences also suggest cell-specific posttranslational modifications . IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents . Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence. J Bacteriol, 1992 Jan, 174(1), 155 - 60 In vitro repair of double-strand breaks accompanied by recombination in bacteriophage T7 DNA; Masker W; A double-strand break in a bacteriophage T7 genome significantly reduced the ability of that DNA to produce viable phage when the DNA was incubated in an in vitro DNA replication and packaging system . When a homologous piece of T7 DNA (either a restriction fragment or T7 DNA cloned into a plasmid) that was by itself unable to form a complete phage was included in the reaction, the break was repaired to the extent that many more viable phage were produced . Moreover, repair could be completed even when a gap of about 900 nucleotides was put in the genome by two nearby restriction cuts . The repair was accompanied by acquisition of a genetic marker that was present only on the restriction fragment or on the T7 DNA cloned into a plasmid . These data are interpreted in light of the double-strand gap repair mode of recombination. Yi Chuan Xue Bao, 1992, 19(5), 475 - 80 {Nucleotide frequency analysis of bacteriophage phi X174 genome}; Yang Z; Detailed analysis was carried out on the nucleotide frequencies in the genome of bacteriophage phi X174 . In coding regions the distribution of four nucleotides at the three codon sites was highly non-random, and the patterns were conspicuously similar among the genes, implying that it might be a feature of the genome . An index c, which was based on the chi 2 statistics of the site by base 3 x 4 table, was introduced to reveal he extra information embedded in the coding region, and also to discriminate between coding and non-coding open reading frames . Doublet deviation analysis showed that deviation patterns were not significantly related to the codon sites in coding regions, and were also very similar in different genes . Possible mechanisms of such similarity are discussed. Faraday Discuss, 1992, (93), 173 - 81 Dissection of protein structure and folding by directed mutagenesis; Baase WA et al.; The lysozyme from bacteriophage T4 is being used as a model system to determine the roles of individual amino acids in the folding and stability of a typical globular protein . One general finding is that the protein is very adaptable, being able to accommodate many potentially destabilizing replacements . In order to determine the importance of 'alpha-helix propensity' in protein stability, different replacements have been made within alpha-helical segments of T4 lysozyme . Several such substitutions of the form Xaa-->Ala increase the stability of the protein, supporting the idea that alanine is a strongly helix-favouring amino acid . It is possible to engineer a protein that has up to ten alanines in succession, yet still folds and has normal activity . This illustrates the redundancy that is present in the amino acid sequence . A number of 'cavity-creating' mutants of the form Leu-->Ala have been constructed to understand better the nature of hydrophobic stabilization . The structural consequences of these mutations differ from site to site . In some cases the protein structure hardly changes at all; in other cases removal of the wild-type side-chain allows surrounding atoms to move in and occupy the vacated space, although a cavity always remains . The destabilization of the protein associated with these cavity-creating mutations also varies from case to case . The results suggest how to reconcile recent conflicting reports concerning the strength of the hydrophobic effect in proteins. Glia, 1992, 6(4), 301 - 9 Determination of the origin and nature of brain macrophages and microglial cells in mouse central nervous system, using non-radioactive in situ hybridization and immunoperoxidase techniques; de Groot CJ et al.; The origin and nature of brain macrophages and microglial cells in the mouse central nervous system (CNS) were investigated . First, the expression and localization of determinants recognized by the different monoclonal antibodies (mAbs) MOMA-1, Mac-1-alpha, and F4/80 (raised against cells of the mononuclear phagocyte system) were immunohistochemically studied in the developing and adult mouse brain . In order to clarify the origin of brain macrophages and microglial cells, we used bacteriophage lambda transgenic mice as donors for bone marrow transplantations in recipient mice of different ages . During ontogeny, numerous MOMA-1-, Mac-1-alpha-, and F4/80-positive blood monocyte-derived brain macrophages (amoeboid microglia) infiltrated the CNS parenchyma . These brain macrophages gradually disappeared from the brain parenchyma at postnatal day 7 (P7) . From P17 on, Mac-1-alpha- and F4/80-positive cells were detected within the brain parenchyma with the morphology of resting microglial cells . Transitional forms between brain macrophages and "resting" microglia were not observed in the developing brain . Combined non-radioactive in situ hybridization and immunohistochemistry revealed many MOMA-1-positive bone marrow-derived brain macrophages that were located in the leptomeninges, the ventricles, and occasionally the blood vessel walls . These results show that brain macrophages are of bone marrow origin . Many "resting" microglial cells were detected in the brain, mainly in the white matter . It appeared that about 10% of these cells displayed the transgenic signal . This result indicates that the majority of "resting" microglial cells are of local, presumably neuroectodermal, origin. Med Microbiol Immunol (Berl), 1992, 181(4), 215 - 26 Determination of the minimal length of preS1 epitope recognized by a monoclonal antibody which inhibits attachment of hepatitis B virus to hepatocytes; Sominskaya I et al.; The minimal amino acid sequence sufficient to be recognized efficiently by virus-attachment inhibiting murine monoclonal anti-preS1 antibody MA18/7 has been determined . We have constructed a recombinant gene library using the cloned coat protein gene of Escherichia coli RNA bacteriophage fr as a carrier . Different fragments of preS1 region from cloned hepatitis B virus (HBV) genomes, subtype ayw and adw, were inserted at position 2 of the 129 amino acid-long fr coat protein gene in the appropriate E . coli expression vectors . Fine mapping of preS1 epitope recognized by MA18/7 was accomplished by bidirectional shortening of the preS1 within original recombinant preS-fr coat protein genes with Bal31 exonuclease . Immunoblot analysis of the obtained recombinant protein library revealed that the tetrapeptide Asp-Pro-Ala-Phe (DPAF), located at the position preS(31-34) and conserved in all known HBV genomes, is sufficient to bind MA18/7 antibody . Recognition of the preS1 region by MA18/7 occurred irrespective of the amino acid context surrounding this DPAF tetrapeptide . Further shortening of this minimal epitope from the left or from the right side completely prevented antibody binding in immunoblots. Gene, 1991 Dec 30, 109(2), 243 - 7 Tagging the genome of the murine leukemia retrovirus SL3-3 by a bacterial lac operator sequence; Jorgensen P et al.; The bacterial lactose operator (lacO) was introduced into the PstI site of the long terminal repeat of the SL3-3 murine leukemia virus, generating a virus, SL3-3lacO, that can replicate in NIH3T3 cell cultures . DNA sequences harboring the lacO sequence might be recovered by molecular cloning in Escherichia coli lac+ lacZ+ using bacteriophage lambda or plasmid vectors . The high copy numbers of the lacO sequence titrate out the lac repressor, leading to the induction of the lac operon in the host . We show here that the lacO and the proviral sequences are carried stably together in the genomes of SL3-3lacO-infected cell cultures and in viral particles . This system is designed to facilitate studies on the provirus and the site of viral integration. J Mol Biol, 1991 Dec 20, 222(4), 983 - 94 Analysis of replicative intermediates produced during bacteriophage phi 29 DNA replication in vitro; Gutierrez C et al.; Replication of bacteriophage phi 29 DNA initiates at either end of its linear double-stranded DNA molecule and proceeds by a strand-displacement mechanism . In the present paper we have used an in vitro phi 29 DNA replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times . Two types of replicative intermediates were observed: type I (full-length double-stranded phi 29 DNA molecules with one or more single-stranded DNA branches) and type II (full-length phi 29 DNA molecules formed by a double-stranded DNA portion of variable length from one end plus a single-stranded DNA portion spanning to the other end) . Thus, the types of replicative intermediates produced in vivo were also formed in the in vitro phi 29 DNA replication system . Analysis of type I intermediates indicated that initiation of DNA replication occurs preferentially at both ends of the same DNA template, in a non-simultaneous manner . Type II intermediates appeared as early as two minutes after the reaction started, well before unit-length single-stranded phi 29 DNA molecules were synthesized . In addition, replication of recombinant phi 29 DNA templates lacking terminal protein at one end did not produce type II intermediates and led to an accumulation of full-length single-stranded phi 29 DNA molecules . These two observations strongly suggest that type II intermediates appear when two growing DNA chains, running from opposite ends, merge. J Mol Biol, 1991 Dec 20, 222(4), 897 - 908 Analysis of the integration function of the streptomycete bacteriophage phi C31; Kuhstoss S et al.; A 2.1 kb (1 kb = 10(3) base-pairs) segment of DNA from the streptomycete bacteriophage phi C31 was found to be sufficient to direct site-specific integration of plasmid vectors in Streptomyces ambofaciens and Streptomyces fradiae in the absence of any streptomycete origin of replication . Sequencing and analysis of phage, chromosomal and junction attachment sites of S . ambofaciens and S . fradiae revealed that recombination is conservative and that crossover takes place within three bases of homology between phage and host . Deletion analysis, sequencing and site-specific mutagenesis of the phi C31 DNA revealed a large open reading frame (ORF 613) whose expression was necessary for integration . This ORF begins near the point of crossover and reads away from the attachment site . A comparison of the predicted amino acid sequence of ORF 613 with known recombinases did not reveal any significant similarities . A genetic analysis of the amino-terminal region of ORF 613 suggested that translation could initiate at any one of three possible start codons . Primer extension experiments showed that transcriptional initiation occurred at a T and a C only four and five bases, respectively, from the site of crossover . This analysis suggested that ORF 613 would be separated from its promoter upon integration. Gene, 1991 Dec 20, 109(1), 21 - 30 Null mutation in the stringent starvation protein of Escherichia coli disrupts lytic development of bacteriophage P1; Williams MD et al.; As initial steps toward understanding the regulation and function of the stringent starvation protein (SSP) of Escherichia coli, we have isolated the ssp gene (encoding SSP), defined the operon in which ssp is found, and created insertion-deletion mutations of the ssp gene in recBC, sbc and recD strains by linear DNA transformation . During attempts to move the insertion-deletion structure to other strains by P1 transduction, we found that P1 was unable to form plaques on hosts lacking an intact ssp gene . The delta ssp mutation, however, did not affect transduction of the delta ssp strains and mutant strains were able to support lysogenic P1 . When P1 lytic growth was induced, an increase in P1 DNA was detected without lysis or plaque formation . Examination of proteins synthesized in the delta ssp host during induction revealed the absence of P1 late gene products . Also, the apparent continued synthesis of early gene products during late time points was observed in the delta ssp host . The results reported here suggest that the defect in P1 lytic growth brought about by the absence of SSP occurs at the point at which bacteriophage P1 shifts from early to late gene expression . We also report the results of experiments on stable RNA synthesis following amino acid (aa) starvation induced by serine hydroxamate, and experiments on stable RNA synthesis following resupplementation of a limiting aa . These experiments show that SSP is not involved in stable RNA synthesis . Additionally, complementation studies have shown that ssp is identical to the previously described pog gene of E . coli. Gene, 1991 Dec 20, 109(1), 13 - 9 Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13; Markland W et al.; Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated . Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein . Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation . Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin . Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand . The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage. Eur J Biochem, 1991 Dec 18, 202(3), 1013 - 20 Cell-free synthesis of rat and human catechol O-methyltransferase . Insertion of the membrane-bound form into microsomal membranes in vitro; Ulmanen I et al.; The protein-coding capacities of rat and human catechol O-methyltransferase (COMT) DNA clones were analysed by in vitro transcription and translation using bacteriophage RNA polymerase and rabbit reticulocyte lysate . Two types of clones corresponding to the structures of human placental cDNA clones were used . The shorter clones, containing the 663-residue open reading frame for the soluble COMT (S-COMT), produced 24-kDa (rat) and 26-kDa (human) polypeptides . Translation of the longer clones, containing 43 (rat) or 50 (human) amino acid amino-terminal extensions to the S-COMT polypeptides, yielded 28-kDa (rat) and 30-kDa (human) putative membrane-bound COMT (MB-COMT) polypeptides as the main products . These clones also yielded low amounts of the S-COMT polypeptides . Labelling time or ionic conditions during translation did not eliminate the shorter products, suggesting translation initiation from the second S-COMT AUG codon . In accordance with this postulation, the relative amount of S-COMT could be affected by changing the translation initiation contexts preceding the first AUG codon . The 28-kDa and 30-kDa products, but not the 24-kDa and 26-kDa products, associated with microsomal membranes cotranslationally, indicating that the amino-terminal extensions were functional signal sequences . However, the presence of membranes did not affect the mobilities of the proteins in SDS/polyacrylamide gels . The MB-COMT polypeptides could not be released from the microsomes by treatments with phospholipase C or alkali and were not protected by the microsomes against proteinase K digestion . These results indicate that MB-COMT synthesized in vitro is an integral membrane protein having an amino-terminal signal-anchor sequence. Gene, 1991 Dec 15, 108(2), 201 - 9 Enhancement of the vaccinia virus/phage T7 RNA polymerase expression system using encephalomyocarditis virus 5'-untranslated region sequences; Vennema H et al.; A recombinant vaccinia virus producing the bacteriophage T7 RNA polymerase was used to express foreign genes in eukaryotic cells . Translation efficiency in this expression system was enhanced significantly by employing the encephalomyocarditis virus (EMCV) 5'-untranslated region (UTR) which confers cap-independent translation by directing internal initiation of translation . The enhancement was accomplished by fusing open reading frames (ORFs) to the N terminus of the EMCV polyprotein coding region, thus utilizing its highly efficient translation initiation site . Expression vectors were constructed to allow cloning in all three reading frames . As reporter genes, we used the lacZ gene and a number of genes encoding coronavirus structural proteins: among others the genes encoding glycoproteins with N-terminal signal sequences . The signal sequences of these glycoproteins are located internally in the primary translation product . We demonstrated that this did not interfere with translocation and glycosylation and yields biologically active proteins . The usefulness of sequences that direct internal initiation was extended by using EMCV UTRs to express two and three ORFs from polycistronic mRNAs. Biophys J, 1991 Dec, 60(6), 1337 - 49 Raman spectroscopy of filamentous bacteriophage Ff (fd, M13, f1) incorporating specifically-deuterated alanine and tryptophan side chains . Assignments and structural interpretation; Aubrey KL et al.; Structural interpretation of the Raman spectra of filamentous bacteriophages is dependent upon reliable assignments for the numerous Raman vibrational bands contributed from coat protein and packaged DNA of the virion . To establish unambiguous assignments and facilitate structural conclusions derived from them, we have initiated a systematic study of filamentous bacteriophage Ff (fd, f1, M13) incorporating protein subunits with specifically deuterated amino-acid side chains . Here, we report and interpret the Raman spectra of fd virions which incorporate: (a) a single deuterio-tryptophan residue per coat protomer {fd(Wd5)}, (b) ten deuterio-alanines per protomer {fd(10Ad3)}, and (c) both deuterio-tryptophan and deuterio-alanine {fd(Wd5 + 10Ad3)} . The unambiguous assignment of coat protein Raman bands in normal and deuterated isotopomers of fd establishes the validity of earlier empirical assignments of many key Raman markers, including those of packaged ssDNA (Thomas et al., 1988) . Present results confirm that deoxyguanosine residues of the packaged ssDNA molecule depart from the usual C2'-endo/anti conformation characteristic of protein-free DNA in aqueous solution, although C2'-endo/anti conformers of thymidine are not excluded by the data . The combined results obtained here on normal fd, and on fd incorporating deuterio-tryptophan {fd(Wd5) and fd(Wd5 + 10Ad3)}, show also that the microenvironment of the single tryptophan residue per coat protomer (W26) can be clearly deduced as follows: (a) The indole 1-NH donor group of each protomer in fd forms a moderately strong hydrogen bond, most likely to a hydroxyl oxygen acceptor . (b) The planar indole ring exists in a hydrophilic environment . (c) The torsion angle describing the orientation of the indole ring (C3-C2 linkage) with respect to the side-chain (C alpha-C beta bond) is unusually large, i.e., magnitude of X2,1 approximately 120 degrees . With respect to alanine isotopomers, the present results show that alanine residues, and possibly other methyl-containing side chains, are significant contributors to the fd Raman spectrum . The present study provides new information on protomer side chains of fd and demonstrates a Raman methodology which should be generally useful for investigating single-site interactions and macromolecular conformations in other nucleoprotein assemblies. Nucleic Acids Res, 1991 Dec 11, 19(23), 6499 - 503 RNA binding properties of the coat protein from bacteriophage GA; Gott JM et al.; The coat protein of bacteriophage GA, a group II RNA phage, binds to a small RNA hairpin corresponding to its replicase operator . Binding is specific, with a Ka of 71 microM -1 . This interaction differs kinetically from the analogous coat protein-RNA hairpin interactions of other RNA phage and also deviates somewhat in its pH and salt dependence . Despite 46 of 129 amino acid differences between the GA and group I phage R17 coat proteins, the binding sites are fairly similar . The essential features of the GA coat protein binding site are a based-paired stem with an unpaired purine and a four nucleotide loop having an A at position -4 and a purine at -7 . Unlike the group I phage proteins, the GA coat protein does not distinguish between two alternate positions for the unpaired purine and does not show high specificity for a pyrimidine at position -5 of the loop. Biochemistry, 1991 Dec 10, 30(49), 11490 - 4 DNA-dependent adenosinetriphosphatase A is the eukaryotic analogue of the bacteriophage T4 gene 44 protein: immunological identity of DNA replication-associated ATPases; Mesner LD et al.; We report the construction of three stable murine hybridomas that secrete monoclonal antibodies which recognize calf thymus DNA-dependent adenosinetriphosphatase A . All three of the antibodies react specifically with calf thymus ATPase A and the gene 44 protein from the bacteriophage T4 DNA-dependent ATPase . Each of the three anti-ATPase A antibodies appears to recognize a different epitope and none of the antibodies inhibit DNA-dependent ATP hydrolysis by ATPase A . Furthermore, one of the antibodies has been shown to react with two different preparations of HeLa cell DNA-dependent ATPases and a yeast DNA-dependent ATPase, all of which have been implicated in the enzymology of DNA replication . These findings provide strong evidence for the role of ATPase A in DNA replication . These observations lead us to conclude that, apart from the nucleotide binding sites, there are at least three epitopes common to both the bacteriophage and eukaryotic DNA-dependent ATPases that we have examined and that the different preparations of the eukaryotic ATPases contain the same DNA-dependent ATPase. J Mol Biol, 1991 Dec 5, 222(3), 479 - 94 RNA polymerase bound to the PR promoter of bacteriophage lambda inhibits open complex formation at the divergently transcribed PRM promoter . Implications for an indirect mechanism of transcriptional activation by lambda repressor; Hershberger PA et al.; We demonstrate that RNA polymerase bound at the PR promoter of bacteriophage lambda can repress transcription initiation from the divergently transcribed PRM promoter in vitro . Using abortive initiation and run-off transcription experiments we show that inactivating mutations introduced into either the -10 or -35 regions of PR result in a significant increase in the rate of formation of transcriptionally competent complexes at the PRM promoter . This is due primarily to an increase in the rate constant for the isomerization of closed to open complexes . Gel shift and DNase I footprinting experiments were employed to further define the mechanism by which PR sequences mediate PRM repression . From these assays we were able to conclude that the formation of an open complex at the PR promoter did not exclude RNA polymerase from binding at PRM . Rather, initiation at PRM was impaired because closed complexes must isomerize in the presence of an open complex already situated at the PR promoter . Extensive evidence has been obtained previously indicating that lambda repressor activates transcription directly by contacting RNA polymerase situated at the PRM promoter . Results presented here raise the possibility that an additional mechanism could be operative, whereby lambda repressor indirectly activates PRM transcription by excluding RNA polymerase from the PR promoter. J Biol Chem, 1991 Dec 5, 266(34), 23191 - 6 Molecular properties of global suppressors of temperature-sensitive folding mutations in P22 tailspike endorhamnosidase; Lee SC et al.; Two global suppressors (Val-331 greater than Ala and Ala-334 greater than Val) have been identified for temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 (Mitraki, A., Fane, B., Haase-Pettingell, C., Sturtevant, J., and King, J . (1991) Science 253, 54-58) . We have introduced 19 different single amino acid substitutions at the two global suppressor sites independently and examined the effects on the tailspike formation in Escherichia coli . Folding and maturation patterns of the various substitutions at the two global suppressor sites in the wild-type background suggest that Val-331 is located on the protein surface and Ala-334 is in the hydrophobic region . In combination with a tsf mutation, tsfH304 (Gly-244 greater than Arg), only Gly at 331 and Ile at 334, the substitutions that have similar side chain properties to the original suppressor sequences, were active as tsf suppressors . The newly identified suppressors of tsfH304 could also alleviate the tsf defect of three other mutations . The mutant carrying both Val-331 greater than Ala and Ala-334 greater than Val substitutions was also a global suppressor and was more active in suppressing the tsf defect than mutants carrying only one substitution . The suppressors may act by increasing the stability of an intermediate in the productive pathway of folding and maturation of the mutant polypeptides. Eur J Biochem, 1991 Dec 5, 202(2), 349 - 60 Sequence-specific 1H-NMR assignment and secondary structure of the Tyr41----His mutant of the single-stranded DNA binding protein, gene V protein, encoded by the filamentous bacteriophage M13; Folkers PJ et al.; Sequence-specific 1H-NMR assignments are reported for the Tyr41----His (Y41H) mutant of the single-stranded DNA binding protein, encoded by gene V of the filamentous bacteriophage M13 (GVP) . The mutant protein was chosen for this purpose because it exhibits significantly improved solubility characteristics over wild-type GVP {Folkers et al . (1991) Eur . J . Biochem . 200, 139-148} . The secondary structure elements present in the protein are deduced from a qualitative interpretation of the nuclear Overhauser enhancement spectra and amide exchange data . The protein is entirely composed of antiparallel beta-structure . It is shown that identical structural elements are present in wild-type GVP . Previously, we have demonstrated that the secondary structure of the beta-loop, encompassing residues 13-31 which is present in GVP in solution, deviates from that proposed for the same amino acid sequence on the basis of X-ray diffraction data {van Duynhoven et al . (1990) FEBS Lett . 261, 1-4} . Now that we have arrived at a complete description of the secondary structure of the protein in solution, other deviations with respect to the crystallographically determined structure became apparent as well . The N-terminal part of the protein is, in solution, part of a triple-stranded beta-sheet while, in the crystal, it is an extended strand pointing away from the bulk of the protein dimer . One of the antiparallel beta-sheets in the protein which had been designated earlier as the complex loop has, in the solution structure, a different pairwise arrangement of the residues in its respective beta-ladders . Residues 30 and 48 are opposite to one another in the solution structure while in the crystal structure residues 32 and 48 are paired . A similar observation is made for the so-called dyad domain of the protein of which the beta-sheet in the solution structure is shifted by one residue with respect to that of the crystal structure. J Biol Chem, 1991 Dec 5, 266(34), 23407 - 15 In vivo restriction . Sequence and structure of endonuclease II-dependent cleavage sites in bacteriophage T4 DNA; Krabbe M et al.; Endonuclease II of bacteriophage T4 is required for in vivo restriction of cytosine-containing DNA from its host, Escherichia coli, (as well as from phage mutants lacking cytosine modification), normally the first step in the reutilization of host DNA nucleotides for synthesis of phage DNA in infected cells . The phage cytosine-DNA is fragmented incompletely to yield genetically defined fragments . This restriction is different from that of type I, II, or III restriction enzymes . We have located seven major endonuclease II-dependent restriction sites in the T4 genome, of which three were analyzed in detail; in addition, abundant sites were cleaved in less than or equal to 5% of all molecules . Sites I, II, and III shared the sequence 5'-CCGNNTTGGC-3' and were cleaved in about 25% (I and III) and 65% (II) of all molecules, predominantly staggered around the first or second of the central unspecified base pairs to yield fragments with one 5' base . The less frequently cleaved sites I and III deviated from site II in predicted helical structure when viewed from the consensus strand, and in sequence when viewed from the opposite strand . Thus, interaction with a particular helical structure as well as recognition of the bases in DNA appears important for efficient cleavage. J Biol Chem, 1991 Dec 5, 266(34), 23240 - 50 Requirements for primer synthesis by bacteriophage T7 63-kDa gene 4 protein . Roles of template sequence and T7 56-kDa gene 4 protein; Mendelman LV et al.; Gene 4 of bacteriophage T7 encodes two proteins, a 63-kDa protein and a colinear 56-kDa protein, that are essential for synthesis of leading and lagging strands during DNA replication . The gene 4 proteins together catalyze the synthesis of oligoribonucleotides, pppACC(C/A) or pppACAC, at the single-stranded DNA sequences 3'-CTGG(G/T)-5' or 3'-CTGTG-5', respectively . Purified 56-kDa protein has helicase activity, but no primase activity . In order to study 63-kDa gene 4 protein free of 56-kDa gene 4 protein, mutations were introduced into the internal ribosome-binding site responsible for the translation of the 56-kDa protein . The 63-kDa gene 4 protein was purified 16,000-fold from Escherichia coli cells harboring an expression vector containing the mutated gene 4 . Purified 63-kDa gene 4 protein has primase, helicase, and single-stranded DNA-dependent dTTPase activities . The constraints of primase recognition sequences, nucleotide substrate requirements, and the effects of additional proteins on oligoribonucleotide synthesis by the 63-kDa gene 4 protein have been examined using templates of defined sequence . A three-base sequence, 3'-CTG-5', is necessary and sufficient to support the synthesis of pppAC dimers . dTTP hydrolysis is essential for oligoribonucleotide synthesis . Addition of a 7-fold molar excess of 56-kDa gene 4 protein to 63-kDa protein increases the number of oligoribonucleotides synthesized by 63-kDa protein 100-fold . The increase in oligonucleotides results predominantly from an increase in the synthesis of tetramers, with relatively little change in the synthesis of dimers and trimers . The presence of 56-kDa protein also causes 63-kDa protein to synthesize "pseudo-templated" pppACCCC pentamers at the recognition sequence 3'-CTGGG-5' . T7 gene 2.5 protein, a single-stranded DNA binding protein, increases the total number of oligoribonucleotides synthesized by 63-kDa gene 4 protein on single-stranded M13 DNA, but has no effect on the ratio of dimers to trimers and tetramers. Virology, 1991 Dec, 185(2), 788 - 94 Analysis of interactions among factors involved in the bacteriophage T3 DNA packaging reaction in a defined in vitro system; Fujisawa H et al.; During head assembly of phage T3, DNA is packaged into the cavity of a preformed protein shell, called the prohead, with the aid of noncapsid, packaging proteins, the products of genes 18 and 19 (gp18 and gp19) . gp18 and gp19 separately form complexes with DNA and proheads, respectively . These complexes associate to form a precursor which can be converted to filled heads by the addition of ATP . Interactions among factors involved in DNA packaging were analyzed . In the presence of ATP, gp19 formed functional complexes with proheads . Formation of gp19-prohead complex showed a sigmoidal dependence on ATP concentration with a half maximal concentration of about 7.5 microM . Six molecules of gp19 bound to the prohead at a saturating amount of gp19 . gp19 did not bind to proheads lacking the connector of gp8 (8- prohead) . In the absence of ATP, proheads were inactivated by gp19 . The gp19-prohead complexes formed in the absence of ATP contained 20-30 gp19 molecules per prohead and formed multimeric aggregates . 8- proheads did not bind gp19 and did not form such aggregates even in the absence of ATP . From these results, we conclude that 6 molecules of gp19 bind to the gp8 connector structure in the portal vertex of the prohead . The cleavage patterns of gp19 by several proteases were altered by the addition of ATP, indicating that ATP induces a conformational change in gp19, gp18 bound only to linear, duplex DNA. J Bacteriol, 1991 Dec, 173(23), 7695 - 7 The Escherichia coli terB sequence affects maintenance of a plasmid with the M13 phage replication origin; Uzest M et al.; Replication initiated at the bacteriophage M13 origin can be affected by interaction of a properly oriented termination signal terB and the Tus protein . The effect can be alleviated by overproduction of the M13 replication gene protein II. Mol Microbiol, 1991 Dec, 5(12), 2953 - 63 RNaselll activation of bacteriophage lambda N synthesis; Kameyama L et al.; The bacteriophage lambda N gene product is one of the first genes expressed during phage development . N protein allows the expression of other phage genes by altering the transcription elongation process so as to prevent transcription termination . We have found that N levels may be modulated soon after induction or infection . Using N-lacZ fusions, we determined that cells containing RNaselll have at least a fourfold greater expression than cells defective for RNaselll . This effect is exerted at the post-transcriptional level . RNaselll processes an RNA stem structure in the N-leader RNA . Removal of the stem structure by deletion increases N expression and prevents further stimulation by RNaselll . The base of this stable stem is adjacent to the N ribosome binding site . We present a model for control of N synthesis in which this stable stem inhibits ribosome access to the N mRNA. Nucleic Acids Res, 1991 Dec, 19(25), 7207 - 14 Nucleotide sequence of the DNA packaging and capsid synthesis genes of bacteriophage P2; Linderoth NA et al.; Overlapping DNA fragments containing the DNA packaging and capsid synthesis gene region of bacteriophage P2 were cloned and sequenced . In this report we present the complete nucleotide sequence of this 6550 bp region . Each of six open reading frames found in the interval was assigned to one of the essential genes (Q, P, O, N, M and L) by correlating genetic, physical and mutational data with DNA and protein sequence information . Polypeptides predicted were: a capsid completion protein, gpL; the major capsid precursor, gpN; the presumed capsid scaffolding protein; gpO; the ATPase and proposed endonuclease subunits of terminase, gpP and gpM, respectively; and a candidate for the portal protein, gpQ . These gene and protein sequences exhibited no homology to analogous genes or proteins of other bacteriophages . Expression of gene Q in E . coli from a plasmid caused production of a Mr 39,000 Da protein that restored Qam34 growth . This sequence analysis found only genes previously known from analysis of conditional-lethal mutations . No new capsid genes were found. Virology, 1991 Dec, 185(2), 901 - 3 Receptor sequence in the terminal protein of bacteriophage M2 that interacts with an RGD (Arg-Gly-Asp) sequence of the primer protein; Kobayashi H et al.; At the initiation of protein-primed DNA replication of bacteriophages M2 and phi 29, the Arg-Gly-Asp (RGD) sequence of primer protein participates in the recognition of terminal protein (TP), where the initiation site for protein-primed DNA replication of template DNA is located . We compared the sequences of M2 and phi 29 TP with those of the members of the integrin superfamily and found the highly homologous sequences Lys-Lys-Ile-Pro-Pro-Asp-Asp (KKIPPDD) in M2 and phi 29 TP and Lys-Lys-Gly-Cys-Pro-Pro-Asp-Asp (KKGCPPDD) in the beta-subunit of fibronectin receptor protein . A synthetic 20mer peptide that contained the KKIPPDD sequence interfered with the inhibitory effect of the RGD peptide on both transfection and the protein-priming reaction in vitro . We propose that the sequence KKIPPDD of M2 TP is the receptor sequence for RGD. J Virol, 1991 Dec, 65(12), 6714 - 23 Rescue of Sindbis virus-specific RNA replication and transcription by using a vaccinia virus recombinant; Li GP et al.; A heterologous system expressing functional Sindbis virus nonstructural proteins (nsPs) has several possible uses for studying Sindbis virus-specific RNA replication and transcription in vivo and in vitro . Of the many possible approaches, vaccinia virus offers an attractive transient expression system given that Sindbis virus replication can occur in cells which have been previously infected by vaccinia virus . In this report, a vaccinia virus recombinant (called vSINNS), which contains the cDNA encoding the Sindbis virus nsPs under the control of either the vaccinia virus 7.5K promoter or the bacteriophage T7 promoter, has been constructed and characterized . Upon infection of several cell types with vSINNS, Sindbis virus nsP precursors and processed forms, including nsP1, nsP2, and both phosphorylated and nonphosphorylated forms of nsP3, were synthesized . Proteins containing the putative RNA-dependent RNA polymerase domain (nsP4 and nsP34), which are normally produced in small amounts by readthrough of an opal termination codon, were not detected in vSINNS-infected cells . However, all nsP functions necessary for Sindbis virus-specific RNA synthesis must have been expressed, since both replication and subgenomic mRNA transcription of an engineered Sindbis virus defective interfering RNA in cells infected with vSINNS was observed . Furthermore, vSINNS could be used as a helper virus to amplify, to relatively high titers, a replication-defective Sindbis virus mutant containing an in-frame deletion in the conserved N-terminal domain of nsP3 . These data, as well as the observation that normal yields of parental Sindbis virus are produced in cells which have been previously infected with vSINNS, indicate that expression of Sindbis virus nsPs, in the absence of Sindbis virus-specific RNA replication, is not sufficient to block the formation of active RNA replication complexes by superinfecting Sindbis virus. Curr Opin Genet Dev, 1991 Dec, 1(4), 478 - 84 The antiquity of group I introns; Shub DA; The recent discovery of self-splicing introns in cyanobacteria has given renewed interest to the question of whether introns may have been present in the ancestor of all living things . The properties of introns in genes of bacteria and bacteriophages are discussed in the context of their possible origin and biological function. Protein Eng, 1991 Dec, 4(8), 955 - 61 Phage-enzymes: expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage; McCafferty J et al.; We have demonstrated that an active enzyme can be expressed on the surface of a bacteriophage . The gene encoding alkaline phosphatase from Escherichia coli was cloned upstream of gene 3, which encodes a minor coat protein of the filamentous bacteriophage, fd . A fusion protein of the correct size was detected from viral particles by Western blotting . Ultrafiltration confirmed that the enzyme fusion behaves as part of a larger structure as would be expected of an enzyme fused to a viral particle . Both wild-type alkaline phosphatase (Arg166) and an active site mutant (Ala166) expressed in this way retain catalytic activity and have qualitatively similar kinetic properties to free enzyme . Values were obtained for Km of 72.7 and 1070 microM respectively whilst relative kcat for the mutant was 36% of that for wild-type . Phage particles expressing alkaline phosphatase were bound to an immobilized inhibitor (arsenate-Sepharose) and eluted with product (20 mM inorganic phosphate) . In this way, the functional enzyme is co-purified with the DNA encoding it . This may permit a novel approach to enzyme engineering based on affinity chromatography of mutant enzymes expressed on the phage surface. J Biomol Struct Dyn, 1991 Dec, 9(3), 525 - 36 Observation of DNA molecules undergoing capillary electrophoresis; Song L et al.; This paper presents a method to observe the motions and configurations of large DNA molecules undergoing capillary electrophoresis (CE) . A simple device to perform CE horizontally under microscopic observation is designed and images of single DNA molecules inside the capillary are obtained using an epi-fluorescence microscope . DNA molecules moved towards the negative electrode when an electric field was applied . The mobilities of three types of DNA (T4 and lambda bacteriophage DNA and PBR322 plasmid DNA) were measured at different electric field strength . The mobility vs . electric field strength curves of these three large DNAs showed that the mobility remained constant at high electric field strength (200-600 Volt/cm) and increased significantly at low electric field strength (less than or equal to 50 Volt/cm.) . The apparent mobilities of the large DNA molecules were independent of molecular weight . At electric field strengths greater than or equal to 400 Volt/cm., big aggregates (snowballs) of DNA molecules formed and moved upstream towards the positive electrode . When the field was turned off, the aggregates dissociated into a cloud of single DNA molecules, and diffused into the solution. New Biol, 1991 Dec, 3(12), 1195 - 205 Bypass of a primase requirement for bacteriophage T4 DNA replication in vivo by a recombination enzyme, endonuclease VII; Mosig G et al.; A primase, the product of phage T4 gene 61, is required to initiate synthesis of Okazaki pieces and to allow bidirectional replication from several T4 origins . However, primase-defective T4 gene 61 mutants are viable . In these mutants, leading-strand DNA synthesis starts at the same time as in wild type infections, but, in contrast to wild type, initiation is unidirectional and the first replicative intermediates are large displacement loops . Rapid double-strand DNA replication occurs later after infection, generating multiple branched concatemers, which are cut and packaged into viable progeny particles, as in wild-type T4 . Evidence is presented that this late double-strand DNA replication requires functional endonuclease VII (endo VII), the product of the T4 gene 49 . We propose that endo VII can provide a backup mechanism when primase is defective, because it cuts recombinational junctions, generating 3' ends . These ends can prime DNA synthesis to copy the DNA strands that had been displaced during the initial origin-dependent replication . We explain the DNA-delay phenotype and the commonly observed temperature dependence of DNA replication in primase-deficient gene 61 mutants as a consequence of temperature-dependent translational control of gene 49 expression . In the presence or absence of functional primase endo VII is essential for correct packaging of DNA . The powerful selection that