Infect Agents Dis, 1993 Jun, 2(3), 118 - 31 Human monoclonal antibody Fab fragments cloned from combinatorial libraries: potential usefulness in prevention and/or treatment of major human viral diseases; Chanock RM et al.; Currently, there is increased interest in the use of human viral antibodies for prophylaxis and therapy because recent advances in molecular immunology have made it possible to generate large numbers of human monoclonal antibodies of desired specificity and functional activity in short order . The strategy, developed particularly at The Scripps Research Institute and at Cambridge University is based on antigen selection of such antibodies from combinatorial libraries that express Fabs on the surface of filamentous DNA bacteriophage . Fabs or their whole immunoglobulin derivatives that exhibit high avidity for the selecting antigen and high neutralizing activity for the corresponding virus have been identified, and many of these human monoclonal antibodies should prove to be useful in prophylaxis or therapy of presently uncontrolled, medically important human viral diseases. J Gen Virol, 1993 Jun, 74 ( Pt 6), 1133 - 40 Infectious RNA transcripts derived from cloned cDNA of papaya mosaic virus: effect of mutations to the capsid and polymerase proteins; Sit TL et al.; Genomic length cDNAs of papaya mosaic virus (PMV) RNA were generated utilizing reverse transcriptase (RNase H-) for first strand synthesis, Sequenase for second strand synthesis and primers specific for the 5' and 3' termini of the viral genome . These cDNAs were cloned into plasmid pUC18 and infectious RNA transcripts were synthesized in vitro from a bacteriophage T7 RNA polymerase promoter incorporated into the 5' specific primer . The infectivity of transcripts was 16% that of native PMV RNA . Increasing the poly(A) tail length from A24 to A71 produced a 43% increase in infectivity . Transcripts synthesized with or without an m7GpppG cap structure were biologically active although uncapped transcripts were much less infectious . The addition of up to 2434 non-viral nucleotides at the 3' end of transcripts decreased but did not abolish infectivity . Insertions of two amino acid residues within the polymerase coding region inactivated viral transcripts . A single amino acid deletion within the capsid protein (CP) produced local lesions of a reduced size as compared to native PMV RNA . Viral particles could not be observed in crude extracts from lesions produced by this deletion mutant suggesting that it exists as a naked RNA species within the host . Mutations to the CP suggest that it is required not only for viral assembly but also for some other unidentified function(s) during the replication cycle. Infect Immun, 1993 Jun, 61(6), 2377 - 82 Cloning and expression of a cDNA encoding epitopes shared by 15- and 60-kilodalton proteins of Cryptosporidium parvum sporozoites; Jenkins MC et al.; A cDNA (CP15/60) encoding epitopes of Cryptosporidium parvum 15- and 60-kDa sporozoite proteins was isolated and expressed in Escherichia coli toward the goal of developing an immunogen for producing high-titer anticryptosporidial colostrum . Antisera prepared in rats to native C . parvum 15-kDa protein and used to identify the CP15/60 bacteriophage clone recognized both 15- and 60-kDa in vitro translation products derived from sporozoite RNA . Antisera specific for recombinant CP15/60 antigen recognized native 15- and 60-kDa C . parvum sporozoite proteins by immunoblotting and identified both surface and internal antigens on C . parvum sporozoites by immunofluorescence staining . Northern (RNA) and Southern blot hybridization experiments using sporozoite RNA and DNA indicated that CP15/60 DNA is transcribed as a single 1.4-kb RNA species from a single-copy gene . Recombinant CP15/60 antigen was recognized by hyperimmune colostrum from cows immunized with C . parvum oocyst-sporozoite protein and by convalescent-phase sera from C . parvum-infected calves. Mutat Res, 1993 Jun, 302(2), 91 - 6 Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene; Shephard SE et al.; 2-Acetylaminofluorene (2-AAF) was administered at levels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic mice bearing the lacI gene in a lambda vector (Big Blue mice) . The lambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E . coli bacteria, forming plaques on agar plates . Approximately 10(5) plaques were screened per animal for the appearance of a blue colour, indicative of mutations in the lacI gene which had resulted in an inactive gene product . Background mutation rate was 2.7 x 10(-5) (pooled results of two animals, 8 mutant plaques/289,530 plaques) . At 300 ppm in the diet, the rate of 3.5 x 10(-5) (8/236,300) was not significantly increased over background . At 600 ppm in the diet, the rate increased approximately 3 fold to 7.7 x 10(-5) (17/221,240) . In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose levels (10-1000 fold lower) . Overt toxicity could thus be avoided . The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2-AAF. Biochim Biophys Acta, 1993 May 28, 1173(2), 201 - 8 Cloning, expression and in vitro characterisation of the M13 gene 5 protein; Turner GP et al.; The gene 5 protein encoded in the genome of bacteriophage M13 is a single stranded DNA binding protein essential for phage replication . We have cloned a fragment of the M13 genome containing gene 5, and investigated the effect of upstream elements on expression of the gene by means of Bal 31 deletion analysis . The gene was also expressed from the lac promoter of the phagemid vector pUC119, and the recombinant protein purified and characterised for DNA binding . The affinity of the recombinant protein for single-stranded DNA was shown to be essentially identical to that of wild type gene 5 protein . Wild type gene 5 protein has a glutamic acid residue at position 30 which, on the basis of the crystal structure, was believed to play a role in maintaining the tertiary structure of the protein through the formation of a salt bridge with arginine-80 . We show that substitution of glutamic acid at position 30 by lysine does not impair DNA binding, suggesting that a salt bridge between glutamate-30 and arginine-80 is not essential for the structural integrity of the gene 5 protein as previously proposed. J Immunol Methods, 1993 May 26, 161(2), 169 - 76 Phage display as a rapid gene expression system: production of bioactive cytokine-phage and generation of neutralizing monoclonal antibodies; Gram H et al.; Proteins, such as hormones, enzymes, or antibody binding sites, can be expressed in an active conformation on the surface of filamentous bacteriophage . Although the phage display technology was originally developed for binding studies, we demonstrate here that this technique can rapidly provide cytokines for studies of biological activity and for raising neutralizing monoclonal antibodies . A phage M13-based cloning vector was constructed that facilitated the expression of human interleukin 3 (hIL-3) on the phage surface . The recombinant phage could stimulate the growth of the hIL-3 dependent cell line M-07, providing evidence for the display of hIL-3 in an active form . Injection of recombinant phage into mice provoked an immune response to hIL-3, and neutralizing monoclonal antibodies directed against native hIL-3 could be established from these mice with a high frequency. J Theor Biol, 1993 May 21, 162(2), 243 - 52 Folding of the MS2 coat protein in Escherichia coli is modulated by translational pauses resulting from mRNA secondary structure and codon usage: a hypothesis; Guisez Y et al.; Possible translational pauses within the coat protein of the RNA bacteriophage MS2 were located on the basis of a distribution plot of rare codons and RNA secondary structure . It appeared that the position of certain codon pauses corresponds with the size of some nascent polypeptide intermediates, which have been isolated from MS2-infected cells . Other accumulated polypeptide intermediates seemed to be related to RNA regions, where double-stranded secondary structures occur, which probably impede the movement of ribosomes during chain elongation . We assume that a discontinuous translation rate is designed to allow optimal folding of this (and other) polypeptide(s). J Mol Biol, 1993 May 20, 231(2), 361 - 74 Studies of bacteriophage P2 DNA replication . The DNA sequence of the cis-acting gene A and ori region and construction of a P2 mini-chromosome; Liu Y et al.; A self-replicating plasmid was constructed from the 76.7 to 91.6% region of bacteriophage P2, which contains the P2 origin or replication (ori) and the genetically defined replication genes B and A . The sequence of the 76.7 to 80.2% has been determined previously, and the sequence of the 80.2 to 91.6% region is now reported . The sequenced region contained gene A, which predicts a 761 amino acid residue polypeptide known to induce a single-strand cut at or near ori, and ori, which has been located by electron microscopy to about 89% from the left end of the phage genome . Analysis of plasmid-encoded proteins in minicells indicated that the A gene product was about 78 kilodaltons . Five previously unknown open reading frames (orf-80, orf-81, orf-82, orf-83 and orf-91) were discovered . They have been cloned and their respective products were identified . The products of orf-80, orf-82 and gene A were found to be lethal to the host when overexpressed . The predicted amino acid sequences of the orf-82 and orf-83 gene products were similar to two early gene products of phage 186; the orf-91 product resembled the hypothetical protein of orfd of retron Ec67 . Similarities between the products of P2 gene A, 186 gene A and orf2 and orf3 of Ec67 were also found . A P2 mini-chromosome has been constructed that contains only the P2 A gene and the beta-lactamase gene as a marker. J Mol Biol, 1993 May 20, 231(2), 343 - 60 Clustered arginine residues of bacteriophage lambda N protein are essential to antitermination of transcription, but their locale cannot compensate for boxB loop defects; Franklin NC; The N protein coded by bacteriophage lambda plays an essential role in the completion of lambda transcription by recognizing the boxB sequence in nascent transcripts and then aggregating with Escherichia coli RNA polymerase and four other E . coli proteins into an unstoppable transcription complex . In order to explore the functionality of N protein and the specific recognition between N and boxB, 14 amino acid positions near the amino-terminal end of N lambda were mutated extensively . The mutant proteins were scored for N function in vivo by a two-plasmid construct that visualizes readthrough transcription as lacZ expression in colonies of E . coli . Mutation was achieved by single TAG replacements, translated through suppression into 13 different amino acids, or by scrambling at assorted three-codon sets . Of the 14 amino acid positions tested (Tables 5 and 6), six remained functional with a wide variety of substitutions, while substitution was sometimes deleterious at one Ala and two Gln positions . At each of the five Arg positions, however, maintenance of Arg occupancy proved important for N function . Despite effective screening for increased N function at boxBs with defective loops, no N mutant, simple or complex, was found to change the order of preference of wild-type N lambda for boxBs with defective loops . Thus, although multiple amino-terminal Arg positions are found to be important for N function, mutations in the region spanning the five Arg residues were not found to compensate for defects in boxB loop. Anal Chem, 1993 May 15, 65(10), 1323 - 8 Rapid purification of double-stranded DNA by triple-helix-mediated affinity capture; Ji H et al.; A simple and rapid method for the preparation of highly pure plasmid DNA has been developed . The DNA is directly captured from bacterial cell lysates by formation of a triple-helical structure between the plasmid dsDNA and a 20-base biotinylated oligonucleotide attached to streptavidin-coated magnetic beads and then eluted from the beads in pH 9 buffer solution . No phenol extraction, ethanol precipitation, RNase digestion, or CsCl gradient centrifugation is required . A general purpose cloning vector, pHJ19, was constructed for this application from pUC19 DNA by insertion of a 40-base sequence suitable for triple-helix formation . The approach was also found suitable for the purification of lambda bacteriophage DNA. J Biol Chem, 1993 May 15, 268(14), 10668 - 75 Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins; Patel SS et al.; The oligomeric structure of bacteriophage T7 gene 4 helicase/primase proteins was investigated using protein cross-linking and high pressure gel-filtration chromatography . Studies were carried out with both 4A' and 4B proteins . 4A' is a M64L mutant of 4A which has similar helicase and primase activities as the wild-type mixture of 4A and 4B proteins (Patel, S . S., Rosenberg, A . H., Studier, F . W., and Johnson, K . A . (1992) J . Biol . Chem . 267, 15013-15021), and 4B is the smaller protein which has only helicase activity . Chemical cross-linking of 4A' and 4B proteins with dimethyl suberimidate resulted in cross-linked species ranging from dimers to hexamers and beyond . The cross-linking time course, however, indicated that hexamers were the predominant species to accumulate in both 4A' and 4B proteins . The effect of MgNTP and DNA binding on oligomerization of the gene 4 proteins was investigated using high pressure gel-filtration chromatography at increasing protein concentrations . In the absence of added ligands, close to 100 microM protein concentrations were required to form stable oligomers beyond dimers . However, in the presence of Mg-beta, gamma-methylene deoxythymidine triphosphate (nonhydrolyzable analog of dTTP), 4A' and 4B protein assembled into stable hexamers at protein concentrations less than 8 microM . Addition of single-stranded DNA further stabilized the hexamer structure . Therefore, in the presence of a 60-nucleotide-long single-stranded DNA, hexamers were observed at protein concentrations as low as 0.2 microM . Nuclease protection experiments indicated that the 4A' and 4B hexamers protect about 60-65 bases of single-stranded DNA. J Biol Chem, 1993 May 15, 268(14), 10282 - 95 Isolation of helicase alpha, a DNA helicase from HeLa cells stimulated by a fork structure and signal-stranded DNA-binding proteins; Seo YS et al.; A DNA helicase, called DNA helicase alpha, was purified from HeLa cells to apparent homogeneity . The helicase and its single-stranded DNA-dependent ATPase activities cosedimented in glycerol gradients with two polypeptides of 110 and 90 kDa with a sedimentation coefficient of 7.4 S . The DNA helicase was markedly stimulated by DNA substrates with a 5'-tailed fork . A DNA substrate with a 3'-tailed fork structure was less stimulatory, although it was more active than substrates without a fork . The directionality of unwinding is 3'-->5' with respect to the single-stranded DNA to which the enzyme was bound . The helicase activity also required a single-stranded DNA-binding protein (SSB) for unwinding activity . The stimulation by SSBs was nonspecific; all SSBs tested, such as human SSB, bacteriophage T4 gene 32, and Escherichia coli SSB, stimulated the DNA helicase activity to a varying extent in the presence of a fork structure . With long duplex substrates (> 500 base pairs), the presence of a fork substantially stimulated the DNA helicase activity in the presence of E . coli SSB . Human SSB stimulated the DNA helicase activity to the greatest extent (> 10-fold) with a substrate containing a fork compared with substrates without a fork . DNA helicase activity required ATP hydrolysis and could be supported by all eight nucleoside triphosphates . The Km values for ATP and dATP in unwinding were 28 and 48 microM, respectively . In general, ribonucleoside triphosphates were better effectors than deoxyribonucleoside triphosphates . The properties of this DNA helicase make it a candidate for a DNA replicative helicase in human cells. Biochem Biophys Res Commun, 1993 May 14, 192(3), 1445 - 52 Expression and biochemical characterization of the DNA binding activity of TcA, the putative transposase of Caenorhabditis elegans transposable element Tc1; Abad P et al.; The TcA protein is one of the proteins essential for Tc1 transposition . In order to study the biochemical parameters of Tc1 transposition mechanism, we used TcA protein overproduced in baculovirus system for DNA binding experiments . We show that, despite its relatively strong non specific affinity for DNA, TcA exhibits a better affinity for its Tc1 specific binding sites . The K0.5 is 3.8 nM for the Tc1 whereas in the same type of experiment the K0.5 is 24 nM for calf thymus DNA . The ratio value between specific and non specific DNA binding activity of the TcA protein was also exhibited by other transposases such as those of the bacteriophage Mu, Tn 10 and the Drosophila P element . This nonspecific DNA binding activity may be involved in determining sites of transposable element insertion. Nucleic Acids Res, 1993 May 11, 21(9), 2131 - 8 Repression of bacteriophage promoters by DNA and RNA oligonucleotides; Skoog JU et al.; We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids rather than polypeptides . An in vitro model system involving repression of bacteriophage T7 RNA polymerase initiation has been employed to demonstrate that certain DNA oligonucleotides can repress transcription by site-specific triple-helix formation at two kinds of homopurine operator sequences {Maher, L . J., III, (1992) Biochemistry 31, 7587-7594} . Recognition in the purine motif is based on antiparallel oligonucleotide binding (G.G.C and T.A.T triplets) . Recognition in the pyrimidine motif is based on parallel oligonucleotide binding (C+.G.C and T.A.T base triplets) . Using this system, we report that the concentration-dependence of repression by DNA oligonucleotides provides triple-helix inhibition constant (Ki) estimates of approximately 2 x 10(-7) M for both purine motif and pyrimidine motif DNA complexes . RNA oligonucleotides are shown to repress promoters overlapping pyrimidine motif operators (Ki = 6 x 10(-7) M), but not purine motif operators . Although competent to hybridize to complementary single strands, RNA oligonucleotides fail to bind the purine motif operator . Partial substitution of deoxyribose residues tends to rescue repressor activity by RNA oligonucleotides in the purine motif . These results suggest prospects for, and constraints on, natural and artificial RNA-based repressors. Biochemistry, 1993 May 11, 32(18), 4708 - 18 Effect of site-specifically located mitomycin C-DNA monoadducts on in vitro DNA synthesis by DNA polymerases; Basu AK et al.; A series of site-specifically modified oligodeoxynucleotides were synthesized that contained either of the two known mitomycin C-DNA monoadducts . In vitro DNA synthesis was carried out on some of these templates using a modified bacteriophage T7 DNA polymerase (Sequenase), AMV reverse transcriptase, and two different varieties of Escherichia coli DNA polymerase I (Klenow fragment)--one that carries the normal 3'-->5' exonuclease activity and a mutant protein that lacks this enzymatic function . Regardless of the type of DNA polymerase being used, DNA synthesis was terminated nearly quantitatively at the nucleotide 3' to each of these two monoadduct sites, although primer extension to full length of the template was noted with the unmodified control template . Substitution of Mn2+ for Mg2+ at a high concentration of the deoxynucleotide triphosphates resulted in incorporation of nucleotides opposite the adduct in the incubations with Sequenase or the 3'-->5' exonuclease-free Klenow fragment; however, primer extension beyond the adduct site did not take place . These studies demonstrated that the mitomycin monoadducts are strong blocks of replication and are likely to be toxic lesions in vivo . Since previous molecular modeling studies and molecular mechanical calculations indicated that the mitomycin adduction does not induce severe distortions at the site of adduction, a lack of base-pairing ability of the modified base in the extended product is unlikely to be the reason for the inhibitory effect . Instead, energy-minimized structural models indicated that additional hydrogen-bonding interactions have been introduced by the mitomycin moiety, and perhaps this increased thermodynamic stabilization of a distorted structure of the replication fork, in turn, may block the replication bypass . Experimental evidence of increased thermodynamic stability was provided by thermal melting of a template/primer complex that presumably a polymerase encounters in a typical replication fork . Consistently higher Tm of the adducted "replication fork" was noted when compared to its unmodified counterpart. Nucleic Acids Res, 1993 May 11, 21(9), 2073 - 9 Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E . coli and T7 RNA polymerases; Hanna MM et al.; A new photocrosslinking CTP analog that functioned as a substrate during transcription was synthesized and used to photoaffinity label E . coli and bacteriophage T7 RNA polymerases . This analog, 5-((4-azidophenacyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 A from the nucleotide base and specifically replaced CTP during synthesis of RNA by both polymerases . Analog was placed at the 3' end or internally within RNA . Both polymerases inefficiently incorporated two 5-APAS-CMP molecules sequentially, as was found for the related 5-APAS-UMP . Analog was placed at the 3' end of RNA in transcription complexes paused at the site of Q-modification of E . coli RNA polymerase, downstream of the lambda PR' promoter (+16), a pause that requires specific DNA sequences but no apparent RNA hairpin . Crosslinking was examined in the presence and absence of the NusA protein, which enhances the transcriptional pause at this site and is required for Q modification of the polymerase . Crosslinking of the 3' end of the RNA to NusA was not observed, consistent with our earlier results involving a NusA-enhanced pause site downstream from an RNA hairpin. J Mol Biol, 1993 May 5, 231(1), 19 - 28 Conformation of the origin of P1 plasmid replication . Initiator protein induced wrapping and intrinsic unstacking; Mukhopadhyay G et al.; The origin of plasmid DNA replication in bacteriophage P1 has five 19 base-pair sites that bind the plasmid-encoded initiator, RepA . Here we show, using a DNA band retardation assay, that RepA can bend DNA that carries one or more of the RepA binding sites . RepA binding to supercoiled DNA carrying the five sites, directly repeated and phased two turns of B-DNA apart, absorbs about one positive superhelical turn of DNA as determined by two-dimensional gel electrophoresis . This indicates that the DNA is wrapped around RepA as a consequence of in-phase bending at the individual binding sites . The RepA-DNA complexes did not show elevated sensitivity to KMnO4, a reagent specific for pyrimidine bases (T >> C) in unstacked DNA . The wrapping of the DNA around RepA, therefore, does not lead to significant unwinding of the double helix . Extensive unwinding, suitable for the initiation of DNA replication, most likely requires participation of factors other than RepA . We also noted that the thymine bases of the sequence 5'-ATC-3', of which there are 20 in the origin, all reacted to KMnO4 strongly whether or not RepA was present . The preferential reactivity of ATC sequences was specific to the origin region, as thymine residues including those in the ATC sequences did not display elevated sensitivity to KMnO4 in a DNA fragment from pBR322 . On one of two strands of the origin the selective reactivity at the ATC sequences was supercoiling dependent . These results indicate that the origin includes unstacked DNA bases, the significance of which remains to be determined. Plant Cell, 1993 May, 5(5), 577 - 86 Analysis of a tobacco mosaic virus strain capable of overcoming N gene-mediated resistance; Padgett HS et al.; The genome of Ob, a tobamovirus that overcomes the N gene-mediated hypersensitive response (HR), was cloned as a cDNA, and its nucleotide sequence was determined . The genomic organization of Ob is similar to that of other tobamoviruses, consisting of 6506 nucleotides and containing at least four open reading frames . These open reading frames encode a 126-kD polypeptide with a 183-kD readthrough product, a 30.6-kD movement protein, and an 18-kD coat protein . A bacteriophage T7 promoter sequence was fused to the full-length cDNA clone to obtain infectious RNA transcripts . These transcripts, when inoculated onto tobacco plants, induced disease symptoms indistinguishable from plants inoculated with Ob viral RNA . To determine which viral factor is responsible for the resistance-breaking character of Ob, a recombinant virus was constructed in which the movement protein gene of tobacco mosaic virus was replaced with that of Ob . Cultivar Xanthi NN tobacco plants infected with this virus responded with an HR, indicating that the Ob movement protein alone does not act to overcome the N gene-mediated response . Following mutagenesis of the infectious Ob cDNA clone with hydroxylamine, populations of transcripts from the mutagenized DNA were inoculated onto Xanthi NN tobacco, and a variant that induced the HR was identified . The mutant was analyzed and found to contain a single nucleotide change in the 126-kD gene . Recreating the mutation in the Ob cDNA clone by site-directed mutagenesis resulted in a virus that caused symptoms identical to the chemically induced mutant. J Bacteriol, 1993 May, 175(10), 2833 - 8 Cloning, expression, and characterization of the icd gene in the immI operon of bacteriophage P1; Riedel HD et al.; The immI operon of P1 contains the genes c4, icd (formerly called orfx), and ant which are constitutively transcribed in that order from a single promoter, P51b . C4 is an antisense RNA which is processed from the precursor transcript . C4 RNA acts as a translational repressor of icd, thereby also inhibiting antirepressor (ant) synthesis . We have cloned the icd and the overlapping icd and ant genes . We show, by means of plasmid deletion analysis, that icd is translationally coupled to ant . An internal in-frame deletion of icd making up 65% of the codons still allows antirepressor synthesis at a reduced rate, indicating that a functionally active icd gene product is dispensable for ant expression . We identify the product of the icd gene as a 7.3-kDa protein which interferes with cell division . The results suggest that constitutive expression of icd, in the absence of a functionally active antirepressor, prevents P1 lysogen formation because of its detrimental effect on the host cell. J Bacteriol, 1993 May, 175(10), 2809 - 17 Identification of a segment of the Escherichia coli Tsx protein that functions as a bacteriophage receptor area; Schneider H et al.; The Escherichia coli outer membrane protein Tsx functions as a nucleoside-specific channel and serves as the receptor for colicin K and a number of T-even-type bacteriophages, including phage T6 . To identify those segments of the Tsx protein that are important for its phage receptor function, we devised a selection and screening procedure which allowed us to isolate phage-resistant strains synthesizing normal amounts of Tsx . Three different Tsx-specific phages (T6, Ox1, and H3) were employed for the selection of phage-resistant derivatives of a strain expressing a tsx(+)-lacZ+ operon fusion, and 28 tsx mutants with impaired phage receptor function were characterized . Regardless of the Tsx-specific phage used for the initial mutant selection, cross-resistance against a set of six different Tsx phages invariably occurred . With one exception, these mutant Tsx proteins could still serve as a colicin K receptor . DNA sequence analysis of 10 mutant tsx genes revealed the presence of four distinct tsx alleles: two point mutations, an 18-bp deletion, and a 27-bp tandem duplication . In three isolates, Asn-249 was replaced by a Lys residue (tsx-504), and in four others, residue Asn-254 was replaced by Lys (tsx-505) . The deletion (tsx-506; one isolate) removed six amino acids (residue 239 to residue 244) from the 272-residue Tsx polypeptide chain, and the DNA duplication (tsx-507; two isolates) resulted in the addition of nine extra amino acids (residue 229 to residue 237) to the Tsx protein . In contrast to the wild-type Tsx protein and the other mutant Tsx proteins the Tsx-507 protein was cleaved by trypsin when intact cells were treated with this protease . The Tsx proteins encoded by the four tsx alleles still functioned in deoxyadenosine uptake in vivo, demonstrating that their nucleoside-specific channel activity was not affected by the alterations that caused the loss of their phage receptor function . HTe changes in the Tsx polypeptide that confer resistance against the Tsx-specific phages are clustered in a small region near the carboxy terminus of Tsx . Our results are discussed in terms of a model for the topological organization of the carboxy-terminal end of the Tsx protein within the outer membrane. Virology, 1993 May, 194(1), 117 - 27 The short tail-fiber of bacteriophage T4: molecular structure and a mechanism for its conformational transition; Makhov AM et al.; Electron microscopy, image processing and computational sequence analysis were used to investigate the structure of the short tail-fiber of bacteriophage T4 . This molecule, an oligomer of gp12, is an adhesin that binds the virion irreversibly to the bacterial surface . Short tail-fibers were isolated from mutant-infected cells in which gp12 is synthesized and assembled correctly, but not incorporated into virions . Visualized in negative stain, these filamentous molecules are approximately 38 nm in total length, with an arrowhead-shaped head (approximately 10 nm long by 6 nm wide), a 24-nm shaft of uniform width (approximately 3.8 nm), and a small, seemingly flexible, tail . The primary sequence contains a domain consisting of tandem quasi-repeats, each about 40 residues long, extending from approximately residue 50 to residue 320 . Molecular mass analyses by scanning transmission electron microscopy confirm that the molecule is a trimer . The masses of the head, shaft, and tail domains are consistent with (trimers of) the carboxy-terminus, the repeat region, and the amino-terminus, respectively . When short tail-fibers are visualized extending from baseplates, their heads are distal, i.e., detached, implying that it is the tail that remains in contact with the baseplate . Analysis of the molecules' curvature properties detects three hinge-sites: these suggest how the short tail-fiber may be initially accommodated in a compact conformation in the "hexagon" state of the baseplate, from which it converts to the extended conformation when the baseplate switches into its "star" state. J Immunol, 1993 May 1, 150(9), 3817 - 24 Strain-dependent leakiness of mice with severe combined immune deficiency; Nonoyama S et al.; Mice with immunodeficiency provide an excellent in vivo model for cell transfer experiments . In this study, we compare the extent of immune deficiency of the original CB17 severe combined immune-deficient (SCID) mice with that of two other strains of immune-deficient mice, the recently developed C3H SCID mice and the beige/nude/X-linked immune-deficient (BNX) mice . Detectable levels of serum lg (higher than 0.4 microgram/ml) were found in 79% of CB17 SCID mice studied (n = 24) and in all BNX mice (n = 12); some leaky CB17 SCID mice had normal levels of Ig . In contrast, only 15% of C3H SCID mice (n = 61) had detectable serum lg; the highest Ig level in this strain was 9.6 micrograms/ml . Age had no effect on serum Ig concentrations of C3H SCID mice; in contrast, all old (> 1-year-old) CB17 SCID mice studied had detectable levels of serum Ig . Transfer of syngeneic, normal, neonatal thymocytes increased serum Ig of SCID mouse origin to near-normal levels in all CB17 SCID mice but had no effect on serum lg concentrations in C3H SCID mice . Treatment with anti-asialo-GM-1 antiserum to abrogate NK cell activity increased serum Ig levels in 37% of CB17 SCID mice but had no effect on Ig production in C3H SCID mice . Flow cytometric analysis failed to identify mature T or B cells in C3H SCID mice; in contrast, some leaky CB17 SCID mice had detectable numbers of T and B cells in the peritoneal cavity . After immunization with bacteriophage phi X 174, neither C3H nor CB17 SCID mice, including leaky mice, produced specific antibody to phage . In contrast, BNX mice produced small but significant amounts of anti-phage antibody . These results indicate that, of the three strains of immune-deficient mice, C3H SCID mice have the most severe immune defect . We predict that C3H SCID mice will be best suited for cell transfer experiments. J Struct Biol, 1993 May-Jun, 110(3), 177 - 9 Liquid crystalline DNA in fowl adenovirus; Ruigrok RW et al.; Fowl adenovirus particles were studied using negative stain electron microscopy . Upon preparation, some particles collapsed and showed their internal structure . The DNA could be observed as thin parallel lines with a spacing of 25 A, very similar to images of the liquid crystalline DNA in bacteriophage heads and in herpes virus. J Bacteriol, 1993 May, 175(10), 3067 - 74 Localization and nucleotide sequences of genes mediating site-specific recombination of the SLP1 element in Streptomyces lividans; Brasch MA et al.; SLP1 is a 17.2-kbp genetic element indigenous to the Streptomyces coelicolor chromosome . During conjugation, SLP1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells . We report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events . A region of SLP1 adjacent to the previously identified site of integration, attP, was found to be sufficient to promote site-specific integration of an unrelated Streptomyces plasmid . Nucleotide sequence analysis of a 2.2-kb segment of this region reveals two open reading frames that are adjacent to and transcribed toward the attP site . One of these, the 1,365-bp int gene of SLP1, encodes a predicted 50.6-kDa basic protein having substantial amino acid sequence similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage lambda integrase . A linker insertion in the 5' end of the cloned int gene prevents integration, indicating that Int is essential for promoting integration . An open reading frame (orf61) lying immediately 5' to int encodes a predicted 7.1-kDa basic peptide showing limited sequence similarity to the excisionase (xis) genes of other site-specific recombination systems. Virology, 1993 May, 194(1), 192 - 9 Analysis of retroviral assembly using a vaccinia/T7-polymerase complementation system; Dong J et al.; The nature of protein-protein interactions during retroviral assembly is not well understood, and mutational analyses of the potential signals involved in the viral assembly process has been difficult, particularly with the avian retroviruses due to the level of viral proteins expressed in the clonal cell lines containing defective viral genomes . We describe here a complementation system in which the retroviral gag/pol and env gene products were expressed independently from different plasmids under the control of the bacteriophage T7 promoter, in avian cells . Coexpression of the T7 polymerase from a vaccinia virus vector resulted in a high level of biosynthesis of retroviral structural proteins and efficient assembly of virus particles . Electron microscopy and protein composition analyses demonstrated that these virions were indistinguishable from those produced from RSV-infected cells . Through the use of mutant glycoprotein genes it was possible to demonstrate the specificity of the assembly process and the applicability of this system to other retroviral systems is described. J Bacteriol, 1993 May, 175(9), 2625 - 31 A role for the Clp protease in activating Mu-mediated DNA rearrangements; Shapiro JA; Bacteriophage Mu, one of the best-characterized mobile genetic elements, can be used effectively to answer fundamental questions about the regulation of biochemical machinery for DNA rearrangement . Previous studies of Mu virulence have implicated the Clp protease in repressor inactivation (V . Geuskens, A . Mhammedi-Alaoui, L . Desmet, and A . Toussaint, EMBO J . 13:5121-5127, 1992) . These studies were extended by analyzing the phenotypic consequences of clp alleles in two Escherichia coli systems: (i) the periodic replication of Mudlac transposons in colonies and (ii) the action of a Mu prophage in forming araB-lacZ coding sequence fusions . The clpP::CM mutation, which removes the proteolytic subunit of Clp protease, caused a drastic reduction in Mu activity in both systems . The clpA::Tn10 mutation, which removes a regulatory subunit of Clp protease, altered the timing of Mu activity in both systems . A clpA deletion reduced the extent of Mudlac replication in colonies . These results point to temporal changes in Clp proteolysis of the Mucts62 repressor as a key molecular event in the regulation of one class of genomic change in E . coli. Photochem Photobiol, 1993 May, 57(5), 819 - 24 Determination of residual 4'-aminomethyl-4,5',8-trimethylpsoralen and mutagenicity testing following psoralen plus UVA treatment of platelet suspensions; Wagner SJ et al.; Psoralens and UVA light have been used in the laboratory to study the inactivation of viruses that may be infrequently present in platelet concentrates that are prepared for transfusion . In order to evaluate safety aspects of the treatment of platelet suspensions with 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), we have investigated the residual levels and mutagenic potential of AMT after UVA phototreatment . 4'-aminomethyl-4,5',8-trimethylpsoralen, at a final concentration of 40 micrograms/mL, was added to platelet suspensions which contained 16% plasma and a synthetic medium . Platelet suspensions containing AMT were irradiated with up to 7.2 J/cm2 UVA light under normal oxygen levels . Residual levels of AMT were determined by HPLC and a bioassay based on bacteriophage phi 6 inactivation . The photodestruction of AMT or its activity by UVA was characterized by a D37 value of 0.6 and 0.3 J/cm2 with HPLC or bioassay, respectively . At 2.4 J/cm2 UVA, which results in approximately 5 log10 inactivation of vesicular stomatitis virus (VSV) and retention of platelet in vitro properties, 12% (HPLC) to 9% (bioassay) AMT remained . Like other psoralens, AMT was found to bind to serum proteins as shown by ultrafiltration . Results are consistent with approximately 36% of the initial drug load binding primarily to serum albumin . It was determined using 3H-AMT that 9 to 18% of radioactivity was bound to platelets in the absence of irradiation . Similar fractions (13 to 18%) of AMT were bound to platelets after 3.6 J/cm2 UVA irradiation, and 8 to 10% of total AMT was associated with saline-washed irradiated platelets and is presumably tightly bound.(ABSTRACT TRUNCATED AT 250 WORDS) Somat Cell Mol Genet, 1993 May, 19(3), 245 - 55 New patterns of bulk DNA repair in ultraviolet irradiated mouse embryo carcinoma cells following differentiation; Rasko I et al.; Mouse embryocarcinoma stem cells differentiate in culture, given the appropriate induction . We examined whether these cells could provide information about the regulation of nucleotide excision repair in relation to differentiation by measuring the rate-limiting incision step, the removal of cyclobutane dimers and (6-4) photoproducts from the genome as a whole and the effect of the bacteriophage T4 endonuclease (denV) gene on repair in differentiated cells . It was found that differentiation is accompanied by a marked decline in the early incision ability after UV irradiation (sixfold for P19, fourfold for PCC7 and twofold for F9), and we measured, in parallel, the loss of two common UV photoproducts {cyclobutane dimers and (6-4) photoproducts} from P19 cells . After differentiation, the excellent overall cyclobutane dimer repair capacity of proliferating cells (84% removal in 24 h) is lost (no removal in 24 h), while removal of (6-4) photoproducts, although normal at 24 h (94%), is much slower than in undifferentiated P19 at 3 h (no removal versus 64%) . The presence of the denV gene greatly stimulates the repair of cyclobutane dimers in undifferentiated P19 cells (94% removal at 3 h versus 40%) and also in differentiated cells (50% removal at 24 h versus no removal) . The denV gene also stimulates the early repair of (6-4) photoproducts in both differentiated and undifferentiated cells. PCR Methods Appl, 1993 May, 2(4), 275 - 87 High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity; Lawyer FC et al.; The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda PL promoter . A truncated form of Taq Pol I was also constructed . The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment . Upon heat induction, the 832-amino-acid construct produced 1-2% of total protein as Taq Pol I . The induced 544-amino-acid construct produced 3% of total protein as Stoffel fragment . Enzyme purification included cell lysis, heat treatment followed by Polymin P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography . For full-length 94-kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste . For the 61-kD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight cell paste . The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCl2 and 10-55 mM KCl . The nature of the substrate determines the precise conditions for maximal enzyme activity . For both proteins, MgCl2 is the preferred cofactor compared to MnCl2, CoCl2, and NiCl2 . The full-length Taq Pol I has an activity half-life of 9 min at 97.5 degrees C . The Stoffel fragment has a half-life of 21 min at 97.5 degrees C . Taq Pol I contains a polymerization-dependent 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity . A comparison is made among thermostable DNA polymerases that have been characterized; specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest reported. Genomics, 1993 May, 16(2), 333 - 41 Chromosomal structure and expression of the human OTF1 locus encoding the Oct-1 protein; Sturm RA et al.; The genomic structure of the POU domain containing oct-1 gene (OTF1 locus) coding region has been determined using human DNA recombinant bacteriophage and a yeast artificial chromosome clone . The gene is encoded by 16 exons spanning over 150 kb, and the Oct-1 protein reading frame has been extended to 766 amino acids . The exonic structure has been compared to the mouse Oct-2 protein and reveals a conservation of exon-intron boundaries as well as protein sequence similarity . To provide insight into Oct-1 control of transcriptional regulation during the cell-cycle the expression of the oct-1 gene was examined during cellular DNA replication and shows that the steady-state level of the oct-1 mRNA is not S-phase regulated. Genes Chromosomes Cancer, 1993 May, 7(1), 15 - 27 Characterization of human bone marrow-derived closed circular DNA clones; Lou Z et al.; Because of interest in mechanisms of recombination involved in chromosomal deletions in neoplastic disease, and their relation to possible rearrangements in normal tissues, we are studying circular DNA molecules from human tissue with a long-term goal of investigating them as possible by-products of physiologically relevant intrachromosomal recombination events . Covalently closed circular (ccc) DNA from human bone marrow was cloned in bacteriophage vectors, and fourteen clones chosen randomly from the cccDNA-derived library were characterized . Five clones originated from chromosome-specific centromeric alpha-satellite DNA; two clones carried highly repetitive sequences probably derived from interspersed repetitive elements; six clones were derived from single-copy chromosome-specific sequences which detected homologous rodent sequences; and one clone (EPM10) was derived from a small chromosome 11-specific sequence family which localized to chromosome regions 11cen and 11q14 . Oligonucleotide primers derived from the cccDNA clones were used in polymerase chain reaction studies to show that (1) the EPM10 clone carried the circular junction, (2) several of the single-copy products could be detected in three different bone marrow cccDNA preparations, and (3) the Alu-PCR profile for bone marrow cccDNA showed distinct bands which were similar in four bone marrow cccDNA preparations. Biotech Histochem, 1993 May, 68(3), 153 - 8 Synthesis of digoxigenin-labeled cRNA probes for nonisotopic in situ hybridization using reverse transcription polymerase chain reaction; Young ID et al.; Nonisotopic methods of mRNA in situ hybridization have distinct advantages over isotopic techniques . Nonisotopically labeled probes are stable and nontoxic, have short detection times, demonstrate excellent spatial resolution of their signals and have sensitivities comparable to radiolabeled probes . We developed a simple method of generating nonisotopically labeled cRNA probes which is based on the reverse transcription polymerase chain reaction (RT-PCR) and used it to synthesize a panel of probes for various murine extracellular matrix genes . Engelbreth-Holm-Swarm (EHS) tumor RNA was reverse transcribed and PCR was used to amplify defined regions of multiple extracellular matrix protein genes from the resulting first strand cDNAs . Bacteriophage promoters which had been incorporated into the PCR products were then used to generate digoxigenin-conjugated antisense and sense cRNAs . The antisense probes were employed to detect the specific extracellular matrix protein mRNAs in the EHS tumor by in situ hybridization . This technique provides a rapid and efficient alternative to conventional transcription systems which use plasmid vectors for the synthesis of digoxigenin-labeled cRNA probes. Mol Biol (Mosk), 1993 May-Jun, 27(3), 561 - 8 {Preparation of a specific immunogen based on bacteriophage M13}; Minenkova OO et al.; Earlier we developed an expression vector on the basis of bacteriophage M13 allowing the exposure of short peptides on the virion surface . It was used to obtain a recombinant phage carrying the antigenic determinant of HIVI gag protein p17 . This phage was tested as immunogen in rabbits . It was shown by ELISA that Ig against the fusion phage reacted with the 17-kDa core protein of the virus and with its polyprotein precursor p55 on strips activated by the transfer of HIVI viral proteins . These data may be used in vaccine development. Mutat Res, 1993 May, 299(3-4), 183 - 91 Damage to plasmid DNA by singlet oxygen and its protection; Sies H; Singlet oxygen, generated by photoexcitation or by chemiexcitation, selectively reacts with the deoxyguanosine moiety in DNA (kq + kr about 5 x 10(6) M-1s-1) . The oxidation products include 8-oxo-7,8-dihydroeoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) . Singlet oxygen also causes strand breaks in DNA, studied in plasmids and bacteriophages . The biological consequences include a loss of transforming activity as well as mutagenicity and genotoxicity . Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet-oxygen-induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells . Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids (kq = 10(9) - 10(10) M-1s-1) and tocopherols . Whether this activity explains the protective effect of carotenoids on neoplastic transformation is still unknown. Mutat Res, 1993 May, 299(3-4), 165 - 82 Mutational specificity of oxidative DNA damage; Retel J et al.; In this paper we describe our studies on the mutagenic consequences of oxidative DNA damage introduced by radiation-induced OH radicals (.OH) and by exposure to singlet oxygen (1O2), released by thermo-dissociation of the endoperoxide 3,3'-(1,4-naphthalidene) dipropionate (NDPO2) . We have made use of M13mp10 bacteriophage and pUC18 plasmid DNA, containing a 144 base pair (bp) insert in the lacZ alpha gene . This 144 bp insert was used as a mutational target sequence . When dilute aqueous solutions of double-stranded (ds) M13mp10 (plus 144 bp insert) were gamma-irradiated in the presence of oxygen (O2; 100% .OH) or nitrous oxide (N2O; 90% .OH, 10% .H), very specific mutation spectra were found . Mainly bp substitutions were observed, of which C/G to G/C transversions are the predominant type . Moreover, the mutations are for the most part concentrated into two mutational hot spots: a minor and major one . Differences between the oxic (O2) and anoxic (N2O) mutation spectra could also be observed . Under N2O-1 bp deletions were detected, which are absent in the presence of O2, and in the anoxic spectrum more C/G to A/T transversions are present . To investigate whether these differences were due to the small amount of H radicals, which are formed under N2O, ds M13mp10 (plus 144 bp insert) was exposed to gamma-rays in phosphate buffer under nitrogen (55% .H, 45% .OH) . Under these conditions a remarkable shift was observed from C/G-->G/C to C/G-->A/T transversions, while the mutations were far more scattered along the 144 bp sequence and no -1 bp deletions were detected . These results strongly suggest that H radicals do not cause -1 bp deletions, but may be responsible for the observed C/G to A/T transversions . The kind of bp substitution not only appeared to be dependent on the type of the water radicals, but also appeared to be strongly influenced by the replicon in which the target sequence is incorporated . When an oxygenated solution of pUC18 plasmid DNA (plus 144 bp insert) is irradiated, mainly C/G to A/T transversions were found at the same major hot spot instead of C/G to G/C transversions when the 144 bp sequence is part of M13mp10 DNA . Finally, in agreement with the observation that 1O2 reacts preferentially with guanine in DNA, a guanine is involved in most of the mutations scored after exposure of single-stranded (ss) M13mp10 DNA to NDPO2-generated 1O2.(ABSTRACT TRUNCATED AT 400 WORDS) J Virol, 1993 May, 67(5), 2879 - 86 Protein P4 of double-stranded RNA bacteriophage phi 6 is accessible on the nucleocapsid surface: epitope mapping and orientation of the protein; Ojala PM et al.; Protein P4, an early protein of double-stranded RNA bacteriophage phi 6, is a component of the virion-associated RNA polymerase complex and possesses a nucleoside triphosphate (NTP) phosphohydrolase activity . We have produced and characterized a panel of 20 P4-specific monoclonal antibodies . Epitope mapping using truncated molecules of recombinant P4 revealed seven linear epitopes . The accessibility of the epitopes on the phi 6 nucleocapsid (NC) surface showed that at least the C terminus and an internal domain, containing the consensus sequence for NTP binding, protrude the NC shell . Four of the NC-binding antibodies distorted the integrity of the NC by releasing protein P4 and the major NC surface protein P8 . This finding suggests a close contact between these two proteins . The dissociation of the NC led to the activation of the virion-associated RNA polymerase . The multimeric status of the recombinant P4 was similar to that of the virion-associated P4, indicating that no accessory virus proteins are needed for its multimerization. Gene, 1993 Apr 30, 126(2), 227 - 35 Human L7a ribosomal protein: sequence, structural organization, and expression of a functional gene; De Falco S et al.; A cDNA coding for the human L7a ribosomal protein (r-protein) was used to isolate the corresponding gene by screening two human genomic libraries constructed in bacteriophage lambda and in a cosmid vector . One of the cosmid clones isolated, cos1.1, contains the whole L7 alpha gene, composed of eight exons and seven introns spanning 3226 bp . As in other mammalian housekeeping genes, the promoter and the first exon of the L7 alpha reside within a CpG-rich island . Furthermore, similar to the other higher eukaryote r-protein-encoding genes characterized so far, the human L7 alpha gene has a C as the major transcriptional start point localized in a pyrimidine-rich region and lacks a canonical TATA sequence . We show that 130 bp of the human L7 alpha gene 5'-flanking region represent the minimal element required to promote its transcription . This element is strikingly conserved between the mouse and human L7 alpha genes . Finally, a comparison of the human L7 alpha gene coding sequence and the predicted amino acid (aa) sequence with the sequences of mouse L7a, rat L7a, and the homologous yeast L4 shows that the aa sequence has been highly conserved during evolution. Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 511 - 7 High-level expression of porcine fructose-1,6-bisphosphatase in Escherichia coli: purification and characterization of the enzyme; Burton VA et al.; A cDNA encoding porcine fructose-1,6-bisphosphatase was isolated from total pig liver RNA . The enzyme's coding sequence was fused to the bacteriophage T7 gene 10 promoter and transcription terminator sequence and expressed in E . coli under control of the T7 RNA polymerase . Induced cells contain elevated levels of fructose-1,6-bisphosphatase enzymatic activity and an abundant 37,000 dalton protein . The enzyme was purified to apparent homogeneity and judged identical to wild-type porcine fructose-1,6-bisphosphatase . The kinetic parameters are similar to those reported for the pig kidney enzyme . The N-terminal amino acid sequence is identical to the predicted sequence and the kinetic parameters are consistent with freedom from proteolysis . As estimated from enzymatic activity and visual inspection of coomassie blue-stained SDS-PAGE gels, porcine fructose-1,6-bisphosphatase constitutes as much as 20% of the soluble protein from the over-expressing E . coli strain. Nature, 1993 Apr 29, 362(6423), 852 - 5 Solution structure of the POU-specific DNA-binding domain of Oct-1; Dekker N et al.; The transcription factor Oct-1 belongs to a family containing a POU DNA-binding domain . This bipartite domain is composed of a POU-specific domain (POUs) and a POU-homeodomain (POUhd) connected by a flexible linker . The left half of the optimal POU binding site, the octamer ATGCAAAT, is recognized by POUs and the right half by POUhd . We have determined the solution structure of POUs by nuclear magnetic resonance . It consists of four alpha-helices connected by short loops . Helices I and IV are in a parallel coiled-coil arrangement . The folding topology appears to be similar to that of the bacteriophage lambda-repressor and 434 repressor . For the well defined parts of the protein (residues 1-71), the average root-mean square deviation for the backbone atoms is 0.9 A . Based on the observed selective exchange broadening in the (15N,1H)-HMQC (heteronuclear multiple quantum coherence) spectrum of the POUs-DNA complex we conclude that DNA-binding is mediated by helix III . We propose a model for the POU-DNA complex in which both recognition helices from the two subdomains have adjacent positions in the major groove. Biochemistry, 1993 Apr 27, 32(16), 4270 - 4 Tests of a model for promoter recognition by T7 RNA polymerase: thymine methyl group contacts; Maslak M et al.; The DNA-dependent RNA polymerase from bacteriophage T7 is highly specific for a 17 base promoter sequence . Interactions between T7 RNA polymerase and its promoter DNA have been probed using modified oligonucleotides and a steady-state kinetic assay . The incorporation of deoxyuridine in place of thymidine at individual sites in the promoter sequence results in the replacement of an exocyclic methyl group by hydrogen (effectively removing the thymine methyl) . This substitution has been placed individually at each of the thymines in the T7 consensus promoter . Many of these substitutions do not affect binding or catalysis; however, the thymine methyl group at position-6 is critical to recognition . The kinetic parameter Km increases approximately 10-fold while kcat is only slightly affected, suggesting that this thymine methyl is critical to binding specificity, but not to the kinetics of initiation . Two methyl groups near the start site on the template strand (at positions -1 and -3) also contribute to promoter specificity, while nearby methyl groups on the nontemplate strand do not . The implications of these results are discussed with respect to recent models for promoter binding. Nucleic Acids Res, 1993 Apr 25, 21(8), 1919 - 25 Ribonuclease III cleavage of a bacteriophage T7 processing signal . Divalent cation specificity, and specific anion effects; Li HL et al.; Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro . The RNase III-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 mM MgCl2) . Mn2+ and Co2+ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal ions exhibit concentration dependences similar to that of Mg2+ . Mn2+ and Co2+ in addition promote enzymatic cleavage of a secondary site in R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity . Neither Ca2+ nor Zn2+ support RNase III processing, and Zn2+ moreover inhibits the Mg(2+)-dependent enzymatic reaction without blocking substrate binding . RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion . First, R1.1 RNA secondary site cleavage increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate . Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs . Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not involve inhibition of substrate binding. J Biol Chem, 1993 Apr 25, 268(12), 8943 - 8 Interference by PR-bound RNA polymerase with PRM function in vitro . Modulation by the bacteriophage lambda cI protein; Hershberger PA et al.; Activation of the weak PRM promoter by cI protein is an essential process in the establishment of lysogeny . Much evidence has accumulated that cI protein binds cooperatively to the operators OR1 and OR2 and that protein at the OR2 site contacts RNA polymerase to facilitate open complex formation at the PRM promoter . We had shown previously in vitro that RNA polymerase situated at the nearby PR promoter could interfere with open complex formation at PRM and that an additional mechanism of PRM activation in vitro involved cI-mediated RNA polymerase exclusion from PR . Here we further characterize this second indirect mode of activation . We demonstrate the addition of cI and inactivation of the PR promoter activate open complex formation at PRM similarly over the temperature range from 37 to 20 degrees C in which the extent of activation decreases from 8- to 2-fold . We also show that the binding of cI protein to OR1 is sufficient to effect an increase in the rate of synthesis of abortive RNA products at PRM . This result is difficult to explain based on direct cI-RNA polymerase contacts alone but is readily interpreted in terms of our previously proposed model involving the exclusion of an interfering RNA polymerase from binding at PR. J Biol Chem, 1993 Apr 25, 268(12), 8908 - 18 DNA determinants of restriction . Bacteriophage T4 endonuclease II-dependent cleavage of plasmid DNA in vivo; Carlson K et al.; Endonuclease II of coliphage T4 is necessary for the in vivo restriction of plasmid DNA in phage-infected cells . Double-stranded restriction cleavage at 12 sites in pBR322 commenced before 10-min postinfection with T4 at 37 degrees C and proceeded more slowly in the presence of competing phage DNA than in its absence, utilizing the same sites in both cases; in a 200-base pair segment of the plasmid, single-stranded nicks also were frequent . The plasmid sites were cleaved with a speed that varied with the site, yielding frequencies of cleavage at different sites varying between 10 and 90%, at 50-min postinfection . All sites contained good matches to a consensus, 5'-GRCCGCNTYGC-3', most frequently cleaved around the variable central base pair, generating fragments with blunt ends or 1-2-base 5' overhangs . Using the frequency of cleavage to determine a weighted consensus, a larger sequence, 5'-CGRCCGCNTTGSYNGC-3', was identified . Thus, DNA sequence elements 3' to the cut site appear important for rapid cleavage . Several models describing the sequence-dependent structure of DNA suggest structural anomalies around the cleavage sites . The endonuclease II restriction system is most similar to type II systems, although it differs from known type II systems in several respects. Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3211 - 5 Assembly of a functional replication complex without ATP hydrolysis: a direct interaction of bacteriophage T4 gp45 with T4 DNA polymerase; Reddy MK et al.; The seven-protein bacteriophage T4 DNA replication complex can be manipulated in vitro to study mechanistic aspects of the elongation phase of DNA replication . Under physiological conditions, the processivity of DNA synthesis catalyzed by the T4 polymerase (gp43) is greatly increased by the interaction of this enzyme with its accessory proteins (gp44/62 and gp45) and the T4 single-stranded DNA binding protein (gp32) . The assembly of this T4 holoenzyme requires hydrolysis of ATP by the gp44/62 complex . We demonstrate here that processive T4 holoenzyme-like DNA synthesis can be obtained without hydrolysis of ATP by simply adding gp45 to the T4 DNA polymerase at extremely high concentrations, effectively bypassing the ATPase subunits (gp44/62) of the accessory protein complex . The amount of gp45 required for the gp43-gp45 heteroassociation event is reduced by addition of the macromolecular crowding agent polyethylene glycol (PEG) as well as gp32 . A chromatographic strategy involving PEG has been used to demonstrate the gp43-gp45 interaction . These results suggest that gp45 is ultimately responsible for increasing the processivity of DNA synthesis via a direct and functionally significant interaction with the T4 DNA polymerase . A corollary to this notion is that the specific role of the gp44/62 complex is to catalytically link gp45 to gp43. Gene, 1993 Apr 15, 126(1), 99 - 104 Overproduction, purification, and characterization of DNA-binding protein P19 of bacteriophage PRD1; Pakula TM et al.; The early protein, P19, of bacteriophage PRD1 was purified after overexpression of the cloned gene, XIX, in Escherichia coli DH5 alpha cells . The purified protein binds as multimers to single-stranded DNA (ssDNA), and with a lower affinity to double-stranded DNA (dsDNA), without sequence-specificity . Two distinct P19-ssDNA complexes were discovered in gel- mobility-shift assays at different protein:DNA ratios . P19 was capable of fully protecting ssDNA against nuclease P1 . Electron microscopy of protein P19-ssDNA complexes showed DNA molecules which were extensively coated with protein and whose contour length was clearly reduced by P19 binding . The results suggest that P19 binds to ssDNA with moderate cooperativity and are consistent with the DNA being wrapped around the P19 multimers. Gene, 1993 Apr 15, 126(1), 25 - 33 A chromosomal expression vector for Escherichia coli based on the bacteriophage Mu; Weinberg RA et al.; A new Escherichia coli expression vector with increased stability was developed based on bacteriophage Mu . Unlike traditional expression vectors, the vector described herein is chromosome based rather than existing as an autonomously replicating plasmid . The chromosomal location resulted in extreme stability of the vector even in the absence of selective pressure . Both replication and heterologous protein synthesis could be induced by temperature shift . Expression of the heterologous gene was controlled by the Mu middle promoter and was dependent on the presence of the transactivator, Mor, of the Mu middle promoter . Four proteins, beta-galactosidase, chloramphenicol acetyltransferase, porcine somatotropin and human growth hormone, were made from this vector at levels ranging from 5 to 20% of total cell protein . Expression from the middle promoter was highest when inductions were done in rich media . The expression of some genes varied in different strains. J Biol Chem, 1993 Apr 15, 268(11), 7721 - 7 Deletion between directly repeated DNA sequences measured in extracts of bacteriophage T7-infected Escherichia coli; Kong D et al.; An in vitro system based upon extracts of bacteriophage T7 infected Escherichia coli was used to study genetic deletions and to examine the importance of DNA replication in the deletion process . When T7 genomes with gene 1.3 inactivated by a 43-bp insert of random sequence DNA bracketed by 11-bp direct repeats were replicated in vitro the inserts were deleted with a frequency of about 10(-5) deletions per genome replication . Under conditions where deletion could take place only by recombination between direct repeats on distinct DNA molecules deletion frequency was at least an order of magnitude lower . These data demonstrate the utility of the in vitro system as a means for examining deletion mechanisms and underscore the importance of DNA replication in deletions. J Biol Chem, 1993 Apr 15, 268(11), 7904 - 11 The role of protein-protein interactions in the assembly of the presynaptic filament for T4 homologous recombination; Jiang H et al.; The presynaptic filament is an obligatory intermediate in general genetic recombination . It is composed of a strand transferase protein polymerized along single-stranded DNA . In bacteriophage T4-infected cells, the presynaptic filament is composed of at least three proteins . In addition to the strand transferase (the uvsX protein), the uvsY (an accessory factor), and gene 32 (a helix-destabilizing factor), proteins also bind to the single-stranded DNA . In this report, we probe the assembly of the T4 presynaptic filament and the organization of the proteins in the complex . We find that interactions between the uvsY protein and the C terminus of the gene 32 protein are required to load UvsY onto gene 32 protein-covered DNA . Efficient binding of the uvsY protein to DNA is shown to be necessary for subsequent loading of the uvsX protein. Biochemistry, 1993 Apr 13, 32(14), 3623 - 8 Identification of a compact DNA-binding domain in the gene 5 protein of Pf1 bacteriophage; Plyte SE et al.; The structure of the gene 5 protein of filamentous bacteriophage Pf1 and its interaction with viral DNA have been investigated by a series of limited proteolysis experiments . The ability of purified proteolytic fragments of the Pf1 gene 5 protein to bind oligonucleotides and polynucleotides was monitored by gel retardation and fluorescence . The results show the presence of a compact DNA-binding "core" domain consisting of residues 1-112 of the protein, which is protected from proteolysis in the nucleoprotein complex . Digestion of the free gene 5 protein with subtilisin produces a smaller fragment (residues 7-102) which can no longer bind DNA . Although the N-terminal "core" domain shows full DNA binding activity by fluorescence, the gel retardation experiments suggest reduced kinetic stability of this domain in complexes with oligonucleotides, resulting from the removal of residues 113-144 from the C-terminus of the protein . The sequence of the C-terminal 32 amino acid residues is unusual, with a high proportion of alanine, glutamine, and proline residues which may be related to the role of this sequence in stabilizing the complex. Biochemistry, 1993 Apr 13, 32(14), 3535 - 9 Kinetics of bisulfite-induced cytosine deamination in single-stranded DNA; Chen H et al.; The rate of bisulfite-induced deamination of cytosine to uracil in single-stranded (ss) DNA at physiological temperature and pH was monitored by a sensitive genetic assay . The assay is based on reversion of a mutation in the lacZ alpha gene of bacteriophage M13mp2 and employs ung- (NR9404) and ung+ (MC1061) bacterial strains which are isogenic except for uracil glycosylase activity . For ss DNA incubated with 1-50 mM bisulfite and transfected into an ung- cell strain, the reversion frequency increased linearly with time of incubation and with concentration of bisulfite . Of 54 revertants sequenced, all were C-->T transitions . Reduction in reversion frequency upon transfecting ss DNA into ung+ cells indicated that the majority of mutations were occurring via a uracil intermediate . Assuming that all revertants arose via uracil, the pseudo-first-order rate constant for deamination in 10 mM sodium bisulfite and 10 mM Hepes-NaOH, pH 7.4, at 37 degrees C as measured by transfecting into an ung- cell strain was 3.5 x 10(-10) s-1, as compared to a spontaneous background rate constant of 0.6 x 10(-10) s-1 in buffer alone. Cell, 1993 Apr 9, 73(1), 193 - 205 The solution structure of the Oct-1 POU-specific domain reveals a striking similarity to the bacteriophage lambda repressor DNA-binding domain; Assa-Munt N et al.; The POU-specific (POUs) domain, in association with a POU-type homeodomain, forms the bipartite DNA-binding POU domain . The solution structure of the Oct-1 POUs domain has been determined by multidimensional nuclear magnetic resonance spectroscopy and consists of four alpha helices surrounding a conserved hydrophobic core . The POUs domain is structurally similar to the DNA-binding domains of the bacteriophage lambda and 434 repressors and 434 Cro . These domains exhibit superimposable helix-turn-helix (HTH) motifs, except that in the POUs domain, the first helix and the linker to the second helix of the motif are extended . The conserved structural features have been used to propose a plausible model for DNA binding by the POUs domain . A human dwarfism mutation that affects positive control in the related POU domain protein Pit-1 maps to the same region of the HTH motif as do positive control mutations in lambda repressor. J Mol Biol, 1993 Apr 5, 230(3), 717 - 21 Expression of bacteriophage T4 gene 25 is regulated via RNA secondary structure in the translational initiation region; Nivinskas R et al.; Analysis of the nucleotide sequence in the 5' flanking region of bacteriophage T4 gene 25 revealed three potential Shine and Dalgarno sequences, SD1, SD2 and SD3, with a spacing of 8, 17 and 27 nucleotides from the initiation codon of this gene, respectively . Results of our experiments in the bacteriophage T7 expression system clearly demonstrate that the SD3 sequence is required for efficient expression of gene 25 . We propose the existence of a stem-loop structure that includes SD1 and SD2 sequences and brings the SD3 sequence to a favourable spacing with the initiation codon of gene 25 . Since the predicted secondary structure in the translational initiation region of gene 25 is relatively unstable and the SD3 sequence, GAGG, is more typical than the SD1 sequence, GAG, we suggest that this structure could control the level of gene expression. J Biol Chem, 1993 Apr 5, 268(10), 7393 - 400 Relationship between alpha subunit ligand occupancy and beta subunit autophosphorylation in insulin/insulin-like growth factor-1 hybrid receptors; Frattali AL et al.; Insulin receptor beta subunit autophosphorylation occurs in an intramolecular trans-reaction in which one beta subunit phosphorylates the adjacent beta subunit within an alpha 2 beta 2 holoreceptor complex (Frattali, A . L., Treadway, J . L., and Pessin, J . E . (1992) J . Biol . Chem . 267, 19521-19528) . To determine the spatial relationship between alpha subunit occupancy and beta subunit autophosphorylation, the vaccinia virus/bacteriophage T7 transient expression system was used to generate insulin/insulin-like growth factor (IGF)-1 hybrid receptors . The extent of hybrid receptor formation was proportional to the molar ratio of the insulin and IGF-1 receptor expression plasmids used for transfection of cultured fibroblasts . Insulin/IGF-1 hybrid receptors displayed high affinity binding for insulin and IGF-1 similar to that observed for homotypic insulin and IGF-1 receptors, respectively . As expected, insulin poorly competed for 125I-IGF-1 binding to the insulin/IGF-1 hybrid receptors compared with IGF-1 . IGF-1, however, competed more efficiently than insulin for 125I-insulin binding, indicating interactions between the alpha subunit binding sites . Furthermore, insulin or IGF-1 stimulated the autophosphorylation of both beta subunits within wild type insulin/IGF-1 hybrid receptors . Ligand-stimulated autophosphorylation of two different mutant/wild type insulin/IGF-1 hybrid receptors also resulted in the labeling of each beta subunit independent of which alpha subunit was occupied with ligand . These data demonstrate that insulin/IGF-1 hybrid receptors bind both ligands with high affinity and that occupancy of either alpha subunit results in a series of intramolecular trans-autophosphorylation reactions between beta subunits. J Biol Chem, 1993 Apr 5, 268(10), 7256 - 60 Covalent catalysis in nucleotidyl transfer . A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases; Cong P et al.; Vaccinia virus RNA capping enzyme, a heterodimer of 95- and 31-kDa subunits, catalyzes transfer of GMP from GTP to the 5'-diphosphate terminus of RNA via a covalent enzyme-guanylate intermediate . The GMP residue is attached to the 95-kDa subunit through a phosphoamide bond to the epsilon-amino group of a lysine residue . The amino acid sequence of the large subunit includes a lysine-containing motif, Tyr-X-X-X-Lys260-Thr-Asp-Gly, that is conserved in the RNA guanylyltransferases encoded by Shope fibroma virus and Saccharomyces cerevisiae . The KXDG motif is also encountered at the sites of covalent adenylylation of bacteriophage T4 RNA ligase and mammalian DNA ligase I (Thogerson, H . C., Morris, H . R., Rand, K . N., and Gait, M . J . (1985) Eur . J . Biochem . 147, 325-329; Tomkinson, A . E., Totty, N . F., Ginsburg, M., and Lindahl, T . (1991) Proc . Natl . Acad . Sci . U . S . A . 88, 400-404) . We find that conservative amino acid substitutions at three out of four positions within the KTDG sequence of vaccinia capping enzyme either prevent or strongly inhibit enzyme-guanylate formation . The conserved motif is therefore an essential component of the guanylyltransferase domain . Lys260 is implicated as the active site . Comparison of the sequences of capping enzymes and polynucleotide ligases from diverse sources suggests that KX(D/N)G may be a signature element for covalent catalysis in nucleotidyl transfer. Curr Opin Immunol, 1993 Apr, 5(2), 263 - 7 Production of human antibodies using bacteriophage; Griffiths AD; The immune system produces antibodies by a process of antigen-driven selection . An in vitro process of antigen-driven selection, based on the display of antibody fragments on filamentous bacteriophage, has recently been developed . This enables human antibody fragments of high affinity and specificity to be produced without immunization. Electrophoresis, 1993 Apr, 14(4), 296 - 303 Two-dimensional motion of DNA bands during 120 degrees pulsed-field gel electrophoresis; Neitzey LM et al.; The position and velocity of a band of double-stranded, linear DNA from bacteriophage G were measured during 120 degrees pulsed-field gel electrophoresis, using a video micrometer . Both the x and y coordinates were determined simultaneously in the plane of a 1% agarose gel; x is the mean drift direction . For pulse durations T greater than the tube renewal time T*, the path traced by the band of 670 kb DNA in the xy plane was in remarkably good accord with that predicted by Southern's ratchet model . However, the measured instantaneous velocity vx showed a sharp backward spike each time the field changed direction, with amplitude about twice the mean drift velocity . This spike is not consistent with models which assume a constant curvilinear velocity of DNA in a tube, nor with the biased reptation model without fluctuations . The corresponding measurements of vy showed a sharp positive spike with amplitude more than 3 times the plateau velocity in the y direction; neither model predicted this . The sharp velocity spikes are consistent with the idea that, for T > T*, a large fraction of the DNA chains are stretched into U-shaped or herniated configurations . When the field changes direction, the arms of the U's and the hernias recoil rapidly in response to intramolecular DNA chain tension . Because the base of a U or hernia is fixed by gel fibers, the center of mass of the chain recoils backward every time the field changes direction. Electrophoresis, 1993 Apr, 14(4), 271 - 7 Pulsed field agarose gel electrophoresis in the study of morphogenesis: packaging of double-stranded DNA in the capsids of bacteriophages; Serwer P et al.; To understand how comparatively simple macromolecular components become biological systems, studies are made of the morphogenesis of bacteriophages . Pulsed field agarose gel electrophoresis (PFGE) has contributed to these studies by: (i) improving the length resolution of both mature, linear, double-stranded bacteriophage DNAs and the concatemers formed both in vivo and in vitro by the end-to-end joining of these mature bacteriophage DNAs, (ii) improving the resolution of circular conformers of bacteriophage DNAs, (iii) improving the resolution of linear single-stranded bacteriophage DNAs, (iv) providing a comparatively simple technique for analyzing protein-DNA complexes, and (v) providing a solid-phase quantitative assay for all forms of bacteriophage DNA; solid-phase assays are both less complex and more efficient than liquid-phase assays such as rate zonal centrifugation . Conversely, studies of bacteriophages have contributed to PFGE the DNA standards used for determining the length of nonbacteriophage DNAs . Among the solid-phase assays based on PFGE is an assay for excluded volume effects. Mol Gen Genet, 1993 Apr, 238(3), 455 - 8 Random mutagenesis of the gene for bacteriophage T7 RNA polymerase; Rechinsky VO et al.; Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme . A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity . It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme . It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases. J Egypt Soc Parasitol, 1993 Apr, 23(1), 305 - 12 Effect of bacteriophage lysogeny on the efficacy of two bacterial larvicide formulations under field conditions; Ali SM; Two types of man-made ditches were selected for carrying out this experiment; one polluted with nitrogenous matters (sewage water) and second filled with accumulated irrigated clear non-chlorinated water . No phages were detected in samples collected from both types of ditches . However, phage(s) specific to only B . sphaericus was (were) detected after spraying the two types with both B . thuringiensis H-14 and B . sphaericus commercial formulations . The detection of phage(s) was observed after 3 days post-spraying in the polluted ditch and after one week in the non-polluted one . This observation was explained by a possible transduction of naturally existed phage(s) on other spore forming bacteria to the sprayed B . sphaericus only, as its commercial formulation is based on spores which germinate to produce vegetative cell, while B . thuringiensis H-14 contains only the O-endotoxin as active ingredient, and also due to the increase of the number of bacteria in the sprayed ditches due to the recycling of B . sphaericus in the aquatic breeding places. Biotechniques, 1993 Apr, 14(4), 624 - 9 Plasmid rescue from transgenic mouse DNA using LacI repressor protein conjugated to magnetic beads; Gossen JA et al.; A method for the efficient rescue of lac operator containing plasmids from transgenic mouse genomic DNA is described . The method is based on the high affinity of the LacI repressor protein for the lac operator sequence . Using the LacI repressor protein conjugated to magnetic beads, more than 95% of plasmid sequences could be purified from restriction enzyme digested genomic DNA . After circularization, the plasmids were introduced into Escherichia coli by means of electroporation . Since the plasmid was cloned into a bacteriophage lambda vector, the efficiency of plasmid rescue could easily be compared with in vitro packaging . Our results indicate that plasmid rescue is about 25 times more efficient . Application of this method should be especially useful with transgenic mouse models harboring LacZ plasmid shuttle vectors for studying spontaneous or induced mutations in vivo. Protein Expr Purif, 1993 Apr, 4(2), 101 - 9 Expression and single-step purification of enzymatically active vaccinia virus thymidine kinase containing an engineered oligohistidine domain by immobilized metal affinity chromatography; Franke CA et al.; A method has been developed for the controlled expression in Escherichia coli and rapid purification of an enzymatically active vaccinia virus (VV) thymidine kinase protein containing an engineered oligohistidine domain . The nucleotide sequence that encodes the VV thymidine kinase open reading frame was inserted into a plasmid expression vector (pET-16b, Novagen Inc., Madison, WI) under the control of a strongly repressed bacteriophage T7 promoter and high efficiency translational signals . The construct (pET-16b:TK) directs the synthesis of a fusion protein (His-TK) with an N-terminal histidine decapeptide fused to the VV thymidine kinase polypeptide . Upon induction of E . coli strain BL21(DE3)pLysS with isopropyl beta-D-thiogalactoside, accumulation of large quantities of a 22-kDa protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This protein reacted with polyclonal antiserum raised against a TrpE-VVTK fusion protein . The predominantly soluble fusion protein (approximately 13% of the total soluble bacterial protein) was purified to homogeneity from crude bacterial extracts in a single-step by immobilized metal chelate affinity chromatography (Ni(2+)-nitrilotriacetic acid-agarose) under nondenaturing conditions and was shown to have thymidine kinase activity . The yield of the purification scheme was about 15 mg recombinant protein/liter of bacterial culture . The availability of purified VV TK protein should greatly facilitate biochemical studies on its enzymatic activity, as well as analyses of its structural and functional domains. J Bacteriol, 1993 Apr, 175(8), 2393 - 9 Sites and gene products involved in lambdoid phage DNA packaging; Smith MP et al.; 21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage . The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence . The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific . DNA packaging by 21 is dependent on a host protein, integration host factor . A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1 . gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase . orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI . orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth . 21 was also not able to complement a lambda FI mutant. EMBO J, 1993 Apr, 12(4), 1303 - 9 The portal protein of bacteriophage SPP1: a DNA pump with 13-fold symmetry; Dube P et al.; Electron microscopy in combination with image processing is a powerful method for obtaining structural information on non-crystallized biological macromolecules at the 10-50 A resolution level . The processing of noisy microscopical images requires advanced data processing methodologies in which one must carefully avoid the introduction of any form of bias into the data set . Using a novel multivariate statistical approach to the analysis of symmetry, we studied the structure of the bacteriophage SPP1 portal protein oligomer . This portal structure, ubiquitous in icosahedral bacteriophages which package dsDNA, is located at the site of symmetry mismatch between a 5-fold vertex of the icosahedral shell and the 6-fold symmetric (helical) tail . From previous studies such 'head-to-tail connector' structures were generally accepted to be homododecamers assembled in a 12-fold symmetric ring around a central channel . Using a new analysis methodology we have found that the phage SPP1 portal structure exhibits 13-fold cyclical symmetry: a new point group organization for oligomeric proteins . A model for the DNA packaging mechanism by 13-fold symmetric portal protein assemblies is presented which attributes a coherent functional meaning to their unusual symmetry. Proc Natl Acad Sci U S A, 1993 Apr 1, 90(7), 2579 - 83 Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity; Frey MW et al.; Bacteriophage T4 DNA polymerase has a proofreading 3'-->5' exonuclease that plays an important role in maintaining the accuracy of DNA replication . We have constructed a T4 DNA polymerase deficient in this exonuclease by converting Asp-219 to Ala . The exonuclease activity of the mutant T4 DNA polymerase has been reduced by a factor of at least 10(7), but it retains a polymerase activity whose kinetic parameters, kcat, Kd DNA, and Kd dATP, are very close to those of the wild-type enzyme . Bacteriophage T4 with the mutant polymerase gene has a markedly increased mutation frequency . Asp-219 in T4 DNA polymerase is within a sequence similar to those surrounding Asp residues previously shown to be essential for the exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I (Asp-424), bacteriophage phi 29 DNA polymerase (Asp-66), and Saccharomyces cerevisiae DNA polymerase delta (Asp-405) . Thus, these studies support the proposal that there are similar sequences in the active sites for the proofreading exonucleases of these and related DNA polymerases. Virology, 1993 Apr, 193(2), 748 - 52 DNA packaging ATPase of bacteriophage T3; Morita M et al.; A defined in vitro DNA packaging system of phage T3, which is composed of purified proheads and two packaging proteins, the products of genes 18 and 19 (gp18 and gp19, respectively), displayed a DNA-dependent ATPase activity . ATP was hydrolyzed to ADP and Pi . The ATPase activity was stimulated by nonpackageable DNA, such as single-stranded or circular DNA, or RNA (nonpac-ATPase) . Among the inhibitors of DNA packaging, actinomycin D specifically inhibited the ATPase activity that was tightly coupled to DNA packaging (pac-ATPase), but did not inhibit the nonpac-ATPase activity . Both activities depended upon a functional packaging complex, but the nonpac-ATPase, once activated, did not require DNA . Unpackageable pUC18 DNA inhibited the pac-ATPase and the phage yield in parallel . Approximately one molecule of ATP was hydrolyzed during the translocation of 1.8 bp of T3 DNA. Diabetes, 1993 Apr, 42(4), 626 - 30 Transcription factor jun-B is target of autoreactive T-cells in IDDM; Honeyman MC et al.; Target antigens defined by autoantibodies in IDDM include insulin, a putative glycolipid that reacts with islet cell antibodies, and a 64,000-M(r) protein recently identified as glutamic acid decarboxylase . In addition, some IDDM sera that contain antibodies to glutamic acid decarboxylase also coprecipitate a 38,000-M(r) protein from islets . This study used a high titer anti-38,000-M(r) serum to screen bacteriophage lambda cDNA expression libraries and identified human islet and placental clones encoding jun-B, the nuclear transcription protein, of predicted 38,000 M(r) . Peripheral blood T-cells exhibited significant proliferation in response to a recombinant fragment of jun-B (amino acids 1-180) in 12 of 17 (71%) recent-onset IDDM subjects, 8 of 16 (50%) ICA-positive first-degree relatives of IDDM subjects who were at risk, 3 of 12 (25%) other autoimmune disease subjects, and 0 of 10 healthy control subjects . Proliferation to tetanus toxoid did not differ significantly between the groups . Responses to jun-B were not related to age, sex, or human leukocyte antigen status . Thus, autoreactive T-cells identify a novel antigen, p38 jun-B, in IDDM and appear to indicate subjects at risk for the development of clinical disease. J Virol, 1993 Apr, 67(4), 2175 - 81 Nucleotide sequence of the primer RNA for DNA replication of filamentous bacteriophages; Higashitani N et al.; We determined the nucleotide sequence of RNA synthesized in vitro by Escherichia coli RNA polymerase at the complementary-strand replication origin on the single-stranded viral DNA of bacteriophages f1 and IKe (ori-RNA) by using chain-terminating ribonucleoside triphosphate analogs . The results indicated that the start site of f1 ori-RNA synthesis is 20 nucleotides downstream from the site previously reported (K . Geider, E . Beck, and H . Schaller, Proc . Natl . Acad . Sci . USA 75:645-649, 1978) and that the RNA sequence {(5')pppAGGGCGAUGGCCCACUACGU-OH(3')} is complementary to the f1 DNA sequence from nucleotides 5736 to 5717, with minor heterogeneity at the 3' end . IKe ori-RNA had a sequence identical to that of f1 ori-RNA, except for a single base substitution, and IKe RNA was complementary to a region of IKe DNA (from nucleotides 6441 to 6422) that was homologous to the f1 sequence . Phenotypes and ori-RNA sequences in the relevant region of the genome of f1 deletion mutants were consistent with the presently determined sequence of ori-RNA . A possibility that ori-RNA synthesis is initiated by a mechanism similar to that for general transcription is suggested as a result of the new assignment of the ori-RNA start site . The double-origin plasmid assay of minus-strand origin activity, a sensitive in vivo method for detecting cis-acting elements for the initiation of DNA replication on a single-stranded DNA template, is described. Proc Natl Acad Sci U S A, 1993 Apr 1, 90(7), 2999 - 3003 Sites of predicted stress-induced DNA duplex destabilization occur preferentially at regulatory loci; Benham CJ; This paper describes a computational method to predict the sites on a DNA molecule where imposed superhelical stresses destabilize the duplex . Several DNA sequences are analyzed in this way, including the pBR322 and ColE1 plasmids, bacteriophage f1, and the polyoma and bovine papilloma virus genomes . Superhelical destabilization in these molecules is predicted to occur at small numbers of discrete sites, most of which are within regulatory regions . The most destabilized sites include the terminator and promoter regions of specific plasmid operons, the LexA binding sites of genes under SOS control, the intergenic control region of bacteriophage f1, and the polyadenylylation sites in eukaryotic viruses . These results demonstrate the existence of close correspondences between sites of predicted superhelical duplex destabilization and specific types of regulatory regions . The use of these correspondences to supplement string-matching techniques in the search for regulatory loci is discussed. J Virol, 1993 Apr, 67(4), 2305 - 16 Analysis of five presumptive protein-coding sequences clustered between the primosome genes, 41 and 61, of bacteriophages T4, T2, and T6; Selick HE et al.; In bacteriophage T4, there is a strong tendency for genes that encode interacting proteins to be clustered on the chromosome . There is 1.6 kb of DNA between the DNA helicase (gene 41) and the DNA primase (gene 61) genes of this virus . The DNA sequence of this region suggests that it contains five genes, designated as open reading frames (ORFs) 61.1 to 61.5, predicted to encode proteins ranging in size from 5.94 to 22.88 kDa . Are these ORFs actually genes? As one test, we compared the DNA sequence of this region in bacteriophages T2, T4, and T6 and found that ORFs 61.1, 61.3, 61.4, and 61.5 are highly conserved among the three closely related viruses . In contrast, ORF 61.2 is conserved between phages T4 and T6 yet is absent from phage T2, where it is replaced by another ORF, T2 ORF 61.2, which is not found in the T4 and T6 genomes . As a second, independent test for coding sequences, we calculated the codon base position preferences for all ORFs in this region that could encode proteins that contain at least 30 amino acids . Both the T4/T6 and T2 versions of ORF 61.2, as well as the other ORFs, have codon base position preferences that are indistinguishable from those of known T4 genes (coefficients of 0.81 to 0.94); the six other possible ORFs of at least 90 bp in this region are ruled out as genes by this test (coefficients less than zero) . Thus, both evolutionary conservation and codon usage patterns lead us to conclude that ORFs 61.1 to 61.5 represent important protein-coding sequences for this family of bacteriophages . Because they are located between the genes that encode the two interacting proteins of the T4 primosome (DNA helicase plus DNA primase), one or more may function in DNA replication by modulating primosome function. Oral Microbiol Immunol, 1993 Apr, 8(2), 100 - 4 Characterization and physical mapping of the genome of bacteriophage phi Aa from Actinobacillus actinomycetemcomitans; Stevens RH et al.; The size, configuration and restriction map of Actinobacillus actinomycetemcomitans bacteriophage phi Aa DNA was determined by means of restriction endonuclease analysis . Digestion of the phi Aa DNA with restriction enzymes Hind III, Eco RI and Sal I produced 6, 5, and 4 fragments, respectively . Based upon the sum of the sizes of the restriction fragments of these enzymes, the DNA was estimated to be 47.2 kilobase pairs in length . A restriction map was constructed using Hind III and Sal I . Incubation with exonuclease Bal 31 for increasing lengths of time resulted in progressive hydrolysis of the DNA, as expected for a linear molecule . No sub-molar fragments or diffuse bands were observed in the agarose gels of the restriction endonuclease digests of the phi Aa DNA . Attempts at ligating the ends of the DNA were consistently unsuccessful . Therefore, we found no evidence for cohesive ends, a circular permutation of the genome or for headful packaging mechanism from a concatameric DNA precursor. Rev Latinoam Microbiol, 1993 Apr-Jun, 35(2), 165 - 9 Herpes simplex virus infection in primary neuronal cultures and antiviral activity of dsRNA; Barron B et al.; Central neurons in culture represent a limitless substratum for research in neurobiology and experimental neurology . Primary cultures of NIH mouse neurons have shown that about 83% of total cells in the cultures are neuron clumps, detected by their reaction with the neuron specific-enolase (NSE) marker . Herpes Simplex Virus type 1 (HSV-1) can grow efficiently in these cultures, as it does in nonneuronal cultures usually used for antiviral drugs testing . For that reason, the primary neuronal cultures were used for testing antiviral activity against HSV-1, after an overnight treatment with different concentrations of dsRNA from phi 6 bacteriophage . The dsRNA started to be toxic for the cells at concentrations of 4 micrograms/ml, but it was found that 1 microgram/ml of this dsRNA protected all the neuronal cultures from HSV-1 infection . The dsRNA value for effective dose (ED50) was 0.27 microgram/ml. J Bacteriol, 1993 Apr, 175(8), 2175 - 83 H-pilus assembly kinetics determined by electron microscopy; Maher D et al.; The kinetics of pilus outgrowth were examined for Escherichia coli containing pDT1942, a TnlacZ insertion derivative of the IncHI1 plasmid R27, which was derepressed for transfer . IncHI1 plasmids are thermosensitive for transfer . The pili specified by pDT1942 were examined by transmission electron microscopy after the pilus had been labeled with the H-pilus-specific bacteriophage Hgal, which had been inactivated with RNase A . H pili were extended by extrusion from the cell surface and not by the addition of pilin subunits to the pilus tip . After pili were removed by vortexing, the outgrowth of full-length pili (2 microns long) required 20 min . H pili expressed at the transfer optimal temperature (27 degrees C) remained stable after incubation at the transfer inhibitory temperature (37 degrees C), but the formation of mating aggregates was inhibited at 37 degrees C . Within 1 min of exposure of the host cell to a heat stimulus of 50 degrees C, pili vanished . Pili were observed in straight and flexible forms with a field emission scanning electron microscope, which may indicate a dynamic role for the pilus in conjugation. Curr Opin Immunol, 1993 Apr, 5(2), 268 - 71 Epitope mapping using bacteriophage peptide libraries; Lane DP et al.; Vast libraries of random peptides displayed on the surface of bacteriophage potentially allow identification of specific ligands for any peptide receptor of interest . The sequence specificity of antibody-peptide interactions allows these libraries to be used to define and localize continuous epitopes . Remarkably clear-cut results have been obtained with some antibodies; however, the technique has not proved to be generally applicable. Biotechniques, 1993 Apr, 14(4), 608 - 17 Surprising lability of biotin-streptavidin bond during transcription of biotinylated DNA bound to paramagnetic streptavidin beads; Fujita K et al.; We investigated the use of immobilized DNA templates as substrates for bacteriophage RNA polymerases in order to develop a simple method for separating template DNA from synthesized RNA . Double-stranded DNA molecules with a T7 or T3 RNA polymerase promoter at one end and a single biotin moiety at the other end were attached to streptavidin-coated paramagnetic beads and used in transcription reactions . When the biotin was attached by a nucleotide base on the nontemplate strand, the DNA-bead complex was moderately stable and could be used for multiple rounds of RNA synthesis . However, when the biotin was attached through a phosphodiester bond on the template strand, the enzymatic activity of RNA polymerase reversibly dissociated up to 80% of biotinylated DNA from the streptavidin beads . Biotinylated DNA bound to streptavidin beads in this system with a binding constant on the order of 10(12) M-1 . These results stress the need for careful evaluation of solid phase adaptations of standard solution reactions in molecular biology. J Gen Virol, 1993 Apr, 74 ( Pt 4), 541 - 8 Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid; Mastico RA et al.; We have produced a plasmid expression vector for the coat protein of RNA bacteriophage MS2 . The vector has been modified to introduce a unique KpnI restriction site within the coat protein gene at a site corresponding to the most radially distant feature of the bacteriophage capsid, namely the top of the N-terminal beta-hairpin (between residues 15 and 16) . Insertion of DNA oligonucleotides at this site allows the production of chimeric MS2 coat proteins having foreign peptide sequences expressed as the central part of the hairpin . We have produced chimeras with a number of different peptide sequences (up to 24 amino acids in length) chosen because of their known antigenic properties . The chimeric coat proteins self-assemble into largely RNA-free phage-like capsids in Escherichia coli and can be easily disassembled and reassembled in vitro . Such peptide-presenting particles may have a number of biotechnological applications, including use as a cost-effective, synthetic vaccine . We have tested the antigenicity of one such construct in vivo in mice and have shown that these particles are immunogenic and that antibody titres against the inserted peptide epitope can be obtained. Biotechnology (N Y), 1993 Apr, 11(4), 503 - 7 Chaperonin assisted phage display of antibody fragments on filamentous bacteriophages; Soderlind E et al.; We have used the GroE chaperonins to assist in the packing of a new phage display vector, pEXmide3 . Titers of the packed phagemid increased almost 200-fold from approximately 4 x 10(11) cfu/ml, without coexpression of the GroE proteins, to approximately 7 x 10(13) cfu/ml with their coexpression . Equal titers of non-assisted and assisted phagestocks exhibited the same antigen specificity and ELISA reactivity, indicating the same frequency of displayed Fab-fragments . While the diversity of antibody libraries depends on the bacterial transformation efficiency, the copy number of each antibody is determined by subsequent amplification of the phage, thus chaperonin assisted phagemid packing in bacteriophage M13 can be used as a general and simple tool to increase the amplification level of expressed Fab fragments . pEXmide3 was developed for display of Fab and single chain Fv-fragments (scFv), using restriction enzymes that do not cut, or cut with low frequencies, in genes encoding immunoglobulin variable domains . The vector allows cloning of genes for the variable domains linking these to predetermined human constant domains or cloning of the entire light and heavy Fab chains . A modification of the pelB leader sequence, with a glutamine to alanine substitution at residue 18, was used for export of the light chain. Mutat Res, 1993 Apr, 286(2), 189 - 97 A proflavin-induced frameshift hotspot in the thymidylate synthase gene of bacteriophage T4; Brown MD et al.; Twenty-one independent thymidylate synthase deficient (td) mutants were isolated after proflavin mutagenesis of T4D0 phage . A strikingly high proportion of these mutations (17 of 21; 80%) mapped in a small 122 nucleotide (nt) region which spans the 5' splice site of this intron-containing gene . This region comprises only 14% of the total td exon sequence . RNA sequence analysis of these mutants identified a series of frameshift insertion/deletion mutations and indicated a hotspot for proflavin-induced mutations in the 3' end of exon I of the td gene . The mutant sequences at the hotspot site fully support a previously proposed mutagenic mechanism for proflavin-induced mutations in which frameshifts are produced as a consequence of exonuclease or DNA polymerase activity at the 3' ends of nicks in the DNA produced by perturbation of the T4-encoded type II topoisomerase activity by the acridine . Sixteen of the seventeen DNA mutations in the hotspot region can be explained by the model as a consequence of enzymatic processing of nicks at two phosphodiester bonds staggered by 4 base pairs (bp) and located on opposite strands of the DNA . Thus, these mutants exhibit precisely the symmetry expected of topoisomerase-mediated mutagenesis . The DNA sequences of the td hotspot mutants, when considered with the sequences of proflavin-induced mutants in the T4 rIIB and lysozyme genes, confirm the view that proflavin-induced mutations in diverse bacteriophage T4 DNA sequences are all produced by the topoisomerase-dependent mechanisms and do not support the view that classical misalignments in DNA repeats are hotspots for proflavin-induced mutagenesis in T4. Philos Trans R Soc Lond B Biol Sci, 1993 Mar 29, 339(1289), 271 - 7; discussion 277-8 The Escherichia coli chaperones involved in DNA replication; Zylicz M; Mutations in the Escherichia coli heat shock genes, dnaK, dnaJ or grpE, alter host DNA and RNA synthesis, degradation of other proteins, cell division and expression of other heat shock genes . They also block the initiation of DNA replication of bacteriophages lambda and P1, and the mini-F plasmid . An in vitro lambda DNA replication system, composed entirely of purified components, enabled us to describe the molecular mechanism of the dnaK, dnaJ and grpE gene products . DnaK, the bacterial hsp 70 homologue, releases lambda P protein from the preprimosomal complex in an ATP- and DnaJ-dependent reaction (GrpE-independent initiation of lambda DNA replication) . In this paper, I show that, when GrpE is present, lambda P protein is not released from the preprimosomal complex, rather it is translocated within the complex in such a way that it does not inhibit DnaB helicase activity . Translocation of lambda P triggers the initiation event allowing DnaB helicase to unwind DNA near the ori lambda sequence, leading to efficient lambda DNA replication . Chaperone activity of the DnaK-DnaJ-GrpE system is first manifested in the selective binding of these heat shock proteins to the preprimosomal complex, followed by its ATP-dependent rearrangement . I show that DnaJ not only tags the preprimosomal complex for recognition by DnaK, but also stabilizes the multi-protein structure . GrpE also participates in the binding of DnaK to the preprimosomal complex by increasing DnaK's affinity to those lambda P proteins which are already with DnaJ.(ABSTRACT TRUNCATED AT 250 WORDS) Biochim Biophys Acta, 1993 Mar 26, 1162(3), 323 - 5 Some properties of restriction endonuclease ApaBI from Acetobacter pasteurianus; Grones J et al.; A new site-specific endonuclease has been isolated from Acetobacter pasteurianus and has been named ApaBI . The enzyme recognizes 35 cleavage sites on bacteriophage lambda DNA, 20 sites on adenovirus-2 DNA and 2 sites on plasmid pBR322 . The recognition sequence for this enzyme is 3'-CGT/NNNNNACG-5' 5'-GCANNNNN/TGC-3'. J Mol Biol, 1993 Mar 20, 230(2), 453 - 60 Phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction; Chen Y et al.; Using phenyl-azide-mediated photocrosslinking, we show that the alpha carbon of amino acid 2 of the helix-turn-helix motif of bacteriophage lambda Cro is within 12 A of the bottom-strand nucleotides at positions 2 and 3 of the DNA half site in the Cro-DNA complex in solution . This result is in excellent agreement with the crystallographic structure of the Cro-DNA complex . The results of phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction, together with the previously reported results of phenyl-azide-mediated photocrosslinking analysis of CAP-DNA interaction, establish that phenyl-azide-mediated photocrosslinking is generalizable and provide information regarding the structural requirements for phenyl-azide-mediated photocrosslinking . Comparison of the results of phenyl-azide-mediated photocrosslinking to the results of EDTA: iron-mediated affinity cleaving indicates that phenyl-azide-mediated photocrosslinking yields superior resolution. Biochemistry, 1993 Mar 16, 32(10), 2469 - 72 5-hydroxytryptophan as a new intrinsic probe for investigating protein-DNA interactions by analytical ultracentrifugation . Study of the effect of DNA on self-assembly of the bacteriophage lambda cI repressor; Laue TM et al.; Pairwise cooperativity between proteins bound to DNA is believed to be important in governing the transcriptional regulation of numerous genes . However, the spectral overlap of normal proteins and DNA has blocked the study of these interactions by many physical methods . As shown recently by Ross et al . (in press), lambda cI repressor spectrally enhanced by 5-hydroxytryptophan (5-OHTrp), expressed in vivo using an Escherichia coli tryptophan auxotroph, exhibits dimer formation and DNA binding properties identical with those of the wild-type repressor . Moreover, the 5-OHTrp provides a spectral signal that allows monitoring of the protein concentration without interference from DNA . In this article, the ability to selectively detect 5-OHTrp-labeled repressor during analytical ultracentrif