Bacteriophage

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 ultracentrifugation is used to study the higher order assembly of repressor dimers in the absence and in the presence of operator DNA . Contrary to the expectation that tetramer might be the limiting oligomer, lambda cI repressor undergoes a definite association to octamer . The relatively narrow concentration range over which transition from predominantly dimer to predominantly octamer occurs makes it unlikely that significant levels of tetramer are formed in the absence of DNA . Moreover, mass measurements reveal that an OR1 oligonucleotide binds to octameric repressor and does not dissociate it to tetramers . The use of the 5-OHTrp spectral enhancement opens a promising new avenue for the exploration of protein-protein and protein-nucleic acid interactions by analytical ultracentrifugation.

Proc Natl Acad Sci U S A, 1993 Mar 15, 90(6), 2522 - 6
Chromosomal structure of Rhodobacter capsulatus strain SB1003: cosmid encyclopedia and high-resolution physical and genetic map; Fonstein M et al.; A combination of cosmid genome walking and pulsed-field gel electrophoresis was used to construct a high-resolution physical and genetic map of the 3.8-megabase (Mb) genome of Rhodobacter capsulatus SB1003 . The mapping was done by hybridization of pulsed-field gel blots and by grouping and further mapping of the cosmids and bacteriophages from genomic libraries . Cosmid clones formed two uninterrupted and ordered groups, one corresponding to the chromosome of R . capsulatus, the other to its 134-kb plasmid . Cos site end-labeling and partial EcoRV digestion of cosmids were used to construct a high-resolution EcoRV map of the genome . Overlapping of the cosmids was confirmed by the resemblance of the cosmid restriction maps and by direct end-to-end hybridization with SP6- and T7-specific transcripts . Twenty-three previously cloned genes and eight groups of repeated sequences, revealed in this work, were located in the ordered gene library and mapped with an accuracy of 1-10 kb . Blots of a minimal set of 192 cosmids, covering the chromosome and the plasmid with the known map position of each cosmid, give to R . capsulatus the same advantages that the Kohara phage panel gives to E . coli.

Proc Natl Acad Sci U S A, 1993 Mar 15, 90(6), 2137 - 40
Atomic force microscopy of long DNA: imaging in air and under water; Lyubchenko Y et al.; We have obtained striking atomic force microscopy images of the intact lambda bacteriophage genome and of several lambda restriction fragments both in air and under water . The DNA is unstained and the images are stable under continuous scanning for up to 30 min . Measured contour lengths of fully imaged restriction fragments and intact lambda DNA are accurate to within a few percent . The key to this development is the use of a process for binding unmodified double-stranded DNA to chemically treated mica surfaces . This procedure leads to strong DNA attachment and yields high-quality images that are stable under repeated scanning, even with the sample submerged in water . This allows normal hydration conditions to be maintained during scanning and in addition leads to a general improvement of image quality . Both the lateral resolution and the contrast increase by a factor of approximately 3 under water.

J Biol Chem, 1993 Mar 15, 268(8), 5496 - 503
The membrane domain of a bacteriophage assembly protein . Membrane insertion and growth inhibition; Guy-Caffey JK et al.; The gene I protein (pI) of the f1 filamentous bacteriophage is a non-capsid protein that is required for the assembly of the bacteriophage . It spans the Escherichia coli inner membrane once with its amino terminus in the cytoplasm and its carboxyl-terminal portion in the periplasm . The presence of moderate amounts of this protein in the membrane results in rapid inhibition of cell growth, probably from a loss of membrane potential . Previous observations defined a 55-amino-acid sequence within pI required for its membrane insertion which includes a 20-residue hydrophobic stretch preceded by a 13-residue positively charged amphiphilic helix . To define the minimal sequence required for membrane translocation and for growth inhibition, a deletion analysis was performed on a tripartite fusion construct containing the 55-residue pI sequence flanked upstream by the amino-terminal portion of EcoRI endonuclease and downstream by the enzymatic portion of alkaline phosphatase . Only the 20-residue hydrophobic stretch immediately preceded by 1 arginine residue is required for membrane insertion of the fusion proteins . This region also sufficed to inhibit cell growth provided it contained protein domains exposed in both the cytoplasm and periplasm . It was not possible to separate the domains required for membrane insertion and cell growth inhibition . No requirement for the positively charged amphiphilic helix was detected either for membrane insertion or growth inhibition, suggesting that it plays a role in phage assembly and not membrane insertion.

J Biol Chem, 1993 Mar 15, 268(8), 5488 - 95
The membrane domain of a bacteriophage assembly protein . Transmembrane-directed proteolysis of a membrane-spanning fusion protein; Guy-Caffey JK et al.; A tripartite fusion construct encoding the amino-terminal half of EcoRI endonuclease followed by amino acids 217-299 of the filamentous bacteriophage gene I protein (pI) attached to the enzymatic portion of alkaline phosphatase results in the production of two proteins . The larger protein, pIf, is the complete tripartite fusion protein while the smaller protein, pIf*, results from internal initiation of translation at pI methionine 241 . Both pIf and pIf* span the Escherichia coli inner membrane via a 20-amino-acid hydrophobic stretch of pI with their amino termini in the cytoplasm and their carboxyl-terminal alkaline phosphatase domains in the periplasm . The alkaline phosphatase moiety of approximately 70% of pIf is released into the periplasm by in vivo proteolysis, but only about 10% of pIf* is cleaved . Neither DegP, OmpT, nor protease III are responsible for the cleavage in vivo, and leader peptidase is unable to cleave the fusion protein in vitro . Deletion and substitution analyses demonstrate that the degree of periplasmic cleavage depends on the sequence of the cytoplasmic domain of the fusion proteins . Possible mechanisms for this transmembrane-directed cleavage event are compared to proposed models for signal transduction.

Nucleic Acids Res, 1993 Mar 11, 21(5), 1117 - 23
Atomic force microscopy of DNA and bacteriophage in air, water and propanol: the role of adhesion forces; Lyubchenko YL et al.; We have developed a chemical treatment for the mica surface which allows biopolymers to be held in place for atomic force microscopy, even under water, using conventional, untreated force sensing tips . We illustrate the procedure with images of lambda DNA and fd phage . The phage adheres well enough to permit in situ imaging of the adsorption process in water . These experiments yield a mean length for the phage of 883 +/- 72 nm . This compares with a measured length of 883 +/- 33 nm when the phage are imaged after drying following adsorption from water, showing that the effect of dehydration is quite small . Adhesion forces between the force sensing tip and the substrate and the sensing tip and the biomolecules are very different in the three media (air, water and propanol) . The apparent height of the phage and the width and height of the DNA depends upon these adhesion forces quite strongly . In contrast, changing the Hookean spring force exerted by the scanning tip makes little difference . These results suggest that the chemical factors involved in adhesion can dominate atomic force images and that the composition of the scanning tip is at least as important a factor as its geometry.

J Mol Biol, 1993 Mar 5, 230(1), 349 - 52
Crystallization of the major coat protein of PRD1, a bacteriophage with an internal membrane; Stewart PL et al.; The major multimeric coat protein, P3, of the bacterial virus PRD1 has been crystallized by vapor diffusion from polyethylene glycol 4000 . The PRD1-P3 crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 121.6 A, b = 123.2 A, c = 128.6 A and diffract to 3.0 A resolution . Density measurements show that there is one trimer (3 x 43.1 kDa) per asymmetric unit and a high solvent content of 67% . A self-rotation function calculation shows a pronounced peak indicating a non-crystallographic threefold axis . This indicates that the major viral capsomer is a trimer and allows the viral T-number to be postulated.

J Mol Biol, 1993 Mar 5, 230(1), 28 - 40
The bacteriophage 434 right operator . Roles of O(R)1, O(R)2 and O(R)3; Bushman FD; Lysogenic induction of bacteriophage lambda is controlled by the action of the phage repressor and Cro proteins at the phage right operator (O(R)) . This study examines the roles of the repressor and Cro proteins of the related phage 434 . The start sites of transcription of the divergently oriented promoters in the 434 O(R) region, PR and PRM, were mapped, and the effects of 434 repressor and Cro on promoter activity were assessed using promoter fusions to lacZ . The effects of repressor or Cro bound to each of the operator subsites (O(R)1, O(R)2 and O(R)3) were assessed by examining regulation in the presence of operator mutations . The binding of Cro to a 434 operator was probed by an ethylation interference experiment which, together with other data, indicates that 434 Cro and repressor probably turn off transcription by blocking binding of RNA polymerase to promoter sequences . In general, the 434 and lambda right operators are controlled in a similar fashion, but differences in detail were also encountered: (1) 434 Cro represses transcription from PR primarily by binding to O(R)1, whereas binding of lambda Cro to O(R)1 and O(R)2 contribute equally to repression . (2) The 434 cI message, unlike that of lambda, has a recognizable homology to the Shine-Dalgarno ribosome binding site . (3) Occupancy of O(R)3 by repressor may be somewhat greater in a 434 lysogen than in a lambda lysogen . (4) The 434 repressor probably activates transcription when bound at O(R)2 by contacting RNA polymerase, as does lambda repressor, but also by influencing competition between PR and PRM . An analysis of the six right operator systems for which data are available indicates that all six repressors may employ the mechanism of transcriptional activation first described for lambda, P22 and 434: apposition of an acidic surface to a particular part of RNA polymerase.

J Mol Biol, 1993 Mar 5, 230(1), 248 - 59
Superhelical path of the DNA in the nucleoprotein complex that activates the initiation of phage phi 29 DNA replication; Serrano M et al.; Initiation of bacteriophage phi 29 DNA replication is activated by protein p6, a viral double-stranded DNA-binding protein that forms a nucleoprotein complex at the viral replication origins . This complex consists of a DNA right-handed superhelix wrapped around a multimeric protein p6 core with protein p6 dimers regularly bound every 24 base-pairs (bp) . In this paper, we have constructed a concatemer formed by direct repeats of a 24 bp sequence previously proposed to act as a signal for protein p6 binding at a phi 29 replication origin . DNase I footprinting shows that protein p6 binds to the concatemer in a similar way to the phi 29 DNA replication origins but with higher affinity, indicating that the 24 bp sequence is a recognition signal for protein p6 . Furthermore, the concatemer was cloned in a plasmid and, by electron microscopy, it was shown to be the highest-affinity protein p6 binding region present in the plasmid . Based on these observations, the linking number change restrained by protein p6 has been measured in a series of plasmids containing concatemers with different numbers of 24 bp repeats; from the values obtained the linking number change restrained by a single protein p6 dimer has been estimated (delta Lkd = 0.1) . In addition, when protein p6-DNA complexes fixed with glutaraldehyde were analysed by electron microscopy, it was observed that protein p6 compacts 4.2-fold the length of naked DNA . These data, together with the previously known value of the surface-related DNA helical repeat in the complex (12 bp), completely define the superhelical path of the DNA in the complex: one superhelical turn approximately involves 63 bp and 2.6 protein p6 dimers, and the DNA superhelix has a diameter of 6.6 nm and a slope of 14 degrees . The data obtained also indicate that the DNA in the protein p6-DNA complex is undertwisted (11.5 bp/turn) and strongly bent (66 degrees/12 bp) . These DNA conformational changes might contribute to the activation of phi 29 DNA initiation of replication by protein p6.

Biochim Biophys Acta, 1993 Mar 5, 1162(1-2), 161 - 70
Properties of an affinity-column-purified human deoxycytidylate deaminase; Maley GF et al.; Deoxycytidylate deaminase was purified about 7000-fold to homogeneity from a human source (HeLa cells) . The final step in the purification employed an affinity column, which increased the specific activity of the enzyme from the previous step by 500-fold . Similar to most other dCMP deaminases, this enzyme is allosterically regulated by microM levels of dCTP and dTTP . However, unlike the other enzymes the most dramatic allosteric responses occur at substrate levels of 0.1 mM dCMP or less, where at least a 10-fold increase in activity is effected by dCTP . The enzyme is particularly sensitive to inhibition by dTTP with 50% inhibition being obtained at 1.5 x (10(-6) M in the absence of dCTP . Antibody to the human enzyme did not cross-react with a dCMP deaminase induced in Escherichia coli by T4-bacteriophage, nor did antibody to the phage-induced enzyme cross-react with the human deaminase . A potential transition-state analogue of the substrate, 2'-beta-D-deoxyribose-pyrimidin-2-one 5'-phosphate was prepared, and found to inhibit dCMP deaminase competitively with a Ki of 1.2 x 10(-8) M.

J Biol Chem, 1993 Mar 5, 268(7), 4821 - 7
Initiation of lambda DNA replication . The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein; Osipiuk J et al.; It is known that the initiation of bacteriophage lambda replication requires the orderly assembly of the lambda O.lambda P.DnaB helicase protein preprimosomal complex at the ori lambda DNA site . The DnaK, DnaJ, and GrpE heat shock proteins act together to destabilize the lambda P.DnaB complex, thus freeing DnaB and allowing it to unwind lambda DNA near the ori lambda site . The first step of this disassembly reaction is the binding of DnaK to the lambda P protein . In this report, we examined the influence of the DnaJ and GrpE proteins on the stability of the lambda P.DnaK complex . We present evidence for the existence of the following protein-protein complexes: lambda P.DnaK, lambda P.DnaJ, DnaJ.DnaK, DnaK.GrpE, and lambda P.DnaK.GrpE . Our results suggest that the presence of GrpE alone destabilizes the lambda P.DnaK complex, whereas the presence of DnaJ alone stabilizes the lambda P.DnaK complex . Using immunoprecipitation, we show that in the presence of GrpE, DnaK exhibits a higher affinity for the lambda P.DnaJ complex than it does alone . Using cross-linking with glutaraldehyde, we show that oligomeric forms of DnaK exhibit a higher affinity for lambda P than monomeric DnaK . However, in the presence of GrpE, monomeric DnaK can efficiently bind lambda P protein . These findings help explain our previous results, namely that in the GrpE-dependent lambda DNA replication system, the DnaK protein requirement can be reduced up to 10-fold.

J Biol Chem, 1993 Mar 5, 268(7), 4584 - 7
Conformational states of mutant M13 coat proteins are regulated by transmembrane residues; Li Z et al.; Mutational and structural analysis of the 28 viable bacteriophage M13 mutants obtained by randomized mutagenesis of the effective transmembrane (TM) segment of the 50-residue major coat (gene VIII) protein (residues 21-39) demonstrated that M13 coat protein functionality, as reflected by phage viability, is incompatible with an increase in Gly + beta-branched residue content in its TM core . SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy performed in membrane environments on purified mutant coat proteins revealed that these proteins exist in a range of state(s), identified as helical monomers and dimers and polymeric (alpha-helical and/or beta-sheet) species, of which relative populations, and thermally induced conformational transitions, were dependent uniquely upon mutation type and locus . Mutations to relatively polar residues (e.g . G23D, Y24D, Y24H, A27E, I32T, and T36S) stabilized principally monomeric species, while mutants with decreased beta-branched content in the protein TM hydrophobic core (e.g . V29A, V30A, V31A, V31L, and V33A) displayed mainly dimeric species . Mutation of Ile37-->Thr within a "Sternberg-Gullick" consensus sequence of the coat protein TM segment led to a highly oligomerized/polymerized protein . The overall results suggest that TM residues in M13 coat protein are not universal components of a hydrophobic anchor segment per se, but are further selected (i) to impart conformational flexibility to the TM segment through helix destabilization and (ii) to retain the capacity to regulate protein-protein association and packing motifs within membranes.

J Mol Biol, 1993 Mar 5, 230(1), 137 - 50
Analysis of establishment phase replication of the plasmid ColE1; Merlin S et al.; The replication regulatory mechanisms by which the small, multicopy plasmid ColE1 maintains a constant steady-state copy number have been extensively characterized by a combination of in vivo genetics and in vitro biochemistry . We have extended the analysis of replication control into the "establishment" phase of replication, when ColE1-directed replicons replicate more than once per cell generation and the intracellular concentrations of plasmid-encoded replication regulatory elements are changing . To study establishment phase replication, in which plasmid-directed replicons amplify from an initially low concentration to the characteristic, steady-state concentration, bacteriophage-plasmid hybrids, termed phasmids, were constructed . Phasmids were shown to exhibit stability, segregation, and incompatibility properties similar to those of the parent plasmid . Establishment phase replication was analyzed by measuring the number of phasmids per cell as a function of time after infection . We observed a linear increase in phasmid concentration until the steady-state concentration characteristic of the ColE1 plasmid component of the hybrid was reached . The number of cell doublings required for the phasmid concentration to reach steady-state was inversely related to cell growth rate . The observed amplification kinetics imply that the frequency of replication initiation per phasmid continually decreases until steady-state is reached . Kinetics of establishment phase amplification were sensitive to rate of expression of RNA II . A phasmid containing an up mutation in the RNA II promoter amplified at a 15-fold faster rate than the wild-type phasmid . Concentration of the ColE1 replication negative regulator (RNA I) was proportional to phasmid concentration throughout the amplification phase . These results suggest that the same elements that regulate steady-state replication also control establishment phase replication.

Mol Biol (Mosk), 1993 Mar-Apr, 27(2), 382 - 91
{Gene 26 of bacteriophage T4 baseplate . II . Synthesis of a smaller peptide of gene 26 is initiated at an unusual AUU-codon}; Vaishkunaite RI et al.; The sequence of gene 26 consisting of 624 nucleotides codes for two peptides whereby the smaller peptide (gp26*) is encoded by the 3'-end of the same open reading frame . Expression of gene 26 from plasmids, containing various fragments from the terminal part of this gene, allowed us to determine the gene 26 internal segment responsible for the initiation of gp26* synthesis . By site-directed mutagenesis the nucleotide sequence of this DNA segment has been changed, and in further expression experiments of the mutagenized gene 26 it was determined that an unusual AUU codon, triplet 114 of gene 26, is used to initiate the synthesis of the smaller peptide . Thus gene 26 encodes two peptides of M(r) 23,880 and M(r) 10,873 . The regulation of the expression of gene 26 in T4-infected cells is discussed.

Chromosoma, 1993 Mar, 102(4), 253 - 66
A combined molecular and cytogenetic approach to genome evolution in Drosophila using large-fragment DNA cloning; Lozovskaya ER et al.; Methods of genome analysis, including the cloning and manipulation of large fragments of DNA, have opened new strategies for uniting molecular evolutionary genetics with chromosome evolution . We have begun the development of a physical map of the genome of Drosophila virilis based on large DNA fragments cloned in bacteriophage P1 . A library of 10,080 P1 clones with average insert sizes of 65.8 kb, containing approximately 3.7 copies of the haploid genome of D . virilis, has been constructed and characterized . Approximately 75% of the clones have inserts exceeding 50 kb, and approximately 25% have inserts exceeding 80 kb . A sample of 186 randomly selected clones was mapped by in situ hybridization with the salivary gland chromosomes . A method for identifying D . virilis clones containing homologs of D . melanogaster genes has also been developed using hybridization with specific probes obtained from D . melanogaster by means of the polymerase chain reaction . This method proved successful for nine of ten genes and resulted in the recovery of 14 clones . The hybridization patterns of a sample of P1 clones containing repetitive DNA were also determined . A significant fraction of these clones hybridizes to multiple euchromatic sites but not to the chromocenter, which is a pattern of hybridization that is very rare among clones derived from D . melanogaster . The materials and methods described will make it possible to carry out a direct study of molecular evolution at the level of chromosome structure and organization as well as at the level of individual genes.

Mol Microbiol, 1993 Mar, 7(6), 975 - 82
Translation across the 5'-splice site interferes with autocatalytic splicing; Ohman-Heden M et al.; The bacteriophage T4 nrdB gene, encoding the ribonucleotide reductase small subunit, contains a self-splicing group IA2 intron with an ochre codon in frame with the preceding exon sequence . The stop codon was changed to an amino acid codon and splicing efficiency was compared with that of the wild type in the presence and absence of translation . In vivo the mutant has a much lower efficiency for producing a mature transcript than the wild type . Also, the relative production of the full-length translation product is correspondingly lower in the mutant than in the wild type . These results confirm the importance of the stop codon, which spans the splice site of the nrdB intron . The occurrence of stop codons in 56 group I introns in protein-encoding genes was investigated . In 33 of those translation is terminated upstream of the first common elements of the catalytic core, of group I introns . In the rest translation is terminated in intron regions outside the heart of the catalytic core, with one exception . Our observations suggest that in situations where transcription and translation are coupled events there has been an evolutionary pressure to preserve stop codons in the 5'-region of these introns or to prevent translational termination from occurring in vital parts of the introns.

Appl Environ Microbiol, 1993 Mar, 59(3), 922 - 3
Quality control of bacterial enumeration; Donnison AM et al.; Standard bacterial suspensions can be used to assess test method performance, via control charts, and inhibition of recovery when analyzing water samples . Variability in standard suspensions prepared from different strains and species and the use of frozen environmental samples for quality control for spore and bacteriophage analyses are also discussed.

Ultramicroscopy, 1993 Mar, 48(3), 347 - 58
Use of radial density plots to calibrate image magnification for frozen-hydrated specimens; Belnap DM et al.; Accurate magnification calibration for transmission electron microscopy is best achieved with the use of appropriate standards and an objective calibration technique . We have developed a reliable method for calibrating the magnification of images from frozen-hydrated specimens . Invariant features in radial density plots of a standard are compared with the corresponding features in a "defocused" X-ray model of the same standard . Defocused X-ray models were generated to mimic the conditions of cryo-electron microscopy . The technique is demonstrated with polyoma virus, which was used as an internal standard to calibrate micrographs of bovine papilloma virus type 1 and bacteriophage phi X174 . Calibrations of the micrographs were estimated to be accurate to 0.35%-0.5% . Accurate scaling of a three-dimensional structure allows additional calibrations to be made with radial density plots computed from two- or three-dimensional data.

J Bacteriol, 1993 Mar, 175(6), 1856 - 9
The filamentous bacteriophage assembly proteins require the bacterial SecA protein for correct localization to the membrane; Rapoza MP et al.; The noncapsid assembly proteins pI and pI of the filamentous bacteriophage f1 are inserted into the inner membrane of Escherichia coli via an internal signal sequence . Inhibition of the activity of SecA with low concentrations of sodium azide results in rapid accumulation of pI and pI proteins in the cytoplasm . However, both proteins are inserted into the membrane under the same conditions when synthesized in bacteria containing a secA azide resistance mutation . The other noncapsid assembly protein, pIV, is an outer membrane protein synthesized with a cleavable signal sequence . Wild-type bacteria accumulate the precursor to pIV when protein synthesis is in the presence of low concentrations of sodium azide . These results suggest that the f1 bacteriophage assembly proteins require SecA and consequently the bacterial Sec system to reach their proper membrane location.

J Bacteriol, 1993 Mar, 175(6), 1844 - 6
Ribosomal protein S1 and NusA protein complexed to recombination protein beta of phage lambda; Venkatesh TV et al.; The red genes of bacteriophage lambda specify two proteins, exonuclease and beta protein, which are essential for general recombination of lambda in recA cells . Earlier studies suggested that these proteins form an equimolar complex (C . M . Radding, J . Rosenweig, F . Richards, and E . Cassuto, J . Biol . Chem . 246:2510-2512, 1971) . A more recent study indicated that beta protein forms a strong complex with an unknown polypeptide of 70 kDa (K . Muniyappa and C . M . Radding, J . Biol . Chem . 261:7472-7478, 1986) . In the present study, in addition to the complex of beta and the 70-kDa protein, a new association of beta protein with a 65-kDa protein was observed . N-terminal sequencing identified these proteins as host-encoded ribosomal protein S1 and transcription terminator protein NusA.

J Bacteriol, 1993 Mar, 175(6), 1785 - 95
Interaction between the Chlamydia trachomatis histone H1-like protein (Hc1) and DNA; Christiansen G et al.; The gene encoding the Chlamydia trachomatis histone H1-like protein (Hc1) from serovar L2 was cloned into Escherichia coli by use of expression vector pET11d . In this vector, transcription of the gene is under the control of a bacteriophage T7 promoter, and T7 RNA polymerase is inducible in the host . Following induction, the E . coli cells were lysed gently . Gel filtration of the lysate revealed comigration of DNA and Hc1 in the voided volume . Electron microscopy revealed the DNA to be complexed with protein in large aggregates, often in the form of spherical bodies . Purified recombinant Hc1 maintained its DNA-binding capacity and was able at high concentrations to form condensed aggregates with DNA (one molecule of Hc1 per base pair) independently of the form or size of the DNA but with a slight preference for supercoiled DNA . Hc1 alone is thus able to package DNA into condensed spherical bodies.

J Bacteriol, 1993 Mar, 175(5), 1272 - 7
Evolution of bacteriophage T7 in a growing plaque; Yin J; The emergence of mutants during the 10(9)-fold amplification of a bacteriophage was spatially resolved in a growing plaque . When wild-type phage T7 was grown on an Escherichia coli host which expressed an essential early enzyme of the phage infection cycle, the T7 RNA polymerase, mutant phage relying on this enzyme appeared in 10(8) phage replications and outgrew the wild type . Spatial resolution of the selection process was achieved by analyzing stab samples taken along a plaque radius . Different mutants were selected at different rates along different radii of the plaque, based on host range and restriction patterns of the isolates . The mutants deleted up to 11% of their genomes, including the gene for their own RNA polymerase . They gained an advantage over the wild type by replicating more efficiently, as determined by one-step growth cultures.

J Bacteriol, 1993 Mar, 175(5), 1239 - 49
Characterization of the binding sites of two proteins involved in the bacteriophage P2 site-specific recombination system; Yu A et al.; Integration of the bacteriophage P2 genome into the Escherichia coli host chromosome occurs by site-specific recombination between the phage attP and E . coli attB sites . The phage-encoded 38-kDa protein, integrase, is known to be necessary for both phage integration as well as excision . In order to begin the molecular characterization of this recombination event, we have cloned the int gene and overproduced and partially purified the Int protein and an N-terminal truncated form of Int . Both the wild-type Int protein and the integration host factor (IHF) of E . coli were required to mediate integrative recombination in vitro between a supercoiled attP plasmid and a linear attB substrate . Footprint experiments revealed one Int-protected region on both of the attP arms, each containing direct repeats of the consensus sequence TGTGGACA . The common core sequences at attP and attB were also protected by Int from nuclease digestion, and these contained a different consensus sequence, AA T/A T/A C/A T/G CCC, arranged as inverted repeats at each core . A single IHF-protected site was located on the P (left) arm, placed between the core- and P arm-binding site for Int . Cooperative binding by Int and IHF to the attP region was demonstrated with band-shift assays and footprinting studies . Our data support the existence of two DNA-binding domains on Int, having unrelated sequence specificities . We propose that P2 Int, IHF, attP, and attB assemble in a higher-order complex, or intasome, prior to site-specific integrative recombination analogous to that formed during lambda integration.

Virology, 1993 Mar, 193(1), 356 - 66
Reovirus protein lambda 3 is a poly(C)-dependent poly(G) polymerase; Starnes MC et al.; Reovirus protein lambda 3 has been isolated from cells infected with two recombinant vaccinia viruses into the TK gene of which the reovirus serotype3 strain Dearing L1 genome segment under the control of the bacteriophage T7 RNA polymerase promoter, or the T7 polymerase gene itself, had been cloned . Highly purified protein lambda 3 does not transcribe double-stranded reovirus RNA into single-stranded RNA, or plus-stranded reovirus RNA into minus-stranded RNA, but it does transcribe poly(C) into poly(G) . It prefers Mn2+ to Mg2+ . A polymer consisting of poly(C) linked linearly to poly(U) provided template activity only for its poly(C) moiety . Protein lambda 3 forms complexes with protein lambda 1, as well as with protein lambda 2, and with both lambda 1 and lambda 2, which are sufficiently stable to be precipitated by monospecific antisera . None of these complexes are capable of transcribing either ds- or ssRNA.

Virology, 1993 Mar, 193(1), 313 - 8
Defective interfering RNA-mediated resistance against cymbidium ringspot tombusvirus in transgenic plants; Kollar A et al.; Defective interfering (DI) RNA of cymbidium ringspot tombusvirus was cloned downstream from the bacteriophage T7 RNA polymerase promoter . In vitro synthesized RNA was biologically active when coinoculated with parental genomic RNA onto Nicotiana benthamiana plants and prevented the occurrence of apical necrosis . N . benthamiana plants were transformed with the DI RNA sequences in both the positive and negative orientations relative to the cauliflower mosaic virus 35S promoter . Integration of DI RNA sequences in the plant genome was verified using PCR amplification of DNA extracts and Northern blot analysis of RNA extracts . DI RNA-related transcripts were detected in uninfected transgenic plants, but inoculation with the parental virus induced replication of the DI RNA only in transgenic plants expressing DI RNA in the positive orientation . Transgenic plants in which DI RNA accumulated were protected from apical necrosis and death.

Mol Biol (Mosk), 1993 Mar-Apr, 27(2), 335 - 41
{Contribution of 3'---5'-exonuclease from rat liver nuclei in precision of DNA synthesis, catalyzed by mammalian DNA polymerase alpha}; Shevelev IV et al.; Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of editing 3'-->5'exonucleolytic activity . Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases . Two 3'-->5'exonucleases with molecular masses of 40 and 50 kDa have been isolated from rat liver nuclei and purified to near homogeneity . They are shown to excise mismatched nucleotides from a poly {d(A-T)} template respectively 10- and 2-fold faster than the matched ones . Upon addition of any of these exonucleases to DNA polymerase alpha from rat liver or calf thymus, the fidelity of in vitro reproduction of primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, the levels of exonuclease and polymerase activities being approximately the same . The extrapolation of replication fidelity to cellular activities of the exonucleases and alpha-polymerase suggests that exonuclease proofreading augments the accuracy of DNA synthesis at least by three orders of magnitude.

J Bacteriol, 1993 Mar, 175(6), 1756 - 66
Properties of Escherichia coli expressing bacteriophage P22 Abc (anti-RecBCD) proteins, including inhibition of Chi activity; Murphy KC et al.; Escherichia coli strains bearing plasmids expressing phage P22 anti-RecBCD functions abc1 and abc2 were tested for the presence of recBC-like phenotypes . Abc2 induces moderate sensitivity to UV light in wild-type and recD mutant strains but severely sensitizes both recF and recJ mutants . Abc1 has little effect on UV sensitivity in wild-type or recF or recJ mutant hosts but increases the sensitivity of recD mutants to a UV dose of 20 J/m2 about 10-fold . Abc2 induces E . coli to segregate inviable cells during growth, interferes with the growth of lambda red gam chi+ and chi 0 phage (the effect is greater with chi+ phage), inhibits Chi and Chi-like activity as measured by lambda red gam crosses, and prevents SOS induction in response to nalidixic acid; Abc1 has no effect in these tests . Abc2, alone or with Abc1, does not allow the growth of lambda red gam in the presence of a P2 prophage but does not kill the P2 lysogenic host (as lambda Gam does) . Finally, Abc2 inhibits conjugational recombination in wild-type cells to the level seen in recBC mutants . These data suggest that Abc2 inhibits the recombination-promoting ability of RecBCD but leaves the exonuclease functions intact.

J Neurochem, 1993 Mar, 60(3), 961 - 71
Antibodies to the human gamma 2 subunit of the gamma-aminobutyric acidA/benzodiazepine receptor; Khan ZU et al.; A gamma-aminobutyric acidA (GABAA) receptor (GABAAR) gamma 2 subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage lambda gt11 . The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors lambda gt11 and pGEX-3X . Both beta-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the gamma 2IL were purified, and a rabbit antibody to the LacZ-gamma 2IL was made . The antibody reacted with the gamma 2IL of both LacZ and GST fusion proteins and immunoprecipitated the GABAAR/benzodiazepine receptor (GABAAR/BZDR) from bovine and rat brain . The antibody reacted in affinity-purified GABAAR/BZDR immunoblots with a wide peptide band of 44,000-49,000 M(r) . Immunoprecipitation studies with the anti-gamma 2IL antibody suggest that in the cerebral cortex, 87% of the GABAARs with high affinity for benzodiazepines and 70% of the GABAARs with high affinity for muscimol contain at least a gamma subunit, probably a gamma 2 . These results indicate that there are {3H}muscimol binding GABAARs that do not bind {3H}flunitrazepam with high affinity . Immunoprecipitations with this and other anti-GABAAR/BZDR antibodies indicate that the most abundant combination of GABAAR subunits in the cerebral cortex involves alpha 1, gamma 2 (or other gamma), and beta 2 and/or beta 3 subunits . These subunits coexist in > 60% of the GABAAR/BZDRs in the cerebral cortex . The results also show that a considerable proportion (20-25%) of the cerebellar GABAAR/BZDRs is clonazepam insensitive . At least 74% of these cerebellar receptors, which likely contain alpha 6, also contain gamma 2 (or other gamma) subunit(s) . The alpha 1 and beta 2 or beta 3 subunits are also frequently associated with gamma 2 (or other gamma) and alpha 6 in these cerebellar receptors.

Proc Natl Acad Sci U S A, 1993 Mar 1, 90(5), 1761 - 5
Gene 5.5 protein of bacteriophage T7 inhibits the nucleoid protein H-NS of Escherichia coli; Liu Q et al.; Gene 5.5 of coliphage T7 is one of the most highly expressed genes during T7 infection . Gene 5.5 protein, purified from cells overexpressing the cloned gene, purifies with the nucleoid protein H-NS of Escherichia coli during three chromatographic steps . A fusion protein of gene 5.5 protein and maltose binding protein also purifies with H-NS . The fusion protein binds to the DNA-H-NS complex and abolishes H-NS-mediated inhibition of transcription by Escherichia coli and T7 RNA polymerases in vitro . Expression of gene 5.5 also relieves the repression of the Escherichia coli proU promoter by H-NS in vivo . The change of leucine to proline at residue 30 of gene 5.5 protein abolishes the interaction between gene 5.5 protein and H-NS.

J Bacteriol, 1993 Mar, 175(5), 1309 - 15
In vivo regulatory responses of four Escherichia coli operons which encode leucyl-tRNAs; Rowley KB et al.; Four Escherichia coli operons, the leuV operon which encodes tRNA(1Leu), the leuX operon which encodes tRNA(6Leu), the metT operon which encodes tRNA(3Leu), and the argT operon which encodes tRNA(1Leu), were examined for the stringent response induced by serine hydroxamate and for growth rate-dependent regulation . In nuclease protection assays, the leuV operon displayed the stringent response in response to leucine starvation, analog inhibition, and growth of a temperature-sensitive leucyl-tRNA synthetase mutant at nonpermissive temperatures . The leuV operon also exhibited the stringent response in multicopy plasmids . The promoters of all four leucyl operons were fused to the gene for beta-galactosidase and inserted into the chromosome by using bacteriophage lambda . All except the leuX promoter displayed growth rate-dependent regulation, consistent with the recent report that the concentration of tRNA(6Leu) actually decreases as growth rate increases . The leuV promoter fused to the beta-galactosidase gene showed a decrease in efficiency in the presence of extrachromosomal copies of rRNA genes . All chromosomal tRNA genes examined showed decreased transcriptional activity following a stringent response, but the leuX gene responded to a lesser extent (3-fold versus 10-fold or more) than the others . Primer extension analysis of this promoter showed little if any response to serine hydroxamate treatment, suggesting that multiple levels of control may exist or that promoter context effects are important in regulation.

J Virol, 1993 Mar, 67(3), 1269 - 77
Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5'-noncoding region and the E2 envelope glycoprotein; Kinney RM et al.; The virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalitis (VEE) virus and its live attenuated vaccine derivative, TC-83 virus, have different neurovirulence characteristics . A full-length cDNA clone of the TC-83 virus genome was constructed behind the bacteriophage T7 promoter in the polylinker of plasmid pUC18 . To identify the genomic determinants of TC-83 virus attenuation, TRD virus-specific sequences were inserted into the TC-83 virus clone by in vitro mutagenesis or recombination . Antigenic analysis of recombinant viruses with VEE E2- and E1-specific monoclonal antibodies gave predicted antigenic reactivities . Mouse challenge experiments indicated that genetic markers responsible for the attenuated phenotype of TC-83 virus are composed of genome nucleotide position 3 in the 5'-noncoding region and the E2 envelope glycoprotein . TC-83 virus amino acid position E2-120 appeared to be the major structural determinant of attenuation . Insertion of the TRD virus-specific 5'-noncoding region, by itself, into the TC-83 virus full-length clone did not alter the attenuated phenotype of the virus . However, the TRD virus-specific 5'-noncoding region enhanced the virulence potential of downstream TRD virus amino acid sequences.

Biochem Biophys Res Commun, 1993 Feb 26, 191(1), 295 - 301
cDNA expression studies of rat liver aryl sulphotransferase; Cruickshank D et al.; A cDNA encoding an isoenzyme of rat liver aryl sulphotransferase was isolated from a rat liver bacteriophage Lambda gt 11 library by the polymerase chain reaction technique . The resulting cDNA was functionally expressed in COS-7 cells and characterised by determining the sulphating capacity of the cells with a range of substrates . The COS-expressed enzyme catalysed the sulphation of both phenol and dopamine with Kms of the same order as those obtained for the high affinity isozyme in rat liver cytosol, while low activity was observed with tyrosine methyl ester . The common food additive vanillin was also a good substrate for sulphate conjugation . The sulphation of vanillin catalysed by the COS-expressed enzyme was consistent with a single enzyme system, in contrast, the kinetics of the reaction catalysed by cytosolic sulphotransferase indicated that vanillin was sulphated by more than one isozyme.

Nucleic Acids Res, 1993 Feb 25, 21(4), 817 - 21
Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids; Boyd AC; The method uses a novel plasmid vector, p9lox5, containing a site-specific recombination sequence lox from the lox/Cre recombinase system of bacteriophage P1 . There are two distinct stages . Firstly, vector and fragment DNAs are ligated intermolecularly under conditions of macromolecular crowding (15% polyethylene glycol 6000) which accelerate blunt-end joining a thousandfold . Secondly, circular recombinant molecules are efficiently excised from the ligation products by Cre recombinase acting on pairs of lox sites within directly repeated vector molecules flanking insert DNA . Recombinants are introduced into cells conventionally by transformation or electroporation . In both a model system and the cloning of PCR products, yields approaching those obtainable in cohesive-end cloning were achieved . Applications of the technique to cDNA library generation and recovery of DNA from archive material are discussed.

Nucleic Acids Res, 1993 Feb 25, 21(4), 863 - 9
Endonuclease-sensitive DNA modifications induced by acetone and acetophenone as photosensitizers; Epe B et al.; Repair endonucleases, viz . endonuclease III, formamidopyrimidine-DNA glycosylase (FPG protein), endonuclease IV, exonuclease III and UV endonuclease, were used to analyse the modifications induced in bacteriophage PM2 DNA by 333 nm laser irradiation in the presence of acetone or acetophenone . In addition to pyrimidine dimers sensitive to UV endonuclease, 5,6-dihydropyrimidines (sensitive to endonuclease III) and base modifications sensitive to FPG protein were generated . The level of the last in the case of acetone was 50% and in the case of acetophenone 9% of the level of pyrimidine dimers . HPLC analysis of the bases excised by FPG protein revealed that least some of them were 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) . In the damage induced by direct excitation of DNA at 254 nm, which was analysed for comparison, the number of FPG protein-sensitive base modifications was only 0.6% of that of the pyrimidine dimers . Mechanistic studies demonstrated that the formation of FPG protein-sensitive modifications did not involve singlet oxygen, as the damage was not increased in D2O as solvent . Hydroxyl radicals, superoxide and H2O2 were also not involved, since the relative number of single strand breaks and of sites of base loss (AP sites) was much lower than in the case of DNA damage induced by hydroxyl radicals and since the presence of SOD or catalase had no effect on the extent of the damage . However, the mechanism did involve an intermediate that was much more efficiently quenched by azide ions than the triplet excited carbonyl compounds and which was possibly a purine radical . Together, the data indicate that excited triplet carbonyl compounds react with DNA not only by triplet-triplet energy transfer yielding pyrimidine dimers, but also by electron transfer yielding preferentially base modifications sensitive to FPG protein, which include 8-hydroxyguanine.

J Mol Biol, 1993 Feb 20, 229(4), 805 - 11
Hierarchy of base-pair preference in the binding domain of the bacteriophage T7 promoter; Diaz GA et al.; The activity of bacteriophage T7 RNA polymerase (RNAP) at a collection of T7 promoter mutants having all possible single base-pair substitutions in the region from -15 to -6 was determined by transcription in vitro, thus establishing a hierarchy of base-pair preference at each position . The tolerance of the RNAP for base-pair substitutions is not uniform across the binding domain . Under stringent conditions (20 mM-MgCl2), T7 RNAP is highly permissive for all base-pair substitutions at -13 and -12 . The RNAP is partially permissive at -15, -14, -11, -10 and -6, and exhibits a clear pattern of base-pair preference at these positions . The RNAP is non-permissive for substitutions at -9 to -7, and will accept only the consensus base-pairs at these positions . Under lower stringency conditions (8 mM-MgCl2, or additionally in the presence of dimethylsulfoxide) a decrease in specificity is observed at most positions except -9 . Analysis of these data suggests potential contacts that may be important for promoter function.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1498 - 502
Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase; Quadt R et al.; The association of host proteins with viral RNA replication proteins has been reported for a number of (+)-strand RNA viruses . However, little is known about the identity or function of these host proteins in viral replication . In this paper we report the characterization of a host protein associated with the RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley . A host protein was specifically and proportionally enriched with BMV RdRp activity through several purification steps . This RdRp-associated host protein reacted with an antiserum prepared against wheat germ eukaryotic translation initiation factor 3 (eIF-3) . The RdRp-associated host protein, the p41 subunit of wheat germ eIF-3, and an antigenically related protein from rabbit reticulocyte lysates were all found to bind with high affinity and specificity to BMV-encoded protein 2a, which is involved in viral RNA replication . Moreover, addition of wheat germ eIF-3 or the p41 subunit from wheat germ to BMV RdRp gave a specific and reproducible 3-fold stimulation of (-)-strand RNA synthesis in vivo . These results suggest that the barley analog of eIF-3 subunit p41, or a closely related protein, associates with BMV RdRp in vivo and is involved in BMV RNA replication . This observation and the established role of translation factors in bacteriophage Q beta RdRp suggest that association with translation factors may be a general feature of RNA replication by (+)-strand RNA viruses.

J Biol Chem, 1993 Feb 15, 268(5), 3056 - 65
Physical and kinetic characterization of the DNA packaging enzyme from bacteriophage lambda; Tomka MA et al.; Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of the viral genome into a preformed capsid . The overexpression of bacteriophage lambda-terminase in Escherichia coli has been previously reported (Chow, S., Daub, E., and Murialdo, H . (1987) Gene (Amst.) 60, 277-289), and we present here a purification scheme for the isolation of milligram quantities of protein which is homogenous ( > 97%) as determined by SDS-polyacrylamide gel electrophoresis . lambda-Terminase is composed of the gene products of Nu1 and A . Using N-terminal amino acid sequence analysis of the purified protein, we have determined a subunit stoichiometry of 2 gpNu1 polypeptides/gpA molecule in terminase holoenzyme . The circular dichroism spectrum for the purified holoenzyme has been obtained and is consistent with a protein complex composed primarily of alpha-helical structure . The endonucleolytic activity of the enzyme (the TER reaction) has been optimized with respect to pH, salt, and polyamine concentrations . Divalent metal ion is strictly required for the reaction and may be satisfied by either magnesium or manganese, but not by any of the other metals examined . E . coli integration host factor in amounts stoichiometric with the DNA substrate stimulates the TER reaction, but only when the enzyme is present in limiting amounts . Increasing the enzyme/DNA ratio attenuates the observed stimulation by integration host factor . A kinetic analysis of the TER reaction suggests that the assembly of multiple terminase promoters is required for efficient cleavage of viral DNA and that this reaction appears to be stoichiometric, rather than catalytic under the reaction conditions utilized . The implications of these results with respect to the packaging of viral DNA by terminase enzymes are discussed.

Arch Biochem Biophys, 1993 Feb 15, 301(1), 91 - 7
Methylene blue and rose bengal photoinactivation of RNA bacteriophages: comparative studies of 8-oxoguanine formation in isolated RNA; Schneider JE Jr et al.; Several reactive oxygen species, including singlet oxygen (1O2) and hydroxyl free radical (.OH), may potentially be involved in the photoinactivation of viruses by agents such as methylene blue (MB) and rose bengal (RB) . Both 1O2 and .OH also mediate the formation of 8-oxoguanine (8-oxoGua) in DNA and RNA . Evidence that MB-or RB-induced bacteriophage (R17 or Q beta) inactivation and 8-oxoGua formation in RNA result from 1O2 rather than .OH was obtained utilizing complementary experimental approaches which show that: (i) the rate of phage photoinactivation by MB was unchanged by the presence of iron chelators or by different temperatures in the 13-37 degrees C range; (ii) MB- and RB-mediated rates of 8-oxoGua formation in isolated RNA have very little, if any, temperature dependence, in contrast to a significant temperature dependence of 8-oxoGua formation by a .OH generating system, the ultraviolet light irradiation of H2O2; and (iii) deuterium oxide (D2O) enhanced the RB-mediated rate of phage photoinactivation and 8-oxoGua formation in isolated RNA . The presence of superoxide dismutase in the RB photoinactivation reaction did not alter the rate of phage inactivation . The data suggest that 8-oxoGua serves as a marker that correlates qualitatively with 1O2-mediated lethal lesions in RNA bacteriophages.

Gene, 1993 Feb 14, 124(1), 21 - 8
Identification of unusual RNA folding patterns encoded by bacteriophage T4 gene 60; Le SY et al.; A 50-nucleotide (nt) untranslated region (coding gap sequence) that interrupts the amino acid coding sequence in T4 gene 60, plus an additional 5 nt upstream and another 3 nt downstream from the gap sequence, shows unusual folding patterns according to RNA structure prediction . A predicted highly stable and significant hairpin structure in the 5' half of the gap sequence and a plausible tertiary structural element computed in the 3' part of the gap sequence seem significant by statistical tests on the wild-type (wt) sequence . This feature is absent in insertion, deletion and substitution variants of the gap sequence, in which template activities are markedly lower than that of the wt . The proposed feature is consistent with currently available data showing that the translational bypass of the coding gap is correlated with a stop codon involved in a stem-loop structure folded in the gap sequence . We suggest that the role of this segment in 'ribosomal bypass' of a portion of the mRNA sequence is a property of its special folded structure.

Nucleic Acids Res, 1993 Feb 11, 21(3), 727 - 32
Structure/function analysis of the Ala116-->Lys121 region of endonuclease V by random targeted mutagenesis; Green AP et al.; Endonuclease V is the product of the denV gene of bacteriophage T4 and is responsible for the recognition and repair of pyrimidine dimers due to UV irradiation of DNA . This is accomplished by a two-step mechanism involving incision at the site of the lesion followed by cleavage of the phosphate backbone . In order to better understand this molecule, and to validate our new mutagenesis procedure, we have constructed a series of random mutations within the region Ala116-->Lys121 using a random targeted mutagenesis procedure developed for this study . The results presented here suggest an important role for this region in the stabilization of the thymine dimer-containing substrate . These mutants also confirm a direct correlation between survival and both DNA binding and pyrimidine dimer-DNA glycosylase activity . No such correlation exists between survival and AP lyase activity . The results are consistent with the recently published X-ray crystal structure.

J Mol Biol, 1993 Feb 5, 229(3), 671 - 84
Role of DNA-protein interactions in bacteriophage phi X174 DNA injection; Ilag LL et al.; Like most bacteriophages, phi X174 transfers its DNA through the cell wall, leaving an empty capsid on the cell surface . The process begins with ejection of the genome at its host-receptor site . The rate of this event can be measured, so detailed structure/function analysis of the mechanism is possible now that an atomic structure of the phi X174 protein shell has been obtained . Amino acid substitutions at two arginine residues near the DNA-binding pocket of F capsid protein decrease the eclipse rate, while deletion of 27 bases from the J-F non-coding region increases the rate . An alanine to serine change in the N-terminal region of the phi X174 H "spike" protein has suppressor activity in that this mutation also increases the eclipse rate when the complete genome is present within both mutant and wild-type F capsids . These results suggest that a portion of H protein is inside the capsid, and disruption of DNA-protein interactions is involved in the ejection mechanism.

J Biol Chem, 1993 Feb 5, 268(4), 2288 - 91
T4-phage deoxycytidylate deaminase is a metalloprotein containing two zinc atoms per subunit; Moore JT et al.; Deoxycytidylate (dCMP) deaminase, a hexameric allosteric enzyme induced on infection of Escherichia coli by bacteriophage T4, was shown to contain two atoms of zinc per subunit by atomic absorption spectroscopy . One zinc appears to be involved in catalysis, as described for adenosine deaminase (Sharaff, A . J., Wilson, D . K., Chang, Z., and Quiocho, F . A . (1992) J . Mol . Biol . 226, 917-921) and cytidine deaminase (Yang, C., Carlow, D., Wolfenden, R., and Short, S . A . (1992) Biochemistry 31, 4168-4174) . This thesis is supported by the finding that the enzyme loses about 80% of its activity in the presence of o-phenanthroline . It has also been found that zinc is released when the enzyme is denatured in the presence of the metallochromic indicator, 4-(2-pyridylazo)resorcinol . Renaturation of the deaminase to an active form occurred in the presence but not in the absence of zinc . The second atom of zinc is proposed to be located in a region of T4-dCMP deaminase that resembles a zinc finger . This region, which has the sequence His-X3-Cys-X14-His-X3-His, would represent a zinc-binding motif that has not been described previously.

Genetika, 1993 Feb, 29(2), 257 - 65
{Characteristics of the bacteriophage N15 lysogenic conversion gene and identification of its product}; Malinin AIu et al.; The plasmids containing EcoRV fragments of N15 phage DNA and inhibiting the adsorption of T1, phi 80 and N15 phages were selected and characterized . The N15 lysogenic conversion gene (cor) was mapped in the SalI-PstI fragment of 700 bp in length which is localized near SalI site with the coordinates 40.1 kb on the N15 plasmid prophage DNA physical map . The cor gene was recloned on a multicopy vector in the both possible orientations and its expression was shown to occur most probably under control of its own promoter in the direction from SalI to the ClaI site of the SalI-PstI fragment . The molecular weight of the Cor protein (7-9 kD) was determined by the analysis in the system of mini-cells . Initiation of transcription to wards the cor gene from the external promoter led to the bacteriostatic effect.

Ultramicroscopy, 1993 Feb, 49(1-4), 235 - 51
Concentration evaluation of chromatin in unstained resin-embedded sections by means of low-dose ratio-contrast imaging in STEM; Bohrmann B et al.; Quantitative STEM with the imaging mode of ratio-contrast was investigated in order to evaluate the local concentration of DNA in situ for different kinds of DNA plasms in terms of intracellular packing densities (p.d.) . The ability of ratio imaging to suppress thickness variations provided the basis to use unstained sections from cryofixed and freeze-substituted material . The DNA p.d . within the nucleoid of E . coli was determined to be about 100 mg ml-1 . Quantitative data concerning the p.d . of DNA in condensed eukaryotic chromatin assuming equal amounts of DNA and protein were evaluated for the first time: approximately 400 mg ml-1 chromatin which corresponds to 200 mg ml-1 DNA . The p.d . of DNA in chromosomes from the dinoflagellate Amphidinium carterae, a eukaryote devoid of histones and with only small relative amounts of histone-like protein, was also found to be of the order of 200 mg ml-1 . The highest p.d . of DNA was measured for the head of the bacteriophage T4 with more than 800 mg ml-1, in fair agreement with previous calculations . The results provide further support for a condensation mode of low protein chromatins that involves a liquid-crystalline organization of the DNA filaments.

Virus Genes, 1993 Feb, 7(1), 89 - 94
Gene A32 product of vaccinia virus may be an ATPase involved in viral DNA packaging as indicated by sequence comparisons with other putative viral ATPases; Koonin EV et al.; Statistically significant sequence similarity was revealed between the gene A32 product of vaccinia virus (VV), gene I products (gpI) of filamentous single-stranded DNA bacteriophages, and IVa2 gene products of adenoviruses . Four conserved sequence motifs were delineated, the two N-proximal of which correspond to the A and B motifs of the purine NTP-binding pattern . Based on the role of gpI and IVa2 proteins in virion morphogenesis, and on the conservation of the NTP-binding pattern in these proteins, we hypothesize that the A32 gene product might be involved in an ATP-consuming function in VV virion formation, e.g., packaging of the DNA in the virus particle.

Mol Microbiol, 1993 Feb, 7(3), 395 - 405
DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics; Hatfull GF et al.; Genetic studies of Mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system . To address this issue we have developed a viral system through a detailed characterization of mycobacteriophage L5, a temperate phage that infects both fast- and slow-growing mycobacteria . We describe here the complete DNA sequence of the L5 genome and initial characterization of L5 virion structure and gene expression . In addition to providing a genetic 'tool-box' for the mycobacteria we find that L5 offers a new paradigm for dsDNA phages, being phenotypically temperate but employing genetic strategies for phage growth usually associated with lytic bacteriophages.

Mol Cell Probes, 1993 Feb, 7(1), 75 - 80
Isolation and ordering of bacteriophage genomic clones corresponding to two YACs from 19q13.3; Buxton J et al.; We describe a method for rapidly isolating overlapping bacteriophage clones corresponding to the genomic region cloned in a yeast artificial chromosome (YAC) that does not require sub-cloning or lambda DNA preparation . Purified YACs from 19q13.3 were used to screen a flow-sorted chromosome 19 library, and the resulting positive clones were characterized using inter-Alu PCR . In addition, aliquots of the lambda stocks were gridded out, and hybridized with probes known to be present in the YACs, thereby avoiding having to perform DNA preparations . The application of this technique in the identification of lambda clones which span the myotonic dystrophy (DM) locus on 19q13.3 is presented, and its general advantages are discussed.

Mol Cell Probes, 1993 Feb, 7(1), 67 - 73
Direct sequencing of lambda DNA from crude lysates using an improved linear amplification technique; Lasham A et al.; We describe an improved method for directly sequencing lambda (lambda) DNA that has been isolated from either crude cleared lysates or plate lysates . This protocol does not require that the DNA be obtained from bacteriophage particles that have been purified by caesium chloride centrifugation . Nanogram quantities of lambda DNA are unidirectionally amplified using a radioactively-labelled oligonucleotide primer, and Thermus aquaticus (Taq) DNA polymerase, in the presence of T4 gene 32 protein (gp 32) . The amplification/sequencing reactions are then incubated with terminal deoxynucleotidyl transferase (TdT) and all four deoxynucleotide triphosphates to elongate any prematurely-arrested products . This procedure, which is a modification of a previously-published method, results in a significant improvement in the quality and amount of DNA sequence information that can be obtained from lambda templates . Although it was developed to sequence DNA directly from lambda EMBL3 recombinants, it can also be used with cosmid DNA, M13 and plasmid DNA, and polymerase chain reaction (PCR) amplification products, yielding excellent ladders in each case . In addition, our method resolves the nucleotide sequences of double-stranded plasmid templates that are difficult to determine by conventional dideoxynucleotide sequencing protocols because of 'stalling', in which bands appear at the same position in all four lanes.

EMBO J, 1993 Feb, 12(2), 595 - 600
The RNA binding site of bacteriophage MS2 coat protein; Peabody DS; The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis . In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated . To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles . Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects . Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured . DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity . Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding . The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.

Genetics, 1993 Feb, 133(2), 143 - 8
The effects of central asymmetry on the propagation of palindromic DNA in bacteriophage lambda are consistent with cruciform extrusion in vivo; Chalker AF et al.; The propagation of lambda phages carrying long perfect palindromes has been compared with that of phages carrying imperfect palindromes with small regions of central asymmetry . The perfect palindromes confer a more deleterious phenotype than those with central asymmetry and the severity of the phenotype declines with the length of asymmetry in the range from O to 27 base pairs . These results argue that a center-dependent reaction is involved in the phenotypic effects of palindromic DNA sequences, consistent with the idea that cruciform extrusion occurs in vivo.

Biotechniques, 1993 Feb, 14(2), 222 - 4
SP6 RNA polymerase containing vaccinia virus for rapid expression of cloned genes in tissue culture; Usdin TB et al.; A hybrid transient expression system, in which tissue culture cells are infected with a recombinant vaccinia virus encoding bacteriophage DNA-dependent RNA polymerase and transfected with a plasmid containing a cloned gene behind the bacteriophage promoter, allows rapid high-level expression in nearly 100% of the cells . In order to extend this system to clones from libraries containing SP6 promoters, a new vaccinia virus was constructed encoding bacteriophage SP6 RNA polymerase.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 1092 - 6
Overexpression, purification, and characterization of SHPTP1, a Src homology 2-containing protein-tyrosine-phosphatase; Pei D et al.; A protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) containing two Src homology 2 (SH2) domains, SHPTP1, was previously identified in hematopoietic and epithelial cells . By placing the coding sequence of the PTPase behind a bacteriophage T7 promoter, we have overexpressed both the full-length enzyme and a truncated PTPase domain in Escherichia coli . In each case, the soluble enzyme was expressed at levels of 3-4% of total soluble E . coli protein . The recombinant proteins had molecular weights of 63,000 and 45,000 for the full-length protein and the truncated PTPase domain, respectively, as determined by SDS/PAGE . The recombinant enzymes dephosphorylated p-nitrophenyl phosphate, phosphotyrosine, and phosphotyrosyl peptides but not phosphoserine, phosphothreonine, or phosphoseryl peptides . The enzymes showed a strong dependence on pH and ionic strength for their activity, with pH optima of 5.5 and 6.3 for the full-length enzyme and the catalytic domain, respectively, and an optimal NaCl concentration of 250-300 mM . The recombinant PTPases had high Km values for p-nitrophenyl phosphate and exhibited non-Michaelis-Menten kinetics for phosphotyrosyl peptides.

J Gen Virol, 1993 Feb, 74 ( Pt 2), 169 - 74
Biologically active transcripts from cloned cDNA of genomic grapevine fanleaf nepovirus RNAs; Viry M et al.; Transcripts were produced in vitro by run-off transcription from full-length cDNA of RNA1 and RNA2 of grapevine fanleaf nepovirus (GFLV; isolate F13) cloned downstream from a bacteriophage RNA polymerase promoter . These transcripts, which possess a 5' terminal cap structure and a non-viral G residue instead of the naturally occurring genome-linked viral protein (VPg), are infectious to Chenopodium quinoa protoplasts when inoculated by electroporation . Synthetic RNA1 alone replicated in protoplasts . Inoculation of C . quinoa plants with synthetic RNA1 plus RNA2 produced symptoms similar to, but weaker, than those observed in plants infected with natural GFLV 6 to 8 days post-inoculation . Co-inoculated RNA1 and RNA2 were able to replicate and spread systemically in plants but RNA1 alone produced no symptoms and was not detected in non-inoculated leaves, suggesting that virus spread requires RNA2 . Analysis of the genomic RNAs in plants infected with transcripts showed that the non-viral G at their 5' ends was not retained in the progeny.

J Protein Chem, 1993 Feb, 12(1), 1 - 5
Specificity of factor Xa in the cleavage of fusion proteins; He M et al.; The precursor protein honey bee prepromelittin has been expressed as a fusion protein in Escherichia coli joined to the C-terminus of a truncated form of the bacteriophage gene 10 protein via an engineered recognition sequence for Factor Xa . Factor Xa was found to cleave poorly at the engineered site, giving a low yield of the required prepromelittin . In contrast, cleavage on the C-terminal side of the sequence VLGR at residue 67 in the gene 10 sequence proceeded in high yield . Factor Xa may be inhibited by adjacent hydrophobic sequences on the C-terminal side of a potential cleavage site.

J Bacteriol, 1993 Feb, 175(3), 642 - 6
Characterization of dinY, a new Escherichia coli DNA repair gene whose products are damage inducible even in a lexA(Def) background; Petit C et al.; Bacteriophage Mu dX(Ap lac) was used to isolate a mutation in an Escherichia coli lexA(Def) strain representing a previously undescribed gene (dinY) which does not seem to be under the direct control of LexA . The insertion created a dinY::lacZ fusion in which beta-galactosidase expression required a DNA-damaging treatment (UV irradiation or mitomycin) and activable RecA protein . This strain showed a decreased Weigle reactivation of bacteriophage lambda . However, it was fully inducible for UV mutagenesis . Two-dimensional gel electrophoresis analysis identified two spots absent in the mutant which were both UV inducible only in the presence of activated RecA protein (RecA*) . This finding suggests that the dinY::lacZ fusion lies in a gene either that is under the direct control of activated RecA or whose product undergoes RecA*-dependent posttranscriptional/posttranslational modification(s) . The dinY gene may also control the expression of some other gene(s) and/or lie in an operon . The fusion was mapped at a position between 41 and 41.5 min on the E . coli chromosome, in the vicinity of the ruv operon.

J Bacteriol, 1993 Feb, 175(4), 1134 - 43
Sequence analysis and phenotypic characterization of groEL mutations that block lambda and T4 bacteriophage growth; Zeilstra-Ryalls J et al.; The groES and groEL genes of Escherichia coli have been shown previously to belong to a single operon under heat shock regulation . Both proteins have been universally conserved in nature, as judged by the presence of similar proteins throughout evolution . The GroEL protein has been shown to bind promiscuously to many unfolded proteins, thus preventing their aggregation . ATP hydrolysis by GroEL results in the release of the bound polypeptides, a process that often requires the action of GroES . In an effort to understand GroEL and GroES structure and function, we have determined the nucleotide changes of nine mutant alleles of groEL . All of these mutant alleles were isolated because they block bacteriophage lambda growth . Our sequencing results demonstrate that (i) many of these alleles are identical, in spite of the fact that they were independently isolated, and (ii) most of the different alleles are clustered in the same region of the gene . One of the mutant alleles was shown to possess two nucleotide alterations in the groEL coding phase, one of which is located in a putative ATP-binding domain . The two nucleotide changes were separated by genetic engineering, and each individual change was shown to exert an effect on bacteriophage growth . But, using genetic analyses, we demonstrate that the restriction on bacterial growth at elevated temperatures is conferred only by the mutation within the putative ATP-binding domain . We have cloned the mutant alleles on multicopy plasmids and overexpressed their products . By testing for the ability of bacteriophage either to propagate or to form colonies at 43 degrees C, we have been able to divide the mutant proteins into those with no activity and those with residual activity under the various conditions tested.

J Bacteriol, 1993 Feb, 175(3), 716 - 22
Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system; Rosenberg AH et al.; A system for testing the effects of specific codons on gene expression is described . Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages . Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli . The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis . Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon . The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA . Production of aberrant proteins was also stimulated at high levels of mRNA . The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons . Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNA(AGG) in the cell to become sequestered in translating peptidyl-tRNA(AGG) -mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome of frameshift, hop, or terminate translation.

Cell, 1993 Jan 29, 72(2), 261 - 8
Recognition of boxA antiterminator RNA by the E . coli antitermination factors NusB and ribosomal protein S10; Nodwell JR et al.; The boxA sequences of the E . coli ribosomal RNA (rrn) operons are sufficient to cause RNA polymerase to read through Rho-dependent transcriptional terminators . We show that a complex of the transcription antitermination factors NusB and ribosomal protein S10 interacts specifically with boxA RNA . Neither NusB nor S10 binds boxA RNA on its own, and neither NusA nor NusG affects the interaction of the NusB-S10 complex with boxA RNA . Mutations in boxA that impair its antitermination activity compromise its interaction with NusB and S10, suggesting that ribosomal protein S10 regulates the synthesis of ribosomal RNA in bacteria . RNA containing the closely related boxA sequence from the bacteriophage lambda nutR site is not stably bound by NusB and S10 . This probably explains why antitermination in phage lambda depends on the phage lambda N protein and the boxB component of the nut site, in addition to boxA.

Biochemistry, 1993 Jan 26, 32(3), 982 - 8
Mutagenicity and genotoxicity of the major DNA adduct of the antitumor drug cis-diamminedichloroplatinum(II); Bradley LJ et al.; The mutagenicity and genotoxicity of cis-{Pt(NH3)2{d(GpG)-N7(1),-N7(2)}} (G*G*), the major DNA adduct of the antitumor drug cisplatin, has been investigated in Escherichia coli . A duplex bacteriophage M13 genome was constructed to contain the G*G* adduct at a specific site in the (-) strand . The singly platinated duplex genome exhibited a survival of 22% relative to that of the unplatinated control genomes, and this value rose to 38% in cells treated with ultraviolet light to induce the SOS response . Singly platinated single-stranded genomes were also produced . Replication of the single- and double-stranded genomes in vivo yielded SOS-dependent, targeted mutations at frequencies of 1.3% and 0.16%, respectively . The mutagenic specificity of G*G* in both single- and double-stranded DNA was striking in that 80-90% of the mutations occurred at the 5'-platinated G . Approximately 80% of the mutations were G-->T transversions at that site . A model of mutagenesis is presented to explain this mutational specificity with respect to current understanding of platinum-DNA adduct structure.

J Biol Chem, 1993 Jan 25, 268(3), 1603 - 9
Chemical modification of bacteriophage T4 deoxynucleotide kinase . Evidence of a single catalytic region; Brush GS et al.; The mechanism underlying the unusual specificity of bacteriophage T4 deoxynucleotide kinase, which catalyzes the phosphorylation of 5-hydroxymethyldeoxycytidylate, dTMP, and dGMP, has been investigated by chemical modification of the protein . Pyridoxal 5'-phosphate inactivates deoxynucleotide kinase by modifying a single lysine out of the 17 per monomer . Lysine 10 has been tentatively identified as the site of modification, although the possibility of mutually exclusive reactive residues has not been eliminated . Diethylpyrocarbonate also inactivates the enzyme, suggesting that histidine plays a role in catalytic function . With either reagent, the three activities are lost at equal rates, supporting the contention that one active site is responsible for the exclusive phosphorylation of three dissimilar deoxynucleotides . These studies also identify two distant regions of the primary sequence that are likely to be closely associated in the active region of the folded protein.

J Mol Biol, 1993 Jan 20, 229(2), 398 - 418
Assembly of the bacteriophage T4 replication machine requires the acidic carboxy terminus of gene 32 protein; Hurley JM et al.; The acidic carboxy-terminal 89-amino acid fragment of bacteriophage T4 gene 32 protein was expressed in Escherichia coli to high levels from an inducible plasmid construct . Infection of induced cells by wild-type T4 phage results in impaired phage DNA synthesis . The time at which DNA synthesis begins and the diminution in DNA synthesis rates correlate with the amount of carboxy-terminal peptide that accumulates intracellularly prior to infection . Correspondingly, when induced cells are infected with viable phage containing a small deletion near the carboxy-terminus of 32 protein (delta PR201), the inhibition of phage DNA synthesis was much more severe . The mutant 32 protein competes less well against overproduced wild-type acid peptide than does wild-type 32 protein . The purified acid peptide, when used as the attached ligand for affinity chromatography, binds several T4 proteins from phage-infected cells, including 43 protein (T4 DNA polymerase), Dda protein (a DNA helicase), and UvsX protein (a Rec-like recombination protein) . Furthermore, at 50- to 100-fold molar excess of acid peptide over intact 32 protein, phage DNA synthesis was specifically inhibited at the initiation step in an in vitro 5-protein DNA replication experiment . We propose that one or more phage replication proteins are titrated as non-productive protein-protein complexes at a site away from the DNA template . This implies that the carboxy-terminal domain of 32 protein is involved in an obligate step of replication machine assembly when the protein is properly attached to ssDNA in the vicinity of a primer-template junction . The assembly defect we observe is strikingly similar to the repression, or "squelching", of the activity of certain eukaryotic transcriptional activators.

Biochemistry, 1993 Jan 19, 32(2), 537 - 43
Subunit conformational changes accompanying bacteriophage P22 capsid maturation; Prevelige PE Jr et al.; In double-stranded DNA bacteriophages, packaging of dsDNA requires the transformation of a precursor procapsid into a mature viral capsid . Lattice expansion and release of scaffolding subunits accompanying DNA packaging . Three-dimensional structures of procapsid and mature phage lattices demonstrate that the capsid transformation involves substantial changes in subunit environment . Since this transformation occurs without subunit dissociation, it represents a transition between at least two stable subunit conformations . Using Raman spectroscopy, we have identified changes in coat protein secondary structure and side-chain environments which accompany the capsid transformation . The subunits of procapsid shells contain only 2.0 +/- 0.4% more alpha-helix and less beta-sheet than those of mature capsids; however, numerous side chains are substantially altered by the transformation, including tyrosines, tryptophans, phenylalanines, and aliphatics, which are widely distributed through the subunit sequence . We propose, therefore, that procapsid expansion is accomplished through the relative motion of coat subunit domains with little change in secondary structure . Such hinge-bending conformational transitions may couple ATP-dependent dsDNA condensation with shell expansion.

FEMS Microbiol Lett, 1993 Jan 15, 106(2), 135 - 8
Expression of beta-galactosidase from a hybrid promoter:operator element in Escherichia coli; Dixon KE et al.; A hybrid trpPO:lacO regulatory sequence was cloned upstream of a promoterless lacZ gene and recombined onto a lambda bacteriophage . Escherichia coli lysogens representing the four possible phenotypes for lacI and trpR were constructed and the synthesis of beta-galactosidase was assayed under various growth conditions . The results illustrated that both control elements could be efficiently and independently regulated by the addition or omission of appropriate accessory molecules.

Eur J Biochem, 1993 Jan 15, 211(1-2), 347 - 55
Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli; Arcone R et al.; Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid- regulated anti-inflammatory factor . cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter . Upon induction with isopropyl thio-beta-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form . The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps . The final yield was about 30 mg/l bacterial culture . Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge . Recombinant human lipocortin 1 reduces the carrageenin-induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose-related response is observed.

Proc Natl Acad Sci U S A, 1993 Jan 15, 90(2), 472 - 6
RNA polymerase idling and clearance in gal promoters: use of supercoiled minicircle DNA template made in vivo; Choy HE et al.; We have developed an in vivo system to engender supercoiled "minicircle" DNA containing a single promoter by using the integrative recombination system of bacteriophage lambda . The resulting minicircle templates allow quantitative analysis of the stages of transcription initiation from a promoter, including synthesis of both full-length and aborted transcripts in the same reactions under physiological conditions . We have used such minicircle DNA templates to study in vitro transcription of the Escherichia coli gal promoter . The full-length transcripts from gal P1 and P2 promoters responded to cAMP-cAMP receptor protein in a manner identical to that observed in vivo . There is a 3.5-fold stimulation of P1 and almost total inhibition of P2 in the presence of cAMP . Thus, the unitary promoter system described here duplicates the in vivo physiology . In spite of the synthesis in equimolar amounts of full-length transcripts from P1 and P2 in the absence of cAMP in vitro, as in vivo, RNA polymerase encountered different rate-limiting steps of transcription initiation at the two promoters.

J Mol Biol, 1993 Jan 5, 229(1), 37 - 51
Effects of a single base-pair deletion in the bacteriophage lambda PRM promoter . Repression of PRM by repressor bound at OR2 and by RNA polymerase bound at PR; Woody ST et al.; We have deleted a single base-pair in the -35 region of the bacteriophage lambda PRM promoter . The deletion (PRM delta 34) creates a better match of PRM to consensus, thereby substantially increasing the activity of the promoter in vitro and in vivo . Since the mutation also increases the overlap between OR2 and the -35 region of PRM, binding of repressor to OR2 no longer activates, but in fact represses PRM . Finally, the mutation decreases the distance between the PRM and PR transcription start sites from 82 to 81 base-pairs . As a consequence, the interaction of RNA polymerase with either promoter in vitro strongly inhibits open complex formation at the other . Kinetic analyses and DNase I protection assays lead to the surprising result that mutual inhibition is not due to steric occlusion . Both promoters can be occupied by RNA polymerase at the same time . Determination of KB and kf revealed that inhibition of PRM delta 34 by PR was manifest in a 100-fold decrease in the value of kf, but at the same time KB was increased tenfold . These data raise the possibility that RNA polymerase molecules bound at the two promoters contact and mutually stabilize each other and that this interaction subsequently inhibits a substep in the isomerization of closed to open complexes . In footprinting assays, each promoter is characterized by sites of enhanced cleavage when that promoter is occupied alone . These enhancements are substantially diminished when both promoters are occupied, suggesting that complexes of each promoter with RNA polymerase alter the structure of complexes formed at the other promoter . Assays of the effects of the delta 34 mutation in vivo indicate that interference between PRM and PR does not limit the rate of open complex formation at PRM in the cell . Apparently, transcription initiation clears the promoter rapidly enough that neither promoter is occupied a significant fraction of the time.

FEBS Lett, 1993 Jan 2, 315(1), 51 - 5
The DNA-binding properties of an artificial 42-residue polypeptide derived from a natural repressor; Hehlgans T et al.; Bacteriophage 434 repressor recognizes the operator sequences ACAAG and ACAAT . As the same or similar sequences occur in the enhancer region of HIV-1, 434 repressor was a potential HIV enhancer-binding protein . We found that the interaction of the DNA-binding domain of 434 repressor with a 57-bp HIV enhancer DNA was very weak whereas a 42-residue construct, comprising the recognition helix and four copies of a positively charged segment of the repressor, bound strongly . The results of footprint and cell-free in vitro transcription studies showed that the 42-residue peptide bound preferably to the enhancer region of HIV-1 and acted as an artificial repressor . Replacement of an essential glutamine of the recognition helix by glutamic acid resulted in a partial shift of the sequence specificity of the 42-residue peptide.

Vet Med (Praha), 1993, 38(4), 223 - 8
{DNA fingerprinting in horses}; Pazdera J et al.; Using a multilocus DNA probe, individual - specific hybridization patterns, the so-called DNA fingerprints (TAB) were determined in six horse families by the DNA fingerprinting method . The probe with evolutionally preserved nucleotide sequence from bacteriophage M13 determines hypervariable regions placed in genomic minisatellite DNA . The use of this probe permits an identification of an individual and execution of paternity relationships with a probability over 99.99 per cent.

Methods Enzymol, 1993, 218, 104 - 21
Automated fluorescent DNA sequencing of polymerase chain reaction products; Du Z et al.; The methods described in this chapter provide some useful approaches for DNA sequencing of templates produced by PCR . These procedures have been employed successfully for large-scale DNA sequencing of cosmid fragments subcloned in plasmid or M13 vectors, and for sequence analysis of cDNAs cloned in bacteriophage lambda vectors . In addition, the method describing direct sequencing from PEG-precipitated PCR product has been used successfully for analysis of Caenorhabditis elegans genomic and cDNA sequences . It is important to reiterate that for every combination of amplification primer pair and target DNA, there is an optimal method for PCR amplification; the ability to sequence the products of any PCR experiment directly will also vary . A coupled PCR/DNA sequencing method that works well for one experimental system may work quite poorly with others . Hence, a few days or hours spent optimizing PCR amplification conditions and selecting the best DNA sequencing method for the target DNA of interest will be time well spent.

Nat Genet, 1993 Jan, 3(1), 44 - 8
A recombination-based assay demonstrates that the fragile X sequence is transcribed widely during development; Hanzlik AJ et al.; To identify transcribed sequences rapidly and efficiently, we have developed a recombination-based assay to screen bacteriophage lambda libraries for sequences that share homology with a given probe . This strategy determines analytically whether a given probe is transcribed in a given tissue at a given time of development, and may also be used to isolate preparatively the transcribed sequence free of the screening probe . We illustrate this technology for the fragile X sequence, demonstrating that it is transcribed ubiquitously in an 11 week fetus, in a variety of 20 week human fetal tissues, including brain, spinal cord, eye, liver, kidney and skeletal muscle, and in adult jejunum.

Mol Biol (Mosk), 1993 Jan-Feb, 27(1), 92 - 102
{Study of the form of bacteriophage T7 RNA polymerase containing point substitutions in the region of functionally important amino acid residues}; Tunitskaia VL et al.; A study was carried out on the influence of point mutations of the functional amino acid residues on the secondary and ternary structure of bacteriophage T7 RNA polymerase as well as on the activity of the enzyme . A change in residue 631 is accompanied by significant changes in secondary structure (alpha-->beta transition) . The substitution Lys-172 Leu changes both the secondary and ternary structures whereas the deletion of residues 172-173 does not lead to such changes . Changes in residues 631-632 do not affect the ability of the enzyme to bind the promoter and/or the synthesis of a full-length transcript but disturbs phosphodiester bond formation.

Biokhimiia, 1993 Jan, 58(1), 43 - 9
{Study of phage T7 DNA-dependent RNA-polymerase using GTP analogs . Affinity modification and study of interaction with matrices using fluorescent markers}; Mishin AA et al.; Interactions of the bacteriophage T7 DNA-dependent RNA polymerase with three GTP analogs have been studied . All of the three analogs tested contained substituted naphthalenesulphamide groups and were shown to be under appropriate conditions irreversible covalent inhibitors of the enzyme, the modified enzyme possessing fluorescent properties . One of these analogs contained the reactive 2-bromoethyl phosphonate group and was shown to cause the loss of the enzyme affinity for polynucleotide templates . The other two modifiers which contained the azide reactive group did not alter the enzyme-template affinity, the polynucleotide binding leading to a notable increase of the enzyme fluorescence intensity . The latter two modifiers are supposed to be convenient for fluorescent labelling of the active site of RNA polymerase for enzyme-template binding studies.

Kidney Int Suppl, 1993 Jan, 39, S20 - 5
Positional cloning approach to the dominant polycystic kidney disease gene, PKD1; Germino GG et al.; Positional cloning is a powerful strategy for identifying the site of disease-producing mutations when the underlying biochemical defect is unknown . The approach also offers new methods for the presymptomatic diagnosis of genetic disease . Using these methods we have localized the PKD1 gene, mutated in the majority of PKD1 families, to a small (500 kb) segment of chromosome 16, band p13.3 . Virtually all of this interval has been cloned in cosmids and lambda bacteriophage . Over 20 sets of non-overlapping cDNA clones have been isolated from the region . Sequence and mutational analyses are currently underway . In addition, a set of polymorphic clones has been identified for presymptomatic diagnosis . Included in this set are several highly variable {CA}n microsatellite repeats . These highly informative markers can be rapidly assayed from a small amount of genomic DNA using the polymerase chain reaction . Despite these advances, presymptomatic diagnosis cannot be established with certainty in many families . However, identification of the PKD1 gene itself will eventually allow diagnosis by direct detection of mutations.

Proteins, 1993 Jan, 15(1), 10 - 25
Insertion of peptide chains into lipid membranes: an off-lattice Monte Carlo dynamics model; Milik M et al.; A combination of dynamic Monte Carlo simulation techniques with a hydropathy scale method for the prediction of the location of transmembrane fragments in membrane proteins is described . The new hydropathy scale proposed here is based on experimental data for the interactions of tripeptides with phospholipid membranes (Jacobs, R.E., White, S.H . Biochemistry 26:6127-6134, 1987) and the self-solvation effect in protein systems (Roseman, M.A . J . Mol . Biol . 200:513-522, 1988) . The simulations give good predictions both for the state of association and the orientation of the peptide relative to the membrane surface of a number of peptides including Magain2, M2 delta, and melittin . Furthermore, for Pf1 bacteriophage coat protein, in accord with experiment, the simulations predict that the C-terminus forms a transmembrane helix and the N-terminus forms a helix which is adsorbed on the surface of the bilayer . Finally, the present series of simulations provide a number of insights into the mechanism of insertion of peptides into cell membranes.

Vet Microbiol, 1993 Jan, 34(1), 19 - 34
Clonal diversity among strains of Escherichia coli incriminated in turkey colisepticemia; White DG et al.; The extent of genetic differentiation among 80 Escherichia coli isolates collected from turkeys with acute colisepticemia was assessed based on allelic variation at 20 enzyme-encoding loci detected by multilocus enzyme electrophoresis . Isolates were polymorphic at 17 loci and were classified into 32 multilocus genotypes, delineating clones, that differed on average at 36% of the loci . In the total sample, 29 (36%) of the isolates belonged to one of two closely related clones, differing only in a single electromorph, and 11 of these isolates were serogroup O78 . Most isolates fell into one of 4 genetically distinct clusters of strains . Three of these clusters represent E . coli clone complexes that have been previously identified in avian diseases and a fourth cluster which is specific to colisepticemia in turkeys . Most (73%) isolates produced aerobactin, whereas none produced hemolysins . Assays for detecting K1 capsules, including the use of polyclonal antisera, monoclonal antibodies, and K1-specific bacteriophages, gave variable results, but showed that overall 18% of the strains from colisepticemia were K1 encapsulated with most of the K1+ isolates found in one clone cluster . The results show that many cases of colisepticemia in turkey flocks are caused by a small number of pathogenic clones representing at least three distinct clone complexes.

Can J Microbiol, 1993 Jan, 39(1), 134 - 9
Cloning of a xylanase gene from the ruminal fungus Neocallimastix patriciarum 27 and its expression in Escherichia coli; Lee JM et al.; An endo-beta-1,4-xylanase gene was cloned from Neocallimastix patriciarum 27 in the bacteriophage vector lambda gtWES lambda B and was subcloned into the plasmid vectors pUC18 and pUC19 in which xylanase activity was expressed in both orientations . The xylanase was located in the periplasmic space of the host, Escherichia coli HB101 . The pH and temperature optima for periplasmic xylanase activity were 6.2 and 40 degrees C, respectively, and the Km for oat spelt xylan hydrolysis was 0.89 mg.mL-1 . It also exhibited hydrolytic activity on carboxymethyl cellulose that was equivalent to 28% of the activity exhibited by the enzyme on xylan . It bound to crystalline cellulose, but lacked hydrolytic activity on amorphous cellulose . SDS-PAGE followed by zymogram analysis showed active bands of 68, 58, and 51 kDa . Isoelectric focusing in gels combined with zymogram analysis showed one band of xylanase activity with a pI of 3.6.

Lasers Surg Med, 1993, 13(1), 18 - 22
Viable bacteriophage in CO2 laser plume: aerodynamic size distribution; Matchette LS et al.; The size of CO2 laser generated plume particles containing viable bacteriophage, phi X174, was determined with 2 models (10-830 and 15-500) of Andersen bioaerosol cascade impactors . Samples were collected during 100 one-second laser exposures (approximately 68 W/cm2) of a bacteriophage-agar substrate with and without a space-confining hood . The hood appeared to facilitate collection of the largest particles (P < 0.1) . In addition, Andersen model 15-500 was a more efficient collector of the largest particles, a phenomenon which is likely a function of the dynamic nature of the laser plume as well as impactor design . We found that laser plume particles containing viable bacteriophage are very large, in one instance exhibiting a count median aerodynamic diameter (CMAD) and mass median aerodynamic diameter (MMAD) of 23 and 55 microns, respectively . Furthermore, the appearance of viable bacteriophage in these plume particles constitutes an extremely rare event . The limitations of cascade impactor design when used to analyze particles with high water concentrations generated in a laser plume are discussed.

J Bacteriol, 1993 Jan, 175(1), 85 - 93
The asiA gene of bacteriophage T4 codes for the anti-sigma 70 protein; Orsini G et al.; The anti-sigma 70 factor of bacteriophage T4 is a 10-kDa (10K) protein which inhibits the sigma 70-directed initiation of transcription by Escherichia coli RNA polymerase holoenzyme . We have partially purified the anti-sigma 70 factor and obtained the sequence of a C-terminal peptide of this protein . Using reverse genetics, we have identified, at the end of the lysis gene t and downstream of an as yet unassigned phage T4 early promoter, an open reading frame encoding a 90-amino-acid protein with a predicted molecular weight of 10,590 . This protein has been overproduced in a phage T7 expression system and partially purified . It shows a strong inhibitory activity towards sigma 70-directed transcription (by RNA polymerase holoenzyme), whereas it has no significant effect on sigma 70-independent transcription (by RNA polymerase core enzyme) . At high ionic strength, this inhibition is fully antagonized by the neutral detergent Triton X-100 . Our results corroborate the initial observations on the properties of the phage T4 10K anti-sigma 70 factor, and we therefore propose that the gene which we call asiA, identified in the present study, corresponds to the gene encoding this T4 transcriptional inhibitor.

J Bacteriol, 1993 Jan, 175(1), 288 - 90
Host RecJ is required for growth of P22 erf bacteriophage; Mahan MJ et al.; Growth of bacteriophage P22 erf is known to require host RecA recombination function . We show that the RecA function is necessary but not sufficient to restore the plaque-forming ability of phage P22 erf; such mutant phage also requires host RecJ function . The residual efficiency of plaquing of P22 erf in a recJ background (0.03%) is completely abolished in recJ recB hosts (< 0.001%), suggesting that the RecBCD nuclease can provide an alternative function allowing phage growth . One tentative explanation is that circularization of P22 erf DNA mostly proceeds through the RecF pathway of recombination; however, less efficient circularization via the RecBCD pathway may also occur . In a recJ background, lysates obtained upon induction of an erf prophage show reduced yield (10%), suggesting that growth of P22 erf may require host RecJ in a step(s) other than circularization of phage DNA.

J Bacteriol, 1993 Jan, 175(1), 277 - 87
Identification and characterization of the Escherichia coli RecT protein, a protein encoded by the recE region that promotes renaturation of homologous single-stranded DNA; Hall SD et al.; Recombination of plasmid DNAs and recombination of bacteriophage lambda red mutants in recB recC sbcA Escherichia coli mutants, in which the recE region is expressed, do not require recA . The recE gene is known to encode exonuclease VIII (exoVIII), which is an ATP-independent exonuclease involved in the RecE pathway of recombination . A 33,000-molecular-weight (MW) protein was observed to be coexpressed with both exoVIII and a truncated version of exoVIII, pRac3 exo, when they were overproduced under the control of strong promoters . We have purified this 33,000-MW protein (p33) and demonstrated by protein sequence analysis that it is encoded by the same coding sequence that encodes the C-terminal 33,000-MW portion of exoVIII . p33 is expressed independently of exoVIII but is probably translated from the same mRNA . p33 was found to bind to single-stranded DNA and also to promote the renaturation of complementary single-stranded DNA . It appears that p33 is functionally analogous to the bacteriophage lambda beta protein, which may explain why RecE pathway recombination does not require recA.

J Bacteriol, 1993 Jan, 175(1), 222 - 8
Role of the carboxyl-terminal domain of TolA in protein import and integrity of the outer membrane; Levengood-Freyermuth SK et al.; The TolA protein is involved in maintaining the integrity of the outer membrane of Escherichia coli, as mutations in tolA cause the bacteria to become hypersensitive to detergents and certain antibiotics and to leak periplasmic proteins into the medium . This protein also is required for the group A colicins to exert their effects and for many of the filamentous single-stranded bacteriophage to infect the bacterial cell . TolA is a three-domain protein, with the amino-terminal domain anchoring it to the inner membrane . The helical second domain is proposed to span the periplasmic space to allow the carboxyl-terminal third domain to interact with the outer membrane . A plasmid that allowed the synthesis and transport of the carboxyl-terminal third domain into the periplasmic space was constructed . The presence of an excess of this domain in the periplasm of a wild-type cell resulted in an increased sensitivity to deoxycholate, the release of periplasmic alkaline phosphatase and RNase into the medium, and an increased tolerance to colicins E1, E2, E3, and A . There was no effect on the cells' response to colicin D, which depends on TonB instead of TolA for its action . The presence of the free carboxyl-terminal domain of TolA in the periplasm in a tolA null mutation did not restore the wild-type phenotype, suggesting that this domain must be part of the intact TolA molecule to perform its function . Our results are consistent with a model in which the carboxyl-terminal domain of TolA interacts with components in the periplasm or on the inner surface of the outer membrane to function in maintaining the integrity of this membrane.

J Virol, 1993 Jan, 67(1), 249 - 57
A micromolar pool of antigenically distinct precursors is required to initiate cooperative assembly of hepatitis B virus capsids in Xenopus oocytes; Seifer M et al.; Assembly of hepatitis B virus capsid-like (core) particles occurs efficiently in a variety of heterologous systems via aggregation of approximately 180 molecules of a single 21.5-kDa core protein (p21.5), resulting in an icosahedral capsid structure with T = 3 symmetry . Recent studies on the assembly of hepatitis B virus core particles in Xenopus oocytes suggested that dimers of p21.5 represent the major building block from which capsids are generated . Here we determined the concentration dependence of this assembly process . By injecting serially diluted synthetic p21.5 mRNA into Xenopus oocytes, we expressed different levels of intracellular p21.5 and monitored the production of p21.5 dimers and capsids by radiolabeling and immunoprecipitation, by radioimmunoassay, or by quantitative enzyme-linked immunosorbent assay analysis . The data revealed that (i) p21.5 dimers and capsids are antigenically distinct, (ii) capsid assembly is a highly cooperative and concentration-dependent process, and (iii) p21.5 must accumulate to a signature concentration of approximately 0.7 to 0.8 microM before capsid assembly initiates . This assembly process is strikingly similar to the assembly of RNA bacteriophage R17 as defined by in vitro studies.

Biophys Chem, 1993 Jan, 45(3), 281 - 303
A comparative study of Scatchard-type and linear lattice models for the analysis of EPR competition experiments with spin-labeled nucleic acids and single-strand binding proteins; Keyes RS et al.; An EPR competition formalism is developed which provides relative affinities of proteins for nucleic acids . Two models for analyzing protein-nucleic acid interactions, one assuming independent binding sites (Model 1) and the other considering site overlap (Model 2), are examined with respect to their validity and limitations . The models are employed to derive affinity ratio relationships which are used to calculate the relative affinities of gene 32, gene 5, and SSB proteins for various nucleic acids . It is determined that although Model 2 must be used when determining absolute binding constants, by taking the ratio of binding constants the site overlap becomes unimportant under conditions of moderate to high cooperativity and relatively small site size . This allows Model 1 to considerably simplify binding analyses . Both models are applied to the single-strand binding proteins of bacteriophage T4 gene 32, bacteriophage fd gene 5, and the Escherichia coli ssb gene, and the results are compared.

J Virol, 1993 Jan, 67(1), 60 - 6
Bacteriophage T4 DNA polymerase mutations that confer sensitivity to the PPi analog phosphonoacetic acid; Reha-Krantz LJ et al.; Mutations that conferred sensitivity to the pyrophosphate analog phosphonoacetic acid in bacteriophage T4 DNA polymerase were identified . The mutations were loosely clustered in four regions of the gene . As found for herpes simplex virus DNA polymerase, T4 mutations that altered sensitivity to phosphonoacetic acid also altered sensitivity to nucleotide analogs . Some of the T4 DNA polymerase mutations also altered the ability of the enzyme to translocate from one template position to the next and affected DNA replication fidelity . Kornberg (A . Kornberg, Science 163:1410-1418, 1969) envisioned a DNA polymerase active center which accommodates primer terminus and template DNAs and the incoming nucleotide . Some mutations identified on the basis of sensitivity to phosphonoacetic acid may be part of such an active center because single amino acid substitutions simultaneously alter several DNA polymerase functions.

J Virol, 1993 Jan, 67(1), 305 - 14
Varicella-zoster virus glycoprotein gpI/gpIV receptor: expression, complex formation, and antigenicity within the vaccinia virus-T7 RNA polymerase transfection system; Yao Z et al.; The unique short region of the varicella-zoster virus (VZV) genome contains two open reading frames which encode glycoproteins designated gpI and gpIV (herpes simplex virus homologs gE and gI, respectively) . Like its herpesviral counterpart gE, the VZV gpI gene product functions as a cell surface receptor (V . Litwin, W . Jackson, and C . Grose, J . Virol . 66:3643-3651, 1992) . To evaluate the biosynthesis of the two VZV glycoproteins and further explore their relationship to one another, the two glycoprotein genes were individually cloned into a pTM1 vector under control of the T7 promoter . Transfection of the cloned gpI or gpIV construct into HeLa cells previously infected with vaccinia recombinant virus expressing bacteriophage T7 polymerase resulted in a much higher level expression of each VZV glycoprotein than previously achieved . Synthesis of both gpI and gpIV included intermediary partially glycosylated forms and mature N- and O-linked final product . Transfections in the presence of 32Pi demonstrated that the mature forms of both gpI and gpIV were phosphorylated, while similar experiments with {35S}sulfate showed that only the mature gpI was sulfated . When gpI and gpIV were coexpressed in the same cell, the two glycoproteins were complexed to each other, as both proteins could be immunoprecipitated by antibodies against either gpI or gpIV . Coprecipitation did not occur as a result of a shared epitope, because gpI expressed alone was not precipitated by antibody to gpIV, and gpIV expressed alone was not precipitated by antibody to gpI . Pulse-chase analysis demonstrated that the gpI-gpIV association occurred early in processing; furthermore, this complex formation interfered with posttranslational modifications and thereby reduced the M(r)s of the mature forms of both gpI and gpIV . Similarly, the molecular masses of the cotransfected gene products corresponded with those of the infected cell glycoproteins, a result which suggested that authentic gpI and gpIV were ordinarily found within a complex . Thus, the adjacent open reading frames 67 and 68 code for two glycoproteins which in turn form a distinctive sulfated and phosphorylated cell surface complex with receptor properties.

Yi Chuan Xue Bao, 1993, 20(3), 272 - 8
{Construct of the stable-produced strain for the thermostable alpha-amylase}; Chen Q et al.; The plasmid pE194 as a vector is used to subclone the thermostable alpha-amylase gene of B . licheniformis and construct recombinant plasmid pNW102 . The plasmid pNW102 was transduced into B . subilis BF 7658 by bacteriophage PBS1 . The B . subtilis BF7658 (pNW102) strain was treated at non-permissive temperature for a long time, and obtained many recombinant strains . This is the homologous recombination results between alpha-amylase genes of pNW102 and BF7658 . The homologous recombination occurs at any site in alpha-amylase gene but there are some heat-spots . We have already screened 2 strains from the recombination strains which can stably produce the thermostable alpha-amylase in B . subtilis BF7658 . The analysis of enzymology shows that the characters of the thermpstable alpha-amylase produced from the recombinants strains are the same with that of B . licheniformis.

J Basic Microbiol, 1993, 33(3), 213 - 5
Influence of the different amino acid substitutions in Escherichia coli thioredoxin on the growth of bacteriophages T7 and f1; Minarik P et al.; We have constructed three mutants in the thioredoxin (trxA) gene changing its catalytic core between Cys-32 and Cys-35 . Oligonucleotide-directed mutagenesis was carried out to replace conservative Gly-33 or Pro-34 by leucine, lysine, glutamine, phenylalanine or tryptophane . The mutants were characterized using an in vivo assay based on the ability of cell (mutants in the chromosomal trxA gene) to support growth of T7 and filamentous f1 phages . The results indicate that the smaller group side-chain in the position 33 and 34 of amino acid residues are indispensable for the growth of phages.

Cell Mol Biol Res, 1993, 39(4), 385 - 91
Structure and function of the bacteriophage T7 RNA polymerase (or, the virtues of simplicity); McAllister WT; A consideration of the properties of a number of mutants of T7 RNA polymerase, together with emerging structural information (Sousa et al., 1993) allows an interpretation of the the mechanics of transcription by this relatively simple RNA polymerase . Evidence indicating features in common with other nucleotide polymerases (such as DNA polymerases and reverse transcriptases) is reviewed.

Biochimie, 1993, 75(7), 517 - 21
Mapping the promoter for subgenomic RNA synthesis on beet necrotic yellow vein virus RNA 3; Balmori E et al.; During infection of Tetragonia expansa leaves, RNA 3 of the quadripartite genome of beet necrotic yellow vein virus directs synthesis of a subgenomic RNA (RNA 3sub) which corresponds to the 3'-terminal 600 residues of the RNA 3 molecule . Biologically active run-off transcripts have been prepared from full-length cDNA of RNA 3 cloned behind a bacteriophage T7-RNA polymerase promoter . RNA 3 transcripts carrying deletions in the vicinity of the RNA 3sub initiation site were produced by site-directed mutagenesis at the cDNA level and then tested for their capacity to direct RNA 3sub synthesis in infected leaves . The cis-acting domain essential for normal levels of RNA 3sub production in planta (the 'core' promoter) did not extend in the 5'-direction beyond position -16 relative to the RNA 3sub transcription initiation site . The 3'-boundary of the core promoter domain was located somewhere between positions +100 and +208 . Displacement of the promoter domain to an upstream site in RNA 3 produced a new subgenomic RNA starting at or near the predicted upstream site.

Med Dosw Mikrobiol, 1993, 45(1), 15 - 8
{Bacteriophages of serologic group A converting synthesis of staphylokinase and beta toxin in S . aureus}; Mlynarczyk A et al.; The properties of the eight S . aureus bacteriophages of the serogroup A converting staphylokinase production were investigated . Three of them were able to a double conversion: production of the staphylokinase and inhibition of beta toxin synthesis . All of the investigated bacteriophages were classified as the I morphological group of the Styloviridae on the basis of the electron microscope analysis . The size of capsids of the examined bacteriophages was 77 +/- 2.8 nm x 43.1 +/- 1.9 nm and the tail length was 272.7 +/- 12.7 nm . Most of them (6 bacteriophages) had the tail terminated in the basal plate . The lytic properties of the investigated bacteriophages were not identical . Seven of them showed features of the III and one of the V (miscellaneous) lytic group.

Arch Microbiol, 1993, 160(3), 229 - 37
Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121; Moser DP et al.; We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121 . A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extracts . Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC . Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site . These data further suggested that cleavage occurs after the first G (5'-G/GNCC-3') in this site to generate a three base 5' overhang . Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I . Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 10(3) viable cells . Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants . Conjugal transfer of pRL25 from E . coli into Nostoc was also possible but at much lower efficiency than by electroporation . These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.

Acta Biochim Pol, 1993, 40(2), 273 - 8
Expression of genes 51, 27, 28 coding for proteins of the central part of bacteriophage T4 baseplate in the bacteriophage T7 promoter/RNA polymerase expression system; Nieradko J et al.; A fragment of T4 DNA (XbaI-HindIII) comprising the genes 51, 27, 28, which encodes the central plug proteins was cloned into plasmid pT7-5 and p7-6 (T7 RNA polymerase expressing system) . The examined genes were only overexpressed when the orientation of cloned DNA to promoter phi 10 was as follows: promoter phi 10 and genes 51, 27, 28 . This was achieved when the fragment (XbaI-HindIII) was cloned into plasmid pT7-5 . Gene 27 and 28 were overexpressed when the intact fragment (XbaI-HindIII) was used . The high rate of the synthesis of proteins 27 and/or 28 had a strong inhibitory effect on the level of synthesis of the product of gene 51 . For the overexpression of gene 51 in this system a deletion derivate which was devoid of gene 28 and a larger fragment of gene 27 was prepared.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1993, 11(2), 116 - 9
{The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum}; Liu KY et al.; The DNA of Plasmodium falciparum has been purified and fragmented with restriction endonuclease BamHI . The fragments have been incorporated in vitro into derivatives of bacteriophage lambda EMBL4 digested with BamHI and Sal I . The recombinant mixture has been ligated and packaged in vitro . The recombinant phages have been identified in E . coli L95 host cell and the libraries have been established in which most of the parasite DNA is represented . The ligation proportion of vector to insert is 3:1 . The recombinant phages of 4 x 10(5) have been obtained . By plaque hybridization, we have been able to recover from these libraries specific clones containing repetitive DNA sequences.

Int J Clin Lab Res, 1993, 23(4), 192 - 8
From cells to genes: how to make antibodies useful in human diagnosis and therapy; Zaccolo M et al.; Monoclonal antibodies (mAb) have great potential value for in vivo diagnosis and therapy in humans . Their antigenic nature is however responsible for severe side effects and successful applications in the clinic have prove to be more limited than was originally hoped . This review summarize both cell biology and molecular biology approaches developed in order to overcome these limitations . Improving methodologies to immortalize cell lines producing human mAbs are reported with a particular attention to the techniques aimed at rescuing B cells expressing high-affinity human antibodies . A major part of this review is devoted to the protein engineering work . Genetic manipulation of mouse monoclonals to produce humanized antibodies and preparation of bacteriophage libraries displaying Ig repertoires are examined . The possibility to extend these approaches to the production of in vitro repertoires and to obviate the in vivo immunization step is also discussed.

Zh Mikrobiol Epidemiol Immunobiol, 1993 Jan-Feb, (1), 45 - 8
{The virological safety and bacterial sterility of a method for fractionating blood plasma proteins with rivanol}; Zhurina NA et al.; The bacterial and virological safety of the method of rivanol fractionation of blood plasma proteins has been evaluated in experiments with samples of donor blood plasma mixed with the suspension of viruses and Escherichia coli used as models . The bacteriostatic action of rivanol and the elimination of bacteriophage and influenza virus from the end product at the stages of rivanol precipitation and adsorption on carbon have been established.

Cancer Biother, 1993 Winter, 8(4), 327 - 37
Recombinant bifunctional molecule FV/IFN-gamma possesses the anti-tumor FV as well as the gamma interferon activities; Xiang J et al.; Recombinant DNA techniques were used to clone, construct and express the bifunctional molecule FV/IFN-gamma . The FV/IFN-gamma is a single-chain 42KD fusion protein expressed in E . coli under control of the strong T7 bacteriophage promoter in the expression vector pT7-7-FV-IFN-gamma . The fused gene fragment FV-IFN-gamma containing a single-chain anti-TAG72 FV gene fragment as well as the human recombinant cDNA fragment of IFN-gamma molecule . The renatured soluble form of FV/IFN-gamma was purified from E . coli inclusion bodies using HTPT chromatography . The yield of this fusion protein was estimated at 10mg/L . Our data showed that the FV/IFN-gamma molecule retained the TAG72 antigen-binding specificity and the IFN-gamma activity as measured in ELISA, Western blotting and up-regulation of CEA expression by IFN-gamma . Therefore, it may prove to be useful in targeting the biological effect of IFN-gamma to tumor cells and stimulating its immune destruction.

Planta, 1993, 190(4), 498 - 10
Cloning and developmental expression of the sucrose-phosphate-synthase gene from spinach; Klein RR et al.; A 561-base-pair (bp) polymerase-chain-reaction (PCR) product of sucrose-phosphate synthase (SPS) was amplified using degenerate oligonucleotide primers corresponding to tryptic peptides of SPS (EC 2.4.1.14) from spinach (Spinacia oleracea L) . Crucial to the primer specificity and the synthesis of the 561-bp product was the use of primer pools in which the number of degenerate primer species was limited . A full-length cDNA was subsequently obtained by screening a cDNA bacteriophage library with the 561-bp product of SPS and 5' PCR-RACE (Rapid Amplification of cDNA Ends) . The 3530-bp cDNA of SPS encoded for a 1056-amino-acid polypeptide of predicted molecular mass of 117 kDa . The deduced amino-acid sequence of spinach SPS showed regions of strong homology with SPS from maize (A.C . Worrell et al., 1991, Plant Cell 3, 1121-1130); amino-acid identity was 54% over the entire protein . Western and Northern analyses of root, petiole and spinach leaf tissue showed that SPS was expressed in an organ-specific manner, being predominantly localized in the leaf . The accumulation of SPS protein and mRNA during leaf development coincided with the early rapid phase of leaf expansion and the apparent transition of the leaf from sink to source status . Levels of SPS mRNA and protein were reduced during the acclimation of leaves to low-irradiance conditions . Transfer of low-irradiance-adapted leaves to higher-irradiance conditions resulted in a gradual increase in SPS protein and mRNA . Diurnal changes in irradiance did not alter SPS protein or transcript levels, indicating that short-term regulation of SPS primarily involves a modulation of enzyme activity.

J Cell Sci, 1993 Jan, 104 ( Pt 1), 77 - 87
Characterization of the nuclear translocation of acidic fibroblast growth factor; Cao Y et al.; The subcellular localization of human acidic FGF (aFGF; FGF-1) expressed to high levels by using a bacteriophage T7 RNA polymerase-driven vaccinia virus expression system was studied in BHK21 and HeLa cells . Acidic FGF was detected by immunoblotting or immunofluorescence using an affinity-purified rabbit polyclonal antibody . The nuclei of most transfected cells, but not nuclei of control cells, were strongly immunoreactive . The nuclear accumulation of aFGF was confirmed by subcellular fractionation and immunoblotting, indicating that about 50% of the expressed protein was located in the nuclei at 12 h after transfection . It has previously been reported that a putative N-terminal nuclear localization sequence (NLS) in aFGF is required for full mitogenic activity (Imamura et al., Science 249, 1567-1570, 1990) . We found that deletion of the first 27 residues including the putative NLS did not prevent the nuclear translocation of aFGF in either cell type . This observation suggests that the putative NLS sequence is not essential for targeting aFGF to the cell nucleus . To analyze further the mechanism of nuclear import, purified aFGF was microinjected into the cytoplasm of growing BHK21 cells under various conditions . In chilled (4 degrees C) or ATP-depleted cells, the injected aFGF entered the nucleus with similar efficiency to that in control cells at 37 degrees C . This suggests that aFGF, which has a molecular mass of only 16,500, enters the cell nucleus by free diffusion, and possibly becomes trapped by binding to some nuclear structures . When added exogenously to growing BHK21 cells, aFGF was not localized to the nucleus . Instead, a punctate staining pattern in the cytosol was observed, reminiscent of that in the endosomal-lysosomal compartments . In addition, a diffuse extracellular surface-staining was evident . This result demonstrates that receptor-mediated endocytosis of aFGF does not result in its translocation to the nucleus, as has been reported for basic FGF.

EXS, 1993, 63, 31 - 47
Heterologous expression of the membrane proteins that control cellular excitability; Karschin A; Versatile and potent expression systems are needed to decipher the structure and functions of the many excitability proteins that have been identified through molecular cloning . This article reviews the use of recombinant vaccinia viruses (VV), which have been recently explored for the heterologous expression of eukaryotic proteins . Vaccinia viruses feature a series of favourable properties, most of all a broad host range and high efficiency of infection, that make them uniquely suited as flexible expression vectors . In one type of experiment, the recombinant virus simply harbors the cDNA for the foreign protein; in a second type the virus harbors the cDNA for the specific and efficient RNA polymerase of bacteriophage T7, which in turn generates RNA from a separate introduced plasmid or virus . Both variations have been successfully applied to the expression and analysis of voltage-dependent ion channels, neurotransmitter receptors and other excitability proteins in many cell lines and postmitotic cells in culture . VV vectors promise to be particularly useful to study membrane proteins that require posttranslational processing, association with cell-specific subunits or coupling to endogenous second messengers pathways.

Biochem Biophys Res Commun, 1992 Dec 30, 189(3), 1674 - 80
Transcriptional inhibition of the bacteriophage T7 early promoter region by oligonucleotide triple helix formation; Ross C et al.; We have identified a purine-rich triplex binding sequence overlapping a -35 transcriptional early promoter region of the bacteriophage T7 . Triplex-forming oligonucleotide designed to bind this target was annealed to T7 templates and introduced into in vitro transcription systems under conditions favoring specific initiation from this promoter . These templates demonstrated significant transcriptional inhibition relative to naked genomic templates and templates mixed with non-triplex-forming oligonucleotide . It is suggested that triplex formation along this target interferes with transcriptional initiation, and this mechanism may hold potential to disrupt bacteriophage T7 early transcription in vivo.

J Biol Chem, 1992 Dec 25, 267(36), 26097 - 103
Identification of amino acid residues at the interface of a bacteriophage T4 regA protein-nucleic acid complex; Webster KR et al.; The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs . To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to {32P}p(dT)16 . The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography . Two tryptic peptides cross-linked to {32P}p(dT)16 were isolated . Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein . Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex . The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36 . The nucleic acid binding domain of regA protein was further examined by chemical cleavage of regA protein into six peptides using CNBr . Peptide CN6, which extends from residue 95 to 122, retains both the ability to be cross-linked to {32P}p(dT)16 and 70% of the nonspecific binding energy of the intact protein . However, peptide CN6 does not exhibit the binding specificity of the intact protein . Three of the other individual CNBr peptides have no measurable affinity for nucleic acid, as assayed by photo-cross-linking or gel mobility shifts.

Nucleic Acids Res, 1992 Dec 25, 20(24), 6713 - 21
Bound Lac repressor protein differentially inhibits the unwinding reactions catalyzed by DNA helicases; Yancey-Wrona JE et al.; A partial duplex DNA substrate containing the Lac repressor binding site, within the duplex region, was constructed to examine the effect of bound Lac repressor on the unwinding reaction catalyzed by several DNA helicases . The substrate contained 90 base pairs of double-stranded DNA and, in the absence of Lac repressor, was effectively unwound by each of the seven helicases tested . The unwinding reactions catalyzed by Escherichia coli Rep protein, bacteriophage T4 Dda protein and E . coli DNA helicase I were not inhibited by the presence of bound Lac repressor . Both SV40 T antigen and E . coli helicase II were partially inhibited by bound repressor at the highest repressor concentrations tested . The helicase reactions catalyzed by E . coli DnaB protein and helicase IV were substantially inhibited by the presence of bound protein . When the length of the duplex region was increased to 323 base pairs the inhibition spectrum caused by bound Lac repressor on the unwinding reactions catalyzed by DnaB protein, helicase I and helicase II was essentially the same as that observed using the shorter partial duplex molecule . Inhibition of the unwinding reaction was due to the presence of bound Lac repressor as evidenced by the substantially weaker inhibition of helicase IV by Lac repressor in the presence of IPTG . In addition, we have shown that Rep protein displaces the bound repressor protein during the course of an unwinding reaction.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 12023 - 7
Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage lambda cI repressor; Ross JB et al.; We have used a tryptophan-requiring Escherichia coli auxotroph to replace the three tryptophan residues of lambda cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp) . By using a nonleaky promoter, we have achieved > 95% replacement of tryptophan in the repressor . We show that the absorbance and fluorescence properties of 5-OHTrp-lambda cI are clearly distinct from lambda cI repressor and that the fluorescence of 5-OHTrp-lambda cI repressor can be observed selectively in the presence of exogenous tryptophan . We also show that the 5-OHTrp-lambda cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor . Based on these results, we anticipate that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 11910 - 4
Deformation of DNA during site-specific recombination of bacteriophage lambda: replacement of IHF protein by HU protein or sequence-directed bends; Goodman SD et al.; Escherichia coli IHF protein is a prominent component of bacteriophage lambda integration and excision that binds specifically to DNA . We find that the homologous protein HU, a nonspecific DNA binding protein, can substitute for IHF during excisive recombination of a plasmid containing the prophage attachment sites attL and attR but not during integrative recombination between attP and attB . We have examined whether IHF and HU function in excisive recombination is mediated through DNA bending . Our strategy has been to construct chimeric attachment sites in which IHF binding sites are replaced by an alternative source of DNA deformation . Previously, we demonstrated that properly phased bends can substitute for the binding of IHF at one site in attP . Although this result is highly suggestive of a critical role of IHF-promoted bending in lambda integration, its interpretation is obscured by the continued need for IHF binding to the remaining IHF sites of these constructs . In the present work, we engineered a population of sequence-directed bends in the vicinity of the two essential IHF sites found in attR and attL . Even in the absence of IHF or HU, pairs of these attachment sites with properly phased bends are active for both in vitro and in vivo excision . This success, although tempered by the limited efficiency of these systems, reinforces our interpretation that IHF functions primarily as an architectural element.

J Immunol, 1992 Dec 15, 149(12), 3914 - 20
Application of a filamentous phage pVIII fusion protein system suitable for efficient production, screening, and mutagenesis of F(ab) antibody fragments; Huse WD et al.; We describe the application of a novel filamentous phage vector system suitable for efficient screening and production of F(ab) antibody fragments . The vector system can concurrently produce free F(ab) fragments and F(ab) displayed on the surface of M13 bacteriophage via a VHCH1-pVIII fusion protein . When expressed in a supO (nonsuppressor) strain of Escherichia coli free F(ab) can be produced . Antibody F(ab) fragments are secreted into culture medium at concentrations up to 0.3 mg/liter and conveniently subjected to detailed analysis with little or no purification . Higher concentrations of F(ab) (approximately 10 mg/liter) were found to accumulate in the periplasmic space . In this report the vector system is shown to produce correctly folded and assembled F(ab) fragments of chimeric L6, a mAb against a tumor-associated Ag expressed by many human carcinomas.

J Biol Chem, 1992 Dec 15, 267(35), 25251 - 5
Structure of the human cellular retinoic acid-binding protein II gene . Early transcriptional regulation by retinoic acid; Astrom A et al.; The gene for human cellular retinoic acid-binding protein II (CRABP-II) has been cloned . It was isolated from a human placenta genomic library and is contained within one bacteriophage clone . The gene spans 6 kilobases and consists of 4 exons . One major transcription initiation site was mapped to an A residue 137 nucleotides upstream of the ATG initiation codon . The upstream region contains a TATA box and potential AP2, Sp1, and Krox-24 binding sites, as well as a direct repeat with homology to a retinoic acid-responsive element . The CRABP-II mRNA was rapidly induced within 2-6 h in cultured human skin fibroblasts by retinoic acid, reaching a plateau after 6 h of treatment . The rapid increase of CRABP-II message was mainly due to an increased rate of transcription as determined by nuclear run-on experiments . Increased transcription could be detected as early as 1 h after addition of retinoic acid, peaked at 2 h, and returned to basal levels within 6 h . On-going protein synthesis was required for this transient increase of transcription, since the induction was blocked by cycloheximide . These data suggest that the human CRABP-II gene is transcriptionally regulated by a newly synthesized regulatory protein.

Biochemistry, 1992 Dec 1, 31(47), 11835 - 42
Secondary structure and interactions of the packaged dsDNA genome of bacteriophage P22 investigated by Raman difference spectroscopy; Aubrey KL et al.; Vibrational spectra of the double-stranded DNA genome of bacteriophage P22 in packaged and unpackaged states are compared by digital difference Raman spectroscopy . The difference Raman spectrum, which is sensitive to structural changes at the level of < 2% of a given nucleotide type, reveals the effects of packaging upon sugar pucker, glycosyl orientation, phosphodiester geometry, base pairing, base stacking, and the electrostatic environment of DNA phosphate groups . For both packaged and unpackaged states, the experiments were performed on aqueous solutions at 25 degrees C containing effective P22 DNA concentrations of 30-50 mg/mL in 200 mM NaCl + 10 mM MgCl2 + 10 mM Tris at pH 7.5 . At the experimental conditions employed, the B-form secondary structure of unpackaged P22 DNA is minimally perturbed by packaging the viral genome in the virion capsid . However, the electrostatic environment of DNA phosphates is dramatically altered with packaging . Specifically, we find the following: (1) C2'-endo sugar pucker and anti glycosyl orientations are conserved for all nucleosides . (2) Watson-Crick base pairing is essentially completely retained . (3) Alternative secondary structures, whether right- (A or C form) or left-handed (Z form), are not evident in either the packaged or unpackaged viral genome . (4) Small Raman hyperchromic effects (< 10%) observed for certain marker bands of dG, dA, and dT in the packaged state of P22 DNA suggest slightly reduced base-stacking interactions with packaging . These are consistent with previously reported UV hyperchromic effects, but the Raman spectrum shows that they are not associated with either base unpairing or strand separation.(ABSTRACT TRUNCATED AT 250 WORDS)

J Theor Biol, 1992 Dec 7, 159(3), 287 - 98
Synonymous codon preferences in bacteriophage T4: a distinctive use of transfer RNAs from T4 and from its host Escherichia coli; Kunisawa T; Codon usage data of bacteriophage T4 genes were compiled and synonymous codon preferences were investigated in comparison with tRNA availabilities in an infected cell . Since the genome of T4 is highly AT rich and its codon usage pattern is significantly different from that of its host Escherichia coli, certain codons of T4 genes need to be translated by appropriate host transfer RNAs present in minor amounts . To avoid this predicament, T4 phage seems to direct the synthesis of its own tRNA molecules and these phage tRNAs are suggested to supplement the host tRNA population with isoacceptors that are normally present in minor amounts . A positive correlation was found in that the frequency of E . coli optimal codons in T4 genes increases as the number of protein monomers per phage particle increases . A negative correlation was also found between the number of protein monomers per phage and the frequency of "T4 optimal codons", which are defined as those codons that are efficiently recognized by T4 tRNAs . From these observations it was proposed that tRNAs from the host are predominantly used for translation of highly expressed T4 genes while tRNAs from T4 tend to be used for translation of weakly expressed T4 genes . This distinctive tRNA-usage in T4 may be an optimization of translational efficiency, and an adjustment of T4-encoded tRNAs to the synonymous codon preferences, which are largely influenced by the high genomic AT-content, would have occurred during evolution.

J Mol Biol, 1992 Dec 5, 228(3), 885 - 92
Terminating a macromolecular helix . Structural model for the minor proteins of bacteriophage M13; Makowski L; Analysis of the results of X-ray diffraction, electron microscopy and s sequence studies of filamentous bacteriophage M13 are used to construct structural models for the minor proteins gp7 and gp9 at the end of the virus assembled first, and a portion of gp6 at the end of the virus that binds host . Comparison of the sequence of the major coat protein, gp8, with those of gp7, gp9 and gp6 indicates that significant portions of these three proteins have sequences similar to that of gp8 . Assuming that sequence similarity is indicative of structural similarity, gp7, gp9 and portions of gp6 are modeled based on what is known about the structure of gp8 . These molecular models are analyzed to predict the packing of the minor proteins with the terminal gp8 proteins (the last gp8 proteins at either end of the helix) . This analysis indicates that the gp8 proteins integrated into the virus first may have a structure distinct from those in the body of the virus particle . The gp8 proteins at the end assembled last appear to have a conformation very similar to that of the integral coat proteins . These models place specific constraints on models for the process of viral assembly.

J Mol Biol, 1992 Dec 5, 228(3), 870 - 84
Conformational changes of a viral capsid protein . Thermodynamic rationale for proteolytic regulation of bacteriophage T4 capsid expansion, co-operativity, and super-stabilization by soc binding; Steven AC et al.; We have used differential scanning calorimetry in conjunction with cryo-electron microscopy to investigate the conformational transitions undergone by the maturing capsid of phage T4 . Its precursor shell is composed primarily of gp23 (521 residues): cleavage of gp23 to gp23* (residues 66 to 521) facilitates a concerted conformational change in which the particle expands substantially, and is greatly stabilized . We have now characterized the intermediate states of capsid maturation; namely, the cleaved/unexpanded, state, which denatures at tm = 60 degrees C, and the uncleaved/expanded state, for which tm = 70 degrees C . When compared with the precursor uncleaved/unexpanded state (tm = 65 degrees C), and the mature cleaved/expanded state (tm = 83 degrees C, if complete cleavage precedes expansion), it follows that expansion of the cleaved precursor (delta tm approximately +23 degrees C) is the major stabilizing event in capsid maturation . These observations also suggest an advantage conferred by capsid protein cleavage (some other phage capsids expand without cleavage): if the gp23-delta domains (residues 1 to 65) are not removed by proteolysis, they impede formation of the stablest possible bonding arrangement when expansion occurs, most likely by becoming trapped at the interface between neighboring subunits or capsomers . Icosahedral capsids denature at essentially the same temperatures as tubular polymorphic variants (polyheads) for the same state of the surface lattice . However, the thermal transitions of capsids are considerably sharper, i.e . more co-operative, than those of polyheads, which we attribute to capsids being closed, not open-ended . In both cases, binding of the accessory protein soc around the threefold sites on the outer surface of the expanded surface lattice results in a substantial further stabilization (delta tm = +5 degrees C) . The interfaces between capsomers appear to be relatively weak points that are reinforced by clamp-like binding of soc . These results imply that the "triplex" proteins of other viruses (their structural counterparts of soc) are likely also to be involved in capsid stabilization . Cryo-electron microscopy was used to make conclusive interpretations of endotherms in terms of denaturation events . These data also revealed that the cleaved/unexpanded capsid has an angular polyhedral morphology and has a pronounced relief on its outer surface . Moreover, it is 14% smaller in linear dimensions than the cleaved/expanded capsid, and its shell is commensurately thicker.

J Mol Biol, 1992 Dec 5, 228(3), 862 - 9
Selection of high affinity RNA ligands to the bacteriophage R17 coat protein; Schneider D et al.; RNA ligands with high affinity for the bacteriophage R17 coat protein were isolated from a pool of random RNA molecules using SELEX . Of the 38 ligands isolated, 36 were found to contain a hairpin very similar to the naturally occurring coat protein binding site in the R17 genome . The common features of these 36 sequences provide a consensus binding site and predict components of a hairpin that promote favorable interaction with the coat protein . These include a tetraloop of primary sequence AUCA and a variable-length stem with a bulged adenosine residue at a specific stem position . The predicted consensus agrees well with the highest-affinity RNA binding site of the R17 coat protein, identified through classical but laborious techniques . These results demonstrate the value of SELEX as a tool for isolating high affinity RNA ligands to a specific target protein, and the further value of those ligands to point the researcher toward natural sequences for that target protein.

J Mol Biol, 1992 Dec 5, 228(3), 720 - 4
Construction of a microphage variant of filamentous bacteriophage; Specthrie L et al.; The intergenic region in the genome of the Ff class of filamentous phage (comprising strains fl, fd and M13) genome constitutes 8% of the viral genome, and has essential functions in DNA replication and phage morphogenesis . The functional domains of this region may be inserted into separate sites of a plasmid to function independently . Here, we demonstrate the construction of a plasmid containing, sequentially, the origin of (+)-strand synthesis, the packaging signal and a terminator of (+)-strand synthesis . When host cells harboring this plasmid (pLS7) are infected with helper phage they produce a microphage particle containing all the structural elements of the mature, native phage . The microphage is 65 A in diameter and about 500 A long . It contains a 221-base single-stranded circle of DNA coated by about 95 copies of the major coat protein (gene 8 protein).

Microbiol Rev, 1992 Dec, 56(4), 577 - 91
Bacteriophage lambda as a cloning vector; Chauthaiwale VM et al.; Extensive research has been directed toward the development of multipurpose lambda vectors for cloning ever since the potential of using coliphage lambda as a cloning vector was recognized in the late 1970s . An understanding of the intrinsic molecular organization and of the genetic events which determine lysis or lysogeny in lambda has allowed investigators to modify it to suit the specific requirements of gene manipulations . Unwanted restriction sites have been altered and arranged together into suitable polylinkers . The development of a highly efficient in vitro packaging system has permitted the introduction of chimeric molecules into hosts . Biological containment of recombinants has been achieved by introducing amber mutations into the lambda genome and by using specific amber suppressor hosts . Taking advantage of the limited range of genome size (78 to 105% of the wild-type size) for its efficient packaging, an array of vectors has been devised to accommodate inserts of a wide size range, the limit being 24 kbp in Charon 40 . The central dispensable fragment of the lambda genome can be replaced by a fragment of heterologous DNA, leading to the construction of replacement vectors such as Charon and EMBL . Alternatively, small DNA fragments can be inserted without removing the dispensable region of the lambda genome, as in lambda gt10 and lambda gt11 vectors . In addition, the introduction of many other desirable properties, such as NotI and SfiI sites in polylinkers (e.g., lambda gt22), T7 and T3 promoters for the in vitro transcription (e.g., lambda DASH), and the mechanism for in vivo excision of the intact insert (e.g., lambda ZAP), has facilitated both cloning and subsequent analysis . In most cases, the recombinants can be differentiated from the parental phages by their altered phenotype . Libraries constructed in lambda vectors are screened easily with antibody or nucleic acid probes since several thousand clones can be plated on a single petri dish . Besides the availability of a wide range of lambda vectors, many related techniques such as rapid isolation of lambda DNA, a high efficiency of commercially available in vitro packaging extracts, and in vitro amplification of DNA via the polymerase chain reaction have collectively contributed to lambda's becoming one of the most powerful and popular tools for molecular cloning.

Cell Mol Biol (Noisy-le-grand), 1992 Dec, 38(8), 885 - 93
{protein synthesis in alkylated bacteriophage T7}; Racine JF et al.; We have studied the toxic effects of alkylating agents with a well characterized model: phage T7 . Treatment of bacteriophage T7 with methyl methanesulfonate led to perturbation of phage-specific protein synthesis . Synthesis of class I and II proteins was prolonged, while production of class II and III proteins was delayed . This delay increased for proteins coded by genes located further to the right on the T7 genetic map . In extracts prepared from cells infected by alkylated phage, the specific activity of T7 RNA polymerase was decreased . These results suggest that the toxic action of methyl methanesulfonate is directed towards viral transcription.

Curr Genet, 1992 Dec, 22(6), 491 - 500
The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase; Hermanns J et al.; The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid . pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp . Exonuclease digestion experiments indicated that proteins are covalently bound at the 5' termini of the plasmid . Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively . The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3'-5' exonuclease activity and the typical consensus sequences for DNA polymerases of the D type . In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein . According to these properties, this first linear plasmid identified in P . anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.

Comput Appl Biosci, 1992 Dec, 8(6), 563 - 7
GeneScape: a relational database of Escherichia coli genomic map data for Macintosh computers; Bouffard G et al.; We present a relational database program developed in FoxBase+/Mac for the viewing and manipulation of ordered restriction maps and associated features of the Escherichia coli genome including sequenced genes and the Kohara miniset of bacteriophage lambda clones . Use of this program allows easy access to the wealth of information being collected in a dataset of DNA sequences, maps and genetic data known as EcoSeq, EcoMap and EcoGene respectively.

J Bacteriol, 1992 Dec, 174(24), 8144 - 7
The carboxy-terminal 14 amino acids of phage lambda N protein are dispensable for transcription antitermination; Franklin NC; The analogous N proteins encoded by lambdoid bacteriophages lambda, 21, and 22 are very different in amino acid sequence, except at their carboxy-terminal ends . Since N lambda remains functional despite the deletion of most of its terminal region of homology to N21, that region of homology cannot represent a region of conserved function.

J Bacteriol, 1992 Dec, 174(24), 8073 - 80
Lysis of lysis-inhibited bacteriophage T4-infected cells; Abedon ST; T4 bacteriophage (phage)-infected cells show a marked increase in latent-period length, called lysis inhibition, upon adsorption of additional T4 phages (secondary adsorption) . Lysis inhibition is a complex phenotype requiring the activity of at least six T4 genes . Two basic mysteries surround our understanding of the expression of lysis inhibition: (i) the mechanism of initiation (i.e., how secondary adsorption leads to the expression of lysis inhibition) and (ii) the mechanism of lysis (i.e., how this signal not to lyse is reversed) . This study first covers the basic biology of the expression of lysis inhibition and lysis of T4-infected cells at high culture densities . Then evidence is presented which implies that, as with the initiation of lysis inhibition, sudden, lysis-associated clearing of these cultures is likely caused by T4 secondary adsorption . For example, such clearing is often observed for lysis-inhibited T4-infected cells grown in batch culture during T4 stock preparation . The significance of this secondary adsorption-induced lysis to wild T4 populations is discussed . The study concludes with a logical argument suggesting that the lytic nature of the T4 phage particle evolved as a novel mechanism of phage-induced lysis.

Genes Dev, 1992 Dec, 6(12A), 2409 - 16
Transcriptional control via translational repression by c4 antisense RNA of bacteriophages P1 and P7; Biere AL et al.; The c4 repressors of bacteriophages P1 and P7 are antisense RNAs that inhibit antirepressor synthesis . This antisense inhibition is unusual in that the c4 repressor and the repressed genes orfx and ant are cotranscribed in that order from the same promoter, and c4 RNA is processed from a precursor RNA . Here, we show that c4 RNA directly represses translation of orfx, a small open reading frame, to which ant is translationally coupled . This translational repression blocks ant transcription via a rho-dependent terminator . Thus, c4 RNA controls expression of the ant gene by a novel indirect mechanism combining translational repression, translational coupling, and rho-dependent termination.

Gene, 1992 Dec 1, 122(1), 77 - 84
Transcription map of the early region of the Streptomyces bacteriophage phi C31; Ingham CJ et al.; Streptomyces coelicolor A3(2), lysogenised by the temperature-sensitive cts1 mutant of phi C31, can be synchronously induced into the lytic cycle by heat treatment . A transcription map of 10 kb of the phi C31 early gene cluster was deduced using low-resolution S1 nuclease mapping of RNA prepared 10 min after induction . At least nine early transcripts, early (e)RNAs 1-9, were localised reading exclusively rightwards with respect to the standard physical map of phi C31 . The mRNAs were extensively overlapping, frequently initiating at the same place but terminating at different sites, and vice versa . Gene expression during the lytic cycle was tightly regulated; no transcription was observed before induction . Transcription was maximal at 10 min post-induction, and at 20 min, eRNAs 5 and 6 persisted whilst eRNAs 7-9 were severely reduced or absent . The pattern of transcription of the early region is consistent with the simultaneous activation of a large number of promoters and differential termination efficiency.

Gene, 1992 Dec 1, 122(1), 1 - 7
Involvement of the Escherichia coli RNA polymerase alpha subunit in transcriptional activation by the bacteriophage lambda CI and CII proteins; Wegrzyn G et al.; Escherichia coli cells harbouring the rpoA341 mutation produce an RNA polymerase which transcribes inefficiently certain operons subject to positive control . Here, we demonstrate that the rpoA341 allele also prevents lysogenization of the host strain by bacteriophage lambda, a process dependent upon the action of two phage-encoded activators . This phenomenon was shown to arise from an inability to establish an integrated prophage rather than a failure to maintain the lysogenic state . The inability of the rpoA341 host to support lysogenization could be completely reversed by CII-independent expression of int and cI in trans . These results led us to propose that the inhibition of lysogenization arises from a defective interaction between the phage lambda transcriptional activator CII and the mutant RNA polymerase at the phage promoters pI and pE . Finally, we also provide genetic evidence for impaired transcription of the cI gene from the CI-activated promoter, pM in the rpoA341 background.

Virology, 1992 Dec, 191(2), 607 - 18
Cloned DNA copies of cowpea severe mosaic virus genomic RNAs: infectious transcripts and complete nucleotide sequence of RNA 1; Chen X et al.; Cowpea severe mosaic virus (CPSMV) is a member of the comovirus group of messenger-sense RNA viruses with bipartite genomes, of which cowpea mosaic virus (CPMV) is the type member . Full-length copies of CPSMV RNA 1 were cloned in plasmids bearing a bacteriophage T7 promoter . Previously, similar clones of CPSMV RNA 2 had been obtained . A 5'-rUAUUAAAAUUUU sequence is common to RNA 1 and RNA 2 . From two RNA 1 clones and four RNA 2 clones we excised non-CPSMV sequences so as to provide templates for in vitro transcripts that have only a single guanylate preceding CPSMV RNA sequences . Transcripts from the most active RNA 1 and RNA 2 clones, when mixed, showed about 5% of the infectivity of unfractionated CPSMV RNAs from virions . The longest, 1858 codon open reading frame of the 5957 nt CPSMV RNA 1 extends from an AUG at nt 257 to a UGA termination codon at nt 5831 . The calculated molecular weight of the polyprotein is 208,000 . Comparisons with the available amino acid residue (aa) sequence information from the complete CPMV RNA 1 sequence and the partial sequence of red clover mottle virus RNA 1 suggest that CPSMV RNA 1 specifies the expected set of five mature proteins: 32K proteinase cofactor, 58K presumed helicase, VPg 5'-linked protein of the genomic RNAs, 24K proteinase, and 87K presumed polymerase, separated by four cleavage sites . Of the determined and deduced cleavage sites of the three RNA 1 polyproteins, only that at the 24K/87K junction has a distinct aa pair in the CPSMV polyprotein . Of the five proteins, VPg and 87K show the greatest similarity between CPSMV and CPMV, with identities of 68 and 55%, respectively . Published mutational analysis of the CPMV 24K proteinase and alignment of aa sequences from three comoviruses suggest that cysteine-168, histidine-40 and glutamic acid-77 form the catalytic triad of the CPSMV 24K proteinase . Results are discussed in the context of the resistance that some cowpea (Vigna unguiculata) lines exhibit against CPMV but not against CPSMV.

J Bacteriol, 1992 Dec, 174(23), 7757 - 61
Differential activities of bacteriophage depolymerase on bacterial polysaccharide: binding is essential but degradation is inhibitory in phage infection of K1-defective Escherichia coli; Pelkonen S et al.; Host range mutants were derived from bacteriophages PK1A and PK1E specific for the K1 polysialic acid capsule of Escherichia coli . The mutants were selected for their ability to infect E . coli bacteria with a low level of the K1 capsule . A specific loss of the cleaving activity of the phage endosialidase was observed in all the mutants, while the ability to bind specifically to the polysialic acid capsule was retained . The results indicate that the polysaccharide-binding activity of the bacteriophage enzyme is essential for the infection process . The cleaving activity, in contrast, is required for the penetration of the dense polysaccharide of wild-type bacteria but is inhibitory in the infection of bacteria with a sparse capsular polysaccharide.

J Virol, 1992 Dec, 66(12), 7232 - 8
Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins through a heterologous RNA binding site; McDonald D et al.; The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins induce cytoplasmic expression of incompletely spliced viral mRNAs by binding to these mRNAs in the nucleus . Each protein binds a specific cis-acting element in its target RNAs . Both proteins also associated with nucleoli, but the significance of this association is uncertain because mutations that inactivate nucleolar localization signals in Rev or Rex also prevent RNA binding . Here we demonstrate that Rev and Rex can function when tethered to a heterologous RNA binding site by a bacteriophage protein . Under these conditions, cytoplasmic accumulation of unspliced RNA occurs without the viral response elements, mutations in the RNA binding domain of Rev do not inhibit function, and nucleolar localization can be shown to be unnecessary for the biological response.

J Virol, 1992 Dec, 66(12), 6960 - 8
Plasmid models for bacteriophage T4 DNA replication: requirements for fork proteins; Benson KH et al.; Bacteriophage T4 DNA replication initiates from origins at early times of infection and from recombinational intermediates as the infection progresses . Plasmids containing cloned T4 origins replicate during T4 infection, providing a model system for studying origin-dependent replication . In addition, recombination-dependent replication can be analyzed by using cloned nonorigin fragments of T4 DNA, which direct plasmid replication that requires phage-encoded recombination proteins . We have tested in vivo requirements for both plasmid replication model systems by infecting plasmid-containing cells with mutant phage . Replication of origin and nonorigin plasmids strictly required components of the T4 DNA polymerase holoenzyme complex . Recombination-dependent plasmid replication also strictly required the T4 single-stranded DNA-binding protein (gene product 32 {gp32}), and replication of origin-containing plasmids was greatly reduced by 32 amber mutations . gp32 is therefore important in both modes of replication . An amber mutation in gene 41, which encodes the replicative helicase of T4, reduced but did not eliminate both recombination- and origin-dependent plasmid replication . Therefore, gp41 may normally be utilized for replication of both plasmids but is apparently not required for either . An amber mutation in gene 61, which encodes the T4 RNA primase, did not eliminate either recombination- or origin-dependent plasmid replication . However, plasmid replication was severely delayed by the 61 amber mutation, suggesting that the protein may normally play an important, though nonessential, role in replication . We deleted gene 61 from the T4 genome to test whether the observed replication was due to residual gp61 in the amber mutant infection . The replication phenotype of the deletion mutant was identical to that of the amber mutant . Therefore, gp61 is not required for in vivo T4 replication . Furthermore, the deletion mutant is viable, demonstrating that the gp61 primase is not an essential T4 protein.

BMJ, 1992 Nov 28, 305(6865), 1348 - 52
Adapting antibodies for clinical use; Hawkins RE et al.; Techniques for antibody engineering are now overcoming the problems that have prevented monoclonal antibodies being used routinely in clinical practice . With chemical and genetic manipulation antibodies can be linked to bacterial toxins, enzymes, radionuclides, or cytotoxic drugs, allowing targeting of treatment . Antigen binding sites from antibodies raised in mice can be jointed with human IgG to reduce immunogenicity . In vitro gene amplification and genetic engineering of bacteriophage have produced large antibody gene libraries and facilitated large scale production of human monoclonal antibodies with high specificity . The trickle of monoclonal antibodies into clinical practice may soon become a flood.

Nucleic Acids Res, 1992 Nov 25, 20(22), 6081 - 90
Inactive O6-methylguanine-DNA methyltransferase in human cells; Zhukovskaya N et al.; A plasmid encoding a recombinant human O6-methylguanine-DNA methyltransferase (MGMT) fused to a fragment of the bacteriophage lambda N protein has been constructed . The fusion protein retained methyltransferase activity when expressed at high levels in E.coli and was purified to essential homogeneity by a simple procedure . Antisera raised against the purified fusion protein recognized MGMT in western blots of extracts of human cells . For most cell lines, there was a quantitative relation between the amount of immunologically detectable MGMT protein and enzyme activity . However, four cell lines contained detectable MGMT protein despite having no measurable methyltransferase activity . Additionally, a HeLa line contained considerably more immunoreactive MGMT protein than could be accounted for by its methyltransferase activity . Thus, some cells contain significant amounts of inactive MGMT . Preliminary characterization of the inactive protein in HeLaS3 cells indicated that it has some properties in common with MGMT methylated at the active cysteine residue.

Nucleic Acids Res, 1992 Nov 25, 20(22), 6023 - 32
Genetic effects of oxidative DNA damage: comparative mutagenesis of 7,8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine in Escherichia coli; Wood ML et al.; A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZ alpha gene of bacteriophage M13 DNA induces a targeted G-->T transversion after replication in Escherichia coli (Biochemistry, 29, 7024-7031 (1990)) . This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO.base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond . A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs . Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation . This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system . A deoxypentanucleotide containing A8-OXO {d(GCT-A8-OXOG)} was synthesized . After 5'-phosphorylation with {gamma-32P} ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site . Genomes containing either A8-OXO (at position 6275, {+} strand) or G8-OXO (position 6276) were denatured with heat and introduced into E.coli DL7 cells . Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-->T transversions at an apparent frequency of approximately 0.3% . The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistiguishable from those generated from transfection of an adenine-containing control genome . We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.

Lancet, 1992 Nov 21, 340(8830), 1252 - 4
Cross-contamination potential with dental equipment; Lewis DL et al.; Some types of reused dental equipment, especially handpieces and their attachments for drilling and cleaning teeth, might be responsible for cross-contamination if patient material were to lodge temporarily in difficult-to-disinfect internal mechanisms . This possibility is worrisome with respect to transmission of hepatitis B and human immunodeficiency viruses (HBV, HIV) . Previous cross-contamination studies have relied on laboratory experiments with bacteria or dye tracers . To assess possible risk more thoroughly, we tested 30 new prophylaxis angles and 12 new high-speed handpieces to see whether they would take up and expel contaminants in laboratory and clinical trials . In treatments of three patients, including two infected with HIV, human-specific DNA (beta-globin, HLA DQ alpha) and HIV proviral DNA were detected inside or coming back from the devices . Similarly, when handpieces were operated in contact with blood pooled from HBV-infected patients, HBV DNA was detected in samples taken from inside the equipment and from their attached air/water hoses . When we used bacteriophage phi X174 as a model virus in laboratory tests, many infective viral particles were recovered from internal mechanisms of handpieces, their connecting air/water hoses, and from water spray expelled when the equipment was reused . We recommend that reused high-speed, air-driven handpieces and prophylaxis angles should be cleaned and heat-treated between patients . Further studies are needed to determine ways of eliminating the risks associated with exhaust hoses and air/water input lines.

J Mol Biol, 1992 Nov 20, 228(2), 596 - 618
Structure of oxidized bacteriophage T4 glutaredoxin (thioredoxin) . Refinement of native and mutant proteins; Eklund H et al.; The structure of wild-type bacteriophage T4 glutaredoxin (earlier called thioredoxin) in its oxidized form has been refined in a monoclinic crystal form at 2.0 A resolution to a crystallographic R-factor of 0.209 . A mutant T4 glutaredoxin gives orthorhombic crystals of better quality . The structure of this mutant has been solved by molecular replacement methods and refined at 1.45 A to an R-value of 0.175 . In this mutant glutaredoxin, the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, to mimic that of Escherichia coli thioredoxin . The main-chain conformation of the wild-type protein is similar in the two independently determined molecules in the asymmetric unit of the monoclinic crystals . On the other hand, side-chain conformations differ considerably between the two molecules due to heterologous packing interactions in the crystals . The structure of the mutant protein is very similar to the wild-type protein, except at mutated positions and at parts involved in crystal contacts . The active site disulfide bridge between Cys14 and Cys17 is located at the first turn of helix alpha 1 . The torsion angles of these residues are similar to those of Escherichia coli thioredoxin . The torsion angle around the S-S bond is smaller than that normally observed for disulfides: 58 degrees, 67 degrees and 67 degrees for wild-type glutaredoxin molecule A and B and mutant glutaredoxin, respectively . Each sulfur atom of the disulfide cysteines in T4 glutaredoxin forms a hydrogen bond to one main-chain nitrogen atom . The active site is shielded from solvent on one side by the beta-carbon atoms of the cysteine residues plus side-chains of residues 7, 9, 21 and 33 . From the opposite side, there is a cleft where the sulfur atom of Cys14 is accessible and can be attacked by a nucleophilic thiolate ion in the initial step of the reduction reaction.

J Mol Biol, 1992 Nov 20, 228(2), 529 - 38
Is the bacteriophage lambda lysozyme an evolutionary link or a hybrid between the C and V-type lysozymes? Homology analysis and detection of the catalytic amino acid residues; Jespers L et al.; The relationship between the bacteriophage lambda lysozyme (lambda L) and the C and V-type lysozymes has been investigated by sequence alignment, secondary structure prediction and pattern recognition methods . The alignment of the amino terminal part of lambda L with that of V-type lysozymes suggests that Glu19 is a residue essential for catalysis . Its mutation to Gln leads to a completely inactive enzyme . In the alignment of the sequence of lambda L with those of the C-type lysozymes a strongly homologous fragment of about 30 amino acid residues is detected . Taking into consideration this observation and the published structural alignments between C and V-type lysozymes, a repetition of the beta-sheet motif in lambda L is proposed . The multiple alignment draws the attention to a possible catalytic role for Asp34 that would be positioned in the middle of the second strand of the beta-sheet as in the C-type lysozymes . This role is confirmed by mutagenesis . The implications of these observations in terms of the evolutionary relationship between lambda L and the other lysozymes is discussed.

J Mol Biol, 1992 Nov 20, 228(2), 506 - 15
Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity; Raskin CA et al.; The bacteriophage T3 and T7 RNA polymerases (RNAP) are closely related, yet exhibit high specificity for their own promoter sequences . In this work the primary determinant of T7 versus T3 promoter specificity has been localized to a single amino acid residue at position 748 in the T7 RNAP . Substitution of this residue (Asn) with the corresponding residue found in T3 RNAP (Asp) results in a switch in promoter specificity, and specifically alters recognition of the base pairs (bp) at positions -11 and, possibly, -10 in the promoter . A complementary mutation in T3 RNAP (T3-D749N) results in a similar switch in promoter preference for that enzyme . The hierarchy of bp preference by the mutant and wild-type enzymes for bp at -10 and -11, and the results of previous experiments, lead to a model for specificity in which it is proposed that N748 in T7 RNAP (and D749 in T3 RNAP) make specific hydrogen bonds with bases at -11 and -10 on the non-template strand in the major groove . The specificity determining region of T7 RNAP does not appear to exhibit homology to any known sequence-dependent DNA binding motif.

J Mol Biol, 1992 Nov 20, 228(2), 488 - 505
Characterization of bacteriophage T7 RNA polymerase by linker insertion mutagenesis; Gross L et al.; Thirty-four mutants of phage T7 RNA polymerase (RNAP) were generated by linker-insertion mutagenesis and characterized with respect to their ability to carry out various steps in the transcription cycle . A number of mutants with interesting biochemical properties were identified . These include: (1) Mutant RNAPs that are catalytically active but that bind weakly to a T7 promoter; one of these mutants is affected in a region of the RNAP that exhibits homology with the sigma subunit of Escherichia coli RNAP . Another is affected in a region that has been previously implicated in the discrimination of T7 versus T3 promoters (Joho, et al., 1990) . (2) Mutant RNAPs that can bind to the promoter but are transcriptionally inactive; some of these RNAPs lack catalytic activity, others are catalytically active but are unable to initiate productive transcription at a T7 promoter . Among the latter class of mutants are enzymes that appear to be weakened in their ability to melt open (or to remain associated with) double-stranded DNA; these RNAPs make only abortive initiation products and are unable to proceed to the formation of a productive elongation complex . The mutations causing this phenotype affect regions of the RNAP that exhibit homology with the catalytic site of DNA polymerase I (Delarue et al., 1990) . (3) A C-terminal insertion mutant with properties similar to a previously characterized "foot" mutant (Mookhtiar et al., 1991) . This RNAP appears to be defective in the very early steps of transcription and may be unable to translocate and/or empty the active site . (4) A mutant that is transcriptionally active, but is unable to complement the growth of T7 gene 1- phage . This phenotype may result from disruption of a function of the RNAP that is distinct from its role in RNA synthesis.

Biochemistry, 1992 Nov 17, 31(45), 11203 - 9
The hydrophobic core of Escherichia coli thioredoxin shows a high tolerance to nonconservative single amino acid substitutions; Hellinga HW et al.; A set of single amino acid substitutions has been constructed at positions Leu42 and Leu78 in the hydrophobic core of Escherichia coli thioredoxin . This protein is required for the in vivo assembly of filamentous bacteriophages such as M13 . Almost all the mutants retain this activity regardless of the change in size, hydrophobic nature, or charge of the substitution . Determination of the free energies of unfolding of the mutants containing charged residues shows that these are significantly destabilized as would be expected from simple considerations of the hydrophobic effect . Thioredoxin therefore represents a class of proteins where the often observed correlation between a particular biological activity and thermodynamic stability is not evident for single mutants in the all-or-none assay used . Native thioredoxin is very stable . Thus, structurally single mutants may not perturb the folding equilibrium or the dynamic behavior sufficiently for the effects to be sensed in vivo.

Biochemistry, 1992 Nov 17, 31(45), 10984 - 94
Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4; Capson TL et al.; The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA . As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation . A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase . The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 microM) followed by rapid conversion (400 s-1) of the T4.13/20-mer.dATP complex to the T4.14/20-mer.PPi product species . A slow step (2 s-1) following PPi release limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer-->17/20-mer) which occur at rates > 400 s-1 . Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors > 10(3) against misincorporation in the nucleotide binding step . The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4.DNA-->T4.(DNA)*, whose rate constants reflect whether the 3'-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s-1, and for mismatched 13T/20-mer, 5 s-1 . Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100s-1) . In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites . Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s-1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s-1/(8 s-1+5 s-1)), ca . 40% . Consequently, a major role for the accessory proteins may be to slow the rate of enzyme.substrate dissociation, thereby increasing overall fidelity and processivity.

Proc Natl Acad Sci U S A, 1992 Nov 15, 89(22), 10638 - 42
Roles of bacteriophage T7 gene 4 proteins in providing primase and helicase functions in vivo; Mendelman LV et al.; The helicase and primase activities of bacteriophage T7 are distributed between the 56- and 63-kDa gene 4 proteins . The 56-kDa gene 4 protein lacks 63 amino acids found at the N terminus of the colinear 63-kDa protein and catalyzes helicase activity . The 63-kDa gene 4 protein catalyzes both primase and helicase activities . A bacteriophage deleted for gene 4, T7 delta 4-1, has been tested for growth by complementation on Escherichia coli strains that contain plasmids expressing either one or both of the gene 4 proteins . T7 delta 4-1 cannot grow (efficiency of plating, 10(-7)) on E . coli cells that express only 56-kDa gene 4 protein . In contrast, T7 delta 4-1 has an efficiency of plating of 0.1 on an E . coli strain that expresses only 63-kDa gene 4 protein in which glycine is substituted for methionine at position 64 . A bacteriophage, T7 4B-, in which methionine at residue 64 is replaced by glycine, expresses only 63-kDa gene 4 protein . The burst sizes, latency periods, and Okazaki fragment sizes of T7 4B- are similar in the presence and absence of the 56-kDa gene 4 protein; however, T7 4B- has a reduced rate of DNA synthesis when compared with a phage that synthesizes both gene 4 proteins.

Science, 1992 Nov 13, 258(5085), 1145 - 8
Bacteriophage lambda PaPa: not the mother of all lambda phages; Hendrix RW et al.; The common laboratory strain of bacteriophage lambda--lambda wild type or lambda PaPa--carries a frameshift mutation relative to Ur-lambda, the original isolate . The Ur-lambda virions have thin, jointed tail fibers that are absent from lambda wild type . Two novel proteins of Ur-lambda constitute the fibers: the product of stf, the gene that is disrupted in lambda wild type by the frameshift mutation, and the product of gene tfa, a protein that is implicated in facilitating tail fiber assembly . Relative to lambda wild type, Ur-lambda has expanded receptor specificity and adsorbs to Escherichia coli cells more rapidly.

Nucleic Acids Res, 1992 Nov 11, 20(21), 5555 - 64
Bacteriophage and spliceosomal proteins function as position-dependent cis/trans repressors of mRNA translation in vitro; Stripecke R et al.; The translational regulation of ferritin expression currently represents the only well characterized example for eukaryotic translational control by high affinity interactions between a specific cytoplasmic protein, iron regulatory factor {IRF}, and an mRNA-binding site, the iron-responsive element {IRE}, located in the 5' untranslated region {UTR} of ferritin mRNAs . To elucidate whether IRE/IRF may represent the first physiological example of a more general mechanism for mRNA-specific translational control, high affinity RNA-binding sites for the bacteriophage MS2 coat protein or the spliceosomal protein U1A were introduced into the 5' UTR of capped chloramphenicol acetyltransferase {CAT} transcripts . In the absence of these RNA-binding proteins, CAT mRNA was efficiently translated . Addition of purified MS2 coat protein or U1A caused a specific, dose-dependent repression of CAT biosynthesis in rabbit reticulocyte and wheat germ in vitro translation systems . The translational blockage imposed by the RNA/protein complex was reversible and did not alter the stability of the repressed mRNAs . Translational repression caused by binding of U1A or MS2 proteins to their target mRNAs is shown to be position-dependent in vitro . Thus, mRNA/protein complexes without an a priori role in eukaryotic mRNA translation function as translational effectors with characteristics resembling those of IRE/IRF.

J Mol Biol, 1992 Nov 5, 228(1), 88 - 100
Role of MotA transcription factor in bacteriophage T4 DNA replication; Benson KH et al.; At least two bacteriophage T4 replication origins, ori(uvsY) and ori(34), contain a T4 middle-mode promoter that is necessary for origin function . We wanted to analyze the requirement of these two replication origins for the MotA protein, which is the phage-encoded activator of middle-mode promoters . To ensure the complete absence of MotA protein, we deleted the motA gene from the T4 genome . Unexpectedly, the deletion mutant was not viable unless the MotA protein was provided from a recombinant plasmid . Therefore, MotA is an essential protein for T4 growth . The motA delta mutation reduced the synthesis of several proteins that are encoded by genes with middle-mode promoters, delayed and reduced the synthesis of late proteins, and substantially reduced phage genomic replication . The motA delta mutation also reduced the replication of an ori(uvsY)-containing plasmid and virtually abolished replication of an ori(34)-containing plasmid . The replication defects of the two origins correlated with transcriptional defects: the motA delta mutation modestly reduced transcription from the plasmid-borne ori(uvsY) promoter and strongly reduced transcription from the ori(34) promoter . These results provide strong evidence that MotA protein is normally involved in origin-dependent replication . However, MotA is not required for origin-directed replication as long as transcription can occur from the origin promoter.

J Mol Biol, 1992 Nov 5, 228(1), 72 - 87
Genetic analysis of mutations affecting terminase, the bacteriophage lambda DNA packaging enzyme, that suppress mutations in cosB, the terminase binding site; Cue D et al.; Terminase, the DNA packaging enzyme of phage lambda, binds to lambda DNA at a site called cosB, and introduces staggered nicks at an adjacent site, cosN, to generate the cohesive ends of virion lambda DNA molecules . Terminase also is involved in separation of the cohesive ends and in binding the prohead, the empty protein shell into which lambda DNA is packaged . Terminase is a DNA-dependent ATPase, and both subunits, gpNu1 and gpA, have ATPase activity . cosB contains a series of gpNu1 binding sites, R3, R2 and R1; between R3 and R2 is a binding site, I1, for integration host factor (IHF), the Escherichia coli DNA bending protein . In this work, a series of mutations in Nu1 have been isolated as suppressors of cosB mutations . One of the Nu1 mutations is identical to the previously described Nu1ms1/ohm1 mutation predicted to cause the change L40F in the 181 amino acid-long gpNu1 . Three other Nu1 missense mutations, the Nu1ms2 (L40I), ms3 (Q97K) and ms4 (A92G) mutations, have been isolated; the relative strengths of suppression of cosB mutations by the Nu1ms mutations are: ms1 > ms2 > ms3 > ms4 . The Nu1 missense mutations all affect amino acid residues that lie outside of the putative helix-turn-helix DNA binding motif of gpNu1 . The Nu1ms1 and Nu1ms2 mutations alter an amino acid residue (L40) that lies directly between two segments of gpNu1 proposed to be involved in ATP binding and hydrolysis; thus these mutations are likely to alter the gpNu1 ATP-binding site . The Nu1ms3 and Nu1ms4 mutations both affect amino acid residues in the central region of gpNu1 that is predicted to form a hydrophilic alpha-helix . To explain how the Nu1ms mutations suppress cosB defects, models involving alterations of the DNA binding and/or catalytic properties of terminase are considered . The results also indicate that terminase occupancy of a single gpNu1 binding site (R3) is necessary and sufficient for the efficient initiation of DNA packaging; the Nu1ms1, ms2 and ms3 mutations permit IHF-independent plaque formation by a phage lacking R2 and R1.

J Mol Biol, 1992 Nov 5, 228(1), 58 - 71
Genetic analysis of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda; Cue D et al.; cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, consists of three binding sites (called R3, R2 and R1) for gpNu1, the small subunit of terminase; and I1, a binding site for integration host factor (IHF), the DNA bending protein of Escherichia coli . cosB is located between cosN, the site where terminase introduces staggered nicks to generate cohesive ends, and the Nu1 gene; the order of sites is: cosN-R3-I1-R2-R1-Nu1 . A series of lambda mutants have been constructed that have single base-pair C-to-T transition mutations in R3, R2 and R1 . A single base-pair transition mutation within any one of the gpNul binding sites renders lambda dependent upon IHF for plaque formation . lambda phage with mutations in both R2 and R3 are incapable of plaque formation even in the presence of IHF . Phages that carry DNA insertions between R1 and R2, from 7 to 20 base-pairs long, are also IHF-dependent, demonstrating the requirement for a precise spacing of gpNu1 binding sites within cosB . The IHF-dependent phenotype of a lambda mutant carrying a deletion of the R1 sequence indicates that IHF obviates the need for terminase binding to the R1 site . In contrast, a lambda mutant deleted for R2 and R1 fails to form plaques on either IHF+ or IHF- cells, indicating terminase binding of R2 is involved in suppression of R mutants by IHF . A fourth R sequence, R4, is situated on the left side of cosN; a phage with a mutant R4 sequence shows a reduced burst size on both an IHF+ and an IHF- host . The inability of the R4- mutant to be suppressed by IHF, plus the fact that R4 does not bind gpNu1, suggests R4 is not part of cosB and may play a role in DNA packaging that is distinct from that of cosB.

J Mol Biol, 1992 Nov 5, 228(1), 101 - 7
Mutational analysis of the bacteriophage P1 late promoter sequence Ps; Lehnherr H et al.; The bacteriophage P1 late promoter sequence Ps controls the expression of the genes in the tail-fibre operon . Transcription from Ps only occurs during the second half of the P1 vegetative growth cycle and is positively regulated by the product of the phage gene 10 . In this study degenerate oligonucleotides were used as primers in site-directed mutagenesis reactions in order to construct a large set of point mutations within the late promoter sequence Ps . A total of 35 independent single point mutations was isolated and the mutants were tested for promoter activity . Mutations in the Escherichia coli-like -10 region and in a late operator sequence, containing a symmetric sequence centred around position -22, resulted in significant reductions in promoter strength . Most of these mutations alter base-pairs that are highly conserved among the known late promoter sequences of the P1 family . In addition, insertion mutants that change the spacing between the -10 and the late operator indicate that a special topological arrangement between the two boxes is crucial for late promoter function . These results suggest that the product of gene 10 binds specifically to a late operator in order to activate transcription from P1 late promoter sequences.

Carbohydr Res, 1992 Nov 4, 235, 211 - 20
The structure of the capsular polysaccharide of Escherichia coli O9:K35:H-; Hackland PL et al.; The annexed structure of the capsular polysaccharide of Escherichia coli O9:K35:H- (A104a) has been determined by glycose and methylation analysis, base-catalysed degradation, and 1D and 2D NMR spectroscopy on the polysaccharide and the products obtained by bacteriophage-mediated depolymerisation . {formula: see text}

Gene, 1992 Nov 2, 121(1), 179 - 80
A vector, pSHT, for the expression and secretion of protein domains in mammalian cells; Madison EL et al.; A phagemid (pSHT) containing the pUC and M13 ori sequences was constructed to facilitate the expression of partial cDNAs or of sequences encoding mammalian membrane- and secretory-protein domains . It provides a start codon and signal sequence flanked upstream by the simian virus 40 and bacteriophage T7 promoters and downstream by cloning sites, stop codons in all three frames, splicing and polyadenylation signals.

Gene, 1992 Nov 2, 121(1), 9 - 15
Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors; Roberts BL et al.; We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product . Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin . A point mutation of BPTI {Lys15-->Leu(K15L)} alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated . Phage were eluted from an immobilized protease with step gradients of decreasing pH . Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix . Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.

Mol Microbiol, 1992 Nov, 6(22), 3415 - 25
Bacteriophage P4 immunity controlled by small RNAs via transcription termination; Deho G et al.; Satellite bacteriophage P4 immunity is encoded within a short DNA region 357 bp long containing the promoter PLE and 275 bp downstream . PLE is active both in the early post-infection phase, when genes necessary for P4 lytic cycle are transcribed from this promoter, and in the lysogenic condition, when expression of the above genes is prevented by prophage immunity . In order to understand how P4 immunity is elicited, we have characterized the transcription pattern during the establishment and the maintenance of the satellite phage P4 lysogenic condition . We found that prophage transcription starting at PLE ends prematurely and the transcripts do not extend beyond 300-400 nucleotides downstream of PLE . Thus P4 immunity acts by causing premature transcription termination rather than by repressing transcription initiation . The P4 immunity region is transcribed in the prophage, but it does not seem to be translated; this region contains two elements (seqA and seqB) of a palindromic sequence . In addition to transcripts about 300 nucleotides long, P4 prophage produces a family of shorter transcripts, about 80 nucleotides long, containing seqA or seqB . Evidence is presented suggesting that SeqB RNA is the trans-acting immunity factor, and that interaction of SeqB RNA with the complementary nascent RNA containing seqA may be involved in bringing about premature transcription termination.

Mol Microbiol, 1992 Nov, 6(22), 3313 - 20
Site-directed mutagenesis of an amino acid residue in the bacteriophage P2 ogr protein implicated in interaction with Escherichia coli RNA polymerase; King RA et al.; The P2 ogr gene encodes a 72-amino-acid protein required for P2 late gene expression . This gene was defined originally by a class of compensatory mutations which overcome the block to P2 late transcription imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of Escherichia coli RNA polymerase . Spontaneous compensatory ogr mutations substitute a Cys for a Tyr residue at amino acid 42 in the Ogr polypeptide . Using suppression of an ogr amber mutation and site-directed oligonucleotide mutagenesis, we have studied the effect of amino acid substitutions at this position in Ogr . Substitution of charged residues at this site renders Ogr protein inactive, in rpoA+ and rpoA109 strains . While 11 different amino acids are capable of replacing the wild-type Tyr-42 to allow P2 growth to varying degrees in a wild-type E . coli strain, only three of these allow phage growth in strains carrying the rpoA109 mutation . Phages carrying Cys or Ala in place of Tyr-42 gave burst sizes at least as high as P2 ogr+ in a rpoA+ strain; a Gly substitution also allowed P2 to grow in either a rpoA+ or rpoA109 background, but markedly reduced the burst size . These results are consistent with a direct interaction between Ogr and the alpha subunit of E . coli RNA polymerase in positive control of P2 late transcription, and indicate that the block imposed by the rpoA109 mutation is due to steric hindrance.

Mol Endocrinol, 1992 Nov, 6(11), 1867 - 73
Thyroid hormone receptor dimerization function maps to a conserved subregion of the ligand binding domain; Lee JW et al.; Thyroid hormone receptors (TRs) bind as dimers to specific DNA response elements . We have used a genetic approach to identify amino acid sequences required for dimerization of the TR beta isoform . Bacteria expressing a chimeric repressor composed of the DNA binding domain of the bacteriophage lambda cl repressor fused to the TR beta ligand binding domain are immune to lambda infection as a consequence of homodimerization activity provided by the receptor sequences . The phenotypes of deletions and point mutations of the TR beta sequences map dimerization activity to a subregion of the ligand binding domain that is highly conserved among all members of the nuclear hormone receptor superfamily . These results confirm and extend previous findings indicating that this subregion plays an important role in the dimerization of TR beta and other superfamily members.

Biophys J, 1992 Nov, 63(5), 1286 - 92
Assembly-associated structural changes of bacteriophage T7 capsids . Detection by use of a protein-specific probe; Khan SA et al.; To detect changes in capsid structure that occur when a preassembled bacteriophage T7 capsid both packages and cleaves to mature-size longer (concatameric) DNA, the kinetics and thermodynamics are determined here for the binding of the protein-specific probe, 1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid (bis-ANS), to bacteriophage T7, a T7 DNA deletion (8.4%) mutant, and a DNA-free T7 capsid (metrizamide low density capsid II) known to be a DNA packaging intermediate that has a permeability barrier not present in a related capsid (metrizamide high density capsid II) . Initially, some binding to either bacteriophage or metrizamide low density capsid II occurs too rapidly to quantify (phase 1, duration < 10 s) . Subsequent binding (phase 2) occurs with first-order kinetics . Only the phase 1 binding occurs for metrizamide high density capsid II . These observations, together with both the kinetics of the quenching by ethidium of bound bis-ANS fluorescence and the nature of bis-ANS-induced protein alterations, are explained by the hypothesis that the phase 2 binding occurs at internal sites . The number of these internal sites increases as the density of the packaged DNA decreases . The accompanying change in structure is potentially the signal for initiating cleavage of a concatemer . Evidence for the following was also obtained: (a) a previously undetected packaging-associated change in the conformation of the major protein of the outer capsid shell and (b) partitioning by a permeability barrier of the interior of the T7 capsid.

Mol Gen Genet, 1992 Nov, 235(2-3), 253 - 8
Lysis protein T of bacteriophage T4; Lu MJ et al.; Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis . Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane . The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations . Expression of the cloned gene t was lethal without causing gross leakiness of the membrane . The functional equivalent of T in phage lambda is protein S . An amber mutant of gene S can be complemented by gene t, although neither protein R of lambda (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts . The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca . 18,000) and a rather basic nature not exhibited by other E . coli cystosolic proteins . The results suggest that T acts as a pore that is specific for this type of enzyme.

J Microsc, 1992 Nov, 168 ( Pt 2), 181 - 201
Artefacts and morphological changes during chemical fixation; Kellenberger E et al.; The normally 'condensed' (darkly stained) chromosomes of dinoflagellates decondense by swelling . This occurs in an increasing number of cells when the concentration of added OsO4 is decreased . With different fixatives other types of disintegration can be observed, which vary with the concentration . With cryofixation and freeze-substitution the chromosomes are most 'condensed' . Escherichia coli infected with bacteriophage T4, with or without active lysozyme production, were studied by optical densitometry for partial lysis and by light and electron microscopy for observing swelling . When active lysozyme is present some of the acrolein (2.5%)-glutaraldehyde (2%)-fixed cells swell at 0 degrees C, but do not in the absence of lysozyme nor when fixed at room temperature . If OsO4 is added at concentrations < or = 0.5%, partial lysis occurs when lysozyme is present . The optical density decreases, the cells lose some matter and swell slightly . The corresponding electron micrographs show gap formation by curdling and/or a decreased concentration of the cytoplasm which reveals certain phage-related particles.

Toxicol Appl Pharmacol, 1992 Nov, 117(1), 58 - 64
Determination of mutagenicity in tissues of transgenic mice following exposure to 1,3-butadiene and N-ethyl-N-nitrosourea; Recio L et al.; 1,3-Butadiene (BD) is carcinogenic in the B6C3F1 mouse in multiple organs, including lung and liver . We conducted a study to measure the frequency of BD mutations in mouse tissues using a transgenic mouse (Muta mouse; MM) . MM is a BALB/c x DBA/2 (CD2F1) mouse that has a bacteriophage lambda shuttle vector with the target gene lacZ integrated into the mouse genome . Mice were exposed by inhalation to 625 ppm BD (6 hr/day) for 5 days and the lacZ- mutant frequency (mf) was determined in lung, bone marrow, and liver . The lacZ- mf in lung increased twofold above air-exposed control animals, but the bone marrow and liver samples did not exhibit an increase above background . N-ethyl-N-nitrosourea (250 mg/kg ip) was mutagenic in all three tissues examined . Studies on the biotransformation of BD using MM liver microsomes showed that the ratio between the rates of BD bioactivation to BD monoepoxide (BMO) and hydrolysis of BMO by epoxide hydrolases was approximately 40% less than this ratio using B6C3F1 mouse liver microsomes . Quantitation of adducts of BMO to N-terminal valine in hemoglobin (Hb) in the MM revealed an adduct level of 3.7 pmol/mg globin . Using this value, the predicted Hb adduct level in MM would be approximately one-half of that measured in the B6C3F1 mouse following similar exposures . These results indicate that BD induces mutations in vivo in a known murine target tissue, but strain differences in the biotransformation of BD should be considered in comparing the susceptibility of transgenic mouse strains to mutation.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10046 - 50
Hepatitis B virus capsid particles are assembled from core-protein dimer precursors; Zhou S et al.; Our studies on the assembly of hepatitis B virus capsids or core particles in Xenopus oocytes have demonstrated that unassembled p21.5 core proteins ("free p21.5") provide a pool of low-molecular-mass precursors for core-particle assembly . Here we have characterized this material . Free p21.5 sedimented through gradients of 3-25% sucrose (wt/vol) as a single protein species of approximately 40 kDa, corresponding to a p21.5 dimer . On nonreducing SDS/polyacrylamide gels, free p21.5 migrated as disulfide-linked p21.5 dimeric species of 35 and 37 kDa . Truncated core proteins lacking most or all of the 36-amino acid protamine region at the p21.5 carboxyl terminus were also found to behave as disulfide-linked dimers with appropriately reduced molecular masses . Our experiments failed to reveal monomeric core proteins or stable intermediates between dimers and capsids along the assembly pathway . We conclude that hepatitis B virus core particles are most likely assembled by aggregating 90 (or possibly 180) disulfide-linked p21.5 dimers . We discuss similarities between the assembly of hepatitis B virus capsids and simple T = 3 plant virus and bacteriophage structures.

Curr Genet, 1992 Nov, 22(5), 385 - 97
Genetic organization and structural features of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa; Court DA et al.; The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined . The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs) . Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid . ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo . The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages . A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages . The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains . The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue . Although maranhar and the senescence-inducing kalilo plasmid of N . intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.

Virology, 1992 Nov, 191(1), 406 - 16
Expression of functional parvoviral NS1 from recombinant vaccinia virus: effects of mutations in the nucleotide-binding motif; Nuesch JP et al.; The gene encoding the major replicative protein, NS1, of minute virus of mice (MVM) was transferred into a recombinant vaccinia virus vector in place of the vaccinia thymidine kinase gene . The NS1 gene was placed under control of a bacteriophage T7 promoter and expressed in cells coinfected with another recombinant vaccinia virus, vTF7-3, which encodes the T7 RNA polymerase . Expression of NS1 was further enhanced by the presence of a 5' untranslated region, derived from encephalomyocarditis virus, which allows efficient cap-independent translation . This system was used to produce and analyze wild-type NS1 and two mutant forms of the protein, NS1K405R and NS1K405M, in which the highly conserved lysine codon located in the putative purine triphosphate binding site of NS1 was changed to arginine and methionine, respectively . Full-length NS1 was expressed efficiently in both human and mouse cells infected with each of the three recombinant viruses, and in each case the NS1 was rapidly and efficiently translocated into the nucleus . Wild-type NS1 expressed in this way was biologically active . It was able to trans-activate an MVM P38 promoter located in a host chromosomal site, whereas the two mutant forms of NS1 showed no significant activity in this assay, and it was capable of resolving palindromic junction fragments cloned from multimeric MVM replicative form DNA molecules . These substrates, representing MVM genomic left-end:left-end and right-end:right-end fusions, were resolved in a DNA synthesis-dependent in vitro reaction supplemented with nuclear extracts containing recombinant wild-type NS1 . Neither of the two mutant forms of the polypeptide had any detectable activity in this assay.

Virology, 1992 Nov, 191(1), 246 - 50
Transcription dependence of DNA packaging of bacteriophages T3 and T7; Hashimoto C et al.; T3 and T7 phages package homologous DNA more efficiently than heterologous DNA and recombinant plasmids carrying DNA sequences necessary for DNA packaging (pac sequence) . The pac sequence contains a promoter for phage RNA polymerase and transcription from the promoter is necessary for DNA packaging . T3 and T7 RNA polymerases are stringently specific for their own promoters . To examine the relationship between DNA packaging and transcription, we constructed a cleared in vitro system for packaging T3 or T7 DNA containing an ammonium sulfate fractionate of a high-speed supernatant of phage-infected cells . In the system, DNA packaging required GTP and was inhibited by the 3'-deoxy analog of GTP, ATP, or CTP . The DNA packaging activity paralleled the transcriptional activity, assayed by incorporation of {32P}UTP into acid-insoluble material . In the system, homologous DNA was packaged more efficiently than heterologous DNA, but heterologous DNA was packaged as efficiently as homologous DNA by the addition of heterologous phage RNA polymerase, demonstrating that the transcriptional specificity determines the DNA packaging specificity of T3 and T7.

Mol Gen Mikrobiol Virusol, 1992 Nov-Dec, (11-12), 29 - 32
{Preparation of single-stranded E1A-oncogene DNA fragments from simian adenovirus SA7 by endonuclease hydrolysis recombinant phage M13 SS-DNA complexed with oligonucleotides, containing restriction sites}; Krutov AA et al.; The ss-DNA of the (+) and (-) chains of Ela DNA fragment was obtained by hydrolysis of the recombinant bacteriophages M13 mp8G and mp9G (where G is 1-1750 bp:, E1a region of oncogene SA7) in complexes with the 16 bp oligonucleotides containing AluI and BspRI sites of restriction and sequences complementary to E1a SA7 . The obtained fragments overlap the E1a zones associated with the immortalizing potential of SA7.

Biochim Biophys Acta, 1992 Oct 27, 1117(3), 237 - 42
The structural and functional organization of the baseplate distal part of T4 bacteriophage; Khusainov AA et al.; The structural organization of the baseplate distal part of bacteriophage T4 has been studied . The investigations resulted in revealing the functional role of gene products '11' and '12' in the course of baseplate reorganization and DNA injection . A new model of 'arrangement' of gp12 in the intact particle is suggested . A functional interrelationship between long fibers of the phage and the protein complex of baseplate distal part has been established.

Biochemistry, 1992 Oct 27, 31(42), 10315 - 21
Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis; Graves KL et al.; The proposed roles of Cys148 and Asp179 in deoxycytidylate (dCMP) hydroxymethylase (CH) have been tested using site-directed mutagenesis . CH catalyzes the formation of 5-(hydroxymethyl)-dCMP, essential for DNA synthesis in phage T4, from dCMP and methylenetetrahydrofolate . CH resembles thymidylate synthase (TS), an enzyme of known three-dimensional structure, in both amino acid sequence and the reaction catalyzed . Conversion of Cys148 to Asp, Gly, or Ser decreases CH activity at least 10(5)-fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys residue in TS) . In crystalline TS, hydrogen bonds connect O4 and N3 of the substrate dUMP to the side-chain amide of an Asn; the corresponding residue in CH is Asp179 . Conversion of Asp179 to Asn reduces the value of kcat/KM for dCMP by (1.5 x 10(4))-fold and increases the value of kcat/KM for dUMP by 60-fold; as a result, CH(D179N) has a slight preference for dUMP . Wild-type CH and CH(D179N) are covalently inactivated by 5-fluoro-dUMP, a mechanism-based inactivator of TS . Asp179 is proposed to stabilize covalent catalytic intermediates, by protonating N3 of the pyrimidine-CH adduct.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9642 - 6
Symmetry in the mechanism of bacteriophage lambda integrative recombination; Burgin AB Jr et al.; During the strand-exchange events of bacteriophage lambda integration, pairs of phosphodiester bonds are broken and then rejoined to form novel DNA linkages . The reaction proceeds in vitro in the absence of an external energy source; the bond energy needed to rejoin broken strands of DNA must therefore be conserved during cleavage . Although some of this conservation involves a covalent intermediate between DNA and the recombinase Int, it is possible that such an intermediate is formed with only one of the two phosphodiesters . In such an asymmetric mechanism, the second phosphodiester would be attacked by a nucleophile that is exposed by cleavage of the first DNA strand . In contrast, a symmetric mechanism hypothesizes nucleophilic attack by Int on both phosphodiesters . We have distinguished these two mechanisms by removing potential nucleophiles from the integrative recombination reaction . Our data are inconsistent with an asymmetric mechanism . We conclude that during strand exchange both phosphodiesters proceed through a covalent protein-DNA intermediate.

Gene, 1992 Oct 21, 120(2), 135 - 41
A selective lambda phage cloning vector with automatic excision of the insert in a plasmid; Maruyama IN et al.; A bacteriophage lambda cloning vehicle has been constructed for the generation of cDNA libraries . The vector has the following properties . (1) It has a unique BamHI site engineered into the lambda gam gene . Segments of DNA can be cloned into this site and clones with an insert can be selected by their ability to grow on an Escherichia coli host lysogenic for phage P2 (Spi- phenotype) . (2) When the recombinant phage infects a Cre-producing E . coli strain, a site-specific recombination event results in the excision of a plasmid replicon with the cloned insert . (3) Single-stranded DNAs can be recovered by growing helper M13 phages on bacteria harboring such plasmids . The vector, lambda MGU2, has been used to construct a nematode (Caenorhabditis elegans) cDNA library.

J Mol Biol, 1992 Oct 20, 227(4), 985 - 90
Stimulation of the phage lambda pL promoter by integration host factor requires the carboxy terminus of the alpha-subunit of RNA polymerase; Giladi H et al.; Escherichia coli integration host factor (IHF) binds with high affinity to two tandem IHF consensus sequences located upstream from the pL promoter of bacteriophage lambda . IHF was shown to stimulate transcription initiation from the pL promoter by increasing close complex formation (KB) . We show here, by the use of reconstituted mutant RNA polymerases, that the C-terminal portion of the alpha subunit of RNA polymerase plays an essential role in the stimulation of transcription by IHF . Our results are in agreement with the hypothesis that IHF, like the cAMP-CRP activator, increases the affinity of RNA polymerase to the promoter by protein-protein interaction.

J Mol Biol, 1992 Oct 20, 227(4), 1086 - 99
Molecular genetic analysis of bacteriophage P22 gene 3 product, a protein involved in the initiation of headful DNA packaging; Casjens S et al.; Bacteriophage P22 DNA packaging events occur in processive series on concatemeric phage DNA molecules . At the point where such series initiate, the DNA is recognized at a site called pac, and most molecular left ends are generated within six short regions called end sites, which are present in a 120 base-pair region surrounding the pac site . The bacteriophage P22 genes 2 and 3 proteins are required for successful generation of these ends and DNA packaging during progeny virion assembly . Mutants lacking the 162-amino-acid gene 3 protein replicate DNA and assemble functional procapsids . In this report we describe the nucleotide changes and DNA packaging phenotypes of a number of missense mutations of gene 3, which give the phage a higher than normal frequency of generalized transduction . In cells infected by these mutants, more packaging events initiate on the host chromosome than in wild-type infections, so the mutations are thought to affect the specificity of packaging initiation . In addition to having this phenotype, these mutations affect the process of phage DNA packaging in detectable ways . They may: (1) alter the target site specificity for packaging; (2) make target site recognition more promiscuous; (3) affect end site utilization; (4) alter the pac site; and (5) cause apparent random DNA packaging series initiation on phage DNA.

J Mol Biol, 1992 Oct 20, 227(4), 1054 - 67
Inhibition of bacteriophage lambda development by the klaA gene of broad-host-range plasmid RK2; Saltman LH et al.; The kil-kor regulon of broad-host-range plasmid RK2 is an unusual array of eight co-regulated operons that express at least 21 genes, including the plasmid replication initiator gene . Some of the operons were first identified as kil loci because uncontrolled expression in the absence of certain kor regulatory genes leads to death of the host cells . The functions of kilA, C and E are unknown, although co-regulation with the replication initiator gene suggests that they may have importance in the maintenance or host range of the plasmid . Here we report studies on the function of klaA, the first of three host-lethal genes in the kilA operon . We found that lambda pklaA-1, a lambda phage containing the klaA gene, is unable to form plaques unless the host expresses the KorA and KorB repressors needed to regulate transcription from the klaA promoter . The failure to form plaques depends on the klaA gene product and results from the inability of infected cells to produce viable phage particles . Transcription of early, delayed early and late genes or processing of lambda DNA are not affected by klaA overexpression, while cell lysis, lambda DNA replication and production of functional phage heads are reduced . However, the failure to produce viable phage is best explained by the inability to synthesize lambda tails . The finding that klaA strongly inhibits a specific morphogenetic step in the assembly of lambda phage particles has significance with respect to the function of klaA on plasmid RK2.

J Mol Biol, 1992 Oct 20, 227(4), 1164 - 72
Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of mutants of 5-fluorouracil-substituted Escherichia coli valine tRNA; Chu WC et al.; In order to utilize 19F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNAVal labeled by incorporation of 5-fluorouracil, we have assigned its 19F spectrum . We describe here assignments made by examining the spectra of a series of tRNAVal mutants with nucleotide substitutions for individual 5-fluorouracil residues . The result of base replacements on the structure and function of the tRNA are also characterized . Mutants were prepared by oligonucleotide-directed mutagenesis of a cloned tRNAVal gene, and the tRNAs transcribed in vitro by bacteriophage T7 RNA polymerase . By identifying the missing peak in the 19F NMR spectrum of each tRNA variant we were able to assign resonances from fluorouracil residues in loop and stem regions of the tRNA . As a result of the assignment of FU33, FU34 and FU29, temperature-dependent spectral shifts could be attributed to changes in anticodon loop and stem conformation . Observation of a magnesium ion-dependent splitting of the resonance assigned to FU64 suggested that the T-arm of tRNAVal can exist in two conformations in slow exchange on the NMR time scale . Replacement of most 5-fluorouracil residues in loops and stems had little effect on the structure of tRNAVal; few shifts in the 19F NMR spectrum of the mutant tRNAs were noted . However, replacing the FU29.A41 base-pair in the anticodon stem with C29.G41 induced conformational changes in the anticodon loop as well as in the P-10 loop . Effects of nucleotide substitution on aminoacylation were determined by comparing the Vmax and Km values of tRNAVal mutants with those of the wild-type tRNA . Nucleotide substitution at the 3' end of the anticodon (position 36) reduced the aminoacylation efficiency (Vmax/Km) of tRNAVal by three orders of magnitude . Base replacement at the 5' end of the anticodon (position 34) had only a small negative effect on the aminoacylation efficiency . Substitution of the FU29.A41 base-pair increased the Km value 20-fold, while Vmax remained almost unchanged . The FU4.A69 base-pair in the acceptor stem, could readily be replaced with little effect on the aminoacylation efficiency of E . coli tRNAVal, indicating that this base-pair is not an identity element of the tRNA, as suggested by others.

J Immunol Methods, 1992 Oct 19, 155(1), 77 - 89
Molecular cloning of murine monoclonal anti-idiotypic Fab; Kasai Y et al.; Anti-idiotypic antibodies (Ab2) binding to idiotopes on antibodies with various antigen binding specificities (Ab1) are potential regulators of immunity in a variety of diseases, such as autoimmunity, cancer, and viral, bacterial, or parasitic infections . Furthermore, Ab2 are useful probes for the characterization of receptor/ligand interactions . Thus far, Ab2 production has been limited to the isolation of polyclonal Ab2 from immune sera or monoclonal Ab2 from hybridoma supernatants . However, both approaches have produced a limited number of Ab2 . As an alternative approach, we demonstrate here the production of Ab2-Fab by using repertoire cloning . Using HIV-1 as a model system, the Ab2-Fab were generated from the spleens of mice immunized with the virus-neutralizing and syncytia-inhibiting anti-HIV-1 monoclonal antibody 0.5 beta . A bacteriophage lambda vector system was used to express a combinatorial library in Escherichia coli . Iodinated 0.5 beta was used to identify 17 Ab2-Fab clones . DNA sequence analysis of five clones revealed three similar kappa and Fd combinations . The Ab2-Fab bound with high affinity (3.5-6.5 x 10(9) liters/mol) specifically to the Ab1 and not to isotype-matched antibodies with unrelated specificities . The three Ab2-Fab probably bind to the same idiotope on the Ab1 as demonstrated in cross-competition binding studies . The Ab2-Fab inhibited binding of the Ab1 to antigen, and therefore, may functionally mimic the epitope defined by the Ab1 . Repertoire cloning of Ab2-Fab may facilitate the generation of Ab2 that have potential as modulators of immune responses against various antigens.

Eur J Biochem, 1992 Oct 15, 209(2), 511 - 21
The asparaginyl-tRNA synthetase gene encodes one of the complementing factors for thermosensitive translation in the Escherichia coli mutant strain, N4316; Aoki H et al.; Escherichia coli strain N4316 is a mutant that exhibits temperature-sensitive growth at 43 degrees C and temperature-sensitive translation in vivo and in vitro . Extracts of the mutant produce an aberrant pattern of translation products of MS2 bacteriophage RNA . Previous work has shown that a protein, called 'rescue', isolated from the parental strain partly corrects the defective translation in vitro . Here we report the purification to homogeneity of a second factor from ribosomal eluates of the wild-type parental strain; the purified protein is a homodimer of 54 kDa . The partial sequence of the second protein was determined, and a recombinant plasmid was isolated based on its ability to complement the temperature-sensitive growth phenotype of the mutant at the non-permissive temperatures . The cloned gene was sequenced, mapped to the 20.9-min region of the E . coli chromosome and shown to code for a 466-amino-acid protein with a molecular mass of 52 kDa . Analysis of the DNA sequence and the correspondence to that of the partial protein sequence has identified the complementing factor as asparaginyl-tRNA synthetase . Marker rescue experiments indicate that the asnS mutation in N4316 resides within the motif 2 domain of the synthetase . A potential role of this synthetase in restoring normal protein synthesis with respect to ribosomal frameshifting, read-through of nonsense codons and protein copy number is discussed.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9774 - 8
Genetic analysis of the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin; Himawan JS et al.; Gene 5 protein of bacteriophage T7 is a nonprocessive DNA polymerase . During infection of Escherichia coli, T7 annexes the host protein thioredoxin for use as a processivity factor for T7 DNA polymerase . We describe here a genetic method to investigate the interaction between T7 gene 5 protein and E . coli thioredoxin . The strategy is to use thioredoxin mutants that are unable to support the growth of wild-type T7 phage to select for T7 revertant phage that suppress the defect in thioredoxin . A thioredoxin mutation that replaces glycine at position 74 with aspartic acid fails to support the growth of wild-type T7 . This mutation is suppressed by six different mutations within T7 gene 5, each of which results in a single amino acid substitution within gene 5 protein . Three of the suppressor mutations are located within the putative polymerization domain of gene 5 protein, and three are located within the putative 3'-to-5' exonucleolytic domain . Each suppressor mutation alone is necessary and sufficient to confer the revertant phenotype.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9579 - 83
Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication; Mendez J et al.; Bacteriophage phi 29 DNA replication is initiated when a molecule of dAMP is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral DNA polymerase . We demonstrate that single-stranded DNA molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 DNA polymerase . Using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 DNA right end to evaluate the effect of nucleotide changes at the replication origin and to determine the precise initiation site . The results indicate that (i) there are no strict sequence requirements for protein-primed initiation on single-stranded DNA; (ii) initiation of replication occurs opposite the second nucleotide at the 3' end of the template; (iii) a terminal repetition of at least two nucleotides is required to efficiently elongate the initiation complex; and (iv) all the nucleotides of the template, including the 3' terminal one, are replicated . A sliding-back model is proposed in which a special transition step from initiation to elongation can account for these results . The possible implication of this mechanism for the fidelity of the initiation reaction is discussed . Since all the terminal protein-containing genomes have some sequence reiteration at the DNA ends, this proposed sliding-back model could be extrapolable to other systems that use proteins as primers.

J Biol Chem, 1992 Oct 15, 267(29), 20804 - 10
Cloning and characterization of the major promoter of the human protein kinase C beta gene . Regulation by phorbol esters; Obeid LM et al.; The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3) . The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage . This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region . Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG . Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island . Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330 . To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene . This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells . Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs . The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta . This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site . These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.

J Biol Chem, 1992 Oct 15, 267(29), 20674 - 81
Overexpression, purification, sequence analysis, and characterization of the T4 bacteriophage dda DNA helicase; Hacker KJ et al.; The bacteriophage T4 dda protein is a 5'-3' DNA helicase that stimulates DNA replication and recombination reactions in vitro and seems to play a role in the initiation of T4 DNA replication in vivo . Oligonucleotide probes based on NH2-terminal amino acid sequence were used to precisely map the location of the dda gene on the T4 chromosome . Using polymerase chain reaction techniques, the dda gene was then cloned into an expression vector, and the overproduced protein was purified in two chromatography steps . Both the genomic and cloned dda genes were sequenced and found to be identical, encoding a protein of 439 amino acids . The dda protein contains amino acid sequences resembling those of other known helicases, and is most homologous to the Escherichia coli recD protein . Protein affinity chromatography was used to show a direct interaction between the dda protein and the T4 uvsX protein (a rec A-type DNA recombinase).

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