J Mol Biol, 1989 Feb 20, 205(4), 751 - 64 Q beta replicase: mapping the functional domains of an RNA-dependent RNA polymerase; Mills DR et al.; We have localized a functional region of the RNA bacteriophage Q beta replicase following an extensive mutational analysis . Using the method of oligonucleotide linker-insertion mutagenesis, we specifically introduced mutations into a cloned DNA copy of the Q beta replicase gene so that the resulting replicase products would putatively contain small amino acid insertions . In a selective phenotypic assay, we screened mutant replicases for RNA-directed replication activity in vivo . Analysis of 37 different mutant clones indicated that Q beta replicase can accept amino acid substitutions and insertions at several sites at the amino and carboxy termini without abolishing functional activity in vivo or in vitro . However, disruption within the internal amino acid sequence resulted almost exclusively in nonfunctional enzyme . The results suggest that the central region of the replicase protein contains a rigid amino acid composition that is required for replicase function, whereas the amino and carboxy termini are much more receptive to small amino acid insertions and substitutions . These experiments should further enable us to analyze the coding function of the Q beta replicase gene independently of other phage RNA functions contained within this nucleotide region. Gene, 1989 Feb 20, 75(2), 261 - 70 A versatile phage lambda expression vector system for cloning in Escherichia coli; Sieg K et al.; By integrating fragments from the expression plasmids pJK2 and pJK4 into a derivative of the bacteriophage lambda, we constructed the phage expression vectors lambda JK2 and lambda JK4, which allow efficient cloning of genomic or cDNA either into the 5' end or the 3' end of the lacZ gene of Escherichia coli . Expression of barrier-free DNA in phase may lead to fusion proteins consisting of active beta-galactosidase (beta Gal) plus an additional polypeptide encoded by the inserted DNA . Analysis of distinct recombinant clones is quick and easy, due to the reversible integration of the plasmid into the genome . As an example, we constructed an expression library of genomic Plasmodium falciparum DNA in lambda JK2 . We polymerised (amplified) and expressed a synthetic DNA fragment, which codes for a potential antigenic determinant of the 11-1 gene of Plasmodium falciparum as a fusion to the N terminus of active beta Gal . We demonstrate that such chimeric molecules can be affinity-purified and that polypeptides can be separated from the beta Gal part by cleavage with the protease factor Xa. J Biol Chem, 1989 Feb 15, 264(5), 3035 - 42 Supercoiling and integration host factor change the DNA conformation and alter the flow of convergent transcription in phage Mu; Higgins NP et al.; Transcription of bacteriophage Mu is modulated by its repressor, by negative supercoiling, and by the Escherichia coli protein integration host factor (IHF) . Two converging Mu promoters regulate lytic and lysogenic development . The influence of IHF on these convergent promoters depended on the DNA conformation . When Mu operator DNA changed from the relaxed to the negative supercoiled form, IHF changed from a stimulatory factor to an inhibitor of the repressor promoter, and the ratio of the lytic transcript relative to the repressor transcript increased by 40-fold . Flexibility in Mu operator DNA was demonstrated by an unusual supercoil-induced DNA conformation, which was detectable by chemical modification with bromoacetaldehyde or digestion with P1 nuclease . IHF binding adjacent to this site induced dramatic bending of Mu DNA . A topological model we call a superloop is proposed to explain the effect of IHF on Mu transcription in vitro and on the lytic-lysogeny decision of the virus grown in IHF and gyrase mutants. J Biol Chem, 1989 Feb 15, 264(5), 2853 - 61 Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication; Mensa-Wilmot K et al.; We have established an in vitro system, composed of highly purified bacteriophage lambda and Escherichia coli proteins, that specifically replicates supercoiled templates bearing the lambda replication origin (ori lambda) . The complete system is composed of three groups of proteins: the virus-encoded initiator proteins (the lambda O and P proteins), the E . coli replication fork propagation machinery (single-stranded DNA-binding protein, dnaB helicase, dnaG primase, DNA polymerase III holoenzyme, and DNA gyrase), and two bacterial heat shock proteins (dnaJ and dnaK proteins) . DNA replication in this system is initiated at or near ori lambda and proceeds unidirectionally rightwards through theta-structure intermediates, ultimately yielding a pair of intertwined daughter circles as the final product . In striking contrast to the situation in vivo and in crude in vitro systems, initiation of lambda DNA replication in the purified protein system does not require "transcriptional activation" of the origin region by E . coli RNA polymerase . We conclude that E . coli primase generates the primers for all leading and lagging strand DNA chains synthesized in this reconstituted lambda replication system. Science, 1989 Feb 10, 243(4892), 792 - 4 Control of enzyme activity by an engineered disulfide bond; Matsumura M et al.; A novel approach to the control of enzyme catalysis is presented in which a disulfide bond engineered into the active-site cleft of bacteriophage T4 lysozyme is capable of switching the activity on and off . Two cysteines (Thr21----Cys and Thr142----Cys) were introduced by oligonucleotide-directed mutagenesis into the active-site cleft . These cysteines spontaneously formed a disulfide bond under oxidative conditions in vitro, and the catalytic activity of the oxidized (cross-linked) T4 lysozyme was completely lost . On exposure to reducing agent, however, the disulfide bond was rapidly broken, and the reduced (non-cross-linked) lysozyme was restored to full activity . Thus an enzyme has been engineered such that redox potential can be used to control catalytic activity. J Mol Biol, 1989 Feb 5, 205(3), 493 - 500 Bent DNA is needed for recombinational enhancer activity in the site-specific recombination system Cin of bacteriophage P1 . The role of FIS protein; Hubner P et al.; A series of recombinational enhancer mutants was constructed by manipulating the ClaI site between the two FIS binding sites of the Hin enhancer . These mutants include insertions from two to 12 base-pairs and two deletions of one or two base-pairs . Recombinational enhancer activity was found only with four mutants carrying either a four base-pair substitution, ten base-pair insertions or a one base-pair deletion, respectively; two other ten base-pair insertion mutants, however, were inactive, although FIS protein binding was unaffected . So, besides binding of FIS protein to its specific sites within the enhancer sequence and the correct helical positioning of these sites on the DNA, another criterion for enhancer activity must be fulfilled . DNA bending assays identify this requirement as a change of the enhancer DNA conformation, which FIS protein is able to induce and to stabilize . This conformational change of the DNA can be blocked by mutations in the central segment between the two FIS binding sites of the Hin enhancer . This sequence has special functions for the recombinational enhancer activity. J Mol Biol, 1989 Feb 5, 205(3), 501 - 9 Analysis of single-stranded DNA stability and damage-induced strand loss in mammalian cells using SV40-based shuttle vectors; Madzak C et al.; The fate and stability of fully or partially single-stranded DNA molecules transfected into mammalian cells have been analysed . For this, we constructed a simian virus 40 (SV40)-based shuttle vector containing the f1 bacteriophage replication origin in the two possible orientations (pi SVF1-A and pi SVF1-B) . This vector contains the SV40 origin of replication, the late viral genes and DNA sequences for replication and selection in Escherichia coli . It also carries the lacO sequence, which permits the analysis of plasmid stability . Single-stranded DNA from pi SVF1-A and pi SVF1-B were produced in bacteria and annealed in vitro to form a heteroduplex molecule . We showed that, in monkey kidney COS7 cells, single-stranded vectors replicate to form duplex molecules . After transfection of the three forms of molecules (single-stranded, heteroduplex or double-stranded), replicated DNA was rescued in E . coli . Vector stability was analysed by checking for plasmid rearrangements and screening for lacO mutants . The single-stranded pi SVF1 has a lower rearrangement level, while the spontaneous mutation frequency (on the lacO target) is in the same range as for the double-stranded vector . In contrast, the level of spontaneous mutagenesis is higher for the heteroduplex than for the single- and double-stranded forms . In addition, we found that replication of heteroduplex with one strand containing ultraviolet light-induced lesions yields progeny molecules in which the irradiated strand is mostly lost . This result indicates for the first time the specific loss of the damaged strand in mammalian cells. Mutat Res, 1989 Feb, 216(1), 19 - 26 Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione; Kortenkamp A et al.; The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems . To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH . The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2 . The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA . No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex . The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH . A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations . Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol . Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate. J Bacteriol, 1989 Feb, 171(2), 1035 - 40 Role of the flagellum in cell-cycle-dependent expression of bacteriophage receptor activity in Caulobacter crescentus; Bender RA et al.; The rate of adsorption of Caulobacter bacteriophage phi CbK to Caulobacter crescentus is dependent on the structural integrity of the flagellum . Cells lacking part or all of the flagellum because of either mutation or mechanical shear were defective in adsorption, and the extent of the defect in adsorption reflected the amount of flagellar structure missing . Maximal adsorption rates were also dependent on cellular motility and energy metabolism, since adsorption to cells with paralyzed flagella was slower than adsorption to motile cells and inhibition of cellular energy metabolism with azide also reduced adsorption rates, even for nonmotile cells . Nevertheless, the flagellum is not the receptor for phage phi CbK, since flagellumless mutants adsorbed phi CbK at detectable rates . While some portion of the fluctuation in the phi CbK receptor activity during the C . crescentus cell cycle can be ascribed to the periodicity of flagellar loss and reappearance, the phage receptor activity remaining in flagellumless mutants was periodic in the cell cycle . Therefore, the periodic expression of phage receptor activity is an intrinsic property of the C . crescentus cell cycle, although the amplitude of the oscillation may be altered by the periodic expression of flagellar motility. J Virol, 1989 Feb, 63(2), 705 - 13 Fine structure mapping of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase; Thompson CL et al.; We have mapped the temperature-sensitive (ts) lesions of three mutants, ts51, ts53, and ts65, and two other mutants, ts7 and ts20, to regions on the vaccinia virus genome that encode the 147- and 22-kilodalton subunits of the viral DNA-dependent RNA polymerase, respectively . Plasmid and bacteriophage clones from the HindIII J region and the region spanning the HindIII J-H junction were used in marker rescue experiments to map the mutations . Sequence analysis of the region encoding the 22-kilodalton subunit in the wild-type, ts7, and ts20 viruses revealed a single base change in the mutants compared with that in the wild-type virus . The identification of these RNA polymerase mutants provides us with tools to understand transcription and its regulation in vaccinia virus. Mol Gen Mikrobiol Virusol, 1989 Feb, (2), 13 - 25 {Three-dimensional reconstruction of bacterial viruses from electron microscopy data . Bacteriophage structure}; Mikhailov AM et al.; The structure of bacteriophages from different groups is presented based on the data obtained by the three-dimensional reconstruction, optical diffraction and filtration and electron microscopy techniques . The study made possible to suggest the scheme for the mechanism of sheath molecules rearrangement at contraction of bacteriophage tail sheath, the model of bacteriophage FI-1 connector . The structural elements for difference and relation of various bacteriophages are demonstrated. Genetics, 1989 Feb, 121(2), 205 - 12 Escherichia coli mutator mutD5 is defective in the mutHLS pathway of DNA mismatch repair; Schaaper RM; We have previously reported that the Escherichia coli mutator strain mutD5 was defective in the correction of bacteriophage M13mp2 heteroduplex DNA containing a T.G mismatch . Here, this defect was further investigated with regard to its interaction with the mutHLS pathway of mismatch repair . A set of 15 different M13mp2 heteroduplexes was used to measure the mismatch-repair capability of wild-type, mutL and mutD5 cells . Throughout the series, the mutD5 strain proved as deficient in mismatch repair as the mutL strain, indicating that the repair defect is similar in the two strains in both extent and specificity . {One exception was noted in the case a T.G mispair that was subject to VSP (Very Short Patch) repair . VSP repair was abolished by mutL but not by mutD.} Variation in the dam-methylation state of the heteroduplex molecules clearly affected repair in the wild-type strain but had no effect on either the mutD or mutL strain . Finally, mutDmutL or mutDmutS double-mutator strains were no more deficient in mismatch repair as were the single mutator strains . The combined results strongly argue that the mismatch-repair deficiency of mutD5 cells resides in the mutH,L,S-dependent pathway of mismatch repair and that the high mutation rate of mutD strains derives in part from this defect. Proc Natl Acad Sci U S A, 1989 Feb, 86(3), 835 - 9 Expression of human ferredoxin and assembly of the {2Fe-2S} center in Escherichia coli; Coghlan VM et al.; A cDNA fragment encoding human ferredoxin, a mitochondrial {2Fe-2S} protein, was introduced into Escherichia coli by using an expression vector based on the approach of Nagai and Thogersen {Nagai, K . & Thogersen, M . C . (1984) Nature (London) 309, 810-812} . Expression was under control of the lambda PL promoter and resulted in production of ferredoxin as a cleavable fusion protein with an amino-terminal fragment derived from bacteriophage lambda cII protein . The fusion protein was isolated from the soluble fraction of induced cells and was specifically cleaved to yield mature recombinant ferredoxin . The recombinant protein was shown to be identical in size to ferredoxin isolated from human placenta (13,546 Da) by NaDodSO4/PAGE and partial amino acid sequencing . E . coli cells expressing human ferredoxin were brown in color, and absorbance and electron paramagnetic resonance spectra of the purified recombinant protein established that the {2Fe-2S} center was assembled and incorporated into ferredoxin in vivo . Recombinant ferredoxin was active in steroid hydroxylations when reconstituted with cytochromes P-450scc and P-450(11) beta and exhibited rates comparable to those observed for ferredoxin isolated from human placenta . This expression system should be useful in production of native and structurally altered forms of human ferredoxin for studies of ferredoxin structure and function. J Clin Microbiol, 1989 Feb, 27(2), 291 - 4 Determination by DNA hybridization of Shiga-like-toxin-producing Escherichia coli in children with diarrhea in Thailand; Brown JE et al.; Specific DNA probes were used to identify Shiga-like-toxin (SLT) I- and II-producing Escherichia coli from children less than 5 years of age with bloody diarrhea, in infants with diarrhea, and in controls of the same age without diarrhea in Thailand . At one hospital, SLT-producing E . coli was identified in 4 (7%) of 54 children with bloody diarrhea from whom other enteric pathogens were not identified and from 3 (6%) of 50 children without diarrhea . In the positive specimens, SLT-producing E . coli constituted only 0.3 to 4% of the 100 to 300 colonies on the replica blots . Non-toxin-encoding 933J and 933W bacteriophagelike DNA sequences were detected by colony hybridization with E . coli isolates from 18 (33%) of 54 children with bloody diarrhea and 23 (46%) of 50 controls . At another hospital, SLT-producing E . coli was not identified in 115 infants with diarrhea and 119 controls without diarrhea . One infant with diarrhea was infected with E . coli O76:H7 that hybridized with the enterohemorrhagic E . coli probe but not with the SLT probes . E . coli producing SLT I or SLT II was isolated in small numbers from a similar proportion of Thai children with bloody diarrhea in whom no other enteric pathogen was identified and from controls without diarrhea. J Bacteriol, 1989 Feb, 171(2), 799 - 806 Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer; Meyer R; R1162 is efficiently comobilized during conjugative transfer of the self-transmissible plasmid R751 . Bacteriophage M13 derivatives that contain two directly repeated copies of oriT, the site on R1162 DNA required in cis for mobilization, were constructed . Phage DNA molecules underwent recombination during infection of Escherichia coli, with the product retaining a single functional copy of oriT . Recombination was strand specific and depended on R1162 gene products involved in mobilization, but did not require the self-transmissible plasmid vector . Two genes were identified, one essential for recombination and the other affecting the frequency of recombination . Recombination of bacteriophage DNA could form the basis of a simple model for some of the events occurring during conjugation without the complexity of a true mating system. J Bacteriol, 1989 Feb, 171(2), 1106 - 17 Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1; Vimr ER et al.; The kps gene cluster of Escherichia coli K1 encodes functions for sialic acid synthesis, activation, polymerization, and possibly translocation of polymer to the cell surface . The size and complexity of this membrane polysaccharide biosynthetic cluster have hindered genetic mapping and functional descriptions of the kps genes . To begin a detailed investigation of the polysialic acid synthetic mechanism, acapsular mutants were characterized to determine their probable defects in polymer synthesis . The mutants were tested for complementation with kps fragments subcloned from two separately isolated, functionally intact kps gene clusters . Complementation was assayed by immunological and biochemical methods and by sensitivity to the K1-specific bacteriophage K1F . The kps cluster consisted of a central 5.8-kilobase region that contained at least two genes coding for sialic acid synthetic enzymes, a gene encoding the sialic acid-activating enzyme, and a gene encoding the sialic acid polymerase . This biosynthetic region is flanked on one side by an approximately 2.8-kilobase region that contains a potential regulatory locus and at least one structural gene for a polypeptide that appears to function in polysialic acid assembly . Flanking the biosynthetic region on the opposite side is a 6- to 8.4-kilobase region that codes for at least three proteins which may also function in polymer assembly and possibly in translocating polymer to the outer cell surface . Results of transduction crosses supported these conclusions and indicated that some of the kps genes flanking the central biosynthetic region may not function directly in transporting polymer to the cell surface . The results also demonstrate that the map position and probable function of most of the kps cluster genes have been identified. J Bacteriol, 1989 Feb, 171(2), 650 - 6 Structure and function of conjugative pili: inducible synthesis of functional F pili by Escherichia coli K-12 containing a lac-tra operon fusion; Grossman TH et al.; In vivo and in vitro recombination methods were used to construct the recombinant plasmid pTG801, in which the F-plasmid DNA transfer (tra) genes required for the formation of functional F pili were placed under the lac transcriptional control sequences of pUC19 . The 20 kilobases of cloned F DNA includes genes traA through the 5'-terminal part of traG; the plasmid lacks the positive regulatory gene traJ and all of the known tra genes required for the DNA transfer stage of conjugation . pTG801 transformants were sensitive to the donor-specific bacteriophages Q beta and f1, as measured by the formation of infectious centers . They were relatively insensitive to bacteriophage R17, as expected from the absence of traD . In the presence of a lacIq allele, sensitivity of pTG801 transformants to f1 and Q beta depended on the concentration of inducer (isopropyl-beta-D-thiogalactopyranoside {IPTG}) . Viewed by electron microscopy, pTG801 transformants elaborated 7- to 10-nm-diameter filaments that could be laterally decorated with RNA bacteriophage particles, consistent with the formation of F pili . In stationary-phase cultures, these filaments formed massive aggregates and could be seen to adhere lengthwise to the cell surface; few pili accumulated in the medium as single filaments. J Biomol Struct Dyn, 1989 Feb, 6(4), 701 - 6 A model for the complex between the helix destabilizing protein GP32 of bacteriophage T4 and single-stranded DNA; Scheerhagen MA et al.; A model for the structure of the complex between the helix-destabilizing protein of bacteriophage T4, GP32, and single-stranded DNA is proposed . In this model the bases are arranged in a helix, that is characterized by a relatively large distance between successive bases, a substantial base tilt, in combination with a small rotation per base . This helix is further organized into a tertiary structure, possibly a superhelix, of which the corresponding protein shell corresponds to the relatively rigid and rod-like structure that is observed in hydrodynamic experiments . It is proposed that similar structural features apply to other single-stranded DNA binding proteins in complex with polynucleotides. Proc Natl Acad Sci U S A, 1989 Feb, 86(4), 1307 - 11 Bacteriophage T4 DNA topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in T4-infected Escherichia coli; Huff AC et al.; The mammalian type II DNA topoisomerase has been proposed to be the intracellular target of a variety of antitumor agents, including m-AMSA {4'-(9-acridinylamino)-methanesulfon-m-anisidide} . Because the bacteriophage T4-encoded topoisomerase resembles the mammalian enzyme, we are using T4 as a simple model system to investigate the mechanism of action of m-AMSA . A mutation that renders T4 growth m-AMSA-resistant is closely linked to an amber mutation in T4 gene 39, which encodes one of the topoisomerase subunits . In addition, the gene 39 subunit from the m-AMSA-resistant mutant phage has an altered net charge, strongly indicating that the drug-resistance mutation is within gene 39 . Topoisomerase purified from mutant phage-infected Escherichia coli exhibits drug-insensitive DNA relaxation and DNA cleavage activities . Because a single mutation results in both drug-resistant phage growth and a drug-insensitive viral topoisomerase, we conclude that the T4-encoded type II DNA topoisomerase is the physiological target of m-AMSA in phage-infected E . coli. J Bacteriol, 1989 Feb, 171(2), 996 - 1001 Isolation of an ammonium or methylammonium ion transport mutant of Escherichia coli and complementation by the cloned gene; Jayakumar A et al.; During nitrogen-limited growth, Escherichia coli expresses a specific ammonium or methylammonium ion transport system (Amt) . Strains carrying defects in Amt have been isolated following Tn10 transposon mutagenesis . These mutants have less than 10% of the transport activity of the parental strain . Glutamate, glutamine, arginine, or high levels (20 mM) of ammonium will serve as the sole nitrogen source for growth of these strains, and glutamine synthetase is normally expressed and repressed by the nitrogen regulatory (Ntr) system . When transformed with plasmid pGln84, containing lacZ fused to an Ntr promoter (glnLp), the Amt mutants expressed a normal level of beta-galactosidase . Furthermore, P1 bacteriophage transduction of the amt mutation into an Ntr mutant, normally constitutive for Amt, gave Amt- transductants . Therefore, the mutations are unlikely to lie within genes affecting Ntr elements . Following transformation with plasmid libraries of E . coli genomic DNA constructed in pUC9, two plasmids conferring the Amt+ phenotype on the amt mutants were isolated . These plasmids were unable to complement the Amt- phenotype of Ntr- mutants . Restriction digestion of these plasmids revealed common fragments, and Southern blot analyses indicated that the Amt-complementing sequence and the site of Tn10 insertion in the genome occur in the same 3.4-kilobase HindIII-SalI fragment . Insertion of TnphoA into this fragment produced amt::phoA fusions which gave high levels of alkaline phosphatase under nitrogen-limiting conditions but low levels during ammonia excess . This suggests that the amt product contains domains which are exported to the periplasm. Anal Biochem, 1989 Feb 1, 176(2), 373 - 4 Large scale preparation of bacteriophage lambda by tangential flow ultrafiltration for isolation of lambda DNA; Rembhotkar GW et al.; A preparative procedure for purifying bacteriophage lambda from large volumes of phage lysates by recirculating tangential flow ultrafiltration is described . Lambda DNA, isolated by deproteinization of the phage, is suitable for use in molecular biology. Mol Gen Mikrobiol Virusol, 1989 Feb, (2), 29 - 35 {The Z-form of bacteriophage lambda DNA, modified in situ}; Ulanov BP et al.; Using the methods of chemical modification, restriction analysis and immune-electron microscopy it has been shown that the definite regions of the bacteriophage lambda DNA contain unpaired bases in situ . The distribution map of such sites along the genome has been constructed . The correlation of the in situ modification and the reaction with anti-Z-DNA antibodies is shown for the 44972 bp site of bacteriophage DNA . The possibility of the existence of Z-form DNA in situ is discussed. Mol Gen Mikrobiol Virusol, 1989 Feb, (2), 26 - 9 {Features of expression of cloned genes under the control of tandem promotors pL and pR of phage lambda}; Zheleznaia LA et al.; The regulatory block P'R from the bacteriophage lambda has been inserted between the promoter and initial part of the gene into the plasmid pCJ55 carrying the gene for the Klenow fragment under the control of pL . As it should be predicted, at the inverted orientation the sharp decrease in the Klenow fragment quantity is registered . However, at the direct orientation there is some decrease in the synthesis of the protein, as compared with the synthesis of the Klenow fragment in the strain harbouring the plasmid pCJ55 . A plausible explanation of the fact may be in the transcriptional interference of the promoters pL and p'R in artificially constructed structures. Photochem Photobiol, 1989 Feb, 49(2), 161 - 7 Induction of lambda prophage near the site of focused UV laser radiation; Matchette LS et al.; DNA damage from photon scatter or beam spread during UV excimer laser irradiation was investigated using the induction of bacteriophage lambda in E . coli BR339 . Prophage induction in these cells leads to the production of beta-galactosidase which can be detected colorimetrically by the application of appropriate substrates . An agar surface overlayed with BR339 cells was placed at various distances from the focal point of a converging lens and exposed to either 193 or 248 nm laser radiation . Energy densities ranging from approximately 5 mJ/cm2 to 30 J/cm2 were used . Ablation with 193 nm laser radiation produced an 800 microns wide clear 'trench' surrounded by a 500 microns zone of cells in which lambda had been induced . Following ablation with 248 nm laser radiation, the zone of induction was several millimeters wide . Exposures to 193 nm radiation at 170 mJ/cm2/pulse produced visible ablation of the agar surface at 1.7 J/cm2 . Lambda induction was observed surrounding cleared ablation areas . The presence of induction in this system suggests that both 248 and 193 nm excimer laser radiation delivered at high energy densities has sufficient spread or scatter to damage DNA in cells surrounding areas of ablation. Virology, 1989 Feb, 168(2), 370 - 7 DNA looping induced by bacteriophage lambda O protein: implications for formation of higher order structures at the lambda origin of replication; Schnos M et al.; A plasmid has been constructed, pOri2, which contains two lambda replication origin sequences separated by 1068 bp; both lambda sequences having the same orientation . When lambda initiation protein O is reacted with linearized pOri2 and examined by electron microscopy it is found to contain a looped area in which two parts of the plasmid are bound together by the O protein complex . Length measurements show that the O protein binds at the expected positions of the lambda origin sequences and that the looped area represents the DNA segment between the two O protein binding domains . Similar looping occurs in reactions with supercoiled pOri2 or if an amino-terminal fragment of O protein is used . When looped molecules are reacted with psoralen, crosslinked by irradiation with uv light, and then denatured, it is found that the looped area is more thermostable than the rest of the molecule . This indicates that the DNA within the looped segment is torsionally constrained while that outside the loop is free to rotate and suggests that simultaneous binding of O to two origins fixes the linkage number of the intervening DNA . The double origin binding ability of O may be diagnostic of the details of the reaction of O with a single origin sequence . A model is presented that rests on the assumption that O can produce microscopic looping between O protein binding sites within a single ori sequence. Biotechniques, 1989 Feb, 7(2), 132 - 4,136 A general method to optimize the amount of enzyme in restriction and DNA modification reactions using the beta galactosidase blue-white plaques assay; Cab-Barrera EL et al.; We propose a simple and economical method for assaying the activity of restriction and other modifying enzymes . The method involves assaying the use of the blue and white colored phenotypes of bacterial colonies obtained by digesting the polylinker sequence of M13 bacteriophage vectors followed by transformation in appropriate strains on X-gal/IPTG plates . In conjunction with restriction enzymes and DNA ligases, the method can evaluate polymerase activity and can be applied to test 3'...5' exonuclease activities such as that of T4 DNA polymerase, without having to use expensive radioisotopes . We describe its application in the assessment of restriction enzymes, DNA ligase and DNA polymerase activities. Can J Microbiol, 1989 Feb, 35(2), 289 - 94 Mapping of transfer and H pilus coding regions of the IncHII plasmid pHH1508a; Yan W et al.; The IncHII plasmid pHH1508a (208 kilobases) encodes resistance to potassium tellurite, trimethoprim, and streptomycin . Conjugative pili encoded by pHH1508a were isolated, purified, and used for preparation of anti-H pilus antiserum . Immuno-gold labelling experiments using H pilus specific antiserum showed that antigenic determinants were located along the entire length of the H pilus . Immuno-gold labelling and lysis studies using pilH alpha, a bacteriophage specific for H pili, were used to investigate transfer-deficient mutants of pHH1508a obtained by Tn5 mutagenesis and an in vitro constructed derivative of 96 kilobases, pDT1178, which also conferred resistance to potassium tellurite, trimethoprim, and streptomycin . The transfer-deficient mutants did not specify H pili, whereas pDT1178, which transferred at low frequency (1 x 10(-4) transconjugants per recipient), specified a small number of H pili . A naturally occurring plasmid, pMG110, was found to encode the production of H pili, but was completely transfer deficient (less than 1 x 10(-7) transconjugants per recipient) . This study suggests that genes required for H pilus production and assembly as well as low level transfer are located separately within the 96-kilobase fragment of pDT1178 and that other genes, located outside this region, are essential for the regulation and full expression of conjugative transfer. Gene, 1989 Jan 30, 75(1), 3 - 11 Structure of the human genomic glutathione S-transferase-pi gene; Morrow CS et al.; The complete human genomic glutathione-S-transferase-pi gene (GST-pi) was isolated from a lambda Charon 4A bacteriophage library which was screened by hybridization to a human GST-pi cDNA . We have sequenced 4261 bp which include the entire GST-pi gene as well as over 1200 bp of the 5' and 200 bp of the 3' flanking regions . The GST-pi gene has 7 exons and 6 introns contained within approximately 2.8 kilobases . Primer extension experiments identified four possible transcription start points closely spaced between 29 and 33 nucleotides (nt) 5' to the start of translation . Analysis of the GST-pi promoter region revealed 4 putative transcription regulatory motifs; these sequences include a 'TATA' box 29 bp upstream from the major transcription start point (nt position -29), 2 Sp1 recognition sequences (GGGCGG, nt positions -46 to -41 and -56 to -51), and an AP-1 recognition sequence (TGACTCA, nt positions -69 to -63) . The first 200 nt 5' to the start point of transcription contain a G + C-rich region (79%) . Additionally, an intriguing A + T-rich region was found between nt positions -505 and -413 which contained 17 AAAAT tandem repeats . Comparison of the GST-pi gene with the homologous rate gene, GST-P, disclosed extensive conservation of genomic organization between the two species. Gene, 1989 Jan 30, 75(1), 39 - 46 Nucleotide sequence of the Stp-1 gene coding for rat spermatid nuclear transition protein 1 (TP1): homology with protamine P1 and assignment of the mouse Stp-1 gene to chromosome 1; Heidaran MA et al.; Spermatid transition protein 1 (TP1) is a 54 amino acid (aa), highly basic chromosomal protein found in mammals during the brief period when histones are being replaced by protamines in the haploid phase of spermatogenesis . Using a cDNA clone as probe, we have isolated the gene (Stp-1) coding for rat TP1 from a population of recombinant bacteriophage lambda . The nucleotide (nt) sequence was established from a point 126 nt upstream from the mRNA cap site to a point about 30 nt downstream from the predicted site of polyadenylation . The gene contains a single intron separating the codon for aa 45 of the mature protein . Comparison of the nucleotide sequences for Stp-1 and the mouse gene coding for protamine P1 suggests a possible evolutionary relationship . Southern blot hybridization to genomic DNA isolated from a panel of mouse-hamster somatic cell hybrids unambiguously mapped Stp-1 to mouse chromosome 1. FEBS Lett, 1989 Jan 30, 243(2), 115 - 8 Prediction of terminal protein and ribonuclease H domains in the gene P product of hepadnaviruses; Khudyakov YuE et al.; By means of comparative analysis of primary and secondary structures, and hydropathy plots of hepadnavirus P proteins new functional domains were revealed additionally to the polymerase domain which had been found earlier in these proteins . The C-terminal part of P proteins was revealed to be significantly similar to ribonuclease H of E . coli . The ribonuclease H functional domain is known to be an integral entity of retrovirus reverse transcriptase as a rule . Availability of this domain indicates once more the putative reverse transcriptase properties of the P products . The proteins of hepadnaviruses were compared to terminal proteins of picornaviruses, adenoviruses and bacteriophages . The data obtained suggested that a conservative N-terminal region of P proteins functions as protein primer for DNA synthesis in hepadnaviruses. Nucleic Acids Res, 1989 Jan 25, 17(2), 659 - 74 Excess information at bacteriophage T7 genomic promoters detected by a random cloning technique; Schneider TD et al.; In our previous analysis of the information at binding sites on nucleic acids, we found that most of the sites examined contain the amount of information expected from their frequency in the genome . The sequences at bacteriophage T7 promoters are an exception, because they are far more conserved (35 bits of information content) than should be necessary to distinguish them from the background of the Escherichia coli genome (17 bits) . To determine the information actually used by the T7 RNA polymerase, promoters were chemically synthesized with many variations and those that function well in an in vivo assay were sequenced . Our analysis shows that the polymerase uses 18 bits of information, so the sequences at phage genomic promoters have significantly more information than the polymerase needs . The excess may represent the binding site of another protein. Biochemistry, 1989 Jan 24, 28(2), 691 - 9 Low-temperature unfolding of a mutant of phage T4 lysozyme . 2 . Kinetic investigations; Chen BL et al.; A disulfide-bridged variant of bacteriophage T4 lysozyme has been found to undergo a low- as well as high-temperature unfolding transition in guanidinium chloride {see Chen and Schellman (1989)} . The kinetics for this process have been followed for several temperatures, a range of guanidinium chloride concentrations, and a number of values of pH . Microscopic rate constants for protein unfolding and refolding were extracted from these data to explore the nature of the cold unfolding transition . The data were interpreted using transition-state theory . It was found that the Arrhenius energy is temperature dependent . The transition state is characterized by (1) a high energy and low entropy compared to the native state, (2) a heat capacity which is closer to the native state than to the unfolded state, and (3) a low exposure to solvent compared to the unfolded state, as judged by its interaction with guanidinium chloride . With increasing concentration of guanidinium chloride, the low-temperature unfolding rate increases strongly, and the refolding rate decreases very strongly. J Theor Biol, 1989 Jan 23, 136(2), 135 - 50 Four strand recombination models; McGavin S; A main point of this paper is to develop the idea that synapsis of DNA duplexes might take place by Watson-Crick base pairing between essentially intact duplex structure to form a four stranded intermediate . This intermediate is the same regular and compact four strand structure already discussed (e.g., McGavin, 1971a, J . molec . Biol., 55, 293-298; 1979, J . theor . Biol., 77, 83-99) and which has been used in several related recombination models (see, for example, McGavin, 1977, Heredity, 39, 15-25; 1984, J . theor . Biol., 107, 37-56; Wilson, 1979, Proc . natn . Acad . Sci . U.S.A., 76, 3641-3645; Nash et al., 1980, Cold Spring Harbor Symposium of Quantitative Biology, 45, 417-428; Nash, 1981, A . Rev . Genetics, 15, 143-167) . These models can also be related to one recently suggested by Hopkins (1986, J . theor . Biol . 120, 215-222) . An immediate stimulus to the development of the idea was the recent work of Griffith & Nash (1985, Proc . natn . Acad . Sci . U.S.A., 82, 3124-3128), on the site specific recombination system of the lambda bacteriophage . They observed trefoil knots with exclusively positive nodes among the products of the interaction of two relatively inverted attachment sites within circular molecules . The model discussed here may though be of interest in itself . The paper also compares several closely related models for recombination which involve formation of either identical or closely related four strand secondary structures. J Mol Biol, 1989 Jan 20, 205(2), 407 - 19 Molecular design of PhoE porin and its functional consequences; Jap BK; The three-dimensional structure of PhoE porin from Escherichia coli, negatively stained with uranyl acetate, has been determined by electron crystallographic techniques to a resolution of about 18 A . The structure shows that PhoE porin consists of trimeric stain-filled channels as the basic unit . The trimeric channels converge as they transverse the membrane but they do not merge . Our three-dimensional structure of PhoE porin indicates that there is a short, narrower segment of channel, which extends beyond the visible strain-filled portion of the channel . The map of glucose-embedded PhoE porin in projection normal to the membrane has also been determined to a resolution of 6.5 A . The projected map shows trimeric ring-like structures, which are presumably cylindrical domains of beta-sheet . At the 3-fold symmetry axis of the trimer, there is a low density region, which is suggested to be a site of lipopolysaccharide that is required for channel and bacteriophage receptor activities . The structural model of the PhoE monomer consists of a flattened cylinder with a large water-filled vestibule about 35 A long with an elliptically shaped opening that is 27 A along the major axis and 18 A along the minor axis . The vestibule has a narrower extension about 10 A long with an average diameter of about 10 A . The vestibule wall is formed by beta-sheet, which may have a large fraction of the beta-strands oriented normal to membrane . Our structural model provides a clue as to how the surface charges on the outer membrane may regulate the permeation of ionic solutes through the channel. J Mol Biol, 1989 Jan 20, 205(2), 397 - 405 Structure and inherent properties of the bacteriophage lambda head shell . VI . DNA-packaging-defective mutants in the major capsid protein; Katsura I; Some amino acid substitutions in the major capsid protein (gene E product) of lambda phage are found to cause a defect in DNA packaging . These substitutions permit initiation of DNA packaging and expansion of the prohead . However, cleavage of the concatemer DNA at the cos site takes place only to a very small extent, and the capsid eventually becomes empty . Interestingly, the mutations are suppressed by a decrease of the DNA length between the cos sites by 8000 to 10,000 bases . These properties are similar to those of amber mutants in gene D, which codes for the capsid outer-surface protein . Studies on the E missense.D amber double mutant show that the E protein and the D protein contribute additively to the stabilization of the condensed form of the DNA molecule in phage heads. Mol Biochem Parasitol, 1989 Jan 15, 32(2-3), 153 - 61 cDNA encoding an immunogenic region of a 22 kilodalton surface protein of Eimeria acervulina sporozoites; Jenkins MC et al.; cDNA encoding an immunogenic region of a 22 kDa surface protein of Eimeria acervulina sporozoites was cloned and expressed in the bacteriophage lambda gt11 vector . The recombinant beta-galactosidase fusion protein, designated MA1, has an apparent molecular size of 125 kDa . Immunofluorescence staining of intact E . acervulina sporozoites and merozoites and immunoblotting of 125I-surface labeled protein from both stages revealed exclusive expression of the cloned cDNA in the sporozoite stage . The gene encoding the 22 kDa surface protein appears to exist as a single copy sequence as revealed by Southern blot hybridization utilizing the cDNA insert as a probe . Although not recognized by immune serum, purified recombinant MA1 antigen induced significant in vitro activation of T lymphocytes obtained from chickens immune to E . acervulina . DNA sequencing and hydropathic analysis of the predicted amino acid sequence revealed a central hydrophilic region surrounded by two hydrophobic areas which may represent exposed and transmembrane regions of the protein. J Biol Chem, 1989 Jan 15, 264(2), 910 - 9 Characterization of mRNA species related to human liver cytochrome P-450 nifedipine oxidase and the regulation of catalytic activity; Bork RW et al.; P-450NF is the major enzyme in human liver involved in the metabolism of the calcium-channel blocker nifedipine . By screening a bacteriophage lambda gt11 expression library, a cDNA clone designated NF 10 with an insert length of 2.8 kilobases (kb) was isolated . This clone was sequenced and found to be highly similar in its overlapping section with sequences of two other cDNA clones previously isolated from the same expression library, NF 25 (Beaune, P . H., Umbenhauer, D . R., Bork, R . W., Lloyd, R . S., and Guengerich, F . P . (1986) Proc . Natl . Acad . Sci . U . S . A . 83, 8064-8068) and HLp (Molowa, D . T., Schuetz, E . G., Wrighton, S . A., Watkins, P . B., Kremers, P., Mendez-Picon, G., Parker, G . A., and Guzelian, P . S . (1986) Proc . Natl . Acad . Sci . U . S . A . 83, 5311-5315) . However, clone NF 10 had an extra 814 or 813 bases of 3'-noncoding sequence relative to NF 25 or HLp, respectively, and this additional sequence contained a second consensus polyadenylation signal . Specific oligonucleotides were synthesized to differentiate between these three clones at the mRNA level . Oligonucleotides specific to the protein coding region of each clone were found to hybridize to mRNAs of 2.2 and 3.0 kb in size at a ratio of approximately 10:1 . The major species of hybridizable mRNA was specific to clone NF 25, and levels of this mRNA could be correlated with levels of immunochemically detectable P-450NF and nifedipine oxidase activity in individual human liver samples . In addition, an oligonucleotide specific to the 3'-noncoding region of clone NF 10 hybridized only with the 3.0-kb mRNA . We conclude that alternative use of the second polyadenylation signal present in clone NF 10 results in production of the 3.0-kb mRNA species and that a pretranslational control mechanism is primarily involved in the regulation of nifedipine oxidase activity. Nucleic Acids Res, 1989 Jan 11, 17(1), 301 - 15 The inconsistent distribution of introns in the T-even phages indicates recent genetic exchanges; Quirk SM et al.; Group I self-splicing introns are present in the td, nrdB and sunY genes of bacteriophage T4 . We previously reported that whereas the td intron is present in T2, T4 and T6, the nrdB intron is present in T4 only . These studies, which argue in favor of introns as mobile genetic elements, have been extended by defining the distribution of all three T4 introns in a more comprehensive collection of T2, T4 and T6 isolates . The three major findings are as follows: First, all three introns are inconsistently distributed throughout the T-even phage family . Second, different T2 isolates have different intron complements, with T2H and T2L having no detectable introns . Third, the intron open reading frames are inherited or lost as a unit with their respective flanking intron core elements . Furthermore, exon sequences flanking sites where introns are inserted in the T4 td, sunY and nrdB genes were determined for all the different T-even isolates studied . Six of eighteen residues surrounding the junction sequences are identical . In contrast, a comprehensive comparison of exon sequences in intron plus and intron minus variants of the sunY gene indicate that sequence changes are concentrated around the site of intron occurrence . This apparent paradox may be resolved by hypothesizing that the recombination events responsible for intron acquisition or loss require a consensus sequence, while these same events result in sequence heterogeneity around the site. Biochemistry, 1989 Jan 10, 28(1), 71 - 6 Specific RNA binding by Q beta coat protein; Witherell GW et al.; The interaction between the bacteriophage Q beta coat protein and its specific binding site on Q beta genomic RNA was characterized by using a nitrocellulose filter binding assay . Q beta coat protein bound to a synthetic 29-nucleotide RNA hairpin with an association constant of 400 microM-1 at 4 degrees C, 0.2 M ionic strength, pH 6.0 . Complex formation had a broad pH optimum centered around pH 6.0 and was favored by both enthalpy and entropy . The salt dependence of Ka revealed that four to five ion pairs may be formed in the complex although approximately 80% of the free energy of complex formation is contributed by nonelectrostatic interactions . Truncation experiments revealed that coat protein binding required only the presence of a hairpin with an eight base pair stem and a three-base loop . Analysis of the binding properties of hairpin variants showed that the sequence of the stem was not important for coat protein recognition and only one of the three loop residues was essential . A bulged adenosine present in the coat protein binding site was not required for coat protein binding . Q beta coat protein binding specific is therefore primarily achieved by the structure and not by the sequence of the operator. J Mol Biol, 1989 Jan 5, 205(1), 127 - 35 ban operon of bacteriophage P1 . Mutational analysis of the c1 repressor-controlled operator; Heinzel T et al.; The repressor of bacteriophage P1, encoded by the c1 gene, represses the phage lytic functions and is responsible for maintaining the P1 prophage in the lysogenic state . The c1 repressor interacts with at least 11 binding sites or operators widely scattered over the P1 genome . From these operators, a 17 base-pair asymmetric consensus sequence, ATTGCTCTAATAAATTT, was derived . Here, we show that the operator, Op72 of the P1ban operon consists of two overlapping 17 base-pair sequences a and b forming an incomplete palindrome . Op72a matches the consensus sequence, whereas Op72b contains two mismatches . The evidence is based on the sequence analysis of 27 operator mutants constitutive for ban expression . They were identified as single-base substitutions at positions 2 to 10 of Op72a (26 mutants) and at position 8 of Op72b (one mutant) . We conclude from gel retardation and footprinting studies that two repressor molecules bind to the operator and that positions 4, 5 and 7 to 10 of the operator play an essential role in repressor recognition. J Mol Biol, 1989 Jan 5, 205(1), 1 - 13 Structure of the Drosophila DNA topoisomerase II gene . Nucleotide sequence and homology among topoisomerases II; Wyckoff E et al.; We have determined the nucleotide sequence of the Drosophila DNA topoisomerase II gene . Data from primer extension and S1 nuclease protection experiments were combined with comparisons of genomic and cDNA sequences to determine the structure of the mature messenger RNA . This message has a large open reading frame of 4341 nucleotides . The length of the predicted protein is 1447 amino acids with a molecular weight of 164,424 . Topoisomerase II can be divided into three domains: (1) an N-terminal region with homology to the B (ATPase) subunit of the bacterial type II topoisomerase, DNA gyrase; (2) a central region with homology to the A (breaking and rejoining) subunit of DNA gyrase; (3) a C-terminal region characterized by alternating stretches of positively and negatively charged amino acids . DNA topoisomerase II from the fruit fly shares significant sequence homology with those from divergent sources, including bacteria, bacteriophage T4 and yeasts . The location and distribution of homologous stretches in these sequences are analyzed. FEBS Lett, 1989 Jan 2, 242(2), 444 - 6 Interaction between bacteriophage T4 coded gene 32 protein and poly(rA); Watanabe F; The cooperative binding of T4 gene 32 protein with polynucleotides, of which the quantitative aspects in the literature have not satisfied the requirements of thermodynamics, is studied by adopting a modified formula of the lattice theory . A moderate value is found for the cooperativity parameter (q approximately 200 at 0.2 M NaCl), which is weakly dependent on salt concentration . The cation effect on the binding suggests that the shielding of negative charges of the protein or a loose cation bridge between the bound protein molecules plays a role in the cooperative binding process. Eur J Biochem, 1989 Jan 2, 178(3), 663 - 74 Regulation of gene expression at the translational level . The rescue factor reverses thermosensitive protein synthesis in N4316, a conditionally-lethal mutant of Escherichia coli defective in translation; Ganoza MC et al.; Extracts of the conditionally-lethal mutant Escherichia coli N4316 are defective in a newly described translation factor, the rescue protein . We have analyzed the in vitro translation products of this mutant by gel electrophoresis during normal and arrested synthesis at the permissive and non-permissive temperatures . Translation programmed with MS2 bacteriophage RNA at the non-permissive temperature results in highly reduced synthesis of the coat protein with no detectable levels of the maturation and replicase products . Thus the relative number of copies of proteins synthesized by the ribosomes is altered in this mutant . In addition, there is mistranslation of the coat gene which results in the overproduction of the phage encoded no . 7 protein . Aberrant synthesis is also reflected in the increased read-through of termination codons during synthesis directed by phage RNAs harbouring amber mutations in the coat cistron . The rescue protein, purified from the parental strain, is able to complement the thermosensitive defect and restore proper synthesis . Biochemical characterization of the defect in the absence of rescue shows no detectable deficiency in the extent of initiation complex formation in reactions inhibited with sparsomycin . Peptidyltransferase is fully active as judged by the kinetics of formylmethionine-puromycin formation . However, rescue does exert an effect at the level of termination . In addition, the thermolability of the mutant can be reversed by dissociating 70S ribosomes into 30S and 50S subunits . Based on these and other observations, we propose tht rescue mediates a novel function in the association/dissociation of ribosomal subunits which is essential to the accuracy and efficiency of translation. Immunogenetics, 1989, 29(3), 186 - 90 Sequence analysis of HLA class II domains: characterization of the DQw3 family of DQB genes; Hiraiwa A et al.; HLA class II allelic variants within the DQw3-related family of genes carry distinct allo-specificities and have been implicated in specific HLA-disease associations, such as insulin-dependent diabetes mellitus . To investigate the nucleotide variations which characterize DQw3 genes, we applied a novel cDNA cloning strategy that uses a single-stranded vector/primer system to facilitate DNA sequencing of allelically variable gene families . Using a DQB-specific primer sequence and M13 bacteriophage as the cloning vector, direct cloning and sequencing of multiple DQB genes was performed without the need for second strand synthesis or for subcloning . Sequence analysis from eight lymphoblastoid cell lines selected to represent different ethnic backgrounds revealed three DQw3-related DQB genes, DQB3.1, 3.2, and 3.3, corresponding to the newly designated HLA-DQw7, w8, and w9 specificities, respectively . An unusual Pro-Pro couplet at codons 55-56 is characteristic of all DQw3-positive sequences and may be contributing to the broad DQw3 allospecificity . Comparisons among ethnically disparate DQw3-related sequences showed no additional expressed or silent nucleotide substitutions among these DQB alleles . Thus, polymorphism within the DQw3 family of genes appears to be extremely limited, with a paucity of nucleotide variations accumulated by evolutionary distance. J Virol, 1989 Jan, 63(1), 460 - 2 In vitro transcription of bacteriophage phi 29 DNA: inhibition of early promoters by the viral replication protein p6; Barthelemy I et al.; The effect of the phi 29 protein p6 on the in vitro initiation of transcription from the main phi 29 promoters was studied . Protein p6 interfered with the transcription from the early promoters C1 and C2, located at the right end of the phi 29 genome . Transcription initiated at the early promoters A1, A2b, and A2c and at the late promoter A3, located at the left region of the viral genome, was not affected by protein p6 . These results suggest that protein p6 might play a dual role, acting as a negative modulator of some phi 29 early promoters apart from being a positive modulator of the viral DNA replication. J Electron Microsc (Tokyo), 1989, 38 Suppl, S105 - 9 Ultrahigh resolution scanning electron microscopy of biological materials; Tanaka K; In recent years, several ultrahigh resolution scanning electron microscopes (SEM) were successively developed . They were all equipped with a field emission electron gun and an objective lens with a short focal length, and showed a resolution better than 1 nm . With such instrument, not only intracellular structures but also virus, bacteriophages, and biological macromolecules were clearly observed . With improvement of the instrumental resolution, some unexpected problems came to the fore in the area of specimen preparation . The first is the metal coating of the specimen, because coated metal particles are plainly seen as rounded "pebbles" at very high magnifications . For observations at the high magnifications, therefore, uncoated and not conductively stained specimens were used . The second problem is contamination by the electron beam . This problem is complicated and remains unsolved . Although the ultra-high resolution scanning electron microscopy has just begun, it will surely open new research fields in biomedicine. Gene, 1989, 76(2), 245 - 54 Specificity of the nick-closing activity of bacteriophage T4 DNA ligase; Wu DY et al.; Bacteriophage T4 DNA ligase effectively joins two adjacent, short synthetic oligodeoxyribonucleotides (oligos), as guided by complementary oligo, plasmid and genomic DNA templates . When a single bp mismatch exists at either side of the ligation junction, the efficiency of the enzyme to ligate the two oligos decreases . Mismatch ligation is approximately five-fold greater if the mismatch occurs at the 3' side rather than at the 5' side of the junction . During mismatch ligation the 5' adenylate of the 3' oligo accumulates in the reaction . The level of the adenylate formation correlates closely with the level of the mismatch ligation . Both mismatch ligation and adenylate formation are suppressed at elevated temperatures and in the presence of 200 mM NaCl or 2-5 mM spermidine . The apparent Km for the oligo template in the absence of salt is 0.05 microM, whereas the Km increases to 0.2 microM in the presence of 200 mM of NaCl . In this report, we demonstrate these properties of T4 DNA ligase for oligo pairs complementary to the beta-globin gene at the sequence surrounding the single bp mutation responsible for sickle-cell anemia . Because of the highly specific nature of the nick-closing reaction, ligation of short oligos with DNA ligase can be used to distinguish two DNA templates differing by a single nucleotide. Biol Cell, 1989, 65(2), 89 - 98 High resolution scanning electron microscopy of the cell; Tanaka K; The scanning electron microscope (SEM) has become a powerful tool for ultrastructural research with improvement of the instrument's resolution and progress in specimen preparation techniques . With regard to resolution, it has been improved step-by-step in this decade and, in 1985, an ultra-high resolution SEM (UHS-T1) was developed, with a resolution of 0.5 nm . Concerning specimen preparation, the osmium-DMSO-osmium method, which is effective for revealing intracellular structures, has come to be widely used . Techniques for observing smaller objects, such as bacteriophages, viruses, and biological macromolecules, have also been devised in recent years . As a result of these preparation techniques and the availability of the ultra-high resolution SEM, the application of SEM in biology is expanding rapidly . In this paper, an outline of the ultra-high resolution SEM, techniques for specimen preparation, findings of some biological materials by these techniques, and guidelines to making the specimens, are described. Lasers Surg Med, 1989, 9(3), 296 - 9 In vitro production of viable bacteriophage in a laser plume; Ediger MN et al.; Bacteriophage phi X 174, present at high concentrations in an agar overlay in vitro system, were used as a target for pulsed 2.94 microns (Er:YAG) laser ablation . In this preliminary experiment, the potential for transport of viable viruses in the photoablation plume was investigated for laser energy densities of approximately 3.5 J/cm2 per pulse in exposures of 75-600 laser pulses . Transport of relatively few numbers of viable viruses from the ablation site to the plate assay detector, a distance of approximately 2 cm, was observed. Bull Math Biol, 1989, 51(1), 79 - 94 Stochastic models for heterogeneous DNA sequences; Churchill GA; The composition of naturally occurring DNA sequences is often strikingly heterogeneous . In this paper, the DNA sequence is viewed as a stochastic process with local compositional properties determined by the states of a hidden Markov chain . The model used is a discrete-state, discrete-outcome version of a general model for non-stationary time series proposed by Kitagawa (1987) . A smoothing algorithm is described which can be used to reconstruct the hidden process and produce graphic displays of the compositional structure of a sequence . The problem of parameter estimation is approached using likelihood methods and an EM algorithm for approximating the maximum likelihood estimate is derived . The methods are applied to sequences from yeast mitochondrial DNA, human and mouse mitochondrial DNAs, a human X chromosomal fragment and the complete genome of bacteriophage lambda. Proteins, 1989, 5(4), 266 - 70 Single crystals of bacteriophage T7 RNA polymerase; Sousa R et al.; Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 x 0.3 x 0.3 mm . The crystals are composed of fully intact T7 RNA polymerase which is enzymatically active upon dissolution . These crystals belong to the monoclinic space group P2(1) and have unit cell parameters a = 114.5 A, b = 139.6 A, c = 125.7 A, and beta = 98.1 degrees . Self-rotation function studies indicate that there are three molecules per asymmetric unit . The crystals diffract to at least 3.0 A resolution . These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination. Ann Ist Super Sanita, 1989, 25(1), 155 - 61 Mutagenic specificity of alkylated and oxidized DNA bases as determined by site-specific mutagenesis; Essigmann JM et al.; This work demonstrates the use of the tools of site-specific mutagenesis to study the mutagenic activity of two DNA adducts, O6-methylguanine and cis-thymine glycol . The former adduct is one of the methylated bases formed by carcinogenic and mutagenic alkylating agents . It was built into the single-stranded genome of bacteriophage M13 and replicated in Escherichia coli (E . coli) . The mutation frequency of O6-methylguanine was 0.4% in physiologically normal cells . In cells in which the repair systems for O6-methylguanine were compromised by challenge with an alkylating agent, the mutation frequency rose to approximately 20% . DNA sequencing revealed that O6-methylguanine induced exclusively G----A transitions, which was most consistent with it pairing with thymine during DNA synthesis . The mutagenic effects also were investigated of cis-thymine glycol isomers, which are major, stable products of ionizing radiation and oxidative damage to DNA . By techniques similar to those employed for the study of O6-methylguanine mutagenesis, a single thymine glycol was situated in an M13 phage genome . The genome was replicated in E . coli that were physiologically normal, induced for SOS functions, or deficient in the nth gene product and, in all cases, the mutagenic processing of thymine glycol in vivo yielded mutant progeny phage at a frequency of 0.3-0.4% . All mutations occurred at the site that originally contained thymine glycol, and all were demonstrated by DNA sequencing to have resulted from targeted T----C transitions . These data suggest that thymine glycol pairs with guanine during replication. Chem Biol Interact, 1989, 70(3-4), 249 - 62 DNA alkylation and formation of DNA interstrand cross-links by potential antitumour 2,5-bis(1-aziridinyl)-1,4-benzoquinones; Lusthof KJ et al.; A series of 3,6-substituted 2,5-bis(1-aziridinyl)-1,4-benzoquinone derivatives was shown to alkylate calf thymus DNA and to form DNA interstrand cross-links . Alkylation and cross-link formation were enhanced after electrochemical reduction of the compounds and increased with lower pH in the pH range from 4.5 to 8.0 . Reduction especially shifts the pH at which cross-linking and alkylation occurs to higher values, which are more physiologically relevant . This shift is probably caused by the increase in pKa value of the aziridine ring after reduction of the quinone moiety . The inactivation of single-stranded bacteriophage M13mp19 DNA to form phages in an E . coli host, by the 3,6-unsubstituted parent compound 2,5-bis(1-aziridinyl)-1,4-benzoquinone (TW13) was dependent upon reduction and pH in a similar way as was alkylation . The compound in our series with the least bulky, 3,6-substituents, TW13, caused a high amount of cross-link formation . Compounds with methyl-substituted aziridine rings showed low cross-linking ability . Our results support the concept that the protonated reduced compound is the reactive species that alkylates DNA, and that steric factors play an important role in the reactivity towards DNA . A correlation is observed between the ability to induce DNA interstrand cross-links and inactivation of M13mp19 bacteriophage DNA . Cross-link formation was also demonstrated in E . coli K12 cells, where the compounds are reduced endogenously by bacterial reductases. Nucleic Acids Res, 1989, 17 Suppl, 283 - 309 Compilation of DNA sequences of Escherichia coli; Kroger M; We have compiled the DNA sequence data for E . coli K12 available from the GENBANK and EMBO databases and over a period of several years independently from the literature . We have introduced all available genetic map data and have arranged the sequences accordingly . As far as possible the overlaps are deleted and a total of 940,449 individual bp is found to be determined till the beginning of 1989 . This corresponds to a total of 19.92% of the entire E . coli chromosome consisting of about 4,720 kbp . This number may actually be higher by some extra 2% derived from the sequence of lysogenic bacteriophage lambda and the various insertion sequences . This compilation may be available in machine readable form from one of the international databanks in some future. Mol Gen Genet, 1989 Jan, 215(2), 245 - 9 A mutational analysis of the bacteriophage P1 cin recombinase gene: intragenic complementation; Haffter P et al.; Bacteriophage P1 encodes a site-specific recombinase, Cin, which regulates the alternate expression of tail fibre genes by inverting a DNA segment . To define regions of Cin important for the recombination process, we have isolated and characterised 24 different mutations of the cin gene . Most of these mutations affected amino acids that are highly conserved in other related recombinases . Some of these mutants complement each other in vivo . This intragenic complementation could be due to the assembly of heteromers containing both mutant proteins, suggesting that the active enzyme is at least a dimer. Lasers Surg Med, 1989, 9(1), 67 - 9 Role of primary photoacceptors in low-power laser effects: action of He-Ne laser radiation on bacteriophage T4-Escherichia coli interaction; Tiphlova O et al.; The effect of He-Ne laser radiation (lambda = 632.8 nm) on bacteriophage T4-Escherichia coli WP2 interactions was studied . Irradiation of bacteria having respiratory chain components as primary photoacceptors accelerated their division in a dose-dependent manner, but irradiation had no effect on the properties of the phage (measured as its ability to infect host cells) . At the same time, exposure of bacteria to stimulating doses of He-Ne laser radiation (from 10(3) to 6 x 10(4) J/m2) increased their ability to promote the growth of unexposed phages . These results clearly indicate that low-power laser effects require primary photoacceptors (phage contains no chromophores for red light). J Bacteriol, 1989 Jan, 171(1), 488 - 97 Cloning and identification of bacteriophage T4 gene 2 product gp2 and action of gp2 on infecting DNA in vivo; Lipinska B et al.; We sequenced bacteriophage T4 genes 2 and 3 and the putative C-terminal portion of gene 50 . They were found to have appropriate open reading frames directed counterclockwise on the T4 map . Mutations in genes 2 and 64 were shown to be in the same open reading frame, which we now call gene 2 . This gene codes for a protein of 27,068 daltons . The open reading frame corresponding to gene 3 codes for a protein of 20,634 daltons . Appropriate bands on polyacrylamide gels were identified at 30 and 20 kilodaltons, respectively . We found that the product of the cloned gene 2 can protect T4 DNA double-stranded ends from exonuclease V action. Curr Stud Hematol Blood Transfus, 1989, (56), 122 - 7 Inactivation of hepatitis viruses and HIV in plasma and plasma derivatives by treatment with beta-propiolactone/UV irradiation; Stephan W; A combined treatment of plasma or plasma derivatives by beta-propiolactone (beta-PL)/UV irradiation is in use at Biotest for the preparation of the virus-safe, stabilized serum (Biseko) and coagulation factor concentrates . The efficacy of this sterilization procedure has been demonstrated for HAV (greater than 8.2 log10), HBV (6.9 log10), NANBHV (greater than 4.5 log10) and HIV (greater than 6.0 log10) . The methods used in these studies (titration in chimpanzees or cell cultures) are not applicable in routine monitoring of sterilization processes . We therefore developed a test system using four types of bacteriophages: phi X174, phi e, Kappa and f2 . Using these bacteriophages in 88 single tests, sterilization efficacy was regularly monitored during the period from 1981 to 1986 . The four types of bacteriophages showed, on average, an inactivation rate of 6.7 log10, independent of size or genome structure . This inactivation is in the range of the inactivation of the relevant pathogenic virus, HBV, by beta-PL/UV . It was shown that under the production conditions of Intraglobin and the other Biotest immunoglobulin preparations, beta-PL (without UV) is as virucidal as the combination of beta-PL/UV in plasma or cryo-poor plasma. Acta Cient Venez, 1989, 40(2), 124 - 6 {Stable recombinants of bacteriophage M13 and plasmid pBR322}; Dagert M; Two recombinants between the phage M13 and the plasmid pBR322 were isolated, analyzing the plasmid content of over one hundred colonies obtained by transduction . The study of the structure of both recombinants indicates that a fragment of the M13 genome has been integrated to pBR322 . In both cases, the fragment contains a part of the phage replication region inserted either in the vicinity or within the pBR322 replicon . The fact that the phage and plasmid replicons seem to be involved in the recombination event suggests that it is helpful when the replication begins . So far it has not been possible to isolate a recombinant taking the whole genomes of pBR322 and M13 . This is, undoubtedly, due to the instability of the recombinant molecule. Anim Genet, 1989, 20(3), 267 - 78 Cloning of the bovine major histocompatibility complex class II genes; Groenen MA et al.; Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library . The library was constructed in the bacteriophage lambda vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome . Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes . Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis . The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail . These analyses showed that these 37 clones were derived from five different A and 10 different B genes . The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes . Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle. Acta Microbiol Hung, 1989, 36(4), 377 - 413 New developments on the generation of mutations in Escherichia coli lysogens; Lieber M; Through the lytic process, P1 is a bacteriophage or virion capable at very low frequency of carrying out generalized transduction between strains of Escherichia coli . When P1 is not involved in lytic functions, it exists as a prophage in the form of circular DNA molecule, persisting in an extra-chromosomal or plasmid state, and not integrating into the host chromosome . E . coli carrying such plasmids have been referred to as lysogens . Earlier research dealt with P1 plasmids carrying drug-resistant factors . Up to the present study, P1 plasmids of any type were not known to have any mutagenic effect on the E . coli chromosome (genome), nor was it known that generalized transduction can be associated with mutagenicity . From P1 plasmids carrying a chloramphenicol resistance-factor, mutant plasmids of a particular type were isolated in the present study . P1 plasmids of this type carried a mutant factor which greatly impaired the capacity of phage derived from such plasmids, upon the completion of the lytic cycle, to lysogenize recA E . coli through phage promoted recombination . The plasmid of this mutant type is referred to as P1CMrec, and lysogens carrying such are referred to as P1CMrec lysogens . This paper describes the history of these P1CMrec lysogens and the genetic mutability within the E . coli chromosome of such P1CMrec lysogens, and the relationship of such genetic mutability (instability) to the incorporation of virion-DNA, following the absorption of P1 phage by these lysogens . As illustrated in the paper, a mutagenic effect was generated within the E . coli chromosome of P1CMrec lysogens by means of the P1CMrec plasmid . Furthermore, this mutagenic effect was found to be greatly, non-locally, and uniformly enhanced as a consequence of P1 virion incorporation by, or likely generalized transduction of, such lysogens . More specifically, the plasmid of this mutant type (P1CMrec) is responsible for the creation of a wide range of genetic mutabilities (instabilities) of differing degree within the E . coli genome (not carrying recA), some mutabilities being very high upon extended incubation . The P1CMrec plasmid was also involved in the creation of new mutant genes within the E . coli genome (not carrying recA), some mutabilities being very high upon extended incubation . The P1CMrec plasmid was also involved in the creation of new mutant genes within the E . coli chromosome, some of which manifested high mutability.(ABSTRACT TRUNCATED AT 400 WORDS) Genome, 1989, 31(2), 594 - 6 Amino acid similarities to other proteins offer insights into roles of UmuD and UmuC in mutagenesis; Battista JR et al.; The products of the umuD and umuC genes are required for most uv and chemical mutagenesis in Escherichia coli . The genes are organized in an operon that is repressed by LexA and regulated as part of the SOS response . The umuD protein shares homology with the carboxyl-terminal domain of LexA . Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis . The COOH-terminal fragment of UmuD is both necessary and sufficient for this role . Similarities of UmuD to gene 45 protein of bacteriophage T4 and of UmuC to gene 44 protein and gene 62 protein suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence. Genome, 1989, 31(1), 53 - 67 Recombination of bacteriophage lambda in recD mutants of Escherichia coli; Thaler DS et al.; RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity . Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme . Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity . However, mutants lacking the D subunit are competent for homologous recombination . We report that the distribution of exchanges along the chromosome of Red-Gam-phage lambda is strikingly altered by recD null mutations in the host . When lambda DNA replication is blocked, recombination in recD mutant strains is high near lambda's right end . In contrast, recombination in isogenic recD+ strains is approximately uniform along lambda unless the lambda chromosome contains a chi sequence . Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on lambda . The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent chi) . The distribution of exchanges in recD mutants is strongly affected by lambda DNA replication . The distribution of exchanges on lambda growing in rec+ cells is not influenced by DNA replication . The exchange distribution along lambda in recD mutant cells is independent of chi in a variety of conditions . Recombination in rec+ cells is chi influenced . Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for lambda recombination via the RecF pathway . We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break--repair, as it does in lambda's Red pathway and E . coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a chi sequence. Biofizika, 1989 Jan-Feb, 34(1), 130 - 2 {DNA-recognizing structure helix-turn-helix in replication initiators from polyomavirus large T antigens}; Shestopalov BV; Evidence is presented which supports a suggestion that in large T antigens from polyomaviruses-serving as replication initiators--there is DNA-recognizing structure helix-turn-helix, firstly observed in transcription regulators of E . coli and some bacteriophages . It is suggested that all these proteins recognize DNA by the same mechanism. J Bacteriol, 1989 Jan, 171(1), 392 - 401 Turning off flagellum rotation requires the pleiotropic gene pleD: pleA, pleC, and pleD define two morphogenic pathways in Caulobacter crescentus; Sommer JM et al.; We have identified mutations in three pleiotropic genes, pleA, pleC, and pleD, that are required for differentiation in Caulobacter crescentus . pleA and pleC mutants were isolated in an extensive screen for strains defective in both motility and adsorption of polar bacteriophage phi CbK; using temperature-sensitive alleles, we determined the time at which the two genes act . pleA was required for a short period at 0.7 of the swarmer cell cycle for flagellum biosynthesis, whereas pleC was required during an overlapping period from 0.6 to 0.95 of the cell cycle to activate flagellum rotation as well as to enable loss of the flagellum and stalk formation by swarmer cells after division . The third pleiotropic gene, pleD, is described here for the first time . A pleD mutation was identified as a bypass suppressor of a temperature-sensitive pleC allele . Strains containing this mutation were highly motile, did not shed the flagellum or form stalks, and retained motility throughout the cell cycle . Since pleD was required to turn off motility and was a bypass suppressor of pleC, we conclude that it acts after the pleA and pleC gene functions in the cell cycle . No mutants defective in both flagellum biosynthesis and stalk formation were identified . Consequently, we propose that the steps required for formation of swarmer cells and subsequent development into stalked cells are organized into at least two developmental pathways: a pleA-dependent sequence of events, responsible for flagellum biosynthesis in predivisional cells, and a pleC-pleD-dependent sequence, responsible for flagellum activation in predivisional cells and loss of motility and stalk formation in progeny swarmer cells. J Virol, 1989 Jan, 63(1), 216 - 25 Purification and properties of poliovirus RNA polymerase expressed in Escherichia coli; Plotch SJ et al.; A cDNA clone encoding the RNA polymerase of poliovirus has been expressed in Escherichia coli under the transcriptional control of a T7 bacteriophage promoter . The poliovirus enzyme was designed to contain only a single additional amino acid, the N-terminal methionine . The recombinant enzyme has been purified to near homogeneity, and polyclonal antibodies have been prepared against it . The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity as well as RNA polymerase activity . In the presence of an oligo(U) primer, the enzyme catalyzes the synthesis of a full-length copy of either poliovirus or globin RNA templates . In the absence of added primer, RNA products up to twice the length of the template are synthesized . When incubated in the presence of a single nucleoside triphosphate, {alpha-32P}UTP, the enzyme catalyzes the incorporation of radioactive label into template RNA . These results are discussed in light of previously proposed models of poliovirus RNA synthesis in vitro. Appl Theor Electrophor, 1989, 1(3), 169 - 73 A hybrid mode of rotating gel electrophoresis for separating linear and circular duplex DNA; Louie D et al.; During agarose gel electrophoresis, alternately applying the electrical potential gradient (E) in two directions (separated by an angle, psi) has been used to enhance the separation by length of linear, double-stranded DNA; the value of psi is usually between 0.5 pi and 0.7 pi radians (Cantor et al . (1988) . Ann . Rev . Biophys . Biophys . Chem . 17, 287-304) . When the direction of E is changed by rotating the gel (rotating gel electrophoresis, or RGE), open circular DNA longer than 48 Kb is found here to form bands that decrease in either sharpness or migration distance as psi is decreased from 1.1 pi to 0.5 pi radians . A satisfactory band is formed only for psi greater than or equal to 1.1 pi radians . In contrast, the previously-demonstrated separation and banding of linear DNAs (48.5-800 Kb) was observed for psi values between 0.6 pi and 1.4 pi radians . The cause of psi's effect on open circular DNA is electrophoresis during the gel's rotation . RGE by use of psi values above 1.0 pi is a hybrid mode of RGE, incorporating favorable characteristics of both the usual mode and a more recently demonstrated (Serwer, P . and Hayes, S.J . (1989) . Applied and Theoretical Electrophoresis, 1, 95-98) mode in which psi is 2 pi radians . To separate linear from open circular DNA and to fractionate by length both of these conformational forms, the following procedure of two-dimensional agarose gel electrophoresis has been developed: (a) a first dimension performed by use of RGE and a psi of 1.4 pi radians, (b) a second dimension performed with an invariant electrical field . Circular DNA has been found among the bacteriophage lambda DNA concatemers (48.5-800 Kb) used as length standards . By use of a psi = 1.4 pi radians, the band of 48.5 Kb monomeric open circular lambda DNA is sometimes accompanied by at least three other bands of open circular DNA; the latter are presumed to be those formed by dimeric, trimeric and tetrameric open circular lambda DNA. Arch Microbiol, 1989, 153(1), 1 - 6 The right end of MudI(Ap,lac); Zieg J et al.; Stable derivatives of the bacteriophage MudI-(Ap,lac) were used to generate operon fusions in S . typhimurium which exhibit a sectoring phenotype with respect to lacZ expression . The Lac- to Lac+ conversion was shown to be the result of small deletions involving the right end of the Mud/I element . DNA sequence analysis of several different fusions revealed that this end of MudI(Ap,lac) contains an assymetric inverted repeat of the attR site found in the wild-type Mu phage . A model is presented which explains how such a structure was formed in the construction of MudI(Ap,lac) . In addition, this model explains the observed deletion formation and the Lac- to Lac+ conversion in the sectoring fusions. Gene, 1989, 76(2), 239 - 43 Organization of the bacteriophage P1 tail-fibre operon; Guidolin A et al.; The revised sequence of a bacteriophage P1 DNA fragment containing the 5' end of the tail-fibre gene, gene 19, revealed that this gene is closely preceded by another open reading frame (ORF) of 432 bp . We have designated this ORF as gene R . The tail-fibre gene and gene R are transcriptionally and translationally coupled . Thus, the tail-fibre operon of bacteriophage P1 consists of three genes: gene R, gene 19 (or gene S) and gene U. Oncogene Res, 1989, 4(1), 9 - 17 Nucleotide sequence and structural organization of the human FMS proto-oncogene; Hampe A et al.; The human proto-oncogene c-fms {FMS} on chromosome 5q33.3 encodes a transmembrane glycoprotein with tyrosine kinase activity that functions as the cell surface receptor for the macrophage colony stimulating factor (CSF-1 or M-CSF) . Overlapping bacteriophage clones that included 35 kb of the FMS locus and contained the complete coding sequence of the CSF-1 receptor were subjected to nucleotide sequencing analysis . Comparison with the cDNA sequence of the human c-fms gene indicated that at least one 5' noncoding exon is located far upstream (ca . 26 kb) from sequences encoding the CSF-1 receptor . The FMS coding sequence consists of 21 small exons and heterogeneously sized introns, ranging from 6.3 kb to less than 0.1 kb in complexity. J Bacteriol, 1989 Jan, 171(1), 588 - 92 Retroregulation of the bacteriophage lambda int gene: limited secondary degradation of the RNase III-processed transcript; Plunkett G 3rd et al.; Expression of the int gene of bacteriophage lambda from two promoters, pI and pL, is differentially regulated through RNA processing . Efficient Int protein synthesis from the pL RNA is inhibited by the action of sib, a cis-acting retroregulator downstream from the int gene . We have used mapping procedures with nuclease S1 to study the pL transcripts produced in vivo after phage lambda infection . We have found an RNase III-dependent processing site within the Int coding sequence, 387 nucleotides upstream from the site of the primary cleavage by RNase III at Sib . This secondary processing site is located at the most stable region of secondary structure in the sib int region, as predicted by computer analysis . We suggest that RNase III cleavage at the Sib site allows processive exonucleolytic degradation of the RNA to proceed to a region of secondary structure within the Int coding sequence, which protects the upstream region of the transcript from further degradation. Transfus Med Rev, 1989 Jan, 3(1 Suppl 1), 27 - 30 Confirmation of HIV infection using gene amplification; Zaia JA et al.; Direct recognition of viral gene sequences can be used to detect human immunodeficiency virus (HIV-1) in clinical specimens . A modification of the polymerase chain reaction (PCR) for amplification of gene sequences was used for detection of HIV-1-specific RNA prepared from peripheral blood mononuclear cells (PBMC) . The RNA served as a template for reverse transcriptase using primers derived from both the 3'ORF and the LTR regions of HIV-1, as well as from the control cellular sequences encoding beta-actin and T cell receptor . The resultant DNA was amplified with DNA polymerase . A transcriptional step using the bacteriophage T7 promoter recognition sequences, incorporated into the primers, was used to enhance the efficiency of the amplification process . This assay detects as few as 100 RNA copies of cloned HIV-1 genome . Starting with 1 microgram RNA isolated from PBMC, we were able to detect HIV-1 sequences in patients with symptomatic and asymptomatic HIV-1 infection . The inclusion of T cell-specific primers permitted simultaneous evaluation of an immunologic parameter . The PCR can be applied to RNA samples for detection of viral and cellular sequences and is a rapid and efficient means for detection of HIV-1 sequences as well as potentially informative cellular sequences. Bioorg Khim, 1989 Jan, 15(1), 18 - 23 {Localization of a lysine residue near the site of initiating substrate binding of T7 bacteriophage RNA polymerase}; Maksimoa TG et al.; A highly selective affinity label was introduced into the T7 phage RNA polymerase by means of GMP ortho-formylphenyl ester and {alpha-32P}UTP nearby the enzyme's active site, which was located using limited cleavage technique . Hydroxylamine, bromine, N-chlorosuccinimide, and cyanogen bromide were employed as the reagents . Analysis of gel-electrophoretic patterns of the cleavage products led to a conclusion that Lys631 is the target of labelling . The region nearby this residue has a high degree of sequence homology with regions of RNA polymerases from T3 and SP6 phages and yeast mitochondria. J Bacteriol, 1989 Jan, 171(1), 162 - 71 Isolation, sequence, and expression in Escherichia coli of an unusual thioredoxin gene from the cyanobacterium Anabaena sp . strain PCC 7120; Alam J et al.; Two sequences with homology to a thioredoxin oligonucleotide probe were detected by Southern blot analysis of Anabaena sp . strain PCC 7120 genomic DNA . One of the sequences was shown to code for a protein with 37% amino acid identity to thioredoxins from Escherichia coli and Anabaena sp . strain PCC 7119 . This is in contrast to the usual 50% homology observed among most procaryotic thioredoxins . One gene was identified in a library and was subcloned into a pUC vector and used to transform E . coli strains lacking functional thioredoxin . The Anabaena strain 7120 thioredoxin gene did not complement the trxA mutation in E . coli . Transformed cells were not able to use methionine sulfoxide as a methionine source or support replication of T7 bacteriophage or the filamentous viruses M13 and f1 . Sequence analysis of a 720-base-pair TaqI fragment indicated an open reading frame of 115 amino acids . The Anabaena strain 7120 thioredoxin gene was expressed in E . coli, and the protein was purified by assaying for protein disulfide reductase activity, using insulin as a substrate . The Anabaena strain 7120 thioredoxin exhibited the properties of a conventional thioredoxin . It is a small heat-stable redox protein and an efficient protein disulfide reductase . It is not a substrate for E . coli thioredoxin reductase . Chemically reduced Anabaena strain 7120 thioredoxin was able to serve as reducing agent for both E . coli and Anabaena strain 7119 ribonucleotide reductases, although with less efficiency than the homologous counterparts . The Anabaena strain 7120 thioredoxin cross-reacted with polyclonal antibodies to Anabaena strain 7119 thioredoxin . However, this unusual thioredoxin was not detected in extracts of Anabaena strain 7120, and its physiological function is unknown. Mutat Res, 1989 Jan, 210(1), 207 - 9 On the nature of suppression by Escherichia coli HF4714; Chambers RW; The growth of 2 well characterized bacteriophage phi X174 mutants, phi XGam3 and phi XGms3 (TCG)Ser, was examined on 3 suppressor strains of Escherichia coli: CQ3 Su1, which inserts serine at amber codons; HF4714, a well known amber suppressor that has been used as the permissive strain for a number of phi X mutants; and CQ2 Su3, which inserts tyrosine . The data demonstrate HF4714 inserts glutamine and is Su2, not Su1 as has been reported in several recent papers. Acta Leiden, 1989, 57(2), 81 - 91 Human cysticercosis and taeniasis: molecular approaches for specific diagnosis and parasite identification; McManus DP et al.; The construction and antibody screening of Taenia solium cDNA libraries, generated in the Escherichia coli bacteriophage lambda gt11, with the identification of clones putatively expressing antigen B, T . solium-specific and other antigens is described . Lysogens were produced from a number of selected clones and beta-galactosidase fusion peptides ranging in Mr of approximately 135,000-150,000 were demonstrated . These proteins were shown by immunoblotting to be reactive with a pool of sera from cysticercotic patients originally used in the cDNA library screening . We report a method whereby Taenia (T . saginata and T . pisiformis) eggs can be detected with high sensitivity in a specific DNA dot-blot hybridisation assay using total parasite DNA as probe . We show also that intra-specific DNA variability occurs in T . solium isolates obtained from different geographical areas and discuss the potential significance of this heterogeneity. Verh Dtsch Ges Pathol, 1989, 73, 372 - 87 {A highly sialylated, embryonic form of the neural cell adhesion molecule in Wilms tumor: identification of a cell adhesion molecule as a onco-developmental antigen}; Roth J et al.; The neural cell adhesion molecule NCAM is a general Ca2+ -independent cell adhesion molecule which exerts important functions during the development of the nervous system . NCAM polypeptides exist in various isoforms all of which have a similar extracellular domain structure containing a homophilic binding site for establishment of cell-cell contacts . A common structural feature of NCAM is the presence of homopolymers of alpha 2,8-linked sialic acid residues, the so-called polysialic acid, which is developmentally regulated . It undergoes a change from a highly less sialylated short chain form from embryonic to adult life . The polysialic acid regulates the homophilic adhesive properties of NCAM . The highly sialylated form of NCAM typically found in developing tissues decreases homophilic NCAM-NCAM interactions and, therefore, cell-cell contacts due to its large excluded volume and electric repulsive forces . We have used a monoclonal antibody against polysialic acid, polyclonal antibodies against NCAM, polysialic acid specific bacteriophage-encoded endoneuraminidases and a NCAM cDNA to investigate this molecule by light and electron microscopic immunolabeling, in situ hybridization and immunochemistry . The highly sialylated form of NCAM was found to be expressed in a developmentally-regulated fashion in embryonic kidney, undetectable in the normal adult kidney and re-expressed in Wilms tumor . In Wilms tumor the blastemal cell masses as well as epithelial differentiations such as tubules and glomeruloid bodies were immunolabeled whereas the stroma did not label for polysialic acid . Combination of immunoprecipitation and immunoblotting using antibodies against polysialic acid and NCAM, respectively, directly demonstrated structural relationship of renal polysialic acid with NCAM polypeptide . By immunoblot analysis two NCAM isoforms of approximately 120 and 140 kD were found in Wilms tumor . Immuno-electron microscopy provided direct morphological evidence for prevention of cell-cell contacts due to the presence of a thick cell surface coat composed of polysialic acid . In nephroblastomatosis complexes only blastemal cells exhibited immunocytochemically detectable polysialic acid . All other investigated kidney tumors, i.e . clear cell sarcoma, malignant rhabdoid tumor, cystic nephroma and renal cell carcinoma, were negative . These data allow the conclusion that the highly sialyated, embryonic form of NCAM is an onco-developmental antigen in human kidney . At the same time this is the first observation that a molecule with a well defined role for controlled cell migration and differentiation during embryonic organ development represents an onco-developmental antigen. Biochemistry, 1988 Dec 27, 27(26), 9210 - 6 High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA . 2 . Effect of pulse time and electric field strength and implications for models of the separation process; Mathew MK et al.; Bacteriophage DNAs annealed into linear oligomeric concatemers were used to examine the quantitative pulsed-field gel electrophoretic behavior of different-sized DNAs as a function of electrical field strength and pulse time . Three zones of resolution are observed for increasingly larger DNAs . In the first two zones, the electrophoretic mobility decreases linearly with increasing DNA size . The separation in zone 2 is roughly twice that in zone 1 . The largest DNA molecules do not resolve at all and migrate in a compression zone . Mobility in zone 1 increases linearly with the electric field strength and decreases with the inverse of the pulse time . The behavior of DNA in zone 2 is qualitatively similar . However, the effect of field strength and pulse time on the separations in each zone is quite different . The results for zone 1 are generally consistent with the predictions of several existing physical models of pulsed-field gel electrophoresis, but no model accounts for all of the observed behavior in the three zones. Biochemistry, 1988 Dec 27, 27(26), 9204 - 10 High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA . 1 . DNA size standards and the effect of agarose and temperature; Mathew MK et al.; Pulsed-field gel electrophoresis (PGF) subjects DNA alternately to two electrical fields to resolve DNA ranging from 10,000 base pairs (10 kb) to 10,000 kb in size . The separations are quite sensitive to a variety of experimental variables . This makes it critical to have a wide range of reliable size standards . A technique is described for preparing mixtures of bacteriophage DNA oligomers that span a size range from monomer to more than 30-mer . The relationship between size and mobility of oligomers of different bacteriophage DNA monomers is generally self-consistent . Thus, these samples can serve as primary length standards for DNAs ranging from 10 kb to more than 1500 kb . They have been used to estimate the size of the chromosomal DNAs from various Saccharomyces cerevisiae strains and to test the effect of gel concentration and temperature on PFG . DNA resolution during PFG is slightly improved in agarose gels with small pore sizes, in contrast to continuous electrophoresis where the opposite is observed . PFG mobility is surprisingly sensitive to changes in the running temperature. Gene, 1988 Dec 25, 74(1), 39 - 42 The SalI (SalGI) restriction-modification system of Streptomyces albus G; Rodicio MR et al.; The salIR and salM genes of Streptomyces albus G specify the SalGI (SalI) restriction enzyme and its cognate methyltransferase, respectively . These enzymes are responsible for restriction and modification of bacteriophages . Some phages carry genes that interfere with SalI-specific modification . The sal genes have been cloned in a Streptomyces host-vector system . Use of the cloned DNA as a hybridization probe reveals that sal mutants frequently arise from transposition of a DNA segment of approx . 1 kb into the sal genes . Some, but not all, other bacteria that produce SalGI isoschizomers contain nucleotide sequences that hybridize with sal DNA. Gene, 1988 Dec 25, 74(1), 271 - 3 Isolation of mutants in a DNA methyltransferase through mcrB-mediated restriction; Blumenthal RM et al.; A procedure has been developed that permits the positive selection of mutants in a DNA methyltransferase (MTase) gene . The stringency of this selection can be varied so as to yield null mutants only, or a mixture of null and partially defective mutants . The procedure was developed with the PvuII MTase gene (pvuIIM), which was subcloned into a bacteriophage lambda vector . Growth of this lambda pvuIIM construct on an mcrB+ host selected for non-methylating mutants, and the stringency of selection was proportional to the number of consecutive lytic cycles . Many cytosine MTases have been found to generate substrates for mcrB-mediated restriction, and this procedure should be applicable to a number of cytosine MTase genes. J Biol Chem, 1988 Dec 25, 263(36), 19788 - 95 Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms; Spruce BA et al.; Monoclonal antibodies have been generated to a chimeric peptide comprised of Escherichia coli beta-galactosidase fused to the amino acid sequence 69-207 of human preproenkephalin A . Two monoclonal antibodies, PE-1 and PE-2, were identified by their ability to recognize the same segment of proenkephalin A fused to the cII gene product of the E . coli bacteriophage lambda . The binding domains of PE-1 and PE-2 have been broadly located, with respect to the primary translation product, within the amino acid sequences 152-207 and 84-131, respectively . Immunoblot analysis of total bovine adrenomedullary chromaffin granule lysate reveals PE-1 and PE-2 immunoreactive forms of observed molecular mass 35, 33, 29, 24, 22, and 15 kDa, and an 18-kDa PE-1 immunoreactive form . Separation of granule membranes from their contents reveals differential membrane association of these high molecular weight polypeptides . There is preliminary evidence that PE-1 may be detecting a subset of polypeptides where shortening from the NH2 terminus has occurred . We postulate that the 35-kDa form represents the intact bovine enkephalin precursor of predicted molecular mass 27.3 kDa . This experimental approach should be generally applicable to the generation of antibodies which will recognize intact peptide precursors together with their post-translational cleavage products. J Mol Biol, 1988 Dec 20, 204(4), 939 - 47 Roles of operator and non-operator RNA sequences in bacteriophage R17 capsid assembly; Beckett D et al.; In order to understand the role of sequences other than the translational operator on bacteriophage R17 assembly, in vitro capsid assembly was studied with R17 coat protein and a variety of RNAs . For a series of RNA oligomers of the same chain length, sequences that bind coat protein dimer with a lower affinity require higher concentrations of RNA and protein for assembly . Among a series of non-specific RNA molecules of differing lengths, lower protein and RNA concentrations are required for assembly of capsids containing longer RNAs . For RNA molecules of any length, the presence of a single high-affinity translational operator sequence lowered the concentration requirements for capsid assembly . However, the advantage for encapsidation provided by the operator sequence is small for large RNA molecules . The experiments indicate that in the overall assembly process the interaction of coat protein with non-specific sequences is at least as important as its interaction with the specific translational operator sequence . In light of the data, a mechanism of achieving selective packaging of the R17 genomic RNA in vivo is discussed. J Mol Biol, 1988 Dec 20, 204(4), 927 - 38 Ribonucleoprotein complexes of R17 coat protein and a translational operator analog; Beckett D et al.; The coat protein of the simple spherical (triangulation no . T = 3) RNA coliphage R17 protects the genomic RNA in the virus particle and acts as a translational repressor of the phage-encoded replicase gene . It has been suggested that these two functions are related and that the translational repression complex serves as a nucleation complex for subsequent assembly of the bacteriophage . We have used a translational operation fragment to examine the relationship between formation of the translational repression complex and the assembly of the protein into T = 3 capsids . In vitro analysis of the aggregation properties of R17 coat protein reveals that binding of the translational operator fragment to the protein dimer triggers polymerization of the protein into T = 3 capsids of well-defined composition . The data further implicate the translational operator in nucleation of assembly and suggest a possible physical-chemical basis of the nucleation step. Biochim Biophys Acta, 1988 Dec 20, 951(2-3), 419 - 24 Protein-primed replication of bacteriophage phi 29 DNA; Salas M et al.; The replication of phi 29 DNA-protein p3 represents a simple model system to study the protein-priming mechanism of initiation of replication . The phi 29 DNA polymerase involved both in the initiation and elongation steps of phi 29 DNA-protein p3 replication, is a very processive enzyme and it is able to produce strand-displacement in the absence of other proteins . To correlate functional and structural domains in the phi 29 DNA polymerase point mutants in the most carboxyl region of amino-acid homology with other DNA polymerases have been constructed . Most of the mutations had a decreased initiation and elongation activity, but normal 3'----5' exonuclease activity, suggesting that this region contributes to the active domain for initiation and elongation . Point and deletion mutants in the terminal protein have allowed the mapping of one DNA-binding region and two DNA-polymerase-binding regions . The viral protein p6, which stimulates the initiation of replication, binds to a set of specific signals present at both phi 29 DNA ends . A good correlation of binding and stimulation of replication has been obtained by using fragments containing phi 29 DNA-terminal sequences and deletion mutants of protein p6 . The viral protein p5 has been shown to bind to single-stranded DNA, to protect the latter against nuclease digetion, and to stimulate phi 29 DNA-protein p3 replication in vitro. Biochim Biophys Acta, 1988 Dec 20, 951(2-3), 280 - 9 Replication of single-stranded porcine circovirus DNA by DNA polymerases alpha and delta; Gassmann M et al.; Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al . (1982) Nature 295, 64-66) . Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al . (1987) Arch . Virol . 96, 39-57) . In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase alpha and delta as well as by a partially purified preparation from pig thymus . The results indicated that three different purification fractions of calf thymus DNA polymerase alpha and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA . It appears that the sites at which DNA primase synthesizes primers are not entirely random . Subsequent DNA elongation by a highly purified DNA polymerase alpha holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al . (1987) Nucleic Acids Res . 15, 4789-4807) is very efficient . Complete conversion to the double-stranded form is obtained in less than 1 min . When the DNA synthesis by DNA polymerase alpha is blocked with the DNA polymerase alpha specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase delta can efficiently replicate from the primers . This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E . coli to study initiation and elongation of DNA replication. Biochim Biophys Acta, 1988 Dec 20, 951(2-3), 330 - 4 Multiple roles of DNA ligase at the replication fork; Montecucco A et al.; The loss of superhelical turns from a covalently closed duplex DNA exposed to bacteriophage T4 DNA ligase in the presence of AMP and Mg2+ has recently been found to be gradual and not sudden (Montecucco, A . and Ciarrocchi, G . (1988) Nucleic Acids Res . 16, 7369-7381) . In this pape