Bacteriophage

Mol Gen Mikrobiol Virusol, 1988 Aug, (8), 12 - 7
{Transactivation of p'R promoter of phage lambda}; Troianovskaia IN et al.; The host-vector system for efficient expression of the cloned genes under the control of transactivated promoter p'R of bacteriophage lambda has been elaborated . The Q protein activating p'R promoter is coded by the defective prophage constructed in vitro by means of excision of the late phage genes between the distant sites of the restriction endonuclease MluI and change of the central SalI fragment carrying the kill gene for the kanamycin resistance gene . The general recombination system is impaired during the change, thus the bacteriophage DNA can be obtained from the induced RecA cells as a plasmid DNA . The induction of the prophage results in a sharp increase of beta-lactamase synthesis (30% of soluble cell protein) under the control of p'R promoter in a plasmid derived of pBR322.

Mol Gen Genet, 1988 Aug, 213(2-3), 325 - 31
In vivo effect of DNA repair on the transition frequency produced from a single O6-methyl- or O6-n-butyl-guanine in a T:G base pair; Chambers RW et al.; We have previously reported some effects of DNA repair on the transition frequencies produced by an O6-methyl-guanine (MeG) or an O6-n-butyl-guanine (BuG) paired with C at the first position of the third codon in gene G of bacteriophage phi X174 form I' DNA (Chambers et al . 1985) . We now report experiments in which the transition is produced from T:MeG or T:BuG, instead of C:MeG or C:BuG, located at this site . The site-modified DNAs were transfected into cells with normal DNA repair as well as into cells with repair defects (uvrA, uvrB, uvrC, recA, uvrArecA) . The lysates were screened for phage carrying the expected transition using a characteristic change in phenotype . The data demonstrate that the transition frequency from T:BuG is low (0.3% of total phage progeny) in cells with normal repair (Escherichia coli AB1157) and increases 7-fold in uvrA cells (E . coli AB1886) . A similar increase is seen in uvrB and uvrC cells (AB1885, AB1884) . These data, like our previous data, indicate BuG is repaired primarily by excision . In contrast to this, the transition frequency from T:MeG is high (5 +/- 2%) in cells with normal repair . After induction of alkyl transfer repair in E . coli AB1157, the transition frequency goes up 5-fold . Compared with cells with normal repair, the transition frequency goes up 2-fold in uvrA, uvrB and uvrC cells; it goes up 1.5-fold in recA cells (E . coli AB2463) . The data reinforce our earlier conclusion that MeG is repaired primarily by alkyl transfer, but the ABC excinuclease as well as RecA protein inhibit this repair process.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1988 Aug, 85(15), 5439 - 43
Translation initiation controls the relative rates of expression of the bacteriophage lambda late genes; Sampson LL et al.; The late operon of bacteriophage lambda contains the genes encoding the morphogenetic proteins of the phage . These genes are transcribed equally from the single late promoter . Although the functional half-lives of the mRNA for the various genes of this operon vary less than 2-fold, their relative rates of expression have been shown to vary by nearly 1000-fold . This variation could result from differing rates of translation initiation, from overlapping upstream translation, or from differential elongation rates due to the presence of codons for which the corresponding tRNAs are rare . To distinguish between these possibilities, we have cloned sequences surrounding the initiator codons of several of these genes and measured their ability to drive synthesis of hybrid lambda-beta-galactosidase proteins . The rates of expression of the hybrid genes thus produced correlate very well with the natural rates of expression of the corresponding phage genes, suggesting that the rate of initiation of translation controls the relative expression rates of these genes.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Aug, 269(2), 147 - 55
Bacteriophages of Bordetella sp.: features of lysogeny and conversion; Holzmayer TA et al.; It has been the purpose of this paper to study molecular-biological features of the Bordetella bacteriophage interaction with the host cell during lysogeny and conversion as well as to determine the degree of homology between genomes of homologous and heterologous bacteriophages . Genomes of bacteriophages from B . pertussis 134, 41405 and B . bronchiseptica 214 were studied . Heteroduplex and restriction analyses revealed a heterogeneity of bacteriophage populations, and their DNAs were found to differ in size and position of inserts . As shown by blot hybridization, the bacteriophage genome is not inserted into the chromosome of the lysogenic cell but apparently exists as an autonomous plasmid replicon . It has been established that during conversion only a part of the phage genome is inserted into the chromosome of the recipient cell.

J Biomol Struct Dyn, 1988 Aug, 6(1), 35 - 49
Resonance Raman spectroscopy of complexes of the helix destabilizing proteins GP32 and GP5 with poly(rA) and poly(dA); Otto C et al.; The bacteriophage T4 helix destabilizing protein (hdp) gp32 and its complexes with poly(rA) and poly(dA) were studied with ultra-violet resonant Raman spectroscopy . The UV-resonant Raman (UV-RR) spectrum of the complex of gp5, the coat protein of bacteriophage M13, with poly(dA) was also measured and is compared with the spectrum of the gp 32/poly(dA) complex . The excitation wavelength was 245.1 nm . This is on the far UV-side of the first absorption bands of adenine and near a "window" in the protein absorption spectrum . The overlap of fluorescence due to chromophores present in the protein and resonance Raman scattering was prevented by this choice of wavelength . The spectra of the protein/polynucleotide complexes are compared with the native nucleotide spectra measured at varying temperatures . The hyperchromicity which is expected when a nucleotide changes from a stacked to an unstacked conformation was not observed for poly(rA), neither upon temperature increase nor on protein binding . In both cases poly(dA) revealed a clear hyperchromicity . This different behavior of poly(rA) and poly(dA) is probably a consequence of their different conformations . The contributions of the proteins to the spectra is weak except for two bands, at 1550 and 1610 cm-1 due to tryptophan (in case of gp32) and one band near 1610 cm-1 due to tyrosine and phenylalanine.

Can J Microbiol, 1988 Aug, 34(8), 1022 - 4
Phage f2 desorption from clay in estuarine water using nonionic detergents, beef extract, and chaotropic agents; Armon R et al.; Experimentally adsorbed bacteriophage f2 was eluted from clay particles in estuarine water using 1% Tween, 80.3% beef extract, and 0.3 M NaNO3 with 54% recovery . Replacing sodium nitrate with tetrasodium pyrophosphate (0.4 M) increased the recovery to 81% . Estuarine sediments treated with 1% Tween 80 revealed significantly higher male-specific phage elutions.

Mol Gen Mikrobiol Virusol, 1988 Aug, (8), 17 - 23
{SOS-induction of the RP4 plasmid tet-determinant}; Skavronskaia AG et al.; Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested . Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light . The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4 . The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes . Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon . The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell . Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.

J Gen Virol, 1988 Aug, 69 ( Pt 8), 2033 - 42
Gene sequence and mapping data from Marek's disease virus and herpesvirus of turkeys: implications for herpesvirus classification; Buckmaster AE et al.; Purified DNAs from Marek's disease virus (MDV) and the herpesvirus of turkeys (HVT) were randomly sheared and cloned into the M13 bacteriophage . Two-hundred and ten MDV and 130 HVT clones were sequenced to give representative samples of the genome sequences . The predicted amino acid sequences from these gammaherpes-viruses were compared to known sequences from other herpesviruses using computer analysis . Thirty-five MDV and 24 HVT genes were identified by comparison with varicella-zoster virus (VZV), an alphaherpesvirus . However, only 14 MDV and seven HVT genes, giving generally lower homology scores, were found by comparison with Epstein-Barr virus (EBV), a gammaherpesvirus, indicating that MDV and HVT sequences bear greater similarity to VZV than to EBV sequences . A number of sequences were mapped by hybridizing labelled M13 clones to Southern blots of restriction fragments of MDV or HVT DNA . The results were consistent with the MDV and HVT genomes being collinear with VZV.

Nucleic Acids Res, 1988 Jul 25, 16(14A), 6385 - 96
Influence of fd gene 2-protein and the viral replication origin on the compatibility of pfd-plasmids; Geider K et al.; Plasmids with the replication origin of bacteriophage fd, the pfd-plasmids, were investigated for compatibility in E . coli cells expressing fd gene 2-protein . This was measured by transformation of Ca-treated cells with and without a residing pfd-plasmid . When the two plasmids contained the complete intergenic region of bacteriophage fd, they were fully compatible in contrast to the situation in which at least one plasmid had a shortened origin for viral strand replication . This incompatibility effect was partially compensated for by a pfd-plasmid with a short origin and with the fd gene 2 . The fd replication origin on a colEl plasmid did not affect compatibility in polA+ cells indicating its idling in the presence of the colEl origin . It can be concluded that a short replication origin requires high amounts of gene 2-protein in contrast to the long origin . Accumulation of replication intermediates severely interferes with host cell metabolism.

Nucleic Acids Res, 1988 Jul 25, 16(14A), 6531 - 46
RNA-primed initiation sites of DNA replication in the origin region of bacteriophage lambda genome; Yoda K et al.; Using DNA molecules synthesized in the early stage of lambda phage infection, deoxynucleotides at the transition sites from primer RNA to DNA synthesis have been mapped in the 1.5 kbase area of the lambda phage genome containing the genetically defined replication origin (ori lambda) . Sites in the 1-strand (the polarity of the 1-strand is 5' to 3' from the left to the right direction of the lambda phage genetic map) were distributed both inside and outside of the ori lambda, whereas the sites in the r-strand (the strand in the opposite polarity) were mainly distributed more than three hundred nucleotides apart from the ori lambda to the right . A CPuPu sequence was found at -12 to -10 region of transition sites of the r- and the 1-strands in the frequency of 80% and 70%, respectively, and over 60% of the CPuPu sequences were CAG . Properties of the transition sites are discussed in relation to the primer synthesis.

Nucleic Acids Res, 1988 Jul 25, 16(14A), 6353 - 60
Detection of an epitope, not required for polymerization, that is conserved between E.coli DNA polymerases I and III and bacteriophage T4 DNA polymerase; Franden MA et al.; Monoclonal antibodies directed against the alpha subunit of the DNA polymerase III holoenzyme (1) of E . coli were tested for cross-reactivity with a variety of polymerases . We found that one monoclonal antibody bound to E . coli DNA polymerase I as well as to DNA polymerase III . A weaker, but specific, interaction was also detected with T4 DNA polymerase . We exploited the proteolysis procedure developed by Setlow, Brutlag and Kornberg (2) to determine which domain of DNA polymerase I contained the conserved epitope . Contrary to expectations, it was not found in the polymerase domain, but in the 5'----3' exonuclease domain . This reveals a sequence or structure, sufficiently important to be conserved among these polymerases, that is not directly involved in the polymerization reaction.

J Mol Biol, 1988 Jul 20, 202(2), 233 - 43
Deletion formation in bacteriophage T4; Singer BS et al.; We have manipulated the dispensable region of the rIIB gene of bacteriophage T4 in order to study the generation of deletions involving direct repeats . We show that recombination between different parental chromosomes is one source of the deletions we have studied . We have also investigated the effects of structure, base composition and distance on deletion formation . We demonstrate that the potential to form structure in single-stranded DNA has variable effects on the frequency of deletion formation and conclude that, in some cases, slipped mispairing during DNA synthesis can make a substantial contribution to deletion frequencies . The G + C richness of the direct repeats involved in deletion formation is an important parameter of the frequency of deletion formation . We have confirmed that increasing the distance between direct repeats decreases deletion frequency.

J Biol Chem, 1988 Jul 15, 263(20), 9831 - 9
The effect of the T7 and Escherichia coli DNA-binding proteins at the replication fork of bacteriophage T7; Nakai H et al.; In this paper we compare the effect of single-stranded DNA-binding proteins of bacteriophage T7 (gene 2.5 protein) and of Escherichia coli (SSB) at the T7 replication fork . The T7 gene 4 protein acts processively as helicase to promote leading strand synthesis and distributively as primase to initiate lagging strand synthesis by T7 DNA polymerase . On a nicked double-stranded template, the formation of a replication fork requires partial strand displacement so that gene 4 protein may bind to the displaced strand and unwind the helix catalytically . Both the T7 gene 2.5 protein and E . coli SSB act stoichiometrically to promote this initial strand displacement step . Once initiated, processive leading strand synthesis is not greatly stimulated by the single-stranded DNA-binding proteins . However, the T7 gene 2.5 protein, but not E . coli SSB, increases the frequency of initiation of lagging strand synthesis by greater than 10-fold . The results suggest a specific interaction of the T7 gene 2.5 protein with the T7 replication apparatus.

J Biol Chem, 1988 Jul 15, 263(20), 9818 - 30
Leading and lagging strand synthesis at the replication fork of bacteriophage T7 . Distinct properties of T7 gene 4 protein as a helicase and primase; Nakai H et al.; Reactions at the replication fork of bacteriophage T7 have been reconstituted in vitro on a preformed replication fork . A minimum of three proteins is required to catalyze leading and lagging strand synthesis . The T7 gene 4 protein, which exists in two forms of molecular weight 56,000 and 63,000, provides helicase and primase activities . A tight complex of the T7 gene 5 protein and Escherichia coli thioredoxin provides DNA polymerase activity . Gene 4 protein and DNA polymerase catalyze processive leading strand synthesis . Gene 4 protein molecules serving as helicase remain bound to the template as leading strand synthesis proceeds greater than 40 kilobases . Primer synthesis for lagging strand synthesis is catalyzed by additional gene 4 protein molecules that undergo multiple association/dissociation steps to catalyze multiple rounds of primer synthesis . The smaller molecular weight form of gene 4 protein has been purified from an equimolar mixture of both forms . Removal of the large form results in the loss of primase activity but not of helicase activity . Submolar amounts of the large form present in a mixture of both forms are sufficient to restore high specific activity of primase characteristic of an equimolar mixture of both forms . These results suggest that the gene 4 primase is an oligomer which is composed of both molecular weight forms . The large form may be the distributive component of the primase which dissociates from the template after each round of primer synthesis.

Biochemistry, 1988 Jul 12, 27(14), 5240 - 5
Thermal denaturation of T4 gene 32 protein: effects of zinc removal and substitution; Keating KM et al.; Gene 32 protein (g32P), the single-stranded (ss) DNA binding protein from bacteriophage T4, is a zinc metalloprotein . The intrinsic zinc is one of the factors required for the protein to bind cooperatively to a ssDNA lattice . We have used differential scanning calorimetry to determine how the thermodynamic parameters characterizing the denaturation of g32P are affected by removal or substitution of the intrinsic zinc . Over a wide concentration range (1-10 mg/mL), the native Zn(II) protein unfolds at a tm of 55 degrees C with an associated mean enthalpy change of 139 kcal mol-1 . Under the same conditions, the metal-free apoprotein denatures over a relatively broader temperature range centered at 49 degrees C, with a mean enthalpy change of 84 kcal mol-1 . Substitution of Zn(II) in g32P by either Cd(II) or Co(II) does not significantly change the enthalpy of denaturation but does affect the thermal stability of the protein . All metallo forms of g32P when bound to poly(dT) undergo highly cooperative denaturational transitions characterized by asymmetric differential scanning calorimetry peaks with increases in tm of 4-5 degrees C compared to the unliganded metalloprotein . Removal of the metal ion from g32P significantly reduces the cooperativity of binding to poly(dT) {Giedroc, D . P., Keating, K . M., Williams, K . R., & Coleman, J . E . (1987) Biochemistry 26, 5251-5259}, and presumably as a consequence of this, apo-g32P shows no change in either the shape or the midpoint of the thermal transition on binding to poly(dT).(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1988 Jul 11, 16(13), 6205 - 21
Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP; Inokuchi Y et al.; We report the nucleotide sequence of the Group IV RNA bacteriophage SP . The entire sequence is 4276 nucleotides long . Four cistrons have been identified by comparison with the related Group III phage Q beta . The maturation protein contains 449 amino acids, the coat protein contains 131 amino acids, the read-through protein contains 330 amino acids and the replicase beta-subunit contains 575 amino acids . SP is 59 nucleotides longer than Q beta . We have analyzed both sequence and structural conservation between SP and Q beta and shown that the sequences for the coat and central region of the replicase are strongly conserved between the two genomes . We also show that the S and M replicase binding sites of Q beta are strongly conserved in SP . Interestingly, the base composition of SP and Q beta differ significantly from one another, and most of the differences can be accounted for by a strong preponderance of U in the third position of each codon of Q beta relative to SP . We also compare conserved hairpins associated with potential coat protein and replicase binding sites.

Nucleic Acids Res, 1988 Jul 11, 16(13), 5895 - 914
Characterization of the origins of replication of bacteriophage phi 29 DNA; Gutierrez J et al.; The origins of replication of phi 29 DNA have been studied by analyzing the activity as templates in the phi 29 in vitro replication system of E . coli recombinant plasmids and M13 derivatives containing phi 29 DNA terminal sequences . Plasmid pITR, containing the 6 bp long inverted terminal repeat of phi 29 DNA, was shown to be essentially inactive . The analysis of a series of deletion derivatives of plasmid pID13, that contains the 73 and 269 bp from the left and right phi 29 DNA ends, respectively, indicated that the minimal origins of replication are comprised within the mutagenesis at these sequences was carried out . Changes of the second or third A into a C completely abolished the template activity . In the case of changes at position from 4 to 12, only 3 out of 14 mutations reduced the template activity; these 3 mutations were double changes and 2 of them affected the inverted terminal repeat . The results suggest that the sequence requirement at the end-proximal region of the origin of replication is more strict than that at the distal region.

Nucleic Acids Res, 1988 Jul 11, 16(13), 5727 - 40
Site-directed mutagenesis in the DNA linking site of bacteriophage phi 29 terminal protein: isolation and characterization of a Ser232----Thr mutant; Garmendia C et al.; By site-directed mutagenesis we have changed the serine residue 232 of the phi 29 terminal protein, involved in the covalent linkage to dAMP for the initiation of replication, into a threonine residue . The mutant terminal protein has been purified to homogeneity and shown to be inactive in the formation of the initiation complex; nevertheless, the mutant protein retains its ability to interact with the phi 29 DNA polymerase and with the DNA . The results obtained indicate a high specificity in the linking site of the terminal protein.

J Mol Biol, 1988 Jul 5, 202(1), 59 - 66
Properties of a mutant Cre protein that alters the topological linkage of recombination products; Abremski K et al.; The bacteriophage P1 Cre-loxP site-specific recombination system consists of two components: the Cre recombinase protein and the loxP DNA sequence where recombination takes place . We report here on the analysis of a mutation in the cre structural gene that produces a mutant protein with altered recombination properties . The mutant protein, Cre111, carries out recombination at a much slower rate than the wild-type Cre protein . To determine why the reaction is slow, we have examined a number of activities associated with Cre-mediated recombination . Our results indicate that the binding of Cre111 to the loxP site is comparable to wild-type Cre . Furthermore, the rate at which Cre111 resolves Holliday structures, an intermediate in this recombination reaction, is also comparable to wild-type Cre . Thus, DNA binding and resolution of the intermediate are not affected, suggesting that either synapsis, the process of bringing two lox sites together, or the first strand exchange event, could be affected in the mutant protein . The types of DNA products formed following recombination of a supercoiled substrate can reflect mechanisms of synapsis . Wild-type Cre generates mainly topologically unlinked and unknotted circular products . This suggests that the wild-type protein brings two lox sites together in a way that excludes the entanglement of supercoils present in the substrate DNA . In contrast, when Cre111 recombines a supercoiled molecule it generates many complicated catenanes and knotted DNA products . Presumably, the supercoils present in the DNA substrate are being trapped in the reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1988 Jul 5, 202(1), 11 - 6
Clone size distributions of mutations induced by ethyl methanesulfonate in bacteriophage T4; Drake JW; Size distributions of mutant clones can reveal important aspects of the mutation process . Previously published data on mutant clones induced by ethyl methanesulfonate (EMS) in bacteriophage T4 generated a distribution that was essentially flat, implying a mutagenic mechanism involving only rare mispairing by reacted bases . Here, methods for estimating the spontaneous component of such a distribution are used to generate a corrected distribution . The corrected distribution is strongly peaked, implying frequent (but not obligatory) mispairing . Frequent mispairing is in accord with current views of the fates of DNA lesions believed to mediate EMS-induced mutagenesis.

J Mol Biol, 1988 Jul 5, 202(1), 17 - 34
Estimation of in-vivo miscoding rates; Ripley LS; The replication of premutagenic DNA lesions generates mutant progeny in patterns that distinguish lesions that rarely produce a mutation per DNA replication from those that frequently do so . The quantitative aspects of this distinction were tested in studies of heat-mutagenized bacteriophage T4 . Previous T4 studies had demonstrated that transition mutations produced at G.C base-pairs depended upon heat-induced DNA lesions distinct from those responsible for transversions at G.C pairs . In this study the transversion mutations are shown to arise in patterns predicted for mutations produced from lesions that miscode rarely (fewer than 10% per replication) . In contrast, the transition mutations arise in patterns predicted for mutations produced from lesions that miscode at about 20 to 60% per replication . The fact that the two classes of DNA lesions are distinguishable as predicted by the quantitative model suggests that such studies may in general be useful in quantifying the behavior of mutation-generating DNA lesions . The method employed also estimates the frequency of premutagenic lesions in DNA.

J Biol Chem, 1988 Jul 5, 263(19), 9427 - 36
The mechanism of homologous DNA strand exchange catalyzed by the bacteriophage T4 uvsX and gene 32 proteins; Kodadek T et al.; A strand exchange reaction between a single-stranded DNA circle and a homologous linear double-stranded DNA molecule is catalyzed by a mixture of two T4 bacteriophage proteins, the uvsX protein (a DNA-dependent ATPase that resembles the recA protein) and the gene 32 protein (a helix-destabilizing protein) . The products are different from those formed in the corresponding recA protein-catalyzed reaction; rather than producing a linear single strand plus a nicked circular double-stranded (form II) DNA molecule as the final products, interlinked DNA networks are rapidly generated . Electron microscopy reveals that these networks form from multiple pairing reactions that involve the recombination intermediates . Since the uvsX protein is present in substoichiometric quantities, it presumably recycles to catalyze these successive pairing events . Recycling of the uvsX protein has been more directly examined in an assay that monitors the rate of uvsX protein-catalyzed branch migration . The branch migration reaction is rapidly inhibited by dilution of the uvsX protein or by the addition of a heterologous competitor DNA, showing that the uvsX protein-DNA filaments that catalyze strand exchange are dynamic structures . The evidence suggests that individual uvsX protein monomers are continuously entering and leaving the cooperatively formed filament in a cycle that is strongly affected by their ATP hydrolysis.

Blood, 1988 Jul, 72(1), 314 - 21
Molecular cloning, expression, and chromosomal localization of a human gene encoding the CD33 myeloid differentiation antigen; Peiper SC et al.; Monoclonal antibodies of the CD33 cluster group recognize a 67-kilodalton (Kd) protein, designated p67, expressed on the surface of normal human myeloid progenitors and leukemic cells from most patients with acute myelogenous leukemia . The human gene encoding p67 was isolated in a mouse genetic background after DNA-mediated gene transfer and fluorescence-activated cell sorting (FACS) for transformants that bound the monoclonal antibody MY9 . After three serial rounds of gene transfer and cell sorting, multiple independently derived tertiary mouse cell transformants were obtained that expressed p67 . Southern blot analysis revealed that these transformants shared restriction fragments containing highly reiterated human DNA sequences . Two shared EcoRI fragments of 3.3-kilobase (kb) and 9.5-kb pairs were molecularly cloned into bacteriophage vectors . A subsegment of the 3.3-kb fragment lacking repeated sequences was then used as a unique sequence probe to isolate two independent cosmid clones . Cells transfected with DNA from both cosmid clones bound MY9, and the human p67 protein was demonstrated by immunoprecipitation . NFS mice inoculated with a mouse cell transformant coexpressing p67 and the v-fms oncogene product produced antisera that specifically immunoprecipitated p67 from human leukemic cell lines, mouse cell transformants, and mouse cells transfected with the biologically active cosmid clones . The human p67 locus was previously assigned to chromosome 19 by screening a panel of rodent X human somatic cell hybrids with the unique sequence probe . The gene was sublocalized to the q13.3 region of chromosome 19 by in situ hybridization . RNA transcripts of approximately 1.6 kb and 1.4 kb were identified in polyadenylated RNA from human myeloid leukemia cell lines using a probe from the genomic locus . Manipulation of the cloned p67 gene may provide insight into the function of its product and mechanisms regulating its expression.

Virology, 1988 Jul, 165(1), 317 - 20
Monoclonal antibodies to the major structural proteins of bacteriophage phi 6; Olkkonen VM et al.; A panel of 38 monoclonal antibodies to the five major structural proteins of phi 6 was generated and characterized . The panel includes antibodies recognizing the receptor recognition protein P3, the major hydrophobic envelope protein P9, the nucleocapsid surface protein P8, and the nucleocapsid proteins P1 and P4, which are involved in the viral RNA polymerase activity and form the internal protein skeleton of the nucleocapsid . Six out of the fourteen antibodies to the receptor recognition protein, P3, showed neutralizing activity, interfering with the adsorption of phi 6 to host cells.

Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4633 - 7
DNA twisting and the affinity of bacteriophage 434 operator for bacteriophage 434 repressor; Koudelka GB et al.; The affinity of the Escherichia coli phage 434 operator for phage 434 repressor is affected by changes in the sequence of the noncontacted base pairs near the operator's center . The results presented here show that base composition near the center of the operator affects the operator's affinity for repressor by altering the ease with which the operator can be overtwisted into the proper configuration for complex formation . We show that both DNA flexibility and repressor flexibility influence the strength of the repressor-operator interaction: an operator with a single-strand nick at its center has a higher affinity for repressor than does the intact operator: and a repressor bearing a mutation that results in a relaxed dimer interaction is less sensitive than is wild type to changes in the flexibility of the operator . We show that the effect of noncontacted base pairs on operator affinity is independent of the slight overall bend of the operator seen in the repressor-operator complex . Central sequence effects on affinity for repressor are independent of the identity of adjacent base pairs, suggesting that the structure of the individual base pairs, not interactions between them, are responsible for the different torsional rigidities of different operators.

Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4610 - 4
One-dimensional diffusion of Escherichia coli DNA-dependent RNA polymerase: a mechanism to facilitate promoter location; Ricchetti M et al.; The mechanism of promoter location by DNA-dependent RNA polymerase of Escherichia coli was investigated . The occupancies of DNA fragments carrying the A1 promoter of bacteriophage T7 were analyzed as a function of the length of flanking sequences adjacent to the promoter . Competition between the promoters on different fragments showed qualitatively that DNA sequences downstream of the promoter enhanced promoter occupancy, whereas upstream flanking sequences had little or no influence on occupancy . This was studied quantitatively by using a set of DNA fragments with four identical A1 promoters (I-IV) equidistant from each other, but with different lengths of flanking sequences upstream from promoter I and downstream from promoter IV . The relative occupancies of these promoters showed that downstream DNA sequences of up to 250 base pairs increased the occupancy of the adjacent promoter, whereas upstream sequences longer than 70 base pairs had little or no effect on occupancy . Promoter occupancies measured as a function of the length of the downstream flanking DNA sequences were fit by a published theory that takes into account an enhancement of signal-sequence location by linear diffusion.

J Gen Microbiol, 1988 Jul, 134 ( Pt 7), 1773 - 8
Characterization of SE-3, a virulent bacteriophage of Saccharopolyspora erythraea; Smorawinska M et al.; SE-3 is a virulent bacteriophage isolated from a large-scale culture of Saccharopolyspora erythraea, an erythromycin producer . The host range of the phage is narrow, limited to some strains of this species . Another strain of Sac . erythraea, and a strain of Sac . hirsuta, are able to adsorb phage particles but do not sustain their complete multiplication . SE-3 is closely related to the phage SE-5 as shown by DNA restriction mapping . The differences between SE-3 and SE-5 genomes are apparently limited to two DNA segments flanked by short inverted repeats, visualized by electron microscopy.

Mol Gen Mikrobiol Virusol, 1988 Jul, (7), 42 - 7
{Complementary addressed modification of single- and double-stranded DNA by alkylating derivatives of oligonucleotides isolated by partial DNA fragmentation}; Gaidamakov SA et al.; Reagents for complementary addressed modification of nucleic acids are proposed to be synthesized on the base of oligonucleotides obtained by partial chemical fragmentation of DNA . The alkylating 4-(N-2 chlorethyl-N-methylamino) benzyl-5'-phosphamide derivatives of 5'-{32P}-labelled oligonucleotides obtained from single and double-stranded DNA cloned in bacteriophage M13 mp9 have been synthesized . The alkylated derivatives of oligonucleotides selectively modify the complementary tracts of single-stranded DNA-target . They are also able to modify the complementary regions in double-stranded supercoiled plasmid DNA.

J Gen Microbiol, 1988 Jul, 134 ( Pt 7), 1765 - 71
Characterization of bacteriophage phi C69 of Saccharopolyspora erythraea and demonstration of heterologous actinophage propagation by transfection of Streptomyces and Saccharopolyspora; Katz L et al.; A bacteriophage, designated phi C69, isolated from a culture of Saccharopolyspora erythraea was characterized . The phage propagates on Sac . erythraea NRRL 2338 but does not infect 10 Streptomyces or 3 Micromonospora species tested . It infects Sac . erythraea NRRL 2359 but does not produce infectious phage particles in this host . phi C69 is approximately 40 kb in length and contains cohesive ends . A cos fragment containing ligated phage DNA ends was cloned in Escherichia coli . Restriction maps of the phage DNA and the cos fragment for several enzymes are shown . Transfection of both Sac . erythraea and Streptomyces lividans with phi C69 resulted in approximately equal titres of infectious phage particles produced from approximately the same number of regenerating cells . Transfection of Sac . erythraea with DNA from Streptomyces phages SH10 and KC404 also resulted in the production of infectious phage particles . The basis for differences among hosts in susceptibility to infection by various actinophages is discussed.

Mol Gen Genet, 1988 Jul, 213(1), 30 - 5
Nucleotide sequence of the rci gene encoding shufflon-specific DNA recombinase in the IncI1 plasmid R64: homology to the site-specific recombinases of integrase family; Kubo A et al.; Shufflon is a novel type of DNA rearrangement in which four DNA segments are flanked by seven 19-bp repeat sequences . The site-specific recombination between any inverted repeats results in an inversion of the DNA segment(s) either independently or in groups . The recombination is mediated by a gene designated rci . We have determined the nucleotide sequence of the rci gene and found that it encodes a basic protein with 384 amino acid residues . The rci gene was fused with lacZ and its gene product was identified by Western blot analysis . The Rci protein shows regional homologies to the site-specific recombinases encoded by the bacteriophage genomes, including those of lambda, phi 80, P22, P2, 186, P4 and P1.

Mikrobiologiia, 1988 Jul-Aug, 57(4), 623 - 8
{Attachment of long fibrils at various stages of assembly of the virus particle of a bacteriophage}; Abuladze NK et al.; Bacteriophage T4 fibrillar structural elements were obtained when the basal plates and their complexes with the core were complemented in vitro with long tail fibrils . The organisation and functioning of the complexes were studied using PAAG electrophoresis, electron microscopy and sedimentation analysis . About 80% of the particles attached fibrils and were biologically active structures, i.e . could participate in the process of infection . Apparently, the organisation of virus particles is not a strictly regulated and consistent process at all the steps, but only at certain stages of their formation.

Genes Dev, 1988 Jul, 2(7), 801 - 6
In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage; Vinson CR et al.; We have used a recombinant bacteriophage that expresses the DNA-binding domain of C/EBP to optimize conditions for a screening technique that may facilitate the cloning of genes that encode sequence-specific DNA-binding proteins . The method relies on the expression of cDNA inserts in bacteriophage lambda gt11 . Fusion protein adsorbed onto nitrocellulose filters is probed with radioactive, double-stranded DNA as a ligand . Two procedures greatly increase the level of binding between ligand and recombinant fusion protein . First, nitrocellulose filters are processed through a denaturation/renaturation regimen using 6 M guanidine hydrochloride . Second, synthetic DNA corresponding to the specific binding site is catenated extensively using DNA ligase . The combination of these procedures leads to remarkably strong detection signals . Specific DNA-binding signals can be detected on duplicate filters, and filters can be washed and reused by repeating the cycle of denaturation/renaturation.

Appl Environ Microbiol, 1988 Jul, 54(7), 1731 - 7
Transduction of Escherichia coli by bacteriophage P1 in soil; Zeph LR et al.; Transduction of Escherichia coli W3110(R702) and J53(RP4) (10(4) to 10(5) CFU/g of soil) by lysates of temperature-sensitive specialized transducing derivatives of bacteriophage P1 (10(4) to 10(5) PFU/g of soil) (P1 Cm cts, containing the resistance gene for chloramphenicol, or P1 Cm cts::Tn501, containing the resistance genes for chloramphenicol and mercury {Hg}) occurred in soil amended with montmorillonite or kaolinite and adjusted to a -33-kPa water tension . In nonsterile soil, survival of introduced E . coli and the numbers of E . coli transductants resistant to chloramphenicol or Hg were independent of the clay amendment . The numbers of added E . coli increased more when bacteria were added in Luria broth amended with Ca and Mg (LCB) than when they were added in saline, and E . coli transductants were approximately 1 order of magnitude higher in LCB; however, the same proportion of E . coli was transduced with both types of inoculum . In sterile soil, total and transduced E . coli and P1 increased by 3 to 4 logs, which was followed by a plateau when they were inoculated in LCB and a gradual decrease when they were inoculated in saline . Transduction appeared to occur primarily in the first few days after addition of P1 to soil . The transfer of Hg or chloramphenicol resistance from lysogenic to nonlysogenic E . coli by phage P1 occurred in both sterile and nonsterile soils . On the basis of heat-induced lysis and phenotype, as well as hybridization with a DNA probe in some studies, the transductants appeared to be the E . coli that was added . Transduction of indigenous soil bacteria was not unequivocally demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene Anal Tech, 1988 Jul-Aug, 5(4), 80 - 2
Small-scale purification of bacteriophage lambda DNA by an airfuge centrifugation step in cesium chloride gradients; Mirande M et al.; A rapid and efficient procedure for purifying bacteriophage lambda DNA is described . This small-scale purification involves isolation of bacteriophage particles on cesium chloride gradients . Using an Airfuge ultracentrifuge, the centrifugation step can be readily achieved in 90 minutes . The method allows a 1-day purification of up to 12 independent lambda DNA (20-40 micrograms each) . The recovered DNA, essentially devoid of RNA and DNA contaminants, is efficiently cut by restriction endonucleases and can serve as starting material for the ligation of DNA fragments in other cloning vehicles.

Genetics, 1988 Jul, 119(3), 477 - 84
Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage lambda terminase; Wu WF et al.; The large subunit of phage lambda terminase, gpA, the gene product of the phage A gene, interacts with the small subunit, gpNul, to form functional terminase . Terminase binds to lambda DNA at cosB to form a binary complex . The terminase:DNA complex binds a prohead to form a ternary complex . Ternary complex formation involves an interaction of the prohead with gpA . The amino terminus of gpA contains a functional domain for interaction with gpNul, and the carboxy-terminal 38 amino acids of gpA contain a functional domain for prohead binding . This information about the structure of gpA was obtained through the use of hybrid phages resulting from recombination between lambda and the related phage 21 . lambda and 21 encode terminases that are analogous in structural organization and have ca . 60% sequence identity . In spite of these similarities, lambda and 21 terminases differ in specificity for DNA binding, subunit assembly, and prohead binding . A lambda-21 hybrid phage produces a terminase in which one of the subunits is chimeric and had recombinant specificities . In the work reported here; a new hybrid, lambda-21 hybrid 67, is characterized . lambda-21 hybrid 67 is the result of a crossover between lambda and 21 in the large subunit genes, such that the DNA from the left chromosome end is from 21, including cosB phi 21, the 1 gene, and the first 48 codons for the 2 gene . The rest of the hybrid 67 chromosome is lambda DNA, including 593 codons of the A gene . The chimeric gp2/A of hybrid 67 binds gp1 to form functional terminase.(ABSTRACT TRUNCATED AT 250 WORDS)

Virology, 1988 Jul, 165(1), 103 - 14
Late stages in bacteriophage lambda head morphogenesis: in vitro studies on the action of the bacteriophage lambda D-gene and W-gene products; Perucchetti R et al.; The in vitro maturation of bacteriophage lambda can be divided into discrete steps . Concatemers of lambda DNA bind terminase to form complex I . This DNA-terminase complex then binds a prohead to form a ternary complex (II) . Complex II in turn can be converted to infectious phage by the addition of extracts containing the products of the phage genes D, W, FII, as well as phage tails . By using in vitro complementation assays gpD and gpW have been partially purified and their interactions with complex II studied . gpD can bind to complex II in vitro to form a new complex (III) which can be isolated by sedimentation on neutral sucrose gradients . This complex requires only the addition of gpW, gpFII, and phage tails to form mature phage particles . The sedimentation of complex III is virtually identical to that of complex II; however, the resistance of the former to inactivation by DNase is higher, likely due to the partial packaging of the DNA . In similar experiments it was shown that gpW cannot bind to complex II but can effectively interact with complex III . This latter reaction converts complex III to a DNase-resistant form which sediments in a manner identical to that of full phage heads (complex IV) . After isolation of the complex IV only gpFII and tails are required for mature phage formation in vitro . gpW is a heat-stable protein of molecular weight approximately 10,000.

J Bacteriol, 1988 Jul, 170(7), 3089 - 93
Replication forks of Escherichia coli are not the preferred sites for lysogenic integration of bacteriophage Mu; Sivan S et al.; The question of whether bacteriophage Mu prefers replication forks for lysogenic integration into Escherichia coli chromosomes was tested by using two different systems . In the first, inactivation of genes was scored in synchronized cultures infected by Mu at various times . No increase in the mutation frequency of a gene was found after infection at the time of its replication . In the second, the composition of colonies formed by bacteria lysogenized by Mu was determined; the newly formed lysogens should give rise to mixed colonies (containing lysogenized as well as nonlysogenized bacteria), uniform colonies, or both, depending on the mode of integration . Both types of colonies were found, and the fraction of uniform colonies was proportional to the relative length of the unreplicated segment of an average chromosome in the culture . The results in both systems clearly preclude the possibility that a lysogenizing Mu integrates with high preference at the chromosome replication forks.

Mol Gen Genet, 1988 Jul, 213(1), 134 - 9
DNA sequence analysis of point mutations in traA, the F pilin gene, reveal two domains involved in F-specific bacteriophage attachment; Frost LS et al.; Six missense point mutations in traA (WPFL43,44,45,46,47 and 51), the gene encoding F pilin in the transfer region of the F plasmid, have been characterized for their effect on the transfer ability, bacteriophage (R17, QB and fl) sensitivity and levels of piliation expressed by the plasmid . The sequence analysis of the first five of these mutations revealed two domains in the F pilin subunit exposed on the surface of the F pilus which mediate phage attachment . These two domains include residues 14-17 (approximately) and the last few residues at the carboxy-terminus of the pilin protein . One of these mutants had a pleiotropic affect on pilus function and was thought to have affected pilus assembly . The sixth point mutant (WPFL51), previously thought to be in traA, was complemented by chimeric plasmids carrying the traG gene of the F transfer region, which may be involved in the acetylation of the pilin subunit . A traA nonsense mutant (JCFL1) carried an amber mutation near the amino-terminus which is well suppressed in SuI+ (supD) and SuIII+ (supF) strains . Neither the antigenicity of the pilin nor the efficiency of plating of F-specific bacteriophages were affected when this plasmid was harbored by either suppressor strain . A second amber mutant (JCFL25) which is not suppressible, carried its mutation in the codon for the single tryptophan in F pilin, suggesting that this residue is important in subunit interactions during pilus assembly . Two other point mutants (JCFL32 and 44) carried missense mutations in the leader sequence (positions 9 and 13) which affected the number of pili per cell presumably by altering the processing of propilin to pilin.

J Gen Virol, 1988 Jul, 69 ( Pt 7), 1683 - 94
Molecular cloning and restriction enzyme mapping of an African swine fever virus isolate from Malawi; Dixon LK; DNA prepared from a field isolate of African swine fever virus, which causes high mortality and severe disease in domestic pigs, was cloned in bacteriophage lambda and plasmid vectors . Clones containing DNA inserts overlapping with each other and together covering the complete genome, apart from short fragments close to the cross-linked termini of the genome, were obtained . A complete restriction enzyme site map of the genome for three enzymes was deduced.

J Bacteriol, 1988 Jul, 170(7), 3016 - 24
A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease; Skorupski K et al.; A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease has been identified . This pin (proteolysis inhibition) gene was selected for its ability to support plaque formation by a lambda Ots vector at 40 degrees C . Southern blot experiments indicated that this T4 gene is included within the 4.9-kilobase XbaI fragment which contains gene 49 . Subcloning experiments showed that T4 gene 49.1 (designated pinA) is responsible for the ability of the Ots vector to form plaques at 40 degrees C . Deficiencies in Lon protease activity are the only changes known in E . coli that permit lambda Ots phage to form plaques efficiently at 40 degrees C . lon+ lysogens of the lambda Ots vector containing pinA permitted a lambda Ots phage to form plaques efficiently at 40 degrees C . Furthermore, these lysogens, upon comparison with similar lysogens lacking any T4 DNA, showed reduced levels of degradation of puromycyl polypeptides and of canavanyl proteins . The lon+ lysogens that contained pinA exhibited other phenotypic characteristics common to lon strains, such as filamentation and production of mucoid colonies . Levels of degradation of canavanyl proteins were essentially the same, however, in null lon lysogens which either contained or lacked pinA . We infer from these data that the T4 pinA gene functions to block Lon protease activity; pinA does not, however, appear to block the activity of proteases other than Lon that are involved in the degradation of abnormal proteins.

J Biol Chem, 1988 Jun 15, 263(17), 8413 - 9
The Nu1 subunit of bacteriophage lambda terminase; Parris W et al.; The maturation and packaging of bacteriophage lambda DNA are catalyzed by the phage terminase enzyme . Terminase is composed of two protein subunits, gpNu1 and gpA . The holoenzyme is multifunctional in vitro; it binds to and cleaves lambda DNA at the cos site (where cos represents cohesive-end site), packages DNA into lambda proheads, and is also a DNA-dependent ATPase . The genes of the two subunits have been cloned separately into powerful expression vectors which allow for very high levels of protein overproduction . The gpNu1 protein has been purified to homogeneity and has a monomeric molecular weight of 21,200, in close agreement with the Mr of 20,444 expected from its amino acid sequence . Both gel filtration and sedimentation velocity centrifugation indicate that the native gpNu1 protein exists as a Mr greater than 500,000 aggregate . The sequence of the first 20 amino acids and the overall composition both match those predicted by the nucleotide sequence of the Nu1 gene . Purified gpNu1 is able to complement gpA-containing extracts in both lambda DNA packaging and cos cleavage assays . The Nu1 gene amino acid sequence predicts DNA binding by the protein, and gpNu1 does show specific binding to lambda DNA by filter binding assays . Also, as predicted from its sequence, gpNu1 exhibits ATPase activity; but in contrast to the holoenzyme, this activity is DNA-independent.

Gene, 1988 Jun 15, 66(1), 121 - 34
Expression vectors permitting cDNA cloning and enrichment for specific sequences by hybridization/selection; Pruitt SC; A set of vectors is described which allow the efficient cloning of full-length cDNAs, using a modification of the method of Okayama and Berg {Mol . Cell Biol . 2 (1982) 161-170}, and enrichment of specific sequences directly from cDNA libraries by hybridization/selection . The vectors pcDpolyB+ and pcDpolyB- are derived from an expression vector described previously {Okayama and Berg, Mol . Cell Biol . 3 (1983) 280-289} and allow expression of cloned cDNAs in eukaryotic cells from the simian virus 40 early region promoter . The vectors BSB+ and BSB- contain convenient priming sites for sequence analysis and the T3 and T7 RNA polymerase promoters, allowing synthesis of transcripts homologous to either strand of the cDNA . Each of these vectors also contains the intergenic region from the bacteriophage f1 permitting synthesis of single-stranded (ss) copies of the cDNA libraries . Enrichment for cDNAs containing sequences homologous to the hypoxanthine phosphoribosyl transferase gene from an ss copy of a cDNA library by hybridization/selection is demonstrated . Levels of enrichment sufficient for the direct cloning of specific sequences without requiring colony or plaque hybridizations were obtained . Libraries constructed from different cell types can be screened against each other to create sublibraries highly enriched in sequences specific to a single cell type . The availability of cDNA expression libraries enriched for cell-type-specific cDNAs should greatly enhance the efficiency with which cDNAs can be identified on the basis of functional assays.

Biochemistry, 1988 Jun 14, 27(12), 4357 - 67
Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-{Pt(NH3)2(d(GpG}}, built into a specific site in a viral genome; Naser LJ et al.; A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-{Pt(NH3)2(d(GpG}} intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II) . The duplex dodecamer d(AGAAGGCCTAGA).d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site . A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant . The dodecamer d(TCTAGGCCTTCT) was synthesized by the solid-phase phosphotriester method and platinated by reaction with cis-{Pt(NH3)2(H2O)2}2+ (yield 39%) . Characterization by pH-dependent 1H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines . The platinated oligonucleotide was phosphorylated in the presence of {gamma-32P}ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome . Approximately 80% of the gapped duplexes incorporated a dodecanucleotide in the ligation reaction . Of these, approximately half did so with the dodecanucleotide covalently joined to the genome at both 5' and 3' termini . The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII . The cis-{Pt(NH3)2(d(GpG}} cross-link completely inhibited StuI cleavage, which was fully restored following incubation of the platinated genome with cyanide to remove platinum as {Pt(CN)4}2- . Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-{Pt(NH3)2(d(GpG}} cross-link induces localized weakening of the DNA double helix . In addition, double- and single-stranded genomes, in which the cis-{Pt(NH3)2(d(GpG}} cross-link resides specifically in the plus strand, were constructed . Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes . In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-{Pt(NH3)2(d(GpG}} cross-link is lethal to the single-stranded bacteriophage.

Biochemistry, 1988 Jun 14, 27(12), 4350 - 7
Sugar pucker and phosphodiester conformations in viral genomes of filamentous bacteriophages: fd, If1, IKe, Pf1, Xf, and Pf3; Thomas GJ Jr et al.; The laser Raman spectra of filamentous viruses contain discrete bands which are assignable to molecular vibrations of the encapsidated, single-stranded DNA genomes and which are informative of their molecular conformations . Discrimination between Raman bands of the DNA and those of the coat proteins is facilitated by analysis of viruses containing deuterium-labeled amino acids . Specific DNA vibrational assignments are based upon previous studies of A-, B-, and Z-DNA oligonucleotide crystals of known structure {Thomas, G.J., Jr., & Wang, A.H.-J . (1988) in Nucleic Acids and Molecular Biology (Eckstein, F., & Lilley, D.M.J., Eds.) Vol . 2, Springer-Verlag, Berlin} . The present results show that canonical DNA structures are absent from six filamentous viruses: fd, If1, IKe, Pfl, Xf, and Pf3 . The DNAs in three viruses of symmetry class I (fd, If1, IKe) contain very similar nucleoside sugar puckers and glycosyl torsions, deduced to be C3'-endo/anti . However, nucleoside conformations are not the same among the three class II viruses examined: Pf1 and Xf DNAs contain similar conformers, deduced to be C2'-endo/anti, whereas Pf3 DNA exhibits bands usually associated with C3'-endo/anti conformers . Conformation-sensitive Raman bands of the DNA 3'-C-O-P-O-C-5' groups show that in all class I viruses and in Pf1 the ssDNA backbones do not contain regularly ordered phosphodiester group geometries, like those found in ordered single- and double-stranded nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1988 Jun 10, 16(11), 5055 - 66
A comparison of two phage coat protein-RNA interactions; Wu HN et al.; The interaction between the coat protein of the group I bacteriophage fr with its translational operator site is compared with the previously studied R17 interaction . The sequence of the two RNA binding sites differ by 2 of 20 nucleotides and two coat proteins by 17 of 129 amino acids . An analysis of the binding of fr coat protein to 24 operator variants revealed that the two proteins recognize operator sequences in virtually the same way . However, fr coat protein binds to nearly every RNA 6 to 14-fold tighter than R17 coat protein . Since the fr operator is a weaker binding variant and the fr coat protein shows a different temperature dependence of binding, it is unlikely that the two systems have different Kas in vivo . RNA fragments containing the operator sequences can initiate the capsid assembly with both fr and R17 coat protein . Surprisingly, the two coat proteins can form a mixed capsid in vitro.

Nucleic Acids Res, 1988 Jun 10, 16(11), 4789 - 800
Expression of the E.coli hemolysin secretion gene hlyB involves transcript anti-termination within the hly operon; Koronakis V et al.; The genes hly A and hly B dictating synthesis and secretion of hemolytic toxin by Escherichia coli are transcribed as part of an operon hly C, hly A, hly B but are separated by an inverted repeat and poly-T sequence characteristic of a rho-independent terminator . The hly A-hly B intergenic sequence caused a reduction of in vivo transcriptional read-through from the gal promotor into gal K when inserted in the pKG1900 terminator-probe vector and also in vitro 3' to the bacteriophage T7 luminal diameter 10 promotor . Hybridisation of mRNA generated by the hly determinant of recombinant DNA pBR202-312 to an antisense RNA probe spanning the hly intergenic sequence revealed specific termination of 80% of in vivo hly transcripts within the poly-T sequence, immediately preceding the hly B translation start . Extension of the hly determinant with 3.5kbp of hly promotor-proximal DNA sequence raised intracellular and cell-free hemolytic activity 3-fold and 20-fold respectively and increased markedly the secretion of the 107Kd hemolysin protein (HlyA) . Parallel hybridisation of in vivo mRNA to hly A and hly B antisense probes revealed that levels of hly A and hly B mRNA were increased by approximately 3-fold and 90-fold . The dramatically enhanced level of hly B mRNA resulted primarily from specific suppression of transcription termination at the intergenic rho-independent terminator from 80% to 1%, thus allowing virtually all hly A transcripts to elongate into hly B.

J Biol Chem, 1988 Jun 5, 263(16), 7604 - 9
Isolation and characterization of the mouse ornithine decarboxylase gene; Katz A et al.; Mouse ornithine decarboxylase (ODC) genomic clones were isolated from a bacteriophage lambda genomic library representing mouse myeloma 653-1 cells which over-produce ODC due to amplification of an active ODC gene . Sequence analysis of the amplified ODC gene revealed that ODC mRNA is encoded by 12 exons, 10 of which (exons 3 to 12) code for the ODC protein . Exon 12 also corresponds to the 3' noncoding region of the two species of ODC mRNA which are formed by alternative utilization of two polyadenylation signals separated from each other by 422 nucleotides . The transcription initiation site was mapped by S1 nuclease protection and by primer extension analysis . The 5' flanking region is extremely rich in G + C and contains typical promoter motifs such as the TATA box and SP1 transcription factor binding sites . Joining the 5' flanking region to the Escherichia coli chloramphenicol acetyltransferase structural gene and its introduction into mouse cells resulted in the expression of a high level of chloramphenicol acetyltransferase activity . Comparing the sequence of the ODC gene to our previously published sequence of ODC cDNA revealed a disagreement between the sequences located 5' to the AvaI site and demonstrated that this region of our previously reported cDNA represents a cloning artifact . The portion of the correct 5' noncoding region encoded by exon 1 is extremely rich in G + C and includes potential secondary structures which may be involved in translational regulation of ODC mRNA.

J Biol Chem, 1988 Jun 5, 263(16), 7478 - 86
Primary structure of T4 DNA polymerase . Evolutionary relatedness to eucaryotic and other procaryotic DNA polymerases; Spicer EK et al.; Bacteriophage T4 gene 43 codes for the viral DNA polymerase . We report here the sequence of gene 43 and about 70 nucleotides of 5'- and 3'-flanking sequences, determined by both DNA and RNA sequencing . We have also purified T4 DNA polymerase from T4 infected Escherichia coli and from E . coli containing a gene 43 overexpression vector . A major portion of the deduced amino acid sequence has been verified by peptide mapping and sequencing of the purified DNA polymerase . All these results are consistent with T4 DNA polymerase having 898 amino acids with a calculated Mr = 103,572 . Comparison of the primary structure of T4 DNA polymerase with the sequence of other procaryotic and eucaryotic DNA polymerases indicates that T4 DNA polymerase has regions of striking similarity with animal virus DNA polymerases and human DNA polymerase alpha . Surprisingly, T4 DNA polymerase shares only limited similarity with E . coli polymerase I and no detectable similarity with T7 DNA polymerase . Based on the location of specific mutations in T4 DNA polymerase and the conservation of particular sequences in T4 and eucaryotic DNA polymerases, we propose that the NH2-terminal half of T4 DNA polymerase forms a domain that carries out the 3'----5' exonuclease activity whereas the COOH-terminal half of the polypeptide contains the dNTP-binding site and is necessary for DNA synthesis.

J Mol Biol, 1988 Jun 5, 201(3), 517 - 35
Autogenous regulatory site on the bacteriophage T4 gene 32 messenger RNA; McPheeters DS et al.; We have identified the binding site on the bacteriophage T4 gene 32 mRNA responsible for autogenous translational regulation . We demonstrate that this site is largely unstructured and overlaps the initiation codon of gene 32 as previously predicted . Co-operative binding of gene 32 protein to this site specifically blocks the formation of 30 S-tRNA(fMet)-gene 32 mRNA ternary complexes and initiation of translation . The translational operator is bound co-operatively by gene 32 protein and this binding is facilitated by a nucleation site far upstream from the initiation codon . A similar unstructured mRNA lacking this nucleation site is also bound co-operatively, but only at concentrations of gene 32 protein higher than those needed to repress binding of ribosomes to the gene 32 mRNA . Some sequence-specific interactions may also influence this binding . Comparison of the bacteriophage T2, T4 and T6 gene 32 operator sequences leads us to propose that the nucleation site is a pseudoknot.

J Mol Biol, 1988 Jun 5, 201(3), 487 - 96
Maltose transport and starch binding in phage-resistant point mutants of maltoporin . Functional and topological implications; Charbit A et al.; The relationships between the bacteriophage lambda binding site, the starch binding site and the pore formed by maltoporin (LamB protein, lambda receptor protein) were investigated . Bacteria with single amino acid substitutions in the maltoporin sequence, which were previously shown to be strongly reduced in phage lambda sensitivity, were assayed for maltose- (and maltodextrin) selective pore functions . Maltose transport assays was performed at low substrate concentrations, under conditions where LamB is limiting for transport . It revealed three classes of mutants . Class A is composed of mutants with no effect on transport (substitutions at amino acid residues 154, 155, 259, 382 and 401); class B corresponds to mutants with a significant but variable reduction in transport (sites 148, 151, 152, 163, 164, 245, 247 and 250); class C is represented by a single mutant for which transport is almost completely abolished (site 18) . Starch binding was assayed by two different methods that gave compatible results . In class A mutants, binding was normal, while no binding was observed in the class C mutant . Binding was impaired to various extents in category B mutants . There was a correlation between the level of impairment of starch binding and impairment of maltose transport, consistent with the notion that the residues influencing starch binding are inside, or in close proximity to, the pore . These results, together with previous data on starch-binding mutants that were not affected in phage binding (substitutions at residues 8, 74, 82, 118 and 121), suggest that the binding sites for starch and phage lambda overlap but are distinct . Mutations affecting transport and starch binding are located in the first third of the protein and in the region of residues 245 to 250 . Mutations affecting phage adsorption are located mainly in the last two-thirds of the protein . The topological constraints suggested by the results with the available mutants altered in the lamB gene were used to propose a revised model of maltoporin folding across the outer membrane as well as to define the outlines of footprints of macromolecular binding sites (phage, starch and monoclonal antibodies) on the surface of the protein.

Appl Environ Microbiol, 1988 Jun, 54(6), 1637 - 41
Morphological diversity of ruminal bacteriophages from sheep and cattle; Klieve AV et al.; Large numbers of bacteriophages (2 x 10(7) to 1 x 10(8)/ml) were present in ruminal fluid from sheep and cattle . Twenty-six distinct types were identified and placed in three morphological groups; several phages possessed unusual structural features . The large numbers and diversity of phages observed indicates a possible role in bacterial lysis and hence in the population dynamics of the ruminal bacteria.

J Bacteriol, 1988 Jun, 170(6), 2866 - 9
Enrichment of the bacteriophage PR4 membrane in phosphatidylglycerol is not essential for phage assembly and infectivity; Vanden Boom T et al.; The membrane phospholipids of bacteriophage PR4 grown on wild-type Escherichia coli are markedly enriched in phosphatidylglycerol (PG) relative to host phospholipids . To investigate the role of PG in phage assembly and infectivity, we propagated PR4 on an E . coli mutant defective in PG synthesis . The PG content of PR4 grown on the mutant host accounted for 0.4% of the total viral phospholipids, representing a 90-fold decrease in PG relative to the PG content of phage grown on a wild-type host . Phosphatidylethanolamine and phosphatidic acid, the two major phospholipid species present in these phage preparations, accounted for 88.4 and 9.4% of the total viral phospholipids, respectively . This drastic alteration of the phage phospholipid composition had little or no adverse effect on either the stability or infectivity of the phage . We conclude that the enrichment of the PR4 virion in PG does not reflect an absolute structural requirement of the phage and is not essential for phage infectivity.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4162 - 5
Mutant 16S ribosomal RNA: a codon-specific translational suppressor; Murgola EJ et al.; We have isolated an unusual codon-specific translational suppressor in Escherichia coli . The suppressor resulted from a spontaneous mutation in a chromosomal gene during a selection for suppressors of the auxotrophic nonsense mutation trpA(UGA211) . The suppressor allows readthrough of UGA mutations at two positions in trpA and at two sites in bacteriophage T4 . It does not, however, suppress amber (UAG) or ochre (UAA) mutations that were tested in both genomes, some of which were at the same positions as the suppressible UGA mutations . The suppressor also does not allow mistranslation of the UGA-related trpA missense mutations UGG at positions 211 and 234, AGA at 211 and 234, CGA at 211, or UGU and UGC at 234 . The suppressor mutation was mapped by genetic procedures to position 89 on the E . coli genetic map . Localization of the suppressor mutation to rrnB was achieved by cloning it in the low-copy-number plasmid pEJM007 by in vivo recombination from the chromosome . Recloning in bacteriophage M13 and subsequent DNA sequence analysis allowed the identification of the suppressor mutation as a deletion of the cytidylic acid residue at nucleotide position 1054 of the 16S ribosomal RNA . The mutant EcoRI-Xba I fragment from the suppressor gene was recloned, from M13, in an otherwise wild-type rrnB in the plasmid pEJM007, and UGA suppression was examined . The UGA-suppressing activity of the reconstructed suppressor-containing pEJM007 was indistinguishable from that of the original recombinant suppressor-containing plasmid . This result demonstrates that the C1054 deletion in 16S rRNA is both necessary and sufficient for UGA suppression . The existence of this mutant suggests an important role for rRNA in codon recognition, at least for accurate polypeptide chain termination.

J Bacteriol, 1988 Jun, 170(6), 2850 - 4
The fadL gene product of Escherichia coli is an outer membrane protein required for uptake of long-chain fatty acids and involved in sensitivity to bacteriophage T2; Black PN; The fadL+ gene of Escherichia coli encodes an outer membrane protein (FadL) essential for the uptake of long-chain fatty acids (C12 to C18) . The present study shows that in addition to being required for uptake of and growth on the long-chain fatty acid oleate (C18:1), FadL acts as a receptor of bacteriophage T2 . Bacteriophage T2-resistant (T2r) strains lacked FadL and were unable to take up and grow on long-chain fatty acids . Upon transformation with the fadL+ clone pN103, T2r strains became sensitive to bacteriophage T2 (T2s), became able to take up long-chain fatty acids at wild-type levels, and contained FadL in the outer membrane.

J Ultrastruct Mol Struct Res, 1988 Jun, 99(3), 189 - 202
Head structure of bacteriophages T2 and T4; Baschong W et al.; The length-to-width ratios of bacteriophage T2 and T4 heads and stereometric angles specifying the prolate icosahedral T2 capsid were evaluated on electron micrographs recorded from samples prepared by a variety of methods . The copy numbers of the major capsid protein, gp23*, of T2 and T4 phages were compared by quantitative gel electrophoresis . Taken together, the resulting values are most compatible with triangulation numbers T = 13 and Q = 21 for both T2 and T4, thus confirming the previously proposed capsid architecture of T4 revealed by indirect measurements and thereby eliminating the repeatedly reported discrepancy between T2 and T4 in favor of a common Q number of 21 corresponding to 960 copies of gp23*.

J Bacteriol, 1988 Jun, 170(6), 2493 - 500
Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli; Moreau PL; Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes . (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected . These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein . However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair . Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA.

J Virol, 1988 Jun, 62(6), 2124 - 33
Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro; Polo JM et al.; Genetic loci affecting Sindbis virus pathogenesis in neonatal mice have been examined by using a full-length cDNA clone of the virus (Toto1101) . The full-length cDNA is linked to a bacteriophage SP6 promoter to facilitate the synthesis of infectious RNA transcripts in vitro . Virus derived from Toto1101 showed reduced virulence (attenuation) in neonatal mice . Replacement of the E1 glycoprotein and 6K genes of Toto1101 with cloned E1 and 6K genes derived from a virulent Sindbis virus strain, AR339 (SB), resulted in a new construct, TR2000, that gave rise to virulent virus . Sequence determinations for the entire substituted regions of TR2000, Toto1101, and related virulent and attenuated strains identified three coding differences in E1 between Toto1101 and TR2000 . These differences, individually or in combination, may be responsible for the attenuated phenotype . Previous studies in this laboratory identified another attenuating mutation at amino acid position 114 of the E2 glycoprotein (N.L . Davis, F.J . Fuller, W.G . Dougherty, R.A . Olmsted, and R.E . Johnston, Proc . Natl . Acad . Sci . USA 83:6771-6775, 1986) . Substitution of Arg-114 in the mutant SB-RL for Ser-114 of SB appears to confer three distinguishing phenotypes: attenuation in neonatal mice, increased sensitivity to specific E2 monoclonal antibodies, and accelerated penetration of BHK cells . Replacement of TR2000 sequences containing the codon for amino acid 114 of E2 with corresponding fragments from cDNA clones of SB or SB-RL produced two strains of Sindbis virus (TR2100 and TR2200) which were isogenic except for the E2 114 codon (Ser and Arg, respectively) . The three diagnostic phenotypes cosegregated according to the origin of the codon for amino acid 114 of E2, confirming the dramatic effect of this single amino acid substitution on these three phenotypes.

Gene, 1988 May 30, 65(2), 259 - 68
Purification and characterization of the DNA-binding protein Ner of bacteriophage Mu; Allet B et al.; The construction is described of a plasmid (pL-ner) which directs the high-level production of the bacteriophage Mu Ner protein in Escherichia coli . The protein, recovered in the soluble cellular fraction, was susceptible to in vivo proteolytic processing, in many host strains, but not in E . coli B, a natural lon- prototroph . A simple purification method is described which takes advantage of the basic nature of the protein . The purified protein was shown to be physically and chemically homogeneous and to have an amino acid sequence identical to that predicted for the authentic protein . The protein was also shown to have in vitro biological activity, as measured by specific binding to a DNA fragment containing the consensus Ner-binding sequence, and in vivo biological activity as the protein produced by the pL-ner plasmid allowed lysogenic-like maintenance of a Mu prophage c mutant unable to synthesise a functional Mu repressor.

Nucleic Acids Res, 1988 May 25, 16(10), 4595 - 605
Integration host factor of Escherichia coli regulates early- and repressor transcription of bacteriophage Mu by two different mechanisms; van Rijn PA et al.; Integration host factor (IHF) of E . coli positively regulates both early and repressor transcription of bacteriophage Mu . In this paper we show that although binding of IHF to the same binding site is responsible for both types of transcription regulation, the mechanisms by which these regulations occur are different: Activation of transcription from the early promoter (Pe) requires a helix-dependent orientation of IHF- and RNA polymerase binding sites on the DNA helix with a limited distance between both sites . Activation of repressor transcription shows no helix dependency between promoter and IHF binding site and the distance between both sites can be enlarged at least by 100 base pairs without affecting the positive control . A possible mechanism for both types of transcription stimulation will be discussed.

J Biol Chem, 1988 May 25, 263(15), 6960 - 3
Inhibition of the RNA polymerase-catalyzed synthesis of RNA by marcellomycin . Preferential interference of the inhibitor with the stabilization of the ternary promoter-RNA polymerase-nascent RNA complex; Kriebardis T et al.; Marcellomycin is a strong inhibitor of the Escherichia coli RNA polymerase-catalyzed synthesis of RNA from the strong A promoters of bacteriophage T7 DNA . Marcellomycin inhibits preferentially the last phase of transcription initiation . During this phase a stabilized ternary complex is formed consisting of RNA polymerase, DNA template, and a nascent RNA oligonucleotide about 11 nucleotides long, resulting from the extension of the RNA dinucleotide component of the corresponding early ternary complex . Marcellomycin is also responsible for minor inhibition of the formation of the open binary RNA polymerase-template complex, which serves as the precursor of the ternary complex . These findings suggest that marcellomycin may be a potentially useful tool in the study of the late stages of transcription initiation . The present findings may also contribute to a better overall understanding of the mode of drug action at the level of individual genes.

Nucleic Acids Res, 1988 May 25, 16(10), 4483 - 98
In vitro transcription and translational efficiency of chimeric SP6 messenger RNAs devoid of 5' vector nucleotides; Jobling SA et al.; A plasmid containing the bacteriophage SP6 promoter, designated pHSTO, permits in vitro transcription of RNAs devoid of vector-derived nucleotides . This vector has been characterized for relative transcriptional activity using constructs which alter the conserved nucleotides extending beyond the SP6 transcriptional initiation site . SP6 polymerase efficiently transcribes cDNA inserts which contain a guanosine (G) nucleotide at position +1 relative to the SP6 promoter; however, inserts with an adenosine (A) or pyrimidine at position +1 are not transcribed . Several cellular and viral cDNAs have been transcribed into translatable messenger RNA using this vector; however, SP6 polymerase will not transcribe the A-T rich untranslated leader from alfalfa mosaic virus RNA 4 efficiently unless the viral mRNA cap site is separated from the transcriptional initiation site by twelve base pairs of vector DNA . Chimeric messenger RNAs were created by linking the untranslated leader sequence of several viral mRNAs to the coding region of barley alpha-amylase, and the resultant mRNAs were translated in a wheat germ extract to determine relative translational efficiencies . The untranslated leader sequences of turnip yellow mosaic virus coat protein mRNA and black beetle virus RNA 2 did not increase translational efficiency, while the tobacco mosaic virus leader stimulated translation significantly . The results indicate that substitution of a cognate untranslated leader sequence with a leader derived from a highly efficient mRNA does not necessarily predict enhanced translational efficiency of the chimeric mRNA.

FEBS Lett, 1988 May 23, 232(2), 308 - 12
Structural requirements of choline derivatives for 'conversion' of pneumococcal amidase . A new single-step procedure for purification of this autolysin; Sanz JM et al.; Tertiary amines appear to be the minimal structure needed to convert in vitro the inactive form (E-form) of pneumococcal amidase to the catalytic active form (C-form) . Diethylethanolamine was one of the compounds that converted the E-form, a finding that has been used successfully to develop an affinity chromatography system in DEAE-cellulose for the rapid and efficient purification of lytic enzymes of pneumococcus and its bacteriophages.

J Mol Biol, 1988 May 20, 201(2), 239 - 46
Oxidative damage in DNA . Lack of mutagenicity by thymine glycol lesions; Hayes RC et al.; Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is a base damage common to oxidative mutagens and the major stable radiolysis product of thymine in DNA . We assessed the mutagenic potential of thymine glycols in single-stranded bacteriophage DNA during transfection of Escherichia coli wild-type and umuC strains . cis-Thymine glycols were induced in DNA by reaction with the chemical oxidant, osmium tetroxide (OsO4); modification of thymines was quantitated by using anti-thymine glycol antibody . Inactivation of transfecting molecules showed that one lethal hit corresponded to 1.5 to 2.1 thymine glycols per phage DNA in normal cells, whereas conditions of W-reactivation (SOS induction) reversed 60 to 80% of inactivating events . Forward mutations in the lacI and lacZ' (alpha) genes of f1 and M13 hybrid phage DNAs were induced in OsO4-treated DNA in a dose-dependent manner, in both wild-type and umuC cells . Sequence analysis of hybrid phage mutants revealed that mutations occurred preferentially at cytosine sites rather than thymine sites, indicating that thymine glycols were not the principal pre-mutagenic lesions in the single-stranded DNA . A mutagenic specificity for C----T transitions was confirmed by OsO4-induced reversion of mutant lac phage . Pathways for mutagenesis at derivatives of oxidized cytosine are discussed.

J Mol Biol, 1988 May 20, 201(2), 327 - 38
Control of cell division by sex factor F in Escherichia coli . III . Participation of the groES (mopB) gene of the host bacteria; Miki T et al.; Cell division of F+ bacteria is coupled to DNA replication of the F plasmid . Two plasmid coded genes, letA (ccdA) and letD (ccdB) are indispensable for this coupling . To investigate bacterial genes that participate in this coupling, we attempted to identify the target of the division inhibitor (the letD gene product) of the F plasmid . Two temperature-sensitive growth defective mutants were screened from bacterial mutants that escaped the letD product growth inhibition that occurs in hosts carrying an FletA mutant . Phage P1-mediated transduction and complementation analysis indicated that the temperature-sensitive mutations are located in the groES (mopB) gene, which is essential for the morphogenesis of several bacteriophages and also for growth of the bacteria . The nucleotide sequence of the promoter region of the gene in which the temperature-sensitive mutations had occurred was virtually identical with that of the groES gene of Escherichia coli; furthermore the sequence of the first five amino acid residues and the overall amino acid composition predicted from the nucleotide sequence of the gene match those of the purified GroES protein . The temperature-sensitive mutants did not allow the propagation of phage lambda at 28 degrees C and formed long filamentous structures without septa at 41 degrees C, as is observed in the case of groES mutants . Growth of the two groES mutants tested was not inhibited by the F plasmid with the letA mutation . These observations suggest to us that the morphogenesis gene groES plays a key role in coupling between replication of the F plasmid and cell division of the host cells.

Anal Biochem, 1988 May 15, 171(1), 192 - 6
Purification by ammonium sulfate precipitation of bacteriophage lambda gt11 DNA for restriction analysis of cloned cDNA inserts; Ziai MR et al.; A rapid and efficient method to purify lambda gt11 DNA is described . This technique involves precipitation of intact bacteriophage particles with ammonium sulfate, followed by phage lysis with sodium dodecyl sulfate, proteinase K, and alkaline treatment . The quality of DNA for subsequent restriction analysis, infectivity, subcloning, and radiolabeling is comparable to that isolated by cesium chloride banding or ion exchange chromatography . The yield of the phage DNA is, however, two to eight times higher than that obtained by other conventional methods of lambda gt11 purification . Furthermore the time required to process the bacteriophage lysate is approximately 2 h and therefore more rapid than other currently used methods.

J Immunol, 1988 May 15, 140(10), 3640 - 5
Characterization of lipopolysaccharide-induced macrophage gene expression; Tannenbaum CS et al.; A cDNA library from LPS-treated murine peritoneal macrophages has been screened by differential hybridization with radiolabeled cDNA from untreated and LPS-treated macrophages . Six clones hybridizing with mRNA sequences present in LPS-treated cells but not in controls were selected for further characterization . When the recombinant bacteriophage DNA from each clone was used as a probe in Northern analysis of total RNA from LPS-treated macrophages, inducible mRNA ranging from 1.45 to 6.4 kb were seen . In five of six cases, the mRNA expression was undetectable in untreated macrophage cultures . All but one clone identified mRNA that were inducible even in the presence of cycloheximide, indicating the independence of such gene expression from protein synthesis; none of the genes were superinduced by this treatment . The time course of expression differed among the individual genes . Four were induced transiently, whereas two showed stable increasing accumulation through an 8-h period after stimulation . In addition, four of the genes were seen within 30 min of stimulation, whereas two were seen only after 2 to 4 h . Two genes were induced only by treatment with LPS, whereas four were also induced in response to other agents, including IFN-gamma, macrophage CSF, and PMA . The insert sequences from these recombinant clones did not hybridize with a set of cDNA encoding other inducible gene products, including TNF, IL-1, ornithine decarboxylase, c-myc, c-fos, JE, or KC . Thus, these six cDNA appear to encode inducible macrophage genes that are distinct from one another as well as from a selection of previously described early genes . Although their functional identity remains indeterminate, they may encode previously described early proteins induced in macrophages treated with LPS.

Nucleic Acids Res, 1988 May 11, 16(9), 3907 - 18
DNA unwinding and inhibition of T4 DNA ligase by anthracyclines; Montecucco A et al.; The ability to alter DNA tertiary structure of ten anthracycline derivatives whose antitumor potency is known was studied by an assay that makes use of nicked circular DNA and bacteriophage T4 DNA ligase . This assay allows the detection of tertiary structure alterations caused by DNA binding of both intercalating and non-intercalating drugs . The determination of these events can be obtained at different temperatures in the range of activity of DNA ligase . The results indicate that anthracyclines alter the DNA tertiary structure but this property does not correlate with their cytotoxic or antitumor activities . An additional interesting finding was that several anthracyclines inhibit T4 DNA ligase . The inhibition can be complete and is a cubic function of drug concentration . The inhibition of DNA ligase does not correlate with the ability of anthracyclines to alter the tertiary structure of DNA but is dependent from the presence of an amino group on the sugar ring.

Nucleic Acids Res, 1988 May 11, 16(9), 4053 - 67
Analysis of the entire nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar L1 . Evidence for involvement in DNA replication; Hatt C et al.; Chlamydia trachomatis serovar L1/440/LN possesses a 7498bp plasmid which was designated pLGV440 . The plasmid was cloned at the BamH1 site of pAT153 into Escherichia coli and the recombinant plasmid was designated pCTL1 . A detailed restriction endonuclease map of pCTL1 was constructed . A fragment of the chlamydial plasmid was shown to function as a promoter in E . coli when placed upstream of the lacZ gene . The entire plasmid was sequenced by the chain termination method . Open reading frames were identified from the resulting consensus sequence together with a candidate for the plasmid origin of replication consisting of four perfect tandem repeats of a 22bp sequence, an A:T rich sequence and an open reading frame which could generate a 34.8kdal product . The predicted polypeptide products of the open reading frames were compared by computer with all reported protein sequences . Homology of the predicted polypeptide product of an open reading frame to the E . coli dnaB protein and the analogous product of gene 12 of bacteriophage P22 is described.

Biochim Biophys Acta, 1988 May 6, 950(1), 75 - 80
Mapping and regulation of the pifC promoter of the F plasmid; Kennedy M et al.; The pif region of the F plasmid, which causes abortive infection of Escherichia coli by T7 bacteriophage, is autogenously controlled by the product of the pifC gene . Here we describe the identification of the pif operon promoter by S1-nuclease mapping, and show that it is autoregulated at the transcriptional level and that its activity is modulated by integration host factor.

J Mol Biol, 1988 May 5, 201(1), 19 - 30
Conformational isomerization of the Holliday junction associated with a cruciform during branch migration in supercoiled plasmid DNA; Dickie P et al.; The variable positions of a branch-migrating cruciform junction in supercoiled plasmid DNA were mapped following cleavage of the DNA with bacteriophage T7 endonuclease I . T7 endonuclease I specifically cleaved, and thereby resolved, the Holliday junction existing at the base of the cruciform in the circular bacterial plasmid pSA1B.56A . Cruciform extrusion of cloned sequences in pSA1B.56A (containing a 322 base-pair inverted repeat insert composed of poxvirus telomeric sequences) topologically relaxed the plasmid substrate in vitro . Thus, numerous crossover positions were identified within the region of cloned sequences, reflecting the range of superhelical densities in the native plasmid preparation . Endonuclease I-sensitive crossover positions, mapped to both strands of the viral insert following the T7 endonuclease I digestion of either plasmid preparations or individual topoisomers, were regularly separated by approximately ten nucleotides . The appearance of sensitive crossovers every ten nucleotides corresponds to a change in linking difference (delta Lk) of +/- 2 in the circular core domain of the plasmid during branch point migration . In contrast, individual topoisomers of a plasmid preparation differ in linking number in increments of +/- 1 . Thus, the observed linearization of each individual topoisomer following enzyme treatment, as a result of resolution of the crossovers associated with each topoisomer, showed that branch point migration to sensitive crossover positions must have occurred facilely . T7 endonuclease I randomly resolved across either axis of the cruciform, though some discrimination (related to the sequence specificity of the enzyme) was observed . The ten-nucleotide spacing between sensitive crossover positions is accounted for by an isomerization of the cruciform junction on branch point migration . An hypothesis is that this isomerization was imposed upon the cruciform junction by the change in helix twist (delta Tw) in the two branches that compose the topologically closed, circular domain of the plasmid . T7 endonuclease I may discriminate between the various isomeric forms and cleave a sensitive conformation that appears with every turn of branch migration which leads to the extrusion, or absorption, of two turns of helix from the circular core.

J Mol Biol, 1988 May 5, 201(1), 91 - 100
Bacteriophage T3 connector: three-dimensional structure and comparison with other viral head-tail connecting regions; Donate LE et al.; The bacteriophage T3 connector, which consists of 12 copies of protein gp8, has been studied by image processing of electron micrographs from negatively stained ordered aggregates . A three-dimensional reconstruction of T3 connectors was obtained by collection of tilted views and using the direct Fourier method, up to 2.3 nm resolution . The reconstructed unit cell contains two connectors whose main structural features are essentially identical, but facing in opposite directions . The T3 connector has a height of about 10.9 nm, with two clearly defined domains: a wider one 14.4 nm in diameter, with 12 morphological units in the periphery, and a narrower one, 9.7 nm in diameter . There is a channel clearly defined in the narrower domain that almost closes along the wider domain . Comparison of the three-dimensional structure obtained for the connector of phages T3 and phi 29, and that of the neck extracted from phage phi 29 particles, reveals striking similarities and significant differences . A model for a general connector to account for the common functions carried out by these viral assemblies is discussed together with the possible role of the channel for DNA translocation.

J Mol Biol, 1988 May 5, 201(1), 115 - 25
Spatial arrangement of DNA-dependent RNA polymerase of Escherichia coli and DNA in the specific complex . A neutron small angle scattering study; Heumann H et al.; In this paper we demonstrate that neutron small angle scattering is a suitable method to study the spatial arrangement of large specific protein-DNA complexes . We studied the complex of DNA-dependent RNA polymerase of Escherichia coli and a 130 base-pair DNA fragment containing the strong promoter A1 of bacteriophage T7 . Contrast variation of the complex with deuterium allowed us to "visualize" either RNA polymerase, or DNA, or both components in situ . From the corresponding scattering curves information was derived about: (1) Conformational changes of RNA polymerase and DNA by complex formation: comparison of the scattering profiles of the isolated and complexed components showed that by specific complex formation the cross-section of RNA polymerase decreases, while the DNA fragment does not undergo a gross conformational change . (2) The spatial arrangement of RNA polymerase and DNA in the specific complex from the cross-sectional radii of gyration of the complex the normal distance dn between the centre of gravity of the RNA polymerase and the axis of the DNA fragment was derived as 5.0 (+/- 0.3) nm . On the basis of these and footprinting data a low resolution model of the RNA polymerase-promoter complex is proposed . The main feature of this model is the positioning of RNA polymerase to only one side of the DNA.

J Biol Chem, 1988 May 5, 263(13), 6202 - 8
Effect of bacteriophage T4 DNA topoisomerase gene 39 on level of beta chain of ribonucleoside diphosphate reductase in a T4 nrdB mutant; Cook KS et al.; Bacteriophage T4 ribonucleoside diphosphate reductase consists of alpha 2 and beta 2 subunits encoded by genes nrdA and nrdB, respectively, and plays a central role in the T4-induced deoxyribonucleotide synthetase complex . The accompanying paper describes the decreased rate of synthesis of deoxyribonucleotides after infection by the T4 mutant, nrdB93, and the suppression of this defect by a second mutation in gene 39, coding for one of the three protein chains of T4 DNA topoisomerase . In this study we examined these effects at the protein level . On infection by nrdB93 not only was the beta 93 protein chain altered, as shown by its migration relative to the wild type protein in electrophoretic gels and by its temperature sensitivity, but the infected cells showed very low levels of the protein . However, on infection with the double mutant of nrdB93 and 39-01 (gene 39) the concentration of beta 93 chain returned to the values of beta protein found with wild type phage . A double mutant bearing nrdB93 and an amber mutation of gene 39 also suppressed the nrdB93 defect . By contrast, a temperature-sensitive mutant of gene 39, A41, did not show suppression at either 30 or 41 degrees C . Amber mutations in the two other genes coding for T4 DNA topoisomerase, 52 and 60, did not suppress the defect . We propose that the deficiency in the quantity of beta 93 chain and the suppression of this defect occur at the transcriptional or translational expression of the nrdB93 gene and that a specific domain of the gene 39 protein, not acting in the capacity of T4 DNA topoisomerase, inhibits the expression.

J Biol Chem, 1988 May 5, 263(13), 6193 - 201
Defect in synthesis of deoxyribonucleotides by a bacteriophage T4 nrdB mutant is suppressed on mutation of T4 DNA topoisomerase gene; Wirak DO et al.; Bacteriophage T4 infection is known to induce the formation of a complex of enzymes effecting the de novo synthesis of deoxyribonucleoside triphosphates, which in turn are channeled into T4 DNA replication . The first step in this pathway is catalyzed by a ribonucleoside diphosphate reductase, comprised of subunits coded by T4 genes nrdA and nrdB . Maximum rates of synthesis of the pyrimidine deoxyribonucleotides and of DNA replication in vivo also require a type II DNA topoisomerase encoded by T4 genes 39, 52, and 60 . We report the identification of a unique mutant, nrdB93, and the suppression of its defective deoxyribonucleotide synthesis by a gene 39 mutation, 39-01 . After infection by 39-01, DNA synthesis and plaque formation were temperature-sensitive, but nearly wild type rates of deoxyribonucleotide synthesis were retained at all temperatures . The nrdB93 mutation had a profound effect on deoxyribonucleotide synthesis at 41 degrees C; even at the permissive temperature of 30 degrees C, synthesis was reduced to 30% of that of wild type or 39-01 . However, on infection at 30 degrees C by the double mutant, 39-01 nrdB93, the level of deoxyribonucleotide synthesis again reached that of wild type phage infections; involvement of the comparable host enzyme in the suppression process has been excluded . Suppression of the effect of nrdB93 by 39-01 implicates the gene 39 product in the regulation of nrdB expression . The accompanying paper (Cook, K . S., Wirak, D . O., Seasholtz, A . F., and Greenberg, G . R . (1988) J . Biol . Chem . 263, 6202-6208) examines the nature of the suppression process at the molecular level.

Biochemistry, 1988 May 3, 27(9), 3210 - 5
Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication; Kodadek T et al.; We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA . The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme . The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA . When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion . In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site . We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.

Genetika, 1988 May, 24(5), 791 - 802
{The casein genes of Bos taurus . II . Isolation and characteristics of the beta-casein gene}; Gorodetskii SI et al.; The expression of the casein genes in mammary gland cells is regulated by peptide and steroid hormones . To study underlying regulatory mechanisms, the bovine beta-casein gene was isolated and characterized from lambda bacteriophage bovine DNA library . The beta-casein gene is 8.6 kb long and is 7.8 times longer than the mature casein mRNA coded for by 9 exons . The genomic clones incorporate additional 8.5 and 4.5 kb of the 5'- and 3'-flanking regions . The nucleotide sequences of 5' and 3' ends of the beta-casein gene are determined . Conserved sequences identical or homologous to potential sites of binding with the nuclear factor CTF/NF-1, glucocorticoid and progesterone receptors were identified . The regulatory region of the casein gene contains two different TATA signals flanking the duplication site in the promoter region.

Virology, 1988 May, 164(1), 81 - 90
The structure of three bacteriophage T4 genes required for tail-tube assembly; Ishimoto LK et al.; Three different protein molecules copurify with T4 tail tubes after the tubes are released from the baseplate by guanidine hydrochloride treatment . These tube-associated proteins (TAPs) are the products of genes 29, 48, and 54 . To further investigate the structural roles that these proteins may play in T4 tail assembly we have cloned and sequenced the genes coding for these proteins and have deduced their predicted amino acid sequences . The sequence data reveal a region of amino acid sequence similarity between gp54 and the T4 tail-tube structural protein, gp19 . We believe that this region of similarity is significant and consistent with the role gp54 may play in initiating T4 tail-tube polymerization.

Mutat Res, 1988 May, 199(1), 183 - 90
Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an 'uncloneable' phenotype; Mita S et al.; Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors . For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome . All of 50 recombinant M13 clones containing this 'uncloneable' fragment had one or more changes in nucleotide sequence . Each clone contained at least one alteration in two nucleotide positions within the tRNAThr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNAThr . These results imply that the HeLa mitochondrial tRNAThr gene is responsible for the 'uncloneable' phenotype of this region of human mitochondrial (mt) DNA . A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNAThr gene . 56 mutations were single-base substitutions; 5 were deletions . Approximately 80% of the base substitution mutations were A:T----G:C transitions . A preference for A:T----G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals . This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin.

J Bacteriol, 1988 May, 170(5), 2276 - 82
Transduction of plasmid DNA in Streptomyces spp . and related genera by bacteriophage FP43; McHenney MA et al.; A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus . The transducing particles contained linear concatemers of plasmid DNA . Lysates of FP43 prepared on S . griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA . Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared . FP43 lysates prepared on S . griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.

Appl Environ Microbiol, 1988 May, 54(5), 1297 - 9
Selective stabilization by the bacteriophage 434 repressor of the plasmid expressing bovine growth hormone in Escherichia coli; Giladi H et al.; The maintenance of a plasmid vector-host system that selects for bacteria carrying the plasmid without the need for antibiotics is described . In this system, the bacteriophage 434 repressor gene cloned on the plasmid protects the host from lysis by a lambda imm434 cI- prophage . Cells that occasionally lose the plasmid are killed by prophage induction and therefore do not accumulate in the growing culture . The presence of the phage 434 repressor in the cells does not interfere with the process of lambda repressor inactivation and the high-level production of bovine growth hormone.

Radiat Res, 1988 May, 114(2), 319 - 30
Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22; Hartman PE et al.; Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C) . T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+ . Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H2O2 or some product derived therefrom was predominant in causing inactivation of plaque formation . Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected . A similar effect was found for the polyamine, spermidine . In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection . L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man . L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals.

J Virol, 1988 May, 62(5), 1723 - 9
Heat cleavage of bacteriophage T4 gene 23 product produces two peptides previously identified as head proteins; Robinson DR et al.; During studies on the intracellular protein pools of bacteriophage T4, we found that amber mutants in gene 23 blocked the synthesis of a 20-kilodalton (kDa) protein . Radiolabeled amino acid pulses showed that the protein appears at 8 min postinfection with kinetics similar to those of other major late species . Pulse-chase experiments demonstrated that the 20-kDa protein behaves like a primary product and also revealed a 29-kDa protein which, like other proteins cleaved during head assembly, appeared only after a long chase . Both species have been identified as constituents of the T4 head and have resisted previous efforts to identify their genetic origin . The dependence of the 20- and 29-kDa head proteins on the presence of gene 23 protein (gp23) and the observation that the sum of their masses equalled that of mature cleaved gp23 suggested that these two proteins were derived from this major capsid species . Evidence is presented demonstrating that heating samples before electrophoresis causes peptide bond cleavages in gp23, leading to the formation of the two peptides . As predicted by the results of Rittenhouse and Marcus (Anal . Biochem . 138:442-448, 1984), the cleavage occurs at Asp-336-Pro-337 and at two other Asp-Pro sites . Limited heat-induced proteolysis followed by two-dimensional gel analysis provided a peptide map of gp23 useful in the characterization of its assembly-related cleavages.

J Gen Microbiol, 1988 May, 134 ( Pt 5), 1333 - 8
Characterization of B278, a phage different from Mu that also produces auxotrophic mutations in Escherichia coli K12; Grinberg DR et al.; Bacteriophage B278 has been characterized and compared with Mu, the only phage known to produce random mutations in E . coli . Although both phages are morphologically indistinguishable and have a similar host range, they clearly differ at both the protein and the DNA level . B278 apparently possesses a DNA protection mechanism that is different from the mom system described for Mu.

J Gen Virol, 1988 May, 69 ( Pt 5), 969 - 74
Formation of complexes between long tail fibres and substructural elements of phage T4D; Abuladze NK et al.; Complexes of substructural elements of bacteriophage T4 (baseplates, baseplate-core complexes) with long tail fibres were obtained for the first time by complementation in vitro . A study of the organization of the complexes was carried out by PAGE, electron microscopy and sedimentation analysis . About 90% of baseplates and baseplate-core complexes were combined with fibres . However, the number of the attached fibres varied from one to six . On the basis of the data obtained, we proposed that the attachment of long tail fibres can occur before the assembly of the whole bacteriophage.

Proc Natl Acad Sci U S A, 1988 May, 85(9), 2914 - 8
A C-terminal, calmodulin-like regulatory domain from the plasma membrane Ca2+-pumping ATPase; Brandt P et al.; A cDNA that encodes what appears to be the inhibitory domain of the plasma membrane calcium-pumping ATPase (Ca2+-ATPase) has been isolated by screening a lambda gt11 bovine brain cDNA library with antibodies prepared against the human erythrocyte membrane Ca2+-ATPase . This screening resulted in isolation of a bacteriophage containing a 1.5-kilobase cDNA insert encoding a 71-residue polypeptide, the remainder being a large 3' terminal noncoding region . A portion of this deduced peptide sequence was identical to that of a peptide isolated from a V8 protease digest of the human erythrocyte Ca2+-ATPase except for 1 residue . Antibodies purified by immunoabsorption to the fusion protein containing this cDNA-encoded polypeptide reacted only with those fragments of a limited trypsin digest of the human erythrocyte Ca2+-ATPase that contain the inhibitory domain . Moreover, these antibodies were able to partially stimulate basal enzyme activity and block further activation by calmodulin . The encoded polypeptide bears homology to the glutamic acid-rich regions N-terminal to the Ca2+-binding loops of calmodulin and to a lesser extent with the loops themselves . This encoded polypeptide also represents the C terminus of the Ca2+-ATPase . Portions of the isolated cDNA were homologous to the 3' noncoding region of the sarcoplasmic reticulum Ca2+-ATPase cDNA, indicating a possible mechanism for the evolution of these distinct membrane Ca2+ pumps.

J Bacteriol, 1988 May, 170(5), 2012 - 21
Modulation of Escherichia coli RecBCD activity by the bacteriophage lambda Gam and P22 Abc functions; Poteete AR et al.; Plasmids that express the bacteriophage lambda gam gene or the P22 abc2 gene (with and without abc1) at controllable levels were placed in Escherichia coli and tested for effects on the activity of RecBCD . Like Gam, Abc2 inhibited the ATP-dependent exonuclease activity of RecBCD, apparently not by binding to DNA . However, Abc2-mediated inhibition was partial, while Gam-mediated inhibition was complete . Both Abc2 and Gam inhibited host system-mediated homologous recombination in a Chi-containing interval in the chromosome of a hybrid lambda phage; Abc2 inhibited it more strongly than Gam . Gam but not Abc2 spared a phage T4 gene 2 mutant from restriction by RecBCD; Abc2 exhibited weak sparing activity in combination with Abc1 and substantial activity in combination with both Abc1 and P22 homologous recombination function Erf . Either Gam or the combination of the lambda recombination functions Exo and Bet was sufficient to induce a mode of plasmid replication that produced linear multimers . The combination of Abc2, Abc1, and Erf also exhibited this activity . However, Erf was inactive, both by itself and in combination with Abc1; Abc2 had weak activity . These results indicate that Gam and Abc2 modulate the activity of RecBCD in significantly different ways . In comparison with lambda Gam, P22 Abc2 has a weak effect on RecBCD nuclease activity but a strong effect on its recombination-promoting activity.

Can J Microbiol, 1988 May, 34(5), 651 - 5
A highly efficient second-step concentration technique for bacteriophages and enteric viruses using ammonium sulfate and Tween 80; Armon R et al.; Addition of Tween 80 to a 1.5% solution of beef extract was found to enhance the elution of bacteriophages adsorbed to electronegative filters . When reconcentration of the eluate was attempted by ammonium sulfate precipitation, a floating layer containing most of the viruses was formed . This floating layer can be obtained with several nonionic detergents including Tween 80 and under a salt saturation of 55% with ammonium sulfate, potassium tartrate, and sodium phosphate . Virus recovery ranged from 91 to 103% and was obtained with several bacteriophage strains . With poliovirus type 1, coxsackievirus B-4, and rotavirus SA-11 the recoveries were 100, 20, and 80%, respectively, but toxicity to cell culture was encountered: after removal of the detergent by a second floating layer method the recovery was 32% for poliovirus . Compared with organic flocculation, this method also had both improved recovery for bacteriophages and protective properties for samples frozen at -70 degrees C.

Mol Gen Mikrobiol Virusol, 1988 May, (5), 30 - 3
{Subcloning of DNA fragments of the simian adenovirus SA7 oncogene in bacteriophage M13}; Zhukova EL et al.; The XmaI/PstI and XmaI DNA fragments of adenovirus SA7 oncogene and the adjacent region (16.7% of the physical map of SA7 left end DNA) were recloned in M13 bacteriophages mp8 and mp9 in order to obtain the singlestranded fragments EIa and EIb from the DNA region of monkey adenovirus SA7 located on the recombinant plasmid pASP carrying the DNA APstI fragment including the adenovirus SA7 oncogene.

Mol Gen Genet, 1988 May, 212(2), 325 - 36
Structural diversity of the patatin gene family in potato cv . Desiree; Twell D et al.; We have used a combined genetical and molecular approach to study the structural diversity of the patatin gene family in tetraploid Solanum tuberosum L . cv . Desiree (2n = 4x = 48) . Nine dihaploid derivatives (2n = 2x = 24) of cv . Desiree were isolated by gynogenesis through prickle pollination with S . phureja Juz et Buk . Patatin DNA sequences in Desiree and in the dihaploids were examined by probing Southern blots of restriction endonucleases HindIII and XbaI digested DNA with patatin cDNA region-specific gene probes and by more detailed examination (restriction endonuclease mapping and partial DNA sequencing) of 10 patatin genomic clones in bacteriophage lambda replacement vector EMBL4 . This provided positive identification for most individual patatin gene family members and some estimate of their organisation and diversity . Most of the 64-72 patatin DNA copies showed little allelic variation based on HindIII and XbaI restriction fragment length polymorphism (RFLP) mapping and did not appear to be very tightly clustered . Four of the 6-8 class I patatin genes (without a characteristic 22 bp insert in their untranslated leader DNA), showed apparent allelic homogeneity, whilst the remaining class I genes comigrated with a single class II patatin gene RFLP subclass . Of the isolated clones, 4 contained apparent pseudogenes lacking 5' control sequences and exon-1 DNA while another clone contained a patatin gene truncated at the 5' region due to the cloning event . The remaining 5 all contained class II genes (with the 22 bp insert) and these showed varying degrees of sequence homology for 400 bp of conserved 5' coding and non-coding DNA (from 77%-95%) . In one case the extent of homology differed, with complete sequence divergence upstream of position -80 from the start of transcription . The structural diversity of the patatin gene family is discussed in relation to expression of individual patatin genes and the use of cv . Desiree as a host for potato transformation experiments.

DNA, 1988 May, 7(4), 227 - 33
Nucleotide sequence of the bovine gene for follicle-stimulating hormone beta-subunit; Kim KE et al.; The gene for the beta-subunit of follicle-stimulating hormone (FSH-beta) was isolated from a library of bovine DNA fragments cloned in bacteriophage gamma and the complete nucleotide sequence of the gene was determined . The bovine FSH-beta gene contains approximately 4000 nucleotides and consists of three exons separated by two intervening sequences . The transcription initiation site of the gene was mapped by nuclease protection experiments . Analysis of RNA species present in pituitary mRNA demonstrated the presence of a 4.0-kb RNA containing FSH-beta sequences, which is the appropriate size for the primary transcript of the gene . Comparison of nucleotide sequence of the 5'-flanking sequence of the FSH-beta gene to the 5'-flanking regions of other pituitary glycoprotein hormone genes reveals little sequence similarity.

J Cell Biol, 1988 May, 106(5), 1489 - 98
Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus; Hull JD et al.; The M2 protein of influenza A virus is a small integral membrane protein of 97 residues that is expressed on the surface of virus-infected cells . M2 has an unusual structure as it lacks a cleavable signal sequence yet contains an ectoplasmic amino-terminal domain of 23 residues, a 19 residue hydrophobic transmembrane spanning segment, and a cytoplasmic carboxyl-terminal domain of 55 residues . Oligonucleotide-mediated deletion mutagenesis was used to construct a series of M2 mutants lacking portions of the hydrophobic segment . Membrane integration of the M2 protein was examined by in vitro translation of synthetic mRNA transcripts prepared using bacteriophage T7 RNA polymerase . After membrane integration, M2 was resistant to alkaline extraction and was converted to an Mr approximately equal to 7,000 membrane-protected fragment after digestion with trypsin . In vitro integration of M2 requires the cotranslational presence of the signal recognition particle . Deletion of as few as two residues from the hydrophobic segment of M2 markedly decreases the efficiency of membrane integration, whereas deletion of six residues completely eliminates integration . M2 proteins containing deletions that eliminate stable membrane anchoring are apparently not recognized by signal recognition particles, as these polypeptides remain sensitive to protease digestion, indicating that in addition they do not have a functional signal sequence . These data thus indicate that the signal sequence that initiates membrane integration of M2 resides within the transmembrane spanning segment of the polypeptide.

J Bacteriol, 1988 May, 170(5), 2352 - 8
A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli; Zhu Y et al.; L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase . The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde . Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted . The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR . The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector . Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA {fucO(Con) fucA(Con)}, but noninducible in fucPIK {fucPIK(Non)} . An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK . Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein . When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA . The fucPIK operon became hyperinducible . The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal . Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose . In contrast, lysogenization of {fucO(Con)fucA(Con)fucPIK(Non)crp-201} cells by the same phage retarded their growth on fucose.

J Bacteriol, 1988 May, 170(5), 2095 - 105
Entry of bacteriophage T7 DNA into the cell and escape from host restriction; Moffatt BA et al.; T7 DNA did not become susceptible to degradation by the host restriction enzymes EcoB, EcoK, or EcoP1 until 6 to 7 min after infection (at 30 degrees C) . During this period, T7 gene 0.3 protein is made and inactivates EcoB and EcoK, allowing wild-type T7, or even a mutant that has recognition sites flanking gene 0.3, to escape restriction by these enzymes . However, T7 failed to escape restriction by EcoP1 even though 0.3 protein was made, evidently because 0.3 protein is unable to inactivate EcoP1 . How T7 DNA can be accessible to transcription but not restriction in the first few minutes of infection is not yet understood, but we favor the idea that the entering DNA is initially segregated in a special place . Entry of T7 DNA into the cell is normally coupled to transcription . Tests of degradation of DNAs having their first restriction sites different distances from the end of the DNA indicated that only the first 1,000 or so base pairs (2.5%) of the molecule enter the cell without transcription . An exception was the only mutant tested that lacks base pairs 343 to 393 of T7 DNA; most or all of this DNA entered the cell without being transcribed, apparently because it lacks a sequence that normally arrests entry . This block to DNA entry would normally be relieved by the host RNA polymerase transcribing from an appropriately situated promoter, but the block can also be relieved by T7 RNA polymerase, if supplied by the host cell . T7 mutants that lack all three strong early promoters A1, A2, and A3 could grow by using a secondary promoter.

J Virol, 1988 May, 62(5), 1697 - 704
Alternative promoters in the development of bacteriophage plasmid P4; Deho G et al.; Infection of Escherichia coli with the satellite virus P4 without its helper bacteriophage P2 leads either to the immune integrated state or to the nonimmune multicopy plasmid condition . We analyzed the transcription pattern of the phage plasmid P4 early and late after infection and during the stable plasmid or lysogenic condition . The early postinfection phase is characterized by the leftward transcription of an operon including the genes cI (P4 immunity) and alpha (replication) . This early transcript starts from the promoter PLE, which shows a good homology with the E . coli sigma 70 promoter . At later times, the transcription of this operon starts from a different promoter, PLL, located 400 base pairs upstream of PLE, and sharing little homology with the canonical E . coli promoter sequence; a longer transcript encoding an additional open reading frame is thus produced . PLL shares two boxes of homology with the P4 late promoter PSID, positively regulated by the P4 delta gene product, and depends on delta function for its full activation . In the multicopy plasmid state, the transcription pattern is similar to that observed at late times after infection . Since in the plasmid state not only is P4 immunity not expressed but its establishment is prevented, even though the P4 cI gene is transcribed, the P4 cI function may be regulated at the posttranscriptional level . In the immune state, transcription starts from PLE but does not continue to cover the P4 alpha gene . This suggests that P4 immunity acts by prematurely terminating transcription initiated at PLE.

J Virol, 1988 May, 62(5), 1687 - 96
Construction and characterization of poliovirus subgenomic replicons; Kaplan G et al.; Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase . Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470) . All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo . Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro . Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA . Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells . Replication of P2/Lansing virion RNA was inhibited by cotransfection with the R1 replicon, as demonstrated by hybridization analysis with a serotype-specific oligonucleotide probe . A higher level of inhibition of RNA replication was observed when P2/Lansing RNA was cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII) that were missing 395 3' nucleotides and a poly(A) tail . These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs . No inhibition of plaque size was observed when replicon RNAs were used that were missing 1,384 or 1,839 3' nucleotides or contained plasmid-derived sequences downstream of the 3' poly(A) . The trans-acting inhibitory effect of R1-PvuII on the replication of poliovirus P2/Lansing RNA did not involve entry of RNA into cells and appeared to reduce viral translation and RNA synthesis late in the infection cycle.

Mol Biol (Mosk), 1988 May-Jun, 22(3), 731 - 40
{Genetically engineered mutants of the envelope protein of the RNA-containing bacteriophage}; Kozlovskaia TM et al.; Expression of the coat protein gene of RNA bacteriophage fr in Escherichia coli cells leads to the formation of capsid-like structures of ca . 25 nm in diameter, which are immunologically indistinguishable from the native phage fr capsids . The modification strategy of the coat protein gene by gene engineering technique was developed in order to localize coat protein regions, which are exposed on the capsid surface and are capable to include foreign amino acid inserts without an appreciable effect on the capsid self-assembly . The oligonucleotide linkers, coding short amino acid sequences and bearing also convenient restriction sites, were synthesized and inserted into different regions of the coat protein gene . The mutant proteins, containing insertions of 2-12 amino acids in potentially exposed regions, were obtained . It was shown that N- and C-terminal insertions, as well as the insertion into codon 51 in the RNA-binding region, do not prevent the self-assembly . The regions (codons 96 and 112) were also revealed, insertions in them decreased drastically the protein yield as a consequence of a block in the self-assembly.

Proc Natl Acad Sci U S A, 1988 May, 85(9), 3165 - 9
Localization of a factor VIII-inhibiting antibody epitope to a region between residues 338 and 362 of factor VIII heavy chain; Ware J et al.; We have used a recombinant DNA epitope library to localize the binding region of a factor VIII (FVIII) monoclonal antibody that neutralizes coagulant activity . The antibody, C5, has previously been described and has been shown to have a FVIII neutralizing potency of 1488 Bethesda units per mg of purified immunoglobulin . A recombinant DNA epitope library was constructed from short, random FVIII cDNA fragments and immunologically screened with C5 to identify bacteriophage expressing the antigenic determinant . The isolation and characterization of immunoreactive bacteriophage restricted the C5 epitope to the overlapping or shared DNA sequence of nine different clones and corresponded to amino acid residues 338-362 of the mature FVIII peptide . The defined epitope is between the proposed activated protein C cleavage site (Arg-336) and thrombin cleavage site (Arg-372) on the amino-terminal 90-kDa FVIII heavy-chain subunit . The identification of the epitope of an inhibiting anti-FVIII antibody between two critical cleavage sites suggests that this amino acid sequence plays a role in regulating FVIII coagulant activity.

J Bacteriol, 1988 May, 170(5), 2056 - 62
The lit gene product which blocks bacteriophage T4 late gene expression is a membrane protein encoded by a cryptic DNA element, e14; Kao C et al.; Escherichia coli lit(Con) mutations cause a severe inhibition of gene expression late in infection by bacteriophage T4 owing to the overproduction of one, and possibly two, proteins (C . Kao, E . Gumbs, and L . Snyder, J . Bacteriol . 169:1232-1238, 1987) . One or both of these proteins interact, either directly or indirectly, with a short sequence about one-quarter of the way into the major capsid protein gene of T4, and the inhibition occurs when this late gene of the virus is expressed . In this report we show that lit(Con) mutations are up-promoter mutations in the cryptic DNA element e14 and that only one of the proteins, gplit, of about 34 kilodaltons, is required for the inhibition . We have sequenced the lit gene and the surrounding regions . From the sequence, and from cell fractionation studies, we conclude that gplit is an inner membrane protein . Since the assembly of T4 heads is thought to occur on the inner face of the inner membrane, we propose that gplit interferes with a normal regulation which coordinates the synthesis of proteins and the assembly of T4 heads.

Infect Immun, 1988 May, 56(5), 1344 - 51
Isolation and characterization of recombinant lambda gt11 bacteriophages expressing eight different mycobacterial antigens of potential immunological relevance; Andersen AB et al.; A genomic lambda gt11 DNA library of Mycobacterium tuberculosis was screened for expression of mycobacterial protein antigens with murine monoclonal antibodies . The reactivity patterns of the monoclonal antibodies ranged from those showing a limited interspecies reactivity to antibodies widely cross-reactive among different mycobacterial species . Twelve recombinant bacteriophages were isolated, containing eight mycobacterial genes (paa, pab, pac, pad, paeA, paeB, pafA, and pafB) encoding protein antigens . Physical maps of the phages were generated and the products of the recombinant genes were analyzed by immunoblotting techniques . PaeA and PaeB are distinct proteins but were shown to share an epitope . A similar condition was observed between PafA and PafB . Among the phages isolated, two groups expressed epitopes specific for M . tuberculosis and Mycobacterium bovis BCG . One group of phages produced an antigenic determinant which is found in M . tuberculosis and Mycobacterium marinum but not in M . bovis BCG.

Infect Immun, 1988 May, 56(5), 1281 - 7
Generation and characterization of monoclonal antibodies to 28-, 35-, and 65-kilodalton proteins of Mycobacterium tuberculosis; Damiani G et al.; Three monoclonal antibodies (H60.15, H61.3, and H105.10) directed to protein antigens of Mycobacterium tuberculosis were obtained and characterized . H60.15 recognizes a protein with a molecular mass of 28 kilodaltons (kDa) with broad cross-reactivity on a panel of 12 species and strains of mycobacteria . H61.3 reacts with a 35-kDa protein present in M . tuberculosis, Mycobacterium bovis BCG, and M . africanum . On the basis of the antigen molecular masses and competition experiments with other monoclonal antibodies, H60.15 and H61.3 seem to be the first described monoclonal antibodies to these M . tuberculosis proteins . H105.10 binds to the cross-reactive 65-kDa protein present in mycobacteria . Epitope mapping of H105.10 was performed by using the M . leprae DNA sublibrary available in bacteriophage lambda gt11 for this antigen and revealed that its epitope resides in the region from amino acids 20 to 54 . The 28-, 35-, and 65-kDa antigens isolated by immunoblotting and presented on nitrocellulose to pleural effusion T cells from tuberculosis patients induced a proliferative response, indicating the presence of T-cell epitopes . These observations indicate that two protein antigens should be added to the list of antigens detectable in M . tuberculosis by monoclonal antibodies . The common feature of such proteins, the elicitation of an immune response of limited or broad cross-reactivity for mycobacteria, encourages the search for their role in the pathogenesis of mycobacterioses.

Science, 1988 Apr 29, 240(4852), 640 - 2
Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro; Imlay JA et al.; Exposure of Escherichia coli to low concentrations of hydrogen peroxide results in DNA damage that causes mutagenesis and kills the bacteria, whereas higher concentrations of peroxide reduce the amount of such damage . Earlier studies indicated that the direct DNA oxidant is a derivative of hydrogen peroxide whose formation is dependent on cell metabolism . The generation of this oxidant depends on the availability of both reducing equivalents and an iron species, which together mediate a Fenton reaction in which ferrous iron reduces hydrogen peroxide to a reactive radical . An in vitro Fenton system was established that generates DNA strand breaks and inactivates bacteriophage and that also reproduces the suppression of DNA damage by high concentrations of peroxide . The direct DNA oxidant both in vivo and in this in vitro system exhibits reactivity unlike that of a free hydroxyl radical and may instead be a ferryl radical.

Nature, 1988 Apr 28, 332(6167), 861 - 3
Uncoupling of the recombination and topoisomerase activities of the gamma delta resolvase by a mutation at the crossover point; Falvey E et al.; In several well-characterized site-specific recombination systems it has been shown that, for efficient recombination, the two recombining sites must have identical DNA sequences across the region between the staggered points of exchange . The precise DNA sequence of this overlap region, however, appears to be of little importance (with the exception of one position in the loxP site of bacteriophage P1 (ref . 6} . In this report we characterize a mutant recombination site for the site-specific recombination enzyme gamma delta resolvase (encoded by the gamma delta transposon), in which the dinucleotide at the crossover point is changed from AT to CT . Our results indicate that identity of the two overlap regions is not sufficient for recombination . Although resolvase binds normally to the mutant site and induces the structural deformation characteristic of the wild-type recombination site, catalysis at the crossover point (cutting and rejoining of DNA strands) is effectively limited to just one of the two strands, allowing resolvase to act as a topoisomerase but not as a recombinational enzyme.

FEBS Lett, 1988 Apr 25, 231(2), 352 - 4
Sequence-specific chemical modification of a hybrid bacteriophage M13 single-stranded DNA by alkylating oligonucleotide derivatives; Vlassov VV et al.; Alkylating oligonucleotide derivatives react with the complementary sequences in hybrid M13mp7 bacteriophage single-stranded DNA and destroy the infecting ability of the DNA . The reagents do not damage M13mp9 single-stranded DNA lacking the target nucleotide sequence.

Nature, 1988 Apr 21, 332(6166), 728 - 31
A group specific anamnestic immune reaction against HIV-1 induced by a candidate vaccine against AIDS; Zagury D et al.; The first experimental immunization of humans against the AIDS retrovirus, HIV-1, was started in a series of HIV seronegative, healthy volunteers in November 1986 . For the primary vaccination recombinant vaccinia virus (V25) expressing the complete gp160 env protein of the HTLV-IIIB strain of HIV-1 was introduced by scarification . This elicited a weak primary response which we subsequently attempted to enhance by additional immunizations (boosting), using four different immunization protocols . We report here that intravenous injection of paraformaldehyde-fixed autologous cells infected in vitro with V25 (individual D.Z.) gave the best results . This individual received second and third boosts of intramuscular gp160 derived from an HTLV-IIIB clone using the hybrid vaccinia virus/bacteriophage T7 expression system . An anamnestic humoral and cellular immune reaction was achieved for over one year after the original vaccination, with high levels of antibodies to the viral envelope, and neutralizing antibodies against divergent HIV-1 strains such as HTLV-IIIB and HTLV-IIIRF (also called HTLV-III HAT) after the first boost . In addition, group-specific cell-mediated immunity and cell-mediated cytotoxicity against infected T4 cells were obtained after the primary vaccine and enhanced by the boosts . Finally, skin tests showed both immediate and delayed hypersensitivity to gp160 in vivo . Although this protocol is not practical for a large scale vaccine trial, our results show for the first time that an immune state against HIV can be obtained in man.

J Mol Biol, 1988 Apr 20, 200(4), 741 - 3
Secondary structure of filamentous bacteriophage coat protein is preserved in lipid environments; Schiksnis RA et al.; 1H nuclear magnetic resonance experiments have shown that the amide hydrogens of residues 30 to 40 of bacteriophage Pf1 coat protein in micelles undergo very slow exchange with solvent deuterons . The amide 1H resonances from these residues were used to monitor the structural stability of the membrane-spanning helix of the coat protein during the transition of the coat protein from its structural form, in the virus particle, to the membrane-bound form, in micelles . The helix was found to remain folded on the 10(-3) second time-scale of the experiment, which indicates that no major disruption or rearrangement of the central part of the protein structure occurs during the process of coat protein solubilization by detergent . The results also suggest that a helical peptide can associate with lipids without reorganization of its secondary structure . However, a general model for the insertion of proteins into membranes cannot be established from these results, because the mechanism of the detergent solubilization process may differ somewhat from that of the membrane insertion process.

J Mol Biol, 1988 Apr 20, 200(4), 735 - 9
Interactions between Escherichia coli RNA polymerase and lambda repressor . Mutations in PRM affect repression of PR; Hwang JJ et al.; The rightward operator, OR, of bacteriophage lambda is part of a complex regulatory region that includes PRM, the promoter for repressor synthesis by a prophage, the rightward early promoter PR, and three repressor-binding sites, OR1, OR2 and OR3 . By binding to OR2, repressor blocks transcription from PR and simultaneously stimulates the formation of open complexes between RNA polymerase and PRM . In this letter, we describe a test of the hypothesis that the interaction between RNA polymerase bound at PRM and repressor bound at OR2 increases the apparent affinity of repressor for OR . One implication of this hypothesis is that the amount of repressor required for repression of PR should be inversely correlated with PRM promoter strength . This is indeed the case . The amount of repressor required for 50% repression of PR is decreased by prmup-1, an "up" mutation of PRM, and is increased by prm- mutations . An unexpected finding is that in addition to their effect on the apparent affinity of repressor for OR, mutations in the -35 region of PRM alter the shape of repressor-titration curves . We propose that these mutations alter the interaction between RNA polymerase bound at PRM and repressor bound at OR2 in such a way that cooperativity in the binding of repressor to OR1 and OR2 is also disrupted.

J Mol Biol, 1988 Apr 20, 200(4), 695 - 708
Characterization of a doubly mutant derivative of the lambda PRM promoter . Effects of mutations on activation of PRM; Hwang JJ et al.; The mutation, prmE37, located at -14 in the PRM promoter of bacteriophage lambda, reduces PRM function dramatically both in vitro and in vivo . In a search for second-site revertants of prmE37, we isolated a double mutant that exhibits a partially restored Prm+ phenotype . The second-site mutation (at -31) is identical to the mutation prmup-1 . The activity of the doubly mutant (pseudo-revertant) promoter, prmE37prmup-1, was investigated in vivo using a PRM-lacZ fusion phage and found to be intermediate between that of prmE37 and wild-type PRM . However, the relative strength of the prmE37prmup-1 promoter was greater than expected following superinfection of a lambda lysogen . Since nalidixic acid was found to preferentially inhibit transcription from the doubly mutant promoter under these conditions, we suggest that DNA supercoiling favors activation of this promoter by repressor . In runoff transcription assays in the absence of repressor, the activity of wild-type PRM and the doubly mutant promoter were the same . However, while addition of repressor significantly stimulated wild-type PRM, it had little or no effect on the activity of the doubly mutant promoter . Values of KB, the equilibrium constant for formation of closed complexes, and kf, the rate constant for isomerization of closed to open complexes, were determined in abortive initiation assays, and the product of kfKB was used as a measure of promoter strength . The results of these assays are in agreement with those obtained in runoff transcription assays . In the absence of repressor, values of kfKB for the doubly mutant promoter and wild-type PRM are the same; however, tau obs, the time required for open complex formation, is significantly greater for the double mutant than for wild-type PRM at all RNA polymerase concentrations used for the abortive initiation analysis . In the presence of repressor, the doubly mutant promoter is stronger than the prmE37 promoter, but much weaker than wild-type PRM . This is due to the fact that kf for the doubly mutant promoter is increased 2.5-fold by repressor, but KB is reduced to the same extent . These two effects counteract each other, so that repressor has no net effect on the strength of the prmE37prmup-1 promoter in vitro . In contrast, repressor increases kf for wild-type PRM eightfold and increases overall promoter strength (KBkf) nearly fivefold . In the presence of repressor, the effects of the two mutations, prmE37 and prmup-1, on kf are independent . This observation is discussed in relation to revised models for open complex formation.

J Mol Biol, 1988 Apr 20, 200(4), 665 - 80
Hotspot sites for acridine-induced frameshift mutations in bacteriophage T4 correspond to sites of action of the T4 type II topoisomerase; Ripley LS et al.; The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations . Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activity . The ability of an acridine to promote topoisomerase-dependent cleavage at specific DNA sites in vitro is correlated to its ability to produce frameshift mutations at those sites in vivo . The specific phosphodiester bonds cleaved in vitro are precisely those at which frameshifts are most strongly promoted by acridines in vivo . The cospecificity of in vitro cleavage and in vivo mutation implicate acridine-induced, topoisomerase-mediated DNA cleavages as intermediates of acridine-induced mutagenesis in T4.

Biochemistry, 1988 Apr 19, 27(8), 2998 - 3004
Calf thymus DNA polymerases alpha and delta are capable of highly processive DNA synthesis; Sabatino RD et al.; We have demonstrated that calf thymus DNA polymerases alpha and delta are capable of highly processive DNA synthesis . Processivity values between 300 and 2000 nucleotides were observed when poly(dA)-oligo(dT) or singly primed single-stranded circular bacteriophage M13 DNA at pH 6.0 and 1 mM magnesium chloride was used . These conditions do not correlate with conditions, pH 7.0 and 5 mM magnesium chloride, that support the maximum synthetic rate . Lowering the pH and magnesium concentration lowers the Km value of the reaction with respect to primer terminus concentration . Furthermore, under these same conditions, both polymerases become insensitive to dissociation from the template as a result of encountering the 5' ends of primers . Overall, these results suggest that the affinity of the polymerases for the primer termini is higher throughout the polymerization reaction of pH and magnesium concentrations are lowered from those favoring maximum synthetic rate . Experiments with short primer templates, however, indicate that this higher affinity does not cause the DNA polymerase to remain stably bound after synthesizing up to the end of the template.

Biochemistry, 1988 Apr 19, 27(8), 2753 - 62
Structure and dynamics of a detergent-solubilized membrane protein: measurement of amide hydrogen exchange rates in M13 coat protein by 1H NMR spectroscopy; O'Neil JD et al.; The coat protein of bacteriophage M13 is inserted into the inner membrane of Escherichia coli where it exists as an integral membrane protein during the reproductive cycle of the phage . The protein sequence consists of a highly hydrophobic 19-residue central segment flanked by an acidic 20-residue N-terminus and a basic 11-residue C-terminus . We have measured backbone amide hydrogen exchange of the protein solubilized in perdeuteriated sodium dodecyl sulfate using 1H nuclear magnetic resonance (NMR) spectroscopy . Direct proton exchange-out measurements in D2O at 24 degrees C were used to follow the exchange of the slowest amides in the protein . Multiple exponential fitting of the exchange data showed that these amides (29 +/- 3 at pH 4.5) exchanged in two kinetic sets with exchange rates {(1.2 +/- 0.4) x 10(-4) s-1 and (4.1 +/- 1.2) x 10(-7) s-1} that differed by more than 100-fold, the slower kinetic set being retarded 10(5)-fold relative to poly(DL-alanine) . The exchange rate constant for the slowest set of amides exhibited an unusual pD dependence, being proportional to {OD-}1/2 . It is shown that this is an artifact of the multiple exponential fitting of the data, and a new method of presentation of exchange data as a function of pD is introduced . Steady-state saturation-transfer techniques were also used to measure exchange . These methods showed that 15-20 amides in the protein are very stable at 55 degrees C and that about 30 amides have exchange rates retarded by at least 10(5)-fold at 24 degrees C . Saturation-transfer studies also showed that the pH dependence of exchange in the hydrophilic termini was unusual . This is explained as being due to long-range electrostatic effects arising both from the protein itself and also from the anionic detergent molecules . Hydrogen exchange studies on the products of proteinase K digestion of the protein localized the slowly exchanging amides to the hydrophobic core of the protein . Relaxation {Henry, G.D., Weiner, J.H., & Sykes, B.D . (1986) Biochemistry 25, 590-598} and solid-state NMR experiments {Leo, G.C., Colnago, L.A., Valentine, K.G., & Opella, S.J . (1987) Biochemistry 26, 854-862} have previously shown that the majority of the protein backbone is rigid on the picosecond to microsecond time scale, except for the extreme ends of the molecule which are mobile.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochim Biophys Acta, 1988 Apr 14, 953(3), 232 - 40
Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate; Handford PA et al.; Asp-362, a potential key catalytic residue of Escherichia coli citrate synthase (citrate oxaloacetate-lyase {pro-3S)-CH2COO- ----acetyl-CoA), EC 4.1.3.7) has been converted to Gly-362 by oligonucleotide-directed mutagenesis . The mutant gene was completely sequenced, using a series of synthetic oligodeoxynucleotides spanning the structural gene to confirm that no additional mutations had occurred during genetic manipulation . The mutant gene was expressed in M13 bacteriophage and produced a protein which migrated in an identical manner to wild-type E . coli citrate synthase on SDS-polyacrylamide gels and which cross-reacted with E . coli citrate synthase antiserum . The mutant gene was subsequently recloned into pBR322 for large scale purification of the protein, and the resulting plasmid, pCS31, used to transform the citrate synthase deletion strain, W620 . The mutant enzyme purified in an analogous manner to wild-type E . coli citrate synthase and expressed less than 2% of wild-type enzyme activity . The activity of the partial reactions catalysed by citrate synthase was similarly affected suggesting that this residual activity may be due to contaminating wild-type enzyme activity . The mutant citrate synthase retains a high-affinity NADH-binding site consistent with the protein preserving its overall structural integrity . Oxaloacetate binding to the protein is unaffected by the Asp-362 to Gly-362 mutation . Binding of the acetyl-CoA analogue, carboxymethyl-CoA, could not be detected in the mutant protein indicating that the lack of catalytic competence is due primarily to the inability of the protein to bind the second substrate, acetyl-CoA.

J Theor Biol, 1988 Apr 7, 131(3), 351 - 85
Movable Finite Automata (MFA) models for biological systems . I: Bacteriophage assembly and operation; Thompson RL et al.; A new class of models, called Movable Finite Automata (MFA) models, is introduced . MFA models are physically realistic, but still share some of the features of cellular automata that make the latter easy to handle mathematically and computationally . They are found to be quite versatile in modeling the self-organization of biological systems . Their use in simulating the interaction of protein molecules in the self-assembly and operation of the T4 bacteriophage is described . The results of these simulations carried out on a microcomputer, are given.

J Mol Biol, 1988 Apr 5, 200(3), 475 - 88
Cloning, overexpression and purification of the terminase proteins gp16 and gp17 of bacteriophage T4 . Construction of a defined in-vitro DNA packaging system using purified terminase proteins; Rao VB et al.; Terminases of double-stranded DNA bacteriophages are required for packaging and generation of terminii in replicated concatemeric DNA molecules . Genetic evidence suggests that these functions in phage T4 are carried out by the products of genes 16 and 17 . We cloned these T4 genes into a heat-inducible cI repressor-lambda PL promoter vector system, and overexpressed them in Escherichia coli . We developed an in-vitro DNA packaging system, which, consistent with the genetic data, shows an absolute requirement for the terminase proteins . The overexpressed terminase proteins gp16 and gp17 appear to form a specific complex and an ATP binding site is present in the gp17 molecule . We purified the terminase proteins either as individual gp16 or gp17 proteins, or as a gp16-gp17 complex . The gp16 function of the terminase complex is dispensable for packaging mature DNA, whereas gp17 is essential for packaging DNA under any condition tested . We constructed a defined in-vitro DNA packaging system with the purified terminase proteins, purified proheads and a DNA-free phage completion gene products extract . All the components of this system can be stored at -90 degrees C without loss of packaging activity . The terminase proteins, therefore, may serve as useful reagents for mechanistic studies on DNA packaging, as well as to develop T4 as a packaging-cloning vector.

J Mol Biol, 1988 Apr 5, 200(3), 489 - 500
Engineered recombinant messenger RNA can be replicated and expressed inside bacterial cells by an RNA bacteriophage replicase; Mills DR; In this paper we describe how an RNA replicase can be "tricked" into recognizing, binding to, and replicating host-cell message RNAs . In our system, the gene encoding Q beta replicase is constitutively expressed from a plasmid vector present in an Escherichia coli host, while a second plasmid directs the transcription of a replication-competent, phage-like template RNA . This 680 nucleotide transcript (N- RNA) contains specific sequences required for Q beta replicase function . Included within this template RNA is a 360-nucleotide sequence that is complementary to the messenger RNA for DNA bacteriophage lambda N protein . The active messenger RNA for lambda N protein is expressed only upon replication of N- RNA by Q beta replicase . By employing an E . coli host strain that requires lambda N protein for growth under specific selective conditions, we were able to select only those cells in which RNA-directed RNA replication occurred . This system provides a potential for in vivo amplification of other heterologous messenger RNAs and their protein products via RNA replication and will enable one to study the evolution of messenger RNA in the live cell under a large variety of physiological pressures.

Biochem Int, 1988 Apr, 16(4), 701 - 12
Identification and expression of the 20 kd structural protein gene of colitis bacteriophage; Subrahmanyam YV et al.; The 2.3 kb BamHI fragment from the colitis bacteriophage DNA was transcribed and translated into a 20 kd structural protein P6, in a coupled transcription-translation system derived from Escherichia coli . This protein was expressed in vivo by the 2.3 kb DNA cloned in pBR322 . The gene with the regulatory elements for this protein was located on the 680 bp AvaII fragment of the insert DNA . It hybridized with two RNAs of sizes 520 and 1630 nucleotides indicating that both are messengers for the 20 kd protein . Dot-blot hybridization showed that the transcripts for P6 reached a maximum level at 12 min after phage infection.

Virology, 1988 Apr, 163(2), 618 - 21
Transient expression and sequence of the matrix (M1) gene of WSN influenza A virus in a vaccinia vector; Baylor NW et al.; A cDNA encoding the entire amino acid sequence of the matrix (M1) protein of influenza A/WSN/33 virus was cloned, sequenced, and expressed in a vaccinia virus system consisting of the T7 bacteriophage RNA polymerase and a plasmid carrying the M1 gene flanked by T7 polymerase promoter and terminator sequences . The transiently expressed M1 gene product comigrated on SDS-polyacrylamide gels with the endogenous WSN virus M1 protein and was recognized in Western blot analysis by three epitope-specific monoclonal antibodies directed to the M1 protein . The nucleotide sequence and the predicted amino acid sequence of the cloned WSN virus M1 coding region was found to be more than 97% homologous to that of the M1 gene of influenza virus A/PR/8/34 reported by G . Winter and S . Fields (Nucleic Acids Res . 8, 1965-1974, 1980).

J Virol, 1988 Apr, 62(4), 1180 - 5
Nucleotide sequence of the large double-stranded RNA segment of bacteriophage phi 6: genes specifying the viral replicase and transcriptase; Mindich L et al.; The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA . We determined the nucleotide sequence of cDNA derived from the largest RNA segment (L) . This segment specifies the procapsid proteins necessary for transcription and replication of the phi 6 genome . The coding sequences of the four proteins on this segment were identified on the basis of size and the correlation of predicted N-terminal amino acid sequences with those found through analysis of isolated proteins . This report completes the sequence analysis of phi 6 . This constitutes the first complete sequence of a double-stranded RNA genome virus.

Vaccine, 1988 Apr, 6(2), 161 - 3
Roles of vaccinia virus in the development of new vaccines; Moss B et al.; Vaccinia virus is an efficient expression vector with broad host range infectivity and large DNA capacity . This vector has been particularly useful for identifying target antigens for humoral and cell-mediated immunity . With increased levels of gene expression, obtained either with stronger vaccinia promoters or through incorporation of the bacteriophage T7 RNA polymerase gene into the vaccinia genome, proteins may be synthesized in mammalian cells for use as subunit vaccines . For use as a live recombinant vaccine, efforts are being made to attenuate vaccinia virus further, either by inactivating genes contributing to virulence or by introducing human lymphokine genes into the vaccinia genome.

J Bacteriol, 1988 Apr, 170(4), 1541 - 7
Construction and characterization of mutations in hupB, the gene encoding HU-beta (HU-1) in Escherichia coli K-12; Storts DR et al.; Plasmid pJMC21 contains Escherichia coli chromosomal DNA encoding Lon protease, HU-beta (HU-1), and an unidentified 67,000-dalton protein . A kanamycin resistance cassette was used in the construction of insertion and deletion mutations in hupB, the gene encoding HU-beta on plasmid pJMC21 . The reconstructed plasmids were linearized and used to introduce hupB chromosomal mutations into JC7623 (recBC sbcBC) . These mutations, as expected, mapped in the 9.8-min region of the E . coli chromosome by P1 transduction (16% linkage to proC+) . Southern blot hybridization of chromosomal fragments verified that hupB+ was replaced by the mutant allele, with no indication of gene duplication . All the mutant strains had growth rates identical to that of wild-type E . coli, were resistant to UV irradiation and nitrofurantoin, and supported the in vivo transposition-replication of bacteriophage Mu, Mu lysogenization, Tn10 transposition from lambda 1098, and lambda replication-lysogenization . The only observable phenotypic variation was a reduced Mu plaque size on the hupB mutant strains; however, the yield of bacteriophage Mu in liquid lysates prepared from the mutant strains was indistinguishable from the yield for the wild type.

J Bacteriol, 1988 Apr, 170(4), 1980 - 3
Isolation of fla-lacZ fusions in Escherichia coli K-12: most fusions result in soluble beta-galactosidase; Komeda Y; A series of fusions of flagellar genes to the lacZ gene was generated by insertion of Mu dII301 (Apr lac) bacteriophage into the genome of Escherichia coli . The beta-galactosidase activity in each resulting mutant was measured, and the location of the activity in the membrane, periplasmic, or cytoplasmic fraction of the cell was determined . There were three classes of mutants: those which had beta-galactosidase activity mainly in the membrane fraction, those which had it distributed in the soluble and membrane fractions, and those which had it in the cytoplasmic fraction only . The last, soluble-fraction-only, class was predominant in fla-lac gene fusions . In particular, the following mutants were shown to have beta-galactosidase activity in the membrane fractions: on the inner membrane, mutants with flaB fusions, and on the inner and outer membranes, mutants with flaA4850, flaM, and flaU4849 fusions . These results suggest that fla-lacZ gene fusions produce proteins which are able to detect the signals of the leader sequence and the membrane-anchoring region of the flagellar system.

EMBO J, 1988 Apr, 7(4), 1229 - 37
The DNA invertase Gin of phage Mu: formation of a covalent complex with DNA via a phosphoserine at amino acid position 9; Klippel A et al.; The DNA invertase Gin encoded by bacteriophage Mu catalyses efficient site-specific recombination between inverted repeat sequences (IR) in vivo and in vitro in the presence of the host factor FIS and the recombinational enhancer . We demonstrate that Gin alone is able to introduce single strand breaks into duplex DNA fragments which contain the IR sequence . Strand cleavage is site-specific and can occur on either strand within the IR . Cleaved molecules contain Gin covalently attached to DNA . The covalent complex is formed through linkage of Gin to the 5' DNA phosphate at the site of the break via a phosphoserine . Extensive site-directed mutational analysis showed that all mutants altered at serine position 9 were completely recombination deficient in vivo and in vitro . The mutant proteins bind to DNA but lack topoisomerase activity and are unable to introduce nicks . This holds true even for a conservative amino acid substitution at position 9 . We conclude that serine at position 9 is part of the catalytic domain of Gin . The intriguing finding that the DNA invertase Gin has the same catalytic center as the DNA resolvases that promote deletions without recombinational enhancer and host factor FIS is discussed.

EMBO J, 1988 Apr, 7(4), 1219 - 27
Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin; Mertens G et al.; Site-specific DNA inversion in phage Mu is catalysed by the phage-encoded DNA invertase Gin and a host factor FIS . We demonstrate that purified Gin protein binds specifically to 34-bp sequences that flank the G segment as inverted repeats . Each inverted repeat (IR) contains two binding sites for Gin which have to be arranged in a specific configuration to constitute a recombinogenic site . While one of these sites is bound when present alone, the other site is bound only in conjunction with the first one, suggesting cooperative binding . In addition to the sites within the IR, Gin binds with lower affinity to AT-rich sequences adjacent to the IR . We demonstrate that these sites do not participate in the inversion reaction . The IR itself can be shortened to 25 bp without effect on inversion frequency . Using gel mobility shift experiments on circular permuted fragments containing the IR we show that Gin bends DNA upon binding . We discuss the possibility that DNA bending is related to the formation of a productive synaptic complex.

Mol Gen Genet, 1988 Apr, 212(1), 142 - 8
Bacteriophage lambda DNA packaging: a mutant terminase that is independent of integration host factor; Feiss M et al.; Lambda+ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF+ cells . In vitro, however, we find that the lambda DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions . The cos59 mutation renders lambda dependent on IHF in vivo . The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of lambda . Variants of lambda cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase . The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine . The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro . The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.

J Bacteriol, 1988 Apr, 170(4), 1683 - 90
Intermediates in bacteriophage Mu lysogenization of Escherichia coli him hosts; Bourret RB et al.; Characterization of a putative intermediate in the Mu lysogenization pathway is possible in a variant Escherichia coli himD strain which exhibits greatly diminished lysogen formation . In this strain, most infecting Mu genomes form stable, transcribable, nonreplicating structures . Many of these genomes can be mobilized to form lysogens by a second Mu infection, which can be delayed by at least 100 min . This intermediate structure can be formed in the absence of Mu A or B function . We suggest that the inferred intermediate could be the previously reported protein-linked circular form of the Mu genome . Providing Mu B function from a plasmid enhances Mu lysogenization in this him strain, and the enhancement is much greater when both Mu A and B functions are provided.

J Bacteriol, 1988 Apr, 170(4), 1672 - 82
Lysogenization of Escherichia coli him+, himA, and himD hosts by bacteriophage Mu; Bourret RB et al.; The possible outcomes of infection of Escherichia coli by bacteriophage Mu include lytic growth, lysogen formation, nonlysogenic surviving cells, and perhaps simple killing of the host . The influence of various parameters, including host himA and himD mutations, on lysogeny and cell survival is described . Mu does not grow lytically in or kill him bacteria but can lysogenize such hosts . Mu c+ lysogenizes about 8% of him+ bacteria infected at low multiplicity at 37 degrees C . The frequency of lysogens per infected him+ cell diminishes with increasing multiplicity of infection or with increasing temperature over the range from 30 to 42 degrees C . In him bacteria, the Mu lysogenization frequency increases from about 7% at low multiplicity of infection to approach a maximum where most but not all cells are lysogens at high multiplicity of infection . Lysogenization of him hosts by an assay phage marked with antibiotic resistance is enhanced by infection with unmarked auxiliary phage . This helping effect is possible for at least 1 h, suggesting that Mu infection results in formation of a stable intermediate . Mu immunity is not required for lysogenization of him hosts . We argue that in him bacteria, all Mu genomes which integrate into the host chromosome form lysogens.

J Virol, 1988 Apr, 62(4), 1186 - 93
Nucleotide sequence of the tail sheath gene of bacteriophage T4 and amino acid sequence of its product; Arisaka F et al.; The nucleotide sequence of gene 18 of bacteriophage T4 was determined by the Maxam-Gilbert method, partially aided by the dideoxy method . To confirm the deduced amino acid sequence of the tail sheath protein (gp18) that is encoded by gene 18, gp18 was extensively digested by trypsin or lysyl endopeptidase and subjected to reverse-phase high-performance liquid chromatography . Approximately 40 peptides, which cover 88% of the primary structure, were fractionated, the amino acid compositions were determined, and the corresponding sequences in DNA were identified . Furthermore, the amino acid sequences of 10 of the 40 peptides were determined by a gas phase protein sequencer, including N- and C-terminal sequences . Thus, the complete amino acid sequence of gp18, which consists of 658 amino acids with a molecular weight of 71,160, was determined.

Mol Gen Mikrobiol Virusol, 1988 Apr, (4), 22 - 5
{Isolation and characteristics of the bacteriophage from the vaccine strain Tohama phase I}; Karataev GI et al.; Some properties of bacteriophage phi T isolated from the vaccine strain Bordetella pertussis Tohama phase I and propagated in Bordetella parapertussis 504 cells are presented . Phage phi T belongs to the IV group in accordance with Tikhonenko classification . The diameter of head and length of noncontractile tail sheath are 49.5 +/- 0.5 and 145 +/- 7 nm, respectively . Diameter of the tail sheath is 3.2 +/- 0.6 nm . Molecular mass of the phage DNA is 37 +/- 3 kb . Population of phi T phage is polymorphous and consists of particles the genomes or which vary from each other by the "insert" located 6.8 +/- 0.6 kb from the end of molecule . The blot hybridization has demonstrated that the bacteriophage genome is not inserted into the chromosome of the lysogenic strain . Autonomous location of the phage genome in the host cell is suggested . The temperature and hydrogen ions concentration effects on bacteriophage phi T stability were studied . The conditions for phage suspension storage are described.

Ann Inst Pasteur Virol, 1988 Apr-Jun, 139(2), 217 - 25
{cRNA probes (riboprobes) synthesized in vitro for detecting enterovirus by molecular hybridization}; Kopecka H et al.; Radioactively labelled RNA transcripts made in vitro of various fragments from cDNA clones of poliovirus type 1 and of hepatitis A virus under the control of bacteriophage T7 or SP6 promoters have been evaluated for diagnostic purposes . The RNA transcripts were 2 orders of magnitude more sensitive as hybridization probes than corresponding cDNA preparations labelled by the nick translation procedure . A combination of hybridization analysis and sequence comparison showed that some regions of the genome of a number of enteroviruses are highly conserved, while others show very little homology; the general order of conservation is: 5'-non-coding greater than 3'-terminal greater than central (2C) greater than VP3 greater than VP1 . The 350 bases of the poliovirus VP1 region were highly specific for that virus, while the 450-bases of the 5'NC region showed extensive cross-reaction with other enteroviruses . However, these probes did not hybridize with HAV, which was detected only by HAV-specific riboprobes . The transcripts have been successfully applied as hybridization probes in diagnostic tests on supernatants of infected cell culture lysates and in clinical samples, mainly in stool extracts.

Mol Gen Genet, 1988 Apr, 212(1), 157 - 65
The bacteriophage lambda cohesive end site: isolation of spacing/substitution mutations that result in dependence on Escherichia coli integration host factor; Miller G et al.; Substitution, insertion and deletion mutations have been constructed at the XmnI restriction site in cos lambda . The XmnI site is located between cosB, the site where terminase binds lambda DNA; and cosN, the site where terminase introduces staggered nicks to generate cohesive ends . Substitution mutations and deletion of a base pair (a -1 change) do not obviously affect lambda growth and DNA packaging . Changes of -2, +2 and -3 render lambda unable to grow on host cells lacking integration host factor (IHF) . The -3 mutant has a reduced burst size in IHF+ cells, due to a defect in the initiation of packaging . A -7 deletion mutation is lethal . Models for the basis of these mutational effects are discussed.

Mol Gen Genet, 1988 Apr, 212(1), 149 - 56
A point mutation in the Nul gene of bacteriophage lambda facilitates phage growth in Escherichia coli with himA and gyrB mutations; Granston AE et al.; A mutant of lambda was isolated that grows in the Escherichia coli himA delta/gyrB-him320(Ts) double mutant at 42 degrees C; conditions which are non-permissive for wild-type lambda growth . The responsible mutation, ohm1, alters the 40th codon of the Nul reading frame . The Nul and A gene products comprise the terminase protein which cleaves concatameric DNA into unit-length phage genomes during DNA packaging . The Nul-ohm1 gene product acts in trans to support lambda growth in the double himA/gyrB mutant, and lambda cos154 growth in the single himA mutant . The observation that an alteration in Nul suppresses the inhibition of growth in the double himA/gyrB mutant implicates DNA gyrase, as well as integration host factor, in the DNA:protein interactions that occur at the initiation of packaging.

Mol Gen Genet, 1988 Apr, 212(1), 11 - 9
Cell division inhibition gene dicB is regulated by a locus similar to lambdoid bacteriophage immunity loci; Bejar S et al.; A mutation (dicA1) of a repressor gene located in the terminus region of the Escherichia coli chromosome has previously been shown to lead to temperature-dependent inhibition of division, and to be complemented by plasmids carrying either dicA or an adjacent gene dicC . In this study, operon fusions in the region coding for the division inhibition gene dicB have been used to show that temperature sensitivity does not result from high temperature inactivation of the dicA repressor . Sequence comparisons indicate that dicA and dicC are similar to genes c2 and cro respectively of bacteriophage P22, and carry similarly organized tandem operators, indicating a common evolutionary origin for dicAC and P22 immC . Nevertheless, the consensus half-operator sequence of dicAC, TGTTA-GYYA, differs significantly from that of P22 immC (ATT-TAAGAN) . An analysis of the in vivo control of promoters dicAp, dicBp and dicCp placed upstream of malQ shows that the dicAC system is functionally similar to that of an immunity region, with the possible exception of an absence of pairwise cooperative binding . Our results also indicate that the dicA1 mutation causes a switch to permanent control by dicC at all temperatures.

J Bacteriol, 1988 Apr, 170(4), 1994 - 8
Expression and DNA sequence of the cloned bacteriophage T4 dCMP hydroxymethylase gene; Thylen C; A plasmid expressing the cloned bacteriophage T4 gene 42 gave the same levels of complementation of gene 42 mutants in a polarity-suppressing rho mutant as in a rho+ host . A reading frame likely corresponding to gene 42 and putative promoter and terminator sequences were identified in the partial sequence of the cloned fragment.

J Bacteriol, 1988 Apr, 170(4), 1730 - 8
Genetic analysis of sequences in maltoporin that contribute to binding domains and pore structure; Heine HG et al.; Maltoporin (LamB protein) is a maltodextrin transport protein in the outer membrane of Escherichia coli with binding sites for bacteriophage lambda and maltosaccharides . Binding of starch by bacteria was found to inhibit swarming of Escherichia coli in soft agar plates; the inhibition was dependent on the maltodextrin affinity of maltoporin . On the basis of this observation, chemotactic cell-sorting techniques were developed for the isolation and analysis of mutants with an altered starch-binding phenotype . Fifteen lamB mutations generated by hydroxylamine and linker mutagenesis, as well as spontaneous mutations, were analyzed . The effects of the mutations on starch and lambda-binding, as well as transport specificity, were assayed . Mutations that affect residues near 8 to 18, 74 to 82, and 118 to 121 were found to affect starch binding and maltodextrin-selective functions strongly, confirming and extending previous results with substitutions at these regions . Substitutions and insertions in two previously undefined regions in the protein, in or near residues 194 and 360, also resulted in defects in maltodextrin-specific functions and indicate that C-terminal parts of the protein also contribute to the discontinuous binding and pore domains . There was a detectable transport defect in all binding-affected mutants, and one mutation caused near-total pore blocking towards both maltose and nonmaltoside . The highly discontinuous phage lambda-binding site was affected by mutations near residues 9 and 10 and 194, as well as previously established regions near residues 18, 148 to 165, 245 to 259, and 380 to 400 . The significance of these mutations is discussed in the context of a model of the functional topology of maltoporin . The additional role of regions near residues 10 and 120 in maltoporin assembly, as well as starch binding, was suggested by the temperature-sensitive biogenesis of maltoporin in strains with one- or two-codon insertion at these sites.

Biochem Biophys Res Commun, 1988 Mar 30, 151(3), 1173 - 9
5-Hydroxymethylcytosine DNA glycosylase activity in mammalian tissue; Cannon SV et al.; The enzymatic release of 5-hydroxymethylcytosine from T2 bacteriophage DNA was effected by an extract of calf thymus . Like the previously described 5-hydroxymethyluracil DNA glycosylase, 5-hydroxymethylcytosine DNA glycosylase was not detectable in bacterial extracts . The phylogenetic distribution of these activities indicates that their primary function is the maintenance of methylcytosine residues in differentiated tissue.

J Biol Chem, 1988 Mar 25, 263(9), 4208 - 14
Kinetics of promoter search by Escherichia coli RNA polymerase . Effects of monovalent and divalent cations and temperature; Singer PT et al.; The rapid mixing/photocross-linking technique developed in our laboratory has been employed in the study of the mechanism of promoter binding by Escherichia coli RNA polymerase (RPase) . We have previously reported on the quantitation of the one-dimensional diffusion coefficient (D1) for RPase along the DNA template (Singer, P . T., and Wu, C.-W . (1987) J . Biol . Chem . 262, 14178-14189) . In this paper, we describe the effect of salt concentration and temperature on the kinetics of promoter search by RPase using plasmid pAR1319 DNA, which contains the A2 early promoter from bacteriophage T7, as template . Over a range of KCl concentrations from 25 to 200 mM, the apparent bimolecular rate constant (ka) for the association of RPase with the A2 promoter on this DNA template varied approximately 2-fold, achieving a maximal value between 100 and 125 mM KCl . More significantly, the transient distribution of RPase among nonspecific DNA binding sites changed markedly as a function of salt concentration, indicative of gross changes in the average number of base pairs covered by sliding during a nonspecific lifetime . Using the mathematical treatment outlined in our earlier report, the nonspecific dissociation rate constant (koff) was calculated from the binding curves for the nonspecific as well as promoter-containing DNA . The observed variations in ka as a function of monovalent cation concentration ({M+}) were due primarily to changes in koff, as D1 was found to be essentially independent of {M+} . Interestingly, D1 decreased by one-third as the concentration of magnesium was lowered from 10 to 1 mM . In addition, the dependence of koff (and consequently the nonspecific equilibrium association constant, keq) on {M+} agreed qualitatively with the results of deHaseth et al . (deHaseth, P.L., Lohman, T . M., Burgess, R . R., and Record, M . T., Jr . (1977) Biochemistry 17, 1612-1622), though we consistently measure a weaker Keq . The association rate constant was also measured between 4 and 37 degrees C, and was found to vary approximately 2-fold over that range . An activation energy for the bimolecular association of RPase to the A2 promoter was calculated to be 2.2 +/- 0.4 kcal/mol, while the activation energy for one-dimensional diffusion was 4.7 +/- 0.8 kcal/mol.

Nucleic Acids Res, 1988 Mar 25, 16(5), 2283 - 94
Recognition sequences of type II restriction systems are constrained by the G + C content of host genomes; McClelland M; I show that the recognition sequences of Type II restriction systems are correlated with the G + C content of the host bacterial DNA . Almost all restriction systems with G + C rich tetranucleotide recognition sequences are found in species with A + T rich genomes, whereas G + C rich hexanucleotide and octanucleotide recognition sequences are found almost exclusively in species with G + C rich genomes . Most hexanucleotide recognition sequences found in species with A + T rich genomes are A + T rich . This distribution eliminates a substantial proportion of the potential variance in the frequency of restriction recognition sequences in the host genomes . As a consequence, almost all restriction recognition sequences, including those eight base pairs in length (Not I and Sfi I), are predicted to occur with a frequency ranging from once every 300 to once every 5,000 base pairs in the host genome . Since the G + C content of bacteriophage DNA and of the host genome are also correlated, the data presented is evidence that most Type II "restriction systems" are indeed involved in phage restriction.

Biochemistry, 1988 Mar 22, 27(6), 2088 - 94
The Mnt repressor of bacteriophage P22: role of C-terminal residues in operator binding and tetramer formation; Knight KL et al.; A set of C-terminal deletion mutants of the Mnt repressor of bacteriophage P22 has been constructed, and the corresponding truncated proteins have been purified . A truncated protein lacking the three C-terminal residues, Lys80-Thr81-Thr82, binds operator DNA with an affinity near wild type and has a normal tetrameric structure . Loss of the next residue, Lys79, causes a 600-fold decrease in operator affinity, but the truncated protein is still tetrameric . Further sequential deletions of Tyr78 and Leu77 cause modest decreases in operator affinity, but the truncated proteins are now dimeric . These results indicate that Lys79 is an important determinant of the high affinity of Mnt repressor for operator DNA and that Tyr78 is an important determinant of tetramer formation by Mnt repressor.

Biochemistry, 1988 Mar 22, 27(6), 1832 - 8
Site-directed mutagenesis of the T4 endonuclease V gene: role of lysine-130; Recinos A 3rd et al.; The DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid lambda promoter OLPR in a plasmid vector . The OLPR-denV sequence was subcloned in M13mp18 and used as template to construct site-specific mutations in the denV structural gene in order to investigate structure/function relationships between the primary structure of the protein and its various DNA binding and catalytic activities . The Lys-130 residue of the wild-type endonuclease V has been postulated to be associated with its apurinic endonuclease (AP-endonuclease) activity . The codon for Lys-130 was changed to His-130 or Gly-130, and each denV sequence was subcloned into a pEMBL expression vector . These plasmids were transformed into repair-deficient Escherichia coli (uvrA recA), and the following parameters were examined for cells or cell extracts: expression and accumulation of endonuclease V protein (K-130, H-130, or G-130); survival after UV irradiation; dimer-specific DNA binding; and kinetics of phosphodiester bond scission at pyrimidine dimer sites, dimer-specific N-glycosylase activity, and AP-endonuclease activity . The enzyme's intracellular accumulation was significantly decreased for G-130 and slightly decreased for H-130 despite normal levels of denV-specific mRNA for each mutant . On a molar basis, the endonuclease V gene products generally gave parallel levels of each of the catalytic and binding functions with K-130 greater than H-130 greater than G-130 much greater than control denV-.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1988 Mar 20, 200(2), 351 - 65
Molecular substructure of a viral receptor-recognition protein . The gp17 tail-fiber of bacteriophage T7; Steven AC et al.; The bacteriophage T7 tail complex consists of a conical tail-tube surrounded by six kinked tail-fibers, which are oligomers of the viral protein gp17 (Mr 61,400) . We have derived a molecular model for the tail-fiber by integrating secondary structure predictions with ultrastructural information obtained by correlation averaging of electron micrographs of negatively stained tail complexes . This model has been further refined by high-resolution scanning transmission electron microscopy of purified fibers, both negatively stained and unstained . Mass measurements made from the latter images establish that the fiber is a trimer of gp17 . The proximal half-fiber is a uniform rod, about 2.0 nm in diameter and 16.4 nm long, which we infer to be a triple-stranded coiled-coil, containing three copies of an alpha-helical domain of about 117 residues, starting at Phe151 . The distal half-fiber is 15.5 nm long, and is made up of four globules, 3.1 to 4.8 nm in diameter, in rigid linear array: it contains the carboxy-terminal halves (residues approximately 268 to 553) of the constituent gp17 chains, arranged with 3-fold symmetry around its long axis . The amino-terminal domains (residues 1 to 149) link the fiber to the tail-tube . We conclude that the three gp17 chains are quasi-equivalent in the proximal half-fiber, equivalent in the distal half-fiber, and non-equivalent in the kink region that separates the two half-fibers: such localized non-equivalence may represent a general mechanism for the formation of kinked joints in segmented homo-oligomeric proteins.

Eur J Biochem, 1988 Mar 15, 172(3), 553 - 63
Deoxycytidylate hydroxymethylase gene of bacteriophage T4 . Nucleotide sequence determination and over-expression of the gene; Lamm N et al.; We describe two approaches to cloning and over-expressing gene 42 of bacteriophage T4, which encodes the early enzyme deoxycytidylate hydroxymethylase . In Bochum a library of sonicated fragments of wild-type phage DNA cloned into M13mp18 was screened with clones known to contain parts of gene 42 . Two overlapping fragments, each of which contained one end of the gene, were cleaved at a HincII site and joined, to give a fragment containing the entire gene . In Corvallis a 1.8-kb fragment of cytosine-substituted DNA, believed to contain the entire gene, was cloned into pUC18 and shown to express the enzyme at low level . The cloned fragment bore an amber mutation in gene 42 . From the DNA sequence of gene 42, the cloned gene was converted to the wild-type allele by site-directed mutagenesis . Both gene-42-containing fragments were cloned into the pT7 expression system and found to be substantially overexpressed . dCMP hydroxymethylase purified from one of the over-expressing strains had a turnover number similar to that of the enzyme isolated earlier from infected cells . In addition, the N-terminal 20 amino acid residues matched precisely the sequence predicted from the gene sequence . The amino acid sequence of gp42 bears considerable homology with that of thymidylate synthase of either host or T4 origin . The gene 42 nucleotide sequences of bacteriophages T2 and T6 were determined and found to code for amino acid sequences nearly identical to that of T4 gp42.

J Immunol, 1988 Mar 15, 140(6), 1939 - 45
Role of C3 in humoral immunity . Defective antibody production in C3-deficient dogs; O'Neil KM et al.; Previous studies have shown that animals pharmacologically depleted of C3 have impaired antibody responses . However, such C depletion is neither complete nor sustained, and the C3 cleavage products generated by C3 depletion can both enhance and inhibit the immune response . To clarify the role of C3 in humoral immunity, the antibody response of dogs with genetically determined total deficiency of C3 (C3D) was examined . Serum IgG levels of the C3D animals were within the normal range, but were significantly lower than levels seen in normal controls or C3D heterozygotes . Specific antibody production was defective: the antibody titers of C3D dogs in response to primary intravenous immunization with two different T cell-dependent Ag (sheep E and bacteriophage phi X-174) were markedly reduced when compared to either normal controls or C3D heterozygotes . After secondary immunization with T-dependent Ag, the total antibody titers were normal, but the C3D dogs made proportionately more IgM and less IgG antibody than did either control group . After i.v . immunization with a T cell-independent Ag (DNP-Ficoll), the C3D dogs had reduced levels of IgM and IgG antibody after primary and secondary immunization . Neither i.m . immunization nor the use of a 20-fold increase in Ag dose i.v . could correct the defect seen in the antibody response of C3D dogs . The results herein demonstrate that C3 plays a critical role in the generation of a normal humoral immune response.

Eur J Biochem, 1988 Mar 15, 172(3), 641 - 6
A maximum of two tryptophan residues in gene-32 protein from phage T4 undergo stacking interactions with single-stranded polynucleotides; Casas-Finet JR et al.; The effect of specific photochemical and radiochemical modification of tryptophyl and cysteinyl residues of the gene 32 protein (gp 32) of bacteriophage T4 on its affinity towards single-stranded polynucleotides has been investigated . Oxidation of Cys residues of gp 32 by the free-radical anion I-.2 induces a partial loss of the protein affinity, probably by affecting the metal-binding domain which includes three of the four cysteine residues of gp 32 . Ultraviolet irradiation of gp 32 in the presence of trichloroethanol results in the modification of three of its five Trp residues and total loss of the protein binding . Analysis of the relative affinity of ultraviolet-irradiated gp 32 for single-stranded polynucleotides suggest that modification of a Trp of enhanced reactivity occurs first and has no effect on the protein binding . Radiochemical modification of three Trp residues of gp 32 by (SCN)-.2 results in total loss of activity . Complexation of gp 32 with denatured DNA prior to gamma-irradiation protects two Trp residues and prevents the protein inactivation . These results suggest that at most two Trp residues are involved in stacking interactions with nucleic acid bases . However, time-resolved spectroscopic methods which allow us to monitor selectively the stacked tryptophan residues have not yielded evidence of more than a single residue undergoing such interactions.

J Biol Chem, 1988 Mar 15, 263(8), 3554 - 7
Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor; Robertson CA et al.; Escherichia coli integration host factor (IHF) is a small basic protein that is required for efficient integrative recombination of bacteriophage lambda . IHF binds specifically to sequences within attP, the site in bacteriophage lambda that undergoes recombination . It has been suggested that the binding of IHF creates bends in DNA so as to help attP condense into a compact structure that is activated for recombination . In this work we show that IHF binding to either of two sites found within attP does indeed produce bending of DNA . In contrast, the other recombination protein needed for integrative recombination, Int, does not appreciably bend the DNA to which it is bound . In agreement with the proposal that IHF bending is important for creating a condensed attP, bending by IHF persists in the presence of bound Int . Our conclusions about protein-directed bends in DNA are based on the study of the electrophoretic mobility of a set of permuted DNA fragments in the presence or absence of IHF and/or Int . To facilitate this study, we have constructed a novel vector that simplifies the generation of permuted fragments . This vector should be useful in studying the bending of other DNA sequences by specific binding proteins.

Nucleic Acids Res, 1988 Mar 11, 16(5), 1657 - 66
Color graphics representations of large sequences in the GEM environment; Douthart RJ et al.; The analytical and descriptive color graphics capabilities (the GEM environment) of the CAGE/GEM(a) software system are described using bacteriophage lambda (48,502 base pairs) and Epstein Barr virus (172,282 base pairs) as examples . Genetics and features, as graphic drawings, and the results of sequence analysis as pseudo-colored representations, are simultaneously displayed at all zooming levels . The present upper limit on sequence size is 5 x 10(5) bases . The overlay editor utility which provides the capability to structure complex sequence associated data and knowledge bases into a manageable color graphics format is also described . Holistic correlations that can be made by the display of bacteriophage lambda and Epstein Barr virus in the GEM environment are also discussed.

Biochemistry, 1988 Mar 8, 27(5), 1722 - 8
Influence of DNA sequence on the nature of mispairing during DNA synthesis; Lai MD et al.; A series of synthetic oligonucleotide primers, annealed at various positions along the lacZ-alpha region of bacteriophage M13mp9 template, were elongated by purified DNA polymerases in the presence of only 3 of the 4 deoxynucleoside triphosphates to achieve misincorporation at a total of 49 different positions along the template . The newly synthesized strands (containing misincorporated bases) were isolated and sequenced to determine the identity of misincorporated deoxynucleoside monophosphates . The results indicate that the kind of mispairing that occurs during DNA synthesis is greatly influenced by the nucleotide sequence of the template . Transition-type base substitutions predominated overall, but at many template positions, transversion-type base substitutions occurred, most commonly via A.A mispairing . The results of parallel determinations made with Escherichia coli DNA polymerase I ("large fragment" form) and DNA polymerase of Maloney murine leukemia virus indicated that, overall, the identity of polymerase had only a small effect on the kind of misincorporation that occurred at different positions along the template . However, at certain template positions, the nature of mispairing during DNA synthesis was reproducibly affected by differing polymerase active-site environment.

Mol Biol (Mosk), 1988 Mar-Apr, 22(2), 369 - 76
{Structural changes in proteins of the bacteriophage T4 basal plate resulting from the attachment of long fibrils}; Abuladze NK et al.; The effect of the attachment of long tail fibers on the structure of proteins of the bacteriophage T4 baseplate was studied by digital processing of electron microscopic images . The attachment of the long fibers was found to result in dramatical changes of the proteins of the baseplate plag, while the wedges, to which the long fibers are attached, undergo only slight changes . We studied the baseplates with one to six attached fibers and found that the attachment of one fiber resulted in the change of the entire baseplate, although the wedge located in the vicinity of the fiber attachment changed to a greater extent . Only after the attachment of three and more fibers the changes of the same kind occurred through the entire baseplate.

J Cell Biochem, 1988 Mar, 36(3), 289 - 95
Uncoupling of translocation across microsomal membranes from biosynthesis of influenza virus hemagglutinin; Chao CC et al.; This communication presents our recent studies on the biosynthesis of influenza virus hemagglutinin (HA) in a mammalian-cell-free system and its translocation across microsomal membranes . RNAs coding for wild-type (full-length) and mutant (truncated) forms of HA were generated by in vitro transcription by using bacteriophage T7 DNA-dependent RNA polymerase . These RNAs were translated in a rabbit reticulocyte system that was supplemented with dog pancreas membranes, either before translation was initiated or after it had been artificially terminated with the antibiotic cycloheximide . All forms of HA could be cotranslationally translocated . However, only truncated molecules (83% of full length) could translocate after protein synthesis had been terminated . Posttranslational translocation was dependent on the presence of a functional N-terminal signal sequence and occurred only in the presence of ribosomes . The molecular mechanism of protein targeting and translocation across the membrane of the endoplasmic reticulum is discussed based on the signal hypothesis.

Biophys J, 1988 Mar, 53(3), 425 - 40
A theory of the symmetries of filamentous bacteriophages; Marzec CJ et al.; A mathematical model is presented which explains the symmetries observed for the protein coats of filamentous bacterial viruses . Three viruses (Ff, IKe, and If1) all have five-start helices with rotation angles of 36 degrees and axial translations of 16 A (Type I symmetry), and three other viruses (Pf1, Xf, and Pf3) all have one-start helices with rotation angles of approximately equal to 67 degrees and translations of approximately 3 A (Type II symmetry) . The coat protein subunits in each group diverge from each other in amino acid sequence, and Type II viruses differ dramatically in DNA structure . Regardless of the differences, both Type I and Type II symmetry can be understood as direct, natural consequences of the close-packing of alpha-helical protein subunits . In our treatment, an alpha-helical subunit is modeled as consisting of two interconnected, flexible tubular segments that follow helical paths around the DNA, one in an inner layer and the other in an outer layer . The mathematical model is a set of algebraic equations describing the disposition of the flexible segments . Solutions are described by newly introduced symmetry indices and other parameters . An exhaustive survey over the range of indices has produced a library of all structures that are geometrically feasible within our modeling scheme . Solutions which correspond in their rotation angles to Type I and Type II viruses occur over large ranges of the parameter space . A few solutions with other symmetries are also allowed, and viruses with these symmetries may exist in nature . One solution to the set of equations, obtained without any recourse to the x-ray data, yields a calculated x-ray diffraction pattern for Pf1 which compares reasonably with experimental patterns . The close-packing geometry we have used helps explain the near constant linear mass density of known filamentous phages . Helicoid, rigid cylinder, and maximum entropy structure models proposed by others for Pf1 are reconciled with the flexible tube models and with one another.

Virology, 1988 Mar, 163(1), 183 - 90
Nucleotide sequence of the middle dsRNA segment of bacteriophage phi 6: placement of the genes of membrane-associated proteins; Gottlieb P et al.; The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA . We have determined the nucleotide sequence of cDNA derived from the middle-size RNA segment . The coding sequences of three proteins on this segment were identified on the basis of size and the correlation of predicted N-terminal amino acid sequences with those found through the analysis of isolated proteins . In contrast to our results with the small phi 6 dsRNA segment, the open reading frames are not tightly clustered . The homologous terminal noncoding regions between the middle and small dsRNA segments are found to be more extensive than RNA sequencing had previously indicated.

Dev Biol, 1988 Mar, 126(1), 141 - 9
Post-translational control of ribosomal protein L1 accumulation in Xenopus oocytes; Baum EZ et al.; A functional ribosomal protein mRNA, encoding the 60 S subunit protein L1, has been synthesized in vitro using bacteriophage SP6 RNA polymerase . This mRNA directs the synthesis of a product indistinguishable from L1 protein purified from Xenopus ovarian ribosomes . Our results show that L1 synthesis in stage VI oocytes increases in response to microinjection of exogenous SP6-L1 mRNA, but excess L1 protein is not stably accumulated . These results indicate that dosage compensation does not occur at the translational level for this ribosomal protein mRNA and that the abundance of this protein in fully grown oocytes is subject to post-translational regulation.

Mutat Res, 1988 Mar, 198(1), 27 - 36
Influence of divalent metal activator on the specificity of misincorporation during DNA synthesis catalyzed by DNA polymerase I of Escherichia coli; Lai MD et al.; To test whether the identity of divalent metal activator affects the specificity of misincorporation during polymerization catalyzed by E . coli DNA polymerase I, we carried out the following procedure . A series of oligonucleotide primers, annealed at different positions along the lacZ region of bacteriophage M13mp9 DNA, were elongated in the presence of 3 of the 4 deoxynucleoside 5'-triphosphates (dNTPs) until one or a few misincorporations occurred in each elongated primer . The elongated primers (containing deoxynucleotide residues that had been misincorporated in the presence of either Mg2+ or Mn2+) were then isolated and sequenced by the 'dideoxy' chain termination method to determine the identity of deoxynucleoside monophosphates (dNMPs) that had been misincorporated at different template positions during the original 'minus' reactions, activated by Mg2+ or Mn2+ . The results obtained by this approach revealed that both the type of misincorporation and the effect of substituting Mn2+ for Mg2+ depended on the nucleotide sequence of the template . At 40% of the template positions at which misincorporation was compared with both metal ions (8 out of 20), the identity of mispairs differed significantly for synthesis activated by Mn2+ versus Mg2+ . Of these 8 sites, 4 exhibited increased transversions in the presence of Mn2+, while 4 exhibited decreased transversions with Mn2+.

Virology, 1988 Mar, 163(1), 209 - 13
Infectious positive- and negative-strand transcript RNAs from bacteriophage Q beta cDNA clones; Shaklee PN et al.; Plasmids containing full-length cDNA copies of the Q beta RNA phage genome and flanking T7 promoters were constructed . Positive-strand Q beta RNA, generated by in vitro transcription of these plasmids with T7 RNA polymerase, was infectious to Escherichia coli spheroplasts . The Q beta replicase gene from the cloned DNA was subcloned and expressed in E . coli cells by means of a thermoinducible plasmid . Full-length, negative-strand Q beta transcripts were infectious when transfected into spheroplasts containing the induced replicase gene.

Mutat Res, 1988 Mar, 193(2), 87 - 96
UV mutagenesis in E . coli with excision repair initiated by uvrABC or denV gene products; Bockrath R et al.; Mutation frequency responses produced by ultraviolet light are compared in 4 closely related strains of E . coli B/r having the same tyr(Oc) allele and different excision-repair capabilities: uvr+ (excision repair initiated by wild-type UvrABC activity), uvrA (excision repair defective), uvrA/pdenV-7 (excision repair initiated by endonuclease V of bacteriophage T4, DenV activity), and uvr+/pdenV-7 (excision repair initiated by UvrABC and DenV activities) . The production of Tyr+ prototrophic mutants is classified into back-mutations and de novo or converted glutamine tRNA suppressor mutations to indicate different mutation events . Cells transformed with the plasmid pdenV-7 require larger exposures than the parent strains to produce comparable mutation frequency responses, indicating that DenV activity can repair mutagenic photoproducts . When damage reduction by UvrABC or DenV is compared for each of the specific categories of mutation, the results are consistent with the idea that pyrimidine dimers infrequently or never target back-mutations of this allele, frequently target the de novo suppressor mutations, and extensively or exclusively target the converted suppressor mutations . This analysis is based on the distinction that UvrABC-initiated excision repair recognizes dimer and non-dimer (pyrimidine (6-4) pyrimidone) photoproducts but that DenV-initiated repair recognizes only pyrimidine dimers.

J Bacteriol, 1988 Mar, 170(3), 1384 - 8
Roles of the Escherichia coli heat shock sigma factor 32 in early and late gene expression of bacteriophage T4; Frazier MW et al.; We have analyzed early and late T4 gene expression at the levels of transcription and translation in rpoH+ (sigma 32+) and rpoH mutant cells infected under heat shock conditions . We found, as expected, that Escherichia coli cells must be adapted before infection to high temperature by the heat shock response to allow early T4 transcription, subsequent late gene expression, and progeny production at 42 degrees C . Unexpectedly, we found in addition that when rpoH mutant (sigma 32 mutant) cells were shifted from 30 to 42 degrees C 10 min after infection, late T4 genes were not expressed, even though DNA synthesis appeared to be normal.

J Bacteriol, 1988 Mar, 170(3), 1245 - 53
ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification; Grodberg J et al.; Bacteriophage T7 RNA polymerase is stable in Escherichia coli but very susceptible to cleavage by at least one endoprotease after cell lysis . The major source of this endoprotease activity was found to be localized to the outer membrane of the cell . A rapid whole-cell assay was developed to screen different strains for the presence of this proteolytic activity . Using this assay, we identified some common laboratory strains that totally lack the protease . Genetic and Southern analyses of these null strains allowed us to conclude that the protease that cleaves T7 RNA polymerase is OmpT (formerly termed protein a), a known outer membrane endoprotease, and that the null phenotype results from deletion of the OmpT structural gene . A recombinant plasmid carrying the ompT gene enables these deletion strains to synthesize OmpT and converts them to a protease-positive phenotype . The plasmid led to overproduction of OmpT protein and protease activity in the E . coli K-12 and B strains we used, but only weak expression in the E . coli C strain, C1757 . This strain-dependent difference in ompT expression was investigated with respect to the known influence of envZ on OmpT synthesis . A small deletion in the ompT region of the plasmid greatly diminishes the amount of OmpT protein and plasmid-encoded protease present in outer membranes . Use of ompT deletion strains for production of T7 RNA polymerase from the cloned gene has made purification of intact T7 RNA polymerase routine . Such strains may be useful for purification of other proteins expressed in E . coli.

Tubercle, 1988 Mar, 69(1), 43 - 6
Bacteriophage typing of Mycobacterium tuberculosis cultures from incidents of suspected laboratory cross-contamination; Jones WD Jr; Bacteriophage typing was performed on 235 Mycobacterium tuberculosis cultures submitted from 31 laboratories . In each instance, either the attending physician questioned the misdiagnosis of tuberculosis or the laboratory supervisor suspected that laboratory cross-contamination had occurred . Phage typing data confirmed these suspicions . Phage typing is a useful adjunct in the investigation of suspected cross-contamination of laboratory cultures of M . tuberculosis.

Comput Appl Biosci, 1988 Mar, 4(1), 79 - 88
Recognition of ill-defined signals in nucleic acid sequences; Grob U et al.; A set of programs has been developed for the definition and handling of nucleic acid sequence consensus information . The sequences of known genetic control signals are combined in a matrix . The origins and positions of the signals are recorded . Old matrices can be updated dynamically: new signals are included and obsolete ones deleted . Matrices of several different types are computed optionally . Several of these matrices can be combined to find possible new signals . The use of matrices allows the exact quantification of signal qualities . The described programs are part of a program library named GENEXPERT . Application examples given are the search for tRNA genes and the search for promoters in the bacteriophage lambda genome.

J Virol, 1988 Mar, 62(3), 882 - 6
Nucleotide sequence of the tail tube structural gene of bacteriophage T4; Arisaka F et al.; The nucleotide sequence of gene 19 of bacteriophage T4, the structural gene of the tail tube protein, was determined by both the dideoxy and the Maxam-Gilbert methods . The predicted Mr of tube protein gene product 19 is 18,842 . The N-terminal amino acid of the tube protein was determined by Edman degradation, and the C-terminal sequence was confirmed by isolation of the C-terminal tryptic peptide . In the noncoding region between genes 18 and 19, there are two late-T4-promoter consensus sequences, 51 bases apart . The implication of the two late promoter sequences was examined by an S1 nuclease protection experiment . Both serve as weak promoters, but the bulk of the transcripts arise from further upstream of the two promoters.

Comput Appl Biosci, 1988 Mar, 4(1), 41 - 51
Algorithms for identifying local molecular sequence features; Karlin S et al.; Efficient algorithms are described for identifying local molecular sequence features including repeats, dyad symmetry pairings and aligned matches between sequences, while allowing for errors . Specific applications are given to the genomic sequences of the Epstein-Barr virus, Varicella-Zoster virus and the bacteriophages lambda and T7.

J Bacteriol, 1988 Mar, 170(3), 1279 - 89
Bacteriophage T4 late transcription from plasmid templates is enhanced by negative supercoiling; Albright LM et al.; Concurrent viral replication is normally required to activate bacteriophage T4 late promoters; replication is thought to provide a template structure which is competent for late transcription . Transcription from plasmid-borne T4 late promoters, however, is independent of replication in vivo and in vitro . In this work, we have shown that, when the late gene 23 promoter is located on a plasmid, its utilization in vivo depends upon the ability of host DNA gyrase to maintain some degree of negative superhelicity . This suggests that an alternative pathway exists for activation of late promoters: DNA which is under sufficient negative torsional stress is already competent for late transcription . We also describe a method for isolating ternary complexes of plasmid DNA, RNA polymerase, and nascent RNA which have initiated transcription in vivo . The topoisomer distribution of such ternary complexes prepared from T4-infected cells showed that, late in infection, transcriptional activity resides primarily in the subset of the plasmid population with the most negatively supercoiled topoisomers . However, the overall transcriptional pattern in these ternary complexes indicated that both vector and T4 sequences are actively transcribed . Much of this transcriptional activity could be independent of gp55, the T4-specific RNA polymerase-binding protein that confers late promoter recognition.

J Virol, 1988 Mar, 62(3), 776 - 82
Expression of the M gene of vesicular stomatitis virus cloned in various vaccinia virus vectors; Li Y et al.; Initial attempts to clone the matrix (M) gene of vesicular stomatitis virus (VSV) in a vaccinia virus expression vector failed, apparently because the expressed M protein, and particularly a carboxy-terminus-distal two-thirds fragment, was lethal for the virus recombinant . Therefore, a transient eucaryotic expression system was used in which a cDNA clone of the VSV M protein mRNA was inserted into a region of plasmid pTF7 flanked by the promoter and terminator sequences for the T7 bacteriophage RNA polymerase . When CV-1 cells infected with recombinant vaccinia virus vTF1-6,2 expressing the T7 RNA polymerase were transfected with pTF7-M3, the cells produced considerable amounts of M protein reactive by Western blot (immunoblot) analysis with monoclonal antibodies directed to VSV M protein . Evidence for biological activity of the plasmid-expressed wild-type M protein was provided by marker rescue of the M gene temperature-sensitive mutant tsO23(III) at the restrictive temperature . Somewhat higher levels of M protein expression were obtained in CV-1 cells coinfected with a vaccinia virus-M gene recombinant under control of the T7 polymerase promoter along with T7 polymerase-expressing vaccinia virus vTF1-6,2.

Mol Biol (Mosk), 1988 Mar-Apr, 22(2), 454 - 8
{Regulation of expression of the Escherichia coli htpR gene by antisense RNA}; Kiselev VI et al.; A vector plasmid directing the synthesis of antisense RNA of Escherichia coli gene htpR has been constructed . For this fragment of htpR gene encoding the synthesis of N-terminal sequence of HtpR protein has been cloned in this plasmid in the opposite orientation under the control of Pr promoter of bacteriophage lambda . Under conditions of Pr transcription the induction the growth of bacterial cells carrying the recombinant plasmid was delayed and the rate of degradation of puromycin polypeptides was decreased 3-fold . Vectors constructed on the basis of such a principle may be used for thermoinducible synthesis of heterologous proteins with a simultaneous correction of the intracellular proteolysis.

Mol Cell Biol, 1988 Mar, 8(3), 1319 - 26
Characterization of a mouse multigene family that encodes zinc finger structures; Chavrier P et al.; The Drosophila segmentation gene Kruppel encodes multiple tandemly repeated units predicted to form DNA-binding zinc fingers . We have isolated 23 bacteriophages, containing nonoverlapping inserts from a mouse genomic DNA library, on the basis of cross-hybridization under nonstringent conditions to a probe corresponding to the Kruppel finger region . Nucleotide sequence analysis of six phage DNAs indicated that they all contained regions with similarity to Kruppel and potentially encoded zinc finger domains . Within these regions, the level of similarity to Kruppel was particularly high between successive fingers . Northern (RNA) blotting analysis suggested that the mouse sequences belonged to different genes, the expression of some of which was modulated during cell differentiation and development . Hybridization experiments suggested that the similarity between some of the genes extended outside of the finger regions . In conclusion, our data suggest that the mouse genome contains a large family of evolutionarily related genes encoding possible trans-acting factors . These genes are likely to play a regulatory role at the transcriptional level.

Mol Gen Genet, 1988 Mar, 211(3), 400 - 6
Change in priming sites for discontinuous DNA synthesis between the monomeric and concatemeric stages of phage T7 replication; Sugimoto K et al.; We have analyzed the transition sites between primer RNA and DNA in a 589 bp segment of the bacteriophage T7 genome . In the monomeric replication stage, RNA-DNA transition sites are predominantly on the light (L) strand (with 5'----3' polarity on the genetic map) but rarely on the heavy (H) strand, indicating that replication proceeds semidiscontinuously with the H and L strands corresponding to the leading and lagging strands, respectively . The direction of replication is that expected from the position of the primary origin and also indicates that secondary origins are seldom if ever used . In the concatemeric stage of replication, RNA-DNA transition sites are instead distributed on both strands of the segment with equally high frequency, showing that initiation occurs within the concatemeric molecule per se and by a different mechanism.

Proc Natl Acad Sci U S A, 1988 Mar, 85(5), 1364 - 8
CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes; Tuerk C et al.; The mRNA of bacteriophage T4 contains a strikingly abundant intercistronic hairpin . Within the 55 kilobases of known T4 sequence, the hexanucleotide sequence CTTCGG is found 13 times in the DNA strand equivalent to mRNA sequences . In 12 of those occurrences, the sequence is flanked by inverted repeats predictive of RNA hairpins with UUCG in the loop . Avian myeloblastosis virus reverse transcriptase, which can traverse hairpins of larger calculated stability, terminates efficiently at these CUUCGG hairpins . Thermal denaturation studies of model hairpins show that the loop sequence UUCG dramatically stabilizes RNA hairpins when compared to a control sequence . These data, when combined with previously described parameters of helix stability, suggest that T4 has utilized this loop sequence to optimize the stability of intercistronic hairpins . The stability of CUUCGG hairpins is also utilized in the RNAs of many organisms besides T4.

Biochim Biophys Acta, 1988 Feb 28, 949(2), 240 - 6
Bacteriophage T2 and T4, dam+ and damh and Eco dam+ methylation: preference at different sites; Doolittle MM et al.; We present a method for determining preference for methylation at minor methylation sites . The target DNA sequence is first subjected to computer-assisted analysis to predict which restriction endonuclease(s) will generate fragments that will contain only one or two likely minor methylation site(s) . The target DNA is then methylated in vitro with a radioactive methyl-group donor and subjected to digestion by the chosen restriction enzyme(s) . The amount of radioactivity in the various fragments is determined, after separating them using polyacrylamide gel electrophoresis . We documented the effect of nearby bases on the methylation preference and the relative preference for methylation at some specific minor methylation sites.

Science, 1988 Feb 26, 239(4843), 1005 - 12
A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60; Huang WM et al.; A 50-nucleotide untranslated region is shown to be present within the coding sequence of Escherichia coli bacteriophage T4 gene 60, which encodes one of the subunits for its type II DNA topoisomerase . This interruption is part of the transcribed messenger RNA and appears not to be removed before translation . Thus, the usual colinearity between messenger RNA and the encoded protein sequence apparently does not exist in this case . The interruption is bracketed by a direct repeat of five base pairs . A mechanism is proposed in which folding of the untranslated region brings together codons separated by the interruption so that the elongating ribosome may skip the 50 nucleotides during translation . The alternative possibility, that the protein is efficiently translated from a very minor and undetectable form of processed messenger RNA, seems unlikely, but has not been completely ruled out.

Nucleic Acids Res, 1988 Feb 25, 16(4), 1517 - 28
An oligopurine sequence bias occurs in eukaryotic viruses; Beasty AM et al.; Twenty four DNA and RNA viral nucleotide sequences, comprising over 346 kilobases, have been analyzed for the occurrence of strings of contiguous purine or pyrimidine residues . On average strings greater than or equal to 10 contiguous purines or pyrimidines are found three and a half times more frequently than would be expected for a random distribution of bases . Detailed analysis of the 172 kilobase Epstein-Barr viral sequence shows that the bias in favor of contiguous purine residues increases with the length of the purine string . These findings are similar to those seen for genomic DNA from higher eukaryotes . In contrast no overrepresentation of oligopurine or oligopyrimidine strings is observed in 52 kilobases from eight bacteriophage and E . coli DNA sequences.

Nucleic Acids Res, 1988 Feb 25, 16(4), 1577 - 91
Accurate in vitro cleavage by RNase III of phosphorothioate-substituted RNA processing signals in bacteriophage T7 early mRNA; Nicholson AW et al.; To test the ability of an RNA processing enzyme to cleave chemically-modified RNA substrates, RNA transcripts containing RNase III cleavage sites were enzymatically synthesized in vitro to contain specific phosphorothioate diester internucleotide linkages . One transcript (R1.1 RNA) was generated using phage T7 RNA polymerase and a cloned segment of phage T7 DNA containing the R1.1 RNase III processing site . The second transcript was the phage T7 polycistronic early mRNA precursor, which was synthesized using E . coli RNA polymerase and T7 genomic DNA . The RNA transcripts contained phosphorothioate diester groups at positions including the scissile bonds . The modified RNAs were stable to incubation in Mg2+-containing buffer, and were specifically cleaved by RNase III . RNA oligonucleotide sequence analysis showed that the modified R1.1 RNA processing site was the same as the canonical site and contained a phosphorothioate bond . Furthermore, RNase III cleaved the phosphorothioate internucleotide bond with 5' polarity . RNase III cleavage of phosphorothioate substituted T7 polycistronic early mRNA precursor produced the same gel electrophoretic pattern as that obtained with the control transcript . Thus, RNase III cleavage specificity is not altered by phosphorothioate internucleotide linkages.

Nucleic Acids Res, 1988 Feb 25, 16(4), 1593 - 601
The genetic structure of mouse ornithine transcarbamylase; Scherer SE et al.; The gene encoding the mouse urea cycle enzyme, ornithine transcarbamylase has been isolated on five partially overlapping bacteriophage lambda clones . We have characterized the gene and found that it is split between ten exons distributed over approximately 70 kb of the X chromosome . The introns range in size from 88 bases to the relatively unusual size of approximately 26 kilobases, while the splice donor/splice acceptor sequences conform to the consensus established for other eukaryotic genes.

J Biol Chem, 1988 Feb 25, 263(6), 2939 - 47
The major apoprotein of rabbit pulmonary surfactant . Elucidation of primary sequence and cyclic AMP and developmental regulation; Boggaram V et al.; The major apoprotein of rabbit pulmonary surfactant is a developmentally and hormonally regulated sialoglycoprotein, Mr congruent to 29,000-36,000 (SP 29-36) . In the present study, specific antibodies were used to isolate cloned cDNA inserts for SP 29-36 from a fetal rabbit lung cDNA library in bacteriophage lambda gt11 . Two species of cDNA of 1.9 and 3.0 kilobases (kb) in size were isolated that are complementary to two species of mRNA of 2.0 and 3.0 kb which differ primarily in the lengths of their 3'-untranslated regions . The 2.0-kb species of mRNA is approximately 5 times more abundant than the 3.0-kb mRNA . The results of Southern hybridization analysis of rabbit genomic DNA cut with a number of restriction enzymes are indicative that the two mRNA species are encoded by a single gene . The two mRNA species appear to be expressed only in rabbit lung tissue and are coordinately regulated during development in vivo and in vitro; hybridizable SP 29-36 mRNA is first detectable in rabbit lung tissue on day 26 of gestation, increases to a maximum on day 31, and declines somewhat after birth . The 3.0-kb cDNA is comprised of 57 nucleotides of 5'-untranslated region, an open reading frame of 741 nucleotides, and a 3'-untranslated region of 2,165 nucleotides that contains three poly(A)-addition signals . The most 5' of the poly(A) addition signals is utilized in synthesis of the 2.0-kb SP 29-36 mRNA, while the most 3' is utilized in synthesis of the 3.0-kb mRNA . The open reading frame of the 3.0-kb cDNA encodes a protein of 247 amino acids which is highly homologous to the major apoproteins of dog and human pulmonary surfactant . The SP 29-36 cDNAs were utilized to evaluate the effects of dibutyryl cyclic AMP (Bt2cAMP) on the levels of this mRNA in fetal rabbit lung tissue in organ culture . Bt2cAMP caused an induction of SP 29-36 mRNA that was detectable as early as 2 h after its addition to the medium . This inductive effect of Bt2cAMP was blocked when cycloheximide was also present in the medium for greater than or equal to 4 h . Cycloheximide treatment also reduced the levels of SP 29-36 mRNA in control explants; this inhibitory effect on control and Bt2cAMP-treated explants was reversed within 12 h of the removal of cycloheximide from the medium . These findings suggest that a labile protein factor mediates the transcription of the SP 29-36 gene and its induction by Bt2cAMP.

Biochemistry, 1988 Feb 23, 27(4), 1367 - 73
Deoxycytidylate hydroxymethylase: purification, properties, and the role of a thiol group in catalysis; Lee MH et al.; Deoxycytidylate (dCMP) hydroxymethylase from Escherichia coli infected with a T-4 bacteriophage amber mutant has been purified to homogeneity . It is a dimer with a subunit molecular weight of 28,000 . Chemical modification of the homogeneous enzyme with N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) leads to complete loss of enzyme activity . dCMP can protect the enzyme against NEM inactivation, but the dihydrofolate analogues methotrexate and aminopterin alone do not afford similar protection . Compared to dCMP alone, dCMP plus either methotrexate or aminopterin greatly enhances protection against NEM inactivation . DTNB inactivation is reversed by dithiothreitol . For both reagents, inactivation kinetics obey second-order kinetics . NEM inactivation is pH dependent with a pKa for a required thiol group of 9.15 +/- 0.11 . Complete enzyme inactivation by both reagents involves the modification of one thiol group per mole of dimeric enzyme . There are two thiol groups in the totally denatured enzyme modified by either NEM or DTNB . Kinetic analysis of NEM inactivation cannot distinguish between these two groups; however, with DTNB kinetic analysis of 2-nitro-5-thiobenzoate release shows that enzyme inactivation is due to the modification of one fast-reacting thiol followed by the modification of a second group that reacts about 5-6-fold more slowly . In the presence of methotrexate, the stoichiometry of dCMP binding to the dimeric enzyme is 1:1 and depends upon a reduced thiol group . It appears that the two equally sized subunits are arranged asymmetrically, resulting in one thiol-containing active site per mole of dimeric enzyme.

J Mol Biol, 1988 Feb 20, 199(4), 597 - 607
Bacteriophage lambda DNA packaging . The product of the FI gene promotes the incorporation of the prohead to the DNA-terminase complex; Becker A et al.; Lambda DNA packaging in vitro can be examined in stages . In a first step, lambda DNA interacts with terminase to form a DNA-enzyme complex, called complex I . Upon addition of proheads, in a second step, a ternary complex, complex II, containing DNA, terminase and the prohead is formed . Finally, upon addition of the rest of the morphogenetic components, complete phages are assembled . We have investigated the effect of the FI gene product (gpFI) in these reactions and found that a stimulation in phage yield is observed when gpFI is included early in the reaction, at the time when DNA, terminase and proheads interact to form complex II . Measurements of complex II formation revealed that gpFI stimulated the rate of formation of this intermediate . gpFI was further shown to stimulate the addition of proheads to preformed complexes I to give complex II, but the protein did not stimulate complex I formation.

J Mol Biol, 1988 Feb 20, 199(4), 587 - 96
Myelin proteolipid protein gene structure and its regulation of expression in normal and jimpy mutant mice; Ikenaka K et al.; The mouse proteolipid protein (PLP) gene was cloned into the lambda bacteriophage Charon 4A . The organization and the nucleotide sequence of the exons of the mouse PLP gene were quite similar to those of their human counterparts, consisting of seven exons . The transcription of the PLP gene started from multiple sites . There was a unique sequence tandemly repeated four times, sharing homology with the herpes simplex virus DR2 sequence, upstream from the transcribed region . Expression of the myelin basic protein (MBP) is also restricted to the oligodendrocytes in the central nervous system as is the PLP expression . Homology search against the mouse MBP gene revealed that several boxes in the 5'-flanking region of PLP show a high degree of homology with the sequence present in the MBP 5'-flanking region, possibly of importance in the concomitant expression of both genes in the central nervous system . PLP-mRNA in jimpy mutant mice does not contain exon 5 and its content is greatly reduced . We analyzed the jimpy PLP-mRNA and showed that the transcription initiated from the same sites as those in normal mice . Cloning and sequencing of the 5'-flanking region of the jimpy PLP gene revealed that there were no mutations in the promoter region of the jimpy PLP gene . Therefore, it is likely that a mutation, presumably existing within the jimpy PLP gene, caused the skipping of exon 5 and directly affected the mRNA level.

J Biol Chem, 1988 Feb 15, 263(5), 2493 - 9
Pore-forming activity of the Tsx protein from the outer membrane of Escherichia coli . Demonstration of a nucleoside-specific binding site; Maier C et al.; The Tsx protein from the outer membrane of Escherichia coli is known to be involved in the permeation of nucleosides across the outer membrane under limiting substrate conditions . We purified Tsx from an E . coli strain that overproduces Tsx . The purified protein was still functional since it could neutralize the Tsx-specific bacteriophage T6 in vitro . When the purified Tsx was reconstituted into a lipid bilayer, there was a large increase of the membrane conductance, indicating pore-forming activity of Tsx in vitro . This increase could be strongly blocked with adenosine and to a much lesser extent with cytidine . Titration of the pore conductance with adenosine or cytidine suggested the presence of a binding site for nucleosides in the Tsx pore, with a Ks of 6 X 10(-4) and 2 X 10(-2) M for adenosine and cytidine, respectively . We propose that the Tsx protein functions in vivo as a pore that specifically facilitates the permeation of nucleosides across the outer membrane due to its binding site for nucleosides.

J Biol Chem, 1988 Feb 15, 263(5), 2469 - 76
Gene 19 of bacteriophage T7 . Overexpression, purification, and characterization of its product; White JH et al.; Gene 18 and 19 proteins of bacteriophage T7 are essential for DNA maturation and packaging . The phage capsid is the site of both maturation and packaging of T7 DNA . Both gene 18 and 19 proteins bind to capsid intermediates during DNA packaging but are not found in mature virions, suggesting that they play a direct role in the enzymatic mechanisms of DNA maturation and packaging . As part of an effort to reconstitute T7 DNA maturation and packaging with purified components, we have cloned and overexpressed T7 gene 19 in Escherichia coli . Gene 19 has been inserted downstream from the bacteriophage PL promoter controlled by the temperature-sensitive lambda repressor encoded by c1857 . Upon thermal induction, most of the overproduced gene 19 protein is insoluble and inactive . However, by attenuation of the expression of gene 19 from the PL promoter, significant levels of soluble and active gene 19 protein are produced . Soluble gene 19 protein can be monitored by its ability to complement extracts of T7-infected cells for packaging of exogenous DNA . We have used this assay to monitor the activity of gene 19 protein during purification . The native protein is a monomer of molecular weight 66,000 . We have also tested for the formation of a stable complex between gene 18 and 19 proteins . Coproduction of gene 18 and 19 proteins has no effect on either the solubility or activity of gene 19 protein, despite the fact that gene 18 protein is produced at at least 10-fold greater rates . Furthermore, we find no evidence for any interaction between soluble gene 18 and 19 proteins in extracts or between the purified proteins.

Biochem Biophys Res Commun, 1988 Feb 15, 150(3), 1244 - 50
Three-alpha-helical coiled-coil, as a proposed model for a thin rod segment of bacteriophage T3 tail fibers; Takahashi S et al.; Three-stranded alpha-helical coiled-coil was considered as a model for a thin proximal rod of T3 phage tail fiber on the basis of amino acid sequence . A segment of residues from ca . 130th to 270th was shown to have a unique feature to satisfy the required conditions of the coiled-coil, and to give the observed geometry.

FEBS Lett, 1988 Feb 15, 228(2), 223 - 7
Sizing of single-stranded regions in double-stranded DNA by preparative benzoylated DEAE-cellulose chromatography; Norris MD et al.; The concentration of caffeine required to elute wholly single-stranded DNA from benzoylated DEAE-cellulose is proportional to the polynucleotide length . The use of benzoylated DEAE-cellulose chromatography for isolating and sizing single-stranded regions in double-stranded DNA has been examined using a series of hybrid molecules . Restriction fragments of the replicating form of bacteriophage luminal diameter X174 were hybridized to the intact 'plus' strand, thereby forming hybrids having single- and/or double-stranded regions in the kilobase range . A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE-cellulose . After logarithmic transformation, a linear relationship (R = 0.94) could be demonstrated between eluting caffeine concentration and single-stranded length, irrespective of the length of associated double-stranded regions or the location, within a given fragment, of unpaired nucleotides . Benzoylated DEAE-cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double-stranded DNA containing single-stranded regions.

Nucleic Acids Res, 1988 Feb 11, 16(3), 1011 - 26
N4 virion DNA dependent-RNA polymerase: initiation sequences utilized by the enzyme on heterologous templates; Markiewicz P et al.; Bacteriophage N4 virion-encapsulated RNA polymerase, the enzyme responsible for transcription of the phage early RNAs, is unable to use duplex linear DNA as a template . In contrast to other RNA polymerases, the enzyme transcribes denatured N4 DNA with in vivo specificity . The promoter sequences for three sites of transcription initiation on the N4 genome have been determined and found to contain conserved sequences and two sets of inverted repeats . In order to define the minimal sequence requirements for N4 virion RNA polymerase activity, we have screened several heterologous DNAs, amounting to 64,328 bases, for their ability to support transcription . Several sequences allowing specific initiation were found . Their location, properties and the relation to N4 virion RNA polymerase promoters are discussed.

J Mol Biol, 1988 Feb 5, 199(3), 467 - 74
Analysis in vivo of the bacteriophage P22 headful nuclease; Casjens S et al.; Bacteriophage P22 packages its double-stranded DNA chromosomes from concatemeric replicating DNA in a processive, sequential fashion . According to this model, during the initial packaging event in such a series the packaging apparatus recognizes a nucleotide sequence, called pac, on the DNA, and then condenses DNA within the coat protein shell unidirectionally (rightward) from that point . DNA ends are generated near the pac site before or during the condensation reaction . The right end of the mature chromosome is created by a cut made in the DNA by the "headful nuclease" after a complete chromosome is condensed within the phage head . Subsequent packaging events on that concatemeric DNA begin at the end generated by the headful cut of the previous event and proceed in the same direction as the previous event . We report here accurate measurements of the P22 chromosome length (43,400( +/- 750) base-pairs, where the uncertainty is the range in observed lengths), genome length (41,830( +/- 315) base-pairs, where the uncertainty represents the accuracy with which the length is known), the terminal redundancy (1600( +/- 750) base-pairs or 3.8( +/- 1.8)%, where the uncertainty is the observed range) and the imprecision in the headful measuring device ( +/- 750 base-pairs or +/- 1.7%) . In addition, we present evidence for a weak nucleotide sequence specificity in the headful nuclease . These findings lend further support to, and extend our understanding of, the sequential series model of P22 DNA packaging.

J Mol Biol, 1988 Feb 5, 199(3), 491 - 502
Secondary structure and thermostability of the phage P22 tailspike . XX . Analysis by Raman spectroscopy of the wild-type protein and a temperature-sensitive folding mutant; Sargent D et al.; The thermostable tailspike endorhamnosidase of bacteriophage P22 has been investigated by laser Raman spectroscopy to determine the protein's secondary structure and the basis of its thermostability . The conformation of the native tailspike, determined by Raman amide I and amide III band analyses, is 52 to 61% beta-sheet, 24 to 27% alpha-helix, 15 to 21% beta-turn and 0 to 10% other structure types . The secondary structure of the wild-type tailspike, as monitored by the conformation-sensitive Raman amide bands, was stable to 80 degrees C, denatured reversibly between 80 and 90 degrees C, and irreversibly above 90 degrees C . The purified native form of a temperature-sensitive folding mutant (tsU38) contains secondary structures virtually identical to those in the wild-type in aqueous solution at physiological conditions (0.05 M-Na+ (pH 7.5}, at both permissive (20 degrees C) and restrictive (40 degrees C) temperatures . This supports previous results showing that the mutational defect at 40 degrees C affects intermediates in the folding pathway rather than the native structure . At temperatures above 60 degrees C the wild-type and mutant forms were distinguishable: the reversible and irreversible denaturation thresholds were approximately 15 to 20 degrees C lower in the mutant than in the wild-type protein . The irreversible denaturation of the mutant tailspikes led to different aggregation/polymerization products from the wild-type, indicating that the mutation altered the unfolding pathway . In both cases only a small percentage of the native secondary structure was altered by irreversible thermal denaturation, indicating that the aggregated states retain considerable native structure.

Proc Natl Acad Sci U S A, 1988 Feb, 85(4), 1151 - 5
Structural conservation among three homologous introns of bacteriophage T4 and the group I introns of eukaryotes; Shub DA et al.; Three group I introns of bacteriophage T4 have been compared with respect to their sequence and structural properties . The introns include the td intervening sequence, as well as the two newly described introns in the nrdB and sunY genes of T4 . The T4 introns are very closely related, containing phylogenetically conserved sequence elements that allow them to be folded into a core structure that is characteristic of eukaryotic group IA introns . Similarities extend outward to the exon sequences surrounding the three introns . All three introns contain open reading frames (ORFs) . Although the intron ORFs are not homologous and occur at different positions, all three ORFs are looped-out of the structure models, with only the 3' ends of each of the ORFs extending into the secondary structure . This arrangement invites interesting speculations on the regulation of splicing by translation . The high degree of similarity between the T4 introns and the eukaryotic group I introns must reflect a common ancestry, resulting either from vertical acquisition of a primordial RNA element or from horizontal transfer.

Mol Gen Mikrobiol Virusol, 1988 Feb, (2), 17 - 20
{Molecular weight and restriction mapping of the DNA of cholera phages}; Degtiarev BM et al.; Molecular masses of cholera bacteriophages 493, 7226 and Eltor II were defined by electron microscopic technique . DNA of these bacteriophages was digested by the restriction endonucleases PstI, BglI, MluI and SalI . The number and molecular masses of the obtained restricts were identified . The physical map of bacteriophage 493 was constructed using three restriction endonucleases . The obtained data can be used for classification and molecular biology research of cholera bacteriophages.

Biophys Chem, 1988 Feb, 29(1-2), 31 - 7
Recognition of DNA sequences by the repressor of bacteriophage 434; Harrison SC et al.; The structure of a complex between the DNA-binding domain of phage 434 repressor and a 14 base-pair synthetic DNA operator reveals the molecular interactions important for sequence-specific recognition . A set of contacts with DNA backbone, notably involving hydrogen bonds between peptide-NH groups and DNA phosphates, position the repressor and fix the DNA configuration . Direct interactions between amino acid side chains and DNA bases involve nonpolar van der Waals contacts as well as hydrogen bonds . The structures of the repressor domain and of the 434 cro protein are extremely similar . There appear to be no major conformational changes in the proteins when they bind to DNA.

Virology, 1988 Feb, 162(2), 397 - 405
Phage T4 DNA codes for two distinct 10-kDa proteins which strongly bind to RNA polymerase; Orsini G et al.; Radioactive proteins have been synthesized in vitro from a coupled transcription-translation system primed with bacteriophage T4 DNA . The labeled proteins were chromatographed on RNA polymerase-Sepharose affinity columns in order to identify a protein which comigrates with the 10-kDa anti-sigma subunit of T4-modified RNA polymerase . When we primed the in vitro system with specific restriction fragments of T4 DNA, we found that there seemed to be two widely separated genes which code for this protein . The products of these two genes were compared by two-dimensional electrophoresis; they were found to have different charges, even though they had the same molecular weight and strong affinity for RNA polymerase . One of these proteins exists in a form which has the same charge as the 10-kDa subunit isolated from purified T4-modified RNA polymerase . We report a preliminary mapping experiment within the 22.5-kb fragment harboring this gene.

Virology, 1988 Feb, 162(2), 328 - 36
Cloning and generation of a genetic map of bacteriophage N4 DNA; Malone C et al.; Analysis of coliphage N4 development has been hindered by the lack of a genetic map . Conventional methods of complementation and recombination have been inadequate because N4 shows very high levels of recombination, which are independent of the host recombination system . We have cloned restriction fragments of the 72-kb genome of N4 and have used these clones to rescue a collection of suppressor-sensitive and temperature-sensitive mutants . After mutations were localized to a small region by marker rescue, complementation groups were defined . In this way several functions essential for transcription and replication have been mapped . Finally, a nonessential 6-kb region of the N4 genome has been identified by characterizing phage deletions isolated after heat and citrate treatment of virions.

J Virol, 1988 Feb, 62(2), 400 - 6
Isolation of bacteriophage T4 baseplate proteins P7 and P8 and in vitro formation of the P10/P7/P8 assembly intermediate; Plishker MF et al.; Two bacteriophage T4 proteins, P7 and P8, which are components of the phage baseplate have been purified to apparent homogeneity . P7 and P8 are the protein products of T4 genes 7 and 8 . A plasmid has been constructed which contains approximately 5 kilobases of T4 DNA, including genes 7 and 8, under the control of the tac promoter . Induction of Escherichia coli W3110iQ cells containing this plasmid resulted in the production of functional P7 and P8 . Standard protein isolation procedures were used to purify both P7 and P8 from extracts of induced cells . In T4-infected cells, these two proteins and P10 interact in a strictly ordered sequential manner (P10 + P7----P10/P7,P10/P7 + P8----P10/P7/P8) to form an intermediate in the baseplate assembly pathway . The three purified proteins assembled in vitro to form a limited number of oligomeric species, as determined by nondenaturing gel electrophoresis . P10 and P7 interacted in vitro to form two assemblies with distinct electrophoretic mobilities, both containing P10 and P7 . Addition of P8 to this mixture resulted in the disappearance of both P10/P7 species and the appearance of a single new assembly with a different electrophoretic mobility . These interactions occurred without the addition of any catalyst or cofactors . Isolated P11 appeared to add as predicted to the in vitro-formed complexes without affecting the formation of the two P10/P7 or the single P10/P7/P8 intermediates . Interactions between P7 and P8 in the absence of P10 or interactions between P10 and P8 in the absence of P7 could not be detected . These data indicate that purified P10, P7, and P8 interact in vitro in a manner completely in accord with the published assembly pathway and thus establish a system for further study of the regulation of the formation of this assembly intermediate in vitro.

Immun Infekt, 1988 Feb, 16(1), 18 - 20
{Monitoring of sterilization procedures for plasma derivatives using bacteriophages}; Dichtelmuller H et al.; Since 1968 the combination of beta-propiolactone (beta-PL) plus UV-inactivation for the sterilization of plasma derivatives is in use at Biotest . In 1981, a bacteriophage test system was established for routine monitoring of the efficacy of this sterilization procedure, using the bacteriophages phi x 174, phi e, Kappa and f2 . In the period of 1981 to 1986, 88 control experiments were performed under production conditions demonstrating a mean inactivation of these test viruses of greater than or equal to 6.7 log10 . This constant and high efficacy of the beta-PL/UV sterilization procedure guarantees the longstanding safety of beta-PL/UV sterilized blood derivatives . Bacteriophages are also useful experimental viruses for monitoring the efficacy of pasteurization processes.

Genetics, 1988 Feb, 118(2), 181 - 91
An in vitro assay for frameshift mutations: hotspots for deletions of 1 bp by Klenow-fragment polymerase share a consensus DNA sequence; de Boer JG et al.; The fidelity of in vitro DNA synthesis catalyzed by the large fragment of DNA polymerase I was examined . The templates, specifically designed to detect shifts to the +1 or to the -1 reading frame, are composites of M13mp8 and bacteriophage T4 rIIB DNA and were designed to assist in the identification of the types of frameshifts that are the specific consequence of DNA polymerization errors . In vitro polymerization by the Klenow fragment produced only deletions, rather than the mixture of duplications and deletions characteristic of in vivo frameshifts . The most frequent frameshifts were deletions of 1 bp opposite a template purine base . Hotspots for these deletions occurred when the template purine immediately preceded the template sequence TT . The highest mutation frequencies were seen when the TTPu consensus sequence was adjacent to G:C rich sequences in the 3' direction . The nature of the consensus sequence itself distinguishes this 1-bp deletion mechanism from those operating in DNA repeats and attributed to the misalignment of DNA primers during synthesis . Deletions that were larger than 1 or 2 bp isolated after in vitro replication were consistent with the misalignment of the primer . Deletions of 2 bp and complex frameshifts (the replacement of AA by C) were also found . Mechanisms that may account for these mutations are discussed.

Genetics, 1988 Feb, 118(2), 173 - 80
Escherichia coli mutations that prevent the action of the T4 unf/alc protein map in an RNA polymerase gene; Snyder L et al.; Bacteriophage T4 has the substituted base hydroxymethylcytosine in its DNA and presumably shuts off host transcription by specifically blocking transcription of cytosine-containing DNA . When T4 incorporates cytosine into its own DNA, the shutoff mechanism is directed back at T4, blocking its late gene expression and phage production . Mutations which permit T4 multiplication with cytosine DNA should be in genes required for host shutoff . The only such mutations characterized thus far have been in the phage unf/alc gene . The product of this gene is also required for the unfolding of the host nucleoid after infection, hence its dual name unf/alc . As part of our investigation of the mechanism of action of unf/alc, we have isolated Escherichia coli mutants which propagate cytosine T4 even if the phage are genotypically alc+ . These same E . coli mutants are delayed in the T4-induced unfolding of their nucleoid, lending strong support to the conclusion that blocking transcription and unfolding the host nucleoid are but different manifestations of the same activity . We have mapped two of the mutations, called paf mutations for prevent alc function . They both map at about 90 min, probably in the rpoB gene encoding a subunit of RNA polymerase . From the behavior of Paf mutants, we hypothesize that the unf/alc gene product of T4 interacts somehow with the host RNA polymerase to block transcription of cytosine DNA and unfold the host nucleoid.

Mol Gen Genet, 1988 Feb, 211(2), 350 - 6
Function of cloned T4 recombination genes, uvsX and uvsY, in cells of Escherichia coli; Minagawa T et al.; Genes uvsX and uvsY of bacteriophage T4 both control genetic recombination and repair of damaged DNA, and their mutant phenotypes bear a striking resemblance to each other . It has been shown recently that the uvsX gene product is analogous to the recA gene product of Escherichia coli (Yonesaki et al . 1985; Yonesaki and Minagawa 1985; Formosa and Alberts 1986), but the function of the uvsY gene is unknown . To obtain further insight into the function of these genes we introduced plasmidborne copies of the two genes separately or together into E . coli . The uvsX gene rendered recA- cells more resistant to UV and raised the recombination frequency of lambda phage and E . coli, but hampered induction of the lambda prophage and the SOS function of E . coli . The uvsY gene had no detectable function when introduced alone into E . coli but significantly enhanced the function of the uvsX gene when the two plasmid-borne genes were introduced together.

Mol Gen Genet, 1988 Feb, 211(2), 335 - 41
The immunity and lysis genes of ColN plasmid pCHAP4; Pugsley AP; Nucleotide sequencing of part of the plasmid pCHAP4, which encodes the ca . 42,000 Da putative poreforming colicin N, confirmed previous results indicating that the colicin N immunity gene (cni) and the colicin release or lysis gene (cnl) are located immediately downstream from the colicin N structural gene (cna) in the order cna-cni-cnl . The cni gene is transcribed in the opposite direction to cna and probably encodes an Mr 15239 Da protein . The putative immunity protein was detected among the {35S}methionine-labelled proteins produced by minicells carrying cni cloned under lac promoter control, and when the gene was subcloned into expression vectors under the control of a bacteriophage T7 promoter . Deletion of the region immediately upstream from cni completely abolished colicin N immunity, presumably because the natural promoter had been deleted . cnl is in the same operon as cna, and encodes a typical Col plasmid pro-lysis protein comprising a signal peptide and a 34 residue mature polypeptide with high homology to all but one of the other known Col lysis proteins, including the fatty acylated amino-terminal cysteine residue which was specifically labelled with 3H-palmitate . Cell fractionation studies indicated that the cnl gene product was located predominantly in the outer membrane.

Microbiologia, 1988 Feb, 4(1), 47 - 53
The effect of rifampicin on the development of the Streptomyces bacteriophage phi C31; Rodriguez A et al.; The production of phi C31 progeny virus was inhibited by rifampicin when it was added at any time before 20 minutes after induction of the thermoinducible lysogen Streptomyces coelicolor 01 . The inhibition was gradually lost as the antibiotic was being added later on until the end of the latent period, which lasts about 45 minutes . This effect was not due to resistance of transcription to rifampicin but to accumulation of intracellular virions from around 20 minutes postinduction . When a rifampicin-resistant lysogen was induced in the presence of the antibiotic, no inhibition of RNA synthesis was detected, although a smaller population of progeny than in control cultures without rifampicin was obtained . Two possible explanations of this fact are discussed.

Mol Endocrinol, 1988 Feb, 2(2), 125 - 32
Hormonal regulation of rat androgen-binding protein (ABP) messenger ribonucleic acid and homology of human testosterone-estradiol-binding globulin and ABP complementary deoxyribonucleic acids; Reventos J et al.; Complementary DNA clones coding for rat androgen-binding protein (rABP) were isolated from a rat testis cDNA library constructed in the bacteriophage lambda gt11 . The library was screened immunochemically, using two different antibodies against rABP . The identity of the isolated clones was confirmed by epitope selection and DNA sequence analysis . The mRNA encoding rABP could be detected in the testes of 20- and 46-day-old-rats, but not in the 10-day-old rats by hybridization with 32P-labeled rABP cDNA in a Northern blot of poly(A)+-RNA fractioned by agarose gel electrophoresis . No hybridization signal was seen with poly(A)+-RNA isolated from kidney and liver . The rABP mRNA appeared as a single species with a size of 1.65 kilobase, sufficient to encode a protein of 42,000 daltons . The concentration of rABP mRNA in the testes of 37-day-old hypophysectomized rats increased after treatment with testosterone and FSH, given alone or in combination . Sequence and hybridization analysis of cDNAs for rABP, human testosterone-estradiol-binding globulin, and human ABP demonstrates that the cDNAs for human testosterone-estradiol binding globulin and human ABP have greater sequence similarity with each other than either has with rABP.

Tsitologiia, 1988 Feb, 30(2), 115 - 26
{Role of the mono(ADP)ribosylation of proteins in cellular regulation}; Nemchinskaia VL; In addition to its well established role as a cofactor in redox reactions, NAD+ serves also as a substrate for a ubiquitous group of enzymes called ADP-ribosyltransferases . These enzymes are found in the cytosol and in the nucleus of eukaryotic cells, they are involved as components of pathogenic bacterial toxins, and as part of a mechanism for inactivating host cell protein biosynthetic machinery in bacteriophage-infected cells . An overview of mono(ADP)ribosylation reactions is provided.

J Mol Recognit, 1988 Feb, 1(1), 48 - 57
T4 phage deoxyribonucleoside triphosphate synthetase: purification of an enzyme complex and identification of gene products required for integrity; Moen LK et al.; We have isolated a highly enriched preparation of the multienzyme complex which synthesizes deoxyribonucleoside triphosphates (dNTPs) from bacteriophage T4-infected bacteria . By a combination of SDS polyacrylamide gel electrophoresis and assays for specific enzyme activities, we have been able to identify in our final preparation ten different gene products which were previously identified as constituents of this complex, based upon studies with crude preparations . The complex dissociates at high concentrations of NaCl and MgCl2 but is stable under ionic conditions thought to exist in vivo . The purified complex catalyzes the efficient five-step conversion of dCTP to dTTP . Experiments with several T4 mutants have demonstrated that gene products encoded by cd, regA, nrdA, and nrdB are necessary to retain physical integrity of the complex throughout the preparative procedure, while gp44, gp55, and gppseT are not required . We conclude from this evidence that the T4 early gene products which function in dNTP biosynthesis are, in fact, physically linked as a multienzyme complex, and that regA contributes to the integrity of this complex . However, the dNTP-synthesizing complex as we isolate it contains no detectable DNA polymerase, nor have other known replication proteins been detected.

J Gen Microbiol, 1988 Feb, 134 ( Pt 2), 359 - 67
Aspects of the regulation of adenylate cyclase synthesis in Escherichia coli K12; Roy A et al.; In Escherichia coli K12 expression of the adenylate cyclase gene is subject to multiple controls . In order to gain understanding of the regulation of adenylate cyclase synthesis, operon and protein fusions were constructed by in vitro recombination either into bacteriophage lambda or low-copy-number plasmids, or directly on the chromosome at the cya locus . The fusions were used in physiological experiments as probes to study transcriptional and translational controls of cya expression . It was found that adenylate cyclase synthesis was insensitive to glucose effects . As already described by other workers, the CAP-cAMP complex had a moderate negative control on cya expression . In addition it was observed that concomitant with a severe slackening of growth rate, specific to the growth of cya strains in rich medium, cya expression was considerably enhanced . This increase of adenylate cyclase synthesis did not appear to be directly dependent on the presence of a functional cAMP receptor (CAP), and seemed to be controlled at the level of transcription . Finally, translation of the cya message was very weak when compared to cya transcription (the mRNA level was the same in protein and operon fusions.

Can J Microbiol, 1988 Feb, 34(2), 190 - 3
Transduction of Escherichia coli in soil; Germida JJ et al.; Bacteriophage P1-mediated generalized transduction of Escherichia coli K-12 was assessed in nonsterile soil . Auxotrophic recipient cells (thr- leu- thi- rpsL) were incubated in a sandy and a silty clay loam soil, and the transducing phage lysates from prototrophic strains carrying transposon 10(Tn10) in either purE or aroL regions were added . At intervals, the bacterial populations derived from the soils were plated on selective-differential media to enumerate prototrophic (thr+, leu+, or Tcr) transductants . Of 100 bacterial isolates obtained on the selective-differential media, 58 (14 thr+; 11, leu+; 33 Tcr) were confirmed E . coli transductants . The frequency of transduction in soil was ca . 10(-6) . These data demonstrate the potential use of bacteriophage P1 to genetically manipulate E . coli in situ.

Mol Endocrinol, 1988 Feb, 2(2), 181 - 5
Cloning of a complementary deoxyribonucleic acid coding for human thyroxine-binding globulin (TBG): existence of two TBG messenger ribonucleic acid species possessing different 3'-untranslated regions; Kambe F et al.; An adult human liver cDNA library constructed in expression vector, bacteriophage lambda gt11, was screened with polyclonal antibody directed against human T4-binding globulin (TBG) . TBG cDNA cloned in the present study was 944 nucleotides in length . It contained approximately 70% of the coding region and complete 3'-untranslated region . When the sequence was compared with that of TBG cDNA recently cloned by I . L . Flint, T . J . Bailey, T . A., Gustafson, B . E . Markham, and E . Morkin, the 3'-untranslated region of our cDNA was 231 nucleotides shorter than their cDNA . These results indicated that two TBG mRNAs with different length of 3'-untranslated regions may exist in human liver . Indeed, Northern blot analysis revealed that two TBG mRNAs differing in the length approximately 200 base pairs were present in normal human liver as well as in human hepatoma cell line (HepG2) . It was demonstrated that this size difference was due to the length of 3'-untranslated region by hybridization with a probe specific to the longer 3'-end . Together with the sequence data, it was suggested that these two TBG mRNA species may be produced by alternative processing and polyadenylation at two different sites.

Genes Dev, 1988 Feb, 2(2), 184 - 95
Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms; Ho YS et al.; The bacteriophage lambda transcriptional activator protein cII is a DNA-binding protein that coordinately regulates transcription from phage promoters important for lysogenic growth . We have genetically and structurally characterized more than 80 different single amino acid substitutions in this 97-amino-acid protein . A subset of 25 of these variant proteins was utilized for detailed biochemical analysis, which allows us to define specific domains critical for sequence-selective DNA recognition, nonspecific DNA binding, and protein oligomerization . The mutation studies also demonstrated the remarkable correlation of oligomeric structure of cII protein to its stability within the bacterial host . An Escherichia coli HtpR- strain has been identified that greatly stabilizes these highly unstable cII mutants.

J Bacteriol, 1988 Feb, 170(2), 972 - 9
The dnaK gene of Escherichia coli functions in initiation of chromosome replication; Sakakibara Y; A newly isolated dnaK mutant of Escherichia coli, which contains the mutation dnaK111, has been found to be conditionally defective in initiation of DNA replication . Mutant cells that were transferred to high temperature exhibited residual DNA synthesis before the synthesis stopped completely . Analysis of the DNA synthesized at high temperature by hybridization with probe DNAs for detection of DNA replicated in the origin (oriC) and terminal (terC) regions has revealed that this mutant is unable to initiate a new round of DNA replication at high temperature after termination of the round in progress . The cells exposed to high temperature were subsequently capable of initiating DNA replication at low temperature in a synchronous manner . DNA synthesis of this mutant became temperature resistant upon inactivation of the rnh gene, similar to that of dnaA mutants, although cell growth of the dnaK mutant with the inactive rnh gene remained temperature sensitive . The dnaK mutation prevented DNA synthesis of lambda bacteriophage at high temperature even in the absence of the rnh gene function.

Anal Biochem, 1988 Feb 1, 168(2), 324 - 31
A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column; Reddy KJ et al.; A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described . The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer . The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells . The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation . This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation . The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing . A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h . This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.

Proc Natl Acad Sci U S A, 1988 Feb, 85(3), 752 - 6
Gin-mediated DNA inversion: product structure and the mechanism of strand exchange; Kanaar R et al.; Inversion of the G loop of bacteriophage Mu requires the phage-encoded Gin protein and a host factor . The topological changes in a supercoiled DNA substrate generated by the two purified proteins were analyzed . More than 99% of the inversion products were unknotted rings . This result excludes synapsis by way of a random collision of recombination sites, because the resulting entrapped supercoils would be converted into knots by recombination . Instead, the recombination sites must come together in the synaptic complex in an ordered fashion with a fixed number of supercoils between the sites . The linking number of the substrate DNA increases by four during recombination . Thus, in three successive rounds of inversion, the change in linking number was +4, +8, and +12, respectively . These results lead to a quantitative model for the mechanism of Gin recombination that includes the distribution of supercoils in the synaptic complex, their alteration by strand exchange, and specific roles for the two proteins needed for recombination.

J Bacteriol, 1988 Feb, 170(2), 961 - 6
Regulation of expression of the cytochrome d terminal oxidase in Escherichia coli is transcriptional; Georgiou CD et al.; The cytochrome d complex is one of the two terminal oxidases in the aerobic respiratory system of Escherichia coli . This enzyme is not present in cells grown with high levels of dissolved oxygen in the culture medium but accumulates after mid-exponential growth, reaching high levels in stationary-phase cells . In this study, the transcriptional activity of the cyd operon, encoding the two subunits of the enzyme, was examined under a variety of growth conditions . This was accomplished by the use of a chromosomal operon fusion, cyd-lacZ, generated in vivo by a lambda plac-Mu hopper bacteriophage and also by the use of a cyd-lacZ protein fusion created in vitro on a plasmid, transferred onto a lambda transducing phage, and examined as a single-copy lysogen . Transcription of the gene fusions was monitored by determination of beta-galactosidase activity . The data clearly show that cyd is transcriptionally regulated and that induction is observed when the culture reaches a sufficient cell density so as to substantially reduce the steady-state levels of dissolved oxygen . The transcriptional activity is also regulated by other growth conditions, including the carbon source . The turn-on of cyd under semianaerobic conditions does not require the fnr gene product, cyclic AMP, or the cyclic AMP-binding protein.

J Bacteriol, 1988 Feb, 170(2), 908 - 15
nusA amber mutation that causes temperature-sensitive growth of Escherichia coli; Tsugawa A et al.; The nusA134 mutation was isolated from a sup0 strain as a temperature-sensitive mutant which grew at 32 degrees C but not at 42 degrees C . Immunoblot analysis showed that this mutant produced a 31,000-dalton nusA-encoded protein instead of the full-size 54,500-dalton product . Sequence and genetic analyses of the mutant nusA gene revealed a substitution of T for C at the PstI site (i.e., CTGCAG to CTGTAG), thereby creating a nonsense UAG codon . These results indicate that nusA134 is an amber mutation and that the 31,000-dalton amber fragment is active for Escherichia coli growth at 32 degrees C but not at 42 degrees C . Most lambda bacteriophage variants tested grew normally on the nusA134 mutant both at permissive and at nonpermissive temperatures . However, lambda r32, which carries an IS2 insertion beyond the tR1 terminator, was restricted at 42 degrees C . Defects in the transcriptional antitermination process, but not in transcription termination, were observed . A comparative study of nusA134 protein and a PstI-truncated protein suggests that truncation of the peptide chain at the PstI site by the amber mutation, rather than the loss of the glutamine residue, is primarily responsible for the defect in antitermination . The mode of the involvement of mutant nusA proteins in the N-mediated antitermination reaction is discussed.

J Bacteriol, 1988 Feb, 170(2), 889 - 94
Temporal control of transposition in Tn5; McCommas SA et al.; IS50R is an insertion sequence associated with the transposon Tn5 . IS50R carries the structural genes for two proteins; one (P1) is the Tn5 transposase, and the other (P2) is an inhibitor of transposition . These two proteins are translated from two different transcripts, m1 and m2 . When bacteriophage lambda::IS50R DNA was introduced into a bacterial cell, m1 and m2 were initially at relative levels of about 1 to 2 . As time progressed the amount of m1 fell, whereas the amount of m2 continued to increase, until after about 3 h the ratio of m1 to m2 was about 1 to 80 . The temporal changes in the levels of these transcripts correlated with temporal changes in P1 and P2 levels and Tn5 transposition that have been documented in other studies . We measured the stability of the messages and showed that the differences in the levels of m1 and m2 must reflect real differences in the strengths of their promoters and that the changes in transcription kinetics are mediated by the dam methylation system of the cell and are not determined by IS50R products . Our results show that the 5' end of m2 is about twice as stable as that of m1, which raises the possibility that differential message stability does, in part, influence the ratio of inhibitor to transposase.

J Virol, 1988 Feb, 62(2), 387 - 92
The Nul subunit of bacteriophage lambda terminase binds to specific sites in cos DNA; Shinder G et al.; The maturation and packaging of bacteriophage lambda DNA are under the control of the multifunctional viral terminase enzyme, which is composed of the protein products of Nu1 and A, the two most leftward genes of the phage chromosome . Terminase binds selectively to the cohesive end site (cos) of multimeric replicating lambda DNA and introduces staggered nicks to regenerate the 12-base single-stranded cohesive ends of the mature phage genome . The purified gpNu1 subunit of terminase forms specific complexes with cos lambda DNA . DNase I footprinting experiments showed that gpNu1 bound to three distinct regions near the extreme left end of the lambda chromosome . These regions coincided with two 16-base-pair sequences (CTGTCGTTTCCTTTCT) that were in inverted orientation, as well as a truncated version of this sequence . Bear et al . (J . Virol . 52:966-972,1984) isolated a mutant phage which contained a CG to TA transition at the 10th position of the rightmost 16-base-pair sequence, and this phage (termed lambda cos 154) exhibits a defect in DNA maturation when it replicates in Escherichia coli which is deficient in integration host factor . Footprinting experiments with cos 154 DNA showed that gpNu1 could not bind to the site which contained the mutation but could protect the other two sites . Since the DNA-packaging specificity of terminase resides in the gpNu1 subunit, these studies suggest that terminase uses these three sites as recognition sequences for specific binding to cos lambda.

Biotechnol Appl Biochem, 1988 Feb, 10(1), 59 - 62
Regulation of proteolysis in Escherichia coli cells by antisense RNA of htpR gene; Kiselev VI et al.; A new vector has been constructed on the basis of plasmid pCQV2 containing thermoinducible regulatory elements of bacteriophage lambda . The vector makes it possible to combine an inducible synthesis of foreign proteins with negative control of intracellular proteolysis . The principle employed for the construction of the plasmid implies the regulation of gene expression by antisense RNAs . In the SalI restriction site of pCQV2 a fragment of htpR gene encoding the N-terminal region of the polypeptide has been cloned in opposite orientation . At 42 degrees C transcription from the PR promoter of the plasmid is initiated, resulting in the synthesis of htpR antisense RNA . The product of htpR gene is known to be responsible for positive regulation of transcription of at least three genes encoding proteases lon, groEL, and groES . Inactivation of the htpR transcript due to its complementary pairing with the antisense RNA hinders the synthesis of proteases under temperature stress conditions, thus leading to the reduction in the rate of proteolysis of abnormal proteins.

Cell, 1988 Jan 29, 52(2), 179 - 84
DNA-bound Fos proteins activate transcription in yeast; Lech K et al.; We constructed genes encoding the DNA binding region of the bacterial LexA repressor fused to the v-fos and c-fos oncogene products . The resulting LexA-Fos fusion proteins activated transcription in yeast . Transcription activation by these proteins was as strong as transcription activation by proteins native to yeast . LexA-Fos fusion proteins only activated transcription of genes when they were bound to LexA binding sites inserted upstream of those genes . Transcription was activated less strongly by similar proteins in which the DNA binding region of LexA was fused to vMyc and cMyc . Transcription was not activated by native LexA or by proteins containing the DNA binding domain of LexA fused to bacteriophage 434 repressor or yeast MAT alpha 2 protein . These results demonstrate that Fos proteins activate eukaryotic gene expression when they are bound to promoter DNA, and thus suggest that Fos proteins exert some of their effects because they stimulate transcription of cellular genes . Regulation of transcription by Fos and Myc proteins in yeast provides a phenotype that may facilitate genetic analysis of the function of these proteins in higher organisms.

Biochemistry, 1988 Jan 26, 27(2), 706 - 11
Raman spectroscopy of mercury (II) binding to two filamentous viruses: Ff (fd, M13, f1) and Pf1; Day LA et al.; Ff and Pf1 are filamentous bacteriophages . Each contains, in a central core region surrounded by protein, a circular single-stranded DNA molecule, and it is known that the DNA bases are sites of Hg(II) binding . In the present study, Raman spectra were obtained for the two viruses in the presence of increasing amounts of Hg(II), with ratios (m) of Hg(II) added per nucleotide residue in the range 0 less than m less than 2.0 . Hg(II) binding to the viruses induces Raman intensity changes in previously assigned Raman lines of viral DNA, demonstrating metal binding to the DNA bases, but also in many lines assigned to protein . The overall structures of the viruses do not change with Hg(II) binding, and the Raman spectra indicate little, if any, change in protein secondary structure . Changes in certain protein Raman lines induced by Hg(II) binding to the DNA for low values of m are attributed to altered interactions between solvent and protein side chains, aliphatic groups being the most affected . The nature of such changes for both viruses suggests DNA-protein linkage . In Pf1, lines assigned to ring vibrations of all four bases are perturbed upon initial addition of Hg(II) to m = 0.25 . In Ff, however, lines assigned to base ring vibrations are not perturbed until m greater than or equal to 0.5 . The results provide additional evidence for fundamentally different DNA structures in Ff and Pf1.

J Biol Chem, 1988 Jan 25, 263(3), 1174 - 81
Late sigma factor of bacteriophage T4 . Formation and properties of RNA polymerase-promoter complexes; Malik S et al.; Bacteriophage T4 late gene promoters do not display sequence homology in the -35 region (Christensen, A . C., and Young, E . T . (1982) Nature 299, 369-371), suggesting an unusual geometry of RNA polymerase-promoter interaction . We have analyzed in vitro utilization of a late T4 promoter by RNA polymerase reconstituted from E . coli core enzyme (E) and bacteriophage T4 late sigma factor (sigma gp55) . The E sigma gp55 holoenzyme forms a stable promoter complex which lacks protein-DNA contacts upstream from position -30 and is sensitive to direct attack by heparin . This complex is capable of reiterative oligonucleotide synthesis (abortive initiation) . Kinetic analysis of complex formation reveals a rapidly forming inactive intermediate (closed complex) which is slowly isomerized into a catalytically active form (open complex) . The results indicate that all components essential for promoter binding, open complex formation, and initiation of transcription are present in the "downstream" part of the RNA polymerase molecule, which is defined by the -30 to +20 footprint . On the basis of these observations and the results of others, we suggest that during transcription initiation at bipartite (Escherichia coli) promoters, an "upstream" DNA-binding domain of RNA polymerase which recognizes specific sequence elements in the -35 region plays an auxiliary role by regulating the rate of productive interactions in the downstream part of the molecule through an allosteric mechanism.

Biochim Biophys Acta, 1988 Jan 25, 949(1), 143 - 7
DNA damage induced by ascorbate in the presence of Cu2+; Kobayashi S et al.; DNA damage induced by ascorbate in the presence of Cu2+ was investigated by use of bacteriophage phi X174 double-stranded supercoiled DNA and linear restriction fragments as substrates . Single-strand cleavage was induced when supercoiled DNA was incubated with 5 microM-10 mM ascorbate and 50 microM Cu2+ at 37 degrees C for 10 min . The induced DNA damage was analyzed by sequencing of fragments singly labeled at their 5'- or 3'-end . DNA was cleaved directly and almost uniformly at every nucleotide by ascorbate and Cu2+ . Piperidine treatment after the reaction showed that ascorbate and Cu2+ induced another kind of DNA damage different from the direct cleavage . The damage proceeded to DNA cleavage by piperidine treatment and was sequence-specific rather than random . These results indicate that ascorbate induces two classes of DNA damage in the presence of Cu2+, one being direct strand cleavage, probably via damage to the DNA backbone, and the other being a base modification labile to alkali treatment . These two classes of DNA damage were inhibited by potassium iodide, catalase and metal chelaters, suggesting the involvement of radicals generated from ascorbate hydroperoxide.

J Biol Chem, 1988 Jan 25, 263(3), 1391 - 7
The c1 repressor of bacteriophage P1 . Isolation and characterization of the repressor protein; Dreiseikelmann B et al.; The c1 repressor gene of bacteriophage P1 is located on P1 DNA EcoRI fragment 7 (Sternberg, N . (1979) Virology 96, 129-142) . Subfragments of P1 DNA EcoRI fragment 7 were cloned into expression vectors, and the c1 repressor protein from P1 wild-type phage and a revertant of a temperature-sensitive repressor mutant were overproduced in Escherichia coli and purified to near-homogeneity . The decreased electrophoretic mobility of P1 DNA BamHI fragment 9 in the presence of appropriate protein fractions was used as an assay for the repressor protein . Highly purified repressor migrates as a single polypeptide on denaturing sodium dodecyl sulfate-polyacrylamide gels, corresponding to a molecular weight of about 33,000 . A molecular weight of about 63,000 for the native repressor molecule was calculated from determinations of the sedimentation coefficient, which was 2.6 s, and the Stokes radius, which was 55 A . Cross-linking the protein with glutaraldehyde yielded two bands . These data and a high frictional coefficient (2.1) suggest that the native repressor exists in solution as an asymmetric dimer molecule.

J Mol Biol, 1988 Jan 20, 199(2), 373 - 7
Nucleotide and deduced amino acid sequence of stp: the bacteriophage T4 anticodon nuclease gene; Chapman D et al.; Pre-existing host tRNAs are reprocessed during bacteriophage T4 infection of certain Escherichia coli strains . In this pathway, tRNALys is cleaved 5' to the wobble base by anticodon nuclease and is later restored in polynucleotide kinase and RNA ligase reactions . Anticodon nuclease depends on prr, a locus found only in host strains that restrict T4 mutants lacking polynucleotide kinase and RNA ligase; and on stp, the T4 suppressor of prr restriction . stp was cloned and the nucleotide sequences of its wild-type and mutant alleles determined . Their comparison defined an stp open reading frame of 29 codons at 162.8 to 9 kb of T4 DNA (1 kb = 10(3) base-pairs) . We suggest that stp encodes a subunit of anticodon nuclease, perhaps one that harbors the catalytic site; while additional subunits, such as a putative prr gene product, impart protein folding environment and tRNA substrate recognition.

J Mol Biol, 1988 Jan 20, 199(2), 241 - 58
Transcriptional activation of bacteriophage T4 middle promoters by the motA protein; Guild N et al.; Transcriptional activation of middle genes in bacteriophage T4 requires the phage-encoded motA protein . Many middle genes are involved in deoxyribonucleotide biosynthesis and phage DNA replication . In the absence of motA, the gene products that are required for DNA synthesis are transcribed from other, upstream promoters . Using primer extension sequencing on RNA templates isolated from T4 motA+ and motA- infected cells, we have characterized 14 motA-dependent transcripts . The T4 middle promoters have a consensus sequence of nine base-pairs, (a/t)(a/t)TGCTT(t/c)A, spaced 11 to 13 nucleotides away from the Escherichia coli--10 consensus sequence, TAnnnT . The motA protein also can act as a transcriptional repressor for at least one early gene . Furthermore, the phage-encoded motA protein can activate in trans a middle promoter resident on a plasmid.

Mol Biochem Parasitol, 1988 Jan 15, 27(2-3), 233 - 9
Expression of diagnostic 31/32 kilodalton proteins of Schistosoma mansoni as fusions with bacteriophage MS2 polymerase; Klinkert MQ et al.; An expression plasmid pEx34b was used to synthesise parts of the 31/32 kDa Schistosoma mansoni antigens as fusions with the amino terminus of the phage MS2 polymerase . Purified MS2-schistosome fusion proteins reacted specifically in an enzyme-linked immunosorbent assay with sera from S . mansoni-infected patients . The observation that the majority of human sera tested recognised schistosome-specific epitopes, but not the MS2 polymerase fragment, suggests that the fusion proteins are useful for the immunodiagnosis of schistosomiasis and might be incorporated in a serological test system based on recombinant antigens.

J Biol Chem, 1988 Jan 5, 263(1), 443 - 7
Cloning and expression of a Streptomyces plicatus chitinase (chitinase-63) in Escherichia coli; Robbins PW et al.; 4-Methylumbelliferyl (4-MU) glycosides of N-acetylglucosamine oligosaccharides were used as substrates to detect expression of a Streptomyces chitinase in Escherichia coli . Low levels of enzyme were detected when S . plicatus DNA was cloned into a bacteriophage lambda vector (EMBL-4) . Subcloning into E . coli plasmids also gave low but detectable levels of enzyme expression . High level expression was achieved by resection of the cloned S . plicatus DNA with Bal31 followed by in-frame fusion to the amino-terminal peptide sequence of beta-galactosidase found in the pUC vectors . The Streptomyces chitinase was secreted into the periplasmic space of E . coli, and its signal sequence was removed . We characterized the activity of the cloned enzyme and compared it to three other purified Streptomyces plicatus chitinases with respect to hydrolysis of the 4-MU oligosaccharides . We found that two of the enzymes form 4-methylumbelliferone much more rapidly from the 4-MU disaccharide than from the trisaccharide . These same enzymes convert the 4-MU trisaccharide primarily to diacetylchitobiose and the 4-MU monosaccharide, a nonfluorescent product . The latter compound is not hydrolyzed appreciably by any of the enzymes . On the basis of these results, we suggest a new definition of "exo" and "endo" chitinase that differs from that found in the literature . We propose that exochitinase activity be defined as processive action starting at the nonreducing ends of chitin chains with release of successive diacetylchitobiose units, and that endochitinase activity be defined as random cleavage at internal points in chitin chains.

J Mol Biol, 1988 Jan 5, 199(1), 171 - 82
Conformation of the coat protein of filamentous bacteriophage Pf1 determined by neutron diffraction from magnetically oriented gels of specifically deuterated virions; Stark W et al.; The structure of filamentous bacteriophage Pf1 has been studied using neutron diffraction from magnetically oriented gels of native and valine-deuterated phage . Neutron diffraction intensities were measured to approximately 8 A resolution along the equator and first six layer-lines, and differences due to the deuterated valine residues were apparent . Analysis of equatorial data indicate that one valine residue is located at a radius of about 13 A, three are in the hydrophobic center of the protein coat at an average of about 22 A radius, and one is near the outer surface of the virion at about 28 A radius . Analysis of the three-dimensional data was initiated using the rod model for the alpha-helices of the coat protein derived from earlier X-ray diffraction studies . This model was refined against the neutron diffraction intensities from native phage to obtain a phase set that was used to calculate a difference map between the valine-deuterated and native phage . The difference map exhibits peaks that correspond to the positions of the five valine residues in the coat protein . From the amino acid sequence and the alpha-helical conformation of the coat protein, the five valine residues can be unambiguously assigned to the difference peaks . This assignment indicates that the two alpha-helices of the coat protein are parallel to one another, connected by a short stretch of non-helical peptide . The valine positions also indicate that the helical surface lattice of the phage particle is right-handed.

J Mol Biol, 1988 Jan 5, 199(1), 95 - 105
Dominance in lambda S mutations and evidence for translational control; Raab R et al.; Phenotypic analysis of a collection of point mutations in the lysis gene S of bacteriophage lambda indicates that many of the S alleles exhibit at least partially dominant character, suggesting that the S gene product (gpS) must oligomerize to achieve its lethal membrane effect . Moreover, mutations found 5' to the coding sequence also show a dominant character and appear to define a site, designated sdi (structure directed initiation) where mRNA secondary structure controls the choice of initiation codons . We propose that formation of the sdi structure occludes the consensus Shine-Dalgarno sequence and results in initiation at the Met3 codon, generating a lethal 105 residue polypeptide . The model predicts that, in the absence of the sdi stem-and-loop, initiation occurs at the Met1 codon, generating a 107 residue polypeptide, which is a non-lethal inhibitor of lysis . In support of the model, alteration of the first codon was achieved using site-directed mutagenesis, resulting in an S allele that is more lethal and induces lysis significantly sooner than the wild-type.

J Mol Biol, 1988 Jan 5, 199(1), 219 - 22
Prediction of an ATP reactive center in the small subunit, gpNu1, of the phage lambda terminase enzyme; Becker A et al.; The small subunit of the bacteriophage lambda terminase enzyme, the product of the phage's Nu1 gene, is shown to contain amino acid segments homologous to those present in a large number of ATPases . In keeping with these predictions, the purified protein has been found to hydrolyze ATP with a relatively low turnover number . Terminase holoenzyme is a known ATPase, and the biochemical significance of an ATP-interactive center situated in the gpNu1 subunit is discussed.

J Biol Chem, 1988 Jan 5, 263(1), 135 - 9
Escherichia coli glycerol kinase . Cloning and sequencing of the glpK gene and the primary structure of the enzyme; Pettigrew DW et al.; The glpK gene, which codes for Escherichia coli K-12 glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase), has been cloned into the HindIII site of pBR322 . The gene was contained in a 2.8-kilobase DNA fragment which was obtained from a lambda transducing bacteriophage, lambda dglpK100 (Conrad, C.A., Stearns, G.W., III, Prater, W.E., Rheiner, J.A., and Johnson, J.R . (1984) Mol . Gen . Genet . 195, 376-378) . The DNA sequence of 2 kilobases of the cloned HindIII fragment was obtained using the dideoxynucleotide method . The start of the open reading frame for the glpK gene was identified from the N-terminal sequence of the first 22 amino acid residues of the purified enzyme, which was determined by automated Edman degradation . The open reading frame codes for a protein of 502 amino acids and a molecular weight of 56,106 which is in good agreement with the value previously determined by sedimentation equilibrium . The primary structure of the protein as deduced from the gene sequence was corroborated by the isolation and sequencing of four tryptic peptides, which were found to occur at the following amino acid locations: 173-177, 203-211, 279-281, 464-468 . The N-terminal sequence of the purified enzyme shows that the enzyme undergoes post-translational processing . Restriction digestion as well as DNA sequencing of the supercoiled plasmid shows that the HindIII fragment is inserted into pBR322 such that the glpK gene is transcribed in a counterclockwise direction . Examination of the upstream DNA sequence reveals two possible promoters of essentially the same efficiency: the P1 promoter of pBR322 and a hybrid promoter which contains both bacterial and pBR322 DNA sequences.

Genetics, 1988 Jan, 118(1), 5 - 12
Effects of DNA heterologies on bacteriophage lambda packaging; Pearson RK et al.; We have examined the impact of DNA heterologies on the packaging of lambda DNA in vitro . Heterology-containing DNA molecules were constructed by denaturing and reannealing a mixture of DNA from cI+ phage and DNA front phage carrying small insertion or deletion mutations in the cI gene . We found that molecules with heterologies of up to 19 base pairs (bp) can be packaged as viable heterozygous phage with approximately the same efficiency as molecules with a base pair mismatch . In contrast, with a heterology of 26-bp heterozygous plaque formers are rare . In principle, the absence of cI heterozygotes among packaged phage may be due either to a failure to encapsulate the DNA or a failure to inject the packaged DNA on infection . Southern blot analysis of DNA isolated from packaged phage indicates that DNA harboring a 26-bp heterology is almost completely absent in packaged phage . Thus, an upper limit has been established for the size of heterology that can be accommodated by the packaging apparatus The size of the connector portal could be the basis for this limit.

Genetics, 1988 Jan, 118(1), 13 - 9
Effects of DNA heterologies on bacteriophage lambda recombination; Pearson RK et al.; Previous studies of bacteriophage lambda recombination have provided indirect evidence that substantial sequence nonhomologies, such as insertions and deletions, may be included in regions of heteroduplex DNA . However, the direct products of heterology-containing heteroduplex DNA--heterozygous progeny phage--have not been observed . We have constructed a series of small insertion and deletion mutations in the cI gene to examine the possibility that small heterologies might be accommodated in heterozygous progeny phage . Genetic crosses were carried out between lambda cI- Oam29 and lambda cI+ Pam80 under replication-restricted conditions . Recombinant O+P+ progeny were selected on mutL hosts and tested for cI heterozygosity . Heterozygous recombinants were readily observed with crosses involving insertions of 4 to 19 base pairs (bp) in the cI gene . Thus, nonhomologies of at least 19 bp can be accommodated in regions of heteroduplex DNA during lambda recombination . In contrast, when a cI insertion or deletion mutation of 26 bp was present, few of the selected recombinants were heterozygous for cI . Results using a substitution mutation, involving a 26-bp deletion with a 22-bp insertion, suggest that the low recovery of cI heterozygotes containing heterologies of 26 bp or more is due to a failure to encapsulate DNA containing heterologies of 26 bp or more into viable phage particles.

Virology, 1988 Jan, 162(1), 38 - 46
Concatemerization and packaging of bacteriophage T7 DNA in vitro: determination of the concatemers' length and appearance kinetics by use of rotating gel electrophoresis; Son M et al.; During its morphogenesis both in intact infected cells (in vivo) and in lysates of infected cells (in vitro), bacteriophage T7 forms end-to-end concatemers of its mature DNA, a linear, nonpermuted, terminally repetitious DNA . During morphogenesis, in vivo T7 concatemers are packaged in preformed capsids and cut to mature size . In the present study the lengths and appearance kinetics of concatemers formed in vitro from mature T7 DNA have been determined . The following procedures are used here for the first time: (a) 20-35% efficient in vitro concatemerization and packaging of T7 DNA; the mixture used for packaging contained two lysates that together had all T7 gene products, and (b) fractionation of concatemers by rotating gel electrophorsis (RGE), which improves the resolution by length of concatemer-length DNA . Concatemerization at 30 degrees was so fast that some other process must be rate limiting for packaging . The concatemers formed were linear and joined left-end to right-end by complementary base pairing, not by blunt-end ligation . Concatemers formed at 30 degrees were reconverted to mature DNA by packaging in vitro . Reducing the temperature to 0 degrees both slowed concatemerization to the time scale (minutes) needed for control of the extent of concatemerization and reduced packaging to insignificant levels, thereby also uncoupling packaging from concatemerization . At both 30 degrees and 0 degrees bands of discrete-length concatemers were observed by RGE . The lengths were n times the length of mature T7 DNA; n was found to be any integer from 2 to 15 . The bands were stronger at 0 degrees than they were at 30 degrees in comparison to a background of heterogeneous DNA . No evidence for the favoring of any value of n was found . In addition, it was found by two-dimensional agarose gel electrophoresis that a comparatively small amount of circular DNA was produced in vitro.

Mutat Res, 1988 Jan, 193(1), 65 - 73
Two DNA endonuclease activities from normal human and xeroderma pigmentosum chromatin active on psoralen plus ultraviolet light treated DNA; Lambert MW et al.; DNA endonuclease activities from the chromatin of normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells were examined on DNA treated with 8-methoxypsoralen (8-MOP) or 4,5',8-trimethylpsoralen (TMP) plus long wavelength ultraviolet (UVA) light, which produce monoadducts and DNA interstrand cross-links, and angelicin plus UVA light, which produces mainly monoadducts . 9 chromatin-associated DNA endonuclease activities were isolated from normal and XPA cells and assayed for activity on PM2 bacteriophage DNA that had been treated with 8-MOP or TMP in the dark and then exposed to UVA light . Unbound psoralen was removed by dialysis and a second dose of UVA light was given . Cross-linking of DNA molecules was confirmed by alkaline gel electrophoresis . In both normal and XPA cells, two DNA endonuclease activities were found which were active on 8-MOP and TMP plus UVA light treated DNA . One of these endonuclease activities, pI 4.6, is also active on intercalated DNA and a second one, pI 7.6, is also active on UVC (254 nm) light irradiated DNA . The major activity against angelicin plus UVA light treated DNA in both normal and XPA cells was found in the fraction, pI 7.6 . The levels of activity of both of these fractions on all 3 psoralen-damaged DNAs were similar between normal and XPA cells . These results indicate that in both normal and XPA cells there are at least two different DNA endonucleases which act on both 8-MOP and TMP plus UVA light treated DNA.

Exp Cell Res, 1988 Jan, 174(1), 199 - 214
Cell cycle phase-specific cDNA libraries reflecting phase-specific gene expression of Ehrlich ascites cells growing in vivo; Lu X et al.; Asynchronous populations of Ehrlich ascites tumor cells grown in vivo were separated by centrifugal elutriation into fractions of G1-, S-, and G2/M-phase cells with less than 10% cross-contamination . Cytoplasmic mRNA from phase-synchronous cells was used to prepare cDNA which was ligated with bacteriophage lambda gt10 arms and amplified in Escherichia coli C600 hfl- . EcoRI digests of DNA isolated from the sublibraries (G1, S, G2/M) were submitted to Southern hybridizations with radiolabeled probes either (a) for genes whose phase-specific expression is clearly documented, thymidine kinase, dihydrofolate reductase, and thymidylate synthase, or (b) for genes whose change of expression during the cell cycle is likely, lamin C, beta-actin, alpha- and beta-tubulin, c-myc, c-fos, p53 . The cDNA sequences for genes of group (a) were found to be significantly enriched in DNA of the S-phase library indicating that the cell cycle phase-specific patterns of the respective mRNA levels are conserved in the sublibraries . Sequences belonging to group (b) were also found to be enriched in DNA isolated from the sublibraries: c-fos in G1 phase, lamin C, beta-actin, tubulins, c-myc in S phase, and p53 in G1/S phase . The unexpected prevalence of c-myc and alpha-tubulin in the S-phase library is supported by Northern analysis of RNA from phase-synchronous cells . Non-phase-specific, randomly chosen sequences hybridized equally strong with DNA isolated from the different sublibraries . No significant changes of the patterns of hybridization signals were observed with DNA from different amplifications of the sublibraries when analyzed with the same DNA probe indicating that the cDNA complexities are well conserved during amplifications . Consequently, the sublibraries are useful to obtain information about the cell cycle phase-specific expression of mRNAs for other genes of interest . Since the sublibraries reflect mRNA levels of the cells growing in vivo they supply data on the physiological in vivo pattern of gene expression undisturbed by potentially unphysiological in vitro conditions.

J Virol, 1988 Jan, 62(1), 181 - 7
Production of a polyhedral particle in Escherichia coli from a cDNA copy of the large genomic segment of bacteriophage phi 6; Gottlieb P et al.; A polyhedral particle that resembles in composition and structure the procapsid of bacteriophage phi 6 was produced in Escherichia coli containing cDNA copies of the entire large genomic segment inserted into expression vector plasmids under the control of lac or tac promoters . The particles were composed of proteins P1, P2, P4, and P7 in the same stoichiometry as in the intact virion . In electron micrographs of negatively stained samples, the particles appeared as hexagons, stars, or rings of 10 knobs, which are characteristic of the five-, three-, and twofold axes of symmetry characteristic of phi 6 procapsids . Stable particles were also produced from cDNA deletions that produce only P1 and P4 . Other cDNA deletions producing P1 and P7 and P1 alone resulted in unstable particles which could only be visualized in electron micrographs of thin sections of E . coli transformed by the recombinant plasmids . Our results indicate that the assembly of the phi procapsid is independent of other phage proteins and of normal phage RNA.

Biotechniques, 1988 Jan, 6(1), 50 - 5
Ultraviolet resonance Raman spectroscopy as a probe of protein structure in the fd virus; Grygon CA et al.; Resonance Raman spectroscopy can provide details of molecular structure via the enhancement of specific vibrational bands in the spectrum of the scattered light when the laser excitation is tuned to electronic absorption wavelengths of the molecule . The availability of lasers operating in the deep ultraviolet region makes it possible to apply this technique to problems of protein structure . The backbone conformation and the environments of aromatic side chains can be probed via appropriate enhancement of selected vibrational modes . In this article we investigate ultraviolet resonance Raman (UVRR) spectra from the coat protein of the filamentous bacteriophage, fd, in the intact virus and in sodium dodecyl sulfate (SDS) suspension . The results indicate that 1) the protein is completely alpha-helical in the mature virus, but loses a large fraction of its helix content in the SDS micelles . 2) The two tyrosine residues appear to behave as H-bond acceptors in the intact phage but this interaction is lost in the micelles . 3) The tryptophan residue is not solvent-exposed in either protein conformation, although in SDS it is accessible to H/D exchange with the solvent . 4) The three phenylalanine residues are involved in stacking interactions in the intact virus; these are disrupted in the SDS micelles . 5) The single proline residue appears to be in a trans conformation both in the virus and in the micelles.

Acta Biochim Pol, 1988, 35(4), 357 - 66
The construction of the hybrid plasmid containing genes of the central part of phage T4 baseplate and gene 29 product separation; Nieradko J et al.; A fragment of E . coli bacteriophage T4 genome including the four genes (genes 51, 27, 28, 29) coding for the central plug proteins was cloned into plasmid pMCC17 . The genes present on this fragment were expressed in E . coli in the absence of phage infection producing hub proteins, which could be identified on polyacrylamide gels . By applying affinity chromatography protein 29 was purified from extracts of E . coli transformed with this hybrid plasmid . The isolated protein had the ability to complement T4 29 amber mutants . The molecular weight of the purified protein was estimated as 75,000 to 85,000 depending on the composition of SDS-polyacrylamide gel used for the assay.

Microbios, 1988, 56(227), 97 - 104
Preliminary studies on some coliphages from Kuwait; Qureshi MA et al.; Seasonal data for raw sewage indicated a higher concentration of bacteriophages in the warmer months compared with the cooler months . Regardless of the amount of phages in the raw sewage and the time of year the efficiency of removal was as high as 83-96% for effluent II and 93-98% for sludge II . Four major types of plaque sizes were identified as Q1, Q2, Q3 and Q4 . Although all isolates contained DNA, they showed variation in host range and heat sensitivity from 50 degrees C to 80 degrees C.

Proteins, 1988, 4(1), 1 - 6
Photochemical crosslinking of bacteriophage T4 single-stranded DNA-binding protein (gp32) to oligo-p(dT)8: identification of phenylalanine-183 as the site of crosslinking; Shamoo Y et al.; Using ultraviolet light, both the 33,000-dalton single-stranded DNA-binding protein from T4 bacteriophage (gp32) as well as a 25,000-dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single-stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8 . After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion-exchange high-performance liquid chromatography . Gas-phase sequencing of this modified peptide gave the following sequence: Gln-Val-Ser-Gly-(X)-Ser-Asn-Tyr-Asp-Glu-Ser-Lys, which corresponds to residues 179-190 in gp32 . Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:p(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.

Intervirology, 1988, 29(3), 162 - 9
Characterization of potato yellow mosaic virus as a geminivirus with a bipartite genome; Roberts EJ et al.; A whitefly-transmitted virus from Venezuela, potato yellow mosaic virus, has been propagated, isolated and characterized as a member of the Geminivirus group . The virus was transmitted to several species of Nicotiana and to Petunia hybrida by mechanical inoculation, and to potato and tomato plants by grafting . Purified virus possessed typical geminate particle morphology and encapsidated both genomic and subgenomic species consisting of single-stranded DNA . The genome of the virus was cloned into both bacteriophage and plasmid vectors following restriction of supercoiled double-stranded DNA species isolated from infected plant extracts . Two distinct classes of cloned DNA were generated, both about 2,500-2,600 base pairs in length and designated A and B, which when inoculated in combination to Nicotiana benthamiana were infectious after excision from the recombinant clones . The subgenomic DNA was shown to be related to DNA B.

Rev Mal Respir, 1988, 5(6), 577 - 81
{Distribution of bacteriophage types of Mycobacterium tuberculosis in France}; Clavel-Seres S et al.; The bacteriophage typing of M . tuberculosis enables separation of the strains of the species according to their sensitivity to certain bacteriophages . A relationship has been observed between the geographical origin of patients and the distribution of the phage types of their strains . This study related to a sample of 450 strains isolated between 1978 and 1987 in patients of French origin coming from different regions in France . An analysis of the data provided by the study indicates that the distribution of the 5 phage types, A, AX, I, B, and C is relatively homogenous in the different regions with a predominance of the phage type A and B . The mean percentage of the lysotypes A, AX, I, B and C in the country as a whole are 43%, 15%, 7%, 30% and 5% respectively . Only in region 3 (Haute and Basse Normandie), 8 (Provence-Cote-d'Azur) and 9 (Rhone-Alpes) is there any perceptible deviation from this distribution . This study also underlines the contribution that can be provided by this type of information in the epidemiology of tuberculosis.

C R Acad Sci III, 1988, 307(6), 323 - 8
{Isolation and preliminary characterization of mutants of Escherichia coli K-12 overproducers of 2 exported proteins: beta-lactamase and alkaline phosphatase}; Magnouloux-Blanc B et al.; Spontaneous mutants of Escherichia coli characterized by the overproduction of two periplasmic proteins, beta-lactamase and alkaline phosphatase were isolated . Such olp (Overproduction of beta-Lactamase and alkaline Phosphatase) mutants were selected for growth in the presence of ampicillin and were identified on the basis of their increased content in alkaline phosphatase activity . Phenotypic analysis of olp mutants (resistance to bacteriophages and colicins) suggest that the organisation of their envelope has been deeply modified . Analysis of their cell envelope protein composition indicated that most mutants have a decreased content of porin proteins OmpF and OmpC . These mutations were mapped near the mtl locus, at minute 81 of the bacterial genetic map.

Acta Biochim Pol, 1988, 35(1), 51 - 5
Cloning of cysE gene of Escherichia coli with a mini-Mu-lac containing a plasmid replicon; Sirko AE et al.; The restriction map of cysE gene of Escherichia coli was established after cloning a mini-Mu-lac bacteriophage containing a plasmid replicon and recloning on pBR322 vector plasmid . Enzyme assays of transformants indicated the lack of autoregulation of cysE gene.

Radiat Environ Biophys, 1988, 27(4), 261 - 75
Lethal modifications of DNA via the transmutation of 32P and 33P incorporated in the genome of the S13 bacteriophage; Cols P et al.; When circular single-stranded DNA of phage S13 is labelled with 32P or 33P, the transmutations very efficiently bring about a loss of phage infectiousness (efficiency = 1 for 32P and 0.73 for 33P) . For both radionuclides, the lethal efficiencies as well as the lethal events are different . In the case of 32P, the lethal event is the loss of the circular integrity of the DNA molecule, occurring as a consequence of a systematic single strand-break caused by each 32P decay (100%) . Conversely, in the case of 33P, the lethal events are either a single strand-break (40%) or a local stereochemical modification (33%) . The same primary event, the substitution at each 33P decay of a phosphate by a sulfate molecule, leads to one of these lethal events in relation to the decay site . Moreover, neither the phage adsorption nor its genome injection into bacteria depends on the physical state of the genome, and thus lethality is revealed at only the genetic level.

Biotechnology, 1988, 10, 85 - 102
Chimeric single-stranded DNA phage-plasmid cloning vectors; Mead DA et al.; A variety of ssDNA phagemid cloning vectors have been constructed that combine the advantages of the filamentous coliphages with a number of plasmid and other bacteriophage-encoded functions . The practical and biological advantages of a chimeric phage-plasmid vector are considerable, and the trend toward converting existing plasmids into ssDNA phagemids and consolidating any number of useful features into one or a few vectors will undoubtedly accelerate . A new helper phage specifically designed for the isolation of large amounts of single-stranded plasmid DNA simplifies the use of these cloning vehicles . Additional refinements in phagemid-helper phage systems should extend the potential of these vectors even further.

Cancer Detect Prev, 1988, 12(1-6), 657 - 62
Mechanism of immune dysfunction associated with minor antigen graft-vs-host disease in mice; Hamilton BL et al.; A well-characterized murine model of graft-vs-host disease (GVHD) that develops in response to minor histocompatibility antigens was used to study the mechanism of an immunodeficiency syndrome that is associated with GVHD . Lethally irradiated mice were transplanted with a combination of bone marrow and spleen cells from H-2 compatible donors that differed at multiple minor histocompatibility antigens, or from syngeneic donors . Four to 12 weeks later, the humoral responses of transplanted and control mice to the T dependent antigens bacteriophage phiX174 and TNP-sheep red blood cells (TNP-SRBC), and to the T independent antigen TNP-Brucella abortus (TNP-BA) were determined . The results demonstrate that mice with GVHD have relatively intact B Cell function and a profound defect in T helper cell function . The immune response to T dependent antigens normalized with repeated immunization . We conclude that immune dysfunction in mice with GVHD is due to a reversible defect in T helper cell function.

J Clin Immunol, 1988 Jan, 8(1), 57 - 63
Abnormal antibody responses in patients with persistent generalized lymphadenopathy; Ochs HD et al.; Persistent, generalized lymphadenopathy (PGL) is a recognized component of human immunodeficiency virus (HIV) infection . We conducted longitudinal studies of B and T cell function in seven homosexual men with HIV infection and PGL . All seven had abnormal antibody-mediated immunity as studied by sequential assessment of in vivo antibody responses after immunization with the T-dependent neoantigens bacteriophage phi X 174 and keyhole limpet hemocyanin (KLH), the T-independent tetradecavalent pneumococcal polysaccharide vaccine, and the recall antigens diphtheria and tetanus toxoid . Compared to HIV-negative heterosexual controls, PGL patients responded with lower antibody titers and, following immunization with phage, failed to develop immunologic memory and to switch from IgM- to IgG-isotype antibody . In vitro antigen-induced antibody production was markedly diminished; and some patients showed depressed mitogen responses . There was a correlation between the degree of compromised immunity and the clinical condition; those with the most severe symptoms showed the most extensive immune deficiency . Yet despite obvious immunologic impairment five of the seven men have remained clinically stable over a 3-year follow-up period.

Gene, 1988, 62(2), 315 - 21
High-level production of hepatitis B viral X protein in Escherichia coli using gene II promoter of bacteriophage M13; Chen ML et al.; Region X is one of the four open reading frames (ORFs) of hepatitis B virus (HBV) and encodes a polypeptide of 154 amino acids (aa) . A 584-bp BamHI-BglII fragment of the HBV DNA containing the major part of ORF X which encodes 145 aa was inserted into the BglII site within the gene II of bacteriophage M13 . The insertion resulted in an in-phase gene II-X fused protein of 174 aa under the control of the gene II promoter . Cells harboring plasmids (pML alpha X.59 and pMLX.12d) derived from the above construct overproduced the 19-kDa fused protein in Escherichia coli at a level of 10%-20% of total cellular protein . The fused protein was recognized by the anti-X antibodies . This is the first demonstration of using gene II promoter of M13 to express a foreign gene efficiently.

Chem Biol Interact, 1988, 65(1), 85 - 95
The relationship between lipophilic-hydrophilic balance, uptake and anti-bacteriophage lambda activity of experimental anti-tumour bisquaternary salts; Robertson IG et al.; The uptake by Escherichia coli of a series of bisquaternary experimental anti-tumour agents (quinolinium 4-{p-9(-pyridylamino)phenylcarbamoyl}-aniline-bisalkyl dibromides) has been measured both by association of radiolabelled compounds and their inhibition of the vegetative replication of bacteriophage lambda (after heat inactivation of the phage repressor) as a measure of biologically effective intracellular drug concentration . Uptake of these compounds was correlated with biological effect, and was a function of both incubation temperature and the lipophilic-hydrophilic balance of the compound . At 30 degrees C uptake was drug concentration-dependent and was not readily reversible . No saturation of uptake was apparent over the concentration range tested . Preliminary experiments indicated that time-dependent drug uptake was also related to growth inhibition in cultured L1210 murine leukaemia cells . These results are consistent with the hypothesis that uptake occurs by diffusion across the plasma membrane followed by strong binding to cell constituents such as DNA . The approximate range of uptake of the most active compounds, using an external drug concentration of 1 microM, are 100 and 2400 molecules/s respectively for bacteria and murine leukemia cells . For bacteria, the uptake of approx . 2 X 10(5) molecules of drug/cell inhibits the yield of phage lambda by 90%.

Mol Gen Genet, 1988 Jan, 211(1), 72 - 7
The J gene of phi X174: isolation and characterization of a J gene mutant; Hamatake RK et al.; The J gene protein of bacteriophage phi X174 is a component of the mature phage . The previous lack of J gene mutants has prevented an in vivo analysis of J protein functions . A phi X174 mutant was constructed by inserting an 11 nucleotide sequence into the J gene . This mutant, designated insJ, was viable only in the presence of a wild-type J gene carried on a plasmid that could provide J protein . An analysis of DNA synthesis during insJ mutant infection under non-permissive conditions confirmed that the J protein is not required for viral DNA synthesis.

J Immunol, 1988 Jan 1, 140(1), 311 - 7
Isolation and characterization of a complementary DNA expressing human U1 small nuclear ribonucleoprotein C polypeptide; Yamamoto K et al.; A cloned complementary DNA, termed pS2, was isolated from a human fibroblast cDNA library in the bacteriophage expression vector lambda gt11 after screening with a patient's serum containing a high titer of anti-ribonucleoprotein (RNP) antibodies . A reasonable amount of cro-beta-galactosidase fusion protein (pS2EX) was obtained through subcloning of the pS2 insert into a plasmid expression vector pEX-2 . Antibody against pS2EX (anti-pS2EX) was purified from this patient's serum by Sepharose 4B conjugated with pS2EX . Immunofluorescent staining of HeLa cells with anti-pS2EX antibody exhibited a typical speckled pattern in the interphase nuclei . In the immunoblot analysis, the anti-pS2EX antibody recognized the 22 kDa protein . Using immunoprecipitation of cell lysate and subsequent RNA analysis, anti-pS2EX antibody was shown to precipitate U1 RNP only . The reactivities of various anti-RNP sera to pS2EX correlated well with the positive reaction to C polypeptide in the immunoblot . These findings indicate that pS2 is a cDNA for C polypeptide of U1 snRNP . In the Northern blot using human RNA and radiolabeled pS2, a single band about 800 base was observed . The nucleotide sequence of pS2 showed no significant homologies to known proteins.

Teratog Carcinog Mutagen, 1988, 8(5), 293 - 301
Mutagenicity of topoisomerase-active agents in bacteriophage T4; DeMarini DM et al.; Recently, the antitumor agent 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) was shown to revert a frameshift mutant of T4 (rFC11), and its mutagenicity was shown to be mediated by T4 DNA topoisomerase II {Ripley et al.: J Mol Biol 200: 665-680, 1988} . Here we report dose-response data on the mutagenicity and toxicity of m-AMSA in T4 rFC11 . We find that m-AMSA is among the most potent frameshift mutagens observed in T4, inducing a 10-fold increase in mutant frequency in the absence of toxicity and a 500-fold increase in mutant frequency at 31% survival . In addition to m-AMSA, the topoisomerase-active agents ellipticine, oxolinic acid, and nalidixic acid also reverted rFC11; however, they required concentrations 10-100 times greater than those required by m-AMSA in order to be mutagenic, and they did not produce mutant frequencies as high as those produced by m-AMSA . Unlike m-AMSA, all three agents were mutagenic only at toxic doses . The other agents evaluated--actinomycin D, adriamycin, 9-aminoellipticine, 9-methoxyellipticine, teniposide (VM-26), and novobiocin--were toxic but not mutagenic to T4 rFC11 . Thus, m-AMSA appears to be distinctly different from the other topoisomerase-active agents in exhibiting such potent mutagenic activity in T4 rFC11 . Because E . coli DNA gyrase may substitute for T4 topoisomerase II, we examined the ability of two inhibitors of E . coli DNA gyrase, novobiocin and nalidixic acid, to inhibit m-AMSA's mutagenicity . Both agents substantially reduced the mutagenicity of m-AMSA in T4 rFC11, further suggesting that topoisomerase mediates the mutagenicity of m-AMSA.

Immunogenetics, 1988, 28(1), 22 - 9
Class II genes of miniature swine . I . Class II gene characterization by RFLP and by isolation from a genomic library; Sachs DH et al.; Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library . For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes . One, or at most two, unique fragments were detected by hybridization with each of the human alpha probes tested . In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human beta probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency . Genomic DNA from the SLAc haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes . The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis . Thus, unique alpha genes were obtained which showed no evidence of cross-hybridization, while beta genes showed extensive cross-hybridization and were frequently detected in the library by more than one human beta gene probe . These data are consistent with early evolutionary divergence of alpha genes, prior to mammalian speciation, and with continuing evolution of beta genes, with possible shared usage of these genes by different alpha loci . The data also imply that alpha genes can readily be assigned to loci homologous to their human counterparts, but that beta genes will require further mapping and/or sequence analysis to confirm assignments.

Proc Natl Acad Sci U S A, 1988 Jan, 85(1), 222 - 6
An extended HLA-D region haplotype associated with celiac disease; Howell MD et al.; Celiac disease has one of the strongest associations with HLA (human leukocyte antigen) class II markers of the known HLA-linked diseases . This association is primarily with the class II serologic specificities HLA-DR3 and -DQw2 . We previously described a restriction fragment length polymorphism (RFLP) characterized by the presence of a 4.0-kilobase Rsa I fragment derived from an HLA class II beta-chain gene, which distinguishes the class II HLA haplotype of celiac disease patients from those of many serologically matched controls . We now report the isolation of this beta-chain gene from a bacteriophage genomic library constructed from the DNA of a celiac disease patient . Based on restriction mapping and differential hybridization with class II cDNA and oligonucleotide probes, this gene was identified as one encoding an HLA-DP beta chain . This celiac disease-associated HLA-DP beta-chain gene was flanked by HLA-DP alpha-chain genes and, therefore, was probably in its normal chromosomal location . The HLA-DP alpha-chain genes of celiac disease patients also were studied by RFLP analysis; 84% of HLA-DR3, -DQw2 patients had a 16-kb Xba I fragment that was present in only 36% of HLA-DR3, -DQw2 controls . Moreover, 79% of these patients had both alpha- and beta-chain polymorphisms in contrast to 27% of controls . Thus, celiac disease is associated with a subset of HLA-DR3, -DQw2 haplotypes characterized by HLA-DP alpha- and beta-chain gene RFLPs . Within the celiac-disease patient population, the joint segregation of these HLA-DP genes with those encoding the serologic specificities HLA-DR3 and -DQw2 indicates: (i) that the class II HLA haplotype associated with celiac disease is extended throughout the entire HLA-D region, and (ii) that celiac-disease susceptibility genes may reside as far centromeric on this haplotype as the HLA-DP subregion.

J Bacteriol, 1988 Jan, 170(1), 409 - 15
Sequential regulation of developmental events during polar morphogenesis in Caulobacter crescentus: assembly of pili on swarmer cells requires cell separation; Sommer JM et al.; Pili, along with the flagellum and DNA bacteriophage receptors, are structural markers for polar morphogenesis in Caulobacter crescentus . Pili act as primary receptors for a number of small, C . crescentus-specific DNA and RNA bacteriophages, and the timing of pilus-dependent adsorption of bacteriophage phiCb5 in synchronized cell populations has led to the general conclusion that pili are formed coordinately with the flagellum and other polar surface structures in the predivisional cell . The use of rotary platinum shadow casting and electron microscopy as a direct assay for formation of flagella and pili in synchronous cell cultures now shows, however, that when expressed as fractions of the swarmer cell cycle, flagella are assembled on the predivisional cells at approximately 0.8 and that pili are assembled on the new swarmer cells at approximately 0.1 of the next cell cycle . Adsorption of pilus-specific bacteriophage phiCb5 prevented the loss of pili from swarmer cells during development, which suggests that these structures are retracted at the time of stalk formation . Examination of temperature-sensitive cell division mutants showed that the assembly of pili depends on completion of cell separation . These results indicate that the stage-specific events required for polar morphogenesis in C . crescentus occur sequentially, rather than coordinately in the cell cycle, and that the timing of these events reflects the order of underlying cell cycle steps.

Eur Biophys J, 1988, 16(5), 279 - 86
OR3 operator of bacteriophage lambda in a 23 base-pair DNA fragment: sequence-specific 1H NMR assignments for the non-labile protons and comparison with the isolated 17 base-pair operator; Grutter R et al.; Sequence-specific 1H NMR assignments are presented for a non-selfcomplementary 23-base-pair DNA duplex of molecular weight 15,000 daltons, containing the OR3 repressor binding site of bacteriophage lambda as the central core . The NMR techniques used were mainly phase-sensitive two-dimensional NOE and 2Q spectroscopy, the latter to overcome overlap problems within the spectral region of the deoxyribose spin-systems . Direct sequential NOE connectivities are observed between adenine 2 H and deoxyribose 1' protons . We propose the use of these connectivities as a check of the assignments of C1' and A2 protons, which have independently been derived via other assignment pathways.

Folia Biol (Praha), 1988, 34(3), 129 - 46
Avian nephroblastomas induced by a retrovirus (MAV-2) lacking oncogene . I . Construction of MAV-1 and MAV-2 proviral restriction maps and preparation of specific proviral molecular subclones; Pecenka V et al.; A 9.8 kb DNA fragment containing the complete MAV-1 provirus was recloned from the recombinant bacteriophage lambda 311411 (Perbal et al., 1985) into the plasmid pAT153 . A detailed and precise restriction map of the obtained clone (pAT-MAV-1) was constructed . From compilation of this map and the known sequence of a variable portion of the MAV-2 env gene was a restriction map of MAV-2 deduced . Knowledge of the detailed pAT-MAV-1 map facilitated the preparation of five specific proviral subclones: pAT-U3 and pUC-U3 (both contain the U3 domain of the proviral LTR, which is MAV-specific and displays no homology with other hitherto known retroviruses including avian endogenous proviruses), pUC-RU5 (containing the R and U5 domains of the proviral LTR), pUC-UT5 (containing untranslated sequences flanking the 5' LTR), and pUC-UT3 (containing untranslated sequences flanking the 3' LTR) . Thus tools for analysis of integrated MAV-2 proviruses in nephroblastomas induced by this virus were formed.

Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 44 - 54
{Identification in vivo of promoter activity in the left site of the att region of bacteriophage lambda DNA}; Gileva IP et al.; The promoter-probing vector (pSK plasmid) was explored for cloning of the fragments from lambda cI857 and lambda b2 DNAs containing different regions of the att site . We have constructed all-tet fusions where the fusions are: 1) HindIII/BamHI-491 base pairs (b . p.) fragment of lambda cI857 DNA containing POP' site (plasmid pSK-PP'); 2) AluI-242 b . p . fragment of lambda cI857 DNA containing the left arm of the POP' site (plasmid pSK-P); 3) AluI-242 b . p . fragment of lambda cI857 DNA with opposite orientation (plasmid pSK-P); 4) EcoRI/BamHI-750 b . p . fragment of lambda b2 DNA containing the right arm of the POP' site (plasmid pSK-P') . These fusions permit us to analyse the effect of various pieces of the attachment site on the expression tet gene as the result of reparation of this gene promoter . We find that expression of tet (tetracycline resistant phenotype) takes place in the pSK-PP' and pSK-P but not in the pSK-P' and pSK-P . These facts permit us to conclude that the left arm of the att site contains a rightward promoter functioning in vivo . We postulate that this promoter activity might correspond to the promoter patt, which was described in previous experiments in vitro.

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