Escherichia coli

J Bacteriol, 1991 Sep, 173(17), 5539 - 45
An essential iteron-binding protein required for plasmid R1162 replication induces localized melting within the origin at a specific site in AT-rich DNA; Kim YJ et al.; The R1162-encoded protein RepIB is essential for replication of the plasmid and binds specifically to iterons within the replicative origin . The protein causes the localized melting of DNA (determined by sensitivity to P1 nuclease) at a site within the AT-rich region of the origin, about 60 bp from the iteron binding sites and separated from them by a GC-rich tract . Point mutations have been isolated in the AT-rich DNA . These mutations interfere with origin activity and also prevent the protein-induced sensitivity to P1 . A second-site suppressor of one of these mutations maps in the repIb gene and restores both origin function and sensitivity to P1 . The results suggest a specific interaction between RepIB and origin DNA at a position distant from its primary binding site.

J Bacteriol, 1991 Sep, 173(17), 5523 - 31
A gene for a new lipoprotein in the dapA-purC interval of the Escherichia coli chromosome; Bouvier J et al.; Cloning and sequence analysis of the region located downstream of the dapA gene of Escherichia coli has revealed the presence of an open reading frame that is cotranscribed with dapA . This gene codes for a 344-amino-acid polypeptide with a potential signal sequence characteristic of a lipoprotein . When this gene, called nlpB, is expressed from a multicopy plasmid in bacteria grown in the presence of {3H}palmitate, a labeled 37-kDa protein is produced . A slightly larger precursor molecule is detected when minicells expressing nlpB are treated with globomycin, a specific inhibitor of lipoprotein signal peptidase . Therefore, the nlpB gene encodes a new lipoprotein, designated NlpB . This lipoprotein is detected in outer membrane vesicles prepared from osmotically lysed spheroplasts and appears to be nonessential, since a strain in which the nlpB gene is disrupted by insertion of a chloramphenicol resistance gene is still able to grow and shows no discernible NlpB phenotype . The putative transcription termination signals of the dapA-nlpB operon overlap the promoter of the adjacent purC gene.

J Bacteriol, 1991 Sep, 173(17), 5516 - 22
Purification and characterization of Escherichia coli heat-stable enterotoxin II; Fujii Y et al.; Escherichia coli heat-stable enterotoxin II (STII) was purified to homogeneity by successive column chromatographies from the culture supernatant of a strain harboring the plasmid encoding the STII gene . The purified STII evoked a secretory response in the suckling mouse assay and ligated rat intestinal loop assay in the presence of protease inhibitor, but the response was not observed in the absence of the inhibitor . Analyses of the peptide by the Edman degradation method and fast atom bombardment mass spectrometry revealed that purified STII is composed of 48 amino acid residues and that its amino acid sequence was identical to the 48 carboxy-terminal amino acids of STII predicted from the DNA sequence (C . H . Lee, S . L . Mosely, H . W . Moon, S . C . Whipp, C . L . Gyles, and M . So, Infect . Immun . 42:264-268, 1983) . STII has four cysteine residues which form two intramolecular disulfide bonds . Two disulfide bonds were determined to be formed between Cys-10-Cys-48 and Cys-21-Cys-36 by analyzing tryptic hydrolysates of STII.

J Bacteriol, 1991 Sep, 173(17), 5396 - 402
Buoyant density studies of several mecillinam-resistant and division mutants of Escherichia coli; Bylund JE et al.; The buoyant density of wild-type Escherichia coli cells has previously been reported not to vary with growth rate and cell size or age . In the present report we confirm these findings, using Percoll gradients, and analyze the recently described lov mutant, which was selected for its resistance to mecillinam and has been suggested to be affected in the coordination between mass growth and envelope synthesis . The average buoyant density of lov mutant cells was significantly lower than that of wild-type cells . Similarly, the buoyant density of wild-type cells decreased in the presence of mecillinam . The density of the lov mutant, like that of the wild type, was invariant over a 2.8-fold range in growth rate . In this range, however, the average cell volume was also constant . Analysis of buoyant density as a function of cell volume in individual cultures revealed that smaller (newborn) lov mutant cells had higher density than larger (old) cells; however, the density of the small cells never approached that of the wild-type cells, whose density was independent of cell size (age) . A pattern similar to that of lov mutant cells was observed in cells carrying the mecillinam-resistant mutations pbpA(Ts) and rodA(Ts) and the division mutation ftsI(Ts) at nonpermissive temperatures as well as in wild-type cells treated with mecillinam, but not in mecillinam-resistant crp or cya mutants.

Genes Dev, 1991 Sep, 5(9), 1635 - 45
Alignment of recombination sites in Hin-mediated site-specific DNA recombination; Moskowitz IP et al.; The Hin site-specific recombination system normally promotes inversion of DNA between two recombination sites in inverted orientation . We show that the rate of deletion of DNA between two directly repeated recombination sites is 10-300 times slower than inversion between sites in their native configuration as measured in vivo and in vitro, respectively . In vitro studies have shown that the deletion reaction has the same requirement for Fis, a recombinational enhancer, and DNA supercoiling as the inversion reaction . These requirements, together with the finding that the deletion products are interlinked once suggest that the deletion synaptic complex is similar to the invertasome intermediate that generates inversion . The inefficiency of the deletion reaction is not a function of a reduced ability to recognize or synapse recombination sites in direct orientation . Not only do these substrates support an efficient knotting reaction, but directly repeated recombination sites with symmetric core sequences also invert efficiently . These findings demonstrate that the recombination sites are preferentially assembled into the invertasome structure with the sites aligned in the configuration for inversion regardless of their starting orientation . We propose that the dynamics of a supercoiled DNA molecule biases the geometric assembly of specific intermediates . In the case of Hin-mediated recombination, inversion is overwhelmingly preferred over deletion because DNA supercoiling favors a specific alignment of DNA strands in the synaptic complex.

Crit Care Med, 1991 Sep, 19(9), 1183 - 7
Liver glutathione and cytochrome P450 activity in experimental infection: study of the relative effects of infectious stress and malnutrition; Lescut D et al.; OBJECTIVE: To study the effects of infection and malnutrition on liver glutathione and cytochrome P450 (P450) in rats . DESIGN: Controlled experimental groups (12 groups) . ANIMALS: Adult male Sprague-Dawley rats . INTERVENTIONS: Experimental endocarditis, pyelonephritis, or peritonitis were caused . Controls included free-fed rats and sham-operated rats, pair-fed to infected animals . Infection was verified by tissue culture . Rats were killed 3 days (acute infection) or 10 days (chronic infection, except endocarditis) after the induction of infection . RESULTS: Sham rats had lower liver weights, liver/body weight, and liver glutathione values than controls . Infected rats had larger liver weights and liver/body weight ratios and liver glutathione content than shams, and larger liver/body weight ratios than controls (acute infection) . Infected rats had lower P450 values than both shams and controls . CONCLUSION: The malnutrition associated with infection caused decreased liver weight and glutathione content . Infection increased the liver weight, and liver glutathione content, but caused severe reduction in liver P450 . If the same finding is true in infected patients, it could have consequences for the management of such patients.

Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7719 - 23
I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target; Bell-Pedersen D et al.; Mobility of the phage T4 td intron depends on activity of an intron-encoded endonuclease (I-TevI), which cleaves a homologous intronless (delta In) target gene . The double-strand break initiates a recombination event that leads to intron transfer . We found previously that I-TevI cleaves td delta In target DNA 23-26 nucleotides upstream of the intron insertion site . DNase I-footprinting experiments and gel-shift assays indicate that I-TevI makes primary contacts around the intron insertion site . A synthetic DNA duplex spanning the insertion site but lacking the cleavage site was shown to bind I-TevI specifically, and when cloned, to direct cleavage into vector sequences . The behavior of the cloned duplex and that of deletion and insertion mutants support a primary role for sequences surrounding the insertion site in directing I-TevI binding, conferring cleavage ability, and determining cleavage polarity . On the other hand, sequences around the cleavage site were shown to influence cleavage efficiency and cut-site selection . The role of cleavage-site sequences in determining cleavage distance argues against a strict "ruler" mechanism for cleavage by I-TevI . The complex nature of the homing site recognized by this unusual type of endonuclease is considered in the context of intron spread.

Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7620 - 4
Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis; Medina JF et al.; Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by site-directed mutagenesis on cDNA . The codons corresponding to His-295, His-299, or Glu-318 were replaced by codons encoding tyrosine, tyrosine, and glutamine, respectively . The mutated cDNAs were expressed in Escherichia coli, and the three mutated proteins were purified to apparent homogeneity . None of these mutants contained significant amounts of zinc, as determined by atomic absorption spectrometry, and all of them were practically devoid of both LTA4 hydrolase and peptidase enzyme activities . Nevertheless, the mutated proteins could be positively identified by their immunoreactivities with an antiserum for human LTA4 hydrolase in immunoblot analysis . Site-directed mutagenesis was also carried out on human LTA4 hydrolase cDNA . Codons encoding His-295, His-299, and Glu-318 were replaced by ones encoding tyrosine, leucine, and alanine, respectively, and the three mutants were expressed in E . coli . The LTA4 hydrolase activities of the total soluble proteins produced in these expressions were less than 10% of that obtained for bacteria harboring nonmutated cDNA . In agreement with earlier predictions, our experimental data demonstrate that His-295, His-299, and Glu-318 constitute the three ligands of the intrinsic zinc atom in LTA4 hydrolase . Additionally, the combined loss of enzyme activities and zinc content in the purified mutated mouse proteins, emphasizes the critical role of the zinc atom for catalysis, whereas the virtually identical chromatographic behaviors of the mutated and nonmutated mouse LTA4 hydrolase proteins suggest that the metal is of limited importance for the maintenance of the enzyme tertiary structure.

Infect Immun, 1991 Sep, 59(9), 3327 - 9
Successive synovial Mycoplasma hominis isolates exhibit apparent antigenic variation; Olson LD et al.; The expression of surface proteins by 14 successive Mycoplasma hominis isolates obtained from the synovial fluid of a chronically infected septic arthritis patient was examined . Marked differences in the expression of surface proteins, as determined by monoclonal antibodies raised against the first isolate, were observed . However, identical restriction patterns and virtually identical hybridization patterns with probes containing the conserved genes of the Mycoplasma capricolum rRNA operon and the Escherichia coli elongation factor Tu suggest that the protein differences might reflect antigenic variation by M . hominis during infection.

Infect Immun, 1991 Sep, 59(9), 3163 - 8
The T-cell-stimulating 17-kilodalton protein of Francisella tularensis LVS is a lipoprotein; Sjostedt A et al.; A T-cell-stimulating, membrane-located 17-kDa protein of the live vaccine strain Francisella tularensis LVS has previously been cloned and sequenced . In the present study, it is shown to be a lipoprotein . When F . tularensis was grown in the presence of {3H}palmitate, several proteins of the organism, including a 17-kDa protein, were radiolabeled . The labeled 17-kDa protein was found by Western blot (immunoblot) analysis to be identical to the cloned protein . It was located in the detergent phase after partitioning with the nonionic detergent Triton X-114, thereby behaving like a hydrophobic integral membrane protein . The protein was predominantly hydrophilic and contained no putative transmembrane domain . The presence of fatty acids is therefore the probable explanation of the membrane location of the 17-kDa protein . The amino acid sequence of the 17-kDa protein contains the tetrapeptide Leu-Ala-Ser-Cys, which is a recognition sequence of the lipoprotein signal peptidase . Globomycin, a specific inhibitor of the peptidase, inhibited maturation of the 17-kDa lipoprotein . The protein incorporated {3H}palmitate also when expressed by Escherichia coli . The 17-kDa lipoprotein was recognized not only by T cells but also by serum antibodies of F . tularensis-primed individuals.

Infect Immun, 1991 Sep, 59(9), 3111 - 8
Susceptibility of inbred mouse strains to infection with Serpula (Treponema) hyodysenteriae; Nibbelink SK et al.; Several inbred strains of mice were inoculated with Serpula (Treponema) hyodysenteriae B204 to determine susceptibility to infection . Challenge doses of 10(7) or 10(8) spirochetes induced cecal lesions in C3H/HeJ mice and other C3H strains of mice . However, more than a 100-fold difference existed between the dose required to induce lesions in 50% of the infected C3H/HeJ mice (8.3 x 10(7)) and that required to induce them in 50% of the infected C3H/HeN mice (5 x 10(5)) . C3H/HeJ mice lack a splenocyte mitogenic response to Escherichia coli lipopolysaccharide but exhibited a mitogenic response comparable to those of other C3H strains of mice when stimulated with S . hyodysenteriae endotoxin (butanol-water extract) . Different inbred strains exhibited different susceptibilities to infection, with the strain C3H/HeN being the most susceptible on the basis of colonization and development of macroscopic cecal lesions . The ity gene had no apparent effect on susceptibility of mice challenged with S . hyodysenteriae . The involvement of the H-2 haplotype with susceptibility is unclear, but the mice bearing H-2k were more susceptible than mice with the H-2b, H-2d, or H-2q haplotype . These data support the hypothesis that the host's responsiveness to lipopolysaccharide influences the susceptibility to infection with S . hyodysenteriae . However, differences in susceptibility between inbred mice exist independent of the lps locus, suggesting that there are other inherent differences between mouse strains that affect susceptibility to infection by S . hyodysenteriae.

Infect Immun, 1991 Sep, 59(9), 2885 - 91
Actinobacillus pleuropneumoniae-induced thymic lesions in mice and pigs; Stine DL et al.; Actinobacillus pleuropneumoniae produces several hemolysins/cytotoxins that may be important in the pathogenesis of acute lesions . Little is known, however, about the role of these virulence factors in chronic disease or the carrier state . We investigated the effects of live bacterial infection and transthoracic injection of a sterile culture supernatant on primary lymphoid organs and lymphocyte populations . Transthoracic inoculation of mice or intranasal inoculation of pigs with virulent A . pleuropneumoniae serotypes 1 and 7 induced thymic cortical lymphoid necrosis . These lesions were reproduced in mice by transthoracic injection of a concentrated sterile culture supernatant . The cytotoxic effect of this culture supernatant was also demonstrated in vitro by using a tetrazolium dye reduction assay . Both porcine and murine thymic lymphocytes as well as splenic T lymphocytes were susceptible to the toxin . Porcine convalescent serum, but not preimmune serum, prevented thymic lesions and neutralized the in vitro cytotoxic effect of the culture supernatant on murine thymic lymphocytes . Thymic lesions also were reproduced in mice by using purified lipopolysaccharide (LPS) from Escherichia coli O111:B4; however, LPS had no in vitro cytotoxic effect on either porcine or murine thymic lymphocytes . These results suggest that secreted A . pleuropneumoniae toxin(s) is capable of affecting host T-lymphocyte populations and may affect host immune function.

Dev Biol, 1991 Sep, 147(1), 32 - 45
Temporal and spatial expression of the yellow gene in correlation with cuticle formation and dopa decarboxylase activity in Drosophila development; Walter MF et al.; The yellow (y) gene of Drosophila is required for the formation of black melanin and its deposition in the cuticle . We have studied by immunohistochemical methods the temporal and spatial distribution of the protein product of the y gene during embryonic and pupal development and have correlated its expression with events of cuticle synthesis by the epidermal cells and with cuticle sclerotization . Except for expression in early embryos, the y protein is only found in the epidermal cells and may be secreted into the cuticle as it is being deposited . The amount of y protein in various regions of the embryo and pupa correlates directly with the intensity of melanization over any section of the epidermis . Expression of the y gene begins in the epidermal cells at 48 hr after pupariation and is well correlated with the beginning deposition of the adult cuticle . At this stage the adult cuticle is unsclerotized and unpigmented and dopa decarboxylase levels, a key enzyme in catecholamine metabolism which provides the crosslinking agents as well as the precursors for melanin, is low . As a separate event 26 hr after the onset of y gene expression, the first melanin deposition occurs in the head bristles and pigmentation continues in an anterior to posterior progression until eclosion . This melanization wave is correlated with elevated dopa decarboxylase activity . Crosslinking of the adult cuticle also occurs in a similar anterior to posterior progression at about the same time . We have shown by imaginal disc transplantation that timing of cuticle sclerotization depends on the position of the tissue along the anterior-posterior axis and that it is not an inherent feature of the discs themselves . We suggest that actual melanization and sclerotization of the cuticle by crosslinking are initiated at this time in pupal development by the availability of the catecholamine substrates which diffuse into the cuticle . Intensity of melanization and position of melanin pigment is determined by the presence or absence of the y protein in the cuticle, thus converting the y protein prepattern into the melanization pattern.

Mol Cell Biol, 1991 Sep, 11(9), 4796 - 803
Multiple CArG boxes in the human cardiac actin gene promoter required for expression in embryonic cardiac muscle cells developing in vitro from embryonal carcinoma cells; Pari G et al.; Chimeric genes composed of the human cardiac actin promoter driving the Escherichia coli lacZ reporter gene were constructed, transfected, and stably integrated into genomes of P19 embryonal carcinoma cells . The transfected constructs were expressed actively in cardiac myocytes formed following dimethyl sulfoxide (DMSO)-induced cell differentiation but poorly in undifferentiated cultures and in cultures treated with retinoic acid to develop into derivatives of the neuroectoderm . A number of deletions of the promoter were constructed and tested . Three regions required for efficient expression in P19-derived cardiac muscle were identified, each containing sequences referred to as CArG boxes (CC{AT-rich}6GG) . This analysis indicated that regulatory sequences important for expression in cardiac muscle were present upstream of the core promoter identified previously by transient assays in skeletal myoblasts . Expression of the cardiac actin promoter was enhanced 10-fold in undifferentiated P19 cells in the presence of the myoD protein . The promoter regions important for expression in P19-derived cardiocytes were similar to those important for myoD-induced enhancement, a result we interpret to be consistent with the idea that cardiac muscle might contain a myoD-like activity.

J Exp Med, 1991 Sep 1, 174(3), 657 - 72
Ultrastructural localization of albumin transport across the cerebral microvasculature during experimental meningitis in the rat; Quagliarello VJ et al.; Injury to the blood brain barrier (BBB) is a fundamental sequela of bacterial meningitis, yet the precise mechanism facilitating exudation of albumin across the endothelium of the cerebral microvasculature remains conjectural . After intracisternal inoculation of Escherichia coli (0111:B4) lipopolysaccharide in rats to elicit a reversible meningitis and BBB injury, we utilized in situ tracer perfusion and immunolabeling procedures to identify by transmission electron microscopy the precise topography and microvascular exit pathway(s) of bovine serum albumin (BSA) . Results revealed that during meningitis there was: (a) an inducible increase in immunodetectable monomeric BSA binding to the luminal membrane of all microvascular segments in the pia-arachnoid and superficial brain cortex; (b) similar uptake of both colloidal Au-BSA (as well as monomeric BSA) by plasmalemmal vesicles but no detectable transcytosis to the abluminal side; and (c) predominant exit of both perfused Au-BSA and immunodetectable monomeric BSA through open intercellular junctions of venules in the pia-arachnoid . This was corroborated in separate experiments documenting focal pial venular leaks of in situ perfused 0.01% colloidal carbon black during experimental meningitis . These results provide precise localization of BBB injury in meningitis to meningeal venules, confirm a paracellular exit pathway of albumin via open intercellular junctions, and suggest an injury mechanism amenable to specific therapeutic intervention.

J Virol, 1991 Sep, 65(9), 4966 - 72
Purification and characterization of human papillomavirus type 16 E7 protein with preferential binding capacity to the underphosphorylated form of retinoblastoma gene product; Imai Y et al.; Human papillomavirus type 16 E7 is considered to be a major viral oncoprotein playing an important role(s) in cervical cancers . E7 protein was shown to bind to the protein product of the retinoblastoma gene (RB), while simian virus 40 large T and adenovirus E1A were also shown to possess binding activity to RB protein . The RB protein is a cell cycle regulator that is highly phosphorylated specifically in S, G2, and M, whereas it is underphosphorylated in G0 and G1 . Recently, large T was demonstrated to bind preferentially to the underphosphorylated RB protein, which is considered to be an active form restricting cell proliferation . However, it is not known whether E7 can bind to phosphorylated RB protein . We successfully purified large quantities of unfused human papillomavirus type 16 E7 protein expressed in Escherichia coli by using a T7 promoter-T7 RNA polymerase system . The purified E7 protein was demonstrated to bind preferentially to the underphosphorylated RB protein.

J Virol, 1991 Sep, 65(9), 4665 - 9
Genetic analysis of the DNA recognition sequence of the P2 Cox protein; Cores de Vries G et al.; The Cox protein of temperate Escherichia coli phage P2 is involved in three important biological processes: (i) excision of the integrated prophage genome (G . Lindahl and M . Sunshine, Virology 49:180-187, 1972), (ii) transcriptional repression of the P2 Pc promoter, which controls the expression of the immunity repressor C and the integrase (S . Saha, E . Haggard-Ljungquist, and K . Nordstrom, EMBO J . 6:3191-3199, 1987), and (iii) transcriptional activation of the late PII promoter of the unrelated satellite phage P4 (S . Saha, E . Haggard-Ljungquist, and K . Nordstrom, Proc . Natl . Acad . Sci . USA 86:3973-3977, 1989) . A comparison of the DNA regions protected by Cox from DNaseI degradation has revealed a presumptive Cox recognition sequence (Saha et al., Proc . Natl . Acad . Sci . USA) . The binding region of Cox in the P2 Pc promoter contains three presumptive recognition sequences, "Cox boxes," located in tandem . P2 vir3 and P2 vir24 are virulent deletion mutants unable to plate on Cox-producing strains, most likely because the deletions locate the new early promoters too close to the Cox-binding region (Saha et al., EMBO J.) . In this report, spontaneous P2 vir3 and vir24 mutants, no longer sensitive to repression by the Cox protein, have been isolated . These mutants plate with equal efficiency on strains with or without a Cox-producing plasmid, and they have been named cor for cox resistance . Three types are recognized; the four P2 vir3 cor mutants have a 1-base deletion in the first Cox box, while the P2 vir24 cor mutants were of two types; four have a base substitution in the first Cox box, and one has a base substitution in the second Cox box . The effect of the Cox protein on the mutated P2 vir3 and vir24 promoters was analyzed in vivo by using fusions to a promoterless cat (chloramphenicol acetyltransferase) gene . The activities of the P2 vir3 and vir24 early promoters, as opposed to the wild-type early Pe promoter, are drastically reduced by the Cox protein, and the cor mutation renders them as resistant to Cox as the wild-type Pe promoter . Thus, at least the first two Cox boxes are essential for binding of the Cox protein.

J Virol, 1991 Sep, 65(9), 4636 - 44
Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage; Vink C et al.; Retroviral integration requires cis-acting sequences at the termini of linear double-stranded viral DNA and a product of the retroviral pol gene, the integrase protein (IN) . IN is required and sufficient for generation of recessed 3' termini of the viral DNA (the first step in proviral integration) and for integration of the recessed DNA species in vitro . Human immunodeficiency virus type 1 (HIV-1) IN, expressed in Escherichia coli, was purified to near homogeneity . The substrate sequence requirements for specific cleavage and integration of retroviral DNA were studied in a physical assay, using purified IN and short duplex oligonucleotides that correspond to the termini of HIV DNA . A few point mutations around the IN cleavage site substantially reduced cleavage; most other mutations did not have a drastic effect, suggesting that the sequence requirements are limited . The terminal 15 bp of the retroviral DNA were demonstrated to be sufficient for recognition by IN . Efficient specific cutting of the retroviral DNA by IN required that the cleavage site, the phosphodiester bond at the 3' side of a conserved CA-3' dinucleotide, be located two nucleotides away from the end of the viral DNA; however, low-efficiency cutting was observed when the cleavage site was located one, three, four, or five nucleotides away from the terminus of the double-stranded viral DNA . Increased cleavage by IN was detected when the nucleotides 3' of the CA-3' dinucleotide were present as single-stranded DNA . IN was found to have a strong preference for promoting integration into double-stranded rather than single-stranded DNA.

J Virol, 1991 Sep, 65(9), 4598 - 608
Sequence analysis, expression, and deletion of a vaccinia virus gene encoding a homolog of profilin, a eukaryotic actin-binding protein; Blasco R et al.; A 4,500-bp BamHI fragment, located within the HindIII A segment of the vaccinia virus genome, was found to contain eight potential coding regions for polypeptides of 78 to 346 amino acids . The open reading frames with 133, 346, and 125 codons were homologous to profilin (an actin-binding protein), 3-beta-hydroxysteroid dehydrogenase, and Cu-Zn superoxide dismutase, respectively . Sequence alignments indicated that the vaccinia virus and mammalian profilins were more closely related to each other than to known profilins of other eukaryotes . The expression and possible role of the profilin homolog in the virus replicative cycle were therefore investigated . Antibody raised to Escherichia coli expressed vaccinia virus profilin was used to demonstrate the synthesis of the 15-kDa polypeptide at late times after vaccinia virus infection of mammalian cells . The protein accumulated in the cytoplasm, but only trace amounts remained associated with highly purified virions . The isolation of vaccinia virus mutants (in strains WR and IHD-J), with nearly the entire profilin gene replaced by the E . coli gpt gene, indicated that the protein is not essential for infectivity . The characteristic vaccinia virus-induced changes in actin fibers, seen by fluorescence microscopy, occurred in cells infected with the mutant . Moreover, the virus-encoded profilin homolog was not required for actin-associated events, including intracellular virus movement to the periphery of the cell, formation of specialized microvilli, or release of mature virions, as shown by electron microscopy and yields of infectious intra- and extracellular virus.

EMBO J, 1991 Sep, 10(9), 2699 - 705
The replication termination signal terB of the Escherichia coli chromosome is a deletion hot spot; Bierne H et al.; Hybrids composed of phage M13, plasmid pBR322 and the termination signal of Escherichia coli chromosome replication terB were used to show that arrest of DNA synthesis creates a very efficient deletion hot spot . Up to 80% of deletions occurring in these hybrids had one deletion end-point at terB provided that (i) terB was oriented to arrest M13 and pBR322 leading strand synthesis; and (ii) the host cells contained the Tus protein necessary for arresting DNA synthesis at terB . The position of terB and the flanking sequences had little effect on deletion hot spot activity . About 90% of the deletions at terB ended 5-6 nucleotides in front of the major replication arrest site . We propose two models to account for deletion formation and speculate that many genome rearrangements may be due to the pausing of DNA replication.

EMBO J, 1991 Sep, 10(9), 2589 - 94
Flexibility of the DNA enhances promoter affinity of Escherichia coli RNA polymerase; Werel W et al.; Two types of mechanisms are discussed for the formation of active protein-DNA complexes: contacts with specific bases and interaction via specific DNA structures within the cognate DNA . We have studied the effect of a single nucleoside deletion on the interaction of Escherichia coli RNA polymerase with a strong promoter . This study reveals three patterns of interaction which can be attributed to different sites of the promoter, (i) direct base contact with the template strand in the '-35 region' (the 'recognition domain'), (ii) a DNA structure dependent interaction in the '-10 region' (the 'melting domain'), and (iii) an interaction which is based on a defined spatial relationship between the two domains of a promoter, namely the 'recognition domain' and the 'melting domain'.

EMBO J, 1991 Sep, 10(9), 2533 - 41
Genomic targets of the serendipity beta and delta zinc finger proteins and their respective DNA recognition sites; Payre F et al.; The closely related Drosophila serendipity (sry) beta (beta) and delta (delta) Cys2-His2 zinc finger proteins show partly overlapping in vitro DNA binding specificities and distinct patterns of binding sites on polytene chromosomes . Using a newly developed procedure, we identified genomic DNA targets for these two proteins . Both the sry beta and delta proteins protect an 18-22 base region from DNase I digestion within each analysed genomic binding site, that includes a 13 bp consensus sequence . The consensus recognition sites sry beta 5'-YCAGAGATGCGCA-3' and sry delta 5'-YTAGAGATGGRAA-3' thus differ by nucleotides at four out of 13 positions . They are determinant for specific binding of the sry beta and delta proteins, respectively, produced in Escherichia coli or present in Drosophila embryos . We further show that sry beta is the major (if not exclusive) Drosophila nuclear protein that specifically binds the 5'-CAGAGTGCGC-3' sequence . The identified sry beta genomic targets are all contained within single-copy DNA in euchromatic regions of the genome . Two out of the five characterized in detail map at cytological positions coincident with binding sites of the native sry beta protein on polytene chromosomes.

J Neurochem, 1991 Sep, 57(3), 1003 - 12
Recombinant human and rat ciliary neurotrophic factors; Masiakowski P et al.; The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA . The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence . The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons . An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E . coli . The rat CNTF gene was also introduced into similar vectors . Both the human and rat proteins were expressed at exceptionally high levels, at 20-40% and 60-70% of total protein, respectively . Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture . The biological activity of both recombinant proteins was comparable to that of native rat CNTF.

Gastroenterology, 1991 Sep, 101(3), 726 - 33
Antitumor effect of synthetic derivatives of lipid A in an experimental model of colon cancer in the rat; Jeannin JF et al.; Colon carcinoma is one of the most frequent causes of cancer death in industrialized countries . The patients generally die of the metastases . In a colon cancer rat model, the authors have shown that lipopolysaccharides from Escherichia coli induced the regression of carcinomatosis and cured 20%-30% of the rats . Some synthetic derivatives of lipid A, which are less toxic than lipopolysaccharides, were injected 14 days after the tumor cells . They induced the complete regression of peritoneal carcinomatosis consisting of numerous nodules measuring 1-5 mm in 20%-30% of rats . Only compounds with three or more hydroxymyristic acid residues were effective . In vivo effects were correlated with the capacity to induce the production of interleukin 1 and tumor necrosis factor but not with the capacity to induce macrophage-mediated cytolysis . It is therefore possible to synthesize weakly toxic derivatives of lipopolysaccharides retaining their antitumoral property in vivo.

Mol Mar Biol Biotechnol, 1991 Sep, 1(1), 73 - 7
Disulfide bonds in native and recombinant fish growth hormones; Vestling M et al.; Disulfide linkages were characterized for the first time in a fish growth hormone . Trypsin digestion of chum salmon growth hormone, followed by mass spectrometry established that Cys-49 is linked to Cys-161, while Cys-178 is linked to Cys-186 . This is analogous to the big loop, little loop pattern found in human growth hormone . Ninety-three percent of the primary structure of a recombinant rainbow trout growth hormone whose cDNA codes for the same amino acid sequence as chum salmon growth hormone was confirmed by mass spectrometric peptide mapping.

Plant J, 1991 Sep, 1(2), 167 - 74
Molecular characterization of an Arabidopsis thaliana cDNA encoding a small GTP-binding protein, Rha1; Anuntalabhochai S et al.; We have isolated a cDNA encoding a small GTP-binding protein from an Arabidopsis thaliana cDNA library using an oligonucleotide probe derived from the most conserved domain of the ras superfamily . The cDNA encodes a 21.8 kDa protein, designated Rha1, which shows high homology to members of the ras superfamily in the regions involved in GTP binding, GTPase activity, and membrane attachment . The amino acid sequence is 60% identical to the sequence of the mammalian Rab5 protein, a small GTP-binding protein which is believed to be involved in endocytosis . Several regions, including the putative effector domain are completely conserved . This high percentage of amino acid identity suggests that the Rha1 protein is the functional plant counterpart of the Rab5 protein . When expressed in E . coli, the Rha1 protein was shown to bind GTP . The rha1 gene is most highly expressed in root and callus tissue, weakly expressed in stems and inflorescences and virtually not expressed in leaves and seed pods . Genomic Southern analysis revealed that rha 1 is part of a small multigene family.

Mol Microbiol, 1991 Sep, 5(9), 2285 - 92
Sequencing, mutational analysis, and transcriptional regulation of the Escherichia coli htrB gene; Karow M et al.; The Escherichia coli htrB gene was originally discovered because its insertional inactivation led to an exquisitely temperature-sensitive phenotype in rich media, i.e . the ability to form colonies at temperatures below 32 degrees C, but not above 33 degrees C . The htrB gene has been sequenced . It can potentially code for two proteins, with Mr values of 35,407 Da and 8669 Da, that are encoded by overlapping, divergent open reading frames . Our data are consistent with the 35,407 Da protein being HtrB . Northern blot analysis clearly shows that the monocistronic htrB message is not under heat-shock regulation . We have also sequenced the flanking DNA and have discovered a new gene, designated orf39.9, located immediately adjacent to htrB, but divergently transcribed.

Agents Actions, 1991 Sep, 34(1-2), 93 - 6
Phospholipase A2 acyl-hydrolytic activity in rat RPAR-induced pleurisy; Berkenkopf JW et al.; Endogenous phospholipase A2 (PLA2) specific activity (SA) (% hydrolysis/min/mg protein) in the rat pleural cavity, measured as the acyl-hydrolysis of {3H}-arachidonic acid E . coli substrate, was quantitated after induction of a reverse passive Arthus reaction (RPAR) . PLA2 SA, derived when the rate of hydrolysis was linear (1-5 min), was 1.9, 1.4, 3.8 and 4.1% h/min/mg at 10 min, 2 h, 4 h and 24 h, respectively, after induction of the RPAR . This time course appeared to correlate with the influx of mononuclear inflammatory cells, although the effect of plasma leakage on changes in exudate PLA2 SA could not be determined . Oral administration of antiinflammatory drugs significantly inhibited pleural fluid exudation and inflammatory cell influx to varying degrees . However, whereas these drugs additionally reduced total exudate protein and PLA2, they had no effect on the concentration of either parameter, implying that pleural PLA2 may be passively linked to fluid and cell movement.

Mol Biochem Parasitol, 1991 Sep, 48(1), 17 - 26
Cloning of a cDNA encoding phosphofructokinase from Haemonchus contortus; Klein RD et al.; Phosphofructokinase (PFK), the key regulatory enzyme in glycolysis, has been cloned from the pathogenic parasitic nematode Haemonchus contortus by functional complementation in Escherichia coli . An E . coli strain deleted for both PFK loci (strain DF1020) was transformed with plasmid DNA from a lambda ZAP II H . contortus cDNA library . Two out of 3 x 10(7) transformants were able to grow on minimal medium with mannitol as the sole carbon source . A plasmid, pPFK, containing a 2.7-kb insert, was isolated from one of these transformants and conferred on DF1020 the ability to grow on mannitol (the PFK phenotype) . The complemented cells contain PFK enzyme activity, absent in the E . coli mutant, at levels considerably higher than in wild type E . coli . Sequence analysis of the 2.7-kb insert shows an open reading frame that predicts a 789-amino acid protein that has approximately 70% similarity to mammalian PFKs . The amino acid sequence around asp182, thought to be the catalytic site, is completely conserved from nematodes to mammals.

Mol Microbiol, 1991 Sep, 5(9), 2133 - 41
Binding of basement-membrane laminin by Escherichia coli; Valkonen KH et al.; An invasive Escherichia coli (EIEC) isolate was found to bind basement-membrane laminin in a saturable and time-dependent manner . Excess of unlabelled laminin inhibited the binding of the radioactively labelled protein . Non-invasive E . coli K-12 exhibited only low-level laminin binding but introduction of the virulence-associated plasmid from the EIEC isolate led to high-level binding . Expression of a receptor for laminin on the bacteria was therefore associated with the presence of the virulence plasmid . Scatchard plot analysis indicated approximately 1000 receptors per bacterial cell, and a Kd of high-affinity binding of 0.5 pM . A laminin-binding protein which correlated with the presence of the plasmid was isolated and characterized . Its sequence of the eight amino-terminal amino acids was identical to that of the LamB protein of E . coli, although the molecular mass of the two in sodium dodecyl sulphate/polyacrylamide gel (SDS-PAGE) appeared to be slightly different . Both proteins reacted in immunoblot assays with polyclonal antisera raised against either protein, and both proteins bound laminin . Southern-blot hybridization analysis established that both the EIEC strain and the K-12 strains with or without the virulence plasmid contained one lamB gene only, and no laminin-binding protein appeared when the virulence plasmid was introduced into bacteria deleted for the lamB gene . On the basis of these results we suggest that native LamB protein of E . coli or a modified variant of it serves as a major receptor for laminin binding and is present at an increased level in invasive E . coli containing the virulence plasmid.

Z Naturforsch {C}, 1991 Sep-Oct, 46(9-10), 759 - 64
Subunit delta of chloroplast F0F1-ATPase and OSCP of mitochondrial F0F1-ATPase: a comparison by CD-spectroscopy; Engelbrecht S et al.; CD spectra have been recorded with subunit delta from chloroplast CF0CF1 and with OSCP from mitochondrial MF0MF1 . These subunits are supposed to act similarly at the interface between proton transport through the F0-portion and ATP-synthesis in the F1-portion of their respective F0F1-ATPase . Evaluation of the data for both proteins revealed a very high alpha-helix content of approximately 85% and practically no beta-sheets . Despite their low homology on the primary structure level (23% identity) and their different electrostatic properties (pI-values differ by 3 units), spinach delta and porcine OSCP are indistinguishable with respect to their secondary structure as measured by CD . Prediction and analysis of consensual alpha-helices even in poorly conserved regions indicate a high degree of structural similarity between chloroplast delta and OSCP . In view of the topology and function of delta and OSCP in intact F0F1 these findings are interpreted to indicate the dominance of secondary and tertiary structure over the primary structure in their supposed function between proton flow and ATP-synthesis.

Oncogene, 1991 Sep, 6(9), 1617 - 22
Accumulation of the c-mos protein is correlated with post-natal development of skeletal muscle; Leibovitch SA et al.; Previously we reported that c-mos proto-oncogene RNA was developmentally up-regulated during post-natal maturation of the rat skeletal muscle . Using two different site-directed affinity-purified antipeptide antibodies we can observe that c-mos product (p43 c-mos) accumulates increasingly during post-natal development of the skeletal muscle and exhibits protein kinase activity . We find that in adult rat p43 c-mos is 10-fold higher in skeletal muscle than in ovaries, and 20- to 40-fold higher than in heart, lung, testis and liver, and may represent about 0.005% of the total soluble proteins . In addition adult skeletal muscle from Xenopus, mouse and man was found to contain p43 c-mos . These data argue in favour of a novel muscle-specific function of c-mos.

Mol Cell Biol, 1991 Sep, 11(9), 4356 - 62
Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa; Kanaan MN et al.; cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation . The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain . The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity . The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene . A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity . The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

Virology, 1991 Sep, 184(1), 423 - 7
Expression of Sindbis virus nsP1 and methyltransferase activity in Escherichia coli; Mi S et al.; We have constructed two plasmids, pSR5-42 and pSR5-Toto, which under lac control expressed the SVLM21 and the SVToto forms, respectively, of the Sindbis virus nonstructural protein, nsP1 . The induced protein, which was the major protein made following induction with IPTG, had an apparent molecular weight of 60,000 and an amino terminal sequence in agreement with that expected for nsP1 . Following induction with IPTG, cells carrying pSR5-42 (which contains the SVLM21 gene sequence) generated much higher RNA methyltransferase activity than cells carrying pSR5-Toto (which contains the SVToto gene sequence) . This result is in agreement with what is observed when methyltransferase is measured in cells infected with SVLM21 and SVSTD (or SVToto), respectively . These results provide strong evidence that nsP1 has methyltransferase activity in the absence of any other viral nonstructural proteins.

Indian J Pediatr, 1991 Sep-Oct, 58 Suppl 1, 23 - 32
Development, production and pharmacodynamics of human growth hormone; Jorgensen JT et al.; With the advent of recombinant DNA technology, it is possible to produce biosynthetic human growth hormone (B-hGH) . Novo Nordisk A/S has developed a method for manufacturing B-hGH which is identical to the 22K fraction of pituitary human growth hormone (P-hGH), using a nonpathogenic strain of Escherichia coli as host . B-hGH has been investigated extensively in physical, chemical and biological studies and found to be identical to P-hGH . Pharmacological studies have revealed that B-hGH possesses the same pharmacokinetic and short-term metabolic profiles as P-hGH . Long term clinical studies have shown that B-hGH induces a significant increase in height velocity in children with growth hormone deficiency (GHD) and is characterized by a low antigenicity.

J Dev Physiol, 1991 Sep, 16(3), 167 - 72
Hemodynamic effects of oleic acid in newborn lambs: role of arachidonic acid metabolites; Schreiber MD et al.; Oleic acid injection produces acute lung injury and pulmonary hypertension in adult animals . In other types of acute lung injury, such as that caused by E . coli endotoxin, metabolites of arachidonic acid are important mediators of pulmonary hypertension . In order to understand the hemodynamic response of newborn animals to oleic acid injection and the contribution of arachidonic acid metabolites to that response, we injected oleic acid into awake, chronically instrumented newborn lambs . The hemodynamic response of lambs to injections of oleic acid alone was compared to their response after pretreatment with either FPL57231, a putative leukotriene receptor antagonist, or indomethacin, a cyclooxygenase synthesis inhibitor . Oleic acid caused acute pulmonary hypertension associated with an increase in protein-rich lung lymph fluid . Systemic hemodynamic effects were variable . FPL57231 completely blocked the oleic acid-induced pulmonary hypertension while indomethacin significantly attenuated the response . Therefore, metabolites of arachidonic acid metabolism appear to be important mediators of oleic acid-induced pulmonary hypertension in newborn lambs.

Mutagenesis, 1991 Sep, 6(5), 385 - 90
Miscoding potential of N2,3-ethenoguanine studied in an Escherichia coli DNA-dependent RNA polymerase in vitro system and possible role of this adduct in vinyl chloride-induced mutagenesis; Mroczkowska MM et al.; The miscoding potential of N2,3-ethenoguanine (epsilon G), one of the carcinogen vinyl chloride adducts to DNA bases, has been evaluated in an Escherichia coli DNA-dependent RNA polymerase in vitro system . Epsilon G present in poly(C) templates causes incorporation of cytosine (C), uridine (U) and adenosine (A) under competitive and non-competitive conditions, and in the presence of either Mn2+ and Mg2+ cations, indicating that this modified base still retains the coding properties of unmodified G and can also act as A or U . The formation of hydrogen bonded pairs between different tautomeric forms of epsilon G and C, U and A is proposed . The possible role of epsilon G, along with a role of other vinyl chloride adducts in causing of GC----AT transitions, the most frequent mutation induced by a vinyl chloride metabolite, is discussed.

Mutagenesis, 1991 Sep, 6(5), 335 - 41
Development of assays for the detection of photomutagenicity of chemicals during exposure to UV light--I . Assay development; Dean SW et al.; Two complementary assay systems have been adapted for the detection of compounds which may form mutagenic photoproducts during exposure to UV light from an Osram Ultra-Vitalux sunlamp as used in the evaluation of the effectiveness of sun filters . The effects of UVA and of UVB were evaluated . A bacterial plate test using Escherichia coli strain WP2 allowed the bacteria, co-plated with test chemical in soft agar, to be irradiated with various doses of UV light . Mutagenesis was assessed by scoring numbers of tryptophan-independent colonies . The chosen reference compound was 8-methoxypsoralen (8-MOP) which was non-mutagenic alone at the highest dose tested (1000 micrograms/plate) . Following simultaneous exposure of bacteria to 8-MOP and doses of UV light which alone had little effect, large numbers of revertants were scored . Numbers of mutants were dependent upon the doses of both 8-MOP and of UV light . The second test system involved the exposure of Chinese hamster ovary cells to UV light in the presence of test chemical to determine the clastogenic effects of photoproducts . Treatment with 8-MOP alone up to 50 micrograms/ml was not clastogenic but concomitant exposure to non-damaging doses of UV light caused large increases in the incidence of chromosome aberrations of all types . Damage was again dependent on the doses of both components . Two additional photoactive compounds, para-aminobenzoic acid and chlorpromazine both show photoclastogenic but not photomutagenic properties . These two complementary assay systems take advantage of using no-effect levels of UV light as a baseline against which photomutagenicity readily can be compared.(ABSTRACT TRUNCATED AT 250 WORDS)

J Neurosci Res, 1991 Sep, 30(1), 154 - 62
Differences in the abilities of human tau isoforms to promote microtubule assembly; Scott CW et al.; Three isoforms of human tau protein were compared for their abilities to induce microtubule assembly . The three isoforms, tau 3 (tau containing three microtubule-binding domains), tau 4 (tau containing four microtubule-binding domains) and tau 4L (tau containing four microtubule binding domains plus a 58-amino-acid insert near the N-terminus) were expressed in E . coli and purified using ammonium sulfate precipitation, ion exchange, and size exclusion chromatography . All three isoforms induced microtubule assembly at micromolar concentrations and showed similar critical concentrations for assembly of 0.4-0.45 microM . However, tau 4 induced microtubule formation at a rate five- to tenfold faster than either tau 3 or tau 4L . The rate of microtubule elongation seen with tau 4 was twofold greater than with tau 3 or tau 4L, suggesting that the faster rate of microtubule assembly seen with tau 4 was due, at least in part, to faster elongation . Tau 4 induced a greater number of microtubules to form at steady state than did tau 3 or tau 4L . The microtubules generated with each tau isoform had similar steady-state length distributions and were equally susceptible to cold-induced disassembly . These results indicate that the additional microtubule-binding domain in tau 4 enhances microtubule assembly, while the 58-amino-acid insert negates the stimulatory effect of the fourth microtubule-binding domain.

Agents Actions, 1991 Sep, 34(1-2), 77 - 80
Antiflammin-2 (HDMNKVLDL) does not inhibit phospholipase A2 activities; Hope WC et al.; A basic nonapeptide P2 (antiflammin-2, HDMNKVLDL) which is identical to a portion of the amino acid sequence (residues 246-254) of lipocortin I, has been described to have antiinflammatory activity in a rat paw edema model (Nature 335: 726-730 {1988}) . P2 (0.05 microM) was also reported to inhibit porcine pancreatic phospholipase A2 (PLA2) . The effect of synthetic P2 (98% pure) on PLA2 was evaluated in two assay systems . Using porcine pancreatic PLA2 and phosphatidylcholine/deoxycholate mixed micellar substrate, P2 (0.005-50 microM) had no effect on PLA2 activity, even in the presence of 2-mercaptoethanol to prevent peptide oxidation . In another assay, using human synovial fluid PLA2 as the enzyme and {14C}-oleate-labelled E . coli substrate, P2 (0.005-50 microM) had no significant effect on PLA2 activity . A reported PLA2 inhibitor, manoalide, was a potent inhibitor of PLA2 in both assay systems . On the basis of these results, we conclude that P2 is devoid of PLA2 inhibitory activity.

Agents Actions, 1991 Sep, 34(1-2), 30 - 1
Effects of the prostaglandin analogue misoprostol on inflammatory mediator release by human monocytes; Widomski DL et al.; The effect of misoprostol (M) on IL-1 beta, TNF-alpha, and lipid mediator release (assessed by RIA) by adherent (assessed by electron microscopy) human monocytes were studied in vitro . Human monocytes stimulated with E . Coli-derived lipopolysaccharide showed an increase in both IL-1 beta and TNF-alpha release . Incubation of the monocytes with LPS and M (18 hrs.), resulted in a reduction of both IL-1 beta and TNF-alpha levels . Leukotriene B4 levels did not increase in response to LPS or M . LPS also caused an increase in thromboxane (TXB2) . M decreased TXB2 levels . 6-keto PGF1 alpha (6KP) . Incubation with LPS and M stimulated release . LPS caused an increase in PGE2 levels . M (100 microM) caused an increase in PGE2 levels, M (1 microM) had no effect on PGE2 . These data suggest a possible immunomodulatory role for misoprostol in inflammatory diseases.

Agents Actions, 1991 Sep, 34(1-2), 28 - 9
Contribution of the monocyte to thrombotic potential; Spillert CR et al.; The circulating monocyte exhibits the capacity to initiate and accelerate the coagulation cascade . We have devised a simple whole blood clotting assay which quantitates the monocyte's contribution of procoagulant (a marker of monocyte activation) to the clotting process and in addition, measures the in vivo activation of the cell . Citrated whole blood is added to saline and the recalcification time (RT) determined without incubation (RT control), with two hours incubation (RT saline), or added to endotoxin with incubation (RT endotoxin) . The reduction in value between RT control and RT saline is a measure of monocyte activation in vivo . The reduction in time between RT saline and RT endotoxin is a marker of monocyte activation in vitro . This simple test enables the measurement of monocyte activation and this cells contribution to the hypercoagulability described in many disease states.

Agents Actions, 1991 Sep, 34(1-2), 203 - 4
Dissociation between LPS-induced bronchial hyperreactivity and airway edema in the guinea-pig; Vincent D et al.; The interactions between LPS-induced bronchial hyper-reactivity (BHR) and lung inflammation (LI) were investigated . LPS-induced LI was assessed with the augmented alveolo-capillary permeability (ACP) and with the increased migration of neutrophils into the broncho-alveolar lavage fluid . BHR was defined as the increase in the response to a standard dose of serotonin . Mepyramine and the PAF antagonist WEB 2170 blocked LPS-induced increase of ACP, whereas aspirin was inactive . By contrast, neither LPS-induced neutrophil attraction to airways, nor LPS-induced HBR were inhibited by these agents . Our results indicate that LPS-induced edema and BHR are dissociated.

Agents Actions, 1991 Sep, 34(1-2), 106 - 9
Phosphonate-phospholipid analogues inhibit human phospholipase A2; Marshall LA et al.; A phosphonate-containing phospholipid (PL) analogue (Compound 1) designed as a transition-state inhibitor competively inhibits non-human extracellular PLA2 at a mole fraction of 0.003 in the kinetic "scooting mode" (Jain et al., Biochem 28:4135 (1989} . To further profile the activity of Compound 1, we examined its activity with purified human enzyme and in whole cell systems . Compound 1 effectively inhibited a 14 kDa human PLA2 purified from joint synovial fluid of patients with rheumatoid arthritis using 3H-AA labeled E . coli as substrate (IC50 = 1.7 microM) and a high MW PLA2 (110 kDa) isolated from the cytosol of a human monocytic cell line, U-937, which selectively hydrolyzes AA-containing PL (IC50 = 165 microM) . It failed to reduce A23187-induced PGE2 or LTC4 production by human adherent monocytes or LTB4 release from human neutrophils which may be due, in part, to poor membrane partitioning.

J Photochem Photobiol B, 1991 Sep, 10(4), 339 - 44
Comparison of the effects of visible femtosecond laser pulses and continuous wave laser radiation of low average intensity on the clonogenicity of Escherichia coli; Karu TI et al.; To evaluate the contribution of local pulsed heating of light-absorbing microregions to biochemical activity, irradiation of Escherichia coli was carried out using femtosecond laser pulses (lambda = 620 nm, tau p = 3 x 10(-13) S, fp = 0.5 Hz, Ep = 1.1 x 10(-3) J cm-2, Iav = 5.5 x 10(-4) W cm-2, Ip = 10(9) W cm-2) and continuous wave (CW) laser radiation (lambda = 632.8 nm, I = 1.3 W cm-2) . The irradiation dose required to produce a similar biological effect (a 160%-190% increase in the clonogenic activity of the irradiated cells compared with the non-irradiated controls) is a factor of about 10(3) lower for pulsed radiation than for CW radiation (3.3 X 10(-1) and 7.8 X 10(2) J cm-2 respectively) . The minimum size of the microregions transiently heated on irradiation with femtosecond laser pulses is estimated to be about 10 A, which corresponds to the size of the chromophores of hypothetical primary photoacceptors--respiratory chain components.

J Photochem Photobiol B, 1991 Sep, 10(4), 329 - 37
uvrB-dependent, recF-independent post-replication (or replication) repair in Escherichia coli; Slezarikova V et al.; In UV-damaged cells, a large fraction of pyrimidine dimers may remain unexcised and may be tolerated by a uvrB recA lexA-dependent non-excisional mode of repair (M . Sedliakova, J . Brozmanova, F . Masek and K . Kleibl, Biophys . J., 36 (1981) 429-441) . We show here that a similar repair pathway operates in the Escherichia coli recF 143 single mutant but not in the recF uvrB double mutant . This indicates that the putative repair pathway is recF independent.

Fiziol Zh, 1991 Sep-Oct, 37(5), 41 - 7
{Ultrastructural alterations in rat brain tissues under the direct action of Escherichia coli endotoxin}; Bardakhch'ian EA et al.; The experiments on the rats with intracisternal injection of endotoxin have revealed essential differences in the mechanisms of its permeability through cerebrospinal fluid-brain and blood-cerebrospinal fluid barriers . As for ependymal cells, endotoxin shunts through the cytoplasm in the areas of tight junctions and then it reaches perineuronal spaces . The mechanism of endotoxin penetration through the epithelium of choroid plexi is associated with receptor-mediated endocytosis.

Biochem Int, 1991 Sep, 25(2), 249 - 59
In vitro studies on mild alkali induced lesions in DNA; Musarrat J et al.; S1 nuclease hydrolysis, benzoylated naphthoylated DEAE cellulose (BND-cellulose) chromatography as well as certain immunological and genetic techniques have been used to evaluate the effect of mild alkali (pH10.0) on the DNA molecule . Native calf thymus DNA after exposure to alkaline pH10.0 when subjected to S1 nuclease hydrolysis released significant amount of acid soluble nucleotides as compared to the untreated control . With pBR322 DNA, the population of linear DNA species increased on S1 digestion with concomitant reduction in the supercoiled form . BND cellulose chromatographic studies also suggested the formation of single strandedness and/or distortions in the alkali treated DNA molecule . Antisera raised against the alkali treated DNA exhibited high cross-reactivity with both single stranded and Z DNA . Moreover, a significant reduction in transformation frequency of the treated DNA molecule compared with the untreated control further ascertained the structural alterations in DNA as a result of exposure to mild alkali.

Indian J Med Res, 1991 Sep, 93, 286 - 8
ELISA for detection of heat stable enterotoxin producing Escherichia coli strains; Ram S et al.; Among 557 strains of Esch . coli isolated from patients with acute diarrhoea, 392 (70.4%) isolates demonstrated ST production by ELISA . Predominant ST producing serogroups were 020 (45), 078 (40), 0128 (21), 061 (19), 0149 (9), 04, 055, 0106 and 0114 (8 each) . The inhibition ELISA range was between 10.5 and 40.5 per cent . Visual difference between a negative and a positive ELISA test was distinct . A comparison of ELISA with classical suckling mouse assay for 100 strains showed 88 and 80 positive strains respectively for ST . ELISA proved a more specific, rapid and sensitive assay which may be useful for screening large number of isolates in epidemiological studies.

J Clin Microbiol, 1991 Sep, 29(9), 1963 - 8
Monoclonal antibodies specific for the Escherichia coli heat-stable enterotoxin STb; Urban RG et al.; Protein A-STb and STb-alkaline phosphatase protein fusions were used as immunogen and antigen, respectively, for the generation and screening of monoclonal antibodies to the Escherichia coli heat-stable enterotoxin STb . Following immunization with immunoglobulin G-Sepharose-purified protein A-STb and hybridoma construction, STb-alkaline phosphatase hybrid protein was used in a labeled antigen capture assay to detect the production of STb-specific monoclonal antibody . STb-specific monoclonal antibodies were characterized by using a combination of immunoblotting and synthetic-peptide-based enzyme immunoassay techniques . Four distinct anti-STb antibodies were identified and characterized.

J Clin Microbiol, 1991 Sep, 29(9), 1893 - 8
Colonization factors of enterotoxigenic Escherichia coli isolated from children with diarrhea in Argentina; Binsztein N et al.; A prospective study was performed to evaluate the presence of colonization factor antigens (CFAs) in enterotoxigenic Escherichia coli (ETEC) strains isolated from 1,211 children with diarrhea in Argentina . One hundred nine ETEC strains that were isolated from seven different laboratories in various regions of the country were tested for CFAs by using monoclonal antibodies against CFA/I and E . coli surface antigens CS1, CS2, and CS3 of CFA/II and CS4 and CS5 of CFA/IV; a polyclonal antiserum against CS6 was used . The CFAs searched for were found in 52% of the ETEC strains: 23% of the strains carried CFA/I, 17% carried CFA/IV, and 12% carried CFA/II . All of the CFA/I strains produced heat-stable enterotoxin, and several of them were of the prevalent serotypes O153:H45 and O78:H12 . Among the 19 strains expressing CFA/IV, 16 expressed CS5 and CS6 and produced the heat-stable enterotoxin and most were of serotype O128:H21; the remaining 3 strains produced CS6 only . No ETEC strains expressing CS4 were found . Most (11 of 13) of the CFA/II-carrying ETEC strains expressed CS1 and CS3, and 10 of them were of the O6:K15:H16 serotype and produced both heat-labile and heat-stable toxins . As many as 24 of the 109 CFA-negative ETEC strains gave mannose-resistant hemagglutination with erythrocytes from different species; 4 strains had high surface hydrophobicity, suggesting the presence of additional, as yet undefined, colonization factors in up to 25% of the ETEC isolates.

Biochem Int, 1991 Sep, 25(1), 19 - 27
DNA polymerase action blocked by adenine adducts induced by 5-hydroxymethylchrysene sulfate; Suzuki M et al.; Modification of M13mp10 single-stranded DNA with 5-hydroxymethylchrysene (5HCR) sulfate, the ultimate carcinogenic metabolite of 5-methylchrysene, resulted in formation of N6{(chrysen-5-yl)methyl}adenine and N2{(chrysen-5-yl)methyl}-guanine at the ratio of 2.7:1 . Measurement of DNA synthesis using this modified template and E.coli DNA polymerase I (Klenow fragment) demonstrated that increasing levels of adducts caused a progressive decline in replication . Analysis of reaction products on DNA-sequence gels revealed DNA elongation to be arrested exclusively at adenine adducts in -AAAGGA- and -AACA- sequences.

J Biochem (Tokyo), 1991 Sep, 110(3), 324 - 7
Activation of the osmoregulated ompC gene by the OmpR protein in Escherichia coli: a study involving synthetic OmpR-binding sequences; Maeda S et al.; Expression of the Escherichia coli outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity . The OmpR and EnvZ proteins are transcriptional factors involved in this osmotic regulation of the ompC and ompF genes . In particular, expression of the ompC gene is activated by the positive regulator, OmpR, in response to high osmolarity of the medium . In this study, we succeeded in defining a functional OmpR-binding sequence by analyzing a set of synthetic oligonucleotides, and propose a consensus motif for OmpR-binding . It was also demonstrated that the asymmetric OmpR-binding sequence, thus identified, can activate the canonical ompC promoter in an orientation independent-manner, providing that this sequence is placed closely and stereo-specifically with respect to the -35 region.

J Biochem (Tokyo), 1991 Sep, 110(3), 315 - 20
Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme; Kanatani A et al.; Protease II gene of Escherichia coli HB101 was cloned and expressed in E . coli JM83 . The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host . The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method . The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing . The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence . The reactive serine residue of protease II was identified as Ser-532 with tritium DFP . The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases . Except for this region, protease II showed no significant sequence homology with E . coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases . However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain . Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.

Appl Environ Microbiol, 1991 Sep, 57(9), 2762 - 3
Recovery of an integration shuttle vector from tandem repeats in Methanococcus maripaludis; Sandbeck KA et al.; Transformation of Methanococcus maripaludis by using an integration vector, pKAS102, is described . Selection and subsequent growth at high concentrations of puromycin caused pKAS102 to develop tandem repeats within the genome . As a result, total DNA isolated from the transformant could be used to recover the intact vector by direct transformation of competent Escherichia coli.

Mol Microbiol, 1991 Sep, 5(9), 2085 - 91
The role of the 'gearbox' in the transcription of essential genes; Vicente M et al.; Regulation of transcription occurs at different levels, one being in the presence of sequences specifically recognized by different forms of RNA polymerase, i.e . the promoters . Three different kinds of promoter are defined according, among other things, to their dependence on the growth rate of the cell: the 'house-keeper' promoter of many metabolic genes, the stringent promoter found at several rRNA and ribosomal protein genes, and the 'gearbox' at genes whose products are required at higher relative amounts at lower growth rates . The identified gearbox promoters of Escherichia coli share specific homologies in the -10, -35 and upstream regions . Although there may be different types of gearbox promoters, the -10 sequence of one of these promoters has been found to be essential for functioning as a gearbox . This suggests the existence of specific sigma factors for its transcription . RpoS (KatF) is a likely candidate for being one of these sigma factors . Computer simulation allows us to predict that such sigma factors should, in turn, be expressed following a gearbox mode, which would then imply the existence of self-regulated loops contributing to the expression of some genes of bacterial division.

Genet Anal Tech Appl, 1991 Sep, 8(6), 181 - 5
A novel approach to the rapid isolation and nucleotide sequencing of genomic clones; Boivin R et al.; In this study, a genomic library subdivided into fractions was rapidly screened by a Southern detection technique . Deletion libraries were obtained from recovered genomic clones by single random cuts with nuclease S1 . These deletion libraries proved useful for localizing genes in the inserts and yielded, after size fractionation, nested deletions suitable for nucleotide sequencing . An heterologous vector (pDB21) carried the insert used as probe for all hybridizations involved in the process of genomic clones isolation and characterization.

Cytokine, 1991 Sep, 3(5), 421 - 7
Differential activity of granulocyte-macrophage and macrophage colony stimulating factors on bone resorption in fetal rat long bone organ cultures; Bertolini DR et al.; In this study, the ability of recombinant human macrophage (M) and murine granulocyte-macrophage (GM) colony stimulating factor (CSF) to affect both basal and stimulated bone resorption in fetal rat long-bone organ cultures was assessed . It was found that M-CSF does not affect basal bone resorption or bone resorption stimulated by parathyroid hormone, recombinant human interleukin 1 beta, prostaglandin E2 (PGE2), and 1,25 dihydroxy vitamin D3 . Specifically, M-CSF at concentrations as high as 30 nM (1 microgram/mL) did not modulate 45Ca release from fetal rat long bones stimulated by these agents . The addition of recombinant murine GM-CSF (at equal molar concentration to M-CSF) also did not affect bone resorption stimulated by parathyroid hormone and interleukin 1 beta . On the other hand, GM-CSF stimulated basal bone resorption over a 120-h period and augmented the resorption mediated by exogenous PGE2 over a 48-h incubation . In addition, GM-CSF was shown to stimulate production of endogenous PGE2 in cultures of bone rudiments . These effects on bone resorption were blocked by the addition of prostaglandin synthesis inhibitors and specific antibodies to murine GM-CSF . These data indicate that M-CSF does not act as a regulator of bone turnover, but GM-CSF may cause bone resorption by stimulating the synthesis of PGE2 in bone.

Biochimie, 1991 Sep, 73(9), 1187 - 93
Characterization of an osmoregulated periplasmic glycine betaine-binding protein in Azospirillum brasilense sp7; Riou N et al.; Azospirillum brasilense is able to use glycine betaine as a powerful osmoprotectant; the uptake of this compound is strongly stimulated by salt stress, but significantly reduced by cold osmotic shock . Non-denaturing PAGE in the presence of {methyl-14C} glycine betaine and autoradiography demonstrated the presence of one glycine betaine-binding protein (GBBP) in periplasmic shock fluid obtained from high-osmolarity-grown cells . The binding activity was absent in periplasmic fractions from cells grown at low osmolarity . SDS-PAGE analysis showed that the osmotically inducible GBBP has an apparent molecular weight of 32,000 . The isoelectric point was between 5.9 and 6.6, as determined by isoelectric focusing . This protein bound glycine betaine with high affinity (KD of 3 microM), but had no affinity for either other betaines (proline betaine, gamma-butyrobetaine, pipecolate betaine, trigonelline, homarine) or related compounds (choline, glycine betaine aldehyde, glycine and proline) . Optimum binding activity occurred at pH 7.0 to 7.5, and was not altered whether or not the binding assays were done at low or high osmolarity . Immunoprecipitation and Western blotting showed that immunoadsorbed anti-GBBP antibody from E coli cross-reacted with the GBBP produced by A brasilense cells grown at high osmolarity.

Mol Gen Mikrobiol Virusol, 1991 Sep, (9), 3 - 6
{Synthesis of the E-antigen of the hepatitis B virus (HBeAg) in eukaryotic cells by a recombinant strain of the vaccinia virus}; Loparev VN et al.; The recombinant plasmids containing the gene for hepatitis B viral core-antigen with the pre-core-sequence controlled by the early-late promoter of the 7.5' K protein gene were constructed . The recombinant strains of vaccinia virus were obtained on their basis (vHBe42-1 and vHBe42-3) selectively expressing HBeAg of hepatitis B virus . The kinetics of HBeAg synthesis was studied in infected cells as well as secretion of the protein into culturing medium . Three proteins were found by blotting technique in the cells infected by vHBe42-3 that react with the specific antiserum to HBeAg and have the mol . masses 25, 22 and 17 kD . The completely processed HBeAg 17 kD was found in the culturing medium . The rabbit serums from the animals immunized by recombinant vHBe42-3 contained antibodies to HBeAg but not to HBcAg . This makes it possible to study the structural and functional organization, immunological properties and role of this antigen in pathogenesis of hepatitis virus B and to construct the specific test systems for screening HBeAg and corresponding antibodies.

Mol Gen Mikrobiol Virusol, 1991 Sep, (9), 17 - 21
{Behavior of Sa plasmid in tularemia pathogen cells}; Pomerantsev AP et al.; The genome of Sa plasmid is shown to be a subject of genetical rearrangements in Francisella tularensis cells . The rearrangements either result in plasmid integration into the host cell genome or intramolecular amplification of cat-gene with the subsequent excision and recombination of the derivative plasmids . Stable inheritance of the plasmid is registered after integration while plasmid elimination occurs in case of extrachromosomal localisation.

J Vet Pharmacol Ther, 1991 Sep, 14(3), 219 - 29
Pharmacodynamics and pharmacokinetics of carprofen, a non-steroidal anti-inflammatory drug, in healthy cows and cows with Escherichia coli endotoxin-induced mastitis; Lohuis JA et al.; The pharmacodynamics of carprofen and its pharmacokinetics in plasma and milk of healthy cows and cows with endotoxin-induced mastitis were studied after a single intravenous dose of 0.7 mg/kg body weight . Carprofen was administered to five clinically healthy cows and to the same cows 3 weeks later, 2 h after intramammary infusion of endotoxin . Mastitis developed in all endotoxin-infused quarters . The pharmacokinetic characteristics of carprofen in healthy cows were a small volume of distribution (0.09 l/kg), a relatively low systemic clearance (2.4 ml/h kg), and a long elimination half-life (30.7 h) . In the mastitic cows, systemic clearance (1.4 ml/h kg) was significantly lower (P less than 0.01), and elimination half-life (43.0 h) was significantly longer (P less than 0.01) than in the normal animals . Concentrations of carprofen in milk from healthy quarters were below the limit of detection for the assay (0.022 micrograms/ml) . In milk from mastitic quarters, concentrations of carprofen increased up to 0.164 micrograms/ml during the first 12 h after induction of mastitis, but were less than 0.022 micrograms/ml at 24 to 48 h . Compared with the untreated mastitic controls, carprofen treatment significantly reduced heart rate (P less than 0.01), rectal temperature (P less than 0.001), quarter swelling (P less than 0.01) and other parameters measured . Local and systemic adverse reactions to carprofen were not observed.

Circ Shock, 1991 Sep, 35(1), 44 - 52
Systemic and mesenteric O2 metabolism in endotoxic pigs: effect of graded hemorrhage; Heard SO et al.; Normally, supply-dependency of oxygen uptake (VO2) is not demonstrable unless oxygen delivery (DO2) is less than a critical value (DO2crit) below which VO2 is linearly dependent upon DO2 . Because recent evidence suggests that VO2 is pathologically supply-dependent in endotoxic or septic animals and humans, we sought to determine whether 1) pathological systemic and/or mesenteric oxygen extraction (O2EXT) defects occur in a porcine model of endotoxicosis and 2) arterial lactate and ileal intramucosal pH (pHI) serve as useful markers of supply-dependency of VO2 in endotoxic animals . Normal (group I, n = 11) and endotoxic (group II, n = 8) anesthetized pigs were subjected to graded hemorrhage . Endotoxicosis was induced by infusing Escherichia coli lipopolysaccharide (150 micrograms/kg bolus at t = 0 min and 20 micrograms/kg-hr at t = 60 min) . From t = 0-60 min, pigs in group II were resuscitated with hetastarch and blood (12 ml/kg each) . Hemorrhage was initiated at t = 0 min or t = 70 min in groups I and II, respectively . DO2crit was determined by a modified "dual-line" regression method . Systemic DO2crit was 12.9 +/- 0.9 ml/kg-min in group I and 16.9 +/- 1.3 ml/kg-min in group II (P less than .05) . Systemic O2EXT at DO2crit was similar in both groups . Arterial lactate concentration at DO2crit was significantly higher in endotoxic pigs (group I, 2.64 +/- 0.29 mM; vs . group II, 3.88 +/- 0.45 mM; P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Circ Shock, 1991 Sep, 35(1), 31 - 6
Cardiac function in an ovine model of endotoxemia; Redl G et al.; Eight awake sheep were monitored with ultrasonic crystals, positioned at the anterior and posterior left ventricular wall . A left-sided intraventricular pressure transducer and a right ventricular ejection fraction catheter were positioned in the right and left hearts, respectively . After administration of endotoxin (Escherichia coli, LPS 1.5 micrograms/kg in 30 min), the hemodynamic variables showed a triphasic course . Phase I, (0-1 hr post LPS) was characterized by an increased pulmonary artery pressure and a decreased right ventricular ejection fraction . The inability of the right ventricle to compensate for the increased preload resulted in a fall of the left ventricular preload, stroke volume, and cardiac output . Three hours after LPS administration a second drop of the cardiac output was noted (phase II) . This occurred as a result of a fall in preload . Eight hours post LPS a hyperdynamic phase (phase III) was distinguished, with a high cardiac output and a low systemic vascular resistance . During this time there was evidence of probable reduced myocardial contractility.

Circ Shock, 1991 Sep, 35(1), 25 - 30
Endothelial-dependent and -independent responses in the thoracic aorta during endotoxic shock; Young JS et al.; Endotoxic shock is characterized by a variety of hemodynamic disturbances which result in tissue hypoperfusion . There is some evidence for endothelial damage caused by endotoxin . The present study addressed the hypothesis that vascular responsiveness to endothelial-dependent vasodilators is altered in endotoxic shock . Dose-response relationships for an endothelial-dependent vasodilator, acetylcholine, and an endothelial-independent vasodilator, adenosine, were determined in guinea pig aortic rings . Rings were examined from either control (untreated) animals or from animals given Escherichia coli endotoxin (4 mg/kg, i.p.) 16 hr prior to functional studies . Dose-response relationships to adenosine were similar in aortic rings from control and shocked animals . However, response to acetylcholine were attenuated by 30% (P less than .05) in the shocked group . To distinguish between a direct, acute effect of endotoxin versus effects produced by systemic changes that occur during shock, rings were isolated from untreated animals and incubated with endotoxin in vitro for 30 min prior to and during dose-response measurements . Incubation with endotoxin caused no change in aortic responses to adenosine or acetylcholine . Electron microscopy revealed a separation of the endothelium from the internal elastic lamina and an increase in inter-endothelial gaps in rings isolated from shocked animals . These structural changes were not observed in rings from untreated animals or in rings incubated with endotoxin in vitro . We conclude that endothelial-dependent vasodilation is attenuated during endotoxic shock . The functional changes are correlated with ultrastructural alterations of the endothelium.

Mikrobiologiia, 1991 Sep-Oct, 60(5), 942 - 3
{Activity of glucose-6-phosphate dehydrogenase and level of nucleic acids in Escherichia coli during various rates of growth}; Krolichenko TP; Nucleic acids content and activity of glucose-6-phosphate dehydrogenase were studied at various growth rates . The activity of glucose-6-phosphate dehydrogenase was shown to correlate with the growth rate and with the DNA content in Escherichia coli.

Biochem Int, 1991 Sep, 25(1), 57 - 65
RNA processing enzymes RNase III, E and P in Escherichia coli are not ribosomal enzymes; Srivastava RA et al.; We recently showed that RNase III can process a small stable RNA, precursor 10Sa RNA, that accumulates in an rne (RNase E) strain at non-permissive temperatures . Precursor 10Sa (p10Sa) RNA is processed to 10Sa RNA in two steps, the first step is catalyzed by RNase III in the presence of Mn2+ but not Mg2+ . It was shown that RNase III cosediments with membrane preparation from wild type as well as RNase III overexpressing cells . However, the possibility of membrane preparation contamination with ribosomes could not be ruled out . Here we show that RNase III, E and P are not associated with ribosomes . E . coli cells were opened either by alumina grinding or by sonication and fractionated into cytosolic and pellet fractions . The characterization of membrane preparations was done by assaying NADH oxidase, a bona fide membrane enzyme . Ribosomes prepared by alumina grinding were found to be contaminated with small fragments of membrane which contained RNase III activity . RNase III and NADH oxidase activities were present in the ribosomal preparations which could be solubilized by reagents that dissolve the inner membrane . Isopycnic sucrose gradient centrifugation of the membrane and ribosomal preparations also confirmed that RNase III fractionated with the inner membrane . Similarly RNase P activity was found in the corresponding fractions when isopycnic centrifugation of membrane and ribosome preparations was carried out . RNase E activity was also found to be present mostly in the post-ribosomal supernatant . These findings show that RNase III, E and P are not ribosomal enzymes.

Mol Microbiol, 1991 Sep, 5(9), 2293 - 301
Genetic mapping of starch- and lambda-receptor sites in maltoporin: identification of substitutions causing direct and indirect effects on binding sites by cysteine mutagenesis; Francis G et al.; Cysteine mutagenesis was used to test the proximity of 16 residues to protein-ligand interaction sites in maltoporin (LamB protein) . LamB protein with additional cysteines was incorporated into the outer membrane of Escherichia coli except with a Ser-30----Cys substitution . Phage Lambda and starch binding was assayed before and after incubation of mutants with six thiol-specific reagents . Four categories of mutation were recognized on the basis of phenotype and modification for each of the Lambda- and starch binding sites . The thiol modification experiments helped to clarify whether the phenotype of a mutation was due to a substitution at the binding site or an indirect perturbation of the structure . This study suggests that the cysteine mutagenesis/thiol modification approach may be usefully applied to the operational mapping of surface-accessible binding sites or epitopes.

J Appl Physiol, 1991 Sep, 71(3), 1106 - 11
Lung to blood passage of different-sized molecules during lung inflammation in the rat; Folkesson HG et al.; The passage of different-sized marker molecules over the lower respiratory tract into the blood circulation during pulmonary inflammation induced by dextran, endotoxin {i.e., lipopolysaccharide from Escherichia coli (LPS)}, or ferritin was assessed in the rat . Bovine immunoglobulin G (BIgG, mol wt = 150,000 Da), bovine serum albumin (BSA, mol wt = 67,000 Da), and the nonapeptide 1-deaminocysteine-8-D-arginine vasopressin (dDAVP, mol wt = 1,067 Da) were used as permeability markers after intratracheal instillation . The pathophysiological indexes of a proceeding lung inflammation were increased total cell number, changed leukocyte proportions and increased total protein content obtained in bronchoalveolar lavage, and lung edema formation shown as an increased lung wet-dry weight difference . Intratracheal instillation of dextran induced a moderate neutrophil invasion into the lungs but had no effect on the passage of the different markers over the lungs (BIgG 1.8 +/- 0.6%, BSA 3.5 +/- 1.2%, dDAVP 26.1 +/- 20.7%) compared with control rats instilled with the markers alone (1.8 +/- 0.4%, 4.1 +/- 1.3%, 20.0 +/- 3.8%, respectively) . Endotoxin administration resulted in markedly higher lavage cell counts and lung edema concomitantly with an increased lung passage of the markers (3.2 +/- 0.9%, 22.0 +/- 6.1%, 33.3 +/- 12.0%, respectively; P less than 0.01-P less than 0.001) . The highest marker passage was obtained when the inflammation was most severe, i.e., after ferritin administration (17.6 +/- 2.3%, 60.0 +/- 6.7%, 41.6 +/- 6.9%, respectively; P less than 0.001), which resulted in markedly elevated lavage cell numbers and protein content as well as edema formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Biol (Mosk), 1991 Sep-Oct, 25(5), 1248 - 57
{Reverse transcriptase of the human immunodeficiency virus: cloning, expression in Escherichia coli, purification of the enzyme, and production of monoclonal antibodies}; Rechinskii VO et al.; To express HIV-1 reverse transcriptase in E . coli a number of genetic constructions containing reverse transcriptase and virus protease nucleotide sequences was obtained . The products of expression were characterized; monoclonal antibodies to reverse transcriptase were produced . The purification of reverse transcriptase was carried out . The substantial proteolysis of reverse transcriptase during purification was shown . The purified preparation is predominantly, an active protein with Mr 57 kDa . Some properties of this protein differed from the reverse transcriptase isolated from HIV.

Biotechniques, 1991 Sep, 11(3), 364 - 6, 368-71
Cloning and expression of antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen in Escherichia coli; Frorath B et al.; Autoantibodies directed against the 68-kDa (U1) ribonucleoprotein antigen are mainly found in sera of patients with mixed connective tissue disease . The corresponding cDNA was fragmented into four regions coding for the major antigenic epitopes A', B', C' and D' . All the epitopes were subcloned and expressed as fusion proteins with the glutathione S-transferase in Escherichia coli using the novel expression system pGEX that allows very high yields of recombinant proteins after a single-step purification . The sera of patients with the autoimmune disease were analyzed for the expressed recombinant proteins by an immunoblotting technique . All positive sera showed a patient-specific behavior and could be divided into four groups regarding recognition of the four antigenic epitopes of the 68-kDa (U1) ribonucleoprotein antigen . The epitope B' was reactive to all patient sera positively tested and classified as the marker antigenic epitope for the mixed connective tissue disease.

Mutat Res, 1991 Sep, 255(2), 143 - 53
Drosophila methyltransferase activity and the repair of alkylated DNA; Guzder SN et al.; The biochemical mechanism and developmental expression for the repair of alkylated DNA has been characterized from Drosophila . As in other organisms, the correction of O6-methylguanine in Drosophila was found to involve the transfer of a methyl group from DNA to a protein cysteine residue . Two methylated proteins with subunit molecular weights of 30 kDa and 19 kDa were identified following incubation with {3H}-methylated substrate DNA and denaturing polyacrylamide gel electrophoresis . Identical molecular weights were found for the unmethylated forms of protein through their reaction to an antibody prepared against the 19 kDa Escherichia coli methyltransferase . Both Drosophila proteins are serologically reactive in adult males and females and most of the other developmental stages tested, with embryos representing the possible exception . The Drosophila proteins do not appear to be induced by sublethal exposures to alkylating agent.

Mol Immunol, 1991 Sep, 28(9), 965 - 73
Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1)--II . Synthetic peptides linked to HIV-1 carrier proteins gag and nef; Neurath AR et al.; Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection . To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier . It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity . The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose . The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS) . Conjugates prepared by the second method elicited approximately 10-100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins . The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nef and the respective conjugates elicited an immune response to these proteins . Despite the low immunization dose of single peptides (10 micrograms) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: greater than 16,000, 1: greater than 80,000 for the nef and gag P18 conjugates, respectively) . Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nef carriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes.

Mol Gen Genet, 1991 Sep, 229(1), 52 - 6
10Sa RNA, a small stable RNA of Escherichia coli, is functional; Oh BK et al.; The ssrA gene, coding for the metabolically stable 10Sa RNA, affects cell growth . A mutant in which the chromosomal 10Sa RNA gene is interrupted by a cat insert does not produce detectable levels of 10Sa RNA, and it grows more slowly than the parental strain.

J Med Microbiol, 1991 Sep, 35(3), 152 - 8
A comparison of probes for restriction fragment length polymorphism (RFLP) typing of Legionella pneumophila serogroup 1 strains; Saunders NA et al.; The use of probes derived from rRNA sequences to detect restriction fragment length polymorphisms (RFLPs) associated with the ribosomal RNA genes for epidemiological typing (ribotyping) is a powerful and readily applicable tool . Different probes and enzymes for ribotyping were compared for a series of 73 unrelated Legionella pneumophila serogroup 1 strains . The probes compared were cDNAs, transcribed from L . pneumophila or Escherichia coli rRNA subunits, and a cloned L . pneumophila rRNA gene . The cloned rRNA gene probe gave the best discrimination and this probe was further compared with cloned probes comprised of randomly selected (non-rRNA) parts of the L . pneumophila chromosome . In this instance the greatest discrimination was achieved when one of the non-ribosomal RNA gene probes was employed . The overall discrimination of RFLP typing was enhanced by combining the data obtained with both rRNA and non-rRNA probes.

Eur J Immunol, 1991 Sep, 21(9), 1973 - 9
Glucocorticoid-dependent and -independent mechanisms involved in lipopolysaccharide tolerance; Evans GF et al.; Injection of bacterial lipopolysaccharide (LPS) into animals results in a transient increase in serum tumor necrosis factor (TNF) . Maximal increases in TNF were detected by 1 h and 3-4 h serum TNF was no longer apparent . These animals were LPS tolerant and a repetitive LPS stimulus did not result in an additional peak in TNF . Regulation of TNF expression in LPS-tolerant animals was at the transcriptional level as TNF mRNA was not apparent in spleen or peritoneal macrophages following a second LPS stimulus . Adrenalectomized (adrex) mice, in contrast, did not become LPS tolerant and sera from these animals demonstrated an additional peak in TNF 1 h following a second LPS stimulus . Concomitant with the secondary rise in serum TNF in adrex mice was an increase in splenic TNF mRNA . The ability of adrex mice to become LPS tolerant was restored by exogenous glucocorticoids . LPS tolerance was also investigated in the galactosamine LPS model which like the adrex model is characterized by a thousandfold increase in the sensitivity of these animals to the lethal effects of LPS . Consistent with the absence of LPS tolerance in adrex mice, galactosamine-sensitized mice were also responsive to a second LPS stimulus and did not become LPS tolerant . While LPS-treated adrex mice had no significant increases in serum corticosterone, corticosterone levels in LPS-treated galactosamine-sensitized mice were comparable to LPS-stimulated normals suggesting that LPS tolerance involves both glucocorticoid-dependent and -independent components . Finally, prophylactic administration of a monoclonal antibody against murine TNF protected normal and galactosamine-sensitized mice from a lethal dose of LPS and yet had no protective effect in adrex animals.

J Bacteriol, 1991 Sep, 173(18), 5732 - 9
Structure and organization of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis; Black DS et al.; High-frequency persistence to the lethal effects of inhibition of either DNA or peptidoglycan synthesis, the Hip phenotype, results from mutations at the hip locus of Escherichia coli K-12 . The nucleotide sequence of DNA fragments which complement these mutations revealed an operon consisting of a possible regulatory region, including sequences with modest homology to an E . coli promoter, and two open reading frames which are translated both in vitro and in vivo . The stop codon of a 264-bp open reading frame, hipB, and the start codon of a 1,320-bp open reading frame, hipA, share an adenine residue . Assays of promoter strength, the location of the probable promoter with respect to the start of transcription, and codon usage all indicate that hipB and hipA are weakly expressed genes . The activity of the promoter is impaired by an adjacent downstream sequence which includes the coding region of hipB . The impairment is partially relieved by insertion of a premature translation termination signal within the coding region of hipB, suggesting involvement of the HipB protein in the regulation of this promoter . The arrangement of hipB and hipA within the operon and the toxicity of hipA for strains defective in or lacking hipB suggest an important interaction between the products of these genes.

J Bacteriol, 1991 Sep, 173(17), 5419 - 30
The cyclic AMP (cAMP)-cAMP receptor protein complex functions both as an activator and as a corepressor at the tsx-p2 promoter of Escherichia coli K-12; Gerlach P et al.; The tsx-p2 promoter is one of at least seven Escherichia coli promoters that are activated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and negatively regulated by the CytR repressor . DNase I footprinting assays were used to study the interactions of these regulatory proteins with the tsx-p2 promoter region and to characterize tsx-p2 regulatory mutants exhibiting an altered response to CytR . We show that the cAMP-CRP activator complex recognizes two sites in tsx-p2 that are separated by 33 bp: a high-affinity site (CRP-1) overlaps the -35 region, and a low-affinity site (CRP-2) is centered around position -74 bp . The CytR repressor protects a DNA segment that is located between the two CRP sites and partially overlaps the CRP-1 target . In combination, the cAMP-CRP and CytR proteins bind cooperatively to tsx-p2, and the nucleoprotein complex formed covers a region of 78 bp extending from the CRP-2 site close to the -10 region . The inducer for the CytR repressor, cytidine, does not prevent in vitro DNA binding of CytR, but releases the repressor from the nucleoprotein complex and leaves the cAMP-CRP activator bound to its two DNA targets . Thus, cytidine interferes with the cooperative DNA binding of cAMP-CRP and CytR to tsx-p2 . We characterized four tsx-p2 mutants exhibiting a reduced response to CytR; three carried mutations in the CRP-2 site, and one carried a mutation in the region between CRP-1 and the -10 sequence . Formation of the cAMP-CRP-CytR DNA nucleoprotein complex in vitro was perturbed in each mutant . These data indicate that the CytR repressor relies on the presence of the cAMP-CRP activator complex to regulate tsx-p2 promoter activity and that the formation of an active repression complex requires the combined interactions of cAMP-CRP and CytR at tsx-p2.

Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7773 - 7
Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein; Stuehr DJ et al.; A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon gamma and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography . SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa . When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NO2- plus NO3- per min per mg . The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 microM, respectively . Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors . Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of approximately 250 kDa but could be dissociated into inactive monomers of approximately 130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin . Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit . Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.

Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7734 - 8
Oligomerization and RNA binding domains of the type 1 human immunodeficiency virus Rev protein: a dual function for an arginine-rich binding motif; Zapp ML et al.; The Rev protein of human immunodeficiency virus type 1 is a sequence-specific RNA binding protein that is essential for viral replication . Here we present evidence that Rev is a stable oligomer both in vitro and in vivo . Analysis of Rev mutants indicates that oligomerization is essential for RNA binding and hence Rev function . The oligomerization and RNA binding domains overlap over 47 amino acids . Within this region is a short arginine-rich motif found in a large class of RNA binding proteins . Substitution of multiple residues within the arginine-rich motif abolishes oligomerization, whereas several single-amino-acid substitution mutants oligomerize but do not bind RNA . Thus, Rev's arginine-rich motif participates in two distinct functions: oligomerization and RNA binding.

Virology, 1991 Sep, 184(1), 428 - 32
Expression of the F glycoprotein gene from human respiratory syncytial virus in Escherichia coli: mapping of a fusion inhibiting epitope; Martin-Gallardo A et al.; A cDNA copy of the gene encoding the entire amino acid sequence of the fusion (F) protein of human respiratory syncytial virus (strain A2) was inserted into a bacterial expression vector containing the lambda PR promoter . Upon heat induction, Escherichia coli cells harboring the vector produced a 45-kDa peptide which reacted with rabbit polyclonal antiserum to the native F protein . Expression of the F gene resulted in severe inhibition of bacterial growth, which was overcome by deletion of the DNA sequences encoding the F signal peptide . The region of the F protein which reacted with a virus-neutralizing and fusion-inhibiting monoclonal antibody was probed by expressing cDNA fragments encoding different protein domains in E . coli and testing antibody reactivity by Western blot analysis . Analysis of six fragments yielded an overlapping antibody-reactive region between amino acids 253 and 298 . Analysis of reactivity with a cassette of synthetic peptides confirmed that the virus-neutralizing epitope mapped between residues 289 and 298 defined by the amino acid sequence M-S-I-I-K-E-E-V-L-A.

EMBO J, 1991 Sep, 10(9), 2489 - 95
Authentic reverse transcriptase is coded by jockey, a mobile Drosophila element related to mammalian LINEs; Ivanov VA et al.; The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms . It is transcribed at different stages of Drosophila ontogenesis . The Drosophila LINE family includes active transposable elements . Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins . As demonstrated here, a 2.23 kb DNA fragment from the region of jockey encoding the putative reverse transcriptase was stably introduced into an expression system under inducible control of the Escherichia coli lac regulatory elements . We describe the expression of the 92 kDa protein and identify this polypeptide alone as the authentic jockey reverse transcriptase based on some of its physical and enzymic properties . The jockey polymerase demonstrates RNA and DNA-directed DNA polymerase activities but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulphydryl reagent . The enzyme prefers poly(rC) and poly(rA) as template and 'activated' DNA is not effective.

Yonsei Med J, 1991 Sep, 32(3), 207 - 14
Identification of mutagenic site of c-H-ras oncogene damaged by N-acetoxyacetylaminofluorene(AAAF); Oh SH et al.; A molecularly cloned human cellular H-ras (c-H-ras) oncogene(pbc N1 plasmid) was treated with N-acetoxyacetylaminofluorene (AAAF) in vitro and subcloned into E.coli . This was done to identify the mutational changes at specific codons of the gene . Guanine nucleotides were identified as the major AAAF binding site of the DNA adduct formed . Base changes in codons 12 and 61 were determined by the analysis of restriction fragment length polymorphism (RFLP) and site specific oligonucleotide hybridization . RFLP was observed due to the loss of the Hpall recognition site at codon 11 and 12 of AAAF-treated c-H-ras gene . Hybridization of AAAF treated c-H-ras with 32P-labeled oligonucleotide probes for the mutant alleles of codon 61 showed no substitutions at codon 61 . From these results, it is assumed that AAAF treatment in vitro caused mutation at codon 12 but not at codon 61 of the c-H-ras oncogene and that codon 12 is the primary target of mutation by AAAF.

Immunology, 1991 Sep, 74(1), 74 - 7
Effect of oestradiol on the secretory immune system in the rat: an increase in biliary IgM antibodies against a T-cell independent antigen; Dahlgren UI et al.; The effect of sex hormones on the secretory immune system was studied in rats ooforectomized and substituted with oestradiol in permeable capsules deposited subcutaneously . Ooforectomized rats and sham-operated rats without oestradiol substitution served as controls . Two weeks after the ooforectomy the rats were immunized in the Peyer's patches with Escherichia coli O6 carrying type 1 fimbriae . Some rats were given a booster dose with the same antigen at the same site 3 weeks later . Bile and serum were taken 7 days after the last immunization . The oestradiol treatment did not influence the total level of IgA or IgG or the level of specific IgA or IgG antibodies in bile or serum . Instead there was a specific increase in biliary IgM antibodies against lipopolysaccharide (LPS) as well as a rise in the total IgM concentration in the bile in the oestradiol-treated rats . Despite this there was no difference in the biliary IgM anti-fimbrial antibody level between the different groups . The oestradiol treatment did not change the levels of total immunoglobulins or antibodies against fimbriae and LPS in serum . An oestradiol-induced increase similar to the one seen in biliary IgM anti-LPS antibodies in primary immunized animals was not seen during the secondary response in booster immunized rats . Thus it seemed as if the effect of oestradiol on the secretory immune system in the bile was mainly due to an influence on primary stimulated B-cell clones in the liver, producing IgM antibodies against a T-cell-independent antigen . The effect may be mediated through a direct action of oestradiol on the B lymphocytes.

J Bacteriol, 1991 Sep, 173(17), 5308 - 14
Roles of fimB and fimE in site-specific DNA inversion associated with phase variation of type 1 fimbriae in Escherichia coli; McClain MS et al.; Evidence obtained with an improved in vivo assay of fimbrial phase variation in Escherichia coli supported a revised understanding of the roles of fimB and fimE in the site-specific DNA rearrangement with which they are associated . A previously proposed model argued that fimB and fimE play antagonistic, unidirectional roles in regulating the orientation of the invertible DNA element located immediately upstream of fimA, the gene encoding the major subunit of type 1 fimbriae . This conclusion, though, is based on an in vivo DNA inversion assay using recombinant plasmid substrates under conditions that, among other things, were incapable of detecting recombination of the fim invertible element from the on to the off orientation . Using a modified system that overcome this and several additional technical problems, we confirmed that fimB acts independently of fimE on the invertible element and that the additional presence of fimE results in the preferential rearrangement of the element to the off orientation . It is now demonstrated that fimE can act in the absence of fimB in this recombination to promote inversion primarily from on to off . In contrast to the previous studies, the effect of fimB on a substrate carrying the invertible element in the on orientation could be examined . It was found that fimB mediates DNA inversion from on to off, as well as from off to on, and that, contrary to prior interpretations, the fimB-associated inversion occurs with only minimal orientational preference to the on phase.

J Bacteriol, 1991 Sep, 173(17), 5298 - 307
Type 1 fimbriation and fimE mutants of Escherichia coli K-12; Blomfield IC et al.; We reexamined the influence of fimE, also referred to as hyp, on type 1 fimbriation in Escherichia coli K-12 . We found that one strain used previously and extensively in the analysis of type 1 fimbriation, strain CSH50, is in fact a fimE mutant; the fimE gene of CSH50 contains a copy of the insertion sequence IS1 . Using a recently described allelic exchange procedure, we transferred the fimE::IS1 allele from CSH50 to our present wild-type strain, MG1655 . Characterization of this IS1-containing strain (AAEC137), together with another fimE mutant of MG1655 (AAEC143), led to two conclusions about the role of fimE . First, the formation of phase variant colony types, reported widely in strains of E . coli, depends on mutation of fimE, at least in K-12 strain MG1655 . Here we showed that this phenomenon reflects the ability of fimE to stimulate the rapid inversion of the fim invertible element from on to off when the bacteria are grown on agar . Second, our analysis of fimE mutants, which is limited to chromosomal constructs, provided no evidence that they are hyperfimbriate . We believe that these results, which are at odds with a previous study using fim-containing multicopy plasmids, reflect differences in gene copy number.

Int J Radiat Biol, 1991 Sep, 60(3), 449 - 52
Can .OH scavengers protect against direct UV-C damage in vivo?
Ewing D.
It has previously been shown that ethanol and tert-butanol protect poly(U) against strand breaks from pulsed laser ultraviolet (UV) photons (lambda = 248 nm) . This protection is believed to be based on a modification of direct DNA damage, not on scavenging hydroxyl radicals . In this paper, several .OH scavengers were tested in vivo with Escherichia coli DNA repair-deficient mutants to see if protection could also be demonstrated with non-laser UV photons (lambda approximately 254 nm) . No protection was observed.

Infect Immun, 1991 Sep, 59(9), 3086 - 93
Heat shock proteins and antigens of Mycobacterium tuberculosis; Young DB et al.; The heat shock response of Mycobacterium tuberculosis has been characterized in detail by one- and two-dimensional polyacrylamide gel electrophoresis after metabolic labeling with {35S}methionine and 14C-amino acids . A temperature increase from 37 to 42 degrees C induced elevated synthesis of three major proteins corresponding to the DnaK, GroEL, and GroES proteins of M . tuberculosis previously identified as prominent antigens . At higher temperatures (45 to 48 degrees C), synthesis of GroEL decreased and novel heat shock proteins with molecular masses of 90, 28, 20, and 15 kDa were observed . These new proteins did not comigrate with known antigens during two-dimensional gel electrophoresis . The heat shock response is discussed with regard to the possible importance of transcriptional regulation of mycobacterial genes in vivo.

J Infect Dis, 1991 Sep, 164(3), 550 - 4
Attaching and effacing enteropathogenic Escherichia coli as a cause of infantile diarrhea in Bangkok; Echeverria P et al.; To identify Escherichia coli that cause infantile diarrhea in Bangkok, Thailand, E . coli isolated in a year-long study of infantile diarrhea were examined for O and H serotypes and virulence determinants . Classic enteropathogenic E . coli (EPEC) were isolated from 28 of 509 infants with diarrhea (cases) and 11 of 509 age-matched controls (P = .009; odds ratio {OR}, 2.64) . Most of this difference was attributable to EPEC adherence factor (EAF)-positive EPEC that produced an attachment and effacement lesion, as identified in the fluorescence actin staining assay, isolated from 13 cases and 1 control (P = .003; OR, 13.3) . EAF-EPEC was isolated from 15 cases and 10 controls (P = .418; OR, 1.52) and EAF+ non-EPEC from 17 cases and 10 controls (P = .242; OR, 1.72) . EAF+EPEC that caused an attachment and effacement lesion was found in 3% of children less than 6 months old with diarrhea who were studied in an outpatient clinic in Bangkok in 1988.

J Biochem (Tokyo), 1991 Sep, 110(3), 443 - 9
Amplification and substantial purification of cardiolipin synthase of Escherichia coli; Hiraoka S et al.; A simple, specific, and sensitive assay procedure for cardiolipin synthase of Escherichia coli has been developed . This measures the radioactivity of glycerol formed from phosphatidyl {2-3H}glycerol and is mainly based on the findings that 400 mM phosphate and 0.015% Triton X-100 markedly activate the enzyme . Cardiolipin synthase was amplified 760-fold upon induction with isopropyl beta-D-thiogalactoside in cells harboring a pBR322 derivative in which the cls gene encoding this enzyme was preceded by the tac promoter . Under these conditions, cardiolipin content increased, membrane potential decreased, spheroplasts became fragile, cells lost viability, and inducer-resistant mutants appeared at a high frequency . The amplification enabled the isolation of an enzyme preparation with a specific activity approximately 10,000-times higher than that of wild-type whole cell lysate . This purification was simply achieved by extraction of the crude membrane fraction with Triton X-100 and a single phosphocellulose column chromatography . This preparation, together with the crude envelope fraction, was used to characterize the basic properties of E . coli cardiolipin synthase, some of which were utilized in setting up the assay conditions.

Mol Microbiol, 1991 Sep, 5(9), 2093 - 8
Energy requirements for protein translocation across the Escherichia coli inner membrane; Geller BL; Both ATP and an electrochemical potential play roles in translocating proteins across the inner membrane of Escherichia coli . Recent discoveries have dissected the overall transmembrane movement into separate subreactions with different energy requirements, identified a translocation ATPase, and reconstituted both energy-requiring steps of the reaction from purified components . A more refined understanding of the energetics of this fundamental process is beginning to provide answers about the basic issues of how proteins move across the hydrophobic membrane barrier.

Circ Shock, 1991 Sep, 35(1), 53 - 9
A PAF receptor antagonist, BN 52021, attenuates thromboxane release and improves survival in lethal canine endotoxemia; Moore JM et al.; Platelet-activating (PAF) is a putative mediator in endotoxemia and sepsis . Administration of a PAF receptor antagonist prior to endotoxin improves survival in rats and attenuates the hypotension of endotoxemia . Both PAF and endotoxin stimulate eicosanoid production . We hypothesized that a PAF receptor antagonist, BN 52021, would alter the hemodynamic events, improve the survival and attenuate the eicosanoid release associated with endotoxemia in a resuscitated, but lethal, canine model . Male dogs were randomzied to two groups (n = 10 each) . Group I received only E . coli endotoxin, 1 mg/kg IV, at time 0, while group II received BN 52021, 5 mg/kg IV, 30 min before and again 240 min after endotoxin treatment . During the 4-h study period, hemodynamics were measured and blood samples were taken at 0, 2, 60, 120, and 240 min . Survival was determined at 24, 48, and 72 h . All group I animals died before 24 h; all group II lived longer than 72 h (P less than 0.05) . In group I, plasma TXB2 values increased from a baseline value of 0.26 +/- .04 ng/ml to 4.38 +/- 1.56 ng/ml at 120 min and then decreased to 2.64 +/- .96 ng/ml by 240 min . For group II, respective plasma TXB2 values were 0.35 +/- 0.13 ng/ml at baseline, 0.58 +/- 0.14 ng/ml at 120 min, and 0.39 +/- .09 ng/ml at 240 min . At the 120-min and 240-min time points, the groups differed at P less than 0.05 . Heart rate tended to be less in group II, but MAP was unaffected . In group I, pH values were more acidotic than those observed in group II.

Antimicrob Agents Chemother, 1991 Sep, 35(9), 1891 - 9
Appearance of a new trimethoprim resistance gene, dhfrIX, in Escherichia coli from swine; Jansson C et al.; A new gene, dhfrIX, coding for a trimethoprim-resistant dihydrofolate reductase (DHFR), was found in porcine isolates of Escherichia coli . The new enzyme, DHFR IX, containing 178 amino acids, showed an amino acid similarity of about 26% with DHFR I and the chromosomal DHFR of E . coli K-12 . The dhfrIX gene was observed to occur on two distinctly different transferable plasmids, although a fragment of about 2.9 kb, including dhfrIX, had an identical restriction enzyme digestion map in each case . The new plasmid-borne dhfrIX gene mediates resistance to a drug level of only about 250 micrograms/ml, as compared with more than 1,000 micrograms/ml for the more frequently encountered dhfrI gene . The new plasmid-borne trimethoprim resistance gene could have been selected and spread as a consequence of the extensive use of trimethoprim in veterinary practice in Sweden . It will be important to try to follow its possible occurrence in human pathogens as well.

Clin Sci (Lond), 1991 Sep, 81(3), 357 - 65
Time course of plasma and pulmonary lymph endothelin-like immunoreactivity during sustained endotoxaemia in chronically instrumented sheep; Morel DR et al.; 1 . Endothelin, a novel vasoconstrictor 21-residue peptide isolated from the supernatant of cultured porcine endothelial cells, has been shown to be increased in plasma in a variety of cardiovascular disease states, including acute myocardial infarction, acute renal failure and essential hypertension . We determined the time course of plasma and pulmonary lymph endothelin-like immunoreactivity in relation to the progressive deterioration of cardiopulmonary function in an ovine septic shock model leading to multi-organ failure syndrome and death within 42 h of a continuous intravenous infusion of Escherichia coli endotoxin (40 ng min-1kg-1) . 2 . Plasma and pulmonary lymph endothelin-like immunoreactivity were measured by r.i.a . using a specific antiserum raised in rabbits against porcine endothelin-1 . Endothelin-like immunoreactivity was further determined in lung tissue and the thoracic duct lymph of endotoxin-treated sheep by reversed-phase h.p.l.c . In control instrumented conscious sheep not infused with endotoxin, there were no significant changes in any of the measured cardiopulmonary and biochemical variables, with plasma and pulmonary lymph endothelin-like immunoreactivity remaining below the detection limit (less than 1 pg/tube) throughout the 72 h study period . 3 . Conscious sheep receiving endotoxin showed a major hypotensive septic syndrome, including persistently decreased systemic blood pressure, systemic vascular resistance, stroke volume, left ventricular stroke work, associated with sustained pulmonary vasoconstriction and protein-rich pulmonary oedema (greater than five-fold increase in pulmonary lymph flow and protein clearance), and marked lactic acidosis, leading to the death of animals within 14-42 h despite institution of mechanical ventilation and adequate intravascular volume replacement.(ABSTRACT TRUNCATED AT 250 WORDS)

Clin Sci (Lond), 1991 Sep, 81(3), 313 - 7
Effect of age on hypothalamic prostaglandin E2 production and fever in response to tumour necrosis factor (cachectin) and endotoxin in rats; Bibby DC et al.; 1 . Decreased febrile responses to interleukin-1 and endotoxin have been noted in a number of species with ageing . 2 . The present study extends these observations by examining the pyrogenic response to intravenous recombinant human tumour necrosis factor-alpha (50 micrograms/kg) using conscious rats aged 7,20 and 80 weeks . 3 . The febrile response decreased in magnitude and duration with age . Fevers of 0.9 degree C and of 5 h duration were observed in the youngest rats, whereas those aged 80 weeks were afebrile . The depression in serum zinc level and the elevation in liver zinc level, which occurred 7 h after injection, were unaffected by age . 4 . The mechanism of the reduced pyrogenic response was examined by assessing prostaglandin E2 production in vitro from hypothalami of rats, aged 10 and 24 weeks, in response to Escherichia coli endotoxin and tumour necrosis factor . 5 . Whereas the production of prostaglandin E2 increased by 47% and 52%, respectively, in hypothalami from 10-week-old rats, no response to either pyrogen was obtained in tissue from rats aged 24 weeks . 6 . Maturity brings about a decreased responsiveness of hypothalamic prostaglandin E2 production to pyrogens, which may explain the decreased febrile responses observed.

Mol Gen Genet, 1991 Sep, 229(1), 10 - 6
Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage; Woodgate R et al.; Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon . RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD') that is active for mutagenesis . Processing of UmuD is therefore a critical step in the fixation of mutations . In this paper we have analyzed the requirements for UmuD processing in vivo . Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E . coli extracts . We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing . Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage . Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions . UmuD was present at approximately 180 copies per uninduced cell and was measured at approximately 2400 copies per cell in strains that lacked a functional repressor . Induced levels of UmuC were approximately 12-fold lower than UmuD with approximately 200 molecules per cell . These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.

J Gen Virol, 1991 Sep, 72 ( Pt 9), 2269 - 74
Immunofluorescent detection of bovine papillomavirus E4 antigen in the cytoplasm of cells permissive in vitro for viral DNA amplification; Jareborg N et al.; The E4 gene of several human papillomavirus types is expressed in association with vegetative viral DNA synthesis in differentiated epidermal cells . To develop reagents to study expression of the bovine papillomavirus type 1 (BPV-1) E4 gene in warts and in virus-transformed cell lines, rabbit polyclonal antiserum was raised to the BPV-1 E4 antigen produced as a fusion polypeptide in Escherichia coli . By immunoblotting analysis of productively infected bovine fibropapilloma tissue, E4-related proteins of 16K, 21K, 30K and 42K were detected . In some but not all C127 cell lines transformed by BPV-1 or by a replication-competent BPV-1 deletion mutant, cytoplasmic E4 antigen with a predominantly perinuclear localization was detected by immunofluorescence analysis in a subpopulation of cells in stationary-phase cultures . The E4-expressing cells were identified by their grossly enlarged size to represent the same cell subpopulation shown earlier to support BPV-1 DNA amplification . The observation of synthesis of the E4 protein in association with viral DNA amplification in this system provides further evidence that there is a switch in viral early region gene expression in a subpopulation of division-arrested cells, which may accurately reflect events occurring during the vegetative phase of BPV-1 replication in terminally differentiated cells in vivo.

Eur J Biochem, 1991 Sep 1, 200(2), 471 - 6
An in vitro model showing different rates of substrate cycle for phosphofructokinases of Escherichia coli with different kinetic properties; Torres JC et al.; An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fructose-bisphosphatase . The model is applied to the study of three PFK of Escherichia coli: two isoenzymes, phosphofructokinase-1 (PFK-1) and phosphofructokinase-2 (PFK-2), and a mutant form of phosphofructokinase-2 (PFK-2*) . Results show that for a variety of conditions the PFK-1/fructose-bisphosphatase pair gives the lowest and the PFK-2*/fructose-bisphosphatase pair the highest rates of substrate cycle, with the PFK-2/fructose-bisphosphatase pair in an intermediate position . The effects of variables such as maximum activity ratios and MgATP concentration were explored . The possible role of MgATP in decreasing the futile cycle of the PFK-2/fructose-bisphosphatase pair is described . The results are discussed in terms of possible metabolic consequences of PFK-2* and of predictions of the model to be tested in vivo.

J Clin Invest, 1991 Sep, 88(3), 811 - 6
Altered transcriptional regulation of phosphoenolpyruvate carboxykinase in rats following endotoxin treatment; Hill M et al.; The molecular mechanism involved in altered regulation of the rate-limiting enzyme in hepatic gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK), during endotoxemia is not completely understood . We examined, therefore, the effect of a nonlethal dose of Escherichia coli endotoxin on PEPCK gene expression in fasted rats . 5 h after endotoxin treatment, the PEPCK transcription rate and the amount of mRNA(PEPCK) were significantly decreased at a time when the insulin/glucagon (I/G) molar ratio and plasma corticosterone levels were significantly increased . Similar results were observed in a time course study, in which altered cAMP induction of PEPCK gene expression paralleled changes in the I/G molar ratio . In diabetic rats treated with endotoxin, PEPCK gene expression was decreased in the absence, however, of an increased I/G molar ratio . This finding indicates that other factors, such as inflammatory mediators or cytokines, alter PEPCK gene transcription during endotoxemia . IL-6, a putative mediator of endotoxin action in the liver, had no effect on PEPCK gene expression in fasted rats, but did decrease cAMP induction of PEPCK gene expression . These results indicate that, during endotoxemia, regulation of PEPCK gene expression is influenced by inflammatory mediators in addition to the classical endocrine hormones . IL-6, however, does not appear to be involved directly in the altered regulation of the PEPCK gene during endotoxemia.

J Bacteriol, 1991 Sep, 173(18), 5901 - 8
Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1; Chistoserdov AY et al.; An isolated 5.2-kb fragment of Methylobacterium extorquens AM1 DNA was found to contain a gene cluster involved in methylamine utilization . Analysis of polypeptides synthesized in an Escherichia coli T7 expression system showed that five genes were present . Two of the genes encoded the large and small subunits of methylamine dehydrogenase, and a third encoded amicyanin, the presumed electron acceptor for methylamine dehydrogenase, but the function of the other two genes is not known . The order on the 5.2-kb fragment was found to be large-subunit gene, the two genes of unknown function, small-subunit gene, amicyanin gene . The gene for azurin, another possible electron acceptor in methylamine oxidation, does not appear to be present within this cluster of methylamine utilization genes.

J Bacteriol, 1991 Sep, 173(18), 5887 - 92
Nucleotide sequence of the gene (ard) encoding the antirestriction protein of plasmid colIb-P9; Delver EP et al.; The IncI1 plasmid ColIb-P9 was found to encode an antirestriction function . The relevant gene, ard (alleviation of restriction of DNA), maps about 5 kb from the origin of transfer, in the region transferred early during bacterial conjugation . Ard inhibits both restriction and modification by each of the four type I systems of Escherichia coli tested, but it had no effect on restriction by either EcoRI, a type II system, or EcoP1, a type III system . The nucleotide sequence of the ColIb ard gene was determined; the predicted molecular weight of the Ard polypeptide is 19,193 . The proposed polypeptide chain contains an excess of 25 negatively charged amino acids, suggesting that its overall character is very acidic . Deletion analysis of the gene revealed that the Ard protein contained a distinct functional domain located in the COOH-terminal half of the polypeptide . We suggest that the biological role of the ColIb Ard protein is associated with overcoming host-controlled restriction during bacterial conjugation.

J Bacteriol, 1991 Sep, 173(18), 5808 - 21
Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme; Murphy KC; The lambda Gam protein was isolated from cells containing a Gam-producing plasmid . The purified Gam protein was found to bind to RecBCD without displacing any of its subunits . Gam was shown to inhibit all known enzymatic activities of RecBCD: ATP-dependent single- and double-stranded DNA exonucleases, ATP-independent single-stranded endonuclease, and the ATP-dependent helicase . When produced in vivo, Gam inhibited chi-activated recombination in lambda red gam crosses but had little effect on the host's ability to act as a recipient in conjugational recombination . These experiments suggest that RecBCD possesses an additional "unknown" activity that is resistant to or induced by Gam . Additionally, the expression of Gam in recD mutants sensitizes the host to UV irradiation, indicating that Gam alters one or more of the in vivo activities of RecBC(D-).

J Bacteriol, 1991 Sep, 173(18), 5771 - 7
Characterization of insertion sequence IS892 and related elements from the cyanobacterium Anabaena sp . strain PCC 7120; Cai Y; IS892, one of the several insertion sequence (IS) elements discovered in Anabaena sp . strain PCC 7120 (Y . Cai and C . P . Wolk, J . Bacteriol . 172:3138-3145, 1990), is 1,675 bp with 24-bp near-perfect inverted terminal repeats and has two open reading frames (ORFs) that could code for proteins of 233 and 137 amino acids . Upon insertion into target sites, this IS generates an 8-bp directly repeated target duplication . A 32-bp sequence in the region between ORF1 and ORF2 is similar to the sequence of the inverted termini . Similar inverted repeats are found within each of those three segments, and the sequences of these repeats bear some similarity to the 11-bp direct repeats flanking the 11-kb insertion interrupting the nifD gene of this strain (J . W . Golden, S . J . Robinson, and R . Haselkorn, Nature {London} 314:419-423, 1985) . A sequence similar to that of a binding site for the Escherichia coli integration host factor is found about 120 bp from the left end of IS892 . Partial nucleotide sequences of active IS elements IS892N and IS892T, members of the IS892 family from the same Anabaena strain, were shown to be very similar to the sequence of IS892.

J Bacteriol, 1991 Sep, 173(17), 5564 - 7
Novobiocin-dependent topA deletion mutants of Escherichia coli; Hammond GG et al.; Previous reports of the transduction of topA deletions in Escherichia coli suggested that delta top A transductants grow normally only if they acquire spontaneous mutations that compensate for the topoisomerase I defect . We show that P1-mediated transduction of delta topA in the presence of sublethal concentrations of novobiocin, an inhibitor of the DNA gyrase B subunit, yields uncompensated Top- isolates which are dependent on novobiocin for optimum growth . In the absence of novobiocin these delta topA strains grow slowly, indicating that topA deletions are deleterious but not lethal to the cell . We propose that inhibitors of DNA gyrase B, presumably by lowering intracellular levels of DNA supercoiling, can phenotypically suppress a topoisomerase I defect in E . coli.

J Bacteriol, 1991 Sep, 173(17), 5551 - 3
Gyrase inhibitors increase the content of knotted DNA species of plasmid pBR322 in Escherichia coli; Ishii S et al.; Treatment of Escherichia coli cells harboring pBR322 with the DNA gyrase inhibitors oxolinic acid and coumermycin A1 led to an increase in the content of knotted pBR322 molecules . This phenomenon was attributed to inhibition of gyrase-catalyzed unknotting of the plasmid DNA knotted by transcription.

J Bacteriol, 1991 Sep, 173(17), 5414 - 8
Conjugational recombination in resolvase-deficient ruvC mutants of Escherichia coli K-12 depends on recG; Lloyd RG; ruvC mutants of Escherichia coli appear to lack an activity that resolves Holliday intermediates into recombinant products . Yet, these strains produce close to normal numbers of recombinants in genetic crosses . This recombination proficiency was found to be a function of recG . A "mini-kan" insertion in recG was introduced into ruvA, ruvB, and ruvC strains . Conjugational recombination was reduced by more than 100-fold in recG ruvA::Tn10, recG ruvB, and recG ruvC strains and by about 30-fold in a recG ruvA strain carrying a ruvA mutation that is not polar on ruvB . The double mutants also proved very deficient in P1 transduction and are much more sensitive to UV light than ruv single mutants . Since mutation of recG alone has very modest effects on recombination and sensitivity to UV, it is concluded that there is a functional overlap between the RecG and Ruv proteins . However, this overlap does not extend to circular plasmid recombination . The possibility that RecG provides a second resolvase that can substitute for Ruv is discussed in light of these findings.

Infect Immun, 1991 Sep, 59(9), 2955 - 62
Superoxide generation by human neutrophils induced by low doses of Escherichia coli hemolysin; Bhakdi S et al.; Escherichia coli hemolysin (Hly) was isolated from bacterial culture supernatants by polyethylene glycol precipitation and centrifugation in glycerol density gradients . The toxin preparations contained less than 1 mol of lipopolysaccharide per 10 mol of protein, and they had no fatty acids . The capacity of purified hemolysin to stimulate superoxide anion production in polymorphonuclear leukocytes was monitored kinetically in a lumimeter by using the lucigenin assay and was correlated with the kinetics of transmembrane pore formation . When applied to leukocytes suspended in protein-free buffer, very low concentrations (0.02 to 0.1 HU/ml) of the toxin strongly stimulated the production of superoxide anions; shortly thereafter, irreversible membrane permeabilization occurred . When the toxin was applied at concentrations exceeding 0.2 to 0.3 HU/ml, membrane permeabilization was so rapid that the cells were unable to mount a respiratory burst . When applied in the narrow range of 0.05 to 0.1 HU/ml, E . coli hemolysin rivaled phorbol myristate acetate in its capacity to stimulate production of superoxide anions . Additionally, hemolysin applied at doses that elicited no pore formation (0.01 to 0.02 HU/ml) primed leukocytes for an augmented response to subsequent challenge by the phorbol ester . These data demonstrate that very low doses of E . coli hemolysin can evoke cellular reactions that appear independent of and precede transmembrane pore formation and cell death.

Mol Cell Biol, 1991 Sep, 11(9), 4786 - 95
Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site; Gibbs JS et al.; Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities . For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide . The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity . This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain . We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase . Two mutants were severely impaired for viral DNA replication and polymerase activity . The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding . The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E . coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.

Virology, 1991 Sep, 184(1), 437 - 40
Genome cloning and analysis of the large RNA segment (segment A) of a naturally avirulent serotype 2 infectious bursal disease virus; Kibenge FS et al.; The genome of infectious bursal disease virus (IBDV) of serotype 2 (strain OH) has been cloned, and 3171 nucleotides of genome segment A cDNA sequence have been determined for the first time . Sequence homology of OH-IBDV with the most distant serotype 1 IBDV at the nucleotide level is 83.1%, and the amino acid sequence homology of the polyprotein is 89.6% . Alignment of the polyprotein amino acid sequences showed the hypervariable region in VP2 to be 151-152 amino acid residues long in IBDV . A second variable region, 37 amino acid residues long, was identified in the N-terminal third of the IBDV VP2 molecule . IBDV strains, like the IPNV strains, also contain inverted repeats that may form stem-and-loop structures in the 5' noncoding sequences . These inverted repeats are variable between the two IBDV serotypes, particularly at the AT basepairs.

Virology, 1991 Sep, 184(1), 370 - 82
Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence; Upton C et al.; Poxviruses are known to contain a large number of open reading frames, particularly near the termini of the viral genome, that are not required for growth in tissue culture . However, many of these gene products are believed to play important roles in determining the virulence of the virus by modulating the host immune response to the infection . Recently it has been shown that Shope fibroma virus encodes, within the terminal inverted repeats, a protein (T2) related to the cellular tumor necrosis factor receptor (TNFR) and which specifically binds both TNF alpha and TNF beta . We have sequenced the terminal regions of two other Leporipoxviruses (myxoma virus and malignant rabbit fibroma virus) that are extremely invasive and capable of inducing extensive immunosuppression in rabbits and demonstrate that they also encode a closely related T2 homolog with all the structural motifs predicted for a secreted TNF binding protein . To investigate the biological role of the T2 protein, we have inactivated the myxoma virus T2 gene within each copy of the viral TIR by the insertion of a dominant selectable marker (Escherichia coli guanosine phosphoribosyltransferase) and selection of the recombinant virus in the presence of mycophenolic acid . The success of the inactivation of both copies of T2 was confirmed by the loss a broad protein band (52-56 kDa) of the predicted size for T2 from the profile of proteins secreted from mutant virus-infected BGMK cells at early times after infection . Although the T2-minus recombinant myxoma virus grew normally in tissue culture, upon infection of susceptible rabbits the viral disease was observed to be significantly attenuated . The majority of infected rabbits were able to mount an effective immune response to the infection and completely recovered . These survivor rabbits became immune to subsequent challenge with wild type myxoma virus . We conclude that the T2 viral protein is an important secreted virulence factor and that it in all likelihood functions by compromising the antiviral effects of TNF . We propose the term "viroceptor" to describe viral-encoded homologs of cellular lymphokine receptors whose function is to intercept the activity of the cognate lymphokine in order to short circuit the host immune response to the viral infection.

Virology, 1991 Sep, 184(1), 330 - 40
Cooperation of EBV DNA polymerase and EA-D(BMRF1) in vitro and colocalization in nuclei of infected cells; Kiehl A et al.; Expression of the Epstein-Barr virus (EBV) DNA polymerase (EBVpol) open reading frame (BALF5) by in vitro transcription-translation yielded a 116-kDa primary translation product . Enzymatic DNA polymerase activity of the in vitro translated polypeptide required the presence of the 47-kDa BMRF1 (EA-D) gene product . Antiserum raised to the BALF5 gene product expressed in Escherichia coli specifically precipitated a 116-kDa polypeptide in extracts of latently infected lymphoblastoid cells induced for EBV replication . Immunofluorescence microscopy revealed colocalization of the EBVpol and EA-D(BMRF1) to discrete foci within the nuclei of induced cells; however, the blockade of viral DNA synthesis resulted in diffuse nuclear staining patterns for both antigens . Bromodeoxyuridine staining of these discrete foci colocalizing with EBVpol suggests that they are sites of early viral DNA synthesis . These observations suggest that EA-D(BMRF1) may be an accessory protein of the EBV DNA polymerase which colocalizes in vivo with EBVpol to sites of viral DNA replication and cooperates in vitro to form an active EBVpol holoenzyme.

Virology, 1991 Sep, 184(1), 33 - 42
Studies on vaccination against papillomaviruses: prophylactic and therapeutic vaccination with recombinant structural proteins; Jarrett WF et al.; The L1 and L2 proteins of BPV-2 have been produced in Escherichia coli as beta-galactosidase fusion proteins . The fusion proteins have been used to vaccinate calves both prophylactically and therapeutically . The L1 fusion protein prevented tumor formation when administered before challenge with BPV-2, while the L2 fusion protein was very effective in promoting tumor rejection, independently from whether it was administered before or after challenge . Animals vaccinated with L1, but not with L2, responded rapidly with production of serum neutralizing antibodies, showing that this peptide contains B-cell-specific epitopes . The massive infiltration of lymphocytes in the tumors of L2-vaccinated animals suggests that the peptide contains epitopes specific for T-cells . The two structural proteins of BPV-2 therefore interact with both efferent arms of the immune system, and this observation allows the choice between two different types of antiviral vaccination.

Virology, 1991 Sep, 184(1), 310 - 8
Adeno-associated viruses having nonsense mutations in the capsid genes: growth in mammalian cells containing an inducible amber suppressor; Smuda JW et al.; When an adeno-associated virus (AAV) genome contained in a recombinant plasmid is transfected into adenovirus-infected cells, infectious AAV particles are efficiently generated . We previously described the construction of a conditional lethal mutant of AAV having an amber termination codon inserted in the rep gene . This mutant was propagated on a monkey kidney cell line (SupD12) having an inducible amber suppressor tRNAser . We now describe the construction and propagation of two additional conditional lethal mutants of AAV having amber codons affecting all three capsid proteins (AAV Capam) or only the VP1 capsid protein (AAV VP1am) . Suppression of the amber mutations in the capsid proteins was demonstrated directly by immunoblot analysis . The efficiency of amber suppression on the SupD12 cell was about 6 to 10% for AAV VP1am and 4 to 5% for AAV Capam . The reversion frequency of either mutant was apparently less than 10(-5) . On nonsuppressing cells AAV VP1am exhibited an Lip (Inf) phenotype, whereas AAV Capam exhibited a Cap phenotype.

J Virol, 1991 Sep, 65(9), 4565 - 72
Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif; Jablonski SA et al.; RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule . Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G . The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity . Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive . Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme . An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity . These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.

EMBO J, 1991 Sep, 10(9), 2689 - 94
Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor; Reygers U et al.; DNA helicase I, the traI gene product of the Escherichia coli F factor, was shown to be associated with endonuclease activity specific for the transfer origin of the F plasmid, oriT . In the presence of Mg2+, the purified enzyme forms a complex, stable in the presence of sodium dodecylsulfate (SDS) with a negatively superhelical chimeric plasmid containing oriT . The enzyme nicks and, after this, apparently binds to the 5' nick terminus when this complex is heated in the presence of SDS and/or EDTA or treated with proteinase K . Dideoxy sequencing locates the nick site in the F DNA strand transferred during bacterial conjugation after nucleotide 138 clockwise of the mid-point of the BglII site at 66.7 kb of the F genetic map . A sequencing stop after nucleotide 137 of this strand (where oriT-nicking seems to occur in vivo) is possibly an artefact caused by helicase I protein attached to the 5' terminal nucleotide . Deletion in the amino-terminal part of the traI polypeptide abolishes the oriT-nicking activity while leaving the strand-separating activity intact . These results confirm the prediction from genetic studies that helicase I is bifunctional with site-specific endonuclease and strand-separating activities.

EMBO J, 1991 Sep, 10(9), 2613 - 20
The location of mRNA in the ribosomal 30S initiation complex; site-directed cross-linking of mRNA analogues carrying several photo-reactive labels simultaneously on either side of the AUG start codon; Dontsova O et al.; Messenger RNA molecules 30-35 bases long, with sequences related to the 5'-region of cro-mRNA from lambda-phage, were prepared by T7 transcription from synthetic DNA templates . Each mRNA contained five or six internal uridine residues, which were transcribed using a mixture of UTP and thio-UTP . Initiation complexes were formed with Escherichia coli 30S ribosomes in the presence or absence of tRNA(fMet), and cross-linking of the thio-U residues was induced by UV irradiation at wavelengths greater than 300 nm . The cross-linked ribosomal proteins were identified immunologically, and cross-linked regions of the 16S RNA were isolated by excision with ribonuclease H and suitable deoxyoligonucleotides . In both cases, the particular thio-U residue involved in the cross-link was identified by ribonuclease T1 fingerprinting of the (radioactive) mRNA in the isolated cross-linked complex . The principal results were that, at thio-U positions upstream of the AUG codon, specific cross-linking occurred to protein S7 and to the 3'-terminus of the 16S RNA, in agreement with similar experiments using 70S ribosomes . Less specific cross-linking was observed to proteins S1, S18 and S21 at various positions within the mRNA . Six bases downstream from the AUG codon, a tRNA-dependent cross-link was found to position approximately 1050 of the 16S RNA, but--in contrast to similar experiments with 70S ribosomes--no cross-linking was found to the 1390-1400 region.

EMBO J, 1991 Sep, 10(9), 2583 - 8
Gyrase-dependent stabilization of pSC101 plasmid inheritance by transcriptionally active promoters; Beaucage SL et al.; The pSC101 plasmid encodes a cis-acting genetic locus termed par that ensures the stable inheritance of plasmids in a population of dividing cells . In the absence of selection, par-defective plasmids are lost rapidly from the bacterial population . We report here that the stability of par-deleted pSC101 derivatives is restored by introducing certain adventitious bacterial promoters onto the plasmid . Stabilization requires active transcription from the inserted promoter and is affected by the site and orientation of the insertion, the length of the nascent transcript and DNA gyrase activity . While a promotor-associated overall increase in negative superhelicity of plasmid DNA was observed, stabilized inheritance appeared to be dependent on localized rather than generalized supercoiling . Our demonstration that promoter-induced DNA supercoiling can mimic the effects of the pSC101 par locus provides evidence that the previously reported superhelicity-generating effects of par are intrinsic to its function.

EMBO J, 1991 Sep, 10(9), 2553 - 8
Vaccinia virus capping enzyme is a transcription initiation factor; Vos JC et al.; It has previously been demonstrated that vaccinia virus capping enzyme is involved both in the formation of a 5' cap structure and in termination of early transcription . Here we show that capping enzyme has an additional activity which is required for transcription of intermediate genes . VITF-A and VITF-B have been defined as two activities which together with RNA polymerase are necessary and sufficient to transcribe intermediate genes in vitro . VITF-A and the viral capping enzyme are shown to copurify to near homogeneity . Direct evidence that capping enzyme is VITF-A was obtained by complementation of a reconstituted transcription system with viral capping enzyme expressed in Escherichia coli . Although capping enzyme is a cofactor in early transcription termination, intermediate transcription is not terminated in response to the early termination signal . Capping enzyme is shown to form a complex with RNA polymerase in the absence of VITF-B . This appears to be a prerequisite for the formation of a stable initiation complex.

Agric Biol Chem, 1991 Sep, 55(9), 2259 - 64
Gene encoding a putative zinc finger protein in Synechocystis PCC6803; Ogura Y et al.; A 5.5-kb HindIII fragment of Synechocystis PCC6803 containing a liverwort (ORF316) homolog encoding a putative zinc finger protein was cloned . Nucleotide sequence analysis showed that the homology of the amino acid sequence deduced from the ORF326 of Synechocystis PCC6803 with the counterparts of a liverwort and tobacco was 50% and 46%, respectively . Synechocystis ORF326 also showed 38% homology with the dedB gene in Escherichia coli . The gene organization of the region in these species of organisms was quite different . This suggests that the Synechocystis ORF326 and liverwort ORF316 genes may be related to a common regulatory gene, but not photosynthetic gene characteristic to chloroplasts.

Enzyme Microb Technol, 1991 Sep, 13(9), 708 - 15
Preliminary assessment of removal of pyrogenic lipopolysaccharides with colloidal zirconia adsorbents; Karl DW et al.; Preliminary evaluation of bare or polymer-coated colloidal monoclinic zirconia of nominal particle size 100 nm indicated that it is an effective adsorbent for pyrogenic lipopolysaccharides (LPS) as measured by chemical and Limulus amebocyte lysate (LAL) assays . Zirconia at 50 micrograms ml-1 adsorbed 99.95% of added E . coli O128 LPS . Residual LPS levels below 0.1 ng ml-1 were easily attained . Colloidal zirconia was able to remove LPS from solution in the presence of bovine albumin (BSA) . Some LPS contaminating BSA lacked affinity for zirconia . Preadsorption of phosphate onto bare zirconia blocked LPS adsorption . However, phosphated-oligomeric glycidyl (epoxy) pentaerythritol-coated colloidal zirconia could be derivatized with imidazole-containing ligands to produce an LPS-binding surface . Preliminary results of adsorption of LPS by the coated particles indicated a reduced level of LPS binding compared to bare zirconia, probably because the particles aggregated during the derivatization process, reducing the effective surface available for LPS adsorption.

Biotechnology (N Y), 1991 Sep, 9(9), 873 - 7
Rapid clonal growth measurements at the single-cell level: gel microdroplets and flow cytometry; Weaver JC et al.; We describe a new, general method for rapidly measuring clonal growth of large numbers of individual members of a cell population . This method is based on microculture of individual colony-forming units in gel microdrops (GMDs; here agarose; 20 to 90 mu in diameter), which are sufficiently robust to be handled much like cells, and diffusionally transparent for molecules of interest . Flow cytometry provides rapid measurements of GMD-entrapped microcolonies, and permits subpopulation analysis . Here the method is demonstrated with mammalian, fungal and bacterial species . Additional results illustrate rapid determination of a drug-resistant subpopulation in a mixed species sample, and nutrient sensitivity for a murine hybridoma.

Biotechnology (N Y), 1991 Sep, 9(9), 869 - 72
Secretion of active bovine somatotropin in Escherichia coli; Klein BK et al.; We have expressed a chimeric protein, comprising the LamB secretion signal sequence fused to mature bovine somatotropin (bST), in Escherichia coli . Plasmid constructs with the recA promoter showed significant protein accumulation prior to induction and cell lysis occurred after induction . In contrast, the lacUV5 promoter was tightly regulated . With the lacUV5 promoter, temperature and inducer concentration had significant effects on the total amount of recombinant protein produced and the fraction processed to mature bST . Quantitation of bST from shake flask cultures showed that 1-2 micrograms/ml/OD550 could be released from the periplasm by osmotic shock . N-terminal sequence analysis of the purified protein indicated that the majority of the secreted bST was correctly processed . The bST present in the osmotic shock fraction was judged to be correctly folded by comigration with oxidized methionyl-bST standard on a non-reducing polyacrylamide gel and activity in a bovine liver radioreceptor assay . These results provide a rapid method to produce bST for use in structure-function studies.

Gene, 1991 Aug 30, 105(1), 23 - 9
Functional analysis of a C-terminally altered TonB protein of Escherichia coli; Anton M et al.; Deletions within the 3'-end of the cloned Escherichia coli tonB gene were constructed and recombined into the chromosome . Deletions affecting the last eight C-terminal amino acids (aa) yielded functionally active TonB, whereas deletion of the last 15 C-terminal aa, which removed the C-terminal hydrophobic region of TonB, abolished TonB function entirely . Distribution of TonB within cell compartments was not significantly affected by any of the deletions . All deletion derivatives were less stable than wild-type TonB . A major degradation product of approx . 29 kDa was observed for all TonB derivatives, indicating that it was an N-terminal fragment . The possibility is discussed that the C-terminal hydrophobic region is involved in the intramolecular interaction rather than in the membrane association of TonB.

Biochem Biophys Res Commun, 1991 Aug 30, 179(1), 683 - 8
Purification of recombinant human tumor necrosis factor precursor from Escherichia coli; Tanabe Y et al.; To study its biological functions, tumor necrosis factor precursor (proTNF) with a molecular size of 26-KDa was obtained as a recombinant protein from Escherichia coli . The recombinant proTNF was successfully accumulated in the insoluble form, corresponding to about 10-15% of total E . coli proteins . Solubilization, gel filtration and anion exchange chromatography were performed under denatured conditions followed by dialysis in phosphate-buffered saline . These processes removed most of the contaminating bacterial proteins, yielding proTNF with a purity of about 70-80% . This recombinant proTNF is expected to be useful for functional studies on activated macrophages with membrane integrated proTNF.

Biochem Biophys Res Commun, 1991 Aug 30, 179(1), 668 - 74
The monomer of pyruvate kinase, subtype M1, is both a kinase and a cytosolic thyroid hormone binding protein; Parkison C et al.; Using a T7 expression system, the monomer of rat pituitary pyruvate kinase, subtype M1 (PKM1), was overexpressed in Escherichia coli and purified to homogeneity . The monomeric p58-M1 has intrinsic enzymatic activity with a Vmax of 79 +/- 20 units/mg and Km's for ADP and PEP of 1.43 +/- 0.76 and 0.14 +/- 0.07 mM, respectively . The monomer binds 3,3',5-triiodo-L-thyronine (T3) with Ka = 1.5 x 10(7) M-1 . The order of analog specificity is L-T3 greater than L-thyroxine greater than D-T3 greater than 3'-isopropyl-3,5-diiodo-L-thyronine greater than or equal to 3',5',3-triiodo-L-thyronine . In contrast, tetrameric PKM1 lacks T3 binding activity . The kinase activity of p58-M1 is inhibited by T3 and its analogs in a concentration-dependent manner with the order of inhibitory activity similar to that of binding activity . This inhibition, however, is reversed by the addition of fructose 1,6-bisphosphate . p58-M1 is the second PK isoenzyme monomer to be identified as having thyroid hormone binding activity.

Biochem Biophys Res Commun, 1991 Aug 30, 179(1), 183 - 9
Cloning and sequencing of cDNA encoding human sepiapterin reductase--an enzyme involved in tetrahydrobiopterin biosynthesis; Ichinose H et al.; A full-length cDNA clone for sepiapterin reductase, an enzyme involved in tetrahydrobiopterin biosynthesis, was isolated from a human liver cDNA library by plaque hybridization . The nucleotide sequence of hSPR 8-25, which contained an entire coding region of the enzyme, was determined . The clone encoded a protein of 261 amino acids with a calculated molecular mass of 28,047 daltons . The predicted amino acid sequence of human sepiapterin reductase showed a 74% identity with the rat enzyme . We further found a striking homology between human SPR and carbonyl reductase, estradiol 17 beta-dehydrogenase, and 3 beta-hydroxy-5-ene steroid dehydrogenase, especially in their N-terminal region.

J Chromatogr, 1991 Aug 30, 555(1-2), 109 - 24
Anion-exchange chromatography of DNA restriction fragments; Strege MA et al.; The abilities of several high-performance liquid chromatography (HPLC) anion-exchange packings to separate DNA restriction fragments, ranging in size from 50 to 23,000 base pairs, were studied . The ion exchangers investigated include the porous packings Protein-Pak DEAE-5PW, Nucleogen-DEAE 4000-7, Poros-Q and BakerBond WP-PEI, and the non-porous packings TSK Gel DEAE-NPR, Gen-Pak FAX, and ProPac PA1 . The results indicated that the non-porous packings could separate all 18 fragments (less than 600 base pairs) in a pBR322 DNA-HaeIII digest, while of the porous packings, only Nucleogen-DEAE 4000-7 could resolve DNA fragments in this size range . Only Gen-Pak FAX and TSK Gel DEAE-NPR could significantly resolve the very large DNA fragments (125-23,000 base pairs) of a lambda DNA-HindIII digest . The chromatographic parameters governing this separation by Gen-Pak FAX were optimized so that six of eight fragments were resolved . Split-peak phenomena were observed at low flow-rates when employing non-poros packings, but were eliminated by the incorporation of organic modifiers or surfactants, suggesting that, under certain conditions, hydrophobicity may play a significant role in separations on this packing . Gen-Pak FAX also separated 21 of 23 fragments in a 1000-base pair DNA ladder, a performance which, in addition to the quantitative capabilities of HPLC, makes anion-exchange chromatography a powerful method complementary to slab-gel electrophoresis, and perhaps preferable over agarose gel electrophoresis for applications such as the confirmation of plasmid integrity.

Gene, 1991 Aug 30, 105(1), 31 - 6
Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes; Hama H et al.; The gene encoding nucleoside diphosphate (NDP) kinase of Escherichia coli was identified by polymerase chain reaction using oligodeoxyribonucleotide primers synthesized on the basis of consensus sequences from Myxococcus xanthus and various eukaryotic NDP kinases . The gene (ndk), mapped at 54.2 min on the E . coli chromosome, was cloned and sequenced . The E . coli NDP kinase was found to consist of 143 amino acid residues that are 57, 45, 45, 42, 43, and 43% identical to the M . xanthus, Dictyostelium discoideum, Drosophila melanogaster, mouse, rat, and human enzymes, respectively . The ndk gene appears to be in a monocistronic operon and, when cloned in a pUC vector, NDP kinase was overproduced at a level of approx . 25% of total cellular proteins . The protein could be labeled with {gamma-32P}ATP and migrated at a 16.5 kDa when electrophoresed in SDS-polyacrylamide gel, which is in good agreement with the Mr of the purified E . coli NDP kinase previously reported.

Science, 1991 Aug 30, 253(5023), 1001 - 7
Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees; Schultz SC et al.; The 3 angstrom resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with a 30-base pair DNA sequence shows that the DNA is bent by 90 degrees . This bend results almost entirely from two 40 degrees kinks that occur between TG/CA base pairs at positions 5 and 6 on each side of the dyad axis of the complex . DNA sequence discrimination by CAP derives both from sequence-dependent distortion of the DNA helix and from direct hydrogen-bonding interactions between three protein side chains and the exposed edges of three base pairs in the major groove of the DNA . The structure of this transcription factor--DNA complex provides insights into possible mechanisms of transcription activation.

Biochem Biophys Res Commun, 1991 Aug 30, 179(1), 251 - 8
Illegitimate recombination in a bovine papillomavirus shuttle vector: a high level of site specificity; Kitamura Y et al.; Recombination in a bovine papillomavirus shuttle vector carrying direct repeats of Moloney murine leukemia virus LTR sequence was examined . Differently from similar vectors carrying direct repeats of SV40 polyA addition signal or neomycin resistance gene, the vector exhibited no homologous recombination between the repeats . Instead, illegitimate recombination took place . There were two major types of recombination products from the restriction cleavage pattern . The plasmids in independent cellular clones belonging to the same recombination type shared the identical crossover point . Thus, in this plasmid, illegitimate recombination occurred at preferential sites involving exactly the same sequences.

Biochim Biophys Acta, 1991 Aug 27, 1090(1), 95 - 101
Expression of LDL receptor, apolipoprotein B, apolipoprotein A-I and apolipoprotein A-IV mRNA in various mouse organs as determined by a novel RNA-excess solution hybridization assay; Srivastava RA et al.; We report expression of LDL receptor, apolipoprotein B (apoB), apolipoprotein A-I (apoA-I) and apolipoprotein A-IV (apoAIV) mRNA in various mouse organs . These mRNA were quantified by an RNA-excess solution hybridization assay . For preparing specific probes, we cloned cDNA fragments of rat LDL receptor, apoB and apoA-I and mouse apoA-IV into the polylinker region of pGEM3Zf(+) and used the recombinant vectors for preparing 32P-labeled cRNA probes as well as RNA standards using the T7 and SP6 promoters flanking the polylinker regions . Preparation of cRNA probes and RNA standards is faster and more convenient than preparing cDNA probes and ssDNA standards . Absolute levels of mRNA were quantified in the liver, intestine, kidney, heart, lung, spleen and adrenals of females of two mouse strains . C3H/HeJ and C57BL/6J . ApoB, apoA-I and apoA-IV genes in mice are expressed in the liver and intestine and LDL receptor gene is expressed mainly in liver, intestine and adrenals . ApoA-I mRNA levels were found to be 730 and 1039 molecules per cell in liver and intestine, respectively, in C3H mice and 762 and 952 molecules per cell in C57BL mice . ApoB mRNA levels were 66 and 170 molecules per cell in the liver and intestine of C3H and 83 and 243 molecules per cell in C57BL, respectively . ApoA-IV mRNA was found to be 3525 and 2964 molecules per cell in the liver and intestine of female C57BL mice, respectively . LDL receptor mRNA levels were 39, 32 and 14 molecules per cell in the liver, intestine and adrenals of C3H.

Biochim Biophys Acta, 1991 Aug 27, 1090(1), 70 - 80
Expression and characterization of recombinant human ciliary neurotrophic factor from Escherichia coli; McDonald JR et al.; The gene for ciliary neurotrophic factor (CNTF) was cloned from a human genomic DNA library by screening with a DNA fragment amplified from human genomic DNA using the polymerase chain reaction . A DNA sequence coding for human CNTF was placed under control of an regulatable promoter in the expression vector pJU1003 and transformed into Escherichia coli strain BL21(DE3) . Induction of expression in cultures of this transformant led to the accumulation of approx . 25 mg/l per A600 unit of human CNTF . CNTF was purified to homogeneity from cell lysates via anion-exchange, cation-exchange and Zn(2+)-affinity chromatography . Purified CNTF contained less than 0.1% contaminating E . coli proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis and reversed-phase high-pressure liquid chromatography (HPLC) . The protein exhibited an ultraviolet absorption maximum at 279 nm with a calculated extinction coefficient of A1%(279) = 9.0 . Peptide map and amino acid sequence analyses confirmed that the expressed protein has the amino acid sequence expected for human CNTF, except for the absence of the amino-terminal methionine . High-purified recombinant human CNTF supported the survival of chick embryo parasympathetic, sympathetic and sensory neurons in culture at low picomolar concentrations . These results indicate that the biological activities previously ascribed to impure CNTF preparations indeed reside in one molecule.

Biochemistry, 1991 Aug 27, 30(34), 8470 - 6
Stepwise improvements in catalytic effectiveness: independence and interdependence in combinations of point mutations of a sluggish triosephosphate isomerase; Blacklow SC et al.; Second-site suppressor changes that improve the catalytic potency of a sluggish mutant of the enzyme triosephosphate isomerase have been examined both individually and in combination . Each of the second-site mutations increases the specific catalytic activity of a triosephosphate isomerase in which the catalytic base, glutamate-165, has been changed to aspartate . These second-site suppressors are G10S, S96P, S96T, E97D, V167D, and G233R . Not one of these changes enhances the value of kcat/Km for the wild-type enzyme, which is consistent with the knowledge that the reaction catalyzed by the wild-type enzyme is already diffusion-controlled . Indeed, two of the changes, S96P and V167D, are catalytically deleterious to the wild-type isomerase . When pairs of second-site suppressors are combined with the primary lesion E165D, six pairs show additive independence while the effects of eight other pairs are less than additive . The sites fall into two clusters: pairs within a cluster always interfere with one another and do not produce additive improvements in catalytic activity, whereas combinations of changes from different clusters tend to be additive in their effects . No combination of second-site suppressor mutations behaves synergistically, though there seems to be no a priori reason to exclude this possibility . Since the catalytic potency of each of the six second-site suppressor mutants can be further improved by the introduction of (at least) one of the other five changes, it is evident that none of the double mutants lies at a local catalytic maximum . In these cases, therefore, the opportunity exists for at least two "steps" of monotonic catalytic improvement along each of six different "paths" in protein space.

Biochemistry, 1991 Aug 27, 30(34), 8477 - 80
Steady-state fluorescence of Escherichia coli phosphofructokinase reveals a regulatory role for ATP; Berger SA et al.; We have investigated the effects of ligands and effectors on the intrinsic fluorescence of Escherichia coli phosphofructokinase (PFK) . We have found that the substrate fructose 6-phosphate (Fru6P) or the allosteric activator ADP can quench the fluorescence up to 35% . The response is hyperbolic with Ks{Fru6P} of 20 microM and Ks{ADP} of 13 microM . The allosteric inhibitor phosphoenolpyruvate (PEP) converts the hyperbolic response with respect to Fru6P to a sigmoidal response . AMP-PNP, a nonhydrolyzable analogue of ATP, also inhibits the Fru6P fluorescence response . PFK mutant KA213, which is insensitive to effectors, has a decreased fluorescence response with respect to ADP, and PEP does not convert the Fru6P response to sigmoidicity . However, its fluorescence response with respect to Fru6P is decreased by ATP or AMP-PNP . Taken together, these results suggest that, in the absence of effectors or ligands, E . coli PFK exists in a state with high affinity for Fru6P ("R" state) . This state can be altered to a low affinity ("T" state) by PEP binding to the allosteric site or by ATP binding to the enzyme.

Biochemistry, 1991 Aug 27, 30(34), 8296 - 305
Alteration of the iron-sulfur cluster composition of Escherichia coli dimethyl sulfoxide reductase by site-directed mutagenesis; Rothery RA et al.; We have used site-directed mutagenesis to alter the {Fe-S} cluster composition of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC) . The electron-transfer subunit (DmsB) of this enzyme contains 16 Cys residues arranged in 4 groups (I-IV) which provide ligands to 4 {4Fe-4S} clusters {Cammack, R., & Weiner, J . H . (1990) Biochemistry 29, 8410-8416} . Strong homologies exist between these Cys groups and the four Cys groups of the electron-transfer subunit (NarH) of E . coli nitrate reductase (NarGHJI), which contains a {3Fe-4S} cluster in addition to multiple {4Fe-4S} clusters . The Cys group primarily involved in providing ligands to the {3Fe-4S} cluster of NarH has a Trp residue at a position equivalent to Cys102 of DmsB . We have mutated Cys102 to Trp, Ser, Tyr, and Phe and have investigated the altered enzymes in terms of their enzymatic activities and EPR properties . The mutant enzymes do not support electron transfer from menaquinol to DMSO, although they retain high rates of electron transport from reduced benzyl viologen to DMSO . The mutations cause major changes in the EPR properties of the enzyme in the fully reduced and oxidized states . In the oxidized state, new species are observed in all the mutants; these have spectral features comprising a peak at g = 2.03 (gz) and a peak-trough at g = 2.00 (gxy) . The temperature dependencies, microwave power dependencies, and spin quantitations of these species are consistent with the Trp102, Ser102, Phe102, and Tyr102 mutations causing conversion of one of the {4Fe-4S} clusters present in the wild-type enzyme into {3Fe-4S} clusters in the mutant enzymes.

Nucleic Acids Res, 1991 Aug 25, 19(16), 4515 - 21
Further sequence requirements for male germ cell-specific expression under the control of the 14 bp promoter element (beta 2UE1) of the Drosophila beta 2 tubulin gene; Michiels F et al.; We have investigated a 14 bp promoter element (beta 2UE1) that is required for testis-specific expression of the Drosophila beta 2 tubulin gene . To further elucidate the role of the 14 bp element, we fused different promoter constructs to the E . coli lacZ gene and established transgenic strains with the aid of the Drosophila P-element transformation system . Germ line transformation experiments with constructs in which the element in the beta 2 tubulin gene promoter was exchanged for a related sequence from the promoter region of the Drosophila beta 3 tubulin gene led to a dramatic reduction in the expression of the lacZ gene in the testis . Exchanging the 14 bp promoter element for a similar sequence from the distal promoter of the Drosophila alcohol dehydrogenase gene abolished expression . This might indicate that the sequence differences between the beta 2UE1 and the beta 2UE1-related elements reflect functional differences between these elements . Constructs in which the beta 2UE1 was fused to the hsp70 promoter revealed that testis-specific expression of a marker gene is obtained only when the element is located at the correct distance from the transcription initiation site . However, constructs in which the beta 2UE1 was inserted at about the correct position (between -41 and -54 bp) upstream of a truncated beta 3 tubulin gene promoter did not show any expression . By making beta 2-beta 3 gene promoter fusions it was found that both the region surrounding the beta 3 transcription initiation site as well as the first 116 b of beta 3 leader sequences independently reduce testis-specific expression . These findings suggest that the testis-specific expression of the Drosophila beta 2 tubulin gene underlies a unique regulatory mechanism.

Nucleic Acids Res, 1991 Aug 25, 19(16), 4503 - 8
Stringent integrity requirements for both trans-activation and DNA-binding in a trans-activator, Oct3; Imagawa M et al.; POU-specific and POU-homeo domains of Oct3 were produced in Echerichia coli for characterization of DNA binding to the octamer sequence . POU domain protein including A, B and H domains could bind to the octamer sequence efficiently and specifically, and DNase I footprint analysis gave an indistinguishable protection pattern between recombinant POU protein of Oct3 and native Oct3 from undifferentiated P19 cells . Truncated mutants, which contained B-specific and H domains or the H domain only, showed no binding activity, indicating that both of POU-specific and POU-homeo domains are essential for binding activity to octamer sequence . Furthermore, a 6 amino acid deletion from the N-terminal region of the A-specific domain is enough to destroy the binding activity . As for trans-activation, the N-terminal region is essential and sufficient . Deletion of the N-terminal proline-rich region rapidly eliminated trans-activating activity . These data strongly indicate the stringent integrity requirements for both trans-activation and DNA-binding domains in Oct3.

J Biol Chem, 1991 Aug 25, 266(24), 16135 - 40
Role of the NADP/thioredoxin system in the reduction of alpha-amylase and trypsin inhibitor proteins; Kobrehel K et al.; Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources . Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor) . This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins . The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5',5'-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E . coli . In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect . When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues . These results also raise the question of the occurrence of glutaredoxin in plants, as E . coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.

J Biol Chem, 1991 Aug 25, 266(24), 16105 - 12
A putative glutathione-binding site in T4 glutaredoxin investigated by site-directed mutagenesis; Nikkola M et al.; A glutathione monomer has been docked into the active site cleft of T4 glutaredoxin (previously called T4 thioredoxin) using molecular graphics . The central part of the cleft is formed by the side chain of Tyr-16 on one side and the residues Thr-64, Met-65, and Pro-66 on the other . The entire glutathione molecule fits well into the cleft . A cis-peptide bond between the residues Met-65 and Pro-66 allows glutathione to bind in an anti-parallel fashion to residues 64-66 . Hydrogen bonds can be formed between Met-65 and the glutathione cysteine . This binding positions the glutathione sulfur atom ideally for reaction with the glutaredoxin disulfide . In the model, glutathione can form a hydrogen bond to the hydroxyl group of Tyr-16 . Charged interactions at opposite ends of the binding cleft are provided by His-12 and Asp-80 . The negatively charged alpha-carboxyl group of glutathione may interact with a positive helix dipole of the protein . Fifteen mutant T4 glutaredoxins have been produced and assayed for glutathione binding by determining thioltransferase activity . Mutant proteins with substitutions in the sides of the cleft (Tyr-16, Pro-66) exhibited the most marked decreases in thioltransferase activity . Mutation of His-12 to a serine decreases the catalytic efficiency whereas substitution of Asp-80 by serine increases the catalytic efficiency . A double mutant, D80S;H12S, has much less affinity for glutathione than either single mutant . Substitution of Cys-14 produces an inactive protein, whereas C17S retains some thioltransferase activity.

J Biol Chem, 1991 Aug 25, 266(24), 15710 - 5
Cloning and expression in Escherichia coli of a human cDNA encoding the DNA repair protein N-methylpurine-DNA glycosylase; Chakravarti D et al.; A 871-base pair cDNA encoding the human N-methylpurine-DNA glycosylase (MPG) was cloned from a HeLa S3 cDNA expression library in a pUC vector by phenotypic screening of MPG-negative (tag- alkA-) Escherichia coli cells exposed to methylmethane sulfonate . The active MPG is expressed as a 31-kDa fusion protein . The human cDNA-encoded MPG releases 3-methyladenine, 7-methylguanine, and 3-methylguanine from DNA and thus has a substrate range similar to that of the indigenous enzyme and the E . coli AlkA protein . The cDNA hybridizes with distinct restriction fragments of mammalian DNAs but not with E . coli or yeast DNA . A search in the GenBank data bank failed to show any other cloned DNA with a similar sequence . Although the human protein has 62% sequence homology with the corresponding rat enzyme, only a few amino acid residues are conserved between the human protein and the E . coli and yeast MPGs . However, a conserved glutamine residue in all MPGs that release 3-alkyladenine and an arginine residue in eukaryotic MPGs and E . coli AlkA that act additionally on N-alkylguanines suggest that these residues are involved in recognition of adenine and guanine adducts in DNA, respectively . Although the 1.1-kilobase mRNAs of MPG from human and rodents are similar in size, liver and cultured cells of rat have much lower levels of MPG mRNA than do human and mouse cells . A hamster cell line variant isolated as being resistant to methylmethane sulfonate does not have a higher level of MPG mRNA than the parent cell line.

J Biol Chem, 1991 Aug 25, 266(24), 15688 - 92
Characterization of purified, reconstituted site-directed cysteine mutants of the lactose permease of Escherichia coli; van Iwaarden PR et al.; lac permease mutated at each of the 8 cysteinyl residues in the molecule was solubilized from the membrane, purified, and reconstituted into proteoliposomes . The transport activity of proteoliposomes reconstituted with each mutant permease relative to the wild-type is virtually identical with that reported for intact cells and/or right-side-out membrane vesicles . Moreover, a double mutant containing Ser in place of both Cys148 and Cys154 exhibits significant ability to catalyze active lactose transport . The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism . The effect of sulfhydryl oxidant 5-hydroxy-2-methyl-1,4-naphthoquinone on lactose transport in proteoliposomes reconstituted with wild-type or mutant permeases was also investigated, and the results indicate that inactivation is probably due to formation of a covalent adduct with Cys148 and/or Cys154 rather than disulfide formation . Thus, it seems unlikely that sulfhydryl-disulfide interconversion functions to regulate permease activity.

Nucleic Acids Res, 1991 Aug 25, 19(16), 4413 - 9
Molecular mechanism of negative autoregulation of Escherichia coli crp gene; Hanamura A et al.; Transcription of the Escherichia coli crp gene encoding cAMP receptor protein (CRP) is negatively regulated by CRP-cAMP complex that binds to a specific site located downstream from the transcription start site . The binding of CRP-cAMP to this site activates transcription from a second divergent overlapping promoter . The mechanism of this negative autoregulation of the crp gene has been investigated by in vitro transcription, gel shift, DNase I footprinting, and exonuclease III protection assays . We demonstrated that the crp and divergent promoters are reciprocally and coordinately regulated by CRP-cAMP . The abortive initiation assay revealed that the divergent RNA itself is not required for the inhibition of crp transcription . Detailed binding studies revealed that CRP-cAMP stimulates the binding of RNA polymerase to the divergent promoter and thus blocks the occupation of the crp promoter by RNA polymerase.

Nucleic Acids Res, 1991 Aug 25, 19(16), 4399 - 403
A comparative study of the ribosomal RNA operons of Streptomyces coelicolor A3(2) and sequence analysis of rrnA; van Wezel GP et al.; S . coelicolor A3(2) contains six ribosomal RNA operons . Here we describe the cloning of rrnA, rrnC and rrnE, thereby completing the cloning of all operons . Southern hybridisation of genomic DNA with a heterologous probe from the E.coli rrnB 16S rRNA gene showed differences in hybridisation among the six rRNA operon-containing bands . The nucleotide sequence of the 16S rRNA gene and the upstream region of rrnA was determined and compared with the corresponding sequence of rrnD, showing that the 16S rRNA genes are 99% identical . Substantial differences were found, however, in the upstream regions corresponding to the P1 and P2 promoters of rrnD . Southern analysis showed that some of the other rRNA operons of S.coelicolor A3(2) also differed in this part of the upstream region.

J Biol Chem, 1991 Aug 25, 266(24), 15698 - 704
Isolation of a cDNA encoding a mammalian multiubiquitinating enzyme (E225K) and overexpression of the functional enzyme in Escherichia coli; Chen ZJ et al.; The ubiquitin (Ub)-conjugating enzyme E2(25K) catalyzes the synthesis of multi-Ub chains in which successive Ub units are linked by an isopeptide bond involving the epsilon-amino group of Lys-48 of Ubn, and the COOH-terminal Gly residue of Ubn+1 (Chen, Z., and Pickart, C . M . (1990) J . Biol . Chem., 265, 21835-21842) . We now describe the polymerase chain reaction (PCR)-based cloning of an E2(25K)-encoding cDNA from a bovine thymus library, using degenerate oligonucleotide primers based on the sequences of two E2(25K) peptides . The cDNA encodes a 200-residue protein whose sequence bears similarities of 66 and 59%, respectively, to the sequences of the Ub-conjugating enzymes encoded by the UBC1 and UBC4/UBC5 genes of the yeast Saccharomyces cerevisiae . These three yeast E2s play key roles in Ub-dependent proteolysis (Seufert, W., McGrath, J . P., and Jentsch, S . (1990) EMBO J . 9, 4535-4541) . Comparison of the amino acid sequence of E2(25K) with other known E2 sequences strongly suggests that Cys-92, one of two E2(25K) Cys residues, forms the Ub thiol ester adduct that is an intermediate in E2-catalyzed multiubiquitination . The E2(25K)-encoding cDNA was overexpressed in Escherichia coli, and the recombinant E2(25K) protein was purified to electrophoretic homogeneity; enzymatic assays showed that its multiubiquitinating activity was quantitatively identical with that of the native protein . The availability of a cloned cDNA will allow us to assess the physiological role of E2(25K).

Nucleic Acids Res, 1991 Aug 25, 19(16), 4377 - 85
High-level ribosomal frameshifting directs the synthesis of IS150 gene products; Vogele K et al.; IS150 contains two tandem, out-of-phase, overlapping genes, ins150A and ins150B, which are controlled by the same promoter . These genes encode proteins of 19 and 31 kD, respectively . A third protein of 49 kD is a transframe gene product consisting of domains encoded by both genes . Specific -1 ribosomal frameshifting is responsible for the synthesis of the large protein . Expression of ins150B also involves frameshifting . The IS150 frameshifting signals operate with a remarkably high efficiency, causing about one third of the ribosomes to switch frame . All of the signals required for this process are encoded in a 83-bp segment of the element . The heptanucleotide A AAA AAG and a potential stem-loop-forming sequence mark the frameshifting site . Similar sequence elements are found in -1 frameshifting regions of bacterial and retroviral genes . A mutation within the stem-loop sequence reduces the rate of frameshifting by about 80% . Artificial transposons carrying this mutation transpose at a normal frequency, but form cointegrates at a approximately 100-fold reduced rate.

J Biol Chem, 1991 Aug 25, 266(24), 16232 - 7
Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT; Matson SW et al.; A site- and strand-specific nick, introduced in the F plasmid origin of transfer, initiates conjugal DNA transfer during bacterial conjugation . Recently, molecular genetic studies have suggested that DNA helicase I, which is known to be encoded on the F plasmid, may be involved in this nicking reaction (Traxler, B . A., and Minkley, E . G., Jr . (1988) J . Mol . Biol . 204, 205-209) . We have demonstrated this site- and strand-specific nicking event using purified helicase I in an in vitro reaction . The nicking reaction requires a superhelical DNA substrate containing the F plasmid origin of transfer, Mg2+ and helicase I . The reaction is protein concentration-dependent but, under the conditions used, only 50-70% of the input DNA substrate is converted to the nicked species . Genetic data (Everett, R., and Willetts, N . (1980) J . Mol . Biol . 136, 129-150) have also suggested the involvement of a second F-encoded protein, the TraY protein, in the oriT nicking reaction . Unexpectedly, the in vitro nicking reaction does not require the product of the F plasmid traY gene . The implications of this result are discussed . The phosphodiester bond interrupted by helicase I has been shown to correspond exactly to the site nicked in vivo suggesting that helicase I is the site- and strand-specific nicking enzyme that initiates conjugal DNA transfer . Thus, helicase I is a bifunctional protein which catalyzes site- and strand-strand specific nicking of the F plasmid in addition to the previously characterized duplex DNA unwinding (helicase) reaction.

J Biol Chem, 1991 Aug 25, 266(24), 16226 - 31
Expression and analysis of Gs alpha mutants with decreased ability to activate adenylylcyclase; Itoh H et al.; We have constructed mutants of the alpha subunit of Gs in an attempt to identify sites in the protein that are important for its interaction with adenylylcyclase . Some residues specific for those G proteins that activate adenylylcyclase were replaced with residues characteristic of Gi alpha . Mutant proteins were expressed in Escherichia coli, and two of these were purified to homogeneity and characterized in detail . Mutation of trp263, leu268, or arg269 caused a significant loss of the capacity of Gs alpha to stimulate adenylylcyclase, and the triple mutant had less than 1% of the ability of wild type Gs alpha to activate the enzyme . Guanine nucleotide binding and GTP hydrolysis by the mutant proteins were unaltered, as was guanosine 5'-3-O-(thio)triphosphate-induced enhancement of intrinsic tryptophan fluorescence . Mutant proteins also appeared to have a reduced affinity for the G protein beta gamma subunit complex . Secondary structure analysis and comparison with the structure of p21ras suggests that the region of Gs alpha that we have identified is part of a loop that may be involved in interaction of the protein with adenylylcyclase . Although these residues are essential for activation of adenylylcyclase, they are not sufficient to do this when placed in the context of another G protein alpha subunit.

J Biol Chem, 1991 Aug 25, 266(24), 16056 - 62
Replication of plasmid R6K origin gamma in vitro . Dependence on dual initiator proteins and inhibition by transcription; MacAllister TW et al.; We have developed a more efficient in vitro replication system for the plasmid R6K with the objective of dissecting the mechanism of activation of replication origins at a distance . Using this in vitro system we have shown that the activation of replication origin gamma of R6K is absolutely dependent on two exogenously added initiator proteins: namely the host-encoded DnaA and the plasmid-encoded Pi proteins . Replication was inhibited by novobiocin, suggesting a requirement for DNA gyrase . Surprisingly, rifampicin stimulated in vitro replication significantly, and this stimulation was manifested in the quantitative enhancement of replication without any noticeable qualitative change in the reaction products . This result suggests that transcription at or near the gamma origin keeps it repressed . Replication intermediates that were allowed to accumulate by dideoxynucleoside triphosphate incorporation were analyzed both by restriction enzyme digestion and by electron microscopy, and both sets of analyses revealed initiation from the gamma origin resulting in theta-type replication intermediates . Further development of this system should help us to understand how DNA-protein interaction at the gamma origin/enhancer activates the distal origins alpha and beta.

J Biol Chem, 1991 Aug 25, 266(24), 15924 - 37
Conformational changes induced by integration host factor at origin gamma of R6K and copy number control; Kelley WL et al.; We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold . Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number . Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites . The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding . Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding . Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry . The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence . We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.

J Biol Chem, 1991 Aug 25, 266(24), 15782 - 9
Molecular cloning and expression of the regulatory (RG1) subunit of the glycogen-associated protein phosphatase; Tang PM et al.; DNA clones encoding the glycogen-binding (RG1) subunit of glycogen-associated protein phosphatase were isolated from rabbit skeletal muscle lambda gt11 cDNA libraries . Overlapping clones provided an open reading frame of 3327 nucleotides that predicts a polypeptide of 1109 amino acids with a molecular weight of 124,257 . Northern hybridization of rabbit RNA identified a major mRNA transcript of 7.5 kilobases present in skeletal, diaphragm, and cardiac muscle, but not in brain, kidney, liver, and lung . Southern analysis of rabbit genomic DNA digested with various restriction endonucleases gave rise to a single hybridizing fragment, suggesting that a single gene is present . Expression of the complete RG1 subunit coding sequence in Escherichia coli generated a protein of apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 160,000, similar to the size of the polypeptide detected by Western immunoblot in rabbit skeletal muscle extracts . The RG1 subunit shares significant homology with the Saccharomyces cerevisiae GAC1 gene product which is involved in activation of glycogen synthase and glycogen accumulation . The homology with GAC1 substantiates the role of this enzyme in control of glycogen metabolism . Hydropathy analysis of the RG1 subunit amino acid sequence revealed the presence of a hydrophobic region in the COOH terminus, suggesting a potential association with membrane . This result suggests that the same phosphatase regulatory component may be involved in targeting the enzyme both to membranes and to glycogen.

J Biol Chem, 1991 Aug 25, 266(24), 15764 - 70
Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase; Sakata J et al.; We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase . Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA fragment . A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli . The expressed protein, purified to homogeneity, showed a Mr of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced Mr of 54,023 . Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same Mr 55,000 was also present in rat testis extract . The active enzyme was a dimer as judged by molecular sieve filtration . The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively . Various kinetic constants of the expressed fructose 6-P,2-kinase were KmFru 6-P = 85 microM and KmATP = 270 microM, and those of fructose 2,6-bisphosphatase were KmFru 2,6-P2 = 21 microM and KiFru 6-P = 3.4 microM . The enzyme was phosphorylated by Fru-2,6{2-32P}P2 and also by protein kinase C, but not by cAMP-dependent protein kinase, which is in contrast to the liver and heart isozymes.

J Biol Chem, 1991 Aug 25, 266(24), 15693 - 7
Expression of rat phosphoribosylpyrophosphate synthetase subunits I and II in Escherichia coli . Isolation and characterization of the recombinant isoforms; Ishijima S et al.; The 34-kDa subunit of rat liver phosphoribosylpyrophosphate synthetase is a mixture of the two highly homologous isoforms, PRS I and PRS II . Heretofore, it was not possible to separate the two . We now describe isolation and characterization of the recombinant isoforms, named rPRS I and rPRS II . The respective rat cDNAs were inserted into vectors constructed from pKK233-2 by replacing its replication origin with that of pGEM-1 and expressed in Escherichia coli . The rPRS I and rPRS II were purified to apparent homogeneity with specific activities of 33,400 and 46,200 milliunits/mg, respectively; these values were at least 2.5-fold higher than the highest value for the mammalian enzyme so far reported . Both isoforms showed a similar dependency on Pi as an absolute activator . Sulfate partially substituted for Pi . The maximal activities of rPRS I and rPRS II with sulfate were 43 and 7%, respectively, of those seen with Pi . The two isoforms differed in sensitivity to inhibition by ADP and GDP . Inhibition of rPRS I and rPRS II by 0.3 mM ADP was 87 and 54%, respectively, and inhibition by 1 mM GDP was 93 and 24%, respectively . rPRS II was 180-fold more sensitive than rPRS I to heat inactivation at 49 degrees C.

J Biol Chem, 1991 Aug 25, 266(24), 15559 - 62
Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme; Langford KG et al.; There are two isozymes of angiotensin-converting enzyme (ACE), one produced by somatic tissues and a smaller protein synthesized by developing spermatozoa (testis ACE) . To investigate the molecular control of testis ACE, we generated mice transgenic for a construct containing a putative testis-specific ACE promoter linked to the Escherichia coli reporter gene encoding beta-galactosidase . The transgenic mice express beta-galactosidase protein and RNA only within the testis . Histochemical analysis of the transgenic mice shows co-localization of beta-galactosidase protein and endogenous ACE within elongating spermatozoa . These studies demonstrate that transcription of testis ACE is controlled by a strong intragenic testis-specific promoter that is contained within a 698-base pair fragment immediately upstream from the transcription start site of testis ACE . Characterization of the testis ACE promoter may provide insights into the molecular mechanisms controlling cell stage-specific gene expression in the male germ line.

Science, 1991 Aug 23, 253(5022), 900 - 2
Related RNA polymerase-binding regions in human RAP30/74 and Escherichia coli sigma 70; McCracken S et al.; RAP30/74 is a heteromeric general transcription initiation factor that binds to mammalian RNA polymerase II . The RAP30 subunit contains a region that is similar in amino acid sequence to the RNA polymerase-binding domain of the Escherichia coli transcription initiation factor sigma 70 (sigma 70) . Mammalian RNA polymerase II specifically protected a serine residue in the sigma 70-related region of RAP30 from phosphorylation in vitro . In addition, human RAP30/74 bound to Escherichia coli RNA polymerase and was displaced by sigma 70 . These results suggest that RAP30 and sigma 70 have functionally related RNA polymerase-binding regions.

Biochemistry, 1991 Aug 20, 30(33), 8268 - 76
Reengineering the catalytic lysine of aspartate aminotransferase by chemical elaboration of a genetically introduced cysteine; Planas A et al.; The active-site essential catalytic residue of aspartate aminotransferase, Lys 258, has been converted to Cys (K258C) by site-directed mutagenesis . This mutant retains less than 10(-6) of the wild-type activity with L-aspartate . The deleted general base was functionally replaced by selective (with respect to the other five cysteines in wild type) aminoethylation of the introduced Cys 258 with (2-bromoethyl)amine following reversible protection of the nontarget sulfhydryl groups at different stages of unfolding . The chemically elaborated mutant (K258C-EA) is 10(5) times more reactive than is K258C and has a kcat value of approximately 7% of that of wild type (WT) . Km and KI values are similar to those for WT . The acidic pKa controlling V/KAsp is shifted from 7.3 (WT) to 6.0 (mutant) . V/K values for amino acids are approximately 3% of those found for WT, whereas they are approximately 20% for keto acids . The value of DV increases from 1.6 for WT to 3.4 for the mutant, indicating that C alpha proton abstraction constitutes a more significant kinetic barrier for the latter enzyme . A smaller, but still significant, increase in D(V/KAsp) from 1.9 in WT to 3.0 in the mutant shows that the forward and reverse commitment factors are inverted by the mutation . The acidic limb of the V/KAsp versus pH profile, is lowered by 1.3 pH units, probably reflecting the similar difference in the basicity of the epsilon-NH2 group in gamma-thialysine versus that in lysine.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Aug 20, 30(33), 8151 - 7
Inactivation mechanism of tetrameric beta-galactosidase by gamma-rays involves both fragmentation and temperature-dependent denaturation of protomers; Potier M et al.; The radiation inactivation method is widely used to estimate the molecular size of membrane-bound enzymes, receptors, and transport systems in situ . The method is based on the principle that exposure of frozen solutions or lyophilized protein preparations to increasing doses of ionizing radiations results in a first-order decay of biological activity proportional to radiation inactivation size of the protein . This parameter is believed to reflect the "functional unit" of the protein defined as the minimal assembly of structure (protomers) required for expression of a given biological activity . We tested the functional unit as a concept to interpret radiation inactivation data of proteins with Escherichia coli beta-galactosidase, where the protomers are active only when associated in a tetramer . Gamma-Irradiation of beta-galactosidase at both -78 and 38 degrees C followed by quantitation of the residual unfragmented promoter band by SDS-polyacrylamide gel electrophoresis yielded the protomer size, indicating that only one protomer is fragmented by each radiation hit . By following the enzyme activity as a function of dose it was found that only the protomer that has been directly hit and fragmented at -78 degrees C was effectively inactivated . In contrast, at 38 degrees C, it was the whole tetramer that was inactivated . beta-Galactosidase cannot have two different functional units depending on temperature . The inactivation of the whole beta-galactosidase tetramer at 38 degrees C is in fact related to protomer fragmentation but also to the production of stable denatured protomers (detected by gel-filtration HPLC and differential UV spectroscopy) due to energy transfer from fragmented protomers toward unhit protomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Aug 20, 30(33), 8201 - 10
Structural dynamics and functional domains of the fur protein; Coy M et al.; Proteolytic enzymes were used to detect metal-induced conformational changes in the ferric uptake regulation (Fur) protein of Escherichia coli K12 . Metal binding results in enhanced cleavage of the N-terminal region of Fur by trypsin and chymotrypsin . Activation of both trypsinolysis sensitivity and DNA binding have similar metal ion specificity and concentration dependencies, suggesting that the conformational change detected is required for operator DNA binding . Isolation and characterization of biochemically generated fragments of Fur as well as other data indicate that the N-terminal region is necessary for the interaction of the repressor with DNA and that a C-terminal domain is sufficient for binding to metal ions.

Biochemistry, 1991 Aug 20, 30(33), 8144 - 51
Investigation of an octapeptide inhibitor of Escherichia coli ribonucleotide reductase by transferred nuclear Overhauser effect spectroscopy; Bushweller JH et al.; Several peptides contained within the C-terminal sequence of the B2 subunit of Escherichia coli ribonucleotide reductase (RNR) were investigated for their ability to inhibit the enzyme, presumably by interfering with association of the B1 and B2 subunits . AcYLVGQIDSE, corresponding by sequence homology to a nonapeptide that inhibits herpes simplex RNR {Gaudreau et al . (1987) J . Biol . Chem . 262, 12413} shows no inhibition of the E . coli enzyme (IC50 greater than 3 mM), whereas AcDDLSNFQL, the C-terminal octapeptide of the E . coli B2 subunit, is a noncompetitive inhibitor (Ki = 160 microM) . Neither bradykinin (RPPGFSPFR) nor the pentapeptide AcSNFQL inhibits the E . coli enzyme . Transferred nuclear Overhauser enhancement spectroscopy was used to probe the conformation of AcDDLSNFQL when it is bound to the B1 subunit . These experiments suggest that the peptide adopts a turn in the region of Asn5 and Phe6 and that a hydrophobic cluster of the phenylalanine and leucine side chains is involved in the interaction surface.

Biochemistry, 1991 Aug 20, 30(33), 8131 - 7
Minimum requirements for protease activation of flavin pyruvate oxidase; Bertagnolli BL et al.; Previous investigations have shown that the catalytic efficiency (kcat/KM) of pyruvate oxidase can be enhanced 450-fold by chymotryptic cleavage of a 23-residue peptide (alpha-peptide) from the carboxy terminus of the enzyme . The minimum requirement for proteolytic activation has been investigated by exposing pyruvate oxidase to a variety of carboxypeptidases, either singly or in combination . The extent of carboxypeptidase hydrolysis was followed by analyzing the release of amino acids and by mass spectral analysis of the truncated alpha-peptides which were derived from the carboxypeptidase-treated preparations . The results indicate that the removal of 7 carboxy-terminal residues does not activate the enzyme whereas the removal of 10 or 11 residues produces activated pyruvate oxidase . Activation of pyruvate oxidase by endoproteinase Glu-C confirms the carboxypeptidase results . Endoproteinase Glu-C specificity predicts hydrolytic cleavage of the peptide bond between Glu-561 and Val-562 with the removal of 11 residues from the carboxy terminus of the enzyme.

J Mol Biol, 1991 Aug 20, 220(4), 855 - 66
Repair of helix-stabilizing anthramycin-N2 guanine DNA adducts by UVRA and UVRB proteins; Tang MS et al.; The transfectivity of anthramycin (Atm)-modified phi X174 replicative form (RF) DNA in Escherichia coli is lower in uvrA and uvrB mutant cells but much higher in uvrC mutant cells compared to wild-type cells . Pretreatment of the Atm-modified phage DNA with purified UVRA and UVRB significantly increases the transfectivity of the DNA in uvrA or uvrB mutant cells . This pretreatment greatly reduces the UVRABC nuclease-sensitive sites (UNSS) and Atm-induced absorbance at 343 nm in the Atm-modified DNA without producing apurinic sites . The reduction of UNSS is proportional to the concentrations of UVRA and UVRB and the enzyme-DNA incubation time and requires ATP . We conclude that there are two different mechanisms for repairing Atm-N2 guanine adducts by UVR proteins: (1) UVRA and UVRB bind to the Atm-N2 guanine double-stranded DNA region and consequently release the Atm from the adducted guanine; (2) UVRABC makes an incision at both sides of the Atm-DNA adduct . The latter mechanism produces potentially lethal double-strand DNA breaks in Atm-modified phi X174 RF DNA in vitro.

Carbohydr Res, 1991 Aug 20, 215(2), 323 - 35
Structural analysis of the heptose/hexose region of the lipopolysaccharide from Escherichia coli K-12 strain W3100; Holst O et al.; The disaccharide L-glycero-D-manno-heptosyl-D-glucose was isolated from the lipopolysaccharide (LPS) of Escherichia coli K-12 strain W3100 after partial hydrolysis with acid, and the structure was determined by methylation analysis, n.m.r . spectroscopy, and comparison with a synthetic standard . In addition, the oligosaccharides L,D-Hep-D-Glc-D-Glc and L,D-Hep-D-Glc-D-Glc-D-Glc were isolated, and their structures were established by g.l.c.-m.s . and methylation analysis . The results indicated that L-glycero-D-manno-heptose, a characteristic constituent of the inner core region, may also occur in the outer core region which, in E . coli, is generally composed of hexoses . A revised structure of the carbohydrate backbone of the hexose/heptose region of the LPS is given.

J Mol Biol, 1991 Aug 20, 220(4), 959 - 73
Osmotic induction of gene osmC expression in Escherichia coli K12; Gutierrez C et al.; osmC, an osmotically inducible gene of Escherichia coli, was physically mapped on the bacterial chromosome, cloned on multicopy plasmids, and its product, OsmC, was identified as a 14 kDa protein in maxicells . The DNA sequence of the gene and its upstream region were determined . The sequence of an osmC-phoA gene fusion confirmed the osmC reading frame . A deletion of osmC from the E . coli chromosome was constructed by gene replacement, demonstrating that it is not an essential gene . The osmCp promoter region was subcloned and a lac operon fusion transcribed under osmCp control was constructed . The expression of this operon fusion demonstrated that osmC regulation occurs at the transcriptional level . S1 nuclease protection experiments and deletion analysis identified two overlapping promoters with transcription start sites separated by ten nucleotides . All the sequences necessary for osmotic regulation of both promoters are located within a 137 base-pair DNA fragment extending from position -95 to +42 with respect to the putative osmC translation start . Two deletions were obtained that abolish the functioning of the upstream promoter . Yet, under our experimental conditions, the subsequent expression of the osmC-lacZ fusion was equivalent to that obtained from the tandem promoters . Mutations leading to constitutive expression of osmC were selected . Two independent mutations were obtained, both affected osmZ, the gene encoding the histone-like protein H1.

Biochemistry, 1991 Aug 20, 30(33), 8181 - 6
Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides; Terlesky KC et al.; Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides . The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography . In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation . The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10 . Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment . When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein . The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R . sphaeroides showed 72% identity to E . coli chaperonin 60 protein, and R . sphaeroides chaperonin 10 showed 45% identity with E . coli chaperonin 10 . R . sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R . sphaeroides chaperonin 10, but not E . coli chaperonin 10 . The E . coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R . sphaeroides and E . coli.

FEBS Lett, 1991 Aug 19, 288(1-2), 219 - 21
6-Hydroxymellein synthetase as a multifunctional enzyme complex in elicitor-treated carrot root extract; Kurosaki F et al.; Synthetic activity of a polyketide compound 6-hydroxymellein was induced in elicitor-treated carrot root tissues . The activity was significantly inhibited by an antiserum raised against the acyl carrier protein (ACP) of fatty acid synthetase, suggesting that the enzyme(s) for 6-hydroxymellein synthesis require(s) a functional unit similar to ACP . However, the synthetic activity was not stimulated by the addition of ACP purified from Escherichia coli and was not lost even after fractionation by gel-filtration chromatography . The active fraction obtained by gel-filtration (136 kDa) was subjected to immunoblot analysis, and a 128 kDa polypeptide in the fraction was found to cross-react with anti-ACP serum . These observations suggest that the biosynthesis of 6-hydroxymellein in carrot cells is catalyzed by an enzyme consisting of a single peptide chain.

FEBS Lett, 1991 Aug 19, 288(1-2), 197 - 200
Internal deletions of amino acids 29-42 of human interleukin-6 (IL-6) differentially affect bioactivity and folding; Arcone R et al.; Internal deletions of the human interleukin-6 (IL-6) cDNA have been generated in the region encoding residues 29 to 42 . Mutant proteins were produced by in vitro transcription-translation or in Escherichia coli and tested for their biological activity using the hybridoma growth factor (HGF) assay or a transcriptional activation assay on human hepatoma cells . The folding of the mutants was also checked by immunoprecipitation with conformation-specific monoclonal antibodies . The results show that only residues 29 to 34 are crucial for IL-6 activity and that the first two amino acids are probably involved in the definition of the IL-6 active site.

FEBS Lett, 1991 Aug 19, 288(1-2), 101 - 4
The lac repressor and its N-terminal headpiece can bind a mini-operator containing a hairpin loop made of a hexaethylene glycol chain; Maurizot JC et al.; The binding of the lac repressor and the lac repressor N-terminal headpiece to a mini-operator with a hairpin loop made of a hexaethylene glycol chain was investigated using circular dichroism spectroscopy . The lac repressor's headpiece binds to the modified mini-operator with the same affinity as to a mini-operator of the same sequence without the hexaethylene glycol loop . The conformational effect due to the binding is not affected by the presence of the hexaethylene loop . It is also shown that the entire lac repressor binds to this modified mini-operator inducing a conformational change.

Anal Biochem, 1991 Aug 15, 197(1), 187 - 90
Standard calibration proteins for Western blotting obtained by genetically prepared protein A conjugates; Lindbladh C et al.; Genetically prepared protein A fusion proteins, having retained antibody binding capacity, were used to design different well-defined standard molecular weight marker proteins for Western blotting . The blotted marker proteins are developed at the same time and with the same reagents as the protein sample of interest.

Clin Chim Acta, 1991 Aug 15, 200(1), 35 - 42
A new sensitive method for determining endotoxin in whole blood; Tamura H et al.; We developed a microplate method for determining endotoxin in whole blood with Endospecy, an endotoxin-specific chromogenic limulus test reagent . The factors in blood that would interfere with the test were successfully removed by exposing samples to 0.66 mol/l HNO3 containing 0.25% Triton X-100 . Recoveries of various endotoxins spiked into whole blood of humans and experimental animals were almost complete, and were not enhanced by sample dilution . Normal endotoxin concentration in human whole blood was less than 10 pg/ml in reference to Escherichia coli 0111:B4 endotoxin . Measurements on paired whole blood and platelet-rich plasma (PRP) samples from 50 normal and 132 diseased subjects showed a good correlation (r = 0.901, P less than 0.01) . This microplate whole blood method has advantages over the conventional PRP method in that it requires less time, less amount of sample and limulus reagent, and less risk of contamination during the procedure.

J Am Vet Med Assoc, 1991 Aug 15, 199(4), 451 - 5
Partial budget analysis of vaccinating dairy cattle against coliform mastitis with an Escherichia coli J5 vaccine; DeGraves FJ et al.; Partial budget analysis of clinical coliform mastitis prevention supported vaccination of dairy cows with an Escherichia coli J5 vaccine . Increased profits of $57/cow lactation were predicted using a computer spreadsheet derived partial budget with generalized herd input data . Herd vaccination programs were predicted to be profitable when greater than 1% of cow lactations resulted in clinical coliform mastitis . Herd vaccination programs were predicted to be profitable at all herd milk production levels.

Gene, 1991 Aug 15, 104(2), 271 - 5
Production of functional rat HMG1 protein in Escherichia coli; Bianchi ME; High-mobility group-1 protein (HMG1) was produced in Escherichia coli under the control of the T7 promoter/T7 RNA polymerase system . The protein can be produced and purified with yields similar to those obtained from animal tissues . HMG1 purified from E . coli is homogeneous and capable of selectively binding cruciform DNA, indicating that post-translational processing of vertebrate HMG1 is not necessary for its DNA-binding ability.

Gene, 1991 Aug 15, 104(2), 247 - 52
Geminivirus-based shuttle vectors capable of replication in Escherichia coli and monocotyledonous plant cells; Kammann M et al.; Shuttle vectors have been constructed that are able to replicate in either Escherichia coli or plant cells . They contain the ColE1 origin of replication and parts of the wheat dwarf virus genome, a geminivirus infecting a variety of species of monocotyledonous plants . Such plasmids are able to replicate in E . coli and wheat cells . The plasmids can be rescued in E . coli and show no changes during their passage through plant cells . Such an E . coli/plant cell shuttle vector system could be used for the amplification of foreign genes in plant cells, for studies on DNA rearrangement or the isolation of plant transposons.

Gene, 1991 Aug 15, 104(2), 147 - 53
A surface expression vector for antibody screening; Breitling F et al.; To select specific antibodies (Ab) from large recombinant libraries using small amounts of antigen, we have constructed a phagemid that expresses a single-chain Ab fused to pIII, a coliphage protein product of gene III that initiates infection by binding to F pili . Surprisingly, the production of the fusion protein (Ab::pIII) was induced by wild-type (wt) phage fd in the absence of IPTG . Ab::pIII was identified by a monoclonal Ab to an epitope in the linker sequence between the heavy and light chains, and by antisera to their N-terminal sequences . It is able to bind antigen and be assembled into infectious phagemid particles that can be enriched on columns of immobilised antigen . The phagemid DNA is even smaller than that of wt fd phages and can easily be propagated in plasmid form . Most importantly, its Ab::pIII-encoding gene can be tightly repressed so that Ab libraries can be amplified without risk of being dominated by deletion mutants . After induction, however, large quantities of the fusion protein can be produced, thus greatly facilitating its analysis.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7356 - 60
Mutant LexA proteins with an increased rate of in vivo cleavage; Smith MH et al.; LexA repressor of Escherichia coli is inactivated by a specific cleavage reaction that requires activated RecA protein in vivo . This cleavage reaction can proceed in vitro in the presence of activated RecA or as an intramolecular RecA-independent reaction, termed autodigestion, that is stimulated by alkaline pH . Here we describe a set of LexA mutant proteins that undergo a greatly increased rate of specific cleavage in vivo, compared with wild-type LexA . Efficient in vivo cleavage of these mutant proteins also took place without RecA . Several lines of evidence suggest that cleavage occurred via a mechanism similar to autodigestion . These mutations changed Gln-92, which lies near the cleavage site, to tyrosine, phenylalanine, or tryptophan . The latter mutation increased the rate of cleavage approximately 500-fold . These findings imply that the rate of wild-type LexA cleavage has been optimized during evolution to make the SOS system properly responsive to DNA-damaging treatments . Availability of these mutants will aid in the understanding of rate-limiting steps in intramolecular reactions.

Biochem J, 1991 Aug 15, 278 ( Pt 1), 203 - 9
Expression of a cDNA clone encoding the haem-binding domain of Chlorella nitrate reductase; Cannons AC et al.; A partial cDNA clone coding for the haem-binding domain of NADH:nitrate reductase (EC 1.6.6.1) (NR) from the unicellular green alga Chlorella vulgaris has been isolated, sequenced and expressed . A 1.2 kb cDNA (pCVNR1) was isolated from a lambda gt11 expression library produced from polyadenylated RNA extracted from nitrate-grown Chlorella cells . pCVNR1 hybridized to a 3.5 kb mRNA transcript that was nitrate-inducible and absent from ammonium-grown cells . The entire sequence of pCVNR1 was obtained and found to have a single uninterrupted reading frame . The derived amino acid sequence of 318 amino acids has a 45-50% similarity to higher-plant NRs, including Arabidopsis thaliana, spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum) . A comparison with the putative domain structure of higher-plant nitrate reductases suggested that this sequence contains the complete haem-binding domain, approximately one-third of the Mo-pterin domain and no FAD-binding domain . A 32% sequence similarity is evident when comparing the Chlorella NR haem domain with that of calf cytochrome b5 . Expression of pCVNR1 in a pET vector synthesized a 35 kDa protein that was antigenic to anti-(Chlorella NR) antibody . The spectral properties of this protein (reduced and oxidized) in the 400-600 nm region are identical with those of native Chlorella NR and indicate that haem is associated with the protein.

Eur J Biochem, 1991 Aug 15, 200(1), 19 - 27
Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei; Allert S et al.; In Trypanosoma brucei (stock 427) genes encoding the glycolytic enzyme pyruvate kinase are present on two homologous chromosomes . We have cloned and characterized one of the alleles . Two large, tandemly arranged open reading frames were found, each coding for a pyruvate kinase polypeptide of 498 amino acids . The gene sequences differ at 15 positions, resulting in five amino acid substitutions . The calculated molecular masses of the polypeptides are 54,378 Da and 54,363 Da . These values are somewhat smaller than those reported for the subunit molecular mass of the purified protein, which is 57-59 kDa . However, in vitro translation of the DNA region corresponding to the open reading frame, and translation of the RNA in a wheat-germ lysate, yielded a product that comigrated exactly with the native polypeptide in SDS/PAGE . The overall identity between the sequences of the trypanosomal enzyme and the enzymes from other sources is 41-51% . The conserved residues are not equally distributed over the polypeptide . The primary structure of domains A and, to a lesser extent, B, which constitute the active site, are rather well conserved . In contrast, the sequence of domain C, which supposedly is involved in the regulation of the enzyme activity, is much more variable . The cytosolically located pyruvate kinase of T . brucei lacks the specific features found in the majority of the glycolytic enzymes of this organism that are sequestered in a microbody-like organelle, the glycosome . It has neither a relatively high subunit molecular mass, due to unique insertions or terminal extensions, nor a high excess of positively charged amino acids . The polypeptide is shorter than that of most other pyruvate kinases and the calculated net charge is only +3.

Eur J Biochem, 1991 Aug 15, 200(1), 131 - 8
Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN-III-ompA system; Auerswald EA et al.; A synthetic gene coding for a chicken egg white cystatin variant was cloned and expressed using the pIN-III-ompA Escherichia coli expression system . After osmotic shock of the E . coli cells, the cysteine proteinase inhibitor was isolated from periplasm and purified by S-carboxymethylpapain affinity chromatography . The resulting inhibitory material was characterized by SDS/PAGE, reversed-phase HPLC, peptide mapping and amino acid sequencing . The recombinant variant chicken AEF-{S1----M, M29----I, M89----L}cystatin shows strong inhibitory activity and displays Ki values in the complex with papain, actinidin and cathepsin B similar to those found for natural chicken cystatin . The purified variant showed a native-chicken-cystatin-like conformational state, as determined by NMR spectroscopy, if the NMR data of 15N-labelled recombinant inhibitor were compared with those of the natural inhibitor.

Biochem Biophys Res Commun, 1991 Aug 15, 178(3), 1472 - 8
An endonuclease activity in human polymorphonuclear neutrophils that removes 8-hydroxyguanine residues from DNA+; Chung MH et al.; An endonuclease that specifically removes 8-hydroxyguanine (oh8Gua) from DNA has been isolated from Escherichia coli . As the amount of oh8Gua produced in DNA of X-ray-irradiated mice is known to decrease with time after irradiation, an attempt was made to find a similar activity in human polymorphonuclear neutrophils (PMNs) using a synthetic dsDNA containing oh8Gua as a substrate . The PMN enzyme was isolated free of other DNases, and found to cleave the substrate DNA simultaneously at 2 sites, the phosphodiester bonds 5' and 3' to oh8Gua, producing free hydroxyl and phosphate groups, respectively . The enzyme showed almost no activity on DNAs containing other kinds of modified base tested or mismatched DNA . Thus human cells also contain an endonuclease that specifically removes oh8Gua residues from DNA.

Biochem Biophys Res Commun, 1991 Aug 15, 178(3), 1280 - 7
Scanning tunnelling microscopy of 16S ribosomal RNA in water; Lesniewska E et al.; The scanning tunnelling microscope has been used to image 16S ribosomal RNA molecules in water electrophoretically deposited on graphite surface . Two kinds of images have been obtained: images showing aggregates of 16S ribosomal RNA molecules similar to those obtained from DNA solutions and others showing individual 16S ribosomal RNA molecules . An interesting characteristic of these images, recorded in constant current mode, is that the 16S ribosomal RNA molecules appear to be located below the graphite surface . The morphology and several structural parameters of the molecules were consistent with the data obtained from electron microscopy.

Biochem Biophys Res Commun, 1991 Aug 15, 178(3), 1176 - 81
Melibiose permease of Escherichia coli: mutation of aspartic acid 55 in putative helix II abolishes activation of sugar binding by Na+ ions; Pourcher T et al.; An aspartic residue (Asp55) located in the putative transmembrane alpha-helix II of the melibiose(mel) permease of Escherichia coli was replaced by Cys using oligonucleotide-directed, site-specific mutagenesis . Although D55C permease is expressed at 0.7 times the level of wild type permease, the mutated mel permease loses the ability to catalyse Na+ or H+ coupled melibiose transport against a concentration gradient . (3H) p-nitrophenyl-alpha-D-galactoside (NPG) binding studies demonstrated that D55C permease binds the sugar co-substrate but Na+ (or Li+) ions do no longer enhance the affinity of D55C permease for the co-transported sugar . In addition sugar binding on D55C permease but not on wild type permease is inactivated by sulfhydryl reagents and the inhibition protected by an excess of melibiose . These observations suggest 1) that the negatively-charged Asp55 residue, expected to be within the membrane embedded domain near the NH2 extremity of mel permease, is in or near the Na(+)-binding site and 2) that the cation and sugar binding sites may be overlapping.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7303 - 7
Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme; Sioud M et al.; Ribozymes are potentially very powerful agents for perturbing intracellular gene expression . However, pilot experiments in eukaryotes have met with mixed success . We now report that a ribozyme designed to cleave the integrase gene of the human immunodeficiency virus (HIV), when transcribed from a plasmid in Escherichia coli, led to destruction of integrase RNA and complete blockage of integrase protein synthesis . These results indicate that ribozymes can be used to study intracellular gene expression in bacteria and that the HIV-1 integrase gene may be a useful target for therapeutic ribozymes.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7271 - 5
Organization and stability of a polytopic membrane protein: deletion analysis of the lactose permease of Escherichia coli; Bibi E et al.; The overall topology of polytopic membrane proteins is thought to result from either the oriented insertion of the N-terminal alpha-helical domain followed by passive insertion of subsequent helices or from the function of independent topogenic determinants dispersed throughout the molecules . By using the lactose permease of Escherichia coli, a well-characterized membrane protein with 12 transmembrane domains and the N and C termini on the cytoplasmic surface of the membrane, we have studied the insertion and stability of in-frame deletion mutants . So long as the first N-terminal and the last four C-terminal putative alpha-helical domains are retained, stable polypeptides are inserted into the membrane, even when an odd number of helical domains is deleted . Moreover, even when an odd number of helices is deleted, the C terminus remains on the cytoplasmic surface of the membrane, as judged by lacY-phoA fusion analysis . In addition, permease molecules devoid of even or odd numbers of putative transmembrane helices retain a specific pathway for downhill lactose translocation . The findings imply that relatively short C-terminal domains of the permease contain topological information sufficient for insertion in the native orientation regardless of the orientation of the N terminus.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7145 - 9
The Rex regulatory protein of human T-cell lymphotropic virus type I binds specifically to its target site within the viral RNA; Unge T et al.; The Rex protein of human T-cell leukemia virus type I (HTLV-I) was expressed in bacteria and partially purified . Rex was shown to bind in vitro specifically to an RNA sequence located in the 3' long terminal repeat of HTLV-I, named Rex-responsive element (RXRE) . Rex also bound in vitro to the human immunodeficiency virus type 1 (HIV-1) Rev-responsive element (RRE), while purified HIV-1 Rev protein did not bind to the RXRE . The binding results obtained in vitro are therefore in agreement with the nonreciprocal function of Rev and Rex in vivo . Rex binds specifically to both RRE and RXRE and activates expression in both HIV-1 and HTLV-I, while Rev binds to RRE and activates only HIV-1 . Binding of Rex to RRE deletion mutants previously shown to lack either the Rev-responsive or the Rex-responsive portion suggested preferential binding of Rex to a distinct target within the RRE . These results demonstrated that Rex, like Rev, acts by binding to a specific RNA target.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 6906 - 10
Molecular cloning and expression of a human heat shock factor, HSF1; Rabindran SK et al.; Human cells respond to heat stress by inducing the binding of a preexisting transcriptional activator (heat shock factor, HSF) to DNA . We have isolated recombinant DNA clones for a human HSF (HSF1) by screening cDNA libraries with a human cDNA fragment . The human HSF1 probe was produced by the PCR with primers deduced from conserved amino acids in the Drosophila and yeast HSF sequences . The human HSF1 mRNA is constitutively expressed in HeLa cells under nonshock conditions and encodes a protein with four conserved leucine zipper motifs . Like its counterpart in Drosophila, human HSF1 produced in Escherichia coli in the absence of heat shock is active as a DNA binding transcription factor, suggesting that the intrinsic activity of HSF is under negative control in human cells . Surprisingly, an independently isolated human HSF clone, HSF2, is related to but significantly different from HSF1 {Schuetz, T . J., Gallo, G . J., Sheldon, L., Tempst, P . & Kingston, R . E . (1991) Proc . Natl . Acad . Sci . USA 88, 6911-6915}.

J Biol Chem, 1991 Aug 15, 266(23), 15511 - 9
Crystal structure of Escherichia coli CheY refined at 1.7-A resolution; Volz K et al.; The three-dimensional structure of wild-type CheY from Escherichia coli has been refined by stereochemically restrained least squares minimization to a crystallographic R-factor of 15.1% at 1.7-A resolution . The structure contains 1165 atoms, including all atoms of the protein, 147 water molecules, and three sulfate ions . The final model has root mean square deviations of 0.018 and 0.049 A from idealized bond lengths and angle distances, respectively . Seven amino acid side chains have been modeled in dual conformations . CheY folds as a compact (beta/alpha)5 globular protein, with the phosphorylation region contained in a cavity on one face of the molecule . This active site area is bordered by the carboxyl termini of the three central beta-strands, by alpha 1, and by the loop connecting beta 5 to alpha 5 . The Lys-109 side chain of this loop extends into the active site by virtue of its cis peptide bond conformation preceding Pro-110 . The epsilon-amino group of Lys-109 is in close bonding contact with the carboxyl group of Asp-57, the residue that is phosphorylated in the activation process of CheY . The details of the hydrogen bonding network in the phosphorylation region indicate that structural rearrangements must accompany the phosphorylation of Asp-57.

J Biol Chem, 1991 Aug 15, 266(23), 15300 - 7
RPC19, the gene for a subunit common to yeast RNA polymerases A (I) and C (III); Dequard-Chablat M et al.; Yeast RNA polymerases A (I) and C (III) share a subunit called AC19 . The gene encoding AC19 has been isolated from yeast genomic DNA using oligonucleotide probes deduced from peptide sequences of the isolated subunit . This gene (RPC19) contains an intron-free open reading frame of 143 amino acid residues . RPC19 is a single copy gene that maps on chromosome II and is essential for cell viability . The amino acid sequence contains a sequence motif common to the Escherichia coli RNA polymerase alpha subunit, the Saccharomyces cerevisiae AC40 and B44.5 subunits, the human hRPB33 product, and the CnjC conjugation-specific gene product of Tetrahymena . The 5'-upstream region contains a sequence element, the PAC box, that has been conserved in at least 10 genes encoding subunits of RNA polymerases A and C.

J Biol Chem, 1991 Aug 15, 266(23), 14918 - 25
Cysteinyl peptides of pig heart NADP-dependent isocitrate dehydrogenase that are modified upon inactivation by N-ethylmaleimide; Smyth GE et al.; Pig heart NADP-specific isocitrate dehydrogenase is inactivated by N-ethylmaleimide (NEM) (Colman, R . F., and Chu, R . (1970) J . Biol . Chem . 245, 601-607), and is completely protected against inactivation, but not against the incorporation of NEM, by isocitrate plus Mn2+ . We have now treated the enzyme with {3H}NEM in the absence and presence of isocitrate plus Mn2+, digested it with trypsin, and isolated and sequenced the labeled Cys peptides . In the inactive enzyme, two major peptides, SSGGFVWACK and DLAGCIHGLSNVK, and two minor peptides, CATITPDEAR and EPIICK, were labeled at Cys . Upon reaction with {3H}NEM in the presence of isocitrate plus Mn2+, full catalytic activity was retained and only DLAGCIHGLSNVK was labeled; the Cys of this peptide is therefore not essential for catalysis . The modification of SSGGFVWACK appears to be the major cause of inactivation by NEM . The Cys in SSGGFVWACK may have a catalytic role, most likely in the strengthened binding of Mn2+ in the presence of isocitrate . Isocitrate dehydrogenase was carboxymethylated under denaturing conditions with {14C}iodoacetate and digested with trypsin; 6 unique labeled Cys peptides, containing 6 unique Cys residues, were purified and sequenced . Six corresponding peptides were isolated from enzyme treated under denaturing conditions with {3H}NEM . These results eliminate the previous uncertainty regarding the number of Cys residues in the enzyme . A comparison of the sequences of the NH2-terminal 30 residues and the 6 Cys peptides of the pig heart NADP-dependent isocitrate dehydrogenase with the Escherichia coli NADP enzyme provides evidence for great dissimilarity between the two enzymes.

J Biol Chem, 1991 Aug 15, 266(23), 14850 - 3
Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2; Sharp JD et al.; Phospholipases A2 (PLA2s) play a key role in inflammatory processes through production of precursors of eicosanoids and platelet-activating factor . Recently, we described the purification of a novel approximately 100-kDa cytosolic PLA2 (cPLA2) from human monoblast U937 cells that is activated by physiological (intracellular) concentrations of Ca2+ (Kramer, R . M., Roberts, E . F., Manetta, J., and Putnam, J . E . (1991) J . Biol . Chem . 266, 5268-5272) . Here we report the isolation of the complementary DNA encoding human cPLA2 and confirm its identity by expression in bacteria and in hamster cells . The predicted 749-amino acid cPLA2 protein has no similarity to the well known secretory PLA2s, but contains a structural element homologous to the C2 region of protein kinase C . The molecular cloning of cPLA2 will allow further studies defining the structure, function, and regulation of this novel PLA2.

Cancer Res, 1991 Aug 15, 51(16), 4131 - 4
O6-methylguanine-DNA methyltransferase in human normal and tumor tissue from brain, lung, and ovary; Citron M et al.; The resistance of human tumor strains in culture to cell killing by alkylating nitrosoureas is correlated with their levels of the DNA repair activity O6-methylguanine-DNA methyltransferase . Strains with the Mer- phenotype have no activity and are extremely sensitive . However, the relationship between the sensitivity of human tumors in vivo and transferase levels is not known, and even the existence of Mer- human tumors in vivo has been questioned . In this study 73 human tumor and normal tissue samples from brain, lung, and ovary were assayed for transferase levels and methylpurine glycosylase activity . For each organ, transferase levels varied over 100-fold, and Mer- tumors were detected in each group . There was no correlation between transferase and glycosylase levels, indicating that the absence of transferase in some tumor samples was not an artifact due to necrosis or inactivation of enzymes in the extract.

J Biol Chem, 1991 Aug 15, 266(23), 14970 - 7
A 25-kDa stretch of the extracellular domain of the human interferon gamma receptor is required for full ligand binding capacity; Fountoulakis M et al.; We investigated which is the shortest fragment of the interferon gamma receptor with ligand binding capacity . A recombinant soluble interferon gamma receptor produced in Escherichia coli was subjected to controlled digestion with several proteolytic enzymes . The fragments generated were assayed by four approaches for interferon gamma binding . A 25-kDa polypeptide comprising residues 6-227 of the mature protein was produced by sequential digestion with trypsin and proteinase K . It was identified as the shortest receptor domain with full interferon gamma binding capacity as judged by ligand blots . The proteolytic fragments were further tested for ligand binding by interferon gamma affinity chromatography . A 15-kDa polypeptide comprising amino acids 94-227 produced by digestion with endoproteinase Glu-C was found to bind with low affinity to immobilized interferon gamma . This fragment, which does not show ligand binding capacity on protein blots, was immunoprecipitated as a complex with interferon gamma by anti-interferon gamma antibodies . It also competed for the binding of radiolabeled interferon gamma to the cell surface receptor when it was assayed as a mixture of the proteolytic products, but not after separation from the cleaved rest of the molecule . The 15-kDa polypeptide probably carries the ligand-binding domain or part of it, but it lacks sequences essential for full interferon gamma binding capacity . The stretch between amino acids 6 and 21 which does not include any disulfide bonds seems to be essential for the receptor to show full activity . The digestion with endoproteinase Glu-C revealed that cysteine residues 60 and 68 of the interferon gamma receptor form a disulfide bond.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7185 - 9
Use of site-directed mutagenesis to enhance the epitope-shielding effect of covalent modification of proteins with polyethylene glycol; Hershfield MS et al.; Modification by covalent attachment of polyethylene glycol (PEG) can reduce the immunogenicity and prolong the circulating life of proteins, but the utility of this approach for any protein is restricted by the number and distribution of PEG attachment sites (e.g., epsilon-amino groups of lysine residues) . We have developed a strategy for introducing additional sites for PEG attachment by using site-directed mutagenesis to selectively replace arginine with lysine codons and tested it with purine nucleoside phosphorylase (PNP) from Escherichia coli, an extremely stable but immunogenic enzyme, that could potentially be used to treat an inherited deficiency of PNP . A triple mutant, RK3, possessing three Arg----Lys substitutions was constructed that increased the number of lysines per PNP subunit from 14 to 17, providing an additional 18 potential PEG attachment sites per hexameric enzyme molecule . The wild-type and RK3 enzymes had similar catalytic activity, antigenicity, and immunogenicity . After PEG modification, both enzymes retained catalytic activity, the plasma half-life of both enzymes in mice increased from approximately 4 hr to 4 days, and the binding of both enzymes by antisera raised against each unmodified enzyme was markedly diminished . However, antibody raised against wild-type PEG-PNP did not bind the PEG-RK3 enzyme . PEG-RK3 PNP was also substantially less immunogenic than wild-type PEG-PNP . Accelerated antibody-mediated clearance of PEG-PNP occurred in 2 of 12 mice treated with PEG-RK3 PNP, compared with 10 of 16 mice treated with the modified wild-type enzyme . This combined use of directed mutagenesis and PEG modification is aimed at permitting the widest choice of proteins, including products of genetic and chemical "engineering," to be used for therapy of inherited and acquired disorders.

Biochem Biophys Res Commun, 1991 Aug 15, 178(3), 1326 - 34
Expression of the p80 region of bovine viral diarrhea virus and identification of specific antibodies to this recombinant protein in bovine sera; Kwang J et al.; A 917-base pair segment of the bovine viral diarrhea virus (BVDV) genome encoding part of the p80 region was cloned into plasmid Gex-2T expression vector for expression as a fusion protein with glutathione-S-transferase (GST) . When the p80 and GST sequences were in the same reading frames, the resulting GST-p80 fusion protein had a molecular mass of 58 kilodaltons (kDa) in SDS-PAGE . Extracts of control E . coli carrying only the vector plasmid (Gex-2T) did not contain this new 58-kDa protein band . Mouse monoclonal antibody specific to BVDV-p80 recognized this recombinant protein . Seventy cattle sera that had an SN titer (to TGAC isolate of cytopathic BVDV) greater than 1:8 reacted with this recombinant protein in Western blots . Of 28 cattle sera that had SN titers less than or equal to 1:8, only one serum tested positive on Western blots.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7381 - 5
Cu,Zn superoxide dismutase is a peroxisomal enzyme in human fibroblasts and hepatoma cells; Keller GA et al.; The intracellular localization of Cu,Zn superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been examined by immunofluorescence using four monoclonal anti-Cu,Zn superoxide dismutase antibodies raised against a recombinant human Cu,Zn superoxide dismutase derivative produced and purified from Escherichia coli . Colocalization with catalase, a peroxisomal matrix enzyme, was used to demonstrate the peroxisomal localization of Cu,Zn superoxide dismutase in human fibroblasts and hepatoma cells . In the fibroblasts of Zellweger syndrome patients, the enzyme is not transported to the peroxisomal ghosts but, like catalase, remains in the cytoplasm . In addition, immunocryoelectron microscopy of yeast cells expressing human Cu,Zn superoxide dismutase showed that the enzyme is translocated to the peroxisomes.

J Biol Chem, 1991 Aug 15, 266(23), 15128 - 34
Crystal structure of ribonuclease T1 complexed with adenosine 2'-monophosphate at 1.8-A resolution; Ding J et al.; Ribonuclease T1 was purified from an Escherichia coli overproducing strain and co-crystallized with adenosine 2'-monophosphate (2'-AMP) by microdialysis against 50% (v/v) 2-methyl-2,4-pentanediol in 20 mM sodium acetate, 2 mM calcium acetate, pH 4.2 . The crystals have orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 48.93(1), b = 46.57(4), c = 41.04(2) A; Z = 4 and V = 93520 A3 . The crystal structure was determined on the basis of the isomorphous structure of uncomplexed RNase T1 (Martinez-Oyanedel et al . (1991) submitted for publication) and refined by least squares methods using stereochemical restraints . The refinement was based on Fhkl of 7,445 reflections with Fo greater than or equal to 1 sigma (Fo) in the resolution range of 10-1.8 A, and converged at a crystallographic R factor of 0.149 . The phosphate group of 2'-AMP is tightly hydrogen-bonded to the side chains of the active site residues Tyr38, His40, Glu58, Arg77, and His92, comparable with vanadate binding in the respective complex (Kostrewa, D., Choe, H.-W., Heinemann, U., and Saenger, W . (1989) Biochemistry 28, 7592-7600) and different from the complex with guanosine 2'-monophosphate (Arni, R., Heinemann, U., Tokuoka, R., and Saenger, W . (1988) J . Biol . Chem . 263, 15358-15368) where the phosphate does not interact with Arg77 and His92 . The adenosine moiety is not located in the guanosine recognition site but stacked on Gly74 carbonyl and His92 imidazole, which serve as a subsite, as shown previously (Lenz, A., Cordes, F., Heinemann, U., and Saenger, W . (1991) J . Biol . Chem . 266, 7661-7667); in addition, there are hydrogen bonds adenine N6H . . . O Gly74 (minor component of three-center hydrogen bond) and adenosine O5' . . . O delta Asn36 . These binding interactions readily explain why RNase T1 has some affinity for 2'-AMP . The molecular structure of RNase T1 is only marginally affected by 2'-AMP binding . Its "empty" guanosine-binding site features a flipped Asn43-Asn44 peptide bond and the side chains of Tyr45, Glu46 adopt conformations typical for RNase T1 not involved in guanosine binding . The side chains of amino acids Leu26, Ser35, Asp49, Val78 are disordered . The disorder of Val78 is of interest since this amino acid is located in a hydrophobic cavity, and the disorder appears to be correlated with an "empty" guanosine-binding site . The two Asp15 carboxylate oxygens and six water molecules coordinate a Ca2+ ion 8-fold in the form of a square antiprism.

J Biol Chem, 1991 Aug 15, 266(23), 14958 - 63
The petite phenotype resulting from a truncated copy of subunit 6 results from loss of assembly of the cytochrome bc1 complex and can be suppressed by overexpression of subunit 9; Schmitt ME et al.; Disruption of the gene for subunit 6 of the yeast cytochrome bc1 complex (QCR6) causes a temperature-sensitive petite phenotype in contrast to deletion of the coding region of QCR6, which shows no growth defect . Mitochondria from the petite strain carrying the disruption allele were devoid of ubiquinol-cytochrome c oxidoreductase activity but retained cytochrome c oxidase and oligomycin-sensitive ATPase activities . Optical spectra of cytochromes in mitochondrial membranes from the petite strain lacked a cytochrome b absorption band and had a reduced amount of cytochrome c1 . Analysis of mitochondrial translation products showed normal synthesis of cytochrome b . Western analysis of mitochondrial membranes from this disruption strain indicates core protein 1 of the cytochrome bc1 complex is present in normal amounts, while cytochrome c1, the Rieske iron-sulfur protein, subunit 6, and subunit 7 were absent or present in very low amounts . Taken together, these findings indicate a loss of assembly of the cytochrome bc1 complex . High copy suppressors of the disruption strain were selected . Two separate families of suppressors were found . The first contained QCR6 . The second family consisted of overlapping clones of a second gene distinct from QCR6 . These plasmids contained QCR9, the gene which codes for subunit 9 of the yeast cytochrome bc1 complex . Suppression of the QCR6 disruption strain by overexpression of QCR9 indicates a critical interaction between these two proteins in the assembly of the cytochrome bc1 complex.

J Biol Chem, 1991 Aug 15, 266(23), 14896 - 902
Characterization of lanthanides as competitors of Na+ and K+ in occlusion sites of renal (Na+,K+)-ATPase; David P et al.; Lanthanides are useful probes in Ca2+ binding proteins, including sarcoplasmic reticulum (Ca2+,Mg2+)-ATPase . Here, we report that lanthanides compete with Rb+ and Na+ for occlusion in renal (Na+,K+)-ATPase . The lanthanides appear to bind at a single site and act as competitive antagonists, without themselves becoming occluded . All lanthanides tested are effective with the order of potencies Pr greater than Nd greater than La greater than Eu greater than Tb greater than Ho greater than Er, but differences are small . The presence of Mg2+ ions does not affect competition of La3+ with Na+ or K+ suggesting that the effects are not exerted via divalent metal sites . Lanthanides compete with Rb+ and Na+ in membranes digested with trypsin so as to produce 19-kDa and smaller fragments of the alpha-chain (Karlish, S.J.D., Goldshleger, R., and Stein, W . D . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 4566-4570), also suggestive of a direct interaction of lanthanides with Na+ and K+ sites . Effects of lanthanides on conformational changes of fluorescein-labeled (Na+,K+)-ATPase are Na(+)-like . They stabilize the E1 state and compete with K+ ions . The Ki for La3+ is 0.445 microM . The apparent affinity in fluorescence assays is proportional to enzyme concentration (Ki = 32.4*{protein} + 0.445 microM La3+), suggesting that lanthanides are also bound nonspecifically (possibly to phospholipids) . Direct assays confirm that Tb3+ binding is nonspecific . Measurements of the rate of various conformational transitions show that the rate of E2(K+)----E1(X) (X = Na+ or La3+) is significantly inhibited by La3+ compared to Na+ . La3+ ions also slightly accelerate the rate of the E1----E2(K+) conformational transition . The dissociation rate of La3+ has been measured by monitoring the rate of E1(La3+)----E2(K+) . It is 1.741 s-1 at 25 degrees C . Based on this value, it is unlikely that La3+ ions are stably occluded, consistent with the conclusion from occlusion experiments . In the future, lanthanides bound to monovalent cation sites with high affinity may become useful probes for location and characterization of sites, although it will be necessary to take into account the large amount of nonspecific binding.

Cancer Res, 1991 Aug 15, 51(16), 4423 - 9
Binding by immunoglobulin to the HPV-16-derived proteins L1 and E4 in cervical secretions of women with HPV-related cervical disease; Snyder KA et al.; Although DNA of the human papillomaviruses (HPV) can be identified in epithelium of a large proportion of patients with genital squamous lesions, relatively little is known about the extent of the local host immune response to this virus . We analyzed cervical secretions from patients undergoing evaluation because of abnormal Papanicolaou smears (cervical biopsy showed nonspecific atypia, flat condyloma, or intraepithelial neoplasia), as well as controls, for immunoglobulin binding to proteins produced in vitro to HPV-16 L1, E4, and E7 open reading frames . Segments of the HPV-16 genome, including portions of the L1 (nucleotides 6153-6794), E4 (nucleotides 3399-3648), and E7 (nucleotides 686-880) open reading frames, were cloned into pATH vectors and expressed as tryptophan synthetase E fusion proteins in Escherichia coli and used as a source of study antigens . Fusion proteins containing the HPV L1, E4, and E7 polypeptides were found to be distinct by molecular weight (59,000; 45,000; and 42,000) as well as by immunological determinants recognized by heterologous immune sera . Of 8 cervical intraepithelial neoplasia lesions tested by RNA-RNA in situ hybridization, 7 were found to be positive for HPV-16-related nucleic acids, in contrast to none (0 of 4) in the condyloma group (three positive for HPV DNA other than type 16) . Immunoglobulin in cervical secretions showed reactivity to HPV type 16 E4 or L1 or both, with highest binding in patients with cervical intraepithelial neoplasia (P less than 0.01 for HPV-16 L1 and E4 compared with controls) . Binding was not tryptophan synthetase E dependent and was, in general, coincident for the HPV-16 E4 and L1 proteins . We conclude that study of cervical secretions, using a quantitative assay for immunoglobulin binding to HPV-16 proteins produced in vitro, may be useful to document the quality and quantity of the immune response of the host to this important human pathogen.

Nature, 1991 Aug 15, 352(6336), 632 - 5
New antiviral strategy using capsid-nuclease fusion proteins; Natsoulis G et al.; Overexpression of dominant-negative mutants of various viral proteins can result in 'intracellular immunization' . Here we describe a new approach to interfering with viral replication in which a nuclease is fused to a capsid component so that the nuclease is encapsidated inside the virion where it can inactivate viral nucleic acid . We used Ty1, a yeast retrotransposon whose transposition closely parallels retroviral replication mechanisms and serves as an easily manipulated model for the retroviral infection process . We constructed fusion genes consisting of the region encoding the N-terminal portion of the TYA/TYB open reading frames of retrotransposon Ty1 and either of two different nuclease genes . Ty1-nuclease fusion proteins are targeted to Ty1 virus-like particles, and are active in degrading nucleic acids . A Ty1-barnase fusion protein causes 98-99% reduction in the efficiency of Ty1 transposition in vivo, presumably by degrading encapsidated Ty1 RNA . This strategy, referred to as capsid-targeted viral inactivation, may be useful for interfering with the replication of retroviruses and other viruses.

Biochemistry, 1991 Aug 13, 30(32), 8083 - 91
C-terminal structure and mobility of rabbit skeletal muscle light meromyosin as studied by one- and two-dimensional 1H NMR spectroscopy and X-ray small-angle scattering; Kalbitzer HR et al.; Intact rabbit myosin and two different C-terminal fragments of rabbit muscle light meromyosin (LMM) expressed in Escherichia coli, LMM-30, and LMM-30C', were studied by 1H NMR spectroscopy . X-ray small-angle scattering shows that at high ionic strength two polypeptide chains of LMM-30 (which consists of the C-terminal 262 amino acids of myosin heavy chain) or LMM-30C' (which corresponds to LMM-30 but lacks the last 17 residues) assemble to form an alpha-helical coiled-coil as it is found also in myosin . The last 12 C-terminal residues of one polypeptide chain of LMM-30 and the last 9 C-terminal residues of the other chain are very mobile . The last 8 residues of the two strands are equivalent from the NMR point of view and unfolded; the valine residues in position 255 in the two strands are not equivalent, suggesting an interaction between the two strands, Ser-252, Arg-253, and Asp-254 are completely immobilized in one of the polypeptide strands and partly mobile in the other . Essentially the same pattern is observed in intact myosin . In spite of the large molecular weights of LMM-30 and LMM-30C', it is possible to resolve almost all aromatic residues and to determine the pK values of all the 4 tyrosine and of 9 (out of 10) histidine residues . The tyrosine residues in the two strands are equivalent in the two polypeptide chains and both have a pK of 10.5 . The pK values of the histidine residues vary between 5.7 and 7.0.

Biochemistry, 1991 Aug 13, 30(32), 8060 - 6
Refolded HIV-1 tat protein protects both bulge and loop nucleotides in TAR RNA from ribonucleolytic cleavage; Harper JW et al.; Substantial evidence indicates that HIV-1 trans-activation by tat protein is mediated through the TAR RNA element . This RNA forms a stem-loop structure containing a three-nucleotide bulge and a six-nucleotide loop . Previous mutagenic analysis of TAR indicates that the bulge residues and a 4 bp segment of the stem constitute, in part, the tat binding site . However, there appears to be no sequence-specific contribution of the six-base loop . We have employed a ribonuclease protection technique to explore the interaction of tat with single-stranded regions of TAR . The results indicate that tat interacts with both the bulge and loop regions of TAR . Treatment of TAR RNA with RNase A results in cleavage at U23 and U31, located in the bulge and loop regions, respectively . High concentrations (approximately 2 microM) of Escherichia coli derived tat protein, prepared by standard procedures, gave complete protection of TAR RNA from RNase A cleavage . However, under these conditions, truncated TAR derivatives in which no stem-loop structure is expected to form were also protected, indicating nonspecific binding . In order to obtain a tat preparation with enhanced specificity toward TAR RNA, methods were developed for refolding the recombinant protein . This treatment enhanced the affinity of tat for TAR by approximately 30-fold {Kd(apparent) less than 25 nM} and markedly increased its specificity for the TAR . Again, tat protected TAR RNA from RNase A cleavage at both U23 and U31 . Protection was also observed with RNase T1 which cleaves TAR RNA at three G residues in the six-base loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Aug 13, 30(32), 8052 - 9
Oligonucleotide site directed mutagenesis of all histidine residues within the T4 endonuclease V gene: role in enzyme-nontarget DNA binding; Augustine ML et al.; In order to evaluate the contributions that histidine residues might play both in the catalytic activities of endonuclease V and in binding to nontarget DNA, the technique of oligonucleotide site directed mutagenesis was used to create mutations at each of the four histidine residues in the endonuclease V gene . Although none of the histidines were shown to be absolutely required for the pyrimidine dimer specific DNA glycosylase activity or the apurinic lyase activity, conservative amino acid changes at His16 produced enzymes with little or no catalytic activity . In addition, the evaluation of conservative and radical amino acid substitutions at positions 34, 56, and 107 is consistent with the interpretation that each of these histidines may be involved in nontarget DNA binding . The data supporting this conclusion are that histidine changes to lysine at positions 34 and 107 enhance the nontarget DNA binding activity of the mutant enzymes while neutralization of charge at His56 reduces nontarget DNA binding.

Biochemistry, 1991 Aug 13, 30(32), 8036 - 40
In vitro transcription analysis of DNA alkylation by nitrogen mustard; Gray PJ et al.; A synchronized in vitro transcription assay has been used to probe the sequence specificity of alkylation of DNA by nitrogen mustard . Transcriptional blockages were detected with use of a 497-base-pair PvuII/SalI restriction fragment of a modified pBR322 vector when initiation of transcription was commenced after the DNA had been alkylated but not if the initiated transcription complex was subjected to alkylation before the elongation phase . The intensity of transcriptional blockages increased with alkylation time and was maximal after 1.5 h at a mustard concentration of 200 microM . There was also evidence of alkylation of the promoter region with increasing mustard concentration . The transcriptional blockage pattern changed at some sites as elongation time was increased and three types of blockages were observed-partial transcription (one or two nucleotides) past an initial blockage site, delayed but normal transcription past some sites, and complete termination at most sites . Eight of the nine blockage sites detected were at G or GG sequences on the template strand, with an apparent specificity for 5'-CTGT sequences of the template strand . Seven of the nine sites were capable of inter- or intrastrand cross-links, including three possible G-G interstrand cross-links spanning an intervening base-pair . In the 103-bp segment probed by this procedure, transcriptional blockages were detected (with one exception) only at sites corresponding to G on the template strand where inter- or intrastrand cross-linking was possible but not for similar sequences on the non-template strand.

Biochemistry, 1991 Aug 13, 30(32), 8026 - 35
DNA unwinding produced by site-specific intrastrand cross-links of the antitumor drug cis-diamminedichloroplatinum(II); Bellon SF et al.; The DNA unwinding produced by specific adducts of the antitumor drug cis-diamminedichloroplatinum(II) has been quantitatively determined . Synthetic DNA duplex oligonucleotides of varying lengths with two base pair cohesive ends were synthesized and characterized that contained site-specific intrastrand N7-purine/N7-purine cross-links . Included are cis-{Pt(NH3)2{d(GpG)}}, cis-{Pt(NH3)2(d(ApG)}}, and cis-{Pt(NH3)2{d(GpTpG)}} adducts, respectively referred to as cis-GG, cis-AG, and cis-GTG . Local DNA distortions at the site of platination were amplified by polymerization of these monomers and quantitatively evaluated by using polyacrylamide gel electrophoresis . The extent of DNA unwinding was determined by systematically varying the interplatinum distance, or phasing, in polymers containing the adducts . The multimer that migrates most slowly gives the optimal phasing for cooperative bending, from which the degree of unwinding can be obtained . We find that the cis-GG and cis-AG adducts both unwind DNA by 13 degrees, while the cis-GTG adduct unwinds DNA by 23 degrees . In addition, experiments are presented that support previous studies revealing that a hinge joint forms at the sites of platination in DNA molecules containing trans-GTG adducts . On the basis of an analysis of the present and other published studies of site-specifically modified DNA, we propose that local duplex unwinding is a major determinant in the recognition of DNA damage by the Escherichia coli (A)BC excinuclease . In addition, local duplex unwinding of 13 degrees and bending by 35 degrees are shown to correlate well with the recognition of platinated DNA by a previously identified damage recognition protein (DRP) in human cells.

Biochemistry, 1991 Aug 13, 30(32), 7901 - 7
Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase; Miran SG et al.; Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine . The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein . The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine . The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme . The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction . However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM . The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged . These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.

Biochemistry, 1991 Aug 13, 30(32), 7883 - 7
Oxygenase side reactions of acetolactate synthase and other carbanion-forming enzymes; Abell LM et al.; Enzymes that mediate carbanion chemistry must protect their reactants from solvent and undesirable electrophiles, such as molecular oxygen . A number of enzymes that utilize carbanionic intermediates were surveyed for O2-consuming side reactions . Several of these enzymes, acetolactate synthase, pyruvate decarboxylase, class II aldolase, and glutamate decarboxylase, catalyze previously undetected oxygen-consuming reactions, while others such as class I aldolase, {(phosphoribosyl)amino}imidazole carboxylase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, and triosephosphate isomerase do not . Prior to this work, only ribulosebisphosphate carboxylase was known to catalyze an oxygenase side reaction . These new example indicate that while O2-consuming side reactions are a more general feature of enzyme-mediated carbanion chemistry than has been previously appreciated, they are not necessarily an inevitable consequence of this chemistry . Expression of an oxygenase activity not only depends on the accessibility of the carbanionic intermediate to molecular oxygen but also may depend on the ability of the enzyme to stabilize the initially formed peroxide anion either through protonation with an appropriate enzymic group or through metal coordination.

J Bacteriol, 1991 Aug, 173(15), 4660 - 7
Genetic and physical analyses of the growth rate-dependent regulation of Escherichia coli zwf expression; Rowley DL et al.; Growth rate-dependent regulation of the level of Escherichia coli glucose 6-phosphate dehydrogenase, encoded by zwf, and 6-phosphogluconate dehydrogenase, encoded by gnd, is similar during steady-state growth and after nutritional upshifts . To determine whether the mechanism regulating zwf expression is like that of gnd, which involves a site of posttranscriptional control located within the structural gene, we prepared and analyzed a set of zwf-lacZ protein fusions in which the fusion joints are distributed across the glucose 6-phosphate dehydrogenase coding sequence . Expression of beta-galactosidase from the protein fusions was as growth rate dependent as that of glucose 6-phosphate dehydrogenase itself, indicating that regulation does not involve an internal regulatory region . The level of beta-galactosidase in zwf-lac operon fusion strains and the level of zwf mRNA from a wild-type strain increased with increasing growth rate, which suggests that growth rate control is exerted on the mRNA level . The half-life of the zwf mRNA mass was 3.0 min during growth on glucose and 3.4 min during growth on acetate . Thus, zwf transcription appears to be the target for growth rate control of the glucose 6-phosphate dehydrogenase level.

Nucleic Acids Res, 1991 Aug 11, 19(15), 4199 - 201
Interaction of ribo- and deoxyriboanalogs of yeast tRNA(Phe) anticodon arm with programmed small ribosomal subunits of Escherichia coli and rabbit liver; Koval'chuke OV et al.; A synthetic ribooligonucleotide, r(CCAGACUGm-AAGAUCUGG), corresponding to the unmodified yeast tRNA(Phe) anticodon arm is shown to bind to poly(U) programmed small ribosomal subunits of both E . coli and rabbit liver with affinity two order less than that of a natural anticodon arm . Its deoxyriboanalogs d(CCAGACTGAAGATCTGG) and d(CCAGA)r(CUGm-AAGA)d(TCTGG), are used to study the influence of sugar-phosphate modification on the interaction of tRNA with programmed small ribosomal subunits . The deoxyribooligonucleotide is shown to adopt a hairpin structure . Nevertheless, as well as oligonucleotide with deoxyriboses in stem region, it is not able to bind to 30S or 40S ribosomal subunits in the presence of ribo-(poly(U} or deoxyribo-(poly (dT) template . The deoxyribooligonucleotide also has no inhibitory effect on tRNA(Phe) binding to 30S ribosomes at 10-fold excess over tRNA . Neomycin does not influence binding of tRNA anticodon arm analogs used . Complete tRNA molecule and natural modifications of anticodon arm are considered to stabilize the arm structure needed for its interaction with a programmed ribosome.

Nucleic Acids Res, 1991 Aug 11, 19(15), 4091 - 6
Isolation of estrogen receptor-binding sites in human genomic DNA; Inoue S et al.; Total genomic DNA digested by restriction enzymes was mixed with the DNA-binding domain of the estrogen receptor (ER-DBD) that was expressed in Escherichia coli and the fragments that bound to it were selected by nitrocellulose filter . These fragments were cloned into a plasmid vector and amplified . This selection process was repeated six times and five fragments ranging from 0.2 to 2 kb were isolated . Interestingly, each of these fragments had a perfect palindromic estrogen responsive element (ERE) (GGT-CANNNTGACC) . More surprisingly, one of the fragments was found to be derived from the same locus as a fragment obtained by another similar but independent experiment . The results indicate that the ER-DBD region can bind by itself specifically to the perfect palindromic ERE with a 3 base pair spacing but it does not bind strongly enough to the half palindromic EREs or to the imperfect palindromic EREs . Chloramphenicol acetyltransferase assay has shown that some of these fragments have estrogen-dependent enhancer activity, suggesting the existence of a target gene near these fragments . The method described here may be generally applicable for screening and isolation of other transcription factor-binding sites in genomic DNA.

Nucleic Acids Res, 1991 Aug 11, 19(15), 4259 - 65
Mutations in 16S rRNA in Escherichia coli at methyl-modified sites: G966, C967, and G1207; Jemiolo DK et al.; Mutations were constructed at three sites in 16S rRNA in E . coli by oligonucleotide-directed mutagenesis, and cloned into the rrnB operon on either pKK3535 or pNO2680 . The mutated bases, G966, C967, and G1207, are located in the 3' major domain of 16S rRNA and are sites post-transcriptionally modified by methylation . We constructed a deletion mutation at C967 (delta 967) and three substitution mutations at each of the following sites: G966, C967, and G1207 . By maxicell analysis, we found that all of the mutations were processed normally and incorporated into 30S subunits and 70S ribosomes . We found that delta 967 was a dominant lethal mutation while the substitution mutations at G966 and C967 had no effects on cell growth rate . The mutants C1207 and U1207 were shown to have dominant lethal phenotypes while A1207 had no effect on cell growth rate . These results help to establish the importance of methyl-modified regions to ribosome function.

Nucleic Acids Res, 1991 Aug 11, 19(15), 4253 - 7
Characterization of the CRPCY core formed after treatment with carboxypeptidase Y; Yang ZH et al.; CRP is resistant to attack by carboxypeptidase Y at 37 degrees C, whereas cAMP-CRP is digested yielding a core terminating at Thr-202 and lacking the seven carboxyl-terminal amino acid residues . A similar core (CRPCY) is formed when CRP is incubated with carboxypeptidase Y at 47 degrees C in the absence of cAMP . CRPCY has a more open conformation than CRP at 37 degrees C . While unliganded CRP is resistant to trypsin, CRPCY is sensitive to tryptic attack . Dithionitrobenzoic acid-mediated intersubunit disulfide crosslinking of CRP requires cAMP, CRPCY subunits are crosslinked in the absence of cAMP . The carboxyl-terminal region of unliganded CRP is conformationally restricted at 37 degrees C . The CRPCY retains cAMP binding activity . The CRPCY which terminates at Thr-202, no longer binds lac P+ DNA nor stimulates abortive initiation by RNA polymerase from the lac P+ promoter . The results indicate that the C-terminal region of CRP participates in the conformational stability of the closed form of CRP and indirectly in DNA binding by the open cAMP-CRP conformer.

Biochim Biophys Acta, 1991 Aug 9, 1079(1), 57 - 64
A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization; Seki S et al.; A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized . The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), Sephadex G-100, single-stranded DNA cellulose and hydroxyapatite . The purified enzyme has an Mr of 34,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escherichia coli exonuclease III . This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities . The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.

J Theor Biol, 1991 Aug 7, 151(3), 359 - 66
Gene regulation under growth conditions . A model for the regulation of initiation of replication in Escherichia coli; Herrick J et al.; A stochastic model is presented to describe gene regulation during growth conditions (non-steady-state), with an emphasis on the distribution of gene activation times . A non-Poissonian distribution, with a smaller variability than in steady-state, is obtained when gene activation by the regulatory protein(s) occurs before the protein(s) reach their saturation concentration . The model is applied to the regulation of initiation of chromosomal replication in Escherichia coli . The rate of initiation is shown to depend linearly on the concentration of the initiator molecule, DnaA, in good agreement with recently published data . It is suggested that the variability of initiation times could be growth rate dependent, with slow growing cells being more synchronized than fast growing ones.

Biochemistry, 1991 Aug 6, 30(31), 7789 - 96
A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity; Xu X et al.; Escherichia coli alkaline phosphatase catalyzes the hydrolysis of a wide variety of phosphomonoesters at similar rates, and the reaction proceeds through a phosphoenzyme intermediate . The active site region is highly conserved between the E . coli and mammalian alkaline phosphatases . The three-dimensional structure of the E . coli enzyme indicates that Lys-328, which is replaced by histidine in all mammalian alkaline phosphatases, is bridged to the phosphate through a water molecule . This water molecule is also hydrogen bonded to Asp-327, a bidendate ligand of the one of the two zinc atoms . Here we report the use of site-specific mutagenesis to convert Lys-328 to both histidine and alanine . Steady-state kinetic studies above pH 7.0 indicate that both mutant enzymes have altered pH versus activity profiles compared to the profile for the wild-type enzyme . At pH 10.3, in the presence of Tris, the Lys-328----Ala enzyme is approximately 14-fold more active than the wild-type enzyme . At the same pH in the absence of Tris the Lys-328----Ala enzyme is still 6-fold more active than the wild-type enzyme . Both mutant enzymes have lower phosphate affinities than the wild-type enzyme at all pH values investigated . Pre-steady-state kinetics at pH 5.5 reveal that the Lys-328----Ala enzyme behaves very similar to the phosphate-free wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Aug 6, 30(31), 7718 - 30
1H and 15N resonance assignments of oxidized flavodoxin from Anacystis nidulans with 3D NMR; Clubb RT et al.; Proton and nitrogen-15 sequence-specific nuclear magnetic resonance assignments have been determined for recombinant oxidized flavodoxin from Anacystis nidulans (169 residues, Mr 19,048) . Assignments were obtained by using 15N-1H heteronuclear three-dimensional (3D) NMR spectroscopy on a uniformly nitrogen-15 enriched sample of the protein, pH 6.6, at 30 degrees C . For 165 residues, the backbone and a large fraction of the side-chain proton resonances have been assigned . Medium- and long-range NOE's have been used to characterize the secondary structure . In solution, flavodoxin consists of a five-stranded parallel beta sheet involving residues 3-9, 31-37, 49-56, 81-89, 114-117, and 141-144 . Medium-range NOE's indicate the presence of several helices . Several 15N and 1H resonances of the flavin mononucleotide (FMN) prosthetic group have been assigned . The FMN-binding site has been investigated by using polypeptide-FMN NOE's.

Biochemistry, 1991 Aug 6, 30(31), 7801 - 9
Effects of point mutations in a hinge region on the stability, folding, and enzymatic activity of Escherichia coli dihydrofolate reductase; Ahrweiler PM et al.; The role of a hinge region in the folding, stability, and activity of Escherichia coli dihydrofolate reductase was investigated with three site-directed mutants at valine-88, the central residue of the hinge . The three mutants, V88A and V88I and a valine-88 deletion, were created to perturb the packing of hydrophobic residues in the interior of a loose turn formed by residues 85-91 . Deleting the valine-88 residue destabilized the protein by 2.93 +/- 0.6 kcal/mol as determined by equilibrium unfolding transitions in urea monitored by circular dichroism at 20 degrees C . Substitution of alanine for valine-88 stabilized the protein by -0.20 +/- 0.02 kcal/mol, and the isoleucine substitution was mildly destabilizing by 1.73 +/- 0.2 kcal/mol . Although there was no clear correlation between side-chain volume and stability, these results suggest that side-chain interactions in the interior of the turn influence the folding and stability of dihydrofolate reductase . The specific activity of the valine deletion mutant was approximately twice that of the wild-type protein while the specific activities of the V88A and V88I proteins were only slightly greater than the wild type . The full time courses of the reactions catalyzed by the mutants were almost identical with that for the wild type, indicating no major changes in the kinetic mechanism . Additionally, the rate constants associated with interconversion between various forms of the apoenzyme were identical for the mutant and wild-type enzymes . The rate constants for refolding transitions were examined by dilution of urea-inactivated protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Aug 6, 30(31), 7796 - 801
Site-directed mutagenesis of Escherichia coli aspartate aminotransferase: role of Tyr70 in the catalytic processes; Inoue K et al.; Site-directed mutagenesis of Tyr70 in the active site of Escherichia coli aspartate aminotransferase (AspAT) followed by kinetic studies has elucidated the roles of the hydroxyl group and benzene ring of Tyr70 . X-ray crystallographic analysis showed that replacement of Tyr70 by Phe did not alter the active-site conformation of the enzyme . Comparison of the kinetic parameters of the four half-transamination reactions (the pyridoxal 5'-phosphate form of the enzyme with L-aspartate or L-glutamate and the pyridoxamine 5'-phosphate form with oxalacetate or 2-oxoglutarate) between the wild-type and {Tyr70----Phe}AspATs showed that the mutation increases the energy level of the transition state by 2 kcal.mol-1 for all the four substrates, suggesting some contribution of the hydroxyl group of Tyr70 to the transition state . When Phe70 was further replaced by Ser, the energy level of the transition state for L-glutamate or 2-oxoglutarate, but not for L-aspartate or oxalacetate, was further increased by 2-3 kcal.mol-1, suggesting that the presence of a benzene ring at position 70 is essential for recognizing the L-glutamate-2-oxoglutarate pair as substrates.

Biochemistry, 1991 Aug 6, 30(31), 7783 - 8
Kinetic studies on chorismate mutase-prephenate dehydrogenase from Escherichia coli: models for the feedback inhibition of prephenate dehydrogenase by L-tyrosine; Turnbull J et al.; Kinetic studies have been undertaken to elucidate the mechanism of the allosteric inhibition by tyrosine of the prephenate dehydrogenase activity of the bifunctional dimeric enzyme chorismate mutase-prephenate dehydrogenase . The effect of tyrosine on the initial velocity of the reactions in the presence of both prephenate and the alternative substrate, 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate, have been determined . In addition, investigations have been made of the effect of tyrosine on the inhibition of the reaction by the inhibitory analogues of prephenate, (4-hydroxyphenyl)pyruvate, and (carboxyethyl)-1,4-dihydrobenzoate . The results of the double inhibition experiments indicate clearly that the enzyme possesses a distinct allosteric site for the binding of tyrosine . The initial velocity data obtained with both substrates have been fitted to the rate equations that describe a wide range of models . From a comparison of the results obtained from studies with the two substrates, and with a knowledge of the value for the dissociation constant of the tyrosine-enzyme complex, definitive conclusions have been reached about the mechanism of the allosteric inhibition . It is concluded that tyrosine combines twice at allosteric sites and in an antisynergistic fashion, while prephenate reacts at both active sites of the dimeric enzyme as well as weakly at one of the allosteric sites . It appears that the latter is simple competition reaction that affects neither the binding of prephenate at the active site nor the rate of product formation . The model also predicts the formation of an active tyrosine-enzyme-prephenate complex that yields product at a much slower rate than does the enzyme-prephenate complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Aug 6, 30(31), 7777 - 82
pH dependency of the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli; Turnbull J et al.; The variation with pH of the kinetic parameters associated with the mutase and dehydrogenase reactions catalyzed by chorismate mutase-prephenate dehydrogenase has been determined with the aim of elucidating the role that ionizing amino acid residues play in binding and catalysis . The pH dependency of log V for the dehydrogenase reaction shows that the enzyme possesses a single ionizing group with a pK value of 6.5 that must be unprotonated for catalysis . This same group is observed in the V/Kprephenate, as well as in the V/KNAD, profile . The V/Kprephenate profile exhibits a second ionizing residue with a pK value of 8.4 that must be protonated for the binding of prephenate to the enzyme . For the mutase reaction, the V/Kchorismate profile indicates the presence of three ionizing residues at the active site . Two of these residues, with similar pK values of about 7, must be protonated, while the third, with a pK value of 6.3, must be unprotonated . It can be concluded that all three groups are concerned with the binding of chorismate to the enzyme since the maximum velocity of the mutase reaction is essentially independent of pH . This conclusion is confirmed by the finding that the Ki profile for the competitive inhibitor, (3-endo,8-exo)-8-hydroxy-2-oxabicyclo{3.3}non-6-ene-3,5-dicarboxylic acid, shows the same three ionizing groups as observed in the V/Kchorismate profile . By contrast, the Ki profile for carboxyethyldihydrobenzoate shows only one residue, with a pK value of 7.3, that must be protonated for binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Aug 6, 30(31), 7711 - 7
1H and 15N NMR resonance assignments and preliminary structural characterization of Escherichia coli apocytochrome b562; Feng YQ et al.; The 1H and 15N resonances of uniformly enriched apocytochrome b562 (106 residues) have been assigned . The assignment work began with the identification of the majority of HN-H alpha-H beta subspin systems in two-dimensional DQF-COSY and TOCSY spectra of unlabeled protein in D2O and in 95% H2O/5% D2O buffer . Intraresidue and interresidue NOE connectivities were then searched for in two-dimensional homonuclear NOESY spectra recorded on unlabeled protein and in the three-dimensional NOESY-HMQC spectrum recorded on uniformly 15N-enriched protein . Those data, combined with the main-chain-directed assignment strategy (MCD), led to the assignment of the main-chain and many side-chain resonances of 103 of the 106 residues . Qualitatively, the helical conformation is found to be the dominant secondary structure in apocytochrome b562 as it is in holocytochrome b562 . The helical segments in apocytochrome b562 overlap extensively with the helical regions defined in the crystal structure of ferricytochrome b562 . In addition, a number of tertiary NOEs have been identified which indicate that the global fold of the apoprotein at least partially resembles the four-helix bundle of the holoprotein . The results presented here, together with the evidence obtained with other methods {Feng and Sligar (1991) Biochemistry (submitted)}, support the notion that the interior of the protein is fluid and may correspond to a molten globule state.

Biochemistry, 1991 Aug 6, 30(31), 7693 - 703
Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy; Kuwajima K et al.; The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 degrees C . The reactions were induced by concentration jumps of a denaturant, urea . The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction . The results can be summarized as follows . (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms) . (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as tau 1, tau 2, tau 3, tau 4, and tau 5 in increasing order of the reaction rate . Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases . (3) The tau 5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region . The absence of the tau 5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the tau 5 phase reflects the formation of a hydrophobic cluster around Trp 74 . From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the tau 5 phase persists during the later stages of folding . (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central beta sheet of the protein . The pairwise similarities in the spectrum between the tau 3 and tau 4 phases, and between the tau 1 and tau 2 phases, also suggest the presence of two parallel folding channels for refolding . (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species . The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1991 Aug 6, 30(31), 7822 - 7
Cooperative protein-DNA interactions: effects of KCl on lambda cI binding to OR; Koblan KS et al.; The effects of monovalent salt activity on the site-specific and cooperative interactions of cI repressor with its three operator sites OR were studied by using quantitative DNase I footprint titration methods . Individual-site binding isotherms were obtained for binding repressor dimers to each site of wild-type OR and to mutant operator templates in which binding to one or two sites has been eliminated . The standard Gibbs energies for intrinsic binding, delta G1, delta G2, and delta G3, and cooperative interactions, delta G12 and delta G23, were determined at each condition (range 50-200 mM KCl) . It is found that the dimer affinity for each of the three sites increases as {KCl} decreases, a striking result given that the monomer-dimer equilibrium shifts toward monomer formation under identical solution conditions {Koblan, K . S., & Ackers, G . K . (1991) Biochemistry (preceding paper in this issue)} . The magnitudes of ion-linked effects are found to differ at the three operator sites, while the intrinsic interaction binding free energies for sites OR1 and OR3 change in parallel over the entire range of {KCl} . The KCl dependencies at OR1 and OR3 represent the average release of 3.7 +/- 0.6 and 3.8 +/- 0.6 apparent ions, respectively . By contrast, the KCl dependency of OR2 binding corresponds to the displacement of 5.2 +/- 0.7 apparent ions . The ability of cI repressor to discriminate between the three operator sites thus appears linked to ion binding/release reactions.

Biochemistry, 1991 Aug 6, 30(31), 7863 - 72
PsaD is required for the stable binding of PsaC to the photosystem I core protein of Synechococcus sp . PCC 6301; Li N et al.; The psaC gene product from Synechococcus sp . PCC 7002 and the psaD gene product from Nostoc sp . PCC 8009 were synthesized in Escherichia coli and purified to homogeneity . Incubation of the PsaC apoprotein with the Synechoccus sp . PCC 6301 photosystem I core protein in the presence of FeCl3, Na2S, and beta-mercaptoethanol resulted in a time-dependent transition in the flash-induced absorption change from a 1.2-ms, P700+ FX- back-reaction to a long-lived, P700+ {FA/FB}- back-reaction . ESR studies showed that FB and FA were photoreduced about equally at 19 K, and while the resonances were shifted upfield, they remained as broad as in the free PsaC holoprotein . When the reconstituted complex was purified in a sucrose gradient containing 0.1% Triton X-100, most of the optical absorption transient reverted to that characteristic of the P700+ FX- back-reaction . Addition of purified PsaD to the incubation mixture led to a greater extent of recovery of electron flow to FA/FB for any given concentration of PsaC . ESR studies showed that FA, rather than FB, became the preferred electron acceptor at 19 K; moreover, the resonances moved upfield and sharpened to become nearly identical with those of a control photosystem I complex . When the sample was purified in a sucrose gradient containing 0.1% Triton X-100, the long-lived P700+ {FA/FB}- optical transient remained stable . Analysis by denaturing polyacrylamide gel electrophoresis showed that the PsaC and PsaD proteins had rebound to the photosystem I core . The data indicate that although PsaC can bind loosely, the presence of PsaD leads to a stable, isolatable photosystem I complex which is spectroscopically indistinguishable from the native complex . Since a PsaC1 fusion protein which contains an amino-terminal extension of five amino acids (MEHSM...) does not bind in the absence of PsaD {Zhao, J., et al . (1990) FEBS Lett . 276, 175-180}, the N-terminus of the PsaC protein could provide a site of interaction with the photosystem I core . We propose that the binding of PsaC to the PsaA/PsaB heterodimer is potentiated by insertion of the FA/FB clusters into PsaC, and stabilized by the presence of PsaD.

J Biol Chem, 1991 Aug 5, 266(22), 14283 - 7
Expression, assembly, and processing of an active plant ferredoxin-NADP+ oxidoreductase and its precursor protein in Escherichia coli; Ceccarelli EA et al.; The flavoprotein ferredoxin-NADP+ reductase (FNR) catalyzes the final step of the photosynthetic electron transport chain, i.e . the reduction of NADP+ by ferredoxin . A cloned FNR cDNA from a pea library (Newman, B., and Gray, J . (1988) Plant Mol . Biol . 10, 511-520) was used to construct plasmids which express the apoenzyme in Escherichia coli . Two recombinant vectors were prepared, one containing the sequence corresponding to the mature enzyme and another including, in addition, the sequence of the transit peptide that directs FNR to the chloroplast . These proteins were expressed as fusion products to the NH2-terminal portion of beta-galactosidase . In both cases, a 35-kDa immunoreactive polypeptide was the major product, suggesting that the proteins were processed in vivo . NH2-terminal sequence determination of the purified recombinant proteins indicate cleavage at positions -1/-2 with respect to the normal processing site in chloroplasts . The processed enzymes showed enzymatic activities and spectral properties that were similar or identical to those of native plant FNR . When a La protease-deficient E . coli strain was used as a host, the expressed FNR precursor was found to be poorly processed, associated to bacterial pellets, and showed no detectable FNR activity . The overall results indicate that acquisition of the native enzyme conformation and assembly of the prosthetic group takes place in the bacterial host, generating an enzyme that is, as far as studied, indistinguishable from plant FNR.

J Biol Chem, 1991 Aug 5, 266(22), 14262 - 9
Subhemolytic doses of Escherichia coli hemolysin evoke large quantities of lipoxygenase products in human neutrophils; Grimminger F et al.; Polymorphonuclear leukocytes (PMN) have been identified as preferred target cells for Escherichia coli hemolysin in human blood (Bhakdi, S., Greulich, S., Muhly, M., Eberspacher, B., Becker, H., Thiele, A., and Hugo, F . (1989) J . Exp . Med . 169, 737-754) . Leukotriene and 5-hydroxyeicosatetraenoic acid generation was investigated in human PMN challenged with E . coli hemolysin in the absence or presence of free arachidonic acid or eicosapentaenoic acid (EPA) . In the absence of exogenous free fatty acids, E . coli hemolysin (0.01-10 hemolytic units/ml) induced moderate generation of leukotriene B4 (LTB4) and its omega-oxidation products . The presence of free arachidonic acid (10 microM) during E . coli hemolysin (0.1 hemolytic unit/ml) challenge evoked the generation of large quantities of these products (greater than 100 pmol/1.5 x 10(7) PMN) . In parallel, large amounts of 5-hydroxyeicosatetraenoic acid and nonenzymatic LTA4 hydrolysis products appeared . Product release peaked or plateaued 5-10 min after E . coli hemolysin challenge . The presence of exogenous EPA upon E . coli hemolysin challenge resulted in the exclusive generation of LTB5 and metabolites, LTA5 decay products and 5-hydroxyeicosapentaenoic acid . Dose and time dependences corresponded to those with arachidonic acid provision, and the total of EPA-derived products surpassed that of arachidonic acid metabolites in corresponding experiments approximately 2-fold . Increasing the time between free fatty acid provision and E . coli hemolysin challenge resulted in a rapid decline in the generation of arachidonic acid or EPA metabolites . Thus, subhemolytic doses of E . coli hemolysin evoke marked PMN eicosanoid generation that is dependent on exogenous free fatty acid supply, with total amounts approximating those found in calcium ionophore-stimulated neutrophils.

FEBS Lett, 1991 Aug 5, 287(1-2), 53 - 6
Molecular cloning of the ecotin gene in Escherichia coli; Lee HR et al.; The nucleotide sequence of a 876 bp region in E . coli chromosome that encodes Ecotin was determined . The proposed coding sequence for Ecotin is 486 nucleotides long, which would encode a protein consisting of 162 amino acids with a calculated molecular weight of 18,192 Da . The deduced primary sequence of Ecotin includes a 20-residue signal sequence, cleavage of which would give rise to a mature protein with a molecular weight of 16,099 Da . Ecotin does not contain any consensus reactive site sequences of known serine protease inhibitor families, suggesting that Ecotin is a novel inhibitor.

FEBS Lett, 1991 Aug 5, 287(1-2), 185 - 8
Uniform labeling of a recombinant antibody Fv-fragment with 15N and 13C for heteronuclear NMR spectroscopy; Riechmann L et al.; The expression of functional antibody fragments in Escherichia coli enables a detailed analysis by NMR spectroscopy . This is demonstrated with the uniform labeling of an Fv-fragment (25 kDa) comprising the antigen binding site of an antibody against 2-phenyloxazolone with 15N and 13C . The antigen-complexed Fv-fragment was analysed for a potential assignment by heteronuclear multi-dimensional NMR spectroscopy . For almost all backbone amides 15N/1H crosspeaks and for 80% of them TOCSY crosspeaks were observed . In a 13C-edited-HCCH-2D experiment 17 out of 18 threonine spin-systems were identified . Thus detailed assignments are possible, but some amino acid specific labeling in addition to uniform labeling will be required for complete assignments of Fv-fragments.

J Mol Biol, 1991 Aug 5, 220(3), 789 - 99
Heterotropic interactions in Escherichia coli aspartate transcarbamylase . Subunit interfaces involved in CTP inhibition and ATP activation; Xi XG et al.; In Escherichia coli aspartate transcarbamylase, each regulatory chain is involved in two kinds of interfaces with the catalytic chains, one with the neighbour catalytic chain which belongs to the same half of the molecule (R1-C1 type of interaction), the other one with a catalytic chain belonging to the other half of the molecule (R1-C4 type of interaction) . In the present work, site-directed mutagenesis was used to investigate the involvement of the C-terminal region of the regulatory chain in the process of feed-back inhibition by CTP . Removal of the two last C-terminal residues of the regulatory chains is sufficient to abolish entirely the sensitivity of the enzyme to CTP . Thus, it appears that the contact between this region and the 240s loop of the catalytic chain (R1-C4 type of interaction) is essential for the transmission of the regulatory signal which results from CTP binding to the regulatory site . None of the modifications made in the R1-C4 interface altered the sensitivity of the enzyme to the activator ATP, suggesting that the effect of this nucleotide rather involves the R1-C1 type of interface . These results are in agreement with the previously proposed interpretation that CTP and ATP do not simply act in inverse ways on the same equilibrium.

J Mol Biol, 1991 Aug 5, 220(3), 649 - 58
Transcriptional organization of the Escherichia coli dnaX gene; Flower AM et al.; We have determined the transcriptional organization of the Escherichia coli dnaX gene, the structural gene for both the gamma and tau subunits of DNA polymerase III holoenzyme . By S1 nuclease protection and primer extension mapping of transcripts encoding the dnaX products, one primary promoter of dnaX has been identified that initiates transcription 37 nucleotides upstream from the first codon . dnaX resides in an operon with two recently sequenced genes, orf12, encoding an unidentified product, and recR, the structural gene for a protein involved in the recF pathway of recombination . Under conditions of balanced growth, a very small amount of transcription from the upstream apt promoter (less than 5%) contributes to the expression of tau and gamma, too low for apt to be considered to be on an operon with dnaX, orf12, and recR are transcribed from an independent promoter as well as from the dnaX promoter, providing a mechanism for orf12 and recR to be regulated independent of dnaX . Transcription of the dnaX-orf12-recR operon is terminated upstream from the previously characterized heat shock gene htpG . The dnaX and orf12-recR promoters, cloned into a promoter detection vector, efficiently direct the expression of the downstream reporter gene, lacZ . These results extend our knowledge of the genetic and transcriptional organization of this region of the E . coli chromosome . The transcriptional organization has been defined as follows: apt, dnaX-orf12-recR, htpG . All of these genes are transcribed in the clockwise direction and only dnaX, orf12 and recR are contained in the dnaX operon.

J Mol Biol, 1991 Aug 5, 220(3), 555 - 68
Factor-independent activation of Escherichia coli rRNA transcription . I . Kinetic analysis of the roles of the upstream activator region and supercoiling on transcription of the rrnB P1 promoter in vitro; Leirmo S et al.; The region from position -154 to position -50 upstream from the start site of transcription of the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), is required for maximal promoter activity in vivo . Maximal activation (20 to 30-fold) requires the binding of Fis protein in vitro and in vivo . However, two- to fourfold activation remains in vivo even in the absence of Fis . Here, we demonstrate that the presence of the UAR increases the rate of formation of E sigma 70-promoter complexes in vitro in the absence of added protein factors (factor-independent activation) . The UAR increases the rate of the RNA polymerase concentration-dependent step in the association pathway to a stable complex formed in the presence of the initiating nucleotides ATP and CTP (RPinit) . The rate of dissociation from RPinit is not affected . In addition, a supercoiled template of native superhelical density increases both the association rate for the formation of RPinit and the lifetime of complexes formed in the absence of nucleotides (RPo or open complex), but does not affect factor-independent activation . The data are consistent with a model whereby the UAR affects only the initial recognition event (closed complex formation) without affecting either the rate or extent of isomerization to the locally denatured open complex . In the accompanying paper, a variety of chemical and enzymatic probes are used to characterize RPinit and RPo both with and without the UAR.

J Mol Biol, 1991 Aug 5, 220(3), 551 - 3
Purification and crystallization of recombinant Escherichia coli malate dehydrogenase; Hall MD et al.; Malate dehydrogenase from Escherichia coli has been crystallized with polyethylene glycol and citrate buffer at pH 5.7 . The enzyme was obtained from an E . coli strain in which the chromosomal malate dehydrogenase gene was contained on a pBR322 vector . Two types of crystals have been observed; a monoclinic C2 form and an orthorhombic C222(1) form, which is found infrequently . Monoclinic crystals were used as seeds in several rounds of crystallization until large crystals suitable for diffraction analysis were available . A complete X-ray data set to 2.0 A has been collected.

Biochim Biophys Acta, 1991 Aug 5, 1067(1), 89 - 96
Affinity-chromatographic purification of sixteen cysteine-substituted maltoporin variants: thiol reactivity and cross-linking in an outer membrane protein of Escherichia coli; Francis G et al.; Wild-type and 16 variant maltoporins with site-directed cysteine substitutions at 14 sites were purified by a novel one-step affinity-chromatographic procedure . The trimer stability of purified proteins with C22S, C38S and G103C substitutions was reduced compared to wild-type maltoporin . Quantitative labelling with N-ethyl{14C}maleimide, cross-linking with bifunctional bismaleimides and disulphide formation was used to test the reactivity of cysteines in the folded protein . The maleimide reactivity of the residues was in the order: 152 approximately equal to 153 greater than 265 greater than 30 approximately equal to 103 approximately equal to 120 approximately equal to 154 approximately equal to 382 greater than 57 approximately equal to 146, with the other sites (22, 38, 97, 184) poorly labelled . Only cysteines at 152 or 153 permitted the formation of inter-subunit disulphide bonds suggesting these residues are located within 0.5-0.9 nm of each other in homotrimers of maltoporin . S152C and S153C as well as S154C permitted the formation of inter-subunit cross-links using bifunctional bismaleimides . The cross-linkability and the high reactivity to N-ethylmaleimide of the 150 region was consistent with the current model of the structure of maltoporin in the outer membrane; the reactivity of the other sites is also discussed within the context of this model.

J Biol Chem, 1991 Aug 5, 266(22), 14562 - 72
Purification, gene cloning, and sequence analysis of an L-isoaspartyl protein carboxyl methyltransferase from Escherichia coli; Fu JC et al.; Mammalian tissues contain protein carboxyl methyltransferases that catalyze the transfer of methyl groups from S-adenosylmethionine to the free carboxyl groups of D-aspartyl or L-isoaspartyl residues (EC 2.1.1.77) . These enzymes have been postulated to play a role in the repair and/or degradation of spontaneously damaged proteins . We have now characterized a similar activity from Escherichia coli that recognizes L-isoaspartyl-containing peptides as well as protein substrates such as ovalbumin . The enzyme was purified by DEAE-cellulose, hydroxylapatite, Sephadex G-100, polyaspartate, and reversed-phase chromatography and was shown to consist of a single 24-kDa polypeptide chain . The sequence determined for the N-terminal 39 residues was used to design an oligonucleotide probe that allowed the precise localization of its structural gene (pcm) on the physical map of the E . coli chromosome at 59 min . Transformation of E . coli cells with a plasmid containing DNA from this region results in a 3-4-fold overproduction of enzyme activity . The nucleotide sequence determined for the pcm gene and its flanking regions was used to deduce a mature amino acid sequence of 207 residues with a calculated molecular weight of 23,128 . This sequence shows 30.8% sequence identity with the human L-isoaspartyl/D-aspartyl methyltransferase and suggests that this enzyme catalyzes a fundamental reaction in both procaryotic and eucaryotic cells.

J Biol Chem, 1991 Aug 5, 266(22), 14539 - 47
Analysis of retroviral protease cleavage sites reveals two types of cleavage sites and the structural requirements of the P1 amino acid; Pettit SC et al.; Retroviruses encode a protease which cleaves the viral Gag and Gag/Pol protein precursors into mature products . To understand the target sequence specificity of the viral protease, the amino acid sequences from 46 known processing sites from 10 diverse retroviruses were compared . Sequence preference was evident in positions P4 through P3' when compared to flanking sequences . Approximately 80% of all cleavage site sequences could be grouped into two classes based on the sequence composition flanking the scissile bond . The sequences at the amino-terminal cleavage site of the major capsid protein of Gag is always a member of one of the two classes while the carboxyl-terminal cleavage site is of the other class, suggesting a biological role for the two classes . Known processing site sequences proved useful in a motif searching strategy to identify processing sites in retroviral protein sequences, particularly in Gag . In all known cleavage sites, the P1 amino acid is hydrophobic and unbranched at the beta-carbon . The sequence requirements of the P1 position were tested by site-directed mutagenesis of the P1 Phe codon in an HIV-1 Pol cleavage site . Mutations were tested for protease-mediated cleavage of the Pol precursor expressed in Escherichia coli.

J Biol Chem, 1991 Aug 5, 266(22), 14478 - 85
RpoB8, a rifampicin-resistant termination-proficient RNA polymerase, has an increased Km for purine nucleotides during transcription elongation; Jin DJ et al.; The rpoB8 allele of Escherichia coli maps to the beta-subunit of RNA polymerase and confers rifampicin resistance as well as increased termination at both intrinsic and rho-dependent terminators in vivo . This phenotype suggests that the mutant is defective in an enzymatic property of RNA polymerase important for all termination events . We analyzed the in vitro transcription properties of this enzyme to determine the nature of the defect . As compared with the wild-type enzyme, RpoB8 exhibits enhanced pausing and a significant reduction in rate of elongation on natural templates . In addition, RpoB8 RNA polymerase has a 3-5-fold higher Km for purine nucleotides during elongation on synthetic templates . In contrast, both the mutant and wild-type enzyme have the same initiation Km for ATP . Kinetic analysis indicates that RpoB8 is likely to be defective in nucleotide binding during elongation, suggesting that the mutational alteration affects the binding site . We show that our data are consistent with the idea that the altered Km underlies the altered pausing and elongation properties of the enzyme, and we discuss the implication of these results for the termination proficiency of the mutant strain.

J Biol Chem, 1991 Aug 5, 266(22), 14406 - 12
A peptide corresponding to an export-defective mutant OmpA signal sequence with asparagine in the hydrophobic core is unable to insert into model membranes; Hoyt DW et al.; We have examined the comparative membrane interaction properties of synthetic peptides corresponding to the wild-type and an export-defective, mutated signal sequence from the Escherichia coli outer membrane protein, OmpA . As part of a collaborative study of the effects of various alterations on the function of the OmpA signal sequence and the biophysical properties of the corresponding synthetic peptides, we incorporated the small, neutral polar residue, asparagine, into the hydrophobic core in place of Ile-8 . This seemingly minor perturbation to the signal sequence caused a complete block of export in vivo (J . Goldstein, S . Lehnhardt, and M . Inouye, following paper) . We now explore in detail the difference in the properties of the wild-type and the Ile-8----Asn OmpA signal peptides . The fluorescent residue Trp was substituted in both peptides in place of the wild-type Phe at position 15 . This mutation is silent phenotypically and provides a superb probe of membrane interaction . We find that the Asn substitution leaves the conformational properties of the signal sequence essentially unchanged, but prevents any significant interaction of the peptide with a lipid bilayer . Asparagines are very underrepresented among known signal sequences . We believe this low frequency to be due to the lowering of mean residue hydrophobicity caused by incorporation of Asn and the consequent reduced ability to bind and insert into membranes.

J Biol Chem, 1991 Aug 5, 266(22), 14328 - 37
The P1 plasmid partition complex at parS . The influence of Escherichia coli integration host factor and of substrate topology; Funnell BE; The P1 ParB protein is required for active partition and thus stable inheritance of the plasmid prophage . ParB and the Escherichia coli protein integration host factor (IHF) participate in the assembly of a partition complex at the centromere-like site parS . In this report the role of IHF in the formation of the partition complex has been explored . First, ParB protein was purified for these studies, which revealed that ParB forms a dimer in solution . Next, the IHF binding site was mapped to a 29-base pair region within parS, including the sequence TAACTGACTGTTT (which differs from the IHF consensus in two positions) . IHF induced a strong bend in the DNA at its binding site . Versions of parS which have lost or damaged the IHF binding site bound ParB with greatly reduced affinity in vitro and in vivo . Measurements of binding constants showed that IHF increased ParB affinity for the wild-type parS site by about 10,000-fold . Finally, DNA supercoiling improved ParB binding in the presence of IHF but not in its absence . These observations led to the proposal that IHF and superhelicity assist ParB by promoting its precise positioning at parS, a spatial arrangement that results in a high affinity of ParB for parS.

J Biol Chem, 1991 Aug 5, 266(22), 14321 - 7
Characterization of active and latent forms of the membrane-associated sn-glycerol-3-phosphate acyltransferase of Escherichia coli; Scheideler MA et al.; The intrinsically active, sn-glycerol-3-phosphate acyltransferase present in membranes prepared from both wild type Escherichia coli and from strains which overproduce the enzyme can be kinetically distinguished from a latent enzyme species which is unmasked by solubilization and reconstitution . Both membrane-associated and solubilized/reconstituted enzyme preparations exhibited cooperativity with respect to sn-glycerol-3-phosphate and palmitoyl-coenzyme A substrates; positive cooperativity in membranes toward palmitoyl-coenzyme A (napp = 4) and negative cooperativity toward sn-glycerol-3-phosphate (napp = 0.75) were significantly altered upon solubilization and reconstitution . Since the degree of alteration increased with the amount of sn-glycerol-3-P acyltransferase present in the membranes, a detergent-dissociable homooligomerization of the sn-glycerol-3-phosphate acyltransferase was considered as an underlying mechanism . This possibility was investigated by changing the protein-to-Triton X-100 ratio of homogeneous enzyme prior to reconstitution and then analyzing the subsequent migration of samples on a Sephacryl S-300 sizing column . The elution positions were consistent with monomeric and dimeric polypeptide bound to micelles of Triton X-100 . Hill coefficients for monomeric, reconstituted enzyme preparations were comparable to those obtained for the active, membrane-associated sn-glycerol-3-phosphate acyltransferase . The reduced cooperativity of dimeric, reconstituted enzyme preparations correlated closely to the Hill coefficient values obtained for latent, solubilized/reconstituted sn-glycerol-3-phosphate acyltransferase from membranes of Escherichia coli which overproduce the enzyme . The physiological significance of these findings is discussed.

J Biol Chem, 1991 Aug 5, 266(22), 14188 - 92
Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif; Carr DW et al.; The type II cAMP-dependent protein kinase is localized to specific subcellular environments through the binding of the regulatory subunit (RII) dimer to RII-anchoring proteins . Computer-aided analysis of secondary structure, performed on four RII-anchoring protein sequences (the microtubule-associated protein 2, P150, and two thyroid proteins Ht 21 and Ht 31), has identified common regions of approximately 14 residues which display high probabilities of forming amphipathic helices . The potential amphipathic helix region of Ht 31 (Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile) lies between residues 494 and 507 . A bacterially expressed 318-amino acid fragment, Ht 31 (418-736), containing the amphipathic helix region, was able to bind RII alpha . Site-directed mutagenesis designed to disrupt the secondary structure in the putative binding helix reduced binding dramatically . Specifically, substitution of proline for Ala-498 significantly diminished RII alpha binding, and similar mutation of Ile-502 or Ile-507 abolished interaction . Mutation of Ala-522 to proline, which is located outside the predicted amphipathic helix region, had no effect on RII alpha binding . These data suggest that anchoring proteins interact with RII alpha via an amphipathic helix binding motif.

J Mol Biol, 1991 Aug 5, 220(3), 613 - 9
Escherichia coli mutations that block transcription termination by phage HK022 Nun protein; Robledo R et al.; The nun gene product of the lambdoid coliphage HK022 provokes premature transcription termination at, or near, the phage lambda nut sites . Termination by Nun and antitermination by lambda N protein both require the nut sites and Escherichia coli NusA, NusB and NusE proteins . To characterize further the host requirements for Nun termination, we selected host mutations that blocked termination at lambda nutR . In addition to mutations in nusA, nusB and nusE, we obtained mutations in rpoC, encoding the RNA polymerase beta' subunit . The nusA and rpoC mutations suppressed Nun termination but not antitermination by lambda N function . The mutations antagonized Nun only at lambda nutR; termination at lambda nutL occurred in all the mutant strains . Thus, nutL is not functionally equivalent to nutR . We conclude that the host requirements for Nun termination overlap but are not identical with those for N antitermination, and, in particular, that the beta' subunit of RNP may be Nun-specific.

J Biol Chem, 1991 Aug 5, 266(22), 14491 - 6
The Escherichia coli DnaK chaperone, the 70-kDa heat shock protein eukaryotic equivalent, changes conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein; Liberek K et al.; The DnaK protein of Escherichia coli and its eukaryotic hsp70 analogues are known to bind some polypeptides and to release or dissociate from them following ATP hydrolysis . Here we demonstrate that hydrolysis (and not simply binding) of nucleotide triphosphates leads to a change in the DnaK protein, from the "closed" to the "open" conformation . A conformational change is not observed with the mutant DnaK756 protein, which is always found in the open conformation . Although ATP is the preferred substrate, the hydrolysis of CTP, GTP, UTP, and dATP also results in DnaK's conversion from a closed to an open conformation . The ability of DnaK to hydrolyze various triphosphates correlates perfectly with its ability to release the bound denatured bovine pancreatic trypsin inhibitor polypeptide.

J Mol Biol, 1991 Aug 5, 220(3), 801 - 18
Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities; Wohrl BM et al.; The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule . In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate . The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA . Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner . The mt enzymes exhibit a severely reduced exonuclease activity . They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity . DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H . The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected . The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme . They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.

J Biol Chem, 1991 Aug 5, 266(22), 14709 - 13
Expression of the heterodimeric form of human immunodeficiency virus type 2 reverse transcriptase in Escherichia coli and characterization of the enzyme; Muller B et al.; A system for the expression of recombinant human immunodeficiency virus type 2 (HIV-2) reverse transcriptase (RT) in Escherichia coli has been developed, which allows purification of the heterodimeric form of the enzyme as well as the separate purification of the two subunits . It is shown that equilibrium formation between monomeric and homodimeric forms of the recombinant 66- and 51-kDa subunits is considerably more rapid than in the case of the corresponding homodimeric forms of HIV-1 RT . In accordance with our previously published studies on HIV-1 RT (Restle, T., Muller, B., and Goody, R.S . (1990) J . Biol . Chem . 265, 8986-8988) RNA-dependent DNA polymerase activity of the HIV-2 RT preparations can be exactly correlated to their dimer content . No significant heterodimer formation can be observed upon coexpression of the 66-kDa subunit of HIV-2 RT with the 51-kDa subunit of HIV-1 RT in the same cell, indicating differences in the dimerization domains of the two proteins . Recombinant HIV-2 RT is not recognized by a set of 23 monoclonal antibodies raised against HIV-1 RT, although it shows weak cross-reactivity with sera from HIV-1-infected patients.

J Biol Chem, 1991 Aug 5, 266(22), 14511 - 8
Structure and expression of TIS21, a primary response gene induced by growth factors and tumor promoters; Fletcher BS et al.; The TIS21 gene is a primary response gene that is induced rapidly and transiently in 3T3 cells by the tumor promoter and mitogen tetradecanoyl phorbol acetate . The predicted open reading frame of the TIS21 cDNA encodes a protein of 158 amino acids with no obvious similarity to any known protein . Antiserum prepared to TIS21 recombinant protein produced in Escherichia coli precipitates a 17-kDa protein from Swiss 3T3 cells . The 2040-nucleotide 3'-untranslated region of the cDNA includes an unusual T18 sequence . The TIS21 gene has a single 1.4-kilobase intron which interrupts the open reading frame and is otherwise identical to the cDNA sequence . The 5'-flanking sequence of the TIS21 gene contains TATA and CAAT box-type sequences, three potential Sp1 sites, two putative cyclic AMP response elements, two potential AP1 binding elements, and an AP2 element . A possible Z-DNA structure of 29 AC repeats is present 660 nucleotides from the start of transcription . Expression from a luciferase reporter construct containing a 460-nucleotide fragment of the TIS21 promoter is induced by tetradecanoyl phorbol acetate, forskolin, epidermal growth factor, and serum, despite the absence of a consensus serum response element.

J Biol Chem, 1991 Aug 5, 266(22), 14497 - 503
Early events in the synthesis of the multicopy single-stranded DNA-RNA branched copolymer of Myxococcus xanthus; Lease RA et al.; Myxobacteria and a variety of strains of Escherichia coli contain an unusual extrachromosomal element, a small single-stranded branched copolymer of DNA and RNA (msDNA) . Interest in msDNA stems from the presence of a 2'-5' linkage between its DNA and RNA moieties and the possible involvement of reverse transcriptase in its synthesis . Two groups have proposed a model for the synthesis of msDNA that involves the following sequence of events: 1) synthesis of an RNA precursor; 2) addition of a dNTP in a 2'-5' linkage to the RNA precursor (branch priming); 3) synthesis by reverse transcriptase of complementary DNA on the primed RNA precursor; 4) concomitant processing of the RNA precursor by RNase H; and 5) further processing of the RNA precursor by RNase H; and 5) further processing of the branched polymer . The branch priming hypothesis (step 2) was originally based on pulse-chase experiments using a lengthy pulse of 30-min duration . Our experiments with shorter pulse durations demonstrate a variety of intermediate forms not predicted by this model . Specifically, we find that early in synthesis, intermediate msDNA forms appear that are apparently not branch-linked to corresponding RNA moieties . The concomitant RNase H hypothesis (step 4) was based in part on intermediate trapping experiments using dideoxy NTPs to interrupt msDNA synthesis . These experiments showed an apparent complementary relationship between DNA length and RNA length in the trapped forms . In contrast, our experiments show that DNA elongation and RNA processing follow different kinetics . These results suggest an alternate model for synthesis of msDNA in which a conventionally primed DNA moiety is joined with the RNA moiety in a branch ligation event taking place several minutes after the synthesis of both moieties.

J Biol Chem, 1991 Aug 5, 266(22), 14163 - 6
Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins; Skinner RH et al.; The C-terminal catalytic domain (residues 704-1047) of the human ras GTPase-activating protein (GAP) has been engineered so as to incorporate the tripeptide, Glu-Glu-Phe, at its C terminus . This motif is recognized by the commercially available YL1/2 monoclonal antibody to alpha-tubulin and has previously been used for the immunoaffinity purification of HIV enzymes engineered to contain this epitope (Stammers, D . K., Tisdale, M., Court, S., Parmar, V., Bradley, C., and Ross, C . K . (1991) FEBS Lett . 283, 298-302) . The engineered GAP catalytic domain (GAP-344) was obtained in high yield and purity from Escherichia coli extracts by means of a single affinity column of immobilized YL1/2, eluted under mild conditions with the dipeptide, Asp-Phe . The protein had similar activity to that previously described for full-length GAP, suggesting that the addition of the epitope did not grossly affect the activity . R903K and L902I mutants of GAP-344 were constructed, and the immunoaffinity purification procedure allowed their rapid characterization . The R903K mutant had less than 3% the activity of the normal protein, whereas the L902I substitution had less than 0.5% of normal activity, suggesting an important role for Leu-902 and Arg-903, residues absolutely conserved among GAP-related proteins . This work exemplifies the general utility of the C-terminal Glu-Glu-Phe motif for the rapid purification of proteins whose function is not altered by C-terminal modification.

FEBS Lett, 1991 Aug 5, 287(1-2), 211 - 4
Site-directed mutagenesis of La protease . A catalytically active serine residue; Amerik AYu et al.; Comparative sequence analysis of Escherichia coli ATP-dependent La protease led to the suggestion that Ser679 is the catalytically active enzyme residue . Site-directed mutagenesis Ser679----Ala, investigation of the cells containing the mutant plasmid, and study of the partially purified mutant protein produced results in favour of this suggestion.

J Mol Biol, 1991 Aug 5, 220(3), 585 - 97
Development of RNA polymerase-promoter contacts during open complex formation; Mecsas J et al.; We have charted the movements of E sigma 32 RNA polymerase at the heat-shock promoter PgroE throughout open complex formation, using hydroxyl radical footprinting . In combination with methylation protection and DNase I experiments, these data suggest the following model for open complex formation . E sigma 32 initially anchors itself in the upstream region of the promoter forming the first closed complex, RPC1; in this complex the enzyme makes backbone contacts in the -35 region of the promoter that are maintained throughout open complex formation . An isomerization follows resulting in a second closed complex, RPC2; in this complex the enzyme makes base-specific and backbone contacts in the -10 region that are almost identical to those found in the open complex . Thus, at the groE promoter, upstream contacts are established in RPC1 and downstream contacts in RPC2 . A similar pattern of backbone contacts was obtained for E sigma 32 bound in the open complex at two additional heat-shock promoters, suggesting that the overall topology of holoenzyme in the open complex is similar regardless of sequence variations in the promoter.

J Mol Biol, 1991 Aug 5, 220(3), 569 - 83
Factor-independent activation of Escherichia coli rRNA transcription . II . characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase; Newlands JT et al.; A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides . We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR . We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex . An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E . coli promoters . We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR . These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.

J Biol Chem, 1991 Aug 5, 266(22), 14367 - 70
Expression, purification, and crystallization of the adipocyte lipid binding protein; Xu ZH et al.; The murine adipocyte lipid binding protein (ALBP/aP2) has been cloned and expressed in Escherichia coli, purified to homogeneity, biochemically characterized, and crystallized for x-ray diffraction study . In the cloning, the ALBP coding region was placed under control of the recA promoter and downstream of the phage T7 g-10 translation enhancer sequence . Nalidixic acid (50 micrograms/ml) induced the expression of ALBP 20-fold over that attained using the pT7 system previously reported (Chinander, L . L., and Bernlohr, D . A . (1989) J . Biol . Chem . 264, 19564-19572) . Recombinant ALBP was purified to homogeneity using a combination of pH fractionation, gel filtration, and immobilized metal affinity chromatography . The fluorescent affinity ligand 12-(9-anthroyloxy)oleic acid bound to homogeneous ALBP with an apparent Kd of 0.5 microM . rALBP was devoid of endogenous fatty acid, and oleic acid inhibited cysteine 117 modification by 5,5' -dithiobis-(2-nitrobenzoic acid) indicating integrity of the binding domain . Recombinant ALBP was phosphorylated by the soluble kinase domain of the insulin receptor with a Vmax of 11 nmol.min.mg of kinase and an apparent Km of 270 microM . Purified protein was crystallized using the hanging drop method with seeding . Crystalline ALBP was orthorhombic with cell dimensions of a = 34.4 A, b = 54.8 A, and c = 76.3 A . The space group was P212121, and there was one molecule per asymmetric unit.

J Biol Chem, 1991 Aug 5, 266(22), 14317 - 20
Zinc (II) coordination in Escherichia coli DNA topoisomerase I is required for cleavable complex formation with DNA; Tse-Dinh YC; Escherichia coli DNA topoisomerase I catalyzes relaxation of negatively supercoiled DNA . The reaction proceeds through a covalent intermediate, the cleavable complex, in which the DNA is cleaved and the enzyme is linked to the DNA via a phosphotyrosine linkage . Each molecule of E . coli DNA topoisomerase I has been shown to have three tightly bound zinc(II) ions required for relaxation activity (Tse-Dinh, Y.-C., and Beran-Steed, R.K . (1988) J . Biol . Chem . 263, 15857-15859) . It is shown here that Cd(II) could replace Zn(II) in reconstitution of active enzyme from apoprotein . The role of metal was analyzed by studying the partial reactions . The apoenzyme was deficient in sodium dodecyl sulfate-induced cleavage of supercoiled PM2 phage DNA . Formation of covalent complex with linear single-stranded DNA was also reduced in the absence of metal . However, the cleavage of small oligonucleotide was not affected, and the apoenzyme could religate the covalently bound oligonucleotide to another DNA molecule . Assay of noncovalent complex formation by retention of 5'-labeled DNA on filters showed that the apoenzyme was not inhibited in noncovalent binding to DNA . It is proposed that zinc(II) coordination in E . coli DNA topoisomerase I is required for the transition of the noncovalent complex with DNA to the cleavable state.

Vet Rec, 1991 Aug 3, 129(5), 94 - 7
Comparison of plasmid profile analysis, antibiogram testing, resistotyping and biotyping in the identification of Escherichia coli isolates from poultry; David BP et al.; Twenty isolates of Escherichia coli from poultry, predominantly from birds with colibacillosis, were collected and plasmid profile analysis, antibiogram testing, resistotyping and biotyping were compared as epidemiological tools for the differentiation and identification of the isolates . Plasmid profile analysis, in conjunction with any one of the other three tests, was found to be more valuable as an epidemiological tool than one test alone.

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