Escherichia coli

Microb Pathog, 1990 Jul, 9(1), 1 - 11
Plasmid-encoded production of coli surface-associated antigen 1 (CS1) in a strain of Escherichia coli serotype O139.H28; Willshaw GA et al.; Production of coli surface-associated antigen 1 (CS1) by Escherichia coli strain E24377 of serotype O139.H28 was controlled by a plasmid that also encoded heat stable and heat labile enterotoxins and CS3 . The presence of a regulatory sequence was detected on this plasmid by hybridization with the cfaD gene that regulates expression of colonization factor antigen I fimbriae and is at least 96% homologous with the rns sequence controlling production of CS1 or CS2 fimbriae by strains of serotype O6.H16 of appropriate biotype . A separate plasmid, pDEP20, carrying the structural genes for CS1 synthesis was identified and transformed into E . coli strain HB101 or a derivative of strain E24377 without large plasmids . Transformants carrying pDEP20 did not produce CS1 fimbrial antigen, but antigen expression was obtained when a cloned cfaD gene or a wild-type plasmid carrying the rns sequence was introduced . Transposon mutagenesis with Tn1000 identified a 3.7 kbp region of pDEP20 essential for production of CS1 fimbriae . Genes encoding production of CS1 fimbriae were cloned on a 9.9 kbp BamHI fragment and were expressed in the presence of the cfaD sequence . A strain producing both CS1 and CS2 antigens was constructed by introduction of the cloned cfaD gene into a strain of serotype O6.H16 biotype C carrying plasmid pDEP20.

Res Microbiol, 1990 Jul-Aug, 141(6), 621 - 31
Genetic analysis of the transfer region of the IncN plasmid N3; Glocker B et al.; Using lambda::Tn5 insertion mutagenesis and screening for conjugation, the boundaries of the IncN plasmid N3 transfer region were determined . Sensitivity to phage IKe infection was used to monitor that part of the N3 transfer region which harbours genes for pilus synthesis and assembly . We cloned this region, creating plasmid pBG21 . Escherichia coli cells transformed with pBG21 became sensitive to phage IKe and produced pili, as shown by electron microscopy . Various plasmid constructions containing parts of the pilus-encoding region were used for expression in a minicell system and for expression in an in vitro translation system, thus characterizing for the first time some of the gene products of domain I (Winans and Walker, 1985a) of the transfer region.

Mol Gen Genet, 1990 Jul, 222(2-3), 317 - 22
Mapping of insertion element IS30 in the Escherichia coli K12 chromosome; Umeda M et al.; We identified seven phage clones containing the insertion element IS30 in a lambda phage library mini-set, which includes 476 clones carrying chromosomal segments that cover almost the entire chromosome of Escherichia coli K12 W3110 (Kohara et al . 1987) . We could assign locations and orientations to four copies of IS30 (named is30A to is30D) on the W3110 chromosome by restriction analysis of phage DNAs containing them . These IS30s were present at the same locations in chromosomes of both W3110 and another E . coli K12 strain JE5519, and thus are assumed to be present in other E . coli K12 derivatives, including early isolates . Among the IS30 copies found, one (is30B) contained a large deletion and possessed only a 181 bp stretch of the right terminal region of IS30.

Mol Microbiol, 1990 Jul, 4(7), 1193 - 8
Immunocytochemical analysis of P-fimbrial structure: localization of minor subunits and the influence of the minor subunit FsoE on the biogenesis of the adhesin; Riegman N et al.; Antibodies recognizing the non-adhesive minor P-fimbral subunit protein E and the P-fimbrial adhesin were used in an immunocytochemical analysis of P-fimbrial structure . It was demonstrated that P-fimbriae of the serotypes F71, F72 and F11 carry their adhesin in a complex with protein E . These complexes are commonly found at the tip of the fimbrial structure . In P-fimbriae of serotype F9, expressed by the uropathogenic Escherichia coli strain 21086, adhesin-protein E complexes are localized at the tips as well as along the shafts of the fimbriae . Protein E of F71 fimbriae (FsoE) plays a catalysing role in the biogenesis of the adhesin, but has no effect on the eventual localization of the adhesin.

Genes Dev, 1990 Jul, 4(7), 1079 - 93
Region-specific recombination and expression are directed by portions of the Drosophila engrailed promoter; Hama C et al.; The Drosophila engrailed gene is expressed in the cells of the posterior developmental compartments . To investigate how the engrailed gene is regulated, chimeric genes consisting of fragments of the engrailed promoter and Escherichia coli lacZ were incorporated into the Drosophila germ line by P-element-mediated recombination . Fusion constructs with 7.5 kb of 5'-flanking sequence contain sufficient information to promote expression in most of the embryonic, larval, and imaginal posterior compartments; transformants with smaller fragments of the 5' region do not . Remarkably, of 20 independent transformants with constructs containing more than 1 kb of 5'-flanking DNA, 7 integrated in or around the engrailed locus . These strains inactivate engrailed function to varying degrees, and some express lacZ with a position- and temporal-specific program that is indistinguishable from the normal engrailed gene . Presumably, in these strains, lacZ is expressed in the context of the engrailed promoter.

Int J Radiat Biol, 1990 Jul, 58(1), 35 - 54
DNA base and strand damage in X-irradiated monkey CV-1 cells: influence of pretreatment using small doses of radiation; Bases R et al.; Base damage in alpha DNA from irradiated monkey CV-1 cells was determined by measuring release of 5'-32P-end labelled DNA fragments after digestion with endonuclease III of E . coli . The frequency and base sequence locations of the enzyme-sensitive sites were determined . Fragments were released from irradiated DNA at sequence sites of pyrimidines and guanines . The time for repair of half the single strand breaks was approximately 1.5 h . Repair of base damage as judged from loss of enzyme-sensitive sites in DNA was slower, with more than half of the damaged bases still detectable after 4 h of repair . Two important changes in the pattern of fragment release from DNA were produced when small radiation doses preceded the large ones needed to produce measurable DNA strand breaks and base damage . 5 Gy to cells incubated several hours before 320 Gy increased by five-fold the abundance of small DNA fragments with 3'-phosphoryl termini detected in high-resolution denaturing gels . These increases were detectable with doses as small as 0.2 Gy and were accompanied by the appearance of new species of DNA fragments of intermediate mobility at specific locations in the base sequence . The patterns resemble those produced by digesting DNA from heavily irradiated cells with endonuclease III.

J Bacteriol, 1990 Jul, 172(7), 3631 - 6
Involvement of gamma-glutamyl peptides in osmoadaptation of Escherichia coli; McLaggan D et al.; Accumulation of K+ ions and glutamate plays a primary role in maintaining osmotic balance in Escherichia coli, as illustrated by the high concentrations of these ions present in cells growing in medium of high osmolality . We found that two gamma-glutamyl peptides and glutamine also accumulated during growth at high osmolarity . In a mutant unable to make trehalose growing in 1.3 osM medium, glutathione, gamma-glutamylglutamine, and glutamine accumulated to levels of 73, 33, and 140 mumol/g of protein, respectively . In such cells, K+ was present at 1,450 mumol/g of protein, indicating that glutathione and gamma-glutamylglutamine accounted for less than 10% of the low-molecular-weight anions accumulated with K+ . However, glutathione is needed for wild-type osmotolerance in this species . A mutant deficient in glutathione because of an insertion in the gshA gene was unable to grow above 1.4 osM, grew more slowly at intermediate osmolarities, and took longer to adapt to growth following osmotic upshock . The involvement of glutathione in osmoregulation was independent of the effect of glutathione on K+ retention.

J Surg Res, 1990 Jul, 49(1), 37 - 44
LY171883 preserves mesenteric perfusion in porcine endotoxic shock; Cohn SM et al.; Superior mesenteric arterial perfusion (Q) decreases and gut intramucosal hydrogen ion concentration, {H+}, increases in resuscitated normodynamic endotoxic pigs . The present study tested the hypothesis that these adverse phenomena can be prevented by pretreatment with LY171883, a specific leukotriene (LT) D4/E4 receptor antagonist . Pentobarbital-anesthetized pigs (14-18 kg) were instrumented to permit measurement of Q (ultrasonic flow probe) and {H+} (tonometer) . Mesenteric O2 delivery (DO2) and consumption (VO2) were calculated from the O2 contents of arterial and superior mesenteric venous blood . At t = -20 min, groups (N = 6) of pigs were pretreated witH LY171883 (10 mg/kg) or vehicle . At t = 0 min, the pigs were infused over 20 min with lipopolysaccharide (LPS; 150 micrograms/kg) and resuscitated for 2 hr with saline (1.2 ml/kg min) . Irrespective of treatment group, mean arterial pressure and systemic vascular resistance index decreased significantly after infusion of LPS . In general, cardiac index (CI) was well preserved, although in controls at t = 20, 100, and 120 min, CI decreased significantly with respect to the t = 0 min value . Normal mesenteric Q and DO2 were maintained in the LY171883 group, whereas, in controls, these parameters decreased significantly . Mesenteric VO2 increased transiently but significantly in controls; this phenomenon was abrograted by the LT receptor antagonist . In controls, intramucosal {H+} increased by almost threefold; this adverse effect was significantly ameliorated by LY171883 . These data suggest that decreased mesenteric Q and increased intramucosal {H+} may be mediated by LT in this porcine endotoxic shock model.

Mol Cell Biol, 1990 Jul, 10(7), 3551 - 61
Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate; Gangloff SP et al.; We have isolated genomic clones complementing the aconitase-deficient strain (glu1-1) of Saccharomyces cerevisiae . Identification of the aconitase gene was established by enzymatic assays and molecular analyses . The corresponding mRNA has been characterized, and its direction of transcription has been determined . The complete nucleotide sequence revealed strong amino acid homologies with the sequences of some peptides isolated from the mammalian protein . Disruption of the gene by deletion-insertion led to glutamate auxotrophy . Expression of the aconitase gene was sensitive to glucose repression and was synergistically down regulated by glucose and glutamate.

AMB Rev Assoc Med Bras, 1990 Jul-Dec, 36(3-4), 153 - 6
{Malacoplakia of the large intestine, bladder and retroperitoneum: a case report}; Vattimo A et al.; The authors present a case of malakoplakia, involving colon, rectum, bladder and retroperitoneum . This rare pathology, generally associated to Escherichia coli infections, result in a granulomatous disease, that can involve one or more organs . Nowadays, it is believed that the illness is due to a failure in the bactericide activity of the macrophage . This case, the first reported in our country, was treated clinically with ascorbic acid and trimethoprim-sulfamethoxazole, and is also unique in the world literature.

Mol Gen Genet, 1990 Jul, 222(2-3), 297 - 303
Expression of foreign epitopes in P-fimbriae of Escherichia coli; van Die I et al.; Hypervariable regions (HRs) of the major subunit of F11 fimbriae were exploited for insertion of foreign epitopes . Two insertion vectors were created that contain a unique cloning site in HR1 or HR4 respectively . Several oligonucleotides, coding for antigenic determinants derived from different pathogens, were cloned in both insertion vectors . Hybrid fimbrial subunits were generally shown to be assembled in fimbriae when the length of the inserted peptide did not exceed 14 amino acids . The inserted peptides appeared to be exposed in the fimbrial filament . One hybrid fimbrial protein induced detectable levels of antibodies against the inserted epitope if injected into mice.

Mol Biol (Mosk), 1990 Jul-Aug, 24(4), 1100 - 8
{Fluorescent analogs of nucleoside-5'-phosphates for the study of nucleic acids by nonradioactive methods}; Aleksandrova LA et al.; The synthesis of 2'-deoxyuridine 5'-triphosphate analogues with fluorescent residues of fluorescein and rhodamine nature at C5 of the uracil base was performed . Reverse transcriptase of avian myeloblastosis virus, DNA polymerase beta of rat liver, terminal deoxynucleotidyl transferase of calf thymus and E . coli DNA polymerase I, Klenow fragment, were shown to be capable to incorporate a nucleotide residue with fluorescent label into 3'-terminus of oligonucleotide . These fluorescent labeled oligonucleotides were used as primers for synthesis of (-)-chain of M13mp10 phage . Fluorescently labeling template-primer complexes were used for DNA sequencing.

Bioorg Khim, 1990 Jul, 16(7), 933 - 40
{Effective synthesis of oligo(poly)deoxyribonucleotides using an H-phosphonate method in plastic microcolumn}; Isaguliants MG et al.; A facile technique of manual oligonucleotide synthesis via H-phosphonate approach is developed . Syntheses carried out in pipette tips with siliconised glasswool filters take 3-3.5 min per cycle with 97-98% yields per condensation . The method was used to synthesize 12-55-mers: T7 and PL promoter regions, gene of the signal peptide of the E . coli OmpA protein, oligonucleotides coding for amino acid sequences 94-105 of preS1- and 133-143 of preS2-regions of hepatitis B virus, hybridisation probes, sequencing primers, oligonucleotides for site-directed mutagenesis, etc.

Mol Microbiol, 1990 Jul, 4(7), 1083 - 90
Molecular aspects of phosphate transport in Escherichia coli; Rao NN et al.; Escherichia coli transports inorganic phosphate (Pi) by the low-affinity transport system, Pit . When the level of the external Pi is lower than 20 microM, another transport system, Pst, is induced with a Kt of 0.25 microM . An outer-membrane porin, PhoE, with a Km of about 1 microM is also induced . The outer membrane allows the intake of organic phosphates which are degraded to Pi by phosphatases in the periplasm . The Pi-binding protein will capture the free Pi produced in the periplasm and direct it to the transmembrane channel of the cytoplasmic membrane . The channel consists of two proteins, PstA and PstC, which have six and five transmembrane helices, respectively . On the cytoplasmic side of the membrane the channel is linked to the PstB protein, which carries a nucleotide (probably ATP)-binding site . PstB probably provides the energy required by the channel to free Pi . The Pst system has two functions in E . coli: (i) the transport of Pi, and (ii) the negative regulation of the phosphate regulon (a complex of 20 proteins mostly related to organic phosphate transport) . It is remarkable that these two functions are not related, since the repressibility of the regulon depends on the integral structure of Pst (PiBP + PstA + PstC + PstB) and not on the Pi transported . Another gene of the pst operon, phoU, produces a protein involved in the negative regulation of the Pho regulon, but the mechanism of this function has not been explained . Thus the regulatory function of the Pst system remains obscure . Its basal level, present when Pi is abundant, is sufficient to repress the Pho regulon but the negative regulatory function is lost upon Pi starvation.

Mol Microbiol, 1990 Jul, 4(7), 1077 - 82
Signal transduction and gene regulation through the phosphorylation of two regulatory components: the molecular basis for the osmotic regulation of the porin genes; Mizuno T et al.; Expression of Escherichia coli outer-membrane porin proteins (OmpF and OmpC) is regulated by the osmolarity of the medium . EnvZ and OmpR, which are positive regulatory factors for the transcriptional osmotic regulation of the ompF and ompC genes, belong to a group of two-component regulatory factors that respond to a variety of environmental stimuli in bacteria . EnvZ-OmpR phosphotransfer was revealed to be involved in signal transduction in response to an osmotic stimulus, and to play a crucial physiological role in the consequent osmotic activation of the porin genes . Based on the various lines of experimental evidence, a model is proposed for the molecular mechanism underlying the osmotic regulation through phosphorylation of the activator (OmpR) by the membrane-located kinase (Env2).

Mol Microbiol, 1990 Jul, 4(7), 1063 - 7
Translation initiation in Escherichia coli: old and new questions; Jacques N et al.; We discuss the features of Escherichia coli mRNAs which determine where and how efficiently translation is initiated . We have shown that DNA fragments comprising 60-80 nucleotides that bracket the initiation codon of real genes generally promote translation when inserted within a foreign mRNA, while those not corresponding to an authentic gene start do not do so even if they include a Shine-Dalgarno-like element followed by AUG or GUG . Therefore, the information that pinpoints the correct start sites, while extending beyond the mere presence of these elements, remains essentially local . The possible nature of this information is discussed . Next, we point out that, in order to remain accessible, translational starts must escape long-range base-pairing within large mRNAs, and we argue that the tight coupling between translation and transcription plays an important role in achieving this . Finally, we discuss two intriguing situations in which the initiation frequency should be dependent upon the rate of translation elongation.

Protein Eng, 1990 Jul, 3(7), 591 - 8
Structural effects induced by removal of a disulfide-bridge: the X-ray structure of the C30A/C51A mutant of basic pancreatic trypsin inhibitor at 1.6 A; Eigenbrot C et al.; The X-ray structure of a variant of basic pancreatic trypsin inhibitor (BPTI) has been analyzed to determine the structural accommodation resulting from removal of a disulfide cross-link in a protein . The disulfide removed, Cys30-Cys51, has been implicated in both the folding pathway of the protein and its overall thermal stability . In the variant studied, C30A/C51A, the disulfide cysteines were replaced by less bulky alanines . The atomic displacements observed for C30A/C51A indicate a set of concerted shifts of two segments of chains, which together significantly diminish a packing defect at the site of the removed cysteine sulfur atoms . The observed structural changes are distributed asymmetrically around the sites of mutation, indicating that the adjacent beta-sheet is more resistant to the perturbation than the alpha-helix on the opposite side of the disulfide bond . The thermal parameters of groups involved in the structural accommodation are not significantly altered . A comparison of the X-ray structures reported for native BPTI determined in three different crystal forms indicates that the magnitude of its conformational variability exceeds that of the structural changes caused by the disulfide removal . This emphasizes the necessity of using isomorphous crystal systems to determine the relatively small effects due to mutation.

Eur J Immunol, 1990 Jul, 20(7), 1541 - 5
Enhanced immunogenicity of recombinant peptide fusions containing multiple copies of a heterologous T helper epitope; Lowenadler B et al.; We have examined the immune response against the nonimmunogenic heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli using recombinant fusion proteins containing the STa-peptide linked to an IgG-binding analogue of protein A and varying numbers of the T helper epitope 323-339 from ovalbumin (ova) . By immunization of inbred strains of mice with a series of STa fusion proteins, containing up to four copies of ova tandemly multiplied, we demonstrated that the anti-STa antibody response is controlled by ova-specific T helper cells in a genetically restricted manner . In the responding mouse strains (2 out of 3 tested), the level of antibody production was increased by addition of multiple ova epitopes, the anti-STa response being considerably higher to fusion proteins containing four than one or two ova epitopes.

Mutat Res, 1990 Jul, 236(1), 9 - 17
9-amino-ellipticine inhibits the apurinic site-dependent base excision-repair pathway; Lefrancois M et al.; The aromatic amine 9-amino-ellipticine is a synthetic DNA intercalating compound derived from the antitumor agent ellipticine, which cleaves at very low doses DNA containing apurinic sites by beta-elimination through formation of a Schiff base . This compound has been shown to potentiate the cytotoxic effect of alkylating drugs, such as dimethyl sulfate, in E . coli through a mechanism involving apurinic sites . We have studied the ability of 9-amino-ellipticine to inhibit an enzymatic repair system mimicking base-excision repair, in which E . coli exonuclease III only presents an endonuclease for apurinic/apyrimidinic site activity . 10 microM of 9-amino-ellipticine inhibits 70% of apurinic site repair . Other intercalating agents with similar affinities for DNA do not induce any inhibition . In another system designed for the direct assay of the exonuclease III-induced incisions 5' to AP sites 10 microM of 9-amino-ellipticine inhibits 65% of the endonuclease for apurinic/apyrimidinic site activity of E . coli exonuclease III . The 9-amino-ellipticine-induced formation of a 2',3'-unsaturated deoxyribose and cleavage at the 3' side of the apurinic site, and possible creation of an adduct, as suggested by Bertrand and coworkers (1989), on the 3' position of the deoxyribose seem to strongly inhibit the endonuclease for apurinic/apyrimidinic site activity . 9-Amino-ellipticine appears therefore to be the first small ligand which can inhibit, by an irreversible modification of the substrate, the repair of apurinic sites through the base excision-repair pathway at a pharmacological concentration.

J Cell Biol, 1990 Jul, 111(1), 153 - 69
Elucidating the early stages of keratin filament assembly; Coulombe PA et al.; Because of extraordinarily tight coiled-coil associations of type I and type II keratins, the composition and structure of keratin subunits has been difficult to determine . We report here the use of novel genetic and biochemical methods to explore the early stages of keratin filament assembly . Using bacterially expressed humans K5 and K14, we show that remarkably, these keratins behave as 1:1 complexes even in 9 M urea and in the presence of a reducing agent . Gel filtration chromatography and chemical cross-linking were used to identify heterodimers and heterotetramers as the most stable building blocks of keratin filament assembly . EM suggested that the dimer consists of a coiled-coil of K5 and K14 aligned in register and in parallel fashion, and the tetramer consists of two dimers in antiparallel fashion, without polarity . In 4 M urea, both end-to-end and lateral packing of tetramers occurred, leading to a variety of larger heteromeric complexes . The coexistence of multiple, higher-ordered associations under strongly denaturing conditions suggests that there may not be a serial sequence of events leading to the assembly of keratin intermediate filaments, but rather a number of associations may take place in parallel.

J Neuroimmunol, 1990 Jul, 28(2), 177 - 84
Recombinant human beta-galactoside binding lectin suppresses clinical and histological signs of experimental autoimmune encephalomyelitis; Offner H et al.; Human placental tissue contains regulatory molecules that may prevent allo-sensitization . Recently, a 14 kDa beta-galactoside binding protein with demonstrated immunoregulatory properties has been cloned using cDNA from human placenta and expressed in Escherichia coli . The present study assesses the ability of this recombinant immunomodulatory lectin (rIML-1), to prevent experimental autoimmune encephalomyelitis (EAE), a paralytic T cell-mediated disease directed against myelin basic protein (BP) . Injection of rIML-1 into Lewis rats inhibited the induction of both clinical and histological signs of EAE, apparently by blocking sensitization of encephalitogenic BP-specific T cells and inducing BP-dependent suppressor cells . Because it is neither immunogenic nor toxic, rIML-1 may have application in humans, and would have distinct advantages over unselective cytotoxic immunosuppressive agents used currently in the treatment of autoimmune diseases and transplantation.

J Bacteriol, 1990 Jul, 172(7), 3952 - 8
The beta-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus; Kovacevic S et al.; Isopenicillin N isomerase (epimerase) has been purified from Streptomyces clavuligerus, and the amino acid sequence of the N-terminus has been determined . By using single oligonucleotide probes based on high GC codon bias ("guessmers"), the translation start codons were determined for two successive genes in the beta-lactam-biosynthetic pathway and mapped within a 3.6-kilobase-pair KpnI restriction fragment . The epimerase gene (cefD) was located immediately upstream of the deacetoxycephalosporin C synthetase (expandase) gene (cefE) that was characterized previously . cefD was sequenced and expressed in Escherichia coli; the resulting cell extracts contained epimerase activity . Western immunoblots demonstrated that a protein comigrated with purified S . clavuligerus epimerase at 44 kilodaltons . cefD and cefE were separated by an 81-base-pair segment . The DNA sequence upstream of the epimerase gene had a high AT content, suggestive of a promoter region . Primer extension analysis of S . clavuligerus mRNA showed that the start of transcription occurred approximately 130 base pairs upstream of the epimerase translation start site; Northern (RNA blot) analysis revealed a hybridization signal large enough to code for both epimerase and expandase, and nuclease S1 protection assays showed that a single message may code for epimerase, expandase, and another unknown protein . When cefD and cefE were placed in an expression vector, concomitant synthesis of both epimerase and expandase occurred in E . coli.

J Bacteriol, 1990 Jul, 172(7), 3577 - 83
Effect of outer membrane permeability on chemotaxis in Escherichia coli; Ingham C et al.; The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC . Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC . A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays . AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II . Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate . Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability . These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E . coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.

EMBO J, 1990 Jul, 9(7), 2331 - 40
Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III; Blomberg P et al.; The replication frequency of IncFII plasmids is regulated through the availability of a rate-limiting protein, RepA . The synthesis of this protein is controlled post-transcriptionally by a small antisense RNA, CopA, which binds to the leader region of the RepA mRNA (CopT) . In this communication we report studies of the IncFII plasmid R1 . We show that the duplex between CopA and CopT is cleaved specifically in vivo . The in vivo cleavage maps to the same position as that resulting from in vitro cleavage of a CopA/CopT duplex by purified RNase III . By introducing plasmids carrying translational repA-lacZ fusions into cells deficient in RNase III we show that the expression of repA is elevated when RNase III activity is severely decreased . Hence, cleavage by RNase III seems to be a key event in the copy number control system of plasmid R1.

EMBO J, 1990 Jul, 9(7), 2101 - 6
The human muscle nicotinic acetylcholine receptor alpha-subunit exist as two isoforms: a novel exon; Beeson D et al.; Analysis of acetylcholine receptor clones isolated from a human leg muscle cDNA library, revealed that the alpha-subunit existed as two isoforms . A novel exon, coding for 25 amino acids, was located in the human genomic DNA sequence; its insertion into the alpha-subunit gives the new isoform of 462 amino acids . In addition, mRNAs for the two isoforms were found in equal proportions in poly(A)+ RNA obtained from three further sources including partially denervated and innervated human muscle and the rhabdomyosarcoma cell line TE671 . Both protein isoforms can be expressed in E . coli . No evidence of a sequence related to that of the new exon was found in cDNA derived from poly(A)+ RNA isolated from fetal calf or embryonic chick muscle or Torpedo marmorata electric organ.

Virology, 1990 Jul, 177(1), 124 - 30
Nucleotide sequence of the leader and nucleocapsid protein gene of mumps virus and epitope mapping with the in vitro expressed nucleocapsid protein; Tanabayashi K et al.; The nucleotide sequence of the leader and the gene encoding the nucleocapsid protein (NP) of mumps virus Miyahara strain have been determined . The leader sequence is 55 nucleotides in length and the NP gene is 1845 nucleotides in length, exclusive of poly(A) . The NP gene codes for a protein of 549 amino acids, with a calculated molecular weight of 61,365 . For epitope mapping, a series of NPs from which C-termini were serially deleted were expressed in vitro from five mRNA constructs and were examined by radioimmunoprecipitation assay (RIPA) with eight nonoverlapping monoclonal antibodies (MoAbs) against the mumps virus NP . It was found that seven out of eight MoAbs reacted with the NP synthesized in vitro . Five recognized the epitopes located within the C-terminal 74 amino acids region and one within the adjacent 64 amino acids upstream . The epitope of the remaining one was in the N-terminal half of the NP.

Biotechnology (N Y), 1990 Jul, 8(7), 644 - 9
Expression in yeast of amino-terminal peptide fusions to hepatitis B core antigen and their immunological properties; Beesley KM et al.; Hepatitis B core protein (HBcAg) is a potent antigen that gives both a T-cell-dependent and a T-cell-independent antibody response . It has been shown that a foreign epitope can be fused to the amino terminus of HBcAg without affecting particle integrity, and that the resulting chimaeric cores retain the immunogenicity of the foreign epitope . Here we describe the efficient expression in yeast of two different chimaeric cores, carrying epitopes of Foot and Mouth Disease Virus (FMDV) or human chorionic gonadotrophin (hCG), which are candidates for FMD and contraceptive vaccines, respectively . These cores could not be produced in E . coli in soluble form but were expressed to high levels in yeast . We constructed a yeast expression vector that allows rapid production of different chimaeric cores by cloning in cassettes encoding foreign epitopes . Both FMDV and hCG-cores were shown to present the epitopes at the surface of the particles . The FMDV-cores produced in yeast were efficient inducers of neutralising antibodies in guinea-pigs after one low dose.

Appl Microbiol Biotechnol, 1990 Jul, 33(4), 429 - 34
Production and characterization of human gamma interferon from Escherichia coli; Perez L et al.; The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, which simplified the purification process, is described . An expression plasmid carrying lipoprotein and the tryptophane promoters in tandem was used . Preparation of highly pure interferon was achieved using high resolution chromatography after denaturation and renaturation steps . Structural characteristics of this protein were verified by mass spectrometric analysis . Additional control tests have shown the suitability of the final product for clinical purposes.

Biotechnol Prog, 1990 Jul-Aug, 6(4), 255 - 61
Sizing biological samples by photosedimentation techniques; Middelberg AP et al.; The performance of the Joyce-Loebl disk centrifuge in the sizing of Escherichia coli cells, protein inclusion bodies, and cell debris is evaluated . The need for a density gradient that extends throughout the entire spin fluid is highlighted, and a set of standard conditions that fulfill this requirement is defined . E . coli cells experience a reduction in their Stokes diameter when exposed to ethanol, indicating that a spin-buffer fluid combination such as glycerol-water is to be preferred for the sizing of bacteria . The instrument baseline is influenced by the presence of particles, and a method of estimating the baseline is described . The sizing of small particles is further complicated by baseline drift due to temperature sensitivity of the optical yoke . An analysis of diffusion in the spin fluid is conducted, and an expression for the sedimentation:diffusive flux ratio is derived . For the current samples, it is shown that diffusion within the spin fluid does not lead to significant errors for 0.15-microns particles, whereas the phenomenon may be significant at the manufacturer's size limit of 0.01 micron.

Appl Microbiol Biotechnol, 1990 Jul, 33(4), 424 - 8
A constitutive expression vector system driven by the deo P1P2 promoters of Escherichia coli; Fischer M et al.; The P1P2 promoters of Escherichia coli K12 deo operon, residing on an AvaII restriction fragment, were used to construct a new expression vector . To evaluate the potential of the P1P2-driven expression system we have inserted the sequence of human superoxide dismutase (hSOD) downstream of the deo ribosome binding site . Expression of hSOD was evaluated by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzyme activity . In crude cell extracts hSOD expression levels were found to be high in hosts possessing no deoR or cytR repressors . Highest levels of hSOD expression were obtained with a high-copy-number plasmid regardless of the host used . Expressed hSOD can account for 35%-40% of total protein in E . coli.

Science, 1990 Jun 29, 248(4963), 1625 - 30
Functional domains and upstream activation properties of cloned human TATA binding protein; Peterson MG et al.; The TATA binding protein, TFIID, plays a central role in the initiation of eukaryotic mRNA synthesis . Here, we present a human cDNA clone for this factor . Comparison of its predicted protein sequence with those from Drosophila and yeast reveals a highly conserved carboxyl-terminal 180 amino acids . By contrast, the amino-terminal region of TFIID has diverged in both sequence and length . A striking feature of the human protein is a stretch of 38 glutamine residues in the NH2-terminal region . Expression of human TFIID in both Escherichia coli and HeLa cells produces a protein that binds specifically to a TATA box and promotes basal transcription; the conserved COOH-terminal portion of the protein is sufficient for both of these activities . Recombinant TFIID forms a stable complex on a TATA box either alone or in combination with either of the general transcription factors, TFIIA or TFIIB . Full-length recombinant TFIID is able to support Sp1 activated transcription in a TFIID-depleted nuclear extract, while a deletion of the NH2-terminal half of the protein is not . These results indicate the importance of the NH2-terminal region for upstream activation functions and suggest that additional factors (co-activators) are required for mediating interactions with specific regulators.

Biochem Biophys Res Commun, 1990 Jun 29, 169(3), 1129 - 37
Expression of simian virus 40 large T antigen in Escherichia coli using vectors based on the regulatable rac promoter; Pistillo JM et al.; Simian Virus 40 large T antigen is a multi-functional protein that is involved in the initiation of viral DNA replication, regulation of viral transcription and cell transformation . Bacterial expression vectors, pER23-1 and pER23-2, that are based on the regulatable rac promoter were used to produce T antigen either as a free protein or as a fusion protein . We have observed efficient transcription of the cloned T antigen gene in most of the recombinants . However, expression of the T antigen protein was inefficient and most of the expressed protein was truncated . This may be due to differences in codon usage in E . coli or to rapid protein degradation.

Science, 1990 Jun 29, 248(4963), 1650 - 3
A cDNA for a protein that interacts with the human immunodeficiency virus Tat transactivator; Nelbock P et al.; The human immunodeficiency virus (HIV) tat protein (Tat) is a positive regulator of virus gene expression and replication . Biotinylated Tat was used as a probe to screen a lambda gt11 fusion protein library, and a complementary DNA encoding a protein that interacts with Tat was cloned . Expression of this protein, designated TBP-1 (for Tat binding protein-1), was observed in a variety of cell lines, with expression being highest in human cells . TBP-1 was localized predominantly in the nucleus, which is consistent with the nuclear localization of Tat . In cotransfection experiments, expression of TBP-1 was able to specifically suppress Tat-mediated transactivation . The strategy described may be useful for direct identification and cloning of genes encoding proteins that associate with other proteins to modulate their activity in a positive or negative fashion.

Science, 1990 Jun 29, 248(4963), 1646 - 50
Cloning of a transcriptionally active human TATA binding factor; Kao CC et al.; Transcription factor IID (TFIID) binds to the TATA box promoter element and regulates the expression of most eukaryotic genes transcribed by RNA polymerase II . Complementary DNA (cDNA) encoding a human TFIID protein has been cloned . The human TFIID polypeptide has 339 amino acids and a molecular size of 37,745 daltons . The carboxyl-terminal 181 amino acids of the human TFIID protein shares 80% identity with the TFIID protein from Saccharomyces cerevisiae . The amino terminus contains an unusual repeat of 38 consecutive glutamine residues and an X-Thr-Pro repeat . Expression of DNA in reticulocyte lysates or in Escherichia coli yielded a protein that was competent for both DNA binding and transcription activation.

Cell, 1990 Jun 29, 61(7), 1199 - 208
Selective inhibition of activated but not basal transcription by the acidic activation domain of VP16: evidence for transcriptional adaptors; Berger SL et al.; The interaction between the chimeric activator GAL4-VP16, consisting of the DNA binding domain of GAL4 and the acidic activation domain of VP16, and its target in the transcriptional machinery was studied in vitro . GAL4-VP16 stimulated transcription from a promoter bearing GAL4 sites, and greatly inhibited transcription from a promoter bearing binding sites for the dA:dT activator and from a basal promoter bearing only a TATA box . Mutations in the acidic domain that reduced activation from the GAL4 site promoter also reduced inhibition from the dA:dT promoter, indicating a similar interaction between VP16 and its target in both processes . Strikingly, if the DNA binding domain of GAL4-VP16 was occupied by a GAL4 site oligonucleotide, the protein inhibited activation by the dA:dT activator but did not inhibit basal transcription . We propose that, under these conditions, GAL4-VP16 acted to titrate an "adaptor" that bridges an interaction between the upstream activator and the basic transcriptional machinery at the TATA box.

Biochemistry, 1990 Jun 26, 29(25), 5994 - 6002
Structural studies on the active site of Escherichia coli RNA polymerase . 2 . Geometrical relationship of the interacting substrates; Beal RB et al.; Since a major function of RNA polymerase must be to bring together substrates in the optimal configuration for internucleotide bond formation, studies have been undertaken to understand the geometrical relationship of the two substrates . A model has been constructed for the geometry of interaction of two ATP molecules poised on the active site of the Escherichia coli enzyme for the formation of the first bond in RNA synthesis . The model is based primarily on the distance, measured by EPR, between the two metals in the i and i + 1 subsites, as well as distances, measured by NMR, from each metal to points on the substrate in the same subsite, in the presence of a poly(dAdT).poly(dAdT) template . Both the Zn(II) in the i site and the Mg(II) in i + 1 are displaced by Mn(II) . The nucleotide bases are not parallel to each other, in line with the reaction of the ATP molecules with DNA within the transcription bubble . The metal in the i site appears too far removed from substrate to participate in catalysis, but the metal in i + 1 is in position to bind to the beta- and gamma-phosphate groups and probably is involved in cleavage of the triphosphate, as has been previously suggested.

Biochemistry, 1990 Jun 26, 29(25), 5987 - 94
Structural studies on the active site of Escherichia coli RNA polymerase . 1 . Interaction of metals on the i and i + 1 sites; Chuknyisky PP et al.; The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion . These ions are therefore useful as probes of substrate interaction during RNA synthesis . We have studied interactions between the metals by EPR spectroscopy . The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II) . The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way . The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals . The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A.

J Biol Chem, 1990 Jun 25, 265(18), 10189 - 92
Regulation of gene expression in vivo by liposome-mediated delivery of a purified transcription factor; Debs RJ et al.; We describe a procedure for assessing the functional activity in vivo of a glucocorticoid receptor derivative, T7X556, a mammalian transcriptional regulator that has been overexpressed in Escherichia coli and purified to homogeneity . The protein was assessed with DOTMA (N-{1-(2,3-dioleyloxy)propyl}-N,N,N-trimethyl-ammonium chloride) liposomes, which are internalized by cultured mammalian cells . T7X556 protein delivered in this manner localized rapidly to the nucleus and selectively enhanced expression from glucocorticoid response element-linked promoters, properties that are characteristic of this receptor derivative when it is synthesized endogenously in mammalian cells . Thus, in vivo activities of T7X556 were not disrupted by expression in bacteria or by biochemical purification . In general, liposome-mediated delivery may permit functional analyses of proteins that have been expressed in heterologous cells and manipulated in vitro.

Nucleic Acids Res, 1990 Jun 25, 18(12), 3515 - 20
Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA; Weitzmann CJ et al.; The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase . At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence . In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence . The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively . Typical transcription reactions yielded 500-700 moles RNA per mole template . This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts . The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100) . Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed.

Nucleic Acids Res, 1990 Jun 25, 18(12), 3479 - 87
Interaction of RNase P from Escherichia coli with pseudoknotted structures in viral RNAs; Mans RM et al.; In a previous study it was shown that RNase P from E . coli cleaves the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA in vitro (Guerrier-Takada et al . (1988) Cell, 53, 267-272) . Cleavage takes place at the 3' side of the loop that crosses the deep groove of the pseudoknot structure present in the aminoacyl acceptor domain . In the present study fragments of TYMV RNA with mutations in the pseudoknot, generated by transcription in vitro, were tested for susceptibility to cleavage by RNase P . Changes in the specificity with respect to the site of cleavage and decreases in the rate of cleavage were observed with most of these substrates . The behaviour of various mutants in the reaction catalyzed by RNase P is in agreement with the present model of the TYMV RNA pseudoknot (Dumas et al . (1987), J . Biomol . Struct . Dyn . 263, 652-657) . Base substitutions in the loop that crosses the shallow groove of the pseudoknot structure resulted, however, in an unexpected decrease in the rate of cleavage, probably due to conformational changes in the substrates . Studies on other tRNA-like structures revealed an important role in the reaction with RNase P for both the nucleotide at the 3' side of the loop that spans the deep groove and the nucleotide at position 4, which correspond to positions--1 and 73, respectively, in tRNA precursors.

Nucleic Acids Res, 1990 Jun 25, 18(12), 3445 - 50
A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase; Mehrpouyan M et al.; A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated . Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min . on the bacterial chromosome . The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44 degrees C . The thermostability at 44 degrees C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme . Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation . A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about 4 fold at 37 degrees C . The role of the alpha dimer in the structure and function of RNA polymerase is discussed.

Nucleic Acids Res, 1990 Jun 25, 18(12), 3439 - 43
Efficient site directed in vitro mutagenesis using ampicillin selection; Lewis MK et al.; A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis . The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance . Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template . The non-mutant DNA strand is selected against by growth in the presence of ampicillin . In tests of the vector, highly efficient (60-90%) mutagenesis was obtained.

J Biol Chem, 1990 Jun 25, 265(18), 10666 - 73
Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid; Hama C et al.; The frequency of replication initiation of the ColIb-P9 plasmid depends on the level of repZ expression, which has been shown to be negatively regulated by inc RNA, the approximately 70-base-long product of the inc gene . To further understand the regulatory mechanism of repZ gene expression, we isolated mutants defective in ColIb-P9 replication using a lambda:ColIb-P9 hybrid phage . Among six mutants isolated, one amber mutant, rep57, failed to synthesize the RepZ protein . The mutation occurred in the repZ leader sequence that encodes a 29-amino-acid reading frame, designated as repY . We also isolated mutants that suppressed the rep57 phenotype . These mutations were single base insertions between the repY initiation codon and the rep57 mutation site and resulted not only in a frame shift of repY but also in the formation of repY-repZ fusions without changing the amino acid sequence of RepZ . Thus, repY is not directly involved in the replication reaction but rather functions as a positive regulator for repZ expression . We propose that repZ expression is coupled with repY translation, which acts to disrupt a secondary structure sequestering the repZ translation initiation signal . The positive and negative regulations of repZ expression were discussed . The other mutants were mapped in repZ, confirming that repZ is essential for ColIb-P9 replication.

J Biol Chem, 1990 Jun 25, 265(18), 10637 - 44
Covalent association of the traI gene product of plasmid RP4 with the 5'-terminal nucleotide at the relaxation nick site; Pansegrau W et al.; Formation of relaxosomes is the first step in the initiation of transfer DNA replication during bacterial conjugation . This nucleoprotein complex contains all components capable of introducing a site- and strand-specific nick at a cognate transfer origin (oriT) on supercoiled plasmid DNA, thus providing the substrate for generation of the strand to be transferred . Characterization of the terminal nucleotides at the oriT nick site revealed that relaxation occurs by hydrolysis of a single phosphodiester bond between a 2'-deoxyguanosyl and a 2'-deoxycytidyl residue . The relaxation nick site and a 19-base pair invert repeat sequence that is recognized by asymmetric binding of the RP4 TraJ protein are interspaced by 8 base pairs . The nicking reaction results in covalent attachment of the RP4 TraI protein to the 5'-terminal 2'-deoxycytidyl residue of the cleaved strand . The arrangement of the TraJ binding site and the relaxation nick site on the same side of the DNA double helix suggests that protein-protein interactions between TraJ and TraI are a prerequisite for oriT specific nicking . In accordance with the current model of transfer DNA replication, the 3' end remains accessible for primer extension by DNA polymerase I, enabling replacement strand synthesis in the donor cell by a rolling circle-type mechanism.

J Biol Chem, 1990 Jun 25, 265(18), 10631 - 6
Molecular cloning of a cDNA for rat hepatic glutaminase . Sequence similarity to kidney-type glutaminase; Smith EM et al.; Mammalian liver possesses a unique isozyme of phosphate-activated glutaminase which plays an important role in the regulation of glutamine catabolism . Antibodies to hepatic glutaminase were used to screen a lambda gt11 rat liver cDNA library . One cDNA to hepatic glutaminase was identified . Changes in the relative abundance of hepatic glutaminase mRNA were determined by hybridization to this cDNA . The mRNA is found only in liver; it is not present prior to birth but its abundance increases dramatically at birth . The abundance of the mRNA is increased approximately 4-fold in diabetes . The sequence of the cDNA was compared to that recently published for kidney (brain)-type glutaminase (Banner, C., Hwang, J.-J., Shapiro, R.A., Wenthold, R.J., Nakatani, Y., Lampel, K.A., Thomas, J.W., Huie, D., and Curthoys, N.P . (1988) Mol . Brain Res . 3, 247-254) . When the predicted amino acid sequences were compared a region of 123 amino acids with greater than 80% identity was found . The presence of scattered amino acid substitutions within stretches of identical amino acids suggests that the glutaminase isozymes are encoded by separate genes . This is the first demonstration of any similarity between the two glutaminases at the molecular level.

J Biol Chem, 1990 Jun 25, 265(18), 10597 - 603
The basement membrane glycoprotein entactin promotes cell attachment and binds calcium ions; Chakravarti S et al.; Mouse entactin derived from the extracellular matrix of M1536-B3 cells and from insect cells infected with a recombinant virus containing entactin sequences were shown to promote the attachment of mouse mammary tumor, human melanoma, and other cells . The cell attachment was inhibited by antibodies against mouse entactin but not by anti-fibronectin or anti-laminin antibodies . On a weight basis entactin was as effective as laminin in promoting the attachment of mouse mammary tumor cells . The attachment of cells to entactin was in part mediated by the integrin recognition RGD peptide sequence . This was demonstrated by the cell attachment properties of peptides derived from entactin which contained this sequence . Furthermore, the peptide RGDS could inhibit the attachment of mouse mammary tumor cells to entactin to approximately 60% of control . It is suggested that additional cell recognition sequences may be present in entactin . The direct binding of calcium ions to entactin was observed . It is probable that the binding sites reside in peptide sequences located toward the NH2 terminus region of entactin . This conclusion was supported by the demonstration that synthetic peptides, containing potential calcium binding sequences derived from entactin, bound calcium . In addition, a recombinant peptide containing the amino-terminal 330 amino acids of entactin also bound calcium ions . The significance of these properties of entactin is discussed.

J Biol Chem, 1990 Jun 25, 265(18), 10424 - 9
Nucleotide sequence of the fadA gene . Primary structure of 3-ketoacyl-coenzyme A thiolase from Escherichia coli and the structural organization of the fadAB operon; Yang SY et al.; The DNA insert of plasmid pK52 contains the fadAB operon coding for the Escherichia coli fatty acid oxidation complex . Studies on the operon's structure and organization revealed that the initiator codon (ATG) of the structural gene for 3-ketoacyl-CoA thiolase, the fadA gene, is located 109 nucleotides 3' to the stop codon (TGA) of the fadB gene that encodes the alpha-subunit, a multifunctional polypeptide . The direction of transcription of this operon is thus from fadB to fadA . The orientation of the fadA and fadB genes is the reverse of what had been published previously . The structural gene for thiolase is 1,164 nucleotides long and starts six nucleotides downstream from a Shine-Dalgarno sequence . 109 nucleotides of 5'-noncoding and 321 nucleotides of 3'-noncoding regions are also reported . The 3-ketoacyl-CoA thiolase beta-subunit is composed of 388 residues and has a calculated Mr of 40,889 . The alpha- and beta-subunits were separated by gel filtration in formic acid, and the sequence of the amino-terminal 10 amino acids of the beta-subunit coincided with that deduced from the nucleotide sequence data . Sequence comparisons suggest that Cys-91 of the E . coli enzyme is the active-site cysteine residue and that the consensus sequence of the active sites of 3-ketoacyl-CoA thiolases is Asn-Arg-X1-Cys-X2-Ser-X3-X4-Gln . Although the quaternary structure of E . coli 3-ketoacyl-CoA thiolase is different from that of other thiolases, the sequence is homologous to rat and human peroxisomal and rat mitochondrial 3-ketoacyl-CoA thiolases, to the extent of 42, 41, and 37%, identity, respectively . An evolutionary tree of thiolases was constructed; it suggests that the genes of E . coli and peroxisomal 3-ketoacyl-CoA thiolases diverged after the appearance of eukaryotic cells.

J Biol Chem, 1990 Jun 25, 265(18), 10196 - 7
Crystallization and preliminary x-ray investigation of colicin E3 in complex with its immunity protein; Frolow F et al.; Crystals of the colicin E3-immunity protein complex have been grown from solutions of citrate at pH 5.6 . The crystals are monoclinic, space group P2(1), with unit cell dimensions a = 67.71, b = 196.67, c = 85.58 A, and beta = 113.67 degrees . The crystals diffract to 3-A resolution and are stable in the x-ray beam for at least a day . Although the stoichiometry of the complex in solution is 1:1 there are two, three, or four such binary complex molecules in the asymmetric unit.

J Biol Chem, 1990 Jun 25, 265(18), 10327 - 33
Biotination of proteins in vivo . A post-translational modification to label, purify, and study proteins; Cronan JE Jr; Post-translational modification of proteins with biotin provides the means to specifically label proteins in vivo and to purify proteins from crude cell lysates . The carboxyl-terminal protein segments modified by reaction with biotin ligase are strongly conserved in nature . We have demonstrated that the proteins encoded by translational gene fusions of a number of heterologous proteins to these carboxyl-terminal sequences become biotinated in vivo . The minimum size of the protein segment needed to allow biotination of fusion proteins is 75 amino acids . This biotination sequence, although of bacterial origin, functions in Saccharomyces cerevisiae as well as in Escherichia coli . Fusion proteins are readily labeled with {3H}biotin in vivo and the labeling is highly specific due to the scarcity (less than 5) of biotinated protein species . Biotinated fusion proteins can be readily purified in native form by binding to columns of monomeric avidin followed by elution with buffers containing biotin . Alternatively, proteins can be purified in a denatured form in presence of 1% sodium dodecyl sulfate or 8 M urea . Thus, this technology allows purification by affinity chromatography of any protein to which a biotination sequence can be attached . The ability to specifically label a protein in vivo should have utility in studies such as intracellular protein trafficking and cytoskeletonal dynamics.

J Biol Chem, 1990 Jun 25, 265(18), 10574 - 81
Transcription of osmB, a gene encoding an Escherichia coli lipoprotein, is regulated by dual signals . Osmotic stress and stationary phase; Jung JU et al.; The osmB gene, which encodes an outer membrane lipoprotein, can be induced by both osmotic and growth phase signals . Construction of two transcriptional fusions, an osmB-lacZ fusion in single copy on the bacterial chromosome and an osmB-cat fusion carried on a multicopy plasmid, demonstrated that induction of osmB by hyperosmolarity and during the stationary phase of growth occurred at the level of transcription . Two transcription initiation sites were identified by RNase protection of in vivo message . The downstream P2 promoter is the primary site for regulation; the basal level of expression is initiated at P2 and transcription from P2 is induced by elevated osmolarity or upon reaching stationary phase . Transcription from the P1 promoter, 150 base pairs (bp) upstream of the P2 promoter, occurred only when both osmotic and growth phase signals were present simultaneously; that is, when cells growing in high osmolarity medium have reached stationary phase . Deletion analysis narrowed the sequences necessary for P2 regulation to the 42-bp region upstream from the transcription start site . A 7-bp sequence just upstream from the -35 region was identified as a cis-acting regulatory element essential for osmotic stimulation of osmB expression . A hexanucleotide sequence within this segment could form the left arm of a region of dyad symmetry, flanking the -35 region of the promoter . Stationary phase induction at P2 does not require the 7-bp element.

J Biol Chem, 1990 Jun 25, 265(18), 10565 - 73
Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase; Huber HE et al.; We have analyzed the processing of the RNA primer for (+) strand DNA synthesis by reverse transcriptase of the human immunodeficiency virus 1 . To test for specific RNA cleavage and primer usage, we constructed a 99-base pair RNA-DNA hybrid containing the viral polypurine tract and flanking viral sequences . Although the RNase H activity of reverse transcriptase cleaves the RNA strand into multiple fragments, only two primers are extended in the presence of nucleoside triphosphates . The major RNA primer includes the entire polypurine tract except for the last adenosine and has the sequence 5'-UUUUAAAAGAAAAGGGGGG-3' . The minor primer has the same 3' end but is two nucleotides shorter . In a subsequent processing step reverse transcriptase releases the primer intact via a cleavage at the RNA-DNA junction . RNA cleavage, primer extension, and primer removal can take place in a single reaction . However, specificity does not require coupling of the three steps and is preserved in the individual reactions . The polypurine primer is generated and removed after its elongation in the absence of DNA synthesis . Furthermore, the polypurine primer is selected among the several RNA fragments available and extended by reverse transcriptase as well as by p51, a short form of reverse transcriptase lacking RNase H activity.

Science, 1990 Jun 22, 248(4962), 1550 - 3
An RNA polymerase II transcription factor shares functional properties with Escherichia coli sigma 70; Conaway JW et al.; A mammalian transcription factor, which, along with other factors, is essential for accurate initiation of transcription from promoters by RNA polymerase II, has been found to regulate the interaction of polymerase and DNA . This factor, designated beta gamma, drastically reduces the affinity of RNA polymerase II for free DNA containing either promoter or nonpromoter sequences . In this respect, beta gamma functions as does the bacterial transcription initiation factor sigma 70, which expedites the binding of Escherichia coli RNA polymerase to promoters in part by accelerating dissociation of the polymerase from nonpromoter sites in DNA.

Biochim Biophys Acta, 1990 Jun 21, 1049(2), 223 - 6
Characterization of a cDNA clone encoding the complete amino acid sequence of cotton isocitrate lyase; Turley RB et al.; A cDNA clone encoding the glyoxysomal enzyme isocitrate lyase (ICL) (EC 4.1.3.1) was isolated from a library prepared from cotton (Gossypium hirsutum L.) cotyledon poly(A)+ RNA . The clone is 1893 basepairs (bp) in length and contains a 1728 bp open reading frame encoding a polypeptide of 576 residues (Mr = 64,741) . The deduced amino acid sequence of cotton ICL is 85.2%, 90.3% and 41.1% identical to ICL from rapeseed, castor bean and E . coli, respectively . Cotton ICL has a C-terminal tripeptide of A-R-M which is a putative trafficking signal for peroxisome (glyoxysome) proteins.

Eur J Biochem, 1990 Jun 20, 190(2), 311 - 8
Protein-decorated micelle structure of sodium-dodecyl-sulfate--protein complexes as determined by neutron scattering; Ibel K et al.; The structure of the complex between sodium dodecyl sulfate (SDS) and a deuterated bifunctional enzyme, N-5'-phosphoribosylanthranilate isomerase/indole-3-glycerol-phosphate synthase (Mr 49,484), has been studied in dilute solution by small-angle neutron scattering . The complex nearly acquired its final size, as shown by molecular-sieve chromatography, at the chosen SDS concentration of 1.6 mM, which is slightly below the critical micelle concentration of 1.8 mM (at the ionic strength of 0.1 M) . The 452 amino-acid residues of the bifunctional enzyme were combined with 216 detergent molecules . The complex was found to be composed of three protein-decorated SDS micelles of unequal size, connected by short flexible polypeptide segments . The largest of the three micelles was the middle one . The SDS-protein complex contained the dodecyl hydrocarbon moieties in three globular cores . Each core was surrounded by a hydrophilic shell, formed by the hydrophilic and amphiphilic stretches of the polypeptide chain, and by the sulfate head groups of the detergent . The average thickness of these shells was 0.7-0.8 nm . The three-micelle complex was cleaved with trypsin at a single site, possibly in a micelle-connecting segment, into a single-micelle fragment at the carboxyl-terminal which comprised 73 SDS molecules and 163 amino-acid residues, and a dual-micelle fragment . One of the micelles within this larger fragment contained 42 SDS molecules and about 90 amino-acid residues; the other micelle contained 101 SDS molecules and about 190 amino-acid residues . The individual micelle sizes seemed to be determined by the amino-acid sequence.

Biochim Biophys Acta, 1990 Jun 20, 1034(3), 253 - 9
Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichia coli; Falkenberg P et al.; The osmoregulatory NAD-dependent betaine aldehyde dehydrogenase (betaine aldehyde:NAD oxidoreductase, EC 1.2.1.8), of Escherichia coli, was purified to apparent homogeneity from an over-producing strain carrying the structural gene for the enzyme (betB) on the plasmid vector pBR322 . Purification was achieved by ammonium sulfate fractionation of disrupted cells, followed by affinity chromatography on 5'-AMP Sepharose, gel-filtration and ion-exchange chromatography . The amino acid composition was determined . The dehydrogenase was found to be a tetramer with identical 55 kDa subunits . Both NAD and NADP could be used as cofactor for the dehydrogenase, but NAD was preferred . The dehydrogenase was highly specific for betaine aldehyde . None of the analogs tested functioned as a substrate, but several inhibited the enzyme competitively . The enzyme was not activated by salts at concentrations encountered during osmotic upshock, but it was salt tolerant, retaining 50% of maximal activity at 1.2 M K+ . It is inferred that salt tolerance is an essential property for an enzyme participating in the cellular synthesis of an osmoprotectant.

J Mol Biol, 1990 Jun 20, 213(4), 705 - 17
Construction of Escherichia coli amber suppressor tRNA genes . II . Synthesis of additional tRNA genes and improvement of suppressor efficiency; Kleina LG et al.; Using synthetic oligonucleotides, we have constructed 17 tRNA suppressor genes from Escherichia coli representing 13 species of tRNA . We have measured the levels of in vivo suppression resulting from introducing each tRNA gene into E . coli via a plasmid vector . The suppressors function at varying efficiencies . Some synthetic suppressors fail to yield detectable levels of suppression, whereas others insert amino acids with greater than 70% efficiency . Results reported in the accompanying paper demonstrate that some of these suppressors insert the original cognate amino acid, whereas others do not . We have altered some of the synthetic tRNA genes in order to improve the suppressor efficiency of the resulting tRNAs . Both tRNA(CUAHis) and tRNA(CUAGlu) were altered by single base changes, which generated -A-A- following the anticodon, resulting in a markedly improved efficiency of suppression . The tRNA(CUAPro) was inactive, but a hybrid suppressor tRNA consisting of the tRNA(CUAPhe) anticodon stem and loop together with the remainder of the tRNA(Pro) proved highly efficient at suppressing nonsense codons . Protein chemistry results reported in the accompanying paper show that the altered tRNA(CUAHis) and the hybrid tRNA(CUAPro) insert only histidine and proline, respectively, whereas the altered tRNA(CUAGlu) inserts principally glutamic acid but some glutamine . Also, a strain deficient in release factor I was employed to increase the efficiency of weak nonsense suppressors.

J Mol Biol, 1990 Jun 20, 213(4), 777 - 88
IncN plasmid replicon . A deletion and subcloning analysis; Krishnan BR et al.; A DNA segment of approximately 2000 base-pairs bounded by restriction enzyme sites for PvuII and containing the minimal replicon of an N group plasmid was characterized . A natural derivative of this miniplasmid was found to have undergone a deletion within one of two tandem iteron families, the group I iterons . Further analysis showed that all plasmid-determined functions essential for stable maintenance in Escherichia coli were localized to a contiguous region of DNA of 1019 nucleotides that excludes entirely these iterons . However, the loss of these iterons led to an increase in plasmid copy number . This indicates that members of the group I iteron-family have a role in determining plasmid copy number perhaps by titrating a plasmid-specified trans-acting product . The 2000 base-pair segment contains six open reading frames of 40 or more amino acid residues . The essential segment contains a 368 nucleotide region that must be present in cis and within which there are three "GATC" sequences and a putative Escherichia coli DnaA protein-binding sequence (dnaA box) . An interesting feature is that the cis-acting region is present entirely within a presumptive rep gene . The essential segment contains four open reading frames, only one of which has an Escherichia coli canonical ribosome-binding site . The 2000 base-pair miniplasmid has two separable regions determining N group plasmid incompatibility.

J Mol Biol, 1990 Jun 20, 213(4), 627 - 30
Induced-fit movements in adenylate kinases; Schulz GE et al.; The high-resolution crystal structures of three homologous adenylate kinases with zero, one and both ( = 2-substrate mimicking inhibitor) bound substrates have been compared . The comparisons are meaningful, because all structures occur in two or three different crystal contact environments indicating that they represent intrinsically stable conformations in solution . Molecular superimpositions revealed that two domains comprising 30 and 38 residues undergo large movements on substrate binding, which can be approximated by rigid-body rotations over 39 degrees and 92 degrees, respectively . Moreover, these movements can be subdivided into two steps: first, a change on binding substrate AMP, which involves only the 30 residue domain (C alpha shifts up to 8.2 A), and second, a change on additional binding of substrate ATP, which again involves the 30 residue domain (C alpha shifts up to 7.6 A) but also the 38 residue domain (C alpha shifts up to 32.3 A) . Taken together, these observations yield a three-picture "moving film" of the induced-fit.

J Mol Biol, 1990 Jun 20, 213(4), 789 - 809
RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates . An ADP-resistant process; Rao BJ et al.; Efficient homologous pairing de novo of linear duplex DNA with a circular single strand (plus strand) coated with RecA protein requires saturation and extension of the single strand by the protein . However, strand exchange, the transfer of a strand from duplex DNA to the nucleoprotein filament, which follows homologous pairing, does not require the stable binding of RecA protein to single-stranded DNA . When RecA protein was added back to isolated protein-free DNA intermediates in the presence of sufficient ADP to inhibit strongly the binding of RecA protein to single-stranded DNA, strand exchange nonetheless resumed at the original rate and went to completion . Characterization of the protein-free DNA intermediate suggested that it has a special site or region to which RecA protein binds . Part of the nascent displaced plus strand of the deproteinized intermediate was unavailable as a cofactor for the ATPase activity of RecA protein, and about 30% resisted digestion by P1 endonuclease, which acts preferentially on single-stranded DNA . At the completion of strand exchange, when the distal 5' end of the linear minus strand had been fully incorporated into heteroduplex DNA, a nucleoprotein complex remained that contained all three strands of DNA from which the nascent displaced strand dissociated only over the next 50 to 60 minutes . Deproteinization of this intermediate yielded a complex that also contained three strands of DNA in which the nascent displaced strand was partially resistant to both Escherichia coli exonuclease I and P1 endonuclease . The deproteinized complex showed a broad melting transition between 37 degrees C and temperatures high enough to melt duplex DNA . These results show that strand exchange can be subdivided into two stages: (1) the exchange of base-pairs, which creates a new heteroduplex pair in place of a parental pair; and (2) strand separation, which is the physical displacement of the unpaired strand from the nucleoprotein filament . Between the creation of new heteroduplex DNA and the eventual separation of a third strand, there exists an unusual DNA intermediate that may contain three-stranded regions of natural DNA that are several thousand bases in length.

J Mol Biol, 1990 Jun 20, 213(4), 719 - 26
Construction of Escherichia coli amber suppressor tRNA genes . III . Determination of tRNA specificity; Normanly J et al.; Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes . In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E . coli dihydrofolate reductase gene fol . The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon . The suppressors can be classified into three groups on the basis of the protein sequence information . Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid . The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase . The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.

J Mol Biol, 1990 Jun 20, 213(4), 617 - 9
Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme; Boulot G et al.; The associated heavy (VH) and light (VL) chain variable domains (Fv) of the monoclonal anti-lysozyme antibody D1.3, secreted from Escherichia coli, have been crystallized in their antigen-bound and free forms . FvD1.3 gives tetragonal crystals, space group P4(1)2(1)2 (or P4(3)2(1)2), with a = 90.6 A, c = 56.4 A . The FvD1.3-lysozyme complex crystallizes in space group C2, with a = 129.2 A, b = 60.8 A, c = 56.9 A and beta = 119.3 degrees . The crystals contain one molecule of Fv or of the Fv-lysozyme complex in their asymmetric units and diffract X-rays to high resolution, making them suitable for X-ray crystallographic studies.

J Mol Biol, 1990 Jun 20, 213(4), 613 - 5
Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli; Glockshuber R et al.; The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0) . The crystals are hexagonal, space group P6(1)22 . The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit . The crystals diffract to 2.0 A . The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI.

Eur J Biochem, 1990 Jun 20, 190(2), 257 - 61
Three human interferon-alpha 2 subvariants disclose structural and functional differences; von Gabain A et al.; The human interferon-alpha 2 subvariants 2a, 2b and 2c differ by only one or two amino acids at positions 23 and/or 34 of the mature protein . In this study, the coding regions of the three interferon-alpha 2 subvariants were derived from the cDNA of interferon-alpha 2c by site-directed in vitro mutagenesis . The interferon-alpha subvariants were synthesized using the same Escherichia coli strain for production and were subsequently purified . Comparative studies revealed that they differ significantly in their biological and antigenic properties . Therefore, amino acid positions 23 and 34 seem to be crucial for structure/function of human interferon-alpha . Furthermore, the study points to the importance of defining, whether such minor structural variants of naturally occurring polypeptides represent functional variants.

J Mol Biol, 1990 Jun 20, 213(4), 607 - 11
Crystallization of genetically engineered active maltose-binding proteins, including an immunogenic viral epitope insertion; Rodseth LE et al.; Three mutants of the maltose- or maltodextrin-binding protein encoded by the malE gene of Escherichia coli, with extensive genetic changes, have been purified and crystallized in different crystal forms . Two of these mutant proteins, MalE178 and MalE341, carry net deletions of seven and 13 residues, respectively, near the surface of the molecule . These mutations have very little effect on either the transport activity of the mutant strains or the sugar-binding activity of the purified mutant proteins . The third mutant protein involves the insertion of an 11-residue peptide of the C3 epitope from type 1 poliovirus VP1 protein into the MalE178 deletion mutant, with retention of essentially all the biological properties of the wild-type and the immunological properties of the C3 epitope . We are undertaking three-dimensional structure analysis in order to understand how the protein accommodates these large changes in its surface structure and how the C3 epitope retains its immunological properties in this new environment . The same system could be used to determine easily the structures of other peptide epitopes, especially those in proteins with unknown structures.

Biochemistry, 1990 Jun 19, 29(24), 5829 - 36
On the nature of the structural change of the colicin E1 channel peptide necessary for its translocation-competent state; Merrill AR et al.; Acidic pH conditions required in vitro for membrane binding and activity of the channel-forming colicin E1 resulted in an increased susceptibility to proteases of the 178-residue thermolytic channel peptide, an increased accessibility to acrylamide of a fluorescence probe linked to cysteine-505 of the peptide, and an increased partition into nonionic detergent . The structural change in the peptide sensed by the fluorescence probe caused by a transition from pH 6.0 to 3.5 occurred in less than 1 s . The presence of low concentrations of detergents (0.001% SDS or 0.44% octyl beta-D-glucoside) or urea (0.2 M) at pH 6 or 4 also increased the susceptibility of the channel peptide to proteases . The increase in protease susceptibility and acrylamide accessibility at low pH, as well as partition of the peptide into nonionic detergent, suggested that acidic pH or the detergents might cause peptide unfolding . However, the hydrodynamic radius of the channel peptide at pH 6, 21-23 A, was not changed at pH 3.5 or by detergents or urea under conditions that increased the susceptibility of the peptide to protease . The activity of the channel peptide at pH 6 measured with liposomes and planar bilayers, which was a factor of 10(3)-10(4) smaller than that at pH 4, was increased by 2-4 orders of magnitude by 0.001% SDS or 0.44% octyl beta-D-glucoside, with an additional small increment of activity on planar bilayers caused by 0.01% SDS . A small increase in Stokes radius of the peptide in the presence of SDS could be detected that was approximately correlated with increased activity.

Biochemistry, 1990 Jun 19, 29(24), 5790 - 6
Glutathione reductase: comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes; Vanoni MA et al.; Kinetic parameters for NADPH and NADH have been determined at pH 8.1 for spinach, yeast, and E . coli glutathione reductases . NADPH exhibited low Km values for all enzymes (3-6 microM), while the Km values for NADH were 100 times higher (approximately 400 microM) . Under our experimental conditions, the percentage of maximal velocities with NADH versus those measured with NADPH were 18.4, 3.7, and 0.13% for the spinach, yeast, and E . coli enzymes, respectively . Primary deuterium kinetic isotope effects were independent of GSSG concentration between Km and 15Km levels, supporting a ping-pong kinetic mechanism . For each of the three enzymes, NADPH yielded primary deuterium kinetic isotope effects on Vmax only, while NADH exhibited primary deuterium kinetic isotope effects on both V and V/K . The magnitude of DV/KNADH at pH 8.1 is 4.3 for the spinach enzyme, 2.7 for the yeast enzyme, and 1.6 for the E . coli glutathione reductase . The experimentally determined values of TV/KNADH of 7.4, 4.2, and 2.2 for the spinach, yeast, and E . coli glutathione reductases agree well with those calculated from the corresponding DV/KNADH using the Swain-Schaad expression . This suggests that the intrinsic primary kinetic isotope effect on NADH oxidation is fully expressed . In order to confirm this conclusion, single-turnover experiments have been performed . The measured primary deuterium kinetic isotope effects on the enzyme reduction half-reaction using NADH match those measured in the steady state for each of the three glutathione reductases.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Jun 19, 29(24), 5752 - 61
1H NMR resonance assignments, secondary structure, and global fold of Apo bovine calbindin D9k; Skelton NJ et al.; The solution structure and dynamics of apo bovine calbindin D9k have been studied by a wide range of two-dimensional 1H nuclear magnetic resonance experiments . Due to the presence of conformational heterogeneity in the wild-type protein, the sequential resonance assignment was carried out on a Pro43----Gly mutant . By use of a combination of scalar correlation experiments acquired from H2O solution, 61 of the 76 1H spin systems could be assigned to particular amino acid types . The remaining resonances were assigned by a parallel series of experiments acquired from 2H2O solution . These spin system assignments provided a basis for complete sequential resonance assignments from interresidue backbone nuclear Overhauser effects (NOEs) . Elements of secondary structure were identified from sequential and medium-range NOEs, backbone spin-spin coupling constants, and slowly exchanging amide protons . Four sections of helix are delineated, together with a short antiparallel beta-sheet interaction between the peptide loops involved in Ca2+ binding . The global fold is provided by combining these elements of secondary structure with a subset of the long-range, interhelix NOEs . Comparison with similar studies on the Ca2(+)-saturated protein indicates that at this crude level the structures are very similar . However, removal of the Ca2+ does dramatically affect the dynamics of the protein, as judged by amide proton exchange rates and aromatic ring rotation . This is particularly evident in the increased flexibility of the residues in the hydrophobic core.

Biochemistry, 1990 Jun 19, 29(24), 5711 - 8
Substrate overlap and functional competition between human nucleotide excision repair and Escherichia coli photolyase and (a)BC excision nuclease; Sibghat-Ullah et al.; Human cell free extract prepared by the method of Manley et al . (1980) carries out repair synthesis on UV-irradiated DNA . Removal of pyrimidine dimers by photoreactivation with DNA photolyase reduces repair synthesis by about 50% . With excess enzyme in the reaction mixture photolyase reduced the repair signal by the same amount even in the absence of photoreactivating light, presumably by binding to pyrimidine dimers and interfering with the binding of human damage recognition protein . Similarly, the UvrB subunit of Escherichia coli (A)BC excinuclease when loaded onto UV-irradiated or psoralen-adducted DNA inhibited repair synthesis by cell-free extract by 75-80% . The opposite was true also as HeLa cell free extract specifically inhibited the photorepair of a thymine dimer by DNA photolyase and its removal by (A)BC excinuclease . Cell-free extracts from xeroderma pigmentosum (XP) complementation groups A and C were equally effective in blocking the E . coli repair proteins, while extracts from complementation groups D and E were ineffective in blocking the E . coli enzyme . These results suggest that XP-D and XP-E cells are defective in the damage recognition subunit(s) of human excision nuclease.

Biochemistry, 1990 Jun 19, 29(24), 5706 - 11
Reconstitution of Escherichia coli photolyase with flavins and flavin analogues; Payne G et al.; Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF) . A procedure was developed for reversible resolution of apophotolyase and its chromophores . To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted . Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase . The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD . Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity.

Biochemistry, 1990 Jun 19, 29(24), 5698 - 706
Active site of Escherichia coli DNA photolyase: mutations at Trp277 alter the selectivity of the enzyme without affecting the quantum yield of photorepair; Li YF et al.; Escherichia coli DNA photolyase repairs pyrimidine dimers by a photoinduced electron-transfer reaction . The enzyme binds to UV-damaged DNA independent of light (the dark reaction) and upon absorbing a 300-500-nm photon breaks the cyclobutane ring of the dimer (the light reaction) and thus restores the DNA . No structural information on the enzyme is available at present . However, comparison of the sequences of photolyases from five different organisms has identified highly conserved regions of homology . These regions are presumably involved in chromophore (flavin and folate) and substrate binding or catalysis . Trp277 (W277) in E . coli photolyase is conserved in all photolyases sequenced to date . We replaced this residue with Arg, Glu, Gln, His, and Phe by site-specific mutagenesis . Properties of the mutant proteins indicate that W277 is involved in binding to DNA but not in chromophore binding or catalysis . Of particular significance is the finding that compared to wild type W277R and W277E mutants have about 300- and 1000-fold lower affinity, respectively, for substrate but were indistinguishable from wild-type enzyme in their photochemical and photocatalytic properties.

Biochemistry, 1990 Jun 19, 29(24), 5694 - 8
Excited-state properties of Escherichia coli DNA photolyase in the picosecond to millisecond time scale; Heelis PF et al.; Escherichia coli DNA photolyase contains a stable flavin radical that is readily photoreduced in the presence of added electron donors . Picosecond, nanosecond, and conventional flash photolysis technique have been employed to investigate the events leading to photoreduction from 40 ps to tens of milliseconds following flash excitation . Direct light absorption by the flavin radical produces the first excited doublet state which undergoes rapid (within 100 ps) intersystem crossing to yield the lowest excited quartet (n pi*) state . In contrast, light absorption by the folate chromophore produces a new intermediate state via interaction of the folate excited singlet state with the ground-state flavin radical, leading to an enhanced yield of the excited radical doublet state and hence quartet state . Subsequent reaction of the excited quartet state involves hydrogen atom abstraction from a tryptophan residue . Secondary electron transfer from added electron donors occurs to the oxidized tryptophan radical with rate constants ranging from 10(4) (dithiothreitol) to 4 x 10(6) M-1 s-1 (n-propyl gallate) . The low value of the latter rate compared to reduction of the tryptophan radical in lysozyme suggests that the reactive tryptophan is highly buried in photolyase . A redox potential diagram has been constructed for the ground and excited states involved . It is concluded that the one-electron reduction potential of the excited quartet state of the flavin radical must be at least 1.23 V more positive than the ground state, in agreement with the value of delta E greater than 1.77 V calculated from spectroscopic data.

Biochemistry, 1990 Jun 19, 29(24), 5761 - 6
A site-directed mutagenesis study on Escherichia coli inorganic pyrophosphatase . Glutamic acid-98 and lysine-104 are important for structural integrity, whereas aspartic acids-97 and -102 are essential for catalytic activity; Lahti R et al.; Analysis of the conservation of functional residues between yeast and Escherichia coli inorganic pyrophosphatases (PPases) suggested that Asp-97, Glu-98, Asp-102, and Lys-104 are important for the action of E . coli PPase {Lahti, R., Kolakowski, L . F., Heinonen, J., Vihinen, M., Pohjanoksa, K., & Cooperman, B . S . (1990) Biochim . Biophys . Acta 1038, 338-345} . We replaced these four residues by oligonucleotide-directed mutagenesis, giving variant PPases DV97, DE97, EV98, DV102, DE102, KI104, and KR104 . PPase variants DV97, DV102, and KI104 had no enzyme activity, whereas PPase variants DE97, EV98, DE102, and KR104 had 22%, 33%, 3%, and 3% of the wild-type PPase activity, respectively . This suggests that Asp-97, Asp-102, and Lys-104 are essential for the catalytic activity of E . coli PPase . PPase variants DV98 and KR104 also had an increased sensitivity to heat denaturation; incubation of these mutant PPases at 75 degrees C for 15 min in the presence of 5 mM magnesium ion decreased the activity to 20% and 1%, respectively, of the initial value while 74% of the activity was observed with wild-type PPase . Furthermore, these thermolabile mutant PPases displayed the most profound conformational changes of the PPase variants examined, as demonstrated by the binding of the fluorescent dye Nile red that monitors the hydrophobicity of protein surfaces . Accordingly, Glu-98 and Lys-104 seem to be important for the structural integrity of E . coli PPase.

Biochemistry, 1990 Jun 19, 29(24), 5665 - 71
Chaperonin-facilitated refolding of ribulosebisphosphate carboxylase and ATP hydrolysis by chaperonin 60 (groEL) are K+ dependent; Viitanen PV et al.; Both the chaperonin- and MgATP-dependent reconstitution of unfolded ribulosebisphosphate carboxylase (Rubisco) and the uncoupled ATPase activity of chaperonin 60 (groEL) require ionic potassium . The spontaneous, chaperonin-independent reconstitution of Rubisco, observed at 15 but not at 25 degrees C, requires no K+ and is actually inhibited by chaperonin 60, with which the unfolded or partly folded Rubisco forms a stable binary complex . The chaperonin-dependent reconstitution of Rubisco involves the formation of a complex between chaperonin 60 and chaperonin 10 (groES) . Formation of this complex almost completely inhibits the uncoupled ATPase activity of chaperonin 60 . Furthermore, although the formation of the chaperonin 60-chaperonin 10 complex requires the presence of MgATP, hydrolysis of ATP may not be required, since complex formation occurs in the absence of K+ . The interaction of chaperonin 60 with unfolded or partly folded Rubisco does not require MgATP, K+, or chaperonin 10 . However, discharge of the complex of chaperonin 60-Rubisco, which leads to the formation of active Rubisco dimers, requires chaperonin 10 and a coupled, K(+)-dependent hydrolysis of ATP . We propose that a role of chaperonin 10 is to couple the K(+)-dependent hydrolysis of ATP to the release of the folded monomers of the target protein from chaperonin 60.

Biochim Biophys Acta, 1990 Jun 19, 1039(2), 197 - 203
A mutational analysis of the epitopes of recombinant human H-ferritin; Arosio P et al.; Murine monoclonal antibodies were elicited by the recombinant human H-ferritin overexpressed in Escherichia coli . They had a specificity analogous to that of the antibodies elicited by natural human H-chain, and all of them showed low additivity in binding the recombinant ferritin . Four antibodies of each group were challenged with four H-ferritin mutants overexpressed in E . coli, altered in different accessible areas of the molecule . They consisted of deletions of the first 13 and last 22 amino acids, a duplication of an 18 amino acid sequence in the loop region, and a substitution of a 5 amino acid stretch in the three-fold symmetry axis region . Double diffusion, immunodot analyses and inhibition plots indicated that: (1) all the mutants were recognized by at least one antibody; (2) the deletion of the N-terminus and the duplication in the loop region had the strongest effect on antibody binding; and (3) epitope boundaries of the various antibodies could not be recognized . The antibodies were tested with H-containing ferritins from rat and hen hearts, and showed low or absent reactivities despite their high structural homology with human ferritin . Comparison of the amino acid sequences of human, mouse, rat and hen H-chains, together with mutational data, suggested that; (i) ferritin epitopes are large, probably encompassing a large portion of the subunit surface and (ii) Thr-5 and Cys-90 have a role in H-ferritin immunogenicity.

Biochim Biophys Acta, 1990 Jun 19, 1039(2), 142 - 8
Substrate specificity for myelin basic protein-specific protein methylase I; Ghosh SK et al.; The substrate specificity of bovine brain myelin basic protein (MBP)-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), which methylates arginine residues of protein, has been studied using various MBPs, several synthetic peptides and heterogeneous nuclear ribonucleoprotein complex protein (hnRNP) . (1) Among MBPs from different species of brain, the carp MBP was found to be the best substrate for MBP-specific protein methylase I . This high degree of methyl acceptability is most likely due to the fact that carp MBP is not in vivo methylated at the arginine residue (Deibler, G.E . and Martenson, R.E . (1973) J . Biol . Chem . 248, 2387-2391) and that the methylatable amino acid sequence is present in this protein . (2) In order to study the minimum chain length of MBP polypeptide which functions as the methyl acceptor, several synthetic polypeptides whose sequences are identical to the region surrounding the residue 107 of bovine MBP (the in vivo methylation site) were synthesized . It was found that the hexapeptide, Gly-Lys-Gly-Arg-Gly-Leu (corresponding to residues 104-109 of bovine MBP), was the shortest methyl accepting peptide, while the tetrapeptide, Gly-Arg-Gly-Leu (corresponding to residues 106-109) was inactive as a substrate . (3) hnRNP protein is known to contain methylarginine at residue 193 (Williams, K.R., Stone, K.L., LoPresti, M.B., Merrill, B.M . and Plank, S.R . (1985) Proc . Natl . Acad . Sci . USA 82, 5666-5670) which is post-translationally modified . Thus, the RNP protein overproduced in Escherichia coli and therefore did not contain methylarginine was examined for its methyl acceptability . It was found that neither MBP-specific nor histone-specific protein methylase I could methylate this methylarginine-less RNP protein . This suggests a possible existence of a distinct protein methylase I specific for this nuclear protein.

FEBS Lett, 1990 Jun 18, 266(1-2), 87 - 90
A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties; Boyot P et al.; We previously reported the production of a fused snake neurotoxin composed of protein A and erabutoxin a in E . coli . The hybrid had much lower toxicity and affinity for the acetylcholine nicotinic receptor than natural erabutoxin . By treating the hybrid with cyanogen bromide we generated a toxin which was purified in a single step by RP-HPLC . This compound, produced in a good yield, recovered all properties of native erabutoxin a, implying that the lower toxic activities of the hybrid were due to the bulky protein A and not to an incorrect folding of the toxin . This work serves as a basis for future studies of toxin-receptor interactions using engineered toxin mutants.

FEBS Lett, 1990 Jun 18, 266(1-2), 67 - 71
Expression of catalytically active radish 3-hydroxy-3-methylglutaryl coenzyme A reductase in Escherichia coli; Ferrer A et al.; Two fragments of a cDNA encoding radish 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were cloned into the vector pET-8c and expressed in Escherichia coli . The large fragment, encoding both the membrane and the cytosolic domains, was expressed at low level, essentially as an insoluble protein without enzymatic activity . In contrast, the fragment encoding only the cytosolic domain was expressed at a high level in a catalytically active form . The amount of soluble active enzyme in cell-free extracts of E . coli dramatically increased when the temperature during the induction was lowered from 37 degrees C to 22 degrees C.

FEBS Lett, 1990 Jun 18, 266(1-2), 63 - 6
Purification, some properties and nucleotide sequence of 5-carboxymethyl-2-hydroxymuconate isomerase of Escherichia coli C; Roper DI et al.; As part of an investigation into the evolution of catabolic pathway enzymes a cloned gene encoding the Escherichia coli C 5-carboxymethyl-2-hydroxymuconate (CHM) isomerase, an enzyme of the homoprotocatechuate catabolic pathway, was used to produce large amounts of the protein . The isomerase was purified to homogeneity and some of its properties determined . The reaction occurred optimally at pH 7.6 and the specificity constant was 5.8 x 10(5) M-1.s-1 with CHM and 6.0 x 10(2) M-1.s-1 with 2-hydroxyhepta-2,4-diene-1,7-dioate, the substrate of a second isomerase in the pathway . The pure protein showed one type of subunit of Mr 14,000 whilst the molecular mass of the native enzyme was 30,000, suggesting that it was a dimer of identical subunits . The first 19 N-terminal amino acids were sequenced and the data used to confirm that the open reading frame of 378 bp, identified from the nucleotide sequence, encoded the CHM isomerase.

Biochem J, 1990 Jun 15, 268(3), 547 - 51
Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+; Cieplak W Jr et al.; The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins . Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+ . Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity . In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule . The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants . Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+ . The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity.

Gene, 1990 Jun 15, 90(2), 215 - 20
Microbody phosphoglycerate kinase of Trypanosoma brucei: expression and complementation in Escherichia coli; Alexander K et al.; In the primitive eukaryotic parasite, Trypanosoma brucei, most of the enzymes of glycolysis are located within microbody organelles called glycosomes . Proteins destined for the glycosome are synthesized on free ribosomes and post-translationally translocated into the organelle . The gene, gPGK, encoding the glycosomal isozyme of phosphoglycerate kinase (gPGK), was cloned adjacent to a T7 promoter and cotransformed with a plasmid encoding T7 RNA polymerase into Escherichia coli Pgk-cells . Functional complementation occurred, but only after the creation of a ribosome-binding site by mutagenesis . This represents the first example of complementation of an E . coli mutant with a gene encoding a microbody protein . Enzymatically active recombinant gPGK was purified to near homogeneity by ion exchange chromatography from highly expressing E . coli . The recombinant protein will aid in studies of glycosomal biogenesis.

FEMS Microbiol Lett, 1990 Jun 15, 58(1), 19 - 22
Cloning of the type VII trimethoprim-resistant dihydrofolate reductase gene and identification of a specific DNA probe; Towner KJ et al.; A 1.3 kb HindIII fragment encoding the type VII trimethoprim-resistant dihydrofolate reductase gene was cloned into pBR322 . Unidirectional deletion of this cloned fragment with exonuclease III identified the start of the dihydrofolate reductase gene . An internal 300bp EcoRV fragment was identified which could be used as a specific non-radioactive DNA probe to distinguish bacteria carrying the type VII gene from those carrying genes encoding other known dihydrofolate reductase types.

J Biol Chem, 1990 Jun 15, 265(17), 9850 - 6
The presence and distribution of reduced folates in Escherichia coli dihydrofolate reductase mutants; Hamm-Alvarez SF et al.; Escherichia coli DNA photolyase was overproduced and purified from each of two mutant E . coli strains lacking dihydrofolate reductase . The extent of over-production in the mutants was comparable to that seen in the wild type strain . Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain . Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives . These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E . coli.

J Biol Chem, 1990 Jun 15, 265(17), 9638 - 44
Identification of valine 177 as a mutation altering specificity for transport of sugars by the Escherichia coli lactose carrier . Enhanced specificity for sucrose and maltose; King SC et al.; A mutant of the Escherichia coli lactose carrier has been selected (in an invertase-positive strain) based on its ability to grow on 6 mM sucrose in a manner dependent upon lactose carrier induction by isopropyl-1-thio-beta-D-galactopyranoside . The mutant was cloned, and DNA sequencing revealed a point mutation in lacY which changed alanine 177 to valine . The valine 177 mutation increased the transport rate for both {14C}sucrose and the maltose analog 4-nitrophenyl-alpha-maltoside . The potency for inhibition of beta-ONPG transport by several sugars containing the glucopyranosyl moiety (maltose, cellobiose, or palatinose) was increased significantly relative to the parental carrier . Similar experiments showed that the mutation did not affect the affinity for such commonly studied substrates as 4-nitrophenyl-alpha-D-galactopyranoside and beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside . These data indicate that gross structural alteration of the galactoside binding site cannot account for increased transport of sucrose and maltose by the valine 177 mutant . We conclude that effects of the valine 177 mutation are not limited strictly to changes in observed sugar affinity and that sugar-specific changes in turnover number may be an important determinant of the altered spectrum of sugar specificities exhibited by the Val-177 carrier . These phenomena may be related to the effect of this mutation on proton recognition (described in King, S.C., and Wilson, T.H . (1990) J . Biol . Chem . 265, 9645-9651).

J Biol Chem, 1990 Jun 15, 265(17), 10164 - 71
On RecA protein-mediated homologous alignment of two DNA molecules . Three strands versus four strands; Lindsley JE et al.; The recA protein from Escherichia coli can homologously align two duplex DNA molecules; however, this interaction is much less efficient than the alignment of a single strand and a duplex . Three strand paranemic joints are readily detected . In contrast, duplex-duplex pairing is detected only when the incoming (second) duplex is negatively supercoiled, and even here the pairing is inefficient . The recA protein-promoted four strand exchange reaction is initiated in a three strand region, with efficiency increasing with the length of potential three strand pairing available for initiation . This indicates that a paranemic joint involving three DNA strands may be an important intermediate in all recA protein-mediated DNA strand exchange reactions and that the presence of three strands rather than four is a fundamental structural parameter of paranemic joints.

J Biol Chem, 1990 Jun 15, 265(17), 10055 - 60
Characterization of the integration host factor binding site in the ilvPG1 promoter region of the ilvGMEDA operon of Escherichia coli; Winkelman JW et al.; The ilvGMEDA operon of Escherichia coli, which encodes four of the five enzyme activities required for the biosynthesis of isoleucine and valine, is preceded by tandem promoters ilvPG1 and ilvPG2 which are separated by 72 base pairs . While both of these promoters are transcriptionally active in vitro, only the operon proximal promoter, ilvPG2, is transcriptionally active in vivo, and upstream DNA sequences encoding the ilvPG1 promoter region enhance the in vivo transcriptional activity of the ilvPG2 promoter 60-fold . The binding of the integration host factor protein (IHF) to this upstream region (Tsui, P., and Freundlich, M . (1989) J . Mol . Biol . 203, 817-820) has been shown to repress transcription from the ilvPG1 promoter both in vivo and in vitro (Pereira, R . F., Ortuno, M . J., and Lawther, R . P . (1988) Nucleic Acids Res . 16, 5972-5989) . Furthermore, E . coli strains deficient for IHF are compromised for isoleucine and valine biosynthesis (Friden, P., Voelkel, K., Sternglantz, R., and Freundlich, M . (1984) J . Mol . Biol . 172, 573-579) . Therefore, in order to further understand this repressor/activator role of IHF, we have undertaken a detailed analysis of the interaction of IHF with the DNA sequences in the ilvPG1 promoter region . The results of hydroxyl radical footprinting, dimethyl sulfate protection, and ethylation interference experiments show that IHF binds to a target site that overlaps the ilvPG1 promoter region . The results of these experiments also demonstrate that IHF interacts primarily with the minor groove of the DNA helix and that the IHF target site in the ilvPG1 promoter region shares a high degree of DNA sequence identity with other high affinity IHF target sites involved in DNA replication and site-specific recombination.

Gene, 1990 Jun 15, 90(2), 207 - 14
Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences; Tsuge T et al.; An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter . Transformants arose on a selective medium containing 100 micrograms Hy/ml . There were two types of transformants, forming large and small colonies on the selective medium . Transformation with one microgram of the vector produced an average of 4.5 large colonies and 600 small ones . In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays . To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A . alternata were used to construct four new vectors for homologous recombination system . Use of these vectors gave higher transformation efficiency than the original plasmid . The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25 . Transformation events in A . alternata with pDH25r1a occurred by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.

J Biol Chem, 1990 Jun 15, 265(17), 9676 - 81
Organization and function of a dioxin-responsive enhancer; Fisher JM et al.; The dioxin-responsive enhancer upstream of the CYP1A1 gene contains four copies of the recognition motif for the liganded Ah receptor . The results of deletion analyses, linker-scanning analyses, and the analysis of individual enhancer subdomains reveal that each copy of the motif contributes to the response of the enhancer to 2,3,7,8-tetrachlorodibenzo-p-dioxin . In the context of the dioxin-responsive enhancer, a GC box, representing the DNA binding site for Sp1 (or a related transcription factor), has no detectable intrinsic activity but enhances gene expression when linked to a recognition motif for the liganded Ah receptor, thereby producing a synergistic effect on enhancer function.

J Biol Chem, 1990 Jun 15, 265(17), 9645 - 51
Characterization of Escherichia coli lactose carrier mutants that transport protons without a cosubstrate . Probes for the energy barrier to uncoupled transport; King SC et al.; The Escherichia coli lactose carrier is an energy-transducing H+/galactoside cotransport protein which strictly couples sugar and proton transport in 1:1 stoichiometry . Here we describe five lactose carrier mutants which catalyze "uncoupled" sugar-independent H+ transport . Symptoms similar to uncoupling by a proton ionophore have been observed in cells expressing these mutant carriers . The mutations occur at two separate loci, encoding substitutions either for alanine 177 (valine) or tyrosine 236 (histidine, asparagine, phenylalanine, or serine) . Compared to the parent, cells expressing the valine 177 carrier grew slowly on minimal media with glucose as carbon source . When washed cells were incubated in the absence of added sugars the mutant showed a reduced protonmotive force compared with the parent . Addition of either thiodigalactoside or alpha-p-nitrophenylgalactoside reduced the defect in protonmotive force . Sugar-independent H+ entry rate into cells expressing either the normal carrier or the Val-177 mutant were measured directly using the pH electrode . Following sudden acidification of the external medium (by either oxygen-pulse or acid-pulse) protons entered more rapidly into cells expressing the Val-177 carrier . This novel sugar-independent mode of H+ transport probably depends on an acquired capacity of the Val-177 carrier to bind the transported proton with higher than normal affinity in a transition state involving the binary carrier/H+ complex.

J Biol Chem, 1990 Jun 15, 265(17), 10012 - 8
Functional cloning of a nucleoside diphosphate kinase from Dictyostelium discoideum; Lacombe ML et al.; A lambda gt11 cDNA library from Dictyostelium discoideum was screened by direct labeling of filter replicas with {35S}guanosine 5'-O-(thiotriphosphate) (GTP gamma S) . A positive clone was obtained and used as probe to isolate additional clones from which a complete cDNA sequence was determined . The cDNA hybridizes to a single copy gene that is expressed as a 0.6-kilobase mRNA in vegetatively growing amoeba . The open reading frame encodes a protein of 155 amino acids (calculated Mr 16,775), devoid of cysteine residues . The protein contains most of the short consensus motifs characteristic of the catalytic domain of protein kinases although the overall homology with this class of enzymes is not greater than 20% . Its size and amino acid composition indicated that it could be the monomer of a nucleoside diphosphate (NDP) kinase, an enzyme which catalyzes the phosphate transfer from triphospho- to diphosphonucleotides . Indeed, specific NDP kinase activity was found in extracts of bacteria transformed with a plasmid expressing the protein . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracts incubated in the presence of {35S}GTP gamma S revealed a single 35S-labeled band of size corresponding to the protein, which likely represents the stable thiophosphorylated reaction intermediate characteristic for the ping-pong reaction mechanism of NDP kinases . The formation of this labeled intermediate probably allowed the detection of the enzyme on the filters during the screening procedure . Although NDP kinases from a great variety of sources have been characterized, the primary structure of the D . discoideum NDP kinase is the first reported for an enzyme of eukaryotic origin.

Int J Cancer, 1990 Jun 15, 45(6), 1028 - 32
Immunological response of nasopharyngeal carcinoma patients to the Epstein-Barr-virus-coded thymidine kinase expressed in Escherichia coli; Littler E et al.; A bacterial expression system which produces large amounts of the Epstein-Barr-virus-coded thymidine kinase has been developed and used to produce protein for Western blot analysis of a number of human antisera . Interestingly, only sera from nasopharyngeal carcinoma (NPC) patients had any detectable IgA antibody which reacted with the EBV TK . These findings provided the basis for ELISA tests using a crude lysate of the E . coli cells expressing the EBV TK as target antigen . Sera from NPC patients showed high levels of IgA reactive antibodies in this test while other sera did not.

J Biol Chem, 1990 Jun 15, 265(17), 10156 - 63
Underwinding of DNA associated with duplex-duplex pairing by RecA protein; Conley EC et al.; Homologous pairing between gapped circular and partially homologous form I DNA, catalyzed by Escherichia coli RecA protein, leads to the formation of nascent synaptic joints between regions of duplex DNA . These duplex-duplex interactions result in underwinding of the form I DNA, as detected by a topoisomerase assay . Underwound DNA species have been studied with regard to their formation, stability, and topological requirements . The synaptic joints are short-lived and of low frequency compared with those formed between single-stranded and duplex DNA . Measurement of the degree of underwinding indicates joints 300-400 base pairs in length, in which the two DNA molecules are presumed to be interwound within the RecA-nucleoprotein filament . Underwound DNA was not detected in reactions between gapped DNA and partially homologous nicked circular or relaxed covalently closed DNA . We have also investigated the requirements for the initiation of strand exchange . Previous results have shown that strand exchange requires a homologous 3'-terminus complementary to the gapped region . We now show that the minimum length of overlap required for efficient initiation of strand exchange is one to two turns of DNA within the RecA-DNA nucleoprotein filament.

Gene, 1990 Jun 15, 90(2), 293 - 7
Cloning and expression of a cDNA encoding mouse kidney D-amino acid oxidase; Tada M et al.; A cDNA encoding D-amino acid oxidase (DAO;EC1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme . Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5'-terminal untranslated region of 68 nt, an open reading frame of 1035 nt that encodes 345 amino acids (aa), and a 3'-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence, ATTAAA . The deduced aa sequence showed 77 and 78% aa identity with the porcine and human enzymes, respectively . Two catalytically important aa residues, Tyr228 and His307, of the porcine enzyme, were both conserved in these three species . RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver . Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence of the mouse DAO cDNA downstream from the tac promoter of plasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-beta-D-thiogalactopyranoside . Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme . Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E . coli cells.

J Biol Chem, 1990 Jun 15, 265(17), 9970 - 7
Identification of epitopes on the human insulin receptor reacting with rabbit polyclonal antisera and mouse monoclonal antibodies; Prigent SA et al.; The sequential epitopes on the human insulin receptor recognized by polyclonal and monoclonal antibodies were investigated using a recombinant DNA technique . Short random fragments of receptor cDNA were cloned and expressed in Escherichia coli as beta-galactosidase fusion proteins by using the expression vector pUEX1 . Immunoreactive peptides were detected by colony blotting and identified by sequencing the corresponding cDNA inserts . Eleven antigenic determinants were located with rabbit antisera, nine of these being on the alpha-subunit and two on the beta-subunit of which one was intracellular . Two human autoantibodies reacted with the alpha-subunit on blots, but no sequential epitopes could be located . In the rabbit sera, antibody reacting with these linear epitopes represented a substantial fraction (approximately 50%) of antibody reacting with reduced denatured receptor on blots, but a generally smaller fraction (5-40%) of antibody reacting with solubilized native receptor . Epitope-specific subfractions of antibodies were purified by binding to an elution from bacterial fusion proteins . All of these subfractions reacted with denatured receptor on nitrocellulose blots, but only three precipitated native receptor (epitopes between amino acids 190 and 231, 654 and 669, 954 and 982) and none inhibited insulin binding . (The numbering system used in this manuscript is that of Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf . L., Clauser, E., Ou, J., Masiarz, F., Kan, Y . W., Goldfine, I . D., Roth, R . A., and Rutter, W . J . (1985) Cell 40, 747-758) . The binding sites of two monoclonal antibodies were also determined . One of these antibodies (83-14) is insulin-mimetic, but inhibits insulin binding and its epitope on the alpha-subunit (between amino acids 469 and 592) may contribute to the insulin binding site in the folded protein . The other antibody (18-44) binds close to the N terminus of the beta-subunit (amino acids 765-770) and does not inhibit insulin binding, but does mimic insulin action . The identification of epitopes therefore provides information on receptor conformation and allows structural domains to be identified which are involved in the functional effects of different antibodies.

J Biol Chem, 1990 Jun 15, 265(17), 9960 - 9
Elongation by Escherichia coli RNA polymerase is blocked in vitro by a site-specific DNA binding protein; Pavco PA et al.; As a means of determining how elongating RNA polymerase responds to a protein in its path, transcription has been carried out in vitro with the purified Escherichia coli enzyme on templates associated with a sequence-specific DNA binding protein . The major RNA species generated is the length expected from RNA polymerase which has transcribed to the position of the bound protein and is unable to elongate further . The binding proteins used are two mutants of the EcoRI endonuclease which are defective in cleavage function but retain high affinity for the wild-type recognition sequence (Wright, D . J., King, K., and Modrich, P . (1989) J . Biol . Chem . 264, 11816-11821) . Blockage of RNA polymerase occurs on linear and circular templates and, although efficient with both proteins, is more effective for the EcoRI derivative with the slower dissociation rate . The protein-blocked transcription complexes are stable over time and remain in an active form, resuming elongation when the blocking protein is displaced by an increase in ionic strength . These paused ternary complexes, if treated with the termination factor rho, undergo release . The 3' ends of the blocked-length RNAs from DNAs of distinct sequences reveal that the ternary complexes are positioned at a constant distance from the protein block, 14 nucleotides upstream of the EcoRI recognition sequence . This information is combined with exonuclease III footprinting data to position the 3' end of the nascent RNA chain in the ternary complex quite near (approximately 7 nucleotides) the leading edge of RNA polymerase.

Biochim Biophys Acta, 1990 Jun 14, 1044(3), 368 - 74
Further characterization of the diacylglycerol-phosphatidylethanolamine exchange reaction catalyzed by cell-free extracts of Escherichia coli; Proulx P et al.; The conditions for phosphatidylethanolamine (PE)-diacylglycerol (DAG) exchange catalysed by cell-free extracts of Escherichia coli were studied using 14C- or 3H-analogues of both these lipids . The reaction, examined with either labelled PE or labelled DAG, occurred without co-factor addition and was inhibited by Ca2+ and Mg2+ . Detergents such as Triton X-100 greatly enhanced the activity; however, the optimal concentration of this agent depended on the lipid substrate concentration . The exchange-catalysing enzyme involved in these extracts appeared to be very specific for DAG and PE, since no other labelled phospholipid or acylglycerol derivative formed radioactive product under the assay conditions tested . Again, endogenous {3H}PE present in the enzyme source, but no other endogenous lipid, was converted to labelled DAG in the presence of added 1,2-dioleoyl-sn-glycerol . The Vmax value for the conversion of labelled PE to DAG was very similar to the Vmax value found for the conversion of labelled DAG to PE as would be expected in the case of an exchange reaction being responsible for both conversions . However, the Km value for PE was appreciably larger than that for DAG . The enzyme involved, displayed a broad acyl chain specificity as could be judged from: (1) the ability of various species of DAG and PE to stimulate the exchange; (2) the suitability of lipid substrates prepared from widely different biological sources; and (3) the interchange of acyl groups that occurred between dimyristoyl PE and dilauroylglycerol . As would be expected for an exchange reaction, the incorporation of lauroyl groups into PE occurred without an increase in the total fatty acid content of this phospholipid . The results of the present study confirm and further characterize the PE-DAG exchange reaction of E . coli.

Nature, 1990 Jun 14, 345(6276), 640 - 2
Activation of transcription by HIV-1 Tat protein tethered to nascent RNA through another protein; Southgate C et al.; The human immunodeficiency virus type I (HIV-1) nuclear protein Tat is a potent activator of viral gene transcription . Activation by Tat requires a cis-acting element, the transactivation response (TAR) site, located immediately downstream of the transcription start site . Several observations suggest that TAR functions as the nascent RNA product of the HIV long-terminal-repeat promoter (for a review, see ref . 6) . Indeed, Tat protein and several cellular proteins bind directly to nascent TAR RNA in vitro . The significance of these in vitro interactions remains to be established . Here we report that Tat can activate transcription when bound to nascent RNA through the RNA-binding domain of another HIV-1 protein, Rev . Rev is a sequence-specific RNA-binding protein, which interacts with the viral RNA element RRE (refs 11-15) . A Tat-Rev fusion protein efficiently activates transcription from an HIV-1 promoter derivative, in which TAR has been replaced by the RRE . We conclude that activation of transcription by Tat can occur by direct binding to nascent RNA, and that the sole function of TAR may be to provide a Tat-binding site . Our results further suggest that cellular proteins that bind specifically to TAR RNA or TAR DNA may not be essential for Tat-responsiveness.

Nature, 1990 Jun 14, 345(6276), 593 - 8
Three-dimensional structure of the free radical protein of ribonucleotide reductase; Nordlund P et al.; The enzyme ribonucleotide reductase furnishes precursors for the DNA synthesis of all living cells . One of its constituents, the free radical protein, has an unusual alpha-helical structure . There are two iron centres that are about 25 A apart in the dimeric molecule . Tyrosine 122, which harbours the stable free radical necessary for the activity of ribonucleotide reductase, is buried inside the protein and is located 5 A from the closest iron atom.

Biochemistry, 1990 Jun 12, 29(23), 5562 - 6
Effect of the distal residues on the vibrational modes of the Fe-CO bond in hemoglobin studied by protein engineering; Lin SH et al.; Using an Escherichia coli gene expression system, we have engineered human hemoglobin (Hb) mutants having the distal histidine (E7) and valine (E11) residues replaced by other amino acids . The interaction between the mutated distal residues and bound carbon monoxide has been studied by Soret-excited resonance Raman spectroscopy . The replacement of Val-E11 by Ala, Leu, Ile, and Met has no effect on the v(C-O), v(Fe-CO) stretching or delta(Fe-C-O) bending frequencies in both the alpha and beta subunits of Hb, although some of these mutations affect the CO affinity as much as 40-fold . The strain imposed on the protein by the binding of CO is not localized in the Fe-CO bond and is probably distributed among many bonds in the globin . The replacement of His-E7 by Val or Gly brings the stretching frequencies v(Fe-CO) and v(C-O) close to those of free heme complexes . In contrast, the substitution of His-E7 by Gln, which is flexible and polar, produces no effects on the resonance Raman spectrum of either alpha- or beta-globin . The replacement of His-E7 of beta-globin by Phe shows the same effect as replacement by Gly or Val . Therefore, the steric bulk of the distal residues is not the primary determinant of the Fe-CO ligand vibrational frequencies . The ability of both histidine and glutamine to alter the v(C-O), v(Fe-CO), or delta(Fe-C-O) frequencies may be attributed to the polar nature of their side chains which can interact with bound CO in a similar manner.

Biochemistry, 1990 Jun 12, 29(23), 5469 - 76
Pre-steady-state kinetics of Escherichia coli aspartate aminotransferase catalyzed reactions and thermodynamic aspects of its substrate specificity; Kuramitsu S et al.; The four half-transamination reactions {the pyridoxal form of Escherichia coli aspartate aminotransferase (AspAT) with aspartate or glutamate and the pyridoxamine form of the enzyme with oxalacetate or 2-oxoglutarate} were followed in a stopped-flow spectrometer by monitoring the absorbance change at either 333 or 358 nm . The reaction progress curves in all cases gave fits to a monophasic exponential process . Kinetic analyses of these reactions showed that each half-reaction is composed of the following three processes: (1) the rapid binding of an amino acid substrate to the pyridoxal form of the enzyme; (2) the rapid binding of the corresponding keto acid to the pyridoxamine form of the enzyme; (3) the rate-determining interconversion between the two complexes . This mechanism was supported by the findings that the equilibrium constants for half- and overall-transamination reactions and the steady-state kinetic constants (Km and kcat) agreed well with the predicted values on the basis of the above mechanism using pre-steady-state kinetic parameters . The significant primary kinetic isotope effect observed in the reaction with deuterated amino acid suggests that the withdrawal of the alpha-proton of the substrates is rate determining . The pyridoxal form of E . coli AspAT reacted with a variety of amino acids as substrates . The Gibbs free energy difference between the transition state and the unbound state (unbound enzyme plus free substrate), as calculated from the pre-steady-state kinetic parameters, showed a linear relationship with the accessible surface area of amino acid substrate bearing an uncharged side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol Methods, 1990 Jun 12, 130(1), 39 - 48
Imaging infections with antibodies . A quantitative autoradiographic analysis; Morrel EM et al.; Radiolabeled IgG has recently been demonstrated to effectively image infections . A potential but unproven mechanism for this localization is the specific binding of IgG to Fc receptors on the surface of inflammatory cells in infections . In an animal model of soft tissue infection, quantitative autoradiography was used to measure 125I-labeled IgG and albumin in tissues with a spatial resolution sufficient to associate these proteins with cellular morphology . Gamma camera images at 24 h localized the infection with target-to-background ratios of 2.2 +/- 0.5 for IgG and 2.3 +/- 1.0 for albumin (mean +/- SD) . Using quantitative autoradiography at 1 h post-injection, significantly higher concentrations were found in infected thighs of 2-4% of initial plasma concentrations (CPo) as compared to 0.2-0.3% of CPo in noninfected thighs (P less than 0.05); at 24 h post-injection, higher concentrations (7-8% of CPo) were found in infected thighs . Radiolabeled proteins were not inflammatory cell associated and were localized primarily within the edematous interstitial spaces of the infection.

Biochemistry, 1990 Jun 12, 29(23), 5458 - 63
A hybrid adenosinetriphosphatase composed of F1 of Escherichia coli and F0 of Propionigenium modestum is a functional sodium ion pump; Laubinger W et al.; Analyses on immunoblots indicated strong binding of the alpha- and beta-subunits of the ATPase of Propionigenium modestum to antibodies raised against the corresponding subunits of the F1F0 ATPase of Escherichia coli . Cross-reactivities of antibodies against the other ATPase subunits were not observed . The use of Na+ or H+ as alternate coupling ions, observed previously for the P . modestum ATPase {Laubinger, W., & Dimroth, P . (1989) Biochemistry 28, 7194-7198}, is not found for the F1F0 ATPase of E . coli, which is specific for protons . However, a hybrid consisting of the F1 moiety of the E . coli ATPase and F0 of that from P . modestum performed Na+ or H+ transport in a reconstituted system . As with the homologous ATPase of P . modestum, H+ pumping of the hybrid was abolished at Na+ concentrations of greater than 1 mM . The F0 sector and not F1, therefore, determines the cation specificity of these F1F0 ATPases.

Biochemistry, 1990 Jun 12, 29(23), 5500 - 8
Mutagenic, electrochemical, and crystallographic investigation of the cytochrome b5 oxidation-reduction equilibrium: involvement of asparagine-57, serine-64, and heme propionate-7; Funk WD et al.; A gene coding for lipase-solubilized bovine liver microsomal cytochrome b5 has been synthesized, expressed in Escherichia coli, and mutated at functionally critical residues . Characterization of the recombinant protein revealed that it has a reduction potential that is approximately 17 mV lower than that of authentic wild-type protein at pH 7 (25 degrees C) . Structural studies determined that the recombinant protein differed in sequence from authentic wild-type cytochrome b5 owing to three errors in amidation status in the published sequence for the protein on which the gene synthesis was based . The structural origin of the lower reduction potential exhibited by the triple mutant has been investigated through X-ray crystallographic determination of the three-dimensional structure of this protein and is attributed to the presence of Asp-57 within 3.3 A of heme vinyl-4 in the mutant . In addition, the model developed by Argos and Mathews {Argos, P., & Mathews, F.S . (1975) J . Biol . Chem . 250, 747} for the change in cytochrome b5 oxidation state has been studied through mutation of Ser-64 to Ala . In this model, Ser-64 is postulated to stabilize the oxidized protein through H-bonding interactions with heme propionate-7 that orients this propionate group 6.2 A from the heme iron . Spectroelectrochemical studies of a mutant in which Ser-64 has been changed to an alanyl residue demonstrate that this protein has a reduction potential that is 7 mV lower than that of the wild-type protein; moreover, conversion of the heme propionate groups to the corresponding methyl esters increases the potential by 67 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1990 Jun 12, 1053(1), 37 - 42
The influence of net surface charge on the interaction of uropathogenic Escherichia coli with human neutrophils; Steadman R et al.; Escherichia coli strains, grown to suppress fimbrial expression, synthesised enhanced quantities of polysaccharide capsule, which significantly lessened their binding to heparin sepharose columns . In the presence of poly-L-lysine, these strains were strongly retained on the columns confirming their highly anionic nature . Uropathogenic strains of E . coli expressing type 1 fimbrial adhesins activated the respiratory burst, the degranulation response and the release of leukotrienes from human neutrophils (PMN) to a significantly greater extent than the same strains grown in a medium to suppress this fimbrial expression . The addition of the poly-cation poly-L-lysine, however, selectively increased neutrophil activation in response to these non-fimbriate strains . This dose-dependent effect was reversed by the addition of heparin suggesting a mechanism dependent on surface charge . The results of this study suggest that non-specific mechanisms involving the neutralisation of surface charge, in addition to specific receptor and adhesin mediated events could affect neutrophil activation at sites of infection.

J Immunol Methods, 1990 Jun 12, 130(1), 141 - 7
Discrimination by rabbit anti-idiotypic antibodies of two murine IgM monoclonal antibodies directed against lipid A; Cornelissen JJ et al.; Two murine IgM monoclonal antibodies (MAs) directed against the lipid A portion of bacterial lipopolysaccharide (LPS) were compared in their binding to Re LPS and lipid A and their idiotypic make-up with rabbit anti-idiotypic sera . Horseradish peroxidase (HRPO)-labelled MAs 8-2 and 26-20 bound equally well to Re LPS . The binding of HRPO-labelled MA 8-2 to synthetic lipid A was low compared to the relatively strong binding of labelled 26-20 . The MAs proved to be competitive in a competition binding assay (CBA) with Re LPS as coating antigen . Rabbit immune sera were raised against individual MAs . Anti-idiotypic antibodies (anti-id Abs) were detected with two sensitive enzyme immunoassays (EIA): a solid-phase EIA and an inhibition EIA . The rabbit antisera proved to be idiotype specific, indicating that both MAs recognize separate epitopes . We expect that anti-id Abs will prove to be of value for the differentiation of panels of LPS specific MAs.

J Bacteriol, 1990 Jun, 172(6), 3208 - 13
Role of the purine repressor in the regulation of pyrimidine gene expression in Escherichia coli K-12; Wilson HR et al.; The pyrC and pyrD genes of Escherichia coli K-12 encode the pyrimidine biosynthetic enzymes dihydroorotase and dihydroorotate dehydrogenase, respectively . A highly conserved sequence in the promoter regions of these two genes is similar to the pur operator, which is the binding site for the purine repressor (PurR) . In this study, we examined the role of PurR in the regulation of pyrC and pyrD expression . Our results show that pyrC and pyrD expression was repressed approximately twofold in cells grown in the presence of adenine {corrected} through a mechanism requiring PurR . A mutation, designated pyrCp926, which alters a 6-base-pair region within the conserved sequence in the pyrC promoter eliminated PurR-mediated repression of pyrC expression . This result indicates that PurR binds to the pyrC (and presumably to the pyrD) conserved sequence and inhibits transcriptional initiation . We also demonstrated that the pyrCp926 mutation had no effect on pyrimidine-mediated regulation of pyrC expression, indicating that pyrimidine and purine effectors act through independent mechanisms to control the expression of the pyrC and pyrD genes.

Nucleic Acids Res, 1990 Jun 11, 18(11), 3363 - 9
Supercoil-induced unusual DNA structures as transcriptional block; Bagga R et al.; The transcriptional activity of pBR322 form V DNA template, a topologically unlinked, highly supercoiled molecule having unusual structures around or within coding regions was studied . Significant transcription was observed in vitro from this template despite high levels of supercoiling . An attenuated transcript, initiated accurately from the P4 promoter of rep gene, was observed which indicated pausing of E . coli RNA polymerase within the gene . This pausing could be removed by relieving the torsional stress implying that a supercoil induced structural alteration within the gene was acting as a transcriptional block . A stabilized unusual structure, most likely a cruciform, was found to be responsible for the elongation block . Absence of initiation from the tetR gene was correlated with the unusual structure present within its promoter region in form V DNA . These in vitro studies show that structural alterations within natural DNA could act as transcriptional blocks both at the level of initiation and elongation.

Nucleic Acids Res, 1990 Jun 11, 18(11), 3287 - 91
Methylase-limited partial NotI cleavage for physical mapping of genomic DNA; Hanish J et al.; Partial cleavage of DNA with the restriction endonuclease NotI (5'...GC/GGCCGC...3') is an important technique for genomic mapping . However, partial genomic cleavage with this enzyme is impaired by the agarose matrix in which the DNA must be suspended . To solve this problem we have purified the blocking methylase M . BspRI (5'...GGmCC...3') for competition digests with NotI . The resulting methylase-limited partial DNA cleavage is shown to be superior to standard techniques on bacterial genomic DNA . Abbreviations: bp, base-pair; kb, one thousand base-pairs; Mb, one million base-pairs; Tris, Tris(hydroxy-methyl)aminomethane; EDTA, (ethylenedinitrilo)tetraacetic; beta-ME, beta-mercaptoethanol; PMSF, phenyl methyl-sulfonyl fluoride; PEG, polyethyleneglycol (MW = 8000); 3H, tritium; SAM, S-adenosylmethionine; KGB, potassium glutamate buffer; DTT, dithiothreitol; IPTG, isopropyl-beta-D-thiogalactopyranoside; BSA, bovine serum albumin.

Nucleic Acids Res, 1990 Jun 11, 18(11), 3293 - 8
Characterization of the biochemical properties of recombinant ribonuclease III; March PE et al.; An Escherichia coli double strand specific endoribonuclease, RNase III, was cloned, expressed in large amounts, and purified to homogeneity . Enzyme activity was monitored by assaying fractions for the ability to correctly process exogenous RNA containing specific RNase III cleavage sites . DEAE-Sepharose ion exchange chromatography in the presence of a linear KCl gradient (from 0.02 M to 0.75 M) demonstrated that RNase III exists as two distinct forms . One form elutes at a KCl concentration of 0.13 M and the other elutes at 0.33 M . The presence of stoichiometric amounts of the GTP-binding protein Era during purification results in the conversion of the low salt form into the high salt form . Size exclusion chromatography demonstrated that both forms exist as a dimer in solution . In order to investigate the nature of the dimer, protein cross-linking was performed and cross-linked products were detected by silver staining . The protein-protein dimer can be visualized at protein:cross-linker molar ratios as low as 1:15 within 1 minute of exposure to cross-linker in 0.1 M KCl . Upon addition of substrate RNA to the cross-linking reaction a second form of the protein-protein dimer (with a slightly smaller apparent molecular weight) becomes prominent . Induction of the new form is absolutely dependent upon the addition of substrate mRNA to the reaction mixture . We postulate that the RNase III dimer undergoes a dramatic conformational change upon recognition of RNA which we are able to trap by cross-linking.

Biochemistry, 1990 Jun 5, 29(22), 5374 - 9
Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes; Buck MA et al.; In previous work we showed that on photolysis of Escherichia coli ribosomes in the presence of {3H}tetracycline (TC) the major protein labeled is S7, and we presented strong evidence that such labeling takes place from a high-affinity site related to the inhibitory action of TC {Goldman, R . A., Hasan, T., Hall, C . C., Strycharz, W . A., & Cooperman, B . S . (1983) Biochemistry 22, 359-368} . In this work we use single protein omission reconstitution (SPORE) experiments to identify those proteins that are important for high-affinity TC binding to the 30S subunit, as measured by both cosedimentation and filter binding assays . With respect to both sedimentation coefficients and relative Phe-tRNAPhe binding, the properties of the SPORE particles we obtain parallel very closely those measured earlier {Nomura, M., Mizushima, S., Ozaki, M., Traub, P., & Lowry, C . V . (1969) Cold Spring Harbor Symp . Quant . Biol . 34, 49-61}, with the exception of the SPORE particle lacking S13 . A total of five proteins, S3, S7, S8, S14, and S19, are shown to be important for TC binding, with the largest effects seen on omission of proteins S7 and S14 . Determination of the protein compositions of the corresponding SPORE particles demonstrates that the observed effects are, for the most part, directly attributable to the omission of the given protein rather than reflecting an indirect effect of omitting one protein on the uptake of another . A large body of evidence supports the notion that four of these proteins, S3, S7, S14, and S19, are included, along with 16S rRNA bases 920-1396, in one of the major domains of the 30S subunit.

Biochemistry, 1990 Jun 5, 29(22), 5339 - 43
Structure of the ATP synthase complex (ECF1F0) of Escherichia coli from cryoelectron microscopy; Lucken U et al.; The structural relationship of the catalytic portion (ECF1) of the Escherichia coli F1F0 ATP synthase (ECF1F0) to the intact, membrane-bound complex has been determined by cryoelectron microscopy and image analysis of single, unordered particles . ECF1F0, reconstituted into membrane structures, has been preserved and examined in its native state in a layer of amorphous ice . Side views of the ECF1F0 show the same elongated bilobed and trilobed projection of the ECF1 views shown previously to be normal to the hexagonal projection . The elongated aqueous cavity of the ECF1 is perpendicular to the membrane bilayer profile in the bilobed view . ECF1 is separated from the membrane-embedded F0 by a narrow stalk approximately 40 A long and approximately 25-30 A thick . The F0 part extends from the lipid bilayer by approximately 10 A on the side facing the ECF1 . There is no clear extension of the protein on the opposite side of the membrane.

J Mol Biol, 1990 Jun 5, 213(3), 465 - 75
Translated translational operator in Escherichia coli . Auto-regulation in the infC-rpmI-rplT operon; Lesage P et al.; The genes coding for translation initiation factor IF3 (infC) and for the ribosomal proteins L35 (rpmI) and L20 (rplT) are transcribed in that order from a promoter in front of infC . The last two cistrons of the operon (rpmI and rplT) can be transcribed from a weak secondary promoter situated within the first cistron (infC) . Previous experiments have shown that the expression of infC, the first cistron of the operon, is negatively autoregulated at the translational level and that the abnormal AUU initiation codon of infC is responsible for the control . We show that the expression of the last cistron (rplT) is also autoregulated at the posttranscriptional level . The L20 concentration regulates the level of rplT expression by acting in trans at a site located within the first cistron (infC) and thus different from that at which IF3 is known to act . This regulatory site, several hundred nucleotides upstream from the target gene (rplT), was identified through deletions, insertions and a point mutation . Thus, the expression of the operon is controlled in trans by the products of two different cistrons acting at two different sites . The localization within an open reading frame (infC) of a regulatory site acting in cis on the translation of a downstream gene (rplT) is new and was unforeseen since ribosomes translating through the regulatory site might be expected to impair either the binding of L20 or the mRNA secondary structure change caused by the binding . The possible competition between translation of the regions acting in cis and the regulation of the expression of the target gene is discussed.

J Biol Chem, 1990 Jun 5, 265(16), 9570 - 4
Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli; Reilly TM et al.; A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns . Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells . The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert . The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1 . A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator . Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C . Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1 . These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.

J Biol Chem, 1990 Jun 5, 265(16), 9563 - 9
cDNA cloning and chromosomal assignment of the human O6-methylguanine-DNA methyltransferase . cDNA expression in Escherichia coli and gene expression in human cells; Rydberg B et al.; The O6-methylguanine-DNA methyltransferases are the most common form of cellular defense against the biological effects of O6-methylguanine in DNA . By screening a cDNA library with oligonucleotide probes derived from the active site amino acid sequence of the bovine methyltransferase, we have isolated a cDNA clone for the human enzyme . The cDNA contains a single open reading frame encoding a protein of Mr 21,700 which exhibits considerable homology to three bacterial methyltransferases . When provided with an Escherichia coli lac promoter, the encoded polypeptide can be expressed in E . coli to produce an active methyltransferase which is indistinguishable in size from the protein from human cells . The enzyme expressed in this way is functional in vivo and protects an E . coli methyltransferase deletion mutant against the mutational and cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine . The methyltransferase gene spans at least 15 kilobases and is located on human chromosome 10 . Alkylating agent-resistant Mex+ cells which express the methyltransferase protein contain a methyltransferase mRNA of about 1 kilobase . However, this mRNA is absent from three alkylation sensitive Mex- human cell lines indicating that the regulation of methyltransferase gene expression in these cell lines may be transcriptional.

J Biol Chem, 1990 Jun 5, 265(16), 9114 - 20
Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity; Lundstrom J et al.; We have demonstrated that calf liver protein disulfide-isomerase (Mr 57,000) is a substrate for calf thymus thioredoxin reductase and catalyzes NADPH-dependent insulin disulfide reduction . This reaction can be used as a simple assay for protein disulfide-isomerase during purification in place of the classical method of reactivation of incorrectly oxidized ribonuclease A . Protein disulfide-isomerase contains two redox-active disulfides/molecule which were reduced by NADPH and calf thioredoxin reductase (Km approximately 35 microM) . The isomerase was a poor substrate for NADPH and Escherichia coli thioredoxin reductase, but the addition of E . coli thioredoxin resulted in rapid reduction of two disulfides/molecule . Tryptophan fluorescence spectra were shown to monitor the redox state of protein disulfide-isomerase . Fluorescence measurements demonstrated that thioredoxin--(SH)2 reduced the disulfides of the isomerase and allowed the kinetics of the reaction to be followed; the reaction was also catalyzed by calf thioredoxin reductase . Equilibrium measurements showed that the apparent redox potential of the active site disulfide/dithiols of the thioredoxin domains of protein disulfide-isomerase was about 30 mV higher than the disulfide/dithiol of E . coli thioredoxin . Consistent with this, experiments using dithiothreitol or NADPH and thioredoxin reductase-dependent reduction and precipitation of insulin demonstrated differences between protein disulfide-isomerase and thioredoxin, thioredoxin being a better disulfide reductase but less efficient isomerase . Protein disulfide-isomerase is thus a high molecular weight member of the thioredoxin system, able to interact with both mammalian NADPH-thioredoxin reductase and reduced thioredoxin . This may be important for nascent protein disulfide formation and other thiol-dependent redox reactions in cells.

J Biol Chem, 1990 Jun 5, 265(16), 9043 - 54
Assembly and disassembly of RecA protein filaments occur at opposite filament ends . Relationship to DNA strand exchange; Lindsley JE et al.; RecA protein primarily associates with and dissociates from opposite ends of nucleoprotein filaments formed on linear duplex DNA . RecA nucleoprotein filaments that are hydrolyzing ATP therefore engage in a dynamic process under some conditions that has some of the properties of treadmilling . We have also investigated whether the net polymerization of recA protein at one end of the filament and/or a net depolymerization at the other end drives unidirectional strand exchange . There is no demonstrable correlation between recA protein association/dissociation and the strand exchange reaction . RecA protein-mediated DNA strand exchange is affected minimally by changes in reaction conditions (dilution, pH shift, or addition of small amounts of adenosine-5'-O-(3-thiotriphosphate) that have large and demonstrable effects on recA protein association, dissociation, or both . Rather than driving strand exchange, these assembly and disassembly processes may simply represent the mechanism by which recA nucleoprotein filaments are recycled in the cell.

J Biol Chem, 1990 Jun 5, 265(16), 9280 - 4
DNA binding activity and transcriptional activator function of the human B-myb protein compared with c-MYB; Mizuguchi G et al.; Three members of the myb gene family have been identified in human cDNA libraries c-myb, A-myb, and B-myb . We compared the DNA binding properties of the B-myb and c-myb proteins (B-MYB and c-MYB) using bacterially synthesized B-MYB and c-MYB in DNase I footprinting . B-MYB bound to most of the c-MYB binding sites examined, including the c-MYB binding site, MBS-I, in the simian virus (SV) 40 enhancer, in which the most frequent sequence was CCTAACTG . The MBS-I site was an enhancer element dependent on B-MYB and c-MYB in a co-transfection assay that used the B-myb or c-myb expression plasmid . Some sites in the SV40 genome, including the MBS-BI site, had high affinity with B-MYB but little or no affinity with c-MYB, in which the most frequent sequence was AGAAANPyrG . The MBS-BI site was an enhancer element dependent on B-MYB and a very weakly dependent on c-MYB . Our results showed that B-MYB is a transcriptional activator, like c-MYB, and that although B-MYB and c-MYB have similar sequence specificity for DNA binding some sequences were recognized by B-MYB preferentially.

J Biol Chem, 1990 Jun 5, 265(16), 9247 - 50
Steady-state measurements of Escherichia coli sodium and proton potentials at alkaline pH support the hypothesis of electrogenic antiport; Pan JW et al.; 31P and 23Na NMR spectroscopy was applied to the measurement of proton and sodium potentials in endogenously respiring Escherichia coli cells over an external pH (pHex) range from mildly acidic (6.4) to fairly alkaline (8.4) . Stable maintenance of alkaline pHex in the face of metabolic acidification was achieved by use of a perfusion system . In the acidic to neutral pHex range, the sodium chemical potential followed the proton chemical potential quite closely, although always exceeding it slightly, as has been reported previously (Castle, A . M., Macnab, R . M., and Shulman, R . G . (1986) J . Biol . Chem . 261, 7797-7806) . Above pHex 7.4, the sodium potential changed abruptly from a decreasing to an increasing function of pHex, whereas the proton potential continued to decrease . As a consequence, the apparent stoichiometry (i.e . the ratio between the sodium and proton electrochemical potentials) took on progressively higher values, increasing from approximately 1.1 at pH 7.4 to approximately 1.3 at pH 7.8 . Thereafter, the sodium chemical potential started to decrease again; however, since the decrease was less steep than that of the proton potential, the apparent stoichiometry continued to increase . At the highest pHex examined (8.4), it had reached a value of approximately 1.4 . These results strongly support the hypothesis of an electroneutral (1:1) H+/Na+ antiporter operating virtually alone under acidic to neutral conditions and then being supplemented to an ever increasing degree by an electrogenic (for example, 2:1) antiporter under more alkaline conditions.

J Biol Chem, 1990 Jun 5, 265(16), 9098 - 104
Spatial constraints on polyadenylation signal function; Heath CV et al.; Efficient cleavage and polyadenylation of eukaryotic messenger RNAs require at least two signal elements: an AAUAAA or closely related sequence located 7-30 base pairs (bp) upstream of the site of processing, and a G/U- or U-rich sequence located 3' to the cleavage site . The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT-rich region . We have shown that the first AATAAA and the GT-rich region are essential for efficient processing, both in vivo and in vitro, whereas the second AATAAA does not appear to play any role in the formation of tk mRNA 3' ends . The failure of a signal containing only the second AATAAA and the GT-rich element to signal cleavage and polyadenylation suggested that these two elements might be too close together to constitute a functional polyadenylation signal . The experiments described in this report were directed at determining the effects on mRNA 3' end formation of alterations in spacing between signal elements . Wild-type tk contains 19 bp between these two elements . Constructs were made in which an AATAAA and the GT-rich region were separated by various distances ranging from 7 to 43 bp . The quantity and location of 3' ends of the tk mRNA produced by these constructs in Cos-1 cells were measured by S1 nuclease protection analysis . Signal efficiency was gradually reduced as the separation between the two signal elements was increased; with a separation of 43 bp, the signal functioned at approximately one-eighth the efficiency of the parental construction . Bringing the two signals closer together resulted in decreased signal efficiency; with a separation of 7 or 9 bp, no tk mRNA polyadenylated within the normal region was produced . Altering the sequences between these two elements without changing the distance had small effects on processing efficiency.

J Biol Chem, 1990 Jun 5, 265(16), 9346 - 50
Purification and biochemical characterization of SELB, a translation factor involved in selenoprotein synthesis; Forchhammer K et al.; The product of the selB gene from Escherichia coli is required for co-translational insertion of selenocysteine into protein . To make the SELB protein accessible to biochemical analysis, the protein was purified from cells that overexpressed the selB gene from a phage T7 promoter plasmid . It was calculated that the overproduced SELB protein was purified 20-fold . The N-terminal amino acid sequence of the purified protein was determined, and it confirmed that the initiation codon of selB mRNA translation overlaps the stop codon of the preceding selA gene by 4 bases . Structural similarity between SELB and elongation factors was demonstrated by limited proteolysis of SELB by trypsin . The cleavage sites within SELB were identified by N-terminal sequencing of the two proteolytic products . The position in the SELB protein of the major cleavage site was homologous to a tryptic cleavage site which is characteristic for elongation factors . Immunological analysis showed that the levels of SELB are equivalent in aerobically and anaerobically grown cells; the amount of the protein was estimated to be approximately 1100 copies/E . coli cell . Upon fractionation of cell extracts, SELB was found to be partially associated with the ribosomes . The results therefore indicate that SELB is the first known elongation factor-like protein that has specificity for a particular charged tRNA.

J Biol Chem, 1990 Jun 5, 265(16), 9011 - 4
Cloning of a cDNA encoding adenylosuccinate lyase by functional complementation in Escherichia coli; Aimi J et al.; Adenylosuccinate lyase was cloned by functional complementation of an Escherichia coli purB mutant using an avian liver cDNA expression library . The derived amino acid sequence is homologous to the bacterial purB-encoded adenylosuccinate lyase which catalyzes the same two steps in purine biosynthesis as the enzyme from animals . Avian adenylosuccinate lyase also shows regions of extensive sequence similarity to the urea cycle enzyme, argininosuccinate lyase . This homology suggests a similar mechanism for catalysis . Homology of adenylosuccinate and argininosuccinate lyases is intriguing because chickens do not utilize the urea cycle in nitrogen excretion . This is the first report of the cloning of a eukaryotic cDNA encoding adenylosuccinate lyase, and it affords a route to isolate the corresponding human gene which has been suggested to be defective in autistic children.

J Mol Biol, 1990 Jun 5, 213(3), 395 - 8
Homeodomain binding sites in the 5' flanking region of the Bombyx mori silk fibroin light-chain gene; Hui CC et al.; Multiple A + T-rich stretches in the 5' flanking region of the Bombyx mori fibroin light-chain gene have been shown to bind two Drosophila homeodomain proteins, EVE (even-skipped) and ZEN (zerknullt), with high affinities . Some of these sites fall into a class that has the established consensus sequence of the binding sites (TCAATTAAAT) for a diverse group of Drosophila homeodomain proteins, while others are quite heterogenous except that they all possess a core TAAT motif . Since clusters of homeodomain binding sites can also be found in the promoters of other silk protein genes, the fibroin gene and the sericin-1 gene, these observations suggest a possible involvement of some homeobox genes in the regulation of a group of silk protein genes.

Biochemistry, 1990 Jun 5, 29(22), 5344 - 51
Structure and function of an OmpC deletion mutant porin from Escherichia coli K-12; Rocque WJ et al.; Escherichia coli K-12 strain RAM122 contains a mutation in the ompC gene that results in an eight amino acid deletion, delta 103-110, in the porin protein . Since this strain is capable of growing on maltodextrins in the absence of a functional lamB gene, the mutant protein is thought to have a larger channel size . The stability and structure/function properties of the mutant OmpC porin were investigated and compared to wild-type porin . Isolated unheated RAM122 porin was characterized as a trimer on sodium dodecyl sulfate-polyacrylamide gels . The RAM122 trimer was less stable to temperature when compared to the wild-type porin . In addition, the overall enthalpy for thermal denaturation was lower for the mutant than the wild-type porin as determined by using differential scanning microcalorimetry . Both the proteins' secondary structures, monitored by circular dichroism, were high in beta-sheet content, but the spectra were slightly different in their crossover points as well as their minima . When the proteins were reconstituted and channel activity was assayed by using a liposome swelling technique, the size-exclusion limit of the mutant porin was twice that of the wild-type porin . Conductance measurements across bilayer lipid membranes showed that the mutant porin was voltage gated at much lower membrane potentials, 50 and 75 mV, than the wild-type sample . The closing events of the mutant porin were predominantly of monomer size . The channels detected by using the mutant protein were larger in size than those measured for the wild-type porin monomer . These data suggest that the OmpC mutant porin has a channel size capable of allowing maltodextrins to enter and that this channel is highly voltage regulated.

FEBS Lett, 1990 Jun 4, 265(1-2), 59 - 62
Functional expression of the mutants of the chloroplast tRNA(Lys) gene from the liverwort, Marchantia polymorpha, in Escherichia coli; Nakahigashi K et al.; The anticodon of the tRNA(Lys) gene (trnK) in the liverwort, Marchantia polymorpha, was artificially converted to an amber anticodon . This mutant tRNA(Lys) (CTA) gene carrying either the intron of the C27-C43 mismatch at the anticodon-stem is not functional in Escherichia coli, but without both of them, it does work as a tRNA(Lys) amber suppressor.

FEBS Lett, 1990 Jun 4, 265(1-2), 17 - 9
Orientation of the carboxyl terminus of the transposon Tn10-encoded tetracycline resistance protein in Escherichia coli; Yamaguchi A et al.; A site-directed antibody was generated against a synthetic polypeptide corresponding to the 14 amino acid residues of the carboxyl terminus of the Tn10 TetA protein . The antibody reacted preferentially with inside-out vesicles, rather than right-side-out vesicles, prepared from Escherichia coli cells harboring transposon Tn10 . When inside-out vesicles were treated with trypsin, the TetA protein was completely digested in the vicinity of the carboxyl terminus, as judged on immunoblot analysis using the antibody . In contrast, when right-side-out vesicles were treated with trypsin, the TetA protein was hardly digested . These results indicate that the carboxyl terminus of TetA is exposed to the cytoplasmic side of the membrane.

FEBS Lett, 1990 Jun 4, 265(1-2), 71 - 4
Secretion of recombinant ribonuclease T1 into the periplasmic space of Escherichia coli with the aid of the signal peptide of alkaline phosphatase; Fujimura T et al.; The ribonuclease T1 (RNase T1) gene was ligated to a synthetic gene for the signal peptide of Escherichia coli alkaline phosphatase . When this fusion gene was expressed in E . coli under the control of the trp promoter, active RNase T1 having the correct N-terminal sequence was secreted into the periplasmic space, indicating that the heterologous signal peptide had been cleaved off correctly . The enzyme could be readily purified from the periplasmic fraction with a yield of 1.8 mg from 1 liter culture . Adopting the same strategy, it was possible to produce a labile mutant of RNase T1 (Glu-58----Ala mutant) in E . coli, the yield of the purified mutant enzyme being 2.0 mg from 1 liter culture.

FEBS Lett, 1990 Jun 4, 265(1-2), 41 - 5
Carboxylterminal deletion mutants of ribulosebisphosphate carboxylase from Rhodospirillum rubrum; Morell MK et al.; The carboxylterminal octapeptide of ribulosebisphosphate carboxylase from Rhodospirillum rubrum, which lacks small subunits, shows homology to a highly conserved region near the amino terminus of the small subunits of hexadecameric ribulosebisphosphate carboxylases, which are composed of large and small subunits . Truncations of the R . rubrum enzyme, which partially or completely deleted the region of homology, demonstrated that the region is not an important determinant of the catalytic efficiency of the enzyme . A further truncation, which replaced the carboxylterminal 19 amino acid residues with a single terminal leucyl residue, yielded a Rubisco whose substrate-saturated catalytic rate resembled that of the wild-type enzyme but which had weaker affinities for ribulose-P2 and CO2.

Vet Microbiol, 1990 Jun, 23(1-4), 221 - 6
Molecular cloning of the mRNA coding for the G protein of the viral haemorrhagic septicaemia (VHS) of salmonids; Thiry M et al.; Viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus, is a major threat for continental European trout fish farming . The development of a recombinant subunit vaccine could solve that problem . The neutralizing epitopes are located on the glycoprotein or G protein, the surface antigen . The G protein has a molecular weight of 65 kDa, reduced to 55 kDa by deglycosylation . cDNA was synthetized from mRNA of VHS virus infected cells, and cloned in E . coli . The viral cDNA was recognized by positive hybridization with a labelled probe made from infected cell RNA, and negative hybridization with labelled cDNA made from cellular RNA . The Northern blot hybridization with different clones on VHS infected cell RNA revealed two VHS mRNA whose lengths, 2.0 and 1.5 kb, were compatible with the mRNA length for G and N proteins respectively . This mRNA must contain about 400 bp of untranslated sequence.

J Gen Microbiol, 1990 Jun, 136 ( Pt 6), 1099 - 107
Characterization and expression of the cbbE' gene of Coxiella burnetii; Minnick MF et al.; A gene which is unique to the QpRS plasmid from chronic isolates of Coxiella burnetii was cloned, sequenced, and expressed in Escherichia coli . This gene, termed cbbE', codes for a putative surface protein of approximately 55 kDa, termed the E' protein . The cbbE' gene is 1485 bp in length, and is preceded by predicted promoter regulatory sequences of TTTAAT (-35), TATAAT (-10), and a Shine-Dalgarno sequence of GGAGAGA, all of which closely resemble those of E . coli and other rickettsiae . The open reading frame (ORF) of cbbE' ends with a UAA codon followed by a second in-frame UAG stop codon and a region of dyad symmetry which may act as a rho-factor-independent terminator . The ORF of cbbE' is capable of coding for a polypeptide of 495 amino acids with a predicted molecular mass of 55893 Da . The E' protein has a predicted pI of approximately 8.7, and contains a distinct hydrophobic region of 12 amino acid residues . In vitro transcription/translation and E . coli expression of recombinant plasmids containing cbbE' produce a protein of approximately 55 kDa . The in vivo expression of cbbE' yields a novel protein that can be detected on immunoblots developed with rabbit antiserum generated against purified outer membrane from C . burnetii . DNA hybridization analysis shows that cbbE' is unique to the QpRS plasmid found in chronic isolates of C . burnetii, and is absent in chromosomal DNA and plasmids (QpH1, QpDG) from other isolates of C . burnetii . A search of various DNA and amino acid sequence data bases revealed no homologies to cbbE'.

Acta Crystallogr B, 1990 Jun 1, 46 ( Pt 3), 370 - 7
Determination of the crystal structure of recombinant pig myoglobin by molecular replacement and its refinement; Smerdon SJ et al.; As part of a protein engineering study, the X-ray crystal structure of recombinant pig myoglobin, prepared and crystallized from E . coli, has been determined . Diffraction data were collected to 2.5 A spacing using a synchrotron X-ray source . The structure was solved using the molecular-replacement method and refined using least-squares minimization procedures to a crystallographic R factor of 18.5% using 14,481 reflections between 10 and 2.5 A . A preliminary comparison of the structure of pig myoglobin with other myoglobin structures is presented.

J Clin Microbiol, 1990 Jun, 28(6), 1329 - 37
Reactivity of human Lyme borreliosis sera with a 39-kilodalton antigen specific to Borrelia burgdorferi; Simpson WJ et al.; Borrelia burgdorferi is the causative agent of Lyme borreliosis, a spirochetal illness with a variety of acute clinical manifestations that may lead to debilitating neurological and arthritic complications . Diagnosis is difficult because symptoms mimic a variety of unrelated clinical conditions, spirochetes cannot always be isolated from infected patients, and current serological tests are frequently inconclusive because of the presence of cross-reacting non-B . burgdorferi antibodies . To identify antigens specific to B . burgdorferi that could be used in the serodiagnosis of Lyme borreliosis, we screened a Borrelia DNA expression library in Escherichia coli for antigens reactive with human Lyme borreliosis sera . One clone carried a 6.3-kilobase EcoRI chromosomal fragment (pSPR33), which encoded two species-specific antigens with molecular masses of 28 (P28) and 39 (P39) kilodaltons (kDa) . These two antigens were immunologically distinct from OspA, OspB, and the 41-kDa flagellin . Ninety-four serum specimens from patients having Lyme borreliosis were tested for reactivity with P39 . All of 33 the serum specimens with immunofluorescence assay titers of greater than or equal to 1:256, 13 of 17 serum specimens with titers of 1:128, and 14 of 44 serum specimens with titers of less than or equal to 1:64 reacted with P39 . Notably, many sera reactive to P39 did not appear to react with the 41-kDa flagellin . Therefore, antibody to P39 could be mistaken for antibody to the 41-kDa flagellin in tests of human sera by Western blot (immunoblot) . Twenty-five control serum specimens, which included sera from syphilitic, relapsing fever, and amyotrophic lateral sclerosis patients as well as from 10 normal individuals, did not react to P39 . Our data suggest that P39 may be a useful antigen for the serological confirmation of Lyme borreliosis.

J Lipid Res, 1990 Jun, 31(6), 1099 - 107
Lipopolysaccharide and tumor necrosis factor cause a fall in plasma concentration of lecithin: cholesterol acyltransferase in cynomolgus monkeys; Ettinger WH et al.; The effects of intravenous injection of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF) were investigated in cynomolgus monkeys (Macaca fascicularis) . Injection of 20 micrograms/kg of LPS from E . coli (serotype 055:B5) into cynomolgus monkeys fed a monkey chow diet caused a twofold increase in plasma triglyceride and a 25% reduction in plasma cholesterol 48 h after injection . Similar results were found with injection of recombinant human TNF at a dose of 20 micrograms/kg into chow-fed animals . However, injection of the same dose of LPS or TNF into animals fed an atherogenic diet containing saturated fat and cholesterol resulted in a 2.4- to 5-fold increase in plasma triglyceride concentrations and no significant change in plasma cholesterol levels . The fall in plasma cholesterol levels observed in chow-fed animals was associated with a 57% decrease in the cholesteryl ester (CE) content in low density lipoprotein (LDL) and 35% decrease in CE in high density lipoprotein (HDL) in LPS-injected animals, and a decrease of 33% in CE concentration of LDL and 41% in CE of HDL in animals injected with TNF . In animals fed the atherogenic diet containing saturated fat and cholesterol, the injection of both LPS and TNF also resulted in a significant decrease in the CE content of LDL and HDL . However, the plasma total cholesterol levels did not change in the animals fed saturated fat and cholesterol because the decrease in CE content of LDL and HDL was offset by an increase in very low density lipoprotein (VLDL)-CE.(ABSTRACT TRUNCATED AT 250 WORDS)

Vaccine, 1990 Jun, 8(3), 237 - 42
Activation by synthetic lipid A subunit analogues (GLA compounds) of tumoricidal properties in human blood monocytes; Maeda H et al.; The authors have determined that synthetic lipid A subunit analogues (GLA compounds), as well as E . coli type lipopolysaccharide (LPS) and synthetic lipid A (compound 506), are able to stimulate human monocytes to become cytotoxic against tumour target cells in vitro . GLA-60, a synthetic lipid A subunit analogue of low toxicity, was found to be more active for the induction of tumoricidal monocytes than GLA-59, and similar to that of LPS . GLA-60 could induce not only the secretion of cytotoxic factor into the culture supernatant but also expression of the membrane-associated form of cytotoxic factor in human monocytes . Supernatant-mediated cytotoxicity was completely inhibited by the addition of monoclonal anti-human TNF antibody . These results indicate that a synthetic lipid A subunit analogue, GLA-60, would be a useful activator of tumoricidal monocytes in spite of its low toxicity.

Inflammation, 1990 Jun, 14(3), 325 - 35
Ocular inflammatory effects of intravitreally injected tumor necrosis factor-alpha and endotoxin; Fleisher LN et al.; Intravitreal injection of human recombinant tumor necrosis factor-alpha (TNF) induced inflammation in the rabbit eye characterized by dilation of blood vessels in the iris, disruption of the blood-ocular barriers, infiltration of inflammatory cells into the anterior chamber, and accumulation of prostaglandin E in intraocular fluids . Inflammation first appeared on day 1, increased on day 2, and remained elevated on day 7 . The inflammatory cell infiltrate in the anterior segment of the eye was largely monocytic on days 1 and 2; by day 7 large numbers of lymphocytes were also present . TNF-induced ocular inflammation therefore differed from that reported for intravitreally injected endotoxin in terms of time course and the types of inflammatory cells in the aqueous humor . In a series of experiments in which combinations of TNF and endotoxin were used, intravitreal injection of TNF, 24 h after a low dose of Escherichia coli endotoxin, produced no more inflammation than that produced by TNF following an injection of endotoxin vehicle . However, if TNF was injected 24 h before endotoxin, the resulting inflammation was greater than that observed in animals given TNF followed by endotoxin vehicle.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4463 - 7
Isolation and expression of the gene for a major surface protein of Giardia lamblia; Gillin FD et al.; To study the interactions between the parasitic protozoan Giardia lamblia and its environment, we have cloned the gene that encodes the two major surface-labeled trophozoite protein species . Sequence analysis of this gene reveals a single open reading frame specifying a hydrophilic, cysteine-rich (11.8%) protein of 72.5-kDa molecular mass with an amino-terminal signal peptide and a postulated hydrophobic membrane-spanning anchor region near the carboxyl terminus . Most of the cysteine residues (58 of 84) are in the motif Cys-Xaa-Xaa-Cys, which is dispersed 29 times throughout the sequence . Antibodies against the recombinant protein react with the entire surface of live trophozoites, including flagella and adhesive disc . These antibodies inhibit trophozoite attachment, prevent growth, and immunoprecipitate the major approximately 66- and 85-kDa proteins from surface-labeled live trophozoites . The recombinant Escherichia coli also expresses polypeptides of approximately 66- and 85-kDa molecular mass, which are not fusion proteins . This suggests that the processing and/or conformational changes that lead to production of these two peptide species in E . coli reflect those that occur in Giardia . The abundance of cysteine residues suggests that the native proteins on the parasite surface may contain numerous disulfide bonds, which would promote resistance to intestinal fluid proteases and to the detergent activity of bile salts and would help to explain the survival of Giardia in the human small intestine.

Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4169 - 73
Primary structure of a key enzyme in plant tetrapyrrole synthesis: glutamate 1-semialdehyde aminotransferase; Grimm B; The formation of delta-aminolevulinate from glutamate 1-semialdehyde (GSA) is catalyzed by glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8) . The active form of the barley enzyme appears to be a dimer of identical subunits with a molecular mass of 46 kDa . From the purified enzyme, amino acid sequences of the N-terminal ends of the mature protein as well as an internal peptide were determined . DNA primers deduced from these peptide sequences were used to amplify with the polymerase chain reaction a cDNA sequence encoding part of the enzyme . Screening a cDNA library with this DNA fragment identified a full-length clone encoding the 49,540-Da precursor of the GSA aminotransferase . The transit peptide for chloroplast import consists of 34 amino acids . GSA aminotransferase and a precursor form were expressed on a multicopy plasmid in Escherichia coli . Both recombinant gene products reacted with an antibody against the barley GSA aminotransferase . Active barley GSA aminotransferase expressed in E . coli was shown to be active in assays of bacterial cell extracts . As a gene symbol for barley GSA aminotransferase, Gsa is proposed.

Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4103 - 7
Effect of genetic modification of tyrosine-185 on the proton pump and the blue-to-purple transition in bacteriorhodopsin; Jang DJ et al.; The retinylidene chromophore mutant (Y185F) of bacteriorhodopsin, in which Tyr-185 is substituted by phenylalanine, is examined and compared with wild-type bacteriorhodopsin expressed in Escherichia coli; both were reinstituted similarly in vesicles . The Y185F mutant shows (at least) two distinct spectra at neutral pH . Upon light absorption, the blue species (which absorbs in the red) behaves as if "dead"--i.e., neither its tyrosine nor its protonated Schiff base undergoes deprotonation nor does its tryptophan fluorescence undergo quenching . This result is unlike either the purple species (which absorbs in the blue) or wild-type bacteriorhodopsin expressed in E . coli . As the pH increases, both the color changes and the protonated Schiff base deprotonation efficiency suggest a blue-to-purple transition of the Y185F mutant near pH 9 . If this blue-to-purple transition of Y185F corresponds to the blue-to-purple transition of purple-membrane (native) bacteriorhodopsin (occurring at pH 2.6) and of wild-type bacteriorhodopsin expressed in E . coli (occurring at pH 5), the protein-conformation changes of this transition as well as the protonated Schiff base deprotonation may be controlled not by surface pH alone, but rather by the coupling between surface potential and the general protein internal structure around the active site . The results also suggest that Tyr-185 does not deprotonate during the photocycle in purple-membrane bacteriorhodopsin.

J Bacteriol, 1990 Jun, 172(6), 3100 - 7
tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon; Gollnick P et al.; Tryptophanase (tna) operon expression in Escherichia coli is induced by tryptophan . This response is mediated by features of a 319-base-pair leader region preceding the major structural genes of the operon . Translation of the coding region (tnaC) for a 24-amino-acid leader peptide is essential for induction . We have used site-directed mutagenesis to investigate the role of the single Trp codon, at position 12 in tnaC, in regulation of the operon . Codon 12 was changed to either a UAG or UGA stop codon or to a CGG arginine codon . Induction by tryptophan was eliminated by any of these changes . Studies with suppressor tRNAs indicated that tRNA(Trp) translation of codon 12 in tnaC is essential for induction of the operon . Reduction of tna expression by a miaA mutation supports a role for translation by tRNA(Trp) in regulation of the operon . Frameshift mutations and suppression that allows translation of tnaC to proceed beyond the normal stop codon result in constitutive tna operon expression . Deletion of a potential site for Rho factor utilization just beyond tnaC also results in partial constitutive expression . These studies suggest possible models for tryptophan induction of tna operon expression involving tRNA(Trp)-mediated frame shifting or readthrough at the tnaC stop codon.

Mol Cell Biol, 1990 Jun, 10(6), 2448 - 57
Purification and cloning of interferon-stimulated gene factor 2 (ISGF2): ISGF2 (IRF-1) can bind to the promoters of both beta interferon- and interferon-stimulated genes but is not a primary transcriptional activator of either; Pine R et al.; Interferon-stimulated gene factor 2 (ISGF2) was purified from HeLa cells treated with alpha interferon . The factor, a single polypeptide of 56 kilodaltons (kDa), bound both to the central 9 base pairs of the 15-base-pair interferon-stimulated response element (ISRE) that is required for transcriptional activation of interferon-stimulated genes and to the PRD-I regulatory element of the beta interferon gene . ISGF2 was a phosphoprotein, and dephosphorylation in vitro reduced its DNA-binding activity . However, conditions that changed the amount of ISGF2 did not change the phosphorylated isoforms in vivo . ISGF2 in unstimulated cells existed in trace amounts and was induced by both alpha interferon and gamma interferon as well as by virus infection . Plasmid-bearing Escherichia coli clones encoding ISGF2 were selected with antibody against purified ISGF2 . Sequence analysis revealed that the ISGF2 protein was the same as that encoded by the cDNA clone IRF-1, which has been claimed to activate transcription of interferon genes . We show that transcription of the ISGF2 gene was induced by alpha interferon, gamma interferon, and double-stranded RNA . However, ISGF2 was neither necessary nor sufficient for induced transcription of the beta interferon gene, while the factor NF kappa B was clearly involved.

Infect Immun, 1990 Jun, 58(6), 1744 - 8
Expression of antigens from chromosomal and linear plasmid DNA of Borrelia coriaceae; Perng GC et al.; Three recombinant plasmids containing DNA from Borrelia coriaceae, the putative agent of epizootic bovine abortion, expressed antigens in Escherichia coli that reacted with antibodies specific for B . coriaceae . Two of the recombinants each expressed a single high-molecular-weight antigen . The third recombinant expressed three smaller antigens . The DNA inserts were sized and mapped . Hybridization of the cloned inserts to pulsed-field electrophoresis samples of B . coriaceae whole-cell DNA revealed the origin of two of the inserts to be located in linear plasmids . One of these, expressing three antigens, was located in a 210-kilobase linear plasmid . A second recombinant expressed a single antigen but hybridized to at least three distinct linear plasmids . The third clone also expressed a single antigen but was demonstrated to be chromosomal in origin.

Jpn J Genet, 1990 Jun, 65(3), 115 - 9
Identification of the mutations in the prfB gene of Escherichia coli K12, which confer UGA suppressor activity; Wu ED et al.; By DNA sequencing and gene dissection, it has been revealed that Su+UGA#11, the mutant prfB of E . coli (Chang et al., 1990) has a double mutation compared with the wild-type LS653: one is a base substitution from T to C at the codon 63 and the other is from G to A at the codon 79 . Both mutations cause amino acid substitution, Leu63----Phe63 (L63F) and Asp79----Gly79 (D79G), and are necessary to confer the efficient UGA suppressor activity.

Mol Gen Genet, 1990 Jun, 222(1), 77 - 80
Par site of the ColN plasmid: structural and functional organization; Kolot MN; A par site involved in the resolution of multimeric plasmid DNA forms was localized in a 679 bp SalI-KpnI fragment of the small colicinogenic plasmid ColN . It was shown that replication of the monomeric pUC19 recombinant plasmid carrying the par region of ColN does not result in the formation of significant numbers of multimers . In order to function properly, the ColN multimer resolution mechanism requires the product of the xerA gene, just as in the case of ColE1 . Nucleotide sequence analysis of the par region of ColN revealed substantial homology with the par locus of the ColE1 plasmid . The results of this study and data from the literature indicate that the par sites of ColE1-type plasmids have substantial homology and the same mechanism of action, and in fact represent a universal stability module for small multicopy colicinogenic plasmids.

Mol Gen Genet, 1990 Jun, 222(1), 33 - 6
Cellular localization of the MalG protein from the maltose transport system in Escherichia coli K12; Dassa E; In Escherichia coli five proteins are directly involved in the high affinity transport system for maltose and maltodextrins . Sequence data and genetic evidence indicate that three of them, MalF, MalG and MalK, are associated with the inner membrane . In order to characterize the MalG protein more thoroughly and to determine its subcellular localization under physiological conditions, we generated well-defined mutations induced in vitro that affect the size and the function of the malG gene product . Wild-type and mutant proteins were detected in total bacterial extracts and on purified subcellular fractions with the help of an antibody prepared against a synthetic peptide based on the predicted N-terminal sequence of MalG . The protein was solubilized from crude membranes by the detergent Triton X-100, indicating that it was localized in the inner membrane . A mutant carrying an in-phase insertion of four amino acids into a region conserved in several inner membrane proteins from periplasmic transport systems displayed a maltose-negative phenotype . This suggested that this region is very probably essential for MalG function.

Mol Gen Genet, 1990 Jun, 222(1), 166 - 8
T-T cyclobutane dimers are misinstructive, rather than non-instructive, mutagenic lesions; Lawrence CW et al.; The lesions produced by SOS-dependent mutagens in Escherichia coli are commonly referred to as nonpairing or non-instructive . Although these terms are likely to be appropriate for some lesions, particularly the abasic site, for others, such as the cyclobutane dimer, their suitability is open to question . To address this question, we have compared the error frequencies and spectra that result when a uniquely located T-T sequence, carried in a single-stranded vector, contains either a cis-syn or a trans-syn cyclobutane dimer, or when either the 5'T or 3'T is converted to an abasic site . The data suggest that the high accuracy with which the dimer-containing templates are replicated is unlikely to be the consequence of polymerase preference for the non-instructive insertion of dAMP . Similarly, mispairing, rather than non-pairing, is likely to cause mutations . Cyclobutane dimers seem therefore to be misinstructive rather than non-instructive lesions, and the common feature shared by SOS-inducing lesions is more their ability to block replication than inability to form correct base pairs.

Mol Gen Genet, 1990 Jun, 222(1), 104 - 11
Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon; Blasco F et al.; The structural genes for NRZ, the second nitrate reductase of Escherichia coli, have been sequenced . They are organized in a transcription unit, narZYWV, encoding four subunits, NarZ, NarY, NarW and NarV . The transcription unit is homologous (73% identity) to the narGHJI operon which encodes the genes for NRA, the better characterized nitrate reductase of this organism . The level of homology between the corresponding polypeptides ranges from 69% for the NarW/NarJ pair to 86% for the NarV/NarI pair . The NarZ polypeptide contains the five conserved regions present in all other known molybdoproteins of E . coli and their relative order is the same . The NarY polypeptide, which contains the same four cysteine clusters in the same order as NarH, is probably an electron transfer unit of the complex . Upstream of narZ, an open reading frame, ORFA, is present which could encode a product which has homology (73% identity) with the COOH-terminal end of NarK . The ORFA-narZ intergenic region, however, is about 80 nucleotides long and does not contain the cis-acting elements, NarL and Fnr boxes, nor the terC4 terminator sequence present in the 500 nucleotide narK-narG intergenic region . This might explain why the narZYWV and the narGHJI operons are regulated differently . Our results tend to support the hypothesis that a DNA fragment larger than that encompassing the narGHJI genes has been duplicated.

Bioorg Khim, 1990 Jun, 16(6), 788 - 800
{Complementary addressed alkylation of Escherichia coli 16S rRNA with 2',3'-O-{4-N-methyl-N-(2-chloroethyl)aminobenzylidene}m derivatives of oligodeoxyribonucleotides . IV . Determination of binding sites of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA, d(pTTACGACT)rU, d(TTTGCTCCCC)rA}; Zenkova MA et al.; By site-directed alkylation of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA (II), d(pTTACGACT)rU (III), d(pTTTGCTCCCC)rA (IV) (reagents (II)--(IV} followed by the RNase H treatment a number of 16S rRNA fragments have been obtained . Hybridisation of these fragments with restriction fragments of plasmid pKK 3535, containing operon rrnB of E . coli rRNAs, led to the identification of all reagents' binding sites in 16S rRNA . Good correlation is found between estimated stability of non-perfect 16S rRNA.oligodeoxyribonucleotide duplexes and the level of modification of this site with alkylating derivative of the same oligodeoxyribonucleotide . With high concentration of the reagents (II)--(IV) ((2-5) x 10(-5) M) the site-directed alkylation proceeds not only at the desired site but also at other sites corresponding to non-perfect duplexes between 16S rRNA and the reagents . It should be noted that the modification mainly occurs in the non-perfect duplexes, carrying mismatched bases at the termini . Influence of the secondary structure of 16S rRNA on the site-directed modification is discussed.

Mol Microbiol, 1990 Jun, 4(6), 943 - 50
Octanoylation of the lipoyl domains of the pyruvate dehydrogenase complex in a lipoyl-deficient strain of Escherichia coli; Ali ST et al.; The overexpression of a subgene encoding a hybrid lipoyl domain of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli has previously been shown to result in the formation of lipoylated and unlipoylated products . Overexpression of the same subgene in a lipoic acid biosynthesis mutant growing under lipoate-deficient conditions has now been shown to produce domains modified by octanoylation as well as unmodified domains . It was concluded from the mass of a lipoyl-binding-site peptide that the modification involves N6-octanoylation of the lysine residue (Lys244) that is normally lipoylated, and this was confirmed by the trypsin-insensitivity of the corresponding Lys244-Ala-245 bond, and the absence of modification in a mutant domain in which Lys244 is replaced by Gln . This novel protein modification raises interesting questions concerning the pathway of lipoic acid biosynthesis and the mechanism of enzyme lipoylation.

Mol Microbiol, 1990 Jun, 4(6), 861 - 5
Recent advances in peptide chain termination; Craigen WJ et al.; Peptide chain termination occurs when a stop codon is decoded by a release factor . In Escherichia coli two codon-specific release factors (RF1 and RF2) direct the termination of protein synthesis, while in eukaryotes a single factor is required . The E . coli factors have been purified and their genes isolated . A combination of protein and DNA sequence data reveal that the RFs are structurally similar and that RF2 is encoded in two reading frames . Frame-shifting from one reading frame to the next occurs at a rate of 50%, is regulated by the RF2-specific stop codon UGA, and involves the direct interaction of the RF2 mRNA with the 3' end of the 16S rRNA . The RF genes are located in two separate operons, with the RF1 gene located at 26.7 min and the RF2 gene at 62.3 min on the chromosome map . Ribosomal binding studies place the RF-binding region at the interface between the ribosomal subunits . A possible mechanism of stop-codon recognition is reviewed.

FEMS Microbiol Lett, 1990 Jun 1, 57(3), 299 - 304
Comparison of coligenoid formation by B subunits of porcine and human Escherichia coli heat-labile enterotoxins; Tsuji T et al.; A hybrid B subunit (coligenoid) of heat-labile enterotoxin could not be made from human heat-labile enterotoxin B subunit(LTh-B) and porcine LTp-B subunit(LTp-B) . LTp-B monomer was able to form coligenoid by reassociation with homologous LTp-B monomer, but not with heterogeneous LTh-B monomer and vice versa . The dissociation of both coligenoids into monomers by SDS treatment occurred in a time-dependent manner, but the dissociation of LTh-B colligenoid was faster than that of LTp-B coligenoid . The association of LTp-B monomer is tighter than that of LTh-B monomer . The pI values of LTp-B coligenoid, LTp-B monomer and denatured LTp-B monomer were similar at 9.6-9.8, while the pI values of LTh-B coligenoid, LTh-B monomer and denatured LTh-B monomer were determined as 5.6-5.8, 9.2-9.6 and 9.2-9.6, respectively . All the ionic amino acids of LTp-B exist on the coligenoid surface . The difference in pI values between LTh-B coligenoid and LTh-B monomer suggests that some basic amino acids are located within the LTh-B coligenoid complex, but are exposed in the LTh-B monomer . These data suggest that the 4 amino acid substitutions between LTh-B and LTp-B result in a three dimensional structure difference and a less stable formation of LTh-B coligenoid compared to LTp-B coligenoid.

Chem Pharm Bull (Tokyo), 1990 Jun, 38(6), 1648 - 52
Effects of various triamines on cell-free polypeptide synthesis of Escherichia coli and on growth of its polyamine auxotrophs; Koumoto Y et al.; The effects of various synthetic triamines having a general structure, H2N(CH2)xNH(CH2)yNH2, where x = 2-5 and y = 2-8 (abbreviated, x-y; with 3-4 being spermidine itself), on poly(U)-directed polypeptide synthesis of Escherichia coli and on growth of its polyamine-requiring mutants were examined in comparison with those of spermidine . Except for 2-2 and 2-3, all of the triamines stimulated more or less polypeptide synthesis at suboptimal Mg2+ concentrations, but the Mg2+ concentration required for the maximal stimulatory effect was different for each triamine . The degree of maximal stimulation caused by 3-3 (norspermidine), 4-4 (homospermidine), or 4-5 was nearly comparable with that by spermidine . The acetylspermidines were inactive, however, they inhibited the spermidine-stimulated polyphenylalanine synthesis . Many of the triamines examined reduced the ratio of leucine to phenylalanine incorporation into polypeptides during poly(U)-directed translation, and the degree of this effect did not necessarily correspond with that of the stimulatory effect . Moreover, 2-4, 2-5, 3-3 and 4-4 could stimulate the growth of a polyamine auxotroph of E . coli, MA 261, as effectively as did spermidine . However, 3-3 was the only triamine which could fully replaced spermidine in promoting growth of a mutant strain, KK 101, which is more dependent on polyamines than MA 261 . Thus, these results demonstrated that some synthetic triamines were as active as spermidine in eliciting these effects, and also that there were some differences among these effects in the structural requirement for triamine.

Vet Microbiol, 1990 Jun, 23(1-4), 245 - 57
Contribution of molecular biology to the study of the porcine interferon system; Lefevre F et al.; We have performed molecular studies on the pig interferon (IFN) system (i) to analyse the role played by endogenous IFN in neonatal viral enteritis such as transmissible gastroenteritis and possibly to obtain, via recombinant DNA technology, a new anti-infectious and immunomodulatory agent in this species, (ii) to characterize the structure and biological functions of the IFN-like antiviral activity produced by the porcine embryo at the time of implantation in the uterus . By probing porcine genomic libraries with human and porcine IFN-alpha probes to isolate related genes, we have shown that the porcine IFN-alpha multigene family included, like several other mammalian species, two subfamilies of related but distinct genes . Class I subfamily contains at least 11 loci, located on chromosome no . 1, among which nine have been cloned and two (potentially functional) sequenced . Class II subfamily, which is specifically expressed by the embryo of ruminants before implantation, contains at least seven loci among which six have been cloned . One of the sequenced class I loci: PoIFN-alpha 1 encodes a 189 amino acids (AA) preprotein . After removal of the sequence encoding the putative signal peptide (23 N-terminal AA) this gene was inserted into an Escherichia coli bicistronic expression vector allowing intracellular synthesis of mature porcine IFN-alpha 1 (methionyl IFN-alpha 1) . Expression of the recombinant protein was optimized by insertion of a seven base pairs long random synthetic sequence in the intercistronic region, followed by cloning in E . coli and immunodetection of clones expressing high amounts of recombinant protein . The E . coli strain obtained produced high levels of a 18,000 Da protein exhibiting the same in vitro overall biological properties as leucocyte derived porcine IFN (LeuIFN) . However, it had a stronger antiviral effect on porcine cells than LeuIFN . After immunoaffinity purification to a specific activity of 5-10 x 10(7) International Units (IU)/mg of protein, pharmacokinetic and pharmacological studies were realized to determine the in vivo half life of this rIFN-alpha in the pig . These experiments revealed no major toxic effects in newborn (given 5 x 10(6) IU/kg) or adult (1 X 10(6) IU/kg) pigs . A significant pyrogenic effect (+ 1.5 degrees C) was noted only in the adults.

Z Naturforsch {C}, 1990 Jun, 45(6), 638 - 44
UVA-induced genetic effects of thioridazine, mesoridazine and sulforidazine: an in vitro study; Schoonderwoerd SA et al.; This in vitro study focuses on the UVA-induced reactions with DNA of thioridazine (TRZ), and two of its major metabolites (TRZ-2-sulfoxide or mesoridazine, MRZ; and TRZ-2-sulfone or sulforidazine, SRZ) . TRZ binds covalently to DNA upon UVA-irradiation . Under comparable irradiation conditions, MRZ binds to a lesser extent and almost no binding was observed with SRZ . Besides, photo-induced genetic effects were investigated by means of a differential DNA repair test in E . coli . The photo-induced genetic effects in E . coli decreased from TRZ, MRZ to SRZ, which corresponds with their capacity for UVA-induced binding to DNA . TRZ, MRZ and SRZ differed in their rate of photodecomposition rather than in the intrinsic reactivity towards DNA of the instable intermediates formed . Irreversible binding to DNA was also observed upon treatment with peroxidase, which is known to oxidize phenothiazines via the formation of reactive radical cation species . As both the colour of the intermediate and its reactivity towards DNA were comparable for peroxidase treatment and exposure to UVA, we assume that the radical cation is the reactive species in the latter case as well.

Mol Biochem Parasitol, 1990 Jun, 41(2), 259 - 68
Expression, purification, biochemical characterization and inhibition of recombinant Plasmodium falciparum aldolase; Dobeli H et al.; The energy metabolism of the blood stage form of the human malaria parasite Plasmodium falciparum is adapted to the host cell . Like erythrocytes, P . falciparum merozoites lack a functional citric acid cycle . Generation of ATP depends therefore fully on the glycolytic pathway . Aldolase is a key enzyme of this pathway and a high degree of sequence diversity between parasite and host makes it a potential drug target . We have expressed the enzyme in its tetrameric form in Escherichia coli and the catalytic constants Vmax and Km of the recombinant enzyme correspond to the constants of parasite-derived aldolase . Rabbit antibodies against the recombinant P . falciparum aldolase inhibit the natural enzyme and no cross-reaction with human aldolase is detectable . Both the recombinant and the natural protein bind to the cytosolic domain of the band 3 membrane protein in vitro . A 19-residue synthetic peptide corresponding to the sequence of the binding domain of band 3 is an inhibitor when included in the binding assay . In addition, this peptide inhibits the catalytic activity of recombinant P . falciparum aldolase when assayed in a buffer system devoid of anions such as chloride or phosphate . The band 3-derived peptides compete with the aldolase substrate fructose-1,6-diphosphate for binding, suggesting that both reagents have a high affinity for the substrate pocket . A similar sequence motif exists in P . falciparum actin II . A 19-residue peptide corresponding to this sequence is also an inhibitor which could suggest that the P . falciparum aldolase can associate with the cytoskeleton of the parasite or of the host.

Mol Biochem Parasitol, 1990 Jun, 41(2), 167 - 76
Characterization, cloning and host-protective activity of a 30-kilodalton glycoprotein secreted by the parasitic stages of Trichostrongylus colubriformis; Savin KW et al.; The helminth Trichostrongylus colubriformis is a parasitic nematode infecting the small intestine of sheep . We report the isolation and characterization of a 30-kDa glycoprotein capable of partially protecting guinea-pigs against the parasite . This glycoprotein is secreted by the L4 and adult parasitic stages of the worm . The sequence of three separate cDNA clones predicts the polypeptide to be about 15 kDa, with four N-linked carbohydrate chains and an internal disulphide bond . The clones also indicate the existence of sequence variability in this antigen . Limited sequence homology to a porcine intestinal peptide suggests an influence on host gut physiology.

Antimicrob Agents Chemother, 1990 Jun, 34(6), 1075 - 8
Inhibition of Pneumocystis carinii dihydropteroate synthetase by sulfa drugs; Merali S et al.; A new reversed-phase high-pressure liquid chromatography assay procedure for dihydropteroate synthetase (DHPS) that involves the elution of the enzyme incubation solution with a series of three solvents of decreasing polarity (ammonium phosphate buffer, 10% methanol, and 50% methanol) was designed . By this procedure DHPS was detected in Escherichia coli and Pneumocystis carinii with specific activities of 450 and 14 U/mg, respectively . A comparison of the effects of five sulfa drugs on P . carinii DHPS activity revealed that dapsone is the most potent of these drugs.

J Bioenerg Biomembr, 1990 Jun, 22(3), 441 - 9
Export and sorting of the Escherichia coli outer membrane protein OmpA; Freudl R et al.; Results of studies, mostly using the outer membrane, 325 residue protein OmpA, are reviewed which concern its translocation across the plasma membrane and incorporation into the outer membrane of Escherichia coli . For translocation, neither a unique export signal, acting in a positive fashion within the mature part of the precursor, nor a unique conformation of the precursor is required . Rather, the mature part of a secretory protein has to be export-compatible . Export-incompatibility can be caused by a stretch of 16 (but not 8 or 12) hydrophobic residues, too low a size of the polypeptide (smaller than 75 residue precursors), net positive charge at the N-terminus, or lack of a turn potential at the same site . It is not yet clear whether binding sites for chaperonins (SecB, trigger factor, GroEL) within OmpA are important in vivo . The mechanism of sorting of outer membrane proteins is not yet understood . The membrane part of OmpA, encompassing residues 1 to about 170, it thought to traverse the membrane eight times in antiparallel beta-sheet conformation . At least the structure of the last beta-strand (residues 160-170) is of crucial importance for membrane assembly . It must be amphiphilic or hydrophobic, these properties must extend over at least nine residues, and it must not contain a proline residue at or near its center . Membrane incorporation of OmpA involves a conformational change of the protein and it could be that the last beta-strand initiates folding and assembly in the outer membrane.

J Bioenerg Biomembr, 1990 Jun, 22(3), 401 - 39
Export of the periplasmic maltose-binding protein of Escherichia coli; Bassford PJ Jr; The export of the maltose-binding protein (MBP), the malE gene product, to the periplasm of Escherichia coli cells has been extensively investigated . The isolation of strains synthesizing MalE-LacZ hybrid proteins led to a novel genetic selection for mutants that accumulate export-defective precursor MBP (preMBP) in the cytoplasm . The export defects were subsequently shown to result from alterations in the MBP signal peptide . Analysis of these and a variety of mutants obtained in other ways has provided considerable insight into the requirements for an optimally functional MBP signal peptide . This structure has been shown to have multiple roles in the export process, including promoting entry of preMBP into the export pathway and initiating MBP translocation across the cytoplasmic membrane . The latter has been shown to be a late event relative to synthesis and can occur entirely posttranslationally, even many minutes after the completion of synthesis . Translocation requires that the MBP polypeptide exist in an export-competent conformation that most likely represents an unfolded state that is not inhibitory to membrane transit . The signal peptide contributes to the export competence of preMBP by slowing the rate at which the attached mature moiety folds . In addition, preMBP folding is thought to be further retarded by the binding of a cytoplasmic protein, SecB, to the mature moiety of nascent preMBP . In cells lacking this antifolding factor, MBP export represents a race between delivery of newly synthesized, export-competent preMBP to the translocation machinery in the cytoplasmic membrane and folding of preMBP into an export-incompetent conformation . SecB is one of three E . coli proteins classified as "molecular chaperones" by their ability to stabilize precursor proteins for membrane translocation.

J Bioenerg Biomembr, 1990 Jun, 22(3), 353 - 67
Structure, function, and biogenesis of SecY, an integral membrane protein involved in protein export; Ito K; The E . coli secY (prlA) gene, located in the operator-distal part of the spc ribosomal protein operon, codes for an integral membrane protein, SecY . The phenotypes of temperature-sensitive and cold-sensitive mutations in secY suggest that the SecY protein plays an essential role in vivo to facilitate protein translocation, whereas the prlA mutations in this gene suggest that SecY may interact with the signal sequence of translocating polypeptides . SecY contains most probably six cytoplasmic and five periplasmic domains, as well as 10 transmembrane segments . Such membrane-embedded structure may confer the SecY protein a "translocator" function, in which it provides a protein-aceous pathway for passage of secreted as well as membrane proteins . Results obtained by in vitro analyses of the translocation reactions, as well as some new phenotypes of the secY mutants, are consistent with this notion . Possible interaction of SecY with other secretion and chaperone-like factors is also discussed.

J Bioenerg Biomembr, 1990 Jun, 22(3), 337 - 51
SecB protein: a cytosolic export factor that associates with nascent exported proteins; Kumamoto CA; Soluble factors participate in protein translocation across a variety of biological membranes . The Escherichia coli soluble protein SecB (the product of the secB gene) is involved in the export of periplasmic and outer membrane proteins . The isolation of secB mutations permitted the demonstration that SecB is required for rapid and efficient export of certain proteins . Consistent with the results of these genetic studies, purified SecB has been shown to stimulate protein translocation across E . coli inner membrane vesicles in vitro . This article presents a review of these past studies of SecB, speculation on the role of SecB in protein translocation, and a comparison of SecB and other factors, trigger factor and GroEL.

J Bioenerg Biomembr, 1990 Jun, 22(3), 291 - 310
The sec and prl genes of Escherichia coli; Bieker KL et al.; Two general approaches have been used to define genetically the genes that encode components of the cellular protein export machinery . One of these strategies identifies mutations that confer a conditional-lethal, pleiotropic export defect (sec, secretion) . The other identifies dominant suppressors of signal sequence mutations (prl, protein localization) . Subsequent characterization reveals that in at least three cases, prlA/secY, prlD/secA, and prlG/secE, both types of mutations are found within the same structural gene . This convergence is satisfying and provides compelling evidence for direct involvement of these gene products in the export process.

J Bioenerg Biomembr, 1990 Jun, 22(3), 271 - 90
Signal peptidases and signal peptide hydrolases; Dev IK et al.; Signal peptidases, the endoproteases that remove the amino-terminal signal sequence from many secretory proteins, have been isolated from various sources . Seven signal peptidases have been purified, two from E . coli, two from mammalian sources, and three from mitochondrial matrix . The mitochondrial enzymes are soluble and function as a heterogeneous dimer . The mammalian enzymes are isolated as a complex and share a common glycosylated subunit . The bacterial enzymes are isolated as monomers and show no sequence homology with each other or the mammalian enzymes . The membrane-bound enzymes seem to require a substrate containing a consensus sequence following the -3, -1 rule of von Heijne at the cleavage site; however, processing of the substrate is strongly influenced by the hydrophobic region of the signal peptide . The enzymes appear to recognize an unknown three-dimensional motif rather than a specific amino acid sequence around the cleavage site . The matrix mitochondrial enzymes are metallo-endopeptidases; however, the other signal peptidases may belong to a unique class of proteases as they are resistant to chelators and most protease inhibitors . There are no data concerning the substrate binding site of these enzymes . In vivo, the signal peptide is rapidly degraded . Three different enzymes in Escherichia coli that can degrade a signal peptide in vitro have been identified . The intact signal peptide is not accumulated in mutants lacking these enzymes, which suggests that these peptidases individually are not responsible for the degradation of an intact signal peptide in vivo . It is speculated that signal peptidases and signal peptide hydrolases are integral components of the secretory pathway and that inhibition of the terminal steps can block translocation.

Hua Xi Yi Ke Da Xue Xue Bao, 1990 Jun, 21(2), 185 - 7
{The changes of beta-glucuronidase in rabbit model with calcium bilirubinate stone}; Li N et al.; The aim of the present study is to determine the beta-glucuronidase changes in bile and hepatobiliary tissue of rabbit model having pigment gallstone by means of biochemical and enzymehistochemical assay methods . The result showed both of the bacterial and non-bacterial beta-glucuronidase take part in the course of pigment gallstone formation . The bacterial beta-glucuronidase level increased quickly before the formation of gallstone, then decreased with control of bacterial infection . Non-bacterial beta-glucuronidase increased slowly in pro-formation stage of gallstone, then kept high level for long period of time . The relationship between beta-glucuronidase and pigment gallstone formation is also discussed.

Zhonghua Liu Xing Bing Xue Za Zhi, 1990 Jun, 11(3), 175 - 8
{A primary study on molecular epidemiology of diarrhea caused by enterotoxigenic Escherichia coli by DNA colony hybridization using three enterotoxigenic gene-probes}; Yu S; 921 isolates of acute-diarrhea-inducing E . Coli strains from several provinces and cities, including Guangzhou, Wuhan, Sichuan, Shenyang and Hunan, have been collected during 1984-1989 and were identified in 1989 for detecting genes coding for these enterotoxins-LT, ST-P, ST-H by DNA colony hybridization using three enterotoxigenic gene probes . Of all the isolates homologous to these genes encoding LT, ST-P, ST-H and both LT and ST were 198 (21.5%), 131 (14.2%), 54 (5.9%) and 18 (2.1%), respectively . It was showed that in our country pathogenic agents of acute infectious diarrheal disease with ETEC strains first might be ETEC-LT strains, secondary ETEC-ST-H strains . Whereas, in rural districts ETEC-ST-P possess higher proportion of ETEC strains . These results provided some essential data and advanced methods for researching further molecular epidemiology of the ETEC diarrheal disease.

J Gen Microbiol, 1990 Jun, 136 ( Pt 6), 1125 - 35
Molecular cloning and nucleotide sequence of another variant of the Escherichia coli Shiga-like toxin II family; Gannon VP et al.; Escherichia coli strain H.I.8 (O128:B12) produces low levels of a Shiga-like toxin (SLT) which we have called SLTIIva because of its close relationship with SLTIIv . The Vero cell cytotoxicity of SLTIIva is neutralized by antisera against SLTII and SLTIIv but not by antisera against SLTI . These data indicate that the SLT of strain H.I.8 is a member of the SLTII family . Since SLTIIva shares with SLTIIv the property of having low cytotoxicity to HeLa cells compared with Vero cells, it is appropriate to consider both toxins as variants of SLTII . SLTIIva differs from SLTIIv in that it is more heat-stable . Further, SLTIIv-producing strains of E . coli have only been isolated from pigs while the SLTIIva-producing E . coli strain examined in this study was isolated from a human infant with diarrhoea . The genes for this SLT were cloned from a cosmid library of total cellular DNA by screening recombinants for Vero cell toxicity and with a DNA probe derived from SLTIIv structural genes . Nucleotide sequence analysis was performed on a 2.0 kb AvaII-HincII fragment which encodes the toxin gene . The nucleotide sequence data confirm the close relationship between SLTIIva and SLTIIv: they have 98% nucleotide sequence homology in the B subunit gene and 70.6% homology in the A subunit gene . Comparison of DNA sequences indicated that SLTIIva was most closely related to SLTIIv, closely related to SLTII and less closely related to SLTI.

J Gen Microbiol, 1990 Jun, 136 ( Pt 6), 1001 - 7
Dynamics of plasmid transfer on surfaces; Simonsen L; A protocol was developed to study the dynamics of growth and plasmid transfer in surface populations of bacteria . This method allows for quantitative estimates of cell population densities over time, as well as microscopic observations of colony growth and interactions . Using this 'surface slide system' (SSS), the dynamics of the plasmid R1 and its permanently derepressed mutant R1drd19 in surface cultures of Escherichia coli K12 was examined . In surface culture, the stationary-phase cell densities were constant over a wide range of initial cell density (= colony density) and comparable to those obtained in liquid culture . For high initial cell densities, where the cells formed a confluent layer at stationary phase, the kinetics of growth and plasmid transfer was similar to that obtained in liquid culture, and the relative yields of R1drd19 and R1 transconjugants were similar in the two habitats . In surface culture, however, R1drd19 transconjugant yield was profoundly affected, and R1 transfer to a lesser extent, by colony density . In contrast, liquid matings were virtually unaffected by initial cell density . The transfer advantage of the permanently depressed over the repressed plasmid was much less apparent for lower colony densities . I propose a hypothesis for plasmid transfer between colonies that explains these observations as a consequence of the geometry of the surface habitat and the effect of transitory derepression of the synthesis of pili.

J Interferon Res, 1990 Jun, 10(3), 255 - 67
Recombinant equine interferon-beta 1: purification and preliminary characterization; Adolf GR et al.; Equine interferon-beta 1 (EqIFN-beta 1) was purified from extracts of recombinant Escherichia coli by sequential chromatography on hydroxylapatite, anion-, and cation-exchangers . The resulting protein was greater than 98% pure as determined by sodium dodecylsulfate gel electrophoresis, gel permeation HPLC, and reverse-phase HPLC . Amino-terminal amino acid sequencing revealed that essentially all molecules contained an additional amino-terminal methionine . The specific antiviral activity of EqIFN-beta 1 determined on equine dermal fibroblasts challenged with vesicular stomatitis virus (VSV) was approximately 5 X 10(8) U/mg . Less than 0.001% of this activity was observed in antiviral assays using human (A549), murine (L-M), ovine (SCP), or bovine (MDBK and BT) cells challenged with VSV or encephalomyocarditis virus . A series of monoclonal murine IgG antibodies were developed which neutralize the antiviral activity of EqIFN-beta 1 . None of these antibodies nor rabbit antiserum to EqIFN-beta 1 were able to neutralize human IFN-beta; antiserum to human IFN-beta did not neutralize EqIFN-beta 1 . Two of the monoclonal antibodies were used to establish a rapid one-step solid-phase enzyme immunoassay for EqIFN-beta 1.

Genes Dev, 1990 Jun, 4(6), 955 - 67
Casein kinase II enhances the DNA binding activity of serum response factor; Manak JR et al.; Serum response factor (SRF) is a mammalian transcription factor that binds to the serum response element in the enhancer of the c-fos proto-oncogene and thus may mediate serum-induction of c-fos transcription . We report here that the DNA binding activity of recombinant SRF made in Escherichia coli can be greatly enhanced by incubation of the protein with HeLa cell nuclear extract . The enhancing activity is ATP or GTP dependent and cofractionates with a protein kinase that phosphorylates SRF on a specific tryptic peptide . Coincubation with phosphatase blocks the enhancing activity, further suggesting that the enhanced binding activity is due to phosphorylation . The specific tryptic phosphopeptide phosphorylated in vitro is also phosphorylated in vivo, demonstrating that this phosphorylation is physiologically important . We have localized the phosphorylation site by a small deletion mutant . Finally, we show that the kinase activity is provided by casein kinase II (CKII) or a close variant . The potential role of CKII as either a regulatory or constitutive modifier of SRF in vivo will be discussed.

Anal Biochem, 1990 Jun, 187(2), 258 - 61
A reactor permitting injection and sampling for steady state studies of enzymatic reactions at high pressure: tests with aspartate transcarbamylase; Hoa GH et al.; A high pressure reactor for steady state studies of enzymes is described . It allows injection, stirring, and sampling without release of the pressure (up to at least 400 MPa) . Thus, either substrate or enzyme can be injected to initiate an enzyme-catalyzed reaction whose progress can then be followed by measurements on samples taken from the reactor . The dead time of sampling is 10-15 s, which allows reactions with pseudo-first-order rate constants smaller than about 1 min-1 to be monitored . It can be used for any enzymatic reaction; unlike previously described high pressure apparatus, it is not limited to the study of enzymes whose activity can be directly followed by spectrophotometry . The use and reliability of this reactor is demonstrated by tests with aspartate transcarbamylase . The activity of this enzyme is enhanced by pressures of the order of 120 MPa.

Anal Biochem, 1990 Jun, 187(2), 220 - 7
Picogram quantitation of total DNA using DNA-binding proteins in a silicon sensor-based system; Kung VT et al.; We report a rapid and reproducible method to quantify total DNA at picogram levels . Two high-affinity DNA-binding proteins are used to construct a sandwich assay and a semiconductor sensor is used for quantitation . Single-stranded DNA-binding protein (SSB) from Escherichia coli is conjugated with a linker molecule, biotin, for specific capture of the DNA complex onto a membrane . Monoclonal anti-DNA antibody is conjugated with an enzyme, urease, for signal generation . To detect DNA, a sample is denatured to form single-stranded DNA and then incubated with a reagent containing both DNA-binding protein conjugates and streptavidin . After incubation of the reagent with the DNA sample for 1 h at 37 degrees C to form a complex of streptavidin--biotin--SSB--DNA--anti-DNA--urease, the mixture is filtered through a biotin-coated nitrocellulose membrane which binds the streptavidin component of the complex . The unbound reagent is washed off the membrane, and then the captured DNA complex is detected with a light-addressable potentiometric sensor which measures the pH change catalyzed by the urease in the complex . This assay can detect 2 pg of DNA with a quantitation coefficient of variation of less than 10% in the range 10 to 200 pg.

Mol Cell Probes, 1990 Jun, 4(3), 193 - 203
A comparison of alkaline phosphatase and radiolabelled gene probes with bioassays for enterotoxigenic Escherichia coli; Bopp CA et al.; Alkaline phosphatase-conjugated oligonucleotide probes (APO), 32P-labelled oligonucleotide (RO) and cloned polynucleotide (RP) probes were evaluated for their ability to detect enterotoxigenic Escherichia coli (ETEC) as defined by bioassay . These three sets of probes were applied to 301 E . coli strains that had previously been defined by the Y1 adrenal cell assay for heat-labile enterotoxin (LT) and the infant mouse assay for heat-stable enterotoxin (ST) . The correlation of the APO probe for LT with the bioassay was 98% with five discrepancies and a positive predictive value (PPV) of 100% . For the APO/ST probe the correlation with the bioassay was 98% with seven discrepancies and a PPV of 96% . The correlation of the RO probe for LT was 99% with four discrepancies and a PPV of 100%, while the overall correlation for the two RO/ST probes was 97% with eight discrepancies and a PPV of 97% . For the RP probes, the correlation for LT was 99% with four discrepancies and a PPV of 100% and for ST was 98% with seven discrepancies and a PPV of 98% . These findings suggest that the APO probes were as sensitive as the RO and RP probes in detecting ETEC by colony hybridization and could be a practical alternative to bioassays and radiolabelled probes for ETEC since they do not require expensive equipment or extensive technical training.

J Clin Microbiol, 1990 Jun, 28(6), 1465 - 8
Identification of enterotoxigenic Escherichia coli by colony hybridization with nonradioactive digoxigenin-labeled DNA probes; Riley LK et al.; Enterotoxigenic Escherichia coli (ETEC) strains were readily identified in pure and mixed cultures with nonradioactive, digoxigenin-labeled DNA probes coding for heat-labile (LTI) and heat-stable (STaI, STaII, and STb) enterotoxins . Digoxigenin-labeled ETEC fragments were more sensitive than and exhibited less nonspecific background contamination than biotinylated ETEC probes.

J Clin Microbiol, 1990 Jun, 28(6), 1139 - 42
Serological evaluation of Escherichia coli-expressed human T-cell leukemia virus type I env, gag p24, and tax proteins; Coates SR et al.; Three proteins (env, gag, and tax) encoded by the human T-cell leukemia virus type I (HTLV-I) genome were cloned and expressed in Escherichia coli . The env protein contained a substantial part of the gp46 domain and a majority of the p21e domain . The gag protein contained all of p24 and portions of p19 and p15 . In addition to these two structural proteins, a full-length tax (p40X) construct was obtained . All three recombinant proteins were purified to near homogeneity . When used in an immunoblot assay, the three recombinant proteins detected antibodies in more HTLV-I antibody-positive patient sera than did the corresponding native proteins . Antibodies to at least two of these three different gene products were detected in 98.4% of adult T-cell leukemia patients, 100% of HTLV-I-associated myelopathy patients, 97.4% of asymptomatic carriers, and 94% of uncharacterized HTLV-I-positive patients . Antibody to recombinant tax was found in 4.9% of adult T-cell leukemia patients, whereas antibody to recombinant env could not be detected . These recombinant proteins from three different gene products may be useful in detecting or confirming the presence of antibodies to HTLV-I.

Genetics, 1990 Jun, 125(2), 341 - 9
Cloning of the DNA repair gene, uvsF, by transformation of Aspergillus nidulans; Oza K et al.; As a first step in the cloning of the DNA repair gene uvsF of Aspergillus nidulans, uvsF pyrG double mutant strains were transformed with a genomic library which carried the complementing Neurospora pyr-4 gene in the vector . Rare pyr+ uvs+ cotransformants were obtained on media lacking pyrimidines, overlayed with MMS (methyl-methane sulfonate) to which uvsF is hypersensitive . Among MMS-resistant transformants, Southerns revealed two types which showed single bands of different sizes when BglII-digested genomic DNA was probed with the vector . Both types produced uvsF- recombinants without vector sequences in homozygous crosses, but only those with the larger band also produced haploid uvs+ progeny . Using BglII-digested genomic DNA to transform Escherichia coli, plasmids of the corresponding two sizes could be rescued . Their inserts had a short internal region in common, giving evidence of rearrangement(s) . In secondary transformation of uvsF mutants, only the plasmids with the larger insert showed complementation and these were used to screen Aspergillus libraries . Three types of genomic and two overlapping cDNA clones were identified . The cDNAs hybridized not only to each other, but also to the common region of the rescued plasmids . Therefore, cDNA subclones were used to map the putative uvsF sequences to a short segment in one genomic clone . In Northerns, the complementing large plasmid hybridized to three mRNAs, while the cDNA subclone identified one of these as the probable uvsF message.

Genetics, 1990 Jun, 125(2), 275 - 80
A set of lacZ mutations in Escherichia coli that allow rapid detection of specific frameshift mutations; Cupples CG et al.; We have used site-directed mutagenesis to alter bases in lacZ near the region encoding essential residues in the active site of beta-galactosidase . The altered sequences generate runs of six or seven identical base pairs which create a frameshift, resulting in a Lac- phenotype . Reversion to Lac+ in each strain can occur only by a specific frameshift at these sequences . Monotonous runs of A's (or of T's on the opposite strand) and G's (or C's) have been constructed, as has an alternating -C-G- sequence . These specific frameshift indicator strains complement a set of six previously described strains which detect each of the base substitutions . We have examined a variety of mutagens and mutators for their ability to cause reversion to Lac+ . Surprisingly, frameshifts are well stimulated at many of these runs by ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-amino-purine, mutagens not widely known to induce frameshifts . A comparison of ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminopurine frameshift specificity with that found with a mutH strain suggests that these mutagens partially or fully saturate or inactivate the methylation-directed mismatch repair system and allow replication errors leading to frameshifts to escape repair . This results in a form of indirect mutagenesis, which can be detected at certain sites.

Genetics, 1990 Jun, 125(2), 261 - 73
Physical analysis of spontaneous and mutagen-induced mutants of Escherichia coli K-12 expressing DNA exonuclease VIII activity; Mahajan SK et al.; We have mapped the extents of two deletion sbcA mutations which result in production of DNA exonuclease VIII (ExoVIII) . One mutation, sbcA8, deletes about 140 kb of DNA which includes most of the Rac prophage and the trg gene . Western blot analysis shows that the protein produced is larger than wild type ExoVIII . The nucleotide sequence shows that a translational gene fusion has occurred . The N-terminal 294 codons of recE have been deleted and the remaining C-terminal codons have been fused to the N-terminal portion of another reading frame we call sfcA . Analysis of the protein sequence encoded by sfcA shows an 83% similarity with rat and mouse NADP-linked malic enzyme . We discuss the possibility that sfcA is identical to maeA which encodes NAD-linked malic enzyme from Escherichia coli . Restriction nuclease analysis of a second deletion, sbcA81, by Southern blot technique indicates that about 105 kb of DNA have been deleted and a transcriptional gene fusion has occurred between recE and the regulatory region of an E . coli chromosomal gene . We also examined eight other sbc mutations that result in ExoVIII production . Five have no effect on restriction nucleotide fragment sizes detected by complementarity to lambda rev as probe . These are presumed point mutations . Three seem to produce additional restriction nucleotide fragments complementary to lambda rev . The possible nature of these sbc mutations is discussed.

Scott Med J, 1990 Jun, 35(3), 87 - 8
"Liver cancer .. . or is it?"; Sridharan GV et al.; There has been a gradual change in the pattern of presentation of pyogenic liver abscess with an increasing incidence in the elderly . At the same time an improvement in mortality with early diagnosis and treatment has been recognised . We describe two patients in whom the diagnosis of liver abscess was not suspected until autopsy in one and aspiration of pus during biopsy of a liver "tumour" in the other.

Mutat Res, 1990 Jun, 230(2), 127 - 34
Effects of vanillin and o-vanillin on induction of DNA-repair networks: modulation of mutagenesis in Escherichia coli; Takahashi K et al.; Vanillin and its isomer o-vanillin have an effect on the adaptive and SOS responses, as well as mutagenesis, induced in Escherichia coli by N-methyl-N-nitrosourea (MNU) and UV irradiation, potentiating in some cases and suppressing in others . o-Vanillin markedly inhibited the MNU-induced adaptive response, while both vanillins potentiated the UV-induced SOS response . These phenomena appear to be responsible for the comutagenic or antimutagenic role of these chemicals in MNU and UV mutagenesis.

Acta Chir Scand, 1990 Jun-Jul, 156(6-7), 423 - 31
Prophylactic treatment with an aerosolized corticosteroid liposome in a porcine model of early ARDS induced by endotoxaemia; Forsgren PE et al.; The effects of prophylactic treatment with an aerosolized corticosteroid liposome (CSL) in high dose were evaluated in a porcine model of early Adult Respiratory Distress Syndrome (ARDS) induced by endotoxaemia . Intermittent positive pressure ventilated (IPPV) pigs under chlormethiazole anaesthesia were infused with E . coli endotoxin (18 micrograms.kg-1.h-1) over 4 h . Eight animals served as controls and were pretreated with aerosolized placebo liposomes, either 15 min or 2 h, before start of the endotoxin infusion . Eight animals were pretreated with CSL in aerosolized form 15 min before start of endotoxin, and eight animals were pretreated 2 h before start of endotoxin . Pretreatment with CSL, both 15 min and 2 h before endotoxin, modified and partly counteracted the late endotoxin-induced impairment in expiratory resistance (EXPres), dynamic compliance (Cdyn) and mean pulmonary artery pressure (MPAP) . The administration of CSL did not seem to have a restrictive influence on the endogenous cortisol production estimated by repeated measurements of serum cortisol levels . These results indicate that CSL, administered prophylactically in an aerosolized form to the lung, might be valuable as a modulator without systemic side effects in regard to some of the endotoxin-induced pulmonary impairments seen in this experimental model of early ARDS.

Pharm Res, 1990 Jun, 7(6), 665 - 9
Characterization of epidermal growth factor receptors on plasma membranes isolated from rat gastric mucosa; Hori R et al.; The binding of human epidermal growth factor (hEGF), beta-urogastrone, to plasma membranes isolated from rat gastric mucosa was studied to characterize gastric EGF receptors . The binding of {125I}hEGF was temperature dependent, reversible, and saturable . A single class of binding sites for EGF with a dissociation constant of 0.42 nM and maximal binding capacity of 42 fmol/mg protein was suggested . There was little change in the binding of {125I}hEGF upon addition of peptide hormones (secretin, insulin), antiulcer drugs (cimetidine), or an ulcer-inducing reagent (aspirin) . Cross-linking of {125I}hEGF to gastric plasma membranes with the use of disuccinimidyl suberate resulted in the labeling of a protein of 150 kDa . These results indicate the presence of EGF receptors on plasma membranes of rat gastric mucosa.

Protein Seq Data Anal, 1990 Jun, 3(2), 157 - 62
Escherichia coli K12 genomic database; Kunisawa T et al.; We have compiled the genomic nucleic acid sequence data of Escherichia coli K12 available from the existing major data collections and from the literature . The collected data are structured as a database for easy access and analysis . The sequence segments in the database are ordered by genetic map position . Sequence redundancy has been completely removed by combining overlapping sequences; therefore, our sequence data are amenable to statistical analysis . We have specified with a plus or minus (+ or -) on which of the two DNA strands the segment exists . The database currently contains a total of 954,392 bp, which corresponds to about 20% of the entire genome size . The sequence data are available on request.

Protein Seq Data Anal, 1990 Jun, 3(2), 149 - 56
Computer-assisted analysis of chromosomal locations and transcriptional directions of Escherichia coli genes; Watanabe H et al.; We present a computer-assisted method for locating and orienting nucleotide sequence segments on a large restriction map by comparing restriction fragment lengths . This method is based on the observation that long restriction fragments are rare and, therefore, the longest restriction fragments serve as effective discriminators . The method was applied to Escherichia coli genes, and chromosomal locations and transcriptional directions for more than 500 genes were determined.

J Surg Res, 1990 Jun, 48(6), 629 - 34
Endotoxin-induced myocardial depression in rats: effect of ibuprofen and SDZ 64-688, a platelet activating factor receptor antagonist; Baum TD et al.; We tested the hypothesis that lipopolysaccharide (LPS)-induced myocardial dysfunction is mediated by cyclooxygenase-derived metabolites of arachidonic acid or platelet activating factor (PAF) . Ether-anesthetized rats were injected iv with normal saline (NS; 2.5 ml/kg), ibuprofen (cyclooxygenase inhibitor; 15 mg/kg), or SDZ 64-688 (PAF receptor antagonist; 5 mg/kg) . Thirty minutes later, the rats were injected iv with NS (5 ml/kg) or Escherichia coli 0111:B4 LPS (20 mg/kg) . Two hours later, atria were harvested, connected to an isometric force transducer-amplifier-recorder apparatus, and maintained in vitro in oxygenated 37.5 degrees C Krebs--Henseleit buffer . Force of contraction indexed to body weight (FOCI; g/kg) was significantly (P less than 0.05) lower in the NS/LPS group (N = 7) than in the NS/NS group (N = 7) . Pretreatment with ibuprofen (ibuprofen/LPS group; N = 8) did not affect the adverse effect of LPS on atrial FOCI . In contrast, pretreatment with SDZ 64-688 (64-688/LPS group; N = 8) ameliorated (P less than 0.05) the deleterious effect of LPS on contractility . The PAF antagonist did not manifest intrinsic positive inotropic activity (64-688/NS group; N = 8) . These results support the notion that LPS-induced myocardial dysfunction in the rat is mediated, at least in part, by PAF.

Biotechnol Appl Biochem, 1990 Jun, 12(3), 284 - 91
High-level expression of complementary DNA encoding rat calmodulin in Escherichia coli; Matsuki S et al.; We report the production and characterization of a rat calmodulin made in Escherichia coli . To express the rat calmodulin cDNA in E . coli, we have employed an expression vector containing the E . coli trp promoter and trpA terminator . The cDNA was modified so as to delete the 5' nontranslated sequence and to incorporate a consensus sequence for the E . coli ribosome-binding site . Several codons for the N-terminal amino acids were selected to fit the E . coli consensus nucleotide sequence around the translational initiation codon . After induction of expression in E . coli, rat calmodulin accounted for over 30% of total cellular proteins . About 100 mg of recombinant rat calmodulin, purified to over 90% homogeneity by extraction from bacterial lysate followed by phenyl-Sepharose column chromatography, was obtained from 1 liter of E . coli culture . This recombinant calmodulin activated rat brain cyclic AMP phosphodiesterase to the same extent as the native calmodulin purified from rat brain . These results indicate that the overproduction system of the recombinant calmodulin in E . coli facilitates the study of the structure-function relationship by site-specific mutagenesis.

Ann Surg, 1990 Jun, 211(6), 663 - 6; discussion 666-8
Organ distribution of radiolabeled enteric Escherichia coli during and after hemorrhagic shock; Redan JA et al.; Translocation of intestinal bacteria to the blood during hemorrhagic shock (HS) has been confirmed in rats and humans . The current study was designed to trace the path of translocated intestinal bacteria in a murine HS model . Thirty-one rats were gavaged with 1,000,000 counts of viable 14C oleic acid-labeled Escherichia coli . Forty-eight hours later the animals were bled to 30 mmHg until either 80% of their maximal shed blood was returned or 5 hours of shock had elapsed and they were resuscitated with Ringer's lactate as previously described . Control animals were cannulated but not shocked . Eight rats immediately after shock and resuscitation, 6 rats 24 hours after shock, 3 rats 48 hours after shock, and 4 animals that died in shock had their heart, lung, liver, spleen, kidney, and serum harvested, cultured, and radioactive content measured . Translocated enteric bacteria are found primarily in the lung immediately after shock with redistribution to the liver and kidney 24 hours later . Animals surviving to 48 hours were capable of eliminating the majority of the bacteria from their major organ systems . Positive cultures for E . coli were also found in the blood, lung, liver, and kidney . We speculate that the inflammatory response stimulated by the bacteria in these organs may contribute to the multiple-organ failure syndrome seen after HS.

Mutat Res, 1990 Jun, 244(2), 115 - 21
Uracil-DNA glycosylase activity affects the mutagenicity of ethyl methanesulfonate: evidence for an alternative pathway of alkylation mutagenesis; Fix DF et al.; Mutagenesis induced by the alkylating agent ethyl methanesulfonate (EMS) is thought to occur primarily via mechanisms that involve direct mispairing at alkylated guanines, in particular, O6-ethyl guanine . Recent evidence indicates that alkylation of guanine at the O-6 position might enhance the deamination of cytosine residues in the complementary strand . To determine whether such deamination of cytosine could play a role in the production of mutations by EMS, the efficacy of this agent was tested in uracil-DNA glycosylase deficient (Ung) strains of Escherichia coli . The Ung- strains showed a linear response with increasing doses of EMS . This response was independent of the umuC gene product . In contrast, the Ung+ strains yielded a dose-squared response that became linear at higher doses of EMS when the cells were defective for the umuC gene product . These results support a model for mutagenesis involving the deamination of cytosines opposite O6-alkylated guanines followed by an error-prone repair event.

J Med Microbiol, 1990 Jun, 32(2), 73 - 81
Detection of cytotoxic necrotising factor (CNF) in extracts of Escherichia coli strains by enzyme-linked immunosorbent assay; Tabouret M et al.; An enzyme-linked immunosorbent assay (ELISA) consisting of a double sandwich technique with rabbit and sheep antibodies, was developed for the detection of cytotoxic necrotising factor (CNF) in extracts of Escherichia coli strains . The assay was evaluated by comparison with the results obtained with an assay based on toxicity for HeLa cell cultures . In a study of extracts of 27 CNF+ and 45 CNF- strains obtained by ultrasonic disintegration, no false positive and only three false negative results were recorded; the latter were obtained with strains that produced less CNF than any of the others examined . Frozen-thawed extracts contained about four times more CNF cytotoxic activity than extracts prepared ultrasonically; the testing of 54 CNF+ and 68 CNF- frozen-thawed extracts resulted in no false positive and only one false negative response . Whichever type of extract was used, no significant cross-reaction was observed with heat-labile (LT) or heat stable (ST) enterotoxin, verotoxin (VT1, VT2), haemolysin, or Vir cytotoxin.

Surgery, 1990 Jun, 107(6), 669 - 76
Effect of a body burn on endotoxin-induced lipid peroxidation: comparison with physiologic and histologic changes; Demling RH et al.; We determined the effect of a 15% total body surface (TBS), full-thickness burn on the physiologic, histologic, and oxidant-induced lipid peroxidation changes produced by endotoxin . The endotoxin-burn response was compared with that of endotoxin alone . Twenty-two adult sheep with chronic lung and flank lymph fistulas were studied . In 11 sheep a burn was produced under anesthesia and 3 days later they were given 2 micrograms/kg of endotoxin . Data were also compared with those of control sheep and those that were given burns alone . Circulating conjugated dienes increased with endotoxin alone but remained at baseline with endotoxin and burn injury . The lung lymph flow response was increased significantly in the endotoxin-burn group (sixfold) compared with that of endotoxin alone (fourfold) . Histologic quantitation of lung neutrophil count was comparable in both groups 6 hours after injury, although mononuclear cells were much more evident in lungs in the endotoxin-burn group . Lipid peroxidation measured by malondialdehyde was significantly increased in the endotoxin group compared with the endotoxin-burn group, despite the greater increase in lymph flow and lung water in the burned group . Oxygen consumption (VO2) remained constant after endotoxin alone compared with baseline . However, VO2 increased twofold immediately after endotoxin in the endotoxin-burn group . This marked increase was followed by a significant decrease in VO2 from baseline . Flank soft-tissue nonburned increased lung lymph flow twofold to threefold with endotoxin and burn, indicating increased soft-tissue permeability, whereas it remained unchanged with endotoxin alone . Liver malondialdehyde increased from a control of 110 +/- 20 to 210 +/- 80 mmol/gm tissue with endotoxin alone and to 450 +/- 54 nmol/gm tissue with endotoxin and burn . We can conclude that burn injury accentuates both the pulmonary and systemic physiologic response to endotoxin, possibly as a result of mediators released from mononuclear cells already activated in the presence of the burn . The increased lung physiologic response does not appear to be caused by greater oxidant-induced lipid peroxidation, as was seen in the liver with the combined injury.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4859 - 63
Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-1-carboxylate synthase in tomato; Van der Straeten D et al.; 1-Aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14), the key enzyme in ethylene biosynthesis, was purified 5000-fold from induced tomato pericarp . ACC synthase activity was unambiguously correlated with a 45-kDa protein by two independent methods . Peptide sequences were obtained both from the N terminus after electroblotting and from tryptic peptides separated by reversed-phase chromatography . Mixed oligonucleotide probes were used to screen a lambda gt11 library prepared from RNA of induced pericarp tissue . Putative ACC synthase clones were isolated with a frequency of 0.01% . One of these contained a 1.9-kilobase insert with a single open reading frame encoding a polypeptide of 55 kDa . A second, partial cDNA clone was found that differed from the first one in 18% of its bases . Genomic Southern blotting suggests possible tandem organization of the two genes in tomato . The entire coding region was expressed in Escherichia coli and the denatured recombinant polypeptide was used to raise polyclonal antibodies . The antibody preparation both immunoinhibits and immunoprecipitates ACC synthase activity from an enriched tomato extract, confirming the identity of the clone . Northern blot analysis demonstrates that the ACC synthase messenger accumulation is coordinated with fruit ripening.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4849 - 53
Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain; Brunger AT et al.; Normal RAS proteins play a key role of molecular switch in the transduction of the growth signal from extracellular to intracellular space . The state of the switch is "on" when GTP is bound and "off" when GDP is bound to the protein . The crystal structure of a complex between a nonhydrolyzable GTP analog and the catalytic domain of a RAS protein has been determined by a rotation-translation search method . The orientations and positions of four independent molecules have been determined using a single molecule as a probe in the search . The crystal structure reveals that the gamma phosphate of the GTP analog induces extensive conformational changes on two loop regions of the protein.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4561 - 5
Sequence and structural similarities between the leucine-specific binding protein and leucyl-tRNA synthetase of Escherichia coli; Williamson RM et al.; A role for the leucyl-tRNA synthetase (EC 6.1.1.4) has been established for regulating the transport of leucine across the inner membrane of Escherichia coli by the leucine, isoleucine, valine (LIV-I) transport system . This transport system is mediated by interactions of periplasmic binding proteins with a complex of membrane-associated proteins, and transcription of the high-affinity branched-chain amino acid transport system genes is repressed by growth of E . coli on high levels of leucine . We now report results from sequence comparisons and structural modeling studies, which indicate that the leucine-specific binding protein, one of the periplasmic components of the LIV-I transport system, contains a 121-residue stretch, representing 36% of the mature protein, which displays both sequence and structural similarities to a region within the putative nucleotide-binding domain of leucyl-tRNA synthetase . Early fusion events between ancestral genes for the leucine-specific binding protein and leucyl-tRNA synthetase could account for the similarity and suggest that processes of aminoacylation and transport for leucine in E . coli may be performed by evolutionarily interrelated proteins.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4413 - 6
Refolding of Escherichia coli dihydrofolate reductase: sequential formation of substrate binding sites; Frieden C; The kinetics of refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3) have been examined upon dilution of unfolded enzyme in 4.5 M urea to 1.29 M urea in 0.02 M phosphate buffer (pH 7.2) at 10 degrees C . Changes in the intrinsic protein fluorescence on refolding are characterized by four phases . Based on changes in the amplitudes of these phases, as a consequence of quenching of the intrinsic fluorescence by ligands, it is possible to determine the step at which a ligand binds during the refolding process . The results show that either NADP or NADPH binds to the last species formed in a sequence involving three intermediates between the unfolded and native states . Dihydrofolate, on the other hand, binds during the formation of the second observed intermediate . When refolding is performed in the presence of methotrexate, an analogue of dihydrofolate, and NADPH, NADPH binds, as determined from changes in NADPH fluorescence, to the third observed intermediate rather than the last (fourth) species formed . Measurements of the recovery of enzymatic activity during refolding suggest that dihydrofolate also induces NADPH binding prior to the final observed folding phase . These results define more closely the formation of structural domains during the folding of dihydrofolate reductase.

Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4401 - 5
Early transcription factor subunits are encoded by vaccinia virus late genes; Gershon PD et al.; The vaccinia virus early transcription factor (VETF) was shown to be a virus-encoded heterodimer . The gene for the 82-kDa subunit was identified as open reading frame (ORF) A8L, based on the N-terminal sequence of factor purified by using DNA-affinity magnetic beads . The 70-kDa subunit of VETF was refractory to N-terminal analysis, and so N-terminal sequences were obtained for three internal tryptic peptides . All three peptides matched sequences within ORF D6R . ORFs A8L and D6R are located within the central region of the vaccinia virus genome and are separated by about 13,600 base pairs . Proteins corresponding to the 3' ends of ORFs A8L and D6R were overexpressed in Escherichia coli and used to prepare antisera that bound to the larger and smaller subunits, respectively, of affinity-purified VETF . Immunoblot analysis of proteins from infected cells indicated that both subunits are expressed exclusively in the late phase of infection, just prior to their packaging in virus particles . The two subunits of VETF have no significant local or overall amino acid sequence homology to one another, to other entries in biological sequence data bases including bacterial sigma factors, or to recently determined sequences of some eukaryotic transcription factors . The 70-kDa subunit, however, has motifs in common with a super-family of established and putative DNA and RNA helicases.

Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4325 - 9
In vivo expression of the lacY gene in two segments leads to functional lac permease; Bibi E et al.; The lacY gene of Escherichia coli was cut into two approximately equal-size fragments with Afl II and subcloned individually or together under separate lac operator/promoters in plasmid pT7-5 . Under these conditions, lac permease is expressed in two portions: (i) the N-terminal portion (the N terminus, the first six putative transmembrane helices, and most of putative loop 7) and (ii) the C-terminal portion (the last six putative transmembrane helices and the C terminus) . Cells harboring pT7-5 encoding both fragments transport lactose at about 30% the rate of cells expressing intact permease to a comparable steady-state level of accumulation . In contrast, cells expressing either half of the permease independently do not transport lactose . As judged by {35S}methionine labeling and immunoblotting, intact permease is completely absent from the membrane of cells expressing lacY fragments either individually or together . Thus, transport activity must result from an association between independently synthesized pieces of lac permease . When the gene fragments are expressed individually, the N-terminal portion of the permease is observed inconsistently, and the C-terminal portion is not observed . When the gene fragments are expressed together, polypeptides identified as the N- and C-terminal moieties of the permease are found in the membrane . It is concluded that the N- or C-terminal halves of lac permease are proteolyzed when synthesized independently and that association between the two complementing polypeptides leads to a more stable, catalytically active complex.

J Clin Invest, 1990 Jun, 85(6), 1746 - 53
Effects of Escherichia coli hemolysin on human monocytes . Cytocidal action and stimulation of interleukin 1 release; Bhakdi S et al.; This study reports on the potent cytocidal and interleukin-1 releasing properties of Escherichia coli hemolysin (ECH) on human monocytes . Nanomolar concentrations of purified ECH (250-2,000 ng/ml) caused rapid and irreversible depletion of cellular ATP to levels below 20% of controls within 60 min . Subcytocidal doses (10-200 ng/ml) of ECH induced rapid release within 60-120 min of large amounts of interleukin 1 beta (IL-1 beta) from cultured monocytes . IL-1 beta release occurred in the presence of actinomycin D and cycloheximide, and was thus probably due to processing and export of intracellular IL-1 beta precursor . Incubation of toxin-producing E . coli at ratios of only 0.3-3 colony-forming units per monocyte evoked approximately 50% depletion of total cellular ATP within 90 min . Toxin producers also stimulated synthesis and release of large amounts of interleukin 1, but not of tumor necrosis factor within the same time span . In contrast, non-toxin producers caused neither cell death nor rapid interleukin 1 release . Stimulation of rapid interleukin 1 release coupled with potent cytocidal effects on cells of monocytic origin may represent pathogenetically significant events incurred by bacterial strains that produce ECH and related cytolysins.

EMBO J, 1990 Jun, 9(6), 1963 - 7
How Trp repressor binds to its operator; Staacke D et al.; We propose that the generally accepted model of a single Trp repressor dimer binding to a center of symmetry in the natural trp operator (Otwinowski et al., 1988) is wrong . We show here that the Trp repressor binds to a sequence whose center is located four base pairs either to the right or to the left of the central axis of symmetry that was previously identified . We show that: (i) the oligonucleotide used by Otwinowski et al . is not retarded by the Trp repressor in a mobility shift assay under conditions wherein a shorter oligonucleotide carrying our consensus sequence is retarded, (ii) that methylation protection experiments on the full natural operator sequence and the short oligonucleotide protect similar patterns and (iii) that by varying every base in the shorter oligonucleotide, we can demonstrate an optimal sequence for Trp repressor binding.

EMBO J, 1990 Jun, 9(6), 1743 - 8
Expression in Escherichia coli of the psbO gene encoding the 33 kd protein of the oxygen-evolving complex from spinach; Seidler A et al.; The cDNA for the 33 kd protein from the oxygen-evolving complex of spinach together with the coding region for the hydrophobic C-terminal part of the transit sequence was cloned into the expression plasmid pDS12/33Ex . The 33 kd protein precursor was expressed in Escherichia coli, secreted into the periplasm and correctly processed to the mature 33 kd protein . Thus the hydrophobic domain of the transit sequence, preceded by a methionine and two lysine residues, can function as a bacterial signal peptide . The periplasmic proteins were released from the cells by osmotic shock and the expressed protein was purified by anion exchange chromatography . The protein was identified by SDS-PAGE and Western blotting . N-terminal sequence analysis showed that the cleavage of the signal peptide occurred at the correct position . The expressed protein could be rebound to CaCl2-washed PSII particles and oxygen evolution was restored in equal amounts by the 33 kd protein from both E . coli and spinach.

J Infect Dis, 1990 Jun, 161(6), 1249 - 51
A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen; Baudry B et al.; The epidemiologic significance of enteroaggregative Escherichia coli (EAggEC) as a diarrheal pathogen has only recently come under study . Although EAggEC has been associated with persistent diarrhea in infants in some developing countries, additional studies are clearly needed . Until now, the only means of identifying EAggEC strains has been the cumbersome HEp-2 cell adhesion assay . The isolation and cloning of a 1-kilobase fragment from the plasmid of EAggEC strain 17-2 is described . This probe is 89% sensitive and 99% specific for EAggEC identification . Thus, this probe should greatly facilitate epidemiologic studies assessing the importance of EAggEC as a diarrheal pathogen.

J Bacteriol, 1990 Jun, 172(6), 3512 - 4
Amino-terminal deletions define a glutamine amide transfer domain in glutamine phosphoribosylpyrophosphate amidotransferase and other PurF-type amidotransferases; Mei BG et al.; A series of deletions was constructed in cloned Escherichia coli purF encoding glutamine phosphoribosylpyrophosphate amidotransferase . These deletions extended into the NH2 terminus of the protein and removed amino acids that are required for glutamine-dependent enzyme activity . Enzyme function, ascribed to the NH3-dependent activity, was retained in deletions that removed up to 237 amino acids . This result supports a model in which PurF-type amidotransferases contain an NH2-terminal glutamine amide transfer domain of approximately 194 to 200 amino acids fused to an aminator domain with NH3-dependent function.

J Bacteriol, 1990 Jun, 172(6), 3500 - 2
Rule governing the division pattern in Escherichia coli minB and wild-type filaments; Jaffe A et al.; Escherichia coli minB mutants form anucleate minicells and multinucleate filaments . We show here that the overwhelming majority of nucleate cells contain 2n (n = 0, 1, 2, ...) nucleoids, as determined by 4',6-diamidino-2-phenylindole staining, and 2n (n = 1, 2, 3, ...) copies of the replication origin, as determined by flow cytometry . This shows that division sites are not chosen randomly among the available sites in minB filaments . Similarly, wild-type cells contain 2n nucleoids, both during cell division inhibition and when furazlocillin-induced filaments are allowed to divide . We conclude that the min+ function is only to prevent septation only at polar sites; the placement of internal cell division sites must obey strict rules, which are the same in minB and wild-type cells.

J Bacteriol, 1990 Jun, 172(6), 3469 - 72
Further electron microscopic studies on the expression of Escherichia coli group II capsules; Kroncke KD et al.; The de novo expression of Escherichia coli K1, K5, and K12 capsules was analyzed with immunoelectron microscopy in temperature upshift experiments, with upshift from 18 degrees C (capsule restrictive) to 37 degrees C (capsule permissive) . Newly produced capsular polysaccharides appeared at the cell surface atop membrane adhesion sites (Bayer's junctions) . After plasmolysis of the bacteria at an early expression stage, the capsular polysaccharides were labeled at discrete sites in the periplasm by the immunogold technique . After temperature upshift in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or chloramphenicol, the polysaccharides were labeled in the cytoplasm.

J Bacteriol, 1990 Jun, 172(6), 3388 - 99
Mutations that affect control of the methylesterase activity of CheB, a component of the chemotaxis adaptation system in Escherichia coli; Stewart RC et al.; Sensory adaptation by the chemotaxis system of Escherichia coli requires adjustments of the extent of methyl esterification of the chemotaxis receptor proteins . One mechanism utilized by E . coli to make such adjustments is to control the activity of CheB, the enzyme responsible for removing receptor methyl ester groups . Previous work has established the existence of a multicomponent signal transduction pathway that enables the chemotaxis receptor proteins to control the methylesterase activity in response to chemotactic stimuli . We isolated and characterized CheB mutants that do not respond normally to this control mechanism . In intact cells these CheB variants could not be activated in response to negative chemotaxis stimuli . Further characterization indicated that these CheB variants could not be phosphorylated by the chemotaxis protein kinase CheA . Disruption of the mechanism responsible for regulating methylesterase activity was also observed in cells carrying chromosomal deletions of either cheA or cheW as well as in cells expressing mutant versions of CheA that lacked kinase activity . These results provide further support for recent proposals that activation of the methylesterase activity of CheB involves phosphorylation of CheB by CheA . Furthermore, our findings suggest that CheW plays an essential role in enabling the chemotaxis receptor proteins to control the methylesterase activity, possibly by controlling the CheA-CheB phosphotransfer reaction.

J Bacteriol, 1990 Jun, 172(6), 3351 - 7
Sulfate and thiosulfate transport in Escherichia coli K-12: nucleotide sequence and expression of the cysTWAM gene cluster; Sirko A et al.; The nucleotide sequence of the sulfate and thiosulfate transport gene cluster has been determined and located 3' to the gene (cysP) encoding the thiosulfate-binding protein . Four open reading frames, designated cysT, cysW, cysA, and cysM, have been identified . Similarities in primary structure were observed between (i) the deduced amino acid sequences of CysT and CysW with membrane-bound components of other binding protein-dependent transport systems, (ii) that of the CysA sequence with the "conserved" component of such systems, and (iii) that of the CysM sequence with O-acetylserine sulfhydrylase A (cysK gene product) and the beta-subunit of tryptophan synthase (coded by trpB) . Expression of the four genes was analyzed in the T7 promoter-polymerase system.

J Bacteriol, 1990 Jun, 172(6), 3278 - 83
Cytosine methylation enhances Z-DNA formation in vivo; Zacharias W et al.; The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays . Both the M.EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo . A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated . Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M.HhaI) in cells also carrying a dC-dG-containing plasmid . Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M.HhaI than in the absence of M.HhaI . These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E . coli . The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.

J Bacteriol, 1990 Jun, 172(6), 3237 - 43
Roles of the two lysyl-tRNA synthetases of Escherichia coli: analysis of nucleotide sequences and mutant behavior; Clark RL et al.; The complete nucleotide sequence of lysU, the gene for the heat-inducible lysyl-tRNA synthetase of Escherichia coli, was determined and compared with the published sequence of lysS (herC), the gene for the constitutive lysyl-tRNA synthetase . These unlinked genes were found to be identical over 72% of their lengths . The deduced amino acid sequences of the respective gene products, LysU and LysS, were identical over 85% and similar over 92% of their lengths . Accumulation of high levels of LysU during growth of strains carrying the wild-type allele of lysU on multicopy plasmids had no observable effect on growth or on the synthesis of LysS . A lysU deletion strain was constructed and was shown to grow normally at low temperature (28 degrees C) but poorly at 44 degrees C; the slow growth (45% of normal) at elevated temperature was fully reversed by plasmids bearing wild-type lysU . The implications of these findings for the existence of two aminoacyl-tRNA synthetases for lysine are discussed.

J Bacteriol, 1990 Jun, 172(6), 3146 - 51
Escherichia coli rna gene encoding RNase I: cloning, overexpression, subcellular distribution of the enzyme, and use of an rna deletion to identify additional RNases; Zhu LQ et al.; The cloning and overexpression of the Escherichia coli rna gene encoding RNase I are described . Only a single copy of the rna gene is present on the E . coli chromosome . Although cells with as much as a 100-fold increase in RNase I activity were constructed, little effect on cell growth was observed . Overexpressed RNase I was found in the periplasmic space to the same degree (approximately 85%) as wild-type enzyme, suggesting no limitation in RNase I transport . The rna clone was used to identify a deletion strain totally lacking the rna gene . The normal growth of this strain showed that RNase I is not essential for cell viability . Extracts from the RNase I deletion strain still retained a low level of RNase activity in the presence of EDTA, conclusively demonstrating the existence of additional EDTA-active RNases in E . coli . The possibility of a RNase I inhibitor is also discussed.

J Bacteriol, 1990 Jun, 172(6), 3060 - 5
Acetohydroxy acid synthase activity from a mutation at ilvF in Escherichia coli K-12; Alexander-Caudle C et al.; Examination of the ilvF locus at 54 min on the Escherichia coli K-12 chromosome revealed that it is a cryptic gene for expression of a valine-resistant acetohydroxy acid synthase (acetolactate synthase; EC 4.1.3.18) distinct from previously reported isozymes . A spontaneous mutation, ilvF663, yielded IlvF+ enzyme activity that was multivalently repressed by all three branched-chain amino acids, was completely insensitive to feedback inhibition, was highly stable at elevated temperatures, and expressed optimal activity at 50 degrees C . The IlvF+ enzyme activity was expressed in strains in which isozyme II was inactive because of the ilvG frameshift in the wild-type strain K-12 and isozymes I and III were inactivated by point mutations or deletions . Tn5 insertional mutagenesis yielded two IlvF- mutants, with the insertion in ilvF663 in each case . These observations suggest that the ilvF663 locus may be a coding region for a unique acetohydroxy acid synthase activity.

J Bacteriol, 1990 Jun, 172(6), 3030 - 6
RecA protein of Escherichia coli has a third essential role in SOS mutator activity; Sweasy JB et al.; The DNA damage-inducible SOS response of Escherichia coli includes an error-prone translesion DNA replication activity responsible for SOS mutagenesis . In certain recA mutant strains, in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity . Like UV mutagenesis, SOS mutator activity requires the products of the umuDC operon and depends on RecA protein for at least two essential activities: facilitating cleavage of LexA repressor to derepress SOS genes and processing UmuD protein to produce a fragment (UmuD') that is active in mutagenesis . To determine whether RecA has an additional role in SOS mutator activity, spontaneous mutability (tryptophan dependence to independence) was measured in a family of nine lexA-defective strains, each having a different recA allele, transformed or not with a plasmid that overproduces either UmuD' alone or both UmuD' and UmuC . The magnitude of SOS mutator activity in these strains, which require neither of the two known roles of RecA protein, was strongly dependent on the particular recA allele that was present . We conclude that UmuD'C does not determine the mutation rate independently of RecA and that RecA has a third essential role in SOS mutator activity.

J Bacteriol, 1990 Jun, 172(6), 3023 - 9
The folding properties of the Escherichia coli maltose-binding protein influence its interaction with SecB in vitro; Weiss JB et al.; It has been proposed that the cytoplasmic SecB protein functions as a component of the Escherichia coli protein export machinery by serving as an antifolding factor that retards folding of the precursor maltose-binding protein (preMBP) into a translocation-incompetent form . In this study, it was found that SecB directly interacts with wild-type preMBP and various mutationally altered MBP species synthesized in vitro to form a SecB-MBP complex that can be precipitated with anti-SecB serum . The association of SecB with wild-type preMBP was relatively unstable; such a complex was formed only when SecB was present cotranslationally or after denaturation of previously synthesized preMBP and was detected with only low efficiency . In marked contrast, MBP species that were defective in the ability to assume the stable conformation of wild-type preMBP or that exhibited significantly slower folding kinetics formed much more stable complexes with SecB . In one case, we demonstrated that SecB did not need to be present cotranslationally for complex formation to occur . Formation of a complex between SecB and MBP was clearly not dependent on the MBP signal peptide . However, we were unable to detect complex formation between SecB and MBP lacking virtually the entire signal peptide but having a completely intact mature moiety . This MBP species folded at a rate considerably faster than that of wild-type preMBP . The propensity of this mutant protein to assume the native conformation of mature MBP apparently precludes a stable association with SecB, whereas an MBP species lacking a signal peptide but exhibiting altered folding properties did form a complex with SecB that could be precipitated with anti-SecB serum.

J Bacteriol, 1990 Jun, 172(6), 2888 - 93
SecY, a multispanning integral membrane protein, contains a potential leader peptidase cleavage site; Akiyama Y et al.; SecY is an Escherichia coli integral membrane protein required for efficient translocation of other proteins across the cytoplasmic membrane; it is embedded in this membrane by the 10 transmembrane segments . Among several SecY-alkaline phosphatase (PhoA) fusion proteins that we constructed previously, SecY-PhoA fusion 3-3, in which PhoA is fused to the third periplasmic region of SecY just after the fifth transmembrane segment, was found to be subject to rapid proteolytic processing in vivo . Both the SecY and PhoA products of this cleavage have been identified immunologically . In contrast, cleavage of SecY-PhoA 3-3 was barely observed in a lep mutant with a temperature-sensitive leader peptidase . The full-length fusion protein accumulated in this mutant was cleaved in vitro by the purified leader peptidase . A sequence Ala-202-Ile-Ala located near the proposed interface between transmembrane segment 5 and periplasmic domain 3 of SecY was found to be responsible for the recognition and cleavage by the leader peptidase, since a mutated fusion protein with Phe-Ile-Phe at this position was no longer cleaved even in the wild-type cells . These results indicate that SecY contains a potential leader peptidase cleavage site that undergoes cleavage if the PhoA sequence is attached carboxy terminally . Thus, transmembrane segment 5 of SecY can fulfill both of the two important functions of the signal peptide, translocation and cleavage, although the latter function is cryptic in the normal SecY protein.

J Bacteriol, 1990 Jun, 172(6), 2839 - 43
Escherichia coli metR mutants that produce a MetR activator protein with an altered homocysteine response; Byerly KA et al.; Using an Escherichia coli lac deletion strain lysogenized with a lambda phage carrying a metH-lacZ gene fusion, we isolated trans-acting mutations that result in simultaneous 4- to 6-fold-elevated metH-lacZ expression, 5- to 22-fold-lowered metE-lacZ expression, and 9- to 20-fold-elevated metR-lacZ expression . The altered regulation of these genes occurs in the presence of high intracellular levels of homocysteine, a methionine pathway intermediate which normally inhibits metH and metR expression and stimulates metE expression . P1 transductions and complementation tests indicate that the mutations are in the metR gene . Our data suggest that the mutations result in an altered MetR activator protein that has lost the ability to use homocysteine as a modulator of gene expression.

Anesth Analg, 1990 Jun, 70(6), 608 - 17
Comparison of halothane, isoflurane, alfentanil, and ketamine in experimental septic shock; Van der Linden P et al.; The effects of four commonly used anesthetic agents, halothane, isoflurane, alfentanil, and ketamine, on cardiovascular function and oxygen balance were studied in a dog model of septic shock . After initial pentobarbital administration, the dogs were given Escherichia coli endotoxin (3 mg/kg) and, after 30 min, fluids to restore cardiac filling pressures to baseline levels . This resulted in a low resistance shock in all animals . Dogs were then given for 2 h either halothane (n = 9, 0.5 MAC), isoflurane (n = 9, 0.5 MAC), or alfentanil (n = 9, 150 micrograms/kg IV plus 2 micrograms.kg-1.min-1) or ketamine (n = 9, 2 mg/kg IV plus 0.2 mg.kg-1.min-1) or no anesthetic (control: n = 9) . Mean arterial pressure increased in the control group (+11 +/- 18 mm Hg) and with ketamine (+10 +/- 20 mm Hg), remained unchanged with isoflurane (-2 +/- 11 mm Hg), and decreased with halothane (-22 +/- 23 mm Hg) and alfentanil (-9 +/- 23 mm Hg) . Heart rate tended to increase in the control group but decreased with the four anesthetic agents, especially with alfentanil and halothane . Cardiac index and left ventricular stroke work index increased in the control group and in each anesthetic group except the halothane group . Systemic vascular resistance decreased in all groups except in the ketamine group . In the control group, the increase in cardiac index was associated with significant increases in oxygen delivery and consumption, and with a significant decrease in blood lactate levels . There was a dramatic decrease in oxygen consumption in all anesthetic groups, whereas oxygen delivery failed to increase only with halothane . Blood lactate increased significantly with halothane (5.0 +/- 1.5 to 6.3 +/- 1.4 mM/L) and isoflurane (4.8 +/- 1.1 to 5.3 +/- 1.2 mM/L), remained unchanged with alfentanil (4.5 +/- 1.5 and 4.6 +/- 0.8 mM/L), and tended to decrease with ketamine (4.9 +/- 1.4 to 4.5 +/- 1.4 mM/L) . In conclusion, among the four anesthetic agents tested, halothane had the least desirable effects . Ketamine best preserved cardiovascular function and appeared to have the least deleterious effects on the hypoxic tissues . Thus, ketamine could be the anesthetic agent of choice in septic shock.

Mol Cell Biol, 1990 Jun, 10(6), 3284 - 8
Lack of introns in the ribosomal protein gene S14 of trypanosomes; Perelman D et al.; Introns are almost always present in ribosomal protein genes, even in organisms in which introns are rare . Although trans spliced, the trypanosome ribosomal protein gene S14 apparently does not have cis introns, which supports the notion that such introns are absent in this organism.

Mol Cell Biol, 1990 Jun, 10(6), 3056 - 66
Chromosomal destabilization during gene amplification; Ruiz JC et al.; Acentric extrachromosomal elements, such as submicroscopic autonomously replicating circular molecules (episomes) and double minute chromosomes, are common early, and in some cases initial, intermediates of gene amplification in many drug-resistant and tumor cell lines . In order to gain a more complete understanding of the amplification process, we investigated the molecular mechanisms by which such extrachromosomal elements are generated and we traced the fate of these amplification intermediates over time . The model system consists of a Chinese hamster cell line (L46) created by gene transfer in which the initial amplification product was shown previously to be an unstable extrachromosomal element containing an inverted duplication spanning more than 160 kilobases (J . C . Ruiz and G . M . Wahl, Mol . Cell . Biol . 8:4302-4313, 1988) . In this study, we show that these molecules were formed by a process involving chromosomal deletion . Fluorescence in situ hybridization was performed at multiple time points on cells with amplified sequences . These studies reveal that the extrachromosomal molecules rapidly integrate into chromosomes, often near or at telomeres, and once integrated, the amplified sequences are themselves unstable . These data provide a molecular and cytogenetic chronology for gene amplification in this model system; an early event involves deletion to generate extrachromosomal elements, and subsequent integration of these elements precipitates a cascade of chromosome instability.

Mol Cell Biol, 1990 Jun, 10(6), 3013 - 9
The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1----6)-beta-D-glucan synthesis and normal cell growth; Meaden P et al.; Yeast kre mutants define a pathway of cell wall (1----6)-beta-D-glucan synthesis, and mutants in genes KRE5 and KRE6 appear to interact early in such a pathway . We have cloned KRE5, and the sequence predicts the product to be a large, hydrophilic, secretory glycoprotein which contains the COOH-terminal endoplasmic reticulum retention signal, HDEL . Deletion of the KRE5 gene resulted in cells with aberrant morphology and extremely compromised growth . Suppressors to the KRE5 deletions arose at a frequency of 1 in 10(7) to 1 in 10(8) and permitted an analysis of deletions which were found to contain no alkali-insoluble (1----6)-beta-D-glucan . These results indicate a role for (1----6)-beta-D-glucan in normal cell growth and suggest a model for sequential assembly of (1----6)-beta-D-glucan in the yeast secretory pathway.

Infect Immun, 1990 Jun, 58(6), 1959 - 64
Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes; Boehm DF et al.; The primary structure of Escherichia coli hemolysin (HlyA) contains a 9-amino-acid sequence which is tandemly repeated 13 times near the C terminus and which is essential for hemolytic activity . Hemolysin also requires an unknown modification by an accessory protein, HlyC, for hemolytic activity . The role of calcium in the interaction of HlyA with erythrocytes was investigated by using recombinant strains which produced inactive hemolysins unmodified by HlyC or deleted of the repeat sequences . 45Ca2+ autoradiography of the recombinant hemolysins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose showed that full-length, active hemolysin bound calcium . The domain involved in binding calcium was identified as the tandemly repeated sequences, since the deletion derivative missing 11 of the 13 repeats did not bind calcium . Inactive hemolysin, unmodified by HlyC, contained the repeated sequences and bound calcium as efficiently as the active, full-length toxin . The binding of the inactive toxins to erythrocytes was investigated by immunoblotting saline-washed, toxin-treated cells with monoclonal antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins . The binding of full-length, active hemolysin to erythrocytes was calcium dependent . Inactive hemolysin deleted of the repeat units did not bind to cells . The inactive hemolysin, unmodified by HlyC, bound calcium but did not bind to erythrocytes . These results highlight the importance of calcium in the binding of hemolysin to erythrocytes and suggest that the binding of hemolysin to cells requires an interaction between the calcium-binding repeat domain and the modification produced by the HlyC protein.

J Virol, 1990 Jun, 64(6), 2796 - 801
Sequence-specific binding of DNA by the Moloney murine leukemia virus integrase protein; Krogstad PA et al.; Genetic studies have indicated that integration of retroviral DNA into the host genome depends on the presence of the inverted repeats at the free termini of the long terminal repeats on the unintegrated DNA and on the product of the 3' end of the pol gene (the integrase {IN} protein) . While the precise function of the Moloney murine leukemia virus IN protein is uncertain, others have shown that it is a DNA-binding protein and functions in the processing of the inverted repeats prior to integration . By using site-directed mutagenesis, we cloned and expressed the IN protein in Escherichia coli . Crude extracts of total cellular protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, denatured in guanidine, renatured, and incubated with oligonucleotide probes . Single- and double-stranded oligonucleotides corresponding to the termini of unintegrated linear viral DNA were specifically bound by the IN protein in this assay . These data suggest that the role of the Moloney IN protein in the early steps of integration involves sequence-specific recognition of the DNA sequences found at the ends of the long terminal repeats.

J Virol, 1990 Jun, 64(6), 2642 - 52
Spleen necrosis virus gag polyprotein is necessary for particle assembly and release but not for proteolytic processing; Weaver TA et al.; The nature of spleen necrosis virus pol gene expression and the role of gag and gag-pol polyproteins in virion assembly was investigated . The DNA sequence of the gag-pol junction revealed that the two genes occupy the same open reading frame but are separated by an in-frame amber stop codon . Biochemical analysis of gag-pol translational readthrough in vitro and in Escherichia coli suggests that, in a manner similar to that in other mammalian type C retroviruses, amber stop codon suppression is required for pol gene expression . Removal of the gag stop codon had little or no effect on synthesis or cleavage of the polyprotein but interrupted particle assembly . This block could be overcome by complementation with wild-type gag protein.

Mol Gen Mikrobiol Virusol, 1990 Jun, (6), 7 - 11
{Cloning of Escherichia coli uridine phosphorylase gene: localization of structural and regulatory regions in the cloned fragment and identification of the protein product}; Brikun IA et al.; Plasmid pUD5 carrying Escherichia coli udp gene was mutagenized with the Tn5 to determine the direction of udp gene transcription . Three independent Tn5 insertions into the plasmid borne udp gene were obtained (udp::Tn5-1, udp::Tn5-4 and udp::Tn5-5) . These insertions cause disappearance of the uridine phosphorylase activity, though the ability of the plasmids to derepress transcription of chromosomal udp gene as a consequence of "titration" of cytoplasmic repressor CytR by operator regions of plasmid udp copies is retained . All the three Tn5 insertions were physically mapped within the 0.6 kb PstI-HindII fragment and transferred into the bacterial chromosome of Escherichia coli recBCsbcB strain . Integrated into the chromosomal udp gene the Tn 5 insertions were genetically mapped (by P1 transduction), with regard to the udp7 point mutation and two markers metE and zif9::Tn10 which flank the udp gene . The position of Tn5 on the constructed genetic map (metE-udp::Tn5-5 - udp::Tn5-1 - udp7 - udp::Tn5-4 - zif9::Tn10) coincides with the positions of Tn5 insertions on the pUD5 . This allows one to predict the direction of transcription of the cloned udp gene from udp::Tn5-5 towards udp::Tn5-4, considering the known fact (Alkhimova et al, 1981) that the udp promoter is located in the vicinity of metE gene.

Genetika, 1990 Jun, 26(6), 1000 - 7
{Isolation and characteristics of mutation pfm affecting the penetration of phage Mu into Escherichia coli K-12 cells}; Oskolkova OB et al.; A mutant of Escherichia coli K-12 strain with the destroyed process of establishment of lysogenic state for phage Mu in the course of zygotic induction has been obtained . The mutation revealed, designated pfm (penetration factor for Mu), interferes with adsorption of phage Mu to the surface of E . coli K-12 cells . On the basis of data obtained, there is every reason to believe that the phage Mu DNA connection with the membrane components of the bacterial cell provides optimizing condition for the primary integrative transposition of phage Mu at the stage of Mu DNA introduction into the cell.

Vet Microbiol, 1990 Jun, 23(1-4), 193 - 201
ELISA detection of bovine viral diarrhoea virus specific antibodies using recombinant antigen and monoclonal antibodies; Lecomte C et al.; A panel of monoclonal antibodies was prepared by immunization of BALB/c mice with Moredun (BD) virus strains . These antibodies were characterized by immunofluorescence and seroneutralization against BD, BVD and hog cholera (HC) virus strains, and radioimmunoprecipitation of BVD-infected cells extracts . The MAbs reacting with the majority of the Pestivirus strains recognize the 80 kDa antigen of the BVD cytophathic strains . The 80 kDa antigen of the BVD/Osloss virus strain has been cloned and expressed in E . coli as a fusion protein with beta-galactosidase . The fusion protein has been purified from inclusion bodies and used successfully as an antigen for ELISA detection of BVDV specific antibodies in bovine sera . A competitive ELISA using MAbs is more specific than a direct assay . These results compare well with the ones obtained with antigen extracted from BVDV-infected cells.

Antimicrob Agents Chemother, 1990 Jun, 34(6), 1271 - 2
Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli; Yoshida H et al.; Nucleotide sequence analysis of the gyrA genes of 10 spontaneous quinolone-resistant gyrA mutants of Escherichia coli KL16, including four mutants examined previously, disclosed that quinolone resistance was caused by a point mutation within the region between amino acids 67 and 106, especially in the vicinity of amino acid 83, of the GyrA protein.

J Bioenerg Biomembr, 1990 Jun, 22(3), 369 - 87
Protein translocation in vitro: biochemical characterization of genetically defined translocation components; Fandl J et al.; Recent years have seen the convergence of both genetic and biochemical approaches in the study of protein translocation in E . coli . The powerful combination of these approaches is exemplified in the use of an in vitro protein synthesis-protein translocation system to analyze the role of genetically defined components of the protein translocation machinery . We describe in this review recent results focusing on the function of the secA, secB, and secY gene products and the demonstration of their requirement for in vitro protein translocation . The SecA protein was recently shown to possess ATPase activity and was proposed to be a component of the translocation ATPase . We present a speculative working model whereby the translocator complex is composed of the integral membrane proteins SecY, SecD, SecE, and SecF, forming an aqueous channel in the cytoplasmic membrane, and the tightly associated peripheral membrane protein SecA functioning as the catalytic subunit of the translocator or "protein-ATPase."

Hua Xi Yi Ke Da Xue Xue Bao, 1990 Jun, 21(2), 117 - 20
{Transformation and identification of recombinant plasmid pAT153 containing HCMV gene Hind III F fragment and its clinical application}; Wu J et al.; Transformation of the recombinant plasmid pAT153 containing human cytomegalovirus (HCMV) gene Hind III F fragment into E.coli strain HB 101 was made by the CaCl2 method with a 24% of transformation efficiency . The bacterial colonies containing the recombinant plasmid were selected in colony hybridization with 32P-labelled HCMV Hind III F fragment . The recombinant plasmid was isolated by the modified alkaline lysis method which is characterized by high quality, great quantity, and time-saving . HCMV Hind III F fragment has no homology to the other herpes virus . The 32P-labelled HCMV Hind III F fragment probe could detect 0.25 pg homological sequences of HCMV DNA . The use of this probe allows the detection of HCMV in clinical specimens with the advantages of quickness, good sensitivity and specificity.

Am J Vet Res, 1990 Jun, 51(6), 855 - 60
Diagnostic complementary DNA probes for genome segments 2 and 3 of epizootic hemorrhagic disease virus serotype 1; Wilson WC et al.; Potential diagnostic complementary DNA (cDNA) clones of gene segments 2 and 3 from epizootic hemorrhagic disease virus serotype 1 (EHDV-1) have been produced . Individual segments of EHDV-1 were isolated, denatured with methylmercury hydroxide, and polyadenylated . The polyadenylated RNA was reverse-transcribed and self-hybridized into duplex structures, and the incomplete ends were repaired . The resulting product was then cloned into the plasmid vector pBR322, using the complementary tailing method . Two clones, 1 from segment 2 (E1-2-10) and 1 from segment 3 (E1-3-16) were isolated, colony-purified, and characterized by cDNA/RNA blot hybridization and endonuclease restriction analysis . The cDNA clones of RNA segment 3 of EHDV-1 cross hybridized with the corresponding segment of EHDV serotype 2 by results of cDNA/RNA blot hybridization, but not with RNA of bluetongue virus serotypes isolated in the United States . After cDNA/RNA dot-blot hybridization analysis of 17 EHDV field strains, the segment-2 clone was found to be serotype-specific, whereas the segment-3 clone was serogroup-specific.

Am J Physiol, 1990 Jun, 258(6 Pt 2), H1674 - 86
Role of platelet-activating factor and eicosanoids during endotoxin-induced lung injury in pigs; Olson NC et al.; We hypothesized that platelet-activating factor (PAF) and eicosanoids might be important mediators of endotoxin-induced respiratory failure in pigs . Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized 10- to 14-wk-old pigs at 5 micrograms/kg the 1st h, followed by 2 micrograms.kg-1.h-1 for 3 h in the presence and absence of SRI 63-675, a specific PAF receptor antagonist . During phase I (i.e., 0-2 h), endotoxin caused pulmonary hypertension and hypoxemia, decreased cardiac index, increased pulmonary vascular resistance, and increased plasma concentrations of thromboxane B2 (TxB2), prostaglandin (PG)F2 alpha, and 6-keto-PGF1 alpha . These phase I effects were attenuated or blocked by SRI 63-675 (10 mg/kg before endotoxin + 3 mg.kg-1.h-1 during endotoxemia) . During phase II endotoxemia (i.e., 2-4 h), the PAF receptor antagonist blocked endotoxin-induced pulmonary edema and hypoxemia and increased relative permeability index of the alveolar-capillary membrane . SRI 63-675 also blocked the endotoxin-induced increases in plasma and bronchoalveolar lavage fluid concentrations of leukotriene B4 (LTB4) . Ex vivo stimulation of whole blood with calcium ionophore caused large increases in plasma concentrations of TxB2 and LTB4 . These increases were not significantly modified in blood derived from pigs treated with SRI 63-675, indicating no inhibition of cyclooxygenase or 5-lipoxygenase and suggesting that the in vivo effects were PAF receptor mediated . We conclude that PAF plays an important role in the release of eicosanoids during endotoxemia and in mediating, either directly or indirectly, endotoxin-induced lung injury in anesthetized pigs.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4717 - 21
Identification of a contact between arginine-180 of the catabolite gene activator protein (CAP) and base pair 5 of the DNA site in the CAP-DNA complex; Zhang XP et al.; We have used site-directed mutagenesis to replace amino acid 1 of the recognition alpha-helix of the catabolite gene activator protein (CAP), Arg-180, with glycine and with alanine . Substitution of Arg-180 of CAP eliminated specificity between G.C, A.T, C.G, and T.A at base pair 5 of the DNA half-site . The effect was position-specific: substitution of Arg-180 did not eliminate specificity between G.C, A.T, C.G, and T.A at base pair 7 of the DNA half-site . We conclude, in agreement with the model for the structure of the CAP-DNA complex {Weber, I . & Steitz, T . (1984) Proc . Natl . Acad . Sci . USA 81, 3973-3977; and Ebright, R., Cossart, P., Gicquel-Sanzey, B . & Beckwith, J . (1984) Proc . Natl . Acad . Sci . USA 81, 7274-7278}, that Arg-180 of CAP makes a specificity-determining contact with base pair 5 of the DNA half-site in the CAP-DNA complex . The identification of the contact by Arg-180 in this report, in conjunction with the identification of the contact by Glu-181 in a previous report {Ebright, R., Cossart, P., Gicquel-Sanzey, B . & Beckwith, J . (1984) Nature (London) 311, 232-235}, provides information sufficient to define the orientation of the helix-turn-helix motif of CAP with respect to DNA in the CAP-DNA complex.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4645 - 9
Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants; Grant SG et al.; Plasmids comprising transgene insertions in four lines of transgenic mice have been retrieved by plasmid rescue into a set of Escherichia coli strains with mutations in different members of the methylation-dependent restriction system (MDRS) . Statistical analysis of plasmid rescue frequencies has revealed that the MDRS loci detect differential modifications of the transgene insertions among mouse lines that show distinctive patterns of transgene expression . Plasmids in mice that express hybrid insulin transgenes during development can be readily cloned into E . coli strains carrying mutations in two of the MDRS loci, mcrA and mcrB . In mice in which transgene expression is inappropriately delayed into adulthood, plasmids can only be cloned into E . coli that carry mutations in all known MDRS activities . Differential cloning frequencies in the presence or absence of the various methylation-dependent restriction genes represent a further way to distinguish regions of mammalian chromosomes . These multiply deficient E . coli strains will also facilitate the molecular cloning of modified chromosomal DNA.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4620 - 4
The priA gene encoding the primosomal replicative n' protein of Escherichia coli; Lee EH et al.; The Escherichia coli gene encoding protein n' has been isolated and named priA for primosomal protein A . Protein n' is absolutely required for the conversion of single-stranded phi X174 DNA to the duplex replicative form in an in vitro-reconstituted system . The gene maps to 88.7 minutes on the chromosome adjacent to the cytR locus . Soluble protein extracts from cells harboring the priA gene on a multicopy plasmid contained 45-fold more n' replication activity than wild-type extracts . Enhanced overproduction of greater than 1000-fold was achieved by replacing the natural Shine-Dalgarno sequence with that of the phage T7 phi 10 gene and placing this priA under the control of the T7 phage promoter and RNA polymerase . The priA sequence reveals a 732-amino acid open reading frame and a nucleotide-binding consensus site consistent with the size and ATPase activity of the purified protein . The gene for protein n has been named priB and the putative gene for protein n", priC.

Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4048 - 52
Role of instability in the cis action of the insertion sequence IS903 transposase; Derbyshire KM et al.; An unusual subset of DNA-binding proteins, termed cis-acting proteins, has been shown to act preferentially at their site of synthesis; the transposases of several bacterial insertion sequences (ISs) fall into this class . The transposase of IS903 exhibits a strong preference for action in cis: complementation of defective transposons in trans occurs at less than 1% . Furthermore, transposition mediated by transposase acting in cis is extremely sensitive to the distance between the 3' end of the transposase gene and the nearest transposon inverted repeat; we find that an insertion of 1 kilobase of DNA reduces transposition to 1-2% of control levels . Here we show that there is a strong correlation between the stability of transposase and its ability to act in trans . We found that the wild-type transposase is a very unstable protein with a physical half-life of about 3 min . However, a transposase-beta-galactosidase fusion protein has a much greater half-life and can act equally well in cis or in trans . In addition, the native transposase is stabilized in lon- strains of Escherichia coli, and, in these protease-deficient strains, trans action of transposase is increased 10- to 100-fold . These results suggest that instability of the IS903 transposase is a major determinant of its cis action and that the La protease, product of the lon gene, is an important determinant of transposase instability.

Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4023 - 7
Interaction and mutual stabilization of the two subunits of vaccinia virus mRNA capping enzyme coexpressed in Escherichia coli; Guo PX et al.; The genes D1 and D2, predicted to encode the 95- and 31-kDa subunits of the vaccinia virus mRNA capping enzyme, were coexpressed from the same plasmid in Escherichia coli . Induction with low concentrations of isopropyl beta-D-thiogalactoside was necessary to obtain soluble enzyme . The active heterodimer was purified by column chromatography and was shown to have both RNA guanylyltransferase and mRNA (guanine-N7-)-methyltransferase activities . Formation of the m7G(5')pppG cap structure was verified by enzyme digestion and thin-layer chromatography . Each subunit was also expressed individually in E . coli . Without the large subunit, the small one was very unstable in some bacterial strains and could only be detected by pulse labeling with radioactive amino acids . The individually expressed large subunit contained the guanylyltransferase domain, but the activity from E . coli was less than 2% of that obtained with both subunits . Two other products of the D1 open reading frame were formed: a 55-kDa subfragment with the GMP binding site and a 38-kDa C-terminal fragment that started at amino acid 498 . Expression of this heterodimeric enzyme in E . coli may facilitate the analysis of its functional domains and provide a useful reagent for the specific 5' labeling of uncapped or capped RNA and for enhancing RNA translatability in eukaryotic systems.

Virology, 1990 Jun, 176(2), 638 - 42
Inhibition of specific binding of EBNA 1 to DNA by murine monoclonal and certain human polyclonal antibodies; Orlowski R et al.; EBNA 1 was expressed as a nonfusion protein in Escherichia coli under control of the lac promoter . The major immunoreactive EBNA 1 proteins migrated as two doublets with molecular masses of about 39/41 and 49/51 kDa . Gel mobility shift experiments showed that these products exhibit the sequence-specific DNA binding on ori P previously characterized for a 28-kDa lambda N-fusion protein encompassing the carboxy third of the EBNA 1 protein . Three monoclonal antibodies previously found to react with EBNA 1 were shown to block binding of DNA by the EBNA 1 products expressed in bacteria . The same monoclonal antibodies also blocked specific DNA binding by EBNA 1 produced in Burkitt lymphoma cells infected by EB virus . Fab fragments of two monoclonal antibodies inhibited DNA binding by EBNA 1, indicating that the antibodies recognize an epitope of the protein that is involved in the recognition of DNA, or another domain crucial for DNA binding such as a dimerization site . Some but not all human antisera with antibody to EBNA 1 neutralized specific binding of EBNA 1 to DNA . These findings will help to map the residues of the EBNA 1 protein which are essential for specific binding of DNA.

Virology, 1990 Jun, 176(2), 439 - 47
Identification and DNA sequence of the Shope fibroma virus DNA topoisomerase gene; Upton C et al.; The Shope fibroma virus (SFV) DNA topoisomerase gene has been identified and mapped to the BamHI D fragment near the midpoint of the genome . The DNA sequence of the SFV BamHI S fragment together with the contiguous BamHI-ClaI subfragment of BamHI D which encompasses the topoisomerase gene and two flanking genes has been determined and analyzed . Both the SFV DNA topoisomerase and the two flanking genes are closely related in terms of sequence and spatial organization to the homologous sequences from the midpoint of the vaccinia virus genome, indicating that these proteins are conserved not only in their sequence but also by position within the poxvirus genome . To confirm the assignment of the SFV gene, the putative SFV DNA topoisomerase has been expressed as an active fusion protein in Escherichia coli and this system should be useful in the analysis of topoisomerase function following the introduction of targeted mutations into the topoisomerase gene . The results of this work shed further light on the evolutionary relationship of the different poxvirus genera and indicate that central unique regions of the poxvirus genomes contain many of the essential viral genes and are thus highly conserved.

J Bacteriol, 1990 Jun, 172(6), 3529 - 33
Activation of expression of the Escherichia coli cir gene by an iron-independent regulatory mechanism involving cyclic AMP-cyclic AMP receptor protein complex; Griggs DW et al.; Synthesis of the colicin I receptor protein, encoded by the cir gene, was determined to be sensitive to control by the catabolite repression regulatory system . Under both high- and low-iron conditions for growth, mutants unable to produce cyclic AMP (cAMP) (cya) or functional cAMP receptor protein (crp) exhibited decreased membrane levels of the receptor relative to those of the wild-type strain . Exogenous addition of cAMP to the cya mutant restored maximal expression . cAMP-dependent changes in steady-state levels of cir mRNA suggested that the effect is mediated by control of transcript synthesis or stability . Potential mechanisms for regulation were examined by deletion and sequence analysis.

J Bacteriol, 1990 Jun, 172(6), 3450 - 61
Trehalose transport and metabolism in Escherichia coli; Boos W et al.; Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can serve as a carbon source at both high and low osmolarity . The elucidation of the pathway of trehalose metabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins and degradative enzymes . The analysis of the phenotypes of these mutants and of the reactions catalyzed by the enzymes in vitro allowed the formulation of the degradative pathway at low osmolarity . Thus, trehalose utilization begins with phosphotransferase (IITre/IIIGlc)-mediated uptake delivering trehalose-6-phosphate to the cytoplasm . It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor . The enzyme catalyzing this reaction was named amylotrehalase . Amylotrehalase and EIITre were induced by trehalose in the medium but not at high osmolarity . treC and treB encoding these two enzymes mapped at 96.5 min on the E . coli linkage map but were not located in the same operon . Use of a mutation in trehalose-6-phosphate phosphatase allowed demonstration of the phosphoenolpyruvate- and IITre-dependent in vitro phosphorylation of trehalose . The phenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system.

J Bacteriol, 1990 Jun, 172(6), 3417 - 26
A new Escherichia coli heat shock gene, htrC, whose product is essential for viability only at high temperatures; Raina S et al.; We identified and characterized a new Escherichia coli gene, htrC . Inactivation of the htrC gene results in the inability to form colonies at 42 degrees C . An identical bacterial phenotype is found whether the htrC gene is inactivated either by Tn5 insertions or by a deletion spanning the entire gene . The htrC gene has been localized at 90 min, immediately downstream of the rpoC gene, and has been previously sequenced . It codes for a basic polypeptide with an Mr of 21,130 . The htrC gene is under heat shock regulation, since it is transcribed actively only in bacteria possessing functional sigma 32 . Inactivation of htrC results in (i) bacterial filamentation at intermediate temperatures, (ii) cell lysis at temperatures above 42 degrees C, (iii) overproduction of sigma 32-dependent heat shock proteins at all temperatures, (iv) overproduction of a few additional polypeptides, (v) underproduction of many polypeptides, and (vi) an overall defect in cellular proteolysis as judged by the reduced rate of puromycyl polypeptide degradation . In addition, the presence of an htrC mutation eliminates the UV sensitivity normally exhibited by lon mutant bacteria.

J Bacteriol, 1990 Jun, 172(6), 3191 - 200
Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions; Metcalf WW et al.; Twenty-four independent phosphate starvation-inducible (psi) transcriptional fusions made with Mu d1(lacZbla) were analyzed by sequencing the psi::lacZ(Mu d1) chromosomal junctions by using DNAs amplified with the polymerase chain reaction or mini-Mu cloning . Our DNA sequence analysis showed that the MuR DNA in Mu d1 has an unexpected structure that is comprised of 104 bases of MuR DNA in the form of a large inverted repeat, which we denoted Mu d1-R . Also, Mu d1s in the phoA and phn (psiD) loci of the phosphate regulon showed regional specificities for the insertion sites despite the randomness of Mu d1 insertions into the genome as a whole . Gene products or open reading frames were identified for seven unknown psi::lacZ(Mu d1) transcriptional fusions by searching DNA data bases with the sequences adjacent and upstream of the Mu d1s . One psiC::lacZ(Mu d1) lies in the ugpB gene of the ugpBAEC operon, which encodes a periplasmic sn-glycerol-3-phosphate-binding protein; two psiQ::lacZ(Mu d1)s lie in the gltB gene, and one psiQ::lacZ(Mu d1) lies in the gltD gene of the gltBDF operon, encoding the large and small subunits of glutamate synthase, respectively; and the psi-51::lacZ(Mu d1) lies in the glpB gene of the glpABC operon, which codes for the anaerobically regulated glycerol-3-phosphate dehydrogenase . psiE and psiF::lacZ(Mu d1)s lie in uncharacterized open reading frames near the xylE and phoA genes, respectively . Six other psi::lacZ(Mu d1)s lie in yet unreported Escherichia coli sequences.

J Bacteriol, 1990 Jun, 172(6), 2844 - 54
Insertional mutagenesis of a plasmid-borne Escherichia coli rpoB gene reveals alterations that inhibit beta-subunit assembly into RNA polymerase; Landick R et al.; A plasmid was constructed that overproduces the Escherichia coli RNA polymerase beta subunit from a lac promoter-rpoB fusion . The overproduced, plasmid-encoded beta subunit assembled into functional RNA polymerase that supplied greater than 90% of the transcriptional capacity of the cells . Excess beta subunit segregated into insoluble inclusion bodies and was not deleterious to cell growth . By insertion of a XhoI linker sequence (CTCGAG) and accompanying deletion of variable amounts of rpoB sequences, 13 structural alterations were isolated in the first and last thirds of the plasmid-borne rpoB gene . Twelve of these alterations appeared to reduce or prevent assembly of plasmid-encoded beta subunit into RNA polymerase . One alteration had no discernible effect on assembly or function of the beta subunit; eight others appeared to inhibit assembly but still produced detectable transcriptional activity . Three of these nine alterations produced beta-subunit polypeptides that inhibited cell growth at 32 degrees C, even though they were present in less than 50% of the cell RNA polymerase . When assembled into RNA polymerase, these three altered beta subunits apparently affected essential RNA polymerase functions . Four of the recovered alterations appeared to inhibit completely or almost completely assembly of the beta subunit into RNA polymerase . The results are consistent with a hypothesis that sequences in the first third of the beta-subunit polypeptide are especially important for proper folding and assembly of the beta subunit.

Circ Shock, 1990 Jun, 31(2), 149 - 58
Failure of endotoxic shock to elicit superoxide anion production in pig brain; Deutschman CS et al.; Endotoxin shock in pigs alters cerebral blood flow regulation . This study sought 1) to evaluate global cerebral electrical function using somatosensory evoked potential and 2) to determine if superoxide anion free radical (O2-) is generated in brain following endotoxin infusion . Five female pigs received E . coli endotoxin (0.1 mg/kg i.v.) and supplemental fluid to maintain cardiac output at control levels . Somatosensory evoked potential was generated before and after endotoxin infusion, and nitroblue tetrazolium (NBT) precipitation in cranial windows was used to determine O2- production by brain . Following endotoxin infusion, mean arterial blood pressure, cerebral perfusion pressure, and systemic vascular resistance fell by 50% and cerebral oxygen extraction increased . The amplitude and the latency of somatosensory evoked potential performed 60 min after endotoxin were unchanged from that generated at control . No precipitation of NBT in cranial windows was observed, indicating that O2- was not generated . Since endotoxin shock did not alter cerebral electrical function and O2- was not generated by brain, we conclude that alterations in cerebral blood flow regulation observed in fluid-resuscitated endotoxic shock do not result from inadequate cerebral oxygen consumption, nor is the extracellular production of O2- involved in the pathogenesis of this disorder.

Mol Cell Biol, 1990 Jun, 10(6), 2882 - 92
Multimeric arrays of the yeast retrotransposon Ty; Weinstock KG et al.; We have identified a novel integrated form of the yeast retrotransposon Ty consisting of multiple elements joined into large arrays . These arrays were first identified among Ty-induced alpha-pheromone-resistant mutants of MATa cells of Saccharomyces cerevisiae which contain Ty insertions at HML alpha that result in the expression of that normally silent cassette . These insertions are multimeric arrays of both the induced genetically marked Ty element and unmarked Ty elements . Structural analysis of the mutations indicated that the arrays include tandem direct repeats of Ty elements separated by only a single long terminal repeat . The Ty-HML junction fragments of one mutant were cloned and shown to contain a 5-base-pair duplication of the target sequence that is characteristic of a Ty transpositional insertion . In addition, the arrays include rearranged Ty elements that do not have normal long terminal repeat junctions . We have also identified multimeric Ty insertions at other chromosomal sites and as insertions that allow expression of a promoterless his3 gene on a plasmid . The results suggest that Ty transposition includes an intermediate that can undergo recombination to produce multimers.

Infect Immun, 1990 Jun, 58(6), 1817 - 20
Heterogeneity of intestinal receptors for Escherichia coli heat-stable enterotoxin; Ivens K et al.; The structure of rat intestinal cell receptors for Escherichia coli heat-stable enterotoxin (ST) was investigated by affinity cross-linking to 125I-ST and analysis by denaturing gel electrophoresis . Cross-linking of labeled toxin to intestinal membranes and analysis by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed five specifically labeled proteins with molecular masses of 160, 136, 78, 71, and 56 (kilodaltons) kDa . Exhaustive reduction of these samples resulted in a similar pattern of labeling . Affinity-labeled proteins were further analyzed by nonreducing SDS-PAGE, reduction of the resulting separated proteins, and further separation by SDS-PAGE in the presence of beta-mercaptoethanol . Thus, the 160-kDa band on nonreducing gels consisted of two different receptors: a 160-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 78-kDa subunit . Similarly, the 136-kDa band on nonreducing gels consisted of a 136-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 71-kDa subunit . The 78-, 71-, and 56-kDa subunits were not further reducible . These data suggest heterogeneity of the ST receptor subunit structure and organization in rat intestinal epithelia.

Infect Immun, 1990 Jun, 58(6), 1565 - 71
Construction and analysis of TnphoA mutants of enteropathogenic Escherichia coli unable to invade HEp-2 cells; Donnenberg MS et al.; Enteropathogenic Escherichia coli (EPEC) strains have recently been shown to invade tissue culture cells . We describe a set of 22 Tn5 IS50L::phoA (TnphoA) insertion mutants of EPEC strain E2348-69 that are unable to invade HEp-2 cells . Each mutant was tested for the ability to adhere to and to induce the polymerization of actin in HEp-2 cells . Southern hybridization of plasmid and total DNA of each strain was performed to determine the location of each TnphoA insert, and each TnphoA insert along with flanking EPEC sequences was cloned . These studies resulted in the grouping of the mutants into five main categories . These include strains with plasmid and chromosomal insertions that alter adherence, chromosomal insertions that alter the ability to induce actin polymerization, and chromosomal insertions that do not affect adherence or actin polymerization . These studies indicate that genes affecting EPEC adherence may be located on both the plasmid and chromosome, that several genes are involved in the induction of actin polymerization in epithelial cells, and that EPEC invasion is a complex process involving multiple genetic loci.

J Virol, 1990 Jun, 64(6), 3108 - 11
Transient dominant selection of recombinant vaccinia viruses; Falkner FG et al.; A general method for constructing and selecting recombinant vaccinia viruses with insertions, deletions, or mutations in any gene that is similar in principle to one originally devised for Saccharomyces cerevisiae (S . Scherer and R . W . Davis, Proc . Natl . Acad . Sci . USA 76:4951-4955, 1979) is described . The selectable marker used, Escherichia coli guanine phosphoribosyltransferase, is not retained within the final recombinant virus, and hence, this procedure may be used serially to introduce several foreign genes or to make multiple site-directed mutations.

J Virol, 1990 Jun, 64(6), 2884 - 94
cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication; Hartl M et al.; Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized . A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced . Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen . Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis . Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity . The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.

J Virol, 1990 Jun, 64(6), 2810 - 8
The Epstein-Barr virus (EBV) ORI1yt enhancer is not B-cell specific and does not respond synergistically to the EBV transcription factors R and Z; Gruffat H et al.; The Epstein-Barr virus DR promoter is located upstream of the PstI repeats, and in addition to the TATA box, it contains an upstream region (positions -69 to -220) responsive to EB1 (Z) (the BZLF1-encoded transcription factor) and an enhancer with two functionally distinct domains, A and B . Domain B has been described as a B-cell-specific EB1-responsive element (P . M . Lieberman, J . M . Hardwick, and S . D . Hayward, J . Virol . 63:3040-3050, 1989) activated synergistically by EB1 and R, an EBV early product encoded by the open reading frame BRLF1 (M . A . Cox, J . Leahy, and J . M . Hardwick, J . Virol . 64:313-321, 1990) . We show here that domain B is an R-responsive element in HeLa cells and is therefore not an EB1-responsive B-cell-specific element . However, there is an EB1-binding site (ZRE-B) located within the R-responsive enhancer region . ZRE-B can be deleted without affecting the R-dependent enhancer activity . Moreover, there is no cooperation or synergy between R and EB1 when activating the B domain (ZRE-B plus the R-responsive element) positioned as an enhancer . ZRE-B is therefore not part of the R-inducible enhancer . We have tested several subregions of the DR enhancer B domain, either alone or in combination, for their capacity to transmit the R-activating signal to the rabbit beta-globin promoter . We found that the R-responsive element is composed of four protoenhancers that span the whole B domain . These protoenhancers alone are weakly or not responsive to R . One of the protoenhancers contains the overlapping palindromes 5'-TTGTCCcgtGGACAAaTGTCC-3' . However, one palindrome, either alone or duplicated, or the overlapping palindromes did not respond to R.

J Virol, 1990 Jun, 64(6), 2560 - 8
In vitro transcriptional activation, dimerization, and DNA-binding specificity of the Epstein-Barr virus Zta protein; Lieberman PM et al.; The Epstein-Barr virus BZLF1 immediate-early gene encodes a transcriptional activator protein, Zta, which acts as a key regulatory switch in the transition between the latent and lytic viral life cycle . In this work, full-length Zta was expressed at high levels in Escherichia coli and purified to homogeneity by DNA affinity chromatography . The bacterial protein bound to specific target sequences (Zta response elements) and activated transcription in vitro from an Epstein-Barr virus early target promoter (BHLF1) . Zta bound to DNA as a dimer . The formation of a heterodimer with a Zta deletion mutant was detected by gel electrophoresis mobility shift assays . Footprinting analysis on the BHLF1, BZLF1, and simian virus 40 control regions revealed multiple binding sites with no simple consensus sequence . Zta bound upstream from its own promoter at low concentrations, while at high concentrations it bound at the transcription start site, suggesting that it may activate and then autoregulate its own expression . These results demonstrate that Zta is a sequence-specific DNA-binding transcription factor.

Biochimie, 1990 Jun-Jul, 72(6-7), 403 - 6
Introduction by site-directed mutagenesis of a tryptophan residue as a fluorescent probe for the folding of Escherichia coli phosphofructokinase; Teschner W et al.; The leucine residue at position 178 in the major allosteric phosphofructokinase from Escherichia coli has been replaced by a tryptophan using site-directed mutagenesis . Transformation by the mutated gene of pfk- bacteria results into the expression of a pfk+ phenotype and the production of an active enzyme . The modified protein has been purified and its fluorescence properties show that it contains 2 tryptophan residues, the original Trp 311 and the new Trp 178 . During unfolding of the protein by guanidine hydrochloride, the changes in the fluorescence of these 2 residues take place at different steps: Trp 311 becomes exposed to solvent when the dimeric form dissociates into monomers, while Trp 178 is exposed only when a folded chain loses its tertiary structure . The mutant enzyme is stabilized by its substrate fructose-6-phosphate against denaturation induced by heat or guanidine hydrochloride.

Genetika, 1990 Jun, 26(6), 1008 - 18
{Pleiotropic effect of the rpoC mutation in Escherichia coli K-12 reducing the frequency of lysogenization by phage lambda}; Gol'denberg DS et al.; We described earlier the isolation of lfl25 mutation reducing the frequency of lysogenization of mutant cells by phage lambda (LycA phenotype) . The mutation is mapped at present in the rpoC gene coding for the beta' subunit of bacterial RNA polymerase and named rpoC90 . It is dominant and causes decrease in pools of some branched amino acids, pyrimidines and thiamine (vitamin B1) in mutant cells . Combination of rpoC90 mutation with some Rif-r mutations strengthens both the LycA phenotype and deficiency in metabolites mentioned above . Such combination was shown to also cause a defect in antitermination (or increases the efficiency of termination) of transcription on the DNA of lambdoid phage phi 434 . This defect may indicate the possible mechanism of changes in the regulation of different bacterial operons induced by rpoC90 mutation.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4660 - 4
Features of the formate dehydrogenase mRNA necessary for decoding of the UGA codon as selenocysteine; Zinoni F et al.; The fdhF gene encoding the 80-kDa selenopolypeptide subunit of formate dehydrogenase H from Escherichia coli contains an in-frame TGA codon at amino acid position 140, which encodes selenocysteine . We have analyzed how this UGA "sense codon" is discriminated from a UGA codon signaling polypeptide chain termination . Deletions were introduced from the 3' side into the fdhF gene and the truncated 5' segments were fused in-frame to the lacZ reporter gene . Efficient read-through of the UGA codon, as measured by beta-galactosidase activity and incorporation of selenium, was dependent on the presence of at least 40 bases of fdhF mRNA downstream of the UGA codon . There was excellent correlation between the results of the deletion studies and the existence of a putative stem-loop structure lying immediately downstream of the UGA in that deletions extending into the helix drastically reduced UGA translation . Similar secondary structures can be formed in the mRNAs coding for other selenoproteins . Selenocysteine insertion cartridges were synthesized that contained this hairpin structure and variable portions of the fdhF gene upstream of the UGA codon and inserted into the lacZ gene . Expression studies showed that upstream sequences were not required for selenocysteine insertion but that they may be involved in modulating the efficiency of read-through . Translation of the UGA codon was found to occur with high fidelity since it was refractory to ribosomal mutations affecting proofreading and to suppression by the sup-9 gene product.

Rev Sci Tech, 1990 Jun, 9(2), 463 - 87
Cestode infections in animals: immunological diagnosis and vaccination; Lightowlers MW; Cestode infections in animals are important because several species are zoonotic, causing cysticercosis and hydatidosis in man, and because of the economic losses incurred due to infections in livestock . Information on immunological diagnosis of and vaccination against cestode infection is restricted almost exclusively to the taeniid cestodes in which two groups of mammalian hosts are concerned: the intermediate host infected with the larval parasite and the definitive host infected with the adult tapeworm parasite . Research towards developing serological tests for the diagnosis of larval cestode infection in animals has been largely unsuccessful . Substantial problems remain, due to the frequent existence of multiple infections with different taeniid species and antigenic crossreactivity between these related parasites, and the low level of specific antibody response to infection . Problems with poor specificity and sensitivity of traditional serological tests for cysticercosis and hydatidosis have prevented the development of any practical test for ante-mortem diagnosis of infection . A recent new approach to the diagnosis of Taenia saginata infection by detecting circulating parasite antigen offers some prospect for the development of a practical diagnostic test for cysticercosis in cattle . The effectiveness of the arecoline purge for detection of Echinococcus granulosus in dogs has been reduced by the widespread availability of praziquantel . A serological method for diagnosis of E . granulosus in dogs has been developed which offers equivalent or superior diagnostic sensitivity compared with arecoline purge . This test should provide a valuable tool in hydatid control campaigns for the diagnosis of existing or recent past infections in dogs . Substantial progress has been made towards developing a practical vaccine for the prevention of T . ovis infection in sheep . An antigen derived from the parasite egg has been identified and produced in Escherichia coli using recombinant DNA techniques . The vaccine, which protects sheep against challenge infection with T . ovis, is the first highly effective defined antigen vaccine against any parasite infection of man or animals . Commercial development of this vaccine is in progress . The success achieved with the T . ovis vaccine augurs well for the rapid development of other recombinant vaccines against cysticercosis caused by other taeniid species and against hydatidosis in animals.

Biochimie, 1990 Jun-Jul, 72(6-7), 407 - 15
Cytoplasmic and periplasmic expression of a synthetic gene for ferredoxin in Escherichia coli; Bourdineaud JP et al.; A synthetic gene coding for a modified ferredoxin II of Desulfovibrio desulfuricans Norway strain was assembled from 10 oligonucleotides . This gene was cloned into various expression vectors allowing either cytoplasmic expression or export to the periplasmic space . In the latter case, two different constructs were made, each of which contained the OmpA signal peptide: one of these constructs contained 3 additional N-terminal amino acids as compared to the wild-type ferredoxin (56 amino acid residues) . The expression of proteins encoded by the 3 constructs was assayed in E coli and the proteins were localized by cell fractionation and immunogold labelling . A low percentage of the periplasmic ferredoxin (approximately 5%) was secreted to the medium in the absence of cell lysis . The recombinant ferredoxin was purified and found to be correctly processed by the leader peptidase . However, due to the high cysteine content intramolecular and intermolecular disulfide bonds were formed and prevented binding of {4Fe-4S} clusters . Reconstitution experiments using these recombinant proteins are in progress.

Genetika, 1990 Jun, 26(6), 981 - 9
{Structural-functional organization of the incompatibility group P plasmid pBS221}; Parfenova OV et al.; Plasmid pBS221 was physically mapped for restriction endonucleases EcoRI, BamHI, BglII, HindIII . The regions essential for the plasmid existence and participating in replication (oriV trfA*) and mobilization (mob) were cloned . The tet determinant and oriV trfA* regions were localized on the physical map of the plasmid . A DNA sequence homologous to genes of Tn501 mer operon was detected in this plasmid . The studies on homology of plasmids RP4 (IncP alpha), R751 (IncP beta) and pBS221 plasmid suggest that the latter belongs to the IncP beta subgroup.

What Is Molecular Microbiology?, What Is Genetic Engineering?, What Is Listeria Monocytogenes?, What Is Anthrax?, What Is Staphylococcus Aureus?, s, Microorganisms, e, Bacteria, o, Microbiology, a, Microbe, e, Microorganism, r, Klebsiella, s, Biodegradation, n, Corynebacter, s, Aeromonades, c, Vibriosis, n, Bacteriological, i, Escherichia coli, e, Prokaryotes, i, Rhizobacter, o, Bacteriological, n, Bactericidal, r, Vibriosis, o, Bactericidal, i, MIC, c, Bacillus, n, Micrococci, s, Vancomycin, n, Antimicrobials, s, Micrococci, o, Staphylococcus aureus, r, Clostridia

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005