Escherichia coli

Genetika, 1991 Feb, 27(2), 210 - 6
{Effectiveness of chemical mutagenesis in multiple damage to one or both strands of plasmid DNA}; Karamysheva TV et al.; Methods for site-directed multiple modification of DNA have been developed and used for modification of either one or two strands of plasmid DNA . Plasmid DNAs modified in the region of the tet gene were transformed into Escherichia coli cells and Tet colonies were screened . It was shown that multiple lesions in one DNA strand performed using either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium bisulfite were effectively repaired in the cell by error-free mechanism . In contrast, modification of two DNA strands led to induction of mutations . The efficiency of mutagenesis in the case of modification of a local region of one DNA strand with sodium bisulfite and modification of the other strand with MNNG was 1.1-7.9% . Mutations were analysed by restriction mapping and sequencing . All of them were G----A transitions.

Immunol Cell Biol, 1991 Feb, 69 ( Pt 1), 51 - 5
Cloning and sequencing of the cDNA for ovine granulocyte-macrophage colony-stimulating factor (GM-CSF); O'Brien PM et al.; Colony-stimulating factors (CSFs) are not only regulators of haemopoiesis but can also enhance the function of mature myeloid cells, and are therefore potential immune adjuvants . By use of the polymerase chain reaction (PCR) with primers based on the bovine granulocyte-macrophage CSF (GM-CSF) sequence, we have amplified the cDNA for ovine GM-CSF, produced from crude mRNA extracted from alveolar macrophages . The PCR product was cloned into pUC119, and electroporated into Escherichia coli . The complete nucleotide sequence of two clones, and the partial sequence of eight others, was determined . At the nucleotide and amino acid levels, the ovine and bovine GM-CSF sequences are 91% and 81% homologous, respectively.

J Biochem (Tokyo), 1991 Feb, 109(2), 294 - 8
Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members; Arai S et al.; Oryzacystatin, a cysteine proteinase inhibitor occurring in rice seeds, contains a particular glycine residue (Gly5) near the NH2-terminal position, and the sequence Gln53-Val54-Val55-Ala56-Gly57 in a central part of the molecule . Both are conserved among most members of the cystatin superfamily . We have found from Escherichia coli expression studies that the NH2-terminal 21 residues of oryzacystatin are not essential for its papain-inhibitory activity, and that the conserved pentapeptide region may be indispensable {Abe, K., Emori, Y., Kondo, H., Arai, S., & Suzuki, K . (1988) J . Biol . Chem . 263, 7655-7659} . Here we present more detailed data based on quantitative analyses of the inhibitory activities of NH2- and COOH-terminally truncated oryzacystatin and site-directed mutants at the Gln-Val-Val-Ala-Gly region . The data indicate the following results . (1) The truncated mutants lacking the NH2-terminal 21 residues or the COOH-terminal 11 residues exhibit potent papain-inhibitory activity equivalent to the activity of wild oryzacystatin . (2) However, neither the mutant lacking the NH2-terminal 38 residues nor that lacking the COOH-terminal 35 residues is completely able to inhibit papain . (3) Site-directed mutants at the Gln residue of the Gln-Val-Val-Ala-Gly region have drastically reduced papain-inhibitory activities: the Gln----Pro mutant is completely inactive and the Gln----Leu mutant has an approximately 150 times higher Ki value than wild-type oryzacystatin.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1991 Feb, 109(2), 288 - 93
Lysophospholipase L1 from Escherichia coli K-12 overproducer; Karasawa K et al.; After screening 900 E . coli strains of the Clarke and Carbon collection for by lysophospholipase L1 activities, we isolated a clone bearing the plasmid pLC6-34, which showed an increased level of lysophospholipase L1 activity . Strains bearing the plasmid pC124, a subclone of pLC6-34 in plasmid vector pUC8, showed approximately 11.4 times higher lysophospholipase L1 activity than that of the parental strain . Starting from those overproducing strains, the lysophospholipase L1 was purified to near homogeneity by sequential use of ammonium sulfate fractionation, Sephacryl S-300, DEAE-cellulose, hydroxyapatite and Sephacryl S-200 column chromatographies . The apparent molecular weight of the purified lysophospholipase L1 was estimated to be 20,500-22,000 both by SDS-polyacrylamide gel electrophoresis and by gel permeation chromatography . The specific activity of the homogeneous lysophospholipase L1 was 10,400 nmol/min/mg protein when 1-acyl-sn-glycero-3-phosphoethanolamine was used as the substrate . The amino acid sequence of the amino-terminal portion of purified lysophospholipase L1 was determined and was different from that of lysophospholipase L2, which had previously been purified from the envelope fraction of E . coli strains bearing its cloned structural gene, pldB {Karasawa, K., Kudo, I., Kobayashi, T., Sa-eki, T., Inoue, K., & Nojima, S . (1985) J . Biochem, 98, 1117-1125} . The gene responsible for overproduction of lysophospholipase L1 activity was designated as pldC (phospholipid degradation C) . Its restriction enzyme map was also different from that of cloned pldB . These results further confirmed that, in E . coli, there are two lysophospholipases with distinct characteristics.

J Biochem (Tokyo), 1991 Feb, 109(2), 262 - 6
Purification and characterization of the Escherichia coli OxyR protein, the positive regulator for a hydrogen peroxide-inducible regulon; Tao K et al.; The Escherichia coli oxyR gene is required for the induction of a regulon that is inducible by hydrogen peroxide and confers resistance to oxidative stresses . We constructed a plasmid system that greatly overproduced OxyR protein and purified the protein . OxyR protein specifically bound to the upstream regulatory regions of the oxyR and katG genes as demonstrated by the gel-retardation assay and the DNase I footprinting experiment, and activated the transcription initiation of the katG gene in vitro . Using a plasmid carrying an oxyR'-'lacZ fusion gene, we studied the regulation of oxyR expression in vivo . The expression of oxyR was not induced by the treatment with a low concentration of hydrogen peroxide which induces the genes in the oxyR regulon . The expression of the oxyR'-'lacZ gene was higher in an oxyR-deletion strain than in the oxyR+ strain, and was repressed by overexpressing the OxyR protein . These results suggest that OxyR protein functions as a repressor for oxyR, in addition to its known function as a transcriptional activator for the genes in the oxyR regulon.

Gut, 1991 Feb, 32(2), 154 - 8
Enteropathogenic Escherichia coli and life threatening chronic diarrhoea; Hill SM et al.; Enteropathogenic Escherichia coli (EPEC) infection is not generally thought to cause severe diarrhoea after the neonatal period . Patients admitted to Queen Elizabeth Hospital for Children over the three years (1984-7) with diarrhoea and EPEC infection were reviewed . Clinical details, features of small intestinal mucosa, and treatment were recorded in those who developed chronic diarrhoea with failure to thrive . Twenty six children with EPEC required hospital admission for diarrhoea and six of these (23%) developed chronic diarrhoea . In contrast only two (5%) of 42 with other serogroups of E coli (p less than 0.01) and 28 (4%) of 764 children without EPEC admitted with acute diarrhoea developed chronic symptoms (p less than 0.01) . EPEC serogroups detected in the stool of the six children with chronic diarrhoea were 0128 in three, 0114 in two, and 0119 in one . The patients' clinical characteristics were: previous good health, no significant immunodeficiency, age 4-10 months, foreign travel (three of six), severe life threatening secretory diarrhoea from 0.5 to 1.5 1 per day (four of six), small intestinal enteropathy (five of six) three of whom showed mucosal adherent, non-invasive E coli of the same serotype as that in the stool, in association with microvillous loss and pedestal formation . All were treated with hypoallergenic feeds, two with parenteral nutrition, and three with parenteral antibiotics . All eventually recovered . EPEC infection is a common treatable cause of life threatening chronic diarrhoea in infancy.

Protein Eng, 1991 Feb, 4(3), 351 - 7
Temperature-sensitive production of rabbit muscle glycogen phosphorylase in Escherichia coli; Browner MF et al.; In order to understand how allosteric switches regulate both the catalytic activity and molecular interactions of glycogen phosphorylase, it is necessary to design and analyze variant proteins that test hypotheses about the structural details of the allosteric mechanism . Essential to such an investigation is the ability to obtain large amounts of variant proteins . We developed a system for obtaining milligram amounts (greater than 20 mg/l) of rabbit muscle phosphorylase from bacteria . Phosphorylase aggregates as inactive protein when a strong bacterial promoter is used under full inducing conditions and normal growth conditions . However, when the growth temperature of bacteria expressing phosphorylase is reduced to 22 degrees C we obtain active muscle phosphorylase . The degree to which the induced expression of phosphorylase protein is temperature sensitive depends on the strain of bacteria used . New assay and purification methods were developed to allow rapid purification of engineered phosphorylase proteins from bacterial cultures . The rabbit muscle phosphorylase obtained from the bacterial expression system is enzymatically identical to the enzyme purified from rabbit muscle . The expressed protein crystallizes in the same conditions used for growing crystals of protein from rabbit muscle and the crystal form is isomorphous . Rabbit muscle phosphorylase is one of the largest oligomeric mammalian enzymes successfully expressed in Escherichia coli . Our results indicate that optimization of a combination of growth and induction conditions will be important in the expression of other heterologous proteins in bacteria.

Biull Eksp Biol Med, 1991 Feb, 111(2), 175 - 7
{Changes in the profile of cytotoxic mediator monocytes in patients with cancer and precancerous conditions of the stomach}; Shepetkin IA et al.; Peripheral blood monocytes from healthy subjects, patients with gastric precancer disease (chronic gastric ulcer, stomach polyps and chronic atrophic gastritis) and different stages of gastric cancer were used . Spontaneous and lipopolysaccharide (LPS)-stimulated TNF-like factors production by monocytes was significantly higher in the precancer gastric disease patients than in the healthy subjects . At the same time the spontaneous capacity of monocytes to produce NTF-like factors was 2.5 lower in the gastric cancer patients compared to the healthy subjects . Moreover, in 5/13 of the gastric cancer patients in TNF-like factors production by the LPS-stimulated and non-stimulated monocytes was 1 unit/ml less . Spontaneous and reactive CL indexes were higher in the cancer patients monocytes than in the healthy subjects . The obtained results suggest that reactive oxygen species production can be an alternative mechanism by which a cytotoxic action of monocytes is regulated.

Anal Biochem, 1991 Feb 1, 192(2), 262 - 7
Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase; Guan KL et al.; Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria . The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D . B . Smith and K . S . Johnson, 1988, Gene 67, 31-40) . This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins . In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly . To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site . This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins . The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein . More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein . The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.

Mol Gen Mikrobiol Virusol, 1991 Feb, (2), 19 - 23
{A comparative study of the functions of recBCD-nuclease from Escherichia coli and "recombinase", encoded by plasmid R1drd-19}; Terent'ev MA et al.; The RecBCD nuclease of Escherichia coli and "recombinase" determined by R1drd-19 plasmid (the latter is able to replace at least partially the indicated cellular enzyme) were shown to differ from each other in some essential features . The product encoded by the plasmid as distinct from RecBCD nuclease practically is not sensitive to inhibition by GamS protein of the lambda phage . Earlier, it was found that the presence of R1drd-19 plasmid in the recBC cells restores the level of the total ATP-dependent exonuclease activity because of appearance in such cells of a new exonuclease activity also ATP-dependent . The exonuclease activity determined by R1drd-19 plasmid was found to differ from the corresponding activity of the RecBCD enzyme . The plasmid enzyme was able to prevent reproduction of T4g2- mutant on recBC cells . The ability of the plasmid "recombinase" to some stimulation of intrachromosomal recombination in recA mutant witness to incomplete RecA-dependence of its function . No significant homology was registered between Escherichia coli DNA fragment containing the recB, recC, recD genes and the EcoRI-C-fragment of R1drd-19 carrying the sequences responsible for recombination and repair functions of the plasmid.

Mol Gen Mikrobiol Virusol, 1991 Feb, (2), 12 - 5
{Design of a gene coding encoding a polypeptide, mixing the enzyme activity of Escherichia coli homoserine kinase and threonine synthetase}; Manucharian KG et al.; The functioning of Escherichia coli threonine operon isolated genes thrB and thrC was studied by using the genetic complementation and enzymatic activity determination techniques . A new gene thrBC was obtained by the genes merging . The genes thrB and thrC were shown to function in Escherichia coli cells independent of the operon and the polipeptide encoded by the thrBC gene combined the functions to express the products of both genes in bacterial cell . At the same time the enzyme coded for by the merged genes demonstrates the level of activity compared with the ones of the isolated genes.

Antimicrob Agents Chemother, 1991 Feb, 35(2), 387 - 9
4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction; Oram M et al.; Seven nalidixic acid-resistant clinical isolates of Escherichia coli were shown to carry resistance mutations in their gyrase A proteins . Six had serine-83 to leucine or tryptophan changes; the seventh had an aspartate-87 to valine substitution . The frequent occurrence of a mutation at serine-83 implies a key role for this residue in quinolone action.

J Chemother, 1991 Feb, 3(1), 16 - 22
Modulation of the intrinsic antiviral activity by Escherichia coli endotoxin in macrophages from patients with neoplasia; Merendino RA et al.; Macrophages from patients with breast cancer showed an impairment of their antiviral activity . The capability to hinder herpes simplex virus type 2 replication of macrophages from healthy donors and from patients with breast cancer was compared to the in-vitro treatment with Escherichia coli lipopolysaccharide (LPS) . The LPS showed a dose-dependent effect on the different macrophage populations studied . Nevertheless, macrophages from healthy donors appeared to be more sensitive to LPS in comparison with macrophages from the patients under our observation . On these cells LPS treatment was not able to modify the antiviral property, when these macrophages were differentiated in autologous serum.

Gene, 1991 Feb 1, 98(1), 77 - 82
Effects of mcr restriction of methylated CpG islands of the L1 transposons during packaging and plating stages of mammalian genomic library construction; Doherty JP et al.; The use of optimally methylation-tolerant mcrA- mcrB- strains has been shown to produce an over tenfold increase in the plating efficiencies of mammalian genomic libraries, compared to a superior conventional phage host strain LE392 which is mcrB+ . However, there is an even more significant effect of mcr restriction . Amongst the recombinants recovered with an mcrB+ host, we have found that there is an additional 30-fold reduction in the frequencies of clones containing the heavily methylated 5'-CpG island sequences of both the human and rat L1 repetitive elements . The mcrA product was also found to restrict clones of these methylated genomic segments, but not as strongly as mcrB . However, the use of packaging extracts made from mcrA+ lysogens did not result in convincing reductions in the recoveries of these dispersed methylated elements . The magnitude of mcr restriction during plating due to methylated dispersed elements is sufficient to make a significant proportion of mammalian genomes unclonable from genomic libraries constructed previously using conventional mcr+ hosts.

Gene, 1991 Feb 1, 98(1), 45 - 52
Gene expression in Deinococcus radiodurans; Smith MD et al.; We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion . However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance . Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E . coli promoter has been deleted, leaving only coding sequences for the marker gene . We find that the insertion of D . radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification . Furthermore, a series of stable E . coli-D . radiodurans shuttle plasmids was devised by inserting fragments of D . radiodurans plasmid pUE10 in an E . coli plasmid directly upstream from a promoterless cat gene . These constructions replicated in D . radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D . radiodurans promoter sequences in the pUE10 fragment . Of three such constructions, none expressed the cat gene in E . coli . Similar results were obtained using a promoterless tet gene . Translational fusions were made between D . radiodurans genes and E . coli 5'-truncated lacZ . Three fusions that produced high levels of beta Gal in D . radiodurans were introduced into E . coli, but beta Gal was produced in only one . The results demonstrate that the E . coli genes cat, tet and lacZ can be efficiently expressed in D . radiodurans if a D . radiodurans promoter is provided, and that D . radiodurans promoters often do not function as promoters in E . coli.

J Bacteriol, 1991 Feb, 173(4), 1492 - 501
Genetic interaction between the beta' subunit of RNA polymerase and the arginine-rich domain of Escherichia coli nusA protein; Ito K et al.; The nusA11 mutation causes reduced transcription termination and temperature-sensitive growth of Escherichia coli . Suppressor mutations that restored growth of nusA11 mutant cells were isolated and named sna mutations . The intergenic suppressor mutation sna-10 was located in the rpoC gene at 90 min, which encodes the beta' subunit of RNA polymerase . sna-10 complemented the defect in tR1 termination caused by nusA11 and by itself stimulated termination of transcription at the lambda tR1 terminator . sna-10 is specific to the nusA11 allele and unable to suppress cold-sensitive growth of the nusA10 mutant . nusA10 carried two base substitutions at positions 311 and 634, causing two amino acid changes from the wild-type sequence . During these studies, we found three -1 frameshift errors in the wild-type nusA sequence; the correct sequence was confirmed by the peptide sequence and gene fusion analyses . The revised sequence revealed that nusA1 and nusA11 are located in an arginine-rich peptide region and substitute arginine and aspartate for leucine 183 and glycine 181, respectively . The intragenic suppressor study indicated that the nusA11 mutation can be suppressed by changing the mutated aspartate 181 to alanine or changing aspartate 84 to tyrosine.

J Gen Virol, 1991 Feb, 72 ( Pt 2), 399 - 404
Immunological studies on the Epstein-Barr virus encoded alkaline deoxyribonuclease found in virus-producing lymphoblastoid cells; Baylis SA et al.; Antisera were raised against a purified recombinant form of the Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) expressed in Escherichia coli . These sera were shown to be reactive with lymphoblastoid cells expressing EBV antigens (B95-8, P3HR-1 and Raji) . Immunostaining studies of cells expressing EBV antigens revealed that the DNase was a component of the restricted early antigen complex of EBV . Western blot analysis of these chemically induced cells revealed that the polypeptide associated with the EBV DNase has an Mr of approximately 55,000, slightly greater than that of the recombinant form, suggesting that the protein undergoes some form of posttranslational modification during virus replication . The DNase enzymic activities observed in B95-8, P3HR-1 and Raji cells following chemical induction were neutralized using the specific antiserum . A detailed examination of protein extracts from the nude mouse-passaged nasopharyngeal carcinoma cell line C-15 failed to detect any antigenic or biochemical evidence for the presence of the DNase . Immunostaining of biopsies of oral 'hairy' leukoplakia with the antisera against EBV DNase revealed high level expression in the more differentiated spinous layers of the epithelium, a pattern of reactivity identical to that observed for other lytic cycle antigens.

J Cell Biol, 1991 Feb, 112(4), 665 - 76
Domain structure in actin-binding proteins: expression and functional characterization of truncated severin; Eichinger L et al.; Severin from Dictyostelium discoideum is a Ca2(+)-activated actin-binding protein that severs actin filaments, nucleates actin assembly, and caps the fast growing ends of actin filaments . Sequence comparison with functionally related proteins, such as gelsolin, villin, or fragmin revealed highly conserved domains which are thought to be of functional significance . To attribute the different activities of the severin molecule to defined regions, progressively truncated severin polypeptides were constructed . The complete cDNA coding for 362 (DS362) amino acids and five 3' deletions coding for 277 (DS277), 177 (DS177), 151 (DS151), 117 (DS117), or 111 (DS111) amino acids were expressed in Escherichia coli . The proteins were purified to homogeneity and then characterized with respect to their effects on the polymerization or depolymerization kinetics of G- or F-actin solutions and their binding to G-actin . Furthermore, the Ca2+ binding of these proteins was investigated with a 45Ca-overlay assay and by monitoring Ca2(+)-dependent changes in tryptophan fluorescence . Bacterially expressed DS362 showed the same Ca2(+)-dependent activities as native severin . DS277, missing the 85 COOH-terminal amino acids of severin, had lost its strict Ca2+ regulation and displayed a Ca2(+)-independent capping activity, but was still Ca2+ dependent in its severing and nucleating activities . DS151 which corresponded to the first domain of gelsolin or villin had completely lost severing and nucleating properties . However, a residual severing activity of approximately 2% was detectable if 26 amino acids more were present at the COOH-terminal end (DS177) . This locates similar to gelsolin the second actin-binding site to the border region between the first and second domain . Measuring the fluorescence enhancement of pyrene-labeled G-actin in the presence of DS111 showed that the first actin-binding site was present in the NH2-terminal 111 amino acids . Extension by six or more amino acids stabilized this actin-binding site in such a way that DS117 and even more pronounced DS151 became Ca2(+)-independent capping proteins . In comparison to many reports on gelsolin we draw the following conclusions . Among the three active actin-binding sites in gelsolin the closely neighboured sites one and two share the F-actin fragmenting function, whereas the actin-binding sites two and three, which are located in far distant domains, collaborate for nucleation . In contrast, severin contains two active actin-binding sites which are next to each other and are responsible for the severing as well as the nucleating function . The single actin-binding site near the NH2-terminus is sufficient for capping of actin filaments.

J Bacteriol, 1991 Feb, 173(3), 1111 - 9
Transcriptional analysis, translational analysis, and sequence of the kilA-tellurite resistance region of plasmid RK2Ter; Walter EG et al.; The tellurite resistance (Ter) determinant of the IncP alpha plasmid RK2Ter, a variant of RK2 (also called RP4), is located between the kilA and korA genes involved in plasmid replication control . Transcriptional and translational fusions were constructed between the gene for beta-galactosidase and the kilA and Ter genes by using the transpositional phage mini-Mu . These fusions indicated that the Ter genes are transcribed in the same direction as kilA and that transcription and translation of the cloned kilA gene are occurring and may not be lethal to the bacterial cell even in the absence of korA . The nucleotide sequence of this region was determined, and three open reading frames (ORFs) were identified . The first ORF codes for KilA, a 28-kDa hydrophilic protein . The second ORF, telA, codes for a hydrophilic protein of 42 kDa . The third ORF, telB, codes for a hydrophobic protein of 32 kDa . This protein appears to be located in the inner membrane of the bacterial cell, since fusions of TelB to alkaline phosphatase were obtained by using TnphoA . All three proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after overproduction using the T7 RNA polymerase/promoter system . The same three proteins were produced when Tes and Ter derivatives of RP4 were expressed in an in vitro transcription-translation system . A single Ser-to-Cys missense mutation in telB was found to be responsible for mutation of RK2 to Ter.

J Bacteriol, 1991 Feb, 173(3), 1027 - 34
traY and traI are required for oriT-dependent enhanced recombination between lac-containing plasmids and lambda plac5; Carter JR et al.; Recombination between F42lac and lambda plac5 is typically 20- to 50-fold more efficient than recombination between chromosomal lac and lambda plac5 . This enhancement of recombination requires trans-acting factors located in the promoter-distal and promoter-proximal regions of the main traY-to-traI (traZ) operon . By testing the ability of deletion mutants of tra to support enhanced recombination, we have identified traY as the only product has been ruled out . We also report that traI is the only gene from the promoter-distal end of the traY to traI operon that is required for recombination enhancement . Of the two proposed domains of traI, we conclude that the oriT-nicking activity is essential, whereas the helicase activity is largely dispensable . The possibility of a third traI activity is also discussed.

J Bacteriol, 1991 Feb, 173(3), 1021 - 6
The Escherichia coli mraY gene encoding UDP-N-acetylmuramoyl-pentapeptide: undecaprenyl-phosphate phospho-N-acetylmuramoyl-pentapeptide transferase; Ikeda M et al.; Amplification of the mraY gene, previously called open reading frame Y (ORF-Y, 1,080 bp), at 2 min in the chromosome map of Escherichia coli enhanced the activity of UDP-N-acetylmuramoyl-pentapeptide: undecaprenyl-phosphate phospho-N-acetylmuramoyl-pentapeptide transferase (EC 2.7.8.13) . This enzyme catalyzes the formation of undecaprenyl-pyrophosphoryl-N-acetylmuramoyl-pentapeptide from UDP-N-acetylmuramoyl-pentapeptide and undecaprenyl-phosphate, the first step in the lipid cycle reactions in biosynthesis of bacterial cell wall peptidoglycans . The enhanced enzyme activity was sensitive to tunicamycin, and the amino tunicamycin-sensitive N-acetylglucosamine-1-phosphate transferase of Saccharomyces cerevisiae . Very probably mraY is the structural gene for the above enzyme.

J Bacteriol, 1991 Feb, 173(3), 1004 - 11
Genetic analysis of the recG locus of Escherichia coli K-12 and of its role in recombination and DNA repair; Lloyd RG et al.; We describe a transposon insertion that reduces the efficiency of homologous recombination and DNA repair in Escherichia coli . The insertion, rec-258, was located between pyrE and dgo at min 82.1 on the current linkage map . On the basis of linkage to pyrE and complementation studies with the cloned rec+ gene, rec-258 was identified as an allele of the recG locus first reported by Storm et al . (P . K . Storm, W . P . M . Hoekstra, P . G . De Haan, and C . Verhoef, Mutat . Res . 13:9-17, 1971) . The recG258 mutation confers sensitivity to mitomycin C and UV light and a 3- to 10-fold deficiency in conjugational recombination in wild-type, recB recC sbcA, and recB recC sbcB sbcC genetic backgrounds . It does not appear to affect plasmid recombination in the wild-type . A recG258 single mutant is also sensitive to ionizing radiation . The SOS response is induced normally, although the basal level of expression is elevated two- to threefold . Further genetic studies revealed that recB recG and recG recJ double mutants are much more sensitive to UV light than the respective single mutants in each case . However, no synergistic interactions were discovered between recG258 and mutations in recF, recN, or recQ . It is concluded that recG does not fall within any of the accepted groups of genes that affect recombination and DNA repair.

Am Rev Respir Dis, 1991 Feb, 143(2), 289 - 93
Early post-treatment with pentoxifylline or dibutyryl cAMP attenuates Escherichia coli-induced acute lung injury in guinea pigs; Hoffmann H et al.; We examined effects of early post-treatment with the methylxanthine pentoxifylline (PTXF), or the cell-permeable adenosine 3', 5'-cyclic monophosphate (cAMP) analog dibutyryl cAMP (db-cAMP) on Escherichia-coli-induced acute lung injury in guinea pigs . Acute lung injury was assessed by measurements of lung water (lung wet/dry weight ratio; W/D ratio), the concentration ratio of 125I-albumin in bronchoalveolar lavage (BAL) fluid and lung tissue compared with plasma (albumin index; BAL-AI or tissue-AI), and total differential leukocyte count in BAL fluid . Mean arterial pressure (Pa) and peripheral WBC counts were monitored continuously over the 8-h experiment . Septicemia was induced by a bolus injection of 2 x 10(9)/kg live E . coli . Thirty minutes later the animals received a bolus injection followed by continuous infusion of PTXF (20 mg/kg + 20 mg/kg/h; n = 8) or db-cAMP (2 mg/kg + 2 mg/kg/h; n = 8) or saline (septic control; n = 8) . Nonseptic control groups were also studied . The lung W/D ratio, BAL-AI, lung tissue-AI, and BAL leukocyte count increased significantly in the septic control group . The PTXF-septic and db-cAMP-septic groups showed no significant increase in lung W/D ratio, BAL-AI, and lung tissue-AI . However, there was no difference in BAL total and differential leukocyte count as compared with the septic control group . PTXF and db-cAMP had no effect on E . coli-induced changes in peripheral WBC count and Pa . Comparison in vitro experiments demonstrated that PTXF and db-cAMP inhibited the endotoxin-induced (E . coli) chemiluminescent response of isolated guinea-pig polymorphonuclear leukocytes (PMN).(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1991 Feb, 11(2), 1114 - 24
Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein; Silve S et al.; Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts . It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus . The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment . Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing . Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase . The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence . In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.

Virology, 1991 Feb, 180(2), 648 - 58
Use of type 1/type 2 chimeric polioviruses to study determinants of poliovirus type 1 neurovirulence in a mouse model; Martin A et al.; We previously described the characteristics of a type 1/type 2 (PV-1/PV-2) chimeric poliovirus, v510, which contains the six amino acids specific for PV-2 in the B-C loop of VP1 . This virus was found to be mouse-adapted, as PV-2 and in contrast with PV-1 . Determinants of host range were studied in detail and are reported here . PV-1/PV-2 chimeras containing partial PV-1----PV-2 substitutions in the B-C loop of VP1 were obtained by making use of a mutagenesis cartridge on PV-1 cDNA . Analysis of mouse neurovirulence of these chimeras, when correlated with the three-dimensional structure of the v510 capsid, revealed that PV-2 residues important for mouse tropism are those which determine the particular conformation of the B-C loop of VP1 in v510 . The mutation of the adenine residue at position 480 of the 5' noncoding region into a guanine residue has been shown to be an important determinant of PV-1 attenuation in monkeys . We show that introduction of this mutation in the v510 genome results in a virus which is partially attenuated for mice . This suggests that analysis of genomic determinants important for PV-1 neurovirulence could be carried out in a mouse model by making use of a mouse-adapted PV-1/PV-2 chimera.

J Virol, 1991 Feb, 65(2), 972 - 5
Upstream promoter elements of the herpes simplex virus type 1 glycoprotein H gene; Steffy KR et al.; To investigate the cis-acting sequence elements that are involved in the regulation of herpes simplex virus type 1 late-gene expression, recombinant viruses were constructed that express the Escherichia coli lacZ gene from the promoter of the glycoprotein H (gH) gene . Deletion experiments established an upstream boundary for the gH promoter of no more than 83 bp from the start of gH transcription and showed that the promoter sequences did not overlap with coding sequences of the upstream thymidine kinase (tk) gene . Sequences of the tk gene previously shown to be required for efficient processing of the tk transcript were essential for expression form the gH promoter and included a TATA-like element . In addition, the gH TATA element was specifically mutagenized to substitute the TATA elements of immediate-early, early, and other late viral promoters for the gH TATA element . The results indicated that the TATA element was an interchangeable component of herpes simplex virus type 1 promoters and did not regulate temporal expression.

Biochimie, 1991 Feb-Mar, 73(2-3), 305 - 12
Genetic control of recombination exchange frequency in Escherichia coli K-12; Lanzov V et al.; The frequency of recombination exchanges per unit length of DNA (Freuld) can be estimated by measuring the scale of the genetic map that is the mean statistical distance between two neighboring crossovers . The scales appear to be equal for the alternative pathways of recombination, RecBCD (wild-type cells) or RecF (recBC- sbcB- sbcC- genotypes) . The absolute value of the scale depends on specific experimental conditions . recR, recQ, ruv, recJ and recN genes of the RecF pathway of recombination (recBC- sbcBC- cell genotypes) do not appear to be silent in wild-type cells where the RecBCD pathway predominates . On the contrary, these genes are responsible for the Freuld . The list recF504::Kmr greater than recQ61::Tn3 greater than ruv-54 greater than recJ284::Tn10 shows decreasing efficiency in inhibiting recombination exchanges by these mutations . The recN264 mutation gives a small, but opposite effect of increasing the frequency of recombination exchanges . The effect of the recF and recQ mutations appears to be additive, but that is not the case in combinations of ruv-54 with recF504::Kmr or recQ61::Tn3.

Eur J Cell Biol, 1991 Feb, 54(1), 95 - 101
Localization of the plasma membrane and mitochondrial H(+)-ATPases in Leishmania donovani promastigotes; Liveanu V et al.; Immunochemical methods were used to characterize the proton-translocating ATPases (H(+)-ATPases) of the plasma membrane and mitochrondrion of Leishmania donovani promastigotes . Antisera directed against the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae reacted with a 66 kDa membrane protein of L . donovani promastigotes . By immunocytochemistry, the antiserum was shown to label the cell and flagellar surface of promastigotes as well as the Golgi apparatus and the membrane of intracellular organelles . The target antigen was shown to possess ATPase activity resembling the leishmanial H(+)-ATPase activity . Antisera raised against the beta-subunit of the F0F1-ATPase of Escherichia coli reacted with a 56 kDa protein in L . donovani promastigotes . Ultrastructurally, the anti-beta-subunit antibody was exclusively associated with the mitochondrion in these cells . This antiserum immunoprecipitates ATP hydrolytic activity typical of the F1 beta-subunit activity of the mitochondria of higher eukaryotes.

Neuron, 1991 Feb, 6(2), 201 - 10
Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice; Lem J et al.; Chimeric gene fusions between 4.4 kb of rod opsin 5' flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5' flanking sequence fused to the E . coli IacZ gene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development . Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death . The 4.4 kb opsin-IacZ transgene followed temporal and spatial gradients of expression that approximate opsin expression . The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression.

Virology, 1991 Feb, 180(2), 668 - 77
Influenza A virus in vitro transcription: roles of NS1 and NP proteins in regulating RNA synthesis; Skorko R et al.; To study the mechanisms by which the influenza A virus RNA-dependent RNA polymerase switches from transcription to replication we have devised a riboprobe protection technique with which we analyzed the 3' end sequence of (+)-strand RNA products of an in vitro transcription reaction containing purified virion-RNP complexes in the presence and the absence of the putative regulatory proteins NP and NS1 . We found that the addition of these proteins did not result in the synthesis of full-length (+)-strand RNA products resulting from read-through of the polyadenylation signal or replication . Because NS1 and NP are both phosphoproteins we searched for protein kinase activity that might play a role in regulating RNA synthesis . We showed that virion RNP complexes phosphorylated NS1 but possessed no autophosphorylating activity . Soluble NP protein derived from RNP complexes did not phosphorylate NS1, but did phosphorylate casein . When NP protein was dephosphorylated, however, it no longer phosphorylated casein . We also showed that NS1 was an ssRNA-binding protein which binds nonspecifically to all ssRNA, and that this activity is not dependent on its state of phosphorylation.

Protein Expr Purif, 1991 Feb, 2(1), 66 - 8
Affinity chromatography of Escherichia coli (m5U54)-methyltransferase on tRNA-agarose; Gu X et al.; tRNA-agarose was prepared by condensing periodate-oxidized tRNA to an agarose matrix containing hydrazide functional groups . The tRNA-agarose was used to take partially purified tRNA (m5U54)-methyltransferase to homogeneity . The method is simple and reproducible and gives high yields.

Protein Expr Purif, 1991 Feb, 2(1), 59 - 62
Rapid desalting of ammonium sulfate solutions by hydrophobic interaction chromatography; O'Hern PA et al.; Open-air hydrophobic interaction chromatography with alkyl carbon functional groups coupled to agarose beads has been used to desalt large volumes of ammonium sulfate-fractionated bacterial cell lysates . The protein of interest can be simultaneously desalted and concentrated in less than 4 h, and the yield is significantly better than that obtained by the standard technique of precipitation, centrifugation, and dialysis.

Protein Expr Purif, 1991 Feb, 2(1), 15 - 23
High-yield purification of HIV-1 proteinase expressed by a synthetic gene in Escherichia coli; Goobar L et al.; A rapid and simple purification procedure for human immunodeficiency virus type 1 (HIV-1) proteinase from a synthetic gene expressed in Escherichia coli has been developed . The synthetic gene was constructed from oligonucleotides containing several restriction enzyme sites in order to allow simple construction of homologous genes . The protein was translated as a precursor which was autocatalytically processed into the mature protein as shown by N-terminal sequence analysis of the purified protein . Immunoblot analysis was used to verify the nature of the expression product and it was found that 2 of 10 anti-peptide antibodies, covering the whole proteinase sequence, were able to react with the enzyme in crude bacterial lysates . These two anti-peptide antibodies represent a continuous sequence partially overlapping the active site . The purification involves two initial precipitation steps followed by cation-exchange and size-exclusion chromatography . A high yield and a high specific activity were achieved.

J Vasc Interv Radiol, 1991 Feb, 2(1), 156 - 8
Percutaneous management of localized emphysematous pyelonephritis; Zagoria RJ et al.; The authors report a case of emphysematous pyelonephritis that was successfully treated with radiologically guided percutaneous drainage . This case illustrates that in certain patients with focal abnormalities, functioning renal tissue can be salvaged and emphysematous pyelonephritis can be eradicated with a combination of antibiotics and radiologically guided percutaneous drainage.

Biochimie, 1991 Feb-Mar, 73(2-3), 251 - 6
Identification of a mouse cDNA fragment whose expressed polypeptide reacts with anti-recA antibodies; Angulo JF et al.; We have previously reported the in vivo detection of a mouse nuclear protein that cross-reacts with antibodies raised against E coli recA protein . Here, we characterize monospecific anti-recA antibodies, their use for the immunological screening of a cDNA expression library and the isolation of a mouse cDNA fragment which codes for a polypeptide recognized by anti-recA antibodies . The cDNA fragment is 601 nucleotide long and was called KIN17(601) . It contains an open reading frame coding for a 200 amino acid polypeptide . In kin17(200) polypeptide, there are amino acids identical to those that form one of the major antigenic determinants of recA protein . Kin17(200) polypeptide also displays a significant similarity with the helix 1 motif of several homeoproteins.

FEMS Microbiol Immunol, 1991 Feb, 3(1), 39 - 45
Identification of a novel B-cell epitope of restricted specificity on the hsp 65-kDa protein of Mycobacterium tuberculosis; Rambukkana A et al.; A B-cell epitope on the carboxy-terminal region of the mycobacterial 65-kDa heat shock protein that distinguishes Mycobacterium tuberculosis/Mycobacterium bovis BCG from Mycobacterium leprae was identified by two novel monoclonal antibodies (mAbs), Ne5 and Nd4 . These mAbs also showed a limited cross reactivity with mycobacterial species belonging to M . tuberculosis complex and Mycobacterium avium complex with the exception of Mycobacterium vaccae . Characterization of the epitope recognized by these mAbs was done with M . bovis BCG 65-kDa fusion proteins expressed in Escherichia coli encoding various segments of the 65-kDa protein . Our results together with those reported in literature indicated that this epitope resides in the highly divergent region of amino acid residues 525 to 540 . This B-cell epitope on the 65-kDa protein of M . tuberculosis/M . bovis BCG has not been recognized by previously reported mAbs, although the analogous epitope sequence of M . leprae 65-kDa has been identified by a known mAb (IIIC8) reported in the literature . Therefore Ne5/Nd4 epitope could be considered important in studying the differential immune response of the host against infections with M . tuberculosis complex/M . avium complex and M . leprae.

Mol Biochem Parasitol, 1991 Feb, 44(2), 213 - 24
Modification of GP63 genes from diverse species of Leishmania for expression of recombinant protein at high levels in Escherichia coli; Button LL et al.; Toward the future development of a defined subunit vaccine against leishmaniasis is, high levels of recombinant GP63 for diverse species of Leishmania were produced in Escherichia coli . Several features of Leishmania GP63 genes were simultaneously modified with the polymerase chain reaction (PCR) using either cloned genes or total genomic DNA from Leishmania as template DNA for the PCR amplification reactions . The PCR products included only the coding region for the predicted mature form of GP63 that occurs on the surface of Leishmania, flanked by the appropriate translation signals and cloning sites for the production of recombinant GP63 as nonfusion protein in E . coli . When the codon usage in the GP63 gene was modified to reduce the guanine and cytosine content for the codons adjacent to the ATG initiation codon, rGP63 represented about 50% of total protein in E . coli . Mouse monoclonal antibodies raised against purified Leishmania major rGP63 had equivalent immunoblotting characteristics for native GP63 and recombinant GP63 with respect to linear determinants on GP63 expressed in diverse species of Leishmania . Human T cell lines and clones were derived from a patient infected with Leishmania braziliensis panamensis using rGP63 purified from an L . major GP63 expression clone as antigen.

Mutat Res, 1991 Feb, 262(2), 79 - 84
In vivo benzo{a}pyrene diol epoxide-induced alkali-labile sites are not apurinic sites; Moran MF et al.; We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo{a}pyrene . DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent . The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV . We conclude that the alkali-labile sites formed in vivo by benzo{a}pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N2-guanine adducts . This finding questions the involvement of apurinic sites in the mutagenic activity of benzo{a}pyrene.

Lab Invest, 1991 Feb, 64(2), 295 - 9
Recruitment of neutrophils in the local endotoxin response: association with de novo endothelial expression of endothelial leukocyte adhesion molecule-1; Munro JM et al.; Endothelium is central to the cellular infiltration that develops during inflammation, and a prominent feature of its involvement is the expression of adhesion molecules for circulating leukocytes . In the present study, we assessed the kinetics of endothelial adhesion molecule expression during the cutaneous endotoxin response in baboons . Immunostained cryostat sections and hematoxylin and eosin-stained paraffin sections of skin biopsies were examined using set scoring systems to provide semiquantitative data on the changes in endothelial phenotype and induced polymorphonuclear leucocyte (PMN) accumulation . Endothelium in control skin did not express endothelial leukocyte adhesion molecule (ELAM)-1 but did show a relatively weak expression of intercellular adhesion molecule (ICAM)-1 . By 2 hours after injection of endotoxin (500 mcg of Escherichia coli-derived lipopolysaccharide), a marked expression of ELAM-1 developed that was associated with concurrent extensive adhesion and extravasation of PMN . The ELAM-1 expression subsequently decreased and was virtually absent by 9 hours . Mean scores for endothelial expression of ICAM-1 changed comparatively little over this time course, and mononuclear cell accumulation was minimal . The response to endotoxin differs from that to tumor necrosis factor injection; the latter causes sustained ELAM-1 expression, and delayed but pronounced increases in ICAM-1, with accompanying mononuclear cell extravasation . Thus, local endotoxin administration provides a model of acute inflammation in which PMN accumulation is associated with striking endothelial expression of ELAM-1 . In this model, appreciable elevations in ICAM-1 expression are unnecessary for PMN infiltration.

Biochem J, 1991 Feb 1, 273 ( Pt 3), 777 - 82
The use of thioglycollate to demonstrate DNA AP (apurinic/apyrimidinic-site) lyase activities . Biological consequences of thiol addition to the 5' product of a beta-elimination reaction at an AP site in DNA; Bricteux-Gregoire S et al.; Thioglycollate reacts with the 5' product of AP lyase activity on apurinic/apyrimidinic (AP) sites in DNA . The 3'-terminal thioglycollate-unsaturated sugar 5-phosphate adduct can be released by the use of Escherichia coli endonuclease IV or endonuclease VI, and identified by DEAE-Sephadex chromatography . In contrast, the mammalian AP endonuclease is unable to excise a 3'-terminal thiol-unsaturated sugar adduct; this lesion, which must sometimes occur in vivo, might be irreparable and have pathological consequences.

J Bacteriol, 1991 Feb, 173(4), 1460 - 70
Suppressors that permit A-signal-independent developmental gene expression in Myxococcus xanthus; Kaplan HB et al.; Progression through the early stages of Myxococcus xanthus fruiting body development requires the cell-to-cell transmission of soluble material called A signal . During these early stages, expression from the gene identified by Tn5 lac insertion omega 4521 increases . A DNA probe of the omega 4521 gene was constructed . Use of this probe showed that accumulation of mRNA corresponding to the omega 4521 gene depends upon A signal . A-signal-deficient (asg) mutants fail to accumulate this RNA, and the external addition of A signal restores accumulation . To identify links between A signal and its responsive gene, omega 4521, suppressors of an asg mutation were generated . All of the suppressor alleles restored lacZ expression from omega 4521 in the absence of A signal, and they were demonstrated to be neither reversions of the asgB mutation nor mutations in the promoter of omega 4521 . Fifteen suppressor mutations map to two loci, sasA and sasB (for suppressor of asg) . sasA and sasB mutants differ phenotypically during growth and development . Mid-logarithmic-phase sasA asgB double mutants, like sas+ asg+ strains, express low levels of lacZ, whereas sasB asgB double mutants express high levels . sasA asg+ mutants form abnormal colonies, are less cohesive than wild type, and are defective in fruiting body formation and sporulation . In contrast, sasB asg+ mutants form normal colonies, are as cohesive as wild type, and appear to develop normally . The characteristics of sasA suppressors implicate the sasA+ product as a negative regulator in the A-signal-dependent regulation of omega 4521.

J Bacteriol, 1991 Feb, 173(4), 1452 - 9
Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product; Gassmann GS et al.; The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli . The structural gene encodes a flagellar protein of 336 amino acids . Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria . The antigenic properties of the B . burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins . No single species-independent immunodominant epitope could be located . However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260) . This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis.

Proc Natl Acad Sci U S A, 1991 Feb 1, 88(3), 1014 - 8
Continuous microspectrophotometric measurement of DNA polymerase activity: application to the Klenow fragment of Escherichia coli DNA polymerase I and human immunodeficiency virus type 1 reverse transcriptase; Baillon JG et al.; Progress of DNA- and/or RNA-directed DNA polymerization reactions can be measured continuously using circular dichroism (CD) or ultraviolet (UV) spectroscopy . In the presence of the Klenow fragment of Escherichia coli DNA polymerase I, a CD change of -0.27 +/- 0.06 millidegree at 248 nm and a UV change of -2.7 +/- 0.3 milliabsorbance units at 275 nm occur upon incorporation of 120 pmol of dTMP in a reaction volume of 120 microliters (1 microM dTMP incorporation) into a synthetic template-primer, p(dA)40-60.p(dT)20 . The transcription of poly(A).p(dT)12-18 by reverse transcriptases can also be monitored using these methods . Kinetic parameters for the polymerization reaction catalyzed by the Klenow fragment were determined from initial velocity measurements using CD or UV assays and were in close agreement with those measured by the standard single point radiochemical filtration assay . The generality of optical techniques for the measurement of DNA polymerase activity was shown by the use of a partially self-complementary hairpin-shaped oligonucleotide substrate for the Klenow fragment . Addition of a single nucleotide residue under steady-state conditions to this 35-mer at a concentration of 1.5-3 microM gave an easily measurable absorbance decrease at 275 nm, and the absorbance changes upon sequential addition of nucleotide units were additive.

J Bacteriol, 1991 Feb, 173(3), 1168 - 74
An unusual correlation between ppGpp pool size and rate of ribosome synthesis during partial pyrimidine starvation of Escherichia coli; Vogel U et al.; Escherichia coli was exposed to partial pyrimidine starvation by feeding a pyrBI strain orotate as the only pyrimidine source . Subsequently, differential rates of synthesis of rRNA and of a few ribosome-associated proteins as well as the pool sizes of nucleoside triphosphates and ppGpp were measured . As the orotate concentration in the medium was reduced, the growth rate decreased and the pools of pyrimidine nucleotides, particularly UTP, declined . We did not observe the normal inverse relation between concentration of ppGpp and growth rate; rather, we observed that the ppGpp pool was low at slow growth rates . Upshifts in growth rate were made by adding uracil to a culture growing slowly on orotate . Downshifts could be provoked by adding aspartate plus glutamate to a culture growing at a high concentration of orotate . Following the upshift, both the rates of synthesis of the ribosomal components and the pool of ppGpp increased rapidly, while they all decreased after the downshift . These results are discussed in relation to the role of ppGpp in the growth rate control and the stringent response.

J Virol, 1991 Feb, 65(2), 904 - 12
Inactivation of the Moloney murine leukemia virus long terminal repeat in murine fibroblast cell lines is associated with methylation and dependent on its chromosomal position; Hoeben RC et al.; The expression of a retroviral vector with the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) promoter after integration into the genome of murine fibroblast cell lines was monitored with the Escherichia coli-derived beta-galactosidase (beta-gal) gene as the reporter . Monoclonal cell lines derived after retroviral infection exhibited a marked heterogeneity in their expression of the reporter gene . We studied two monoclonal cell lines with a single unrearranged copy of the vector provirus integrated into their genome . The first, BB10, expressed the marker enzyme in only 8% of its cell population, whereas in the second, BB16, beta-gal expression could be detected in over 98% of the cells . Treatment of BB10 with the DNA-demethylating agent 5-azacytidine raised the number of beta-gal-positive cells to over 60% . Transfection experiments showed that the Mo-MuLV LTR promoter-enhancer is potentially fully functional in both the BB10 and BB16 cell lines . The inactivated provirus from BB10 cells was cloned and subsequently used to generate retrovirus stocks . The promoter-enhancer activity of its LTR after infection with these BB10-derived viruses showed a variation similar to that of the original virus stocks . Our data showed that (1) inactivation of the Mo-MuLV LTR is a frequent event in murine fibroblast cell lines, (2) inactivation is associated with de novo methylation of cytidine residues, (3) the frequency of inactivation of the provirus must be determined by its chromosomal position, (4) the process of methylation of sequences within the LTR is not necessarily the same as the transcription-repression mechanism that is operating in undifferentiated embryonal carcinoma cells.

Infect Immun, 1991 Feb, 59(2), 514 - 20
Immunoreactive epitopes on an expressed recombinant flagellar protein of Borrelia burgdorferi; Collins C et al.; A recombinant Borrelia burgdorferi flagellin protein expressed in Escherichia coli is bound by a murine monoclonal antiflagellin antibody (H9724) and by antibodies in the sera of patients with Lyme disease . Immunoreactive epitopes on the flagellar protein were identified by immunoblot analysis of antibody binding to expressed truncated flagellar proteins . The epitope recognized by the murine monoclonal antibody is within the central heterologous region of the flagellar protein (amino acids 90 to 266) . However, antiflagellin antibodies in the sera of patients with Lyme arthritis bound an epitope entirely within, or whose conformation was partly formed by, the 90 NH2-terminal amino acids of the flagellar protein . The binding of antibodies in the sera of patients with Lyme arthritis to the NH2-terminal region of the flagellar protein, a region with sequence homology to the flagellar proteins of other bacterial species, suggests the possibility that antigenic mimicry contributes to the immunopathogenesis of Lyme disease . The fact that human antibodies bind to a highly conserved and hence shared portion of the flagellin reduces the specificity of serological assays for the diagnosis of Lyme disease which use the flagellar protein as antigen.

Biochem J, 1991 Feb 1, 273 ( Pt 3), 787 - 90
Expression in Escherichia coli, purification and characterization of two mammalian thioesterases involved in fatty acid synthesis; Naggert J et al.; Thioesterase I, a constituent domain of the multifunctional fatty acid synthase, and thioesterase II, an independent monofunctional protein, catalyse the chain-terminating reaction in fatty acid synthesis de novo at long and medium chain lengths respectively . The enzymes have been cloned and expressed in Escherichia coli under the control of the temperature-sensitive lambda repressor . The recombinant proteins are full-length catalytically competent thioesterases with specificities indistinguishable from those of the natural enzymes.

Genes Dev, 1991 Feb, 5(2), 265 - 77
Autoregulation of a segmentation gene in Drosophila: combinatorial interaction of the even-skipped homeo box protein with a distal enhancer element; Jiang J et al.; Autoregulation has been implicated in the expression of many patterning genes in Drosophila, but the molecular details of this process are largely unknown . In the case of the segmentation gene even-skipped (eve), autoregulation is important for the specification of sharp stripes of gene expression at the onset of gastrulation . Here, we use a combination of DNA binding and P-transformation assays to characterize the cis- and trans-acting factors responsible for autoregulation . We show that eve autoregulation is mediated, at least in part, by a 100-bp minimal autoregulatory sequence (MAS) located approximately 5 kb upstream from the eve transcription start site . Multimerization of a 200-bp DNA fragment that encompasses the MAS drives optimal autoregulatory activity, comparable to that obtained with the native distal enhancer element located between -5.9 and -5.2 kb . The MAS contains two eve protein-binding sites, as well as binding sites for two nuclear factors present in early embryos . Directed mutagenesis of these binding sites suggests that both the eve protein and nuclear factors are essential for autoregulation . These results provide evidence that the eve protein acts combinatorially with other transcription factors to enhance its own expression.

J Bacteriol, 1991 Feb, 173(3), 1230 - 40
Genetic analysis of 987P adhesion and fimbriation of Escherichia coli: the fas genes link both phenotypes; Schifferli DM et al.; The 987P fimbrial gene cluster has recently been shown to contain eight genes (fasA to fasH) clustered on large plasmids of enterotoxigenic Escherichia coli and adjacent to a Tn1681-like transposon encoding the heat-stable enterotoxin STIa . Different genetic approaches were used to study the relationship between 987P fimbriation and adhesion . TnphoA mutagenesis, complementation assays, and T7 RNA polymerase-promoted gene expression indicated that all of the fas genes were involved in fimbrial expression and adhesion . In contrast to other fimbrial systems, the lack of expression of any single fas gene never resulted in the dissociation of fimbriation and adhesion, indicating that the adhesin is required for fimbrial expression and suggesting that FasA, the fimbrial structural subunit itself, is the adhesin . In addition, fimbrial length was shown to be modulated by the levels of expression of different fas genes.

EMBO J, 1991 Feb, 10(2), 239 - 45
Heat-shock proteins can substitute for SecB function during protein export in Escherichia coli; Altman E et al.; In this study we have shown that (i) induction of the heat-shock response can substitute for SecB function in Escherichia coli, (ii) SecB itself is not a heat-shock protein and (iii) a basal level of heat-shock proteins is required for cells to grow in the absence of a functional SecB protein . Overproduction of DnaK, or GroEL/ES, which were candidates for the heat-shock proteins that could substitute for SecB function, did not rescue the export defect caused when SecB was limiting or absent . In an attempt to identify the heat-shock protein(s) which could substitute for SecB function, unlinked suppressors of secB were isolated and characterized . Interestingly, most of the suppressors mapped to the rpoH locus . Since rpoH encodes sigma 32, the heat-shock transcription factor, it is likely that these suppressors affect the synthesis levels of heat-shock proteins that can substitute for SecB function . The remaining suppressors did not map to any known heat-shock or export genes . Collectively, our data suggest that these suppressors may represent unidentified heat-shock proteins or export factors that act in a manner similar to SecB in facilitating the export process in E . coli.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 289 - 94
Complex phenotypes of null mutations in the htr genes, whose products are essential for Escherichia coli growth at elevated temperatures; Karow M et al.; Transposon insertion, followed by screening, has allowed the identification of a set of genes, called htr, whose products are required for Escherichia coli growth at elevated temperatures . The htrB gene has been shown to map at 23.5 min on the E . coli genetic map . It codes for a very basic, hydrophobic, 35,000-Mr polypeptide, possessing a putative membrane-spanning domain . At the non-permissive temperature, htrB mutant bacteria stop dividing, followed by the formation of bulges and eventual lysis . The htrC gene maps at 90 min, is under sigma 32 regulation and codes for a 21, 130-Mr polypeptide . At 43 degrees C, htrC mutant bacteria gradually lyse, whereas at intermediate temperatures they filament extensively . Finally, the htrM gene maps at 81 min, is under sigma 32 regulation and codes for a 35,000-Mr polypeptide . The HtrM null phenotype included inability to grow above 42 degrees C, extreme mucoidness and sensitivity to bile salts, even at the permissive temperatures . The htrM gene is identical to the rfaD gene, whose product is required for the biosynthesis of the lipopolysaccharide precursor ADP-L-glycero-D-mannoheptose (Pegues et al., J . Bact., 1990, 172, 4652-4660).

Res Microbiol, 1991 Feb-Apr, 142(2-3), 269 - 77
Functional analysis of the fic gene involved in regulation of cell division; Komano T et al.; We have shown that cAMP may be a regulation factor in cell division of Escherichia coli (Utsumi et al., 1982, 1989) . The fic (filamentation induced by cAMP) gene of this system found previously (Utsumi, et al., 1982) was analysed in this study . The open reading frame of the fic gene coded for 200 amino acids . The pabA (p-aminobenzoate synthase) gene was found downstream from the fic gene . The distance between the end of fic and the start of pabA was 31 base pairs . To deduce the function of Fic protein, the fic gene was destroyed by the kanamycin-resistant (Kmr) gene and the fic gene was shown to be essential for growth of E . coli . Such mutants required PAB (p-aminobenzoate) or folate for growth . These data suggested that the Fic protein is involved in the synthesis of PAB or folate and the fic gene could be part of a pab operon . In cells starved of them, cell division was inhibited . Addition of folate also repressed the filamentation induced by cAMP at 43 degrees C in the fic-1 mutant . These results would indicate that Fic protein and cAMP are involved in a new regulatory mechanism of cell division via folate metabolism . Furthermore, it is also shown that cell division could be controlled by coordination of cAMP, Fic and Fts proteins.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 223 - 7
DNA replication in Escherichia coli gyrB(Ts) mutants analysed by flow cytometry; von Freiesleben U et al.; We have investigated the initiation of DNA replication in Escherichia coli gyrB (Ts) mutants and find that initiations in single cells, even those that occur at non-permissive temperature, are synchronous . Furthermore, our results indicate that the gradual arrest of DNA replication at non-permissive temperature reflects a general decrease in transcription activity and not an initiation-specific function of the DNA gyrase . At an intermediate temperature, the only effect observed was a lack of segregation (decatenation) of replicated chromosomes in a sizeable fraction of the cell population.

J Toxicol Sci, 1991 Feb, 16 Suppl 1, 95 - 105
8-Hydroxyguanine, a DNA adduct formed by oxygen radicals: its implication on oxygen radical-involved mutagenesis/carcinogenesis; Kasai H et al.; Oxygen radicals have been suggested to be involved in mutation/carcinogenesis . The C-8 position of guanine residues in DNA is hydroxylated to produce 8-hydroxyguanine (8-OH-Gua) in DNA in vitro by various oxygen radical producing agents . The formation of 8-OH-Gua was also observed in cellular DNA in vivo by radiation or oxygen radical forming carcinogens . The 8-OH-Gua residue in DNA is often misread in the position of 8-OH-Gua residue itself but also at neighboring residues next to 8-OH-Gua . When second guanine in codon 12 was specifically replaced with 8-OH-Gua and transferred to NIH3T3, the recipient cells were transformed to malignant cell type . E . coli was found to contain an endonuclease which specifically recognizes 8-OH-Gua residue and cleave DNA strand before and after the modified base . The data obtained imply that 8-OH-Gua formed in DNA in vivo is recognized as an abnormal modified base which, if not repaired, play a role in the mediation of oxygen radical-involved mutation/carcinogenesis.

Biochimie, 1991 Feb-Mar, 73(2-3), 187 - 90
Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA; Muniyappa K et al.; The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected . The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction . The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA . We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I . These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination.

J Virol Methods, 1991 Feb-Mar, 31(2-3), 229 - 38
Influence of the helper virus on expression of beta-galactosidase from a defective HSV-1 vector, pHSVlac; Geller AI; Defective herpes simplex virus type 1 (HSV-1) vectors can deliver genes into both mitotic and postmitotic cells, including neurons and these vectors, therefore, have great potential use . pHSVlac, the prototype HSV-1 vector, expresses the E . coli Lac Z gene from the HSV-1 immediate early 4/5 promoter . pHSVlac can stably express beta-galactosidase in a range of mammalian cell lines and in neurons from throughout the nervous system, both in culture and in the adult rat brain . Thus, HSV-1 vectors may be useful for studying HSV-1 latency, neuronal physiology, and performing gene therapy for neurological conditions . A virus stock of pHSVlac consists of identical HSV-1 particles containing either pHSVlac DNA or the HSV-1 helper virus DNA . Thus, a cell can be infected with the pHSVlac virus, the helper virus, or both; consequently, it is important to determine if the helper virus influences the behavior of pHSVlac . The effect of the helper virus on expression of beta-galactosidase from pHSVlac was investigated: it was demonstrated first, that pHSVlac can be efficiently packaged into HSV-1 virus particles using each of five HSV-1 temperature sensitive (ts) mutants as helper virus . Second, pHSVlac grown with each of the five HSV-1 ts mutants expressed high levels of beta-galactosidase . Third, pHSVlac grown with HSV-1 strain 17 ts K as helper virus expresses the same amount of beta-galactosidase in the absence or presence of ts K . Thus, pHSVlac can efficiently express a gene independent of the HSV-1 helper virus.

Protein Eng, 1991 Feb, 4(3), 245 - 50
Structure of alpha-alpha-hairpins with short connections; Efimov AV; This paper presents the results of detailed stereochemical analysis of structures and sequences of alpha-alpha-hairpins with short connections . It is shown that alpha-alpha-hairpins of each given type have very similar patterns of hydrophobic, hydrophilic and glycine residues in their amino acid sequences . These results can be used in the prediction of alpha-alpha-hairpin conformation as well as in protein design and engineering.

Vaccine, 1991 Feb, 9(2), 89 - 96
Genetically-engineered subunit vaccine against feline leukaemia virus: protective immune response in cats; Marciani DJ et al.; A recombinant retroviral subunit vaccine has been developed that successfully protects cats from infectious feline leukaemia virus (FeLV) challenge . The antigen used is a non-glycosylated protein derived from the envelope glycoprotein of FeLV subgroup A, expressed in Escherichia coli . This recombinant protein, rgp70D, includes the entire exterior envelope protein gp70, plus the first 34 amino acids from the transmembrane protein p15E . The vaccine consists of purified rgp70D absorbed on to aluminium hydroxide and used in conjunction with a novel saponin adjuvant . Cats immunized with this formulation developed a strong humoral immune response, including neutralizing and feline oncornavirus-associated cell membrane antigen antibodies . Vaccinated animals showed an anamnestic response upon intraperitoneal challenge with FeLV-A, and were protected from viral infection . In contrast, the control animals developed viraemia shortly after the challenge, which in most cases became chronic . Formulation of the same antigen with other widely used adjuvants elicited poor protective immune responses in cats.

Mol Microbiol, 1991 Feb, 5(2), 433 - 7
Design of cAMP-CRP-activated promoters in Escherichia coli; Valentin-Hansen P et al.; We have studied the deoP2 promoter of Escherichia coli to define features that are required for optimal activation by the complex of adenosine 3',5' monophosphate (cAMP) and the cAMP receptor protein (CRP) . Systematic mutagenesis of deoP2 shows that the distance between the CRP site and the -10 hexamer is the crucial factor in determining whether the promoter is activated by cAMP-CRP . Based on these observations, we propose that cAMP-CRP-activated promoters can be created by correctly aligning a CRP target and a -10 hexamer . This idea has been successfully tested by converting both a CRP-independent promoter and a sequence resembling the consensus -10 hexamer to strongly cAMP-CRP-activated promoters.

Biotechnology (N Y), 1991 Feb, 9(2), 170 - 2
A genetic system to elicit and monitor antipeptide antibodies without peptide synthesis; Martineau P et al.; We present a simple and flexible procedure to elicit and assay anti-peptide antibodies without peptide synthesis . It consists of expressing the peptide of interest in the form of a genetic insert within two different "recipient" bacterial proteins . One hybrid protein is used as immunogen for the induction of antibodies against the inserted peptide and the other as antigen for monitoring the anti-peptide antibodies raised . The two "recipient" proteins used are the MalE and the LamB proteins from E . coli . The MalE hybrid proteins can be affinity purified on an amylose column using mild nondenaturing conditions and can be crystalized for structural studies; LamB hybrid proteins express the inserted peptide on the cell surface so that intact bacteria can be used as a reagent . We chose, as a model peptide, a B-cell epitope from the pre-S(2) region of Hepatitis B virus . With both MalE and LamB hybrid proteins, high titres of anti-preS antibodies, able to react with native HBsAg particles, were induced in mice . The anti-peptide antibody titres recorded by ELISA were comparable to those obtained when either a synthetic peptide, or the hybrid proteins, were used as immobilized antigen.

Biotechnology (N Y), 1991 Feb, 9(2), 157 - 62
Renaturation, purification and characterization of recombinant Fab-fragments produced in Escherichia coli; Buchner J et al.; Cytoplasmatic expression of murine antibody chains in Escherichia coli results in the formation of insoluble and inactive protein aggregates (inclusion bodies) . By systematic variation of the parameters influencing the folding, formation of disulfide bonds and association of the constituent polypeptide chains, we have designed a renaturation procedure allowing the production of microbially expressed Fab-fragments at yields up to 40 percent of the total amount of recombinant protein . The strategy of optimization is generally applicable for disulfide containing proteins produced as inclusion bodies in bacteria . The purified recombinant antibody fragments obtained are identical with the native murine Fab in all functional and physicochemical parameters tested.

Appl Microbiol Biotechnol, 1991 Feb, 34(5), 632 - 6
Heterologous gene expression in continuous cultures of budding yeast; Porro D et al.; In a previous paper we have studied the expression of beta-galactosidase from Escherichia coli, driven from the inducible GAL1-10/CYC1 hybrid promoter, in batch cultures of budding Saccharomyces cerevisiae and have described operating conditions for maximal productivity . In this paper we show that the plasmid instability in continuous cultures can be overcome by utilizing appropriate selection markers and a high copy number vector . The maximal level of expression is influenced by the dilution rate . Moreover, enzyme accumulation appears to depend also upon the degree of oxygenation . A possible explanation of these modulations is discussed, taking into account the interactions of the UAS-GAL and TATA-CYC1 elements.

Appl Microbiol Biotechnol, 1991 Feb, 34(5), 623 - 7
Application of the tryptophanase promoter to high expression of the tryptophan synthase gene in Escherichia coli; Terasawa M et al.; The application of an inducible regulation system using the tryptophanase operon promoter (TPase promoter; Ptna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli . The main problem in the application of Ptna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source . However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed . Under these conditions, L-tryptophan was also used to induce tryptophan synthase . Thus, the specific activity of TS in E . coli strain no . 168 harbouring pBR322F-Ptna TS was increased 500-fold compared to that of the cultured host strain . About 1 mol L-tryptophan/l reaction mixture was formed from indole and L-serine at 37 degrees C for 3.5 h.

Enzyme Microb Technol, 1991 Feb, 13(2), 127 - 33
Design of two immobilized cell catalysts by entrapment on gelatin: internal diffusion aspects; Castillo E et al.; Experimental results obtained during the design of two immobilized cell catalysts by entrapment on gelatin are presented . Strong diffusional limitations are found and explained with the usual parameters and models, introducing an empirical correlation between substrate concentration and effectiveness factor . The effect of particle size, enzyme load, and specific activity in the system is discussed in terms of cooperation between bioengineers and geneticists.

Lett Appl Microbiol, 1991 Feb, 12(2), 39 - 42
Electroporation-induced transformation of Escherichia coli: evaluation of a square waveform pulse; Elvin S et al.; Four strains of Escherichia coli were tested for electroporation-induced plasmid transformation using a high voltage pulse in a square waveform . Conditions for optimal transformation were determined for each strain and transformation frequencies found to be comparable to those reported previously for E . coli with a sinusoidal waveform of exponential decaying pulse.

J Biotechnol, 1991 Feb, 17(2), 177 - 88
Cloning of three endoglucanase genes from Thermomonospora curvata into Escherichia coli; Presutti DG et al.; A BamHI genomic library from Thermomonospora curvata was constructed in E . coli using cosmid vector pHC79 . Four clones able to hydrolyze CMC were isolated . Restriction digests and Southern gel analysis revealed the presence of three different endoglucanase genes . DNA fragments contained in all of the endoglucanase cosmids hybridized to T . curvata chromosomal DNA . The cellulase genes were expressed in E . coli, but at rather low levels.

Biochem Biophys Res Commun, 1991 Jan 31, 174(2), 898 - 902
A model for histone H5-DNA interaction: simultaneous minor and major groove binding; Segers A et al.; Using the tertiary structure of the globular domain of H5 (GH5) and based on an alternative sequence homology between GH5 and DNA-binding proteins containing the helix-turn-helix motif, a model for H5-DNA interaction is proposed . From molecular graphics it follows that helix II recognizes the major groove of the DNA, as does the second helix of the helix-turn-helix motif, while helix III makes minor groove contacts, in agreement with the hypothesis of Turnell et al . (FEBS letters 232, 263-268) . In the resulting model GH5 makes contact with a full turn of DNA.

Biochem Biophys Res Commun, 1991 Jan 31, 174(2), 529 - 35
Molecular cloning and expression of a human brain inositol 1,4,5-trisphosphate 3-kinase; Takazawa K et al.; A human hippocampus cDNA library was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA . Sequencing of three overlapping clones identified a 1383 bp open reading frame encoding a 461 amino acid protein with a calculated molecular weight of 50988 . The coding amino acid sequence showed an overall 93% similarity with the sequence of rat brain InsP3 3-kinase . The cDNA insert of one isolated partial clone (i.e . hh 26) was in frame with the beta galactosidase fragment fused to it as a Bluescript plasmid; it displayed InsP3 3-kinase activity when expressed in Escherichia coli (E . coli) . Biochemical characterization of human brain InsP3 3-kinase by SDS polyacrylamide gel electrophoresis and regeneration of enzyme activity reveals three active fractions with apparent Mr of 58,000-64,000, 45,000-50,000 and 37,000-39,000.

Biochem Biophys Res Commun, 1991 Jan 31, 174(2), 785 - 9
Crosslinking of substrates occurs exclusively to the p66 subunit of heterodimeric HIV-1 reverse transcriptase; Cheng N et al.; Photoaffinity labeling of the hetero- and homodimeric forms of HIV-1 reverse transcriptase has been carried out using {32P}rA12-18.dT10 as a representative template-primer and {alpha-32P}dTTP as a representative 2'-deoxynucleoside-5'-triphosphate . UV irradiation produces stable, covalent crosslinks between each of the reactants and both the hetero-(p66/p51) and homodimeric (p66/p66, p51/p51) forms of the enzyme . In the case of the p66/p51 heterodimer, the form of the enzyme believed to be involved in viral replication, crosslinking occurs exclusively to the p66 subunit . These results suggest that the polymerase activity of the heterodimer residues on p66.

Biochim Biophys Acta, 1991 Jan 30, 1061(2), 171 - 4
Reconstitution of the L-leucine-H+ cotransporter of the plasma membrane from Chang liver cells into proteoliposomes; Mitsumoto Y et al.; L-Leucine is cotransported with H+ in the plasma membrane of Chang liver cells (Mitsumoto, Y . et al . (1986) J . Biol . Chem . 261, 4549) . The leucine transport system was solubilized from the plasma membrane of the cells with ocytl glucoside and reconstituted in proteoliposomes prepared by a rapid dilution of a mixture of the solubilized proteins, octyl glucoside and liposomes . The proteoliposomes exhibited H(+)-gradient and electrical potential-stimulated leucine uptake . The H(+)-gradient-stimulated leucine uptake could be completely inhibited by carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) and 2-aminobicyclo{2,2,1}heptane-2-carboxylic acid (BCH) . The stimulatory effect of H+ gradient on leucine uptake was shown to be mainly due to decrease of the Km, but not to change of the Vmax, of the transport kinetics . These results suggest that the leucine-H+ cotransporter is solubilized and reconstituted into proteoliposomes.

Biochim Biophys Acta, 1991 Jan 29, 1076(2), 289 - 97
Possible regulation of the in vitro assembly of bovine brain tubulin by the bovine thioredoxin system; Khan IA et al.; Microtubule assembly in vitro and in vivo is highly sensitive to a variety of sulfhydryl-reactive reagents, raising the question of the possible existence of a physiological sulfhydryl-mediated system for regulating microtubule assembly . However, the specific reagents which have previously been used to inhibit microtubule assembly in vitro are either nonphysiological or, if physiological, effective only at concentrations much higher than their physiological ones . Because of reports of association in vivo between microtubules and the sulfhydryl-reactive proteins thioredoxin and thioredoxin reductase, we decided to examine the interaction in vitro between microtubules and the thioredoxin system, comprising thioredoxin, thioredoxin reductase and NADPH . At pH 6.8, both the mammalian and the Escherichia coli thioredoxin systems inhibited microtubule assembly by 4-35% (19 +/- 9%) by reducing one intra-subunit disulfide bond in the tubulin dimer . The thioredoxin-reducible disulfide of the tubulin dimer remains protected from thioredoxin in the assembled microtubules . Thioredoxin or thioredoxin reductase alone, or together in the absence of NADPH, were incapable of either reducing tubulin or inhibiting microtubule assembly . Microtubules formed from reduced tubulin were found to be stable and morphologically identical to those obtained from native tubulin dimers . Since the components of the thioredoxin system were used at concentrations similar to their physiological ones, our results suggest a potential role of the thioredoxin system in regulation of microtubule assembly in vivo.

Biochim Biophys Acta, 1991 Jan 29, 1076(2), 266 - 72
Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy; Altamirano MM et al.; The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods . The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme . From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-sheet, 15% turns and 35% of non-repetitive structures, were estimated . A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23% . The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic . Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure.

Biochemistry, 1991 Jan 29, 30(4), 1141 - 8
An alkaline phosphatase protection assay to investigate trp repressor/operator interactions; Marmorstein RQ et al.; We have used an alkaline phosphatase protection assay to investigate the interaction of the trp repressor with its operator sequence . The assay is based on the principle that the trp repressor will protect a terminally 5'-32P-labeled operator DNA fragment from attack by alkaline phosphatase . The optimal oligonucleotide for investigating the trp repressor/operator interaction extends two base pairs from each end of the genetically defined target sequence predicted by in vivo studies {Bass et al . (1987) Genes Dev . 1, 565-572} . The assay works well over a 10,000-fold range of protein/DNA affinity and is used to show that the corepressor, L-tryptophan, causes the liganded repressor to bind a 20 base pair trp operator duplex 6400 times more strongly than the unliganded aporepressor . The affinity of the trp repressor for operators containing symmetrical mutations was interpreted in terms of the trp repressor/operator crystal structure as follows: (1) Direct hydrogen bonds with the functional groups of G-9 of the trp operator and the side chain of Arg 69 of the trp repressor contribute to DNA-binding specificity . (2) G-6 of the trp operator is critical for DNA-binding specificity probably because of the two water-mediated hydrogen bonds between its functional groups and the N-terminus of the trp repressor's E-helix . (3) Sequence-dependent aspects of the trp operator's conformation help stabilize the trp repressor/operator complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Jan 29, 30(4), 1091 - 7
32P-postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini; Weinfeld M et al.; A 32-P-postlabeling assay has been developed that permits detection of several radiogenic base and sugar lesions of DNA at the femtomole level . The technique is based on the inability of DNase I and snake venom phosphodiesterase to cleave the internucleotide phosphodiester bond immediately 5' to the site of damage so that complete digestion of irradiated DNA with these nucleases and alkaline phosphatase yields lesion-bearing "dinucleoside" monophosphates . Because these fragments contain an unmodified nucleoside at the 5'-end of each molecule, they can be readily phosphorylated by T4 polynucleotide kinase and {gamma-32P}ATP and analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC . We observed a linear induction of total damage in DNA irradiated with 5-50 Gy . Virtually no damage was detected when the DNA was irradiated in solution containing 1 M DMSO, implicating hydroxyl radicals in the formation of these lesions . Evidence for the presence of thymine glycols and phosphoglycolate groups came from (i) a comparison of the radiation-induced products with those produced by OsO4 and KMnO4 and (ii) incubation of irradiated DNA with Escherichia coli endonuclease III and exonuclease III before analysis by the postlabeling procedure . This was confirmed by comigration of the radiogenic products with chemically synthesized markers . G values of 0.0022 and 0.0105 mumol J-1 were obtained for thymine glycol and phosphoglycolate production, respectively . The identity of the 5'-nucleotide of each isolated compound was obtained by nuclease P1 digestion . This analysis of nearest-neighbor bases to thymine glycols and phosphoglycolates indicated a nonrandom interaction between radiation-induced hydroxyl radicals and DNA.

Biochim Biophys Acta, 1991 Jan 29, 1076(2), 225 - 32
Sequence similarities within the family of dihydrolipoamide acyltransferases and discovery of a previously unidentified fungal enzyme; Russell GC et al.; A composite protein sequence database was searched for amino acid sequences similar to the C-terminal domain of the dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli . Nine sequences with extensive similarity were found, of which eight were E2 subunits . The other was for a putative mitochondrial ribosomal protein, MRP3, from Neurospora crassa . Alignment of the MRP3 and E2 sequences showed that the similarity extends through the entire MRP3 sequence and that MRP3 is most closely related to the E2p subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae, with 54% identical residues and a further 36% that are conservatively substituted . Other features of the MRP3 gene and protein are also consistent with it being the acyltransferase subunit of a 2-oxo acid dehydrogenase complex . A multiple alignment of 13 E2 sequences indicated that 120 (34%) of 353 equivalenced residues are identical or show some degree of conservation . It also identified residues that are potentially important for the structure, catalytic activity and substrate-specificity of the acyltransferases.

Biochemistry, 1991 Jan 29, 30(4), 1097 - 118
A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli; Yager TD et al.; In the first part of this paper we present a thermodynamic analysis of the elongation phase of transcription in Escherichia coli . The stability of the elongation complex is described by a "free energy of formation" function (delta G zero f) that is a sum of terms for forming (i) a locally denatured 17-base-pair DNA "bubble"; (ii) a constant-length hybrid between the 3'-terminal 12-nucleotide residues of the RNA transcript and the corresponding region of the DNA template strand; and (iii) a set of binding interactions between the polymerase and certain DNA and RNA residues within and near the "transcription bubble" . The transcriptional elongation complex is very stable at most positions along a natural DNA template and moves in a highly processive fashion . At these positions, the delta G zero f function provides a quantitative measure of the stability of the elongation complex . Besides allowing for the polymerization of the RNA transcript, the elongation complex also serves to define the context within which transcript termination occurs . In the second part of the paper the thermodynamic analysis is extended to discriminate between template positions at which the elongation complex is stable and positions at which it is rendered relatively unstable by the presence of a string of rU residues at the 3'-terminus of the RNA together with the formation of a specific RNA hairpin just upstream of this point . Most factor-independent (intrinsic) termination events are thermodynamically disallowed at the former positions and are thermodynamically allowed at the latter positions . The extended form of the analysis closely predicts the exact sites of termination at a number of intrinsic terminators (and attenuators) in the E . coli genome . It also correctly predicts bidirectional function for a number of bidirectional terminators . In some cases it may identify terminators that are similar to the intrinsic type but that require additional protein factors, unusual polymerase-nucleic acid interactions, or rate-limiting conformational changes in order to function . Finally, it successfully locates intrinsic terminators within a number of E . coli operons and discriminates between these terminators and the surrounding DNA sequence.

FEBS Lett, 1991 Jan 28, 278(2), 225 - 8
Examination of elongation factor Tu for aluminum fluoride binding sites using fluorescence and 19F-NMR methodologies; Hazlett TL et al.; This article reports on a comparison of the interaction of Al3+ and F- with two GTP-binding proteins, elongation factor Tu (EF-Tu) and the hormone sensitive regulatory protein (G protein) G0 alpha . The methodologies chosen to elucidate possible interactions between protein and aluminum fluoride were fluorescence spectroscopy and nuclear magnetic resonance (19F-NMR) . Both proteins have tryptophan residues near their nucleotide binding sites, the purported site of aluminum fluoride interaction . It has been assumed for G proteins (including G0 alpha) that aluminum fluoride, in the presence of Mg2+ mimics the magnesium coordinated gamma-phosphate group for the GDP-form of the protein and shifts the protein's conformation toward the active GTP-form . Indeed, changes in intrinsic fluorescence of G0 alpha effected by aluminum fluoride are observed . The presence of aluminum fluoride did not affect the intrinsic fluorescence, spectra or lifetimes, of EF-Tu.GDP 19F-NMR was then used to directly test for bound F- . Fluoride alone or in the presence of either protein gave a single 19F-NMR peak at -10 ppm, characteristic of free F- . With the addition of aluminum to the protein and F- samples a second peak, shifted upfield from the first to -29 ppm, was observed for G0 alpha.GDP . This second peak, which has been assigned to protein-bound F-, was not observed for EF-Tu.GDP . These observations show that the interaction of Al3+ and F-, in the presence of Mg2+, may be quite different between the hormone-sensitive G proteins, which bind aluminum fluoride, and the GTP-binding proteins as a whole, which include EF-Tu . Care must therefore be exercised when structural data on the elongation factor, specifically on the nucleotide site, are used to interpret data or compose models intended to describe the hormone-sensitive regulatory G proteins.

Nucleic Acids Res, 1991 Jan 25, 19(2), 313 - 8
Training back-propagation neural networks to define and detect DNA-binding sites; O'Neill MC; A three layered back-propagation neural network was trained to recognize E . coli promoters of the 17 base spacing class . To this end, the network was presented with 39 promoter sequences and derivatives of those sequences as positive inputs; 60% A + T random sequences and sequences containing 2 promoter-down point mutations were used as negative inputs . The entire promoter sequence of 58 bases, approximately -50 to +8, was entered as input . The network was asked to associate an output of 1.0 with promoter sequence input and 0.0 with non-promoter input . Generally, after 100,000 input cycles, the network was virtually perfect in classifying the training set . A trained network was about 80% effective in recognizing 'new' promoters which were not in the training set, with a false positive rate below 0.1% . Network searches on pBR322 and on the lambda genome were also performed . Overall the results were somewhat better than the best rule-based procedures . The trained network can be analyzed both for its choice of base and relative weighting, positive and negative, at each position of the sequence . This method, which requires only appropriate input/output training pairs, can be used to define and search for any DNA regulatory sequence for which there are sufficient exemplars.

Nucleic Acids Res, 1991 Jan 25, 19(2), 307 - 11
Molecular cloning and characterization of the gene encoding the adenine methyltransferase M.CviRI from Chlorella virus XZ-6E; Stefan C et al.; The gene encoding the DNA methyltransferase M.CviRI from Chlorella virus XZ-6E was cloned and expressed in Escherichia coli . M.CviRI methylates adenine in TGCA sequences . DNA containing the M.CviRI gene was sequenced and a single open reading frame of 1137 bp was identified which could code for a polypeptide of 379 amino acids with a predicted molecular weight of 42,814 . Comparison of the M.CviRI predicted amino acid sequence with another Chlorella virus and 14 bacterial adenine methyltransferases revealed extensive similarity to the other Chlorella virus enzyme.

J Biol Chem, 1991 Jan 25, 266(3), 1728 - 32
Modified enterotoxin signal sequences increase secretion level of the recombinant human epidermal growth factor in Escherichia coli; Morioka-Fujimoto K et al.; Amino acid substitutions were made in the heat-labile enterotoxin signal sequence of Escherichia coli by recombinant DNA techniques, and their influence on the secretion of recombinant human epidermal growth factor by E . coli was examined . The heat-labile enterotoxin signal sequence is an amino-terminal extension of the octadecapeptide chain and is comprised of three distinct regions: a positively charged amino-terminal region, a central hydrophobic region, and a carboxyl-terminal region with the cleavage site recognized by the signal peptidase . Some alterations in the signal sequence caused a 1.5-3.5-fold increase in the secretion of recombinant human epidermal growth factor . These were the introduction of: (i) polar and small residues into the carboxyl-terminal region (replacement of Pro-1 Leu-3 with Asn-Ala or Ser-Ala), which may give a favorable structure for the recognition and cleavage by the signal peptidase; and (ii) a polar residue into the central hydrophobic region (replacement of Ile-9 with Ser), which may cause an increase of the affinity to the cytoplasmic membrane . In the latter case, a large amount of the unprocessed "precursor" was accumulated . The combination of these modifications, however, did not work additively . An increase in the amino-terminal positive charge (insertion of Lys) had no effect on secretion . These results prove that the level of protein secretion is greatly dependent on the polarity of the carboxyl-terminal region and the hydrophobicity and/or the amphiphilicity of the central region . Moreover, the overall balance of the physicochemical properties of respective regions is important.

J Biol Chem, 1991 Jan 25, 266(3), 1692 - 6
Yeast thioredoxin genes; Gan ZR; Based on the conserved protein sequence of thioredoxins from yeast and other organisms, two primers were synthesized for polymerase chain reaction of yeast genomic DNA . A 34-base pair (bp) sequence around the active site of yeast thioredoxin was obtained from the polymerase chain reaction product . This specific sequence was used as a probe in Southern blot analysis of total yeast genomic DNA digested with various restriction enzymes . Under conditions of high stringency, more than one DNA species hybridized with the probe, suggesting that more than one gene encodes yeast genomic library . Two Sau3A1 fragments, 825 and 2045 bp, respectively, from two different clones were cloned into pUC13 . Sequence analysis of these fragments gave two different open reading frames without introns . The 825-bp Sau3A1 fragment encodes a 103-amino acid residue protein named thioredoxin I . The 2045-bp Sau3A1 fragment contains a sequence encoding thioredoxin II which has 102 amino acid residues . This is the first report of the cloning and sequencing of eukaryotic thioredoxin genes from any source . Both yeast thioredoxins contain a dithiol active site sequence, Cys-Gly-Pro-Cys . Thioredoxins I and II show 78% amino acid sequence identity . They display more amino acid sequence similarity with mammalian thioredoxin than with Escherichia coli and plant chloroplast thioredoxins.

J Biol Chem, 1991 Jan 25, 266(3), 1616 - 26
Monomer-tetramer equilibrium of the Escherichia coli ssb-1 mutant single strand binding protein; Bujalowski W et al.; The Escherichia coli single strand binding (SSB) protein is an essential protein required for DNA replication and involved in recombination and a number of repair processes . It is a stable homotetramer in solution; however the ssb-1 mutation (His-55 to Tyr) destabilizes the tetramer with respect to monomers and this defect seems to explain the observed phenotype (Williams, K . R., Murphy, J . B., and Chase, J . W . (1984) J . Biol . Chem . 259, 11804-11811) . We report a quantitative study of the SSB-1 monomer-tetramer equilibrium in vitro as a function of temperature, pH, NaCl, MgCl2, urea, and guanidine hydrochloride concentrations . The self-assembly equilibrium was monitored by the increase in intrinsic protein fluorescence anisotropy accompanying the formation of the tetramer . The experimental isotherms indicate that SSB-1 dimers are not highly populated at equilibrium, hence the formation of the tetramer is well-described as a one-step association of four monomers . At 25 degrees C, pH 8.1, the monomer concentration for 50% tetramer dissociation is (MT)1/2 = 0.87 microM, corresponding to a monomer-tetramer equilibrium constant, KT = 3 +/- 1 x 10(18) M-3 . The tetramerization constant, KT, is highly dependent upon temperature and pH, with delta H0 = -51 +/- 7 kcal/mol (pH 8.1) and delta H0 = -37 +/- 5 kcal/mol (pH 6.9) . There is no effect of NaCl on the monomer-tetramer association in the range from 0.20 to 1.0 M; however, MgCl2 decreases the stability of the SSB-1 tetramer . In the presence of high concentrations of the single-stranded oligonucleotide, dT(pT)15, the tetramerization constant is slightly increased indicating that binding of the oligonucleotide to the SSB-1 monomer promotes the assembly process, although not dramatically . The large negative delta H0 that is associated with formation of the tetramer provides a likely explanation for the temperature sensitivity of the ssb-1 mutation.

J Biol Chem, 1991 Jan 25, 266(3), 1543 - 50
5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase; Stover P et al.; The interaction of the mono- and triglutamate forms of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate with serine hydroxymethyltransferase were determined by several methods . These methods included: determining dissociation constants by observing the absorbance at 502 nm of a ternary complex of the enzyme, glycine, and the folate compounds; determining inhibition constants from steady-state reactions; and determining the rate of formation and breakdown of the enzyme inhibitor complex by rapid reaction kinetics . Studies of the dissociation and inhibitor constants showed that both 5-methyltetrahydrofolate and 5-formyltetrahydrofolate have essentially the same affinity for the enzyme-glycine binary complex . However, rapid reaction and steady-state kinetic studies showed that the triglutamate form of 5-formyltetrahydrofolate both binds and is released much more slowly from the enzyme-glycine binary complex, compared with the triglutamate form of 5-methyltetrahydrofolate . The results also showed that only one rotamer of 5-formyltetrahydrofolate binds at the active site of serine hydroxymethyltransferase . The results are discussed in terms of the possible role of 5-formyltetrahydrofolate polyglutamates in regulation of one-carbon metabolism.

J Biol Chem, 1991 Jan 25, 266(3), 1469 - 77
Processing of DNA base damage by DNA polymerases . Dihydrothymine and beta-ureidoisobutyric acid as models for instructive and noninstructive lesions; Ide H et al.; The processing of unrepaired DNA lesions is a key to understanding and predicting the biological end points of particular DNA damages . In this study, we prepared single-stranded f1 phage (f1-K12) DNA containing dihydrothymine or beta-ureidoisobutyric acid as models for instructive or noninstructive base lesions and assessed the potential biological consequences of these lesions in vitro and in vivo . To determine the effect of the two lesions on in vitro DNA synthesis, the extent of DNA synthesis was measured by 3H-labeled nucleotide incorporation, and the newly synthesized DNA was analyzed by DNA sequencing gels . The results showed that dihydrothymine in the template was at most a weak block to in vitro DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment (Pol I) and T4 DNA polymerase . In contrast, beta-ureidoisobutyric acid constituted a very strong (probably absolute) replicative block in vitro . With Pol I, termination bands were observed either opposite or one base prior to (3' to) the putative beta-ureidoisobutyric acid depending on its position in the template . However, when DNA synthesis was catalyzed by Pol I lacking a 3'----5' exonuclease activity, termination bands were only observed opposite beta-ureidoisobutyric acid, with purine nucleotides being incorporated preferentially opposite the lesion . With T4 DNA polymerase that contains a very active 3'----5' exonuclease activity, DNA synthesis was arrested almost exclusively one base prior to (3' to) the putative beta-ureidoisobutyric acid site in the template . We also measured survival of transfecting DNA containing dihydrothymine or beta-ureidoisobutyric acid in an attempt to correlate the in vitro data with in vivo processing . In keeping with the results obtained in vitro, dihydrothymine present in transfecting f1-K12 DNA did not constitute an inactivating lesion . On the other hand, it took 0.9 beta-ureidoisobutyric acid residues per molecule to inactivate transfecting f1-K12 DNA, indicating that the lesion was an absolute replicative block in vivo . When host cells were ultraviolet-irradiated to induce the SOS response, a slight increase (about 2-fold) in survival of transfecting f1-K12 DNA containing beta-ureidoisobutyric acid was observed . The potential effects of the structures of base lesions on lesion-polymerase interactions are discussed.

Nucleic Acids Res, 1991 Jan 25, 19(2), 365 - 70
Sequence effect on incision by (A)BC excinuclease of 4NQO adducts and UV photoproducts; Thomas DC et al.; Nucleotide excision repair in Escherichia coli is initiated by (A)BC excinuclease, an enzyme which incises DNA on both sides of bulky adducts and removes the damaged nucleotide as a 12-13 base long oligomer . The incision pattern of the enzyme was examined using DNA modified by 4-nitroquinoline 1-oxide (4NQO) and UV light . Similar to the cleavage pattern of UV photoproducts and other bulky adducts, the enzyme incises the 8th phosphodiester bond 5' and 5th phosphodiester bond 3' to the 4NQO-modifed base, primarily guanine . The extent of DNA damage by these agents was determined using techniques which quantitatively cleave the DNA or stop at the site of the adduct . By comparison of the intensity of gel bands created by (A)BC excinuclease and the specific cleavage at the damaged site, the efficiency of (A)BC excinuclease incision at 13 different 4NQO-induced adducts and 13 different photoproducts was determined by densitometric scanning . In general, incisions made at 4NQO-induced adducts are proportional to the extent of damage, though the efficiency of cutting throughout the sequence tested varies from 25 to 75% . Incisions made at pyrimidine dimers are less efficient than at 4NQO-adducts, ranging from 13 to 65% incision relative to modification, though most are around 50% . The two (6-4) photoproducts within the region tested are incised more efficiently than any pyrimidine dimer.

J Biol Chem, 1991 Jan 25, 266(3), 1672 - 8
Characterization of recombinant human Kirsten-ras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems; Lowe PN et al.; Kirsten-ras is the oncogene most frequently activated in human tumors . Studies of its biological function have been limited by the nonavailability of significant amounts of the major protein product, Kirsten-ras (4B) p21 . When expressed in Escherichia coli K12, the recombinant protein was rapidly cleaved upon cell lysis in the lysine-rich C terminus region, probably by the ompT protease . However, soluble full-length protein was obtained when the Kirsten-ras gene was expressed in an E . coli strain lacking the ompT gene, and also in a baculovirus/insect cell expression system . Additionally, the baculovirus/insect cell system produced about half of the Kirsten-ras protein in a membrane-associated form, which was post-translationally modified by polyisoprenylation and carboxyl-methylation . A C-terminally truncated form (residues 1-166) was also expressed at high levels in E . coli for x-ray crystallographic studies . The kinetics of GDP release and of GTP hydrolysis of the purified proteins are similar to those of the corresponding Harvey-ras proteins, though there are small differences in the relative affinities for GDP and GTP . Biological activity of full-length Kirsten Val-12 p21 was demonstrated by microinjection into Swiss 3T3 cells, resulting in morphological transformation, with a lower potency than that of Harvey Val-12 protein.

Nucleic Acids Res, 1991 Jan 25, 19(2), 371 - 7
Replication of a geminivirus derived shuttle vector in maize endosperm cells; Ugaki M et al.; A maize (Zea mays L.) endosperm cell culture has been shown to efficiently replicate DNA sequences derived from wheat dwarf virus (WDV), a monopartite monocot geminivirus . To analyze sequences necessary for viral replication and to verify their application for a plant gene expression vector, we have developed a 3.7 kilobase pairs Escherichia coli--plant cell shuttle vector, pWI-11 . The p15A origin of replication, functional in E . coli, was introduced into the viral sequences . We have replaced the coding region of the coat protein gene by that of bacterial neomycin phosphotransferase II (NPT II) gene . The resulting NPT II gene fusion can serve as a selectable marker in both plant and E . coli systems . Into a unique cloning site in this pWI-11 vector, we introduced a gene fusion carrying the bacterial beta-glucuronidase (GUS) coding region under control of the cauliflower mosaic virus 35S (CaMV35S) gene promoter and terminator . By transferring these viral sequences into protoplasts derived from maize endosperm cell cultures, we have demonstrated that the plasmid pWI-11 can replicate in maize endosperm cells, that the GUS reporter gene introduced into pWI-11 can be expressed at high level in the transformed cells, and that the replicating viral DNA can be rescued from endosperm cells by transforming E . coli in the presence of kanamycin . The level of GUS gene expression increased progressively in transformed endosperm cells during a prolonged culture period, coinciding with replication of the viral sequences in these cells.

J Biol Chem, 1991 Jan 25, 266(3), 1478 - 83
Superoxide sensitivity of the Escherichia coli 6-phosphogluconate dehydratase; Gardner PR et al.; The activity of 6-phosphogluconate dehydratase was significantly lower in extracts of aerobically grown Escherichia coli deficient in superoxide dismutase (sodAsodB) and in mutants lacking the inducible manganese-containing superoxide dismutase (sodA), exposed to the redox-cycling agent paraquat, than in the parental strain . Growth of these strains on a gluconate minimal medium was also impaired under these conditions . The enzyme was most susceptible to dioxygen in superoxide dismutase (SOD)-free extracts, and exogenous SOD afforded a concentration-dependent protection against inactivation . The amount of SOD necessary for full protection was comparable to the amount normally present in extracts of aerobic E . coli (7-36 units/mg protein), and the rate of reaction of O2- with the dehydratase was estimated to be approximately 2.0 x 10(8) M-1 s-1 . The dehydratase was much less sensitive to O2 or H2O2 than to O2- . The virtual substrate, alpha-glycerophosphate, provided partial protection . Iron chelators, thiol-reactive reagents, and oxidants, including nitrite and diamide, inactivated the enzyme . Fluoride ions stabilized the dehydratase and blocked the effect of oxidants . The O2(-)-sensitive target site is proposed to be an iron-sulfur cluster which is readily destroyed by oxidation.

Cell, 1991 Jan 25, 64(2), 459 - 70
An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers; Sun XH et al.; The kappa E2 sequence binding proteins, E12 and E47, are generated by alternative splicing of the E2A gene, giving closely related basic and helix-loop-helix structures crucial for DNA binding and dimerization . Measurements of dimerization constants and binding strengths to the optimal DNA sequence (the kappa E2 site or its near relatives) showed that E47 homodimers and MyoD heterodimers with E12 or E47 dimerized and bound avidly, but E12 homodimerized efficiently and bound to DNA poorly; MyoD homodimerized poorly and bound strongly . An inhibitory domain N-terminal to the basic region of E12 prevents E12 homodimers but not E12/MyoD heterodimers from binding to DNA . Thus, E47 binds to DNA both as a heterodimer with MyoD and as a homodimer, while E12 and MyoD bind to DNA efficiently only as heterodimers.

Nucleic Acids Res, 1991 Jan 25, 19(2), 353 - 8
A computer method for finding common base paired helices in aligned sequences: application to the analysis of random sequences; Chan L et al.; We describe a new computer program that identifies conserved secondary structures in aligned nucleotide sequences of related single-stranded RNAs . The program employs a series of hash tables to identify and sort common base paired helices that are located in identical positions in more than one sequence . The program gives information on the total number of base paired helices that are conserved between related sequences and provides detailed information about common helices that have a minimum of one or more compensating base changes . The program is useful in the analysis of large biological sequences . We have used it to examine the number and type of complementary segments (potential base paired helices) that can be found in common among related random sequences similar in base composition to 16S rRNA from Escherichia coli . Two types of random sequences were analyzed . One set consisted of sequences that were independent but they had the same mononucleotide composition as the 16S rRNA . The second set contained sequences that were 80% similar to one another . Different results were obtained in the analysis of these two types of random sequences . When 5 sequences that were 80% similar to one another were analyzed, significant numbers of potential helices with two or more independent base changes were observed . When 5 independent sequences were analyzed, no potential helices were found in common . The results of the analyses with random sequences were compared with the number and type of helices found in the phylogenetic model of the secondary structure of 16S ribosomal RNA . Many more helices are conserved among the ribosomal sequences than are found in common among similar random sequences . In addition, conserved helices in the 16S rRNAs are, on the average, longer than the complementary segments that are found in comparable random sequences . The significance of these results and their application in the analysis of long non-ribosomal nucleotide sequences is discussed.

J Biol Chem, 1991 Jan 25, 266(3), 1985 - 96
Integration host factor-mediated expression of the ilvGMEDA operon of Escherichia coli; Pagel JM et al.; The structural genes of the ilvGMEDA operon of Escherichia coli are preceded by two promoters, ilvPG1 and ilvPG2, and a leader-attenuator region . Alkylation protection and hydroxyl radical footprinting techniques have been used to demonstrate that integration host factor (IHF) interacts with the nucleotides in a consensus-like DNA sequence located immediately downstream of the RNA polymerase transcriptional pause site in the leader-attenuator region . In the presence of purified IHF protein, in vitro transcriptional pausing of RNA polymerase at the leader-attenuator pause site is increased 2-fold and, concomitantly, a 2-fold increase in transcriptional termination at the attenuator is observed . Strains containing chromosomal transcriptional fusions of various segments of the ilvGMEDA promoter-attenuator region to the galK gene were used to show that IHF also decreases the in vivo basal level of transcriptional readthrough at the attenuator 2-fold . The binding of IHF to another target site in the ilvPG1 promoter region represses transcription from this promoter and causes a 4-fold stimulation of transcription initiation from the downstream ilvPG2 promoter 4-fold . This IHF-mediated control of transcription initiation from the upstream promoter region is independent of the regulation of transcription termination effected by IHF interaction at the attenuator site . Thus, IHF is capable of regulating the expression of the ilvGMEDA operon in opposing manners; it can activate transcription initiation of this operon from the ilvPG2 promoter 4-fold and increase the termination of this transcription at the downstream attenuator 2-fold.

J Biol Chem, 1991 Jan 25, 266(3), 1898 - 902
Differential localization of two epitopes of Escherichia coli ribosomal protein L2 on the large ribosomal subunit by immune electron microscopy using monoclonal antibodies; Olson HM et al.; Two monoclonal antibodies (mAb), directed toward different epitopes of Escherichia coli ribosomal protein L2, have been used as probes in immune electron microscopy . mAb 5-186 recognizes an epitope within residues 5-186 of protein L2; it is seen to bind to 50 S subunits at or near the peptidyl transferase center, beside the subunit head on the L1 shoulder . mAb 187-272 recognizes an epitope within residues 187-272 . This antibody binds to the face of the 50 S subunit, below the head and slightly toward the side with the stalk; this site is near the translocation domain . Both antibodies can bind simultaneously to single subunits . This indicates that protein L2 is elongated, reaching from the peptidyl transferase center to below the subunit head and approaching the translocational domain . The different locations of the two epitopes are consistent with previous biochemical results with the two antibodies (Nag, B., Tewari, D . S., Etchison, J . R., Sommer, A., and Traut, R . R . (1986) J . Biol . Chem . 261, 13892-13897).

J Biol Chem, 1991 Jan 25, 266(3), 1721 - 7
Transcriptional terminator is a positive regulatory element in the expression of the Escherichia coli crp gene; Aiba H et al.; Plasmids were constructed that contain deletions in the stem region of the presumed rho-independent terminator of the cloned crp gene of Escherichia coli . The level of cyclic AMP binding activity and the amount of CRP in cells harboring the deletion plasmids were found to be significantly lower than those in cells harboring the wild-type crp plasmid . Quantitative S1 assays indicated that the steady-state levels of crp mRNA were markedly reduced in cells harboring the deletion plasmids . Evidence was also presented to show that the crp mRNAs produced from deletion plasmids are less stable than that from the intact crp gene . In vitro transcription assays revealed that the putative crp terminator is indeed a rho-independent terminator . Using the galK expression system and Northern blot analysis we showed that the crp terminator is functional in vivo . Moreover it was shown that the deletion mutations in the stem region of the crp terminator cause a significant readthrough . We conclude that the 3'-flanking sequence of the crp gene acts to stabilize its own mRNA as well as to terminate transcription.

J Biol Chem, 1991 Jan 25, 266(3), 1597 - 604
Dihydroorotase from Escherichia coli . Substitution of Co(II) for the active site Zn(II); Brown DC et al.; Treatment of Escherichia coli dihydroorotase (a homodimer of subunit molecular weight 38,729) containing only the 1 active site Zn(II) ion per subunit with the sulfhydryl reagent N-(ethyl)-maleimide (NEM) blocks the two external Zn(II) sites per subunit and dramatically lessens the precipitation caused by high concentrations of Zn(II); stabilizes the enzyme partially against air oxidation and dilution inactivation; makes the active site Zn(II) easier to remove; and lowers Km and increases kcat . Treatment of NEM-blocked dihydroorotase ((NEM)dihydroorotase) with the chelator 2,6-pyridinedicarboxylic acid at pH 5.0 in the absence of oxygen and trace metal ions removes the active site Zn(II) with a half-life of 15 min, allowing the production of milligram amounts of moderately stable apo-(NEM)dihydroorotase in about 80% yield . Treatment of apo-(NEM)dihydroorotase with Co(II) at pH 7.0 produces (NEM)dihydroorotase completely substituted at the active site with Co(II) in 100% yield: analysis gives 0.95-1.1 g atoms of Co(II) per active site and 0.03-0.05 g atoms of Zn(II) per active site . This Co(II)-(NEM)dihydroorotase is hyperactive at pH 8 . The electronic absorption spectrum of Co(II)-(NEM)dihydroorotase at pH 6.5 implicates an active site thiol group as a ligand to the metal ion . The spectrum is inconsistent with tetrahedral coordination of the active site metal ion and is most consistent with a pentacoordinate structure.

Biochim Biophys Acta, 1991 Jan 23, 1073(1), 149 - 54
The effect of hydrophobic interaction on endotoxin adsorption by polymeric affinity matrix; Hou KC et al.; Endotoxin, a major pyrogen of concern to the biological industry, is a lipopolysaccharide containing a highly hydrophobic region, lipid A, in its structure . The effect of hydrophobic interaction on endotoxin adsorption from an aqueous solution was studied by covalently bonding aminoalkyl groups with varying hydrocarbon lengths to a cellulose and acrylic composite matrix . The amount of endotoxin adsorbed on the matrix increased with the increasing length of alkyl groups, demonstrating the contribution of hydrophobic interaction between endotoxin and the solid matrix . Both the hydrophobic and the charge interaction prove to be effective for endotoxin adsorption, and a synergistic effect from the dual chemical forces is achievable under specified conditions . The effect of solvent, pH and salts on endotoxin adsorption provides further evidence for the importance of hydrophobic force as a means of removing endotoxin from aqueous solutions.

Biochemistry, 1991 Jan 22, 30(3), 813 - 22
Upstream sequence activation of Escherichia coli argT promoter in vivo and in vitro; Hsu LM et al.; Escherichia coli argT promoter in a galK fusion construct is shown by BAL 31 deletion to require its upstream region for high in vivo activity . The extent of activation conferred by the upstream sequence from -130 to -38 is 25-fold . A spontaneous mutant containing a T to G transversion at -37 (i.e., the T-37G promoter) shows a similar requirement; however, the upstream sequence producing a 10-fold effect spans only -130 to -60 . The difference in upstream sequence boundaries between the wild-type and T-37G promoters suggests the possible existence of two activating elements . Gel mobility investigation points to the presence of bent DNA in the argT promoter, and the bent center was localized to the -90 to -95 region by circular permutation analysis . The role of the upstream activating sequence (UAS) in promoter function was probed by competitive transcription experiments in vitro . Results of this type of analysis indicate that the full UAS activates transcription through a combined effect on KB and k2 . Of these, KB is significantly strengthened by the proximal element, and k2 is stimulated to a smaller extent by the distal element . The evidence from deletion analysis, gel mobility investigation, and competitive transcription together support a "two-element" model of UAS function for the argT promoter.

Biochemistry, 1991 Jan 22, 30(3), 788 - 96
A novel combined chemical-enzymatic synthesis of cross-linked DNA using a nucleoside triphosphate analogue; Cowart M et al.; A novel method using combined chemical and enzymatic reactions to allow the preparation of covalently cross-linked DNA duplexes has been described . The method can be used to specifically link two complementary bases of a DNA duplex containing all four natural bases . The modified nucleotide 9-(2-deoxy-5-O-triphospho-beta-D-ribofuranosyl)-N6,N6-ethano -2,6-diaminopurine (6edDTP) was prepared by total chemical synthesis and was found to be incorporated into DNA duplexes in the place of 2'-deoxyguanosine 5'-O-triphosphate by the Klenow fragment of Escherichia coli DNA polymerase I, T4 and T7 DNA polymerases, avian myeloma virus reverse transcriptase, and rat DNA polymerase beta . Once incorporated, the aziridine of the nucleotide is rapidly opened by the N4 of the cytosine on the complementary strand to give cross-linked DNA, where the modified nucleotide is covalently joined to the complementary base by an ethano linkage . The duplexes produced were found to be recognized as substrates by various DNA polymerases . The Km for the incorporation of the 6edDTP into DNA catalyzed by the Klenow fragment of E . coli DNA polymerase I was found to be 29 microM, and the kcat was found to be 0.014 s-1 . The modified nucleoside also served as a substrate for terminal deoxynucleotidyltransferase, where it was added to single-stranded DNA and then hybridized to a complementary strand, after which cross-linking of the two strands occurred within 1 min.

Biochemistry, 1991 Jan 22, 30(3), 745 - 54
Effect of flavin structure and redox state on catalysis by and flavin-pterin energy transfer in Escherichia coli DNA photolyase; Chanderkar LP et al.; 5-DeazaFAD bound to a hydrophobic site in apophotolyase and formed a stable reconstituted enzyme, similar to that observed with FAD . Although stoichiometric incorporation was observed, the flavin ring modification in 1-deazaFAD interfered with normal binding, decreased protein stability, and prevented formation of a stable flavin radical, unlike that observed with FAD . The results suggest that an important hydrogen bond is formed between the protein and N (1) in FAD, but not N (5), and that there is sufficient space at the normal flavin binding site near N (5) to accommodate an additional hydrogen but not near N (1) . Catalytic activity was observed with enzyme containing 5-deazaFADH2 (42% of native enzyme) or 1-deazaFADH2 (11% of native enzyme) as its only chromophore, but no activity was observed with the corresponding oxidized flavins, similar to that observed with FAD and consistent with a mechanism where dimer cleavage is initiated by electron donation from excited reduced flavin to substrate . The protein environment in photolyase selectively enhanced photochemical reactivity in the fully reduced state, as evidenced by comparison with results obtained in model studies with the corresponding free flavins . Phosphorescence was observed with free or photolyase-bound 5-deazaFADH2, providing the first example of a flavin that exhibits phosphorescence in the fully reduced state . Formation of an enzyme-substrate complex resulted in a nearly identical extent of quenching of 5-deazaFADH2 phosphorescence (85.1%) and fluorescence (87.5%) . The data are consistent with a mechanism involving exclusive reaction of substrate with the excited singlet state of 5-deazaFADH2, analogous to that proposed for FADH2 in native enzyme . Direct evidence for singlet-singlet energy transfer from enzyme-bound 5-deazaFADH2 to 5,10-CH(+)-H4folate was provided by the fact that pterin fluorescence was observed upon excitation of 5-deazaFADH2, accompanied by a decrease in 5-deazaFADH2 fluorescence . On the other hand, the fluorescence of enzyme-bound pterin was quenched by 5-deazaFADox, consistent with energy transfer from pterin to 5-deazaFADox . In each case, the spectral properties of the chromophores were consistent with the observed direction of energy transfer and indicated that transfer in the opposite direction was energetically unlikely . Unlike 5-deazaFAD, energy transfer from pterin to FAD is energetically feasible with FADH2 or FADox . The results indicate that the direction of flavin-pterin energy transfer at the active site of photolyase can be manipulated by changes in the flavin ring or redox state which alter the energy level of the flavin singlet.

Biochemistry, 1991 Jan 22, 30(3), 663 - 7
Trifluoperazine binding to mutant calmodulins; Massom LR et al.; Trifluoperazine (TFP) binding by 14 calmodulins, including 12 produced by site-directed mutagenesis, was determined . While vertebrate calmodulin binds 4.2 +/- 0.2 equiv of TFP, Escherichia coli expressed but unmutated calmodulins bind about 5.0 +/- 0.5 equiv of TFP . The cause for this difference is not known . The E . coli expressed proteins consist of two different series expressed from different calmodulin genes, CaMI and SYNCAM . The wild-type genes code for proteins that differ by nine conservative amino acid substitutions . Both these calmodulins bind 5 equiv of TFP with similar affinities, thus none of these conservative substitutions has any additional effect on TFP binding . Some altered calmodulins (deletion of EE83-84 or SEEE81-84, changing DEE118-120----KKK, M124----I,E120----K, or E82----K) have no appreciable effect on TFP binding . Other mutations affect either the binding of one TFP (deletion of E84) or about two TFP (changing E84----K, EEE82-84----KKK, E67----A, DEQ6-8----KKK, or E11----K) . The mutations that affect TFP binding are localized to three regions of calmodulin: The amino-terminal alpha-helix, the central helix between the two globular ends of calmodulin, and a calcium-binding site in the second calcium-binding domain . The results are consistent with each of these regions either directly participating in drug binding or involved structurally in maintaining or inducing the correct conformation for TFP binding in the amino-terminal half of calmodulin.

Biochemistry, 1991 Jan 22, 30(3), 804 - 13
A mutant of DNA polymerase I (Klenow fragment) with reduced fidelity; Carroll SS et al.; The kinetic parameters governing incorporation of correct and incorrect bases into synthetic DNA duplexes have been investigated for Escherichia coli DNA polymerase I {Klenow fragment (KF)} and for two mutants, Tyr766Ser and Tyr766Phe . Tyr766 is located at the C-terminus of helix O in the DNA-binding cleft of KF . The catalytic efficiency for correct incorporation of dNTP is reduced 5-fold for Tyr766Ser . The catalytic efficiencies of all 12 possible misincorporations have been determined for both KF and Tyr766Ser by using single-turnover kinetic conditions and a form of the enzyme that is devoid of the 3'-5' exonuclease activity because of other single amino acid replacements . Tyr766Ser displays an increased efficiency of misincorporation (a reduction in fidelity) for several of the 12 mismatches . The largest increase in efficiency of misincorporation for Tyr766Ser occurs for the misincorporation of TMP opposite template guanosine, a 44-fold increase . In contrast, the efficiencies of misincorporation of dAMP opposite template A, G, or C are little affected by the mutation . A determination of the kinetic parameters associated with a complete kinetic scheme has been made for Tyr766Ser . The rate of addition of the next correct nucleotide onto a preexisting mismatch is decreased for Tyr766Ser . The fidelity of Tyr766Phe was not substantially different from that of KF for the misincorporations examined, indicating that it is the loss of the phenolic ring of the side chain of Tyr766 that leads to the significant decrease in fidelity . The results indicate that KF actively participates in the reduction of misincorporations during the polymerization event and that Tyr766 plays an important role in maintaining the high fidelity of replication by KF.

Biochemistry, 1991 Jan 22, 30(3), 733 - 9
Role of MgATP in the activation and reassociation of cAMP-dependent protein kinase I: consequences of replacing the essential arginine in cAMP binding site A; Neitzel JJ et al.; The type I form of cAMP-dependent protein kinase binds MgATP with a high affinity, and binding of MgATP decreases the affinity of the holoenzyme for cAMP {Hofmann et al . (1975) J . Biol . Chem . 250, 7795} . Holoenzyme was formed here with a mutant form of the bovine recombinant type I regulatory subunit where the essential arginine in site A, Arg-209, was replaced with Lys . Although this mutation does not significantly change the high-affinity binding of MgATP to the holoenzyme, it does abolish high-affinity binding of cAMP to site A . In the absence of MgATP, binding of cAMP to site B is sufficient to promote dissociation of the holoenzyme complex and activation of the catalytic subunit {Bubis et al . (1988) J . Biol . Chem . 263, 9668} . In the presence of MgATP however, holoenzyme formed with this mutant regulatory subunit is very resistant to cAMP . The Kd(cAMP) was greater than 1 microM, and the Ka(cAMP) increased 60-fold from 130 nM to 6.5 microM in the presence of MgATP . Thus, MgATP serves as a lock that selectively stabilizes the holoenzyme and inhibits activation . Both site A and site B are shielded from cAMP in the presence of MgATP . These results suggest that Arg-209 may play a role in stabilizing the MgATP.holoenzyme complex in addition to its role in binding the exocyclic oxygens of cAMP when cAMP is bound to the regulatory subunit . The catalytic subunit also reassociates rapidly with this mutant regulatory subunit, and in contrast to the wild-type regulatory subunit, holoenzyme formation does not require MgATP.

Biochemistry, 1991 Jan 22, 30(3), 668 - 75
Quenching of tryptophan phosphorescence in Escherichia coli alkaline phosphatase by long-range transfer mechanisms to external agents in the rapid-diffusion limit; Mersol JV et al.; Quenching of the room-temperature phosphorescence of Escherichia coli alkaline phosphatase by several freely diffusing molecules was studied, each of whose absorption spectrum overlaps the long-lived emission of this protein and which therefore can quench the excited triplet state by diffusion-enhanced Forster energy transfer . The presence of additional nonresonance transfer mechanisms was also detected, from a lack of linear dependence of quenching rate on spectral overlap . The quenching agents used were the dye molecules methyl red, methyl orange, and 2-{(4-hydroxyphenyl)azo}benzoic acid, as well as the embedded heme groups of myoglobin, metmyoglobin, and the reduced and oxidized forms of cytochrome c . Quenching was found to be greatly diminished upon reduction of each acceptor, indicating that electron transfer occurs efficiently from the excited tryptophan to the oxidized form of the acceptors . The elimination of this electron transfer in the reduced form affords the opportunity to separately measure the Forster transfer rates for the heme proteins . When the transfer rate constant thus measured for myoglobin is applied to a model where both donor and acceptor proteins are taken to be spherical with both tryptophan and the heme group placed off center (a model whose quenching rate equation is newly presented here), the depth of the phosphorescent tryptophan beneath the surface of alkaline phosphatase is found to be 16 A . This value is close to the depth of tryptophan 109 (which is known to be the phosphorescent residue in alkaline phosphatase), showing that with properly chosen probes this technique is indeed valuable for distance determinations in protein structure studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Jan 22, 30(3), 641 - 7
An internal cysteine plays a role in the maintenance of the latency of human fibroblast collagenase; Windsor LJ et al.; The cDNA that encodes the proenzyme form of human fibroblast collagenase has been expressed in Escherichia coli . It has been shown by a number of criteria to be functionally identical with the enzyme isolated from human sources . Mutations of each of three cysteine residues found in procollagenase were constructed by site-directed mutagenesis of the cDNA . The relative activities of these mutants were compared to the wild-type enzyme . All of the mutants retained proteolytic activity, but not necessarily on collagen . Mutations that interfere with the formation of the sulfhydryl bridge in the carboxy-terminal domain in some cases increased and in other cases decreased the rate of casein cleavage . On the basis of extensive autolysis within E . coli of a mutant with a replacement of cysteine-73, the procollagenase molecule produced appeared to be either spontaneously active or perhaps more susceptible to autolytic activation, despite the continued presence of the propeptide . Experiments designed to capture the active forms of the mutant by use of the irreversible inhibitor alpha 2-macroglobulin showed that some degree of latency still persisted in the autolytic mutant . These findings suggest that the cysteine at position 73 is important for maintaining the proenzyme in an inactive state but that the maintenance of latency in MMPs may be a complex process, involving a number of interactions between the propeptide domain and the remainder of the collagenase molecule.

Int J Cancer, 1991 Jan 21, 47(2), 267 - 73
Preparation and characterization of monoclonal antibodies specific to synthetic peptide of carcinoembryonic antigen; Tsujisaki M et al.; Monoclonal antibodies (MAbs) were prepared to a synthetic peptide (PI: 119-140 amino acids) in domain 1 of the carcino-embryonic antigen (CEA) molecule . The majority of the amino acids in peptide PI show a hydrophilic character, and similar sequences are repeated in domains I, II and III of this molecule . These MAbs appear to recognize new epitopes of CEA, since representative conventional MAbs to CEA do not show reactivity to synthetic peptide PI . The resulting MAbs were divided into 2 groups; group 1 (MAb PI-706) reacted with peptide PI, but not with purified CEA preparation, while group 2 (MAb PI-234 and PI-255) reacted with either type of antigen, in the immunoblotting assay using purified CEA and in immunostaining of colonic carcinoma tissues . Furthermore, group-1 MAb PI-706 was reactive to purified CEA after treatment with periodate.

J Mol Biol, 1991 Jan 20, 217(2), 239 - 40
Crystallization and preliminary X-ray diffraction studies of nucleoside diphosphate kinase from Dictyostelium discoideum; Dumas C et al.; Nucleoside diphosphate kinase from the slime mold Dictyostelium discoideum is highly homologous to gene products that are involved in development in Drosophila and in oncogenesis in human cells . The cloned protein expressed in Escherichia coli has been purified and crystallized in a hexagonal space group with a = b = 74.9 A, c = 211.4 A . The asymmetric unit contains either one or two 17,000 Mr subunits of the hexamer.

J Mol Biol, 1991 Jan 20, 217(2), 283 - 92
Decay of mRNA encoding ribosomal protein S15 of Escherichia coli is initiated by an RNase E-dependent endonucleolytic cleavage that removes the 3' stabilizing stem and loop structure; Regnier P et al.; The transcripts of the rpsO-pnp operon of Escherichia coli, coding for ribosomal protein S15 and polynucleotide phosphorylase, are processed at four sites in the 249 nucleotides of the intercistronic region . The initial processing step in the decay of the pnp mRNA is made by RNase III, which cuts at two sites upstream from the pnp gene . The other two cleavages are dependent on the wild-type allele of the rne gene, which encodes the endonucleolytic enzyme RNase E . The cuts are made 37 nucleotides apart at the base of the stem-loop structure of the rho-independent attenuator located downstream from rpsO . The cleavage downstream from the attenuator generates an rpsO mRNA.nearly identical with the monocistronic attenuated transcript, while the cleavage upstream from the transcription attenuator gives rise to an rpsO mesage lacking the terminal 3' hairpin structure . The rapid degradation of the processed mRNA in an rne+ strain, compared to the slow degradation of the transcript that accumulates in an rne- strain, suggests that RNase E initiates the decay of the rpsO message by removing the stabilizing stem-loop at the 3' end of the RNA.

Biochim Biophys Acta, 1991 Jan 17, 1088(1), 31 - 5
Regulation of glpD and glpE gene expression by a cyclic AMP-cAMP receptor protein (cAMP-CRP) complex in Escherichia coli; Choi YL et al.; The glpE gene of E . coli was found to be transcribed divergently with respect to glpD, which is adjacent to glpE head-to-head on the E . coli chromosome . We constructed glpD- and/or glpE-lacZ fusion plasmids, which provided glpD and lacZ as reporter genes . The expression of glpD and glpE, under the control of the cAMP-CRP complex, was examined by measuring the activities in E . coli cells of beta-galactosidase encoded by lacZ and glycerol-3-phosphate dehydrogenase encoded by glpD . In the double-reporter-gene system, the expression of glpD and glpE was found to be positively regulated by cAMP-CRP . We also confirmed that intracellular levels of the translation products and the transcripts from glpD and glpE were positively regulated by cAMP-CRP . The cAMP-mediated induction of gene expression of glpD and glpE was significantly affected by structural alterations of the single CRP-binding site between glpD and glpE . These results indicate that the single CRP-binding site is a cis-acting element involved in the positive regulation of the expression of both glpD and glpE at the transcriptional level.

J Biol Chem, 1991 Jan 5, 266(1), 303 - 8
In vivo competition between iron and manganese for occupancy of the active site region of the manganese-superoxide dismutase of Escherichia coli; Beyer WF Jr et al.; Three forms of the dimeric manganese superoxide dismutase (MnSOD) were isolated from aerobically grown Escherichia coli which contained 2 Mn, 1 Mn and 1 Fe, or 2 Fe, respectively . These are designated Mn2-MnSOD, Mn,Fe-MnSOD, and Fe2-MnSOD . Substitution of iron in place of manganese, eliminated catalytic activity, decreased the isoelectric point, and increased the native electrophoretic anodic mobility, although circular dichroism, high performance liquid chromatography gel exclusion chromatography, and sedimentation equilibrium revealed no gross changes in conformation . Moreover, replacement of iron by manganese restored enzymatic activity . Fe2-MnSOD and the iron-superoxide (FeSOD) of E . coli exhibit distinct optical absorption spectra . These data indicate that the active site environments of E . coli MnSOD and FeSOD must differ . They also indicate that competition between iron and manganese for nascent MnSOD polypeptide chains occurs in vivo, and copurification of these variably substituted MnSODs can explain the substoichiometric manganese contents and the variable specific activities which have been reported for this enzyme.

Gene, 1991 Jan 15, 97(2), 267 - 72
Cloning of trichosanthin cDNA and its expression in Escherichia coli; Shaw PC et al.; Several cDNA clones coding for trichosanthin (TCS) have been isolated from a cDNA library prepared from the poly(A)+RNA of the root tuber of Trichosanthes kirilowii Maximowicz . The nucleotide sequence codes for a protein of 289 amino acids (aa) including a putative signal peptide of 23 aa and an extra 19 aa at the C terminus; the latter two have not been found in TCS obtained from the natural product {Collins et al., J . Biol . Chem . 265 (1990) 8665-8669} . Recombinant TCS (reTCS) was synthesized in Escherichia coli, in which the cDNA without the signal sequence was expressed under the control of the trc promoter; reTCS was detected by a rabbit anti-TCS antiserum.

Gene, 1991 Jan 15, 97(2), 259 - 66
Use of a dual-origin temperature-controlled amplifiable replicon for optimization of human interleukin-1 beta synthesis in Escherichia coli; Mashko SV et al.; A new dual-replicon recombinant plasmid, pPR53-tsr, has been constructed; it is a derivative of the expression vector pPR-TGATG-1 {Mashko et al., Gene 88 (1990) 121-126} . In contrast to its progenitor, pPR53-tsr is a low-copy-number (low-Cop) plasmid amplifiable in temperature-dependent fashion . In addition to both the replicon and the par locus from plasmid pSC101, providing segregational stability and a low Cop at 28 degrees C, the new plasmid contains a mutant ColE1 replicon whose RNAII is synthesized under the control of the pL promoter . The presence of a thermolabile repressor, cIts857, allows the thermo-inducible amplification of pPR53-tsr; the increased plasmid Cop is estimated at approx . 200 per genome 6 h after thermal induction at 42 degrees C . Thus, pPR53-tsr can be used as a donor of the thermo-inducible dual-replicon fragment for recombinant plasmids . Here, we employ such an approach for optimization of production of human interleukin-1 beta (hIL-1 beta) in Escherichia coli at a high level . The thermo-induced level of recombinant hIL-1 beta (re-hIL-1 beta) biosynthesis was around 9% of total cellular protein when the dual-replicon high-Cop vector was used . A method based on acidification of the water-soluble protein fraction to pH 4.0 has been developed that allows for the isolation of 80%-pure re-hIL-1 beta . The homogeneous material was obtained by two subsequent hydrophobic sorbent chromatographies . The protein yield ranged between 3-5 mg of re-hIL-1 beta/g of wet cells . The re-hIL-1 beta specific activity was about 2 x 10(8) units/mg, coinciding with that of the authentic hIL-1 beta.

Biochem J, 1991 Jan 15, 273(Pt 2), 311 - 6
Identification of valine/leucine/isoleucine and threonine/alanine/glycine proton-spin systems of Escherichia coli adenylate kinase by selective deuteration and selective protonation; Bock-Mobius I et al.; Adenylate kinase from two types of Escherichia coli strains, a wild-type and a leucine-auxotrophic strain, was purified . On the one hand, growing the leucine-auxotrophic bacteria on a medium containing deuterated leucine yielded E . coli adenylate kinase with all leucine residues deuterated . On the other hand, by growing the wild-type bacteria on deuterated medium with phenylalanine, threonine and isoleucine present as protonated specimens, 80% randomly deuterated enzyme with protonated phenylalanine, threonine and isoleucine residues could be prepared . Use of these proteins enabled identification of the spin systems of these amino acid residues in the n.m.r . spectra of the protein.

Biochem Biophys Res Commun, 1991 Jan 15, 174(1), 282 - 6
The leader sequence of streptokinase is responsible for its post-translational carboxyl-terminal cleavage; Park SK et al.; When the expression of streptokinase from two tac promoter-controlled expression vectors, one of these deleted a putative leader sequence of streptokinase and the other not, was compared, both normal and degraded streptokinase were detected in proteins expressed from the leader-contained vector, but only normal streptokinase was detected from the leader-deleted vector . These findings indicate that the characteristic carboxyl-terminal cleavage of streptokinase is correlated with its leader sequence and occurs during the defective secretion . The homogeneous preparation of streptokinase was facilitated by expressing from this leader-deleted vector.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 507 - 11
Formation of DNA triplexes accounts for arrests of DNA synthesis at d(TC)n and d(GA)n tracts; Baran N et al.; To study the mechanism of arrest of DNA synthesis at d(TC)n and d(GA)n sequences, single-stranded DNA molecules including d(TC)27 or d(TC)31 tracts or a d(GA)27 tract were used as templates for in vitro assays of complementary DNA synthesis performed by extension of a primer with the Klenow polymerase or the Taq polymerase (Thermus aquaticus DNA polymerase) . Electrophoresis of the products revealed that arrests occurred around the middle of these tracts . The arrests in the d(TC)n sequences were eliminated when dATP or dGTP was replaced with the analogue 7-deaza dATP or 7-deaza dGTP, respectively, or when the templates were preincubated with the Escherichia coli single-strand binding protein (SSB) . Preincubation of the template including a d(GA)27 tract with SSB has also eliminated the arrests at this sequence . Furthermore, arrests did not occur at d{G(7-deaza A)}27 or d{(7-deaza G)A}27 tracts when molecules including such tracts were used as templates . These results are compatible with the notion that the arrests were caused by formation of d(TC)i.d(GA)i.d(TC)i and d(GA)i.d(GA)i.d(TC)i triplexes, in which the bases in the uncopied portions of the d(TC)n tracts, or of the d(GA)27 tract, and the purine bases in the newly synthesized d(TC)i.d(GA)i duplexes were bound by hydrogen bonds . In the assays performed with the Taq polymerase, the pH dependence (in the range of 6.0-9.0) and the temperature dependence of the arrests were determined . As the pH was lowered, the arrests in the d(TC)27 tract were enhanced, in line with the expected properties of d(TC)i.d(GA)i.d(TC)i triplexes . The arrests in the d(GA)27 tract were enhanced by an increase in the pH . At pH 7.2 the arrests in the d(GA)27 tract persisted up to 80 degrees C, whereas the arrests in the d(TC)27 tract were eliminated at 50 degrees C; these results presumably reflect the relative stabilities of the two triplexes mentioned above at this physiological pH value and could be biologically significant.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 502 - 6
Crystal structure of interleukin 8: symbiosis of NMR and crystallography; Baldwin ET et al.; The crystal structure of a host defense system chemotactic factor, interleukin 8, has been solved by molecular replacement using as a model the solution structure derived from nuclear magnetic resonance experiments . The structure was refined with 2 A x-ray data to an R factor of 0.187 (0.217 at 1.6 A) . A comparison indicates some potential differences between the structure in solution and in the crystalline state . Our analysis also predicts that residues 4 through 9 on the amino terminus and the beta-bend, which includes His-33, may be important for receptor binding.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 468 - 72
IRA2, an upstream negative regulator of RAS in yeast, is a RAS GTPase-activating protein; Tanaka K et al.; The ras GTPase-activating protein (GAP), identified and characterized in mammalian cells, stimulates the intrinsic GTPase activity of ras proteins . We have previously proposed that the IRA genes, negative regulators of RAS genes in Saccharomyces cerevisiae, encode yeast homologs of the mammalian GAP . In this paper, we present the following evidence that a product of the IRA2 gene exhibits GAP activity similar to that of the mammalian GAP protein . (i) Extracts of yeast cells overexpressing IRA2 stimulated the GTPase activity of the yeast RAS2 protein . (ii) An epitope for a monoclonal antibody (12CA5) was added to the N terminus of the IRA2 protein . The GAP activity of extracts prepared from cells expressing this fusion protein was shown to be immunoprecipitable by 12CA5 . (iii) An IRA2 protein fused to glutathione S-transferase (GST) was produced and partially purified from Escherichia coli cells . GAP activity was detected with this purified GST-IRA2 fusion protein . (iv) The GAP activity of IRA2 proteins described above did not stimulate the GTPase activity of the RAS2Val19 protein, a protein having an amino acid alteration analogous to that found in mammalian oncogenic ras proteins . This result parallels studies showing that mammalian GAP is incapable of stimulating the GTPase activity of mammalian oncogenic proteins . The remarkable conservation between the GAP activity in mammalian and yeast cells supports the idea that the function of GAP is to negatively regulate ras proteins in mammalian cells.

Biochemistry, 1991 Jan 15, 30(2), 319 - 24
Assembly of a class I tRNA synthetase from products of an artificially split gene; Burbaum JJ et al.; The aminoacyl-tRNA synthetases arose early in evolution and established the rules of the genetic code through their specific interactions with amino acids and RNA molecules . About half of these tRNA charging enzymes are class I synthetases, which contain similar N-terminal nucleotide-fold-like structures that are joined to variable domains implicated in specific protein-tRNA contacts . Here, we show that a bacterial synthetase gene can be split into two nonoverlapping segments . We split the gene for Escherichia coli methionyl-tRNA synthetase (a class I synthetase) at several sites near the interdomain junction, such that one segment codes for the nucleotide-fold-containing domain and the other provides determinants for tRNA recognition . When the segments are folded together, they can recognize and charge tRNA, both in vivo and in vitro . We postulate that an early step in the assembly of systems to attach amino acids to specific RNA molecules may have involved specific interactions between discrete proteins that is reflected in the interdomain contacts of modern synthetases.

J Immunol, 1991 Jan 15, 146(2), 643 - 7
Monoclonal antibodies as probes to investigate the molecular changes of C5 associated with the different stability of the molecule on sheep erythrocytes and Escherichia coli 0111:B4; Rottini G et al.; The fifth C component (C5) exhibits a different stability when bound to sheep E or Escherichia coli 0111:B4, being fairly stable on the bacterial intermediate sensitized E . coli 0111:B4 coated with C components up to C5 (BAC1-5) and extremely labile on the RBC intermediate sensitized sheep E coated with C components up to C5 (EAC1-5) . We examined the possibility that molecular changes of membrane-bound C5 might be responsible for the different functional behavior of the two intermediates using mAb to C5 and sensitive immunoassays to detect bound C5 . The decay of EAC1-5 over 30 min of incubation at 37 degrees C was associated with a significant drop in the reactivity of bound C5 with three of four mAb used . These results contrasted with those obtained with BAC1-5, which showed unchanged reactivity with all mAb tested over the same period of incubation . The effect of mAb on the activity of C5 was then investigated in an attempt to relate the change of the reactivity pattern of EAC1-5 with the functional modification of bound C5 . MAb 1.5 and 1.6 were the only antibodies that interfered with the functional activity of C5, although through a different mechanism . In particular, mAb 1.5 was active both on fluid-phase and on membrane-bound C5 and is therefore likely to interact with the binding site for the late components on C5 . Conversely, mAb 1.6 was only effective on fluid-phase C5 and acted by promoting a decay of BAC1-5 similar to the spontaneous decay of EAC1-5 . We suggest that the bacterial outer membrane may protect C5 from functional decay and that mAb 1.6 interferes with the stabilizing effect of the bacteria in an as yet unclear manner.

J Immunol, 1991 Jan 15, 146(2), 585 - 91
Effects of site-specific mutations on biologic activities of recombinant human IL-6; Snouwaert JN et al.; To examine structure-activity relationships of human IL-6, we have determined the effects of specific mutations on the biologic activity of a human rIL-6 expressed in bacteria . Three types of mutants were examined: 1) a variant that contains serines in place of the four naturally occurring cysteines; 2) a series of cysteine-containing deletion mutants, each having a single internal 20 amino acid deletion; and 3) a cysteine-free variant containing a single 20 amino acid deletion . The mutants of the second type constitute a set of nonoverlapping, adjacent deletions spanning amino acids 4 through 183 of the 184 amino acids in natural human IL-6 . All of the mutants were expressed, along with the full length, cysteine-containing analogue, in Escherichia coli as fusion proteins, joined to beta-galactosidase through a collagen linker . This system allows microgram quantities of the rIL-6 variants to be partially purified from small bacterial cultures without chromatographic or refolding steps . Each of the rIL-6 variants was released from the beta-galactosidase fusion protein with collagenase, and the recovered rIL-6 was quantitated by laser densitometry of Coomassie-stained, SDS polyacrylamide gels . The sp . ac . of each of the rIL-6 variants was determined using four assays: induction of IgM secretion from an EBV transformed human B cell line, induction of fibrinogen secretion from a human hepatoma cell line, induction of fibrinogen secretion from a rat hepatoma cell line, and induction of proliferation of a murine hybridoma cell line . Replacement of cysteines with serines reduced activity relative to cysteine-containing rIL-6 to about 20% in the rat hepatoma assay and about 3% in the mouse hybridoma assay, whereas activity in both of the human cell lines was reduced to less than 0.1% . These data suggest that the murine and rat cell lines are less selective than the human cell lines in their requirements for recognition of biologically active IL-6 . Each of the deletions, except that of amino acids 4 through 23, resulted in loss of activity in all four assays . These results suggest that the information necessary for activity is not contained within any one portion of the IL-6 molecule, but rather that multiple segments of the protein are required for each of the biologic activities that we tested.

J Biol Chem, 1991 Jan 15, 266(2), 1304 - 11
RNA polymerase activity may regulate transcription initiation and attenuation in the rplKAJLrpoBC operon in Escherichia coli; Downing W et al.; The relationship between global RNA transcription capacity and transcript initiation, attenuation, and stability in the rplKAJLrpoBC operon of Escherichia coli has been examined . The rplKAJLrpoBC operon encodes in order the four large ribosome subunit proteins, L11, L1, L10, and L12, and the two large beta and beta' subunits of RNA polymerase . Operon transcripts are initiated at two promoters, PL11 and PL10 . The L12-beta intergenic space contains a transcription attenuator which, during balanced growth, terminates about 80% of the transcripts exiting the L12 gene; the remaining transcripts read through into the beta and beta' encoding genes . The capacity for global transcription initiation was modulated using a strain carrying a temperature-sensitive, initiation-defective mutation in rpoC . Following a shift to 39 degrees C, the global transcription initiation capacity was reduced to about one-half the level at 30 degrees C . This partial restriction resulted in a decrease in the stability of distal beta mRNA, whereas the stability of proximal L11-L1 and L10-L12 mRNA was not changed . Measurements of the synthesis rates of L11-L1, L10-L12, and beta mRNAs relative to total RNA synthesis indicated that this operon was selectively transcribed when the initiation capacity of RNA polymerase was limited . The synthesis rates of L11-L1 and L10-L12 mRNA increased about 2-fold, whereas the synthesis rate of beta mRNA increased nearly 5-fold . The relative transcription of other ribosome component genes and the alpha subunit gene exhibited only a modest increase during the partial restriction . Protection from S1 nuclease was used to demonstrate that the preferential transcription within the operon of beta mRNA was the consequence of active regulation of termination-antitermination at the attenuator structure in the L12-beta intergenic space . These results demonstrate that global transcription capacity may be an important parameter in determining both initiation and attenuation of transcription of the rplKAJLrpoBC ribosomal protein-RNA polymerase operon.

J Biol Chem, 1991 Jan 15, 266(2), 1064 - 70
Structural and immunological comparison of indigenous human O6-methylguanine-DNA methyltransferase with that encoded by a cloned cDNA; von Wronski MA et al.; O6-Methylguanine-DNA methyltransferase, a ubiquitous and unusual DNA repair protein, eliminates mutagenic and cytotoxic O6-alkylguanine from DNA by transferring the alkyl group to one of its cysteine residues in a second-order suicide reaction . This 22-kDa protein was immunoaffinity-purified to homogeneity from cultured human lymphoblasts (CEM-CCRF line) and compared with the O6-methylguanine-DNA methyltransferase purified to homogeneity from Escherichia coli expressing a cloned human cDNA . The cellular and recombinant proteins were identical in size, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of intact molecules and their peptides . Immunoprobing of Western blots with three monoclonal antibodies specific for human cellular O6-methylguanine-DNA methyltransferase further indicated identity of the two proteins . The amino acid sequence of the cellular protein was experimentally determined for 87 out of a total of 207 residues and was found to be identical to that deduced from the cDNA sequence . A unique cysteine residue at position 145 was identified as the methyl acceptor site by autoradiographic analysis of peptides and sequence analysis of 3H-methylated O6-methylguanine-DNA methyltransferase . These observations establish that the cloned O6-methylguanine-DNA methyltransferase cDNA encodes the full-length O6-methylguanine-DNA methyltransferase polypeptide that is normally present in human cells . Moreover, the cellular protein does not appear to be significantly modified by posttranslational processes.

J Biol Chem, 1991 Jan 15, 266(2), 1058 - 63
Primary sequence of the glucanase gene from Oerskovia xanthineolytica . Expression and purification of the enzyme from Escherichia coli; Shen SH et al.; A 2.7-kilobase fragment of DNA from Oerskovia xanthineolytica containing the gene for a beta-1,3-glucanase has been isolated and its complete nucleotide sequence determined . The sequence was found to contain two large open reading frames . Purification of the mature native enzyme and subsequent amino-terminal sequencing defined the glucanase gene in one reading frame which potentially encodes a protein of 548 amino acids . We have expressed this glucanase gene in Escherichia coli under control of the lacUV5 promoter and found the product to be secreted into the periplasm as a mature enzyme of about the same molecular weight as that of the native protein . The recombinant enzyme was purified to near homogeneity by a single step of high performance liquid chromatography . The ability of the recombinant enzyme to digest beta-glucan substrates and to lyse viable yeast cells was found to be indistinguishable from that of the native protein . Deletion of the cysteine-rich carboxyl-terminal 117 amino acids of the enzyme, which also contain two duplicated segments, abolished the lytic activity but did not significantly affect the glucanase function of the protein . The possible involvement of this domain in interaction with the yeast cell wall is discussed.

Cancer Res, 1991 Jan 15, 51(2), 499 - 503
Release of 7-alkylguanines from N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA by 3-methyladenine DNA glycosylase II; Habraken Y et al.; Purified bacterial 3-methyladenine DNA glycosylase II releases four 7-alkylguanines from {3H}N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA: 7-(2-hydroxyethyl)guanine,1,2-bis(7-guanyl)ethane, 7-(2-chloroethyl)guanine, and 7-(2-ethoxyethyl)guanine . 7-(2-Ethoxyethyl)guanine, a new compound, is formed as a result of an interaction with ethanol, a common solvent for the 2-haloethylnitrosoureas . Of the four 7-alkylguanines which are released from {3H}N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA, 7-(2-hydroxyethyl)guanine is released at a rate very much slower than the other three . As shown by a study of the spontaneous decomposition of the corresponding 7-alkyl-deoxyguanines, differences in chemical stability do not appear to explain the slow release of 7-(2-hydroxyethyl)guanine . In view of previous results showing a difference in the distribution of alkylation products between sensitive and resistant glial cell lines, the broad specificity of this enzyme suggests that glycosylase activity could play a role in cellular resistance to 2-haloethylnitrosoureas.

FEMS Microbiol Lett, 1991 Jan 15, 61(2-3), 277 - 82
Detection and purification of the free A subunit of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli; Tsuji T et al.; After removal of total B subunit and heat-labile enterotoxin (LT) from crude cell extracts of enterotoxigenic Escherichia coli (HB 101-EWD 299) by Bio-gel A 5 m column chromatography, the crude cell extract was shown to contain a free A subunit (A' subunit) that did not bind to the coligenoid of the B subunits . The A' subunit was found to be immunologically identical to the A subunit of holo-LT and was purified to show only one band in SDS-poly-acrylamide gel electrophoresis (PAGE) . The mobility of the A' subunit was identical to that of the A subunit of holo-LT . The pI value of the A' subunit was also the same as that of the A subunit of holo-LT . These data suggest that in enterotoxigenic E . coli there is free A subunit which may be involved in formation of holo-LT, analogously to free B subunit (coligenoid), and that the free A subunit is physicochemically and immunologically identical to the A subunit of holo-LT.

FEMS Microbiol Lett, 1991 Jan 15, 61(2-3), 251 - 6
Analysis of Bordetella pertussis cya operon regulation by use of cya-lac fusions; Goyard S et al.; In Bordetella pertussis virulence-associated genes, including adenylate cyclase toxin (Cya), are coordinately regulated in response to environmental signals by proteins coded by the bvg-locus . We have constructed cya-lac fusions in Escherichia coli and have shown that the cya operon is not expressed in E . coli, neither is it activated by bvg, when introduced in trans . The cya-lac fusion is fully active when returned to B . pertussis by homologous recombination and responds to bvg-dependent activation and environmental regulation . These results indicate that in B . pertussis the activation of the cya operon by bvg is indirect.

Carbohydr Res, 1991 Jan 15, 209, 83 - 7
Immobilisation of beta-D-galactosidase from Escherichia coli on cellulose beads and its use for the synthesis of disaccharide derivatives; Kery V et al.; beta-D-Galactosidase, isolated from cloned E . coli, was immobilised on cellulose beads via oxidation with sodium periodate, activation by cyanuric chloride, or diazotisation . beta-D-Galactosidase immobilised via azo bonds showed the highest relative activity and thermostability, and was used for synthesis of disaccharide methyl glycosides.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 458 - 62
Role of a carboxyl-terminal helix in the assembly, interchain interactions, and stability of aspartate transcarbamoylase; Peterson CB et al.; The six individual catalytic polypeptide chains within the two catalytic trimers of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) are folded into two discrete structural domains interconnected in part by helix 12, which comprises residues 285-305 and is located near the carboxyl terminus of the chain . The essential role of this helix in folding of the chains and their assembly into ATCase was demonstrated by introducing a stop codon at the position corresponding to amino acid 284, 291, or 299 . Cells containing these mutations are pyrimidine auxotrophs lacking ATCase-like protein in cell extracts . In contrast, stable active enzyme is formed from chains truncated at position 306 or 307, showing that all 310 amino acids are not required for assembly . Replacements of Gln-288, Asn-291, Arg-296, and Ala-298 were introduced to assess the effect of alterations within helix 12 on protein stability . Stability of the trimers was measured both by differential scanning microcalorimetry and by the rate of exchange of chains at 4 degrees C when mutant trimers were incubated with succinylated wild-type trimers . Melting temperatures of the mutant trimers spanned a range of more than 20 degrees C, with a few higher and others lower than that of wild-type trimers . Large changes in interchain interaction energies were observed for the trimers, but there was no direct correlation between the ease of dissociation of the trimers and their thermal stability . Calorimetry on the mutant holoenzymes revealed alterations in the interactions between trimers and regulatory subunits within the intact enzymes . The striking changes in stability of both trimers and holoenzymes demonstrated that effects of relatively localized amino acid replacements in helix 12 are manifested by indirect global alterations propagated throughout the structure.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 439 - 43
Ribosomal RNA introns in archaea and evidence for RNA conformational changes associated with splicing; Kjems J et al.; The single 23S rRNA gene of the archaeon Staphylothermus marinus exhibits two introns which, at the RNA level, are located in highly conserved regions of domains IV and V . The RNA introns, which are 56 and 54 nucleotides long, respectively, can form single hairpin structures . In vivo, RNA splicing occurs efficiently, whereas in vitro pre-rRNA transcripts containing each intron were cleaved efficiently when incubated with archaeal cell extracts but were poorly ligated . The introns are cleaved by a mechanism which differs from the mechanisms of eukaryotic rRNA introns but resembles those of the rRNA intron of Desulfurococcus mobilis and the archaeal tRNA introns . The cleavage enzyme recognizes and cuts a putative bulge-helix-bulge structure that can form at the archaeal exon-intron junctions . Using a phylogenetic sequence comparison approach, we define the parts of this structural feature that are essential for cleavage . We also provide evidence for conformational changes occurring in the S . marinus 23S RNA, after cleavage, at both exon-exon junctions, which may account for the low yields of ligation observed in vitro.

Biochemistry, 1991 Jan 15, 30(2), 479 - 84
Mechanism of the physiological reaction catalyzed by tryptophan synthase from Escherichia coli; Lane AN et al.; The physiological synthesis of L-tryptophan from indoleglycerol phosphate and L-serine catalyzed by the alpha 2 beta 2 bienzyme complex of tryptophan synthase requires spatial and dynamic cooperation between the two distant alpha and beta active sites . The carbanion of the adduct of L-tryptophan to pyridoxal phosphate accumulated during the steady state of the catalyzed reaction . Moreover, it was formed transiently and without a lag in single turnovers, and glyceraldehyde 3-phosphate was released only after formation of the carbanion . These and further data prove first that the affinity for indoleglycerol phosphate and its cleavage to indole in the alpha subunit are enhanced substantially by aminoacrylate bound to the beta subunit . This indirect activation explains why the turnover number of the physiological reaction is larger than that of the indoleglycerol phosphate cleavage reaction . Second, reprotonation of nascent tryptophan carbanion is rate limiting for overall tryptophan synthesis . Third, most of the indole generated in the active site of the alpha subunit is transferred directly to the active site of the beta subunit and only insignificant amounts pass through the solvent . Comparison of the single turnover rate constants with the known elementary rate constants of the partial reactions catalyzed by the alpha and beta active sites suggests that the cleavage reaction rather than the transfer of indole or its condensation with aminoacrylate is rate limiting for the formation of nascent tryptophan.

Biochemistry, 1991 Jan 15, 30(2), 472 - 8
Reciprocal communication between the lyase and synthase active sites of the tryptophan synthase bienzyme complex; Kirschner K et al.; It is important to understand how the cleavage of indoleglycerol phosphate, which is catalyzed by the alpha subunits in the alpha 2 beta 2 bienzyme complex of tryptophan synthase, is modulated by the presence of L-serine in the beta subunits . Steady-state kinetic data, including the dependence of kcat on pH, allowed values to be assigned to each of the eight rate constants of the minimal catalytic mechanism . An ionizing group having an apparent pK value near 7.5 must be protonated for activity . The alpha active site ligands indolepropanol phosphate, glyceraldehyde 3-phosphate, and glycerol 3-phosphate increase both the affinity and the molar absorbance of L-serine and L-tryptophan bound to the beta active site . These effects prove that the alpha sites communicate with the beta sites over a distance of 30 A . 6-Nitroindole readily condenses with glyceraldehyde 3-phosphate, but not with L-serine . The turnover numbers for 6-nitroindoleglycerol phosphate and 6-nitroindole increased about 10-fold in both directions in the presence of L-serine bound to the beta 2 subunits . These data prove that the alpha and beta active sites communicate reciprocally and explain why the turnover number for the physiological reaction of indoleglycerol phosphate with L-serine greatly exceeds that of the cleavage reaction of indoleglycerol phosphate.

Biochemistry, 1991 Jan 15, 30(2), 362 - 6
Substitution of histidine-84 and the GTPase mechanism of elongation factor Tu; Cool RH et al.; Mutation of His84, a residue situated in one of the loops forming the guanine nucleotide binding pocket, was introduced in the G domain, the isolated N-terminal half molecule of bacterial elongation factor Tu (EF-Tu), in order to investigate the role of this residue on the basic activities of EF-Tu: the interaction with GDP and GTP and the hydrolysis of GTP . Substitution of His84 by Gly reduces the GTPase activity of the G domain to 5%; this activity can still be stimulated by raising the KCl concentration as the activity of wild-type G domain or the intact molecule . Since the affinities of the mutant protein for GDP and GTP are essentially the same as those of the wild-type G domain, His84 is apparently not involved in the binding of the substrates . Calculations of the change in free energy of activation of the GTPase reaction following substitution of His84 by Gly point to the disruption of a weak hydrogen bond, involved in the catalytic reaction . This probably concerns an interaction via a water molecule . The possible mechanism underlying the GTPase reaction is discussed in light of the three-dimensional structure of EF-Tu, taking into account the situation of Ha-ras p21.

Biochemistry, 1991 Jan 15, 30(2), 555 - 61
Nitroxides block DNA scission and protect cells from oxidative damage; Samuni A et al.; The protective effect of cyclic stable nitroxide free radicals, having SOD-like activity, against oxidative damage was studied by using Escherichia coli xthA DNA repair-deficient mutant hypersensitive to H2O2 . Oxidative damage induced by H2O2 was assayed by monitoring cell survival . The metal chelator 1,10-phenanthroline (OP), which readily intercalates into DNA, potentiated the H2O2-induced damage . The extent of in vivo DNA scission and degradation was studied and compared with the loss of cell viability . The extent of DNA breakage correlated with cell killing, supporting previous suggestions that DNA is the crucial cellular target of H2O2 cytotoxicity . The xthA cells were protected by catalase but not by superoxide dismutase (SOD) . Both five- and six-membered ring nitroxides, having SOD-like activity, protected growing and resting cells from H2O2 toxicity, without lowering H2O2 concentration . To check whether nitroxides protect against O2.(-)-independent injury also, experiments were repeated under hypoxia . These nitroxides also protected hypoxic cells against H2O2, suggesting alternative modes of protection . Since nitroxides were found to reoxidize DNA-bound iron(II), the present results suggest that nitroxides protect by oxidizing reduced transition metals, thus interfering with the Fenton reaction.

Biochem Biophys Res Commun, 1991 Jan 15, 174(1), 211 - 6
SecA, an essential component of the secretory machinery of Escherichia coli, exists as homodimer; Akita M et al.; Size exclusion chromatography of the cytosolic fraction of SecA-overproducing cells of Escherichia coli suggested that SecA, an essential component of the secretory machinery, exists as an oligomer . The subunit structure of SecA was then studied using a purified specimen . Estimation of the molecular mass by means of ultracentrifugation and chemical crosslinking analysis revealed that SecA exists as a homodimer . The purified SecA was denatured in 6 M guanidine-HCl and renatured to a dimer, which was fully active in terms of translocation, even in the presence of 1 mM dithiothreitol . It is suggested that the dimeric structure is not critically maintained by disulfide bonding between the two subunits, each of which contains four cysteine residues.

J Biol Chem, 1991 Jan 15, 266(2), 844 - 50
Disruption of an ATP-dependent isomerization of the recA protein by mutation of histidine 163; Muench KA et al.; We have used site-directed mutagenesis to replace histidine 163 of the recA polypeptide with an alanine residue . The new {Ala-163}recA protein catalyzes single-stranded (ss) DNA-dependent ATP hydrolysis with a turnover number that is similar to that of the wild-type recA protein . Despite being proficient in ssDNA-dependent ATP hydrolysis, the {Ala-163}recA protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions, pH 7.5 . The {Ala-163}recA protein does exhibit three-strand exchange activity at pH 6.0-7.0, however, and the induction of strand exchange activity at low pH correlates directly with the activation of an ATP-dependent isomerization of the mutant protein . Thus, the {Ala-163}recA protein is functionally similar to our previously described mutant {Asn-160}recA protein (Bryant, F.R . (1988) J . Biol . Chem . 263, 8716-8723; Muench, K.A., and Bryant, F . R . (1990) J . Biol . Chem . 265, 11560-11566) . Trypsin proteolysis studies indicate that the {Ala-163}recA and {Asn-160}recA proteins, like the wild-type recA protein, are organized into carboxyl-terminal and amino-terminal domains of nearly equal size . According to this structural model, the {Ala-163}recA and {Asn-160}recA mutations may lie in a linker region joining these two domains . We speculate that the {Ala-163}recA and {Asn-160}recA mutations interfere with an ATP-dependent conformational change of the recA protein that perhaps involves a change in the relative orientation of the carboxyl-terminal and amino-terminal domains.

Biochem Biophys Res Commun, 1991 Jan 15, 174(1), 56 - 62
Inhibition of HIV-reverse transcriptase activity by asterriquinone and its analogues; Ono K et al.; Asterriquinone (ARQ; 2,5-bis-{1'-(1", 1"-dimethyl-2"-propenyl)- indol-3'-yl}-3,6-dihydroxy-1,4-benzoquinone) and its three analogues {i.e., 3,6-dihydroxy-2-{2'-(1", 1"-dimethyl-2"-propenyl)-indol-3'-yl}-5-{1', 7'- (1",1"-dimethylpropano)-indol-3'-yl}-1,4-benzoquinone (B1-4), 3,6-dihydroxy-2-{2'-(1", 1"-dimethyl-2"-propenyl)-indol-3'-yl}-5-indol-3'-yl-1,4-benzoquinone (C1-1) and 3,6-dihydroxy-2,5-diindol-3'-yl-1,4-benzoquinone (D-1)} were found to be strong inhibitors of the activity of reverse transcriptase from human immunodeficiency virus type-1 . Under the reaction conditions employed, the enzyme activity was inhibited by more than 70% in the presence of 10 microM each of these compounds . The mode of inhibition by these compounds was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive with respect to the triphosphate substrate, dTTP . The Ki values of HIV-1 reverse transcriptase were determined to be 2.3, 1.5, 0.1 and 0.3 microM for ARQ, B1-4, C1-1 and D-1, respectively.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 627 - 31
The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity; Mayer BJ et al.; Several proteins implicated in the regulation of cell proliferation contain a common noncatalytic domain, src homology region 2 (SH2) . We have used the bacterially expressed SH2 domain of abl protein-tyrosine kinase to evaluate the ability of this domain to bind to cellular proteins . ablSH2 specifically bound to a number of tyrosine-phosphorylated proteins from cells transformed by tyrosine kinase oncogenes in a filter-binding assay and to a subset of those proteins in solution . The SH2 probe bound almost exclusively to tyrosine-phosphorylated proteins, and binding was eliminated by dephosphorylation of cell proteins . Free phosphotyrosine could partially disrupt SH2 binding, suggesting that phosphotyrosine is directly involved in the binding interaction . These results demonstrate that an SH2 domain is sufficient to confer direct, high-affinity phosphotyrosine-dependent binding to proteins and suggest a general role for SH2 domains in cellular signaling pathways.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 405 - 9
Quantitation of ColE1-encoded replication elements; Brenner M et al.; Replication of the Escherichia coli plasmid ColE1 initiates from an RNA primer . This primer is formed by a ColE1 RNA II molecule that remains hybridized to its DNA template in the origin region after transcription . Continued hybridization is inhibited by prior binding to RNA II of another ColE1 transcript, RNA I; and this interaction is regulated by the plasmid-encoded Rom protein . To understand the quantitative aspects of regulation of ColE1 synthesis, we have measured the levels of RNA I, RNA II, and Rom protein in vivo, as well as the half-lives of the RNAs . The intracellular concentrations of RNA I, RNA II, and Rom protein were found to be about 1 microM, 7 nM, and 1 microM, respectively; and the RNAs had half-lives of about 2 min . A simple model derived from these results indicates that the plasmid copy number is little affected by the rate of RNA II synthesis but is strongly dependent on that of RNA I.

Gene, 1991 Jan 15, 97(2), 289 - 93
Isolation and sequence of a cDNA encoding mouse IMP dehydrogenase; Tiedeman AA et al.; Inosinic acid (IMP) dehydrogenase (IMPD) catalyzes the conversion of IMP to XMP as the first committed step in GMP biosynthesis de novo . We have isolated a cDNA containing the complete coding region of mouse IMPD by its ability to complement a bacterial mutant lacking IMPD activity . Two independent cDNA clones were isolated by complementation, of which the longest was 1.7 kb in length . Northern analyses, using the IMPD cDNA as a probe, indicated that mature IMPD mRNA was a single species approx . 2.0 kb in size . Mouse IMPD is almost identical to Chinese hamster and human IMPDs and is highly conserved between Escherichia coli and mouse, with a direct amino acid (aa) identity of 39%, which increases to 60% if conserved aa are considered . The leader region of our longest cDNA clone is G + C-rich and contains two tandem copies of a G + C-rich direct repeat.

FEBS Lett, 1991 Jan 14, 278(1), 98 - 102
Reduction of biological activity of murine recombinant interleukin-1 beta by selective deamidation at asparagine-149; Daumy GO et al.; A biologically active preparation of murine recombinant interleukin-1 beta (mIL-1 beta) from Escherichia coli cell lysates contained tow forms of mIL-1 beta with pI 8.7 and pI 8.1, respectively . Treatment with 0.1 M Tris, pH 8.5, at 37 degrees C for 35 h converted the pI 8.7 form to the pI 8.1 form by the selective deamidation of an asparagine residue (Asn149) in the mIL-1 beta molecule . Deamidated mIL-1 beta had 3- to 5-fold lower co-mitogenic activity and receptor affinity than the unmodified form.

FEBS Lett, 1991 Jan 14, 278(1), 79 - 83
Hemolytic activity of adenylate cyclase toxin from Bordetella pertussis; Ehrmann IE et al.; Adenylate cyclase (AC) toxin from B . pertussis enters eukaryotic cells where it produces supraphysiologic levels of cAMP . Purification of AC toxin activity {(1989) J . Biol . Chem . 264, 19279} results in increasing potency of hemolytic activity and electroelution of the 216-kDa holotoxin yields a single protein with AC enzymatic, toxin and hemolytic activities . AC toxin and E . coli hemolysin, which have DNA sequence homology {(1988) EMBO J . 7, 3997} are immunologically cross-reactive . The time courses of hemolysis elicited by the two molecules are strikingly different, however, with AC toxin eliciting cAMP accumulation with rapid onset, but hemolysis with a lag of greater than or equal to 45 min . Finally, osmotic protection experiments indicate that the size of the putative pore produced by AC toxin is 3-5-fold smaller than that of E . coli hemolysin.

FEBS Lett, 1991 Jan 14, 278(1), 115 - 9
The histone H1-lacZ' fusion protein produced in Escherichia coli binds to the 5'-TTGGCAnnnTGCCAA-3' motif on DNA; Mannermaa RM et al.; The coding region of the chicken histone H1.03 gene was cloned to a bacterial expression vector, and the 291-amino acid H1-beta-galactosidase fusion protein was isolated after induction with IPTG . The fusion protein recognizes the 5'-TTGGCAnnnTGCCAA-3' motif on DNA . The H1 globular domain was initially shown to be responsible for the sequence-specific binding by functional deletion analysis . This function may be indispensable for the role of H1 as a determinant of nucleosome positioning and as a eukaryotic repressor.

FEBS Lett, 1991 Jan 14, 278(1), 120 - 2
A chromophore in glutamate decarboxylase has been wrongly identified as PQQ; De Biase D et al.; Pyrroloquinoline quinone (PQQ) has been claimed to be a component of glutamate decarboxylase from Escherichia coli on the basis of a frequently used procedure in which the protein is extracted with hexanol . We demonstrate that if pyridoxal phosphate (PLP) is not added during the preparation, the apoenzyme prepared from glutamate decarboxylase contains no chromophore absorbing above 280 nm . Full enzyme activity and the original holoenzyme spectrum are restored by the addition of PLP alone . A 340 nm-absorbing band, similar to that which prompted analysis for PQQ, is produced by exposure of the enzyme to solutions of PLP.

FEBS Lett, 1991 Jan 14, 278(1), 31 - 4
ompC mutants which allow growth on maltodextrins show increased channel size and greater voltage sensitivity; Lakey JH et al.; Misra and Benson {(1988) J . Bacteriol . 170, 3611-3617} showed that point mutations in the ompC gene can allow Escherichia coli to grow on maltotriose in the absence of LamB . This report shows that these mutants produce OmpC porins with increased single channel conductance compared to the wild type . The mutants showed similar voltage dependence to each other and to PhoE by being totally closed at 200 mV . The wild type from various sources was largely insensitive to voltages below 200 mV and thus 6 point mutations at 3 sites appear to increase the voltage dependence of OmpC channels.

J Surg Res, 1991 Jan, 50(1), 77 - 81
Attenuation of the pulmonary vascular response to endotoxin by a thromboxane synthesis inhibitor (UK-38485) in unanesthetized sheep; Henry CL Jr et al.; Previous studies have documented the phasic pulmonary vascular response to infused Escherichia coli endotoxin in unanesthetized sheep . Cyclooxygenase inhibition attenuates the initial vasoconstrictive phase (phase I) but not the late phase of increased microvascular permeability (phase II) . We undertook to selectively inhibit thromboxane A2 synthesis and assess the pulmonary microvascular response to endotoxin . Twelve paired studies were carried out in six sheep prepared with chronic lung lymph fistulas and pressure monitoring catheters . Each sheep received E . coli endotoxin (0.5 microgram/kg) at time 0, both alone (control group) and 1 hr after pretreatment with a thromboxane synthetase inhibitor (UK-38485, 2 mg/kg) . The animals were monitored for 1-2 hr prior to and 5 hr following endotoxin infusion to ensure a steady-state baseline and a complete late response . The pairs of studies were done in random order . In the presence of UK-38485, endotoxin caused significantly less pulmonary hypertension and shorter duration of leukopenia and lower lung lymph flow and lymph protein clearance rates than did endotoxin alone . The differences in lymph protein clearance were more pronounced in phase II . These data suggest that both the vasoconstrictive and permeability phases of the pulmonary vascular response to endotoxin may be modified on endogenous thromboxane A2.

Arch Surg, 1991 Jan, 126(1), 70 - 3
Tumor necrosis factor and endotoxin can cause neutrophil activation through separate pathways; Moore FD Jr et al.; We investigated the possibility that tumor necrosis factor (TNF) mediates neutrophil activation by endotoxin . The number of C3b receptors on the neutrophil cell-surface was used as the indicator of activation, as assessed by indirect immunofluorescence . Incubation of buffy-coat neutrophils with TNF-alpha for 30 minutes at 37 degrees C caused neutrophil activation, increasing C3b receptor-dependent fluorescence from 340 with buffer alone to 580 with TNF (250 pg/mL) . Increasing amounts of anti-TNF IgG progressively inhibited neutrophil activation by TNF (250 pg/mL) . Addition of the active dose range of anti-TNF to neutrophils incubating in endotoxin (10 ng/mL) did not affect the degree of endotoxin-mediated neutrophil activation . Mixtures of neutrophils with the 50% suppressive dose of anti-TNF and varying endotoxin concentrations showed the same degree of neutrophil activation as mixtures without the antibody . Thus, an antibody that can inhibit TNF-mediated neutrophil activation does not inhibit endotoxin-mediated neutrophil activation . We conclude that endotoxin and TNF can activate neutrophils through separate pathways.

Arch Surg, 1991 Jan, 126(1), 59 - 62
Alterations in host defense associated with anesthesia and blood transfusions . II . Effect on response to endotoxin; Waymack JP et al.; The effect of blood transfusions and anesthesia on host response to endotoxin was evaluated in multiple Lewis rat models . The rats were randomized to receive A'Sogaloff Cancer Institute rat blood, pentobarbital sodium, or lactated Ringer's solution and, at either 2 or 7 days following administration of these agents, were challenged with intravenous endotoxin . Neither blood transfusions nor anesthesia altered mortality when administered 2 days before endotoxin challenge . However, blood transfusions administered 7 days before endotoxin challenge were found to prolong survival, to prevent endotoxin-induced alterations in T-lymphocyte subsets, and to decrease plasma tumor necrosis factor levels . In conclusion, blood transfusions appear to depress immune function in a beneficial manner in endotoxin shock.

Methods Enzymol, 1991, 208, 291 - 343
Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA; Record MT Jr et al.; The concentration and nature of the electrolyte are key factors determining (1) the equilibrium extent of binding of oligocations or proteins to DNA, (2) the distribution of bound protein between specific and nonspecific sites, and (3) the kinetics of association and dissociation of both specific and nonspecific complexes . Salt concentration may therefore be used to great advantage to probe the thermodynamic basis of stability and specificity of protein-DNA complexes, and the mechanisms of association and dissociation . Cation concentration serves as a thermodynamic probe of the contributions to stability and specificity from neutralization of DNA phosphate charges and/or reduction in phosphate charge density . Cation concentration also serves as a mechanistic probe of the kinetically significant steps in association and dissociation that involve cation uptake . In general, effects of electrolyte concentration on equilibrium constants (quantified by SKobs) and rate constants (quantified by Skobs) are primarily cation effects that result from the cation-exchange character of the interactions of proteins and oligocations with polyanionic DNA . The competitive effects of Mg2+ or polyamines on the equilibria and kinetics of protein-DNA interactions are interpretable in the context of the cation-exchange model . The nature of the anion often has a major effect on the magnitude of the equilibrium constant (Kobs) and rate constant (kobs) of protein-DNA interactions, but a minor effect on SKobs and Skobs, which are dominated by the cation stoichiometry . The order of effects of different anions generally follows the Hofmeister series and presumably reflects the relative extent of preferential accumulation or exclusion of these anions from the relevant surface regions of DNA-binding proteins . The question of which anion is most inert (i.e., neither accumulated nor excluded from the relevant regions of these proteins) remains unanswered . The characteristic effects of temperature on equilibrium constants and rate constants for protein-DNA interactions also serve as diagnostic probes of the thermodynamic origins of stability and specificity and of the mechanism of the interaction, since large changes in thermodynamic and activation heat capacities accompany processes with large changes in the amount of water-accessible nonpolar surface area.(ABSTRACT TRUNCATED AT 400 WORDS)

J Chromatogr, 1991 Jan 11, 537(1-2), 181 - 99
Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins; Welinder BS et al.; The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile . The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation . Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC . Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks . The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%) . The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA . The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid) . Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water . Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis) . Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.

Nucleic Acids Res, 1991 Jan 11, 19(1), 155 - 62
Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1; Boni IV et al.; Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles . Using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinitiation complexes . Targets for S1 on Q beta and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences . In the case of Q beta RNA, this S1 binding site overlaps the S-site for Q beta replicase and the site for S1 binding within a binary complex . It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs . To test this idea we have used E . coli ssb mRNA prepared in vitro with the T7 promoter/RNA polymerase system . By the methods of toeprinting, enzymatic footprinting, and UV crosslinking we have shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent . The oligo(U)-sequence preceding the SD domain was found to be the target for S1 . We propose that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome.

Nucleic Acids Res, 1991 Jan 11, 19(1), 125 - 9
Sequencing and expression of the rne gene of Escherichia coli; Chauhan AK et al.; RNase E is a major endonucleolytic RNA processing enzyme in Escherichia coli . We have sequenced a 3.2 kb EcoRI-BamHI fragment encoding the rne gene, and identified its reading frame . Upstream from the gene, there are appropriate consensus sequences for a putative promoter and a ribosome binding site . We have translated this gene using a T7 RNA polymerase/promoter system . We determined 25 amino acids from the N-terminal of the translated product and they are in full agreement with the DNA sequence . The translated product of the rne gene migrates in SDS containing polyacrylamide gels as a 110,000 Da polypeptide, but the open reading frame found in the sequenced DNA indicates a much smaller protein . The entity that migrates as a 110,000 Da contains RNA, which could account, at least partially, for the migration of the rne gene product in SDS containing polyacrylamide gels.

Cell, 1991 Jan 11, 64(1), 29 - 37
Periplasmic interaction between two membrane regulatory proteins, ToxR and ToxS, results in signal transduction and transcriptional activation; DiRita VJ et al.; ToxR is a transmembrane, DNA-binding protein that can activate transcription of genes encoding cholera toxin (ctxAB) . Here we characterize ToxS, a 19 kd transmembrane regulatory protein that interacts with ToxR and stimulates its activity . If a portion of the periplasmic domain of ToxR is deleted, productive interaction with ToxS is abolished . A ToxR-PhoA fusion protein that retains most of the ToxR periplasmic region remains dependent on ToxS for its ToxR activity . ToxS protects this fusion from proteolytic cleavage, suggesting that these two proteins interact within the periplasm . Mutations in a short cytoplasmic domain of ToxR were isolated that disrupt the periplasmic interaction between ToxR and ToxS . This domain is shared by other bacterial transcriptional activators, suggesting that it may play a common role in function of these proteins and in the molecular mechanism of signal transduction.

Biochim Biophys Acta, 1991 Jan 9, 1061(1), 26 - 32
Beta-galactosidase fused to the hydrophobic domain of cytochrome b5 spontaneously associates with liposomes; George SK et al.; Since liver microsomal cytochrome b5 spontaneously associates with liposomes and membranes by means of its C-terminal hydrophobic domain (HP), chimeric proteins containing HP prepared by genetic fusion might also spontaneously associate with liposomes or cellular membranes . Synthetic DNA corresponding to the hydrophobic domain of cytochrome b5 was enzymatically fused in-frame to cloned DNA corresponding to the C-terminus of the Escherichia coli enzyme, beta-galactosidase . This protein, LacZ:HP, synthesized in E . coli and purified from a crude E . coli membrane extract, was shown to spontaneously associated with liposomes, as does cytochrome b5 . Association is rapid and stable in the presence of salt and high pH and the fusion protein behaves as an integral membrane protein . LacZ:HP can be readily and extensively purified from crude extracts by association with liposomes and this procedure may provide a convenient purification scheme for proteins not otherwise readily purified, for example polypeptides from cloned gene fragments to be used for antibody production . These hybrid proteins may represent a new potentially useful class of polypeptides capable of hydrophobic interactions with membranes.

Biochemistry, 1991 Jan 8, 30(1), 305 - 12
The tyrosine-225 to phenylalanine mutation of Escherichia coli aspartate aminotransferase results in an alkaline transition in the spectrophotometric and kinetic pKa values and reduced values of both kcat and Km; Goldberg JM et al.; Tyrosine-225 is hydrogen-bonded to the 3'-hydroxyl group of pyridoxal 5'-phosphate in the active site of aspartate aminotransferase . Replacement of this residue with phenylalanine (Y225F) results in a shift in the acidic limb of the pKa of the kcat/KAsp vs pH profile from 7.1 (wild-type) to 8.4 (mutant) . The change in the kinetic pKa is mirrored by a similar shift in the spectrophotometrically determined pKa of the protonated internal aldimine . Thus, a major role of tyrosine-225 is to provide a hydrogen bond that stabilizes the reactive unprotonated form of the internal aldimine in the neutral pH range . The Km value for L-aspartate and the dissociation constant for alpha-methyl-DL-aspartate are respectively 20- and 37-fold lower in the mutant than in the wild-type enzyme, while the dissociation constant for maleate is much less perturbed . These results are interpreted in terms of competition between the Tyr225 hydroxyl group and the substrate or quasi-substrate amino group for the coenzyme . The value of kcat in Y225F is 450-fold less than the corresponding rate constant in wild type . The increased affinity of the mutant enzyme for substrates, combined with the lack of discrimination against deuterium in the C alpha position of L-aspartate in Y225F-catalyzed transamination {Kirsch, J . F., Toney, M . D., & Goldberg, J . M . (1990) in Protein and Pharmaceutical Engineering (Craik, C . S., Fletterick, R., Matthews, C . R., & Wells, J., Eds.) pp 105-118, Wiley-Liss, New York}, suggests that the rate-determining step in the mutant is hydrolysis of the ketimine intermediate rather than C alpha-H abstraction which is partially rate-determining in wild type.

Biochemistry, 1991 Jan 8, 30(1), 222 - 30
Transcription by eucaryotic and procaryotic RNA polymerases of DNA modified at a d(GG) or a d(AG) site by the antitumor drug cis-diamminedichloroplatinum(II); Corda Y et al.; We have investigated whether DNA modified at a d(GG) or a d(AG) site by the chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP) can be used as template by wheat germ RNA polymerase II . The templates used in the present study were obtained by ligation of double-helical oligodeoxyribonucleotides, containing 18 pyrimidine bases and 2 central dG, or dA and dG, bases on one strand and 18 purine bases and 2 central dC, or dT and dC, bases on the complementary strand . Therefore, the cis-DDP adducts are only present on one strand of each of the two templates and are regularly spaced by 18 pyrimidine bases . These constructs allowed us to investigate the effect of cis-DDP on transcription of the platinated strand and of the complementary unplatinated sequence . Transcription experiments were carried out in the presence of dinucleotide primers and either a single triphosphate substrate (abortive elongation) or the full set of triphosphate substrates dictated by the template sequence (productive elongation) . The results show that the eucaryotic RNA polymerase can catalyze dinucleotide-primed reactions on platinated DNA . However, the eucaryotic enzyme behaved very differently depending on which strand was transcribed . Thus, transcription elongation was completely blocked on the strand carrying the metal complex, whereas transcription elongation was not blocked on the complementary template strand . However, on this latter strand and with the platinated polymers, productive elongation was slightly inhibited . Furthermore, abortive elongation leading to dinucleotide-primed trinucleotide formation was enhanced on the template strand complementary to that carrying the cis-DDP adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Jan 8, 30(1), 198 - 206
Nonspecific interaction of Escherichia coli pyrenyl RNA polymerase holoenzyme with synthetic polynucleotides as monitored by fluorescence spectroscopy; Johnson RS; A derivative of RNA polymerase containing approximately 2 pyrene equiv per enzyme molecule has been used to study the interaction of RNA polymerase with poly{d(A-T)}.poly{d(A-T)} and poly{d-(G-C)}.poly{d(G-C)} . As monitored by fluorescence spectroscopy, pyrenyl RNA polymerase displays a unique set of conformational changes with each synthetic polynucleotide as a function of temperature . An increase in the fluorescence intensity was observed for both polynucleotides at 5 degrees C . A decrease was observed in the case of poly{d(A-T)}.poly{d(A-T)} at 25 and 37 degrees C, whereas no discernible perturbation was observed in the case of poly{d(G-C)}.poly{d(G-C)} . Different salt dependencies were observed for the interaction of pyrenyl RNA polymerase with these polynucleotides at 5 and 25 degrees C . Further characterization of these interactions as well as correlation of the observed fluorescence changes to the corresponding open and closed complexes was carried out with heparin . The interaction between pyrenyl RNA polymerase and poly{d-(A-T)}.poly{d(A-T)} at 25 degrees C was quantified by using two different methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Jan 8, 30(1), 106 - 11
Inhibition of HIV protease activity by heterodimer formation; Babe LM et al.; The dimeric nature of the HIV protease has been exploited to devise a novel mode of inhibiting the enzyme . The use of defective monomers or nonidentical subunits to exchange with wild-type homodimers produces catalytically defective heterodimers . Incubation of the HIV1 or HIV2 protease with a 4-fold molar excess of an inactive mutant of HIV1 leads to 80 and 95% inhibition of enzyme activity, respectively . Incubating HIV1 and HIV2 proteases at a 1:5 ratio results in a 50% reduction of activity of the mixed enzymes . The HIV1/HIV2 heterodimer was identified by ion-exchange HPLC . The heterodimer may display a disordered dimer interface, thereby affecting the catalytic potential of the enzyme . This mechanism of inactivation is an example of a dominant negative mutation that can obliterate the activity of a naturally occurring multisubunit enzyme . Furthermore, it provides an alternative to active-site-directed inhibitors for the development of antiviral agents that target the dimeric interface of the HIV protease.

Biochim Biophys Acta, 1991 Jan 8, 1076(1), 86 - 90
Functional consequences of substitution of the active site (phospho)histidine residue of Escherichia coli succinyl-CoA synthetase; Majumdar R et al.; Succinyl-CoA synthetase (EC 6.2.1.5, succinate:CoA ligase (ADP-forming} of Escherichia coli is an alpha 2 beta 2 tetramer, with the active site believed to be located at the point of contact between the two subunit types . It has been previously established that the reaction involves the intermediate participation of a phosphorylated enzyme form in the process of catalysis . The site of phosphorylation (His-246) and the binding sites for the substrates ADP and ATP are located in the alpha subunit, and the succinate and CoA binding sites are in beta . A mutant form of this enzyme, with the active site histidine residue replaced by aspartate, has been produced in large quantities and purified to homogeneity . This form appears to be indistinguishable from the native enzyme with respect to its subunit assembly, but has no ability to catalyze the overall reaction . As expected, the His-246 alpha----Asp mutant is incapable of undergoing phosphorylation . We have developed an assay based upon the arsenolysis of succinyl-CoA that effectively isolates the partial reaction that occurs in the portion of the active site contributed by the beta subunit; this reaction does not involve covalent participation of His-246 alpha . We have found that the His-246 alpha----Asp mutant is also devoid of activity in this arsenolysis reaction, indicating that an intact His-246 alpha is required for the establishment of the microenvironment in this portion of the active site that is required for the corresponding step of the overall reaction.

Biochemistry, 1991 Jan 8, 30(1), 207 - 13
Mutagenic spectrum resulting from DNA damage by oxygen radicals; McBride TJ et al.; Oxygen free radicals are highly reactive species that damage DNA and cause mutations . We determined the mutagenic spectrum of oxygen free radicals produced by the aerobic incubation of single-stranded M13mp2 DNA with Fe2+ . The Fe2(+)-treated DNA was transfected into component Escherichia coli, and mutants within the nonessential lac Z alpha gene for beta-galactosidase were identified by decreased alpha-complementation . The frequency of mutants obtained with 10 microM Fe2+ was 20- to 80-fold greater than that obtained with untreated DNA . Mutagenesis was greater after the host cells were exposed to UV irradiation to induce the SOS "error-prone" response . The ability of catalase, mannitol, and superoxide dismutase to diminish mutagenesis indicates the involvement of oxygen free radicals . The sequence data on 94 of the mutants establish that mutagenesis results primarily from an increase in single-base substitutions . Ninety-four percent of the mutants with detectable changes in nucleotide sequence were single-base substitutions, the most frequent being G----C transversions, followed by C----T transitions and G----T transversions . The clustering of mutations at distinct gene positions suggests that Fe2+/oxygen damage to DNA is nonrandom . This mutational spectrum provides evidence that a multiplicity of DNA lesions produced by oxygen free radicals in vitro are promutagenic and could be a source of spontaneous mutations.

J Mol Biol, 1991 Jan 5, 217(1), 93 - 112
Structural analysis of the peptidyl transferase region in ribosomal RNA of the eukaryote Xenopus laevis; Stebbins-Boaz B et al.; Accessible single-strand bases in Xenopus laevis 28 S ribosomal RNA (rRNA) Domain V, the peptidyl transferase region, were determined by chemical modification with dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl-carbodiimide metho-p-toluene sulfonate and kethoxal, followed by primer extension . The relative accessibilities of three rRNA substrates were compared: deproteinized 28 S rRNA under non-denaturing conditions (free 28 S rRNA), 60 S subunits and 80 S ribosomes . Overall, our experimental results support the theoretical secondary structure model of Domain V derived by comparative sequence analysis and compensatory base-pair changes, and support some theoretical tertiary interactions previously suggested by covariation . The 60 S subunits and 80 S ribosomes generally show increasing resistance to chemical modification . Bases which are sensitive in free 28 S rRNA but protected in 60 S subunits may be sites for ribosomal protein binding or induced structural rearrangements . Another class of nucleotides is distinguished by its sensitivity in 60 S subunits but protection in 80 S ribosomes; these nucleotides may be involved in subunit-subunit interactions or located at the interface of the ribosome . We found a third class of bases, which is protected in free 28 S rRNA but sensitive in 60 S subunits and/or 80 S ribosomes, suggesting that structural changes occur in Domain V as a result of subunit assembly and ribosome formation . One such region is uniquely hypersensitive in eukaryotic ribosomes but is absent in Escherichia coli ribosomes . Sites that we determined to be accessible on empty 80 S ribosomes could serve as recognition sites for translation components.

J Mol Biol, 1991 Jan 5, 217(1), 63 - 74
Monomers of the Escherichia coli SSB-1 mutant protein bind single-stranded DNA; Bujalowski W et al.; The Escherichia coli wild-type single strand binding (SSB) protein is a stable tetramer that binds to single-stranded (ss) DNA in its role in DNA replication, recombination and repair . The ssb-1 mutation, a substitution of tyrosine for histidine-55 within the SSB-1 protein, destabilizes the tetramer with respect to monomers, resulting in a temperature-sensitive defect in a variety of DNA metabolic processes, including replication . Using quenching of the intrinsic SSB-1 tryptophan fluorescence, we have examined the equilibrium binding of the oligonucleotide, dT(pT)15, to the SSB-1 protein in order to determine whether a ssDNA binding site exists within individual SSB-1 monomers or whether the formation of the SSB tetramer is necessary for ssDNA binding . At high SSB-1 protein concentrations, such that the tetramer is stable, we find that four molecules of dT(pT)15 bind per tetramer in a manner similar to that observed for the wild-type SSB tetramer; i.e . negative co-operativity is observed for ssDNA binding to the SSB-1 protomers . As a consequence of this negative co-operativity, binding is biphasic, with two molecules of dT(pT)15 binding to the tetramer in each phase . However, the intrinsic binding constant, K16, for the SSB-1 protomer-dT(pT)15 interaction is a factor of 3 lower than for the wild-type protomer interaction and the negative co-operativity parameter, sigma 16, is larger in the case of the SSB-1 tetramer, indicating a lower degree of negative co-operativity . At lower SSB-1 concentrations, SSB-1 monomers bind dT(pT)15 without negative co-operativity; however, the intrinsic affinity of dT(pT)15 for the monomer is a factor of approximately 10 lower than for the protomer (50 mM-NaCl, pH 8.1, 25 degrees C) . Therefore, an individual SSB-1 monomer does possess an independent ssDNA binding site; hence formation of the tetramer is not required for ssDNA binding, although tetramer formation does increase the binding affinity significantly . These data also show that the negative co-operativity among ssDNA binding sites within an SSB tetramer is an intrinsic property of the tetramer . On the basis of these studies, we discuss a modified explanation for the temperature-sensitivity of the ssb-1 phenotype.

J Biol Chem, 1991 Jan 5, 266(1), 66 - 70
Structural role of serine 127 in the NADH-binding site of human NADH-cytochrome b5 reductase; Yubisui T et al.; Serine 127 of human NADH-cytochrome b5 reductase was replaced by proline and alanine by site-directed mutagenesis . The former mutation has been found in the genes of patients with hereditary deficiency of the enzyme . Both the mutant enzymes (Ser-127----Pro mutant and Ser-127----Ala mutant) were overproduced in Escherichia coli and purified to homogeneity . The two purified mutant enzymes showed indistinguishable spectral properties which differed from those of the wild-type enzyme . The mutant enzymes showed higher molecular extinction coefficients at 462 nm than that of the wild-type enzyme . Quenching of FAD fluorescence in these mutant enzymes was significantly less than that in the wild-type enzyme . Furthermore, circular dichroism spectra of the mutant enzymes were different, in both the visible and ultraviolet regions, from that of the wild-type enzyme . The spectra of the mutant enzymes in the visible region were restored to almost the same spectrum as the wild type upon reduction with NADH . Ser-127----Pro mutant and Ser-127----Ala mutant showed very low Kcat/Km (NADH) values (5 x 10(7) and 3.5 x 10(7) s-1 M-1, respectively) with cytochrome b5 as an electron acceptor, than that of the wild-type enzyme (Kcat/Km (NADH) = 179 x 10(7) s-1 M-1), while the Kcat/Km (cytochrome b5) value for each enzyme was similar . The mutant enzymes were less thermostable than the wild-type enzyme . These results indicate that serine 127 plays an important role to maintain the structure of the NADH-binding site in the enzyme.

J Mol Biol, 1991 Jan 5, 217(1), 15 - 7
Preliminary crystallographic analysis of the ATP-hydrolysing domain of the Escherichia coli DNA gyrase B protein; Jackson AP et al.; The 43 kDa N-terminal ATPase domain of the Escherichia coli DNA gyrase B protein has been purified from an over-expressing strain . This protein has been crystallized in two crystal forms, both in the presence of the non-hydrolysable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate . The first crystal form is monoclinic P2(1), with cell dimensions a = 76 A, b = 88 A, c = 82 A, beta = 105.5 degrees, and diffracts to at least 2.7 A resolution using synchrotron radiation . Crystal density measurements suggest that there are two molecules in the asymmetric unit (Vm = 3.08 A3/Da) . The second crystal form is orthorhombic C222(1), with cell dimensions a = 89.2 A, b = 143.1 A and c = 79.8 A . The crystals diffract to beyond 3 A and are stable for at least 100 hours when exposed to X-rays from a rotating anode source . The asymmetric unit of this crystal form appears to contain one molecule (Vm = 2.96 A3/Da) . Data have already been collected to 5 A resolution from native crystals of this second form, and to 6 A resolution from three heavy-atom derivatives . Electron density maps calculated using phases obtained from these derivatives show features consistent with secondary structural elements, and have allowed the molecular boundary to be determined . Higher resolution native and derivative data are being collected.

J Biol Chem, 1991 Jan 5, 266(1), 615 - 22
Structural and functional conservation of synaptotagmin (p65) in Drosophila and humans; Perin MS et al.; Synaptotagmin (p65) is an abundant synaptic vesicle protein that contains two copies of a sequence that is homologous to the regulatory region of protein kinase C . Full length cDNAs encoding human and Drosophila synaptotagmins were characterized to study its structural and functional conservation in evolution . The deduced amino acid sequences for human and rat synaptotagmins show 97% identity, whereas Drosophila and rat synaptotagmins are only 57% identical but exhibit a selective conservation of the two internal repeats that are homologous to the regulatory region of protein kinase C (78% invariant residues in all three species) . The two internal repeats of synaptotagmin are only slightly more homologous to each other than to protein kinase C, and the differences between the repeats are conserved in evolution, suggesting that they might not be functionally equivalent . The cytoplasmic domains of human and Drosophila synaptotagmins produced as recombinant proteins in Escherichia coli specifically bound phosphatidylserine similar to rat synaptotagmin . They also hemagglutinated trypsinized erythrocytes at nanomolar concentrations . Hemagglutination was inhibited both by negatively charged phospholipids and by a recombinant fragment from rat synaptotagmin that contained only a single copy of the two internal repeats . Together these results demonstrate that synaptotagmin is highly conserved in evolution compatible with a function in the trafficking of synaptic vesicles at the active zone . The similarity of the phospholipid binding properties of the cytoplasmic domains of rat, human, and Drosophila synaptotagmins and the selective conservation of the sequences that are homologous to protein kinase C suggest that these are instrumental in phospholipid binding . The human gene for synaptotagmin was mapped by Southern blot analysis of DNA from somatic cell hybrids to chromosome 12 region cen-q21, and the Drosophila gene by in situ hybridization to 23B.

J Biol Chem, 1991 Jan 5, 266(1), 355 - 61
Structural and functional relationships between h- and l-caldesmons; Hayashi K et al.; Two different Mr forms of caldesmon as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr values in the range of 120,000-150,000, h-caldesmon and 70,000-80,000, l-caldesmon) have been already identified . h-Caldesmon is predominantly expressed in smooth muscle cells, whereas l-caldesmon widely distributes in non-muscle cells . Most recently, the molecular cloning of h-caldesmon has been reported (Hayashi, K., Kanda, K., Kimizuka, F., Kato, I., and Sobue, K . (1989) Biochem . Biophys . Res . Commun . 164, 503-511; Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R . G., and Lin, W-G . (1989) J . Biol . Chem . 264, 13873-13879) . The calculated Mr of this protein from its primary structure is 88,743 . Here, the nucleotide and deduced amino acid sequences of l-caldesmon have been determined by cloning and sequencing the cDNA from chick brain and compared with those of h-caldesmon . The l-caldesmon cDNA encodes a sequence of 517 amino acids with the calculated Mr of 58,844 . Two isoforms of caldesmon conserve the completely identical sequences in the NH2- and COOH-terminal domains except for the insertion of Ala-508 in l-caldesmon . Interestingly, the central repeating sequence of h-caldesmon (residues 201-447) is deleted in the l-caldesmon molecule . The short NH2-terminals of two caldesmons individually show the unique sequences . The results of Northern and Southern blot analyses suggest that two mRNAs (4.8 and 4.1 kilo-bases) coding for caldesmon isoforms may be generated from a single gene by alternative splicing . Using a series of truncated caldesmons expressed in Escherichia coli, the common calmodulin-, tropomyosin-, and actin-binding sites and the minimum regulatory domains, which are involved in the Ca2(+)-dependent regulation of actin-myosin interaction, have been identified within the limited consensus sequences (residues 381-433 for l-caldesmon and residues 636-688 for h-caldesmon).

J Biol Chem, 1991 Jan 5, 266(1), 506 - 9
Expression of human IMP dehydrogenase types I and II in Escherichia coli and distribution in human normal lymphocytes and leukemic cell lines; Konno Y et al.; Two distinct cDNAs encoding proteins with 84% sequence identity have been isolated for human IMP dehydrogenase (EC 1.1.1.205) (Natsumeda, Y., Ohno, S., Kawasaki, H., Konno, Y., Weber, G., and Suzuki, K . (1990) J . Biol . Chem . 265, 5292-5295), an important target in antileukemic chemotherapy . We constructed expression plasmids containing these cDNAs in full length with pUC plasmids and produced lacZ'-IMP dehydrogenase fusion proteins in Escherichia coli . Both synthesized proteins exhibited IMP dehydrogenase activity and were partially separated from endogenous E . coli IMP dehydrogenase . By injecting the fusion proteins into mice we generated a polyclonal antibody specific to type I IMP dehydrogenase and an antibody which reacted with both types . Immunoblot analysis revealed that the total amounts of types I and II enzymes increased in human leukemic cell lines K562 and HL-60 in agreement with the increase in IMP dehydrogenase activity to 7.8- and 9.4-fold, respectively, above that of normal lymphocytes . The extent of expression of type I IMP dehydrogenase was similar in these cells, however, indicating that the increase in IMP dehydrogenase amount in leukemic cells was due to specific up-regulation of type II enzyme . Northern blot analysis also showed specific and predominant expression of type II in the leukemic cells . Thus, de novo GTP biosynthesis may be controlled differently in normal and neoplastic cells by different IMP dehydrogenase molecular species.

Eur J Obstet Gynecol Reprod Biol, 1991 Jan 4, 38(1), 19 - 24
Ginger treatment of hyperemesis gravidarum; Fischer-Rasmussen W et al.; Thirty women participated in a double-blind randomized cross-over trial of the efficacy of a natural product, the powdered root of ginger (Zingiber officinale), and placebo in hyperemesis gravidarum . Three patients had to be withdrawn . Each woman swallowed capsules containing either 250 mg ginger or lactose q.i.d . during the first 4 days of the treatment period . Interrupted by a 2 days wash-out period the alternative medication was given in the second 4-day period . The severity and relief of symptoms before and after each period were evaluated by two scoring systems . The scores were used for statistical analyses of possible differences . Subjectively assessed, 19 women (70.4%) stated preference to the period in which ginger, as was later disclosed, had been given (P = 0.003) . More objectively assessed by relief scores a significantly greater relief of the symptoms was found after ginger treatment compared to placebo (P = 0.035) . No side effects were observed . The possible mutagenic and antimutagenic characters of ginger reported in a study of E . coli have not been evaluated with respect to any significance in humans . Powdered root of ginger in daily doses of 1 g during 4 days was better than placebo in diminishing or eliminating the symptoms of hyperemesis gravidarum.

Science, 1991 Jan 4, 251(4989), 81 - 5
Whole animal cell sorting of Drosophila embryos; Krasnow MA et al.; Use of primary culture cells has been limited by the inability to purify most types of cells, particularly cells from early developmental stages . In whole animal cell sorting (WACS), live cells derived from animals harboring a lacZ transgene are purified according to their level of beta-galactosidase expression with a fluorogenic beta-galactosidase substrate and fluorescence-activated cell sorting . With WACS, incipient posterior compartment cells that express the engrailed gene were purified from early Drosophila embryos . Neuronal precursor cells were also purified, and they differentiated into neurons with high efficiency in culture . Because there are many lacZ strains, it may be possible to purify most types of Drosophila cells . The same approach is also applicable to other organisms for which germ-line transformation is possible.

Biochim Biophys Acta, 1991 Jan 3, 1056(1), 76 - 84
Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli; Houssin C et al.; Osmotic upshock of E . coli cells in NaCl or sucrose medium resulted in a large decrease in the cytoplasmic volume and the inhibition of growth, of the electron transfer chain and of four different types of sugar transport system: the lactose proton symport, the glucose phosphotransferase system, the binding-protein dependent maltose transport system and the glycerol facilitator . In contrast to NaCl and sucrose, the permeant solute glycerol had no marked effect . These inhibitions could be partially relieved by glycine betaine . Despite these inhibitions, the internal pH, the protonmotive force and the ATP pool were maintained . It is concluded that inhibition of electron transfer and of sugar transport is the consequence of conformational changes caused by the deformation of the membrane . It is also concluded that the arrest of growth observed upon osmotic upshock is not due to energy limitations and that it cannot be explained by the inhibition of carbohydrate transport.

J Chromatogr, 1991 Jan 2, 562(1-2), 377 - 89
Structural analysis of biologically active peptides and recombinant proteins and their modified counterparts by mass spectrometry; Pramanik BN et al.; The structural characterization of the Escherichia coli-expressed human interferon alpha-2b (rh-IFN alpha-2b) was carried out by employing the fast atom bombardment (FAB) and plasma desorption (PD) mapping methods . The mass spectral data of the rh-IFN alpha-2b and the trypsin-generated peptide mixture allowed rapid and facile confirmation of the cDNA-derived sequence and determination of the existing disulfide pattern in the protein molecule . The same PD/FAB mapping approach was successfully employed in the structural determination of the iodination reaction product of rh-IFN alpha-2b and the potent vasoconstrictor peptide endothelin.

Gene, 1991 Jan 2, 97(1), 21 - 7
Sequence and transcriptional activity of the Escherichia coli K-12 chromosome region between rrnC and ilvGMEDA; Coppola G et al.; We previously identified a protein related to the expression of the ilvGMEDA cluster of Escherichia coli K-12 . It was observed that this ilv-related protein was produced at higher levels in UV irradiated cells infected with lambda dilvGMEDA phage with specific ilvG mutations (ValR), compared to phage carrying the wild-type(ValS) ilvG allele . The gene encoding this protein was further localized to a region between rrnC and ilvGMEDA by analyzing restriction fragment subsets in maxicells . We have now determined the nucleotide (nt) sequence of the 3.5-kb segment between rrnC and ilvGMEDA, and two open reading frames (ORFs) are present in the region expected to contain the ilv-related gene . These ORFs predicts Mrs of 18,751 (ORFI) and 20,085 (ORFII) Da, and both ORFs have a strong probability to encode proteins based on codon frequency analysis . Maxicell analysis revealed that a 1319-bp HindIII-SmaI fragment containing ORFI encodes the ilv-related peptide . We deleted a ClaI fragment that removed a portion of ORFI encoding the C-terminal region of the peptide, and maxicell analysis revealed a decrease in the size of the protein produced in accord with the prediction . RNA slot blots and Northern blots were used to characterize transcripts encoding ORFI . A transcript initiated 112 nt from the ilvGp2 promoter, but proceeding in the opposite direction, may encode the ORFI peptide.

Gene, 1991 Jan 2, 97(1), 131 - 6
Ultraviolet light-induced plasmid-chromosome recombination in Escherichia coli: the role of recB and recF; Mudgett JS et al.; Bacterial host cells of different rec genotypes were used to investigate genetic requirements of ultraviolet light (UV)-induced homologous plasmid-chromosome recombination . Plasmid DNAs which contained a wt or mutant lacY gene were irradiated with UV prior to transformation into Escherichia coli host strains which contained the complementary lacY allele . Surviving transformants were screened to determine the directions of UV-induced recombinational exchange between the bacterial and plasmid lacY genes, by assaying lactose utilization . Nonreciprocal chromosome-to-plasmid recombination was 100% dependent on the recA gene and greater than 80% dependent on the recF gene, but not dependent upon the recB gene of E . coli . In contrast, reciprocal plasmid-chromosome recombination was strictly dependent on the recA gene, greatly dependent (approx . 80%) on the recF gene, and moderately dependent on the recB gene . Nonreciprocal plasmid-to-chromosome recombination was only induced at very low frequencies, and appeared to be moderately dependent on the recB gene, but not dependent on the recF gene . UV-induced plasmid-chromosome recombination appeared to proceed by a two-step mechanism . In this model, the initial step is recF-dependent, recB-independent, and either resolves to become a nonreciprocal chromosome-to-plasmid recombinant, or proceeds to the second step . The second step is moderately recB-dependent and results in the reciprocal exchange of plasmid-chromosome sequences.

Gene, 1991 Jan 2, 97(1), 119 - 23
Rapid filter assay for the detection of DNA polymerase activity: direct identification of the gene for the DNA polymerase from Thermus aquaticus; Sagner G et al.; A method for rapid identification of DNA polymerase activity employing an activated DNA substrate covalently bound to nitrocellulose membranes is described . Samples containing DNA polymerase are spotted and the membranes are incubated in an appropriate polymerization buffer containing radioactively labelled dNTPs . By autoradiography of the dried filters, DNA polymerase activity can be directly identified . The method can be used for fast and large-scale screening of chromosomal expression libraries for heterologous DNA polymerases characterized by activity optima different from those of the host organisms . We have identified the gene of the thermostable DNA polymerase from Thermus aquaticus in an expression library of Escherichia coli.

Gene, 1991 Jan 2, 97(1), 113 - 7
A vector for directional cloning and expression of polymerase chain reaction products in Escherichia coli; Brandt ME et al.; This paper describes the construction of a modified vector for the cloning and expression of protein-encoding genes in Escherichia coli . The vector, pfXblue, is derived from the system originally developed by Nagai and Thogerson {Nature 309 (1984) 810-812}, but contains a modified multiple cloning site (MCS) from M13mp18 to allow directional insertion of foreign coding sequences . The MCS is located within the M13mp18 lacZ' gene and thus allows blue/white screening of colonies for inserts . The inserted gene is expressed as a fusion protein, which, when cleaved by the coagulation factor Xa protease, yields the mature product . This vector was successfully used for the production of a mitochondrial {2Fe-2S}ferredoxin using polymerase chain reaction products generated from a chick kidney cDNA library.

Gene, 1991 Jan 2, 97(1), 69 - 75
Cloning and analysis of the gene encoding the cytadherence phase-variable protein HMW3 from Mycoplasma pneumoniae; Ogle KF et al.; We have cloned the gene encoding the Mycoplasma pneumoniae cytadherence-accessory protein HMW3 into Escherichia coli to study its phase-variable expression . A truncated HMW3 protein (HMW3'; 113 kDa), identified using HMW3-specific, affinity-purified antibodies, was expressed under the control of the lacZ promoter in lambda gt11 . The protein did not react with beta-galactosidase (beta Gal)-specific antibodies, however, indicating that HMW3' was not a beta Gal fusion protein . The direction of transcription was determined by examining gene expression from inserts in opposite orientations with respect to the lacZpo in pUC18 and pUC19, to generate pKV5 and pKV6 . Amino acid sequence data were obtained from an enzymatically generated HMW3 peptide fragment and used to create a degenerate 17-mer probe . The degenerate 17-mer hybridized to the mycoplasma DNA insert in pKV6; both the 17-mer and the pKV6 insert hybridized to a 9.4-kb EcoRI fragment from wild-type (wt) M . pneumoniae chromosomal DNA . This EcoRI fragment was cloned from wt M . pneumoniae and an HMW3-deficient variant in both orientations into pUC18 . The HMW3'-encoding region was localized to the center of the 9.4-kb EcoRI fragment, and no differences were observed in restriction patterns between the wt and variant . Although the 9.4-kb EcoRI fragment included the DNA segment encoding HMW3', neither this protein, nor derivatives thereof, were detected in IPTG-induced E . coli containing the EcoRI fragment from either wt or variant M . pneumoniae, in either orientation in pUC18.

Gene, 1991 Jan 2, 97(1), 77 - 85
A Mycoplasma hyorhinis protein with sequence similarities to nucleotide-binding enzymes; Notarnicola SM et al.; We have determined the nucleotide (nt) and deduced amino acid (aa) sequence of a unique 115-kDa Mycoplasma hyorhinis protein (P115) with an N-terminal region containing a highly conserved consensus sequence characteristics of nt-binding domains of several ATPase and GTPase enzymes . However, P115 lacked additional conserved features characteristic of some classes of nt-binding proteins . Based on the hydropathy profile of the deduced aa sequence, the absence of a leader peptide, its exclusive partitioning into the hydrophilic phase during Triton X-114 phase fractionation of M . hyorhinis, and immunofluorescence analysis indicating no surface-exposed domains, it was concluded that P115 is a cytoplasmic protein lacking intrinsic membrane interaction . M . hyorhinis P115 appears to be a species-specific protein, since it was not detected in any other mycoplasmal or bacterial species examined with specific antibody or genomic probes . Since genetic systems for direct mutational analysis are currently unavailable in this organism, sequence analysis provides critical information in establishing the possible function of this protein . Moreover, the nt sequence encoding P115 reported here supports a previously proposed model, based on synthesis of P115-related proteins in Escherichia coli, suggesting that multiple polypeptide products can be generated from mycoplasma genes by promiscuous translation initiation in this heterologous expression system.

Arch Virol Suppl, 1991, 3, 149 - 56
Identification and production of pestivirus proteins for diagnostic and vaccination purposes; Lecomte C et al.; Using a panel of monoclonal antibodies (MAbs) previously characterized by seroneutralization, immunofluorescence and radioimmunoprecipitation, we have identified Pestivirus proteins useful for diagnostic purposes from the cytopathic Osloss isolate of bovine viral diarrhea virus (BVDV) . Proteins that should be useful for vaccination have also been analysed . Cell-free translation of RNA from glycoprotein-coding cDNA fragments produced, when synthesized in the presence of canine pancreatic microsomes, two glycosylated proteins that were independently recognized and immunoprecipitated by two distinct classes of neutralizing MAbs . A similar in vitro procedure was carried out on nonstructural protein-coding sequences and allowed to identify a viral translation product that specifically reacted with MAbs directed against the 80 kDA protein of a number of Pestivirus strains . Its positioning within the polyprotein encoded by the viral genome was refined by epitope scanning using synthetic hexameric peptides . This viral antigen was further expressed in E . coli, produced as inclusion bodies and used successfully as an ELISA antigen in both competitive and indirect assays for the detection of BVD antibodies in cattle sera.

Int J Immunopharmacol, 1991, 13(2-3), 125 - 8
Inhibition of antibody-dependent allergic autocytotoxicity in rheumatoid arthritis by OM-89; Podleski WK et al.; Rheumatoid arthritis (RA) is a disease of multiple etiologies and clinical evidence suggests that a separate variant called "allergic arthritis" induced by food antigens could exist . A missing link in the confirmation of such an observation is a relative lack of a reliable in vitro assay which can confirm the in vivo oral ingestion challenge . Therefore, white blood cells (WBC) from 33 rheumatoid arthritis patients were separated and their disintegration was measured in the presence of specific IgE RAST positive sera and gluten-gliadin antigens . This assay was called the antibody-dependent allergic autocytotoxicity (ACT) test which represents an equivalent of an oral ingestion challenge with food antigens . Control WBC expressed 10-45% disintegration as compared to 75-95% in RA . Preincubation of WBC with OM-89 (immunomodulating fractions of Escherichia coli, OM Laboratories Ltd, Geneva, Switzerland) inhibited significantly antibody-dependent ACT in a dose-related manner (P less than 0.001) in our patients.

Free Radic Res Commun, 1991, 12-13 Pt 1, 419 - 28
Anaerobic inductions of active forms of superoxide dismutases in Escherichia coli; Privalle CT et al.; Escherichia coli growing anaerobically respond to NO3- with a approximately 3-fold induction of active FeSOD and a approximately 5.5-fold induction of an inactive, but activatable form of MnSOD (pro-MnSOD) . Paraquat, which mediates anaerobic electron flow to NO3-, increased the induction of pro-MnSOD to approximately 2.5-fold . Strains with defects in the SOD genes or which lacked nitrate reductase activity failed to accumulate active or pro-forms of SODs in response to NO3- +/- PQ++ . Diamide caused anaerobic induction of active MnSOD and this effect was also observed in a glutathione-negative strain . These inductions required de novo synthesis of protein, even when cell content of pro-MnSOD had been elevated by exposure to NO3- +/- PQ++ prior to addition of diamide . These results indicate that oxidation of a cell component increases biosynthesis of the SOD gene product and this postulated oxidation can be caused by terminal electron acceptors, such as dioxygen or NO3- . In addition, it appears that insertion of the correct metal can be rate-limiting, leading to competition by other metals and to the accumulation of inactive, incorrectly substituted pro-forms . Metal insertion may be dependent upon the valence of the metal, which may be influenced, in turn, by the redox status of the cells . Diamide and redox active agents such as ferricyanide may thus allow anaerobic production of active MnSOD by favoring the production of a complexed form of Mn(III) which can compete favorably with other metal cations for the active site of nascent MnSOD.

Free Radic Res Commun, 1991, 12-13 Pt 1, 401 - 10
Recombinant human superoxide dismutases: production and potential therapeutical uses; Gorecki M et al.; In many pathological situations, tissue damage is caused by cellular generation of superoxide free radicals (O2-) . These active species are generated during post-ischemic reperfusion of organs, in hyperoxic tissue, during acute and chronic inflammation and during exposure to ionizing radiation . Exogenous superoxide dismutase (SOD) was shown to significantly prevent such damage . The genes for human cytosolic Cu/ZnSOD and mitochondrial MnSOD were cloned and introduced into an E . coli expression system . The proteins were expressed in high yields and purified to homogeneity, yielding pharmaceutical-grade materials . These enzymes were used in a variety of in vivo animal models for the demonstration of their protective effects against oxidative damage . Comparative pharmacokinetic studies in rats have revealed that the half-life of Cu/ZnSOD was 6-10 min., while that of MnSOD was 5-6 hours, thus indicating that MnSOD may be superior to Cu/ZnSOD for the treatment of chronic diseases . Indeed, MnSOD was found to be effective as an anti-inflammatory agent in the rat carrageenan induced paw edema acute inflammation model . Both enzymes were also effective in ameliorating post-irradiation damage in mice exposed to whole-body or localized chest X-ray radiation.

Free Radic Res Commun, 1991, 12-13 Pt 1, 335 - 48
Biochemical and stability properties of recombinant human MnSOD; Werber MM et al.; The light absorption spectral properties of recombinant human MnSOD, which contains an N-terminal additional methionyl residue, were investigated as a function of pH in the range 4.5-10.5 . Whereas the extinction coefficient, epsilon M, at the UV maximum (282 nm) was essentially independent of pH, the epsilon M values of the visible spectrum maximum (482 nm) displayed a bell-shaped dependence with a plateau between pH 6.5 and 8 . Those spectral changes were reversible and the enzymatic activity was not affected by exposure to buffered solutions at 25 degrees C in the pH range 5-10.5 . The stability of MnSOD was determined between 25 and 60 degrees C at two different pH: 6.5 and 8.2 . The enzyme was found to be considerably more stable at pH 6.5 than at pH 8.2, both toward aggregation and degradation . The gel permeation properties of MnSOD were investigated: the enzyme is a tetramer, with a subunit of 22.2 kD; however, it elutes from a Superose 12 column (Pharmacia) with an apparent molecular weight of approximately 60 kD . Under dissociative conditions (such as guanidine-HC1), molecular weights corresponding to the dimer and monomer could also be demonstrated . It thus appears that the tetramer adopts a non-globular shape, which causes the deviation from the Stokes radius corresponding to its molecular weight.

Agents Actions Suppl, 1991, 32, 45 - 9
Antipyretic activity of tebufelone (NE-11740) in man; Powell JH et al.; Tebufelone (formerly NE-11740) is a member of the new class of di-tert-butyl-phenol anti-inflammatory agents . It has previously been reported that this new agent has potent analgesic, antipyretic, and anti-inflammatory effects using in vitro, in vivo, and ex vivo experimental models . A randomized, active- and placebo- controlled double-blinded study in 120 healthy males, 20 to 55 years old, was conducted to clinically assess tebufelone's antipyretic activity . Subjects received a single peroral dose of placebo, 650 mg aspirin (ASA), or tebufelone at doses of 25, 50, 100, or 200 mg . Thirty minutes later, E . coli endotoxin (2 ng/kg) was administered intravenously . Oral temperatures were recorded at 15 minute intervals from 30 minutes post dosing to 8 hours post endotoxin administration . Areas under the temperature curves (AUCs), adjusted for baseline, were significantly lower than placebo for ASA and all but the 25 mg tebufelone groups . An AUC dose-response equation estimates 60 mg tebufelone as equivalent to 650 mg ASA, with 50 mg tebufelone not significantly greater than 650 mg ASA . Side effects, attributable to the endotoxin, included mild flu-like symptoms and were worse in the placebo group and the non-efficacious 25 mg tebufelone group . Doses of 100 and 200 mg tebufelone had onset characteristics indistinguishable from 650 mg ASA, whereas 50 mg tebufelone showed significantly slower onset while suppressing temperature for a longer period than ASA . These results provide an important early demonstration of tebufelone's biological activity in man.

Adv Exp Med Biol, 1991, 283, 225 - 33
Gene specific damage and repair after treatment of cells with UV and chemotherapeutical agents; Bohr VA; We have previously demonstrated preferential DNA repair of active genes in mammalian cells . The methodology involves the use of a specific endonuclease or other more direct approaches to create nicks at sites of damage followed by quantitative Southern analysis and probing for specific genes . Initially, we used pyrimidine dimer specific endonuclease to detect pyrimidine dimers after UV irradiation . We now also use the bacterial enzyme ABC excinuclease to examine the DNA damage and repair of a number of adducts other than pyrimidine dimers in specific genes . We can detect gene specific alkylation damage by creating nicks via depurination and alkaline hydrolysis . In our assay for preferential repair, we compare the efficiency of repair in the DHFR gene to that in the 3' flanking, non-coding region to the gene . In CHO cells, UV induced pyrimidine dimers are efficiently repaired from the active DHFR gene, but not from the inactive region . We have demonstrated that the 6-4 photoproducts are also preferentially repaired and that they are removed faster from the regions studied than pyrimidine dimers . Using similar approaches, we find that DNA adducts and crosslinks caused by cisplatinum are preferentially repaired in the active gene compared to the inactive regions and to the inactive c-fos oncogene . Also, nitrogen mustard and methylnitrosurea damage is preferentially repaired whereas dimethylsulphate damage is not . NAAAF adducts do not appear to be preferentially repaired in this system.(ABSTRACT TRUNCATED AT 250 WORDS)

Vet Res Commun, 1991, 15(2), 95 - 105
The effects of experimentally induced fever on the estimated blood flow to and oxygen utilization by the liver and the viscera drained by the portal vein in sheep; Kisauzi DN et al.; Intrajugular injection of a purified E . coli lipopolysaccharide induced a biphasic fever in sheep after a latent period of 12 to 20 min . The changes in the blood flow from the liver and from the viscera drained by the portal vein were: (a) in the latent period, decreases in total hepatic blood flow (THF) due to decreased portal venous blood flow (PVF); (b) during the first febrile phase, increases in THF due to increased hepatic arterial blood flow and, (c) in the second febrile phase, decreases in THF due to decreased PVF . Although there were large variations in the oxygen supply to the viscera drained by the portal vein and to the liver, there were relatively small or no changes in their oxygen consumption.

Bioorg Khim, 1991 Jan, 17(1), 81 - 7
{Design of recombinant-stable plasmids of the pFH series}; Daniliuk NK et al.; By cloning synthetic oligonucleotides into pUC18 plasmid, pFH123--pFH127 plasmids have been constructed . Their polylinker area, along with sites of widely used restriction endonucleases, contains two pairs each of FokI and HgaI sites in the opposite orientation to provide subfragment with unique predetermined 5'-ends . Comparative stability of the new plasmids and their derivatives has been studied and compared with that of the earlier constructed pMB plasmids.

J Clin Lab Anal, 1991, 5(3), 197 - 205
Use of inactive beta-D-galactosidase for elimination of interference by anti-beta-D-galactosidase antibodies in immune complex transfer enzyme immunoassay for anti-thyroglobulin IgG in serum using beta-D-galactosidase from Escherichia coli as label; Kohno T et al.; A novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG using beta-D-galactosidase from Escherichia coli as label was reported previously . This immunoassay was highly sensitive in demonstrating anti-thyroglobulin IgG not only in all patients with Graves' disease and chronic thyroiditis but also in a large proportion of healthy subjects . However, the detection of anti-thyroglobulin IgG at low levels in some serum samples was difficult, probably due to the presence of anti-beta-D-galactosidase antibodies . In the present study, the use of inactive beta-D-galactosidase was tested for elimination of interference by anti-beta-D-galactosidase antibodies . Preincubation of serum samples with excess of inactive beta-D-galactosidase resulted in sufficiently low backgrounds to detect low levels of anti-thyroglobulin IgG with little effect on the dose-response of anti-thyroglobulin IgG . As a result, it was revealed that anti-thyroglobulin IgG was present in almost all healthy subjects as well as all patients with Graves' disease and chronic thyroiditis.

Free Radic Res Commun, 1991, 12-13 Pt 2, 691 - 6
Protection against free radical-induced and transition metal-mediated damage: the use of "pull" and "push" mechanisms; Chevion M; Free radicals have been incriminated in a variety of injurious processes including the toxicity of the herbicide paraquat and the damage following ischemia and reperfusion of different organs . Based on the assumption that iron and copper could serve as mediators for the transformation of relatively low reactive species (such as superoxide radicals, hydrogen peroxide, ascorbate, and others) to the highly reactive species, in the site-specific metal-mediated mechanism, two new modes for intervention have been tried out . The first is the introduction of specific chelators that "pull" out redox-active and available metals, and by this reduce the apparent damage . Desferrioxamine was shown to protect bacterial cells and mammals against the poisonous effects of paraquat . Using the retrogradly perfused isolated rat heart, we have demonstrated that the chelator neocuproine, which effectively binds both iron and copper provides a major protection against hydrogen peroxide-induced cardiac damage and against ischemia/reperfusion-induced arrhythmias . Likewise, TPEN a heavy metal chelator, provides almost total (greater than 90%) protection against ischemia/reperfusion-induced arrhythmias . The other mode of intervention is the use of redox-inactive metal ions that could compete for the binding sites of iron and copper, and by this "push" these metal ions out, lead to their displacement, and divert the site of free radical attack . Applying Zn(II) complexes provided a marked protection against metal mediated free radical-induced damage in the copper-mediated paraquat toxicity to E . coli, and in the arrhythmias induced by ischemia and reperfusion . It is proposed that the complex zinc-desferrioxamine would be the ultimate protector being effective by both the "pull" and "push" mechanisms.

Free Radic Res Commun, 1991, 12-13 Pt 2, 629 - 32
Free radicals scavengers attenuate platelet-activating factor (PAF)- and endotoxin-induced intestinal myoelectric disturbances in rats; Pons L et al.; Pretreatment with radical scavengers significantly reduced the intestinal myoelectric disturbances following either E . coli endotoxin or platelet-activating factor (PAF) injection in the rat indicating that free radicals might be involved in the intestinal motor alterations observed in endotoxin shock and that PAF acts partially via free radical production . Moreover, dimethylsulfoxide (DMSO) was found to be more effective in inhibiting the endoxotin-induced intestinal motor alterations, than superoxide dismutase (SOD) and allopurinol . BN 52021, a specific PAF antagonist, was able to reduce the effects of endotoxin on intestinal motility . However, when BN 52021 was combined with free radical scavengers, no additive effect was observed . It is concluded that free radicals involved in endotoxin-induced intestinal motility alterations are at least in part produced in response to PAF.

Mutat Res, 1991 Jan, 254(1), 45 - 53
Inhibitory effect of cadmium and mercury ions on induction of the adaptive response in Escherichia coli; Takahashi K et al.; Cadmium and mercury ions inhibited the promotion of ada and alkA gene expression in the adaptive process induced by methylating agents such as N-methyl-N-nitrosourea (MNU), methyl methanesulfonate (MMS) and methyl iodide in Escherichia coli . In fact, the induction of O6-methylguanine-DNA methyl-transferase (MGTase) by MNU was suppressed in E . coli in the presence of these metal ions . These ions potentiated mutagenesis induced by methylating agents such as MNU and MMS, but not that induced by ethylating agents, UV irradiation, or N4-aminocytidine . These comutagenic effects were observed in wild-type and umuC36 strains of E . coli but not in the ada-5 strain, which is unable to induce the adaptive response . These results suggest that the comutagenic effects of Cd2+ and Hg2+ are due to inhibition of ada and alkA gene expression promoted by methylated MGTase.

Gen Pharmacol, 1991, 22(2), 381 - 8
Inhibition of pyrogen Escherichia coli fever with intracerebral administration of prazosin, dihydrobenzperidol and nifedipin in the rabbits; Szreder Z et al.; 1 . The thermoregulatory, effector processes were investigated after treatment with prazosin (PRA), dihydrobenzperidol (DHBP) and nifedipin (ADA) applied to the thermosensitive zone of the anterior hypothalamus (PO/AH) on normothermic and feverish rabbits (LPS, lipopolysaccharide E . coli; 1 meg/kg, i.v.) . 2 . The alpha 1-noradrenergic receptor antagonists, PRA and DHBP, applied to the PO/AH produced an abolishment of fever elicited by pyrogen i.v . injection mainly because of vasodilation of ear skin vessels and attenuation of metabolic rate . 3 . Calcium channel blocker, ADA, also induced a decline in the rabbit's core temperature in the same manner . 4 . All these drugs given to the PO/AH did not change the body temperature in normothermic rabbits . 5 . These results, therefore, strongly suggest that alpha 1-noradrenergic receptors subserve the coordinated thermoregulatory mechanisms in PO/AH which are required for antipyresis . The inhibition of Ca2+ turnover is discussed as a possible mechanism of antipyretic action of these drugs given to the PO/AH.

Methods Enzymol, 1991, 197, 3 - 23
Assay strategies and methods for phospholipases; Reynolds LJ et al.; Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous . One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions . In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options . Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed . The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay . The TLC assay is probably the most accurate, sensitive assay available . These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive . The radioactive E . coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases . The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required . Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed . With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used . This assay is approximately as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially . Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous . The spectrophotometric SIBLINKS assay and some of the fluorescent assays show promise of filling this need.

Electrophoresis, 1991 Jan, 12(1), 22 - 7
Structural analysis of recombinant proteins prepared by semi-dry electroblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Fausset PR et al.; Proteins that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were electroblotted onto polyvinylidene difluoride membranes in procedures to prepare homogeneous recombinant proteins for direct N-terminal sequence analysis . A semi-dry blotting procedure was employed to immobilize protein bands on the membranes for subsequent sequence analysis . This method has been used routinely to evaluate the quality of recombinant proteins, which are present in crude cell extracts produced by different expression systems or under different expression conditions . N-Terminal processing, amino acid misincorporation, as well as the inefficient secretion of recombinant proteins can be detected by direct N-terminal sequence analysis of the purified electroblotted samples . Consequently, time-consuming chromatographic procedures can be eliminated . These procedures are also especially valuable for determining degradation sites of a purified recombinant protein, as well as evaluating multiple gene products expressed by isolated cluster genes.

Chem Pharm Bull (Tokyo), 1991 Jan, 39(1), 150 - 3
Activity of artificial mutant variants of human growth hormone deficient in a disulfide bond between Cys53 and Cys165; Uchida E et al.; In order to understand the role of Cys53 and Cys165 of human growth hormone (hGH) in receptor-binding and biological activity, artificial mutant variants of hGH were prepared in Escherichia coli by in vitro mutagenesis . Variants of hGH were constructed by replacement of Cys165 with Ala ({Ala165}hGH) or Ser ({Ser165}hGH), by replacement of Cys53 with Ala ({Ala53}hGH), by replacement of Cys53 and Cys165 with Ala ({Ala53, Ala165}hGH), or by replacement of Cys53 with Ala and Cys165 with Ser ({Ala53,Ser165}hGH) . All of the variants constructed as well as reduced hGH exhibited less biological activity than that of intact hGH, and the decreases in biological activity were almost equal, as measured by a sensitive biological assay for growth hormone: adipose conversion assay using 3T3-F442A cells . These variants also showed less receptor-binding activity than that of intact hGH . These results suggest that it is possible neither the residue Cys53 nor Cys165 is directly involved in the receptor binding, and that the disulfide bridge between Cys53 and Cys165 in hGH may not always be crucial for the biological activity, though necessary to express full hGH activity.

Arzneimittelforschung, 1991 Jan, 41(1), 60 - 5
Stimulatory effect of romurtide on hematopoiesis in monkeys; Nakajima R et al.; Repeated subcutaneous injections of romurtide (N2-{(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl}-N6-stearoyl-L-lysine, MDP-Lys(L18), muroctasin; CAS 78113-36-7), a synthetic muramyl dipeptide derivative, increased significantly the number of peripheral neutrophils and monocytes in a dose-dependent fashion in healthy cynomolgus monkeys (Macaca fascicularis) . The number of platelets was also increased significantly in monkeys with repeated dosing of romurtide . After single dosing of romurtide (1 mg/head), neutrophils counts showed a marked increase within 24 h and at 120 h after romurtide injection . Monocytes counts were decreased transiently until 8 h, but were increased persistently from 48 to 120 h after injection . Lymphocytes counts were stable throughout the experimental period except for a significant decrease until 24 h . In addition, romurtide stimulated blood neutrophils and monocytes in vivo for enhanced chemiluminescence (CL) responses to opsonized Escherichia coli . When peripheral monocytes from monkeys were incubated with various concentrations of romurtide in vitro, production of colony-stimulating factors (CSFs), interleukin-1 (IL-1) and interleukin-6 (IL-6) by the cells was enhanced significantly, indicating that the augmenting effects of romurtide on the production of various monokines including CSFs by monocytes are involved in the mechanisms of hematopoiesis and enhanced CL generation by phagocytic cells in vivo.

Arch Microbiol, 1991, 155(4), 391 - 5
Futile cycling of ammonium ions via the high affinity potassium uptake system (Kdp) of Escherichia coli; Buurman ET et al.; Escherichia coli Frag1 was grown under various nutrient limitations in chemostat culture at a fixed temperature, dilution rate and pH both in the presence and the absence of a high concentration of ammonium ions by using either ammonium chloride or DL-alanine as the sole nitrogen source . The presence of high concentrations of ammonium ions in the extracellular fluids of potassium-limited cultures of E . coli Frag1 caused an increase of the specific rate of oxygen consumption of these cultures . In contrast, under phosphate-, sulphate- or magnesium-limited growth conditions no such increase could be observed . The presence of high concentrations of ammonium ions in potassium-limited cultures of E . coli Frag5, a mutant strain of E . coli Frag1 which lacks the high affinity potassium uptake system (Kdp), did not increase the specific rate of oxygen consumption . These results indicate that ammonium ions, very similar to potassium ions both in charge and size, are transported via the Kdp leading to a futile cycle of ammonium ions and ammonia molecules (plus protons) across the cytoplasmic membrane . Both the uptake of ammonium ions and the extrusion of protons would increase the energy requirement of the cells and therefore increase their specific rate of oxygen consumption . The involvement of a (methyl)ammonium transport system in this futile cycle could be excluded.

Anal Biochem, 1991 Jan, 192(1), 251 - 3
Gel electrophoresis system for the micropreparation of plasmid DNA; Cao TM et al.; An easy-to-build gel electrophoresis system with continuous elution is described . The design requires only inexpensive materials and common equipment available in any laboratory . The system is used to isolate supercoiled plasmid DNA.

Res Vet Sci, 1991 Jan, 50(1), 54 - 63
Effects of an infusion of dopamine on the cardiopulmonary effects of Escherichia coli endotoxin in anaesthetised horses; Trim CM et al.; Horses with colic may be endotoxaemic and subsequently develop hypotension during anaesthesia for surgical operation . The aim of this study was to evaluate the efficacy of dopamine as a means to improve cardiovascular function in anaesthetised endotoxaemic horses . Nine horses (five in group 1 and four in group 2) were anaesthetised with thiopentone and guaifenesin and anaesthesia was maintained with halothane . After approximately one hour, facial artery pressure, heart rate, pulmonary artery pressure, cardiac output, temperature, pHa, PaCO2, PaO2, base excess, packed cell volume, plasma protein concentration and white cell count were measured (time 0) . Escherichia coli endotoxin was infused intravenously over 15 minutes in both groups . Group 2 horses were given an intravenous infusion of dopamine (5 micrograms kg-1 min-1) starting five minutes after the start of the endotoxin infusion and continuing for 60 minutes . Measurements were made at 15 minute intervals for 120 minutes . In group 1, one horse died during the endotoxin infusion and in two other horses mean facial artery pressures decreased to 50 mm Hg . Total pulmonary vascular resistance and packed cell volume were significantly increased . Cardiac output, cardiac index and change in mean arterial pressure were significantly greater in group 2 horses than in group 1 horses . Conversely, diastolic pulmonary artery pressure, total vascular resistance and total pulmonary resistance were significantly less in group 2 than in group 1 . PaO2, base excess and white blood cell count were significantly decreased in both groups . It was concluded that dopamine improved cardiovascular function in the presence of endotoxaemia and attenuated the rate of haemoconcentration, but had no effect on the development of decreased PaO2 or metabolic acidosis.

Life Sci, 1991, 48(24), 2341 - 8
Endotoxin induced changes in serum estrogen in male rats: influence of testicular maturation; Christeff N et al.; The influence of acute endotoxin (Endo) administration on adrenal and testicular serum hormones, corticosterone (B), progesterone (P4), 17 alpha OH progesterone (17 alpha OH P4), androstenedione (delta 4), testosterone (T), estrone (E1) and estradiol (E2) was studied in male rats aged 8, 12 and 15 weeks . The present study confirms that the concentrations of circulating steroid hormones in male rats vary with age, and indicate that the adrenal glands mature before the testes . The steroid response to Endo is age-dependent . B, P4, 17 alpha OH P4 was increased and T decreased in all animals . But, there was a very significant increase in estrogens (E1 and E2) and a decrease in delta 4 only in male rats aged 12 weeks and over . The lack of an estrogen response to Endo injection in 8 week-old rats may indicate that the reduced sensitivity (refractory period) to Endo (which has been reported to last until 21 days of age) continues longer . The reduced sensitivity to Endo which occurs in young rats could be due in part to the absence of adrenal-testicular cooperation as a result of partial testicular immaturity.

J Gen Microbiol, 1991 Jan, 137 ( Pt 1), 91 - 100
Cloning and characterization of the ColE7 plasmid; Chak KF et al.; The 6.2 kb ColE7-K317 plasmid was mapped and the DNA fragments of the colicin E7 operon subcloned into pUC18 and pUC19 . The size of the functional colicin E7 operon deduced by subcloning was 2.3 kb . The colicin E7 gene product was purified by carboxymethylcellulose chromatography . Both colicin E7 and E9 were demonstrated to exhibit a non-specific DNAase-type activity by in vitro biological assay . The molecular mass of colicin E7 was 61 kDa, as determined by SDS-PAGE . From DNA sequence data, the estimated sizes of the E7 immunity protein and the E7 lysis protein were 9926 Da and 4847 Da, respectively . Comparison of restriction maps and DNA sequence data suggests that ColE7 and ColE2 are more closely related than other E colicin plasmids.

J Gen Microbiol, 1991 Jan, 137 ( Pt 1), 81 - 9
Phospholipase-A-independent damage caused by the colicin A lysis protein during its assembly into the inner and outer membranes of Escherichia coli; Howard SP et al.; The requirement for the activation of phospholipase A by the colicin A lysis protein (Cal) in the efficient release of colicin A by Escherichia coli cells containing colicin A plasmids was studied . In particular, we wished to determine if this activation is the primary effect of Cal or whether it reflects more generalized damage to the envelope caused by the presence of large quantities of this small acylated protein . E . coli tolQ cells, which were shown to be leaky for periplasmic proteins, were transduced to pldA and then transformed with the recombinant colicin A plasmid pKA . Both the pldA and pldA+ strains released large quantities of colicin A following induction, indicating that in these cells phospholipase A activation is not required for colicin release . This release was, however, still dependent on a functioning Cal protein . The assembly and processing of Cal in situ in the cell envelope was studied by combining pulse-chase labelling with isopycnic sucrose density gradient centrifugation of the cell membranes . Precursor Cal and lipid-modified precursor Cal were found in the inner membrane at early times of chase, and gave rise to mature Cal which accumulated in both the inner and outer membrane after further chase . The signal peptide was also visible on these gradients, and its distribution too was restricted to the inner membrane . Gradient centrifugation of envelopes of cells which were overproducing Cal resulted in very poor separation of the membranes . The results of these studies provide evidence that the colicin A lysis protein causes phospholipase A-independent alterations in the integrity of the E . coli envelope.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Cell Biol, 1991 Jan, 69(1), 79 - 83
Specific inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of their second subunit; Cosentino G et al.; Previous studies have shown that herpes virus ribonucleotide reductase can be inhibited by a synthetic nonapeptide whose sequence is identical to the C-terminal of the small subunit of the enzyme . This peptide is able to interfere with normal subunit association that takes place through the C-terminal of the small subunit . In this report, we illustrate that inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of subunit R2 is also observed for the enzyme isolated from Escherichia coli, hamster, and human cells . The nonapeptide corresponding to the bacterial C-terminal sequence was found to inhibit E . coli enzyme with an IC50 of 400 microM, while this peptide had no effect on mammalian ribonucleotide reductase . A corresponding synthetic peptide derived from the C-terminal of the small subunit of the human enzyme inhibited both human and hamster ribonucleotide reductases with IC50 values of 160 and 120 microM, respectively . However, this peptide had no inhibitory activity against the bacterial enzyme . Equivalent peptides derived from herpes virus ribonucleotide reductase had no effect on either the bacterial or mammalian enzymes . Thus, subunit association at the C-terminal of the small subunit appears to be a common feature of ribonucleotide reductases . In addition, the inhibitory phenomenon observed with peptides corresponding to the C-terminal appears not only to be universal, but also specific to the primary sequence of the enzyme.

Jpn J Surg, 1991 Jan, 21(1), 38 - 42
Endotoxin-induced lung hemorrhages in obstructive jaundiced rats; Uchino R et al.; Endotoxemia in patients with biliary obstruction contributes to the high morbidity and mortality rates following surgery . We developed an animal model of disseminated intravascular coagulation (DIC) in rats on which bile duct ligation was performed following an injection of endotoxin (200 micrograms/kg bw) . Macroscopic hemorrhages and microthrombi in the lung were found in jaundiced rats 6 hours after the injection of endotoxin and yet these phenomena were not found in non-jaundiced rats following an injection of the same amount of endotoxin . Coagulation studies also revealed characteristic findings of DIC in the jaundiced rats following the injection of endotoxin . This study confirms that obstructive jaundice is one of the main predisposing factors in the course of endotoxin-induced DIC.

New Biol, 1991 Jan, 3(1), 57 - 62
Dimer-to-tetramer assembly of Lac repressor involves a leucine heptad repeat; Alberti S et al.; The C-terminus of Lac repressor is responsible for the formation of repressor tetramers from active dimers . If properly grafted, the C-terminus of Lac repressor (amino acids 331 to 360) converts Gal repressor dimers into tetramers . Amino acids 342 to 356 of Lac repressor contain a 4-3 hydrophobic repeat of four leucines and one valine . Systematic amino acid replacements of all residues in this region show that the protein-protein interaction between repressor dimers depends mainly on the hydrophobic residues of the 4-3 repeat, which is constitutive for coiled coils . Thus the tetramerization site of Lac repressor resembles the leucine zipper motif found in a family of eukaryotic transcription factors.

Genetika, 1991 Jan, 27(1), 27 - 38
{Unequal crossing-over in conjugated crossings in Escherichia coli K-12}; Sukhodolets VV; A genetic model has been developed allowing to study non-equal crossingover in conjugative crossings of Escherichia coli . The model is based on obtaining tandem duplications in the region of the deo operon of E . coli in conjugative crosses Hfr x Hfr . It was shown that when using transposon (Tn5, Tn10) genetic markers the majority of recombinant progeny phenotypically corresponding to rare 2-crossover and 4-crossover recombinants for the deo operon constitute direct tandem duplications resulting from non-equal interchromosomal exchange . Apart from the deo operon, the duplications obtained cover, in some cases, linked thr and leu loci . In the crosses, where point mutations for genes of the deo operon served as genetic markers, the frequency of duplications' formation was lower than that of rare 2-crossover and 4-crossover recombinants' formation . Evolutionary role of the non-equal crossingover phenomenon in E . coli is discussed.

Exp Pathol, 1991, 41(2), 98 - 109
The glycosaminoglycan metabolism of chondrocyte monolayer cultures under normal and pathological conditions . A methodic study; Kittlick PD et al.; Chondrocyte cultures may serve as a model in investigating changes of the cartilage metabolism . Adherent chondrocytes in vitro maintain polygonal morphology at high cell density in the primary and secondary culture . Collagen type II is only clearly detected in multilayered or nodular areas . The differentiation of the chondrocytes is also indicated by a low HA concentration of the cultural medium . It depends on high cell density, a low number of subcultures and their duration . However, the medium GAG of chondrocyte cultures does not exactly mirror the state of cell differentiation but can partly be used to check it . Subcultures of chondrocytes on small cover slides (minicultures) are used to determine proteoglycan synthesis and degradation for 48 h each . Both synthesis and degradation of cell-associated GAG or proteoglycans, resp., follow similar complex kinetics . The half lives of sulfated GAG or proteoglycans are initially 10 h (T-1 for O-6 h of chase), later 39 h or 95 h (T-2 for 6-48 h of chase) . Conditioned medium of casein-elicited rat peritoneal macrophages reduce the sulfate incorporation into chondrocyte proteoglycans and their degradation rates increase . In the additional presence of E . coli endotoxin (0.5 microgram/ml) the synthesis of proteoglycans is only little affected; the degradation rate is stronger increased . To peritoneal macrophages of rats manifold pretreated with BCG and perhaps desensitized, LPS is added in vitro . Conditioned medium of these MP does not affect the chondrocyte proteoglycan synthesis but enhances the degradation rates in a concentration-dependent manner . Thus it can be demonstrated that chondrocyte monolayer miniscale cultures may serve to elucidate changes in the proteoglycan synthesis and different degradative steps.

Curr Genet, 1991 Jan, 19(1), 43 - 7
A group I intron in the chloroplast large subunit rRNA gene of Chlamydomonas eugametos encodes a double-strand endonuclease that cleaves the homing site of this intron; Gauthier A et al.; During interspecific crosses between Chlamydomonas eugametos and Chlamydomonas moewusii, an optional group I intron of 955 base pairs (CeLSU.5) in the C . eugametos chloroplast large subunit rRNA gene undergoes a duplicative transposition event which is associated with frequent co-conversion of flanking cpDNA sequences . In the present study, we show that the basic protein of 218 amino acids encoded by CeLSU.5 could mediate the phenomenon of intron transposition, also called intron homing . We overexpressed the ORF specifying this protein in E . coli using expression vectors that contain a C . moewusii cpDNA sequence encompassing the intron homing site . The expression product was found to exhibit a double-strand DNA endonuclease activity that is specific for the homing site . This activity was detected in vivo by self-linearization of the expression plasmids.

Appl Environ Microbiol, 1991 Jan, 57(1), 183 - 9
Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes; Stamm LV et al.; A recombinant plasmid carrying the recA gene of Leptospira biflexa serovar patoc was isolated from a cosmid library of genomic DNA by complementation of an Escherichia coli recA mutation . The cloned serovar patoc recA gene efficiently restored resistance to UV radiation and methyl methanesulfonate . Recombination proficiency was also restored, as measured by the formation of Lac+ recombinants from duplicated mutant lacZ genes . Additionally, the cloned recA gene increased the spontaneous and mitomycin C-induced production of lambda phage in lysogens of an E . coli recA mutant . The product of the cloned recA gene was identified in maxicells as a polypeptide with an Mr of 43,000 . Antibodies prepared against the E . coli RecA protein cross-reacted with the serovar patoc RecA protein, indicating structural conservation . Southern hybridization data showed that the serovar patoc recA gene has diverged from the recA gene of L . interrogans, Leptonema illini, and E . coli . With the exception of the RecA protein of L . interrogans serovar hardjo, the RecA protein of the Leptospira serovars and L . illini were synthesized at elevated levels following treatment of cells with nalidixic acid . The level of detectable RecA correlated with previous studies demonstrating that free-living cells of L . biflexa serovars and L . illini were considerably more resistant to DNA-damaging agents than were those of parasitic L . interrogans serovars . RecA protein was not detected in cells of virulent Treponema pallidum or Borrelia burgdorferi.

Int J Pancreatol, 1991 Jan, 8(1), 23 - 33
Serum ribonuclease activity in the diagnosis of pancreatic disease; Kemmer TP et al.; The present study evaluated serum ribonuclease activity (SRA) in patients with inflammatory and neoplastic pancreatic diseases . RNase determination was carried out using t-RNA (T) from E . coli MRE 600 at pH 7.4 and polycytidylic acid (poly-C) (P) at pH 6.6 as RNA substrates with RNase A from bovine pancreas as reference enzyme . Healthy volunteers had a SRA of T: 160 +/- 12 and P: 482 +/- 24 ngeq/mL (mean +/- SEM (n} . In patients with acute interstitial pancreatitis (AIP), SRA was similar to healthy controls (T: 166 +/- 14; P: 474 +/- 30 ngeq/mL) . Patients with acute necrotizing pancreatitis (ANP) had increased SRA (T: 278 +/- 49; P: 791 +/- 145 ngeq/mL, p less than 0.01, compared to controls) . SRA values were also increased in patients with chronic pancreatitis (CP) with T: 224 +/- 15 ngeq/mL (p less than 0.01) and in patients with pancreatic carcinoma (PCA) with T: 331 +/- 35 (p less than 0.001 vs controls, p less than 0.01 vs CP) . Increased SRA was detected in patients with renal insufficiency (T: 2576 +/- 195 ngeq/mL, p less than 0.001) . Diagnostic discrimination between AIP and ANP was achieved in 69% using T-SRA (sensitivity 31%, specificity 88%), and in 78% using P-SRA (sensitivity 54%, specificity 92%) . Discrimination between CP and pancreatic carcinoma was possible in 68% (sensitivity 67%, specificity 71%) . The diagnostic value of serum RNase is limited because of its low sensitivity, but increased T-SRA above a cutoff of 250 ngeq/mL and increased P-SRA above a cutoff of 620 ngeq/mL are specific for detecting pancreatic necrosis in the absence of renal impairment . The kidney is a major site for SRA clearance.

Int J Artif Organs, 1991 Jan, 14(1), 43 - 50
Removal of endotoxin and cytokines by adsorbents and the effect of plasma protein binding; Nagaki M et al.; High serum levels of endotoxin and cytokines, through which its activity is mediated, have been shown to be associated with disease severity in septic shock and in fulminant hepatic failure . In the present study, we have investigated the ability of activated charcoals (DHP-1 and Adsorba 150C) and uncharged resin (Amberlite XAD-7) to adsorb lipopolysaccharide (LPS) and various cytokines, namely tumour necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) . The capacities of the adsorbents were assessed by measurement of their equilibrium adsorption isotherms for these substances labelled with 125I . There was no single adsorbent that uniformly adsorbed LPS and the cytokines from phosphate buffered saline or human plasma . DHP-1 charcoal was superior to Adsorba 150C for all substances and was the most effective adsorbent for binding LPS, IL-1 alpha and IFN-gamma . Amberlite XAD-7 resin was most effective for TNF, IL-6 and IFN-alpha, but bound little LPS, particularly from human plasma . Ultrafiltration through a membrane which retains substances of molecular weight greater than 50 kD did not filter the cytokines from human plasma, although the molecular weight of the cytokines range from 17 to 22 kD . This demonstrated that, TNF, IL-1, IL-6, IFN-alpha and IFN-gamma readily bind to proteins and/or other large molecules in plasma.

Yi Chuan Xue Bao, 1991, 18(1), 73 - 81
{recA gene dependence of chromosome replication of Escherichia coli initiated by mini-F plasmids integrated at predetermined sites}; Mao YM et al.; mini-F plasmids carrying particular chromosomal fragments were constructed . Through homologous recombination they were expected to integrate onto predetermined sites of the chromosome of dnaA46 strain LC381 . Chromosome replication of suppressive integration (Sin) strains were recA dependent or independent according to the site of integration of the plasmid: chromosome replication of those with the plasmid integrated close to oriC were found to be recA dependent at 40 degrees C; those with the plasmid integrated between oriC and terC were recA dependent on rich medium but independent on minimal medium; those with the plasmid integrated close to terC were recA dependent even on minimal medium.

Avian Dis, 1991 Jan-Mar, 35(1), 62 - 9
Polymerase chain reaction for detection of Mycoplasma gallisepticum; Nascimento ER et al.; A species-specific 760-base pair (bp) BamHI to EcoRI DNA fragment (fMG-2) was isolated from a Mycoplasma gallisepticum (MG) genomic library constructed in plasmid pUC8 . Based on the DNA sequence data of fMG-2, a pair of 25 base primers, designated amplification (Amp) left (L) and right (R) primers, was synthesized . When used in the polymerase chain reaction (PCR), the Amp L and R primers directed amplification of DNA of 16 MG strains yielding an expected 732-bp product, but did not amplify DNA of Escherichia coli, calf thymus, lambda phage, pUC8 plasmid, or 16 other species of avian mycoplasmas . As low as 10(-6) picogram of MG DNA, a fraction of the total chromosomal content of one cell, was detected following amplification by PCR . PCR amplification products were visualized by either ethidium bromide/ultraviolet exposure or hybridization with a 481-bp probe (fMG-3) prepared from the central region of fMG-2.

Avian Dis, 1991 Jan-Mar, 35(1), 17 - 22
A vaccine against avian colibacillosis based on ultrasonic inactivation of Escherichia coli; Melamed D et al.; Ultrasonic inactivation of Escherichia coli followed by irradiation was found to be the most efficient method for preparation of an effective vaccine against colibacillosis . Challenge experiments revealed that this vaccine provided the best protection compared with other methods of inactivation: heat, formaldehyde, and irradiation . Preparing the ultrasonicated vaccine from O2:K1 strain increased its range and also supported adequate protection against homologous strain O78:K80 . The degree of protection conferred by the vaccine was positively correlated with the antibody titer against E . coli as measured on day of challenge . Low antibody titers detected 5 days post-vaccination resulted in only 20% protection . High antibody titers detected at 8 and 15 days post-vaccination correlated with a low number of chicks with lesions . In each challenged group, the live chicks that did not develop lesions had higher antibody titers than chicks with lesions, revealing a correlation between numbers of chicks with lesions and antibody titers as measured by enzyme-linked immunosorbent assay.

Photochem Photobiol, 1991 Jan, 53(1), 143 - 8
4,4',5'-trimethyl-8-azapsoralen, a new-photoreactive and non-skin-phototoxic bifunctional bioisoster of psoralen; Vedaldi D et al.; Photochemical and photobiological properties of a new isoster of psoralen, 4,4',5'-trimethyl-8-azapsoralen (4,4',5'-TMAP), have been studied . This compound shows a high DNA-photobinding rate, higher than that of 8-methoxypsoralen (8-MOP), forming both monoadducts and inter-strand cross-links . The yield of cross-links, however, is markedly lower than that of 8-MOP . Antiproliferative activity of 4,4',5'-TMAP, in terms of DNA synthesis inhibition in Ehrlich ascites tumor cells, is higher than that of 8-MOP . Mutagenic activity on E . coli WP2 R46+ cells appeared similar to or even lower than that of 8-MOP . This new compound applied on depilated guinea pig skin and irradiated with UVA did not show any skin-phototoxicity . On the basis of these properties 4,4',5'-TMAP appears to be a potential photochemotherapeutic agent.

Mol Gen Mikrobiol Virusol, 1991 Jan, (1), 16 - 8
{Cloning and expression of the CD4 receptor gene from human T-lymphocytes in Escherichia coli cells}; Zverev VV et al.; The gene for the CD4-membrane glycoprotein-receptor for HIV has been cloned . The 179 amino acids fragment of the CD4-receptor responsible for binding of gp120 HIV glycoprotein has been fused with beta-galactosidase and shown to be expressed in Escherichia coli cells . The recombinant protein in ELISA and immunoblotting techniques reacts with the monoclonal antibodies OKT4A and Leu3A known to block the interaction between the CD4 and gp120 HIV glycoprotein . The recombinant protein can be used for different scientific and practical purposes including studying of the mechanisms for HIV interaction with the sensitive cells as well as for viral gp120 protein purification, etc.

Int J Biochem, 1991, 23(1), 107 - 14
Expression and partial purification of human prolactin in Escherichia coli; Gilbert MS et al.; 1 . Human prolactin has been expressed in Escherichia coli . A cDNA fragment coding for the signal sequence and the full length prolactin molecule was cloned into the expression vector pUR291 which directs the synthesis of a beta-galactosidase prolactin fusion protein when expressed in E . coli . 2 . Cultures of E . coli harbouring the recombinant plasmid pJMBG62 produced a fusion protein of the appropriate molecular weight which was detected by Western blot analysis using a polyclonal antibody raised against pituitary-derived human prolactin . 3 . The fusion protein was isolated from inclusion bodies in a partially pure form and it was used as immunogen to raise antibodies against human prolactin . 4 . When this partially purified fusion protein was injected into rabbits it generated antisera with good prolactin titres in animals which were rested for one year following a disappointing primary immunization with purified human prolactin.

Clin Exp Allergy, 1991 Jan, 21(1), 33 - 7
cDNA encoding the major mite allergen Der f II; Trudinger M et al.; cDNA encoding the major house dust mite allergen Der f II from Dermatophagoides farinae was amplified using the polymerase chain reaction and cloned into E . coli . It encoded a 129-residue protein with a calculated molecular weight of 14,021 D and had the expected high homology (88%) with Der p II including the absence of N-glycosylation sites and conserved cysteine residues . These results are consistent with the high degree of antibody crossreactivity and may help identify the differences in T-cell epitopes revealed for these molecules so far.

Am J Vet Res, 1991 Jan, 52(1), 45 - 9
Relationship between virulence and adherence of various enterotoxigenic Escherichia coli strains to isolated intestinal epithelial cells from Chinese Meishan and European large white pigs; Bertin AM et al.; In vitro adherence to intestinal epithelial cells by enterotoxigenic Escherichia coli strains bearing K88, K99, F41, or 987P adhesins and of their variants not bearing adhesins (K88-, K99-, or F41-) was investigated in European Large White and Chinese Meishan pigs . Possible relationship between adherence and virulence was also examined . The K88-positive (K88+) strain strongly adhered to intestinal epithelial cells from 26 of 28 Large White pigs . This strain had previously been found to be highly virulent for Large White pigs . The only surviving pig was of nonadherent phenotype, and cells from 4 dehydrated moribund pigs had strong adherence . By contrast, the same K88+ strain found previously to have little pathogenicity for Meishan pigs adhered with variable intensity to cells from 17 of 23 Meishan pigs; correlation was not evident between adherence and virulence . The K99+ F41+ strain of porcine origin and the F41+ strain generally adhered strongly to cells from 24 and 23 Meishan pigs, respectively, and to cells from 25 of 26 Large White pigs . Correlation was not found between adherence and virulence for the 2 strains . A K99+ F41+ strain of bovine origin adhered to cells from 20 of 22 Meishan and 22 of 23 Large White pigs, and a K99- F41+ variant adhered to cells from 19 of 23 Meishan and 23 of 24 Large White pigs . The adhesin-negative variants never adhered to intestinal epithelial cells . Strain 987 known not to readily produce 987P adhesin after in vitro growth never adhered to cells during the test.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Vet Res, 1991 Jan, 52(1), 40 - 4
Susceptibility of Chinese Meishan and European large white pigs to enterotoxigenic Escherichia coli strains bearing colonization factor K88, 987P, K99, or F41; Duchet-Suchaux MF et al.; Conventionally raised Chinese Meishan and European Large White pigs were intragastrically challenge exposed with 2.1 x 10(10) enterotoxigenic Escherichia coli strains bearing colonization factor K88, 987P, F41, or F41 plus K99 . In response to challenge exposure with the K88-positive (K88+) organisms, 96% of Large White pigs died within 48 hours, whereas none of the Meishan pigs died . Both breeds of pigs had similar susceptibility to strains bearing 987P or F41 . Lastly, Meishan pigs were found to be more susceptible than Large White pigs to a strain expressing K99 and F41 . In pigs with diarrhea, challenge-exposure strains intensively colonized the jejunum (10(8) to 10(10) bacteria/g of tissue) and, to less extent, the duodenum (except K88+ strain, which comprised 10(8)/g) . In most cases, jejunal concentrations of the challenge-exposure strains were substantially lower in pigs that did not have diarrhea . Half the resistant Meishan pigs eliminated the K88+ strain from the intestines . Colostral antibody titer that agglutinated challenge-exposure strains did not differ between Meishan and Large White gilts . Results indicate that resistance of pigs to the K88+ strain did not extend to enterotoxigenic strains bearing other well-known factors . They indicate, in addition, that genetic resistance to K88+ strains described in pigs in Europe may exist in pigs in China.

Yeast, 1991 Jan, 7(1), 25 - 36
Scarcity of ars sequences isolated in a morphogenesis mutant of the yeast Yarrowia lipolytica; Fournier P et al.; Previous attempts is isolate autonomously replicating sequences (ars) from the dimorphic yeast Yarrowia lipolytica have been unsuccessful . We isolated a Fil- mutant unable to produce hyphae and growing only in a yeast form to facilitate ars isolation . This mutant was transformed with a Y . lipolytica DNA bank and several unstable clones were obtained . Extrachromosomal plasmids were evidenced in yeast, recovered in Escherichia coli and characterized by restriction mapping . They were able to retransform Fil- and Fil+ yeast strains at high frequency and transformants displayed a slightly unstable phenotype . The detailed analysis of the plasmids showed that only two different ars sequences had been isolated, each of them corresponding to a unique sequence in the Y . lipolytica genome . We concluded that functional ars sequences that can be cloned on plasmids are rare in this yeast.

Arch Virol, 1991, 117(3-4), 297 - 304
In vitro expression of a chimeric coat protein gene from Grapevine Fanleaf virus (strain F 13); Serghini MA et al.; The coat protein (CP) cistron of Grapevine Fanleaf virus strain F13 (GFLV-F13) has been located in the C-terminal region of the 122k polyprotein encoded by the genomic RNA 2 {Serghini et al . (1990) J . Gen . Virol . 71: 1433-1441} . A chimeric CP gene of GFLV-F13 including a short sequence corresponding to 3 restriction sites, the leader sequence of the GFLV-F13 satellite RNA and an initiation codon was constructed . Transcripts from this construct were translated in wheat germ extract with equal efficiency to form a 56k protein which comigrates on PAGE with the GFLV-F13 CP and a protein of 52k . Both species react with GFLV-F13 CP-specific antibodies . Deletions in the 5' region of the CP gene show that the 56k protein is initiated at the first AUG after the satellite leader and the 52k protein at the second in-frame AUG . Transcripts with a 142 nt deletion including the two AUG codons from the 5' end of the CP gene are not efficiently expressed in vitro, no major translation product being detected.

Proteins, 1991, 9(1), 28 - 36
Assignment of the nucleotide binding sites and the mechanism of substrate inhibition of Escherichia coli adenylate kinase; Liang P et al.; Site-directed mutagenesis of key amino acids of adenylate kinase has been used to suggest a new model for the location of the AMP and ATP binding sites . Phe-86 and Tyr-133, which are in close contact with the inhibitor Ap5A according to previous crystallographic results, have been independently changed to tryptophan and other amino acids . The Phe-86----Trp mutant had a 3- to 6-fold change in the Km for ATP and a 44-fold increase in the Km for AMP with a simultaneous loss of AMP substrate inhibition . Thus Phe-86 is probably in close contact with bound AMP . The Tyr-133----Trp mutant showed no large effects on enzyme kinetics and suggests that the previous assignment of Ap5A occupying natural adenosine binding sites is probably incorrect . A temperature-sensitive Leu-107----Gln mutant showed a 6-fold decrease in the Km for ATP and no effect on AMP binding, suggesting that this amino acid is near the ATP binding site . Changes in the fluorescence of single tryptophan-containing mutant enzymes provided specific information about AMP and ATP binding . The fluorescence results are consistent with the kinetic studies, and also suggest that AMP substrate inhibition is caused by the formation of an abortive complex that prevents the release of product.

J Med Virol, 1991 Jan, 33(1), 33 - 8
Efficient cloning of hepatitis B virus DNA from single-stranded replicative intermediates and its application to S1 mapping of viral RNA in human liver tissue; Kajino K et al.; An efficient method for cloning subgenomic fragments of the hepatitis B virus (HBV) was developed, utilizing its abundant single-stranded replicative intermediates . The total genomic DNA obtained from the liver tissue of patients with chronic HBV infection was treated by using the Klenow fragment of E . coli DNA polymerase I without adding any exogenous primers . Single-stranded replicative intermediates were efficiently converted to double-stranded linear DNA, one end of which terminated at (or near to) the direct repeat 1 (DR1) sequence of the HBV genome . By screening less than 1,000 recombinants from a DNA library after this treatment, we obtained a subgenomic HBV fragment of 2.0 kilobases . We then analysed HBV RNA in human liver tissue by S1 mapping . It was possible to map HBV RNA only when a DNA probe from the same tissue was used.

J Biochem (Tokyo), 1991 Jan, 109(1), 6 - 7
Crystallization of allosteric L-lactate dehydrogenase from Thermus caldophilus and preliminary crystallographic data; Koide S et al.; L-Lactate dehydrogenase of Thermus caldophilus GK24 was purified from Escherichia coli containing an overexpression plasmid . The enzyme was crystallized from polyethylene glycol 6000 solutions without ligands by the hanging drop vapor diffusion method . Two forms of crystals were obtained . The crystals grown at pH 6.0 were characterized by means of an X-ray diffraction experiment, while those grown at pH 6.5 and 7.0 did not give detectable diffraction spots . The crystals grown at pH 6.0 belonged to monoclinic space group P2(1), the cell dimensions being a = 54.8 A, b = 138.2A, c = 86.1 A, and beta = 93.3 degrees . These crystals diffract to beyond 2.5 A spacing and are stable on X-ray irradiation.

J Biochem (Tokyo), 1991 Jan, 109(1), 190 - 7
A site-directed mutagenesis study of yeast calmodulin; Matsuura I et al.; A site-directed mutagenesis study was carried out in order to understand the regulatory mechanism of calmodulin . We started from the yeast (Saccharomyces cerevisiae) calmodulin gene since it has many differences in amino acid sequence and inferior functional properties compared with the vertebrate calmodulin . Recombinant yeast calmodulins were generated in Escherichia coli transformed by constructed expression plasmids . Three recombinant calmodulins were obtained . The first two were YCM61G, in which the Ca2(+)-binding site 2 (the four Ca2(+)-binding EF-hand structures in calmodulin were numbered from the N-terminus) was converted to the same as that in vertebrate calmodulin, and YCM delta 132-148, in which the C-terminal half sequence of site 4 was deleted . These two recombinant calmodulins had the same maximum Ca2+ binding (3 mol/mol) as yeast calmodulin, which indicates that site 4 of yeast calmodulin was the one losing Ca2+ binding capacity . YCM delta 132-148 could not activate target enzymes, whereas its Ca2+ binding profile was similar to those of yeast calmodulin and YCM61G . Therefore, the structure in site 4 which cannot bind Ca2+ is indispensable for the regulatory function of yeast calmodulin . The complete regulatory function of vertebrate calmodulin can be attained by the combination of 4 Ca2+ binding structures . The negative charge cluster in the central alpha-helix region is suggested to stabilize the active conformation of calmodulin, since the third yeast calmodulin mutant, YCM83E, which had the negative charge cluster, increased the maximum activation of myosin light chain kinase.

J Biochem (Tokyo), 1991 Jan, 109(1), 113 - 9
The Mr 90,000 heat shock protein-free androgen receptor has a high affinity for steroid, in contrast to the glucocorticoid receptor; Ohara-Nemoto Y et al.; Transformed and bacterially expressed glucocorticoid receptors free from Mr 90,000 heat shock protein (hsp90) have a 100-fold lower steroid-binding affinity than the hsp90-bound nontransformed receptor, suggesting that hsp90 is needed for high-affinity steroid binding {Nemoto, T., Ohara-Nemoto, Y., Denis, M., & Gustafsson, J.-A . (1990) Biochemistry 29, 1880-1886} . To investigate whether or not this phenomenon is common to all steroid receptors, we investigated the steroid-binding affinities of bacterially expressed and transformed androgen receptors . The C-terminal portion of the rat androgen receptor containing the putative steroid-binding domain was expressed as a fusion protein of protein A in Escherichia coli . The recombinant protein bound a synthetic androgen, {3H}R1881, with high affinity (Kd = 0.8 +/- 0.3 nM) . Glycerol gradient analysis revealed that the recombinant protein sedimented at around the 3S region irrespective of the presence of molybdate, indicating that the receptor is present in monomeric form . The steroid-free transformed androgen receptor was obtained by exposure of rat submandibular gland cytosol to 0.4 M NaCl in the absence of steroid . High-performance ion-exchange liquid chromatography analysis showed that the transformed androgen receptor bound to {3H}R1881 with high affinity . Thus these observations indicate that, in contrast to the glucocorticoid receptor, hsp90 is not required for the high-affinity steroid binding of the androgen receptor . In addition, the hsp90-free androgen receptor prebound with radioinert R1881 was efficiently relabeled with {3H}R1881, while the triamcinolone acetonide-bound, transformed glucocorticoid receptor failed in ligand exchange . The inability to achieve ligand exchange probably reflects the low steroid-binding affinity of this entity.

Genetics, 1991 Jan, 127(1), 21 - 30
Isolation and characterization of Escherichia coli mutants with altered rates of deletion formation; Whoriskey SK et al.; Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli . After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies . One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail . The mutR gene maps at 38.5 min on the E . coli genetic map . Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9 . Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system . These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions . It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes.

Antimicrob Agents Chemother, 1991 Jan, 35(1), 53 - 6
Delta pH-dependent accumulation of tetracycline in Escherichia coli; Yamaguchi A et al.; The effects of ionophores on tetracycline accumulation in Escherichia coli cells were investigated in the presence of polymyxin B nonapeptide . Accumulation was inhibited by nigericin but not by valinomycin . Tetracycline accumulation was stimulated by decreasing the pH of the medium and inhibited by the addition of magnesium ions . These results indicated that tetracycline enters cells through diffusion as a protonated form (TH2) and is accumulated as a membrane-impermeable magnesium-tetracycline chelate complex (THMg+) . This noncarrier diffusion hypothesis was confirmed by the fact that tetracycline accumulated in protein-free liposomes through an artificially imposed pH difference.

Mol Microbiol, 1991 Jan, 5(1), 39 - 47
Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum; Rossbach S et al.; A Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max) . These ineffective, nitrogen non-fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development . Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyltransferase (SHMT; E.C.2.1.2.1.) . SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one-carbon unit to tetrahydrofolate . The B . japonicum glyA region was able to fully complement the glycine auxotrophy of an E . coli glyA deletion strain . Although the Tn5 insertion in B . japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e . a leaky auxotroph) . Thus, B . japonicum may have an additional pathway for glycine biosynthesis . Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants . It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1-metabolism are indispensable for the establishment of a fully effective, nitrogen-fixing root nodule symbiosis.

Mol Microbiol, 1991 Jan, 5(1), 33 - 7
Evolution of the MIP family of integral membrane transport proteins; Pao GM et al.; Six integral membrane proteins of bacterial, animal, and plant origin, which are believed to function in solute transport, share sequence identity and are grouped together as members of the MIP family . These include the Escherichia coli glycerol facilitator, the major intrinsic protein from bovine lens fibre junction membranes, a plant tonoplast membrane protein, a soybean protein from the peribacteroid membrane, and a Drosophila neurogenic protein . These proteins, each of which appears to consist of six transmembrane helical segments per subunit, apparently arose by internal duplication of a three-transmembrane segment . Phylogenetic 'trees' interrelating these proteins and segments are presented.

Exp Lung Res, 1991 Jan-Feb, 17(1), 77 - 89
Calcium ionophore-activated neutrophils prestimulated with endotoxin increase pulmonary arterial pressure and vascular leakage in isolated perfused rat lungs: role of platelet-activating factor; Lindahl M et al.; The influence of stimulated polymorphonuclear neutrophils on pulmonary arterial pressure and vascular leakage in isolated perfused rat lungs was investigated . We exposed isolated neutrophils to various stimuli in vitro, instilled the cells in the lung perfusate, and studied the effects on pulmonary arterial pressure and passage of fluorescently labeled dextran (4100 dalton) from the pulmonary circulation into the lung . We found that neutrophils stimulated with the calcium ionophore A23187 or with E . coli endotoxin had no significant influence on the pressure or the passage of dextran . On the other hand, neutrophils preincubated with endotoxin and then stimulated with A23187 caused significant increases, both in pulmonary arterial pressure and accumulation of dextran in the lung . Both these effects were attenuated by BN 52021, a specific platelet-activating factor antagonist, and by nordihydroguaiaretic acid, an agent that inhibited the generation of platelet-activating factor in A23187-stimulated neutrophils . These findings demonstrate that activated neutrophils can increase pulmonary arterial pressure and lung fluid accumulation and suggest that endotoxin-stimulated activated neutrophils exert at least some of their action via generation of platelet-activating factor.

Mol Biochem Parasitol, 1991 Jan, 44(1), 53 - 61
The diagnostic value and molecular characterisation of an Echinococcus multilocularis antigen gene clone; Hemmings L et al.; A diagnostic Echinococcus multilocularis antigen gene (EM4) has been expressed using the Escherichia coli expression vector pGEX-1 resulting in the high level synthesis of a readily purifiable, soluble, non-denatured peptide fused to the 26-kDa glutathione-S-transferase of Schistosoma japonicum . This recombinant antigen, on testing by enzyme-linked immunosorbent assay (ELISA) with heterologous human antisera, demonstrated 100% E . multilocularis specificity . Specific anti-EM4 antibody immunoprecipitated a single 66-kDa protein from protoscolex total RNA directed in vitro translation products indicating the probable involvement of post-translational modification in the production of the native EM4 antigens . Southern blotting analysis suggests that the EM4 native antigens are coded for by a single-copy gene and that the genomic organisation of the EM4 related genes in other parasites is not conserved . The nucleotide sequence of the cloned EM4 cDNA molecule has been obtained and the derived amino acid sequence shows no significant homology with other existing protein sequences.

Proteins, 1991, 9(2), 135 - 42
The isolation, purification, and preliminary crystallographic characterization of UDP-galactose-4-epimerase from Escherichia coli; Bauer AJ et al.; Uridine diphosphogalactose-4-epimerase from E . coli has been crystallized in a form suitable for a high-resolution X-ray crystallographic structural analysis . The enzyme complexed with a substrate analogue, uridine diphosphobenzene (UDP-benzene), crystallizes readily using polyethylene glycol 8000 as the precipitant . The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions, a = 76.3 A, b = 83.1 A, and c = 132.1 A . Based on still setting photographs, the crystals diffract to a nominal resolution of 2.3 A and are stable in the X-ray beam . The enzyme used in these experiments was produced by a new expression system and a modified purification scheme.

Proteins, 1991, 9(3), 191 - 206
Comparative modeling of mammalian aspartate transcarbamylase; Scully JL et al.; Mammalian aspartate transcarbamylase (ATCase) is part of a 243 kDa multidomain polypeptide, called CAD, that catalyzes the first three steps in de novo pyrimidine biosynthesis . The structural organization of the mammalian enzyme is very different from E . coli ATCase, a dodecameric, monofunctional molecule comprised of six copies of separate catalytic and regulatory chains . Nevertheless, sequence similarities and other properties suggested that the mammalian ATCase domain and the E . coli ATCase catalytic chain have the same tertiary fold . A model of mammalian ATCase was built using the X-ray coordinates of the E . coli catalytic chain as a tertiary template . Five small insertions and deletions could be readily accommodated in the model structure . Following energy minimization the RMS difference in the alpha carbon positions of the mammalian and bacterial proteins was 0.93 A . A comparison of the hydrophobic energies, surface accessibility index, and the distribution of hydrophilic and hydrophobic residues of the CAD ATCase structure with correctly and incorrectly folded proteins and with several X-ray structures supported the validity of the model . The mammalian ATCase domain associates to form a compact globular trimer, a prerequisite for catalysis since the active site is comprised of residues from adjacent subunits . Interactions between the clearly defined aspartate and carbamyl phosphate subdomains of the monomer were largely preserved while there was appreciable remodeling of the trimeric interfaces . Several clusters of basic residues are located on the upper surface of the domain which account in part for the elevated isoelectric point (pI = 9.4) and may represent contact regions with other more acidic domains within the chimeric polypeptide . A long interdomain linker connects the monomer at its upper surface to the remainder of the polypeptide . The configuration of active site residues is virtually identical in the mammalian and bacterial enzymes . While the CAD ATCase domain can undergo the local conformational changes that accompany catalysis in the E . coli enzyme, the high activity, closed conformation is probably more stable in the mammalian enzyme.

Proteins, 1991, 9(3), 174 - 9
The three-dimensional structure of glutathione reductase from Escherichia coli at 3.0 A resolution; Ermler U et al.; The structure of glutathione reductase from Escherichia coli has been solved at 3 A resolution using multiple isomorphous replacement, solvent flattening, and molecular replacement on the basis of the homologous (53% identical residues) and structurally well-established human enzyme . The structures of both enzyme species agree with each other in a global way; there is no domain rearrangement . In detail, clear structural differences can be observed . The structure analysis of the E . coli enzyme was tackled in order to understand site-directed mutants, the most spectacular of which changed the cofactor specificity of this enzyme from NADP to NAD (Scrutton et al., 1990, Nature 343:38-43).

Comput Appl Biosci, 1991 Jan, 7(1), 9 - 19
Knowledge-based simulation of DNA metabolism: prediction of enzyme action; Brutlag DL et al.; We have developed a knowledge-based simulation of DNA metabolism that accurately predicts the actions of enzymes on DNA under a large number of environmental conditions . Previous simulations of enzyme systems rely predominantly on mathematical models . We use a frame-based representation to model enzymes, substrates and conditions . Interactions between these objects are expressed using production rules and an underlying truth maintenance system . The system performs rapid inference and can explain its reasoning . A graphical interface provides access to all elements of the simulation, including object representations and explanation graphs . Predicting enzyme action is the first step in the development of a large knowledge base to envision the metabolic pathways of DNA replication and repair.

Biotechniques, 1991 Jan, 10(1), 84 - 93
Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA; Hultman T et al.; A solid-phase approach is described for manual and automated sequencing of plasmid DNA obtained directly from bacterial colonies through the polymerase chain reaction . The DNA fragment is selectively immobilized to magnetic beads and after strand-specific elution, the eluted strand, as well as the remaining immobilized strand, is used for bidirectional dideoxy sequencing . The solid-phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions . The approach is exemplified by fluorescent sequencing of a cloned Streptomyces curacoi gene having a G + C content of more than 70%.

Biotechniques, 1991 Jan, 10(1), 68 - 75
Rapid colorimetric quantification of PCR-amplified DNA; Lundeberg J et al.; A diagnostic system for rapid colorimetric quantification of the initial amount of DNA template amplified by the polymerase chain reaction is described . The method is based on co-amplification of target DNA with a cloned DNA fragment, in which a lac operator sequence has been introduced by in vitro mutagenesis . The in vitro-amplified material is immobilized on magnetic beads using the biotin-streptavidin system, and the ratio of target DNA to cloned mutated DNA can be determined using a fusion protein consisting of the Escherichia coli LacI repressor and beta-galactosidase . This method for quantitative detection of immobilized amplified nucleic acids is well adapted for rapid automated or semi-automated assays . Here, we show that it can be used to detect and quantify Plasmodium falciparum genomic DNA in clinical samples.

Biotechniques, 1991 Jan, 10(1), 62 - 6
A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction; Jones DH et al.; We have developed a novel polymerase chain reaction (PCR) method that permits the rapid generation of site-specific mutants and recombinant DNA constructs with a minimum number of steps and primers . DNA segments are modified by using amplifying primers that add homologous ends to the polymerase chain reaction product(s) . These homologous ends undergo recombination in vivo following transformation of recA-E . coli strains used routinely in cloning . In vivo circularization of PCR products containing plasmid sequences with a selective marker permits the rapid cloning of the desired mutant or recombinant . In the mutagenesis protocol, 7 of the 12 clones contained the product of interest, and 6 of these clones had no detected error (50% of the clones without detected errors) . In each of several recombination protocols, at least 50% of the clones tested contained the insert of interest without detected errors.

Radiat Environ Biophys, 1991, 30(1), 21 - 31
After X-irradiation a transient arrest of L929 cells in G2-phase coincides with a rapid elevation of the level of O6-alkylguanine-DNA alkyltransferase; Nehls P et al.; Following X-irradiation of exponentially growing L929 cells two major phenomena have been observed . First, there was a delay in cell division which can be ascribed to the arrest of cells in the G2-phase (G2-block), and, second, the cellular content of the O6-alkylguanine-DNA alkyltransferase (AGT) was markedly increased . Flow cytometrical DNA-measurements revealed that cells began to accumulate in the G2-phase 4 h after irradiation (p.r.) irrespective of the X-ray dose, while both the fraction of cells blocked in G2 and the time period the cells persisted in G2 increased with the radiation dose . About 24 h past release from the G2-block the distribution of cells in the cell cycle was similar to that of untreated control cells . In comparison with control cells the AGT content in irradiated cells (4 Gy) was highest at about 48 h p.r . (3.4-fold increase) . The highest ratio of increase in AGT was, however, observed to occur between about 4 and 13 h p.r . (2.6-fold increase) . As shown by flow cytometrical measurements using a BrdUrd/DNA double labeling technique, this rapid primary increase in AGT coincides very well with the entrance of cells into the G2-phase . This indicates that the cellular AGT content in X-irradiated (parental) cells started to exceed the basal level at the beginning of the G2-phase, but not before or during the S-phase . Once the AGT level was elevated it continued to increase for 2 to 3 cell doubling times.

Mol Gen Genet, 1991 Jan, 225(1), 81 - 93
Intron enhancement of gene expression and the splicing efficiency of introns in maize cells; Luehrsen KR et al.; The inclusion of the alcohol dehydrogenase 1-S(Adh 1-S) intron 1 in the transcription unit of maize gene constructs has been shown to increase gene expression in cultured maize cells . We have extended these studies with Adh1-S intron 1 using the firefly luciferase, Escherichia coli beta-glucuronidase and chloramphenicol acetyltransferase reporter genes adjoined to different plant promoters and find enhancement of transient gene expression in all cases but one . We also show that the enhancement phenomenon can be mediated by the third intron of the maize actin gene . In all cases tested, the inclusion of an intron results in increased levels of steady-state RNA . The degree of enhancement depends on the exon sequences flanking the intron; flanking exons also influence the efficiency of intron splicing . Unexpectedly, unspliced RNAs accumulate during the transient assay.

J Steroid Biochem Mol Biol, 1991 Jan, 38(1), 27 - 33
Identification of residues essential for progesterone binding to uteroglobin by site-directed mutagenesis; Peter W et al.; In order to identify amino acids directly involved in progesterone binding to rabbit uteroglobin we have mutated Phe 6, Tyr 21 and Thr 60 by site-directed mutagenesis of the uteroglobin cDNA . These residues have been postulated previously to participate in progesterone binding . High-level expression of the mutated uteroglobin cDNAs in Escherichia coli yields recombinant protein mutants that, like natural uteroglobin, form stable dimers, suggesting that the tertiary structure of the protein has not been altered . Substitution of Phe 6 by Ser or Ala does not change the progesterone binding characteristics . In contrast, replacement of Tyr 21 by Phe or Ala, drastically decreases progesterone binding . In addition, replacement of Thr 60 by Ala reduces the affinity for progesterone by a factor of three . These data suggest a direct interaction of progesterone with these two amino acids and support the idea of direct hydrogen bonding of the carbonyl (C3 and C20) of progesterone with the hydroxyl groups of Tyr 21 and Thr 60, respectively.

Anat Rec, 1991 Jan, 229(1), 86 - 102
Endotoxin-induced endothelial injury and subendothelial accumulation of fibronectin in rat aorta; Kang YH et al.; Endotoxin (lipopolysaccharide, LPS) induces endothelial injury in arterial vessels . Fibronectin is known to be involved in cell attachment and wound repair . The present study was designed to elucidate the effect of LPS on the production and distribution of fibronectin in relation to injury and repair in rat aortic endothelium . Male Sprague-Dawley rats were sacrificed for ultrastructural and immunocytochemical evaluations at 1, 3, 6, 24, and 48 hr after a single intravenous injection of 1.5 or 3 mg/kg body weight E . coli LPS . Apparent morphological signs of endothelial injury, including cell detachment, denudation, cell death, and edema were observed 1-48 hr after injection . Parietal thrombosis and leukocyte diapedesis were also observed in the aorta . A profound increase in subendothelial fibronectin was found following LPS treatment . However, no distinct change in intracellular fibronectin was observed in the same endothelium until 24 hr after injection . Using horseradish peroxidase (HRP) and anti-fibronectin-HRP antibody as tracers, LPS was also found to increase permeability and extravasation of plasma proteins (fibronectin) of the aortic endothelium . The increase of subendothelial fibronectin possibly resulted from increased influx and sequestration of plasma fibronectin . This increase may provide a firm substratum for reendothelialization after vascular injury.

Scand J Immunol, 1991 Jan, 33(1), 37 - 49
A tumour necrosis factor beta gene polymorphism in relation to monokine secretion and insulin-dependent diabetes mellitus; Pociot F et al.; HLA-class III region genes may be associated with susceptibility to insulin-dependent diabetes mellitus (IDDM) . In this study an NcoI polymorphism of the tumour necrosis factor beta (TNF-beta) gene, which is positioned next to the tumour necrosis factor alpha (TNF-alpha) gene in the HLA class III region, was detected by restriction fragment length polymorphism (RFLP) . This polymorphism has previously been reported to be located in the TNF-alpha gene . Caucasian HLA-DR3,4 heterozygous IDDM patients (n = 26) and DR-matched healthy controls (n = 19), as well as randomly selected IDDM patients (n = 27) and controls (n = 25) were studied . In addition four multiplex families (49 individuals) and eight HLA-non-identical sibpairs concordant for IDDM were analysed . The TNF-beta gene RFLP analysis showed fragments of 5.5 kb and 10.5 kb, which behaved as alleles . In all groups there was a haplotype assignment of the TNF-beta 5.5-kb allele to B8,DR3 haplotypes, and of the TNF-beta 10.5-kb allele to B15,DR4-positive haplotypes . The allelic and genotypic frequencies differed between DR3,4 IDDM patients and DR3,4 controls, and the DR3,4 control group differed significantly from the randomly selected control group (P less than 0.0079) . In HLA-DR3,4- and DQw8-positive persons, the DR3 haplotypes carried the 10.5-kb allele three times more frequently in IDDM patients than in controls, suggesting that the 10.5-kb allele when present on DR3 haplotypes may contribute to susceptibility to IDDM in DR3,4 heterozygous individuals . A contributory role of the 10.5-kb allele in genetic IDDM susceptibility was supported by the sibpair analysis, in which all were TNF-beta identical . Five were 10.5 kb homozygous, and the remaining three pairs were 5.5/10.5 kb heterozygous . Twenty-five healthy and eight newly diagnosed IDDM patients were randomly selected to study the Escherichia coli lipopolysaccharides (LPS)-purified protein derivate (tuberculin) (PPD)-, and phytohaemagglutinin (PHA)-stimulated monocyte (Mo) secretions of interleukin 1 beta (IL-1 beta) and TNF-alpha in relation to the NcoI TNF-beta gene polymorphism . The LPS- and PHA-stimulated Mo IL-1 beta and TNF-alpha secretions were significantly lower for the TNF-beta 5.5/10.5 kb heterozygous individuals than for TNF-beta 10.5 kb homozygous individuals . Furthermore, the Mo IL-1 beta and TNF-alpha secretions of IDDM patients were significantly higher than the Mo secretions of TNF-beta genotype-matched healthy controls . This study suggests an association between the 10.5 kb TNF-beta allele and IDDM, and demonstrates an association between monokine responses and TNF-beta genotypes.(ABSTRACT TRUNCATED AT 400 WORDS)

Oncogene, 1991 Jan, 6(1), 93 - 102
Myc protein structure: localization of DNA-binding and protein dimerization domains; Kerkhoff E et al.; Wildtype and mutant v-Myc proteins were overexpressed in Escherichia coli using the T7 RNA polymerase system, and the in vitro DNA-binding activities of partially or highly purified proteins were analysed by native DNA-cellulose chromatography . For the construction of the expression plasmids, cloned proviral DNA from wildtype MC29 or from its spontaneous deletion mutant Q10C was used, the latter lacking internal v-myc sequences . Both the wildtype (p59) and the mutant (p42) recombinant protein contain at their amino termini 12 amino acids encoded by the vector, followed by 11 gag amino acids and 9 amino acids encoded by v-myc sequences derived from noncoding c-myc sequences . In addition, p59 contains 416 amino acids encoded by v-myc sequences derived from the complete chicken c-myc coding region, whereas p42 lacks 120 amino acids from the central region of the Myc protein including the highly acidic domain . Two additional proteins were engineered which contain the first 309 (p53) or the last 107 (p16) amino acids, respectively, of the Myc protein sequence in addition to vector-encoded amino acids . The p16 protein represents the carboxyl terminus of the Myc protein sequence containing both a muscle determination gene (MyoD1) homology region, including a basic motif and an amphipathic helix-loop-helix motif, and a leucine heptad repeat . All proteins, except p53 which lacks the carboxyl-terminal Myc protein sequences, bound to native DNA-cellulose and were eluted with 200-500 mM NaCl . Based on the DNA-binding activities of recombinant or spontaneous mutant v-Myc proteins extracted from bacterial or from transformed avian cells, we conclude that the DNA-binding domain of avian Myc proteins is confined within the last 86 carboxyl-terminal amino acids . The same region is also shown to be necessary and sufficient for Myc protein dimerization . This 86-amino acid region essentially encompasses a putative basic DNA contact surface and a tandem array of two presumed protein dimerization motifs, helix-loop-helix and leucine repeat.

Eur J Biochem, 1991 Jan 1, 195(1), 177 - 82
The human leukocyte-adhesion ligand, intercellular-adhesion molecule 2 . Expression and characterization of the protein; Gahmberg CG et al.; The leukocyte cell-adhesion receptors, complexes of the cluster of differentiation antigen 11a with cluster of differentiation antigen 18 (CD11a/CD18), cluster of differentiation antigen 11b with cluster of differentiation antigen 18 (CD11b CD18) and cluster of differentiation antigen 11c with cluster of differentiation antigen 18 (CD11c CD18), are of major importance in several leukocyte functions . Previously a cellular ligand named intercellular-adhesion molecule 1 (ICAM-1) was identified, isolated and extensively characterized . Recently a second similar molecule, intercellular-adhesion molecule 2 (ICAM-2), was found by a functional DNA-cloning method . We have now synthesized the ICAM-2 DNA by the polymerase chain reaction (PCR), sequenced it, and transferred it into mammalian and bacterial expression vectors . A functional leukocyte-binding glycoprotein was obtained by transfection of COS-1 cells . A soluble protein-A - ICAM-2 fusion protein was made in Escherichia coli, purified and used for antiserum production . The antiserum precipitated a cell-surface protein with an apparent molecular mass of 55 kDa from ICAM-2 transfected COS-1 cells, leukocytes and endothelial cells, and inhibited leukocyte binding to transfected COS-1 cells . The bacterial fusion protein, lacking carbohydrate, specifically bound to leukocyte receptors.

Genes Dev, 1991 Jan, 5(1), 74 - 82
DNA-protein interaction at the replication termini of plasmid R6K; Sista PR et al.; Understanding the molecular mechanism of specific and polarized termination of DNA replication at a sequence-specific replication terminus requires detailed analyses of the interaction of terminator protein (ter) with specific DNA sequences (tau), constituting the replication terminus . Such analyses should provide the structural basis of the functional polarity of replication inhibition observed in vivo and in vitro at tau sites . With this objective in mind, we have purified the replication terminator protein of Escherichia coli to homogeneity and have analyzed the interaction of the protein with the replication termini of R6K, using chemical probes and by site-directed mutagenesis . The results show that one monomer of ter protein binds to a single tau site with an equilibrium dissociation constant of 5 x 10(-9) moles/liter . Furthermore, a combination of alkylation interference and protection, hydroxyradical footprinting, and site-directed mutagenesis has revealed the phosphate groups and base residues of the tau core sequence that make contacts with ter protein and those residues that are important for both DNA-protein interaction and for termination of replication in vivo . The overall picture that emerges from these analyses reveals that ter forms an asymmetric complex with a tau sequence . Thus, the asymmetric ter-tau complex provides a structural basis for the functional polarity of the arrest of a moving replication fork at a tau site.

EMBO J, 1991 Jan, 10(1), 25 - 33
Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism; Beese LS et al.; The refined crystal structures of the large proteolytic fragment (Klenow fragment) of Escherichia coli DNA polymerase I and its complexes with a deoxynucleoside monophosphate product and a single-stranded DNA substrate offer a detailed picture of an editing 3'-5' exonuclease active site . The structures of these complexes have been refined to R-factors of 0.18 and 0.19 at 2.6 and 3.1 A resolution respectively . The complex with a thymidine tetranucleotide complex shows numerous hydrophobic and hydrogen-bonding interactions between the protein and an extended tetranucleotide that account for the ability of this enzyme to denature four nucleotides at the 3' end of duplex DNA . The structures of these complexes provide details that support and extend a proposed two metal ion mechanism for the 3'-5' editing exonuclease reaction that may be general for a large family of phosphoryltransfer enzymes . A nucleophilic attack on the phosphorous atom of the terminal nucleotide is postulated to be carried out by a hydroxide ion that is activated by one divalent metal, while the expected pentacoordinate transition state and the leaving oxyanion are stabilized by a second divalent metal ion that is 3.9 A from the first . Virtually all aspects of the pretransition state substrate complex are directly seen in the structures, and only very small changes in the positions of phosphate atoms are required to form the transition state.

EMBO J, 1991 Jan, 10(1), 227 - 36
UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme; McGrath JP et al.; All known functions of ubiquitin are mediated through its covalent attachment to other proteins . The post-translational formation of ubiquitin--protein conjugates is preceded by an ATP-requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin-activating enzyme, also known as E1 . We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin-activating enzyme of the yeast Saccharomyces cerevisiae . UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide-binding sites . Expression of catalytically active UBA1 protein in E . coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin-activating enzyme . Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin--protein conjugates is essential for cell viability.

EMBO J, 1991 Jan, 10(1), 17 - 24
The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction; Derbyshire V et al.; We have used site-directed mutagenesis to change amino acid side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease active site of Klenow fragment . Exonuclease assays of the resulting mutant proteins indicate that the largest effects on exonuclease activity result from mutations in a group of carboxylate side chains (Asp355, Asp424 and Asp501) anchoring two divalent metal ions that are essential for exonuclease activity . Another carboxylate (Glu357) within this cluster seems to be less important as a metal ligand, but may play a separate role in catalysis of the exonuclease reaction . A second group of residues (Leu361, Phe473 and Tyr497), located around the terminal base and ribose positions, plays a secondary role, ensuring correct positioning of the substrate in the active site and perhaps also facilitating melting of a duplex DNA substrate by interacting with the frayed 3' terminus . The pH-dependence of the 3'-5' exonuclease reaction is consistent with a mechanism in which nucleophilic attack on the terminal phosphodiester bond is initiated by a hydroxide ion coordinated to one of the enzyme-bound metal ions.

EMBO J, 1991 Jan, 10(1), 1 - 9
Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity; Coll M et al.; Procarboxypeptidase B is converted to enzymatically active carboxypeptidase B by limited proteolysis catalysed by trypsin, removing the long N-terminal activation segment of 95 amino acids . The three-dimensional crystal structure of procarboxypeptidase B from porcine pancreas has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 0.169 . The functional determinants of its enzymatic inactivity and of its activation by limited proteolysis have thus been unveiled . The activation segment folds in a globular region with an open sandwich antiparallel-alpha antiparallel-beta topology and in a C terminal alpha-helix which connects it to the enzyme moiety . The globular region (A7-A82) shields the preformed active site, and establishes specific interactions with residues important for substrate recognition . AspA41 forms a salt bridge with Arg145, which in active carboxypeptidase binds the C-terminal carboxyl group of substrate molecules . The connecting region occupies the putative extended substrate binding site . The scissile peptide bond cleaved by trypsin during activation is very exposed . Its cleavage leads to the release of the activation segment and to exposure of the substrate binding site . An open-sandwich folding has been observed in a number of other proteins and protein domains . One of them is the C-terminal fragment of L7/L12, a ribosomal protein from Escherichia coli that displays a topology similar to the activation domain of procarboxypeptidase.

Arch Biochem Biophys, 1991 Jan, 284(1), 9 - 16
Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli; Kimlova LJ et al.; The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion . A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site . The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site . This product could complement the folC mutation in E . coli strain SF4 as well as a strain deleted in the folC gene . The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity . The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type . Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant . Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene . None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme . The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants . These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.

Mutat Res, 1991 Jan, 259(1), 89 - 93
A comparison of the relative activities of 8 radiosensitizers in the SOS chromotest; Widdick DA et al.; Misonidazole, and RSU 1069 and 6 of its analogues are all reported to show increased cytotoxicity towards hypoxic cells compared to oxic cells . DNA is considered to be the target through which these drugs exert their cytotoxic activity . Therefore we monitored induction of the SOS response in uvrABC excinuclease proficient and deficient strains of E . coli, under oxic and hypoxic conditions, as an indirect method of assessing the activity of these drugs towards DNA in a biological system . This was done using the SOS chromotest which utilizes E . coli strains which possess a sfiA::lacZ fusion allowing induction of the SOS response to be monitored by assaying beta-galactosidase activity . All of the drugs tested here show some induction of the SOS response in both uvrABC excinuclease proficient and deficient strains . Data shown here suggests that the uvrABC excinuclease is important in the production of a SOS induction signal from RSU 1069-induced DNA lesions and that RSU 1069 may act as a crosslinking agent . The data also shows that SOS induction activity and toxicity do not necessarily correlate and that production of a SOS induction signal may occur via a different pathway for RSU 1069 than for its analogues.

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