Escherichia coli

Eur J Immunol, 1990 Apr, 20(4), 819 - 24
New B cell epitopes in the Plasmodium falciparum malaria circumsporozoite protein; Stuber D et al.; Most antibodies directed against the Plasmodium falciparum circumsporozoite (CS) protein react with its central domain, which contains about 40 repeats of the tetrapeptide Asn-Ala-Asn-Pro (NANP) . To search for new epitopes in the non-repetitive part of the CS protein, we expressed the non-repetitive regions of the protein in E . coli as fusion proteins with mouse dihydrofolate reductase linked to six adjacent histidine residues . These fusion proteins were obtained at greater than 70% purity by a single Ni-chelate affinity chromatography step . Of the new epitopes defined in the C-terminal portion of the CS protein, three are located in a stretch of 65 amino acids immediately C-terminal of the protein's central repetitive domain . Pooled sera from inhabitants of a malaria-endemic area reacted with epitopes in this region of the molecule, and four mouse monoclonal antibodies to this region also reacted with the native CS protein on sporozoites . Two of the monoclonal antibodies reacted with a peptide PNDPNRNVD derived from a conserved region of the CS protein . The other two antibodies showed different reactivities to sporozoites of the NF54 and Ro59 parasite isolates . One, which reacted with a peptide ENANANNAV, recognized Ro59 but not NF54 sporozoites, while the other reacted with a small percentage of NF54 but not Ro59 sporozoites . Antibodies which react with non-repetitive regions of the CS protein could contribute to maintaining its genetic variability.

Biol Reprod, 1990 Apr, 42(4), 693 - 701
Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10; Wright RM et al.; cDNAs coding for the intra-acrosomal protein SP-10 were cloned and characterized as a first step in understanding the expression of this antigen during spermatogenesis . Three overlapping SP-10-specific cDNAs were isolated from a human testes cDNA expression library . These cDNAs hybridized to a 1.35-kb mRNA that was present in human testes but was not found in liver or placenta . Complete sequencing of these cDNAs, designated SP-10-5, SP-10-8, and SP-10-10, produced an 1117-bp sequence containing a 265-amino acid-coding region for the SP-10 protein . Hydrophobicity plots generated from the deduced amino acid sequence showed a very hydrophobic amino terminus characteristic of a signal peptide . Sequence data showed that three different amino acid repeats occurred a total of 16 times in the central third of the SP-10 protein . Interestingly, cDNA SP-10-10 has an internal 57-base pair (19 amino acids) in-frame deletion that is not present in SP-10-5, suggesting that alternative splicing generates more than one SP-10 mRNA . The SP-10 protein appears to be a unique acrosomal protein, based on previous immunohistological data and the observation that SP-10 cDNA sequences did not show any significant homology to other sequences found in the Genbank, National Biomedical Research Foundation, or Swiss sequence banks . A recombinant SP-10 fusion protein was produced in an Escherichia coli expression vector and used to generate a polyclonal antiserum . This antiserum stained the acrosomal cap in situ and reacted with a similar set of peptides on Western blots as did a monoclonal antibody to SP-10.

AIDS Res Hum Retroviruses, 1990 Apr, 6(4), 443 - 54
Prevalence of antibodies to the core protein P17, a serological marker during HIV-1 infection; Mehta SU et al.; Studies on monitoring the immune response to viral structural proteins during human immunodeficiency virus (HIV-1) infection have established the significance of antibodies to the core protein p24 during the progression of the disease . We have studied the prevalence of antibodies to the core protein p17 in order to study their diagnostic and prognostic significance in the pathogenesis of HIV-1 . Full-length HIV-1 p17, molecularly cloned and expressed in Escherichia coli was purified by immunoaffinity chromatography using an HIV-1 p17-specific monoclonal antibody . A highly sensitive enzyme-linked immunoassay was developed using the purified recombinant p17 as the serological target to detect antibodies to p17 . The results indicated that antibodies to p17 decline during progression of disease, with the decline being more dramatic as patients moved from asymptomatic to AIDS-related complex (ARC) . Patient specimens deficient in p24 antibody, but having detectable levels of antibody to p17 were almost always positive for p24 antigen . Under these conditions, p17 antibody is an important serological marker because it provides a more consistent marker for core antigens during HIV-1 infection.

Virology, 1990 Apr, 175(2), 575 - 80
Mutational analysis of the ribonuclease H activity of human immunodeficiency virus 1 reverse transcriptase; Hizi A et al.; We have constructed a series of plasmids that, when introduced into Escherichia coli, induce the expression of high levels of either wild-type or mutated forms of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) . Mutant forms of RT that had been previously analyzed for their RNA-dependent DNA polymerase activity were tested for RNase H activity using an in situ polyacrylamide gel assay . Mutations affecting the RNase H are not clustered in a single region of the 66-kDa RT molecule . With only few exceptions, mutations that affect the RNase H activity also cause a substantial decrease in the DNA polymerase function . This suggests that, unlike the RT from murine leukemia virus (MuLV), it is difficult to genetically separate the catalytic domains responsible for the RNase H and DNA polymerase functions of HIV-1 RT . Those few mutations that differentially affect the RNase H and the polymerase activities of HIV-1 RT suggest that, as in MuLV, the polymerase domain is in the amino-terminus and the RNase H domain is in the carboxy-terminus . We have also generated chimeric molecules that are composed of sequences from the RT of HIV-1 and MuLV and these hybrid RTs were analyzed for their enzymatic properties . Two of these chimeric RTs possess RNase H activity but lack detectable DNA polymerase activity.

Proc Natl Acad Sci U S A, 1990 Apr, 87(8), 3097 - 101
Cloning of a chicken liver cDNA encoding 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase by functional complementation of Escherichia coli pur mutants; Chen ZD et al.; We have used functional complementation of Escherichia coli pur mutants to clone avian cDNA encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase-5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase, the bifunctional enzyme catalyzing steps 6 and 7 in the pathway for de novo purine nucleotide synthesis . Mutational analyses have been used to establish the structure-function relationship: NH2-SAICAR synthetase-AIR carboxylase-COOH . The amino acid sequence of the SAICAR synthetase domain is homologous to that of bacterial purC-encoded enzymes, and the sequence of the following AIR carboxylase domain is homologous to that of bacterial purE-encoded enzymes . In E . coli, AIR carboxylase is the product of genes purEK with the purK subunit postulated to have a role in CO2 binding . The avian enzyme lacks sequences corresponding to purK yet functions in E . coli . Functional complementation of E . coli pur mutants can be used to clone additional avian cDNAs for de novo purine nucleotide synthesis.

J Gen Virol, 1990 Apr, 71 ( Pt 4), 881 - 7
Epitope analysis of glycoprotein I of pseudorabies virus; Jacobs L et al.; A panel of 11 monoclonal antibodies (MAbs) raised against pseudorabies virus (PRV) was used to map epitopes on the virus glycoprotein I (gI) . We employed three approaches to map epitopes on gI . By a competition binding assay, six groups of MAbs were defined as reacting with distinct antigenic domains on gI . To identify regions along the gI polypeptide chain encompassing the domains recognized by these MAbs, DNA fragments derived from the gI-coding region were cloned into pEX expression plasmids . The antigenic reactivity of the fusion proteins expressed in Escherichia coli was analysed by immunoblotting . Five antigenic domains were mapped within the first 238 amino acids of gI: domains A, B and D were mapped between amino acids 52 and 123 and domains C and E between amino acids 78 and 238 . One MAb, representing domain F, did not react with the expressed fusion proteins . To assess the precise location and amino acid sequences of the epitopes, overlapping nonapeptides covering the amino acid sequence 52 to 238 were synthesized . The antibody-binding activity of these peptides was tested by an ELISA (Pepscan-method) . Three antigenic domains were mapped: domain A was localized to amino acids 64 to 73 and 75 to 84, domain B to amino acids 52 to 67 and domain D to amino acids 68 to 82 . Four MAbs representing antigenic domains C, E and F did not react in the Pepscan . Finally, sera from pigs infected experimentally with PRV reacted with the fusion protein of plasmid ps 1 (amino acids 52 to 238).

J Cell Biol, 1990 Apr, 110(4), 1199 - 210
The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression; Hatzfeld M et al.; Recombinant DNA technology has been used to analyze the first step in keratin intermediate filament (IF) assembly; i.e., the formation of the double stranded coiled coil . Keratins 8 and 18, lacking cysteine, were subjected to site specific in vitro mutagenesis to change one amino acid in the same relative position of the alpha-helical rod domain of both keratins to a cysteine . The mutations lie at position -36 of the rod in a "d" position of the heptad repeat pattern, and thus air oxidation can introduce a zero-length cystine cross-link . Mutant keratins 8 and 18 purified separately from Escherichia coli readily formed cystine homodimers in 2 M guanidine-HCl, and could be separated from the monomers by gel filtration . Heterodimers with a cystine cross-link were obtained when filaments formed by the two reduced monomers were allowed to oxidize . Subsequent ion exchange chromatography in 8.5 M urea showed that only a single dimer species had formed . Diagonal electrophoresis and reverse phase HPLC identified the dimer as the cystine containing heterodimer . This heterodimer readily assembled again into IF indistinguishable from those obtained from the nonmutant counterparts or from authentic keratins . In contrast, the mixture of cystine-stabilized homodimers formed only large aberrant aggregates . However, when a reducing agent was added, filaments formed again and yielded the heterodimer after oxidation . Thus, the obligatory heteropolymer step in keratin IF assembly seems to occur preferentially at the dimer level and not during tetramer formation . Our results also suggest that keratin I and II homodimers, once formed, are at least in 2 M guanidine-HCl a metastable species as their mixtures convert spontaneously into heterodimers unless the homodimers are stabilized by the cystine cross-link . This previously unexpected property of homodimers explains major discrepancies in the literature on the keratin dimer.

EMBO J, 1990 Apr, 9(4), 1267 - 74
The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site; Ma C et al.; Transposase (tnp) expression from insertion sequence IS10 is controlled, in part, by an antisense RNA, RNA-OUT, which pairs to the translation initiation region of the tnp mRNA, RNA-IN . Genetic experiments suggest that control occurs post-transcriptionally . Here, we present evidence that bears on the control mechanism . Specific ribosome binding at the tnp translation initiation site is demonstrated in vitro . Two mutations that alter tnp translation in vivo are shown to have corresponding effects in vitro . Most importantly, RNA-OUT/RNA-IN pairing is shown to block ribosome binding . In conjunction with the work described in the accompanying paper, we propose that inhibition of ribosome binding also occurs in vivo, and that it is sufficient to account for control . Implications for translational control in analogous systems are discussed.

EMBO J, 1990 Apr, 9(4), 1259 - 66
The IS10 transposase mRNA is destabilized during antisense RNA control; Case CC et al.; RNA stability is an important component of gene expression, and antisense RNAs have been proposed to alter target RNA stability . We show here that the IS10 transposase mRNA, RNA-IN, is rendered unstable during control by the IS10 antisense RNA, RNA-OUT . Destabilization requires RNA-OUT/RNA-IN pairing and ribonuclease III cleavage . Independent of such cleavage, RNA-OUT is rendered unstable through disruption of its secondary structure . Pairing has no other obvious effects on RNA-IN transcription or stability . Nevertheless, RNA-IN destabilization is not required for antisense control in vivo . In the accompanying paper {Ma,C . and Simons, R.W . (1990) EMBO J., 9, 1267-1274 we show that pairing blocks ribosome binding to RNA-IN . Were it not for control at this level, destabilization would play a more prominent role.

EMBO J, 1990 Apr, 9(4), 1229 - 35
Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein; Buvoli M et al.; The human hnRNP core protein A1 (34 kd) is encoded by a 4.6 kb gene split into 10 exons . Here we show that the A1 gene can be differentially spliced by the addition of an extra exon . The new transcript encodes a minor protein of the hnRNP complex, here defined A1B protein, with a calculated mol . wt of 38 kd, that coincides with a protein previously designated as B2 by some authors . In vitro translation of the mRNAs selected by hybridization with A1 cDNA produced two proteins of 34 and 38 kd; Northern blot analysis of poly(A)+ RNA from HeLa cells revealed that the abundance of the A1B mRNA was approximately 5% that of A1 . The A1B protein was detected by Western blotting with an anti-A1 monoclonal antibody both in enriched preparations of basic hnRNP proteins and in 40S hnRNP particles . The A1B protein exhibits a significantly higher affinity than A1 for ssDNA . The recombinant A1B protein, expressed in Escherichia coli, shows the same electrophoretic mobility and charge as the cellular one.

Clin Exp Immunol, 1990 Apr, 80(1), 38 - 43
A recombinant topoisomerase I used for autoantibody detection in sera from patients with systemic sclerosis; Verheijen R et al.; We report the expression of a cDNA clone encoding 695 carboxyl-terminal amino acids of human DNA topoisomerase I (topoI) in Escherichia coli . More than 96% of the anti-HeLa topoI-positive sera from patients with a connective tissue disease displayed also an immunoreactivity with this recombinant protein (the HTopoA protein) . Sera from patients with a definite diagnosis systemic sclerosis and reacting with HeLa topoI, all reacted with the HTopoA protein as well . Sera from patients with systemic sclerosis that did not contain anti-topoI antibodies (about 30% of the systemic sclerosis sera), as concluded from HeLa immunoblot, displayed also no immunoreactivity with our recombinant antigen . By expressing different fragments of HTopoA, we were able to assign at least three different autoimmune epitope regions on the HTopoA protein and we show that over a period of 5 years the amount of anti-topoI antibodies against these regions may fluctuate.

Proc Natl Acad Sci U S A, 1990 Apr, 87(7), 2638 - 42
Association of the v-crk oncogene product with phosphotyrosine-containing proteins and protein kinase activity; Mayer BJ et al.; An antiserum specific for P47gag-crk, the oncogene product of avian sarcoma virus CT10, was used to investigate possible crk-binding proteins . In in vitro kinase assays, four proteins were phosphorylated in anti-crk immunoprecipitates . Tyrosine, serine, and threonine residues were phosphorylated . A number of tyrosine-phosphorylated proteins were identified in anti-crk immunoprecipitates of 32P-labeled cells, including the three major phosphotyrosine-containing proteins of CT10-infected cells . These three proteins also bound to bacterially synthesized crk protein . These results suggest that the crk transforming protein can stably associate with both endogenous kinases and cellular kinase substrates.

J Bacteriol, 1990 Apr, 172(4), 2013 - 9
Coupling of DNA replication to growth rate in Escherichia coli: a possible role for guanosine tetraphosphate; Chiaramello AE et al.; Two promoters for the Escherichia coli operon that contains the four genes dnaA, dnaN, recF, and gyrB were found to be growth rate regulated and under stringent control . Transcript abundance relative to total RNA increased with the growth rate . Changes in transcription from the dnaAp1 and dnaAp2 promoters that were induced by amino acid starvation and chloramphenicol and were relA dependent were correlated with the stringent response . The abundance of these transcripts per total RNA also decreased in spoT mutants as the severity of the mutation increased (guanosine 5'-diphosphate 3'-diphosphate {ppGpp} basal levels increased) . Because expression of these promoters appears to be inhibited by ppGpp, it is proposed that one mechanism for coupling DNA replication to the growth rate of bacteria is through ppGpp synthesis at the ribosome.

J Bacteriol, 1990 Apr, 172(4), 1992 - 7
Posttranscriptional control of plasmid ColIb-P9 repZ gene expression by a small RNA; Shiba K et al.; The replication frequency of plasmid ColIb-P9 depends on the level of repZ gene expression, which is negatively regulated by the action of the inc gene (C . Hama, T . Takizawa, H . Moriwaki, Y . Urasaki, and K . Mizobuchi, J . Bacteriol . 172:1983-1991, 1990) . To further understand the mechanism of this regulation, we analyzed transcripts of the ColIb-P9 replication control region . Four RNA species, designated RNAI to RNAIV, were observed in plasmid pCH11, which contained the whole inc gene region and the 5' portion of the repZ gene . RNAII, RNAIII, and RNAIV, with sizes of approximately 200, 500, and 1,500 bases, respectively, were identified as rightward transcripts that shared common transcription initiation sites; RNAIV was determined to be equivalent to a part of repZ mRNA, which was observed in pCH10, a plasmid that contained sufficient information for replication and control of ColIb-P9 . Conversely, RNAI, with a size of about 70 bases, was transcribed leftward and was identified as the product of the inc gene and hence equivalent to inc RNA detected by in vitro RNA synthesis . This small RNA was found to be complementary to a part of repZ mRNA . These results and quantitative analyses of the transcripts in Inc- mutants indicate that the inc RNA negatively regulates repZ expression mainly at the posttranscriptional level through the possible formation of an inc RNA-repZ mRNA hybrid in the host cells.

J Bacteriol, 1990 Apr, 172(4), 1983 - 91
Organization of the replication control region of plasmid ColIb-P9; Hama C et al.; We identified a 1,845-base-pair sequence that contains essential information for the autonomous replication and regulation of the 93-kilobase-pair IncI alpha group ColIb-P9 plasmid . Biochemical and genetic analyses revealed that this sequence specifies at least two structural genes, designated repZ and inc . The repZ gene encodes a protein with a molecular weight of 39,000, which probably functions as an initiator for the ColIb-P9 replicon . The inc gene that phenotypically governs the incompatibility encodes an RNA with a size of about 70 bases . This small RNA acts in trans to repress the expression of repZ, thereby functioning to maintain a constant copy number of the ColIb-P9 replicon in host cells.

Agric Biol Chem, 1990 Apr, 54(4), 949 - 55
Expression of recombinant bovine beta-lactoglobulin in Escherichia coli; Batt CA et al.; Bovine beta-lactoglobulin A was expressed in Escherichia coli in its mature form . The gene was constructed using a cDNA clone which coded for amino acid residues Leu-11 to Ile-162 and a synthetic oligonucleotide coding for the initial 10 amino acids preceded by a translational start . The met-beta-lactoglobulin was expressed using a tac promoter vector, pTTQ18, and accounted for approximately 15% of the total cellular protein . The recombinant met-beta-lactoglobulin migrated with the same molecular weight as native beta-lactoglobulin A on SDS-PAGE . The majority of the met-beta-lactoglobulin produced was found in an insoluble form but could be solubilized using guanidine-HCl . The renatured preparation was greater than 80% pure and migrated similarly to purified beta-lactoglobulin A under nondenaturing conditions.

Trends Biotechnol, 1990 Apr, 8(4), 88 - 93
Engineering proteins for purification; Sassenfeld HM; Over the past decade, a new protein purification technique has emerged as a result of recombinant DNA technology . DNA, encoding additional polypeptide or protein tags, is fused to the gene of interest . Expression of these gene fusions results in protein fusions which may be purified by techniques using the properties of the additional polypeptide tag . This has eliminated the need for extensive screening and optimization procedures previously required for purification.

Gene, 1990 Mar 30, 88(1), 87 - 95
Extracellular metalloprotease gene of Streptomyces cacaoi: structure, nucleotide sequence and characterization of the cloned gene product; Chang PC et al.; The gene (npr) encoding an extracellular neutral metalloprotease (Npr) from Streptomyces cacaoi YM15 was cloned in Streptomyces lividans using pIJ702 as a vector . The nucleotide (nt) sequence of npr was determined . The deduced open reading frame encoded 550 amino acids (aa) (60 kDa) with a putative signal sequence of 34 aa at the N terminus . High-resolution S1 mapping identified the transcriptional start point at about 132-134 nt upstream from the start codon . The nt sequences at both -10 and -35 regions resemble the consensus sequence of typical Escherichia coli promoters and a fragment containing the promoter was functional in an E . coli promoter probe plasmid . In vitro transcription and translation of the cloned npr sequence revealed a 60-kDa protein product, correlated with the sequence data but not with the size (35 kDa) of the extracellular Npr . The N-terminal aa sequence in conjunction with the aa composition analyses on the purified mature Npr led to the conclusion that it was processed from the 60-kDa pre-proenzyme form encoded by npr . The Npr protease contained putative zinc ligand-binding regions and two repeated motifs, Asp-Ser-Gly, similar to the active site residues of the aspartic acid and retroviral proteases.

Gene, 1990 Mar 30, 88(1), 97 - 9
Heat-labile phosphatase simplifies the preparation of dephosphorylated vector DNA; Hoffman LM et al.; A novel enzyme for DNA dephosphorylation, HK phosphatase, is completely and irreversibly inactivated at 65 degrees C . HK phosphatase treatment of BamHI-digested pBR322 reduces nonrecombinants in cloning experiments to approx . 5% of transformant colonies.

Gene, 1990 Mar 30, 88(1), 121 - 6
TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells; Mashko SV et al.; A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli . In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter . The cloned gene is the distal cistron of this hybrid operon ('overlappon') . The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter . The coding region of this artificial fused cistron {the length of the corresponding open reading frame is about 120 amino acids (aa)} includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E . coli (72 aa) and 3' C-terminal codons of the E . coli trpE gene product . At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E . coli trpD gene and the overlapping stop and start codons (TGATG) . In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E . coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1990 Mar 30, 188(3), 523 - 8
Cloning and sequencing of mammalian glutathione reductase cDNA; Tutic M et al.; The molecular cloning of a partial cDNA to mouse glutathione reductase mRNA and of a full-length cDNA to the mRNA of the human enzyme is described . An initial cDNA clone designated lambda GRM-B11 was isolated by plaque-screening of an induced mouse cDNA expression library in the lambda gt11 vector with a rabbit antibody probe to human glutathione reductase . 125Iodine-labelled whole anti-rabbit immunoglobulin was used as second antibody . EcoRI digestion of the lambda GRM-B11 clone released a 720-bp fragment which was identified as a partial mouse glutathione reductase cDNA by the following techniques . (a) Escherichia coli Y1089 lysogenized with lambda GRM-B11 could be induced to synthesize a recombinant polypeptide whose antigenicity to anti-(glutathione reductase) serum was established by SDS/polyacrylamide gel electrophoresis and subsequent immunoblotting . (b) The GRM-B11 sequence, recloned in the Bluescript vector to give the plasmid pGRM-B11, was found to code for a polypeptide consisting of 242 amino acid residues exhibiting 82% identities with the known amino acid sequence of the human glutathione reductase from position 77 to 318 . The insert of the pGRM-B11 plasmid was used as a bona fide nucleic acid probe to screen mouse and human cDNA libraries prepared in the lambda gt11 or in the lambda gt10 vector . The first full-length cDNA clone (lambda GRH-Mev10) was identified in a human cDNA library based on RNA of human placental cells . Its insert was composed of three EcoRI fragments of 720, 613 and 336 bp . The three fragments were recloned in the Bluescript vector and sequenced . The largest fragment (pGRH-B) is colinear with the mouse sequence cloned in the pGRM-B11 plasmid . The fragment of intermediate size (pGRH-CT) comprises the 3' end of the mRNA and the poly(A) tail while the short fragment (pGRH-NT) corresponds to the 5' region of the mRNA . The amino acid sequence deduced from the nucleotide sequences of the three subclones is identical with the known sequence of the mature glutathione reductase from human erythrocytes in all 478 positions.

Biochem Biophys Res Commun, 1990 Mar 30, 167(3), 962 - 9
Analysis of the Escherichia coli K-12 cysB gene and its product using the method of gene fusion; Tei H et al.; The amino-terminal structure and the essential functional region of the cysB gene product of Escherichia coli K-12 were analyzed by the method of gene fusion . The translational start codon of the cysB gene was located by determining the amino-terminal sequence of a hybrid protein containing the first 31 amino acid residues of the CysB protein at the amino terminus of beta-galactosidase(LacZ protein) . The fact that two other CysB'-'LacZ hybrid polypeptides expressed a normal CysB activity indicated that the functional region of the CysB protein was located within the first 215 amino acid residues of the total 324 amino acids deduced from the nucleotide sequence.

Eur J Biochem, 1990 Mar 30, 188(3), 679 - 87
Purification and further characterization of the second nitrate reductase of Escherichia coli K12; Iobbi-Nivol C et al.; Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli . The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11% . It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule . The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme . Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors . Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin . An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z . Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.

Eur J Biochem, 1990 Mar 30, 188(3), 605 - 14
Characterization of the translational start site for IF2 beta, a short form of Escherichia coli initiation factor IF2; Morel-Deville F et al.; The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo . In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA . Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta . Three types of experiments were carried out . First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2 alpha synthesis . Under these conditions, IF2 beta is still formed . Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ . Any synthesis of a fused protein with beta-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment . It was consequently possible to locate the IF2 beta initiation site within an 18-base region containing an in-phase GUG codon . Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region . A hybrid protein with beta-galactosidase activity was synthesized . Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.

Biochem Biophys Res Commun, 1990 Mar 30, 167(3), 948 - 55
Structure and expression of cysX, the second gene in the Escherichia coli K-12 cysE locus; Tei H et al.; Protein products of the cysE region at 81 min on the chromosome of Escherichia coli K-12 (1) were analyzed by the maxicell method . Two kinds of polypeptides of molecular weight 33 K and 16 K were the products . The open reading frame (ORF) of the 33 K polypeptide consisted of 273 amino acids (Mr = 29,261) . On the other hand, the 16 K ORF was overlapped by the opposite 33 K ORF and specified an extremely basic protein of 130 amino acids (Mr = 15,233) . The gene coding for the 16 K polypeptide was named cysX . The expression of cysE and cysX in vivo was confirmed further by constructions of the cysE'-'lacZ and cysX'-'lacZ hybrid genes.

Gene, 1990 Mar 30, 88(1), 37 - 45
Outer-membrane PhoE protein of Escherichia coli K-12 as an exposure vector: possibilities and limitations; Agterberg M et al.; The phosphate-limitation-inducible outer-membrane protein (PhoE) of Escherichia coli K-12 can be used in an expression system as a carrier for foreign antigenic determinants, facilitating their transport to the bacterial cell surface . The system is very flexible, since insertions varying in length and nature can be made in different cell-surface-exposed regions of PhoE protein, without interfering with the assembly process into the outer membrane . Multiple insertions of an antigenic determinant can be made in the second and eighth exposed regions, resulting in a total insert length of up to 30 and 50 amino acid (aa) residues . Insertions can be made in two exposed regions, simultaneously . However, some limitations were encountered, e.g., insertion of eight or more hydrophobic aa residues affected both the translocation process across the inner membrane and the assembly process into the outer membrane . Also, the insertion of sequences containing many charged residues resulted in accumulation of precursor protein in the cytoplasm.

Biochim Biophys Acta, 1990 Mar 29, 1038(1), 90 - 7
Characterization of the hydrogen peroxide-enzyme reaction for two cytochrome c peroxidase mutants; Vitello LB et al.; The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region . Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide . A second mutant enzyme, E . coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI) . The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide . The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers . The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes . The enzymes react with hydrogen peroxide when the ionizable group is unprotonated . Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis . The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.

Nature, 1990 Mar 29, 344(6265), 461 - 3
Binding of the Drosophila sex-lethal gene product to the alternative splice site of transformer primary transcript; Inoue K et al.; Somatic sexual differentiation in Drosophila melanogaster is accomplished by a hierarchy of genes of which one, Sex-lethal (Sxl), is required for the functional female-specific splicing of the transcripts of the immediately downstream regulatory gene, transformer (tra) . The first exon of the tra primary transcript is spliced to one of two acceptor sites . Splicing to the upstream site yields a messenger RNA which is neither sex-specific nor functional, but that produced after splicing to the downstream acceptor site yields a functional female-specific mRNA . Here we address the question of how the Sxl gene product determines the alternative splicing of tra primary transcripts . One suggestion is that non-sex-specific splicing to the upstream acceptor is blocked in female flies by sex-specific factors, but neither the identity of the female-specific factors nor the mechanism of the blockage has been specified . We have now performed co-transfection experiments in which Sxl complementary DNA and the tra gene are expressed in Drosophila Kc cells . Moreover, we find that female Sxl-encoded protein binds specifically to the tra transcript at or near the non-sex-specific acceptor site, implying that the female Sxl gene product is the trans-acting factor that regulates the alternative splicing.

Mol Cell Biochem, 1990 Mar 27, 93(2), 167 - 72
Effect of Escherichia coli lipopolysaccharide on phosphatidylcholine biosynthesis by rat lung and alveolar type II cells; Bosch MA et al.; Alterations in pulmonary surfactant are partly responsible for the respiratory insufficiency seen under septic shock process . We have used an experimental model of LPS-induced shock in rats to examine the cells responsible for the pulmonary surfactant synthesis and its relationship to lung injury . (14C)Choline incorporation into phosphatidylcholine was significantly reduced in lung homogenates or type II cells obtained from LPS-treated animals . Addition of LPS in vitro fails to increase (14C)choline incorporation in type II cells obtained from LPS-treated animals . We suggest that this depression of pulmonary phosphatidylcholine synthesis may partly explain the occurrence of respiratory failure with septic shock.

Biochemistry, 1990 Mar 27, 29(12), 2023 - 9
The DNA binding domain of GAL4 forms a binuclear metal ion complex; Pan T et al.; The transcription factor GAL4 from Saccharomyces cerevisiae requires Zn(II) or Cd(II) for specific recognition of the UASG sequence (Pan & Coleman, 1989) . An N-terminal fragment consisting of the first 63 amino acid residues of GAL4 {GAL4(63)} has been obtained by partial tryptic proteolysis of a cloned and overproduced N-terminal domain of 149 residues, GAL(149) . We show that GAL4(63) contains the minimal GAL4 DNA binding domain . GAL4(63) binds tightly 1-2 mol of Zn(II) or 2 mol of Cd(II) . 113Cd NMR of 113Cd(II)-substituted GAL4(63) reveals structural identity between the metal binding domains of GAL4(63) and that of the larger precursor GAL4(149) . 113Cd(II) can be substituted for the Zn(II) in GAL4(63), and two 113Cd NMR signals are observed at 706 and 669 ppm, both suggesting coordination of 113Cd(II) to three or four -S- ligands . With the exception of the N-terminal methionine, the only sulfur-containing residues are the six highly conserved cysteines . High-resolution 1H NMR of Zn(II)-GAL4(63) and Cd(II)-GAL4(63) show the two proteins to have almost identical conformations and to be present as monomers in solutions up to millimolar concentration . This leads us to postulate that GAL4 does not possess a TFIIIA-like "Zn-finger" but forms a binuclear metal cluster involving all six cysteines in a "cloverleaf"-like array . GAL4(63) contains about 60% alpha-helix, estimated from circular dichroism . Removal of the native Zn(II) causes substantial unfolding of the secondary structure . Unlike GAL4(149), the resultant apoprotein is not induced to refold by readdition of Zn(II) at low concentrations.

Mol Cell Biochem, 1990 Mar 27, 93(2), 141 - 6
Inhibition of transcription by adriamycin is a consequence of the loss of negative superhelicity in DNA mediated by topoisomerase II; Tarr M et al.; Adriamycin is commonly used as a chemotherapeutic agent and is known to intercalate into the major groove of DNA and inhibit DNA and RNA synthesis . Results presented in this communication suggest that adriamycin affects topoisomerase cleavage of DNA . The resultant change in negative superhelicity (decrease) is responsible for the decrease in transcription . This process is not dependent on the continued presence of adriamycin . The reaction between topoisomerases, DNA and adriamycin is dose-dependent . The results help to explain the relatively enhanced cytotoxicity of this drug to tumor cells.

Biochemistry, 1990 Mar 27, 29(12), 3039 - 46
Low-resolution structure of the tetrameric phenylalanyl-tRNA synthetase from Escherichia coli . A neutron small-angle scattering study of hybrids composed of protonated and deuterated protomers; Dessen P et al.; Escherichia coli phenylalanyl-tRNA synthetase is a tetrameric protein composed of two types of protomers . In order to resolve the subunit organization, neutron small-angle scattering experiments have been performed in different contrasts with all types of isotope hybrids that could be obtained by reconstituting the alpha 2 beta 2 enzyme from the protonated and deuterated forms of the alpha and beta subunits . Experiments have been also made with the isolated alpha promoter . A model for the alpha 2 beta 2 tetramer is deduced where the two alpha promoters are elongated ellipsoids (45 x 45 x 160 A3) lying side by side with an angle of about 40 degrees between their long axes and where the two beta subunits are also elongated ellipsoids (31 x 31 x 130 A3) with an angle of 30 degrees between their axes . This model was obtained by assuming that the two pairs of subunits are in contact in an orthogonal manner and by taking advantage of the measured distance between the centers of mass of the alpha 2 and beta 2 pairs (d = 23 +/- 2 A).

Biochemistry, 1990 Mar 27, 29(12), 3053 - 61
Folding of ribonuclease T1 . 1 . Existence of multiple unfolded states created by proline isomerization; Kiefhaber T et al.; It is our aim to elucidate molecular aspects of the mechanism of protein folding . We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds . Also, an efficient mutagenesis system is available to produce protein molecules with defined sequence variations . Here we present a preliminary characterization of the folding kinetics of ribonuclease T1 . Its unfolding and refolding reactions are reversible, which is shown by the quantitative recovery of the catalytic activity after an unfolding/refolding cycle . Refolding is a complex process, where native protein is formed on three distinguishable pathways . There are 3.5% fast-folding molecules, which refold within the millisecond time range, and 96.5% slow-folding species, which regain the native state in the time range of minutes to hours . These slow-folding molecules give rise to two major, parallel refolding reactions . The mixture of fast- and slow-folding molecules is produced slowly after unfolding by chain equilibration reactions that show properties of proline isomerization . We conclude that part of the kinetic complexity of RNase T1 folding can be explained on the basis of the proline model for protein folding . This is supported by the finding that the slow refolding reactions of this protein are accelerated in the presence of the enzyme prolyl isomerase . However, several properties of ribonuclease T1 refolding, such as the dependence of the relative amplitudes on the probes, used to follow folding, are not readily explained by a simple proline model.

J Immunol Methods, 1990 Mar 27, 128(1), 81 - 7
Parasite antigens expressed in Escherichia coli . A refined approach for epidemiological analysis; Scherf A et al.; A simple method is described to generate carrier-free recombinant antigens following their expression in Escherichia coli . A plasmid, called pMSgt11, has been constructed such that the cleavage site for the protease factor Xa separates the recombinant antigen from an enzymatically active beta-galactosidase . Thus, rapid purification of the active beta-galactosidase recombinant protein, followed by digestion with factor Xa, releases the antigen of interest . The pMSgt11 plasmid is compatible with the phage expression vector, lambda gt11 and the feasibility of applying this system has been demonstrated using malarial recombinant antigens . Inserts from lambda gt11 recombinant Plasmodium falciparum clones have been recloned into the EcoRI site of pMSgt11 and the expressed soluble fusion proteins have been purified from crude extracts using a one step affinity chromatography . After protease digestion, the fusion protein cleavage products were analysed by immunoblot with a panel of different human immune sera . We were able to successfully demonstrate specific antibody titers to the parasite-derived carrier-free antigen, without interference from anti-Escherichia coli-specific antibodies . The general application of this approach to epidemiological analysis is discussed.

FEBS Lett, 1990 Mar 26, 262(2), 245 - 8
Lack of inhibition by colicin M suggests bactoprenol independence of MDO biosynthesis; Harkness RE et al.; Biosynthesis of membrane-derived oligosaccharides (MDO), located in the periplasmic space of Escherichia coli, was not inhibited by colicin M, an inhibitor of bactoprenyl phosphate regeneration . This result suggests that bactoprenol does not serve as a lipid carrier of MDO oligosaccharides across the cytoplasmic membrane.

FEBS Lett, 1990 Mar 26, 262(2), 241 - 4
Interactions of lipoyl domains with the E1p subunits of the pyruvate dehydrogenase multienzyme complex from Escherichia coli; Graham LD et al.; Equilibrium binding experiments were carried out with lipoyl domains and the pyruvate decarboxylase {pyruvate dehydrogenase (lipoamide), E1p, EC 1.2.4.1)} component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli . The dissociation constant (Ks) was estimated to be not less than 0.3 mM, exceeding the Km value (33 microM) for reductive acetylation of the domains by an order of magnitude . Thus, the lipoyl domain, which is required to promote reductive acetylation of the lipoyl group, does not appear to do this simply by enhancing the binding to E1p . The difference between Ks and Km suggests that the formation and release of reductively acetylated lipoyl domains from the enzyme may be a relatively rapid step in the mechanism.

FEBS Lett, 1990 Mar 26, 262(2), 155 - 8
Sensitivity of the retinal circular dichroism of bacteriorhodopsin to the mutagenetic single substitution of amino acids: tyrosine; Du JJ et al.; Bacteriorhodopsin (bR) in the native purple membrane, in wild type expressed in E . coli and reconstituted in lipid vesicles, and its constituted mutants with substitutions of Tyr-185 by Phe all are found to have different visible retinal CD spectra . The results strongly suggest that the environment of the retinal in bR determines the sign and heterogeneity of its visible retinal CD spectrum . This supports the recent proposal that the observed biphasic CD spectrum of bR is due to the superposition of the CD spectra having opposite signs of more than one type of bR rather than due to exciton coupling.

FEBS Lett, 1990 Mar 26, 262(2), 310 - 2
Processing of Ada protein by two serine endoproteases Do and So from Escherichia coli; Lee CS et al.; Two soluble serine proteases Do and So from Escherichia coli were found to distinctively cleave the purified, 39 kDa Ada protein into fragments with sizes of 12-31 kDa . Protease So appears to generate a C-terminal 19 kDa polypeptide, similarly to OmpT protease . In addition, the purified 19 kDa C-terminal half of Ada protein can be further processed mainly to an 18 kDa fragment by protease So and to a 12 kDa by protease Do . These results suggest that proteases Do and So are involved in endogenous cleavage of Ada protein, which may play a role in down-regulating the adaptive response to alkylating agents.

FEBS Lett, 1990 Mar 26, 262(2), 205 - 8
Fluoride is a strong and specific inhibitor of (asymmetrical) Ap4A hydrolases; Guranowski A; Fluoride acts as a noncompetitive, strong inhibitor of (asymmetrical) Ap4A hydrolases (EC 3.6.1.17) . The Ki values estimated for the enzymes isolated from seeds of some higher plants (yellow lupin, sunflower and marrow) are in the range of 2-3 microM and I50 for the hydrolase from a mammalian tissue (beef liver) is 20 microM . The anion, up to 25 mM, does not affect the following other enzymes which are able to degrade the bis(5'-nucleosidyl)-oligophosphates: Escherichia coli (symmetrical) Ap4A hydrolase (EC 3.6.1.41), yeast Ap4A phosphorylase (EC 2.7.7.53), yellow lupin Ap3A hydrolase (EC 3.6.1.29) and phosphodiesterase (EC 3.1.4.1) . None of halogenic anions but fluoride affects the activity of (asymmetrical) Ap4A hydrolases . Usefulness of the fluoride effect for the in vivo studies on the Ap4A metabolism is shortly discussed.

FEBS Lett, 1990 Mar 26, 262(2), 194 - 6
Tightly bound pyrophosphate in Escherichia coli inorganic pyrophosphatase; Shestakov AA et al.; Hexameric inorganic pyrophosphatase of Escherichia coli contains about 1 mol/mol of 'structural' pyrophosphate, which survives gel filtration and prolonged incubation with Mg2+, does not exchange with medium phosphate and pyrophosphate but is removed with 0.8 M perchloric acid . The site of pyrophosphate binding seems to be another than the active site . An additional 0.9 mol of enzyme-bound pyrophosphate is formed in the presence of phosphate and Mg2+ but this pyrophosphate is in fast equilibrium with medium phosphate and appears to be bound to the active site.

FEBS Lett, 1990 Mar 26, 262(2), 185 - 8
A second consensus sequence of ATP-requiring proteins resides in the 21-kDa C-terminal segment of myosin subfragment 1; Burke M et al.; Previous comparisons of sequence homologies of ATP-requiring enzymes have defined three consensus sequences which appear to be involved in the binding of the nucleotide . One of these was identified in the N-terminal 27-kDa segment of the myosin heavy chain but the other two sequences have not hitherto been located in myosin . The present paper proposes that one of these other two consensus sequences is in the 21-kDa C-terminal portion of S1 and that it may contribute to the ATP binding domain.

Carbohydr Res, 1990 Mar 25, 197, 197 - 204
Structure of the K16 antigen from Escherichia coli O7:K16:H-, a Kdo-containing capsular polysaccharide; Lenter M et al.; The K16-antigen from E . coli Rk 21510 (O7:K16:H-) is shown to consist of the repeating unit ----2)-beta-D-Ribf-(1----3)-beta-D-Ribf-(1----5)-alpha-Kd op-(2---- of which approximately 33% is O-acetylated at position 3 of the 2-linked ribose.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1513 - 6
Retropseudogenes constitute the major part of the human elongation factor 1 alpha gene family; Madsen HO et al.; The elongation factor 1 alpha (EF-1 alpha) is a protein which promotes the GTP-dependent binding of aminoacyl-tRNA to ribosomes in the protein synthesis process . A human gene coding for EF-1 alpha has previously been cloned and sequenced along with a pseudo-gene . Here, we have further analyzed the family of human EF-1 alpha genes . Using an EF-1 alpha cDNA as probe twelve genomic EF-1 alpha-like clones were isolated and analyzed . Four of these were sequenced and found to contain EF-1 alpha retropseudogenes . A Southern blot analysis indicated that the remaining eight clones also contained retropseudogenes . Genomic Southern blot analysis revealed at least twenty loci in the human genome with sequence homology to the EF-1 alpha cDNA . Besides the already described active gene only one potentially active locus was found . The others appeared to be retropseudogenes . EF-1 alpha retropseudogenes were also found to be abundant in the mammalian species mouse and pig, while the chicken contained only one presumably active EF-1 alpha gene.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1475 - 9
Structure-function relationship of arginyl-tRNA synthetase from Escherichia coli: isolation and characterization of the argS mutation MA5002; Eriani G et al.; The Escherichia coli K12 argS MA5002 mutant appears to have a functionally altered arginyl-tRNA synthetase (ArgRS) . The gene coding for this enzyme was isolated from E . coli genomic DNA using the PCR procedure and inserted into a pUC18 multicopy vector . Sequencing revealed that it differs from the wildtype ArgRS structural gene only by one mutation: a replacement of a C by an A residue which results in substitution of an arginine by a serine at position 134, located two residues downstream from the HVGH consensus sequence . As compared to the genomic enzyme level, this recombinant vector, containing the mutated gene, produces in E . coli JM103, about 100 times as much modified ArgRS . This enzyme was obtained nearly pure after only two chromatographic steps; it exhibits a 4-6 times as low activity and a 5 times as high Km value for ATP as the wildtype enzyme in the aminoacylation and ATP-PPi reactions; Km values for arginine and tRNAArg remained unaltered . The position of this mutation and its effect on enzymatic properties suggest the implication of arginine 134 in ATP binding as well as in the activation catalytic process.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1441 - 6
Specific transcription of cloned Methanobacterium thermoautotrophicum transcription units by homologous RNA polymerase in vitro; Knaub S et al.; Specific in vitro transcription by partially purified RNA polymerase from Methanobacterium thermoautotrophicum of DNA sequences cloned in between the promoter and terminator regions of the methyl CoM reductase transcription unit of the same organism is described . The 5'-terminus of the product has been mapped . Deletion analyses of the promoter region show the limits of the sequences essential for the promoter function.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1407 - 13
Purification and characterization of the in vitro activity of I-Sce I, a novel and highly specific endonuclease encoded by a group I intron; Monteilhet C et al.; Group I intron encoded proteins represent a novel class of site specific double strand endonucleases . The endonuclease activity of this class of proteins has been first demonstrated in vivo for I-Sce I which is encoded by a mitochondrial intron of Saccharomyces cerevisiae . Assays using crude cell extracts have shown that I-Sce I can be used in vitro as a restriction endonuclease potentially useful for recombinant DNA technology owing to its large recognition sequence (18 nucleotides) . We report here the purification and the first detailed analysis of the in vitro activity and properties of I-Sce I.

J Biol Chem, 1990 Mar 25, 265(9), 5161 - 5
Hormone and DNA binding activity of a purified human thyroid hormone nuclear receptor expressed in Escherichia coli; Lin KH et al.; Using a T7 expression system, large amounts of the human placental c-erbA protein (h-TR beta 1) were expressed . From 1 liter of Escherichia coli culture, approximately 50-100 micrograms of purified h-TR beta 1 were obtained . Analysis of the binding data indicated that the purified h-TR beta 1 binds to 3,3',5-triiodo-L-thyronine (T3) with a Ka = 2.8 x 10(9) M-1 . It binds to 3,3',5-triiodo-L-thyropropionic acid, 3,3',5-triiodo-L-thyroacetic acid, D-T3, L-thyroxine (T4), and 3',5',3-triiodo-L-thyronine with 475, 120, 39, 7, and 0.1%, respectively, of the activity of L-T3 . This order of binding activity to T3 analogs is similar to that reported for the T3 nuclear receptor identified in tissues or cultured cells . Furthermore, the purified h-TR beta 1 binds to the T3 response element of the rat growth hormone gene . Thus, the purified h-TR beta 1 is active . To identify the hormone binding domain, the purified h-TR beta 1 was affinity labeled with underivatized {3',5'-125I}T4 . A partial digestion by trypsin yielded a 125I-labeled 25-kDa fragment which was identified to be the domain Phe240-Asp456 by amino acid sequencing . Thus, the purified h-TR beta 1 appears suitable for other structural and functional studies.

J Biol Chem, 1990 Mar 25, 265(9), 5110 - 2
Crystallization and preliminary X-ray analysis of beta-hydroxydecanoyl thiol ester dehydrase from Escherichia coli; Sharma A et al.; Escherichia coli beta-hydroxydecanoyl thiol ester dehydrase, a key enzyme for the biosynthesis of unsaturated fatty acids in E . coli, has been crystallized by the vapor diffusion method at pH 5.0-5.5 using 20% (w/v) polyethylene glycol (molecular weight 8000) as a precipitant . Two crystal forms have been characterized, and both diffract to at least 1.6 A . The orthorhombic crystals belong to space group P2(1)2(1)2(1), with cell constants of a = 68.4 A, b = 87.3 A, and c = 60.3 A . Monoclinic crystals are of space group C2, with a = 131.9 A, b = 71.5 A, c = 92.5 A, and beta = 103.5 degrees.

J Biol Chem, 1990 Mar 25, 265(9), 4953 - 7
Ca2(+)-dependent interactions between the C-helix of troponin-C and troponin-I . Photocross-linking and fluorescence studies using a recombinant troponin-C; Wang ZY et al.; We have used in vitro mutagenesis to synthesize in Escherichia coli a recombinant rabbit skeletal troponin-C (designated as TnC57) in which Cys-98 was replaced with leucine, and Ala-57 in the C-helix of the N-terminal domain was replaced with cysteine . TnC57 labeled with the bifunctional photocross-linker benzophenone-4-maleimide could be photocross-linked with troponin-I in both the binary complex with troponin-I and in the ternary complex with troponin-I and troponin-T . The fluorescence lifetime of TnC57 labeled with the probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine decreased from 13.2 +/- 0.1 to 11.8 +/- 0.1 ns when Ca2+ bound to the low affinity triggering sites . Complexation with either troponin-I or both troponin-I and troponin-T resulted in significant increases in this lifetime both in the absence and the presence of Ca2+ . In either the binary or the ternary complex, this lifetime increased from 15.5 to 18.0 ns upon Ca2+ binding to the low affinity sites . Complementary acrylamide-quenching studies yielded results that are consistent with the fluorescence lifetime results . Our results show that the C-helix of troponin-C interacts with troponin-I, in confirmation of recent zero-length cross-linking results (Leszyk, J., Grabarek, Z., Gergely, J., and Collins, J.H . (1990) Biochemistry 29, 299-304) . Moreover, they are in support of a model (Herzberg, O., Moult, J., and James, M.N.G . (1986) J . Biol . Chem . 261, 2638-2644) in which the binding of Ca2+ to the triggering sites in the N-terminal domain of troponin-C results in the movement of the B- and C-helices away from the central helix, thereby exposing a putative hydrophobic binding site for troponin-I.

J Biol Chem, 1990 Mar 25, 265(9), 4821 - 7
Photoaffinity labeling of the Klenow fragment with 8-azido-dATP; Rush J et al.; The photoaffinity compound 8-azido-dATP was used as a probe for the deoxyribonucleoside triphosphate-binding site of the large fragment of DNA polymerase I . Azido-dATP specifically modified a saturable binding site within the Klenow fragment, and each of the four natural deoxyribonucleoside triphosphate substrates competed with labeling at this site in proportion to its binding constant, as previously defined by equilibrium dialysis . Analysis of tryptic peptides after azido-dATP modification revealed five major cross-linking products, which apparently arose from five distinct photoadducts formed near Tyr-766.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1625 - 8
Prediction of oligonucleotide frequencies based upon dinucleotide frequencies obtained from the nearest neighbor analysis; Hong J; A statistical method of predicting hexanucleotide frequencies is presented . The method requires dinucleotide frequencies which can be readily obtained by nearest neighbor analysis . The frequencies of 64 hexanucleotides of E . coli were estimated and compared well with those predicted by a third order Markov chain.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1603 - 7
Cleavage at the twelve-base-pair sequence 5'-TCTAGATCTAGA-3' using M.Xbal (TCTAGm6A) methylation and DpnI (Gm6A/TC) cleavage; Patel Y et al.; The DNA methylase M.Xbal was isolated from an E . coli recombinant clone . We deduce that the enzyme methylates at the sequence 5'-TCTAGm6A-3' . In combination with the methylation-dependent restriction endonuclease, DpnI (5'-Gm6A/TC-3'), DNA cleavage occurs at the sequence 5'-TCTAGA/TCTAGA-3' . This twelve-base-pair site should occur once every 16,000,000 base pairs in a random sequence of DNA . The exceptional rarity of the M.XbaI/DpnI sequence makes it an ideal candidate for transpositional integration of a unique cleavage site into bacterial genomes . Retrotransposition into mammalian genomes is also an attractive possibility.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1457 - 64
Lysine 188 of the catabolite gene activator protein (CAP) plays no role in specificity at base pair 7 of the DNA half site; Ebright RH et al.; Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('model I,' Weber, I . and Steitz, T., Proc . Natl . Acad . Sci . USA, 81, 3973-3977, 1984; 'model II,' Ebright, et al., Proc . Natl . Acad . Sci . USA, 81, 7274-7278, 1984) . The most important difference between the two models involves Lys188 of CAP . Model I predicts that Lys188 of CAP makes a specificity determining contact with base pair 7 of the DNA half site . In contrast, model II predicts that Lys188 makes no contact with base pair 7 of the DNA half site . In the present work, we have used site-directed mutagenesis to replace Lys188 of CAP by Asn, an amino acid unable to make the putative contact . We have assessed the specificities of the following proteins, both in vitro and in vivo: wild-type CAP, {Asn188}CAP, {Val181}CAP, and {Val181;Asn188}CAP . The results indicate that Lys188 makes no contribution to specificity at base pair 7 of the DNA half site . We propose, contrary to model I, that Lys188 makes no contact with base pair 7 of the DNA half site.

J Biol Chem, 1990 Mar 25, 265(9), 5166 - 9
The Na,K-ATPase beta 2 subunit is expressed in rat brain and copurifies with Na,K-ATPase activity; Shyjan AW et al.; We have used a cloned fusion protein as antigen to generate an antiserum specific for the rat Na,K-ATPase beta 2 subunit . Utilizing this antiserum, we analyzed some of the structural features and tissue distribution of the beta 2 subunit . Treatment of a rat brain microsomal membrane fraction with N-glycanase F revealed that the beta 2 subunit is composed of an approximately 32 kDa core protein and at least two N-linked carbohydrate chains . The beta 2 subunit also was found to copurify with ouabain-inhibitable Na,K-ATPase activity from rat brain . Western blot analysis of rat tissue microsomes showed that beta 2 subunits were expressed in brain, pineal gland, and thymus . However, no beta 2 subunits were detected in kidney, heart, spleen, liver, mammary gland, or lung . These results suggest that the beta 2 subunit is a functional component of the rat brain Na,K-ATPase . The restricted tissue distribution of beta 2 subunits may reflect important differences in the functions of individual beta subunit isoforms.

J Biol Chem, 1990 Mar 25, 265(9), 4809 - 13
Transport of divalent cations with tetracycline as mediated by the transposon Tn10-encoded tetracycline resistance protein; Yamaguchi A et al.; Tetracycline uptake into inverted membrane vesicles from Tn10-bearing Escherichia coli cells required divalent cations . The degree of the stimulation of tetracycline uptake by various divalent cations showed the following decreasing order: Co2+ greater than Mn2+ greater than Mg2+ greater than Cd2+ greater than Ca2+ . This order is consistent with the increasing order of the dissociation constants for metal chelate complexes of tetracycline . The Hill constants for the tetracycline uptake rate with various divalent cation concentrations were one . These observations strongly suggested that a 1:1 complex of tetracycline and a divalent cation was transported by a tetracycline resistance protein . This notion was confirmed by our observations that 60Co2+ was actively taken up with tetracycline by the membrane vesicles prepared from resistant cells . In the absence of tetracycline, no uptake of 60Co2+ was observed . It is clear that the 60Co2+ uptake was mediated by the tetracycline resistance protein, because the membrane vesicles from tetracycline-sensitive cells did not show the uptake of 60Co2+ and tetracycline . The 60Co2+ uptake was inhibited in the presence of other divalent cations, without any significant effect on tetracycline uptake, indicating that these cations are also transported with tetracycline by the tetracycline resistance protein.

J Biol Chem, 1990 Mar 25, 265(9), 5043 - 8
H(+)-ATPase gamma subunit of Escherichia coli . Role of the conserved carboxyl-terminal region; Iwamoto A et al.; Cloned uncG genes (wild-type or in vitro mutagenized) for the Escherichia coli gamma subunit were introduced into the uncG mutant Gln-14----end), and the functions of the mutant subunits were studied . The F1's with Ala-283----end and Thr-277----end mutant gamma subunits had 63 and 14% of the wild-type ATPase activity, respectively, and mutants with these subunits showed reduced growth by oxidative phosphorylation, indicating that the 10 residues at the carboxyl terminus (286th residue) are important, but dispensable, for catalysis . On the other hand, F1 with a Gln-269----end gamma subunit was inactive . Replacement of conserved residues (Gln-269, Thr-273, or Glu-275) between Gln-269 and Leu-276 gave enzymes with significantly reduced ATPase activity (2-41% of that of the wild-type) and lower ATP-driven proton conduction . Thus these residues are required for the normal catalytic activity of F1, although they are not absolutely essential . Membranes with amino acid replacements (Thr-277----end, Gln-269----Leu, or Glu-275----Lys) and the frameshift mutation (downstream of Thr-277) had about 15% of the wild-type ATPase activity, but showed different degrees of ATP-dependent H+ translocation and growth yield by oxidative phosphorylation, suggesting that the gamma subunit, especially its carboxyl-terminal region, functions in coupling between catalysis and H+ translocation.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1465 - 73
Suppression of the negative effect of minor arginine codons on gene expression; preferential usage of minor codons within the first 25 codons of the Escherichia coli genes; Chen GF et al.; AGA and AGG codons for arginine are the least used codons in Escherichia coli, which are encoded by a rare tRNA, the product of the dnaY gene . We examined the positions of arginine residues encoded by AGA/AGG codons in 678 E . coli proteins . It was found that AGA/AGG codons appear much more frequently within the first 25 codons . This tendency becomes more significant in those proteins containing only one AGA or AGG codon . Other minor codons such as CUA, UCA, AGU, ACA, GGA, CCC and AUA are also found to be preferentially used within the first 25 codons . The effects of the AGG codon on gene expression were examined by inserting one to five AGG codons after the 10th codon from the initiation codon of the lacZ gene . The production of beta-galactosidase decreased as more AGG codons were inserted . With five AGG codons, the production of beta-galactosidase (Gal-AGG5) completely ceased after a mid-log phase of cell growth . After 22 hr induction of the lacZ gene, the overall production of Gal-AGG5 was 11% of the control production (no insertion of arginine codons) . When five CGU codons, the major arginine codon were inserted instead of AGG, the production of beta-galactosidase (Gal-CGU5) continued even after stationary phase and the overall production was 66% of the control . The negative effect of the AGG codons on the Gal-AGG5 production was found to be dependent upon the distance between the site of the AGG codons and the initiation codon . As the distance was increased by inserting extra sequences between the two codons, the production of Gal-AGG5 increased almost linearly up to 8 fold . From these results, we propose that the position of the minor codons in an mRNA plays an important role in the regulation of gene expression possibly by modulating the stability of the initiation complex for protein synthesis.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1351 - 9
Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine; Major GN et al.; DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA . The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT . We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT . Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-{3H-CH3}methylguanine-DNA substrate . One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E . coli) and dat (B . subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue . Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme . The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1) . The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).

J Biol Chem, 1990 Mar 25, 265(9), 5292 - 5
Two distinct cDNAs for human IMP dehydrogenase; Natsumeda Y et al.; IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, is a promising target in antileukemic chemotherapy . We have isolated two distinct cDNA clones (types I and II) encoding IMP dehydrogenase from a human spleen cDNA library . Both clones encode closely related proteins of 514 residues showing 84% sequence identity . Northern hybridization analyses of poly(A)+ RNA from human normal leukocytes and human ovarian tumors demonstrated a striking contrast in mRNA expression in that type I mRNA is the main species in normal leukocytes and type II predominates over type I in the tumor . This is the first report suggesting the existence of two distinct types of human IMP dehydrogenase molecular species which may have different sensitivities to the drugs targeted against IMP dehydrogenase.

J Biol Chem, 1990 Mar 25, 265(9), 5232 - 6
Plasminogen activation with single-chain urokinase-type plasminogen activator (scu-PA) . Studies with active site mutagenized plasminogen (Ser740----Ala) and plasmin-resistant scu-PA (Lys158----Glu); Lijnen HR et al.; The mechanism of the activation of plasminogen by single-chain urokinase-type plasminogen activator (single-chain u-PA, scu-PA) was studied using rscu-PA-Glu158, a recombinant plasmin-resistant mutant of human scu-PA obtained by site-specific mutagenesis of Lys158 to Glu, and rPlg-Ala740, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active-site Ser740 to Ala . Conversion of 125I-labeled single-chain plasminogen to two-chain plasmin was quantitated on reduced sodium dodecyl sulfate-gel electrophoresis combined with autoradiography and radioisotope counting of gels bands . The efficiencies of both rscu-PA-Glu158 and rscu-PA for the activation of rPlg-Ala740 and of natural plasminogen were comparable and were 250-500-fold lower than that of recombinant two-chain u-PA (rtcu-PA) for rscu-PA-Glu158 and 100-200-fold lower for rscu-PA . Pretreatment of rscu-PA-Glu158 or rscu-PA with excess alpha 2-antiplasmin, which efficiently neutralizes all contaminating rtcu-PA, did not significantly reduce the catalytic efficiency of these single-chain moieties, indicating that they have a low but significant intrinsic plasminogen activating potential . The low intrinsic catalytic efficiency of rscu-PA for the conversion of plasminogen to plasmin may be sufficient to generate trace amounts of plasmin, which may regulate plasminogen activation by converting poorly active rscu-PA to very active rtcu-PA.

Science, 1990 Mar 23, 247(4949 Pt 1), 1461 - 5
Engineering human prolactin to bind to the human growth hormone receptor; Cunningham BC et al.; A strategy of iterative site-directed mutagenesis and binding analysis was used to incorporate the receptor-binding determinants from human growth hormone (hGH) into the nonbinding homolog, human prolactin (hPRL) . The complementary DNA for hPRL was cloned, expressed in Escherichia coli, and mutated to introduce sequentially those substitutions from hGH that were predicted by alanine-scanning mutagenesis and other studies to be most critical for binding to the hGH receptor from human liver . After seven rounds of site-specific mutagenesis, a variant of hPRL was obtained containing eight mutations with an association constant for the hGH receptor that was increased more than 10,000-fold . This hPRL variant binds one-sixth as strongly as wild-type hGH, but shares only 26 percent overall sequence identity with hGH . These studies show the feasibility of recruiting receptor-binding properties from distantly related and functionally divergent hormones and show that a detailed functional database can be used to guide the design of a protein-protein interface in the absence of direct structural information.

Science, 1990 Mar 23, 247(4949 Pt 1), 1465 - 8
Direct gene transfer into mouse muscle in vivo; Wolff JA et al.; RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo . Protein expression was readily detected in all cases, and no special delivery system was required for these effects . The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions . In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector . After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.

Biochemistry, 1990 Mar 20, 29(11), 2831 - 41
Use of a site-directed triple mutant to trap intermediates: demonstration that the flavin C(4a)-thiol adduct and reduced flavin are kinetically competent intermediates in mercuric ion reductase; Miller SM et al.; A mutant form of mercuric reductase, which has three of its four catalytically essential cysteine residues replaced by alanines (ACAA: Ala135Cys140Ala558Ala559), has been constructed and used for mechanistic investigations . With disruption of the Hg(II) binding site, the mutant enzyme is devoid of Hg(II) reductase activity . However, it appears to fold properly since it binds FAD normally and exhibits very tight binding of pyridine nucleotides as is seen with the wild-type enzyme . This mutant enzyme allows quantitative accumulation of two species thought to function as intermediates in the catalytic sequence of the flavoprotein disulfide reductase family of enzymes . NADPH reduces the flavin in this mutant, and a stabilized E-FADH- form accumulates . The second intermediate is a flavin C(4a)-Cys140 thiol adduct, which is quantitatively accumulated by reaction of oxidized ACAA enzyme with NADP+ . The conversion of the Cys135-Cys140 disulfide in wild-type enzyme to the monothiol Cys140 in ACAA and the elevated pKa of Cys140 (6.7 vs 5.0 in wild type) have permitted detection of these intermediates at low pH (5.0) . The rates of formation of E-FADH- and the breakdown of the flavin C(4a)-thiol adduct have been measured and indicate that both intermediates are kinetically competent for both the reductive half-reaction and turnover by wild-type enzyme . These results validate the general proposal that electrons flow from NADPH to FADH- to C(4a)-thiol adduct to the FAD/dithiol form that accumulates as the EH2 form in the reductive half-reaction for this class of enzymes.

Biochemistry, 1990 Mar 20, 29(11), 2824 - 30
Oxalyl hydroxamates as reaction-intermediate analogues for ketol-acid reductoisomerase; Aulabaugh A et al.; N-Hydroxy-N-isopropyloxamate (IpOHA) is an exceptionally potent inhibitor of the Escherichia coli ketol-acid reductoisomerase . In the presence of Mg2+ or Mn2+, IpOHA inhibits the enzyme in a time-dependent manner, forming a nearly irreversible complex . Nucleotide, which is essential for catalysis, greatly enhances the binding of IpOHA by the reductoisomerase, with NADPH (normally present during the enzyme's rearrangement step, i.e., conversion of a beta-keto acid into an alpha-keto acid, in either the forward or reverse physiological reactions) being more effective than NADP . In the presence of Mg2+ and NADPH, IpOHA appears to bind to the enzyme in a two-step mechanism, with an initial inhibition constant of 160 nM and a maximum rate of formation of the tight, slowly reversible complex of 0.57 min-1 (values that give an association rate of IpOHA, at low concentration, of 5.9 X 10(4) M-1 s-1) . The rate of exchange of {14C}IpOHA from an enzyme-{14C}IpOHA-Mg2(+)-NADPH complex with exogenous, unlabeled IpOHA has a half-time of 6 days (150 h) . This dissociation rate (1.3 X 10(-6) s-1) and the association rate determined by inactivation kinetics define an overall dissociation constant of 22 pM . By contrast, in the presence of Mn2+ and NADPH, the corresponding association and dissociation rates for IpOHA are 8.2 X 10(4) M-1 s-1 and 3.2 X 10(-6) s-1 (half-time = 2.5 days), respectively, which define an overall dissociation constant of 38 pM . In the presence of NADP or in the absence of nucleotide (both in the presence of Mg2+), the enzyme-IpOHA complex is far more labile, with dissociation half-times of 28 and 2 h, respectively . In the absence of Mg2+ or Mn2+, IpOHA does not exhibit time-dependent inhibition of the reductoisomerase.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Mar 20, 29(11), 2703 - 13
Active site of mercuric reductase resides at the subunit interface and requires Cys135 and Cys140 from one subunit and Cys558 and Cys559 from the adjacent subunit: evidence from in vivo and in vitro heterodimer formation; Distefano MD et al.; Mercuric reductase catalyzes the two-electron reduction of Hg(II) to Hg(0) using NADPH as the reductant; this reaction constitutes the molecular basis for detoxification of Hg(II) by bacteria . The enzyme is an alpha 2 homodimer and possesses two pairs of cysteine residues, Cys135 and Cys140 (redox-active pair) and Cys558 and Cys559 (C-terminal pair), which are known to be essential for catalysis . In the present study, we have obtained evidence for an intersubunit active site, consisting of a redox-active cysteine pair from one subunit and a C-terminal pair from the adjacent subunit, by reconstituting catalytic activity both in vivo and in vitro starting with two inactive, mutant enzymes, Ala135Ala140Cys558Cys559 (AACC) and Cys135Cys140Ala558Ala559 (CCAA) . Genetic complementation studies were used to show that coexpression of AACC and CCAA in the same cell yielded an HgR phenotype, some 10(4)-fold more resistant than cells expressing only one mutant . Purification and catalytic characterization of a similarly coexpressed protein mixture showed the mixture to have activity levels ca . 25% those of wild type; this is the same as that statistically anticipated for a CCAA-AACC heterodimeric/homodimeric mixture with only one functional active site per heterodimer . Actual physical evidence for the formation of active mutant heterodimers was obtained by chaotrope-induced subunit interchange of inactive pure CCAA and AACC homodimers in vitro followed by electrophoretic separation of heterodimers from homodimers . Taken together, these data provide compelling evidence that the active site in mercuric reductase resides at the subunit interface and contains cysteine residues originating from separate polypeptide chains.

J Mol Biol, 1990 Mar 20, 212(2), 253 - 7
Crystallization of the globular domain of histone H5; Graziano V et al.; The globular domain of histone H1/H5 binds to the nucleosome and is crucial for the formation of chromatin higher order structure . We have expressed in Escherichia coli a gene that codes for the globular domain of H5 . The protein produced in E . coli is functional in nucleosome binding assays . We have obtained crystals of the protein that diffract to beyond 2.5 A (1 A = 0.1 nm) resolution . The crystals are orthorhombic with unit cell dimensions of a = 80.1 A, b = 67.5 A and c = 38.0 A.

J Mol Biol, 1990 Mar 20, 212(2), 247 - 52
Adenovirus serotype 3 fibre protein is expressed as a trimer in Escherichia coli; Albiges-Rizo C et al.; The adenovirus serotype 3 (Ad3) fibre has been expressed in Escherichia coli as an insoluble protein . The protein was solubilized by extraction with urea . Slow removal of urea during the purification procedure resulted in a soluble Ad3 fibre preparation . Polyacrylamide gel analysis of the purified fibre protein, as well as cross-linking experiments performed on cellular debris of expressing cells, suggest that the recombinant Ad3 fibre self-assembles as a trimer from identical polypeptide chains . Gel filtration gave the same exclusion volume for the purified recombinant fibre and for the native fibre in the protein mixture extracted from the Ad3-infected cells . The recombinant fibre was partially resistant to proteolytic degradation, suggesting a folded structure.

J Mol Biol, 1990 Mar 20, 212(2), 241 - 5
Localization of the release factor-2 binding site on 70 S ribosomes by immuno-electron microscopy; Kastner B et al.; In protein synthesis Escherichia coli release factor-2 binds to 70 S ribosomes when the termination codon UAA or UGA appears at the decoding site . The weak interaction between factor and ribosome has been stabilized in vitro by chemical cross-linking . Factor so bound can still be recognized by a specific antibody to release factor-2 . Examination of the resulting immuno-complexes by electron microscopy revealed 70 S ribosomes in different projection forms, and the occasional dissociated subunit labelled with antibody . The antibody-binding site was localized on previously characterized 70 S projection forms, and its three-dimensional localization on the 70 S model established . The release factor-2-binding site was found to be positioned at the ribosomal subunit interface, comprising the stalk-protuberance region of the large subunit and the head-neck region of the concave side of the small subunit.

Science, 1990 Mar 16, 247(4948), 1333 - 5
Molecular cloning of the Bombyx mori prothoracicotropic hormone; Kawakami A et al.; Prothoracicotropic hormone (PTTH), a brain secretory polypeptide of insects, stimulates the prothoracic glands to produce and release ecdysone, the steroid essential to insect development . The complementary DNAs encoding PTTH of the silkmoth Bombyx mori were cloned and characterized, and the complete amino acid sequence was deduced . The data indicated that PTTH is first synthesized as a 224-amino acid polypeptide precursor containing three proteolytic cleavage signals . The carboxyl-terminal component (109 amino acids) that follows the last cleavage signal represents one PTTH subunit . Two PTTH subunits are linked together by disulfide bonds, before or after cleavage from prepro-PTTH, to form a homodimeric PTTH . When introduced into Escherichia coli cells, the complementary DNA directed the expression of an active substance that was functionally indistinguishable from natural PTTH . In situ hybridization showed the localization of the prepro-PTTH mRNA to two dorsolateral neurosecretory cells of the Bombyx brain.

Biochem Biophys Res Commun, 1990 Mar 16, 167(2), 554 - 60
Substitutions at the P2' site of gag p17-p24 affect cleavage efficiency by HIV-1 protease; Margolin N et al.; Heptapeptide substrates containing a single amino acid substitution at the p2' position of the gag p17-p24 junction (S-Q-N-Y-P-X-V where X = G, A, L, I, F, and W) were compared as substrates for HIV-1 protease . Binding of the Ile-, Leu-, and Ala- containing peptides was about equal although hydrolysis was 20-fold lower for the Ala and Leu peptides compared to Ile . Insertion of Gly or Phe at the p2' position resulted in significantly lower cleavage of the peptide while a Trp-containing peptide was not cleaved . These data suggest that a relatively small hydrophobic amino acid is important for hydrolysis and binding at this site . Structure-activity studies such as those described here may be useful in the design of specific inhibitors for HIV-1 protease.

Biochem Biophys Res Commun, 1990 Mar 16, 167(2), 504 - 6
Polymerase chain reaction (PCR) amplification with a single specific primer; Kalman M et al.; A method is described for amplification of DNA fragments flanking a single known sequence that is sufficiently long to enable synthesis of a functional primer in polymerase chain reactions.

Biochem Biophys Res Commun, 1990 Mar 16, 167(2), 407 - 12
Effects of replacement of tryptophan-140 by phenylalanine or glycine on the function of Escherichia coli aspartate aminotransferase; Hayashi H et al.; Trp140 of E . coli aspartate aminotransferase has been converted to Phe or Gly by site-directed mutagenesis . As compared to the wild-type enzyme, either of the mutant enzymes showed 10- to 100-fold increase in Km's for natural dicarboxylic substrates, but did not show appreciable changes in Km's for aromatic substrates . Teh kcat values for dicarboxylic and aromatic substrates were greatly decreased by {Trp140----Gly} mutation, but were decreased to lesser extents by {Trp140----Phe} mutation . These findings suggested that N(1) of Trp140 may not be essential for catalysis, but may be partly involved in the binding of the distal carboxylate group of the dicarboxylic substrates.

Biochem Biophys Res Commun, 1990 Mar 16, 167(2), 716 - 21
Protease-specificity of Kunitz inhibitor domain of Alzheimer's disease amyloid protein precursor; Kido H et al.; The putative inhibitor domain of Alzheimer's disease amyloid protein precursor was purified from E . coli containing a synthetic gene encoding the Kunitz domain . The purified protein (A4 inhibitor) inhibited the activity of trypsin, forming a 1:1 molar complex with the enzyme . It also strongly inhibited plasmin (Ki = 7.5 x 10(-11) M) from human serum and tryptase (Ki = 2.2 x 10(-10) M) from rat mast cells (tryptase M) . In addition, it inhibited rat pancreatic trypsin, alpha-chymotrypsin and kallikrein and human serum kallikrein, but did not inhibit rat chymase, pancreatic elastase, alpha-thrombin, urokinase, papain or cathepsin B.

J Biol Chem, 1990 Mar 15, 265(8), 4711 - 7
The gene encoding the phosphatidylinositol transfer protein is essential for cell growth; Aitken JF et al.; Phosphatidylinositol transfer proteins (PI-TPs) catalyze the transfer of phosphatidylinositol and phosphatidylcholine between membranes in vitro . However, the in vivo function of these proteins is unknown . In this paper, we use a combined biochemical and genetic approach to determine the importance of PI-TP in vivo . An oligonucleotide based on the amino-terminal sequence of the PI-TP from Saccharomyces cerevisiae was used to screen a yeast genomic library for the gene encoding PI-TP (PIT1 gene) . Positive clones showed overproduction of transfer activities and transfer protein in the 100,000 x g supernatants . The 5' terminus of the PIT1 gene correlates with the predicted codons for residues 3-30 of the determined protein sequence . A putative intron is located between the codons for residues 2 and 3 of the protein sequence . The codons for the first two amino acids of the protein and the presumptive initiation methionine precede the intron . Tetrad analysis of a heterozygous diploid (PIT1/pit1::LEU2) revealed that the PIT1 gene is essential for cell growth . Nonviable spores could be rescued by transformation of the above diploid prior to sporulation, with a plasmid-borne copy of the wild type gene.

J Biol Chem, 1990 Mar 15, 265(8), 4527 - 33
Functional analysis of Arg-308 mutants of Flp recombinase . Possible role of Arg-308 in coupling substrate binding to catalysis; Parsons RL et al.; The arginine residue at position 308 in the Flp recombinase corresponds to the only invariant arginine within the Int family of recombinases . Alterations of this residue result in Flp variants that retain substrate recognition, but form weaker protein-DNA complexes than wild type Flp . Furthermore, their DNA cleavage activity is significantly diminished . A conservative change of R308K results in a functional Flp variant; however, this protein has a lowered temperature optimum for recombination . The Arg-308 mutants can be stabilized on the DNA substrate through cooperativity with a partner Flp mutant that is tight binding . Thus, interactions between Flp monomers must be a relevant feature of the normal recombination reaction.

J Biol Chem, 1990 Mar 15, 265(8), 4278 - 83
Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine; Som S et al.; Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct . This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either {methyl-3H}AdoMet or {35S}AdoMet . The extent of photolabeling is low . Under optimal conditions, 4.5 pmol of {3H}AdoMet is incorporated into 100 pmol of enzyme . Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold . However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid . A catalytically active conformation of the enzyme is needed for AdoMet photolabeling . Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation . Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation . The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S . (1986) Nucleic Acids Res . 14, 4543-4556) . The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor . These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule . The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.

J Biol Chem, 1990 Mar 15, 265(8), 4233 - 9
Fatty acid monooxygenation by cytochrome P-450BM-3; Boddupalli SS et al.; Cytochrome P-450BM-3 is a catalytically self-sufficient enzyme which monooxygenates saturated and unsaturated fatty acids, alcohols, and amides . The protein has two domains: one which contains heme and is P-450-like and the other which contains FAD and FMN and is P-450 reductase-like . Both domains are on a single polypeptide chain . Utilizing a plasmid containing the gene encoding P-450BM-3, we have transformed the Escherichia coli strain DH5 alpha . This clone overexpresses P-450BM-3 to make approximately 20% of the soluble protein of this organism under optimal conditions . P-450BM-3 can be purified to homogeneity from the soluble fraction of the protein of these cells with a recovery of 50% making this cell line an excellent source of this important enzyme . Purified preparations of P-450BM-3 hydroxylate palmitic acid at a rate of 1600 mol/min/mol of heme at 25 degrees C . The stoichiometry of NADPH to oxygen utilized was 1 for all conditions; however, the ratio of oxygen or NADPH utilized per molecule of fatty acid substrate metabolized was different for different homologs of saturated fatty acids, when low concentrations (less than 100 microM) of substrate were used . Lauric and myristic acids were metabolized to two hydroxylated products, irrespective of the initial concentration of fatty acid in the reaction mixture, and the ratio of oxygen consumed to fatty acid hydroxylated was 1 . High concentrations of palmitic acid (greater than 200 microM) led to the formation of three polar metabolites and a stoichiometry of 1:1 was observed for oxygen and palmitic acid utilization . These results indicate that a single hydroxyl group was inserted into each of these molecules . Lower concentrations (less than 50 microM) of palmitic acid were metabolized to additional polar metabolites, and the ratio of oxygen consumed to fatty acid substrate consumed approximated 3:1 . These results can be explained best by a hypothesis that the initial hydroxylated compounds, which accumulate during the oxidation of palmitic acid by P-450BM-3, can be further oxidized by this enzyme to polyhydroxy- or hydroxy-ketone products.

FEMS Microbiol Lett, 1990 Mar 15, 56(3), 279 - 84
A single protein of 110 kDa is associated with the multinucleating and necrotizing activity coded by the Vir plasmid of Escherichia coli; Oswald E et al.; A lethal and necrotic factor which causes cell multinucleation in HeLa cell cultures has previously been shown to be coded by the Vir plasmid of Escherichia coli . Using an absorbed rabbit antiserum which neutralized the Vir toxic properties, we have compared the SDS-PAGE immunoblots from laboratory and field strains which either produce or do not produce Vir toxicity . A single band of 110 kDa was found to be specifically associated with vir toxicity in E . coli strains . This antiserum also recognized the 115 kDa protein band which was previously identified as the cytotoxic necretozing factor (CNF) of certain E . coli strains . These results suggest that the toxin coded by the Vir plasmid is a protein of 110 kDa distinct from, but immunologically related to CNF.

Gene, 1990 Mar 15, 87(2), 273 - 7
Nucleotide sequence of the zucchini yellow mosaic virus capsid-encoding gene and its expression in Escherichia coli; Gal-On A et al.; Zucchini yellow mosaic virus (ZYMV) RNA was purified and used as a template for the synthesis of cDNA . A partial restriction map covering 9.4 kb of the ZYMV genome was constructed from three clones designated ZYKS-22, ZYKS-16 and ZYKS-3 . Sequencing the 3'-end region of the ZYMV genome indicates the presence of (A)48 chain . This is followed by an untranslated region of 210 nucleotides (nt) and a coding region of 837 nt corresponding to the putative virus coat protein (Cp) gene (cp) . The predicted amino acid (aa) sequence of Cp derived from the cDNA showed about 50% to 62% homology with the known aa sequence for Cp of six other potyviruses . A construct of the putative cp was subcloned in frame with the lacZp gene promoter in a Bluescript plasmid and expressed in Escherichia coli cells . The fusion polypeptides (34 and 41 kDa), positively reacted in Western blots with an antiserum prepared against the native virus Cp.

Clin Chim Acta, 1990 Mar 15, 187(3), 265 - 72
Sensitive enzyme immunoassay for human aldolase A; Okajima K et al.; A sensitive sandwich-type enzyme immunoassay for the aldolase isozyme, A4, was developed using purified antibodies specific to the A subunit of aldolase . The antibodies were raised in sheep being immunized with purified aldolase A4 and then purified by immunoaffinity chromatography on a column of aldolase A4-coupled Sepharose . The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody F(ab')2 fragments labeled with beta-D-galactosidase from Escherichia coli . The assay was sensitive enough to detect 10 pg/tube of aldolase A4 . The assay was specific to the A subunit of aldolase (aldolase A) . It cross-reacted about 40% to aldolase A3C, 7% to A2C2 and 0.3% to AC3, but not cross-reacted with C4 nor B4 . Coefficients of variation in intra- and inter-assay were less than 16% . Serum aldolase A levels were determined in healthy adults, which were about 200 ng/ml . The distribution and concentrations of immunoreactive aldolase A in various human tissues were also determined . High concentrations of aldolase A were found in skeletal muscle, heart muscle, cerebrum and lymphatic tissue.

J Am Vet Med Assoc, 1990 Mar 15, 196(6), 897 - 901
Attaching and effacing Escherichia coli infection as a cause of diarrhea in young calves; Janke BH et al.; A form of enteric Escherichia coli infection was identified in 60 calves from 59 farming operations . The E coli responsible for these infections principally colonized the colon, inducing a distinctive lesion described as attaching and effacing . Hemorrhagic enterocolitis or blood in the feces was observed on 40% of the farms . Of affected calves, 86.6% were dairy calves (average age, 11.8 days) . Forty-four calves were infected concurrently with other enteropathogens (cryptosporidia, rotavirus, coronavirus, enterotoxigenic E coli, bovine viral diarrhea virus, coccidia) . Verotoxin-producing E coli was recovered from 31 calves; 8 were serotype O111:NM isolates, 3 were serotype O5:NM, and 1 was serotype O26:NM.

Blood, 1990 Mar 15, 75(6), 1290 - 5
Biologic and biochemic properties of recombinant platelet factor 4 demonstrate identity with the native protein; Park KS et al.; Platelet factor 4 (PF4) is a 70 amino acid protein released from the alpha-granules of platelets after activation . The exact biologic function of this protein is unknown . We have constructed an expression vector for recombinant PF4 (rPF4) in the T7-based promoter vector pT7-7 to better study the relationship between PF4 structure and function . The protein was expressed in Escherichia coli and purified to homogeneity by heparin-agarose affinity chromatography and reverse-phase high-performance liquid chromatography . Purity of protein was confirmed by immunoblot analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which resulted in a single component with a molecular weight of 8,000 daltons . The amino acid composition and sequence of the N-terminal 20 residues showed that rPF4 is identical to PF4 prepared from human platelets (hPF4), except for an N-terminal initiating methionine residue . Immunoblots revealed that rPF4 and hPF4 bound polyclonal anti-hPF4 equally well, while chemotaxis experiments demonstrated similar potencies as neutrophil attractants . Dose-dependent neutrophil chemotactic responses and competitive studies with polyclonal anti-hPF4 antiserum further demonstrate similar chemotactic properties of the two PF4 species . In conclusion, our data show that this recombinant protein and the native protein appears to have similar immunologic, heparin-binding, and chemotactic properties . The chemotactic properties of hPF4 appear to be entirely intrinsic to the protein and not due, in part, to any contaminating protein . Furthermore, our expression vector should prove useful for the construction of recombinant forms of PF4 to investigate structure/function relationships of this biologically important protein.

Biochim Biophys Acta, 1990 Mar 15, 1016(1), 63 - 70
Orientation of subunit c of the ATP synthase of Escherichia coli--a study with peptide-specific antibodies; Hensel M et al.; Antibodies were raised against a peptide of subunit c of the ATP synthase from Escherichia coli obtained by cleavage with cyanogen bromide . This peptide comprises the amino acid residues Gly-18 to Met-57 and contains the highly conserved, hydrophilic stretch of subunit c . Several conformation-specific populations of antibodies recognized this region both in isolated subunit c and in the intact F0 complex . In antibody binding studies with membrane vesicles of different orientations, recognition occurred only after incubation with everted membrane vesicles, independent of the presence or absence of F1, although a higher membrane protein concentration was necessary to observe the same antibody binding in the presence of the F1 part . From these results we conclude that the hydrophilic region of subunit c is exposed to the cytoplasmic side of the membrane.

FEMS Microbiol Lett, 1990 Mar 15, 56(3), 341 - 6
The effect of the minor groove binding agent DAPI (4,6-diamidino-2-phenyl-indole) on DNA-directed enzymes: an attempt to explain inhibition of plasmid expression in Escherichia coli {corrected}; Parolin C et al.; The activity of DAPI, on a number of DNA-directed enzymes involved in DNA topology, transcription, replication and repair, is reported in this paper . DAPI was always more inhibitory than ethidium bromide, in particular against RNA polymerase and DNA ligase, which seemed to be specifically affected . While the effect on RNA polymerase is likely due to a preferential occupancy of the promoter region, that on DNA ligase could rely upon a mechanism of steric hindrance in the minor groove . These phenomena, independently from an alteration of the tertiary structure of DNA by the ligand, can account for the previously reported inhibition of plasmid expression in Escherichia coli.

FEMS Microbiol Lett, 1990 Mar 15, 56(3), 307 - 11
Cyclic AMP stimulates transcription of the structural gene of the outer-membrane protein OmpA of Escherichia coli; Gibert I et al.; To analyze the effect of cyclic AMP on the expression of the ompA gene of Escherichia coli, encoding the outer-membrane protein OmpA, a fusion between this gene and the lacZ gene was constructed in vitro by using a promoter-probe plasmid . The results obtained indicated that the presence of glucose in the culture medium decreased the transcription of the ompA gene . Likewise, cya and crp mutants exhibited lower levels of ompA gene expression than the wild-type strain . Furthermore, the addition of cyclic AMP increased the expression of the ompA gene in both cya and wild-type strains but not in a crp mutant . All these data show that the cyclic AMP receptor protein-cyclic AMP complex positively modulates ompA transcription in E . coli K-12.

FEMS Microbiol Lett, 1990 Mar 15, 56(3), 295 - 9
Osmoregulatory expression of the ompC gene in Escherichia coli K-12; IS1 insertion in the upstream regulatory region results in constitutive activation of the promoter; Ozawa Y et al.; A novel type of osmoregulatory mutant of Escherichia coli K-12 exhibiting constitutive expression of the ompC gene was isolated and characterized at the molecular level . In this particular mutant (cec; constitutive expression of OmpC), an insertion sequence (IS-1) was found to be located at right upstream of the regulatory sequence for the ompC promoter . We demonstrate that the IS1 insertion observed in the cec mutant does not provide the ompC gene with an artificial promoter, but rather perturbs normal regulation of the ompC promoter, which is mediated by the regulatory gene, ompR.

Gene, 1990 Mar 15, 87(2), 243 - 8
High-level synthesis of recombinant HIV-1 protease and the recovery of active enzyme from inclusion bodies; Cheng YS et al.; A complete chemical synthesis and assembly of genes for the production of human immunodeficiency virus type-I protease (HIV-PR) and its precursors are described . The T7 expression system was used to produce high levels of active HIV-PR and its precursors in Escherichia coli inclusion bodies . The gene encoding the open reading frames of HIV-PR was expressed in E . coli as a 10-kDa protein, while the genes encoding HIV-PR precursors were expressed as larger proteins, which were partially processed in E . coli to the 10-kDa form . These processing events are autoproteolytic, since a single-base mutation, changing the active-site aspartic acid to glycine, completely abolished the conversion . HIV-PR can be released with 8 M urea from washed cellular inclusion bodies, resulting in a preparation with few bacterial host proteins . After refolding, this preparation contains no nonspecific protease or peptidase activities . The recombinant HIV-PR isolated from inclusion bodies cleaves HIV-PR substrates specifically with a specific activity comparable to column-purified HIV-PR.

J Biol Chem, 1990 Mar 15, 265(8), 4646 - 51
Assembly of an in vitro synthesized Escherichia coli outer membrane porin into its stable trimeric configuration; de Cock H et al.; The folding of in vitro synthesized outer membrane protein PhoE of Escherichia coli was studied in immunoprecipitation experiments with monoclonal antibodies which recognize cell surface-exposed conformational epitopes . The signal sequence appears to interfere with the formation of these conformational epitopes, since a mutant PhoE protein which lacks the majority of the signal peptide could be precipitated four times better than the wild type precursor . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitated PhoE protein revealed that part of the immunoprecipitated PhoE was present as a heat-modifiable form of the protein which migrated faster in the gels than the completely denatured protein . This form of the protein probably represents a folded monomer which might be an intermediate in the assembly of the protein . Outer membrane vesicles were required to induce the formation of small amounts of heat-stable trimers, the functional form of the protein in vivo.

J Biol Chem, 1990 Mar 15, 265(8), 4552 - 9
The role of cysteine 41 in the enzymatic activities of the pertussis toxin S1 subunit as investigated by site-directed mutagenesis; Locht C et al.; The S1 subunit (Mr 28,000) of pertussis toxin expresses thiol-dependent enzymatic ADP-ribosyltransferase and NAD-glycohydrolase activities . Site-directed mutagenesis experiments were performed on the codon for Cys-41 of this subunit to investigate the role of this residue in both enzymatic activities . Deletion of Cys-41 caused a decrease in both activities below detectable levels, whereas replacement of this residue by serine, glycine, proline, or asparagine only slightly reduced the activities . The enzymatic activities of these mutants were thiol-independent . The deletion of Ser-40, adjacent to Cys-41, again caused reduction of the enzymatic activities to undetectable levels . Steady-state kinetic experiments showed that the kcat of the mutant protein in which Cys-41 was replaced by glycine was nearly identical to the kcat of the parent version . However, the Km for NAD of the mutant was significantly higher relative to that of the wild type version . These results indicate that the side-chain of Cys-41 is not essential for enzymatic activities and that Cys-41 is not involved in the rate of catalysis but is probably located at or close to the NAD-binding site . The introduction of a negative charge at position 41 through the replacement of Cys-41 by either aspartate or glutamate reduced the enzymatic activities to very low but measurable levels, suggesting a charge-charge repulsive interaction between these residues and possibly one or both of the phosphates of NAD . Cys-41 may therefore be located close to the phosphate subsite of the NAD-binding site.

J Biol Chem, 1990 Mar 15, 265(8), 4483 - 91
Granulocyte-macrophage colony stimulating factor from human lymphocytes . The effect of glycosylation on receptor binding and biological activity; Cebon J et al.; Native human granulocyte-macrophage colony stimulating factor (hGM-CSF) has previously been purified using methods which typically required several sequential chromatographic steps and only yielded small amounts of hGM-CSF . We have purified and characterized hGM-CSF using monoclonal antibodies raised against bacterially synthesized hGM-CSF . Activated donor T-lymphocytes grown in interleukin-2 and then reactivated with phytohemagglutinin produce several forms of hGM-CSF which can be purified using immunoaffinity absorption followed by reversed phase high performance liquid chromatography . The purified hGM-CSF consisted of at least nine species ranging in molecular weight (Mr) from 14,500 to 32,000 . The higher Mr forms contained one or two N-linked carbohydrate moieties and were more acidic by two-dimensional Western blot analysis, consistent with increasing sialation . N-terminal sequence analysis of high and low molecular weight hGM-CSF fractions corresponded to that predicted by the cDNA sequence . Using the AML 193 {3H}thymidine incorporation assay the specific activity of the heavily glycosylated hGM-CSF was 1 x 10(8) units/mg compared with 6 x 10(8) units/mg for the non-glycosylated hGM-CSF produced by Escherichia coli . The different hGM-CSF forms induced neutrophil superoxide anion production by a variable amount depending on the extent of N-linked glycosylation . Receptor binding studies demonstrated lower receptor affinity for the heavily glycosylated form (KD = 820 pM) compared to less heavily glycosylated (KD = 78 pM) and non-glycosylated hGM-CSF produced by E . coli (KD = 30 pM) . These differences are due to differences in the kinetic association rate.

J Biol Chem, 1990 Mar 15, 265(8), 4461 - 71
Molecular biology of carbon-phosphorus bond cleavage . Cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli B; Chen CM et al.; Whereas bacteria such as Escherichia coli have been known for some time to cleave carbon-phosphorus (C-P) bonds in unactivated alkylphosphonates, the enzymes responsible for C-P lyase activity have resisted detection or purification . Genes from E . coli B that support growth on alkylphosphonates as the sole phosphorus source have now been cloned (B . L . Wanner and J . A . Boline, unpublished data) . Deletion analysis demonstrated that at least 13 kilobases of DNA information is required for E . coli to express the phosphonate utilization phenotype (Phn+) . The complete nucleotide sequence of 15,611 bases has been determined, and the gene structures were examined . Seventeen open reading frames (phnA to phnQ) were identified in one transcriptional direction and five open reading frames in the divergent direction . Sequence homology searches identify PhnC, PhnK, PhnL, and, possibly, PhnN proteins as members of nucleotide-binding proteins of the binding protein-dependent transport systems . Candidates for other membrane components and regulatory proteins are also identified . A Pho box-like promoter sequence is also found upstream of the gene cluster starting at phnA, which is consistent with the observation of phosphate regulation of the Phn+ response . Fourteen repetitive extragenic palindromic sequences are found in the phn DNA: 10 exist in the extragenic region between phnA and phnB, two between phnD and phnE, and two between phnK and phnL . An unusual finding is that one of the repetitive extragenic palindromic sequences actually overlaps with the reading frame of the phnE gene.

J Biol Chem, 1990 Mar 15, 265(8), 4374 - 81
sn-1,2-diacylglycerol kinase of Escherichia coli . Diacylglycerol analogues define specificity and mechanism; Walsh JP et al.; A detailed structure/function analysis of the substrate specificity of Escherichia coli sn-1,2-diacylglycerol kinase was performed with three goals in mind: (a) to define the substrate specificity; (b) to discover inhibitors; and (c) to elucidate the specificity of diacylglycerol-dependent inactivation . Forty-seven structural analogues of sn-1,2-diacylglycerol were prepared and examined as substrates, inhibitors, and irreversible inactivators of the enzyme using mixed micellar assay methods . Modification of the acyl chains or the sn-2 ester affected the apparent Km but had only small effects on Vm; modifications of the sn-1 ester, sn-3 methylene, or sn-3 hydroxyl had large effects on the apparent Vm and smaller effects on Km . Consistent with these observations, diacylglycerol analogues modified only in the acyl chains or sn-2 ester were not diacylglycerol kinase inhibitors, whereas analogues with substitutions of the sn-1 ester or sn-3 hydroxyl frequently caused inhibition . A hydrogen bond-donating group was required for an analogue to be a diacylglycerol kinase inhibitor . Studies of diacylglycerol kinase inactivation by the various analogues were consistent with the previous conclusion that this process involves an interaction of diacylglycerols with an enzyme conformation different from that active in catalysis (Walsh, J . P., and Bell, R . M . (1986) J . Biol . Chem . 261, 15062-15069) . Studies with a water-soluble diacylglycerol, sn-1,2-dibutyrylglycerol, allowed direct comparison of diacylglycerol kinase activity in mixed micelles with that in native membranes . The results are discussed in relation to the structural requirements of other diacylglycerol-dependent enzymes.

J Biol Chem, 1990 Mar 15, 265(8), 4254 - 60
Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli; Davidson AL et al.; Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system . We observed a 10-fold increase in the level of transport activity in assays with membrane vesicles when the three membrane-associated components of the transport system (the MalF, MalG, and MalK proteins) were overproduced . In addition, we have successfully reconstituted maltose transport activity in proteoliposome vesicles from solubilized proteins using a detergent dilution procedure . The addition of ATP as an energy source was sufficient to obtain transport, and this activity was dependent on the presence of maltose binding protein and was not seen in proteoliposomes prepared from a strain with a deletion of the maltose genes . We determined that hydrolysis of ATP was directly coupled to maltose uptake . In the majority of these experiments, an average of 1.4 mol of ATP was hydrolyzed for each mole of maltose accumulated . However, in the remaining experiments, ATP hydrolysis was observed to be much higher and averaged 17 mol of ATP hydrolyzed per mol of maltose transported . Possible explanations for a variable stoichiometry are discussed . These results provide strong evidence that it is the hydrolysis of ATP by a component of the transport complex that provides the energy required for active maltose transport.

J Biol Chem, 1990 Mar 15, 265(8), 4664 - 9
Mitochondrial ATP synthase . cDNA cloning, amino acid sequence, overexpression, and properties of the rat liver alpha subunit; Lee JH et al.; The predicted amino acid sequence of the alpha subunit of the rat liver mitochondrial ATP synthase has been obtained by sequencing a cDNA for the alpha subunit . Analysis of the sequence shows that it contains the A and B consensus sequences found in many nucleotide-binding proteins . Twelve amino acids of the rat liver alpha subunit differ from the sequence of the bovine heart alpha subunit; four of these involve differences in charge . The rat liver alpha subunit, from arginine 15 to the C-terminal proline 510, has been overexpressed in Escherichia coli using the alkaline phosphatase promoter (phoA) and leader peptide to direct the export of the expressed protein to the bacterial periplasm . By treating the cells with lysozyme, osmotic shock, and alkaline pH washes, the alpha subunit can be extracted in high yield (greater than 25 mg/liter) and in a high state of purity . The expressed alpha subunit remains soluble at pH 9.5 or greater and precipitates when treated with Mg2+ ions at low millimolar concentration . The bacterially expressed alpha subunit interacts with 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), resulting in a marked fluorescence enhancement upon binding . An enhancement of fluorescence is also observed upon the interaction of the alpha subunit with TNP-ADP . Preincubating the alpha subunit with 1.5 mM ATP significantly reduces the fluorescence enhancement seen with TNP-ATP . The alpha subunit binds TNP-ATP with an apparent Kd in the low micromolar range (1-5 microM) and binds TNP-ADP with an affinity at least 10-fold lower . This work shows that the rat liver alpha subunit can be overexpressed in E . coli to yield a large amount of functional protein . With the acquisition of the overexpressed alpha subunit, it is now possible to test the reconstitution of ATPase activity from a mixture of recombinant and rat liver-derived subunits and to test the formation of complexes by the overexpressed alpha and beta subunits of the rat liver F1-ATPase.

J Biol Chem, 1990 Mar 15, 265(8), 4402 - 10
Thermodynamic analyses of the catalytic pathway of F1-ATPase from Escherichia coli . Implications regarding the nature of energy coupling by F1-ATPases; al-Shawi MK et al.; Thermodynamic properties of 12 different F1-ATPase enzymes were analyzed in order to gain insights into the catalytic mechanism and the nature of energy coupling to delta mu H+ . The enzymes were normal soluble Escherichia coli F1, a group of nine beta-subunit mutant soluble E . coli F1 enzymes (G142S, K155Q, K155E, E181Q, E192Q, M209I, D242N, D242V, R246C), and both soluble and membrane-bound bovine heart mitochondrial F1 . Unisite activity was studied by use of Gibbs free energy diagrams, difference energy diagrams, and derivation of linear free energy relationships . This allowed construction of binding energy diagrams for both the unisite ATP hydrolysis and ATP synthesis reaction pathways, which were in agreement . The binding energy diagrams showed that the step of Pi binding is a major energy-requiring step in ATP synthesis, as is the step of ATP release . It is suggested that there are two major catalytic enzyme conformations, and ATP- and an ADP-binding conformation . The effects of the mutations on the rate-limiting steps of multisite as compared to unisite activity were correlated, suggesting a direct link between the rate-limiting steps of the two types of activity . Multisite activity was analyzed by Arrhenius plots and by study of relative promotion from unisite to multisite rate . Changes in binding energy due to mutation were seen to have direct effects on multisite catalysis . From all the data, a model is derived to describe the mechanism of ATP synthesis . ATP hydrolysis, and energy coupling to delta mu H+ in F1F0-ATPases.

J Biol Chem, 1990 Mar 15, 265(8), 4358 - 63
In vitro kinetic analysis of the role of the positive charge at the amino-terminal region of signal peptides in translocation of secretory protein across the cytoplasmic membrane in Escherichia coli; Sasaki S et al.; By using an in vitro system for the translocation of secretory proteins in Escherichia coli, detailed and quantitative studies were performed as to the function of the positively charged amino acid residues at the amino terminus of the signal peptide . Uncleavable OmpF-Lpp, a model secretory protein carrying an uncleavable signal peptide, and mutant proteins derived from it were used as translocation substrates . When the positive charge, +2 (LysArg) for the wild-type, was changed to 0, -1, or -2, little or no translocation was observed . The number of the positive charge was altered by introducing different numbers of Lys or Arg residues into the amino terminus . The rate of translocation was roughly proportional to this number, irrespective of whether the charged amino acid residues were Lys or Arg . When the amino-terminal LysArg was replaced by His residues, translocation took place more efficiently at pH 6.5 than pH 8.0, whereas that of the wild-type was about the same as the two pH values . We conclude that the signal peptide requires a positive charge at its amino-terminal region to function in the translocation reaction and that the rate of translocation is roughly proportional to the number of the positively charged group, irrespective of the amino acid species that donates the charge . Evidence suggesting that the positive charge is involved in the binding of precursor proteins to the membrane surface to initiate translocation is also presented.

FEMS Microbiol Lett, 1990 Mar 15, 56(3), 255 - 60
Structural and regulatory genes for coli surface associated antigen 4 (CS4) are encoded by separate plasmids in enterotoxigenic Escherichia coli strains of serotype 0.25.H42; Willshaw GA et al.; Two enterotoxigenic Escherichia coli strains of serotype 0.25.H42 that produced coli surface associated antigens CS4 and CS6 hybridized with a probe containing the cfaD sequence that regulates expression of colonization factor antigen CFA/I . Transformation of a cloned cfaD gene into some derivatives of the strains that were negative for CS4 and CS6 resulted in expression of CS4 but not CS6 . By hybridization the sequence that regulated CS4 production in the wild type 025 strains was located on a plasmid that also encoded the CS6 antigen . The structural genes for the CS4 antigen were on a separate plasmid . The 025 strains carried a third plasmid encoding enterotoxin production which was therefore unlinked to regulation sequences or genes encoding CS antigens.

J Biol Chem, 1990 Mar 15, 265(8), 4615 - 21
Identification of the amino acid residues involved in an active site of Escherichia coli ribonuclease H by site-directed mutagenesis; Kanaya S et al.; The amino acid residues essential for the catalytic activity of ribonuclease H (RNase H) from Escherichia coli (E . coli) were identified by site-directed mutagenesis . It has been proposed by computer analysis that E . coli RNase H has homologous amino acid sequence with the RNase H domains of various retroviral reverse transcriptases (RTs) (Johnson, M . S., McClure, M . A., Feng, D . F., Gray, J., and Doolittle, R . F . (1986) Proc . Natl . Acad . Sci . U . S . A . 83, 7648--7652) . Of the eight highly conserved residues examined, Asp10, Glu48, and Asp70 were found to be crucial for RNase H activity . Determination of the kinetic parameters for the mutated enzymes using the chemically synthesized nonanucleotide duplex as a substrate demonstrated that these residues are involved in the catalytic site rather than the substrate-binding site . These residues are fully conserved in the amino acid sequences of not only retroviral RTs but also hepadnaviral, plant viral and retrotransposon RTs . This strongly suggests that they are also involved in the active site of these RTs and RT related enzymes.

J Biol Chem, 1990 Mar 15, 265(8), 4515 - 26
Identification of Neurospora mitochondrial promoters and analysis of synthesis of the mitochondrial small rRNA in wild-type and the promoter mutant {poky}; Kubelik AR et al.; Using an in vitro transcription assay, we previously identified promoters in Neurospora mtDNA at the 5' ends of the genes encoding the mitochondrial (mt) small and large rRNAs and cob pre-mRNA . Here, we identified two additional promoters in mtDNA restriction fragment EcoRI-6, 3.8 and 5.5 kilobases upstream of the 5' end of the mt small rRNA . By comparing the two new promoters with the three identified previously, we derived a modified promoter consensus sequence (AT-rich)15-27TTAG(A/T)RR(G/T)(G/C)N(A/T) . The mt small rRNA in Neurospora is transcribed from at least two promoters, a major promoter at the 5' end of the small rRNA and one or both of the newly identified promoters in EcoRI-6 . The latter gives a series of putative pre-rRNAs that contain 5' end extensions of various sizes . The 5' ends of a number of these RNAs map at or near hairpin structures . The {poky} mutant, which is grossly deficient in the mt small rRNA, has a 4-base pair deletion in the major promoter at the 5' end of the mt small rRNA . The residual small rRNAs in {poky} appear to be synthesized via the upstream promoter(s), but are missing 37-44 nucleotides from their 5' ends, indicating either that pre-rRNAs are processed abnormally or that abnormal 5' RNA ends are unstable . The effect of the promoter mutation in {poky} on other transcripts suggests that the mt small rRNA is cotranscribed with downstream genes encoding tRNAs, coIII and ND6 . Seven nonallelic nuclear suppressors of {poky} result in increased concentrations of the mt small rRNA and pre-rRNAs, but do not restore the ability to synthesize small rRNAs having the correct 5' ends . The suppressor mutations could act by increasing transcription, processing, or stability of the mt small rRNA or its precursors . The suppressors provide a genetic approach for identifying components that affect transcription and processing of the mt small rRNA.

J Biol Chem, 1990 Mar 15, 265(8), 4273 - 7
Epitopes of monoclonal antibodies which inhibit ubiquinol oxidase activity of Escherichia coli cytochrome d complex localize functional domain; Dueweke TJ et al.; The aerobic respiratory chain of Escherichia coli contains two terminal oxidases: the cytochrome d complex and the cytochrome o complex . Each of these enzymes catalyzes the oxidation of ubiquinol-8 within the cytoplasmic membrane and the reduction of molecular oxygen to water . Both oxidases are coupling sites in the respiratory chain; electron transfer from ubiquinol to oxygen results in the generation of a proton electrochemical potential difference across the membrane . The cytochrome d complex is a heterodimer (subunits I and II) that has three heme prosthetic groups . Previous studies characterized two monoclonal antibodies that bind to subunit I and specifically block the ability of the enzyme to oxidize ubiquinol . In this paper, the epitopes of both of these monoclonal antibodies have been mapped to within a single 11-amino acid stretch of subunit I . The epitope is located in a large hydrophilic loop between the fifth and sixth putative membrane-spanning segments . Binding experiments with these monoclonal antibodies show this polypeptide loop to be periplasmic . Such localization suggests that the loop may be close to His186, which has been identified as one of the axial ligands of cytochrome b558 . Together, these data begin to define a functional domain in which ubiquinol is oxidized near the periplasmic surface of the membrane.

Cancer Res, 1990 Mar 15, 50(6), 1671 - 4
Receptor binding of human granulocyte colony-stimulating factor to the blast cells of myeloid leukemia; Piao YF et al.; Human granulocyte colony-stimulating factor (G-CSF) rapidly loses the biological activity and the receptor binding capacity following radioiodination . We have made a mutein of human G-CSF, KW-2228, in which Thr-1, Leu-3, Gly-4, Pro-5, and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg, and Ser; showed more potent G-CSF activity; and retained full biological activity and receptor binding capacity at least 2 weeks of radioiodination . G-CSF is an effective growth factor for the blasts of myeloid leukemia . Radioiodinated KW-2228 was prepared using solid-phase glucose oxidase-lactoperoxidase . Human leukemia cell lines and the blast cells from leukemia patients were examined for binding . High affinity binding sites were identified on myeloid cell lines and on the blasts obtained from acute myeloid leukemia patients . Scatchard analysis showed that a single binding site for G-CSF was observed (361-1688 receptors/cell; Kd 128-1400 pM) . In contrast, specific binding of 125I-KW-2228 was not demonstrated on lymphoblastic cell lines or the blast cells of acute lymphoid leukemia or lymphoma . This difference was reflected in the effectiveness of G-CSF to stimulate colony formation in acute myeloid leukemia blasts, while G-CSF did not stimulate colony formation of the blast cells from acute lymphoid leukemia.

Biochemistry, 1990 Mar 13, 29(10), 2515 - 23
Lead-catalyzed cleavage of yeast tRNAPhe mutants; Behlen LS et al.; Yeast tRNA(Phe) lacking modified nucleotides undergoes lead-catalyzed cleavage between nucleotides U17 and G18 at a rate very similar to that of its fully modified counterpart . The rates of cleavage for 28 tRNA(Phe) mutants were determined to define the structural requirements of this reaction . The cleavage rate was found to be very dependent on the identity and correct positioning of the two lead-coordinating pyrimidines defined by X-ray crystallography . Nucleotide changes that disrupted the tertiary interactions of tRNAPhe reduced the rate of cleavage even when they were distant from the lead binding pocket . However, nucleotide changes designed to maintain tertiary interactions showed normal rates of cleavage, thereby making the reaction of a useful probe for tRNA(Phe) structure . Certain mutants resulted in the enhancement of cleavage at a "cryptic" site at C48 . The sequences of Escherichia coli tRNA(Phe) and yeast tRNA(Arg) were altered such that they acquired the ability to cleave at U17, confirming our understanding of the structural requirements for cleavage . This mutagenic analysis of the lead cleavage domain provides a useful guide for similar analysis of autocatalytic self-cleavage reactions.

Biochemistry, 1990 Mar 13, 29(10), 2572 - 7
Isolation and functional reconstitution of soluble melibiose permease from Escherichia coli; Roepe PD et al.; By use of techniques described recently for lac permease {Roepe, P.D., & Kaback, H.R . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 6087}, the melibiose permease from Escherichia coli, another polytopic integral plasma membrane protein, has been purified in a metastable soluble form after overexpression of the melB gene via the T7 RNA polymerase system . As demonstrated with lac permease, soluble melibiose permease is dissociated from the membrane with 5.0 M urea and appears to remain soluble in phosphate buffer at neutral pH after removal of urea by dialysis, although the protein aggregates in a time- and concentration-dependent fashion . Moreover, soluble melibiose permease behaves as a monomer during purification by size exclusion chromatography in the presence of urea . Circular dichroism of purified soluble melibiose permease reveals that the protein is highly helical in potassium phosphate buffer and that secondary structure is disrupted in 5.0 M urea . Finally, purified melibiose permease can be reconstituted into proteoliposomes, and the preparations catalyze membrane potential driven H+/melibiose or Na+/methyl 1-thio-beta,D-galactopyranoside symport . The results provide further support for the notion that hydrophobic transmembrane proteins may be able to assume a nondenatured conformation in aqueous solution and extend the implication that the approach described may represent a general method for rapid isolation and reconstitution of this class of membrane proteins.

Biochemistry, 1990 Mar 13, 29(10), 2482 - 7
Effect of oligosaccharides and chloride on the oligomeric structures of external, internal, and deglycosylated invertase; Reddy AV et al.; External invertase exists in an oligomeric equilibrium of dimer, tetramer, hexamer, and octamer, the concentrations of which vary with pH, time, and concentration of enzyme {Chu, F.K., Watorek, W., & Maley, F . (1983) Arch . Biochem . Biophys . 223, 543-555; Tammi, M., Ballou, L., Taylor, A., & Ballou, C.E . (1987) J . Biol . Chem . 262, 4395-4401} . To assess the influence of carbohydrate on this equilibrium, we investigated the self-association of external invertase (10 oligosaccharides per subunit), deglycosylated external invertase (2 oligosaccharides per subunit), and internal invertase (no carbohydrate) under various conditions . In addition, the effect of carbohydrate on the interaction of the subunits of these various invertases to form heterooligomers was studied . Chloride ion was found to promote subunit association in the various invertases irrespective of their glycosylation status . However, external invertase was less responsive to chloride ion relative to the internal and deglycosylated invertases . The higher oligomers of deglycosylated invertase were stable at 47 degrees C whereas those of external invertase dissociated rapidly into dimers, suggesting that the additional oligosaccharides in external invertase destabilize subunit interaction . Hybridization experiments with the various invertases showed that the dimers of internal invertase formed heterooligomers with either external or deglycosylated invertase . By contrast, the monomers of external and internal invertases formed their respective homodimers, but not heterodimers . These results suggest that the oligosaccharide content of invertase not only influences the extent of self-association but also affects heterooligomer formation.

Oncogene, 1990 Mar, 5(3), 377 - 86
Preparation and characterization of monoclonal antibodies specific for human transforming growth factor alpha; Sorvillo JM et al.; A series of monoclonal antibodies (mAbs) against transforming growth factor alpha (TGF alpha) have been produced . The generation of these reagents, as well as their biochemical and immunochemical characterization is described . TGF alpha peptides, mutant recombinant TGF alpha proteins and two-site immunoradiometric assays were used to identify the epitopes recognized by each antibody . This approach has allowed the specific localization of immunodominant domains on the molecule . Certain mAbs were found to be useful for selected procedures . mAb 134A-2B3 was used for immunoblotting both the precursor and mature forms of TGF alpha from conditioned media of tumor cells . One mAb 189-2130.1, which reacted with the carboxyl terminal seventeen amino acids, was able to block TGF alpha binding to the EGF receptor . mAb 213-4.4 was used for immunohistochemical detection of TGF alpha in fixed tumor cells . mAbs 137-178 and 134A-2B3 were used to develop a two-site immunoradiometric immunoassay which was sensitive to 1 ng ml-1 and detected TGF alpha from a variety of tumor cells . A series of mAbs such as these could prove useful in studying the biochemical properties as well as the immunochemical localization of TGF alpha in normal tissues and tumors.

Nucleic Acids Res, 1990 Mar 11, 18(5), 1243 - 8
Insertional gene synthesis, a novel method of assembling consecutive DNA sequences within specific sites in plasmids . Construction of the HIV-1 tat gene; Ciccarelli RB et al.; The construction of the HIV-1 tat gene using a novel method termed insertional gene synthesis (IGS) is described . IGS is used to assemble a gene or any DNA sequence in a stepwise manner within a plasmid containing a single stranded DNA phage origin of replication . The IGS method is based upon consecutive targeted insertions of long DNA oligonucleotides (greater than 100 bases) within the plasmid by oligonucleotide-directed mutagenesis . IGS therefore involves synthesis of only a few oligonucleotides corresponding to one strand of a gene . Furthermore, the gene is synthesized directly adjacent to bacterial gene regulatory sequences for direct expression . Using this approach, the 261 bp tat gene was assembled in three successive cycles adjacent to the lac promoter in the pEMBL-derivative, pKH125 . The 15 kD tat protein was produced from this synthetic gene in E . coli upon IPTG induction . However, it was necessary to tightly control the expression of tat by including the lac I gene directly within the tat expression vector.

Nucleic Acids Res, 1990 Mar 11, 18(5), 1115 - 9
The cyanelle genome of Cyanophora paradoxa, unlike the chloroplast genome, codes for the ribosomal L3 protein; Evrard JL et al.; We describe a 1132 bp sequence of the cyanelle genome of Cyanophora paradoxa containing the rpl3 gene . This gene, which is not chloroplast encoded in plants, is the first of a long cyanelle ribosomal operon whose organization resembles that of the S10 operon of E . coli . We have shown that the rpl3 gene is transcribed in cyanelles as a 7500 nucleotide precursor and that the 5'-end of the mRNA starts approximately 90 nucleotides upstream from the initiation codon . However, no typical procaryotic promoter could be found for this gene . We have detected, using anti E . coli L3 antibodies, the cyanelle L3 protein in cyanelle extracts and in E . coli cells transformed with the cyanelle rpl3 gene.

Eur J Biochem, 1990 Mar 10, 188(2), 347 - 54
Impaired in vitro kinetics of EF-Tu mutant Aa; Tapio S et al.; The kirromycin-resistant EF-Tu mutant Aa, previously shown to be an antisuppressor for nonsense and missense suppressor tRNAs, has been characterised in a poly(U)-primed translation system in vitro . Two major defects were found in the function of the mutant . First, the dissociation constant for Aa binding to Phe-tRNA(Phe) was increased tenfold compared to wild-type EF-Tu . Second, kcat/Km for the interaction between the EF-Tu.GTP.aa-tRNA complex and the ribosome was decreased by the mutation to one third of its wild-type value . No differences were observed between mutant and wild-type factor in the regeneration of EF-Tu.GTP from EF-Tu.GDP via EF-Ts or in the mistranslation frequency by Leu-tRNA(4Leu) . The relation between the in vitro results and the mutant phenotype in vivo is discussed.

Eur J Biochem, 1990 Mar 10, 188(2), 339 - 46
Antisuppression by mutations in elongation factor Tu; Tapio S et al.; Two slow-growing kirromycin-resistant Escherichia coli mutants with altered EF-Tu (Ap and Aa) were studied in vivo in strains with an inactive tufB gene . Mutant form Aa was isolated as an antisuppressor of the tyrT(Su3) nonsense suppressor, as described here . Ap, the tufA gene product of strain D2216 (from A . Parmeggiani), has previously been shown to give an increased GTPase activity . The slow cellular growth rates of both EF-Tu mutants are correlated with decreased translational elongation rates . Ap and Aa significantly decrease suppression levels of both nonsense and missense suppressor tRNAs {tyrT(Su3), trpT(Su9), glyT(SuAGA/G)}, but have only little or no effect on misreading by wild-type tRNAs . A particular missense suppressor, lysT(SuAAA/G), which acts by virtue of partial mischarging as the result of an alteration in the amino acid stem, is not significantly affected by the EF-Tu mutations . The combination of tufA(Aa) and a rpsD12 ribosomal mutation is lethal at room temperature and the double-mutant strain has an elevated temperature optimum (42 degrees C) for growth rate, translation rate and nonsense suppression . Our data indicate an alterated interaction between Aa and the ribosome, consistent with our in vitro results.

Eur J Biochem, 1990 Mar 10, 188(2), 239 - 45
Carboxyl-terminal structure of basic fibroblast growth factor significantly contributes to its affinity for heparin; Seno M et al.; The carboxyl-terminal sequence of basic fibroblast growth factor (bFGF) is rich in basic amino acid residues, a common characteristic amongst fibroblast growth factors, and is considered to contribute greatly to the binding to negatively charged extracellular matrixes such as heparin . To study the relationship between the affinity for heparin and the carboxyl-terminal structure of bFGF, amino- or carboxyl-terminal truncated molecules were produced in Escherichia coli using recombinant DNA techniques . These terminally truncated bFGFs were applied to a heparin-affinity HPLC column . Truncation of more than six amino acid residues from the carboxyl-terminal made the bFGF produced in E . coli markedly difficult to solubilize and weakened its affinity for heparin, though bFGF having up to 46 amino acids removed showed significant stimulation of the DNA synthesis of BALB/c3T3 cells . This stimulation of the DNA synthesis was also recognized by the bFGF having 40 amino acids removed from its amino-terminal, while the affinity of this peptide for heparin has been shown to be equal to that of the mature bFGF (146 amino acids) . These results show that the affinity of bFGF for heparin depends significantly on its carboxyl-terminal structure and that the essential part for receptor binding is present between Asp41 and Ser100 . Moreover, it suggests that the Phe139Leu140Pro141, present in all members of the FGF family, contributes greatly to the stable structure of the intact molecule.

Nature, 1990 Mar 8, 344(6262), 175 - 7
Dependence of the torsional rigidity of DNA on base composition; Fujimoto BS et al.; The Escherichia coli phage 434 repressor binds as a dimer to the operator of the DNA helix . Although the centre of the operator is not in contact with protein, the repressor binding affinity can be reduced at least 50-fold by changing the sequence there: operators with A.T base pairs near their centre bind the repressor more strongly than do operators with G.C base pairs at the same positions . To explain these observations, it has been proposed that the base composition at the centre of the operator affects the affinity of the operator for repressor by altering the ease with which operator DNA can undergo the torsional deformation necessary for complex formation . In this model, the variation in binding affinity would require the torsion constant to have specific values and to change in a sequence-dependent manner . We have now measured torsion constants for DNAs with widely different base compositions . Our results indicate that the torsion constants depend only slightly on the overall composition, and firmly delimit the range of values for each . Even the upper-limit values are much too small to account for the observed changes in affinity of the 434 repressor . These results rule out simple models that rely on substantial generic differences in torsion constant between A.T-rich sequences and G.C-rich sequences, although they do not rule out the possibility of particular sequences having abnormal torsion constants.

Biochemistry, 1990 Mar 6, 29(9), 2430 - 6
Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator; Flentke GR et al.; UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5'-diphosphate chloroacetol (UDC) and uridine 5'-diphosphate bromoacetol (UDB) . Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively . The reaction of UDC with the enzyme follows saturation kinetics characterized by a KD of 0.110 mM and kinact of 0.84 min-1 at pH 8.5 and ionic strength 0.2 M . The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent binding of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD+ . The inactivation of epimerase by uridine 5'-diphosphate {2H2}chloroacetol proceeds with a primary kinetic isotope effect (kH/kD) of 1.4 . The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD+ at the active site by the chloroacetol enolate . The resulting adduct between UDC and NAD+ is proposed to be the chromophore with lambda max at 325 nm . The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.

Biochemistry, 1990 Mar 6, 29(9), 2421 - 9
Allosteric effects acting over a distance of 20-25 A in the Escherichia coli tryptophan synthase bienzyme complex increase ligand affinity and cause redistribution of covalent intermediates; Houben KF et al.; The reactions of L-histidine (L-His) and L-tryptophan (L-Trp) with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase are introduced as probes both of beta-subunit catalysis and of ligand-mediated alpha-beta allosteric interactions . Binding of DL-alpha-glycerol 3-phosphate (GP), an analogue of 3-indole-D-glycerol 3'-phosphate (IGP), to the alpha-catalytic site increases the affinity of alpha 2 beta 2 for L-His 4.5-fold and the affinity for L-Trp 17-fold and brings about a redistribution of beta-bound intermediates that favors the quinonoids derived from each amino acid . Inorganic phosphate (Pi) (presumably via binding to the alpha-catalytic site) influences the distribution of L-His intermediates as does GP . Previous binding studies {Heyn, M . P., & Weischet, W . O . (1975) Biochemistry 14, 2962-2968} indicate that when the phosphoryl group subsite of the alpha-catalytic site is occupied by GP or Pi, a high-affinity indole subsite is induced at the alpha-catalytic site . Interaction of benzimidazole (BZ), an analogue of indole, with this site also shifts the distribution of beta-bound L-His intermediates in favor of the L-His quinonoid . In the absence of Pi or GP, BZ interacts primarily at the beta-catalytic site and competes with L-His for the beta-subunit indole subsite . Since L-His and GP (or Pi) are substrate analogues and L-Trp is the physiological product, these allosteric effects likely take place with the natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Mar 6, 29(9), 2379 - 86
Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli; Duncan K et al.; The Escherichia coli D-alanyl-D-alanine-adding enzyme, which catalyzes the final cytoplasmic step in the biosynthesis of the bacterial peptidoglycan precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D- Ala, has been purified to homogeneity from an E . coli strain that harbors a recombinant plasmid bearing the structural gene for this enzyme, murF . The enzyme is a monomer of molecular weight 49,000, and it has a turnover number of 784 min-1 for ATP-driven amide bond formation . Experiments monitoring the fate of radiolabeled UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelate and D-trifluoroalanine proved that the preceding enzyme in the D-alanine branch pathway, D-alanine:D-alanine ligase (ADP), is capable of synthesizing fluorinated dipeptides, which the D-Ala-D-Ala-adding enzyme can then incorporate to form UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelyl-D-++ +trifluoroAla-D- trifluoroAla.

Biochemistry, 1990 Mar 6, 29(9), 2220 - 5
Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase; Ghosh G et al.; We have previously shown that the anticodon of methionine tRNAs contains most, if not all, of the nucleotides required for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase {Schulman, L . H., & Pelka, H . (1988) Science 242, 765-768} . Previous cross-linking experiments have also identified a site in the synthetase that lies within 14 A of the anticodon binding domain {Leon, O., & Schulman, L . H . (1987) Biochemistry 26, 5416-5422} . In the present work, we have carried out site-directed mutagenesis of this domain, creating conservative amino acid changes at residues that contain side chains having potential hydrogen-bond donors or acceptors . Only one of these changes, converting Trp461----Phe, had a significant effect on aminoacylation . The mutant enzyme showed an approximately 60-100-fold increase in Km for methionine tRNAs, with little or no change in the Km for methionine or ATP or in the maximal velocity of the aminoacylation reaction . Conversion of the adjacent Pro460 to Leu resulted in a smaller increase in Km for tRNA(Mets), with no change in the other kinetic parameters . Examination of the interaction of the mutant enzymes with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interactions between the Phe461 mutant and different anticodons . Km values were highest for tRNA(mMet) derivatives containing the normal anticodon wobble base C . Base substitutions at this site decreased the Km for aminoacylation by the Phe461 mutant, while increasing the Km for the wild-type enzyme and for the Leu460 mutant to values greater than 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Mar 6, 29(9), 2330 - 5
Identification of the protein kinase C phosphorylation site in neuromodulin; Apel ED et al.; Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C . Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin {Alexander, K . A., Cimler, B . M., Meier, K . E., & Storm, D . R . (1987) J . Biol . Chem . 262, 6108-6113}, and we have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin . The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate . Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar Km values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose . Recombinant neuromodulin was phosphorylated by using protein kinase C and {gamma-32P}ATP and digested with trypsin, and the resulting peptides were separated by HPLC . Only one 32P-labeled tryptic peptide was generated from phosphorylated neuromodulin . The sequence of this peptide was IQASFR . The serine in this peptide corresponds to position 41 of the entire protein, which is adjacent to or contained within the calmodulin binding domain of neuromodulin . A synthetic peptide, QASFRGHITRKKLKGEK, corresponding to the calmodulin binding domain with a few flanking residues, including serine-41, was also phosphorylated by protein kinase C . We conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmodulin to neuromodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Mar 6, 29(9), 2409 - 17
Kinetic characterization of early immunoreactive intermediates during the refolding of guanidine-unfolded Escherichia coli tryptophan synthase beta 2 subunits; Blond-Elguindi S et al.; This paper deals with stopped-flow studies on the kinetics of the regain of immunoreactivity toward five distinct monoclonal antibodies during the folding of the guanidine-unfolded beta 2 subunit of Escherichia coli tryptophan synthase and of two complementary proteolytic fragments of beta, F1 (N-terminal; Mw = 29,000) and F2 (C-terminal; Mw = 12,000) . It is shown that, while selected as being "specific" for the native protein, these antibodies are all able to recognize early folding intermediates . The two antigenic determinants carried by the F2 domain and the antigenic site carried by the hinge peptide linking F1 and F2 are present so early during the folding process that their kinetics of appearance could not be followed . On the contrary, the rate constants of appearance of two "native-like" epitopes, carried by F1, could be determined during the folding of beta chains . The rate constant of appearance of the epitope to antibody 19 was found to be k = 0.065 s-1 at 12 degrees C . This value is very similar to that we reported previously for the appearance of an early epitope to the same antibody during the folding of acid-denatured beta chains . Thus, in spite of the important structural differences between guanidine-unfolded and acid-denatured beta chains, the same early folding events seem to be involved in the appearance of this epitope . The rate constant was found to be significantly smaller (k = 0.02 s-1 at 12 degrees C) for the appearance of the epitope to antibody 9 . This shows that the regain of immunoreactivity is not concerted within the F1 domain.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1990 Mar 5, 265(7), 4155 - 60
Characterization of the double mutant, Val-177/Asn-322, of the lactose permease; Brooker RJ; The double mutant, Val-177/Asn-322, was investigated with regard to its ability to transport H+ and galactosides . In downhill lactose transport assays, the wild-type strain had a Km value for lactose uptake of 0.9 mM and a Vmax of 0.65 mumol lactose/min.mg protein while the mutant had a significantly higher Km value of 1.9 mM but a similar Vmax of 0.49 mumol/min.mg protein . In spite of its moderate ability to transport lactose downhill, the Val-177/Asn-322 mutant exhibited the striking property of being completely defective in the uphill accumulation of lactose or methyl-beta-D-thiogalactopyranoside . Direct measurements of H+ transport, however, showed that the mutant's defect in active accumulation is not due to a defect in the ability to transport H+ with lactose or methyl-beta-D-thiogalactopyranoside . The Val-177/Asn-322 mutant strain had a H+:lactose stoichiometry of 0.84 which was similar to that measured in the wild-type strain (0.68) . These results are discussed with regard to the role His-322 plays in H+ transport, active accumulation of sugars, and sugar recognition.

J Biol Chem, 1990 Mar 5, 265(7), 4111 - 5
Studies on the mechanism of formation of the pyruvate prosthetic group of phosphatidylserine decarboxylase from Escherichia coli; Li QX et al.; Phosphatidylserine decarboxylase from Escherichia coli uses a pyruvate group as the enzyme cofactor (Satre, M., and Kennedy, E . P . (1978) J . Biol . Chem . 253, 479-483) . Comparison of the DNA sequence of the psd gene with the partial amino acid sequence of the mature gene product suggests that the two nonidentical subunits of the mature enzyme are formed by cleavage of a proenzyme resulting in the conversion of Ser-254 to an amino-terminal pyruvate residue (Li, Q.-X., and Dowhan, W . (1988) J . Biol . Chem . 263, 11516-11522) . The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min . When Ser-254 is changed to cysteine (S254C), threonine (S254T), or alanine (S254A) by site-directed mutagenesis, the rate of processing of the proenzyme and the production of the functional enzyme are drastically affected . Proenzymes with S254C or S254T are cleaved with a half-time of around 2-4 h while the S254A proenzyme does not undergo processing . The reduced processing rate for the mutant proenzymes is consistent with less of the functional enzyme being made . Mutants encoding the S254C and S254T protein produce 16 and 2%, respectively, of the activity of the wild-type allele but can still complement a temperature-sensitive mutant in the psd locus . There is no detectable activity or complementation observed with the S254A protein . These results are consistent with the hydroxyl group of Ser-254 playing a critical role in the cleavage of the peptide bond between Gly-253 and Ser-254 of the prophosphatidylserine decarboxylase and support the mechanism proposed by Snell and coworkers (Recsei and Snell (1984) Annul Rev . Biochem . 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases.

J Biol Chem, 1990 Mar 5, 265(7), 4098 - 104
Murine DNA polymerase alpha fills gaps to completion in a direct assay . Altered kinetics of de novo DNA synthesis at single nucleotide gaps; Davey SK et al.; DNA polymerase alpha was studied in a direct gap-filling assay . Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer . DNA polymerase alpha filled this gap to completion . A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min . In another 35% of the molecules the final nucleotide was lacking after 10 min . This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h . The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM) . DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells . T4 DNA polymerase filled gaps to completion in this assay . Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides . Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.

J Biol Chem, 1990 Mar 5, 265(7), 4091 - 7
Site-directed mutagenesis of the -10 region of the lacUV5 promoter . Introduction of dA4.dT4 tract suppresses open complex formation; Chan PT et al.; Homopolymeric dAn.dTn sequences, where n is 4 or greater, have special properties leading to increased duplex stability and DNA bending . The lacUV5 promoter was used to examine the functional consequences of changing the -10 TATAAT consensus sequence to the sequence TAAAAT . The transversion mutation at the underlined site was accomplished with site-directed mutagenesis using translation termination as the selection procedure . For free DNA, structural differences at the 5' and 3' junction regions of the dA4.dT4 tract can be readily detected by DNase I digestion . However, site binding by Escherichia coli RNA polymerase appeared unaltered by the TAAAAT sequence since identical DNase I footprints were obtained for the lacUV5 and mutant promoters . Binding competition studies under different ionic strengths revealed a significant reduction in mutant promoter open complex formation relative to the lacUV5 promoter . Mutant promoter open complexes also dissociated faster and to a greater extent than the corresponding lacUV5 promoter open complexes when challenged with heparin or a combination of heparin and increased KCl concentration . Consequently, mutant promoter open complexes appear less stable than lacUV5 promoter open complexes.

J Biol Chem, 1990 Mar 5, 265(7), 4004 - 10
Properties of recA441 protein-catalyzed DNA strand exchange can be attributed to an enhanced ability to compete with SSB protein; Lavery PE et al.; We have investigated the recombinase activity of recA441 protein by comparing its in vitro DNA strand exchange activity to that of wild-type recA protein . Consistent with its proficiency in recombination in vivo, recA441 protein is able to catalyze the in vitro exchange of a circular single-stranded DNA molecule for a homologous strand in a linear double-stranded DNA molecule . Under conditions optimal for wild-type recA protein, the rates of joint molecule formation are the same for the two recA proteins, but the wild-type protein converts these intermediate species to gapped circular heteroduplex DNA product molecules more rapidly than recA441 protein . In the recA441 protein reaction, joint molecules are instead converted to extensive homology-dependent DNA networks via presumed reinitiation reactions . Under some conditions, the DNA strand exchange activity of recA441 protein is enhanced relative to the wild-type . These conditions include when single-stranded DNA.SSB protein (where SSB is Escherichia coli single-stranded DNA-binding protein) complexes are formed prior to the addition of recA protein, at low magnesium ion concentration in the presence of spermidine, and at low ATP concentrations . Under the conditions examined, recA441 protein competes more effectively with SSB protein for DNA-binding sites; thus, the differences between the strand exchange activities of the wild-type and recA441 proteins can be attributed to this enhanced ability in SSB protein competition.

J Biol Chem, 1990 Mar 5, 265(7), 3851 - 8
Comparison of helix stability in wild-type and mutant LamB signal sequences; Bruch MD et al.; Previous studies of isolated peptides corresponding to the wild-type signal sequence of the LamB protein of Escherichia coli and to several export-impaired mutants demonstrated that a high tendency to adopt an alpha-helical conformation in low dielectric environments was a property of functional sequences . We have now used nuclear magnetic resonance to establish further characteristics of the helical conformation of these signal peptides in a solvent mixture (50% trifluoroethanol, by volume, in water) which mimics the conformational distribution of these peptides in lipid vesicles . The interactions of signal sequences in vivo may depend on the location of the helix in the sequence, on the length of the helical segment, and on the stability of the helix . We find that the hydrophobic core has the most persistent helix conformation and that the stability of this helix correlates with in vivo function of different mutants of the LamB signal sequence . In the family of signal peptides studied here, the length of the helix required for function appears to be less rigidly restricted since a signal peptide from a functional pseudorevertant with 4 residues deleted from the hydrophobic core takes up helix as stably as wild type but incorporates fewer residues in the helix.

J Biol Chem, 1990 Mar 5, 265(7), 3679 - 84
Template primer-dependent binding of 5'-fluorosulfonyl-benzoyldeoxyadenosine by Escherichia coli DNA polymerase I . Identification of arginine 682 as the binding site and its implication in catalysis; Pandey VN et al.; We have labeled a template primer-dependent substrate deoxynucleoside triphosphate binding domain in Escherichia coli DNA polymerase I using an affinity labeling analogue of dATP, the 5'-fluorosulfonylbenzoyldeoxyadenosine (FSBdA) . Using enzyme-template primer complex as a test system, we find that FSBdA-mediated inactivation occurs only when the template in the enzyme-template primer complex is poly(dT).(dA)10 . A ribonucleotide analogue, 5'-fluorosulfonylbenzoyladenosine (FSBA) is not an effective inactivator under these conditions . In the absence of template primer, however, deoxyribonanalogue (FSBdA) irreversibly inactivates polymerase activity with characteristics similar to those reported for FSBA (Pandey, V.N., and Modak, M.J . (1988) J . Biol . Chem . 263, 6068) . Binding stoichiometric studies in the presence and absence of template primer revealed that only 1 mol of FSBdA is incorporated per mol of enzyme which results in complete inactivation . The site of FSBdA action was investigated by comparative tryptic peptide mapping, followed by amino acid composition analysis of the modified peptide . Arginine 682 was found to be the target of FSBdA reactivity . We therefore conclude that the domain containing Arg-682 plays a major role in template-dependent dNTP binding and polymerization.

J Biol Chem, 1990 Mar 5, 265(7), 3661 - 8
Analysis of clathrin light chain-heavy chain interactions using truncated mutants of rat liver light chain LCB3; Scarmato P et al.; Clathrin light chains are extended molecules located along the proximal segment of each of the three heavy chain legs of a clathrin trimer . All mammalian light chains share a central segment with 10 repeated heptad motifs believed to mediate the interaction with clathrin heavy chains . In order to test this model in more detail, we have expressed intact rat liver clathrin light chain LCB3 in Escherichia coli and find that it binds tightly to calf clathrin heavy chains . Using a set of expressed truncated mutants of LCB3, we show that the presence of seven to eight heptads is indeed necessary for a successful interaction . More extensive deletions of the central segment completely abolish the ability to bind to heavy chains . Neither the amino- nor the carboxyl-terminal domain is essential for binding, but competition experiments show that the presence of the carboxyl-terminal domain does enhance the interaction with heavy chains.

J Biol Chem, 1990 Mar 5, 265(7), 3599 - 602
Regulation of isocitrate dehydrogenase by phosphorylation involves no long-range conformational change in the free enzyme; Hurley JH et al.; The structure of the phosphorylated form of isocitrate dehydrogenase from Escherichia coli has been solved and refined to an R-factor of 16.9% at 2.5-A resolution . Comparison with the structure of the dephosphorylated enzyme shows that there are no large scale conformational changes and that small conformational changes are highly localized around the site of phosphorylation at serine 113 . Tyrosine 160 rotates by 15 degrees, and there is a local rearrangement of water structure . There is an 0.2-A net movement of loop 230-234, and side chain shifts of 0.2 A root mean square for isoleucine 159 and lysine 199 . The lack of large conformational changes, the observation of a possible isocitrate binding site close to serine 113, and the demonstration that the phosphorylated enzyme is unable to bind isocitrate suggest that this enzyme is inactivated by a direct electrostatic interaction between the substrate and the serine phosphate.

J Mol Biol, 1990 Mar 5, 212(1), 167 - 84
Crystal structure of thioredoxin from Escherichia coli at 1.68 A resolution; Katti SK et al.; The crystal structure of thioredoxin from Escherichia coli has been refined by the stereochemically restrained least-squares procedure to a crystallographic R-factor of 0.165 at 1.68 A resolution . In the final model, the root-mean-square deviation from ideality for bond distances is 0.015 A and for angle distances 0.035 A . The structure contains 1644 protein atoms from two independent molecules, two Cu2+, 140 water molecules and seven methylpentanediol molecules . Ten residues have been modeled in two alternative conformations . E . coli thioredoxin is a compact molecule with 90% of its residues in helices, beta-strands or reverse turns . The molecule consists of two conformational domains, beta alpha beta alpha beta and beta beta alpha, connected by a single-turn alpha-helix and a 3(10) helix . The beta-sheet forms the core of the molecule packed on either side by clusters of hydrophobic residues . Helices form the external surface . The active site disulfide bridge between Cys32 and Cys35 is located at the amino terminus of the second alpha-helix . The positive electrostatic field due to the helical dipole is probably important for stabilizing the anionic intermediate during the disulfide reductase function of the protein . The more reactive cysteine, Cys32, has its sulfur atom exposed to solvent and also involved in a hydrogen bond with a backbone amide group . Residues 29 to 37, which include the active site cysteine residues, form a protrusion on the surface of the protein and make relatively fewer interactions with the rest of the structure . The disulfide bridge exhibits a right-handed conformation with a torsion angle of 81 degrees and 72 degrees about the S-S bond in the two molecules . Twenty-five pairs of water molecules obey the noncrystallographic symmetry . Most of them are involved in establishing intramolecular hydrogen-bonding interactions between protein atoms and thus serve as integral parts of the folded protein structure . Methylpentanediol molecules often pack against the loops and stabilize their structure . Cu2+ used for crystallization exhibit a distorted octahedral square bipyramid co-ordination and provide essential packing interactions in the crystal . The two independent protein molecules are very similar in conformation but distinctly different in atomic detail (root-mean-square = 0.94 A) . The differences, which may be related to the crystal contacts, are localized mostly to regions far from the active site.

J Mol Biol, 1990 Mar 5, 212(1), 79 - 96
Nature of the SOS-inducing signal in Escherichia coli . The involvement of DNA replication; Sassanfar M et al.; The SOS genes of Escherichia coli, which include many DNA repair genes, are induced by DNA damage . Although the central biochemical event in induction, activation of RecA protein through binding of single-stranded DNA and ATP to promote cleavage of the LexA repressor, is known, the cellular event that provides this activation following DNA damage has not been well understood . We provide evidence here that the major pathway of induction after damage by a typical agent, ultraviolet light, requires an active replication fork; this result supports the model that DNA replication leaves gaps where elongation stops at damage-induced lesions, and thus provides the single-stranded DNA that activates RecA protein . In order to detect quantitatively the immediate product of the inducing signal, activated RecA protein, we have designed an assay to measure the rate of disappearance of intact LexA repressor . With this assay, we have studied the early phase of the induction process . LexA cleavage is detectable within minutes after DNA damage and occurs in the absence of protein synthesis . By following the reaccumulation of LexA in the cell, we detect repair of DNA and the disappearance of the inducing signal . Using this assay, we have measured the LexA content of wild-type and various mutant cells, characterized the kinetics and conditions for development of the inducing signal after various inducing treatments and, finally, have shown the requirement for DNA replication in SOS induction by ultraviolet light.

J Mol Biol, 1990 Mar 5, 212(1), 127 - 33
Effects of mutagenesis of a conserved base-paired site near the decoding region of Escherichia coli 16 S ribosomal RNA; De Stasio EA et al.; Eleven of 15 possible single and double mutations were constructed in a cloned copy of Escherichia coli 16 S rDNA at a base-paired site, 1409-1491 . Expression of any of these mutations was detrimental to the growth of E . coli . Mutations that substituted unpaired purine bases were lethal in the system described . Otherwise, the degree of detrimental effect on growth-rate was not directly correlated with specific rRNA primary or secondary structures . Using reverse transcription of rRNA isolated from subunits or 70 S ribosomes, we were able to determine the amount of mutant rRNA used in translation . From these experiments, we found that the lethal mutations appeared to be selectively excluded from the pool of 70 S ribosomes following expression from a repressible plasmid . In contrast, a non-lethal mutation was present in subunits, ribosomes and polysomes in approximately equal amounts . Mutations that disrupted base-pairing were found to confer varying levels of resistance to nine aminoglycosides, including four neomycins, two kanamycins, gentamicin, apramycin and hygromycin . A high frequency of reversion from resistant and slow-growth phenotypes due to a host mutation was observed.

J Biol Chem, 1990 Mar 5, 265(7), 3916 - 22
Homogeneous Escherichia coli FPG protein . A DNA glycosylase which excises imidazole ring-opened purines and nicks DNA at apurinic/apyrimidinic sites; Boiteux S et al.; The repair of 2,6-diamino-4-hydroxy-5-N-methyl-formamidopyrimidine (Fapy) residues in DNA is performed by a Fapy-DNA glycosylase activity which is encoded for by the fpg gene in Escherichia coli . Besides DNA glycosylase activity, this protein, the FPG protein, is endowed with an EDTA-resistant activity nicking DNA at apurinic/apyrimidinic (AP) sites . To overproduce the FPG protein, the fpg gene was placed under the control of the tac promoter in the expression vector pKK223-3 yielding the pFPG230 plasmid . The production of the FPG protein in cells harboring the pFPG230 plasmid was 800-fold higher than that of the wild type strain after induction by isopropyl-beta-D-thio-galactopyranoside . From these cells, the FPG protein was purified to homogeneity in sufficient quantity to study its physical and catalytic properties . In its active form, the FPG protein is a globular monomer of 31 kDa and has an experimentally measured isoelectric point of 8.5 . When the FPG protein is heat-denatured in the presence of EDTA the two activities are more rapidly inactivated than when heated in the absence of EDTA, suggesting that the FPG protein possesses a tightly bound metal ion . Atomic absorption spectrophotometric analysis shows that there is one zinc/FPG protein molecule . The FPG protein is different from previously described DNA glycosylases and AP-nicking enzymes in E . coli . The contribution of the AP-nicking activity associated with the FPG protein represents 10-20% of the total EDTA-resistant AP-nicking activities in E . coli.

Virology, 1990 Mar, 175(1), 247 - 54
Cloning of the human parvovirus B19 genome and structural analysis of its palindromic termini; Deiss V et al.; We describe the molecular cloning of the entire 5.6-kb single-stranded DNA genome of the human parvovirus B19 in bacterial plasmids . Stable amplification of the recombinant plasmid DNA was achieved in Escherichia coli JC8111 but not in HB101 cells . Sequence analysis of the cloned DNA shows that the terminal 383 nucleotides at each end of the genome are identical inverted repeats . The distal 365 nucleotides of the repeat represent an imperfect palindrome which presumably folds over to form a hairpin structure . The sequence of the hairpin occurs in two distinct configurations which are related in that one is the inverted complement of the other . Such alternative configurations of the terminal hairpins have been found for all parvoviruses analyzed so far and are referred to as flip and flop.

Virology, 1990 Mar, 175(1), 222 - 31
Purification and characterization of wild-type and ts 112 mutant protein IIIa of human adenovirus 2 expressed in Escherichia coli; Cuillel M et al.; The expression of the protein IIIa gene from human adenovirus type 2 (Ad2) in Escherichia coli has been described previously (M . Cuillel, M . Milleville, and J . C . D'Halluin, 1987, Gene 55, 295-301) . The same construct has now been used to express a protein IIIa gene from an Ad2 mutant ts 112 whose functional mutation occurs in this gene . The mutant virus is defective at nonpermissive temperatures in the latest stage of virus maturation . Both the wild-type and ts 112 recombinant proteins are produced in E . coli in an insoluble form, but are readily solubilized in urea . They have the same molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), they sediment as a monomeric species in sucrose gradient centrifugation, and proteolytic digestion reveals a similar pattern for both proteins . Hydrodynamic studies and electron microscopy show that both proteins have an elongated shape, which can be approximated to a cylinder of 20 nm in length and 2.8 nm in diameter . The only well-established difference between the mutant and the wild-type recombinant protein is the higher solubility of the mutant.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1960 - 4
Reconstitution of protein translocation from detergent-solubilized Escherichia coli inverted vesicles: PrlA protein-deficient vesicles efficiently translocate precursor proteins; Watanabe M et al.; Proteoliposomes were reconstituted by detergent dialysis of a sodium cholate extract of inverted vesicles derived from Escherichia coli plasma membrane . The translocation of precursor proteins into reconstituted vesicles occurred at high efficiency and was SecB dependent . The protein composition of the reconstituted vesicles differed markedly from that of native vesicles . Immunoblot analysis of the sodium cholate extract and of the reconstituted vesicles indicated that PrlA (SecY) protein remained largely unsolubilized under the described conditions and was virtually absent from the reconstituted vesicles, suggesting that PrlA may not be required for in vitro translocation.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1950 - 4
Prediction of the three-dimensional structure of Escherichia coli 30S ribosomal subunit: a molecular mechanics approach; Malhotra A et al.; We introduce a computer-assisted procedure for folding large RNA chains into three-dimensional conformations consistent with their secondary structure and other known experimental constraints . The RNA chain is modeled using pseudoatoms at different levels of detail--from a single pseudoatom per helix to a single pseudoatom for each nucleotide . A stepwise procedure is used, starting with a simple representation of the macromolecule that is refined and then extrapolated into higher resolution for further refinement . The procedure is capable of folding different random-walk chains by using energy minimization, allowing generation of a range of conformations consistent with given experimental data . We use this procedure to generate several possible conformations of the 16S RNA in the 30S ribosomal subunit of Escherichia coli by using secondary structure and the neutron-scattering map of the 21 proteins in the small subunit . The RNA chain is modeled using a single pseudoatom per helix . RNA-RNA and RNA-protein crosslinks, reported in current literature, are included in our model . Footprinting data for different ribosomal proteins in the 16S RNA are also used . Several conformations of the 16S RNA are generated and compared to predict gross structural features of the 30S subunit as well as to identify regions of the 16S RNA that cannot be well-defined with current experimental data.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1937 - 41
Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction; MacFerrin KD et al.; We report an efficient, general approach for the construction of protein-overproducing strains of Escherichia coli . The method, expression-cassette polymerase chain reaction (ECPCR), allows the insertion of virtually any contiguous coding sequence between sequences that direct high-level protein biosynthesis in E . coli . The gene expression cassettes obtained by ECPCR are inserted into a regulated overexpression plasmid, and the resulting construct is used to transform E . coli . By effecting simultaneous 5' and 3' modification of a coding sequence, ECPCR permits the facile generation of mutant proteins having N- and/or C-terminal truncations . The method is a highly efficient way to dissect a multidomain protein into its component domains . The efficiency of the ECPCR approach is demonstrated in this study by construction of permuted overexpression vectors for the first two extracellular domains of the human CD4 protein.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1879 - 83
Modular mutagenesis of human placental ribonuclease inhibitor, a protein with leucine-rich repeats; Lee FS et al.; Human placental ribonuclease inhibitor (PRI) is a potent protein inhibitor of pancreatic ribonucleases and the homologous blood vessel-inducing protein angiogenin . Although inhibition by PRI occurs with a 1:1 stoichiometry, its primary structure is composed predominantly of seven internal leucine-rich repeats . These internal repeats were systematically deleted either singly or in combination by "modular" mutagenesis . Deletion of repeat units 3 plus 4 or repeat unit 6 results in mutants that both bind to and inhibit ribonuclease A . Therefore, the angiogenin/ribonuclease binding site in PRI must reside primarily or entirely in repeats 1, 2, 5, or 7, the short N- or C-terminal segments, or a combination of these . Deletion of repeat units 3-5, 5-6, or 5 alone results in mutants that exhibit only binding activity . Hence, the binding site cannot reside exclusively in repeat 5 . Other internal deletions or N- or C-terminal deletions of 6-86% of the protein all abolish activity . These results suggest that PRI has a modular structure, with one primary structural repeat constituting one module . The approach taken may be applicable to other proteins with repeat structures.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1870 - 3
lac repressor: crystallization of intact tetramer and its complexes with inducer and operator DNA; Pace HC et al.; The intact lac repressor tetramer, which regulates expression of the lac operon in Escherichia coli, has been crystallized in the native form, with an inducer, and in a ternary complex with operator DNA and an anti-inducer . The crystals without DNA diffract to better than 3.5 A . They belong to the monoclinic space group C2 and have cell dimensions a = 164.7 A, b = 75.6 A, and c = 161.2 A, with alpha = gamma = 90 degrees and beta = 125.5 degrees . Cocrystals have been obtained with a number of different lac operator-related DNA fragments . The complex with a blunt-ended 16-base-pair strand yielded tetragonal bipyramids that diffract to 6.5 A . These protein-DNA cocrystals crack upon exposure to the gratuitous inducer isopropyl beta-D-thiogalactoside, suggesting a conformational change in the repressor-operator complex.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1638 - 42
Design of a membrane transport protein for fluorescence spectroscopy; Menezes ME et al.; To modify the lac permease of Escherichia coli for fluorescence spectroscopy, six tryptophan residues at positions 10, 33, 78, 151, 171, and 223 were first replaced individually with phenylalanine by using oligonucleotide-directed site-specific mutagenesis . None of the tryptophan residues is critical for activity, as evidenced by the finding that the mutant permease molecules catalyze lactose/H+ symport almost as well as wild-type permease . Subsequently, a permease molecule was designed in which all of the tryptophan residues were replaced with phenylalanine . Remarkably, the lac permease harboring all six mutations catalyzes active lactose transport about 75% as well as wild-type permease . The fluorescence emission spectrum of purified wild-type permease solubilized in octyl beta-D-glucopyranoside and phospholipid exhibits a broad maximum centered at 350 nm, and the peak is almost completely absent from the spectrum of permease devoid of tryptophan . Furthermore, a new maximum centered at about 306 nm is apparent in the spectrum of the modified permease, suggesting that tyrosine fluorescence in the native protein is quenched by internal energy transfer to tryptophan residues . By using site-directed mutagenesis to replace specified residues in the molecule without tryptophan, it should now be possible to utilize tryptophan fluorescence spectroscopy to study static and dynamic aspects of permease structure and function.

Mutat Res, 1990 Mar, 235(2), 147 - 56
UvrABC nuclease complex repairs thymine glycol, an oxidative DNA base damage; Kow YW et al.; The UvrABC nuclease complex recognizes a wide spectrum of DNA lesions including pyrimidine dimers, bulky chemical adducts and O6-methylguanine . In this study we have demonstrated that the UvrABC complex is also able to incise PM2 DNA containing the oxidative DNA lesion, thymine glycol . However, DNA containing dihydrothymine, a lesion with a similar structure to thymine glycol, was not incised . The UvrABC complex was also able to incise DNA containing reduced apurinic sites or apurinic sites modified with O-alkyl hydroxylamines, but not DNA containing apurinic sites or urea residues . In vivo, in the absence of base-excision repair, nucleotide excision repair was operable on phi X-174 RF transfecting DNA containing thymine glycols . The level of the repair was found to be directly related to the level of the UvrABC complex . Thus, UvrABC-mediated nucleotide excision repair appears to play a role in the repair of thymine glycol, an oxidative DNA-base lesion that is produced by ionizing radiation or formed during oxidative respiration.

J Bacteriol, 1990 Mar, 172(3), 1509 - 15
Molecular cloning of the C-terminal domain of Escherichia coli D-mannitol permease: expression, phosphorylation, and complementation with C-terminal permease deletion proteins; White DW et al.; We have subcloned a portion of the Escherichia coli mtlA gene encoding the hydrophilic, C-terminal domain of the mannitol-specific enzyme II (mannitol permease; molecular mass, 68 kilodaltons {kDa}) of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system . This mtlA fragment, encoding residues 379 to 637 (residue 637 = C terminus), was cloned in frame into the expression vector pCQV2 immediately downstream from the lambda pr promoter of the vector, which also encodes a temperature-sensitive lambda repressor . E . coli cells carrying a chromosomal deletion in mtlA (strain LGS322) and harboring this recombinant plasmid, pDW1, expressed a 28-kDa protein cross-reacting with antipermease antibody when grown at 42 degrees C but not when grown at 32 degrees C . This protein was relatively stable and could be phosphorylated in vitro by the general phospho-carrier protein of the phosphotransferase system, phospho-HPr . Thus, this fragment of the permease, when expressed in the absence of the hydrophobic, membrane-bound N-terminal domain, can apparently fold into a conformation resembling that of the C-terminal domain of the intact permease . When transformed into LGS322 cells harboring plasmid pGJ9-delta 137, which encodes a C-terminally truncated and inactive permease (residues 1 to ca . 480; molecular mass, 51 kDa), pDW1 conferred a mannitol-positive phenotype to this strain when grown at 42 degrees C but not when grown at 32 degrees C . This strain also exhibited phosphoenolpyruvate-dependent mannitol phosphorylation activity only when grown at the higher temperature . In contrast, pDW1 could not complement a plasmid encoding the complementary N-terminal part of the permease (residues 1 to 377) . The pathway of phosphorylation of mannitol by the combined protein products of pGJ9-delta 137 and pDPW1 was also investigated by using N-ethylmaleimide to inactivate the second phosphorylation sites of these permease fragments (proposed to be Cys-384) . These results are discussed with respect to the domain structure of the permease and its mechanism of transport and phosphorylation.

J Bacteriol, 1990 Mar, 172(3), 1491 - 8
Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins; Jennings MP et al.; Escherichia coli contains two L-asparaginase isozymes: L-asparaginase I, a low-affinity enzyme located in the cytoplasm, and L-asparaginase II, a high-affinity secreted enzyme . A molecular genetic analysis of the gene (ansA) encoding the former enzyme has previously been reported . We now present a molecular study of the gene, ansB, encoding L-asparaginase II . This gene was isolated by using oligonucleotide probes, whose sequences were based on the previously determined amino acid sequence . The nucleotide sequence of ansB, including 5'- and 3'-untranslated regions, was determined . The amino acid sequence of L-asparaginase II, deduced from this nucleotide sequence, contains differences at 11 positions when compared with the previously determined amino acid sequence . The deduced amino acid sequence also reveals a typical secretory signal peptide of 22 residues . A single region of sequence similarity is observed when ansA and ansB are compared . The transcriptional start site in ansB was determined, allowing the identification of the promoter region . The regulation of ansB was studied by using ansB'-'lacZ fusions, together with a deletion analysis of the 5' region upstream of the promoter . Regulation by cyclic AMP receptor protein and anaerobiosis (FNR protein) was confirmed, and the presence of nucleotide sequence motifs, with homology to cyclic AMP receptor protein and FNR protein-binding sites, investigated.

J Bacteriol, 1990 Mar, 172(3), 1385 - 91
Purified Escherichia coli F-factor TraY protein binds oriT; Lahue EE et al.; The traY gene of the Escherichia coli F plasmid has been shown by genetic studies (R . Everett and N . Willetts, J . Mol . Biol . 136:129-150, 1980) to be involved in the site-specific nicking reaction at oriT required for the initiation of DNA transfer during bacterial conjugation . In order to assign a biochemical function to TraY protein, the traY gene was cloned in a plasmid vector which utilizes the strong T7 phi 10 promoter to overproduce the protein . The plasmid-encoded TraY protein was specifically labeled with {35S}methionine, and purification of the polypeptide was accomplished by monitoring the radioactive label . Purified TraY protein had a relative molecular mass of approximately 17,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . The amino terminus of the purified protein was sequenced to confirm that the protein was encoded by the traY gene . The protein sequence revealed that the start codon for the TraY protein was a UUG codon 36 base pairs upstream of the AUG start site originally deduced from the DNA sequence (T . Fowler, L . Taylor, and R . Thompson, Gene 26:79-89, 1983) . This start sequence confirmed the premise of Inamoto et al . that the F-plasmid TraY polypeptide-coding sequence would begin with UUG, creating a reading frame which renders a large degree of amino acid sequence identity with the TraY polypeptide from R100 (S . Inamoto, Y . Yoshioka, and E . Ohtsubo, J . Bacteriol . 170:2749-2757, 1988) . The purified TraY protein from F bound specifically to the origin of transfer region of the F plasmid . However, no nicking activity was detected at oriT by using TraY protein or TraY protein in conjunction with helicase I.

Infect Immun, 1990 Mar, 58(3), 761 - 5
Protein phosphorylation by protein kinase C in HEp-2 cells infected with enteropathogenic Escherichia coli; Baldwin TJ et al.; Infection of HEp-2 monolayers with enteropathogenic Escherichia coli 2036-80 (O119) stimulated phosphorylation of several target cell proteins, the most prominent of which had apparent molecular weights of 21,000 and 29,000 . Proteins of the same size were phosphorylated in response to known activators of the calcium-phospholipid-dependent protein kinase C . Screening of clinical isolates of various O serogroups revealed that all strains able to form the characteristic attaching and effacing lesion of enteropathogenic E . coli showed elevated phosphorylation of 21,000- and 29,000-dalton protein species.

Infect Immun, 1990 Mar, 58(3), 732 - 9
Construction and characterization in vivo of Bordetella pertussis aroA mutants; Roberts M et al.; A DNA fragment encoding a kanamycin resistance determinant was used to insertionally inactivate the cloned aroA gene of Bordetella pertussis in Escherichia coli K-12, and a conjugative shuttle vector system based on the suicide vector pRTP1 was used to deliver the mutations from E . coli back into B . pertussis CN2992FS and BP1 . The aroA mutation was introduced by allelic exchange into the chromosome of B . pertussis, resulting in otherwise isogenic parental and aroA mutant pairs . The B . pertussis aroA mutants grew well on laboratory medium supplemented with aromatic compounds but failed to grow on unsupplemented medium . The B . pertussis aroA mutants expressed the normal B . pertussis extracellular, virulence-associated proteins; inactivated, whole-cell vaccines prepared from the mutants protected mice as efficiently as vaccines made from the parent strains against intracerebral challenge with the virulent B . pertussis 18323 . Live B . pertussis aroA bacteria inefficiently colonized the lungs of NIH/S mice after they were challenged with aerosol, unlike the wild-type B . pertussis organism . Mice exposed to three separate aerosols of live B . pertussis aroA bacteria were protected against lung colonization after being exposed to an aerosol containing the virulent parental B . pertussis strain . High-level antibodies against B . pertussis rapidly appeared in the sera of mice immunized by aerosol with the B . pertussis aroA strains and challenged with the virulent parent.

FASEB J, 1990 Mar, 4(5), 1450 - 9
Cobalamin-dependent methionine synthase; Banerjee RV et al.; Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from N5-methyltetrahydrofolate to homocysteine, producing tetrahydrofolate and methionine . Insufficient availability of cobalamin, or inhibition of methionine synthase by exposure to nitrous oxide, leads to diminished activity of this enzyme . In humans, severe inhibition of methionine synthase results in the development of megaloblastic anemia, and eventually in subacute combined degeneration of the spinal cord . It also results in diminished intracellular folate levels and a redistribution of folate derivatives . In this review, we summarize recent progress in understanding the catalysis and regulation of this important enzyme from both bacterial and mammalian sources . Because inhibition of mammalian methionine synthase can restrict the incorporation of methyltetrahydrofolate from the blood into cellular folate pools that can be used for nucleotide biosynthesis, it is a potential chemotherapeutic target . The review emphasizes the mechanistic information that will be needed in order to design rational inhibitors of the enzyme.

Chest, 1990 Mar, 97(3), 751 - 2
Cholecystopleural fistula with cholelithiasis presenting as a right pleural effusion; Cunningham LW et al.; At autopsy, multiple gallstones were recovered from the right pleural space of an elderly patient who presented with a massive right pleural effusion and septic shock . The mechanisms of gallstone migration and fistula formation between the gallbladder and right pleural space are described . Despite atypical presentations, gallbladder disease remains an important differential consideration of right pleural effusion in the elderly.

Southeast Asian J Trop Med Public Health, 1990 Mar, 21(1), 21 - 7
Specific DNA probe for the sensitive detection of Trypanosoma evansi; Viseshakul N et al.; Trypanosoma evansi is the parasitic protozoon that causes "Surra", a wasting disease of domestic animals . Detection of T . evansi plays an important role in epidemiology and animal health . DNA probes were constructed from T . evansi genomic DNA and kinetoplast DNA for sensitive detection of the parasite in infected blood . A 6.5 kb DNA insert of pMUT ec6 plasmid derived from the genomic DNA of T . evansi Npl isolate, selected from 575 recombinant E . coli exhibited the strongest nucleic acid hybridization signal to the T . evansi DNA . Using as the DNA probe, pMUT ec6 could detect as little as 60 pg T . evansi DNA and it did not hybridize to the DNA of cattle, waterbuffalo and two related blood parasites . A simple detection procedure by spotting 10 microliters infected blood onto nylon membrane could sense as little as 1000 parasites . The kinetoplast DNA was cloned in E . coli and found to show a comparable sensitivity to that of the pMUT ec6 . However, the kinetoplast DNA exhibited variation in copy number among parasite isolates thus pMUT ec6 should be the DNA probe of choice for sensitive detection of T . evansi.

Rinsho Byori, 1990 Mar, 38(3), 288 - 93
{A parathyroid hormone related protein (PTHrP) implicated in hypercalcemia associated with malignancy: research of the PTHrP for novel hormonal tumor marker}; Wada C et al.; A cDNA complementary to the parathyroid hormone related protein (PTHrP), the humoral hypercalcemia factor in malignancy, was recently isolated and sequenced . PTHrP expression in human carcinomas was examined (determined) by Northern blot hybridization, Southern blot hybridization and radioimmunoassay (RIA) . Expression of PTHrP mRNA was detected in three out of four lung squamous cell carcinomas, two out of ten breast carcinomas and the one adenosquamous carcinoma of the maxilla . No clinical hypercalcemia was found in these PTHrP mRNA positive carcinomas . No expression of PTHrP was detected in normal human or rat tissues . In Southern blot hybridization, no amplification of PTHrP gene was found in PTHrP positive cases . An insertion in one allele of promotor region of the gene was identified in one PTHrP positive lung squamous cell carcinoma . The serum level of PTHrP was examined using human PTHrP (1-34) RIA in lung carcinomas . We found no correlation between the level of PTHrP and clinical hypercalcemia or the histopathological diagnosis . We discuss some problems of the PTHrP assay as a novel tumor marker for malignancies . A new RIA assay study using recombinant human PTHrP expressed in Escherichia coli is also reported.

J Med Virol, 1990 Mar, 30(3), 163 - 8
Speed of progression to AIDS and degree of antibody response to accessory gene products of HIV-1; Reiss P et al.; Antibodies to E . coli-produced HIV-1 nef, rev, tat, vpu, and vpr proteins were measured by enzyme immunoassay in serial sets of sera from 72 men seroconverting for antibodies to HIV-1 structural proteins, and from 190 initially symptom-free men who were seropositive for these antibodies at entry into the study . In the men seroconverting for antibodies to structural proteins the levels of nef-, rev-, and tat-specific antibodies, but not of vpu-, and vpr-specific antibodies, within 3 months of seroconversion, appeared to be lower in the five men progressing to AIDS, compared with the men remaining symptom-free during follow-up . Analysis of the prevalence of previously described antibody profiles to these accessory gene products was carried out . In all HIV-1 antibody seroconverters and in those HIV-1 antibody seropositive men with 15 or more months of follow-up who progressed to AIDS, there was a shift from predominantly nef- and vpu-specific antibody negative profiles in the men developing AIDS in the early years of the study to predominantly nef- and vpu-specific antibody positive profiles in men who developed AIDS later . Rev- and tat-specific antibody negative profiles were dominant in men progressing to AIDS throughout follow-up . No vpr-specific antibody profile occurred preferentially in the men progressing to AIDS throughout follow-up . Low antibody reactivity to accessory gene products nef, rev, and tat appears, like low anti-core antibody reactivity, to be associated with progression to AIDS relatively rapidly after infection with HIV-1.

J Dairy Sci, 1990 Mar, 73(3), 627 - 32
Physiological responses to intramammary or intravenous treatment with endotoxin in lactating dairy cows; Jackson JA et al.; Twenty-one, middle to late lactation Holstein cows were assigned to one of three treatments in a completely randomized design to examine physiological changes associated with intramammary or intravenous administration of Escherichia coli endotoxin . Treatments were 1) Hank's balanced salt solution infusion in two contralateral quarters (control), 2) E . coli endotoxin infusion in two contralateral quarters, and 3) intravenous infusion of E . coli endotoxin . Blood was sampled and rectal temperature was measured at 30-min intervals . Endotoxin treatment was at 0900 h and sampling continued until 1700 h . Serum prolactin, cortisol, and 13,14-dihydro-15-keto-prostaglandin F2 alpha were measured . A pyretic response was observed in intravenous and intramammary treatment groups after endotoxin treatment . Response peak was higher (41.1 vs . 40.3 degrees C) and occurred later (6 vs . 4.5 h posttreatment) in the intramammary than the intravenous treatment group . Significant prolactin peaks were observed also in intravenous and intramammary endotoxin treatment groups . Prolactin peaked higher (288 vs . 112 ng/ml) and occurred sooner (1 vs . 4 h posttreatment) in the intravenous than in the intramammary treatment group . Cortisol followed a trend similar to prolactin . Cortisol peaked higher (100 vs . 82 ng/ml) and sooner (2.5 vs . 4.5 h posttreatment) in the intravenous than in the intramammary treatment group . Concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha increased rapidly posttreatment in the intravenous group only.

Biol Chem Hoppe Seyler, 1990 Mar, 371(3), 215 - 22
Synthesis of bioactive seminalplasmin by expression of a newly constructed fusion gene; Preuss KD et al.; A synthetic DNA, carrying the coding sequence for seminalplasmin (SAP), the major basic protein of bull semen, was cloned into the C-terminal part of a shortened, mutated fragment of the lacZ gene (lacZ-MF) of vector pLZPWB1 . As a result of the mutation, all methionine as well as cysteine residues are replaced by other amino-acid residues . In the fusion gene lacZ-MF-SAP of the resulting construct pSAP4 the two proteins are linked through a methionine residue . Expression of pSAP4 in E . coli W3110 in the presence of the inducer isopropylthiogalactoside (IPTG) led to production of fusion protein with a yield of approximately 50% of the total proteins synthesized . All SAP-immunoreactive fusion protein was found within the insoluble protein fraction and represented 40% of total proteins produced during expression . The fusion protein was subjected to cyanogen bromide cleavage . The overall yield of crude SAP with a purity of 80% was 10 mg/l of culture . The crude SAP was further purified by calmodulin-Sepharose affinity absorption . Characterisation by protein chemical analysis indicated the identity of recombinant SAP with authentic SAP purified from bull semen.

Mol Reprod Dev, 1990 Mar, 25(3), 302 - 8
Molecular cloning of an acrosomal sperm antigen gene and the production of its recombinant protein for immunocontraceptive vaccine; Liu MS et al.; A monoclonal antibody, HS-63, which reacts specifically with a highly conserved sperm acrosome antigen, was shown to inhibit in vitro fertilization of mouse and human . The corresponding sperm antigen designated as MSA-63 was purified to homogeneity from mouse testes and used as an immunogen to generate polyclonal antisera in rabbits . The cDNA fragments of MSA-63 gene were cloned from mouse testis cDNA library by an immunoscreening method using polyclonal antisera specific for MSA-63 . Using the established cDNA clone as a probe, the gene encoding for MSA-63 protein was found to be conserved among different mammalian species . Only one specific mRNA 1.5 kb in size was identified from the adult mouse testis among different mouse tissues . The recombinant fusion protein containing MSA-63 protein fragment was produced in Escherichia coli and used to immunize female mice . Similar to the original HS-63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition to the in vitro fertilization of mouse oocytes . The results of this preliminary study suggest that it is feasible to mass produce sperm-specific antigens or their antigenic fragments by recombinant DNA technology for the development of sperm antigen-based immunocontraceptive vaccines.

Neuron, 1990 Mar, 4(3), 469 - 76
Visinin: a novel calcium binding protein expressed in retinal cone cells; Yamagata K et al.; Visinin is a retinal cone cell-specific protein (molecular weight 24,000, pI 5.1) . To investigate its function, visinin cDNA was isolated from a chick retinal lambda gt11 cDNA library, using anti-visinin serum . The beta-galactosidase-visinin fusion protein was used for purifying epitope-selected antibody . The purified visinin antibody reacted only with a 24 kd protein in retinal cone cells . Visinin mRNA was expressed only in the retinal photoreceptor layer . The nucleotide sequence of the cDNA revealed that visinin has three E-F hand structures and is a Ca2+ binding protein . Visinin protein expressed in E . coli exhibited Ca2+ binding activity . These results suggest that visinin is a photoreceptor-specific Ca2+ binding protein and may be involved in phototransduction in the cone cells.

Proc Natl Acad Sci U S A, 1990 Mar, 87(6), 2369 - 73
FlbD of Caulobacter crescentus is a homologue of the NtrC (NRI) protein and activates sigma 54-dependent flagellar gene promoters; Ramakrishnan G et al.; The periodic transcription of flagellar genes in the Caulobacter crescentus cell cycle is controlled, in part, by their organization in a regulatory hierarchy . The flbG (hook operon), flaN, and flagellin gene operons, which are at the lowest levels of the hierarchy and expressed late in the cell cycle, contain Ntr-like promoters . We report that flbD, one of the early genes required in trans for expression of these operons, codes for a 52-kDa protein homologous to the transcriptional activators NtrC (NRI), NifA, DctD, HydG, and XylR . Our results show that in Escherichia coli flbD partially complements glnG (ntrC) mutations and stimulates transcription of the C . crescentus sigma 54 RNA polymerase-dependent flbG gene . Additionally, the sequence predicts that FlbD protein, along with NtrC, DctD, and HydG proteins, is structurally related at the amino-terminal domain to a larger family of response regulators that mediate cellular responses to environmental stimuli . FlbD may be a singular member of this large protein family in that its function is tied to an internal cell-cycle signal . FlbD is also unusual in that its amino-terminal domain contains only one of the three residues conserved in previously described members of this family of response regulators.

Proc Natl Acad Sci U S A, 1990 Mar, 87(6), 2047 - 51
Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector; Casadei J et al.; A fusion protein has been expressed from the relevant genes in mammalian cells consisting of the photoprotein aequorin and an anti-4-hydroxy-3-nitrophenacetyl antibody gene . This chimeric antibody has allowed the development of a sensitive luminescent immunoassay . Initially the cDNA of the photoprotein aequorin from Aequorea victoria was cloned and expressed in Escherichia coli . The gene was expressed as apoaequorin and, by using luciferin isolated from Renilla reniformis, its activity was found essentially identical to native aequorin . The aequorin gene was subcloned into a mammalian expression vector to produce a fusion protein directing secretion of apoaequorin; the aequorin gene was fused to the 3' terminus of an immunoglobulin heavy-chain gene that directed expression of an anti-4-hydroxy-3-nitrophenacetyl antibody . The gene fusion contained the variable region, the constant region domain 1, and part of domain 2 for the IgG2b mouse immunoglobulin, followed by the aequorin gene . Transfection of the chimeric gene into a cell line expressing the complementary lambda 1 light chain, J558L, allowed recovery of a chimeric antibody with binding specificity for the 4-hydroxy-3-nitrophenacetyl group and the related 4-hydroxy-3-iodo-5-nitrophenacetyl hapten . The Ca2(+)-dependent bioluminescent activity of aequorin was also recovered.

J Surg Res, 1990 Mar, 48(3), 249 - 53
Muramyl dipeptide improves mononuclear phagocyte system function in obstructive jaundice; Dunn CW et al.; Previous studies have demonstrated depression of the mononuclear phagocyte system (MPS) of which the liver comprises 80-85% in animals subjected to 21 days of obstructive jaundice . This study examined the ability of a macrophage stimulant, muramyl dipeptide (MDP), to reverse MPS dysfunction in an obstructive jaundice rat model . Sixty-two male Sprague-Dawley rats underwent sham (n = 29) laparotomy or common duct ligation (CDL) (n = 33) and were studied after 21 days . Animals were injected with 1-3.5 X 10(6) Escherichia coli via a lateral tail vein, and colony-forming units (CFU) of the liver, lung, and spleen were determined at two time intervals: 30 min postinjection to determine the phagocytic activity of MPS (sham, n = 16; CDL, n = 20) and 24 hr postinjection to determine cytotoxic activity of MPS (sham, n = 13; CDL, n = 13) . MDP (3 micrograms/g) was administered subcutaneously 24 hr prior to E . coli injection in 6 sham and 10 CDL rats studied at the 30-min time interval and 7 sham and 7 CDL rats studied at the 24-hr time interval . Pretreatment with MDP appeared to reverse the impairment of phagocytic activity in the liver of CDL rats returning it to the level of sham animals (P less than 0.05) . However, pretreatment with MDP did not enhance the cytotoxic activity of the MPS as evidenced by higher CFU of E . coli in the liver, lung, and spleen of CDL animals pretreated with MDP as compared to CDL animals that did not receive MDP pretreatment . This increase was only significant in the spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

J Surg Res, 1990 Mar, 48(3), 196 - 203
Kinetics of leukocyte sequestration in the lungs of acutely septic primates: a study using 111In-labeled autologous leukocytes; Hangen DH et al.; To further clarify the role of leukocytes in the pathogenesis of ARDS, we studied the localization and kinetics of leukocyte migration using 111In-labeled autologous white cell scans (111In wbc scans) in four primates made acutely septic with infusions of Escherichia coli . Whole body images were obtained with a gamma camera and were acquired on computer every 15 min beginning immediately after the E . coli infusion . Simultaneous measurements of C5a and peripheral blood leukocyte count were also obtained . Within 5 min of initiating sepsis, three major events occurred: complement activation as measured by the production of C5a, a profound fall in peripheral leukocyte count, and a significant increase in the sequestration of leukocytes in the lungs . The pulmonary sequestration reached a peak at 15 min with a mean of 152% of baseline activity . This sequestration consisted of a population that was predominantly neutrophils . Damage to the pulmonary capillary endothelium was demonstrated by an increase in extravascular lung water . The results support a role for neutrophils and complement as mediators in the pathogenesis of ARDS.

Carcinogenesis, 1990 Mar, 11(3), 431 - 6
Studies in gastric carcinogenesis . IV . O6-methylguanine and its repair in normal and atrophic biopsy specimens of human gastric mucosa . Correlation of O6-alkylguanine-DNA alkyltransferase activities in gastric mucosa and circulating lymphocytes; Kyrtopoulos SA et al.; DNA extracted from biopsies of normal or atrophic gastric mucosa obtained from 20 individuals was analysed for the presence of the precarcinogenic alkylation lesion O6-methylguanine by a recently developed, highly sensitive assay based on repair by the Escherichia coli O6-alkylguanine-DNA alkyltransferase (AGT) enzyme in competition with a radiolabelled oligonucleotide containing O6-methylguanine (O6-meG) . With a limit of detection of 0.5 fmol O6-meG in 10 micrograms DNA, only one DNA sample (derived from a region of the stomach with advanced chronic atrophic gastritis) was found marginally positive, containing 0.52 fmol/10 micrograms DNA (8.3 X 10(-8) mol O6-meG/mol guanine) . Measurements of AGT in 49 biopsies of normal, atrophic, hyperplastic or dysplastic mucosa obtained from the gastric antrum or corpus of 18 individuals did not reveal any significant effects of mucosal histology on AGT . The average AGT value found was 6.9 +/- 3.5 (SD) fmol/micrograms DNA, which is lower than the values reported for a number of other human tissues (liver, small intestine and lung) . Measurement of AGT levels in gastric mucosa and circulating lymphocytes of the same individuals revealed a positive correlation (P less than 0.005), suggesting that lymphocytes may serve as a useful surrogate marker for AGT activity in gastric mucosa in studies of the epidemiology of this important repair enzyme.

Virology, 1990 Mar, 175(1), 19 - 29
A 14,500 MW protein is coded by region E3 of group C human adenoviruses; Tollefson AE et al.; There is an ORF in the early region E3 transcription unit of human adenovirus 5 (Ad5) which could encode a protein of 14,500 MW (14.5K) . This ORF is conserved in Ad5 and Ad2, both group C adenoviruses, and also in Ad3 and Ad7, both group B adenoviruses . To address whether the 14.5K protein is synthesized, we prepared antisera against synthetic peptides corresponding to residues 19-34 or 118-132 in the Ad5 version of 14.5K, and also against a TrpE-14.5K fusion protein expressed in Escherichia coli . These antisera immunoprecipitated the {35S}Met-labeled 14.5K protein from KB cells infected with rec700 (an Ad5-Ad2-Ad5 recombinant), Ad2, and a variety of E3 mutants . Mutants in the 14.5K ORF did not produce the 14.5K protein . The 14.5K is coded in large part, although probably not exclusively, by E3 mRNA f, as indicated by immunoprecipitation of 14.5K from cells infected with mutants that overproduce or underproduce mRNA f . The 14.5K migrated as five to six bands on SDS-PAGE after immunoprecipitation or Western blot, suggesting that it undergoes post-translational modification . Two bands of 14.5K were obtained by cell-free translation of 14.5K from mRNA purified by hybridization from infected cells.

J Exp Med, 1990 Mar 1, 171(3), 889 - 96
Association between protective efficacy of anti-lipopolysaccharide (LPS) antibodies and suppression of LPS-induced tumor necrosis factor alpha and interleukin 6 . Comparison of O side chain-specific antibodies with core LPS antibodies; Baumgartner JD et al.; Two-core LPS antibodies, the rabbit J5 polyclonal antiserum and the human anti-lipid A IgM mAb HA-1A, did not improve the survival of mice challenged with E . coli O111 or P . aeruginosa 3, or with the LPS extracted from them, and did not decrease the incidence of Shwartzman reactions in rabbits challenged with O111 LPS . In contrast, O side chain-specific rabbit antisera were protective in these models . The protection afforded by O side chain-specific antisera against endotoxin lethality was associated with decreased LPS-induced serum TNF and IL-6 levels, whereas core LPS antibodies had no effect on TNF or IL-6 levels . The absence of reduction of LPS-induced cytokines levels by core LPS antibodies suggests that these antibodies are not able to prevent the interactions between LPS and target cells.

Mol Cell Biol, 1990 Mar, 10(3), 1084 - 94
Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression; Kapler GM et al.; To delineate segments of the genome of the human protozoan parasite Leishmania major necessary for replication and expression, we developed a vector (pR-NEO) which can be reproducibly introduced into L . major . This DNA was derived from a 30-kilobase extrachromosomal amplified DNA bearing the dihydrofolate reductase-thymidylate synthase gene, with the coding region for neomycin phosphotransferase substituted for that of dihydrofolate reductase-thymidylate synthase and a bacterial origin of replication and selectable marker added . G418-resistant lines were obtained at high efficiency by electroporation of pR-NEO (approaching 10(-4) per cell), while constructs bearing an inverted neo gene or lacking Leishmania sequences did not confer resistance . pR-NEO replicated in L . major and gave rise to correctly processed transcripts bearing the trans-spliced miniexon . Molecular karyotype analysis showed that in some lines pR-NEO DNA exists exclusively as an extrachromosomal circle, a finding supported by the rescue of intact pR-NEO after transformation of Escherichia coli . These data genetically localize all elements required in cis for DNA replication, transcription, and trans splicing to the Leishmania DNA contained within pR-NEO DNA and signal the advent of stable transfection methodology for addressing molecular phenomena in trypanosomatid parasites.

HPB Surg, 1990 Mar, 2(1), 7 - 12; discussion 12-3
Bile peritonitis in acute cholecystitis; Andersson R et al.; A review of all patients treated for acute cholecystitis (n = 5848) during an 18-year period (1969-1986) at two hospitals (one practising early surgery in patients with acute cholecystitis and the other not) disclosed that 104 (1.8%) had bile within the abdominal cavity at surgery; 71 with a visible perforation of the gallbladder and 33 without . The bile was infected in 82% of performed cultures (most commonly with Escherichia coli) . Mortality was 7.7% (8/104 patients), being 20% (4/20) in the hospital practising delayed surgery and 5% (4/84) in the hospital practising early surgery (p less than 0.10) . Infectious complications were responsible for the deaths by leading to multiple organ failure with pulmonary or renal insufficiency or gastro-intestinal bleeding . The timing of surgery was the only factor that had prognostic significance, i.e . the longer the hospital delay before surgery the higher the mortality, although elderly patients or patients with perforation tended to have a worse prognosis . In conclusion, the results of this study indicated that early surgery is important in patients with acute cholecystitis as a means of lowering mortality in bile peritonitis in this condition.

Res Microbiol, 1990 Mar-Apr, 141(3), 374 - 83
A consensus structure for membrane transport; Maloney PC; Combined information from biochemical and molecular biological experiments reveals a consistent structural rhythm that underlies the construction of all membrane carriers and perhaps all transport systems . Biochemical work shows that while some carrier proteins function as monomers, others operate as dimers . But despite this variation, all examples can be modelled as having a pair of membrane-embedded domains, each of which contains an array of (about) six transmembrane helical elements . This pattern is best documented among membrane carriers, where the minimal functional unit is known in a reasonable number of cases . Nevertheless, the same conclusion is likely to characterize other solute transporters . These unexpected correlations suggest that all membrane carriers, including those that take part in "energy coupling", have a uniform structural design on which is superimposed a variety of kinetic and biochemical mechanisms.

J Gen Microbiol, 1990 Mar, 136 ( Pt 3), 489 - 93
Biological properties of lipopolysaccharides from Bordetella species; Watanabe M et al.; Biological activities of lipopolysaccharides (LPS) extracted from Bordetella pertussis, B . parapertussis and B . bronchiseptica were compared with those of Escherichia coli LPS . The LPS preparations from B . pertussis showed biological activities comparable to those of E . coli LPS in terms of lethal toxicity in galactosamine-sensitized mice, pyrogenicity in rabbits, mitogenicity in C3H/He spleen cell cultures, macrophage activation, and induction of tumour necrosis factor . All the activities of LPS preparations from B . parapertussis, except mitogenicity, were lower than those of E . coli LPS . LPS from B . parapertussis gave the greatest mitogenic action of all those tested . Biological activities stronger than or comparable to those of E . coli LPS were observed for LPS from B . bronchiseptica.

Actas Urol Esp, 1990 Mar-Apr, 14(2), 139 - 42
{Primary abscess of the psoas}; Martinez-Sagarra Oceja JM et al.; We present in this paper a series of cases from our Service of primary abscesses in psoas . We consider the appearance of immunosuppressed patients as a relevant factor of its etiopathogenicity . We believe that a C.A.T . is the best diagnostic method . All three patients that underwent open surgery have had complications . The patients treated with percutaneous puncture has shown a favourable evolution.

Mikrobiol Zh, 1990 Mar-Apr, 52(2), 66 - 9
{A toxicological evaluation of micromycetes isolated from salmon roe}; Lemeshchenko GP et al.; Three species of micromycetes (Aspergillus clavatus Desm., Cladosporium herbarum (Pers) Lk., Penicillium canescens Sopp.) isolated from the cultivated salmon spawn have been studied for their toxigenic properties and pathogenicity for warm-blooded animals . LD50 are determined for mice perorally administered mycelium suspension of the first two species; the third of the studied species proved to be nontoxicogenic . The possible pathogenic action of mycotoxins on fish spawn in aquarium is discussed.

Zh Mikrobiol Epidemiol Immunobiol, 1990 Mar, (3), 20 - 3
{The transformation of legionellae by plasmid DNA}; Marakusha BI et al.; For the first time the possibility of the genetic transformation of L . pneumophila and L . bozemanii strains with the use of purified DNA of plasmids pUC19, pUC4K, pSC101 and RSF1010-pBR322 was shown . The frequency of transformation varied from 5.2 x 10(-6) to 5.8 x 10(-7), depending on the strain used in the experiment and plasmid DNA . In some of the transformants obtained in this investigation plasmid DNA whose molecular weight was similar to that of the plasmid DNA used for transformation was detected . The relatively stable preservation of plasmids pSC101 and RSF1010 in Legionella strains and the loss of plasmids pUC19, pUC4K and pBR322 in 80% of transformants during storage were shown.

Plasmid, 1990 Mar, 23(2), 159 - 62
A yeast-Escherichia coli shuttle vector containing the M13 origin of replication; Rhodes N et al.; A yeast-Escherichia coli shuttle vector containing the M13 origin of replication has been constructed . This vector allows selection and replication in both Saccharomyces cerevisiae and E . coli, as well as single-stranded packaging from E . coli upon infection with a helper phage . The presence of a polylinker with various unique restriction sites facilitates the cloning of desired genes.

Plasmid, 1990 Mar, 23(2), 149 - 54
Diversity of the Chlamydia trachomatis common plasmid in biovars with different pathogenicity; Comanducci M et al.; The 7.5-kb plasmid of Chlamydia trachomatis (CT) is believed to encode essential genes and might have a role in CT pathogenicity . Accordingly, analysis of plasmid-linked mutations in isolates from biovars with different pathogenic properties should help in identifying which plasmid-encoded genes, if any, may be involved in modulating virulence . For this purpose, the plasmid present in a low-virulence isolate (trachoma biovar, serotype D) was cloned and sequenced . Nucleotide changes were experimentally checked against the sequence of the plasmid variant from the highly virulent strain L2/434/Bu (LGV biovar) . By aligning our data with two published sequences of different trachoma and LGV variants a general consensus structure was determined, comprising eight major open reading frames (ORF) and a number of points where there is consensus only between isolates of the same biovar (biovar-specific mutations) . The degree of variation between different isolates is less than 1% . In particular, comparison of serotype-D and -L2 plasmids shows mutations which are generally silent or lead to few (one to four), often conservative, amino acid changes in ORFs 1, 2, 4, 5, 6, and 7 . The protein encoded by ORF8 is completely conserved . In contrast, the polypeptide variants encoded by ORF3 show nine amino acid changes, seven of which are due to biovar-specific mutations.

Mol Biol (Mosk), 1990 Mar-Apr, 24(2), 495 - 500
{Structural-functional organization of the par region of the ColN plasmid}; Kolot MN; In the 679 b.p . SalI-KpnI-fragment of the small colicinogenic plasmid Co1N, the par-region has been localized, functioning at the expense of resolution of plasmid DNA multimer forms . It has been shown that the replication process of the monomeric form of the recombinant plasmid containing the Co1N par-region do not result in formation of a considerable number of multimers . Gene xer A product is necessary for the functioning of the multimer resolution mechanism of Co1N as well as Co1E1 . Nucleotide sequence analysis of the Co1N par-region revealed the presence of essential homology with the par-locus of plasmid Co1E1 . Results obtained in this work and data from literature indicate that par-regions of the Co1E1-type plasmids possess considerable homology, function according to a similar mechanism and represent the universal stability module of multicopy colicinogenic plasmids.

Mol Biol (Mosk), 1990 Mar-Apr, 24(2), 460 - 6
{Study of the mechanism of mutagenesis directed by phosphotriester analogs of oligonucleotides . The role of repair in mutagenesis}; Petrenko VA et al.; The mutation system has been suggested in an effort to test insertion and deletion mutants by changing the Lac-phenotype of bacterial colonies transformed by mutant DNA . This system also makes possible to determine heterozygotes and homozygotes among the mutants . The yield of mutants in shown to depend on the structure of the DNA heteroduplex region . The yield of deletion mutants is greater than that of insertion mutants . Heterozygotes prevail in mutant colonies (greater than 90%).

Mol Biol (Mosk), 1990 Mar-Apr, 24(2), 438 - 47
{Cloning and regulation of gene expression of EcoRV restriction- modification system}; Kravets AN et al.; A number of recombinant plasmids, containing EcoRV restriction-modification genes have been constructed . Individual genes of this system were introduced into plasmids of various incompatibility groups . Promoter regions of genes encoding methylase and restrictase have been cloned and studied . With the use of specialized vector pVE8 it was shown that the efficiency of the endonuclease gene promoter is comparable with early lambda phage promoters and produced about 70% of PL efficiency . The efficiency of the methylase gene promoter region was twice less than the efficiency of the restriction endonuclease gene promoter . Plasmid with restriction endonuclease gene promoter located downstream in relation to the additional regulatable phage lambda promoter PL has been obtained . It enabled us to construct strains 30-40 fold overproducing this enzyme under conditions of inactivation of the temperature sensitive phage repressor c1857 . This construction directs the production of a high level (10%) of the total cellular soluble proteins) of the EcoRV restriction enzyme . The factors that influenced the level of enzyme synthesis under induction are discussed.

Z Urol Nephrol, 1990 Mar, 83(3), 129 - 31
{Fournier gangrene}; Sponholz F et al.; It is reported on symptoms, diagnostics and treatment of Fourniers gangrene . The early diagnosis is important for effective and successful treatment of this rare condition.

Mol Microbiol, 1990 Mar, 4(3), 505 - 11
Identification of a 180 kD protein in Escherichia coli related to a yeast heavy-chain myosin; Casaregola S et al.; A high molecular-weight protein from Escherichia coli sharing structural homology at the protein level with a yeast heavy-chain myosin encoded by the MYO1 gene is described . This 180 kD protein (180-HMP) can be enriched in cell fractions following the procedure normally utilized for the purification of non-muscle myosins . In Western blots this protein cross-reacts with a monoclonal antibody against yeast heavy-chain myosin . Moreover, antibodies raised against the 180 kD protein cross-react with the yeast myosin and with a myosin heavy chain from chicken . Recognition by anti-180-HMP antibodies of an overexpressed fragment of yeast myosin encoded by MYO1 allows the localization of one of the shared epitopes to a specific region around the ATP binding site of the yeast myosin heavy chain . The existence of a high molecular-weight protein with structural similarity to myosin in E . coli raises the possibility that such a protein might generate the force required for movement in processes such as nucleoid segregation and cell division.

Mol Microbiol, 1990 Mar, 4(3), 405 - 12
Activation of potassium efflux from Escherichia coli by glutathione metabolites; Elmore MJ et al.; The mechanism by which N-ethylmaleimide (NEM) elicits potassium efflux from Escherichia coli has been investigated . The critical factor is the formation of specific glutathione metabolites that activate transport systems encoded by the kefB and kefC gene products . Formation of N-ethyl-succinimido-S-glutathione (ESG) leads to the activation of potassium efflux via these transport systems . The addition of dithiothreitol and other reducing agents to cells reverses this process by causing the breakdown of ESG and thus removing the activator of the systems . Chlorodinitrobenzene, p-chloromercuribenzoate and phenylmaleimide provoke similar effects to NEM . lodoacetate, which leads to the formation of S-carboxymethyl-glutathione, does not activate the systems but does prevent the action of NEM . It is concluded that the KefB and KefC systems are gated by glutathione metabolites and that the degree to which they are activated is dependent upon the nature of the substituent on the sulphydryl group.

Mol Microbiol, 1990 Mar, 4(3), 381 - 92
Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus; Flannigan KA et al.; The de novo purine biosynthetic enzymes 5-amino-4-imidazolecarboxamide-ribonucleotide (AICAR) transformylase (EC 2.1.2.3), IMP cyclohydrolase (EC 3.5.4.10) and glycineamide-ribonucleotide (GAR) synthetase (EC 2.1.2.2) are encoded by the purHD locus of Escherichia coli . The DNA sequence of this locus revealed two open reading frames encoding polypeptides of Mr 57,335 and 45,945 (GAR synthetase), respectively, that formed an operon . The DNA sequence, maxicell and complementation analyses all supported the concept that the Mr 57,335 polypeptide is the product of the purH gene and encodes a bifunctional protein containing both AICAR transformylase and IMP cyclohydrolase activities . The 5' end of the purHD mRNA was determined by primer extension mapping and contains two regions of dyad symmetry capable of forming 'hairpin' loops where the formation of the one would prevent the formation of the other but not vice versa . Regulation by the purR gene product was explained by the discovery of a purR binding site in the purHD control region.

Mol Microbiol, 1990 Mar, 4(3), 355 - 63
Transcriptional analysis of the gene encoding pyruvate formate-lyase-activating enzyme of Escherichia coli; Sauter M et al.; The act gene of Escherichia coli encodes the pyruvate formate-lyase-activating enzyme which is necessary for the post-translational modification of pyruvate formate-lyase . The gene is located 191 bp downstream from the pfl structural gene . Northern blot analysis revealed that the act transcript is monocistronic and that transcription is independent of pfl gene expression . Through mapping of the 5' and 3' ends of the act transcript, sequences could be identified showing similarity to both an Escherichia coli sigma 70 promoter and to a rho-independent transcription terminator . Expression of the act gene was analysed with the aid of chromosomally integrated transcriptional and translational lacZ fusions . The results verified that the act gene is transcribed from its own promoter and that expression of the gene is essentially constitutive . Anaerobiosis led only to a two-fold increase in expression over that observed in aerobically grown cells and this elevated expression was independent of the transcriptional regulator, Fnr . Moreover, effectors such as pyruvate and nitrate, which substantially influence anaerobic transcription of the pfl gene, did not affect act gene expression.

Genetika, 1990 Mar, 26(3), 557 - 9
{Increase in stability of the recombinant phage M13 carrying Escherichia coli genes rpIJL by reducing expression of cloned genes}; Vudmaska MI et al.; A recombinant phage mp9MW/rpoB containing the BglII-B fragment of the Escherichia coli rplJL-rpoBC gene cluster was constructed on the basis of filamentous phage M13 . Stability of the phage was increased by insertion of a transcription terminator t beta' which blocked transcription of cloned genes from the Plac of the vector.

Genetika, 1990 Mar, 26(3), 433 - 42
{The effect of specific mutation crp a in the genetic locus of receptor protein cAMP (CRP) on the expression of Escherichia coli K-12 deo-operon}; Nechaeva GD et al.; Expression of the deo operon of Escherichia coli is subjected to double negative control by DeoR and CytR repressors and to the positive control by cAMP-CRP complex . However, sensitivity of the deo operon to catabolite repression is only revealed in bacteria with disrupted synthesis of the CytR protein, since the function of the latter is to prevent CRP activation of the deo operon transcription . In the present work we have studied the influence of crpa specific mutation at the genetic locus of CRP protein on the expression of the deo operon of E . coli . It has been found that the presence of crpa mutation in bacterial genome completely eliminates CytR repression of the deo operon, so that activation of Deo enzymes synthesis by cAMP-CRPa complex becomes possible, even in the presence of CytR . Besides, the modified CRPa protein appears to block the activity of catabolite-sensitive deoPO2 promoter of the deo operon under conditions of cAMP deficiency, which is manifested in the two-fold decrease of deoR derepression in the crpa cells, as compared to the wild type bacteria during the growth on glucose containing medium . It has been supposed that both effects are due to increased affinity of the modified CRPa protein to the specific sites of catabolite sensitive promoters, as compared to the wild type CRP protein.

Environ Health Perspect, 1990 Mar, 84, 75 - 81
Use of photoproteins as intracellular calcium indicators; Blinks JR; The calcium-regulated photoproteins, of which aequorin is the best known, continue to be one of the most useful groups of intracellular Ca2+ indicators . They are self-contained bioluminescent systems that emit blue light in the presence of Ca2+ ions, can readily be purified intact, and are nontoxic when introduced into foreign cells . They have been used successfully as Ca2+ indicators in almost every kind of cell, but are most widely used in muscle cells because of their relative freedom from motion artifacts . Photoproteins have also been used in conjunction with microscopic image intensification to localize Ca2+ in cells . Their large molecular size makes them difficult to introduce into cells, but once there, they have the advantage of staying in the cytoplasm . Aequorin can be microinjected satisfactorily into single cells of almost any size, but a number of alternative methods for introducing photoproteins into cells have been developed in recent years . Disadvantages of the photoproteins for some applications include the nonlinear relation between {Ca2+} and light intensity, the modest speed with which they respond to sudden changes in {Ca2+}, and the fact the Mg2+ antagonizes the effect of Ca2+ . Native photoproteins consist of a mixture of isospecies, and there are differences in Ca2+ sensitivity and in kinetic properties--both among photoproteins and among the isospecies of a given photoprotein . The genes for several of the isospecies of aequorin have been cloned and expressed in E . coli . It seems reasonable to hope that genetic engineering techniques may soon make it possible to consider using, as Ca2+ indicators, rare isospecies or rare photoproteins that have optimal properties for particular applications.

Eur J Epidemiol, 1990 Mar, 6(1), 88 - 90
Demonstration of enterotoxigenic Escherichia coli in diarrheic broiler chicks; Joya JE et al.; An investigation was made to survey the possible presence of enterotoxigenic Escherichia coli (ETEC) in the stools of diarrheal chicks . We analyzed two outbreaks of diarrhea in broiler chicks at two independent farms in the Philippines, from which no pathogens other than Escherichia coli were found . In one outbreak at Farm #1, all 42 isolates produced heat-labile enterotoxin (LT), with 3 of these isolates also producing heat-stable enterotoxin (ST) . The O serotypes of 15 strains tested randomly could not be identified as any known serotype (0-antigen; 1-170) . In another outbreak at Farm #2, 7 out of 52 isolates produced only LT, their subtypes being identified as O-149 or O-8, common serotypes in pig ETEC . Strains from Farm #1 did not produce any pili usually found in human ETEC . We believe this to be the first isolation of ETEC from diarrheal chicks.

DNA Cell Biol, 1990 Mar, 9(2), 129 - 37
Tryptophan promoter derivatives on multicopy plasmids: a comparative analysis of expression potentials in Escherichia coli; Latta M et al.; A collection of variant plasmids expressing either Escherichia coli galactokinase or human serum albumin under the control of several E . coli trp promoter derivatives were constructed and studied for both efficiency of expression and regulation by tryptophan . Several variables, including the length of the upstream region, tandem duplications of a core promoter, and the insertion of the trp repressor trpR gene onto the expression vector, were studied . It is shown that derivatives containing sequences upstream from the -35 region or multiple copies of the trp promoter produce twofold higher levels of protein than plasmids with a minimal trp promoter truncated at -40 . We show that the expression of a heterologous protein such as albumin can be significantly improved (13% vs . 7% of total proteins) if both the upstream trp promoter region, which enhances promoter strength, and an intact trpR gene, are included on the plasmids.

Virus Res, 1990 Mar, 15(3), 197 - 211
Antigenic parvovirus B19 coat proteins VP1 and VP2 produced in large quantities in a baculovirus expression system; Brown CS et al.; Two baculovirus expression vectors derived from Autographica californica nuclear polyhedrosis virus (AcNPV) were prepared containing the complete 2.5 kb coding region for parvovirus B19 coat protein VP1 (AcB19VP1L) and the 1.8 kb coding region for VP2 (AcB19VP2L), placed under the control of the polyhedrin promoter . The recombinant viruses were used to infect Spodoptera frugiperda cells and the proteins expressed were analysed using appropriate antibodies . AcB19VP1L-infected cells produced B19 VP1 as shown by its reaction with 13 human sera containing B19-specific antibodies in Western blot analysis and indirect immunofluorescence . The signal seen with VP1 in immunofluorescence makes it suitable for the development of a diagnostic assay based on this technique . VP1 also reacted with two monoclonal antibodies (mAbs) specific for the B19 protein part of a 196 kDa beta-galactosidase B19 fusion protein expressed in E . coli . Cells infected with AcB19VP2L produced B19 VP2 which reacted with the same human sera in indirect immunofluorescence and with five of the 13 sera in Western blots . VP2 did not react with the fusion protein-specific mAbs . The large amounts of viral antigen produced in this system means the development of widely available diagnostic tests for B19 infection and the further characterization of the B19 structural proteins are within reach.

Protein Eng, 1990 Mar, 3(4), 273 - 7
Construction of rat aldolase C expression plasmid and the hybrid formation between rat aldolase C and human aldolase A or B co-expressed in Escherichia coli; Takasaki Y et al.; Rat aldolase C cDNA was inserted in an Escherichia coli expression vector to construct the rat aldolase C expression plasmid, pRAC42 . This plasmid produces active rat aldolase C in the transfected E . coli host cells . The characteristics of the purified enzyme, e.g . mol . wt, electrophoretic mobilities and kinetic parameters, are indistinguishable from those of authentic rat brain aldolase C . Three different tetrameric hybrid forms, C3A, C2A2 and CA3, in addition to C4 and A4, were found to be produced in the host cell when E.coli was co-transfected with expression plasmids for rat aldolase C and for human aldolase A . Similarly, the hybrid forms, C3B, C2B2 and CB3, in addition to C4 and B4, were also produced in the cells when co-transfected with the plasmids for rat aldolase C and for human aldolase B.

Protein Eng, 1990 Mar, 3(4), 259 - 66
Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the alpha/beta-barrel proteins; Goraj K et al.; We have attempted to construct an artificial polypeptide that folds like the eight-stranded parallel beta-barrel structures . Our approach consists of repeating eight times a unit peptide designed to adopt a 'beta-strand/alpha-helix' pattern . A first 'test' sequence for this structural unit was deduced from a series of parameters defined after an analysis of three natural alpha/beta-barrel proteins and including principally the lengths of the secondary structure elements, the alpha/beta packing and the fitting on average Garnier profiles . The gene encoding this structural unit was synthesized, cloned and expressed in Escherichia coli either as a monomer or as direct repeats of 2-12 units . Preliminary structural characterization of the 7-, 8- and 9-fold unit polypeptides by circular dichroism measurements indicates the presence of the predicted amount of alpha-helix in the three proteins . Further analysis by urea-gradient gel electrophoresis demonstrates that, in the conditions tested, only the 8-fold unit polypeptide forms a compact structure through a cooperative and rapid two-state folding transition involving long-range molecular interactions.

Protein Eng, 1990 Mar, 3(4), 249 - 58
An 8-fold beta alpha barrel protein with redundant folding possibilities; Luger K et al.; Protein sequences containing redundant segments of secondary structure at both termini have the choice a priori of folding into several possible circularly permuted variants of the wild-type tertiary structure . To test this hypothesis the gene of phosphoribosyl anthranilate isomerase from yeast, which is a single-domain 8-fold beta alpha barrel protein, was modified to produce a 10-fold beta alpha homologue in Escherichia coli . It contained a duplicate of the two C-terminal beta alpha units of supersecondary structure fused to its N-terminus . Most of the protein was recovered from the insoluble fraction of disrupted cells by dissolution in guanidinium chloride solutions and refolding . Pristine protein was purified from the soluble fraction . The purified (beta alpha)10 proteins were enzymically almost fully active . Absorbance, fluorescence and circular dichroism spectra as well as the reversible unfolding behaviour of both proteins were also very similar to the properties of the original (beta alpha)8 protein . Digestion with endopeptidases converted both the pristine and the refolded (beta alpha)10 variant to the same large fragment that had the N-terminal sequence and mol . wt of the wild-type (beta alpha)8 protein . The data suggest that the folding of the (beta alpha)10 variant is controlled thermodynamically both in vivo and in vitro.

Mutagenesis, 1990 Mar, 5(2), 173 - 7
Induction of frameshift mutations by caffeine in Escherichia coli K12; Pons FW et al.; Caffeine induced reversions of five different frameshift mutations in Escherichia coli K12 . Only those mutations which were very sensitive to reversion-induction by 9-amino-acridine (9AA) were also sensitive to caffeine . Caffeine mutagenesis was not due to inhibition of cellular repair functions, but to the production of replication errors, which were corrected very efficiently by mismatch repair . From the results reported here we conclude that the targets for caffeine mutagenesis are long runs of GC base pairs, and that this mutagen induces -1 frameshifts.

Mutagenesis, 1990 Mar, 5(2), 127 - 30
The influence of excision repair on the distribution of N-propyl-N'-nitro-N-nitrosoguanidine-induced mutation in Escherichia coli; van der Vliet GM et al.; To determine the influence of excision repair on the distribution of N-propyl-N'-nitro-N-nitrosoguanidine (PNNG)-induced mutation, we have analysed the DNA sequence changes of mutation induced at the lacI gene of Escherichia coli in a UvrB- strain . The mutagenic specificity was similar to that found in the wild-type strain . Base substitutions predominate; G:C----A:T transitions accounted for almost 75% of the mutational events, while G:C----T:A transversions constituted approximately 20% of the mutations . In both strains the distribution of G:C----A:T transitions was nonrandom, but the site specificity of the G:C----A:T transitions appear dissimilar, with the hot and cold sites occurring at different positions . A single - 1 frameshift was recovered in each strain but deletion events were absent in the excision-repair-deficient strain . The complexity of the sequence-specificity of PNNG might reflect the differential repair of n-propyl and iso-propyl adducts.

J Biochem (Tokyo), 1990 Mar, 107(3), 457 - 63
Isolation of a yeast gene, SRH1, that encodes a homologue of the 54K subunit of mammalian signal recognition particle; Amaya Y et al.; A 1.7 kilobase HindIII fragment of Saccharomyces cerevisiae DNA was cloned by cross-hybridization with the Escherichia coli secY gene . The complete nucleotide sequence of the 2.6 kb fragment of the yeast genomic DNA containing the cross-hybridizing HindIII fragment was determined . The sequence showed no apparent similarity with that of the E . coli secY gene with the exception of a completely matched sequence of 21 bp, but it contained a 1,623 nucleotide open reading frame coding for a protein of 541 amino acids with a calculated Mr of 59,600 . The N-terminal portion of 303 residues of the predicted sequence was homologous to the cytosolic domain of the alpha-subunit of the signal recognition particle receptor (SR alpha), including consensus sequence elements for a GTP binding site, whereas the C-terminal portion of 238 residues had an unusual methionine-rich domain containing several repetitive sequences . An mRNA of 2.0 kb was detected on Northern blotting analysis . The predicted sequence was 48% identical with the reported sequences of the 54K subunit of the mammalian signal recognition particle (SRP54) (Romisch K . et al . (1989) Nature 340, 478-483; Bernstein, H.D . et al . (1989) Nature 340, 482-486) . We designated this gene as SRH1 (SRP54 homologue) . Gene disruption experiments showed that the SRH1 gene product is essential for cell growth.

J Biochem (Tokyo), 1990 Mar, 107(3), 369 - 76
DNA binding properties of purified replication initiator protein (Rep) encoded by plasmid pSC101; Sugiura S et al.; We have purified the replication initiator protein (Rep) coded by plasmid pSC101 . The purified protein was confirmed to be Rep by its amino-terminal sequence . Rep exists as a dimer and has a sequence-specific DNA-binding property . Protection experiments with DNA against cleavage by DNase I or exonuclease III showed that Rep bound preferentially to two nearly dyad-symmetric sequences overlapping the promoter of the rep gene, a structure gene of Rep . Transcripts in vitro from the rep promoter were identified and the precise initiation sites were determined by the primer-extension method . Rep represses the transcription from the rep promoter but not that from the bla gene promoter in the same reaction mixture, that is the rep gene is autoregulated . The replication origin (ori) of the plasmid contains directly repeated sequences similar to the symmetric sequences . However, a one order of magnitude higher concentration of the protein is required to bind to the origin repeats.

Biol Chem Hoppe Seyler, 1990 Mar, 371(3), 265 - 72
Purification, assay and kinetic features of HIV-1 proteinase; Billich A et al.; 1) The aspartic proteinase of the human immunodeficiency virus type 1 (HIV-1) was purified from cultures of recombinant E . coli . The enzyme preparation is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing . 2) A rapid assay procedure for the proteinase was established which makes use of the cleavage of a radiolabeled decapeptide and the separation of substrate and labeled product by ion-exchange resin . 3) Activity of the enzyme is optimal at an ionic strength of 2.5-3.5M; also, the inhibitor pepstatin is a more potent inhibitor at higher ionic strength . This can be attributed to a tighter binding of both substrate and inhibitor in high-salt buffer . 4) The Km value of the decapeptide substrate is independent of the pH in the range of 3.5-7.5, while kcat shows a bell-shaped curve with a maximum at pH 5.2 . The shape of the curve can be attributed to pKa values of 4.2 and 6.2 of groups on the enzyme . Pepstatin inhibition is optimal below pH 5.5, but becomes weak above pH 6.

Anal Biochem, 1990 Mar, 185(2), 286 - 93
Specific enzymatic assay for D-glucarate in human serum; Blumenthal HJ et al.; A sensitive and specific spectrophotometric assay was developed to determine levels of D-glucarate in human serum . This assay makes use of the Escherichia coli glucarate catabolic enzymes D-glucarate dehydrase, alpha-keto-beta-deoxy-D-glucarate aldolase, and tartronate semialdehyde (TSA) reductase, to convert D-glucarate to equimolar quantities of pyruvate and TSA . In a one-tube reaction that included NADH, lactate dehydrogenase, and the three E . coli enzymes, 1 mumol of D-glucarate was quantitatively converted to 1 mumol each of D-glycerate and L-lactate with concomitant utilization of 2 mumol of NADH . Using this method, D-glucarate in serum was measured, along with quantitative recovery of authentic D-glucarate from duplicate serum samples to which it had been added . Glucarate is a major serum organic acid, approximating blood pyruvate levels previously determined by others.

Genes Dev, 1990 Mar, 4(3), 410 - 8
DNA looping in cellular repression of transcription of the galactose operon; Mandal N et al.; Communication between distant DNA sites is a central feature of many DNA transactions . Negative regulation of the galactose (gal) operon of Escherichia coli requires repressor binding to two operator sites located on opposite sides of the promoter . The proposed mechanism for regulation involves binding of the repressor to both operator sites, followed by a protein-protein association that loops the intervening promoter DNA (double occupancy plus association) . To assess these requirements in vivo, we have previously converted gal operator sites to lac and shown that both operator sites must be occupied by the homologous repressor protein (Lac or Gal) for negative regulation of the gal operon . We have now addressed more directly the need for protein-protein association by the use of the converted operator sites and a mutant Lac repressor defective in association of the DNA-binding dimers . We have compared the biological and biochemical activity of two Lac repressors: the wild-type (tetramer) I+ form, in which the DNA-binding dimer units are tightly associated; and the mutant Iadi repressor, in which the dimer units do not associate effectively . The I+ repressor is an efficient negative regulator of the gal operon in vivo, but the Iadi mutant is an ineffective repressor . Purified I+ repressor efficiently forms DNA loops between operator sites that we have visualized by electron microscopy; the Iadi repressor fails to form DNA loops, although the protein binds effectively to both operator sites . From the clear correlation between looping in vitro and repression in vivo, we conclude that regulation of the gal operon depends on the association of repressor proteins bound to the two operator sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Clin Microbiol Infect Dis, 1990 Mar, 9(3), 229 - 32
Comparison of the synthetic oligonucleotide gene probe and infant mouse bioassay for detection of enterotoxigenic Escherichia coli; Cryan B; A commercial DNA/DNA hybridisation kit for the detection of Escherichia coli heat stable enterotoxin gene sequences was compared to the suckling mouse bioassay using 183 isolates of Escherichia coli from clinical specimens . The gene probe assay had a specificity of 99% and a sensitivity of 90.4% compared to the infant mouse method . Using the colony blot method of preparing the bacterial DNA and a hybridisation temperature of 50 degrees C optimal results were obtained . The gene probe method is not affected by the incubation conditions of the test organisms . It is technically straightforward and can be applied to large numbers of specimens with fewer logistic difficulties than with the bioassay.

FEMS Microbiol Lett, 1990 Mar 1, 56(1-2), 65 - 8
A bifunctional Streptomyces-E . coli promoter-probe vector; Asturias JA et al.; A bifunctional Streptomyces-E . coli promoter probe vector, pULJA30, has been developed to isolate and characterize nucleotide sequences involved in transcription initiation and regulation . The vector is derived from plasmid pIJ486, carries the pIJ101 replicon and utilizes the promoterless aminoglycoside phosphotransferase (neo) as indicator gene . Important features of the new vector include: wide Streptomyces host range and as high a plasmid copy number as the parental pIJ486, an upstream transcriptional terminator (toop) and a polylinker sequence with unique sites for BamHI and BglII for flexible cloning, fragment re-isolation and direct sequencing of promoter-active inserts . pULJA30 also has an E . coli replicon (from pBR322) and the possibility of selection in Streptomyces and E . coli by using the tsr, neo and bla genes, which makes it very convenient to test the comparative functionality of Streptomyces promoters in E . coli.

FEMS Microbiol Lett, 1990 Mar 1, 56(1-2), 213 - 5
Elimination of ColE1 group (pBR322 and pBR329) plasmids in Escherichia coli by alpha-santonin; Bharathi A et al.; alpha-Santonin, a compound extracted from the flower heads of Arthemisia maritima plant, was effective in elimination of small, multicopy, relaxed plasmids (pBR322 and pBR329 with CoLE1 origin of replication in Escherichia coli, whereas plasmids of IncF1, H1 and X group were totally refractory under similar conditions, suggesting that this agent was specific in curing the CoLE1 group of plasmids.

FEMS Microbiol Lett, 1990 Mar 1, 56(1-2), 19 - 25
Regulation of breakdown of canavanyl proteins in Escherichia coli by growth conditions in lon+ and lon- cells; Rosenberger RF et al.; In vivo rates of proteolysis of canavanyl proteins were compared in lon+ and lon- Escherichia coli strains following growth in a variety of media . Both lon+ and lon- cells grown rapidly in complex media possessed higher levels of constitutive degradative activity than when cultured in minimal media . Pre-growth of lon+ cells in the presence of canavanine induced proteolytic activity following growth in minimal media as did stress agents such as heat, alcohol and puromycin: the lon mutant did not show the increased activity following canavanine treatment . The results suggest the presence of a proteolytic activity which selectively degrades aberrant proteins which does not involve protease La, the product of the lon gene, and which furthermore is regulated in part by growth conditions independently of the stress response.

Pharmacol Res, 1990 Mar-Apr, 22(2), 161 - 70
Endotoxin inhibition of drinking behaviour in the rat; Calapai G et al.; Intravenous (i.v . 320 and 640 micrograms/kg) and intracerebroventricular (i.c.v.; 1 microgram/rat) injection of Escherichia coli lipopolysaccharide (LPS) powerfully inhibited drinking induced by 24 h water deprivation . Pretreatment with acetylsalicylic acid (ASA) into the preoptic area (POA) completely abolished the effect induced by i.v . LPS, but did not modify that elicited by i.c.v . LPS . Intraperitoneal ASA injections significantly reduced the antidipsogenic effect of i.c.v . LPS . Electrolytic ablation of the subfornical organ (SFO) did not modify the effect induced by either i.v . or i.c.v . LPS . Present findings indicate that: (1) the antidipsogenic effect of i.v . LPS is mediated by prostaglandin synthesis into the POA, (2) the SFO is not involved in this effect, and (3) prostaglandins in other brain areas, besides POA, modulate the effect of i.c.v . LPS . It is suggested that at least two different brain sites, inside the blood-brain barrier, might be involved in the antidipsogenic effect of LPS.

J Clin Chem Clin Biochem, 1990 Mar, 28(3), 163 - 8
Alveolar cell pattern and chemiluminescence response of blood neutrophils and alveolar macrophages in sheep after endotoxin injection; Dwenger A et al.; In order to study the pathomechanisms of the Adult Respiratory Distress Syndrome in an acute animal model, we monitored the alveolar cell pattern and the stimulatory chemiluminescence responses of blood neutrophils and alveolar macrophages in sheep after Escherichia coli endotoxin injection (2 micrograms/kg of body weight) . Using appropriate bronchoalveolar lavage techniques, thereby avoiding local inflammation, it was demonstrated that endotoxin injection did not cause any recruitment of neutrophils into the alveoli for a period of up to 24 hours . Following endotoxin injection, blood neutrophils showed a maximal stimulatory response after 5 minutes, and alveolar macrophages after 4 hours . It is concluded that if neutrophils are responsible for initiating the increase in microvascular permeability, then this action must be purely intravascular.

Mol Gen Genet, 1990 Mar, 221(1), 87 - 93
The Escherichia coli minB mutation resembles gyrB in defective nucleoid segregation and decreased negative supercoiling of plasmids; Mulder E et al.; Nucleoid segregation in the Escherichia coli minB mutant and in cells that over-produce minB gene products appeared defective as measured from fluorescence micrographs . Electrophoretic resolution of topoisomers of plasmid isolates from the minB strain revealed a decreased level of negative supercoiling; in addition, multimerization was observed . Over-production of the minB gene product also resulted in a decreased level of negative supercoiling . This phenotype is typical of the gyrB(ts) mutant, which is known to be affected in chromosome decatenation and supercoiling . We propose that the minB mutation and over-production of the minB gene products cause a defect in nucleoid segregation, which may be related to the decrease in negative supercoiling . As in the gyrB(ts) mutant, retardation of nucleoid segregation is proposed to inhibit constriction initiation in the cell centre and to give rise to nucleoid-free cell poles . As a consequence, these cells divide between nucleoid and cell pole, resulting in minicell and (sometimes) in anucleate cell formation.

J Clin Microbiol, 1990 Mar, 28(3), 469 - 72
Nationwide surveillance program to identify diarrhea-causing Escherichia coli in children in Thailand; Sunthadvanich R et al.; Escherichia coli strains isolated from children with diarrhea were collected from 16 hospitals in different districts in Thailand during 1985 and 1986 and submitted to the National Reference Laboratory . Isolates were identified by serogrouping or as enterotoxigenic E . coli (ETEC), enteroinvasive E . coli (EIEC), enteropathogenic E . coli (EPEC) adhesin factor (EAF) E . coli, or Shiga-like-toxin (SLT)-producing E . coli by DNA hybridization . EPEC strains of known serogroups were isolated from 10%, ETEC strains were isolated from 6%, EAF E . coli strains were isolated from 4%, EIEC strains were isolated from less than 1%, and SLT-producing E . coli strains were isolated from none of 393 children with diarrhea . Among 278 children whose ages were recorded, the highest rate of isolation of EAF E . coli was 11% (9 of 85) from children less than 6 months old . ETEC was isolated from 5% (4 of 85) of children less than 6 months old, from 10% (12 of 118) of children 6 to 23 months old, and from 1% (1 of 75) of children greater than 23 months old . EPEC strains of known serogroups were isolated from 18% (15 of 85) of children less than 6 months old, from 11% (13 of 118) of children 6 to 23 months old, and from 9% (7 of 75) of children greater than 23 months old . E . coli strains that hybridized with the EIEC probe were isolated from three children who were 20, 36, and 48 months old . Examining E . coli for hybridization with DNA probes for virulence determinants is a practical way of conducting nationwide surveillance of diarrhea-causing E . coli . Since only 33% (13 of 39) of EPEC serogroups hybridized with the EAF probe and none hybridized with the SLT probes, identification of EPEC by serogroups analysis, followed by serotyping, should continue to be used in the identification of EPEC.

Somat Cell Mol Genet, 1990 Mar, 16(2), 173 - 84
Analysis of GPT activity in mammalian cells with a chromosomally integrated shuttle vector containing altered gpt genes; Gelbert LM et al.; The molecular mechanisms of reversion in mammalian cells were studied utilizing the pZipGptNeo shuttle vector, with the bacterial gpt gene in the vector integrated into the chromosomal DNA of mouse cells . From mutant cell lines containing gpt genes with single base changes, revertants were selected for the reappearance of GPT activity . The copy number and expression of the gpt genes in such revertants were analyzed, and the GPT activity encoded by revertant genes in both mammalian cells and bacteria characterized . Revertants with wild-type amino acid sequence had, on average, the highest levels of GPT activity . Revertants with amino acid sequences different from the original mutants but not corresponding to wild-type had, on average, approximately half the level of GPT activity as wild-type revertants . Revertants that still contained the original mutation in the gpt gene had even lower levels of activity . These revertants were found to have amplified mutant gpt genes, which, when transferred into bacteria, were seen to encode for GPT polypeptides with partial enzymatic activity . A revertant in which the original mutation that destroyed the AUG translational start codon was retained but in which there was a secondary mutation upstream of the start codon also was characterized . The second mutation generated an in-frame CUG codon that apparently functioned as an alternative, upstream translational start codon.

Microbiol Rev, 1990 Mar, 54(1), 18 - 51
Nucleotide excision repair in Escherichia coli; Van Houten B; One of the best-studied DNA repair pathways is nucleotide excision repair, a process consisting of DNA damage recognition, incision, excision, repair resynthesis, and DNA ligation . Escherichia coli has served as a model organism for the study of this process . Recently, many of the proteins that mediate E . coli nucleotide excision have been purified to homogeneity; this had led to a molecular description of this repair pathway . One of the key repair enzymes of this pathway is the UvrABC nuclease complex . The individual subunits of this enzyme cooperate in a complex series of partial reactions to bind to and incise the DNA near a damaged nucleotide . The UvrABC complex displays a remarkable substrate diversity . Defining the structural features of DNA lesions that provide the specificity for damage recognition by the UvrABC complex is of great importance, since it represents a unique form of protein-DNA interaction . Using a number of in vitro assays, researchers have been able to elucidate the action mechanism of the UvrABC nuclease complex . Current research is devoted to understanding how these complex events are mediated within the living cell.

J Exp Zool, 1990 Mar, 253(3), 263 - 70
Effects of starvation and neuroactive drugs on feeding in Caenorhabditis elegans; Avery L et al.; Caenorhabditis elegans concentrates its food, bacteria, by pharyngeal pumping . The rate of pumping is affected by the presence of bacteria . Using a new assay that allows measurement of pumping rate in a population of worms suspended in liquid by measuring their uptake of microscopic iron particles, we have confirmed and quantitated this effect . Furthermore, we demonstrated that starvation stimulates pumping . Worms that had been deprived of bacteria for more than 4 hours pumped in the absence of bacteria under conditions in which well-fed worms did not . Furthermore, starved worms responded to lower amounts of bacteria than did fed worms . The assay was also useful for measuring effects of drugs on pumping . Of about 30 chemicals screened, 5 had clear effects . The neurotransmitter serotonin and the serotonin uptake inhibitor imipramine stimulated pumping, while the serotonin antagonist gramine inhibits . Imipramine stimulation is greatly decreased in cat-1 and cat-4 mutants, which have low levels of serotonin . Muscimol, an agonist for the neurotransmitter GABA, and ivermectin, whose site of action may also be the GABA receptor, both inhibit pumping . Qualitative observations suggested a role for acetylcholine in the regulation of pumping.

Proc Natl Acad Sci U S A, 1990 Mar, 87(6), 2309 - 13
The regulatory subunit of Escherichia coli aspartate carbamoyltransferase may influence homotropic cooperativity and heterotropic interactions by a direct interaction with the loop containing residues 230-245 of the catalytic chain; Newton CJ et al.; A recent x-ray structure of aspartate carbamoyltransferase (carbamoyl-phosphate: L-aspartate carbamoyl-transferase, EC 2.1.3.2) with phosphonoacetamide bound {Gouaux, J . E . & Lipscomb, W . N . (1990) Biochemistry 29, 389-402} shows an interaction between Asp-236 of the catalytic chain and Lys-143 of the regulatory chain . Asp-236 is part of the loop containing residues 230-245 (240s) of the catalytic chain that undergoes a significant conformational change between the tight and the relaxed states of the enzyme . Furthermore, side-chain interactions between the 240s loop and other portions of the enzyme have been shown to be important for the low activity and low affinity of the tight state and the high activity and high affinity of the relaxed state . To determine whether the intersubunit link between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain is important for either homotropic cooperativity and/or the heterotropic interactions in aspartate carbamoyltransferase, site-specific mutagenesis was used to replace Asp-236 with alanine . The mutant enzyme exhibits full activity and a loss of both homotropic cooperativity and heterotropic interactions . Furthermore, the aspartate concentration at half the maximal observed specific activity is reduced by approximately 8-fold . The mutant enzyme exhibits normal thermal stability but drastically altered reactivity toward p-hydroxymercuribenzoate . The catalytic subunit of the mutant and wild-type enzymes have very similar properties . These results, in conjunction with previous experiments, suggest that the intersubunit link involving Asp-236 is involved in the stabilization of the 240s loop in its tight-state position and that the regulatory subunits exert their effect on the catalytic subunits by influencing the position of the 240s loop.

Proc Natl Acad Sci U S A, 1990 Mar, 87(6), 2304 - 8
Cloning, expression, and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines by site-directed mutagenesis; Liu J et al.; The cDNA encoding human cyclophilin from the Jurkat T-cell lymphoma line has been cloned by the expression cassette polymerase chain reaction and sequenced, and an expression vector has been constructed under control of the tac promoter for efficient expression in Escherichia coli . Active cyclophilin is produced at up to 40% of soluble cell protein, facilitating a one-column purification to homogeneity . Wild-type cyclophilin was characterized for binding of the potent immunosuppressant agent cyclosporin A (Kd = 46 nM) by tryptophan fluorescence enhancement and for inhibition (IC50 = 19 nM) of cyclophilin's peptidyl-prolyl cis-trans isomerase (rotamase) activity . With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrate, recombinant human cyclophilin has a high catalytic efficiency; kcat/Km is 1.4 X 10(7) M-1.S-1 at 10 degrees C . To test the prior suggestion that a cysteine residue may be essential for catalysis and immunosuppressant binding, the four cysteines at positions 52, 62, 115, and 161 were mutated individually to alanine and the purified mutant proteins were shown to retain full affinity for cyclosporin A and equivalent catalytic efficiency as a rotamase . Clearly the cysteines play no essential role in catalysis or cyclosporin A binding . These results rule out the recently proposed mechanism {Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T . & Schmid, F . X . (1989) Nature (London) 337, 476-478)} involving the formation of tetrahedral hemithioorthoamide . Whereas mechanisms that embody other tetrahedral intermediates may be operative, an alternative mechanism is considered that involves distortion of bound substrate with a twisted (90 degrees) peptidyl-prolyl amide bond.

Proc Natl Acad Sci U S A, 1990 Mar, 87(6), 2122 - 6
Purification and lipid-layer crystallization of yeast RNA polymerase II; Edwards AM et al.; Yeast RNA polymerase II was purified to homogeneity by a rapid procedure involving immunoaffinity chromatography . The purified enzyme contained 10 subunits, as reported for conventional preparations, but with no detectable proteolysis of the largest subunit . In assays of initiation of transcription at the yeast CYC1 promoter, the enzyme complemented the deficiency of an extract from a strain that produces a temperature-sensitive polymerase II . Mammalian RNA polymerase II was inactive in this initiation assay . The purified yeast enzyme formed two-dimensional crystals on positively charged lipid layers, as previously found for Escherichia coli RNA polymerase holoenzyme . Image analysis of electron micrographs of crystals in negative stain, which diffracted to about 30-A resolution, showed protein densities of dimensions consistent with those of single polymerase molecules.

Proc Natl Acad Sci U S A, 1990 Mar, 87(6), 2087 - 91
Stable nuclear transformation of Chlamydomonas reinhardtii by using a C . reinhardtii gene as the selectable marker; Mayfield SP et al.; We have developed a stable nuclear transformation system for the unicellular green alga Chlamydomonas reinhardtii . Transformation was accomplished by introducing the cloned C . reinhardtii oxygen-evolving enhancer protein 1 (OEE1) gene into C . reinhardtii cells by bombardment with DNA-coated tungsten particles . The recipient strain was an OEE1-deficient, nonphotosynthetic, acetate-requiring mutant, which recovered photosynthetic competence after transformation, and was therefore able to grow in the absence of acetate . Analysis of several transformants indicates that transformation has proceeded via second-site integration of the cloned gene, leaving the endogenous mutant gene intact . In genetic crosses of transformants with wild type, both mutant and wild-type phenotypes were recovered, showing that the photosynthetic competence of transformants was due not to reversion of the original locus but rather to expression of the introduced gene . We suggest that the success of the present system is largely due to using a homologous C . reinhardtii gene, leading to stable maintenance and expression of the gene . Transformation with heterologous genes may be problematic because of poor expression due to an unusual codon bias in C . reinhardtii.

Protein Seq Data Anal, 1990 Mar, 3(1), 11 - 9
Examination of protein sequence homologies . VII . The complementary molecular coevolution of ribosomal proteins equivalent to Escherichia coli L7/L12 and L10; Otaka E et al.; Recently reported P1, P2 and metabacteria line sequences of transposition-type 'A' proteins, equivalent to Escherichia coli ribosomal protein L7/L12, were examined using a correlation method which evaluates the sequence similarity quantitatively . As the sequences could be aligned along the alignment previously constructed for 25 various 'A' proteins, the inclusive alignment further supports the previous claims concerning the rule of "preservation units" and the transpositional regeneration for metabacterial and eukaryotic 'A' proteins . Yeasts contain multispecies of P1 and P2 line genes and their P1 line sequences show low correlation coefficient values compared to other P1 line sequences, indicating a great evolutionary distance between lower and higher eukaryotes . Five sequences of protein P0 from metabacteria, yeast, and human, of which about 20 residues at the C termini are homologous with those of their own transposition-type 'A' proteins, were similarly examined . The N-terminal three-quarters of the sequences align naturally and the first two-thirds of the alignment could involve the E . coli L10 (EL10) sequence . An alignment of the remaining sequences at the C termini was established, relying on the well-matching sequence similarities between the metabacteria 'A' protein and their P0 protein sequences . Finally, the C-terminal halves of P0 protein sequences corresponded with almost overall sequences of the transposition-type 'A' proteins . The gene fusion of a protein might have resulted in the formation of the P0 proteins . A coupling of this gene fusion and the transposition of prototype 'A' proteins may have given rise to the complementary molecular transformations required for the development toward higher organism cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lab Invest, 1990 Mar, 62(3), 355 - 62
Acute endotoxin-induced lymphocyte subset sequestration in sheep lungs; Duke SS et al.; Endotoxemia is associated with an early phase of pulmonary hypertension and a later increase in microvascular permeability . These physiologic changes are attended by peripheral blood and lung lymph leukopenia and a rapid accumulation of both granulocytes and lymphocytes in the peripheral lung . In the present study, the numbers of lymphocytes in blood, lung lymph, and lung tissue after infusion of endotoxin were determined by fluorescent labeling of lymphocyte populations with monoclonal antibodies to sheep T1, T4, T8, or leukocyte common antigen and with rabbit anti-sheep immunoglobulins (B cells) . Peripheral blood, lung lymph, and lung tissue samples were collected at baseline and 15, 30, 60, 120, 180, and 240 minutes after the start of intravenous infusion of E . coli endotoxin (1.25 micrograms/kg, N = 6) or saline (N = 4) from open-chest anesthetized sheep . Pulmonary artery pressure and lung lymph flow were also monitored at these times . Endotoxin caused marked reductions in the number of T and B lymphocytes in blood and lung lymph . As compared with baseline, total blood leukocytes and granulocytes were significantly reduced below control levels from 30 minutes of endotoxin, and lymphocyte numbers were reduced from 60 minutes . T1, T4, T8 and B lymphocyte subsets contributed to the fall in blood lymphocytes . Endotoxin caused a significant fall in number of lung lymph leukocytes (T1, T4 and T8 cells) from 120 minutes; numbers of B lymphocytes were also reduced . Counts of the number of lymphocytes in the biopsy tissue revealed a significant rise in T lymphocytes sequestered in the lung . We conclude endotoxemia in sheep causes a reduction in lymphocytes in blood and lung lymph and an increase in these cell types in peripheral lung tissue . These findings suggest that lymphocyte subpopulations accumulate in the lungs during periods of pulmonary hypertension and increased permeability and may participate in endotoxin-induced lung injury.

J Med Microbiol, 1990 Mar, 31(3), 185 - 90
Differences in susceptibility of inbred and outbred infant mice to enterotoxigenic Escherichia coli of bovine, porcine and human origin; Duchet-Suchaux M et al.; Infant mice from outbred Swiss OF1 and from inbred DBA/2, C57BL/6, BALB/cBy and CBA strains were screened for usefulness in the diarrhoea model with enterotoxigenic Escherichia coli (ETEC) strains of bovine, porcine and human origin . Mouse strains were either weakly susceptible or not susceptible to ETEC strains of porcine or human origin bearing antigen K88, 987P, CFA/I or CFA/II . In contrast, some mouse strains were highly susceptible to bovine and porcine ETEC strains bearing K99 or F41 or both antigens . Swiss OF1 and CBA infant mice were highly susceptible to one bovine ETEC strain bearing antigen K99, whereas DBA/2, BALB/cBy and C57BL/6 mice exhibited nearly complete resistance to the same ETEC strain . Except DBA/2, all mouse strains were highly susceptible to bovine and porcine ETEC strains bearing antigen F41 alone or in combination with antigen K99 . Challenge ETEC strains colonised intestines of all infant mice, but they reached very high levels soon after inoculation in the diarrhoeic ones only.

J Comput Assist Tomogr, 1990 Mar-Apr, 14(2), 272 - 5
Cerebral ventriculitis: MR findings; Barloon TJ et al.; Cerebral ventriculitis is an uncommon site and manifestation of infection of the central nervous system . We report the magnetic resonance findings in two patients with cytologic and CSF culture proof of ventriculitis . The findings included abnormal signal intensity of the ependyma and CSF on T2-weighted images . In addition, ependymal, meningeal, and dural enhancement was seen in one case, on T1-weighted images, after administration of Gd-diethylenetriamine pentaacetic acid.

Genetics, 1990 Mar, 124(3), 573 - 84
Mismatch repair-induced meiotic recombination requires the pms1 gene product; Borts RH et al.; The presence of multiple heterologies in a 9-kilobase (kb) interval results in a decrease in meiotic crossovers from 26.0% to 10.1% . There is also an increase from 3.5% to 11.1% in gene conversions and ectopic recombinations between the flanking homologous MAT loci . The hypothesis that mismatch repair of heteroduplex DNA containing several heterologies would lead to a second round of recombination has now been tested by examining the effect of a mutation that reduces mismatch correction . The repair-defective pms1-1 allele restores the pattern of recombination to nearly that seen in congenic diploids without the heterologies . Mismatch repair-induced recombination causes a significant increase in MAT conversions and ectopic recombination events with as few as two heterozygosities separated by 0.3-0.7 kb, but not when the mismatches are separated by greater than 1 kb . The frequency of these events depends on both the number and position of the heterozygosities relative to the flanking homologous MAT loci used to detect the events . The creation of recombinogenic lesions by mismatch repair in yeast could be analogous to the creation of recombinogenic lesions in dam- Escherichia coli . We suggest that the repair of heteroduplex DNA containing multiple mismatches may produce chromosomal rearrangements and gamete inviability when naturally polymorphic chromosomes undergo meiotic recombination.

Genetics, 1990 Mar, 124(3), 473 - 82
Mechanisms of activation of the cryptic cel operon of Escherichia coli K12; Parker LL et al.; The cel (cellobiose utilization) operon of Escherichia coli K12 is not expressed in the wild-type organism . However, mutants that can express the operon and thereby utilize the beta-glucoside sugars cellobiose, arbutin and salicin are easily isolated . Two kinds of mutations are capable of activating the operon . The first involves mutations that allow the repressor to recognize the substrates cellobiose, arbutin and salicin as inducers . We have identified the sequence changes in five different active alleles and found those differences to be single base pair changes at one of two lysine codons in the repressor gene . The second kind of mutation involves the integration of the insertion sequences IS1, IS2 or IS5 into a 108-bp region 72-180 bp upstream of the start of transcription . Integration occurs at several different sites and in different orientations . Transcription of the cel operon begins at the same base pair in all mutants examined . Of 44 independent cel+ mutants, 27 were activated by point mutations and 17 were activated by insertion sequences . The preferred mechanism of activation appears to be strain dependent, since one of the parents yielded 94% insertionally activated alleles, while another yielded 100% point mutation activated alleles.

EMBO J, 1990 Mar, 9(3), 815 - 9
Induction of non-bilayer lipid structures by functional signal peptides; Killian JA et al.; Using 31P NMR and freeze-fracture electron microscopy we investigated the effect of several synthetic signal peptides on lipid structure in model membranes mimicking the lipid composition of the Escherichia coli inner membrane . It is demonstrated that the signal peptide of the E . coli outer membrane protein PhoE, as well as that of the M13 phage coat protein, strongly promote the formation of non-bilayer lipid structures . This effect appears to be correlated to in vivo translocation efficiency, since a less functional analogue of the PhoE signal peptide was found to be less active in destabilizing the bilayer . It is proposed that signal sequences can induce local changes in lipid structure that are involved in protein translocation across the membrane.

EMBO J, 1990 Mar, 9(3), 615 - 21
Mutant lac repressors with new specificities hint at rules for protein--DNA recognition; Lehming N et al.; Proteins which recognize specific sequences of DNA play a fundamental role in the regulation of protein synthesis in all organisms . A particular helix of the bacterial protein lac repressor recognizes the bases in the major groove of the lac operator . We show that the first two residues of this recognition helix interact independently with two base pairs . This allows us in many cases to predict repression as an indicator of strength of the repressor-operator complex . Rules of recognition can be derived for 16 symmetric operators . They also apply to the gal repressor and possibly to other bacterial repressors.

Circ Shock, 1990 Mar, 30(3), 279 - 92
Survival of primates in LD100 septic shock following therapy with antibody to tumor necrosis factor (TNF alpha); Hinshaw LB et al.; The purpose of this study was to determine the efficacy of treatment with anti-TNF monoclonal antibody in preventing the deleterious effects of sepsis in a nonhuman primate . Experiments were carried out on anesthetized baboons intravenously infused with a lethal dose of Escherichia coli (E . coli) . Twelve baboons (six control and six experimental) received 2 hr infusions of E . coli . The experimental group was administered a bolus of anti-TNF antibody, 15 mg/kg, 30 min after beginning the E . coli infusion . Control baboons lived an average of 19 hr (12-34 hr) . All antibody-treated baboons survived more than 7 days with a significantly improved quality of life compared to the control group . Although some adverse changes occurred during the monitoring period in surviving baboons, they maintained nearly normal arterial pressures, and serum urea nitrogen and creatinine concentrations . The severe histopathologic changes in lungs, liver, adrenals, kidneys, and spleen documented at death in baboons receiving E . coli only were absent after 7 days in baboons given E . coli and early post-treatment with antibody to TNF.

Circ Shock, 1990 Mar, 30(3), 255 - 63
Bone marrow failure after hemorrhagic shock; Livingston DH et al.; The proliferation of white blood cells is an important and necessary response to bacterial infection . The effect of hemorrhagic shock and LPS administration on myelopoiesis was investigated . Rats subjected to hemorrhagic shock and resuscitation were injected IP with 100 micrograms E . coli LPS or saline 24 hr following shock . Twenty-four hours later, myelopoiesis was assessed by the growth of granulocyte-macrophage progenitor cells (CFU-GM) in both bone marrow (BM) and spleen (SPL) . CFU-GM were cultured in the presence of no additional serum or normal rat serum or shock serum obtained 6 hr after hemorrhage . Shock resulted in a peripheral leukocytosis although BM and SPL cellularity was unaffected by either shock or LPS . BM and SPL CFU-GM from unshocked rats significantly increased after LPS administration (BM 47 +/- 6 vs . 70 +/- 8; SPL 40 +/- 4 vs . 72 +/- 14; both P less than 0.05) . Shock had no effect on BM or SPL CFU-GM . In contrast, LPS given to shocked rats decreased BM CFU-GM compared to saline-treated rats (50 +/- 3 vs . 34 +/- 4 P less than 0.05) . The addition of normal serum to the culture system had no effect on BM CFU-GM but the addition of shock serum reduced CFU-GM by 50% in all groups (P less than 0.05) . These data demonstrate that shock markedly alters the myelopoietic response to LPS and may also result in the production or release of inhibitors of CFU-GM growth.

Circ Shock, 1990 Mar, 30(3), 221 - 8
Dichloroacetate administration in the treatment of endotoxin shock; Preiser JC et al.; Dichloroacetate (DCA), an activator of the pyruvate dehydrogenase complex, has been shown to reduce blood lactate levels effectively in various conditions . DCA administration has also sometimes resulted in beneficial cardiovascular effects . To assess its potential value in the routine management of septic shock, we studied the effects of DCA on a canine endotoxic shock model associated with moderate lactic acidosis . Eighteen dogs were pentobarbitone anesthetized, intubated, and mechanically ventilated . Thirty minutes after the administration of 3 mg/kg of Escherichia coli endotoxin, 10 dogs received 100 mg/kg followed by 100 mg/kg/hr of DCA, and eight dogs served as control . In all animals, fluid administration was titrated according to the left-sided filling pressures . In the DCA-treated animals, lactate levels rapidly fell from 3.1 +/- 1.2 to 1.3 +/- 0.8 mEq/liter after 30 min . The bolus of DCA was usually followed by a very transient increase in arterial pressure, but no sustained hemodynamic change was noted . Oxygen consumption (measured from the exhaled gases) was not affected . Four dogs in the DCA group and one dog in the control group survived the next morning (difference not significant) . The present study confirms that DCA can effectively reduce blood lactate levels in endotoxic shock and might therefore be useful in severe lactic acidosis related to septic shock . However, the routine use of DCA in septic shock to improve hemodynamic status is not supported by the present findings.

Circ Shock, 1990 Mar, 30(3), 185 - 205
Effects of N-acetylcysteine and terbutaline treatment on hemodynamics and regional albumin extravasation in porcine septic shock; Groeneveld AB et al.; We studied the therapeutic effects of continuously infused N-acetylcysteine, an O2 radical scavenger (N, n = 6), and terbutaline, a beta 2-agonist (T, n = 6), versus dextrose (controls C, N = 6) on hemodynamics and regional albumin extravasation in porcine septic shock . After instrumentation, injection of 99mTc-labeled red blood cells, and baseline measurements, pigs received a 90 min infusion of 11 +/- 9 X 10(8).kg-1 live Escherichia coli bacteria . Thereafter, therapy was started, and 131I human serum albumin was injected . Images were obtained hourly using a gammacamera and a computer until 5 hours after baseline . Regions of interest were drawn in the 99mTc images, yielding regional 131I/99mTc radioactivity ratios, with blood samples as reference . From these ratios, an albumin leak index, a rate constant of transvascular albumin transport, was calculated . Control pigs developed pulmonary hypertension, arterial hypotension, hemoconcentration, and lactic acidemia . In spite of tachycardia and unchanged filling pressures, cardiac output fell . In arterial blood, white cell count, PO2, albumin level, and colloid osmotic pressure fell . The albumin leak index (X10(-3).min-1) measured 1.56 +/- 0.59 over the lungs and 2.87 +/- 1.19 over the abdomen in C (P less than .05 vs . lung), confirming previously found increased albumin flux in both lung and abdomen, the latter exceeding the former . Neither N nor T significantly affected hemodynamic and biochemical changes . The drugs neither decreased the regional albumin leak index nor attenuated the formation of albumin-rich ascites found at autopsy . However, the lung albumin index obtained at autopsy was significantly reduced with N (P less than .01 vs . C), at similar gravimetrically determined extravascular lung water (EVLW) . EVLW positively correlated with pulmonary albumin extravasation in C (rs = 0.94, P less than .05) and T (rs = 0.89, P = .05) but not in N (rs = -0.37, n.s.) . We conclude that neither N nor T treatment beneficially influences the course of septic shock in pigs . Although neither drug combats noninvasively assessed regional microvascular albumin leakage and formation of albumin-rich ascites, N may ameliorate increased pulmonary microvascular permeability as judged from post mortem data.

Ann Surg, 1990 Mar, 211(3), 323 - 8
Reduced amino acid transport in skeletal muscle caused by a circulating factor during endotoxemia; Warner BW et al.; The present study was designed to determine whether reduced amino acid uptake in skeletal muscle during endotoxemia is due to associated hypotension or is caused by a factor present in plasma . Three series of experiments were performed . In the first series of experiments, mean arterial pressure (MAP), heart rate, and amino acid uptake in incubated soleus muscles were measured after intravenous injection of endotoxin (1 mg/kg) in male Sprague-Dawley rats (40 to 60 g) . Amino acid transport was measured by determining intracellular uptake of {3H}-alpha-amino-isobutyric acid (AIB) during 2 hours of incubation . In the second series of experiments, hypotension was induced by bleeding and muscle amino acid uptake was measured . In the third series of experiments, whole plasma or a low molecular weight fraction (less than 10,000 d) of plasma from endotoxin-injected rats was added in vitro to incubated muscles and amino acid uptake was determined . One hour after injection of endotoxin, MAP was reduced from 80 +/- 2 mmHg to 54 +/- 4 mmHg (p less than 0.05) . AIB uptake was reduced by 20% (p less than 0.05) 2 hours after endotoxin injection . When MAP was maintained at 50 mmHg for 1 hour by bleeding, no changes in muscle AIB uptake were noted . When plasma obtained from rats 2 hours after endotoxin injection was added to incubated soleus muscles, AIB uptake was reduced by 22% . This effect was duplicated by a fraction of endotoxic plasma containing substances with a molecular weight less than 10,000 d . The present results suggest that reduced muscle amino acid uptake during endotoxemia is not due to associated hypotension, but may be caused by a circulating factor(s) with a molecular weight less than 10,000 d.

Ann Surg, 1990 Mar, 211(3), 312 - 6
Platelet activating factor receptor antagonist improves survival and attenuates eicosanoid release in severe endotoxemia; Fletcher JR et al.; Exogenous platelet activating factor (PAF) causes hypotension, plasma extravasation, metabolic acidosis, and death . These effects are similar to those of endotoxin as well as the eicosanoids . A specific PAF receptor antagonist, BN52021, was used to determine its effects on the hemodynamic events, the eicosanoid production, and on survival in severe rat endotoxemia . Endotoxin alone significantly produced hypotension, prostaglandins (TxB2, PGE2) release, and death . In contrast pretreatment with BN52021, a specific PAF receptor antagonist, significantly altered the hypotension, significantly attenuated the eicosanoid release, and improved the survival rate (p less than 0.01) . These findings suggest that PAF receptor activation is an early event in endotoxemia . Eicosanoid release in endotoxemia could be related to PAF synthesis and PAF receptor activation . These findings support the hypothesis that there may be an intimate relationship between PAF and the eicosanoids and that in endotoxemia some of the effects of PAF may be mediated via the cyclo-oxygenase pathway.

Am Rev Respir Dis, 1990 Mar, 141(3), 631 - 9
Sequential assessment of pulmonary epithelial diethylene triamine penta-acetate clearance and intrapulmonary transferrin accumulation during Escherichia coli peritonitis; Ishizaka A et al.; The individual roles of pulmonary capillary endothelial and alveolar epithelial permeability in the pathogenesis of the adult respiratory distress syndrome (ARDS) are unclear . We developed a method for the sequential assessment of pulmonary macromolecule accumulation and small solute clearance in vivo using a gamma camera . We measured the exponential clearance coefficient of 111In-labeled diethylene triamine penta-acetate (111In-DTPA) to assess airway clearance of small solutes . We also calculated the exponential equilibration coefficient of 111In-labeled transferrin (111In-TF) to assess intrapulmonary accumulation of transferrin . We determined these parameters in guinea pigs with Escherichia coli peritonitis and compared them with a saline-treated control group, oleic-acid-treated groups, and a group treated with low molecular weight dextran Ringer solution . The pulmonary DTPA clearance and the intrapulmonary transferrin accumulation were significantly increased in the peritonitis group (29.4 +/- 8.2 x 10(-3) min-1, p less than 0.02, and 15.1 +/- 3.1 x 10(-3) min-1, p less than 0.02) when compared with the control group (3.1 +/- 0.8 x 10(-3) min-1 and 4.5 +/- 0.5 x 10(-3) min-1) . These changes developed within 5.5 h of the initial insult . Neither increased extravascular lung water nor elevated pulmonary artery and left atrial pressures were detected in the peritonitis group . The low molecular weight dextran Ringer group did not show a significant increase in the pulmonary DTPA clearance and the intrapulmonary transferrin accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Res Microbiol, 1990 Mar-Apr, 141(3), 290 - 308
The lac permease of Escherichia coli: site-directed mutagenesis studies on the mechanism of beta-galactoside/H+ symport; Roepe PD et al.; In this communication, we summarize site-directed mutagenesis studies of the lac permease from Escherichia coli, a prototypic H(+)-coupled active transport protein . We classify mutant permeases by phenotype, and suggest possible roles for some individual residues in the mechanism of H+/lactose symport . Although high-resolution structural information is not presently available, kinetic analysis of the partial reactions catalysed by the mutant permeases, as well as biophysical studies, suggest an evolving model for the mechanism of H+/lactose symport.

Zh Mikrobiol Epidemiol Immunobiol, 1990 Mar, (3), 89 - 93
{An experimental study of human recombinant gamma interferon with a proteolytically reduced C-end area of the protein}; Vorob'ev AA et al.; The work presents the results of experimental study of gamma interferon obtained by gene engineering techniques on the basis of Escherichia coli producer strains . The study has revealed that gamma interferon, whose molecular weight is 15 KD, due to intracellular proteolytic degradation shows the absence of some amino acids at the C-end of protein and is electrophoretically homogeneous, while its antiviral, antiproliferative and immunomodulating effects are less pronounced than those of gamma interferon with a molecular weight of 18 KD.

Mol Gen Mikrobiol Virusol, 1990 Mar, (3), 18 - 21
{Study of the regulation of crp gene expression in Escherichia coli K12}; Lisenkov AF et al.; The regulation of crp gene expression by CRP-cAMP complex was studied in E . coli strain by the crp-lac operon fusion . F'141 crp+ episome decreased 5-7 fold the high level of crp-lac expression in crp strains while F'141 crp episome had no effect . The hybrid plasmid pCAP2 crp+ with the intact crp gene did not affect the crp gene expression level in crp mutants, though they had acquired the Crp+ phenotype just as they did in F'141 crp+ presence . The F'141 crp+ and pCAP2 crp+ combination in crp mutants also resulted in decrease of the crp gene expression comparable to the registered in the presence of the F'141 crp+ plasmid . Similar repression occurred only in cya+ strains but not in cya strains . The crp gene is supposed to possess negative regulation by CRP-cAMP complex with a complementary factor also necessary . The latter is evidently located in an E . coli chromosome site overlapped by F'141 episome.

Mol Microbiol, 1990 Mar, 4(3), 479 - 88
Transcriptional regulation of the cytR repressor gene of Escherichia coli: autoregulation and positive control by the cAMP/CAP complex; Gerlach P et al.; The Escherichia coli cytR-encoded repressor protein (CytR) controls the expression of several genes involved in nucleoside and deoxynucleoside uptake and metabolism . The cytR promoter was identified by determining the transcriptional initiation site of the cytR gene . A chromosomal cytR-lacZ+ operon fusion was isolated and used to study the regulation of cytR . We show that cytR expression is negatively controlled by the CytR protein and positively affected by the cAMP/CAP complex . Footprinting studies with purified CAP protein revealed two CAP binding sites upstream of the cytR promoter . A previously described mutation (cytR*) in the cloned cytR gene, which results in the phenotypic suppression of a CytR operator mutation in the tsx P2 promoter, was analysed . DNA sequence analysis of the cytR* mutation revealed a G-C to an A-T base pair transition at position -34 bp relative to the translational initiation site of cytR . This point mutation activates a cryptic promoter that is stronger than the wild-type cytR promoter and leads to overproduction of the CytR repressor.

Mol Microbiol, 1990 Mar, 4(3), 471 - 7
The regulatory role of the IS1-encoded InsA protein in transposition; Zerbib D et al.; We show here that the protein InsA, which is encoded by IS1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS1 transposition activity . We demonstrate that it inhibits both IS1-mediated cointegrate formation and transposition of a synthetic IS1-based transposon ('omegon'; omega-on) . These results also indicate that the omega-on which does not itself encode IS1 transposition functions can be complemented in trans, presumably by the copies of IS1 resident in the Escherichia coli chromosome . Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS1-encoded genes both in cis or in trans . The experiments involving omega-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS1.

Mol Microbiol, 1990 Mar, 4(3), 427 - 37
Sequence of the fhuE outer-membrane receptor gene of Escherichia coli K12 and properties of mutants; Sauer M et al.; The fhuE gene of Escherichia coli codes for an outer-membrane receptor protein required for the uptake of iron(III) via coprogen, ferrioxamine B and rhodotorulic acid . The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues . The mature form, composed of 693 residues, has a calculated molecular weight of 77,453, which agrees with the molecular weight of 76,000 determined by polyacrylamide gel electrophoresis . The FhuE protein contains four regions of homology with other TonB-dependent receptors . A valine to proline exchange in the 'TonB box' abolished transport activity . Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited increasing iron-coprogen transport rates . Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order) . A truncated FhuE protein lacking 24 amino acids at the C-terminal end was exported to the periplasm but failed to be inserted into the outer membrane.

J Membr Biol, 1990 Mar, 114(2), 143 - 51
Mechanism of Na+/proline symport in Escherichia coli: reappraisal of the effect of cation binding to the Na+/proline symport carrier; Yamato I et al.; The proton and sodium ion dependences of the proline binding and transport activities of the proline carrier in Escherichia coli were investigated in detail . The binding activity in cytoplasmic membrane vesicles from a carrier over-producing strain (PT21/pTMP5) was absolutely dependent on the presence of Na+, but did not necessarily require protonation of the carrier, in contrast to the model previously reported (Mogi, T., Anraku, Y . 1984 . J . Biol . Chem . 259:7797-7801) . Based on this and previous observations, we propose a modified model of the proline binding reaction of the proline carrier, in which a proton is supposed to be a regulatory factor for the binding activity . The apparent Michaelis constant of proline (Kt) of the transport activity of cytoplasmic membrane vesicles from the wild type E . coli strain driven by a respiratory substrate, ascorbate, showed dependence on a low concentration of sodium ion . The Michaelis constant of sodium ion for transport (KtNa) was estimated to be 25 microM . The proline transport activities in membrane vesicles and intact cells were modulated by H+ concentration, the inhibitory effect of protons (pKa approximately equal to 6) being similar to that observed previously (Mogi, T., Anraku, Y . 1984 . J . Biol . Chem . 259:7802-7806) . Based on these observations and the modified model of substrate binding to the proline carrier, a model of the proline/Na+ symport mechanism is proposed, in which a proton is postulated to be a regulatory factor of the transport activity.

FEMS Microbiol Lett, 1990 Mar 1, 56(1-2), 13 - 7
IS30 activation of an smp'-lacZ gene fusion in Escherichia coli; Neuwald AF et al.; The Escherichia coli serB gene is divergently transcribed from the gene . Six independently isolated IS30 insertions within the 5' end of the serB structural gene resulted in increased expression of an smp'-lacZ gene fusion . DNA sequence analysis of two of the insertions suggests that a promoter was created upon IS30 insertion.

Biochim Biophys Acta, 1990 Mar, 1022(3), 373 - 80
Mechanism of enhanced melibiose transport rate catalyzed by an Escherichia coli lactose carrier mutant with leucine substituted for serine-306 . The pH-dependence of melibiose efflux; King SC et al.; The mechanism of melibiose transport was studied in cells containing plasmid pAA22 which expresses the mutant lactose carrier (serine-306 to leucine) cloned from Escherichia coli AA22 . These studies were of interest because several lines of evidence suggested that the AA22 mutation conferred novel properties upon the lactose carrier, decreasing turnover with several beta-galactoside substrates, increasing turnover with melibiose, and abolishing active accumulation even though equilibration occurred via symport with H+ . Although severely defective in active melibiose accumulation, the present study indicates that in cells poisoned with azide the AA22 carrier does in fact equilibrate melibiose across the membrane more rapidly than the normal lactose carrier . Similarly, melibiose efflux from cells preloaded with melibiose was more rapidly catalyzed by the AA22 carrier than by the normal carrier (pH 7.0) . Furthermore, although external H+ did reduce net melibiose efflux to a rate slower than seen in equilibrium exchange, a lower than normal pH was required to achieve this effect . Therefore, at pH 7.0, the AA22 carrier (but not the normal carrier) catalyzed net efflux at a rate approaching that for the exchange process (which was pH-resistant in both the mutant and the parent) . At pH 8.0 both the AA22 carrier and the normal carrier catalyzed net melibiose efflux at a rate identical to the equilibrium exchange rate . We suggest (i) that the sensitivity of melibiose efflux to external pH indicates that during efflux the AA22 carrier interacts with protons in a manner similar to the normal carrier (i.e., sugar is cotransported with H+) and hence the absence of accumulation is not explained by internal leak via a binary carrier-melibose complex; and (ii) that the modest increase in rate constants for melibiose exit reflect small changes in activation energy (1 kcal/mol) consistent with a steric mechanism possibly involving van der Waals contacts.

Biochem J, 1990 Mar 1, 266(2), 545 - 52
19F-n.m.r . studies of ligand binding to 5-fluorotryptophan- and 3-fluorotyrosine-containing cyclic AMP receptor protein from Escherichia coli; Sixl F et al.; Two fluorine-containing analogues of the cyclic AMP receptor protein (CRP) from Escherichia coli were prepared by biosynthetic incorporation of 5-fluorotryptophan (5-F-Trp) and 3-fluorotyrosine (3-F-Tyr) . The 19F-n.m.r . spectrum of the {5-F-Trp}CRP showed two signals corresponding to the two tryptophan residues, and that of the {3-F-Tyr}CRP showed six signals (two overlapping) corresponding to the six tyrosine residues: these results are as expected for a symmetrical dimer . A comparison of the 19F-n.m.r . spectra of the CRP analogues in the presence and in the absence of cyclic AMP reveals that the chemical shifts of both tryptophan residues and of two of the six tyrosine residues show differences . Since none of these residues is in direct contact with the bound nucleotide (although Trp-85 is fairly close), these shift changes must arise from induced conformational effects . The 19F-n.m.r . spectra of complexes with cyclic GMP showed chemical-shift perturbations different from those caused by cyclic AMP, indicating that different conformational changes are induced by the binding of cyclic GMP . The 19F-n.m.r . spectrum of the complex of {3-F-Tyr}CRP with tubercidin 3',5'-(cyclic)monophosphate (which can activate transcription) showed essentially the same chemical-shift changes as seen for the cyclic AMP complex, indicating that similar conformational changes have been induced by the nucleotide binding . {3-F-Tyr}CRP in the presence of an equimolar amount of the 20 bp self-complementary DNA oligomer 5'-AATGTGAGTTAACTCACATT-3' and excess cyclic AMP gave an 19F-n.m.r . spectrum that was almost identical with that for the {3-F-Tyr}CRP-cyclic AMP complex, indicating that the binding of DNA does not induce significant conformational changes involving the tyrosine residues . Proteolysis of {3-F-Tyr}CRP with chymotrypsin produced a 31 kDa fragment that is a dimer containing the cyclic AMP-binding domain . This fragment contains five of the six tyrosine residues, and its 19F-n.m.r . chemical shifts were essentially the same as those of the intact protein except for one missing signal (signal F): this signal could be assigned to Tyr-206 and shown to be unperturbed by the binding of cyclic nucleotide to the intact {3-F-Tyr}CRP . The similarity of the 19F-n.m.r . chemical shifts in the alpha-fragment and the intact CRP indicates that the alpha-fragment retains the same structure as found in the intact protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Oncogene, 1990 Mar, 5(3), 423 - 6
The p53 nuclear localisation signal is structurally linked to a p34cdc2 kinase motif; Addison C et al.; We have identified a region of human p53 protein with striking homology to a sequence motif on Simian Virus 40 T antigen which includes the nuclear localisation signal . Mutation of basic amino acid residues in this region of p53 (residues 312 to 323; SSSPQPKKKP) compromises transport of p53 protein to the nucleus . The sequence functions efficiently as a nuclear localisation signal when fused to E . coli beta galactosidase . Serine 315 within this p53 structural motif is phosphorylated in vitro by the cell cycle kinase p34cdc2 . Thus in both T antigen and p53, nuclear localisation signal and p34cdc2 kinase acceptor residue map to a contiguous region of primary amino acid sequence.

Virology, 1990 Mar, 175(1), 1 - 9
Immunological cross-reactivity to laboratory-produced HPV-11 virions of polysera raised against bacterially derived fusion proteins and synthetic peptides of HPV-6b and HPV-16 capsid proteins; Christensen ND et al.; Polysera raised in rabbits to bacterially derived fusion proteins and synthetic peptides of the L1 and L2 ORFs of HPV-6b and -16 were tested for cross-reactivity to laboratory-produced infectious HPV-11 virions . The polysera were analyzed in a series of five different immunological assays including immunoperoxidase staining of the koilocytotic nuclei in sections of formalin-fixed, paraffin-embedded as well as fresh frozen sections of HPV-11 experimental condylomas generated in the athymic nude mouse xenograft system, ELISA, Western blots, and neutralization of infectious HPV-11 virions . ELISA and Western blot assays were used to determine whether the polysera identified external or internal epitopes on HPV-11 virions, and whether there was cross-reactivity to BPV-1 or laboratory-produced CRPV virions . Seven of a total of 12 sera were positive for reactivity to HPV-11 in one or more assays, but none of the reactivity was directed to external epitopes on the intact virions as determined by ELISA . None of the L1 products generated group-specific antigen (GSA) antisera including a synthetic peptide spanning the GSA site . The combination of assays clearly demonstrated that apparent false positive and false negative reactivities of different antisera were obtained for each assay system tested . Thus, no single assay could be used reliably to determine the true antiviral reactivity of a given polysera.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1744 - 8
Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction; Heyduk T et al.; A fluorescence method was developed to study DNA-protein interactions in solution . A 32-base-pair (bp) DNA fragment of the lac promoter containing the primary binding site for Escherichia coli cAMP receptor protein (CRP) was chemically synthesized and labeled specifically at the 5' end with fluorescent probe . Binding of cAMP receptor protein to this fragment can be conveniently followed by measuring changes in polarization of fluorescence of the labeled DNA or by measuring fluorescence energy transfer from protein tryptophan residues to the DNA label . Formation of protein-DNA complex was monitored as a function of cAMP concentration . Various equilibrium constants can be resolved to characterize the binding of cAMP to CRP and the subsequent binding of CRP-cAMP and CRP-(cAMP)2 to DNA . These binding studies showed that the two ligated forms of CRP have significantly different affinities for specific-site DNA . These results show that, in principle, the fluorescence technique can yield thermodynamically valid equilibrium constants under essentially any solution conditions . This technique also has the potential of providing information regarding the structure of protein-DNA complexes.

Mutat Res, 1990 Mar, 235(2), 85 - 92
The sir locus of Escherichia coli: a gene involved in SOS-independent repair of mitomycin C-induced DNA damage; Kumaresan KR et al.; A Mud1 (lac Apr) insertion has been isolated in a delta (lac)recA+ lexA3(Ind-)rpoB87 gyrA87 mutant of Escherichia coli resulting in a decrease in mitomycin C tolerance and an increase in post-mitomycin C DNA degradation . The mitomycin C sensitivity of the insertion mutant is not further increased by substituting either the rpoB87 or the gyrA mutation by the respective wild-type alleles . However, when both rpoB87 and gyrA87 mutations are replaced by rpoB+ and gyrA+ the strain becomes hypersensitive to mitomycin C . Inactivation of recA in the insertion mutant has no effect on its mitomycin C sensitivity provided both rpoB87 and gyrA87 are present . When either or both of the mutations is/are replaced by the wild-type allele inactivation of recA renders the strain hypersensitive to mitomycin C . The locus of Mud1 (lac Apr) insertion, designated sir (SOS-independent repair), has been mapped between 57 and 61 min on the E . coli linkage map . Expression of the sir gene seems to be constitutive and not enhanced by mitomycin C . These results are discussed in relation to the SOS-independent repair of mitomycin C-induced DNA damage reported earlier.

Mutat Res, 1990 Mar, 235(2), 101 - 9
Thiol and hydrogen peroxide modification of recA induction in UV-irradiated wild-type and catalase-deficient Escherichia coli K12; Claycamp HG et al.; Induction of recA in Escherichia coli, monitored as beta-D-galactosidase (beta-Gal) activity in recA-lacZ fusion strains, was shown to be elevated and prolonged by dithiothreitol (DTT) treatment after far-UV radiation . Pretreatment of UV-irradiated cultures using DTT led to a shortened recA response and little increase of beta-Gal yield . Similar studies were performed using a catalase-deficient recA-lacZ strain in which the major feature was elevated levels of recA-lacZ induction . Catalase activity in UV-irradiated wild-type cells was reduced by DTT treatment to levels as low as in a katE mutant strain, leading to similar recA-lacZ induction patterns between the strains . Neither DTT nor H2O2 treatment of cells could induce significant recA transcription in the absence of UV-radiation, implying that both agents modify recA activity primarily by interfering with repair of recA-inducing DNA lesions . The results confirm previous studies suggesting that modification of DNA repair is probably a significant portion of thiol radiation protection.

J Bacteriol, 1990 Mar, 172(3), 1562 - 8
Identification and characterization of gyrB mutants of Escherichia coli that are defective in partitioning of mini-F plasmids; Ogura T et al.; hopA mutants, which have been suggested to be defective in mini-F plasmid partitioning (H . Niki, C . Ichinose, T . Ogura, H . Mori, M . Morita, M . Hasegawa, N . Kusukawa, and S . Hiraga, J . Bacteriol . 170:5272-5278, 1988), were found to carry mutations in the gyrB gene, coding for the B subunit of DNA gyrase . In gyrB(HopA) mutants, relaxation of the superhelicity of plasmids, increased IncG incompatibility, and increased SopB protein production were observed . It is suggested that altered expression of the sop genes, which is due to relaxation of the mini-F plasmid DNA, causes both defective partitioning of the mini-F plasmids and increased IncG incompatibility in gyrB(HopA) mutants.

J Bacteriol, 1990 Mar, 172(3), 1368 - 73
Integration host factor plays a role in IS50 and Tn5 transposition; Makris JC et al.; In Escherichia coli, the frequencies of IS50 and Tn5 transposition are greater in Dam- cells than in isogenic Dam+ cells . IS50 transposition is increased approximately 1,000-fold and Tn5 transposition frequencies are increased about 5- to 10-fold in the absence of Dam methylation . However, in cells that are deficient for both integration host factor (IHF) and Dam methylase, the transposition frequencies of IS50 and Tn5 approximate those found in wild-type cells . The absence of IHF alone has no effect on either IS50 or Tn5 transposition . These results suggest that IHF is required for the increased transposition frequencies of IS50 and Tn5 that are observed in Dam- cells . It is also shown that the level of expression of IS50-encoded proteins, P1 and P2, required for IS50 and Tn5 transposition and its regulation does not decrease in IHF- or in IHF- Dam- cells . This result suggests that the effects of IHF on IS50 and Tn5 transposition are not at the level of IS50 gene expression . Finally, IHF is demonstrated to significantly retard the electrophoretic mobility of a 289-base-pair segment of IS50 DNA that contains a putative IHF protein-binding site . The physiological role of this IHF binding site remains to be determined.

J Bacteriol, 1990 Mar, 172(3), 1186 - 96
Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli; Wanner BL et al.; The Escherichia coli phn (psiD) locus encodes genes for phosphonate (Pn) utilization, for phn (psiD) mutations abolish the ability to use as a sole P source a Pn with a substituted C-2 or unsubstituted hydrocarbon group such as 2-aminoethylphosphonate (AEPn) or methylphosphonate (MPn), respectively . Even though the E . coli K-12 phosphate starvation-inducible (psi) phn (psiD) gene(s) shows normal phosphate (Pi) control, Pn utilization is cryptic in E . coli K-12, as well as in several members of the E . coli reference (ECOR) collection which are closely related to K-12 . For these bacteria, an activating mutation near the phn (psiD) gene is necessary for growth on a Pn as the sole P source . Most E . coli strains, including E . coli B, are naturally Phn+; a few E . coli strains are Phn- and are deleted for phn DNA sequences . The Phn+ phn(EcoB) DNA was molecularly cloned by using the mini-Mu in vivo cloning procedure and complementation of an E . coli K-12 delta phn mutant . The phn(EcoB) DNA hybridized to overlapping lambda clones in the E . coli K-12 gene library (Y . Kohara, K . Akiyama, and K . Isono, Cell 50:495-508, 1987) which contain the 93-min region, thus showing that the phn (psiD) locus was itself cloned and verifying our genetic data on its map location . The cryptic phn(EcoK) DNA has an additional 100 base pairs that is absent in the naturally Phn+ phn(EcoB) sequence . However, no gross structural change was detected in independent Phn+ phn(EcoK) mutants that have activating mutations near the phn locus.

Infect Immun, 1990 Mar, 58(3), 611 - 8
Comparison of the glycolipid receptor specificities of Shiga-like toxin type II and Shiga-like toxin type II variants; Samuel JE et al.; The antigenically distinct Shiga-like toxins (SLTs) SLT-1 and SLT-II are cytotoxic for both Vero and HeLa cells and use Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb3) molecules as functional receptors . SLT-II-related variants SLT-IIvp and SLT-IIvh, produced by a porcine isolate and a human isolate, respectively, are cytotoxic for Vero but not HeLa cells . To investigate the basis for these differences in cytotoxic specificity among SLTs, the nature of the receptor for the SLT-II variants was examined . First, the patterns of binding of SLT-II and the SLT-II variants to Gb3 receptor analogs Gal alpha 1-4Gal-bovine serum albumin and Gal alpha 1-4Gal beta 1-4Glc-bovine serum albumin were compared . SLT-IIvp bound the trisaccharide neoglycoprotein preferentially, while SLT-IIvh bound both analogs equally but with less affinity than did SLT-II . Next, the glycolipids to which the SLT-II variants bound in Vero and HeLa cells were identified by thin-layer chromatography . SLT-IIvp bound to Gb3, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb4), and Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb5) in Vero cells but only Gb3 in HeLa cells . However, SLT-IIvh bound to Gal alpha 1-4Gal beta 1-1Cer (Gb2) and Gb3 in HeLa cells but only Gb3 in Vero cells . In addition, hybrid toxins (SLT-IIvp subunit A with SLT-II subunit B or SLT-II subunit A with SLT-IIvp subunit B) were used to show that the receptor specificities of the SLTs was B subunit specific . These differences in receptor specificities are important in vivo, as evidenced by a 400-fold difference in the 50% lethal doses of purified SLT-IIvp and SLT-II (200 versus 0.5 ng, respectively) for mice . These data indicate that SLT-II-cytotoxic variants can occur as a consequence of differences in receptor specificity and affinity.

J Virol, 1990 Mar, 64(3), 1201 - 6
An antigen chimera of poliovirus induces antibodies against human papillomavirus type 16; Jenkins O et al.; It has been established that the surface of poliovirus type 1 can be extensively modified to incorporate antigenic domains from other poliovirus serotypes and from unrelated viruses . The fact that the modified (chimeric) viruses exhibit dual antigenicity and immunogenicity led us to explore the possibility of using the Sabin vaccine strain of poliovirus type 1 as a vector for the presentation of antigenic domains from human papillomavirus type 16 (HPV-16), a virus associated with the development of cervical carcinoma . We report here the construction and characterization of a chimeric poliovirus containing a 16-residue sequence derived from the major capsid protein (L1) of HPV-16 . This virus chimera stimulated the production in rabbits of antibodies which recognized the HPV-16-derived peptide and an L1 fusion protein synthesized in Escherichia coli and detected HPV-16 in human biopsy material by immunoperoxidase staining . The possibility that poliovirus-HPV chimeras could be used as vaccines against HPV-16 is discussed.

J Virol, 1990 Mar, 64(3), 1171 - 81
Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional study of the corresponding gene promoter; Horisberger MA et al.; The human protein p78 is induced and accumulated in cells treated with type I interferon or with some viruses . It is the human homolog of the mouse Mx protein involved in resistance to influenza virus . A full-length cDNA clone encoding the human p78 protein was cloned and sequenced . It contained an open reading frame of 662 amino acids, corresponding to a polypeptide with a predicted molecular weight of 75,500, in good agreement with the Mr of 78,000 determined on sodium dodecyl sulfate gels for the purified natural p78 protein . The cloned gene was expressed in vitro and corresponded in size, pI, antigenic determinant(s), and NH2 terminus sequence to the natural p78 protein . A second cDNA was cloned which encoded a 633-amino-acid protein sharing 63% homology with human p78 . This p78-related protein was translated in reticulocyte lysates where it shared an antigenic determinant(s) with p78 . A putative 5' regulatory region of 83 base pairs contained within the gene promoter region upstream of the presumed p78 mRNA cap site conferred human alpha interferon (IFN-alpha) inducibility to the cat reporter gene . The p78 protein accumulated to high levels in cells treated with IFN-alpha . In contrast, the p78-related protein was not expressed at detectable levels . The rate of decay of p78 levels in diploid cells after a 24-h treatment with IFN-alpha was much slower than the rate of decay of the antiviral state against influenza A virus and vesicular stomatitis virus, suggesting that the p78 protein is probably not involved in an antiviral mechanism . Furthermore, we showed that these proteins, as well as the homologous mouse Mx protein, possess three consensus elements in proper spacing, characteristic of GTP-binding proteins.

Plant Cell, 1990 Mar, 2(3), 225 - 33
Nopaline synthase promoter is wound inducible and auxin inducible; An G et al.; The activity of the nopaline synthase (nos) promoter is differentially regulated in several plant organs . In this article we demonstrate that the nos promoter is wound inducible in both vegetative and reproductive organs . The induction of the nos promoter was observed in leaves, stems, cotyledons, and various reproductive organs, suggesting that the response is not organ specific . The wound response was further enhanced by addition of auxins . Other growth substances had no effect on the wound-inducible nos promoter activity . Deletion analysis of the nos promoter indicated that the 10-base pair (GCACATACGT) Z element located between -123 and -114 or an element overlapping with this sequence is essential for the wound and auxin responses.

Curr Genet, 1990 Mar, 17(3), 223 - 7
Cloning of the pyr4 gene encoding orotidine-5'-phosphate decarboxylase in Cephalosporium acremonium; Vian A et al.; We have cloned the Cephalosporium acremonium pyr4 gene by cross-hybridization with the equivalent gene from Neurospora crassa, the closest relative from which this gene is available . The C . acremonium pyr4 gene complements an E . coli pyrF mutant lacking orotidine-5'-phosphate decarboxylase (OMPdecase), and most probably does not contain introns . Maxicell analysis in E . coli shows that it encodes a 46 kDa polypeptide . The C . acremonium OMPdecase contains a highly conserved pentadecapeptide characteristic for this category of enzyme . Extensive sequence comparison suggests an important role of this region in enzymatic activity.

Gene, 1990 Mar 1, 87(1), 97 - 103
Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3' end in mRNA stability; Kaul R et al.; The major outer membrane protein (MOMP)-encoding gene (omp1) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced . This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP . The recombinant omp1 is expressed in either insertion orientation, indicating that it utilizes its own promoter system . The endogenous omp1 promoter possesses a relatively low activity despite the high level of MOMP expression . Deletion of a 520-bp fragment at the 3' end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis . When the MOMP-encoding plasmid was introduced into E . coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein . These data indicate that the unprocessed omp1 gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein . The recombinant MOMP was localized to the outer membrane E . coli fraction, comparable to the location of the native C . trachomatis protein.

Vet Clin North Am Food Anim Pract, 1990 Mar, 6(1), 69 - 75
Fluid therapy with specific mucopolysaccharides . A new approach to control diarrhea; Verschoor J et al.; An evaluation is given of the shortcomings of existing oral fluid therapies and their consequences: lack of protection of the intestinal wall and the supply of unabsorbed carbohydrates to the large intestine . The inclusion of specific mucopolysaccharides having a polyxylose backbone and galactose end residues in the side chains seems to offer effective protection of the intestinal wall . A trial conducted in experimentally infected calves (E . coli) shows that this concept of oral fluid therapy limits the number of diarrhea days and induces better growth in calves; a more complete glucose absorption and limitation of the supply of unabsorbed carbohydrates to the large intestine was shown in a trial conducted in human patients with a rotavirus enteritis.

Arch Biochem Biophys, 1990 Mar, 277(2), 283 - 9
Mutations in alpha-subunit of Escherichia coli F1-ATPase obtained by hydroxylamine-mutagenesis of plasmids carrying the uncA gene; Pagan J et al.; In order to generate mutants randomly in the Escherichia coli uncA gene (encoding the alpha-subunit of F1-ATPase), plasmids carrying uncA were treated in vitro with hydroxylamine . Restriction fragments of the mutated uncA gene were then reconstructed into plasmid pDP34, which expresses all of the F1F0 structural genes, and the reconstructed mutant plasmids were expressed in a strain carrying a deletion of chromosomal uncA . Each of the mutations was characterized by DNA sequencing, growth assays, and biochemical assays of membrane preparations . Three nonsense and one frameshift mutation were identified and their properties were studied briefly . Eight new missense mutations were identified and characterization of their properties is described . These eight mutations were R139H, A177V, R210C, R303C, A306V, T343I, G351S, and P370L.

J Bacteriol, 1990 Mar, 172(3), 1621 - 7
Sequence and transcriptional pattern of the essential Escherichia coli secE-nusG operon; Downing WL et al.; Two genes, secE and nusG, situated between the tufB and ribosomal protein rplKAJL operons in the rif region at 90 min on the Escherichia coli chromosome, have been sequenced and characterized . The secE gene encodes a 127-amino-acid-long polypeptide, which is an integral membrane protein essential for protein export (P . J . Schatz, P . D . Riggs, A . Jacq, M . J . Fath, and J . Beckwith, Genes Dev . 3:1035-1044, 1989) . The nusG gene encodes a 181-amino-acid-long polypeptide and is involved in transcription antitermination . The protein product of nusG is essential for bacterial viability . The secE-nusG genes are cotranscribed, with transcripts initiated at the PEG promoter and terminated at the Rho-independent terminator in the region of the rplK promoter . The majority of transcripts are processed at a number of sites in the 5' untranslated leader region by RNase III and are possibly also processed by a second unidentified nuclease . The role of transcript processing in the regulation of secE and nusG has not yet been established . The juxtaposition and coregulation of a protein export factor and a transcriptional factor raise questions concerning a functional connection between the two processes.

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