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