Escherichia coli

J Biol Chem, 1988 Dec 15, 263(35), 18810 - 5
The ATP binding site on rho protein . Affinity labeling of Lys181 by pyridoxal 5'-diphospho-5'-adenosine; Dombroski AJ et al.; We have labeled the nucleoside triphosphate-binding domain of Escherichia coli rho factor with the ATP affinity analog {3H}pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) . PLP-AMP completely inactivates the RNA-dependent ATPase activity of rho upon incorporation of 3 mol of reagent/mol of hexameric rho protein . Although the potency of PLP-AMP is enhanced when an RNA substrate such as poly(C) is present, the stoichiometry for inhibition remains the same as in the absence of poly(C) . The nucleotide substrate ATP competes very effectively for the binding site and protects against PLP-AMP inactivation . A domain of rho called N2, which comprises the distal two-thirds of the molecule (residues 152-419) and encompasses the region proposed to bind ATP, is labeled specifically in the presence of poly(C) . Amino acid sequence analysis of the single {3H}PLP-AMP labeled proteolytic fragment showed Lys181 to be the site of modification, suggesting that this residue normally interacts with the gamma-phosphoryl of bound ATP . These results agree with our proposed tertiary structure for the ATP-binding domain of rho that places this lysine residue in a flexible loop above a hydrophobic nucleotide-binding pocket comprised of several parallel beta-strands, similar to adenylate kinase, F1-ATPase, and related ATP-binding proteins . Parallel studies of rho structure and function by site-directed mutagenesis and chemical modification support this interpretation.

Gene, 1988 Dec 15, 73(1), 141 - 52
Structure of yeast glucokinase, a strongly diverged specific aldo-hexose-phosphorylating isoenzyme; Albig W et al.; Saccharomyces cerevisiae glucokinase (GLK) is the only described hexose-phosphorylating enzyme specific for aldo-hexoses . The gene was cloned by complementation of a triple mutant lacking all hexose-phosphorylating isoenzymes . Restriction sites were confirmed by genomic hybridization and GLK1 was mapped on chromosome III by ROFAGE, a method derived from the orthogonal field alteration gel electrophoresis . The mapping data were in agreement with previous genetic data . The open reading frame was established by two transcription start points in front of the initial ATG codon and by C-terminal beta-galactosidase fusions . The mRNA is 1.75 kb long and codes for 500 amino acid (aa) residues . Diversity of GLK from hexokinases PI and PII is very marked, with only 26 and 28% overall aa homology . A central core of about 350 aa shows 39% homology . No cross-hybridization could be observed by Southern hybridization . However, strong homologies were found over a range of 11 aa between glucokinase, yeast hexokinases (PI, PII) and rat hexokinase with 8 aa in common . These strongly conserved homologies give support to the view that this aa region corresponds to the binding site for glucose . Unlike all other hexose-phosphorylating enzymes, there is no proline residue indicating a conformational turn next to this glucokinase region . This finding may explain the failure of fructose phosphorylation . In both GLK and the hexokinases, a lysine residue is also conserved at aa position 110 which probably corresponds to the ATP-binding site . Additionally, a consensus sequence of 8 aa residues which is common for ATP-binding enzymes is conserved within the C-terminal part of GLK . The codon bias index for GLK1 is 0.25, which is very low compared with other glycolytic enzymes described so far . The gene is moderately expressed and constitutive on different carbon sources investigated . GLK1 null alleles had no detectable effects on sporulation and growth . Hence, a physiological role for GLK, which might explain its preservation, could not be detected under our laboratory test conditions.

Biochem J, 1988 Dec 15, 256(3), 741 - 9
Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli; Allison N et al.; A 'split-gene' technique for the overexpression and mutagenesis of the gene encoding the lipoamide dehydrogenase of Escherichia coli was developed in order to overcome the instability problems encountered when attempting to mutate the intact gene . The lipoamide dehydrogenase gene, lpd, was dissected into two fragments which were separately subcloned into M13 vectors for mutagenesis in vitro followed by reconstitution in the pJLA504 expression vector under the transcriptional control of the lambda PR and lambda PL promoters and a temperature-sensitive lambda repressor . After thermo-induction, E . coli cells transformed with the plasmid carrying the reconstituted lpd gene contained 4-5 times more lipoamide dehydrogenase activity than is normally found in the wild-type organism . The strategy was used to engineer a Glu-188----Asp replacement in lipoamide dehydrogenase, and this generated an enzyme with markedly different kinetic properties.

Eur J Biochem, 1988 Dec 15, 178(2), 445 - 50
Catalytic-site mapping of pyruvate formate lyase . Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate); Plaga W et al.; Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state . The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage {J . Knappe et al . (1984) Proc . Natl Acad . Sci . USA 81, 1332-1335} . Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-{14C}acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423) . Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418 . The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine . This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl . These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase . The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.

Eur J Biochem, 1988 Dec 15, 178(2), 351 - 5
Interaction of Escherichia coli translation-initiation factor IF-1 with ribosomes; Celano B et al.; The interaction between Escherichia coli translation-initiation factor IF-1 and ribosomes was studied in binding experiments by Airfuge centrifugation . IF-1 binds to the 30S, but not to the 50S, ribosomal subunit and its binding is strongly stimulated by IF-3 and IF-2, either alone or in combination . From the dependence of the Kd of the 30S-subunit--IF-1 complex on ionic strength, it can be concluded that IF-1 binds primarily via an ionic interaction, most likely with the 16S rRNA, with the minimum number of ion pairs involved being 2.7-3.6 . The 30S-subunit--IF-1 interaction is unaffected by temperature changes between 11 degrees C and 44 degrees C and is thus accompanied by a negligible enthalpy change . It is concluded that the interaction is an entropy-driven process triggered mainly by the release of counter ions from the RNA phosphates . Titration of 30S-subunit--IF-1 complexes with 50S subunits causes the ejection of the factor indicating that IF-1 is released from the ribosomes during the subunit association step which marks the transition from a 30S-initiation-complex to a 70S initiation complex.

J Biol Chem, 1988 Dec 15, 263(35), 19174 - 80
Proteolytic cleavage of Ada protein that carries methyltransferase and transcriptional regulator activities; Yoshikai T et al.; The 39-kDa Ada protein that carries two distinct methyltransferase activities and an activity to promote transcription of the ada and alkA genes is cleaved to smaller polypeptides in the case of incubation with a crude extract of Escherichia coli . A protease that specifically cleaves the Ada protein was partially purified and characterized . The enzyme showed maximal activity at pH 7.8-8.0 and did not require specific ions or ATP for the reaction . The activity was inhibited by p-chloromercuribenzoic acid, thereby suggesting that it is a thiol protease . When a purified preparation of Ada protein was incubated with the protease preparation, definite sizes of cleavage products were formed; at the initial stage of proteolysis, the 20- and 19-kDa proteins were formed as the major products . When each of these products was purified by successive column chromatography, the 20-kDa protein was found to catalyze transfer of a methyl group from methylphosphotriester of methylated polynucleotides, whereas the 19-kDa protein transfers a methyl group from O6-methylguanine of methylated DNA to each of the molecules . Neither the 20- nor 19-kDa protein, even after methyl acceptance, exhibited an activity to promote transcription of the ada gene . However, alkA transcription was promoted by the 20-kDa protein provided that it was methylated.

J Biol Chem, 1988 Dec 15, 263(35), 19147 - 53
Nucleotide sequence and analysis of the purA gene encoding adenylosuccinate synthetase of Escherichia coli K12; Wolfe SA et al.; Adenylosuccinate synthetase (EC 6.3.4.4), encoded by the purA gene of Escherichia coli K12, catalyzes the synthesis of adenylosuccinate (SAMP) from IMP, the first committed step in AMP biosynthesis . The E . coli K12 purA gene and flanking DNA was cloned by miniMu-mediated transduction, and the nucleotide sequence was determined . The mature SAMP synthetase subunit, as deduced from the DNA sequence, contains 427 amino acid residues and has a calculated Mr of 47,277 . The size of the purA mRNA was determined by Northern blotting to be approximately 1.5 kilobase pairs . The 5'-end of the purA mRNA was identified by primer extension and is located 23 nucleotides upstream of the ATG translational initiation codon . Comparison of the purA control region with the guaBA control region revealed a common region of dyad symmetry which may suggest mutual elements of regulation . The purA control region did not resemble the control regions of the other known pur loci.

J Biol Chem, 1988 Dec 15, 263(35), 19077 - 82
Inhibition and resumption of processing of the staphylokinase in some Escherichia coli prlA suppressor mutants; Iino T et al.; Escherichia coli strains carrying certain prlA mutations (prlA4 and prlA401) could not support the processing and export of staphylokinase, resulting in the accumulation of the precursor form under high-level synthesis conditions . In order to clarify the cause of the defect in the structure of staphylokinase, we constructed signal peptide mutations of sak which suppressed the processing defect in the prlA4 cells by site-directed mutagenesis . The processing defect was suppressed when glycine or asparagine was introduced in place of the serine residue at position 17 from the amino terminus of the signal peptide . Substitutions of glycine for the leucine residue at position 15 and for the serine residue at position 19 were also effective . Other mutations we constructed had no suppression activity . Taking account of the correlation between the suppression activity and the parameter value of each substituted amino acid for the beta-turn probability, we predict that the staphylokinase signal peptide requires a more bending structure at the end of the hydrophobic core to act efficiently in the prlA4 cells than in the prl+ cells and that a function of the PrlA protein necessary to recognize the staphylokinase signal peptide has become deficient through the prlA4 mutation.

J Biol Chem, 1988 Dec 15, 263(35), 19053 - 9
An artificial hydrophobic sequence functions as either an anchor or a signal sequence at only one of two positions within the Escherichia coli outer membrane protein OmpA; MacIntyre S et al.; The 325-residue outer membrane protein, OmpA, of Escherichia coli, like most other outer membrane proteins with known sequence, contains no long stretch of hydrophobic amino acids . A synthetic oligonucleotide, encoding the sequence Leu-Ala-Leu-Val, was inserted four times between the codons for amino acid residues 153 and 154 and two, three, or four times between the codons for residues 228 and 229, resulting in the OmpA153-4, OmpA-228-2, -3, and -4 proteins, respectively . In the first case, the lipophilic sequence anchored the protein in the plasma membrane . In the OmpA228 proteins, 16 but not 12 or 8 lipophilic residues most likely also acted as an anchor . By removal of the NH2-terminal signal peptide, the function of the insert in OmpA153-4 was converted to that of a signal-anchor sequence . Possibly due to differences in amino acid sequences surrounding the insert, no signal function was observed with the insert in OmpA228-4 . Production of the OmpA153-4 protein, with or without the NH2-terminal signal sequence, resulted in a block of export of chromosomally encoded OmpA . Clearly, long hydrophobic regions are not permitted within proteins destined for the bacterial outer membrane, and these proteins, therefore, have had to evolve another mechanism of membrane assembly.

J Biol Chem, 1988 Dec 15, 263(35), 18946 - 52
Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA; Bonner CA et al.; We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus . A DNA polymerase activity has been purified from E . coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid . Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E . coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-) . The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis . Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III) . In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit) . Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III . Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein . Pol X has a molecular mass of 84 kDa . Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E . coli.

Gene, 1988 Dec 15, 73(1), 11 - 20
Structural analysis of mouse S-antigen; Tsuda M et al.; Mouse S-antigen clones were isolated from a mouse retinal cDNA library using a bovine S-antigen cDNA probe . The largest clone (MSC-242) comprised 1532 bp and contained the entire coding sequence . The nucleotide sequence homology between the mouse and bovine coding regions was 84%, while non-coding regions appeared to be more divergent . The deduced amino acid sequence indicated that the mouse S-antigen had 403 residues and its molecular ratio was 44,930 . An overall amino acid sequence similarity of 84% was observed between the mouse and bovine proteins . This degree of similarity dropped to 60% and 47% at the N and the C termini, respectively . The local homology with alpha-transducin observed in the bovine proteins, including the putative phosphoryl and rhodopsin binding sites, was conserved in the mouse as well . There was no overall sequence similarity with other proteins listed in the National Biomedical Research Foundation (NBRF) protein sequence database . Among the uveitopathogenic sites for experimental autoimmune uveitis (EAU), peptides N and M were identical to their bovine counterparts . Peptides 3 and K, however, were more divergent . The short repeats within these peptides were conserved.

J Biol Chem, 1988 Dec 15, 263(35), 18857 - 63
Transcriptional mapping and nucleotide sequence of the Escherichia coli fepA-fes enterobactin region . Identification of a unique iron-regulated bidirectional promoter; Pettis GS et al.; The iron-controlled fepA and fes-entF transcripts from the Escherichia coli enterobactin gene complex are expressed divergently from a limited genetic region, thereby suggesting the existence of a single, possibly overlapping promoter junction for these genes . The nucleotide sequence of a 1,997-base pair HpaI fragment specific for this genetic region allowed for the identification of an 1,122-base pair open reading frame as the previously uncharacterized fes gene . Its product, Fes (approximately Mr 42,573) plays an essential but as yet ambiguous role in the release of ferric iron from the ligand . An additional small open reading frame of 216 nucleotides (encoding a potential product of calculated Mr 8,271) was also identified between fes and entF . A portion of the remaining nucleotide sequence defined a 320-base pair control region for both the fepA and fes-entF messages . Primer extension analyses placed the major in vivo transcription initiation sites to within 18 nucleotides of one another, thereby revealing a novel, extensively overlapping bidirectional promoter as well as long dual leader transcripts . This promoter region contains multiple overlapping nucleotide stretches which show strong homology to the consensus Fur repressor-binding sequence, forms of which are found in all E . coli iron-regulated promoters characterized to date.

Cancer Lett, 1988 Dec 15, 43(3), 191 - 5
Human renal carcinoma: asparagine independence with asparaginase susceptibility in culture; Prager MD et al.; A human renal carcinoma cell line (Caki-1) was examined for asparagine (Asn) dependence and susceptibility to Escherichia coli asparaginase . Because this enzyme hydrolyzes glutamine (Gln) as well as Asn, even though at only 2-3% the rate, Asn- Gln+ and Asn- Gln- media were prepared . Only the former supported Caki growth . The Asn- Gln- medium was then repleted with Asn, Gln, or both . Although Asn repletion failed to promote growth, addition of Gln alone or the combination supported growth as well as complete medium . With {3H}leucine and {3H}mannose incorporation to indicate protein and glycoprotein synthesis, respectively, the Gln repleted medium supported these processes as well as complete medium . Asparaginase added to complete medium was highly toxic to the Caki cells, but this is a reflection of Gln depletion rather than Asn depletion.

Biochem Biophys Res Commun, 1988 Dec 15, 157(2), 816 - 20
3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate; Hedstrom L et al.; The mechanism of 3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P) synthase was investigated . When {18O}-PEP specifically labeled in the enolic oxygen is a substrate for KDO8P synthase, the 18O is recovered in Pi . This indicates that the KDO8P synthase reaction proceeds with C-O bond cleavage of PEP similar to that observed in the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase catalyzed condensation of PEP and erythrose-4-phosphate (1) . No evidence for a covalent enzyme-PEP intermediate could be obtained . No {32P}-Pi exchange into PEP nor scrambling of bridge 18O to non-bridging positions in {18O}-PEP was observed in the presence or absence of arabinose-5-phosphate or its analog ribose-5-phosphate . Bromopyruvate inactivated KDO8P synthase in a time dependent process . It is likely that bromopyruvate reacts with a functional group at the PEP binding site since PEP, but not arabinose-5-phosphate, protects against inactivation.

Gene, 1988 Dec 15, 73(1), 245 - 50
Jekyll, a family of phage-plasmid shuttle vectors; Burmeister M et al.; A series of shuttle vectors has been constructed, which consist of a plasmid carrying a polylinker sequence and an M13 origin integrated into a lambda vector . A short direct repeat flanking the plasmid allows plasmid excision by homologous recombination . Sequences are cloned into unique restriction sites within the plasmid, and can be recovered either in phage or plasmid form, or can be packaged further as single-stranded DNA phage . These vectors therefore combine the efficiency of phage lambda cloning and screening with the ease of handling or analysing plasmid or M13 clones.

J Biol Chem, 1988 Dec 15, 263(35), 18802 - 9
Site-directed alterations in the ATP-binding domain of rho protein affect its activities as a termination factor; Dombroski AJ et al.; We have utilized oligonucleotide site-directed mutagenesis to test our prediction that Escherichia coli rho factor has an ATP-binding domain separate from its RNA-binding domain and similar to that of adenylate kinase . Single amino acid substitutions were generated in regions thought to be within the active site and catalytically important for the ATPase activity, changing lysine 181 and/or lysine 184 to glutamine, and aspartate 265 to valine and asparagine . The altered proteins were purified and characterized in vitro for RNA- and ATP-binding ability, ATPase activity, helicase activity, and ability to catalyze transcription termination . Our results indicate that 1) these amino acid alterations in the proposed ATP-binding domain do not interfere with RNA binding; 2) substitution of lysine 184 by glutamine actually improves the ATPase and related activities while the same substitution at lysine 181 reduces but does not eliminate activity; 3) the double mutation changing both lysine 181 and lysine 184 to glutamine eliminates ATPase activity; and 4) the aspartate at 265 is also required for ATP hydrolysis but not for ATP binding . These results are consistent with our proposal that the general tertiary structure of rho's ATP-binding domain is similar to that of adenylate kinase.

Biochemistry, 1988 Dec 13, 27(25), 9020 - 30
Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase; Warren MJ et al.; The formation of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was shown to depend on the presence of 5-aminolevulinic acid . A hemA- mutant formed inactive deaminase when grown in the absence of 5-aminolevulinic acid since this strain was unable to biosynthesize the dipyrromethane cofactor . The mutant formed normal levels of deaminase, however, when grown in the presence of 5-aminolevulinic acid . Porphobilinogen, the substrate, interacts with the free alpha-position of the dipyrromethane cofactor to give stable enzyme-intermediate complexes . Experiments with regiospecifically labeled intermediate complexes have shown that, in the absence of further substrate molecules, the complexes are interconvertible by the exchange of the terminal pyrrole ring of each complex . The formation of enzyme-intermediate complexes is accompanied by the exposure of a cysteine residue, suggesting that substantial conformational changes occur on binding substrate . Specific labeling of the dipyrromethane cofactor by growth of the E . coli in the presence of 5-amino{5-14C}levulinic acid has confirmed that the cofactor is not subject to catalytic turnover . Experiments with the alpha-substituted substrate analogue alpha-bromoporphobilinogen have provided further evidence that the cofactor is responsible for the covalent binding of the substrate at the catalytic site . On the basis of these cumulative findings, it has been possible to construct a mechanistic scheme for the deaminase reaction involving a single catalytic site which is able to catalyze the addition or removal of either NH3 or H2O . The role of the cofactor both as a primer and as a means for regulating the number of substrates bound in each catalytic cycle is discussed.

Biochemistry, 1988 Dec 13, 27(25), 8915 - 23
Evidence for a singlet intermediate in catalysis by Escherichia coli DNA photolyase and evaluation of substrate binding determinants; Jordan SP et al.; Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyl-tetrahydrofolate (5,10-CH+-H4folate) . Both chromophores are fluorescent, and either can function as a sensitizer in catalysis . At 77 K separate fluorescence emission bands are observed for FADH2 (lambda max = 505 nm, shoulder at 540 nm) and 5,10-CH+-H4folate (lambda max = 465, 440 nm) whereas at 5 degrees C only a shoulder at 505 nm is attributable to FADH2 . Formation of an enzyme-substrate complex with various dimer-containing oligothymidylates {UV-oligo(dT)n} quenches the fluorescence due to FADH2 at 5 degrees C or 77 K and also stabilizes FADH2 against air oxidation . The fluorescence of 5,10-CH+-H4folate is unaffected by substrate . Reduction of the pterin chromophore eliminates the chromophore's fluorescence but does not affect catalytic activity or the ability of substrate to quench FADH2 fluorescence . Quenching of FADH2 fluorescence is fully reversible upon dimer repair . The results are consistent with the proposal that the singlet state of FADH2 functions as an intermediate in catalysis . Fluorometric titrations indicate that the enzyme has a similar affinity for dimers in UV-oligo(dT)4 (KD = 2.5 X 10(-7) M, delta G = 8.4 kcal/mol at 5 degrees C) or UV-oligo(dT)6, except for dimers located at the unphosphorylated 3' end of the oligomers where binding is considerably weaker.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Dec 13, 27(25), 9062 - 70
Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates; Banerjee RV et al.; The transfer of 17O and/or 18O from (COOH-17O or -18O) enriched substrates to inorganic phosphate (Pi) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation . COOH-18O-labeled folate, methotrexate, and dihydropteroate, in addition to {17O}-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E . coli . Pi was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for 17O and 18O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy . In the reactions catalyzed by the E . coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate 18O-enriched carboxyl group to Pi occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions . Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes . The small amounts of Pi obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques . However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that 18O transfer had occurred.

Gene, 1988 Dec 10, 72(1-2), 141 - 9
The differential stability of the Escherichia coli ompA and bla mRNA at various growth rates is not correlated to the efficiency of translation; Lundberg U et al.; Using two monocistronic gene transcripts, bla and ompA, we have studied the relationship between mRNA stability and translational efficiency . It was found that changes in the ompA mRNA stability are not correlated with an alteration in translational efficiency . In addition, at slow bacterial growth rates, the ompA transcript is translated ten times more efficiently than the bla messenger although the stability of the two transcripts is about equal . At rapid bacterial growth rate, chloramphenicol slightly stabilises both the bla and ompA transcripts without affecting their characteristic difference in half-life . Thus, control of mRNA stability seems not necessarily to be mediated either by the efficiency of loading ribosomes on a transcript, or by the arrest or slowing down of translating ribosomes.

Gene, 1988 Dec 10, 72(1-2), 131 - 9
Post-transcriptional control in Escherichia coli: translation and degradation of the atp operon mRNA; McCarthy JE et al.; An attractive subject for investigations of post-transcriptional control is the atp operon, whose nine genes are differentially expressed . The primary mode of control of atp gene expression is exercised at the translational level . It has been clearly demonstrated for almost all of the atp genes that the primary and secondary structures of their respective translational initiation regions direct translational initiation rates that correspond well to the requirements for these subunits in the cell . The relationship between the structure of the translational initiation region, including bases upstream from the Shine-Dalgarno region and downstream from the start codon, and the rates of initiation that it determines, has been investigated in more detail using various polycistronic and monocistronic systems . No evidence could be found for a role of codon usage bias in controlling overall translation rates . The functional half-lives of atpE and of the other six cistrons downstream from it are similar . The chemical stabilities of the first two cistrons of the polycistronic atp mRNA may, however, be lower, and we are investigating the possibility that there may also be control of atp gene expression exercised at the level of mRNA stability . The effects of manipulations of the intercistronic regions of at least the plasmid borne atp operon are consistent with a model of mRNA decay in which rate control is associated with endonucleolytic cleavages within individual cistrons . The experimental data are discussed in relation to the possible ways in which primary and secondary structures of the mRNA might control translational efficiency and stability.

Gene, 1988 Dec 10, 72(1-2), 349 - 60
Alpha-anomeric DNA: beta-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase; Bloch E et al.; Nuclease-resistant alpha-anomeric DNA:beta-RNA hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H . RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A)12:d{G-G-(T)12-G-G}, or by acid-solubility techniques, using a long substrate, poly(A):poly(dT) . Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange . Our results suggest that RNase H, for which DNA:RNA duplexes are the natural substrates, binds to non-physiological alpha-DNA:RNA hybrids and is consequently inhibited . These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities . However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded alpha-DNA itself . Unexpectedly, polymerase activity is highly stimulated by alpha-oligos, analogous in their sequence to the beta primer used at a concentration unable to generate a detectable synthesis . These results suggest that the inhibition of reverse transcriptase activity with the alpha:beta may occur at different levels.

Gene, 1988 Dec 10, 72(1-2), 247 - 52
Antisense RNA does not significantly affect expression of the galK gene of Escherichia coli or the N gene of coliphage lambda; Hasan N et al.; The effect of antisense RNA on the expression of genes galK and N was studied in vivo . These two genes were either present in the Escherichia coli chromosome, as single copies, or were cloned on plasmid vectors . Antisense RNA was supplied from multicopy vectors where the entire galK or N gene, or only their N-proximal portions, were cloned in the antisense orientation downstream from the strong PL, PR or lacZp promoters . In all of the experiments there was no significant inhibition of the galK or N expression by up to a 50-fold excess of the specific antisense RNAs, for both the in cis and in trans experimental designs . The excess of the antisense RNA was calculated as based on respective copy numbers, but was not experimentally measured . The apparent five-fold regulatory effect observed in one of the experiments was found to be artifactually caused by unexpected creation of a terminator in one of our constructs . To avoid such artifacts, all our constructs were equipped with the nut-N antitermination system . We conclude that the reported antimessenger-mediated inhibition of gene expression is not a general phenomenon, but must require some special features which are not present in the galK and N systems.

Gene, 1988 Dec 10, 72(1-2), 219 - 36
Analysis of the promoters and transcripts involved in IS10 anti-sense RNA control; Case CC et al.; Genetic analysis of eleven mutations affecting the IS10 promoters, pIN and pOUT, involved in anti-sense RNA control of transposase gene expression, and characterization of the transcripts, reveal that: (i) The transposase message (RNA-IN) and the anti-sense RNA (RNA-OUT) have been unambiguously identified in vivo . (ii) Five mutations affect pIN activity, and establish that pIN is the only IS10 promoter transcribing the tnp gene, and the only such IS10 promoter that responds to DNA-adenine methylation . (iii) Six mutations alter pOUT activity, and establish that pOUT is the only IS10 promoter specifying the anti-sense RNA-OUT . (iv) The latter, however, need not be so: heterologous promoters, if properly positioned, can also specify active anti-sense RNAs . (v) These heterologously promoted anti-sense RNAs are processed to species closely resembling native RNA-OUT.

Gene, 1988 Dec 10, 72(1-2), 179 - 86
Unexpected translation initiation within the coding region of eukaryotic genes expressed in Escherichia coli; Preibisch G et al.; When expressing several eukaryotic genes in Escherichia coli, we observed N-terminally truncated proteins which were attributed to translation initiation at downstream AUG codons . These AUG codons are located between 4 and 20 nucleotides 3' from sequences resembling bacterial SD elements . Although the presence of such downstream SD sequences is not sufficient for downstream initiation to occur, in two cases their removal abolishes synthesis of the truncated proteins . In one construct, a potential hairpin-loop structure is likely to inhibit translation initiation at the correct site and favor downstream initiation.

Nucleic Acids Res, 1988 Dec 9, 16(23), 11339 - 54
Dihydrothymidine and thymidine glycol triphosphates as substrates for DNA polymerases: differential recognition of thymine C5-C6 bond saturation and sequence specificity of incorporation; Ide H et al.; The ability of dihydrothymidine (DHdTTP) and thymidine glycol (dTTP-GLY) 5'-triphosphates to serve as substrates for different DNA polymerases was investigated . DHdTTP but not dTTP-GLY was used as a substrate by E . coli DNA polymerase I (Pol I) . Within the detection limit of the assay used, neither T4 DNA polymerase nor avian myeloblastosis virus (AMV) reverse transcriptase used DHdTTP or dTTP-GLY as substrates . The ability of DHdTTP and dTTP-GLY to undergo enzyme-catalyzed turnover to the monophosphate paralleled their ability to serve as substrates for polymerization . These results, along with kinetic parameters for the incorporation of DHdTTP with Pol I, strongly suggest that the saturation of thymine C5-C6 bond and the substituent groups at C5 and C6 differentially exert effects on binding to DNA polymerases . DNA sequencing gel analysis of the polymerization products revealed that most single adenine sites were capable of templating DHdTTP, however, DNA synthesis was partially arrested at multiple adenine sites, suggesting that sequential incorporation of DHdTTP produced significant disorder in the primer terminus.

Biochim Biophys Acta, 1988 Dec 9, 972(3), 249 - 56
Increase of histidine decarboxylase activity in murine myelomonocytic leukemia cells (WEHI-3B) in parallel to their differentiation into macrophages; Kawaguchi-Nagata K et al.; When cells of mouse myelomonocytic leukemia cell line, WEHI-3B, were cultured in the presence of actinomycin D plus the serum which was obtained from mice injected with bacterial endotoxin, i.e., lipopolysaccharide, their histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22) (HDC) activity increased about 100-fold with a peak at 48 h . According to the increase in HDC activity, the expression of surface antigens associated with macrophages, such as Mac II, Mac III and Iad, increased markedly on WEHI-3B cells as well as their morphological changes to macrophages . Histamine levels in the culture medium increased concomitantly with the increase in the HDC activity in WEHI-3B cells, whereas the histamine contents inside the cells did not increase remarkably . Furthermore, the addition of lipopolysaccharide to the culture medium caused an additional 2-fold increase in the HDC activity of WEHI-3B cells . These results indicate that the increase in HDC activity in WEHI-3B cells may represent an event in the process of the differentiation to macrophages.

J Biol Chem, 1988 Dec 5, 263(34), 18086 - 92
Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site . A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants; Levi S et al.; To study the functional differences between human ferritin H- and L-chains and the role of the protein shell in the formation and growth of the ferritin iron core, we have compared the kinetics of iron oxidation and uptake of ferritin purified from human liver (90% L) and of the H-chain homopolymer overproduced in Escherichia coli (100% H) . As a control for iron autocatalytic activity, we analyzed the effect of Fe(III) on the iron uptake reaction . The results show that the H-chain homopolymer has faster rates of iron uptake and iron oxidation than liver ferritin in all the conditions analyzed and that the difference is reduced in the conditions in which iron autocatalysis in high: i.e . at pH 7 and in presence of iron core . We have also analyzed the properties of two engineered H-chains, one lacking the last 22 amino acids at the carboxyl terminus and the other missing the first 13 residues at the amino terminus . These mutant proteins assemble in ferritin-like proteins and maintain the ability to catalyze iron oxidation . The deletion at the carboxyl terminus, however, prevents the formation of a stable iron core . It is concluded that the ferritin H-chain has an iron oxidation site which is separated from the sites of iron transfer and hydrolysis and that either the integrity of the molecule or the presence of the amino acid sequences forming the hydrophobic channel is necessary for iron core formation.

J Biol Chem, 1988 Dec 5, 263(34), 18043 - 51
Genomic structure and amino acid sequence domains of the human La autoantigen; Chambers JC et al.; La is an autoimmune RNA-binding protein of 47 kDa that plays a role in the transcription of RNA polymerase III . Both genomic and complementary DNAs were isolated that encompass the coding sequence of the human La molecule . The genomic clones encompass 11 exons and a putative G/C-rich promoter upstream of the mRNA start site . The cDNA sequence encodes a protein of 408 amino acids and can be divided into two structural domains based upon amino acid content and protease sensitivity . An unusually long stretch of 130 amino acids, much of which was predicted to form a stable alpha-helix, was found near the middle of the protein between the two domains . A ribonucleoprotein (RNP) consensus sequence was found just NH2-terminal to the long alpha-helix . The RNP consensus sequence is split into two exons by the fifth intron . Expression of three separate fragments of the La protein in Escherichia coli showed that a strongly autoimmune-reactive portion resides in the fragment containing the RNP consensus sequence and most of the long alpha-helical core . Autoantibodies from La patients also reacted with the terminal regions of the protein, but the extent of reactivity varied among patients . Differences in reactivity of autoantibodies to each portion of La protein may reflect an evolution of recognition of different epitopes during the development of the autoimmune response . These findings support an antigen-driven mechanism for autoimmune reactivity.

J Biol Chem, 1988 Dec 5, 263(34), 18452 - 8
Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine; Fox GM et al.; We have chemically synthesized the gene encoding bovine basic fibroblast growth factor (bFGF) and cloned it into a plasmid vector . This gene was then used as a template for site-directed mutagenesis to produce the human bFGF gene and a gene coding for an analog in which serine residues were substituted for the cysteine residues at positions 70 and 88 . All three constructs were cloned and expressed in Escherichia coli and the proteins purified . The recombinant human and bovine bFGFs exhibited the potent mitogenic activity toward both fibroblasts and endothelial cells, which characterizes natural bFGF . The serine-70,88 analog and natural sequence bovine and human forms were equally active in all assays . Sulfhydryl titration of the purified recombinant bovine bFGF in 4.8 M guanidine hydrochloride indicated the presence of approximately two free sulfhydryl groups . This was consistent with the sequence analysis of peptides derived from trypsin digestion, which suggests that cysteines 70 and 88 exist in free sulfhydryl form while cysteines 26 and 93 form a disulfide bond . Circular dichroism shows that the protein has little ordered structure but is folded into a rigid tertiary configuration . Carboxymethylation of the free sulfhydryl groups resulted in no change in the mitogenic activity or conformation . These results are consistent with previous suggestions that, for tissue-derived bFGF, at least 2 of the 4 cysteines in the molecule are not involved in a disulfide bond.

J Biol Chem, 1988 Dec 5, 263(34), 18343 - 9
In vitro attachment of bilins to apophycocyanin . I . Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C-phycocyanin; Arciero DM et al.; Expression of cloned alpha and beta subunit genes of Synechococcus sp . PCC7002 C-phycocyanin in Escherichia coli led to the production of large amounts of apophycocyanin . The apophycocyanin was purified to homogeneity and shown to be an alpha beta monomer . The reactivity of the apoprotein toward a number of open chain and cyclic tetrapyrroles was examined . Phycocyanobilin (PCB), phycoerythrobilin, and biliverdin all formed covalent adducts with apophycocyanin in 50 mM sodium phosphate buffer at pH 7.0 . Mesobiliverdin, bilirubin, PCB dimethyl ester, protoporphyrin IX, and hemin did not react with the apoprotein . None of these tetrapyrroles reacted with 2 mM 2-mercaptoethanol, cysteine, or reduced glutathione under the same conditions . The adduct with PCB was investigated in greater detail . Its visible absorption spectrum, with a maximum at 646 nm, is more similar to that of allophycocyanin than phycocyanin . Two PCBs are bound per alpha beta monomer when the reaction is performed with excess bilin . While tryptic digestion of the adduct generates numerous bilin peptides, amino acid analysis of these chromopeptides revealed that PCB reacted specifically at alpha-Cys-84 and beta-Cys-82, two of the three cysteinyl residues that serve as the attachment sites for PCB in native phycocyanin . The major bilin peptides arising from in vitro adduct formation at each of these sites differed both in chromatographic behavior and in spectroscopic properties from the corresponding PCB peptides isolated from tryptic digests of native C-phycocyanin.

J Mol Biol, 1988 Dec 5, 204(3), 725 - 47
Complex of N-phosphonacetyl-L-aspartate with aspartate carbamoyltransferase . X-ray refinement, analysis of conformational changes and catalytic and allosteric mechanisms; Ke HM et al.; The allosteric enzyme aspartate carbamoyltransferase of Escherichia coli consists of six regulatory chains (R) and six catalytic chains (C) in D3 symmetry . The less active T conformation, complexed to the allosteric inhibitor CTP has been refined to 2.6 A (R-factor of 0.155) . We now report refinement of the more active R conformation, complexed to the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) to 2.4 A (R-factor of 0.165, root-mean-square deviations from ideal bond distances and angles of 0.013 A and 2.2 degrees, respectively) . The antiparallel beta-sheet in the revised segment 8-65 of the regulatory chain of the T conformation is confirmed in the R conformation, as is also the interchange of alanine 1 with the side-chain of asparagine 2 in the catalytic chain . The crystallographic asymmetric unit containing one-third of the molecule (C2R2) includes 925 sites for water molecules, and seven side-chains in alternative conformations . The gross conformational changes of the T to R transition are confirmed, including the elongation of the molecule along its threefold axis by 12 A, the relative reorientation of the catalytic trimers C3 by 10 degrees, and the rotation of the regulatory dimers R2 about the molecular twofold axis by 15 degrees . No changes occur in secondary structure . Essentially rigid-body transformations account for the movement of the four domains of each catalytic-regulatory unit; these include the allosteric effector domain, the equatorial (aspartate) domain, and the combination of the polar (carbamyl phosphate) and zinc domain, which moves as a rigid unit . However, interfaces change, for example the interface between the zinc domain of the R chain and the equatorial domain of the C chain, is nearly absent in the T state, but becomes extensive in the R state of the enzyme; also one catalytic-regulatory interface (C1-R4) of the T state disappears in the more active R state of the enzyme . Segments 50-55, 77-86 and 231-246 of the catalytic chain and segments 51-55, 67-72 and 150-153 of the regulatory chain show conformational changes that go beyond the rigid-body movement of their corresponding domains . The localized conformational changes in the catalytic chain all derive from the interactions of the enzyme with the inhibitor PALA; these changes may be important for the catalytic mechanism . The conformation changes in segments 67-72 and 150-153 of the regulatory chain may be important for the allosteric control of substrate binding . On the basis of the conformational differences of the T and R states of the enzyme, we present a plausible scheme for catalysis that assumes the ordered binding of substrates and the ordered release o

J Mol Biol, 1988 Dec 5, 204(3), 581 - 91
Construction and characterization of the deletion mutant of hupA and hupB genes in Escherichia coli; Wada M et al.; Insertion and deletion mutations of the hupB and hupA genes, which encode the HU-1 and HU-2 proteins, respectively, of Escherichia coli, have been constructed in vitro and transferred to the hup loci on the bacterial chromosome . The mutations were constructed by inserting a gene encoding chloramphenicol resistance or kanamycin resistance into the coding region of the hupB or hupA gene, respectively . A complete deletion of the hupA gene was constructed by replacing the entire hupA coding region with the kanamycin resistance gene . Cells in which either the hupB or the hupA gene is defective grow normally, but cells in which both of the hup genes are defective exhibit phenotypes different from the wildtype strain . The hupA-hupB double mutants are cold-sensitive, although their growth rate is normal at 37 degrees C . Furthermore, the viability of the hupA-hupB double mutants is severely reduced when the cells are subjected to either cold shock or heat shock, indicating that the hup genes are essential for cell survival under some conditions of stress . The double mutants also exhibit filamentation when grown in the lower range of permissive growth temperature.

FEBS Lett, 1988 Dec 5, 241(1-2), 46 - 50
Engineering of an intersubunit disulphide bridge in glutathione reductase from Escherichia coli; Scrutton NS et al.; By site-directed mutagenesis, Thr-75 was converted to Cys-75 in the glutathione reductase (EC 1.6.4.2) of Escherichia coli . This led to the spontaneous formation of an intersubunit disulphide bridge across the 2-fold axis of the dimeric enzyme . The disulphide bridge had no deleterious effect on the catalytic activity, but nor did it increase the thermal stability of the enzyme, possibly because of local conformational flexibility on the dimer interface . The T75C mutant, like the wild-type enzyme, was inactivated by NADPH, proving that this inactivation cannot be due to simple dissociation of the dimer.

FEBS Lett, 1988 Dec 5, 241(1-2), 33 - 7
Spectroscopic studies on the mode of binding of ATP, UTP and alpha-amanitin with yeast RNA polymerase II; Bhargava P et al.; The binding affinity between the substrates ATP and UTP with the purified yeast RNA polymerase II have been studied here in the presence and absence of Mn2+ . In the absence of template DNA, both ATP and UTP showed tight binding with the enzyme without preference for any specific nucleotide, unlike Escherichia coli RNA polymerase . Fluorescence titration of the tryptophan emission of the enzyme by nucleoside triphosphate substrates gave an estimated Kd value around 65 microM in the absence of Mn2+ whereas in the presence of Mn2+, the Kd was 20 microM . The effect of substrates on the longitudinal relaxation of the HDO proton in enzyme-substrate complex also yielded a similar Kd value.

FEBS Lett, 1988 Dec 5, 241(1-2), 141 - 4
Flagellin parts acquiring a regular structure during polymerization are disposed on the molecule ends; Kostyukova AS et al.; Flagellins of two Escherichia coli strains have been investigated by limited proteolysis and scanning microcalorimetry . It has been shown that a monomer flagellin consists of two parts: a central one cooperatively melting, rather resistant to proteases, and the other without a stable tertiary structure and thus easily degrading terminals . Just these terminals acquire a regular structure during polymerization . Core fragments of the two strains have been isolated and characterized.

J Biol Chem, 1988 Dec 5, 263(34), 18437 - 42
Reconstitution of mitochondrial protein transport with purified ornithine carbamoyltransferase precursor expressed in Escherichia coli; Murakami K et al.; Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues and imported posttranslationally into the mitochondrial matrix . The rat pOTC was synthesized in Escherichia coli using an expression vector containing a thermoinducible lambda pL promoter . The recombinant pOTC represented 5-10% of the total bacterial protein and was present in the precipitate of the disrupted bacteria . The precipitate was washed and pOTC was extracted with 8 M urea or 0.1% cetyltrimethylammonium bromide . The extracted pOTC was essentially homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The purified pOTC was cleaved to the intermediate-sized product of 37,000 Da by a processing protease partially purified from the matrix fraction of rat liver mitochondria . The purified recombinant pOTC, but not the mature form of OTC synthesized in E . coli and purified, competed with the in vitro-synthesized, radiolabeled pOTC for uptake and processing by the isolated rat liver mitochondria . The radiolabeled and purified recombinant pOTC could be imported into the isolated mitochondria and processed to the mature form in an energy- and rabbit reticulocyte lysate-dependent manner . When the purified pOTC was subjected to sucrose gradient centrifugation, it sedimented as a large aggregate of greater than 60 S in the absence of reticulocyte lysate, whereas it sedimented as a complex of about 5 S in the presence of the lysate . These observations together with our previous results indicate that a protein factor(s) present in the lysate interacts with pOTC and holds it in an import-competent form.

J Biol Chem, 1988 Dec 5, 263(34), 18277 - 85
The role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro . Significance for SOS-targeted mutagenesis; Shwartz H et al.; The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated . RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs . In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication . The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed . Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level (Livneh, Z . (1986) J . Biol . Chem . 261, 9526-9533), inhibition at the DNA level also did not increase bypass of photodimers . Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs . Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association . Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected . However, reactivation of lesion-terminated primers was achieved by pretreatment with a 3'----5' exonuclease which excised 200 nucleotides or more, generating new 3'-OH termini located away from the UV lesions . When these exonuclease-treated products were subjected to a second round of replication, an increased level of DNA synthesis was observed including additional bypass of photodimers . These results suggest the possibility that 3'----5' exonuclease processing might be required at least transiently during one of the stages of trans-lesion DNA replication, which is believed to be the mechanism of SOS-targeted mutagenesis.

J Biol Chem, 1988 Dec 5, 263(34), 18017 - 22
Yeast DNA 3'-repair diesterase is the major cellular apurinic/apyrimidinic endonuclease: substrate specificity and kinetics; Johnson AW et al.; DNA strand breaks with damaged 3' termini are potentially toxic lesions caused by free radicals . The purified yeast diesterase that removes small nucleotide fragments from such 3' termini in oxidized DNA has been further characterized with respect to its substrate specificity . In addition to the 3'-phosphoglycolaldehyde esters used to monitor the activity during purification, the enzyme efficiently hydrolyzed a variety of other 3'-esters in DNA . These included 3'-phosphates, 3'-(2,3-didehydro-2,3-dideoxyribose phosphates), and the 3'-blocking damages formed in vivo in Escherichia coli by H2O2 or in vitro by DNA treatment with bleomycin . This same transition metal-dependent enzyme also constitutes the major yeast endonuclease for apurinic/apyrimidinic sites in DNA, hydrolyzing these damages to yield normal 3'-hydroxyl nucleotides and 5'-phosphoryl base-free sugar termini (a Type II apurinic/apyrimidinic endonuclease) . Yeast 3'-phosphoglycolaldehyde diesterase therefore appears to be involved in two distinct pathways of DNA repair: initiation of the repair of oxidative strand breaks in DNA and the restoration of sites of base loss caused by many types of DNA-damaging agents.

J Biol Chem, 1988 Dec 5, 263(34), 17933 - 41
Trypanosoma brucei ornithine decarboxylase: enzyme purification, characterization, and expression in Escherichia coli; Phillips MA et al.; Ornithine decarboxylase from the African trypanosome is an important target for antitrypanosomal chemotherapy . Despite this, the enzyme had not been previously purified or extensively characterized as it is a very low level protein . In this paper we describe the purification of Trypanosoma brucei brucei ornithine decarboxylase from bloodstream form trypomastigotes by 107,000-fold to a specific activity of 2.7 x 10(6) nmol CO2/h/mg of protein in the parasite . T . brucei ornithine decarboxylase had a native molecular weight of 90,000 and a subunit molecular weight of 45,000 . The isoelectric point of the protein was 5.0 . The Km for ornithine was 280 microM and the Ki for the irreversible inhibitor alpha-difluoromethylornithine (DFMO) was 220 microM with a half-time of inactivation at saturating DFMO concentration of 2.7 min . T . brucei ornithine decarboxylase appears similar to mouse ornithine decarboxylase, further supporting our previous suggestion that the selective toxicity of DFMO to the parasite is not due to catalytic differences between the two proteins . Although a small quantity of T . brucei ornithine decarboxylase was purified from T . brucei, extensive structural and kinetic studies will require a more ample source of the enzyme . We therefore expressed our previously cloned T . brucei ornithine decarboxylase gene in Escherichia coli using a vector that contains an inducible lambda promoter . T . brucei ornithine decarboxylase activity was induced in E . coli to levels that were 50 to 200 fold of that present in the long-slender bloodstream form of T . brucei . Ornithine decarboxylase activity in the crude E . coli lysate was 1500-6000 nmol of CO2/h/mg of protein and represented 0.05-0.2% of the total cell protein . The recombinant T . brucei ornithine decarboxylase was purified to apparent homogeneity from the transformed E . coli . The purified recombinant enzyme had kinetic and physical properties essentially identical to those of the native enzyme.

J Biol Chem, 1988 Dec 5, 263(34), 17909 - 12
Transfer RNA is a substrate for RNase D in vivo; Zhang JR et al.; RNase D is a 3'-exonuclease whose in vitro specificity has suggested a role in tRNA processing . However, since mutant Escherichia coli strains devoid of RNase D display a normal phenotype, it has not been possible to ascertain the enzyme's function or even to determine which RNA is its substrate in vivo . Here we show that transformation of strains devoid of tRNA nucleotidyltransferase with a multicopy plasmid carrying the rnd+ gene leads to extremely slow growth due to elevated levels of RNase D activity . Analysis of such a slow-growing strain revealed that less tRNA is present in the cell and that the tRNA that could be recovered is substantially damaged . These studies demonstrate that RNase D can act at the 3' terminus of tRNA in vivo, and they support the conclusion that RNase D participates in tRNA metabolism.

J Biol Chem, 1988 Dec 5, 263(34), 17905 - 8
Synthetic peptides as substrates and inhibitors of human immune deficiency virus-1 protease; Billich S et al.; Retroviruses code for a virus-specific protease which is essential for polyprotein processing and viral infectivity . The human immune deficiency virus-1 protease is an aspartic protease of 9 kDa which was synthesized by recombinant DNA technology and arises by autocatalytic processing from a polyprotein precursor which has recently been demonstrated by use of a protease-specific monoclonal antibody . The protease was shown to form dimers . Here we demonstrate that synthetic peptides can be used as both model substrates as well as inhibitors for investigation of the protease . 14 synthetic peptides, 7-18 amino acids in length, containing putative protease cleavage sites of the viral polyprotein gag and pol precursors, have been analyzed with the partially purified protease by the use of high performance liquid chromatography . In seven cases, where cleavage was observed, the length of the peptides did not significantly influence the cleavage efficiencies, heptapeptides being large enough as model substrates . No cleavage was observed with a protein preparation purified in parallel from control bacteria not expressing the human immune deficiency virus-1 protease . The protease was not only able to cut next to a proline but also between other peptides indicating that the proline is not a prerequisite . Three peptides with either reduced bonds at the cleavage site or a substitution by statin were inhibitory while another uncleaved substrate was not . The usefulness of small model substrates for characterization of the protease is further demonstrated by determination of a kinetic optimum pH (3.5-5.5) and incubation temperature (37 degrees C).

FEBS Lett, 1988 Dec 5, 241(1-2), 251 - 6
Renaturation of guanidine-unfolded tryptophan synthase by multi-mixing stopped-flow dilution in D2O; Blond-Elguindi S et al.; Guanidine hydrochloride (GdnHCl) at high concentrations, e.g . 4 to 8 M, has been used extensively to promote reversible denaturation of several proteins . The refolding is induced by removal of the denaturing agent by diluting the denatured protein at least 50-100-fold in a 'renaturation buffer' . Fast kinetic studies, using a stopped-flow apparatus to achieve such dilutions, are difficult for two reasons: firstly, injecting widely different volumes of the two reagents does not afford a proper mixing . Secondly, the density differences existing between the concentrated GdnHCl solution and the renaturation buffer often causes important mixing and redistribution artefacts particularly in vertical stopped-flows . Here, it is shown that these difficulties can be overcome by using a multi-mixing stopped-flow to achieve 2 successive 7-fold dilutions and by using heavy water (D2O) to adjust the density of the renaturation buffer . This enabled us to study the appearance of a short-lived intermediate during the folding of the beta 2-subunit of Escherichia coli tryptophan synthase.

J Biol Chem, 1988 Dec 5, 263(34), 18099 - 103
Mutants of translational components that alter reading frame by two steps forward or one step back; Falahee MB et al.; External suppressors, sufS, of a -1 frameshift mutant cause ribosomes to shift into the -1 frame when reading the sequence CAG GGA GUG . The resulting product is not Gln-Gly-Val but Gln-Gly-Ser with Ser being encoded by the underlined AGU . The alleles investigated are approximately 2% efficient in causing frameshifting . Two other suppressors, hopR and hopE of the same -1 frameshift mutant, cause some ribosomes reading the sequence GUG UG to decode a single amino acid, Val, from the five nucleotides . The possibility is considered that peptidyl-tRNA(Val) dissociates from the mRNA, but re-pairs in a triplet manner after the mRNA slips forward by two bases.

J Mol Biol, 1988 Dec 5, 204(3), 507 - 22
Domain VI of Escherichia coli 23 S ribosomal RNA . Structure, assembly and function; Leffers H et al.; Domain VI at the 3' end of the 23 S ribosomal RNA from Escherichia coli was prepared using the in vitro T7 RNA polymerase system . Its structure was examined by probing with ribonucleases and chemical reagents, including a psoralen derivative, of various nucleotide specificities, using a reverse transcriptase procedure for analysis . The data provided support for the most recent secondary structure derived from phylogenetic sequence comparisons and for additional structuring that was inferred from earlier experimental data . Moreover, the structure was essentially the same in the free domain, in renatured 23 S RNA and in 50 S subunits . Protein L3 bound to the isolated domain and its binding site was located at a long-range double helix containing a large internal loop . This structure is unusual for a protein-RNA binding site and it may characterize a new (third) class of site . Protein L3 has been implicated, together with L24, in initiating assembly of the 50 S subunit and it shares the exceptional property with L24 that it binds adjacent to the junction of two RNA domains from where it can maximally influence RNA folding . Protein L6 also assembled to domain VI and, in a control experiment, protein L2 bound to isolated domain IV . Domain VI was largely inaccessible in the 50 S subunit and the few accessible RNA sites occurred mainly within conserved sequence regions that constitute potential functional sites . alpha-Sarcin inactivates ribosomes by cutting at one of these sites in 50 S subunits; it also recognized the same site in the free 23 S RNA and in the free domain . Both the EF-Tu ternary complex, and the EF-G ternary complex stabilized by fusidic acid or by a non-hydrolyzable GTP derivative, inhibited alpha-sarcin attack while non-enzymatically bound tRNA did not, thus providing evidence, more direct than before, for the involvement of the RNA region in a common elongation factor binding site.

Science, 1988 Dec 2, 242(4883), 1290 - 5
Sugar and signal-transducer binding sites of the Escherichia coli galactose chemoreceptor protein; Vyas NK et al.; D-galactose-binding (or chemoreceptor) protein of Escherichia coli serves as an initial component for both chemotaxis towards galactose and glucose and high-affinity active transport of the two sugars . Well-refined x-ray structures of the liganded forms of the wild-type and a mutant protein isolated from a strain defective in chemotaxis but fully competent in transport have provided a molecular view of the sugar-binding site and of a site for interacting with the Trg transmembrane signal transducer . The geometry of the sugar-binding site, located in the cleft between the two lobes of the bilobate protein, is novel in that it is designed for tight binding and sequestering of either the alpha or beta anomer of the D-stereoisomer of the 4-epimers galactose and glucose . Binding specificity and affinity are conferred primarily by polar planar side-chain residues that form intricate networks of cooperative and bidentate hydrogen bonds with the sugar substrates, and secondarily by aromatic residues that sandwich the pyranose ring . Each of the pairs of anomeric hydroxyls and epimeric hydroxyls is recognized by a distinct Asp residue . The site for interaction with the transducer is about 18 A from the sugar-binding site . Mutation of Gly74 to Asp at this site, concomitant with considerable changes in the local ordered water structures, contributes to the lack of productive interaction with the transmembrane signal transducer.

Cell, 1988 Dec 2, 55(5), 849 - 56
Supercoiling of intracellular DNA can occur in eukaryotic cells; Giaever GN et al.; The supercoiling of 2 micron DNA in yeast by a process or processes that generate positively and negatively supercoiled domains was shown by the use of yeast DNA topoisomerase mutants expressing Escherichia coli DNA topoisomerase I, an enzyme that relaxes negative supercoils specifically . Intracellular 2 micron DNA becomes positively supercoiled in yeast top1 top2 ts strains expressing the E . coli enzyme when neither one of the yeast DNA topoisomerases I and II is functional . Examination of the linking number distributions of plasmids bearing the inducible promoters of GAL1 and GAL10 genes indicates that the generation of supercoiled domains of opposite signs is related to transcription.

J Clin Invest, 1988 Dec, 82(6), 1934 - 8
Interleukin 1 administration in mice produces hypoferremia despite neutropenia; Gordeuk VR et al.; To determine whether the hypoferremic response to inflammation requires neutrophils, we administered human recombinant IL-1 to mice made neutropenic with cyclophosphamide . With single intraperitoneal injections of IL-1 the plasma iron concentrations decreased significantly in mice with either normal neutrophil counts or neutropenia . After single injections transferrin concentrations were not significantly changed, but the decrease in serum iron lowered mean transferrin saturations from a baseline of 45 to 24-30% in nonneutropenic mice, and from 99 to 70-77% in neutropenic mice . Similar changes were observed after intraperitoneal injections of Escherichia coli . 4-d continuous infusions of IL-1 also led to reductions in serum iron concentrations, but transferrin concentrations doubled . The combination of a decrease in serum iron and an increase in transferrin concentration after chronic infusion in neutropenic mice led to a greater decline in mean transferrin saturations, from a baseline of 110 to 25% . In mice not given cyclophosphamide, chronic IL-1 infusion was associated with a reduction in mean hemoglobin concentrations from 14.7 to 13.5 g/dl, consistent with restricted availability of iron for erythropoiesis associated with low saturation of transferrin . We conclude that IL-1 can decrease the serum iron despite profound peripheral neutropenia and that transferrin in a positive acute phase reactant in the mouse.

Genes Dev, 1988 Dec, 2(12B), 1812 - 23
Gene activities and segmental patterning in Drosophila: analysis of odd-skipped and pair-rule double mutants; Coulter DE et al.; The odd-skipped (odd) gene is required to generate anterior regions of the odd-numbered segments in Drosophila; homozygous embryos show pattern deletions that are always less than a segment in width and are associated with mirror-image duplications of adjacent regions . To define further the role of odd and determine how it interacts with other segmentation genes, we have described the effects of combining odd with mutations at other pair-rule loci . We have observed phenotypic suppression in double-mutant combinations with even-skipped (eve), paired, sloppy paired, and engrailed (en) . In the most thoroughly characterized combination (odd eve), both naked cuticle and specific denticle rows are restored that would normally have been deleted by one of the two mutants alone . In the odd en double mutant, we observe nearly complete suppression of the odd phenotype, such that the mirror image duplications are eliminated and the odd-numbered denticle bands are restored . We conclude that the requirements of pattern elements for specific gene activities are not absolute, and propose mechanisms by which these genes interact to specify cell fates.

Biophys J, 1988 Dec, 54(6), 983 - 93
Particle counting by fluorescence correlation spectroscopy . Simultaneous measurement of aggregation and diffusion of molecules in solutions and in membranes; Meyer T et al.; A method for simultaneous determination of molar weights (M) and lateral diffusion constants (D) of particles in three- and two-dimensional systems is described . Spontaneous concentration fluctuations in space and time are analyzed, by monitoring fluctuations in the fluorescence from fluorescein-labeled molecules (1 dye/molecule is sufficient), excited by a rotating laser spot . For particles in solution, M values are determined over the range of 3 x 10(2) to 3 x 10(11) daltons, and D values can be determined from approximately 10(-7) to 10(-10) cm2/s . The time for a determination is approximately 1 min . Aggregation can be followed by changes of either M or D . This method is used to study the calcium dependence of vesicle aggregation or fusion, and the time course of aggregate formation of porin (an Escherichia Coli outer membrane protein) in lipid monolayers . Essential parameters for the development of the method are described . Equations to estimate the signal-to-noise ratios and to find the optimal free parameters for a specific application are derived . The theoretical predictions for the correlation function of the signal and for the signal-to-noise ratio are compared with observed values.

Epidemiol Infect, 1988 Dec, 101(3), 611 - 21
A study of intranasally administered interferon A (rIFN-alpha 2A) for the seasonal prophylaxis of natural viral infections of the upper respiratory tract in healthy volunteers; Tannock GA et al.; The efficacy of interferon A (rIFN-alpha 2A), an Escherichia coli-derived interferon, in the prophylaxis of acute upper respiratory tract infection, was evaluated in a community-based double-blind placebo-controlled study in the Australian winter of 1985 . The trial population of 412 healthy volunteers (190 males and 222 females, aged 18-65 years) self-administered 1.5, 3.0 and 6.0 megaunits (MU) of interferon A per day or a placebo, intranasally for 28 days . The period of study coincided with an outbreak of H3N2 influenza A (detected in 35 of the 107 acute specimens) as well as substantial numbers of respiratory syncytial virus and adenovirus infections . Rhinoviruses were isolated from only three specimens . In many cases, subjects had laboratory and clinical evidence of having had more than one respiratory tract infection during the period of the study . Viruses were detected in 54 or 107 acute specimens (49%) . No statistically significant differences were noted between the various treatment groups in the incidence of laboratory-proven viral infection (virus isolation and/or antibody response) . Analysis of reported symptoms indicated that blood-tinged mucus and nasal stuffiness occurred more frequently with higher doses of interferon . There appeared to be no clinical benefit from the use of interferon A in the amelioration of symptoms.

Am J Med Sci, 1988 Dec, 296(6), 381 - 6
Endotoxin-induced neutrophilic alveolitis and macrophage chemotaxin production in sheep; Duke SS et al.; The mechanism(s) responsible for endotoxin-induced pulmonary leukostasis has not yet been elucidated . We studied ten unanesthetized sheep by performing serial bronchoalveolar lavages (BAL) before and after infusing Escherichia coli endotoxin to determine whether or not neutrophils appeared in the airways and whether or not endotoxemia stimulated alveolar macrophages to produce neutrophil chemotactic activity . The absolute number and percentage of neutrophils recoverable by BAL increased significantly at 24 hours after infusion of endotoxin . Alveolar macrophages isolated from the BAL of sheep after endotoxemia produced a substance that is chemotactic for neutrophils, a response that is also maximal 24 hours after endotoxin infusion . We conclude that endotoxemia causes migration of neutrophils into lung air spaces and that this response may result from in vivo production of a chemotactic factor(s) by activated alveolar macrophages.

EMBO J, 1988 Dec 1, 7(12), 3975 - 82
The Escherichia coli LexA repressor-operator system works in mammalian cells; Smith GM et al.; We have demonstrated the use of the Escherichia coli LexA repressor-operator system to down-regulate gene expression in mouse cells . The LexA gene was placed downstream of the RSVLTR promoter with polyadenylation and splice signals from SV40 . This expression unit was introduced into mouse Ltk- cells by calcium phosphate transfection and stable transfectants selected which express LexA protein . We have used the bacterial chloramphenicol acetyltransferase gene (CAT) as our reporter gene . Transcription of this gene was driven by the HSV tk promoter, into which we have introduced one or two synthetic LexA operator sequences in various positions throughout the promoter . Necessary 3' signals were from the HSV tk gene . Repression by LexA was assessed by comparing the transient expression of tkCAT target constructs, containing LexA operator sequences in the promoter, in cells expressing LexA protein with that in control cells not expressing the repressor . We have observed up to 10-fold repression of CAT expression in LexA+ cells from promoters containing LexA operator sequences.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8924 - 8
Cocrystal structure of an editing complex of Klenow fragment with DNA; Freemont PS et al.; High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site . Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus . Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes . We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus . Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.

Mol Gen Mikrobiol Virusol, 1988 Dec, (12), 33 - 8
{Mapping of "nonessential" regions in the genome of vaccinia virus}; Loparev VN et al.; The special molecular probe for mapping the "nonessential" regions in the genome of vaccinia virus has been obtained by the genetic engineering methods . The probe included the gene for beta-galactosidase of E . coli under the control of vaccinia virus 7.5 K protein promoter as well as the gene for kanamycin resistance . In its final version the probe is obtainable from the plasmid pUCZ beta using the restriction endonucleases SalI, BamHI, EcoRI . The probe included by the BamHI fragment of DNA was inserted into the HindIII-E-fragment of the vaccinia virus (cloned into a plasmid) in 8 of the existing 9 BglII cleavage sites . The latter plasmids were introduced into the chicken embryo cells infected by the vaccinia virus . The plasmid having the probe inserted into the 5th BglII site (from the left end) of the HindIII-E fragment permitted to obtain the live vaccinia strain expressing the beta-galactosidase . Thus, the "nonessential" region of vaccinia virus, that was not described previously, is mapped.

Biochimie, 1988 Dec, 70(12), 1807 - 14
Allosteric transition of aspartokinase I-homoserine dehydrogenase I studied by time-resolved fluorescence; Jullien M et al.; The allosteric transition of threonine-sensitive aspartokinase I-homoserine dehydrogenase I from Escherichia coli has been studied by time-resolved fluorescence spectroscopy . Fluorescence decay can be resolved into 2 distinct classes of tryptophan emitters: a fast component, with a lifetime of about 1.5 ns; and a slow component, with a lifetime of about 4.5 ns . The fluorescence properties of the slow component are modified by the allosteric transition . In the T-form of the enzyme stabilized by threonine, the lifetime of the slow component is longer, with a red-shifted spectrum; its accessibility to quenching by acrylamide becomes slightly higher without any decrease of fluorescence anisotropy . These results indicate a change in polarity of the slow component environment . The quaternary structure change associated with the allosteric transition probably involves global movements of structural domains without leading to any local mobility on the nanosecond time-scale . We suggest that the slow component corresponds to the unique tryptophan of the buried kinase domain.

J Photochem Photobiol B, 1988 Dec, 2(4), 491 - 501
Effect of UV irradiation at defined wavelengths on the tertiary structure of double-stranded covalently closed circular DNA; Boullard A et al.; Double-stranded, covalently closed, supercoiled circular DNA from phage fd (replicative form) was irradiated with increasing doses of UV light at 254 nm, 290 nm, 313 nm and 365 nm, and subjected to electrophoresis on agarose slab gels . Increasing the doses of UV light at 254 and 290 nm promotes a smooth reduction in the electrophoretic mobility of the sample, as would be expected if the major effect of light at these two wavelengths were to induce the formation of photoproducts leading to the unwinding of the double strand . At high doses, UV light at 290 nm introduces single-strand breaks (1.2 kJ m-2 per nick per million phosphodiester bonds) . UV light at 313 nm promotes an abrupt change in the electrophoretic mobility, as would be expected if the effect of this wavelength were to induce single-strand breaks, leading to the transformation of the supercoiled molecules in their relaxed form (23 kJ m-2 in order to introduce one nick per million phosphodiester bonds) . UV light at 365 nm also promotes single-strand breaks in DNA (140 kJ m-2 per nick per million phosphodiester bonds).

AIDS, 1988 Dec, 2(6), 477 - 80
A new second-generation anti-HIV-1 enzyme immunoassay using recombinant envelope and core proteins; Backer U et al.; In a multicenter collaborative study a new second-generation HIV-1 antibody enzyme immunoassay (Abbott recombinant HIV-1 EIA) using Escherichia coli-expressed recombinant p24 and p41 proteins as solid-phase antigens was compared with the first-generation H9 cell-line-based Abbott HIV-1 EIA . The results of the confirmatory assays (Western blot, immunofluorescence), combined with clinical information, were used as the reference standard for the detection of HIV-1 antibodies in 10,676 random blood donor serum specimens, in a panel of 840 specimens from symptomatic and asymptomatic patients and a total of 63 serial blood specimens from 23 people at risk . With fresh blood donor sera, the specificity of the first-generation assay ranged between 99.54 and 99.76% (95% confidence limits, CL) compared with 99.81-99.95% (95% CL) for the second-generation EIA . With panel specimens the recombinant HIV-1 EIA achieved an overall sensitivity of 100% and a specificity range of 98.3-99.7% (95% CL); the corresponding sensitivity and specificity ranges observed for the first-generation EIA were 98.0-99.5% (95% CL) and 94.3-96.8% (95% CL), respectively . The improved sensitivity for the second-generation assay was confirmed by testing serial samples from seroconverting patients . The use of recombinant proteins eliminated non-specific reactions due to class II human leukocyte antigen (HLA)-directed antibodies.

Mol Gen Genet, 1988 Dec, 215(1), 161 - 4
The isolation and preliminary characterisation of a novel Escherichia coli mutant rorB with enhanced sensitivity to ionising radiation; Debenham PG et al.; Escherichia coli K803 cells were mutagenized and screened for the presence of clones sensitive to gamma-rays but not to ultraviolet light . One new mutant of this type, named rorB, was isolated . This mutant is both cross-sensitive to mitomycin C and shows reduced conjugal recombination frequencies, but to a lesser extent than the phenotypically similar mutant recN . Unlike previously reported mutants of E . coli or yeast with an enhanced sensitivity to ionising radiations, rorB appears to be near wild type in ability to rejoin DNA double-strand breaks . The rorB gene maps close to ilvGEDAC at 84.5 min of the E . coli chromosome.

Mol Gen Genet, 1988 Dec, 215(1), 156 - 60
The cloning of the rorB gene of Escherichia coli; Debenham PG et al.; A segment of the Escherichia coli genome which complements the ionising radiation sensitivity of the rorB mutation was cloned into pBR322 . This DNA segment also complements the mitomycin C sensitivity of the rorB mutation . The gene was subcloned until defined in a fragment of 1.05 kb . Only one gene product, a protein of approximately 16.5 kDa, was found on maxicell analysis of the various subclones . Iso-electric focusing of this gene product suggests it may function in a complex.

Genes Dev, 1988 Dec, 2(12B), 1851 - 8
Escherichia coli heat shock gene mutants are defective in proteolysis; Straus DB et al.; Heat shock proteins in Escherichia coli are relatively abundant and some are essential for growth, but the function that they provide is unknown . The observation that heat shock proteins are induced by some abnormal, rapidly degraded polypeptides, and that strains with mutations in the rpoH gene, the positive regulator of heat shock gene expression, are defective in proteolysis, has led to the proposal that heat shock proteins are required for normal degradation of polypeptides . We have investigated this hypothesis by examining the degradation of polypeptide fragments generated by puromycin and the degradation of a nonsense fragment of beta-galactosidase . Mutations in the dnaK, dnaJ, grpE, and groEL heat shock genes result in defective proteolysis . Furthermore, overproduction of heat shock proteins results in enhanced rates of puromycyl fragment decay . The proteolysis defect of the heat shock gene mutants primarily affects energy-dependent protein degradation . These results indicate that at least one general function of heat shock proteins is to contribute to the ability of the cell to degrade abnormal polypeptides.

Vet Microbiol, 1988 Dec, 18(3-4), 313 - 25
Conservation of antigenicity in a 31-kDa Brucella protein; Bricker BJ et al.; A 31-kilodalton (kDa) protein extracted from Brucella abortus was previously cloned into Escherichia coli and expressed at high levels . The E . coli-derived protein can be purified by a simple 2-step procedure entailing detergent extraction followed by ion-exchange chromatography . Subsequent analyses show that the E . coli-derived protein is identical to the Brucella-derived protein in molecular weight and isoelectric point . A partial amino acid sequence of the N-terminus of the protein of E . coli origin matches the predicted sequence, based on DNA sequence data . Using specific antiserum raised against the E . coli-derived protein, 34 strains of Brucella, representing all 6 recognized species, were examined for expression of the 31-kDa protein by Western blotting . This protein was detectable in all, but one Brucella species (B . ovis), including all 8 biovars of B . abortus tested . This degree of conservation supports further study of the 31-kDa protein for potential exploitation as a vaccine or diagnostic component.

Genetics, 1988 Dec, 120(4), 863 - 73
Mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position; Sambamurti K et al.; In order to examine the mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position, the replicative form DNA of phage M13AB28 (mp8 without the amber codons in phage genes) was modified in vitro with aflatoxin B1-2,3-dichloride and transfected into appropriate Escherichia coli cells . Forward mutations in the lacZ alpha-complementing gene segment were identified as light blue or colorless plaques on appropriate indicator plates, isolated, and defined by DNA sequencing . Transfection of modified DNA into uvrA-/mucAB+ cells without prior UV (SOS) induction increased mutation frequency eight-fold over untreated DNA, whereas this increase was 12-fold upon SOS induction . Transfection of modified DNA after conversion of the primary guanine-aflatoxin lesions to the stable imidazole ring-opened formamidopyrimidine-aflatoxin suggested that these lesions were nearly equally mutagenic . A majority of point mutations under all conditions affected G:C bp . Base substitutions were in the majority, but significant frameshift mutagenesis was also detected in SOS-induced cells . Both G-to-T transversions and G-to-A transitions were produced at equal efficiency and together accounted for virtually all of the base substitutions induced by the primary lesions . Point mutations occurred predominantly at predicted damage hotspots . The characteristics of base substitution and frameshift mutations, together with available information point to multiple mechanisms of mutagenesis by this class of mutagens . The data indicate that primary lesions have the properties of both a noninstructional and pseudo-instructional lesion . In addition, the sequence context appears to play a role in determining whether a frameshift or a base substitution is induced by this bulky lesion.

Eur J Immunol, 1988 Dec, 18(12), 1881 - 7
Specificity of proliferative response of human CD8 clones to mycobacterial antigens; Rees A et al.; Human CD8 T lymphocyte clones (TLC) were generated from the pleural effusion of patients with tuberculosis using a protocol that required, in addition to antigen, coculture of purified CD8+ T cells, accessory cells, interleukin 2 (IL2) and anti-CD3-Sepharose . The TLC obtained were stimulated by mycobacterial soluble extracts in an IL2-dependent and MHC class I-restricted manner . When antigen-responsive TLC were screened with extracts from the recombinant mycobacterial library they were found to respond to either the Y3125 (100-kDa) or the Y3111 (71-kDa) lambda gt11 clones . Polyacrylamide gel immunoblot analysis demonstrated that the CD8 TLC responded to fractions with the molecular mass range 27-45 kDa in the Y3125 lysogen and 60-90 kDa in the mycobacterial soluble extract . The specificity of TLC reactive with the Y3111 clone was confirmed using the 71-kDa antigen purified from the same lysogen . These TLC recognized sequences common to the 71-kDa protein derived from mycobacteria, E . coli or a human cell line . Studies of three TLC using antigen-presenting cells of known genetic haplotype indicated that stimulation with both the Y3125 and the 71-kDa antigens were restricted by determinants encoded by HLA-B8.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8968 - 72
Dual mechanism of repression at a distance in the lac operon; Flashner Y et al.; The mechanism by which the internal lacZ gene sequence O2 influences lac repression was investigated by using in vivo footprinting of operon mutants . Quantitative in vivo binding curves show that O2 strengthens by approximately 3-fold repressor binding to O1 that is located 400 base pairs upstream at the transcription start site . The internal O2 sequence also contributes to repression by a second mechanism: repressor bound internally blocks elongation of beta-galactosidase gene expression . This secondary mechanism of repression is facilitated by the remote O1 operator that strengthens binding to O2 12-fold . Thus, lac repression involves two mechanisms, both of which involve cooperation between remote operator elements . During mild repression only the initiation mechanism applies, but more severe repression favors formation of the presumptive O1-O2 repression loop that allows both mechanisms to act simultaneously.

J Bacteriol, 1988 Dec, 170(12), 5949 - 52
Analysis of the diphtheria tox promoter by site-directed mutagenesis; Boyd J et al.; By oligonucleotide-directed mutagenesis, we introduced alterations in the two putative -10 regions of the diphtheria tox promoter which are positioned at -50 and -56 from the GUG tox initiation signal . The -10 region positioned at -50 is favored in the expression of ADP-ribosyltransferase activity from the wild-type tox promoter in recombinant Escherichia coli; however, the promoter down mutation at position -50 is compensated for by increased activity of the -10 region positioned at -56.

J Bacteriol, 1988 Dec, 170(12), 5890 - 4
Molecular cloning and high-level expression of a bromoperoxidase gene from Streptomyces aureofaciens Tü24; van Pee KH; A bromoperoxidase gene was cloned from Streptomyces aureofaciens Tu24 into Streptomyces lividans TK64 by using the promoter-probe vector pIJ486 . Subcloning of DNA from the original, unstable clone allowed the gene to be localized to a 1.7-kilobase (kb) fragment of DNA . Southern blotting showed that the cloned 1.7-kb insert hybridized to a 4.3-kb fragment in an SstI digest of S . aureofaciens Tu24 total DNA . The 1.7-kb insert was shown to code for a protein with the electrophoretic properties of the subunits of the nonheme bromoperoxidase isolated from S . aureofaciens Tu24 . The protein produced by S . lividans TK64 transformed with pHM621, which contained an 8.0-kb insert, was shown to be identical to the S . aureofaciens Tu24 bromoperoxidase in terms of its electrophoretic mobility on denaturing and nondenaturing polyacrylamide gels and its NH2-terminal amino acid sequence . The bromoperoxidase was overproduced (up to 180 times) by S . lividans TK64 containing pHM621 . Based on the heat stability of the S . aureofaciens Tu24 bromoperoxidase, a new and simple purification procedure with very high yields was developed.

J Bacteriol, 1988 Dec, 170(12), 5460 - 7
Regulation of gene expression in plasmid ColE1: delayed expression of the kil gene; Zhang SP et al.; cea, imm, and kil are a cluster of three functionally related genes of the plasmid ColE1 . The cea and kil genes are in the same inducible operon, with transcription being initiated from a promoter adjacent to the cea gene . The imm gene is located between the cea and kil genes, but it is transcribed in the opposite direction . Complementary interaction between the imm mRNA and the anti-imm sequences in the middle of the cea-kil transcript causes a pronounced delay in expression of the kil gene when the cea-kil operon is induced . A segment in the overlapping region between the cea and imm genes causes delayed expression of the kil gene in the absence of imm gene transcription . This delay effect increases the yields of colicin synthesized in induced cells.

FEMS Microbiol Immunol, 1988 Dec, 1(3), 117 - 25
Non-replicating oral whole cell vaccine protective against enterotoxigenic Escherichia coli (ETEC) diarrhea: stimulation of anti-CFA (CFA/I) and anti-enterotoxin (anti-LT) intestinal IgA and protection against challenge with ETEC belonging to heterologous serotypes; Evans DG et al.; An oral killed (non-replicating) whole-cell anti-ETEC vaccine was prepared by treating enterotoxigenic Escherichia coli strain H-10407 (ST + LT +; 078: H11: CFA/I) with a 100%-lethal amount of colicin E2 . Colicin E2 is a potent DNA endonuclease which enters the target bacterial cells without disrupting cellular integrity . Thus the vaccine consists of intact cells lacking chromosomal and plasmid DNA but possessing a normal complement of antigens, including CFA/I and enterotoxin(s), unaltered by chemical- or heat-treatment . Young healthy volunteers were administered two oral doses, one month apart, of approximately 3 x 10(10) vaccine cells . Of 22 vaccinees, 17 (77.3%) showed an intestinal anti-CFA/I IgA response and 19 (86.4%) showed an increase in intestinal anti-LT IgA . Twenty of 22 (90.9%) vaccinees had antibody responses to either CFA/I, LT, or both antigens, demonstrating that colicin E2-treated CFA-positive E . coli cells are an efficient vehicle in terms of delivery of antigens to the gut immune system . We previously demonstrated protection of vaccinees against challenge with the living homologous ETEC (strain H-10407) . In this study, two groups of 8 vaccinees were challenged with a diarrheagenic dose of virulent ST + LT + ETEC of heterologous serotype; one group was challenged with a CFA/I-positive 063: H- strain and the other group was challenged with a CFA/II-positive 06: H16 strain . Approximately 75% efficacy was achieved in both challenge groups . None of the 16 vaccinees who had responded to both CFA/I and LT became ill upon challenge while both of the vaccinees who had not responded to either antigen did.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Microbiol, 1988 Dec, 134 ( Pt 12), 3129 - 39
Mutants of Escherichia coli specifically deficient in respiratory formate dehydrogenase activity; Mandrand-Berthelot MA et al.; Escherichia coli K12 mutants lacking phenazine-methosulphate-linked formate dehydrogenase (FDH-PMS) activity, but still capable of producing normal levels of benzyl-viologen-linked formate dehydrogenase (FDH-BV) and nitrate reductase activities, have been isolated following P1 localized mutagenesis . The relevant mutations mapped with the same cotransduction frequency close to the rhaD gene, at 88 min on the E . coli chromosome . They were further subdivided into two classes . Class I consisted of six fdhD mutants which synthesized an inactive FDH-PMS protein with the same subunit composition as the wild-type enzyme . In contrast, class II contained four fdhE mutants totally devoid of this antigen . Construction of merodiploid strains harbouring various combinations of the mutated alleles, fdhE on the episome and fdhD on the chromosome, led to the restoration of FDH-PMS activity by complementation of the products encoded by the respective wild-type alleles . Difference spectroscopy suggested that both fdhD and fdhE mutants contained normal amounts of the cytochrome b559 associated with FDH-PMS although the cytochrome had lost its capacity for formate-dependent reduction.

Tsitologiia, 1988 Dec, 30(12), 1463 - 6
{Heterogeneous packing of the Escherichia coli chromosome and its decompaction in in vitro experiments}; Likhoshvai EV et al.; The chromatin organization of E . coli cells, taken on various growth stages of the culture (active, stationary, grown with heightened density), displays different characters when examined by the Miller method . In the active phase of growth, the cell chromatin is released as threads and loops of DNA, threads of nucleosome-like particles and granules 25-38 nm in size . The chromatin from cells in the stationary phase of growth, grown in heightened density conditions, contains not only granules of average size 30 and 100 nm, but also larger conglomerates consisting of several 100 nm particles . The chromatin decompaction of cells grown under heightened density, in conditions of weak alkali medium and low salt buffer, was in general of two types: creation of diffusion cloud with no clear-cut outlines, and spherical structure of 1.5-2 microns in diameter with electron dense centre . In one chromosome both the types of chromatin decompaction can be found at the same time with regions of compact chromatin, which undoubtedly shows different functional activity of some regions of the chromosome.

Mol Cell Probes, 1988 Dec, 2(4), 255 - 60
Modification of the DNA colony hybridization technique for multiple filter analysis; Kaysner CA et al.; Inexpensive fiberglass mesh window screens were used as spacers between colony blot filters to increase the number of bacterial isolates that could be tested by DNA colony hybridization . Sixty filters with up to 48 isolates per filter were tested at one time . This modified technique reduced the amount of radiolabelled probe and other materials needed for simultaneous analysis of up to 2880 test isolates.

Mol Cell Biol, 1988 Dec, 8(12), 5425 - 31
Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector; Keyse SM et al.; Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189) . We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation . Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced . In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm . These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength . Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously . Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.

Microb Pathog, 1988 Dec, 5(6), 419 - 26
Cloning and nucleotide sequence analysis of the genes determining verocytotoxin production in a porcine edema disease isolate of Escherichia coli; Gyles CL et al.; The structural genes determining the edema disease principle were cloned from the total cellular DNA of Escherichia coli strain 412 (O139:K82) isolated from a case of porcine edema disease . An assay for cytotoxicity in Vero cells was used to detect the edema disease principle . A 7.5 kb EcoRI-SalI fragment specifying cytotoxin production was subcloned in pUC18 . Sequences which specified production of cytotoxin were localized to a 0.9 kb region by transposon Tn5 mutagenesis . A 2.4 kb EcoRI-BglII fragment encompassing this region was subcloned into pUC18 . Using nucleotide sequence analysis, two open reading frames separated by 12 bp were identified . They encoded proteins of 319 (A subunit) and 87 (B subunit) amino acids which both had N-terminal sequences typical of E . coli signal peptides . Comparison of these with the published sequence for the Shiga-like toxin II (SLT-II) showed 91% overall nucleotide sequence similarity . The nucleotide sequence similarity extended to 200 base pairs upstream of the putative A subunit translational start site suggesting a common regulatory mechanism . The deduced amino acid sequences of the processed A and B subunits had 94% and 84% similarity, respectively . These findings confirm the close genetic relationship between SLT-II and edema disease principle.

Mol Gen Genet, 1988 Dec, 215(1), 38 - 45
Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein; Kavanagh TA et al.; We have constructed chimaeric genes consisting of sequences encoding the transit peptide and 4, 16, 24, 53 or 126 amino-terminal residues of the mature chlorophyll a/b binding (Cab) apoprotein fused to the Escherichia coli gene encoding beta-glucuronidase (GUS) . These genes were introduced into tobacco plants and the fate of the fusion proteins they encode was analysed . Less than 1% of the total activity of fusion proteins containing the transit peptide and 4 (FP4) or 16 (FP16) amino-terminal amino acids of the mature Cab protein was associated with chloroplasts . Moreover, FP4 appears to be unprocessed . This is in striking contrast to fusion proteins containing the transit peptide and 24 (FP24), 53 (FP53) or 126 (FP126) amino-terminal residues of the mature Cab polypeptide . Approximately 98%, 96% or 75%, respectively, of the total activity of these fusion proteins was associated with purified intact chloroplasts, and protease protection experiments showed that of this, approximately 98%, 87% or 50%, respectively, was located within this organelle . Furthermore, both FP24 and FP53 appear to be processed . However, less than 10% of the activity of those fusion proteins translocated into chloroplasts was associated with thylakoid membranes.

Mol Gen Genet, 1988 Dec, 215(1), 146 - 51
Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1; Bravo A et al.; The proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1 . It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression . This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein . These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein . The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed.

Am J Vet Res, 1988 Dec, 49(12), 2030 - 3
Effects of weaning on diarrhea caused by enterotoxigenic Escherichia coli in three-week-old pigs; Sarmiento JI et al.; We attempted to determine whether weaning is required for induction of diarrhea in pigs with postweaning enterotoxigenic Escherichia coli infection . Three-week-old newly weaned pigs and their suckling littermates were inoculated with the K88+ enterotoxigenic E coli strain M1823B . Fourteen of 21 weaned and 12 of 20 suckling pigs were genetically resistant to intestinal adhesion by the K88+ strain of E coli; they remained healthy, and gained weight at similar rates . Both groups of K88-resistant pigs gained weight faster, and shed fewer bacteria of strain M1823B in their feces, than did their K88-susceptible counterparts . Diarrhea developed in K88-susceptible pigs in the weaned (6 of 7 pigs) and suckling (4 of 8 pigs) groups, and 1 of the 4 affected suckling pigs died from complications resulting from diarrhea . The incidences of diarrhea, weight gain rates, and the numbers of strain M1823B shed in feces of susceptible weaned and suckling pigs were not significantly (P greater than 0.05) different . Diarrhea scores of susceptible weaned pigs were significantly (P less than 0.02) higher than those of susceptible suckling pigs on the second day after inoculation . In this experimental model, it was concluded that weaning is not required for induction of diarrhea, but may modestly increase its severity.

Anal Biochem, 1988 Dec, 175(2), 373 - 85
An efficiently mutagenizable recombinant plasmid for in vitro transcription of the Escherichia coli 16 S RNA gene; Krzyzosiak WJ et al.; The portion of the rrnB operon coding for 16 S RNA was modified to permit efficient in vitro transcription by T7 RNA polymerase of full-length, correctly terminated, biologically active 16 S RNA (W . Krzyzosiak et al., 1987, Biochemistry 26, 2353-2364) . The 5'-end of the gene was fused to the class III T7 promoter and the 3'-end was modified so that cleavage with MstII would generate correctly terminated RNA upon runoff transcription . The modified gene was placed in pUC19 by a four-way ligation reaction involving linearized pUC19, a 1490-bp fragment of 16 S rDNA, and two synthetic oligodeoxynucleotides . Because of the cohesive end design, phosphorylation of the synthetic oligomers was not necessary . Single and tandem cassette insertions were used to generate single base changes in the C-1400 region of 16 S RNA . Three examples are described . This method is generally applicable to the 16 S RNA molecule as suitable singlecleavage restriction sites allow all regions to be mutated by this approach.

Mol Immunol, 1988 Dec, 25(12), 1291 - 8
Purification and characterization of radiolabelled biosynthetic human insulin from Escherichia coli . Kinetics of processing by antigen presenting cells; Semple JW et al.; An Escherichia coli strain transfected with a plasmid containing four linked human proinsulin genes was grown in the presence of 35S and 3H labelled amino acids to gain access to human insulin that was radiolabelled at 19 evenly distributed sites throughout the amino acid sequence . The multi-proinsulin precursor was cleaved at methionine residues with cyanogen bromide, then the individual proinsulin units were folded via their S-cysteine sulfonate derivative and converted to insulin by enzymatic digestion . Purification steps were carried out by ion-exchange and reverse-phase HPLC techniques . The final radiolabelled biosynthetic human insulin was produced at a specific activity of up to 300 Ci/mmol, and was shown to be indistinguishable from commercially available human insulin according to HPLC behavior, amino acid analysis, immunoreactivity and biological activity . A comparison of the kinetics of processing of 35S/3H-labelled biosynthetic human insulin and 125I-labelled commercial human insulin by murine TA3 hybridoma antigen presenting cells demonstrated that radiolabelled biosynthetic insulin was processed approximately 16 times slower than its iodinated counterpart . Measurable 125I TCA soluble radioactivity was detected extracellularly within 15 min whereas the same amount of extracellular TCA soluble 3H/35S radioactivity was not seen until 240 min . These results begin to address the importance of using a biosynthetically labelled protein as opposed to an iodinated protein to study how an APC handles antigen in a physiological manner.

DNA, 1988 Dec, 7(10), 729 - 34
Gene manipulation by homologous recombination in Escherichia coli; Crouse GF et al.; The use of homologous recombination in Escherichia coli is described as a tool for DNA manipulation . The utility of the method is illustrated by the addition of 3'-flanking sequences to a dhfr minigene by plasmid-phage recombination involving a supF-containing dhfr minigene plasmid and a lambda Charon4A phage containing the 3' end of the dhfr gene . In addition, other uses of both plasmid-phage and phage-phage recombination in gene manipulation are described.

Scand J Immunol, 1988 Dec, 28(6), 783 - 9
Avidity of IgA antibody to Escherichia coli polysaccharide and diphtheria toxin in breast milk from Swedish and Pakistani mothers; Roberton DM et al.; The avidity of breast milk IgA antibody was studied with the aid of thiocyanate elution of antibody from solid-phase bound E.coli polysaccharides and diphtheria toxoid . The relative avidity index for each sample was determined by the molarity of thiocyanate required to elute 50% of the bound IgA antibody under conditions of antigen excess . Milk samples collected from Pakistani mothers during early lactation (2-4 weeks after delivery; n = 12) had a significantly lower median relative avidity index of IgA antibody to E.coli antigens than did early lactation samples from Swedish mothers (n = 11; avidity indices 1.78 M and 2.65 M; P less than 0.02) . Samples collected from Pakistani mothers in mid-lactation showed a significant rise in the relative avidity index to a median of 2.50 M (P less than 0.01), with a subsequent fall in late lactation (28-36 weeks after delivery) to 1.75 M (P less than 0.01) . Milk samples from Pakistani mothers in mid-lactation (n = 12) also had a lower median relative avidity index of IgA antibody to diphtheria toxoid than did samples from Swedish mothers (n = 14; avidity indices 2.35 M and 4.30 M; P less than 0.002) . The lower avidity of breast milk IgA in Pakistani mothers in comparison with Swedish mothers may arise from differences in antigen exposure or nutritional status or could possibly be genetically determined.

Pathol Res Pract, 1988 Dec, 184(1), 60 - 8
Induction of lymph follicle formation with endotoxin lipopolysaccharide in the draining lymph node of athymic nude mice; Hoshi H et al.; Formation of lymph follicles in draining popliteal lymph nodes in athymic nude mice and their phenotypically normal littermates (PN mice) was investigated after footpad injection with either endotoxin lipopolysaccharide (LPS) or phytohemagglutinin in soluble and precipitated forms (PHA) . The dose of LPS injected ranged from 10 to 100 micrograms, and that of PHA was 50 micrograms . After any dose of LPS, draining nodes of nude mice produced new lymph follicles in the peripheral cortex outside pre-existing follicles, though the number of follicles induced was somewhat less than in the case of PN mice treated with LPS . After injection of PHA in soluble or precipitated form, PN mice developed a significant number of new follicles in the draining nodes, but nude mice failed to do so . The present results are consistent with the view that nude mice have the ability to develop lymph follicles by way of a thymus-independent mechanism in response to exogenous stimuli.

J Clin Microbiol, 1988 Dec, 26(12), 2682 - 4
Rapid identification of Escherichia coli by Fluorocult media and positive indole reaction; Heizmann W et al.; To assess the specificity and sensitivity of Fluorocult media for the identification of Escherichia coli, the beta-glucuronidase activities of 1,258 bacterial strains, as well as 20 strains of Candida spp., were investigated . Fluorescence of colonies combined with positive indole reaction resulted in specificities of 99.6 to 99.8% . Sensitivities were 59.1% (MacConkey agar), 69.9% (brolacin agar), 85.5% (Columbia agar), and 85.8% (ECD agar).

J Clin Microbiol, 1988 Dec, 26(12), 2572 - 5
Rapid isolation of K88+ Escherichia coli by using immunomagnetic particles; Lund A et al.; Superparamagnetic polymer particles precoated with sheep anti-mouse immunoglobulin G were coated with immunoglobulin G2 monoclonal antibodies to the K88 antigen of Escherichia coli (MAb-K88) . These immunomagnetic particles (IMP) were used for isolation and identification of K88 antigen-positive (K88+) E . coli . The bacteria presenting the K88 antigen were easily isolated in almost pure culture from a mixed culture of five different O serogroups of E . coli . Nonspecific binding of K88 antigen-negative (K88-) E . coli to the IMP was not observed . The sensitivity of the test to detect K88+ E . coli was found to be 4,000 CFU/ml with fluorescence microscopy . When bacteria attached to the MAb-K88 IMP were grown on blood agar, about 20% of the initial number of CFU was recovered . The test is promising as a rapid method for isolation and identification of K88+ E . coli from a mixed culture.

J Clin Microbiol, 1988 Dec, 26(12), 2564 - 6
Heat-stable-enterotoxin-producing Escherichia coli strains isolated from dogs; Wasteson Y et al.; Five strains of hemolytic Escherichia coli isolated from dogs suffering from diarrhea were shown by radioactive and enzyme-labeled oligonucleotide probes to possess genes coding for heat-stable enterotoxin (STIa) . Four of the strains were shown by immunoassay (enzyme-linked immunosorbent assay) and bioassay (infant mouse test) to produce STI in vitro . All five strains, however, were able to induce fluid accumulation in ligated dog intestinal loops . The four STI-producing strains all possessed the K99 fimbrial antigen (F5) and belonged to serotype O42:H37 . In these strains, genes encoding STI were located on a 98-megadalton plasmid . In the fifth strain, which produced STI in vitro only after several subcultivations, the STI gene was located on an 80-megadalton plasmid . This strain was nontypable.

Appl Environ Microbiol, 1988 Dec, 54(12), 3142 - 6
Restoration of colony-forming activity in osmotically stressed Escherichia coli by betaine; Roth WG et al.; Exposure of Escherichia coli to 0.8 M NaCl caused a rapid and large decrease in colony-forming activity . When such osmotically upshocked cells were exposed to betaine, colony-forming activity was restored . Betaine was able to restore colony-forming activity even when chloramphenicol inhibited protein synthesis . Thus, restoration was not the result of cell turnover . The cells were not killed by exposure to 0.8 M NaCl, because during exposure they accumulated ATP intracellularly . Betaine treatment caused this cellular ATP to decrease to a lower level . This work may provide the foundation for a simple plating procedure to quantitatively detect nonculturable E . coli in ocean beach recreational waters.

Br J Exp Pathol, 1988 Dec, 69(6), 805 - 12
Induction of reversible shock by Escherichia coli lipopolysaccharide in rats . Changes in serum and cell membrane parameters; Bosch MA et al.; Reversible endotoxic shock was induced in adult rats by i.v . injection of Escherichia coli O111:B4 lipopolysaccharide (1.6 mg/100 g) . The shock progression was evaluated by measuring serum glucose levels as well as activities of aspartate aminotransferase (GOT) and alkaline phosphatase in serum . A rapid increase of serum glucose levels occurs, after LPS injection, followed by hypoglycaemia (minimum values at 6 h) with progressive reversion to control values . Serum GOT activity increased (twofold) 6 h after endotoxin administration and returned to control values at 72 h . No appreciable changes occurred in serum alkaline phosphatase activity . Endotoxaemia produced a decrease in the cytochrome P-450 levels in all target organs considered: lung, adrenal glands and liver . The progressive decrease in the serum albumin concentration as well as changes of the physical properties of the plasma membranes observed in vivo, can not be explained only by direct interaction of endotoxin with the target organs, underlining the importance of serum mediators in the induction of the shock response.

AIDS Res Hum Retroviruses, 1988 Dec, 4(6), 419 - 31
Expression and purification of protein segments encoded by the envelope and 3'-orf genes of human immunodeficiency virus type 1; DuBois GC et al.; The pJL6 expression vector and its derivatives, pJLA16 and pANH-1, have been used for the synthesis and high-level expression in Escherichia coli of restriction enzyme fragments derived from the envelope and 3'-orf genes of the BH10 and BH8 clones, respectively, of the human immunodeficiency virus (HIV-1) . These bacterially expressed proteins have been purified to apparent homogeneity by sequential detergent extraction, gel filtration, and reverse-phase high-performance liquid chromatography . The recombinant proteins have been used for the production of polyclonal and monoclonal antibodies, and the fusion proteins from the envelope gene are currently being evaluated for use as immunodiagnostic assay reagants.

Genes Dev, 1988 Dec, 2(12A), 1557 - 69
Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements; Williams T et al.; Human AP-2 is a sequence-specific DNA-binding protein that interacts with inducible viral and cellular enhancer elements to stimulate transcription of selected genes . Here, we report the isolation and characterization of a human cDNA clone containing the entire protein-coding region of AP-2 . The deduced primary amino acid sequence of AP-2 does not contain a domain resembling any previously identified DNA binding motif . However, an interesting feature of the AP-2 protein is a clustered arrangement of proline and glutamine residues that have been found recently within the activation domains of other transcription factors . Expression of the AP-2 clone in bacteria yields a protein that binds to DNA and activates transcription in vitro in a comparable manner to native human AP-2 . Transfection of cDNA clones into Drosophila cells indicates that the AP-2 gene product can also activate gene expression in vivo in a DNA template-dependent manner . Expression of endogenous AP-2 is repressed in a hepatoma cell line and stimulated following retinoic-acid-induced differentiation of a human teratocarcinoma cell line . This indicates that AP-2 may be a transcription factor involved in the control of developmentally regulated gene expression.

EMBO J, 1988 Dec 1, 7(12), 4005 - 10
A novel replicon occurring naturally in Escherichia coli is a phage-plasmid hybrid; Seufert W et al.; A novel DNA replicon in Escherichia coli was identified . It is the smallest natural isolate (1282 bp) found so far . In the presence of phage M13 it grows as a filamentous single-stranded DNA phage . Contrary to previously identified mini-phages this replicon displays sequence homology only to parts of the M13 viral and complementary strand origin . In the absence of M13 this DNA replicates autonomously . The only gene (arp) of the replicon encodes a 32-kd protein, which is essential for autonomous replication . The host rep gene required for replication of single-stranded DNA phages is dispensable . Distinct replication mechanisms are thus involved during growth as defective phage or as autonomous plasmid.

EMBO J, 1988 Dec 1, 7(12), 3957 - 62
Messenger RNA recognition in Escherichia coli: a possible second site of interaction with 16S ribosomal RNA; Petersen GB et al.; Examination of the nucleotides following the ATG or GTG initiation codons of a file of 251 genes from Escherichia coli has shown that 247 (98.4%) of them contain a sequence of at least three and 168 (66.9%) of them a sequence of at least four consecutive nucleotides that is complementary to some part of the 16 nt at the 5' terminus of the bacterial 16S rRNA . It is proposed that this sequence, which falls within the first 24 nt coding for the genetic message, might be involved in mRNA recognition through a mechanism analogous to the well-established 'Shine--Dalgarno' interaction with the 3' terminus of the 16S rRNA . Comparison of these data with data derived from a file of 117 'false' gene starts that have a Shine--Dalgarno-like sequence followed by a suitably spaced ATG or GTG triplet but which are believed not to lie at the beginnings of genetic messages shows the association that we have found to be statistically significant at the 99.9% level.

EMBO J, 1988 Dec 1, 7(12), 3949 - 55
Photo-affinity labelling at the peptidyl transferase centre reveals two different positions for the A- and P-sites in domain V of 23S rRNA; Steiner G et al.; Photo-reactive 3-(4'-benzoylphenyl)propionyl-Phe-tRNA bound to the A- or the P-site was crosslinked to 23S RNA upon irradiation at 320 nm . The sites of reaction were identified as U-2584 and U-2585 at the A-site and A-2451 and C-2452 at the P-site . Minor crosslinks from both sites were observed at nucleotides A-2503 to U-2506 . All sites identified lie in close proximity according to the secondary structure model and constitute part of the highly conserved loop region of domain V . Antibiotics known to inhibit peptidyl transferase activity had a pronounced effect on photo-crosslinking . In addition, tetracycline was also shown to photo-crosslink to this region . These experiments permit a dissection of the peptidyl transferase region on the 23S RNA into two distinct areas for the A- and P-site.

EMBO J, 1988 Dec 1, 7(12), 3817 - 21
Functional domains of the RNA component of ribonuclease P revealed by chemical probing of mutant RNAs; Shiraishi H et al.; The higher-order structure of the RNA component of ribonuclease P from Escherichia coli was analyzed using chemical probes . The secondary structure model which had been constructed from the comparative sequence analysis of the RNA was refined using the experimental data . In a mutant RNA (A89 RNA), which contains a G----A substitution at nucleotide 89, we detected a number of conformational alterations clustered between nucleotides 90 and 239 . In view of the fact that A89 RNA is as catalytically active as wild-type RNA, but defective in association with the protein component, it is clear that the catalytic function of the RNA component resides on the structure which is not disrupted by the A89 mutation and that the structures altered by the mutation represent the region(s) interacting with the protein component . Another mutant (A329 RNA), which has a G----A substitution at nucleotide 329 and is defective in catalytic function, showed no detectable change in higher-order structure.

Biull Eksp Biol Med, 1988 Dec, 106(12), 698 - 701
{The stimulating action of lipopolysaccharide and muramyl dipeptide on the activation of mononuclear cells in human peripheral blood by recombinant interleukin-2 in vitro}; Rakhmilevich AL et al.; Muramyl dipeptide (MDP) and lipopolysaccharide (LPS) were effective in augmentation of killer cells generation from human peripheral blood mononuclear cells (PBMC) in response to recombinant human interleukin-2 (IL-2) . Pretreatment of PBMC with combination of LPS and MDP resulted in most significant their proliferation stimulated by IL-2 . Thus our results show the enhancement of PBMC sensitivity to IL-2 by action of LPS, MDP and most of all by their combination in vitro.

Eur J Biochem, 1988 Dec 1, 178(1), 235 - 42
Purification of the yeast mitochondrial methionyl-tRNA synthetase . Common and distinctive features of the cytoplasmic and mitochondrial isoenzymes; Schwob E et al.; Yeast-mitochondrial methionyl-tRNA synthetase was purified 1060-fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four-step procedure based on affinity chromatography (heparin-Ultrogel, tRNA(Met)-Sepharose, Agarose-hexyl-AMP) to yield to a single polypeptide of high specific activity (1800 U/mg) . Like the cytoplasmic methionyl-tRNA synthetase (Mr 85,000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size (Mr 65,000) . In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152,000) made up of identical subunits . The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNA(Met) are similar to those of the cytoplasmic isoenzyme . However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E . coli tRNA(Met) . Furthermore, polyclonal antibodies raised against the two proteins did not show any cross-reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants . Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases are different proteins coded for by two distinct nuclear genes . Like the yeast cytoplasmic aminoacyl-tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin . This distinguishes them from the corresponding enzymes of E . coli . Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.

Eur J Biochem, 1988 Dec 1, 178(1), 101 - 7
An IncY plasmid-encoded single-stranded DNA-binding protein from Escherichia coli shows the identical pattern of stacked tryptophan residues as the chromosomal ssb gene product; Casas-Finet JR et al.; In an extension of earlier studies on the Escherichia coli plasmid-encoded single-stranded DNA-binding proteins pIP71a SSB, F SSB and R64 SSB {Khamis, M . I., Casas-Finet, J . R., Maki, A . H., Ruvolo, P . P . & Chase, J . W . (1987) Biochemistry 26, 3347-3354; Casas-Finet, J . R., Khamis, M . I., Maki, A . H., Ruvolo, P . P . & Chase, J . W . (1987) J . Biol . Chem . 262, 8574-8593}, we have investigated the binding of pIP231a SSB to natural and heavy-atom-derivatized single-stranded homopolynucleotides . Fluorimetric equilibrium binding isotherms indicate that pIP231a SSB has a greater solubility at low ionic strength than any other plasmid SSB protein investigated . Furthermore, its complex with mercurated poly(uridylic acid) {poly(Hg5U)} shows a greater resistance to disruption by salt than the other plasmid SSB complexes . Essentially complete binding of pIP231a SSB to poly(Hg5U) could be achieved, and time-resolved optically detected triplet-state magnetic resonance (ODMR) techniques could be applied to the complex . These methods allowed complete resolution of the three Trp chromophores of pIP231a SSB . Comparison of wavelength-selected ODMR results with those obtained for the poly(Hg5U) complex of a point-mutated chromosomal ssb gene product (Eco SSB) carrying substitutions of Phe for Trp {Khamis, M . I., Casas-Finet, J . R., Maki, A . H., Murphy, J . B . & Chase, J . W . (1987) J . Biol . Chem . 262, 10938-10945} confirm that Trp40 and Trp54 of pIP231a SSB are stacked in the complex, while Trp88 is not . This is the same distribution of stacked Trp residues found in Eco SSB . These results are confirmed further by specific effects observed on the ODMR signals of pIP231a SSB upon binding to poly(Br5U) and poly(dT), which are known to be caused by the stacking of Trp54 with nucleic acid bases.

Virology, 1988 Dec, 167(2), 634 - 8
Expression of the Moloney murine leukemia virus and human immunodeficiency virus integration proteins in Escherichia coli; Hizi A et al.; We have constructed expression plasmids containing the genes for the MuLV and the HIV integration proteins . When introduced into Escherichia coli, these plasmids cause the production of proteins of the expected molecular weight (43K for MuLV, 31K for HIV) . The wild-type MuLV coding region induces the synthesis of large amounts of integration protein; to obtain large amounts of the full-length HIV integration protein in E . coli, it was necessary to modify the coding region to disrupt a sequence that promotes efficient internal translational initiation in E . coli . It is possible to disrupt this sequence without altering the encoded amino acids; the modified plasmid makes large amounts of the full-length protein and small amounts of the internally initiated protein . Both the MuLV and HIV integration proteins require SDS to be solubilized in E . coli extracts; however, following solubilization with SDS and transfer to a nitrocellulose filter, both integration proteins bind DNA.

Proc Natl Acad Sci U S A, 1988 Dec, 85(24), 9683 - 7
Interaction of spatially separated protein-DNA complexes for control of gene expression: operator conversions; Haber R et al.; Two operators, spatially separated from each other and from the promoters, repress the gal operon when bound to Gal repressor . Conversion of either gal operator to a lac operator results in derepression, although both Gal and Lac repressors are present, suggesting that mere occupation of operator sites is not sufficient to cause repression . Conversion of both operators to lac operators restores normal repression in the presence of Lac repressor protein . We propose that normal repression requires interaction between operator-bound like repressor molecules; this generates a DNA loop, which is part of a higher order structure . RNA polymerase and cyclic AMP receptor protein are present in this complex but unable to initiate transcription because of the higher order structure . Such higher order DNA-multiprotein complexes could occur in a variety of genetic regulatory systems that are controlled from distal sites by regulatory proteins.

Proc Natl Acad Sci U S A, 1988 Dec, 85(24), 9451 - 5
Osmotaxis in Escherichia coli; Li C et al.; The escape of motile organisms from high concentrations of chemicals was studied in Escherichia coli . We have found all chemicals tested to be osmorepellents . It was shown in both a spatial assay and a temporal assay that the known sensory receptors for chemotaxis are not used for osmotaxis, so a different sensory mechanism appears to be employed . According to the temporal assay, the mechanism between sensory receptors and flagella is also not used for tumbling response (at least in solutions above 0.4 osmolar).

Proc Natl Acad Sci U S A, 1988 Dec, 85(24), 9426 - 30
High-level expression of a bioengineered, cysteine-free hepatocyte-stimulating factor (interleukin 6)-like protein; Jambou RC et al.; Hepatocyte-stimulating factor, interferon-beta 2, B-cell stimulation factor 2, and hybridoma/plasmacytoma growth factor are identical proteins presently referred to as interleukin 6 (IL-6) . Through the use of synthetic oligonucleotide technology, we have constructed a biologically active recombinant IL-6 (rIL-6) gene based on the sequence of a human IL-6 cDNA . The synthetic gene encodes a cysteine-free, bioengineered rIL-6 protein that is expressed at high levels in Escherichia coli as a tripartite fusion protein . Cleavage of the fusion protein with collagenase releases a 23-kDa rIL-6 protein that can be easily purified to homogeneity . We show that the rIL-6 protein displays a range of biological activities similar to those of natural human IL-6, as demonstrated by its ability to (i) protect cells from viral infection, (ii) stimulate the synthesis of fibrinogen in rat FAZA 967 cells, and (iii) induce the terminal differentiation of B cells, resulting in elevated secretion of immunoglobulin.

Proc Natl Acad Sci U S A, 1988 Dec, 85(24), 9391 - 5
Detection and possible role of two large nondivisible zones on the Escherichia coli chromosome; Rebollo JE et al.; Inversion of many predetermined segments of the Escherichia coli chromosome was attempted by using a system for in vivo selection of genomic rearrangements . Two types of constraints on these inversions were observed: (i) a sensitivity to rich medium when the distance between oriC and the 86- to 91-min region (which carries loci essential for transcription and translation) is increased; (ii) a poor viability or inviability of inversions having at least one endpoint in the one-third of the chromosome around replication terminators (with an exception for some inversions ending between these terminators) . Although the first constraint is simply explained by a decreased dosage of the region involved, the second one may result from disruption of two long-range chromosomal organizations . The nondivisible zones thus disclosed coincide remarkably well with the two zones that we have previously described, which are polarized with respect to their replication . It is proposed that the two phenomena result from a sequence-dependent and polarized organization of the terminal region of the chromosome, which defines chromosome replication arms and may participate in nucleoid organization.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 9199 - 203
Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor; Lindley I et al.; The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharide-stimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA ENS . Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues . A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli . Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF . Recombinant NAF purified to homogeneity had identical amino- and carboxyl-terminal sequences to the 72-amino acid natural NAF . Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 9163 - 6
Escherichia coli mutY gene product is required for specific A-G----C.G mismatch correction; Au KG et al.; A-G mispairs are subject to correction by two distinct pathways in cell-free extracts of Escherichia coli {Su, S.-S., Lahue, R.S., Au, K.G . & Modrich, P . (1988) J . Biol . Chem . 263, 6829-6835; Lu, A.-L . & Chang, D.Y . (1988) Genetics 118, 593-600} . One is the mutHLS-dependent, methyl-directed pathway that recognizes a variety of mismatches and repairs the unmethylated strand of DNA heteroduplexes that are hemimethylated at d(GATC) sequences . The other pathway appears to be specific for A-G mispairs, yields C.G base pairs exclusively, and is independent of the presence of d(GATC) sites . Analyses of cell-free extracts prepared from E . coli mutY strains and isogenic parents have demonstrated that the mutY gene product is involved in the methyl-independent pathway, which converts A-G mispairs to C.G pairs . The specificity of this activity is consistent with the mutator phenotype associated with the mutY locus, which generates G.C----T.A transversions {Nghiem, Y., Cabrera, M., Cupples, C.G . & Miller, J.H . (1988) Proc . Natl . Acad . Sci . USA 85, 2709-2713} . We propose that the mutY product functions at a late stage of a pathway that excludes A-G mispairs during chromosome replication and that involves the function of the mutT gene product . This model suggests that the mutT function acts at an early stage of this pathway to exclude A-G mismatches where the adenine resides on the template DNA strand . A-G mispairs that persist after passage of the replication fork would contain guanine on the template strand and thus be processed to C.G base pairs by the mutY-dependent repair system.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 9143 - 7
A sensitive molecular assay for mutagenesis in mammalian cells: reversion analysis in cells with a mutant shuttle vector gene integrated into chromosomal DNA; Gelbert LM et al.; We have developed a system for the molecular analysis of mutations in mammalian cells . This system is based upon the use of mammalian cell lines containing mutant shuttle vector genes integrated into chromosomal DNA . The target for mutation was the Escherichia coli gpt gene, coding for the enzyme xanthine (guanine) phosphoribosyltransferase (GPT; EC 2.4.2.22) . We have previously isolated a large number of cell lines containing mutant gpt genes with single base changes . From these lines, revertants were selected on the basis of the reappearance of GPT activity . In general, the frequency of revertants was below 10(-7) . The gpt genes were recovered from 32 revertants and sequenced to determine the nature of the base changes associated with reversion . In the majority of the revertants, there was a base change within the originally mutated codon, leading to either restoration of the wild-type amino acid sequence or substitution of a different amino acid at the original mutated site . In no case did reversion of a base substitution mutant involve an amino acid residue other than that affected by the original mutation . The results have demonstrated a number of sites in the GPT polypeptide at which amino acid substitutions are compatible with enzyme activity and one site at which the loss of an amino acid is compatible with enzyme activity . This study establishes reversion analysis as a sensitive molecular assay for mutagenesis in mammalian cells.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 9124 - 7
Overproduction of the epsilon subunit of DNA polymerase III counteracts the SOS mutagenic response of Escherichia coli; Jonczyk P et al.; It has been found that the mutator phenotype of the recA441 and recA730 strains that express the SOS response constitutively is suppressed by pIP1, a high-copy plasmid carrying the dnaQ gene encoding the 3'----5' exonuclease subunit (epsilon) of DNA polymerase III . We have constructed plasmid pIP11, in which the dnaQ gene is fused to the strong tac (trp-lac) promoter . Enhanced synthesis of the epsilon subunit stimulated by isopropyl beta-D-thiogalactopyranoside, the inducer of tac, prevents expression of the mutator phenotype of recA441 and markedly decreases the frequency of UV-induced mutations . These results strongly suggest that a loss of editing capacity by the epsilon subunit of DNA polymerase III holoenzyme plays a crucial role in generation of mutations during the SOS response.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8973 - 7
Promoters largely determine the efficiency of repressor action; Lanzer M et al.; Operator sequence and repressor protein regulate the activity of the lac promoter over a greater than 1000-fold range . Combinations of the lac operator with other promoter sequences, however, differ vastly in the level of repression . The data presented show that the extent of repression is determined largely by the rates of complex formation of the competing systems operator-repressor and promoter-RNA polymerase and by the rate at which RNA polymerase clears the promoter . Moreover, up to 70-fold differences in the level of repression were found when the operator was placed in different positions within the promoter sequence . A kinetic model is proposed that explains the observed effects and that allows predictions on promoters controlled by negatively acting elements.

J Bacteriol, 1988 Dec, 170(12), 5971 - 3
EnvZ, a transmembrane environmental sensor of Escherichia coli K-12, is phosphorylated in vitro; Igo MM et al.; By fusing the transcriptional and translational start signals of lacZ to envZ, we have obtained high-level synthesis of a truncated EnvZ protein (EnvZ115) in which the first 38 amino acids of EnvZ are replaced with the first 8 amino acids of LacZ . Using this construct, we have partially purified the EnvZ115 protein and demonstrated that this protein can be phosphorylated in vitro . We suggest that phosphorylation may be an important feature of EnvZ function.

J Bacteriol, 1988 Dec, 170(12), 5943 - 5
A single base pair change in proline biosynthesis genes causes osmotic stress tolerance; Dandekar AM et al.; A single base pair change has been found in a site corresponding to a regulatory region of the first enzyme in the proline biosynthetic pathway . This change alters feedback inhibition and is responsible for the synthesis of high levels of proline that enable Escherichia coli to withstand osmotic stress.

J Bacteriol, 1988 Dec, 170(12), 5928 - 30
Two cellular components, PrlA and SecB, that recognize different sequence determinants are required for efficient protein export; Trun NJ et al.; We exploited the conditional-lethal phenotype of secB null mutations to demonstrate that SecB function was required for PrlA-mediated suppression of signal sequence mutations . The results of these experiments provide information about the functions performed and the sequence determinants recognized by each of these components of the protein export machinery of Escherichia coli.

J Bacteriol, 1988 Dec, 170(12), 5916 - 8
Identity of the 17-kilodalton protein, a DNA-binding protein from Escherichia coli, and the firA gene product; Aasland R et al.; The 17-kilodalton protein, a DNA-binding protein encoded by the skp gene of Escherichia coli, was found to be identical to histonelike protein I, the product of the firA gene . This conclusion was reached after chromosomal localization, using the recently constructed high- and low-resolution E . coli restriction maps, and by direct comparison of the N-terminal amino acid sequence of histonelike protein I and the 17-kilodalton protein.

J Bacteriol, 1988 Dec, 170(12), 5806 - 13
Analysis of recombination occurring at SLP1 att sites; Lee SC et al.; SLP1int is a conjugative Streptomyces coelicolor genetic element that can transfer to Streptomyces lividans and integrate site specifically into the genome of the new bacterial host . Recombination of SLP1 previously has been shown to occur within nearly identical 112-base-pair att sequences on the plasmid and host chromosome . We report here that both integrative recombination and intermolecular transfer of SLP1int require no more than a 48-base-pair segment of the att sequence and that SLP1 transfer occurs by a conservative rather than a replicative mechanism . The functions responsible for the excision of the element as a discrete DNA segment are induced during the conjugal transfer of SLP1.

J Bacteriol, 1988 Dec, 170(12), 5797 - 805
Analysis of the recE locus of Escherichia coli K-12 by use of polyclonal antibodies to exonuclease VIII; Luisi-DeLuca C et al.; Exonuclease VIII (exoVIII) of Escherichia coli has been purified from a strain carrying a plasmid-encoded recE gene by using a new procedure . This procedure yielded 30 times more protein per gram of cells, and the protein had a twofold higher specific activity than the enzyme purified by the previously published procedure (J . W . Joseph and R . Kolodner, J . Biol . Chem . 258:10411-10417, 1983) . The sequence of the 12 N-terminal amino acids was also obtained and found to correspond to one of the open reading frames predicted from the nucleic acid sequence of the recE region of Rac (C . Chu, A . Templin, and A . J . Clark, manuscript in preparation) . Polyclonal antibodies directed against purified exoVIII were also prepared . Cell-free extracts prepared from strains containing a wide range of chromosomal- or plasmid-encoded point, insertion, and deletion mutations which result in expression of exoVIII were examined by Western blot (immunoblot) analysis . This analysis showed that two point sbcA mutations (sbcA5 and sbcA23) and the sbc insertion mutations led to the synthesis of the 140-kilodalton (kDa) polypeptide of wild-type exoVIII . Plasmid-encoded partial deletion mutations of recE reduced the size of the cross-reacting protein(s) in direct proportion to the size of the deletion, even though exonuclease activity was still present . The analysis suggests that 39 kDa of the 140-kDa exoVIII subunit is all that is essential for exonuclease activity . One of the truncated but functional exonucleases (the pRAC3 exonuclease) has been purified and confirmed to be a 41-kDa polypeptide . The first 18 amino acids from the N terminus of the 41-kDa pRAC3 exonuclease were sequenced and fond to correspond to one of the translational start signals predicted from the nucleotide sequence of radC (Chu et al., in preparation).

J Bacteriol, 1988 Dec, 170(12), 5728 - 38
N-terminal half of CheB is involved in methylesterase response to negative chemotactic stimuli in Escherichia coli; Stewart RC et al.; The chemotactic receptor-transducer proteins of Escherichia coli are responsible for directing the swimming behavior of cells by signaling for either straight swimming or tumbling in response to chemostimuli . The signaling states of these proteins are affected not only by the concentrations of various stimuli but also by the extent to which they have been methylated at specific glutamyl residues . The activities of a chemotaxis-specific methyltransferase (CheR) and a chemotaxis-specific methylesterase (CheB) are regulated in response to chemotactic stimuli to enable sensory adaptation to unchanging levels of stimuli by appropriately shifting the signaling states of the transducer proteins . For CheB this regulation involves a feedback loop that requires some of the components making up the chemotactic signal transduction machinery of the cell . This feedback loop causes the methylesterase activity of CheB to decrease transiently in response to attractant stimuli and to increase transiently in response to negative stimuli (repellent addition or attractant removal) . In this report we demonstrate that the methylesterase response to negative stimuli involves the N-terminal half of the CheB protein, whereas the response to positive stimuli does not require this segment of the protein . Both aspects of the methylesterase response to positive stimuli does not require this segment of the protein . Both aspects of the methylesterase response require CheA . In addition, we demonstrate that mutant forms of CheB lacking methylesterase activity can adversely affect the swimming behavior and chemotactic ability of cells and can markedly diminish modulation of the wild-type methylesterase activity in response to negative stimuli . The significance of these results is discussed in relation to the recent demonstration of phosphoryl transfer from CheA to CheB (J . F . Hess, K . Oosawa, N . Kaplan, and M . I . Simon, Cell 53:79-87, 1988) and the discovery of sequence homology between the N-terminal half of CheB and CheY (A . Stock, D . E . Koshland, Jr., and J . Stock, Proc . Natl . Acad . Sci . USA 82:7989-7993, 1985).

J Bacteriol, 1988 Dec, 170(12), 5654 - 61
Role of the leader peptide of maltose-binding protein in two steps of the export process; Thom JR et al.; During the process of export of maltose-binding protein to the periplasm of Escherichia coli, the leader peptide is involved in at least two steps . The presence of the leader portion of maltose-binding protein was shown to be necessary to mediate initial binding of the precursor to the membrane . However, the presence of a mutationally altered leader which does not sustain export in vivo was sufficient to allow this interaction . Thus, the defect in export which is manifested in vivo by this mutational substitution occurs at a step that follows membrane association, most likely the translocation step . Translocation occurs at discrete sites that are not uniformly distributed over the cytoplasmic membrane . A large proportion of the membrane involved in translocation has a higher density than that of bulk cytoplasmic membrane.

J Bacteriol, 1988 Dec, 170(12), 5625 - 32
Purification, characterization, and primary structure of Escherichia coli protease VII with specificity for paired basic residues: identity of protease VII and OmpT; Sugimura K et al.; Escherichia coli cells were found to contain a novel outer membrane-associated protease, designated protease VII (K . Sugimura and N . Higashi, J . Bacteriol . 170:3650-3654, 1988) . This enzyme was purified to homogeneity and exhibited an apparent molecular weight of 36,000 on sodium dodecyl sulfate gels and 180,000 on a TSK G-3000SW column in the presence of Triton X-100 . It was capable of cleaving several peptides at the center of paired basic residues but not at single basic residues, implying that it is distinct from trypsinlike proteases . Protease VII was most active at pH 6.0 and was sensitive to a serine protease inhibitor, diisopropylfluorophosphate, and to the bivalent cations Zn2+, Cu2+, and Fe2+ . The nucleotide sequence of a protease VII gene-carrying DNA fragment, which had been cloned by complementation analysis (K . Sugimura, Biochem . Biophys . Res . Commun . 153:753-759, 1988) was determined . It carried two putative promoter regions and a putative Shine-Dalgarno sequence in addition to the complete structural gene, which encoded pre-protease VII of 317 amino acid residues, with the N-terminal 20 residues being a signal peptide . By comparing their amino acid sequences, protease VII and OmpT, which specifically cleaves ferric enterobactin receptor protein, were found to be identical.

J Bacteriol, 1988 Dec, 170(12), 5607 - 12
Characterization of a unique methyl-specific restriction system in Streptomyces avermitilis; MacNeil DJ; Streptomyces avermitilis contains a unique restriction system that restricts plasmid DNA containing N6-methyladenine or 5-methylcytosine . Shuttle vectors isolated from Escherichia coli RR1 or plasmids isolated from modification-proficient Streptomyces spp . cannot be directly introduced into S . avermitilis . This restriction barrier can be overcome by first transferring plasmids into Streptomyces lividans or a modification-deficient E . coli strain and then into S . avermitilis . The transformation frequency was reduced greater than 1,000-fold when plasmid DNA was modified by dam or TaqI methylases to contain N6-methyladenine or by AluI, HhaI, HphI methylases to contain 5-methylcytosine . Methyl-specific restriction appears to be common in Streptomyces spp., since either N6-methyladenine-specific or 5-methylcytosine-specific restriction was observed in seven of nine strains tested.

J Bacteriol, 1988 Dec, 170(12), 5446 - 51
Hydrogen metabolism in Escherichia coli: biochemical and genetic evidence for a hydF gene; Sankar P et al.; A new gene whose product is essential for production of all three hydrogenase isoenzymes in Escherichia coli has been identified . This gene, termed hydF, mapped at 59 min in the E . coli chromosome and resided next to the hydB gene . The map order of these genes was hydE, hydF, hydB, fhlA, and fdv . The hydF gene was transcribed from its own promoter and coded for a protein with an apparent molecular weight of 43,000 to 44,000 . Expression of the hydF operon was enhanced by anaerobic growth conditions . Partial products of the hydF gene were capable of supporting various levels of hydrogenase activity in a hydF mutant in the presence of the fhlA gene product, also produced from multicopy plasmids . In the presence of a second mutation in an unidentified, unlinked gene, hydrogenase activity in a hydF mutant was restored by plasmids which carried incomplete hydF and hydB+ genes . These results suggest that the products of hydF and fhlA interact with each other and with yet one other gene product.

J Bacteriol, 1988 Dec, 170(12), 5440 - 5
Gene-product relationships of fhlA and fdv genes of Escherichia coli; Sankar P et al.; Synthesis of formate dehydrogenase coupled to formate hydrogenlyase activity in Escherichia coli was found to require the product of the fhlA gene . Transcription of fdhF, the gene coding for the 80-kilodalton (kDa) selenopeptide of formate dehydrogenase, was not detected in an fhlA genetic background . Mutations in the fhlA gene also abolished production of the hydrogenase activity associated with formate hydrogenlyase activity . The fhlA gene resides next to the hydB gene at 59 min in the E . coli chromosome, and the two genes are transcribed in opposite directions . The fhlA gene codes for a 78-kDa protein . A neighboring gene, fdv, codes for an 82-kDa protein, and the physiological role of this gene product is unknown, although a role in H2 metabolism can be detected.

Biophys Chem, 1988 Dec, 32(2-3), 211 - 27
The internal dynamics of gene 32 protein-DNA complexes studied by quasi-elastic light scattering; Kuil ME et al.; The hydrodynamic properties of large homodisperse single stranded DNAs complexed with the helix destabilizing protein of phage T4, the product of gene 32 (GP32), have been measured . The results suggest a size of the binding site between 8 and 10 nucleotides/GP32 molecule, in reasonable agreement with earlier work on a complex between GP32 and single stranded 145 base DNA . From static light scattering experiments it is concluded that the persistence length of these complexes is about 30 nm, distinctly smaller than the generally accepted value for double stranded DNA . The quasi-elastic light scattering properties of the DNA-GP32 complexes were determined . The variation of the apparent translation diffusion coefficient Dapp with the scattering vector q was analyzed using the discrete ISMF and Rouse-Zimm models {S.C . Lin et al., Biopolymers 17 (1978) 425} . The model parameters that followed from the fit of Dapp versus q2 and from an extensive global analysis of the actually measured autocorrelation functions agreed with the notion that these DNA-protein complexes are indeed rather flexible . The continuous Soda model {K . Soda, Macromolecules 17 (1984) 2365} could successfully explain the variation of Dapp versus q2, assuming a persistence length of 30 nm and a base-base distance in the complex of 0.44 nm.

J Biochem (Tokyo), 1988 Dec, 104(6), 924 - 6
Chemical synthesis and expression of copper metallothionein gene of Neurospora crassa; Sugimoto M et al.; The gene coding for the Neurospora crassa copper metallothionein (MT) was synthesized and inserted in the lacZ' gene of pUC18 plasmid to give the same translational reading frame as the latter gene . The MT-beta-galactosidase fused gene was expressed in Escherichia coli to produce a fused protein in which the amino and carboxy termini of MT are linked to the beta-galactosidase through methionine residues . An MT derivative containing an extra homoserine residue at the carboxy terminus was prepared by cyanogen bromide cleavage of the fused protein followed by a reverse-phase HPLC separation . The spectral features of the MT derivative and its copper complex were similar to those of the corresponding native MTs.

Arch Biochem Biophys, 1988 Dec, 267(2), 690 - 700
Molecular characterization of four forms of phosphofructokinase purified from potato tuber; Kruger NJ et al.; Four forms of phosphofructokinase (PFK) have been purified to apparent homogeneity from tubers of potato (Solanum tuberosum cv . Record) . Each had a final specific activity of about 200 mumol.min-1.mg-1 protein . Similar forms of PFK were found in partially purified extracts from tubers and leaves of other potato cultivars and related wild species . The relative molecular masses of three forms of PFK were about 200,000 whereas that of the fourth PFK was greater than 800,000 . The four forms of PFK contained different proportions of four polypeptides which had apparent relative molecular masses of 46,300, 49,500, 50,000, and 53,000 . These polypeptides gave different patterns of peptide fragments after chemical and proteolytic cleavage . Western blots and immunoprecipitation studies using antibodies raised against the individual polypeptides showed that all four are associated with PFK . Thus, potato tubers contain four distinct forms of PFK that differ in their subunit composition.

EMBO J, 1988 Dec 1, 7(12), 3983 - 9
Isolation and characterization of unusual gin mutants; Klippel A et al.; Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS . Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis . Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur . Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype . This mutant phenotype is caused by single amino acid changes at five different positions of gin . The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FIS-independent manner . The mutants differ in recombination activity . The most active mutant protein was analysed in vitro . The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates . We discuss these results in connection with the role of the enhancer and FIS protein in the wild-type situation.

Int J Pept Protein Res, 1988 Dec, 32(6), 512 - 8
Structural and functional integrity of specificity and catalytic sites of trypsin; Graf L et al.; The aspartic acid residue at the bottom of the substrate-binding pocket of trypsin was replaced by glutamic acid through site-directed mutagenesis . The wild-type (Asp-189) and mutant (Glu-189) trypsinogens were expressed in E . coli, purified to homogeneity, activated by enterokinase, and tested on a series of fluorogenic tetrapeptide substrates . The substrates were of the general formula succinyl-Ala-Ala-Pro-X-AMC, where AMC is 7-amino-4-methylcoumarin and X is Lys, Arg, or Orn (ornithine) . As compared to Asp-189 trypsin, the activity of Glu-189 trypsin on lysyl and arginyl substrates decreased by 3-4 orders of magnitude while its Km values did not significantly change . Lengthening the side-chain of Asp-189 by one methylene group could not be compensated for by shortening the side-chain of the substrate, since Glu-189 trypsin had no measurable activity on the ornithyl substrate . The replacement of Asp-189 with glutamic acid at the base of the substrate-binding pocket of trypsin appears to distort the structure of the critical transition-state complex . This could happen by disrupting interactions normally associated with Asp-189, and by altering the relative position of the scissile peptide bond in the active site of the enzyme.

Mol Cell Biol, 1988 Dec, 8(12), 5417 - 24
Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation; Banks GR et al.; The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations . PYR3 transformants of E . coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity . PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained . A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U . maydis itself . Linear DNA carrying the PYR3 gene transformed a U . maydis pyr3-1 pyrimidine auxotroph to prototrophy . Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used . The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences . A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event . The third type had integrated transforming DNA sequences at multiple sites in the U . maydis genome . In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated . All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation . They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

Genes Dev, 1988 Dec, 2(12B), 1824 - 38
Molecular analysis of even-skipped mutants in Drosophila development; Frasch M et al.; The homeo box gene even-skipped (eve) plays a key role in the regulation of the Drosophila segmentation pattern . eve- embryos lack segment borders and show altered activities of several segmentation genes, including fushi tarazu (ftz), engrailed (en), and wingless (wg) . Here, we present evidence that eve influences its own expression in a tissue-specific manner . Each of four different eve mutations disrupts the normal eve expression pattern, and null mutations cause a premature loss of eve products in ectodermal, but not mesodermal, tissues . Molecular characterization of eve mutations indicates that disruptions of the eve pattern are not due to alterations in the eve promoter but, instead, involve abnormal eve proteins . Two different eve mutations cause single amino acid substitutions within the homeo box, and we discuss the implications of these changes with regard to homeo box gene function . We also present evidence that eve+ gene activity is not only required for the activation of the odd-numbered en stripes but also for the correct positioning of each ftz stripe . We present a model for the loss of en expression in eve- embryos, based on the concentration-dependent regulation of the ftz pattern by eve+ products.

Am J Vet Res, 1988 Dec, 49(12), 2068 - 71
Effects of an orally administered live Escherichia coli pilus vaccine on duration of lacteal immunity to enterotoxigenic Escherichia coli in swine; Moon HW et al.; Primigravid swine were vaccinated orally with a live enterotoxigenic Escherichia coli (ETEC) strain that produces pilus antigen K99 . The titers of K99 antibody in colostrum and milk of vaccinates remained higher than those of nonvaccinated controls through the first lactation after vaccination (4 weeks) . Some control swine had low titers of K99 antibody in colostrum or developed low titers of K99 antibody in milk during lactation . Lacteal K99 antibody titers of vaccinates dropped to control levels during the second lactation, 6 months after vaccination . Pigs suckling vaccinates and controls were equally susceptible to challenge exposure to K99+ ETEC during the second lactation . Orally vaccinated swine given a parenteral booster vaccination (with killed K99+ ETEC) during their second gestation had K99 antibody in milk through their second lactation . During the second lactation, these orally vaccinated parenterally revaccinated swine had higher titers of K99 antibody in postcolostral milk than did nonvaccinated controls, controls given only the parenteral booster injection, or controls vaccinated parenterally during both gestations.

Genes Dev, 1988 Dec, 2(12A), 1600 - 14
Control elements of the P2 promoter of the Antennapedia gene; Boulet AM et al.; Antennapedia (Antp), a homeotic gene of Drosophila required for proper differentiation of the thorax of the fly, is expressed in complex spatial patterns during development . The gene is greater than 100 kb long and has two independently regulated promoters . To characterize cis-acting regulatory elements responsible for the expression pattern, fusions of the Antp promoter 2 cap site and upstream sequences to an Adh-lacZ gene were introduced into flies . A 10-kb sequence directs beta-galactosidase production in a pattern that closely resembles the endogenous P2 pattern . Transcription from the 10-kb fusions is regulated by three genes that regulate Antp transcription . Control elements, including a target of action of homeo-domain-containing proteins, were mapped by deleting parts of the 10-kb sequence.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8934 - 8
Probing the Escherichia coli glnALG upstream activation mechanism in vivo; Sasse-Dwight S et al.; In vivo "footprints" of the glnA regulatory region under activating conditions demonstrate that the three most upstream activator sequences bind the protein NRI in the cell . Together, protections at these sites span six of seven consecutive major grooves and lie on the same helix face . E sigma 54 protects two major grooves of DNA approximately 60 base pairs downstream at the glnAp2 promoter and primarily on the opposite helix face . Experiments using potassium permanganate to probe open complex formation in vivo demonstrate that NRI is absolutely required for E sigma 54 to open the promoter DNA . Together, the dimethyl sulfate and permanganate studies verify {Reitzer, L . J., Bueno, R., Cheng, W . D., Abrams, S . A., Rothstein, D . M., Hunt, T . P., Tyler, B . & Magasanik, B . (1987) J . Bacteriol . 169, 4279-4284} that E sigma 54 occupies the glnAp2 promoter in a closed complex in vivo even in the presence of excess nitrogen and the absence of NRI . Furthermore, the slow step in transcriptional activation is shown to be an NRI-dependent conformational change in the downstream promoter DNA, which results in DNA melting . These observations place interesting restrictions on models describing the mechanism by which NRI activates transcription from glnAp2 at a distance.

J Bacteriol, 1988 Dec, 170(12), 5870 - 6
Genetic manipulation of major P-fimbrial subunits and consequences for formation of fimbriae; Van Die I et al.; The influence of genetic manipulation of the structural genes coding for major P-fimbrial subunits on the formation of fimbriae in Escherichia coli was studied . Deletion of two regions that code for hypervariable parts of the P fimbrillin resulted in strong reduction or total absence of fimbria production . Replacement of deleted amino acids by other amino acid residues restored the formation of fimbriae . The hypervariable regions may be important for biogenesis of fimbriae by imposing correct spacing between conserved regions of the protein . The potential for substituting amino acids in the P-fimbrial subunit opens interesting possibilities for use of fimbriae as carriers of foreign antigenic determinants . An antigenic determinant of foot-and-mouth disease virus (FMDV) was incorporated in the F11 fimbrial subunit . Hybrid fimbriae, recognized by an FMDV-specific neutralizing monoclonal antibody directed against FMDV, were formed.

J Bacteriol, 1988 Dec, 170(12), 5500 - 6
Mutational analysis of the catalytic and feedback sites of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli; Ray JM et al.; The nucleotide sequence of aroH, the structural gene for the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase {DAHPS(Trp)}, is presented, and the deduced amino acid sequence of AroH is compared with that of the tyrosine-sensitive (AroF) and phenylalanine-sensitive (AroG) DAHPS isoenzymes . The high degree of sequence similarity among the three isoenzymes strongly indicates that they have a common evolutionary origin . In vitro chemical mutagenesis of the cloned aroH gene was used to identify residues and regions of the polypeptide essential for catalytic activity and for tryptophan feedback regulation . Missense mutations leading either to loss of catalytic activity or to feedback resistance were found interspersed throughout the polypeptide, suggesting overlapping catalytic and regulatory sites in DAHPS(Trp) . We conclude that the specificity of feedback regulation of the isoenzymes was probably acquired by the duplication and divergent evolution of an ancestral gene, rather than by domain recruitment.

Infect Immun, 1988 Dec, 56(12), 3297 - 300
Nucleotide sequence of the gene encoding the major subunit of CS3 fimbriae of enterotoxigenic Escherichia coli; Boylan M et al.; The complete nucleotide sequence of a 612-base-pair DNA fragment containing the gene for the major fimbrial subunit of CS3 of enterotoxigenic Escherichia coli is presented . A possible promoter region, a ribosome-binding site, and two potential signal peptidase cleavage sites are indicated . Unlike the best-studied fimbrial proteins, the predicted CS3 sequence has no Cys residues.

Biochimie, 1988 Dec, 70(12), 1831 - 9
Escherichia coli 3'-terminal 16S rRNA sequence modulated fidelity during translation; Latif FA et al.; The ribosome is a central component of the protein synthetic apparatus . Although progress has been made in characterizing the functional role of many of the ribosomal proteins, the properties of ribosomal RNA and its role in ribosome structure and function are not well understood . To investigate the working properties of the highly conserved 3'-end of 16S rRNA, a site-specific deletion was made directly within the 16S rRNA molecule . The terminal deletion did not impair in vitro 30S subunit assembly, but the particles produced lost translational fidelity in an in vitro translation system primed with natural mRNA.

J Biochem (Tokyo), 1988 Dec, 104(6), 927 - 33
Carboxyl-terminal truncation and site-directed mutagenesis of the EF hand structure-domain of the small subunit of rabbit calcium-dependent protease; Minami Y et al.; A mutant of the small subunit of rabbit calcium-dependent protease lacking the amino-terminal one-fourth produced in Escherichia coli could associate with the native large subunit to exert protease activity . Deletion of a few carboxyl-terminal residues of this variant small subunit caused a significant decrease in the protease activity after reconstitution with the native large subunit . Loss of the fourth EF hand loop region by further truncation of the variant small subunit made interaction with the large subunit impossible . The calcium binding assay revealed that the fourth EF hand structure of the rabbit small subunit, which has been previously demonstrated to possess two calcium-binding sites, can bind calcium ions . Furthermore it was established by site-directed mutagenesis that the first EF hand structure, in addition to the fourth one, is capable of binding calcium ions . Replacement of amino acids in the EF hand structure affected interaction with the native large subunit or the calcium sensitivity of the reconstituted product . These findings indicate that the EF hand structure-domain of the small subunit is essential for the full protease activity.

Mol Gen Genet, 1988 Dec, 215(1), 69 - 75
Probing FhuA'-'PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12; Gunter K et al.; The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12 . Fusions between fhuA and phoA genes were constructed . They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%-90% of the mature FhuA protein . The fusion sites were nearly randomly distributed along the FhuA protein . The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane . The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies . The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively . The larger fusion proteins above a molecular weight of 64,000 dalton were predominantly found in the outer membrane fraction . They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm . In contrast, FhuA protein in the outer membrane was largely resistant to trypsin . It is concluded that the larger FhuA'-'PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.

Mol Gen Genet, 1988 Dec, 215(1), 139 - 45
Integrative suppression of dnaA46 by broad host-range plasmid R1162; Brasch MA et al.; Replication of plasmid R1162 DNA does not require the product of the dnaA gene . An integrated copy of the plasmid can suppress the temperature-sensitive dnaA46 allele when (1) additional plasmid copies are present in the cytoplasm and (2) an inactive replication origin, generated by deletion, is also present in the chromosome . We propose that the inactive origin sets the rate of initiation of chromosome replication at a level compatible with cell viability, possibly by providing additional binding sites for an R1162-encoded protein that is rate-limiting for plasmid replication.

Genetics, 1988 Dec, 120(4), 887 - 97
Adaptive evolution that requires multiple spontaneous mutations . I . Mutations involving an insertion sequence; Hall BG; Escherichia coli K12 strain chi 342LD requires two mutations in the bgl (beta-glucosidase) operon, bglR0----bglR+ and excision of IS103 from within bglF, in order to utilize salicin . In growing cells the two mutations occur at rates of 4 x 10(-8) per cell division and less than 2 x 10(-12) per cell division, respectively . In 2-3-week-old colonies on MacConkey salicin plates the double mutants occur at frequencies of 10(-8) per cell, yet the rate of an unselected mutation, resistance to valine, is unaffected . The two mutations occur sequentially . Colonies that are 8-12 days old contain from 1% to about 10% IS103 excision mutants, from which the Sal+ secondary bglR0----bglR+ mutants arise . It is shown that the excision mutants are not advantageous within colonies; thus, they must result from a burst of independent excisions late in the life of the colony . Excision of IS103 occurs only on medium containing salicin, despite the fact that the excision itself confers no detectable selective advantage and serves only to create the potential for a secondary selectively advantageous mutation.

Biochem J, 1988 Dec 1, 256(2), 665 - 8
Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain; Pant HC; The degradation of phosphorylated and dephosphorylated neurofilament proteins by the Ca2+-activated neutral proteinase calpain was studied . Neurofilaments were isolated from bovine spinal cord, dephosphorylated by alkaline phosphatase (from Escherichia coli) and radioiodinated with {125I}-Bolton-Hunter reagent . The radioiodinated neurofilament proteins (untreated and dephosphorylated) were incubated in the presence and absence of calpain from rabbit skeletal muscle, and the degradation rates of large (NF-H), mid-sized (NF-M) and small (NF-L) neurofilament polypeptides were analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography . The degradation of dephosphorylated neurofilament proteins occurred at a higher rate, and to a greater extent, than did that of the phosphorylated (untreated) neurofilament proteins . The dephosphorylated high-molecular-mass neurofilament (NF-HD) was proteolyzed 6 times more quickly than the untreated NF-H . The degradation rate of the NF-M and NF-L neurofilament proteins was also enhanced after dephosphorylation, but less than that of NF-H . This indicates that the dephosphorylation of neurofilament proteins can increase their sensitivity to calpain degradation.

Mol Endocrinol, 1988 Dec, 2(12), 1311 - 9
Identification of a cyclic adenosine monophosphate-responsive element in the rat corticotropin-releasing hormone gene; Seasholtz AF et al.; The molecular mechanisms involved in the regulation of expression of the rat CRH gene have been examined in rat pheochromocytoma (PC-12) cells transiently transfected with a chimeric gene containing 1.4 kilobases of rat CRH 5'-flanking DNA fused to the bacterial reporter gene encoding chloramphenicol acetyltransferase . Cyclic AMP analogs and activators of adenylate cyclase positively regulate the expression of this chimeric gene in PC-12 cells, inducing chloramphenicol acetyltransferase activity more than 15-fold . The DNA sequence required for this response to cAMP has been localized to a 59 base pair region located between 238 and 180 base pairs 5' to the putative CRH mRNA cap site . This sequence can confer cAMP-responsiveness on a heterologous promoter in an orientation independent fashion and has homology to cAMP regulatory regions from a number of other eukaryotic genes.

Circ Shock, 1988 Dec, 26(4), 431 - 41
Effect of the leukotriene receptor antagonist LY-171883 on endotoxemia in awake sheep; Krausz MM et al.; The effect of the leukotriene D4, leukotriene E4 (LTD4/E4) receptor antagonist LY-171883 was studied in endotoxemia . Eighteen awake sheep were divided into three groups . In Group (n = 4) 4 mg/kg LY-171883 was twice injected intravenously . In Group II (n = 9) 1 microgram/kg E . coli endotoxin was administered intravenously . In Group III (n = 5) 4 mg/kg LY-171883 was given 15 min before, and 30 min after endotoxin . Infusion of LY-171883 in Group I did not alter baseline hemodynamic and pulmonary measurements . Infusion of endotoxin in Group II was followed by an initial rise of pulmonary artery pressure (PAP) to 51 torr (P less than 0.001), pulmonary microvascular pressure (Pmv) to 25 torr (P less than 0.005), pulmonary vascular resistance (PVR) to 1,019 dynes sec . cm-5 (P less than 0.001), systemic vascular resistance (SVR) to 2,830 dynes sec . cm-5 (P less than 0.001), plasma thromboxane B2 (TXB2) to 4,971 pg/ml (P less than 0.001), lymph TXB2 to 5,500 pg/ml (P less than 0.001), plasma 6-Keto PGF1 alpha to 1,469 pg/ml (P less than 0.005), and lymph 6-Keto PGF1 alpha to 2,518 pg/ml (P less than 0.005) . The cardiac index (CI) fell to 100 ml/min . kg (P less than 0.01), PaO2 to 61 torr (P less than 0.01), and circulating WBC to 2,800 microliter (P less than 0.001) . This was followed by a rise in pulmonary lymph flow (QL) to 35 ml/h (P less than 0.01) and lymph protein clearance (L/P.QL) to 23 ml/h (P less than 0.01) . Pretreatment with LY-171883 in Group III resulted in rise of PAP to 35 torr (P less than 0.005), PmV to 18 torr (P less than 0.05), PVR to 398 dynes sec . cm-5 (P less than 0.01), SVR to 1,732 dynes sec . cm-5 (P less than 0.05), and CI increased to 170 ml/min.kg (P less than 0.005) . L/P.QL, QL, Hgb, WBC, PaO2, PaCO2, Qs/QT, plasma and lymph TXB2, and plasma and lymph 6-Keto PGF1 alpha were not significantly changed by LY-171883 . It is concluded that LY-171883 inhibited the smooth muscle effects of endotoxin, namely reduced PAP, Pmv, PVR, and SVR and increased cardiac output . Hypoxemia and increased pulmonary vascular permeability were unaffected by this leukotriene receptor antagonist.

Eur J Biochem, 1988 Dec 1, 178(1), 131 - 40
The K+-translocating Kdp-ATPase from Escherichia coli . Purification, enzymatic properties and production of complex- and subunit-specific antisera; Siebers A et al.; The Kdp system from Escherichia coli is a derepressible high-affinity K+-uptake ATPase . Its membrane-bound ATPase activity was approximately 50 mumol g-1 min-1 . The Kdp-ATPase complex was purified from everted vesicles by solubilization with the nonionic detergent Aminoxid WS 35 followed by DEAE-Sepharose CL-6B chromatography at pH 7.5 and pH 6.4 and gel filtration on Fractogel TSK HW-65 . The overall yield of activity was 6.5% and the purity at least 90% . The isolated KdpABC complex had a high affinity for its substrates K+ (Km app . = 10 microM) and Mg2+-ATP (Km = 80 microM) and a narrow substrate specificity . The ATPase activity was inhibited by vanadate (Ki = 1.5 microM), fluorescein isothiocyanate (Ki = 3.5 microM), N,N'-dicyclohexylcarbodiimide (Ki = 60 microM) and N-ethylmaleimide (Ki = 0.1 mM) . The purification protocol was likewise applicable to the isolation of a KdpA mutant ATPase which in contrast to the wild-type enzyme exhibited an increased Km value for K+ of 6 mM and a 10-fold lowered sensitivity for vanadate . Starting from the purified Kdp complex the single subunits were obtained by gel filtration on Bio-Gel P-100 in the presence of SDS . Both the native Kdp-ATPase and the SDS-denatured polypeptides were used to raise polyclonal antibodies . The specificity of the antisera was established by immunoblot analysis . In functional inhibition studies the anti-KdpABC and anti-KdpB sera impaired ATPase activity in the membrane-bound as well as in the purified state of the enzyme . In contrast, the anti-KdpC serum did not inhibit enzyme activity.

Virology, 1988 Dec, 167(2), 477 - 84
Insertional mutagenesis of the murine cytomegalovirus genome: one prominent alpha gene (ie2) is dispensable for growth; Manning WC et al.; We have utilized insertional mutagenesis to investigate the functional importance of murine cytomegalovirus (MCMV) immediate early (IE or alpha) region 2 (ie2) in replication . We constructed a recombinant virus, RM408, that carries the Escherichia coli lacZ gene under transcriptional control of the MCMV alpha promoter/enhancer inserted within the ie2 transcription unit . RM408 carries the first defined genetic mutation in a specific CMV gene . After infection of NIH3T3 cells, RM408 expressed beta-galactosidase in abundance and with kinetics indistinguishable from those of the natural ie1 gene product which is left intact in the recombinant . We detected no expression from the ie2 region in RM408-infected cells . Growth kinetics, yield, and expression of other viral genes in RM408 was similar to wild-type MCMV, indicating that the ie2 gene product was nonessential for growth in cell culture.

Proc Natl Acad Sci U S A, 1988 Dec, 85(24), 9416 - 20
Transcription induces gyration of the DNA template in Escherichia coli; Figueroa N et al.; We show that transcription modulation of a plasmid sequence in exponentially growing Escherichia coli cells leads to a rapid change in the linking number of plasmid DNA . Activation of transcription is accompanied by an increase in the plasmid's level of negative supercoiling . The added superhelical turns, whose number is proportional to the strength of the promoter and to the length of the transcript, are promptly removed when transcription is turned off . The transcription-induced increase of template supercoiling can still be detected in the presence of an inhibitor of ATP-dependent DNA gyrase {DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3} . Altogether, our results indicate that, in addition to being under a general control, DNA superhelicity can be modulated locally in response to the topological perturbations associated with DNA tracking processes . We discuss a model in which supercoiling changes are produced by differential swiveling activities on the opposite sides of a transcriptional flow during transcriptional modulation.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8978 - 82
Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro; Weiss JB et al.; The efficient export of a subset of Escherichia coli envelope proteins is dependent upon the product of the secB gene . Previous studies indicated that SecB promotes the export of the periplasmic maltose-binding protein (MBP) by preventing premature folding of the precursor MBP in the cytoplasm into an export-incompetent form . In this study, SecB has been purified to homogeneity and shown to be a soluble, cytoplasmic, multimeric protein composed of identical 17-kDa subunits . SecB was required for efficient in vitro translocation of MBP into inverted membrane vesicles . The addition of purified SecB to an in vitro system prepared from SecB- cells significantly enhanced MBP translocation . The purified protein also quantitatively retarded folding of precursor MBP into a stable, protease-resistant conformation in the absence of membranes . Finally, the inclusion of excess purified SecB in a SecB+ in vitro system significantly prolonged the time in which precursor MBP remained competent for posttranslational import into membrane vesicles.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8953 - 7
SecA suppresses the temperature-sensitive SecY24 defect in protein translocation in Escherichia coli membrane vesicles; Fandl JP et al.; Genetic analysis of protein secretion in Escherichia coli has identified secY/prlA and secA as components of the secretory apparatus . We have examined the roles of the secY(prlA) gene product (an integral membrane protein) and the soluble secA gene product in translocation of OmpA and alkaline phosphatase precursors in an in vitro system . The protein translocation defect of the secY24 mutation was recently demonstrated in vitro as was its suppression by an S300 extract . We show here that the extract was essentially inactive in SecY24 suppression when SecA protein was removed from it by immunoaffinity chromatography . Furthermore, purified SecA protein suppressed the SecY24 defect . Preincubation of the inactivated SecY24 membrane vesicles either with S300 containing SecA or with purified SecA protein reconstituted the membranes and restored the translocation activity when assayed in the absence of additional soluble proteins . These results suggest that the SecY24 translocation defect is suppressed by SecA interacting, directly or indirectly, with SecY24 on the cytoplasmic membrane.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8919 - 23
Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli; Weiss V et al.; It has previously been shown that phosphorylated nitrogen regulator I (NRI-phosphate) is the activator responsible for increasing the transcription of glnA, the structural gene for glutamine synthetase, and that NRII catalyzes the transfer of the gamma-phosphate of ATP to NRI . We have now shown that the reaction of ATP with NRII results in the reversible transfer of the gamma-phosphate of ATP to a histidine residue of NRII . In turn, NRII-phosphate transfers its phosphate reversibly to an aspartic residue of NRI . NRI-phosphate is hydrolyzed to NRI and inorganic phosphate in a divalent cation-requiring autocatalytic reaction.

J Bacteriol, 1988 Dec, 170(12), 5901 - 7
Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12; Lahti R et al.; Escherichia coli K-12 gene ppa encoding inorganic pyrophosphatase (PPase) was cloned and sequenced . The 5' end of the ppa mRNA was identified by primer extension mapping . A typical E . coli sigma 70 promoter was identified immediately upstream of the mRNA 5' end . The structural gene of ppa contains 528 base pairs, from which a 175-amino-acid translation product, Mr 19,572, was deduced . The deduced amino acid composition perfectly fitted with that of PPase as previously determined (P . Burton, D . C . Hall, and J . Josse, J . Biol . Chem . 245:4346-4351, 1970) . Furthermore, the partial amino acid sequence (residues 1 to 108) of E . coli PPase determined by S . A . Cohen (Ph.D . thesis, University of Chicago, 1978) was the same as that deduced from the nucleotide sequence . This is the first report of the cloning of a PPase gene.

J Bacteriol, 1988 Dec, 170(12), 5759 - 64
Role of the 5' upstream sequence and tandem promoters in regulation of the rpsU-dnaG-rpoD macromolecular synthesis operon; Nesin M et al.; Bal31 exonuclease deletion analysis and transposon Tn5 mutagenesis of the 5' regulatory region of the rpsU-dnaG-rpoD macromolecular synthesis operon fused to the chloramphenicol acetyltransferase gene (pGLR301) demonstrated that sequences 5' to the operon promoters were not involved in operon transcriptional regulation and that the three tandem promoters P1, P2, and P3 were functionally independent . P2 was the strongest promoter, and P3 was the weakest . P1, P2, and P3 acting in combination appeared to be stronger than the individual promoters.

J Bacteriol, 1988 Dec, 170(12), 5433 - 9
Biochemical and genetic analysis of hydrogen metabolism in Escherichia coli: the hydB gene; Sankar P et al.; Production of active hydrogenase by Escherichia coli requires several gene products . One of the essential genes, hydB, is encoded by a DNA fragment of approximately 1.0 kilobase . The hydB gene produced a protein with an apparent molecular weight of 32,000 . The hydB gene was transcribed only under anaerobic conditions . Oxygen and nitrate repressed transcription of this gene . hydB gene transcription also required sigma 60, the product of the rpoN gene.

J Bacteriol, 1988 Dec, 170(12), 5409 - 15
Nucleotide sequence of plasmid NAH7 gene nahR and DNA binding of the nahR product; You IS et al.; The nah and sal operons of the 80-kilobase-pair (kb) NAH7 plasmid specify catabolism of naphthalene and salicylate under positive regulation by gene nahR . A 1.75-kb fragment (PstI-HindIII) cloned into the pCP13 derivative of vector RK2 complemented in trans five nahR mutations . The fragment sequence contained a 1,122-base-pair open reading frame with a predicted sequence of 374 residues that was rich in basic amino acids with regions similar to known DNA-binding proteins . Clones from the nahR gene region were expressed in mexicells . Plasmid pY1923, carrying the 1.75-kb PstI-HindIII fragment, expressed a protein of Mr ca . 35,000 which bound to the upstream region of gene nahR in a gel electrophoresis DNA-binding assay . Other clones expressed proteins of currently unknown function; pY1311, with the 1.1-kb HindIII fragment, produced a polypeptide with an Mr of 23,000, and pY1812, with the 1.2-kb PstI-SphI fragment, produced a polypeptide (Mr 41,000) which appeared to be a fused nahR-lacZ product.

Arch Surg, 1988 Dec, 123(12), 1491 - 5
Superoxide production by neutrophils in a model of adult respiratory distress syndrome; Holman RG et al.; Neutrophils (polymorphonuclear leukocytes {PMNs}) are thought to contribute to the pathophysiology of adult respiratory distress syndrome (ARDS) by the release of toxic oxygen metabolites . This study investigated superoxide production by circulating and bronchoalveolar lavage (BAL) PMNs in a rat model of ARDS induced by chronic Escherichia coli (lipopolysaccharide) endotoxemia . Superoxide production was stimulated by fmet-leu-phe, opsonized zymosan, and phorbol myristate acetate . Circulating and BAL PMNs from lipopolysaccharide-infused rats compared with PMNs from control rats are primed for nonselective increased superoxide production . The BAL PMNs are not only partially primed to release superoxide on adherence, they concomitantly have a depressed superoxide response to a phagocytic (opsonized zymosan) stimulus . These PMN responses may partially explain both the pulmonary injury and the increased susceptibility to pulmonary infection seen in patients with ARDS.

Arch Surg, 1988 Dec, 123(12), 1454 - 8
Tumor necrosis factor-enhanced leukotriene B4 generation and chemotaxis in human neutrophils; Meyer JD et al.; In an in vivo study of five normal volunteers infused with endotoxin (20 U/kg of US reference endotoxin lot EC-5), increased neutrophil (PMN) generation of leukotriene B4 and chemotaxis to leukotriene B4 were found concomitantly with elevated plasma tumor necrosis factor (TNF) levels . To clarify the role of TNF in PMN activation, neutrophil responsiveness after in vitro treatment with TNF was examined . Neutrophils from seven normal subjects were incubated with TNF for 30 minutes and tested for chemotaxis to leukotriene B4, formyl-methionyl-leucyl-phenylalanine and zymosan-activated serum, or the calcium ionophore A23187 to assess leukotriene B4 generation . A range of 10(-13) to 10(-9) mol/L of TNF was used for these assays . When 10(-9) mol/L of TNF was used, the amount of leukotriene B4 that was produced was significantly greater than in control cells . The effect of TNF on PMN chemotaxis was uniformly inhibitory for the three stimuli at 10(-10) mol/L compared with untreated cells . At a picomolar range, PMN migration to leukotriene B4, but not to zymosan-activated serum or formyl-methionyl-leucyl-phenylalanine, was significantly increased over that of PMNs not exposed to TNF . This suggests that TNF has a specific facilitatory effect on PMN responsiveness for both leukotriene B4 production and chemotaxis to leukotriene B4 and may be the same signal for this phenomenon in endotoxemic patients.

J Virol, 1988 Dec, 62(12), 4586 - 93
Poliovirus proteinase 3C: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro; Nicklin MJ et al.; Proteinase 3C of poliovirus type 2 (Sabin) was expressed at 4% total protein in Escherichia coli . The protein was soluble and could be purified by a simple scheme . It was weakly active on the capsid precursor P1 (expressed in vitro), which contains two cleavage sites . The products of processing P1 were 1ABC and 1D (VP1) . The activity was insensitive to Triton X-100 . Crude extracts of cells infected with poliovirus type 1 (Mahoney) gave strong processing and yielded 1AB (VP0), 1C (VP3), and 1D in the same assay system but were sensitive to detergent . 3C from cell extracts that was separated from its precursors resembled the recombinant proteinase in its activity . Recombinant 3C cleaved the peptide dansyl-Glu-Glu-Glu-Ala-Met-Glu-Gln-Gly-Ile-Thr-Asn-Lys-NH2 at the Gln-Gly bond . We conclude that 3C is merely the core of the Gln-Gly-cleaving activity which processes P1 in vivo and that there is probably a hydrophobic contact between a larger 3C precursor and its P1 substrate which allows the second processing reaction: 1ABC, 1D----1AB, 1C, 1D.

Arch Biol Med Exp (Santiago), 1988 Dec, 21(3-4), 393 - 401
The role of 16S RNA in ribosome function: single base alterations and their effect on in vitro protein synthesis; Cunningham PR et al.; An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes (Krzyzosiak et al., Biochemistry 26, 2353-2364, 1987) was used to make 10 single base changes around C1400, the residue known to be at the decoding site . C1400 was replaced by U, A, or G, 5 single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400 . Another mutant possessed 7 additional nucleotides at the 3' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form . Another series of 8 mutants tested hypothetical base-pairing between C1404, G1405 and C1496, G1497 . The activity of these 19 mutants in A and P site binding, and in initiation-dependent and initiation-independent peptide synthesis was determined . None of the base substitutions of C1400 were strongly inhibitory . The insertions and deletions completely blocked initiation-dependent peptide synthesis but markedly stimulated the initiation-independent reaction . The effects of tRNA binding were variable . The only alteration to block all ribosomal function was the deletion of G1401 . The extra stem and loop at the 3'-end blocked initiation-dependent peptide synthesis, but all other assays were normal . The mutants which break and reform the hypothetical base-pairs had a functional pattern that suggest the contiguous base-pairs do exist and are functionally important.

Bioorg Khim, 1988 Dec, 14(12), 1633 - 40
{Use of inorganic pyrophosphatase as a marker in enzyme immunoassays}; Baikov AA et al.; A technique of heterogeneous enzyme immunoassay with the E . coli inorganic pyrophosphatase as marker enzyme and Malachite green dye and acidic molybdate as color reagent is developed . Color change (light-yellow/greenish blue) is extremely suitable for visual perception, in some cases making unnecessary the measuring device . Assays with pyrophosphatase are 5-10 times more sensitive than with peroxidase . Further advantages of pyrophosphatase include high thermostability, insensitivity to sodium azide, low value of Michaelis constant (5 microM), substrate stability . Examples are given of use of the pyrophosphatase for assays of human alpha-fetoprotein and immunoglobulin.

J Biochem (Tokyo), 1988 Dec, 104(6), 968 - 72
Expression of rat alpha-fetoprotein cDNA in Escherichia coli and in yeast; Nishi S et al.; Rat alpha-fetoprotein (AFP) cDNA spanning the complete coding region was cloned and expressed in Escherichia coli as well as in yeast, Saccharomyces cerevisiae . The recombinant AFPs (rAFPs) were purified and characterized . The molecular weights determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were 65,000 for E . coli rAFP and 69,000 for yeast rAFP . Amino acid and N-terminal sequence analyses indicated that yeast cells produced mature AFP by processing the signal peptide properly but E . coli rAFP lacked the N-terminal 53 amino acid residues of preAFP . The yeast rAFP was found to be indistinguishable from authentic AFP in the Ouchterlony immunodiffusion test, radioimmunoassay and estradiol-binding assay while E . coli rAFP was less reactive in these tests . These observations indicated that the rAFP expressed in yeast emulated the properties of authentic AFP.

Can J Microbiol, 1988 Dec, 34(12), 1288 - 96
Attenuation of transcription of biotin genes in Escherichia coli; Nath SK; The nature of RNA synthesis from the divergent bioA:BFCD operon has been studied in vivo in Escherichia coli minicells . Minicells containing bio-recombinant plasmids produce 96 nucleotide long RNA . This small RNA was characterized by blotting on diazobenzyloxymethyl cellulose paper and hybridization with the appropriate bio-probe DNA . Determination of beta-galactosidase, using biotin requiring mutant cells of E . coli JM101 infected with a bio-M13 recombinant phage containing the functional bioB region of the operon, demonstrated the presence of a transcription termination site in the bioBFCD segment . There was a three- to five-fold reduction in beta-galactosidase activity in this system at an appropriate d-biotin concentration which indicated that the early termination of transcription is biotin dependent . From the nucleotide sequence of the promoter proximal region of the bioB gene, a 13-oligonucleotide sequence believed to be the early transcription termination site has been located . A model of regulation at termination has been proposed on the basis of the not-too-stable RNA secondary structure at the site of early termination of transcription.

AIDS Res Hum Retroviruses, 1988 Dec, 4(6), 409 - 17
Conserved immunogenic region of a major core protein (p24) of human and simian immunodeficiency viruses; Koito A et al.; A murine monoclonal antibody (MoAb), VAK 4, has been known to specifically react with a major core protein (p24) as well as with its precursor (p55-57) and intermediate precursor (p40) of human immunodeficiency virus strain IIIB (HTLV-IIIB) . Radioimmunoprecipitation assays revealed that VAK 4 MoAb precipitated a major core protein and its precursors from a variety of strains of HIV and also from simian immunodeficiency virus (SIV), although the molecular weights of the precursor proteins in each viral strain were slightly different . A protein synthesized by transfected Escherichia coli containing amino acid sequences corresponding to residues 121-436 of the HTLV-IIIB gag gene was reactive with VAK 4 MoAb, but the protein carrying only residues 121-309 was not reactive, suggesting that the epitope recognized by VAK 4 MoAb resides at the carboxyl terminus of p24 protein . A competitive enzyme-linked immunosorbent assay showed that patient sera containing anticore protein antibody inhibited the binding of VAK 4 to HTLV-IIIB . These findings suggested that VAK 4 MoAb recognized an immunogenic and conserved epitope belonging to a major core protein of HIV-related viruses.

Parasitology, 1988 Dec, 97 ( Pt 3), 373 - 82
A hybrid gene to express protein epitopes from both sporozoite and merozoite surface antigens of Plasmodium falciparum; Holder AA et al.; The DNA coding for parts of the repetitive amino acid sequence of Plasmodium falciparum circumsporozoite protein has been spliced to a sequence encoding part of the precursor to the major merozoite surface antigens, to produce a hybrid gene . Expression in Escherichia coli produces a protein with antigenic determinants from both malaria proteins . Antibodies raised against the expressed material react with both a peptide derived from the circumsporozoite repeat sequence, and the merozoite surface molecule . Hybrid molecules of this type may be the basis of a malaria vaccine.

Circ Shock, 1988 Dec, 26(4), 419 - 30
Dose dependence of endotoxin-induced activation of the plasma contact system: an in vitro study; Roeise O et al.; The dose and time dependence of endotoxin-induced activation of the plasma contact system have been studied . Citrated pool plasma was incubated at 37 degrees C with endotoxin doses of 2.10(5), 2.10(6), 2.10(7), and 2.10(9) ng/l (lipopolysaccharide B, E . coli 026: B6, Difco Laboratories, Detroit, MI) for 24 hr . Samples for determination of components of the contact system were obtained prior to incubation and at 1, 2, 4, 6, 12, and 24 hr . Plasma kallikrein (KK) activity markedly increased at 12 hr in test plasma containing the highest dose of endotoxin (2.10(9) ng/l) . Coincident with the elevated KK activity, reductions of both plasma prekallikrein (PKK) and functional kallikrein inhibition (KKI) were seen as assayed by chromogenic peptide substrate analyses . Also, functionally determined alpha 2-macroglobulin (alpha 2-M) and C1 inhibitor (C1INH) values were decreased, confirming the reduction of KKI values . Changes of Hageman factor (FXII), PKK, and high molecular weight kininogen (HMWK) values were also found at the same time point when assayed by immunochemical techniques . The same pattern of changes was seen in test plasma containing 2.10(7) and 2.10(6) ng/l of endotoxin . These changes, however, were less pronounced and not seen until 24 hr after beginning incubation . In control plasma and in plasma containing the lowest dose of endotoxin (2.10(5) ng/l), no changes were seen in any factors of the contact system . Our study shows that in vitro endotoxin-induced activation of the contact system is a slow process that is both time and dose dependent.

Virology, 1988 Dec, 167(2), 361 - 9
Molecular characterization of a prominent antigen of the vaccinia virus envelope; Gordon J et al.; During vaccinia virus (VV) assembly a major polypeptide migrating with an apparent MW of 35K, designated Ag35, is expressed as an early function and becomes an integral component of the lipoprotein envelope surrounding the mature virion . In a previous study evaluating humoral immunity to VV, a prominent response against Ag35 was invariably detected in immunized mice . In the context of our continuing investigations of the structure and function of the vaccinia envelope, with a view to alteration in antigenicity of this agent when used as a vaccine vector for foreign antigens, we carried out detailed mapping of the Ag35 gene, as well as determination of the nucleotide sequence . Use of hybridization-arrested translation, coupled with immunoprecipitation, located this gene within a 2.7-kbp EcoRI fragment of the larger 8.7-kbp HindIII H fragment . By means of S1 endonuclease resistance analysis a viral transcript was identified at the site of the Ag35 gene, where the occurrence of an open reading frame (ORF), corresponding to the transcript, was deduced from DNA sequence determination . However, the ORF encodes a polypeptide of only 22,300 Da predicted MW, which is much lower than the apparent MW estimated from SDS-polyacrylamide gel electrophoresis . The size discrepancy is not due to glycosylation or phosphorylation of Ag35 but may result from a proline-rich sequence which occurs in this polypeptide . To confirm that the ORF recognized in this study does, indeed, encode Ag35, the gene was expressed as a beta-galactosidase fusion protein in pUC19; Escherichia coli transformed with the relevant clones expressed a polypeptide of the appropriate molecular weight and antigenicity, when tested by Western blots . Regarding secondary structure and hydropathicity it can be predicted from the DNA sequence that Ag35 is highly hydrophilic but contains a hydrophobic region at the carboxy terminus, perhaps providing the stretch involved in membrane insertion . Computer search of a bank of protein sequences revealed an unusually strong similarity of 68% between the Ag35 at amino acid positions 44-121 and the G glycoprotein of respiratory syncytial virus at positions 189-264.

Nature, 1988 Dec 1, 336(6198), 496 - 8
An eIF-4A-like protein is a suppressor of an Escherichia coli mutant defective in 50S ribosomal subunit assembly; Nishi K et al.; The assembly of ribosomes in bacterial cells is a complex process that remains poorly characterized . The in vitro assembly of active ribosomal subunits from purified RNA and protein components indicates that all of the information for proper assembly resides in the primary sequences of these macromolecules . On the other hand, the in vitro requirement of unphysiological heating steps suggests that this pathway may not accurately reflect the in vivo pathway, and that other proteins may be required . One approach to identify any additional proteins is to isolate second-site revertants of mutants defective in ribosome assembly . Ribosomal protein L24 is essential in the assembly of 50S subunits . We have identified an Escherichia coli gene, srmB, that, when expressed at high copy number, can suppress the effect of a temperature-sensitive lethal mutation in L24 . The SrmB amino-acid sequence has sequence identity with mouse translation initiation factor eIF-4A and with the human nuclear protein, p68 . The purified SrmB protein is a nucleic acid-dependent ATPase, like eIF-4A, but can also bind RNA in the absence of ATP and other auxiliary protein factors . The RNA dependent ATPase activity of SrmB suggests that like, eIF-4A, it could be involved in specific alterations of RNA secondary structure.

Exp Parasitol, 1988 Dec, 67(2), 247 - 56
Plasmodium falciparum: analysis of B epitopes of a polypeptide antigen expressed in Escherichia coli, using monoclonal antibodies; Blisnick T et al.; Monoclonal antibodies were raised against a recombinant molecule corresponding to the polypeptide 72 kDa, previously described as possibly related to protection in Plasmodium falciparum infection . Selection of hybridoma cell lines was done by immunofluorescence to guarantee the reactivity of the monoclonal antibodies both against the recombinant and the native molecule of the parasite . Monoclonal antibodies were characterized by serological and immunochemical techniques . Competitive binding assays between monoclonal antibodies defined four different B epitopes . One epitope is specific for P . falciparum, a second is also present in P . vivax, while the two others seem to be ubiquitous and are also present in the rodent parasite P . chabaudi . The ubiquitous epitope 72.C is apparently the only one recognized by squirrel monkey sera presenting protective antibodies against the asexual blood infection by P . falciparum.

J Virol, 1988 Dec, 62(12), 4805 - 8
Mapping of antigenic domains of Sendai virus nucleocapsid protein expressed in Escherichia coli; Gill DS et al.; Several nonoverlapping epitopes were mapped on the primary sequence of the Sendai virus NP protein . After a complete cDNA clone of the Sendai virus NP gene was expressed in Escherichia coli, deletion constructs were used to generate a series of overlapping NP fragments deleted at their C termini . Immunoblot analyses with 11 monoclonal antibodies identified four antigenic sites . All of these sites resided in the C-terminal half of NP and were also the only sites detected with a polyclonal serum . These findings confirm and extend the evidence that the C terminus of the NP protein represents the domain exposed on the surface of the nucleocapsid . One of the monoclonal antibodies reacted with a site, comprising only 6 amino acids, lying with a hinge between an alpha-helix and a beta-strand in the predicted secondary structure of NP . Since this antibody is a potent inhibitor of in vitro viral RNA synthesis (K . L . Deshpande and A . Portner, Virology 139:32-42, 1984), the epitope may be critical to the flexibility of the NP molecule that makes the RNA template accessible during RNA synthesis.

J Virol, 1988 Dec, 62(12), 4786 - 90
Hepatitis B virus particles contain a polypeptide encoded by the largest open reading frame: a putative reverse transcriptase; Mack DH et al.; A segment of the largest open reading frame of hepatitis B virus (HBV) was inserted into an open reading frame vector directing the expression in Escherichia coli of a fusion molecule containing 143 HBV-encoded amino acids . The fusion protein was used to generate antiserum which served in immunoblots to identify a polypeptide with a molecular mass of 65 kilodaltons in HBV particles . Because of the small number of molecules in virus particles, unambiguous detection required the development of a highly sensitive immunoblot procedure.

J Neurochem, 1988 Dec, 51(6), 1858 - 67
Cross-homologies and structural differences between human cholinesterases revealed by antibodies against cDNA-produced human butyrylcholinesterase peptides; Dreyfus P et al.; To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed . Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE) . Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE . In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation . Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies . To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve . Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining . The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not . These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.

Gene, 1988 Nov 30, 71(2), 413 - 20
Structure of a multihormonally regulated rat gene; Tindal MH et al.; Gene 33, a rat gene transcriptionally enhanced by glucocorticoids, insulin, or cyclic AMP, was isolated from a library of rat genomic DNA and characterized by sequence comparison to a full-length cDNA . The structural gene spans 13,500 bp encoding 2970 bp of exon sequences interrupted by three introns of about 9600, 101 and 811 bp, respectively . Exons (5' to 3') are 198, 194, 77 and 2501 bp in length; the first of these initiates at the transcriptional start point determined by S1 nuclease mapping . The 5'-flanking DNA contains several putative transcriptional control elements including TATA and CAAT boxes and a binding site for the Sp1 transcription factor in the usual locations proximal to the start point . Sequences resembling known glucocorticoid and cyclic AMP regulatory elements are also found upstream . A chimeric plasmid was constructed containing putative gene 33 regulatory elements fused to the Escherichia coli gene cat, encoding the enzyme chloramphenicol acetyltransferase, and transfected into cultured fibroblasts . Transient expression assays established that this gene 33 DNA is effective in promoting transcription.

Gene, 1988 Nov 30, 71(2), 349 - 58
Signal sequence of preproglycinin affects production of the expressed protein in Escherichia coli; Utsumi S et al.; The effect of the signal peptide portion on the bacterial production of preproglycinin, a precursor of soybean storage protein, was examined . Nucleotide sequences corresponding to the signal peptide and the mature N-terminal region were deleted stepwise from the cDNA encoding the glycinin A1aB1b subunit precursor, and the deleted cDNAs were placed under the control of trc promoter in an expression vector pKK233-2 . When the amounts of the protein products in Escherichia coli from each expression plasmid were determined, no accumulation of preproglycinin was observed from the plasmids with the full length or the five amino acids of the signal sequence . However, significant accumulation of the preproglycinin homologue proteins was noted from the plasmids retaining less than three amino acids of the signal sequence depending on the extent of deletion . N-terminal amino acid sequences of the products coincided with those predicted from the deleted cDNAs . The preproglycinin homologue proteins expressed from the mutant plasmids assembled into trimers of about 8S.

Gene, 1988 Nov 30, 71(2), 307 - 14
Molecular cloning and sequence analysis of a developmentally regulated cysteine-rich outer membrane protein from Chlamydia trachomatis; Clarke IN et al.; Two overlapping genomic fragments have been cloned from Chlamydia trachomatis serovar L1 DNA . Sequence determination of 2530 bp has revealed two open reading frames coding for 'cysteine-rich' (Cr) proteins . One of these proteins was confirmed, by analysis of the inferred amino acid sequence, as the 60-kDa Cr outer membrane protein associated with differentiation of reticulate bodies (RBs) into elementary bodies (EBs) . The other smaller 15-kDa protein contained a high percentage of methionine and cysteine and may correspond to a reported smaller and co-ordinately synthesised Cr outer-membrane protein also associated with RB to EB differentiation . Sequencing showed three potential stem-loop structures within the 5', 3' and intergenic regions of the cloned fragment . Southern-blot analysis revealed that the cloned fragment is conserved in ten serovars of C . trachomatis and that a strongly cross-hybridising fragment is also present in Chlamydia psittaci.

Gene, 1988 Nov 30, 71(2), 331 - 7
Cloning of Thermomonospora fusca genes coding for beta 1-4 endoglucanases E1, E2 and E5; Hu YJ et al.; Thermomonospora fusca chromosomal DNA was partially digested with EcoRI and fragments in the size range from 4 to 15 kb were isolated, ligated into lambda gtWES.lambda B arms, packaged, and the recombinant phages plated on Escherichia coli . The plaques were screened for carboxymethyl cellulase (CMCase) activity by a gel overlay procedure, and 25 plaques were positive among the 15,000 plaques that were screened . Positive phages were amplified and used to prepare infected E . coli extracts which were assayed for CMCase activity before and after treatment with antisera prepared against five purified T . fusca beta 1-4 endoglucanases (E1-E5) . One phage produced an enzyme that was inhibited by E1 antiserum, nine of the phages produced enzymes that were inhibited by E2 antiserum, 14 produced enzymes that were inhibited by E5 antiserum and the enzyme produced by the other phages was not inhibited by any of the five antisera . The DNA insert present in the phage coding for E1 was cut into a number of different fragments which were subcloned into E . coli first using lambda gtWES.lambda B and then plasmid pBR322 . The smallest active subclone, pTE12, contained a 3.1-kb insert . The insert present in one of the phages coding for E2 was also subcloned and the smallest active subclone pTE23 contained a 2-kb insert . E . coli HB101 containing plasmid pTE12 or pTE23 produced enzymes that were identical to E1 and E2, respectively, in all the properties tested.

Biochemistry, 1988 Nov 29, 27(24), 8823 - 32
Site-directed mutagenesis of Escherichia coli ornithine transcarbamoylase: role of arginine-57 in substrate binding and catalysis; Kuo LC et al.; In the carbamoyl-transfer reaction catalyzed by ornithine transcarbamoylase, an arginine residue in the active site of the Escherichia coli enzyme has been suggested to bind the phosphate moiety of the substrate carbamoyl phosphate . With the application of site-specific mutagenesis, the most likely arginine residue among three candidates at the binding site of carbamoyl phosphate, Arg-57, has been replaced with a glycine . The resultant Gly-57 mutant enzyme is drastically inefficient in catalysis . In the synthesis of L-citrulline from carbamoyl phosphate and L-ornithine with the release of inorganic phosphate, the turnover rate of the mutant is 21,000-fold lower than that of the wild type . However, the mutation of Arg-57 affects only moderately the binding of carbamoyl phosphate; the dissociation constant of this substrate, measured under steady-state turnover condition, is increased from 0.046 to 3.2 mM by the mutation . On the other hand, ornithine binding is substantially affected as estimated by the change in the dissociation constant of its analogue L-norvaline . The dissociation constant of L-norvaline increases about 500-fold from 54 microM for the wild type to 25 mM for the mutant . Since Arg-57 is expected to be distal from the ornithine site and the amino acid (both ornithine and norvaline) binds only after carbamoyl phosphate in the wild-type reaction, the poor norvaline affinity to the mutant suggests that Arg-57 is involved in interactions essential for productive addition of the amino acid . This interpretation is supported by difference ultraviolet absorption spectra which show that the conformational changes induced in the wild type by carbamoyl phosphate upon binding are absent in the mutant . Furthermore, steady-state kinetic data reveal that the ordered binding mechanism of the wild-type enzyme is transformed into a random binding mechanism in the mutant . Thus, the presence of carbamoyl phosphate in the mutant active site is no longer a requisite for ornithine binding . In the 5-50 degrees C temperature range, transcarbamoylation catalyzed by either the wild type or the mutant observes the Arrhenius rate law with almost identical enthalpies of activation, 11 and 10 kcal/mol, respectively . The entropy of activation is -5.5 eu for the wild-type reaction and -29 eu for the mutant reaction, accounting for a loss of 6-7 kcal/mol in the rate-determining step of the enzymic reaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Nucleic Acids Res, 1988 Nov 25, 16(22), 10833 - 47
A related moderately repetitive DNA family in the nematodes Ascaris lumbricoides and Panagrellus silusiae; Warren T et al.; Digestion of genomic DNA from the nematodes Panagrellus silusiae and Ascaris lumbricoides with restriction endonuclease BamH1 releases a 0.7 kilobase (kb) fragment . The 0.7 kb fragment from both nematodes was cloned onto E . coli plasmid pUC19 . Using representative clones as DNA hybridization probes, it was found that (i) the BamH1 fragments cross-hybridize; (ii) a ladder-effect with multiples of 0.7 kb was evident in both species after hybridization to genomic DNA and (iii) the genomic copy number of BamH1 elements is 150 and 195 for P . silusiae and A . lumbricoides respectively . DNA sequence analysis of the inserts, AL700-1 and PS700-1, revealed nucleotide blocks with over 85% similarity . No open reading frames are present in either DNA fragment . Neither fragment hybridizes to genomic DNA from Caenorhabditis elegans . Northern blot hybridization indicated that the 0.7 kb element is transcribed into poly(A)(-)-RNA in P . silusiae; but, is not transcribed in adult Ascaris muscle . Thus, P . silusiae and A . lumbricoides share a homologous, tandemly arrayed, moderately repetitive DNA family.

J Biol Chem, 1988 Nov 25, 263(33), 17658 - 62
Molecular cloning and sequencing of the psaD gene encoding subunit II of photosystem I from the cyanobacterium, Synechocystis sp . PCC 6803; Reilly P et al.; Photosystem I reaction center was isolated from the cyanobacterium, Synechocystis sp . PCC 6803, in a form which contains seven different polypeptide subunits . One of the subunits, with a molecular mass of about 16 kDa, was isolated, and protein sequence information was obtained for the amino terminus and several tryptic peptides . Oligonucleotide probes, corresponding to these sequences, were used to probe a genomic library, and the gene, designated psaD, encoding subunit II was cloned and sequenced . The gene encodes a polypeptide with a mass 15,644 Da, which exhibits a high degree of similarity to subunit II from tomato, as well as amino acid sequences reported from barley photosystem I . In addition to this gene, three large open reading frames were identified . Two remain unidentified, and the third is highly homologous to anthranilate synthase, component 1 from Escherichia coli and Saccharomyces cerevisiae.

Nucleic Acids Res, 1988 Nov 25, 16(22), 10903 - 12
Potential role of proteolysis in the control of UvrABC incision; Caron PR et al.; UvrB is specifically proteolyzed in Escherichia coli cell extracts to UvrB* . UvrB* is capable of interacting with UvrA in an apparently similar manner to the UvrB, however UvrB* is defective in the DNA strand displacement activity normally displayed by UvrAB . Whereas the binding of UvrC to a UvrAB-DNA complex leads to DNA incision and persistence of a stable post-incision protein-DNA complex, the binding of UvrC to UvrAB* leads to dissociation of the protein complex and no DNA incision is seen . The factor which stimulates this proteolysis has been partially purified and its substrate specificity has been examined . The protease factor is induced by "stress" and is under control of the htpR gene . The potential role of this proteolysis in the regulation of levels of active repair enzymes in the cell is discussed.

Nucleic Acids Res, 1988 Nov 25, 16(22), 10817 - 31
Probing the alpha-sarcin region of Escherichia coli 23S rRNA with a cDNA oligomer; White GA et al.; The cause of 50S ribosomal subunit collapse reportedly triggered by hybridization of a 14-base cDNA probe to the alpha-sarcin region of 23S rRNA was investigated by physical measurement of probe-subunit complexes in varying buffer conditions . The results reported here show that this probe was unable to hybridize to its target site in the intact 50S subunit and the physical characteristics of 50S subunits remained unchanged in its presence . Subunit collapse was induced in buffer containing 20mM Tris-HCl (pH 7.5), 600 mM NH4Cl, 1 mM MgCl2, 1 mM DTT, and 0.1 mM EDTA in the absence of probe . The probe bound specifically to its target site in the collapsed particle, but did not promote further unfolding . The results demonstrate that a DNA probe bound to the alpha-sarcin region cannot cause the 50S subunit to unfold or cause 23S rRNA to degrade . We suggest that the previously reported collapse was most probably the result of the ionic conditions used.

Nucleic Acids Res, 1988 Nov 25, 16(22), 10717 - 32
Exploration of the L18 binding site on 5S RNA by deletion mutagenesis; Gewirth DT et al.; Several deletion variants of E . coli 5S RNA have been constructed and produced either in vivo or in vitro using T7 RNA Polymerase . Their structures and ribosomal protein L18 binding properties have been examined . All of them are similar to wild-type 5S RNA in their helix II-III regions, where L18 binds {Huber, P.W . and Wool, I.G . (1984) Proc . Natl . Acad . Sci . (USA) 81, 322-326; Douthwaite, S., Christensen, A., and Garrett, R.A . (1982) Biochemistry 21, 2313-2320.}, by NMR criteria . However, none of the molecules examined that lack the helix IV-helix V stem bind L18 efficiently, even though that portion of 5S RNA is outside the L18 footprint . The L18 binding site is clearly more than a simple hairpin loop.

Nucleic Acids Res, 1988 Nov 25, 16(22), 10681 - 97
Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with synthetic DNA containing a promoter for T7 RNA polymerase; Lee BL et al.; The interaction of 4'-N(2-aminoethyl)aminomethyl-4,5',8-trimethylpsoralen-modified oligonucleoside methylphosphonates with synthetic ds-DNA containing a T7 RNA polymerase promoter was studied . The oligomers effectively crosslinked with either coding or noncoding ss-DNA when irradiated at 365 nm, but not with ds-DNA . The extent of the crosslinking reaction, which was complete within 16 min: (a) reached its maximum at an oligomer concentration of 3 microM; (b) remained constant below the Tm of the duplex and then rapidly decreased; and (c) appeared to depend upon the sequence surrounding the psoralen crosslinking site . An oligomer crosslinked to the template strand inhibited transcription by T7 RNA polymerase whereas an oligomer crosslinked to the non-template strand had only a small inhibitory effect . Oligomers did not crosslink to ds-DNA undergoing transcription nor did they inhibit the transcription reaction.

Nucleic Acids Res, 1988 Nov 25, 16(22), 10529 - 45
Mutational analysis of the L1 binding site of 23S rRNA in Escherichia coli; Said B et al.; The L11 ribosomal protein operon of Escherichia coli contains the genes for L11 and L1 and is feedback regulated by the translational repressor L1 . Both the L1 binding site on 23S rRNA and the L1 repressor target site on L11 operon mRNA share similar proposed secondary structures and contain some primary sequence identity . Several site-directed mutations in the binding region of 23S rRNA were constructed and their effects on binding were examined . For in vitro analysis, a filter binding method was used . For in vivo analysis, a conditional expression system was used to overproduce a 23S rRNA fragment containing the L1 binding region, which leads to specific derepression of the synthesis of L11 and L1 . Changes in the shared region of the 23S rRNA L1 binding site produced effects on L1 binding similar to those found previously in analysis of corresponding changes in the L11 operon mRNA target site . The results support the hypothesis that r-protein L1 interacts with both 23S rRNA and L11 operon mRNA by recognizing similar features on both RNAs.

Nucleic Acids Res, 1988 Nov 25, 16(22), 10453 - 67
Design, synthesis and expression of a human interleukin-2 gene incorporating the codon usage bias found in highly expressed Escherichia coli genes; Williams DP et al.; A synthetic gene encoding human interleukin-2 (IL-2) was designed such that the codon usage bias resembled that found in highly expressed Escherichia coli genes . The percentage of preferred codons was increased from 43% in the native cDNA sequence to 85% in the synthetic sequence . The cDNA and synthetic IL-2 genes were placed under the control of the trc promoter and expressed in E . coli JM101 . While Northern blot analysis of IL-2 mRNA from each genetic construct demonstrated equivalent message half-lives, immunoblot and bioactivity analyses showed the synthetic gene to direct the synthesis of up to 16 times more IL-2 than the native cDNA sequence.

J Biol Chem, 1988 Nov 25, 263(33), 17780 - 4
Radical formation in the dimeric small subunit of ribonucleotide reductase requires only one tyrosine 122; Larsson A et al.; The small subunit of ribonucleoside-diphosphate reductase (EC 1.17.4.1) is a homodimer . Its catalytic site contains one tyrosyl radical, which is localized to Tyr-122 in one of its polypeptide chains . The engineered Tyr-122----Phe protein was used to demonstrate that it is possible to form a correct ferric iron center in vitro in the absence of Tyr-122 . Heterodimers, consisting of one Tyr-122-containing polypeptide chain and one Phe-122-containing polypeptide chain, were constructed . The heterodimer population contained one-half the amount of tyrosyl radical as compared to a homodimer with Tyr-122, i.e . every second heterodimer contains a tyrosyl radical . Thus, one Tyr-122 is sufficient for radical formation . Radical-containing heterodimers are catalytically competent.

J Biol Chem, 1988 Nov 25, 263(33), 17715 - 23
Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli; Levin MS et al.; Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are 132-residue cytosolic proteins which have 56% amino acid sequence identity and bind all-trans-retinol as their endogenous ligand . They belong to a family of cytoplasmic proteins which have evolved to bind distinct hydrophobic ligands . Their patterns of tissue-specific and developmental regulation are distinct . We have compared the ligand binding properties of rat apo-CRBP and apo-CRBP II that have been expressed in Escherichia coli . Several observations indicate that the E . coli-derived apoproteins are structurally similar to the native rat proteins: they co-migrate on isoelectric focusing gels; and when complexed with all-trans-retinol, their absorption and excitation/emission spectra are nearly identical to those of the authentic rat holoproteins . Comparative lifetime and acrylamide quenching studies suggest that there are differences in the conformations of apo-CRBP and apo-CRBP II . The interaction of E . coli-derived apo-CRBP and apo-CRBP II with a variety of retinoids was analyzed using spectroscopic techniques . Both apoproteins formed high affinity complexes with all-trans-retinol (K'd approximately 10 nM) . In direct binding assays, all-trans-retinal bound to both apoproteins (K'd approximately 50 nM for CRBP; K'd approximately 90 nM for CRBP II) . However, all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP . These observations suggests that there is a specific yet distinct interaction between these two proteins and all-trans-retinal . Apo-CRBP and apo-CRBP II did not demonstrate significant binding to either retinoic acid or methyl retinoate, an uncharged derivative of all-trans-retinoic acid . This indicates that the carboxymethyl group of methyl retinoate cannot be sterically accommodated in their binding pockets and that failure to bind retinoic acid probably is not simply due to the negative charge of its C-15 carboxylate group . Finally, neither all-trans-retinol nor retinoic acid bound to E . coli-derived rat intestinal fatty acid-binding protein, a homologous protein whose tertiary structure is known . Together, the data suggest that these three family members have acquired unique functional capabilities.

J Biol Chem, 1988 Nov 25, 263(33), 17627 - 31
Expression of an enzymatically active Yb3 glutathione S-transferase in Escherichia coli and identification of its natural form in rat brain; Abramovitz M et al.; Glutathione S-transferases containing Yb3 subunits are relatively uncommon forms that are expressed in a tissue-specific manner and have not been identified unequivocally or characterized . A cDNA clone containing the entire coding sequence of Yb3 glutathione S-transferase mRNA was incorporated into a pIN-III expression vector used to transform Escherichia coli . A fusion Yb3-protein containing 14 additional amino acid residues at its N terminus was purified to homogeneity . Recombinant Yb3 was enzymatically active with both 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates but lacked glutathione peroxidase activity . Substrate specificity patterns of recombinant Yb3 were more limited than those of glutathione S-transferase isoenzymes containing Yb1- or Yb2-type subunits . Peptides corresponding to unique amino acid sequences of Yb3 as well as a peptide from a region of homology with Yb1 and Yb2 subunits were synthesized . These synthetic peptides were used to raise antibodies specific to Yb3 and others that cross-reacted with all Yb forms . Immunoblotting was utilized to identify the natural counterpart of recombinant Yb3 among rat glutathione transferases . Brain and testis glutathione S-transferases were rich in Yb3 subunits, but very little was found in liver or kidney . Physical properties, substrate specificities, and binding patterns of the recombinant protein paralleled properties of the natural isoenzyme isolated from brain.

J Biol Chem, 1988 Nov 25, 263(33), 17499 - 507
Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein; Orlean P et al.; Dolichol phosphate mannose (Dol-P-Man) synthase (EC 2.4.1.83) catalyzes the formation of Dol-P-Man from Dol-P and GDP-Man . The structural gene for yeast Dol-P-Man synthase (DPM1) was isolated by screening a yeast genomic DNA library for colonies that overexpressed Dol-P-Man synthase activity . This approach relied on a method to screen for Dol-P-Man synthase activity in lysed yeast colonies and used a yeast mutant with very low Dol-P-Man synthase activity in colony lysates . Transformants isolated using this technique expressed Dol-P-Man synthase activity 9-14-fold higher than that of a wild type strain, and all seven plasmids conferring this overproduction had a common region in their yeast genomic DNA insert . DPM1 is the structural gene for yeast Dol-P-Man synthase since Escherichia coli transformants harboring this gene express Dol-P-Man synthase activity in vitro . DNA sequencing of the DPM1 gene revealed an open reading frame of 801 bases . The 30-kDa size of the predicted protein is in excellent agreement with the size of the purified yeast enzyme (Haselbeck, A., and Tanner, W . (1982) Proc . Natl . Acad . Sci . U . S . A . 79, 1520-1524) . Analysis of the predicted amino acid sequence reveals the protein has a potential membrane spanning domain of 25 amino acids at its COOH terminus . The protein's NH2 terminus, though not hydrophobic, meets existing criteria for yeast signal sequences, but there is no site for cleavage by signal peptidase . If the NH2 terminus is a functional signal sequence, the protein is predicted to be oriented toward the lumen of the endoplasmic reticulum with both NH2 and COOH termini serving as membrane anchors . If there is no signal sequence, the enzyme is predicted to face the cytoplasm and be anchored only by its COOH terminus . The DPM1 gene is essential for viability in yeast since disruption of the gene is lethal . We suspect Dol-P-Man synthase is not an essential protein due to its role in N-glycosylation since mutations in other genes that affect the late steps in lipid-linked oligosaccharide synthesis do not affect cell growth . Instead, DPM1 may be an essential gene because its product is required for O-glycosylation in yeast or because Dol-P-Man synthase is needed in some unidentified pathway.

J Biol Chem, 1988 Nov 25, 263(33), 17229 - 32
Site-directed mutagenesis of the tryptophan residues in yeast eukaryotic initiation factor 4E . Effects on cap binding activity; Altmann M et al.; Initiation factor 4E is a 24-kilodalton polypeptide that binds specifically to the 5' cap structure of eukaryotic mRNAs . Sequence analysis of cDNA clones of initiation factor 4E from several species revealed a high tryptophan content (8 residues) . Strikingly, all tryptophans are conserved evolutionarily in number and position between yeast and mammals . Here we show, using site-directed mutagenesis, that two of the tryptophans (those referred to as numbers 1 and 8) are absolutely required for the cap binding activity of an Escherichia coli expressed initiation factor 4E.

Nucleic Acids Res, 1988 Nov 25, 16(22), 10891 - 902
Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair; Caron PR et al.; The incision of damaged DNA by the Escherichia coli UvrABC endonuclease requires ATP hydrolysis . Although the deduced sequence of the UvrB protein suggests a putative ATP binding site, no nucleoside triphosphatase activity is demonstrable with the purified UvrB protein . The UvrB protein is specifically proteolyzed in E . coli cell extracts to yield a 70 kD fragment, referred to as UvrB*, which has been purified and is shown to possess a single-strand DNA dependent ATPase activity . Substrate specificity and kinetic analyses of UvrB* catalyzed nucleotide hydrolysis indicate that the stimulation in DNA dependent ATPase activity following formation of the UvrAB complex results from the activation of the normally sequestered UvrB associated ATPase . Using nucleotide analogues, it can be shown that this activity is essential to the DNA incision reaction carried out by the UvrABC complex.

J Biol Chem, 1988 Nov 25, 263(33), 17437 - 42
Functional domains of epsilon subunit of Escherichia coli H+-ATPase (F0F1); Kuki M et al.; Mutants of the uncC gene for the epsilon subunit (138 amino acid residues) of Escherichia coli H+-ATPase were isolated: strain KF53 (Gln-72----end) and KF148(SD-) (two base substitutions in the Shine-Dalgarno sequence, GGAGG----AAAGG) . These strains did not have F1 bound to membranes and were unable to grow by oxidative phosphorylation . A series of plasmids carrying truncated uncC genes were constructed and introduced into strain KF148(SD-) . Analyses of KF148(SD-) cells with different plasmids indicated that the amino-terminal fragment of the epsilon subunit of 78-80 amino acid residues was capable of forming active membrane-bound F1-ATPase, whereas that of 73 residues was not, indicating that the carboxyl-terminal half of the epsilon subunit is not necessary for the active enzyme . Furthermore, results indicated that residues between 73 and 78-80 may have a critical role(s) in binding F1 to F0 . Truncated epsilon subunits of 80 and 93 residues were identified in purified F1 from cells carrying the respective uncC genes, and only the latter subunit had intrinsic activity to inhibit ATPase of F1, suggesting that residues between 80 and 93 are essential for the inhibitory activity.

Nucleic Acids Res, 1988 Nov 25, 16(22), 10803 - 16
The existence of two genes between infB and rpsO in the Escherichia coli genome: DNA sequencing and S1 nuclease mapping; Sands JF et al.; A number of genes encoding proteins involved in transcription and translation are clustered between 68 and 69 minutes on the Escherichia coli genome map and are transcribed clockwise as two operons: the metY operon, containing metY, P15A, nusA, infB; and about a kilobase further downstream, the rpsO and pnp operon . The DNA sequence between infB and rpsO was determined and two open reading frames were detected which code for proteins of 15,200 (P15B) and 35,091 (P35) daltons . Maxicell analysis showed a relatively strong expression of P15B whereas P35 was synthesized more weakly . An overlap of the termination codon of P15B and the initiator codon for P35 suggests that translation of P15B and P35 may be coupled . S1 nuclease mapping of in vivo transcripts between infB and rpsO provided no evidence for major promoters but detected a moderately efficient rho-independent terminator between infB and P15B . The results indicate that P15B and P35 are expressed as part of the metY operon, but that some transcriptional read through into the rpsO operon also occurs, thereby, functionally linking the expression of these two complex systems.

J Biol Chem, 1988 Nov 25, 263(33), 17237 - 8
Crystallization and preliminary diffraction data of Escherichia coli ADP glucose pyrophosphorylase; Mulichak AM et al.; ADP glucose pyrophosphorylase from Escherichia coli has been crystallized from polyethylene glycol 8000 solutions . The crystals are: orthorhombic, a = 155(2), b = 153(2), c = 174(2) A, space group P2(1)2(1)2(1), four tetrameric molecules/unit cell . This gives a solvent fraction of about 75% consistent with the relatively poor diffraction quality of crystals (5.0-A resolution) and their sensitivity to x-ray exposure damage . Ways of circumventing the former and improving the latter are proposed.

Nucleic Acids Res, 1988 Nov 25, 16(22), 10751 - 64
Analysis of potential expression of highly related members of the ribosomal protein L32 gene family; Jacks CM et al.; The processed gene L32', a member of the mouse gene family for ribosomal protein L32, could encode a 135 amino acid protein nearly identical to L32 . The 5'-flanking region of the gene contains CAAT and TATA sites at positions commonly found in expressed genes . The L32' gene lies within highly methylated, DNase I-insensitive chromatin of mouse L1210 cells . Although S1 nuclease digestion studies suggested that an L32' transcript might be produced, an oligonucleotide probe specific for L32' mRNA, and RNase digestion of a cRNA probe to L32', indicated fewer than 0.1 L32' transcripts/cell . These results demonstrate that extreme caution is required when measuring transcription from related genes.

Science, 1988 Nov 25, 242(4882), 1171 - 3
The accuracy of reverse transcriptase from HIV-1; Roberts JD et al.; A study was conducted to determine the fidelity of DNA synthesis catalyzed in vitro by the reverse transcriptase from a human immunodeficiency virus type 1 (HIV-1) . Like other retroviral reverse transcriptases, the HIV-1 enzyme does not correct errors by exonucleolytic proofreading . Measurements with M13mp2-based fidelity assays indicated that the HIV-1 enzyme, isolated either from virus particles or from Escherichia coli cells infected with a plasmid expressing the cloned gene, was exceptionally inaccurate, having an average error rate per detectable nucleotide incorporated of 1/1700 . It was, in fact, the least accurate reverse transcriptase described to date, one-tenth as accurate as the polymerases isolated from avian myeloblastosis or murine leukemia viruses, which have average error rates of approximately 1/17,000 and approximately 1/30,000, respectively . DNA sequence analyses of mutations generated by HIV-1 polymerase showed that base substitution, addition, and deletion errors were all produced . Certain template positions were mutational hotspots where the error rate could be as high as 1 per 70 polymerized nucleotides . The data are consistent with the notion that the exceptional diversity of the HIV-1 genome results from error-prone reverse transcription.

Biochim Biophys Acta, 1988 Nov 23, 957(2), 222 - 9
Cd2+ activation of L-threonine dehydrogenase from Escherichia coli K-12; Craig PA et al.; Homogeneous preparations of L-threonine dehydrogenase (L-threonine:NAD+ oxidoreductase, EC 1.1.1.103) from Escherichia coli K-12, after having been dialyzed against buffers containing Chelex-100 resin, have a basal level of activity of 10-20 units/mg . Added Cd2+ stimulates dehydrogenase activity approx . 10-fold; this activation is concentration-dependent and is saturable with an activation Kd = 0.9 microM . Full activation by Cd2+ is obtained in the absence of added thiols . The pH-activity profile of the Cd2+-activated enzyme conforms to a theoretical curve for one-proton ionization with a pKa = 7.85 . Mn2+, the only other activating metal ion, competes with Cd2+ for the same binding site . Km values for L-threonine and NAD+ as well as the Vmax for 'demetallized', Cd2+-activated, and Mn2+-activated threonine dehydrogenase were determined and compared.

FEBS Lett, 1988 Nov 21, 240(1-2), 49 - 54
Production of a truncated human c-myc protein which binds to DNA; Naoe T et al.; Two kinds of truncated human c-myc proteins were produced in Escherichia coli . The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids . 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3 . One of the proteins (p42) produced in E . coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus . The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus . The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3 . We investigated the DNA-binding activity in p42 and p23 proteins . DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not . This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23 . This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.

FEBS Lett, 1988 Nov 21, 240(1-2), 115 - 7
Immunogenic protein MPB57 from Mycobacterium bovis BCG: molecular cloning, nucleotide sequence and expression; Yamaguchi R et al.; Using a single-probe method, we have cloned the gene for an immunogenic MPB57 protein of Mycobacterium bovis BCG . The nucleotide sequence includes an ORF of 300 base pairs encoding a protein of 99 amino acids with an Mr of 10,818 . This cloned gene was expressed in an Escherichia coli expression vector to give a mature protein which reacted with a polyclonal antibody raised against MPB57.

J Mol Biol, 1988 Nov 20, 204(2), 331 - 43
Unidirectional replication of plasmid R100; Miyazaki C et al.; We isolated a 284 base-pair BamHI fragment of plasmid R100 that supports initiation of replication of a plasmid regardless of the orientation of the fragment . Analysis of the specific radioactivity of restriction fragments from 32P-labeled replication intermediates synthesized in vitro shows that replication of the plasmid carrying the 284 base-pair fragment is unidirectional . The direction of replication depends on the orientation of the fragment present in the plasmid . The 5' ends of the leading-strand DNA formed in the early stage of replication were mapped to a region downstream from the 284 base-pair fragment in the direction of replication . The lagging-strand DNA products were also identified and their 3' ends mapped to unique sites within the 284 base-pair fragment causing unidirectional replication of R100.

J Mol Biol, 1988 Nov 20, 204(2), 345 - 56
Complementation of mutants of the stability locus of IncFII plasmid NR1 . Essential functions of the trans-acting stbA and stbB gene products; Min YN et al.; A series of unstable mutants of the stability (stb) locus of IncFII plasmid NR1 was subjected to a complementation analysis . The mutant collection included plasmids with point, insertion and deletion mutations in stb . These mutations affected the tandem genes stbA and stbB, which encode stability proteins StbA and StbB, or the PAB transcription promoter, which is located upstream from stbA in a region that contains an essential cis-acting site . Deletion mutants that lacked the region containing promoter PAB could not be complemented (stabilized) by providing StbA and StbB in trans . Deletion mutants that lacked stbA and stbB but retained the PAB region were complemented in trans but required both StbA and StbB, indicating that both proteins were essential for stable inheritance . stbA- point mutants were complemented in trans by either wild-type or stbA+ stbB- clones of the stability region . However, mutants with insertions in stbA were complemented only by wild-type clones, which suggested the insertions were polar on expression of the downstream stbB gene . A plasmid with a stbB- point mutation was complemented in trans by wild-type but not by stbA- stbB+ clones . In addition, plasmid clones that expressed StbB in the absence of StbA caused destabilization of (were incompatible with) stb+ derivatives of NR1 in trans, whereas clones that expressed only wild-type StbA or both StbA plus StbB did not . Plasmid clones that contained only the essential cis-acting PAB region did not cause destabilization of stb+ plasmids in trans . These results suggest that an excess of StbB protein provided in trans may cause a depletion of the essential StbA protein . Therefore, these results may be consistent with the hypothesis that StbB is an autorepressor of the stbAB operon.

J Mol Biol, 1988 Nov 20, 204(2), 283 - 94
The mechanism of loading of the FLP recombinase onto its DNA target sequence; Beatty LG et al.; The FLP recombinase interacts with its target sequence with the formation of three distinct DNA-protein complexes . The first complex leaves neither a DNase footprint nor is the DNA protected from methylation by dimethyl sulfate . We have found, however, that the FLP protein is bound predominantly to only one of the three 13 base-pair (bp) symmetry elements . This asymmetric loading of the FLP site seems to require the presence of an adjacent directly repeated 13 bp element . We speculate that this asymmetric filling of the target site may be accompanied by the unique order of cleavage and exchange of DNA strands.

J Mol Biol, 1988 Nov 20, 204(2), 447 - 81
Model for the three-dimensional folding of 16 S ribosomal RNA; Stern S et al.; We have derived a model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA, using interactive computer graphic methods . It is based on (1) the secondary structure derived from comparative sequence analysis, (2) the three-dimensional co-ordinates for the centers of mass of the 30 S subunit proteins, and (3) the locations of sites in 16 S rRNA that interact with specific ribosomal proteins, from footprinting and crosslinking studies . We present a detailed description of the derivation of the model . About 75% of the RNA chain is sufficiently constrained to provide a useful model . This contains most of the universally conserved core of the molecule . In all but a few instances, protected and crosslinked sites can be placed within or very close to their cognate proteins, while obeying stereochemical rules . The overall shape of the model and locations of specific regions of the RNA correspond well to data derived from electron micrographs of 30 S subunits, although such data were not used to construct the model . Phylogenetic variations in the structure are readily accommodated; as an example, we have modeled the 950-nucleotide mammalian mitochondrial 12 S rRNA by superimposing it on the E . coli structure . The three major RNA domains, as defined by secondary structure, appear to exist as autonomous structural units in three dimensions, for the most part . There is an extensive interface between the 5' and central domains, whereas the 3' major domain has relatively little apparent contact with the rest of the structure . The 5', central and 3' major domains form structures that resemble the body, platform and head, respectively, seen in electron micrographs of 30 S subunits . We discuss possible roles for the ribosomal proteins in stabilizing specific structural features of the RNA during ribosome assembly . The decoding site, as deduced from footprinting and crosslinking studies involving the tRNA anticodon stem-loop, is well-localized . Bases protected from chemical probing by the anticodon stem-loop line the cleft of the subunit . The conserved loop at position 530, which contains some of the bases protected by A site-bound tRNA, is remote (approx . 80 A) from the decoding site . Protection of these bases by the anticodon stem-loop is thus unlikely to be due to direct contact.

J Mol Biol, 1988 Nov 20, 204(2), 295 - 307
Interaction of Escherichia coli ribosomal protein S8 with its binding sites in ribosomal RNA and messenger RNA; Gregory RJ et al.; The ability of ribosomal protein S8 from Escherichia coli to interact with 12 variants of its 16 S rRNA binding site, as well as with a regulatory sequence within spc operon mRNA, has been assessed . Single-site alterations were introduced into the appropriate segment of the E . coli 16 S rRNA gene by mutagenesis in vitro . Their effects on S8-rRNA interaction were measured via a filter-binding assay, utilizing S8 binding sites transcribed in vitro from the altered 16 S rRNA gene fragments . Of the 12 rRNA mutants, six were unable to bind S8 . Significantly, five of these occur within a small, phylogenetically conserved internal loop, defined by nucleotides 596-597 and 641-643, suggesting that this structure plays a major role in S8-16 S rRNA recognition . The reduced affinity of S8 for its binding site in these cases was closely correlated with growth defects that resulted from expression of the same mutations in vivo . Alterations at other positions in the S8 binding site had little influence on complex formation or cell growth, as long as they did not disrupt rRNA secondary structure . The specific interaction of S8 with a segment of the spc operon mRNA containing a putative site of translational feedback regulation was demonstrated using appropriate in vitro transcripts in conjunction with the filter-binding assay . The apparent association constant for the S8-mRNA interaction was determined to be approximately 5 x 10(6) M-1, about five times lower than for the interaction of S8 with wild-type 16 S rRNA . The structure of the regulatory binding site, determined by sequence analysis of spc operon mRNA protected by S8 from RNase digestion, was found to contain all of the characteristic features of the 16 S rRNA binding site, demonstrating that the protein associates with structurally similar domains in both RNAs.

J Mol Biol, 1988 Nov 20, 204(2), 247 - 61
Effects of rifampicin resistant rpoB mutations on antitermination and interaction with nusA in Escherichia coli; Jin DJ et al.; Rifampicin resistant (Rifr mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase . We have used our collection of 17 sequenced Rifr mutations to investigate the involvement of E . coli RNA polymerase in the antitermination systems enhancing expression of delayed early lambda genes or stable RNA . We have found that Rifr mutations affect both lambda N-mediated antitermination and the cellular antitermination system involved in synthesis of stable RNA . Because NusA is involved in antitermination and termination, we also investigated the interaction of NusA and RNA polymerase by determining whether Rifr mutations alter NusA-dependent termination or antitermination in cells with defective nusA alleles . We have shown that Rifr mutations can either enhance or suppress the phenotypes of defective nusA alleles . Most Rifr mutations alter the temperature range over which the nusA1 allele supports lambda N-mediated antitermination . In addition, a number of Rifr alleles restore termination to the nusA10(Cs) and the nusA11(Ts) mutants defective in this process . Our results indicate that the region of the rpoB gene defined by the Rifr mutations is involved in the antitermination process and affects the activity of the NusA protein directly or indirectly.

Science, 1988 Nov 18, 242(4881), 1053 - 6
The presence of fibroblast growth factor in the frog egg: its role as a natural mesoderm inducer; Kimelman D et al.; A complementary DNA clone corresponding to a 4.2-kilobase transcript that is present in the Xenopus oocyte and newly transcribed in the neurula stages of development has been isolated . This messenger RNA encodes a 155-amino acid protein that is 84% identical to the human basic fibroblast growth factor (bFGF) . When expressed in Escherichia coli and purified, the Xenopus FGF induced mesoderm in animal cell blastomeres as measured by muscle actin expression . Immunoblots with an antibody to a Xenopus FGF peptide show that the oocyte and early embryo contain a store of the FGF polypeptide at high enough concentrations to induce mesoderm . The presence of FGF in the oocyte, together with the apparent lack of a secretory signal sequence in the protein, suggest that the regulation of mesoderm induction may involve novel mechanisms that occur after the translation of FGF.

J Chromatogr, 1988 Nov 18, 432, 137 - 51
Free-flow electrophoretic apparatus for separation and concentration of proteins; Shukun SA et al.; Protein separation in the free-flow electrophoretic apparatus with 48 channels at both the inlet and outlet using "artificial" pH gradients was investigated . The separation was carried out in borate-mannitol pH gradients and in pH gradients created by the concentration gradient of boric acid in the solutions of borax and mannitol . The separation pattern depended on the ratio of the rate of sample injection to the flow-rate of solutions in the separation chamber and did not change when the protein concentration in the sample was changed . The protein separation in the pH gradient was better than in case of conventional free-flow electrophoresis . The free-flow electrophoretic apparatus with multi-channel inlet of the separation chamber is suitable for concentration of biological materials, e.g . proteins and bacterial cells.

Nature, 1988 Nov 17, 336(6196), 265 - 6
The role of the distal histidine in myoglobin and haemoglobin; Olson JS et al.; The distal E7 histidine in vertebrate myoglobins and haemoglobins has been strongly conserved during evolution and is thought to be important in fine-tuning the ligand affinities of these proteins . A hydrogen bond between the N epsilon proton of the distal histidine and the second oxygen atom may stabilize O2 bound to the haem iron . The proximity of the imidazole side chain to the sixth coordination position, which is required for efficient hydrogen bonding, has been postulated to inhibit sterically the binding of CO and alkyl isocyanides . To test these ideas, engineered mutants of sperm whale myoglobin and the alpha- and beta-subunits of human haemoglobin were prepared in which E7 histidine was replaced by glycine . Removal of the distal imidazole in myoglobin and the alpha-subunits of intact, R-state haemoglobin caused significant changes in the affinity for oxygen, carbon monoxide and methyl isocyanide; in contrast, the His-E7 to Gly substitution produced little or no effect on the rates and extents of O2, CO and methyl isocyanide binding to beta-chains within R-state haemoglobin . In the beta-subunit the distal histidine seems to be less significant in regulating the binding of ligands to the haem iron in the high affinity quaternary conformation . Structural differences in the oxygen binding pockets shown by X-ray crystallographic studies account for the functional differences of these proteins.

Biochim Biophys Acta, 1988 Nov 17, 967(2), 326 - 30
The use of high field NMR to determine homogeneity in a preparation of human C5a produced by Escherichia coli; Daumy GO et al.; The polypeptide backbone of human C5a was prepared by recombinant DNA techniques . Standard biochemical analysis guided the protein separation to give a sample of C5a which was deemed homogeneous . However, nuclear magnetic resonance (NMR) studies showed the material to be significantly heterogeneous . Reanalysis by high performance liquid chromatography (HPLC) corroborated the NMR results . Further separation by HPLC and analysis by NMR spectroscopy guided the isolation of rC5a to greater than 92% purity . NMR analysis, immunochemical and biological evaluation of the impurities showed them to be C5a structural variants . These results indicate that conventional methods of protein chemistry can fail to reveal heterogeneity in recombinant proteins, and in some circumstances NMR spectroscopy can aid in their purification.

Nature, 1988 Nov 17, 336(6196), 254 - 7
Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein; Bochkareva ES et al.; It has been suggested that newly synthesized proteins are maintained in their unfolded state by cellular ATP-driven factors which may prevent or reverse the formation of misfolded structures or promote the correct assembly of oligomeric proteins or post-translational secretion . Using a photocross-linking approach, we have identified the 20S heat-shock GroEL protein as the major cytosolic component which forms a complex with the unfolded newly synthesized pre-beta-lactamase or chloramphenicol acetyltransferase in Escherichia coli . Dissociation of these complexes is ATP-dependent . The unfolded state of pre-beta-lactamase, maintained by the transient interaction with GroEL, may be essential for the secretion of this protein.

Anal Biochem, 1988 Nov 15, 175(1), 319 - 26
The analysis of recombinant interleukin-2 by thermospray liquid chromatography-mass spectrometry; Blackstock WP et al.; The complete sequence of recombinant human interleukin-2 expressed in Escherichia coli has been confirmed by thermospray liquid chromatography-mass spectrometry (TS-LC-MS) of a tryptic digest derived from 100 micrograms (7 nmol) of reduced carboxymethylated interleukin-2 . The preparation was shown by this method to contain predominantly unprocessed N-terminal initiator Met, with some authentic N-terminal Ala; the rest of the protein was as predicted from the DNA sequence, though some deamidated material was noted . TS-LC-MS proved to be a rapid and efficient method for surveying the protein tryptic peptide products allowing all the data to be collected in one chromatographic run; all tryptic fragments were identified by their molecular ions including those for the larger peptides (Mr 1500-3500) which, due to the presence of doubly and triply charged molecular ions, were brought within the mass range of the instrument (1800 Da) . It is proposed that TS-LC-MS is a good general method for analyzing recombinant protein digests with respect to sequence confirmation, processing, and post-translational modification, and since each chromatographic peak is identified allows for subsequent monitoring of the protein by LC using uv detection . The method suffers from the disadvantages that all the sample is consumed during the experiment and that no fragment (sequence) ions are generally observed.

J Immunol, 1988 Nov 15, 141(10), 3523 - 31
The physical-chemical characterization and biologic activity of serum released lipopolysaccharides; Tesh VL et al.; As a result of the incubation of Escherichia coli in normal human serum, a finite fraction of LPS is released from the bacterial membrane . Approximately half of the LPS released by the action of serum (S-LPS) exists in association with serum proteins in a lower m.w . form than that manifest in phenol-water extracted LPS preparations . The two major LPS-serum protein complexes have apparent Mr of 68 and 32 kDa . The LPS subunit heterogeneity of S-LPS, however, does not appear to differ significantly from LPS retained on the bacteria after serum treatment, or from LPS derived by lysis of whole cells . The biologic activities of S-LPS and phenol-water extracted LPS examined in these studies, differed significantly . In contrast to phenol-water extracted LPS, S-LPS was 1) reduced in lethal toxicity for sensitized mice; 2) reduced in Limulus reactivity; 3) a more potent murine splenocyte mitogen; 4) reduced in the capacity to elicit extracellular, but not membrane-associated IL-1; and 5) reduced in the ability to mediate TNF production . These data suggest that humoral "detoxification" of LPS may involve, in part, the formation of LPS-serum protein complexes with reduced capacities to elicit extracellular cytokine production, whereas the immunomodulatory effects of LPS appear to be enhanced.

Gene, 1988 Nov 15, 71(1), 29 - 40
An alternative approach in gene synthesis: use of long selfpriming oligodeoxynucleotides for the construction of double-stranded DNA; Uhlmann E; A novel approach for the synthesis of double-stranded DNA fragments from only one long oligodeoxynucleotide (oligo) is presented . The basic strategy is to use oligos which possess a short inverted repeat at their 3' end resulting in the formation of a hairpin structure . The 3' end of this hairpin then serves as a primer in the Klenow (large) fragment of E . coli DNA polymerase I-mediated synthesis of the second DNA strand . Removal of the loop structure as well as generation of sticky ends for subsequent cloning is achieved by digestion with restriction enzymes . Several oligos ranging in size from 130 to 147 nt were synthesized and successfully used in the cloning of gene fragments of up to 120 bp in length . Furthermore, a strategy for the simultaneous cloning of two synthetic DNA fragments is outlined yielding even larger gene fragments . By sequential cloning of these gene fragments the methodology presented here will allow the synthesis of genes of any size . The proposed methodology should also be useful for site-directed mutagenesis as well as saturation mutagenesis.

Gene, 1988 Nov 15, 71(1), 19 - 27
Mulcos: a vector for amplification and simultaneous expression of two foreign genes in mammalian cells; Ikeda H et al.; A method was developed for amplification and expression of foreign genes in mammalian cells . This procedure exploits the fact that an SfiI cleavage site, GGCCGCCT/CGGCC (the recognition sequences are underlined), is present at the SV40 replication origin and the cleaved ends, CCT-3' and AGG-3', are not rotationally equivalent . Thus DNA fragments flanked by the SfiI sites can be ligated in head-to-tail tandem arrays and cloned in cosmids; the resulting construct is called a mulcos . The cosmid vector we have used, pCHD2L, contains the single SfiI site as well as HmBR and dhfr genes, selectable markers in mammalian cells . Cassette plasmid pmoRH contains two expression units, each of which consists of SV40 early promoter, EcoRI or HindIII cloning site, small T splicing region, and poly(A) signal, and the two units as a whole are flanked by the SfiI sites . A set of alpha- and beta-chain cDNAs of a human major histocompatibility class-II antigen were inserted into the EcoRI and HindIII sites, respectively . The purified SfiI fragment, containing both expression units, was then ligated with SfiI-linearized cosmid vector pCHD2L at a molar ratio of 20:1 . A mulcos containing eight pairs of the alpha- and beta-chain expression units was isolated by in vitro packaging in phage lambda heads and subsequent transfection into Escherichia coli . Drug-selected cells transfected with the mulcos contained significantly higher copy numbers of the expression units and higher expression levels than those obtained using conventional plasmids . More than 85% of these cells expressed class-II antigen on their cell surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Nov 15, 27(23), 8653 - 60
Function of arginine-234 and aspartic acid-271 in domain closure, cooperativity, and catalysis in Escherichia coli aspartate transcarbamylase; Middleton SA et al.; Two mutant versions of Escherichia coli aspartate transcarbamylase were created by site-specific mutagenesis . Arg-234 of the 240s loop was replaced by serine in order to help deduce the function of the interactions that normally occur between Arg-234 and both Glu-50 and Gln-231 in the R state of the enzyme . The other mutation involved the replacement of Asp-271 by asparagine to further test the functional importance of the Tyr-240-Asp-271 link that has previously been proposed to stabilize the T state of the enzyme {Middleton, S . A., & Kantrowitz, E . R . (1986) Proc . Natl . Acad . Sci . U.S.A . 83, 5866-5870} . The Arg-234----Ser holoenzyme exhibits no cooperativity, a 24-fold reduction in maximal velocity, normal affinity for carbamyl phosphate, and substantially reduced affinity for aspartate and N-(phosphonoacetyl)-L-aspartate (PALA) . Unlike the wild-type enzyme, the heterotropic effectors ATP and CTP are able to influence the activity of the Arg-234----Ser enzyme at saturating aspartate concentrations . The Arg-234----Ser catalytic subunit exhibits a 33-fold reduction in maximal activity, an aspartate Km of 261 mM, compared to 5.7 mM for the wild-type catalytic subunit, and only a small alteration in the Km for carbamyl phosphate . Together these results provide additional evidence that the interdomain bridging interactions between Glu-50 of the carbamyl phosphate domain and both Arg-167 and Arg-234 of the aspartate domain are necessary for the stabilization of the high-activity-high-affinity configuration of the active site of the enzyme . Furthermore, without the interdomain bridging interactions, the holoenzyme no longer exhibits homotropic cooperativity.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem . 1988 Nov 15;263(32):16531.
Crystallization of methionine aminopeptidase from Escherichia coli; Roderick SL et al.; Crystals of methionine aminopeptidase from Escherichia coli have been obtained . The crystals are of space group P2(1) with unit cell dimensions a = 39.3 A, b = 62.6 A, c = 54.3 A, beta = 107.8 degrees and diffract to 2.1-A resolution . They contain one polypeptide chain in the asymmetric unit and are suitable for a high resolution crystallographic study.

Biochemistry, 1988 Nov 15, 27(23), 8661 - 9
Detection and identification of transient intermediates in the reactions of tryptophan synthase with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan . Evidence for a tetrahedral (gem-diamine) intermediate; Roy M et al.; The reactions of 2,3-dihydro-L-tryptophan (DHT) and oxindolyl-L-alanine (OXA) with tryptophan synthase have been investigated by rapid-scanning stopped-flow (RSSF) spectroscopy and by the concentration dependence of rates measured by single-wavelength stopped-flow (SWSF) spectroscopy . The RSSF spectral changes for DHT and OXA show the disappearance of the internal aldimine (lambda max 412 nm), the formation and decay of intermediates absorbing less than or equal to 340 nm, and the appearance of the quinonoid (lambda max 492 and 480 nm, respectively) . Rate constants determined by SWSF were either well resolved (i.e., k1{DHT}, k-1 greater than k2, k-2 greater than k3, k-3) or indicative of a tightly coupled system (i.e., k1{OXA}, k-1 greater than or equal to k2, k-2 greater than k3, k-3) . The RSSF spectral changes and SWSF kinetic studies together with computer simulations of the kinetic time courses are consistent with a mechanism that includes formation of a bleached species . Detection of these shorter wavelength species in the reactions of OXA and DHT indicates that substrate analogues with tetrahedral geometry at C-3 induce new protein-substrate interactions that result in the accumulation of species not previously detected in the tryptophan synthase system . The bleached species with lambda max less than or equal to 340 nm are proposed as the gem-diamine intermediates.

Gene, 1988 Nov 15, 71(1), 9 - 18
Tandem gene amplification in vitro for rapid and efficient expression in animal cells; Takeshita S et al.; Plasmid vectors pHSG293 and pHSG747, suitable for in vitro gene amplification for subsequent animal-cell expression, were developed . A cosmid vector pHSG293 confers Km resistance to Escherichia coli host cells and G418 resistance to animal cells and contains a single BstXI recognition/cleavage site, CCACGGGG/CTGG, near the cos site (the recognition site is underlined) . The cassette vector plasmid pHSG747 contains a multiple cloning site (MCS) between the simian virus 40 early promoter and the poly(A) signal sequence flanked by the same BstXI sites and confers Cm resistance to E . coli host cells . After inserting a coding fragment for human protein C or its derivative in the appropriate orientation in the MCS of pHSG747, the BstXI expression unit fragment was purified, mixed with BstXI-digested pHSG293 DNA at a molecular ratio of 20 to 40:1 and ligated . This allowed for tandem gene amplification due to asymmetric cohesive ends . Ligation products were packaged in lambda phage particles, amplified in E . coli cells as large cosmid molecules, and then introduced into CHO cells . G418R transformants were found to produce and secrete recombinant protein molecules at a high level . The plasmid vectors developed in this work will provide a rapid screening system useful for protein engineering in animal cells.

Gene, 1988 Nov 15, 71(1), 49 - 56
Enrichment for 5'-TG termini: a method for subcloning structural genes into expression vectors; D'Alessio JM et al.; We describe a method for creating a population of randomly digested, blunt-ended DNA fragments with 5'-TG.. . 3'-AC.. . (TG) at their termini . When these fragments were ligated to a blunt-ended vector that contains ...CATA-3' ...GTAT-5' at its termini, as high as 84% of the clones obtained after transformation contained plasmids with a reconstructed Nde I site .. . CATATG.. . ...GTATAC... . When the DNA vector is prepared from an appropriate plasmid {Gross et al., Mol . Cell . Biol . 5 (1985) 1015-1024; Kotewicz et al., Gene 35 (1985) 249-258}, the ATG within the restriction site corresponds to a start codon positioned downstream from a strong ribosome-binding site and controllable promoter . If a gene has been digested to the TG contained within its authentic initiation codon, the endogenous translation-initiation control sequences are deleted and expression can be controlled using the plasmid-derived promoter . In addition, a gene digested to other in-frame TGs can potentially express proteins with altered N termini . Using this method, we have placed the structural gene of SP6 RNA polymerase, trimmed precisely to its authentic start codon, under the control of the tac promoter.

Gene, 1988 Nov 15, 71(1), 1 - 8
Amplification of cloned DNA as tandem multimers using BspMI-generated asymmetric cohesive ends; Kim SC et al.; By generating totally asymmetric and complementary cohesive ends it is possible to amplify any cloned DNA fragment, while assuring that all repeating units are ligated in the same orientation . Starting with plasmid pUC18, which contains a unique BspMI site, an amplification plasmid, pSK3, was constructed in which a multiple cloning site (MCS) is flanked by two BspMI recognition sites with identical cut sites, creating the complementary 5'-ATGC and 5'-GCAT single-stranded ends . Any DNA fragment cloned into the MCS could be amplified by (i) excision with BspMI, (ii) fragment isolation, (iii) self-ligation of the fragments using T4 DNA ligase, (iv) selection of multimers of desired length, and (v) cloning them into the BspMI-digested original plasmid . Using this procedure, plasmids carrying either 30 copies of the 60-bp MCS fragment (a control experiment) or ten copies of the 1.2-kb luxA gene fragment were constructed . The plasmids were stable since all the repeat units were in the same orientation, as determined by restriction analysis . Potentially, not only BspMI but other class-IIS restriction enzymes (with recognition sites separated by a fixed distance from the staggered cut points) could be applied, preferably those that create 4-to-5-nucleotide-long cohesive ends and utilize rather rare recognition sites.

Arch Biochem Biophys, 1988 Nov 15, 267(1), 110 - 8
Purification and properties of the catalytic domain of human 3-hydroxy-3-methylglutaryl-CoA reductase expressed in Escherichia coli; Mayer RJ et al.; Three fragments of the cDNA encoding human 3-hydroxy-3-methylglutaryl-CoA reductase, all incorporating the majority of the catalytic domain of the protein, were subcloned into Escherichia coli expression vectors containing the pL promoter . The two larger expressed fragments (58 and 52 kDa) were soluble and had enzymatic activity, while the smallest (48 kDa) was insoluble . The two active fragments were purified by a combination of conventional techniques and affinity chromatography . A number of properties of the two enzymes were compared including specific activity, kinetic parameters, relative solubility, and cold lability . The 52-kDa enzyme was observed to change from a dimeric to monomeric form and to lose activity at 4 degrees C . In contrast, the 58-kDa enzyme was found to be much less cold labile, and was dimeric at both 20 and 4 degrees C . In order to resolve the number of subunits required to form an active site, the number of inhibitor binding sites for a known inhibitor was determined to be one per subunit in the 58-kDa enzyme.

J Biol Chem, 1988 Nov 15, 263(32), 16934 - 41
The glycine-rich region of Escherichia coli D-serine dehydratase . Altered interactions with pyridoxal 5'-phosphate produced by substitution of aspartic acid for glycine; Marceau M et al.; Replacement of glycine by aspartic acid at either of two sites in a conserved, glycine-rich region inactivates the pyridoxal 5'-phosphate-dependent enzyme D-serine dehydratase (DSD) from Escherichia coli . To investigate why aspartic acid at position 279 or 281 causes a loss of activity, we measured the affinity of the G----D variants for pyridoxal 5'-phosphate and a cofactor:substrate analog complex and compared the UV, CD, and fluorescence properties of wild-type D-serine dehydratase and the inactive variants . The two G----D variants DSD(G279D) and DSD (G281D) displayed marked differences from wild-type D-serine dehydratase and from each other with respect to their affinity for pyridoxal 5'-phosphate and for a pyridoxal 5'-phosphate:glycine Schiff base . Compared to the wild-type enzyme, the cofactor affinity of DSD(G279D) and DSD(G281D) was decreased 225- and 50-fold, respectively, and the ability to retain a cofactor:glycine complex was decreased 765- and 1970-fold . The spectral properties of the inactive variants suggest that they form a Schiff base linkage with pyridoxal 5'-phosphate but do not hold the cofactor in a catalytically competent orientation . Moreover, the amount of cofactor aldamine in equilibrium with cofactor Schiff base is increased in DSD(G279D) and DSD(G281D) relative to that in wild-type DSD . Collectively, our findings indicate that introduction of a carboxymethyl side chain at G-279 or G-281 directly or indirectly disrupts catalytically essential protein-cofactor and protein-substrate interactions and thereby prevents processing of the enzyme bound cofactor:substrate complex . The conserved glycine-rich region is thus either an integral part of the D-serine dehydratase active site or conformationally linked to it.

J Biol Chem, 1988 Nov 15, 263(32), 16926 - 33
D-serine dehydratase from Escherichia coli . DNA sequence and identification of catalytically inactive glycine to aspartic acid variants; Marceau M et al.; We have identified two glycyl residues whose integrity is essential for the catalytic competence of a model pyridoxal 5'-phosphate requiring enzyme, D-serine dehydratase from Escherichia coli . This was accomplished by isolating and sequencing the structural gene from wild type E . coli and from two mutant strains that produce inactive D-serine dehydratase . DNA sequencing indicated the presence of a single glycine to aspartic acid replacement in each variant . The amino acid replacements lie in a glycine-rich region of D-serine dehydratase well removed from pyridoxal 5'-phosphate-binding lysine 118 in the primary structure of the enzyme . The striking effect of these two glycine to aspartic acid replacements on catalytic activity, the conservation of the glycine-rich region in several pyridoxal 5'-phosphate-dependent enzymes that catalyze alpha/beta-eliminations, and the placement of similar glycine-rich sequences in well-characterized active site structures suggest that the glycine-rich region interacts with the cofactor at the active site of the enzyme.

J Biol Chem, 1988 Nov 15, 263(32), 16553 - 60
Analysis of sequential steps of nucleotide excision repair in Escherichia coli using synthetic substrates containing single psoralen adducts; Van Houten B et al.; Escherichia coli ABC excinuclease initiates the removal of dodecanucleotides from damaged DNA in an ATP-dependent reaction . Using a synthetic DNA fragment containing a psoralen adduct at a defined position we have investigated the interaction of the components of the enzyme with substrate by DNase I footprinting . We find that the UvrA subunit binds to DNA specifically in the absence of cofactors and that the binding affinity is stimulated about 4-fold by ATP and only marginally inhibited by ADP . The UvrA.DNA complexes formed in the absence of co-factors or in the presence of either ATP or ADP are remarkably similar . In contrast, adenosine 5'-O-(thiotriphosphate) increases nonspecific binding and completely abolishes the UvrA footprint . The UvrB subunit can associate with the UvrA subunit on DNA in the absence of ATP, but this ternary UvrA.UvrB.DNA complex is qualitatively different from that formed in the presence of ATP . The UvrC subunit elicits no additional change in the UvrA-UvrB footprint . Helicase II (UvrD protein) does not alter the UvrA-UvrB footprint but does appear to interact at the 5'-incision site of the postincision complex . DNA polymerase I fills in the excision gap in the presence or absence of helicase II and apparently releases the ABC excinuclease from the repaired DNA . Nearly 90% of the repair patches are 12 nucleotides long, and this length is not affected by helicase II . We see no evidence by DNase I footprinting for the formation of a multiprotein complex encompassing the UvrA, -B, -C, and -D proteins and DNA polymerase I.

J Biol Chem, 1988 Nov 15, 263(32), 16527 - 30
Evidence for interaction of an aminoacyl transfer RNA synthetase with a region important for the identity of its cognate transfer RNA; Park SJ et al.; Recent experiments showed that a single base pair (G3:U70) in the amino acid acceptor helix is a major determinant for the identity of Escherichia coli alanine transfer RNA . Experiments reported here show that bound alanine tRNA synthetase protects (from ribonuclease attack) seven consecutive phosphodiester linkages on the 3'-side of the acceptor-T psi C helix (phosphates 65-71) and a few additional sites that are in scattered locations . There is no evidence for interaction of the enzyme with the anticodon, a sequence which can be varied without effect on recognition by alanine tRNA synthetase.

Anal Biochem, 1988 Nov 15, 175(1), 196 - 201
Filter-supported preparation of lambda phage DNA; Jakobi R et al.; A rapid and simple method is described for the isolation of DNA from phage lambda which requires neither special equipment nor expensive material such as cesium chloride for ultracentrifugation nor extractions with organic solvents or ethanol precipitation . Microgram quantities of lambda DNA are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures . The method allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries . Lambda phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed centrifugation onto glass fiber filters positioned in disposable syringes . The DNA of phages is released by a 50% formamide/4 M sodium perchlorate solution, washed in filter-bound form, eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation . Comparison of the DNA isolated by this method with that obtained by two conventional procedures reveals both a similar recovery and a similar suitability for restriction enzyme digestion and subcloning.

Gene, 1988 Nov 15, 71(1), 201 - 5
Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome; Chalker AF et al.; Recombinant DNA libraries generated in vitro should in theory contain all of the sequences of the genomes from which they are derived . However, the literature is dotted with reports of sequences that cannot be recovered, are under-represented, or are highly unstable . In particular, long palindromic nucleotide sequences of perfect or near-perfect symmetry are either lethal to the vector or suffer deletions or other rearrangements that remove symmetry {Collins, Cold Spring Harbor Symp . Quant . Biol . 45 (1981) 409-416; Collins et al., Gene 19 (1982) 139-146; Hagan and Warren, Gene 24 (1983) 317-326} . We report here that mutation of a single gene, namely sbcC, can overcome this inviability and allow for the stable propagation of a 571-bp nearly perfect palindrome in Escherichia coli . This has implications for the choice of strains used for the recovery and analysis of cloned nucleotide sequences.

Carbohydr Res, 1988 Nov 15, 183(1), 111 - 22
Escherichia coli serotype K44: an acidic capsular polysaccharide containing two 2-acetamido-2-deoxyhexoses; Dutton GG et al.; The structure of the capsular polysaccharide from Escherichia coli O8:K44 (A):H- (K44 antigen) has been established using the techniques of methylation, beta-elimination, deamination, and Smith degradation . N.m.r . spectroscopy (13C and 1H) was used extensively to establish the nature of the anomeric linkages of the polysaccharide and of oligosaccharides derived through degradative procedures . The K antigen is comprised of repeating units of the linear tetrasaccharide shown . This acidic polysaccharide represents the first instance of an E . coli K antigen in this series (group A) that has been found to contain two different 2-acetamido-2-deoxyhexoses.

Biochemistry, 1988 Nov 15, 27(23), 8554 - 61
Construction and characterization of mutant iso-2-cytochromes c with replacement of conserved prolines; Wood LC et al.; Oligonucleotide-directed mutagenesis has been used to construct two mutant forms of iso-2-cytochrome c . In one, Pro-30 is replaced by threonine; in the other, Pro-76 is replaced by glycine . Both prolines are fully conserved among mitochondrial cytochromes c and play important structural and functional roles . Yeast with either the Pro-30 or the Gly-76 mutation has appreciable levels of mutant protein in vivo and grows on media containing nonfermentable carbon sources . Thus, neither mutation blocks protein targeting to mitochondria, uptake by mitochondria, covalent attachment of heme, or in vivo function . As judged by ultraviolet-visible spectrophotometry and proton nuclear magnetic resonance spectroscopy, the nativelike conformation of purified Gly-76 iso-2 at pH 6 is almost indistinguishable from that of the normal protein at pH 6 . Ultraviolet second-derivative spectrophotometry, however, suggests an increase in the average number of exposed tyrosine side chains, with 2.25 out of 5 residues exposed for the mutant compared to 1.95 for normal iso-2 . Above neutral pH, the protein folds to a mutant conformation possibly related to alkaline cytochrome c . Nuclear Overhauser difference spectroscopy of the reduced nativelike conformation allows assignment of several proton resonances and comparison of side-chain conformations of the heme ligand Met-80 in the mutant and the normal proteins . The proton chemical shifts for the assigned resonances are the same within errors for Gly-76 iso-2 and normal iso-2 at pD 6, 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Biochem Biophys, 1988 Nov 15, 267(1), 280 - 4
Diphosphonates are potent inhibitors of mammalian inorganic pyrophosphatase; Smirnova IN et al.; Methanediphosphonate and 12 analogs thereof with different substituents at the carbon atom are potent competitive inhibitors of highly purified rat liver and bovine heart inorganic pyrophosphatases . The inhibition constants for the most effective diphosphonates, which contain an NH2 or OH group at the bridge carbon atom, are in the micromolar range . Yeast and Escherichia coli pyrophosphatases are markedly less sensitive to the diphosphonates . Pyrophosphatase inhibition may be related to the numerous biological effects exerted by diphosphonates.

J Biol Chem, 1988 Nov 15, 263(32), 16652 - 60
The Tsr chemosensory transducer of Escherichia coli assembles into the cytoplasmic membrane via a SecA-dependent process; Gebert JF et al.; The Tsr protein of Escherichia coli is a chemosensory transducer that mediates taxis toward serine and away from certain repellents . Like other bacterial transducers, Tsr spans the cytoplasmic membrane twice, forming a periplasmic domain of about 150 amino acids and a cytoplasmic domain of about 300 amino acids . The 32 N-terminal amino acids of Tsr resemble the consensus signal sequence of secreted proteins, but they are not removed from the mature protein . To investigate the function of this N-terminal sequence in the assembly process, we isolated translational fusions between tsr and the phoA and lacZ genes, which code for the periplasmic enzyme alkaline phosphatase and the cytoplasmic enzyme beta-galactosidase, respectively . All tsr-phoA fusions isolated code for proteins whose fusion joints are within the periplasmic loop of Tsr, and all of these hybrid proteins have high alkaline phosphatase activity . The most N-terminal fusion joint is at amino acid 19 of Tsr . Tsr-lacZ fusions were found throughout the tsr gene . The beta-galactosidase activity of the LacZ-fusion proteins varies greatly, depending on the location of the fusion joint . Fusions with low activity have fusion joints within the periplasmic loop of Tsr . The expression of these fusions is most likely reduced at the level of translation . In addition, one of these fusions markedly reduces the export and processing of the periplasmic maltose-binding protein and the outer membrane protein OmpA, but not of intact PhoA or of the outer membrane protein LamB . A temperature-sensitive secA mutation, causing defective protein secretion, stops expression of new alkaline phosphatase activity coded by a tsr-phoA fusion upon shifting to the nonpermissive temperature . The same secA mutation, even at the permissive temperature, increases the activity and the level of expression of LacZ fused to the periplasmic loop of Tsr relative to a secA+ strain . We conclude that the assembly of Tsr into the cytoplasmic membrane is mediated by the machinery responsible for the secretion of a subset of periplasmic and outer membrane proteins . Moreover, assembly of the Tsr protein seems to be closely coupled to its synthesis.

Anal Biochem, 1988 Nov 15, 175(1), 327 - 33
Separation of formyl-methionyl transfer RNA, methionyl transfer RNA, and transfer RNAfmet using mixed-mode high-performance liquid chromatography on C6-modified aminopropylsilyl-hypersil; Ricker RD et al.; Preparative amounts of formyl-methionyl-tRNAfmet, methionyl-tRNAfmet, and tRNAfmet were separated from each other with baseline resolution in 30 min using mixed-mode HPLC on hexanoic anhydride-modified aminopropylsilyl-Hypersil 2 . Pure tRNAfmet was aminoacylated with {35S}methionine in the presence or absence of a formyl donor and was immediately fractionated on the column . Two isoacceptors, tRNA1fmet and tRNA2fmet, as well as aminoacyl-tRNA synthetases were clearly separated from each other . The purified f{35S}-methionyl-tRNA was biologically active in that as much as 98% could be bound to ribosomes in response to AUGUAA in vitro . Formyl-methionine was released from this complex by the action of termination factor and greater than 92% of bound formyl-methionine was released by puromycin.

Biochem J, 1988 Nov 15, 256(1), 213 - 8
Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF); Wingfield P et al.; Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110 . About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20% . The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75 . Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity . Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities . In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.

Eur J Biochem, 1988 Nov 15, 177(3), 467 - 75
Purification of transfer RNA (m5U54)-methyltransferase from Escherichia coli . Association with RNA; Ny T et al.; tRNA (m5U54)-methyltransferase (EC 2.1.1.35) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to transfer ribonucleic acid (tRNA) and thereby forming 5-methyluridine (m5U, ribosylthymine) in position 54 of tRNA . This enzyme, which is involved in the biosynthesis of all tRNA chains in Escherichia coli, was purified 5800-fold . A hybrid plasmid carrying trmA, the structural gene for tRNA (m5U54)-methyltransferase was used to amplify genetically the production of this enzyme 40-fold . The purest fraction contained three polypeptides of 42 kDa, 41 kDa and 32 kDa and a heterogeneous 48-57-kDa RNA-protein complex . All the polypeptides seem to be related to the 42/41-kDa polypeptides previously identified as the tRNA (m5U54)-methyltransferase . RNA comprises about 50% (by mass) of the complex . The RNA seems not to be essential for the methylation activity, but may increase the activity of the enzyme . The amino acid composition is presented and the N-terminal sequence of the 42-kDa polypeptide was found to be: Met-Thr-Pro-Glu-His-Leu-Pro-Thr-Glu-Gln-Tyr-Glu-Ala-Gln-Leu-Ala-Glu-Lys- . The tRNA (m5U54)-methyltransferase has a pI of 4.7 and a pH optimum of 8.0 . The enzyme does not require added cations but is stimulated by Mg2+ . The apparent Km for tRNA and S-adenosyl-L-methionine are 80 nM and 17 microM, respectively.

J Chromatogr, 1988 Nov 11, 454, 205 - 15
Structural characterization of recombinant consensus interferon-alpha; Klein ML et al.; Recombinant consensus interferon-alpha is derived from genetically modified Escherichia coli containing a synthetic gene constructed from a consensus of interferon sequences . The purified and biologically active protein has been subjected to detailed structural characterization including sequence determination and peptide isolation and identification . The homogeneous consensus interferon-alpha preparation contains two chromatographically indistinguishable homologous polypeptides with one containing an extra methionyl residue at the amino terminus . The delineated amino acid sequence of the protein is identical to that expected from the coding sequence of the gene . Correct oxidation of the molecule has been confirmed with two intramolecular disulfide linkages observed at Cys(1)-Cys(99) and Cys(29)-Cys(139).

Nucleic Acids Res, 1988 Nov 11, 16(21), 10099 - 108
Zone-interference gel electrophoresis: a new method for studying weak protein-nucleic acid complexes under native equilibrium conditions; Abrahams JP et al.; A new and general electrophoresis method is described for the determination of dissociation constants of weak macromolecular complexes in the range of 10(-6) to 10(-4) M . The method is based on the measurement of the migration distance of a macromolecular complex in rapid dynamic equilibrium as a function of the interacting ligand concentration in a surrounding zone . Special advantages of the method are: its high sensitivity (dependent on the autoradiography, immunoblotting or staining technique used), its speed (electrophoresis time 20 min), and the independence of the Kd determination on the sample concentration of macromolecules . The latter is of great value for labile macromolecules: unknown partial inactivation does not influence the measurement . Studying the interactions between elongation factor EF-Tu and tRNA from E . coli we found for EF-Tu.GTP.aurodox.aminocyl-tRNA a Kd of 3 microM and for EF-Tu.GDP.aurodox.aminoacyl-tRNA a Kd of 11 microM at 9 degrees C.

Nucleic Acids Res, 1988 Nov 11, 16(21), 9961 - 77
Mutagenesis induced by site specifically placed 4'-hydroxymethyl-4,5',8-trimethylpsoralen adducts; Piette J et al.; Closed circular double stranded M13mp19 DNA containing a site-specifically placed HMT (4'-hydroxymethyl-4-5'-8-trimethylpsoralen) monoadduct or crosslink was synthesized in vitro . The damaged DNA were scored for loss of infectivity by transfection into repair proficient or deficient E . coli and into SOS induced E . coli . Mutant phages were detected by the loss of alpha-complementation between the viral and the host Lac Z genes or by the acquisition of resistance to kpn I digestion . Our results indicate that HMT mutagenesis is targeted and that deletion or transversion of the modified thymidine is the predominant sequence change elicited by a monoadduct or a crosslink . Transfection of the monoadducted DNA into a Uvr A deficient strain did not change the mutation pattern but did increase the respective mutation frequencies . Transfection of the crosslinked DNA into a SOS induced host resulted in the appearence of other types of mutations attributable to an increase in both targeted and untargeted mutations.

Nucleic Acids Res, 1988 Nov 11, 16(21), 10053 - 67
Sequence analysis and regulation of the htrA gene of Escherichia coli: a sigma 32-independent mechanism of heat-inducible transcription; Lipinska B et al.; Previous work has established that the E . coli htrA gene product is essential for bacterial survival at temperatures above 42 degrees . We have sequenced the htrA gene region and found an open reading frame (ORF) coding for a protein of 491 amino acids with a calculated molecular weight of 51,163 daltons . This molecular weight corresponds well with that seen following electrophoresis on SDS-polyacrylamide gels . This protein has an amino-terminal sequence typical for a leader peptide and undergoes post-translational modification by cleavage of an amino-terminal portion . The insertional mutations which affect the function of the htrA gene map inside this ORF . The levels of htrA mRNA increase rapidly and transiently upon heat shock in a manner independent of the rpoH gene, which encodes the sigma 32 RNA polymerase subunit and is known to regulate transcription of typical heat shock genes . Using S1 mapping and RNA primer extension, we have identified the htrA promoter and found that it is similar to the P3 promoter of the rpoH gene . The P3 promoter is especially active at high temperatures and is recognized by a recently identified transcriptional factor, sigma E.

Biochim Biophys Acta, 1988 Nov 10, 951(1), 230 - 4
Expression of the mouse metallothionein-I gene in Escherichia coli: increased tolerance to heavy metals; Hou YM et al.; The cDNA of mouse metallothionein, a small metal-binding protein rich in cysteine, has been cloned downstream from a bacterial inducible promoter and expressed in Escherichia coli . Upon induction, E . coli harboring this cDNA clone contained a protein species readily labelled by {35S}cysteine in vivo and incorporated 10-times as much 109Cd from the medium than would otherwise be the case . We show that expression of metallothionein endows resistance in E . coli to heavy metal ions such as mercury, silver, copper, cadmium and zinc by sequestering rather than exclusion or conversion, common mechanisms of metal resistance in bacteria.

Nature, 1988 Nov 10, 336(6195), 179 - 81
Codon and amino-acid specificities of a transfer RNA are both converted by a single post-transcriptional modification; Muramatsu T et al.; An Escherichia coli isoleucine transfer RNA specific for the codon AUA (tRNA(2Ile) or tRNA(minorIle} has a novel modified nucleoside, lysidine in the first position of the anticodon (position 34), which is essential for the specific recognition of the codon AUA . We isolated the gene for tRNA(2Ile) (ileX) and found that the anticodon is CAT, which is characteristic of the methionine tRNA gene . Replacement of L(34) of tRNA(2Ile) molecule enzymatically with unmodified C(34) resulted in a marked reduction of the isoleucine-accepting activity and, surprisingly, in the appearance of methionine-accepting activity . Thus, both the codon and amino-acid specificity of this tRNA are converted by a single post-transcriptional modification of the first position of the anticodon during tRNA maturation.

Biochim Biophys Acta, 1988 Nov 10, 951(1), 78 - 84
Mapping of the monoclonal antibody 80L5C4 epitope on the cell adhesion molecule gp80 of Dictyostelium discoideum; Kamboj RK et al.; EDTA-resistant cell-cell binding sites are expressed on Dictyostelium discoideum cells at the aggregation stage of development . A cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate these binding sites via homophilic interaction . We have previously raised a monospecific monoclonal antibody 80L5C4 against gp80, which blocks the cell binding site of gp80 (Siu, C.-H., Lam, T.Y . and Choi, A.H.C . (1985) J . Biol . Chem . 260, 16030-16036) . To map the 80L5C4 epitope, gp80 was digested with protease V8, and the smallest proteolytic fragment that retained immunoreactivity with 80L5C4 was about 27,000 Da, corresponding to the amino-terminal fragment predicted from the cleavage sites . In addition, cDNA fragments containing different gp80 coding regions were used to construct trpE/gp80 gene fusions in the expression vector pATH10 . An analysis of these fusion proteins led to the mapping of the 80L5C4 epitope to a 51 amino-acid segment between residues 123 and 173.

FEBS Lett, 1988 Nov 7, 239(2), 299 - 304
Recombinant interferon-beta 2 (interleukin-6) induces myeloid differentiation; Chen L et al.; Human IFN-beta 2 cytokine produced in E . coli was purified to homogeneity by immunoaffinity and ion-exchange chromatography . The cytokine inhibits the growth of myeloleukemic M1 cells and induces their morphological and functional differentiation into macrophages . Differentiation was also observed in the histiocytic lymphoma U937 cells . The effect on U937 was synergized by IFN-gamma and under these conditions IFN-beta 2 produced the induction of (2'-5') oligo(A) synthetase typical to IFN action and to differentiation.

FEBS Lett, 1988 Nov 7, 239(2), 190 - 4
UV-induced cross-linking of proteins to plasmid pBR322 containing 8-azidoadenine 2'-deoxyribonucleotides; Meffert R et al.; An efficient method of cross-linking DNA to protein is described . The method is based on the incorporation of photoactive 8-azidoadenine 2'-deoxyribonucleotides into DNA . We have found that 8-N3dATP is a substrate for E . coli DNA polymerase I and that 8-N3dATP can be incorporated into plasmid pBR322 by nick-translation . Subsequently we were able to cross-link a set of different proteins to 8-azido-2'-deoxyadenosine-containing pBR322 by UV irradiation (366nm) . No DNA-protein photocross-linking was observed under the same conditions when the non-photoactive plasmid pBR322 was used.

J Biol Chem, 1988 Nov 5, 263(31), 16063 - 8
Molecular cloning and nucleotide sequence of the gene for the ribosomal protein S11 from the archaebacterium Halobacterium marismortui; Arndt E et al.; The gene encoding ribosomal protein S11 (Escherichia coli S15 homologue) from Halobacterium marismortui was cloned employing two synthetic oligonucleotide mixtures, 23 and 32 bases in length, as hybridization probes . The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (1300 base pairs) were then determined by the dideoxy chain termination method . Comparison of the nucleotide sequence of the H . marismortui S11 gene with that of the E . coli S15 gene (rpsO) showed that the 3'-end of the S11 gene can be aligned with the entire E . coli S15 gene, sharing 44% identical nucleotides . It has been found that the S11 gene has a higher G + C content (G + C = 65%) than that of the E . coli S15 gene (G + C = 53%) . This increase in G + C content specifically shows up as a preference for G + C in the 3rd position of the codon . Upstream of the S11 gene, an archaebacterial promoter sequence (GGACTTTCA) and a putative ribosomal binding site (GCGGT) have been found, 88 and 15 (or 24) base pairs from the initiation codon of the gene . In addition, an open reading frame could be identified immediately after the stop codon for the S11 gene . Northern blotting analysis using the S11 coding region as probe has shown that the S11 gene is located on a 2.4-kilobase mRNA, suggesting that it is cotranscribed with other downstream gene(s).

J Mol Biol, 1988 Nov 5, 204(1), 69 - 77
Stimulation of RecA-mediated cleavage of phage phi 80 cI repressor by deoxydinucleotides; Eguchi Y et al.; The presence of either deoxyguanylyl-(3'----5')-deoxyguanosine (d(G-G} or deoxyadenylyl-(3'----5')-deoxyguanosine (d(A-G} greatly stimulates cleavage of the phage phi 80 cI repressor mediated by the Escherichia coli RecA protein in vitro . No other deoxydinucleoside monophosphate or riboguanylyl-(3'----5')-guanosine (r(G-G} affects the cleavage reaction . Neither the cleavage site of the phi 80 cI repressor nor the requirement for single-stranded DNA and ATP for cleavage is altered by d(G-G) . Photoaffinity labeling experiments with 32P-labeled 5'-phosphoryl deoxyguanylyl deoxyguanosine (pd(G-G}, which also stimulates cleavage, show that pd(G-G) bound to the repressor under the conditions in which the repressor is cleaved by RecA protein . The binding increases the affinity of the repressor for RecA protein and thus greatly stimulates repressor cleavage . The cleavage reactions of LexA and lambda cI repressors by RecA protein are not affected by d(G-G).

J Mol Biol, 1988 Nov 5, 204(1), 61 - 7
Two forms of RecA-single-stranded DNA-adenosine 5'-O-(3-thiotriphosphate) complexes with different activities for cleavage of phage phi 80 cI repressor; Eguchi Y et al.; The kinetics of cleavage of the phage phi 80 cI repressor by Escherichia coli RecA protein were studied . The rate of cleavage in the presence of single-stranded DNA (ssDNA) and either adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ATP or dATP is very low in the first hour at 37 degrees C and then increases sharply as incubation continues . The initial rate of cleavage of the repressor is greatly increased by incubating the RecA protein with ssDNA prior to addition of ATP gamma S and the repressor . However, when ATP gamma S is present during preincubation of RecA protein with ssDNA, the stimulatory effect of preincubation is greatly reduced . This difference in the effect of preincubation in two different conditions can be explained by formation of RecA-ssDNA-ATP gamma S complexes with different activities for cleavage of the repressor . The active complex is formed by binding of ATP gamma S to a complex of RecA protein and ssDNA . However, when the RecA protein binds to ATP gamma S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor.

J Mol Biol, 1988 Nov 5, 204(1), 41 - 50
Development of a trpE promoter-strength measuring system and its use in comparison of the trpEDCBA, trpR and aroH promoters; Cho KO et al.; An expression system was developed for measuring in vivo promoter strength at the single copy level and this system was used to compare the trp, aroH and trpR promoters . This system employs trpE enzyme activity as a measure of promoter strength and lacZ expression for internal copy number reference . Promoter-containing fragments are inserted into a cloning vector and subsequently recombined on to phage lambda by genetic exchange . Single lysogens are then prepared and used in promoter-strength analyses . The strength of several promoters was determined using this system . Among the promoters tested, the Escherichia coli trpEDCBA promoter was the strongest; it was four times more active than the lacUV5 promoter and about ten times stronger than the trpR and aroH promoters . To validate measurement of trpE enzyme activity as an indicator of promoter strength, trpE enzyme activity was compared with the level of trpE mRNA . There was excellent correspondence between the two, suggesting that with this system trpE enzyme activity accurately reflects promoter strength . We also examined a homologous promoter-strength measuring system in which the promoter-cloning plasmid lacked a 104 base-pair DNA spacer that was present immediately downstream from the promoter-cloning site in our preferred system . We found that the spacer was essential; the transcribed region accompanying a cloned promoter apparently affected trpE translational efficiency and/or trpE mRNA stability.

J Biol Chem, 1988 Nov 5, 263(31), 16156 - 62
Roles of the narJ and narI gene products in the expression of nitrate reductase in Escherichia coli; Sodergren EJ et al.; Nitrate reductase, released and purified from membrane fractions of Escherichia coli, is composed of three subunits . Formation of the enzyme depends on induction of the nar operon, narGHJI, which is composed of four open reading frames (ORF) . Previous studies established that the first two genes in the operon narG and narH encode the alpha and beta subunits, respectively, while formation of the gamma subunit, cytochrome bNR, depends on expression of the promoter distal genes . The narJ and narI genes were subcloned separately into plasmids where each was under the control of the nar promoter . Expression of these plasmids in a mutant which forms only alpha and beta subunits revealed that expression of the narI gene is sufficient to restore normal levels of cytochrome bNR, but expression of both genes is required for assembly of fully active, membrane-bound nitrate reductase . The amino acid composition, the N-terminal sequence, and the sequence of cyanogen bromide fragments derived from the isolated gamma subunit corresponds to that expected for a protein produced by the narI ORF . A protein corresponding to the narJ ORF did not appear to be associated with the purified nitrate reductase complex or with the complex immunoprecipitated from Triton X-100-solubilized membrane preparations . We conclude that narI encodes the gamma subunit (cytochrome bNR) and that while the product of the narJ gene is required for assembly of fully active membrane-bound enzyme it is not tightly associated with the active enzyme.

J Biol Chem, 1988 Nov 5, 263(31), 16055 - 7
Site-specific alteration of cysteine 281, cysteine 344, and cysteine 349 in the proline carrier of Escherichia coli; Yamato I et al.; Cys-281, Cys-344, or Cys-349 in the proline carrier of Escherichia coli was changed to a serine residue by site-specific mutagenesis . The activities of the resultant mutants for uptake of proline were as great as that of the wild-type strain . These mutant carriers were all as sensitive as the wild-type carrier to the proline analogue azetidine 2-carboxylate . However, the mutant carriers with Ser-281 and Ser-344 were resistant to N-ethylmaleimide, whereas the mutant carrier with Ser-349 was as sensitive as the wild-type carrier to this reagent . These results indicate that these cysteine residues are not essential for proline transport and that Cys-281 and Cys-344 may be close to the substrate-binding site that contains an N-ethylmaleimide-sensitive residue.

J Biol Chem, 1988 Nov 5, 263(31), 15906 - 14
Topography of lactose permease from Escherichia coli; Page MG et al.; The topography of lactose permease, in native membrane vesicles and after reconstitution of the purified protein into proteoliposomes, has been investigated by labeling the membrane-embedded portions of the protein using photoactivatable, hydrophobic reagents and by labeling the exposed portions of the protein with water-soluble, electrophilic reagents . Some sites of modification have been localized in fragments of the protein produced by chemical and enzymatic cleavage . These define a number of hydrophilic loops and membrane-spanning regions and give some substance to topographic models of the permease . The N-terminal third of the molecule was labeled by three photoactivatable reagents (3-(trifluoromethyl)-3-m-iodophenyldiazirine and the phospholipid analogues 2-(aceto-(4-benzoylphenylether}-1-palmitoylphosphatidylcholine) and 2-(4-azido-2-nitrophenylaminoacetyl)-1-palmitoylphosphatidylcholin e) as well as the water soluble, electrophilic reagents . The C-terminal part of the molecule is labeled by the diazirine and, to a lesser extent, by the phospholipid analogues . It apparently has more nucleophilic groups accessible to water-soluble reagents than the N-terminal domain, in which the density of apparently unreactive ionizable residues proved to be unexpectedly high . The apparent lack of reactivity of some of these residues may be explained either by their being buried in the protein moiety within the membrane domain, or by their close association with other ionizable residues on the surface of the protein.

J Biol Chem, 1988 Nov 5, 263(31), 15897 - 905
The effects of pH on proton sugar symport activity of the lactose permease purified from Escherichia coli; Page MG et al.; The lactose permease, which catalyzes galactoside-proton symport into Escherichia coli, has been purified and reconstituted in active form into artificial lipid vesicles . The roles of many detergents and phospholipids in solubilization and stabilization of the activity of the permease have been examined with a view to its eventual crystallization . Initial rates of uptake into reconstituted proteoliposomes determined by rapid mixing techniques proved that the activity of the permease can be comparable to that observed in the intact cell, while the best values for uptake rates obtained with conventional techniques were comparable to those reported for vesicles . The activity of the purified protein has been monitored over time periods of hours to weeks . It is shown that, under the best current conditions, the permease retains full activity for 1 to 2 weeks . Although this is still marginal for its crystallization, future improvements can now be assayed by rather stringent criteria . The mechanism of galactoside transport into reconstituted proteoliposome has been investigated by examining the effects of pH on influx into the vesicles . It is shown that the observed effects are entirely consistent with the predictions of a simple model of proton symport . The apparent increase in rate of uptake that is observed in the presence of a pH gradient is not so much due to an acceleration by a component of the protonmotive force as to the relaxation of inhibition by a product (internal protons) of the symport reaction.

J Biol Chem, 1988 Nov 5, 263(31), 16443 - 51
Studies on the role of the phi X174 gene A protein in phi X174 viral strand synthesis . III . Replication of DNA containing two viral replication origins; Goetz GS et al.; Supercoiled plasmid bearing two wild-type phi X origin sequences on the same strand supported the phi X A protein-dependent in vitro formation of two smaller single-stranded circles, the lengths of which were equivalent to the distance between the two origins . Additional double origin plasmids were utilized to determine whether origins defective in the initial nicking event (initiation) could support circularization (termination) . In all cases tested, the presence of a mutant origin on the same strand with a wild-type origin affected the level of replication in a manner consistent with the previously determined activity of the mutant origin . When a functional mutant origin was present on the same strand as a wild-type origin, the efficiency of replication and the DNA products formed were almost identical to those of the plasmid containing two wild-type origins . Plasmid DNA bearing both a wild-type origin and a mutant origin that did not support phi X A protein binding or nicking activity, on the other hand, supported efficient DNA synthesis of only full-length circular products, indicating that the origin defective for initiation was incapable of supporting termination . In contrast, the presence of a wild-type origin and an origin that did bind the phi X A protein but was not cleaved resulted in a marked decrease in DNA synthesis along with the production of only full-length products . This suggests that the phi X A protein stalls when it encounters a sequence to which it can bind but cannot cleave . Replication of double origin plasmids containing one functional phi X origin on each strand of the supercoiled DNA was also examined . With such templates, synthesis from the wild-type origin predominated, indicating preferential cleavage of the intact origin sequence . Replication of such substrates also produced a number of aberrant structures, the properties of which suggested that interstrand exchange of the phi X A protein had occurred.

J Biol Chem, 1988 Nov 5, 263(31), 16106 - 12
Fo portion of Escherichia coli H+-ATPase . Carboxyl-terminal region of the b subunit is essential for assembly of functional Fo; Takeyama M et al.; Six chromosomal uncF mutants of Escherichia coli defective in the b subunit of H+-ATPase (156 amino acid residues) were identified (KF92, Met-1----Val; KF164, Gln-64----end; KF61 and KF144, Gln-104----end; KF138, Gln-106----end; and KF79, Gln-123----end) . The membranes of all these mutants had low ATPase activities (less than 5% of that of the wild type), and no functional H+ pathway, although the truncated b subunits were integrated into these membranes . These findings suggest that about 30 carboxyl-terminal amino acid residues of the b subunit are essential for formation of the F1-binding site and H+ pathway . For examination of the role(s) of the carboxyl-terminal region(s) or residue(s) of the b subunit, recombinant plasmids carrying truncated uncF genes of various lengths were constructed by in vitro muta-genesis and introduced into a recA1 derivative of strain KF92 (Met-1----Val) . Analyses of the membranes from the resulting strains demonstrated that almost the entire carboxyl-terminal region of the b subunit is necessary for formation of functional Fo, since loss of the carboxyl-terminal residue resulted in significant reduction of both F1 binding and H+ translocation, and loss of two or more residues abolished both activities completely.

J Biol Chem, 1988 Nov 5, 263(31), 15957 - 63
Directed mutagenesis of the strongly conserved lysine 175 in the proposed nucleotide-binding domain of alpha-subunit from Escherichia coli F1-ATPase; Rao R et al.; The alpha-subunit of Escherichia coli F1-ATPase contains an adenine-specific noncatalytic nucleotide-binding domain . A recent proposal (Maggio, M . B., Pagan, J., Parsonage, D., Hatch, L., and Senior, A . E . (1987) J . Biol . Chem . 262, 8981-8984) suggested that this domain is formed by residues 160-340, approximately, in alpha-subunit . Within this proposed domain is a sequence Gly-X-X-X-X-Gly-Lys which is conserved in a large and diverse group of nucleotide-binding proteins and is thought to interact with phosphate groups of bound nucleotide . In this work, residue alpha Lys-175, the terminal residue of the above conserved sequence in F1-alpha-subunit, was mutagenized to Ile or Glu . The specific activity of purified mutant F1-ATPase was reduced by 2.5-fold (Ile) or 3-fold (Glu) . Apparent binding of ATP to alpha-subunit, as measured by the centrifuge column procedure, was strongly impaired and ATP-induced conformational change in alpha-subunit, as measured by protection against trypsin proteolysis, was nearly abolished in both mutants . The results suggest that residue alpha Lys-175 is located within the nucleotide-binding domain of alpha-subunit, and that this residue is functionally involved in nucleotide binding . The results support previous suggestions that the alpha-subunit nucleotide-binding site is not involved, directly or indirectly, in catalysis.

J Mol Biol, 1988 Nov 5, 204(1), 27 - 40
Specific duplications fostered by a DNA structure containing adjacent inverted repeat sequences; McClain WH; This paper describes the nucleotide sequences of three spontaneous mutations in a suppressor gene of phage T4 tRNA(Ser) . They are duplications of the anticodon and variable arms of the tRNA(Ser) molecule . One is a 34-nucleotide direct repeat of the wild-type sequence . The remaining two have reciprocal structures, with each containing 35-nucleotide inverted and direct repeats of the wild-type sequence . One of the latter mutations is frequent and was present in multiple isolates . All three duplications are unstable, and several revertants of each were sequenced . Most of the revertants had the wild-type nucleotide sequence; however, one had imprecisely removed the duplicated residues, leaving four new nucleotides compared to the wild-type sequence . These mutations represent significant genetic events with regard to their high rates and their gross structural alterations . As to their origin, the mutations can be described as the end-products of endonuclease cleavage of DNA at regions of potential secondary structure and subsequent DNA synthesis . The secondary structure contains four base-paired stems that emerge from duplex DNA . These stems encode the anticodon and variable arm regions of the tRNA(Ser) molecule . The cleavage sites mimic the known substrate of T4 endonuclease VII, an enzyme previously noted for its ability to resolve Holliday-like DNA intermediates.

J Biol Chem, 1988 Nov 5, 263(31), 16364 - 71
Ubiquitin-metallothionein fusion protein expression in yeast . A genetic approach for analysis of ubiquitin functions; Butt TR et al.; We have established a Saccharomyces cerevisiae genetic system that expresses the fusion protein ubiquitin-metallothionein . We have evaluated the effects of amino-terminal ubiquitination of metallothionein on the stability and function of metallothionein . The fusion protein of wild type ubiquitin and metallothionein was rapidly processed in vivo to release free ubiquitin and metallothionein . Site-directed mutants of ubiquitin-metallothionein expressed in yeast were used to study the specificity of the (alpha-NH2-ubiquitin) protein endopeptidases . The data suggest that amino-terminal ubiquitination is not a signal for the proteolysis of yeast metallothionein in yeast . We have also discovered that expression of selected ubiquitin mutants blocked the growth of yeast . The data suggest that in addition to its function as a proteolytic signal, ubiquitination of proteins plays multiple roles in the cell.

J Biol Chem, 1988 Nov 5, 263(31), 15928 - 37
Coupling of heme attachment to import of cytochrome c into yeast mitochondria . Studies with heme lyase-deficient mitochondria and altered apocytochromes c; Dumont ME et al.; Cytochrome c is synthesized in the cytoplasm as apocytochrome c, lacking heme, and then imported into mitochondria . The relationship between attachment of heme to the apoprotein and its import into mitochondria was examined using an in vitro system . Apocytochrome c transcribed and translated in vitro could be imported with high efficiency into mitochondria isolated from normal yeast strains . However, no import of apocytochrome c occurred with mitochondria isolated from cyc3- strains, which lack cytochrome c heme lyase, the enzyme catalyzing covalent attachment of heme to apocytochrome c . In addition, amino acid substitutions in apocytochrome c at either of the 2 cysteine residues that are the sites of the thioether linkages to heme, or at an immediately adjacent histidine that serves as a ligand of the heme iron, resulted in a substantial reduction in the ability of the precursor to be translocated into mitochondria . Replacement of the methionine serving as the other iron ligand, on the other hand, had no detectable effect on import of apocytochrome c in this system . Thus, covalent heme attachment is a required step for import of cytochrome c into mitochondria . Heme attachment, however, can occur in the absence of mitochondrial import since we have detected CYC3-encoded heme lyase activity in solubilized yeast extracts and in an Escherichia coli expression system . These results suggest that protein folding triggered by heme attachment to apocytochrome c is required for import into mitochondria.

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