Escherichia coli

Carbohydr Res, 1992 Nov 4, 235, 199 - 209
The capsular antigen of Escherichia coli serotype O8:K102:H-; de Bruin AH et al.; The structure of the capsular antigen of E . coli O8:K102:H- was investigated by methylation analysis, beta-elimination of the methylated polysaccharide, lithium-ethylenediamine mediated degradation, and by 1D and 2D 1H and 13C NMR spectroscopy of the lithium-degraded and native polysaccharides . The capsular antigen was shown to have the following branched pentasaccharide repeating unit: {formula: see text}

Biochem Pharmacol, 1992 Nov 3, 44(9), 1849 - 58
Demonstration of similar calcium dependencies by mammalian high and low molecular mass phospholipase A2; Marshall LA et al.; The in vitro Ca2+ dependencies of arachidonyl (AA)-selective high molecular mass phospholipase A2 (HMM, 85 kDa-PLA2) and human low molecular mass (LMM-Type II, 14 kDa)-PLA2 were compared . When the LMM-PLA2 and HMM-PLA2 enzymes were examined for hydrolysis against {3H}AA Escherichia coli in an ethyleneglycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA)-free buffer system, neither enzyme demonstrated activity below 10 microM free Ca2+ . Beyond 11 microM Ca2+ both enzyme activities increased steadily exhibiting 50% of maximal activity at 0.1 and 1.0 mM, respectively . Using EGTA-regulated free Ca2+ buffers, both enzymes responded in a biphasic manner, achieving 50% of the maximum response by 0.5 microM Ca2+, stabilizing up to 0.1 mM, then further increasing with exposure to millimolar Ca2+ concentrations . Replacement of {3H}AA-labeled phosphatidylethanolamine vesicles for {3H}AA E . coli or using Tris-HCl buffer instead of HEPES buffer did not alter these findings significantly . The presence of EGTA had a pronounced concentration-dependent effect on the activity of both the HMM- and LMM-PLA2 enzymes but only in the range of 0 to 100 microM free Ca2+ . EGTA (EC50 approximately 200 microM) reduced the concentration of Ca2+ required by PLA2 to achieve 50% of maximal acylhydrolysis . In contrast, the Type I bovine pancreatic PLA2 required millimolar Ca2+ concentrations to elicit 50% of the maximal response in both EGTA-free or EGTA-containing systems, which is concordant with its extracellular role as a digestive enzyme . These data suggest that the LMM-Type II PLA2 and HMM-PLA2 are both activated at submicromolar, intracellularly relevant, Ca2+ concentrations and therefore have the ability to contribute to cellular lipid metabolism.

Biochemistry, 1992 Nov 3, 31(43), 10502 - 9
Passage of RNA polymerase from open complex to elongation mode at the Escherichia coli lacUV5 promoter: nucleolytic hypersensitivity as a probe for complex conformational changes; Spassky A; In transcriptionally active complexes between RNA polymerase and promoters, the center of the melted region is hyperreactive to the nucleolytic activity of the cuprous complex of 1,10-phenanthroline (OP-Cu) . In the first part of this work, using synthetic oligonucleotides and exploiting gel retardation assays, I demonstrate that DNA unpairing is not the only determinant of this hyperreactivity . Polymerase binding is directly implicated, presumably participating in the stabilization of an intermediate required for the cutting . In the second part of the work, I show that, from fine analysis of the nucleolytic pattern of lacUV5 promoter DNA towards OP-Cu and Phe OP-Cu, it is possible to locate polymerase and to characterize its contacts at any time during the early stages of transcription . This analysis provides a description of the passage from the "open complex" to the elongation mode in terms of, first, release of the upstream contacts, and second, loss of sigma subunit . Occupancy of the overlapping promoter, P2, has a positive effect on the escape of polymerase from abortive cycling . The involvement of sigma and beta subunits in the reactivity pattern is discussed with respect to previous cross-linking studies.

Biochemistry, 1992 Nov 3, 31(43), 10483 - 90
Molecular cloning of porcine alveolar macrophage-derived neutrophil chemotactic factors I and II; identification of porcine IL-8 and another intercrine-alpha protein; Goodman RB et al.; Alveolar macrophages (AM) mediate lung inflammation by producing lipid and peptide molecules that attract neutrophils (PMN) to the lung . Recently we described two porcine proteins called alveolar macrophage-derived chemotactic factors, AMCF-I and -II, that are potent, efficacious, and specific PMN chemoattractants both in vitro and in vivo . We report here the cloning of the full-length cDNAs which code for each protein . Porcine AM were stimulated for 4 h in vitro with Escherichia coli endotoxin (LPS), and a cDNA library was created from poly(A)(+)-selected mRNA . Specific oligonucleotide probes for AMCF-I and AMCF-II were amplified from the porcine AM cDNA library by the polymerase chain reaction using degenerate oligonucleotide primer pairs derived from the N-terminal amino acid sequences of the proteins . These probes were used to isolate 2 full-length cDNAs of 1466 (AMCF-I) and 1515 (AMCF-II) base pairs . Both cDNAs code for proteins with four cysteine residues containing the C-X-C sequence characteristic of the intercrine-alpha family of neutrophil chemoattractants . AMCF-I shares 74% identity with human IL-8 and 84% identity with rabbit IL-8, and likely represents the porcine homologue of IL-8 . By contrast, AMCF-II has no obvious human homologue . AMCF-II shares 53% identity with human neutrophil activating peptide 2 . Its shared identity with the GRO-related proteins is as high as 61% (rat CINC/GRO), and its shared identity with the 78 amino acid epithelial cell-derived neutrophil activator (ENA-78) is 67% . AMCF-II may represent a new member of the intercrine-alpha family of neutrophil chemoattractants.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Nov 3, 31(43), 10438 - 42
Spectroscopic studies of arsenic(III) binding to Escherichia coli RI methyltransferase and to two mutants, C223S and W183F; Lam WC et al.; The interactions of an arsenic (III) reagent, (CH3)2AsSCH2CONH2, with two Escherichia coli RI methyltransferase mutants, W183F and C223S, have been studied by phosphorescence, optically detected magnetic resonance, and fluorescence spectroscopy . The phosphorescence spectrum of the W183F mutant containing only one tryptophan at position 225 reveals a single 0,0-band that is red-shifted by 9.8 nm upon binding of As(III) . Fluorescence titration of W183F with (CH3)2AsSCH2CONH2 produces a large tryptophan fluorescence quenching . Analysis of the quenching data points to a single high-affinity As(III) binding site that is associated with the fluorescence quenching . Triplet-state kinetic measurements performed on the perturbed tryptophan show large reductions in the lifetimes of the triplet sublevels, especially that of the T chi sublevel . As(III) binding to the enzyme at a site very close to the Trp225 residue induces an external heavy-atom effect, showing that the perturber atom is in van der Waals contact with the indole chromophore . In the case of the C223S mutant, a single tryptophan 0,0-band also is observed in the phosphorescence spectrum, but no change occurs upon addition of the As(III) reagent . Fluorescence titration of C223S with As(III) shows essentially no quenching of tryptophan fluorescence, in contrast with W183F . These results, along with previous triplet-state and biochemical studies on the wild-type enzyme {Tsao, D . H.H., & Maki, A . H . (1991) Biochemistry 30, 4565-4572}, show that As(III) binds with high affinity to the Cys223 residue and that the Trp225 side chain is located close enough to that of Cys223 to produce a heavy-atom perturbation when As(III) is bound.

Gene, 1992 Nov 2, 121(1), 95 - 102
The rate of decay of Rhodobacter capsulatus-specific puf mRNA segments is differentially affected by RNase E activity in Escherichia coli; Klug G et al.; In Rhodobacter capsulatus the puf operon encodes proteins of the photosynthetic apparatus . The polycistronic puf mRNA is comprised of segments that show differential stability . Here, we show that the rate of decay of the 2.7-kb pufBALMX mRNA species in Escherichia coli depends on the activity of ribonuclease E (RNase E), whereas the degradation of the 0.5-kb pufBA mRNA segment is not affected by a mutation in the rne gene . The RNase E-promoted decay of the pufLMX mRNA depends on the presence of a 1.4-kb pufLM mRNA segment, in which rate-limiting endonucleolytic cleavage was postulated to occur in R . capsulatus . The insertion of 185 bp of this 1.4-kb segment into pufB results in an RNase E-dependent decay of the modified pufBA mRNA segment in E . coli . Our findings suggest that in R . capsulatus an RNase E-like activity is responsible for the rate-limiting endonucleolytic cleavage occurring within the pufLM mRNA segment, whereas the 0.5-kb pufBA mRNA segment is degraded by a different RNase E-independent decay mechanism.

Gene, 1992 Nov 2, 121(1), 79 - 86
Cloning and sequence analysis of the gene encoding an NADP-dependent alcohol dehydrogenase in Mycobacterium bovis BCG; Stelandre M et al.; The nucleotide sequence of a 1619-bp fragment of Mycobacterium bovis BCG containing the gene that encodes an alcohol dehydrogenase (ADH) has been determined . The M(r) calculated from the deduced amino acid (aa) sequence, as well as the N terminus, are in good accordance with those determined for the ADH purified from M . bovis BCG extracts . The M . bovis BCG cloned adh gene was expressed in Escherichia coli by its own promoter and the synthesized product shows ADH activity in the butane-1-ol-NADP system . Based on comparison of the aa sequence, this enzyme belongs to the zinc-containing, long-chain alcohol/polyol dehydrogenase family, which has been primarily described in eukaryotes . Of the 22 strictly conserved residues in this group, 19 are also conserved in M . bovis BCG ADH (BCGADH).

Gene, 1992 Nov 2, 121(1), 127 - 32
Cloning and characterization of genes involved in the biosynthesis of delta-aminolevulinic acid in Escherichia coli; Ikemi M et al.; Several mutants of Escherichia coli that had lost their ability to synthesize delta-aminolevulinic acid (ALA) via the C5 pathway were isolated . Their defective loci were classified into two groups, AlaA- and AlaB- . The genes that complemented these mutations were cloned . Nucleotide sequencing indicated that the gene that complemented AlaA- was identical to hemL which is located at 4 min on the E . coli chromosome and encodes glutamate 1-semialdehyde aminotransferase . The gene complementing AlaB- contained an open reading frame (ORF) encoding a polypeptide of 207 amino acids that was found to be a new gene involved in the synthesis of ALA via the C5 pathway . Thus, we designated the gene hemM . The hemM gene was adjacent to hemA that is located at 27 min and previously thought to encode glutamyl-tRNA dehydrogenase . However, we found that hemA complemented both the AlaA- (hemL) and AlaB- (hemM) mutants defective in the C5 pathway although the transformants showed small colonies on the selective medium without ALA . These results suggest that hemA is not involved in the C5 pathway, but controls a second, minor pathway for the synthesis of ALA.

Gene, 1992 Nov 2, 121(1), 121 - 6
Amino acid substitution in the C-terminal arm domain of HU-2 results in an enhanced affinity for DNA; Goshima N et al.; Three mutants of the Escherichia coli hupA gene, encoding the HU-2 protein, were constructed by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors . The resulting HupAN10, HupAN11 and HupAN12 proteins contained Thr59-->Lys, Gln64-->Lys and Asn53-->Arg substitutions, respectively . These amino acid (aa) changes increased the positive charge of the N-terminal half of the two-strand, antiparallel beta-ribbon of the arm structure, which is believed to be a domain for DNA binding . The three mutant proteins bound to DNA more tightly than wild-type HU-2, and their affinities for DNA increased in the order of HupAN10, HupAN11, HupAN12 . The mutant proteins showed a slightly increased HU activity for supporting Mu phage development . A mutant HU-2 protein with increased basicity, but with an altered aa sequence in the arm region due to a frameshift mutation, was also constructed . This mutant protein showed a reduced affinity to DNA and was unable to support Mu growth, suggesting that a unique aa sequence of the arm domain, rather than mere basicity of this domain, is required for efficient binding to DNA.

Gene, 1992 Nov 2, 121(1), 1 - 7
Cloning, sequencing, overproduction, and purification of M . CviBI (GANTC) methyltransferase from Chlorella virus NC-1A {corrected}; Kan TN et al.; We have cloned and sequenced the cvibIM gene from Chlorella virus NC-1A by selecting for the modification phenotype . The modification gene was cloned on a 7-kb BamHI fragment inserted into the BamHI site of the pUC13 plasmid . The cvibIM gene was localized at the 3' end of this fragment . Sequencing of this region revealed a large open reading frame that codes for methyltransferase (MTase; symbol M.) (predicting 260 amino acids) . M.CviBI (GANTC) aa sequence is homologous to M.Dam(GATC), M.DpnII(GATC), and M.T4 (GATC), and not so to M.HinfI(GANTC), M.HhaII (GANTC), and M.DpnA(GATC) . We also describe the use of the polymerase chain reaction technique to alter transcriptional and translational signals surrounding this gene so as to achieve overexpression in Escherichia coli . This construct yields M.CviBI at 2-3% of the total cellular protein . The MTase was purified by phosphocellulose, DEAE, and gel filtration chromatography . Its size by SDS-PAGE is approx . 28 kDa, in good agreement with that predicted from the nucleotide sequence.

FEBS Lett, 1992 Nov 2, 312(1), 61 - 5
Escherichia coli DNA gyrase: genetic analysis of gyrA and gyrB mutations responsible for thermosensitive enzyme activity; Oram M et al.; Escherichia coli gyrA43 and gyrB203 alleles conferring temperature-sensitive (ts) growth encoded Gly751-->Asp and Pro171-->Ser substitutions in the DNA gyrase A and B subunits, respectively . A plasmid-borne gyrA43 allele was genetically dominant over a chromosomal quinolone-resistant gyrA gene at 30 degrees C but not at 42 degrees C . These results and others confirm the ts phenotype of the mutation, the first to be identified in the C-terminal DNA binding/complex stabilizing domain of gyrase A protein . By contrast, the Pro171-->Ser mutation is located near the ATP-binding site of gyrase B protein and could interfere with energy coupling during DNA supercoiling . These data are discussed in regard to recently described gyrA(ts) mutations that affect the control of chromosome segregation.

Neuroendocrinology, 1992 Nov, 56(5), 680 - 6
Alteration of endotoxin fever and release of arginine vasopressin by dehydration in the guinea pig; Roth J et al.; Arginine vasopressin (AVP), synthesized in hypothalamic neurons, is transported in axons either to the pituitary for release into the circulation or to different brain areas . In our previous experiments we documented central antipyretic AVP pathways from the hypothalamus to the ventrolateral septal area in the limbic system . In the present study we investigated if osmotic stimulation is able to activate peripheral and central release of AVP concurrently and if the antipyretic pathways are influenced by this kind of stimulation . In dehydrated animals (24 h water deprivation) the arterial blood plasma level of AVP doubled causing antidiuretic effects . Also the concentration of AVP in push-pull perfusates of the limbic septal area was significantly higher in dehydrated (5.6 pg AVP/ml perfusate) than in control animals (2.6 pg AVP/ml perfusate) . The febrile response to bacterial endotoxin was reduced by 50% in dehydrated guinea pigs compared to controls, statistically significant between 30 and 180 min after pyrogen application . A microinfusion of AVP antiserum into the limbic septal area enhanced the fever reaction of dehydrated guinea pigs compared to the effects of a microinfused preimmune serum, in this case statistically significant between 180 and 360 min after application . From these data we assume a simultaneous activation of peripheral and central release of AVP with antidiuretic and antipyretic effects by dehydration.

Environ Health Perspect, 1992 Nov, 98, 45 - 51
Intragenomic repair heterogeneity of DNA damage; Scicchitano DA et al.; The mutagenic and carcinogenic consequences of unrepaired DNA damage depend upon its precise location with respect to the relevant genomic sites . Therefore, it is important to learn the fine structure of DNA damage, in particular, proto-oncogenes, tumor-suppressor genes, and other DNA sequences implicated in tumorigenesis . Both the introduction and the repair of many types of DNA lesions are heterogeneous with respect to chromatin structure and/or gene activity . For example, cyclobutane pyrimidine dimers are removed more efficiently from the transcribed than the nontranscribed strand of the dhfr gene in Chinese hamster ovary cells . In contrast, preferential strand repair of alkali-labile sites is not found at this locus . In mouse 3T3 cells, dimers are more efficiently removed from an expressed proto-oncogene than from a silent one . Persistent damage in nontranscribed domains may account for genomic instability in those regions, particularly during cell proliferation as lesions are encountered by replication forks . The preferential repair of certain lesions in the transcribed strands of active genes results in a bias toward mutagenesis owing to persistent lesions in the nontranscribed strands . Risk assessment in environmental genetic toxicology requires assays that determine effective levels of DNA damage of producing malignancy . The existence of nonrandom repair in the mammalian genome casts doubt on the reliability of overall indicators of carcinogen-DNA binding and lesion repair for such determinations . Tissue-specific and cell-specific differences in the coordinate regulation of gene expression and DNA repair may account for corresponding differences in the carcinogenic response to particular environmental agents.

Mol Microbiol, 1992 Nov, 6(22), 3405 - 13
Genetic analysis of the immunity region of phage-plasmid P4; Ghisotti D et al.; In the prophage P4, expression of the early genes is prevented by premature termination of transcription from the constitutive promoter PLE . In order to identify the region coding for the immunity determinant, we cloned several fragments of P4 DNA and tested their ability to confer immunity to P4 superinfection . A 357 bp long fragment (P4 8418-8774) is sufficient to confer immunity to an infecting P4 phage and to complement the immunity-defective P4 cl405 mutant, both in the presence and in the absence of the helper phage P2 . The immunity region covers PLE and the cl locus . We were unable to obtain evidence of translation of the region, thus we suggest that P4 immunity is not elicited by a protein but by a transcript (or transcripts) encoded by the region downstream of the promoter PLE . The promoter PLE appears to be necessary for the expression of P4 immunity: fragments in which the PLE region is deleted did not complement P4 cl405 for lysogenization, although they still interfered with P4 growth . Two complementary sequences downstream of PLE (seqA and seqB) at the 5' and 3' ends of the immunity region play an essential role in the control of P4 immunity.

Mol Microbiol, 1992 Nov, 6(22), 3283 - 8
Role of the RNA polymerase alpha subunit in transcription activation; Ishihama A; The N-terminal two-thirds of the alpha subunit of Escherichia coli RNA polymerase plays an essential role in the initiation of subunit assembly, by gathering two large subunits, beta and beta', together into a core-enzyme complex . One group of RNA polymerase mutants deficient in response to transcription activation carries mutations in the C-terminal region of the alpha subunit, indicating that the C-terminal region of the alpha subunit is involved in protein-protein contact in positive control of transcription . A set of activators (class I transcription factors) which make contact with this contact site I region on RNA polymerase alpha subunit bind in most cases to DNA upstream of the promoter -35 signal . Genetic fine mapping indicates that a cluster of subsites exists in the contact site I region, each interacting with a set of the class I factors and each consisting of a structure formed by only 5-10 amino acid residues.

J Cell Sci, 1992 Nov, 103 ( Pt 3), 719 - 31
Further characterisation of the talin-binding site in the cytoskeletal protein vinculin; Gilmore AP et al.; The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin . Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and alpha-actinin . In this study, we further characterise the talin-binding domain in vinculin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli . Thus 125I-talin bound to a fusion protein spanning residues 1-398, but not to those spanning residues 399-881 or 881-1066 in an SDS-PAGE gel-blot assay . We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167-207 . Interestingly, a fusion protein spanning residues 1-398, but lacking residues 167-207, was unable to bind talin . However, further analysis showed that residues 167-207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity . The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathione-agarose beads . The smallest vinculin fusion protein able to bind talin contained residues 1-258 . This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells . Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect . However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1-398 to target to cell-matrix junctions in Cos cells . We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5' portion of the chicken vinculin gene, and by analysing vinculin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction . Although residues 167-207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.

J Biochem (Tokyo), 1992 Nov, 112(5), 652 - 7
Efficient production of a small peptide by expression as a multimeric form fused with the dihydrofolate reductase affinity handle; Takasuga A et al.; A pentapeptide which potently inhibits primary IgE antibody formation, Asp-Ser-Asp-Gly-Lys (DSDGK), has been efficiently produced with the aid of the dihydrofolate reductase (DHFR) handle {M . Iwakura, et al . (1992) J . Biochem . 111, 37-45} . The genes coding fused proteins comprising DHFR and multimeric forms of DSDGK, namely, DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, were constructed and expressed in Escherichia coli . The C-terminal peptides attached to DHFR did not affect the expression or the function of the DHFR handle, even when the length of the C-terminal peptide was as long as 160 amino acid residues . The fused proteins were easily purified by methotrexate affinity chromatography, one of the major advantages of the DHFR handle . The fused proteins were digested with trypsin and the monomeric peptide, DSDGK, was purified by HPLC . The yields of the peptide were estimated to be 11, 43, and 99 mg per 1 gram of the total cell proteins from E . coli cells producing DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, respectively.

J Biochem (Tokyo), 1992 Nov, 112(5), 616 - 23
Modification of immunopharmacological activities of synthetic monosaccharide lipid A analogue, GLA60, by lysozyme; Tanida N et al.; Recent studies by our group suggested that lysozyme has a high affinity for bacterial lipopolysaccharide (LPS) of both the smooth and rough forms, and inhibits various immunomodulatory activities of LPS . GLA60 is a synthetic monosaccharide analogue of bacterial lipid A well known as having most of the activities of lipid A with very low toxicity . In this study, we characterized the interaction of lysozyme with GLA60 in comparison to that with Escherichia coli 0111 LPS (smooth form) by means of an immunopharmacological approach . Using dansylated lysozyme (DNS-LZM) as a probe, LZM was found to bind to GLA60 . The mitogenic and polyclonal B-cell activating activities were significantly reduced by complex formation . However, there was no inhibitory effect on GLA60 induced production of IL-1 and TNF of macrophages . Interestingly, the activities of macrophages induced by the complex were found to be significantly higher than those induced by GLA60 itself . In contrast, the activities of 0111 LPS were significantly inhibited by LZM . Since the GLA60-LZM complex produced a turbid suspension but the 0111 LPS-LZM complex remained soluble, we consider that the activities of GLA60 alone were mediated by the common functional LPS receptor for dispersed form in both macrophages and B-lymphocytes, but activation of macrophages by the complex was mediated either by another LPS receptor not present in B-lymphocytes or through the phagocytic function of macrophages.

J Biochem (Tokyo), 1992 Nov, 112(5), 590 - 7
Hyperproduction of human interferon gamma by rat cells maintained in low-serum medium using the fibronectin gene promoter; Nakajima T et al.; An expression vector, pF1900M, which expresses a cloned gene at a high level in quiescent mammalian cells was constructed using the rat fibronectin (FN) promoter . Human interferon gamma (HuIFN-gamma) cDNA inserted downstream of the FN promoter in pF1900M was introduced into rat 3Y1 cells and several IFN-producing cell lines were established . These cells secreted a low level of IFN when they were growing but secreted at a high level after they had reached confluence . The level was further increased when the confluent cells were maintained in low-serum medium and a cell line, I7, produced 4 x 10(5) IU/ml of IFN, comparable to that produced by genetically engineered Escherichia coli in 2 days . The IFN-producing ability of I7 cells could be maintained by successive replacements of low-serum medium for at least 2 weeks . HuIFN-gamma secreted into the medium had a molecular weight range of 22,000 to 25,000, similar to that of IFN-gamma produced by human lymphocytes . The N-linked glycosylation of HuIFN-gamma seemed to occur properly, since treatment of the IFN with N-glycanase resulted in a reduction of molecular weight to 17,000, which corresponds to that calculated from the deduced amino acid sequence of HuIFN-gamma.

Biol Reprod, 1992 Nov, 47(5), 751 - 9
Role of human sperm phospholipase A2 in fertilization: effects of a novel inhibitor of phospholipase A2 activity on membrane perturbations and oocyte penetration; Fry MR et al.; Phospholipase A2 was isolated from human sperm and its potential role in the membrane fusion events of fertilization was examined . Highly purified enzyme hydrolyzed the phospholipids of {1-14C}oleate-labeled Escherichia coli optimally at neutral to alkaline pH with 5 mM CaCl2 and 150 mM NaCl (specific activity = 20 mumol/min/mg) . Activity was inhibited in a dose-dependent manner by an oligomer of prostaglandin B1 (IC50 = 1.5 microM) reported to inhibit human phospholipases A2 in vitro and in situ . Sperm phospholipase A2 injected into mouse foot pad induced a dose-dependent edema that was inhibited by oral administration of prostaglandin Bx (IC50 < or = 10 mg/kg) or by pretreatment of the enzyme with 4-bromophenacyl bromide . Human sperm phospholipase A2 (10 micrograms) induced fusion of phosphatidylserine vesicles in the presence of 1 mM calcium chloride by approximately 80% (+/- 10%) as determined by monitoring turbidity (O.D.400) and efficiency of fluorescence resonance energy transfer . This enzyme-induced fusion was accompanied by phospholipid hydrolysis, and both fusion and phospholipid degradation were inhibited by more than 60% when enzyme was preincubated with 5 microM prostaglandin Bx . Sperm penetration of zona pellucida-free hamster oocytes was inhibited in a dose-dependent fashion when sperm were incubated with prostaglandin Bx (IC50 approximately 15 microM) during capacitation; sperm motility was not affected by this treatment . Capacitation in the presence of prostaglandin Bx had little to no effect on the in vitro acrosome reaction . These results suggest that sperm phospholipase A2 and its modulators may contribute to membrane fusion events in mammalian fertilization.

Mol Biochem Parasitol, 1992 Nov, 56(1), 117 - 27
Sequence homology and absence of mRNA defines a possible pseudogene member of the Trypanosoma cruzi gp85/sialidase multigene family; Takle GB et al.; A genomic clone, pTt21, containing DNA apparently transcribed specifically in Trypanosoma cruzi trypomastigotes, was obtained by differentially screening a genomic library with trypomastigote and epimastigote cDNA . This 3444-bp clone contained open reading frames at each end, separated by a 1.8-kb non-coding region . The translated polypeptide from the 3' open reading frame (ORF2) of 1037 bp had 25-30% identity with 5 recently published T . cruzi gp85/sialidase sequences, and 20-25% identity with bacterial sialidases . Rabbit antiserum raised against an Escherichia coli fusion protein derived from the 5' open reading frame (ORF1) identified a surface antigen of 160 kDa, specifically expressed in trypomastigotes . A probe containing the first 211 bp from ORF1 was used to obtain a complete copy (c1821) of a gene that was closely related to ORF1, and encoded another member of the gp85/sialidase family . c1821 encodes a protein of 897 amino acids, but assignment of the N-terminus of the polypeptide was not possible . The 5'-most start codon is an unfavourable context to act as a translation initiator, it does not align with the initiator methionines of other gp85/sialidase sequences, nor is it followed by a signal peptide sequence characteristically found in other gp85/sialidase sequences . Although homology with the 5' ends of other gp85/sialidase sequences decays towards the 5' end of c1821, alignment of c1821 with 4 other gp85/sialidases indicated that the coding sequence should extend upstream at least 160 amino acids . In this region of c1821 there are multiple stop codons in each frame . The presence of the stop codons, the alignment data and our inability to amplify reverse transcribed mRNA using four internal primers, suggest that c1821 may not be present as a mature mRNA and is a pseudogene . Comparison of the apparently non-repetitive 3' coding domain of c1821 with the corresponding repetitive domains of two other members of the gp85/sialidase family revealed a high degree of similarity in nucleotide but not in amino acid sequence, and c1821 may thus represent an evolutionary intermediate between sub-families of the gp85/sialidase superfamily.

Mutagenesis, 1992 Nov, 7(6), 461 - 9
DNA repair modifies the site and strand specificity of ethyl methanesulfonate mutagenesis in yeast; Kunz BA et al.; The influence of DNA repair on the specificity of ethyl methanesulfonate (EMS) mutagenesis in a plasmid-borne copy of the Saccharomyces cerevisiae SUP4-o gene was investigated . Isogenic yeast strains that are repair-proficient (RAD) or defective for nucleotide excision (rad1), postreplication (rad18) or recombinational repair (rad52) were treated with EMS . Compared to the RAD wild-type, the maximum SUP4-o mutation frequency was 2-fold greater in the rad1 background whereas it was approximately 50% less in the rad18 and rad52 strains . The majority (779/788) of SUP4-o mutations characterized by DNA sequencing were single base pair changes, primarily (> 91%) G.C-->A.T transitions in the RAD, rad1 and rad18 strains . In the rad52 background, only 57% of the substitutions were G.C-->A.T transitions with transversions at G.C pairs accounting for almost all of the remaining changes . Comparisons of the distributions of single base pair substitutions in SUP4-o revealed that there was no excision repair-dependent bias for G.C-->A.T events to occur at sites flanked by a 5' or 3' A.T pair as observed previously for EMS mutagenesis of the lacIgene in Escherichia coli (Burns et al., 1986) . These transitions also did not occur more often at sites where the guanine was flanked by a 5' purine than by a 5' pyrimidine . However, they exhibited a small preference for sites having the guanine on the transcribed strand in the RAD and rad52, but not rad1 or rad18, strains.(ABSTRACT TRUNCATED AT 250 WORDS)

J Endocrinol, 1992 Nov, 135(2), 333 - 41
A polyclonal antibody to the rat oestrogen receptor expressed in Escherichia coli: characterization and application to immunohistochemistry; Okamura H et al.; A rat oestrogen receptor-beta-galactosidase fusion protein was expressed using a pEX2/rat oestrogen receptor cDNA construct . Scatchard analysis of {3H}oestradiol-17 beta binding to the cell lysate revealed that the fusion protein had functional binding sites specific for oestradiol with a dissociation constant of 1.49 nmol/l . The relative molecular weight (M(r)) of the fusion protein was determined as 180,000 by immunoblot analysis of the cell lysate employing a monoclonal antibody to the human oestrogen receptor . The protein was isolated by means of SDS-PAGE and subsequent electroblotting . By immunization with the purified materials on nitrocellulose membrane, a polyclonal antibody to the rat oestrogen receptor was raised in a rabbit . Binding of {3H}oestradiol to the oestrogen receptor from the rat uterus was inhibited by the antibody in a dose-dependent manner . The antibody was also able to recognize the oestrogen receptor occupied by {3H}oestradiol . Thus, the antibody could react with both forms of the receptor molecule, either occupied or unoccupied by the hormone . In immunoblot analysis of the cytosol fraction of the rat uterus, a single band of M(r) 67,000, the size of the oestrogen receptor, was detected by the antibody . Moreover, when the antibody was applied to immunohistochemical examination of paraffin-embedded pituitary and brain sections of the rat, immunostaining was observed in cells of the anterior pituitary and in neurones in specific regions of the brain . The immunoreactivity was restricted exclusively to cell nuclei in both tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1992 Nov, 235(2-3), 373 - 80
A umuDC-independent SOS pathway for frameshift mutagenesis; Maenhaut-Michel G et al.; The chemical carcinogen N-acetoxy-N-2-acetylaminofluorene induces mainly frameshift mutations, which occur within two types of sequences (mutation hot spots): -1 frameshift mutations within contiguous guanine sequences and -2 frameshift mutations within alternating GC sequences such as the NarI and BssHII restriction site sequences . We have investigated the genetic control of mutagenesis at these sequences by means of a reversion assay using plasmids pW17 and pX2, which contain specific targets for contiguous guanine and alternating GC sequences, respectively . Our results suggest that mutations at these hot spot sequences are generated by two different genetic pathways, both involving induction of SOS functions . The two pathways differ both in their LexA-controlled gene and RecA protein requirements . In the mutation pathway that acts at contiguous guanine sequences, the RecA protein participates together with the umuDC gene products . In contrast, RecA is not essential for mutagenesis at alternating GC sequences, except to cleave the LexA repressor . The LexA-regulated gene product(s), which participate in this latter mutational pathway, do not involve umuDC but another as yet uncharacterized inducible function . We also show that wild-type RecA and RecA430 proteins exert an antagonistic effect on mutagenesis at alternating GC sequences, which is not observed either in the presence of activated RecA (RecA*), RecA730 or RecA495 proteins, or in the complete absence of RecA as in recA99 . It is concluded that the -1 mutation pathway presents the same genetic requirements as the pathway for UV light mutagenesis, while the -2 mutation pathway defines a distinct SOS pathway for frameshift mutagenesis.

Mol Gen Genet, 1992 Nov, 235(2-3), 166 - 72
The SEG1 element: a new DNA region promoting stable mitotic segregation of plasmids in the zygomycete Absidia glauca; Burmester A et al.; A series of new vectors for the model zygomycete Absidia glauca was constructed on the basis of the structural neomycin resistance (Neor) gene controlled by the promoter of the gene for elongation factor 1 (TEF) . In order to select for transformed colonies with a stable Neor phenotype, spores from primary transformants were pooled and grown for two sporulation cycles under non-selective conditions . Southern blot analysis of DNA from single spore isolates originating from independent transformant pools allowed the identification of two autonomously replicating plasmids . Retransformation of Escherichia coli and restriction analysis of the two plasmids provided evidence for spontaneous in vivo insertion of a new DNA element (SEG1) from the A . glauca genome . The inserted regions in both plasmids are essentially identical and do not represent repetitive DNA . Compared with other autonomously replicating vectors, these SEG1-containing plasmids are mitotically extremely stable and are passed on to the vegetative spore progeny of a retransformed A . glauca strain . We assume that SEG1 contains structural elements involved in partitioning and stable segregation of plasmids . For the construction of stable transformants of A . glauca, the SEG1 element may be regarded as a major breakthrough, because stabilization of transformed genetic traits by integration is difficult to achieve in all mucoraceous fungi and all known replicating plasmids are mitotically unstable.

Mol Gen Genet, 1992 Nov, 235(2-3), 157 - 65
Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides; Caetano-Anolles G et al.; DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints . To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers . Primers of varying length, constructed by removing nucleotides from the 5' terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length . Larger primers produced either identical or related fingerprints, depending on the sequence . Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3' terminus . Increasing annealing temperatures from 15 degrees to 70 degrees C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers . Our observations define a 3'-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it . Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF . A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.

Bioconjug Chem, 1992 Nov-Dec, 3(6), 540 - 8
Preparation and characterization of biologically active 6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A and its conjugated derivative; Myers KR et al.; N-tert-butyloxycarbonyl (t-Boc) protected 6-aminocaproic (Cap) anhydride was reacted with unprotected hexaacyl-4'-O-monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Escherichia coli J5 to yield t-Boc-Cap-MLA . After a column purification step, the t-Boc group was removed by incubating the sample at low temperature in the presence of acid to yield Cap-MLA . This product was analyzed by californium plasma desorption mass spectrometry (PDMS) . Purified t-Boc-Cap-MLA was further fractionated by reverse-phase high-performance liquid chromatography as its methyl ester and characterized by laser desorption mass spectrometry, PDMS, and proton nuclear magnetic resonance spectroscopy . These analyses revealed that the Cap group was selectively introduced into the 6'-position of MLA . To demonstrate that Cap-MLA can be conjugated to other compounds, it was reacted with biotin-Cap N-hydroxysuccinimide ester to yield biotin-(Cap)2-MLA . Analysis of this product by PDMS confirmed its expected molecular weight of 2171 and showed the presence of fragments containing the biotin and Cap groups . Monoclonal antibodies and streptavidin were used to show the presence of both lipid A and biotin in this conjugated product . These two novel lipid A derivatives were then tested for their bioactivities . Although both Cap-MLA and biotin-(Cap)2-MLA showed mitogenic activity using murine splenocytes, they were about 4-8 times less active than MLA at 20 micrograms/mL or less and only one-half as active at 100 micrograms/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmid, 1992 Nov, 28(3), 258 - 61
Promoter probe and shuttle plasmids for Deinococcus radiodurans; Masters CI et al.; Two improved Deinococcus radiodurans-Escherichia coli shuttle vectors have been constructed . pI3 is a 16-kb plasmid that confers chloramphenicol resistance in D . radiodurans (CmR, cat) and ampicillin resistance in E . coli (ApR) and contains a multiple cloning site that does not interrupt sequences necessary for replication or drug resistance in either host . pI304 is a promoter-probe plasmid that is similar to pI3, but lacks the D . radiodurans promoting sequence for the cat gene, while retaining sequences necessary for replication.

Plasmid, 1992 Nov, 28(3), 183 - 93
The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons; Hales BA et al.; Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a "common" plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon . A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E . coli, but not in random environmental coliform isolates . Enteropathogenic E . coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC . The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1 . Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with > 80% showing homology to the regions encoding the rep and par genes . Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site . Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages . This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative . These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function . The evolutionary ramifications of this finding are considered.

FEMS Microbiol Lett, 1992 Nov 1, 77(1-3), 191 - 6
Enterotoxicity and immunological properties of two mutant forms of Escherichia coli STIp with lysine or arginine substituted for the asparagine residue at position 11; Okamoto K et al.; Two variants of Escherichia coli heat-stable enterotoxin Ip, in which the amino acid residue at position 11 was substituted with lysine or arginine, were purified to near homogeneity from the culture supernatants of toxin-producing mutant strains . Neither the purified heat-stable enterotoxin Ip(Lys-11) nor the purified heat-stable enterotoxin Ip(Arg-11) showed a positive response in the suckling mouse assay or in the mouse intestinal loop assay . Furthermore, live bacteria producing these mutant heat-stable Ip enterotoxins did not cause fluid accumulation in mouse intestinal loops, in contrast to bacteria producing native heat-stable enterotoxin Ip . Nevertheless, antisera raised against both heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) neutralized the enterotoxic activity of native heat-stable enterotoxin Ip . These results demonstrate that heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) lose enterotoxicity but retain epitopes which are common to native heat-stable enterotoxin Ip.

FEMS Microbiol Lett, 1992 Nov 1, 77(1-3), 181 - 6
Temperature-sensitive mutants of the Mycobacterium plasmid pAL5000; Guilhot C et al.; Two plasmids were isolated as thermosensitive replicons following in vitro mutagenesis of pB4, a pAL5000 derivative mycobacteria/Escherichia coli shuttle plasmid . Plasmids pCG59 and pCG63 replicate at 30 degrees C but not at 39 degrees C . This will allow their utilisation for transposon delivery, site-specific integration, or allele exchange.

Vet Immunol Immunopathol, 1992 Nov, 34(3-4), 291 - 305
The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus; Campbell GA et al.; Although the immune response to Brucella abortus is multifaceted, the key event in contending with this pathogen appears to be the interaction of the organism with cells of the mononuclear phagocyte system . A cell culture system was developed which allowed the long-term maintenance of blood monocyte-derived macrophages in Teflon culture vessels in a relatively unstimulated state . The assay system was optimized for timing of bacteria-macrophage interaction and numbers of bacteria and macrophages used in each assay . Interaction of B . abortus strain 2308 with bovine mononuclear phagocytes from animals phenotypically resistant and susceptible to infection with B . abortus was investigated . This cell culture and assay system should provide a useful model for the investigation of intracellular parasitism in cattle.

Mol Microbiol, 1992 Nov, 6(21), 3251 - 6
The HsdS polypeptide of the type IC restriction enzyme EcoR124 is a sequence-specific DNA-binding protein; Kusiak M et al.; The HsdS and HsdM polypeptides of the type IC restriction enzyme EcoR124 have been purified independently and used in a set of gel retardation experiments to determine the minimum requirements for sequence-specific recognition of DNA by this enzyme . The HsdS polypeptide alone is able to bind to DNA in a sequence-specific manner . In addition, whilst the presence of the HsdM polypeptide gives rise to a stimulation of DNA binding by the HsdS subunit it is not clear whether, under the conditions of the experiments reported here, the HsdS subunit maintains the same interactions with the HsdM subunits observed in the absence of DNA.

Mol Microbiol, 1992 Nov, 6(21), 3187 - 98
Cloning, mapping and nucleotide sequencing of a gene encoding a universal stress protein in Escherichia coli; Nystrom T et al.; The response of non-differentiating bacteria to nutrient starvation is complex and includes the sequential synthesis of starvation-inducible proteins . Although starvation for different individual nutrients generally provokes unique and individual patterns of protein expression, some starvation stimulons share member proteins . Two-dimensional polyacrylamide gel electrophoresis revealed that the synthesis of a small (13.5 kDa) cytoplasmic protein in Escherichia coli was greatly increased during growth inhibition caused by the exhaustion of any of a variety of nutrients (carbon, nitrogen, phosphate, sulphate, required amino acid) or by the presence of a variety of toxic agents including heavy metals, oxidants, acids and antibiotics . To determine further the mode of regulation of the protein designated UspA (universal stress protein A) we cloned the gene encoding the protein by the technique of reverse genetics . We isolated the protein from a preparative two-dimensional polyacrylamide gel, determined its N-terminal amino acid sequence, and used this sequence to construct a degenerate oligonucleotide probe . Two phages of the Kohara library were found to contain the gene which then was subcloned from the DNA in the overlapping region of these two clones . The amino acid sequence, deduced from the nucleotide sequence of the uspA gene, shows no significant homology with any other known protein . The uspA gene maps at 77 min on the E . coli W3110 chromosome, and is transcribed in a clockwise direction . The increase in the level of UspA during growth arrest was found to be primarily a result of transcriptional activation of the corresponding gene . The induction was independent of the RelA/SpoT, RpoH, KatF, OmpR, AppY, Lrp, PhoB and H-NS proteins during stress conditions that are known to induce or activate these global regulators . The -10 and -35 regions upstream of the transcriptional start site of the uspA gene are characteristic of a sigma 70-dependent promoter.

Int J Biochem, 1992 Nov, 24(11), 1785 - 92
Protein kinase NII from calf thymus chromatin . Isolation, characterization and some functional properties; Angiolillo A et al.; 1 . A protein kinase type II was purified from calf thymus chromatin using ammonium sulphate fractionation, ion exchange chromatography on DEAE and phosphocellulose and affinity chromatography on phosvitin- and casein-sepharose columns . 2 . The enzyme moves as a single band in non-denaturing gel electrophoresis at pH 8.3, which coincides with the enzyme activity assayed on gel slices . 3 . Sodium dodecyl sulphate gel electrophoresis shows three separate polypeptide chains having M(r) of 40,000, 38,000 and 25,000, respectively . The native M(r) was about 130,000, as measured by HPLC on Superose 12 column, suggesting a subunit structure of alpha, alpha', beta 2 type . The enzyme incubated with {gamma 32P}ATP or {gamma 32P}GTP as phosphoryl donors undergoes autophosphorylation in the M(r) = 25,000 subunit . 4 . The enzyme phosphorylates casein (Km = 7 microM) and phosvitin (Km = 5 microM) but not histones and was strongly deactivated by Zn2+ ions (I50 = 0.05 mM) and heparin (I50 = 0.1 micrograms/ml) . 5 . The enzyme seems to be the major phosphorylating system present in the 0.35 M NaCl chromatin extract of calf thymus . The RNA polymerase II from calf thymus and RNA polymerase from E . coli are both phosphorylated by protein kinase NII . The effect of phosphorylation, which causes a remarkable increase of DNA transcription rate, was studied in vitro and extensively discussed.

Plant Mol Biol, 1992 Nov, 20(4), 715 - 31
Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts; Mazzolini L et al.; A hammerhead ribozyme designed against the mRNA coding for the Escherichia coli beta-glucuronidase (GUS) reporter enzyme was constructed . The synthetic ribozyme appeared able to correctly cleave in vitro the target RNA . This catalytic molecule was then assayed for in vivo activity in plant protoplasts . Plasmids coding either for the ribozyme or for the GUS target gene were cotransfected into the cells by the PEG-calcium procedure and GUS gene expression monitored following transient expression by measuring the intracellular GUS enzymatic activity . Expression of the ribozyme to high molar excess over the GUS transcript did not lead to any significant decrease of GUS activity in the transfected protoplasts . Insertion of the ribozyme sequence in the 3'-untranslated region of the GUS mRNA also had no detectable effect on GUS reporter gene expression whereas the corresponding RNA appeared able to self-cleave in vitro . These results indicate that the ability of ribozymes to perform catalytic cleavage of their substrate mRNA in vitro is essential but clearly not sufficient to ensure that efficient inhibition of the corresponding target gene will occur upon endogenous expression of this catalytic RNA in the plant cell.

Plant Mol Biol, 1992 Nov, 20(4), 653 - 62
Novel protein kinase of Arabidopsis thaliana (APK1) that phosphorylates tyrosine, serine and threonine; Hirayama T et al.; During the course of characterizing polymerase chain reaction products corresponding to protein kinases of a higher plant, Arabidopsis thaliana, we found a DNA fragment that potentially codes for a polypeptide with mosaic sequences of two classes of protein kinases, a tyrosine-specific and a serine/threonine-specific one . Overlapping complementary DNA (cDNA) clones coinciding with this fragment were isolated from an A . thaliana cDNA library . From their sequence analyses a protein kinase was predicted composed of 410 amino acid residues (APK1, Arabidopsis protein kinase 1), in which the kinase domain was flanked by short non-kinase domains . Upon expression of APK1 in Escherichia coli cells, several bacterial proteins became reactive with anti-phosphotyrosine antibody but not with the same antibody preincubated with phosphotyrosine, convincing us that APK1 phosphorylated tyrosine residues . APK1 purified from an over-producing E . coli strain showed serine/threonine kinase activity, and no tyrosine kinase activity, towards APK1 itself, casein, enolase, and myosin light chains . APK1 was thus concluded to be a novel type of protein kinase, which could phosphorylate tyrosine, serine, and threonine residues, though tyrosine phosphorylation seemed to occur only on limited substrates . Since the structure of the APK1 N-terminal portion was indicative of N-myristoylation, APK1 might associate with membranes and thereby contribute to signal transduction . The A . thaliana genome contained two APK1 genes close to each other (APK1a and APK1b).

Biochem J, 1992 Nov 1, 287 ( Pt 3), 995 - 9
The Caenorhabditis elegans unc-13 gene product is a phospholipid-dependent high-affinity phorbol ester receptor; Ahmed S et al.; The Caenorhabditis elegans unc-13 mutant is a member of a class of mutants that have un-coordinated movement . Mutations of the unc-13 gene cause diverse defects in C . elegans, including abnormal neuronal connections and modified synaptic transmission in the nervous system . unc-13 cDNA encodes a protein (UNC-13) of 1734 amino acid residues with a predicted molecular mass of 198 kDa and sequence identity to the C1/C2 regions but not to the catalytic domain of the ubiquitously expressed protein kinase C family {Maruyama & Brenner (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 5729-5733} . To characterize the phorbol ester binding site of the UNC-13 protein, cDNA encoding the C1/C2-like regions (amino acid residues 546-940) was expressed in Escherichia coli and the 43 kDa recombinant protein was purified . Phorbol ester binding to the 43 kDa protein was zinc- and phospholipid-dependent, stereospecific and of high affinity (Kd 67 nM) . UNC-13 specific antisera detected a protein of approx . 190 kDa in wild-type (N2) but not in mutant (e1019) C . elegans cell extracts . We conclude that UNC-13 represents a novel class of phorbol ester receptor.

Biochem J, 1992 Nov 1, 287 ( Pt 3), 943 - 9
Site-directed mutagenesis of the leech-derived factor Xa inhibitor antistasin . Probing of the reactive site; Hofmann KJ et al.; Antistasin (ATS) is a leech-derived 119-amino-acid protein which exhibits potent and highly selective inhibition of coagulation Factor Xa . It inhibits Factor Xa according to a common mechanism of serine-proteinase inhibitors in which a conformationally rigid substrate-like reactive site is presented to the enzyme . In this study a recombinant version of ATS was expressed and purified utilizing a yeast expression system in order to probe the reactive site P1 (Arg-34) and P1' (Val-35) residues by site-directed mutagenesis . The results demonstrate the requirement for a positively charged residue in the P1 position of ATS, with an arginine residue preferred over a lysine, yielding K1 values of 61 pM and 1.28 nM respectively . Mutation of the P1 arginine residue to the non-polar amino acid leucine abolished its inhibitory potency toward Factor Xa . The role of the C-terminal domain of ATS, which shares significant amino acid sequence identity with the N-terminal domain, was investigated by creating a second reactive site in the corresponding position of the C-terminal domain . The inhibitory activity of this mutant demonstrated that the C-terminal domain of ATS is not folded into the proper conformation necessary to create a functional inhibitory domain.

Biochem J, 1992 Nov 1, 287 ( Pt 3), 937 - 41
Kinetics of activation of the P4 promoter of pBR322 by the Escherichia coli cyclic AMP receptor protein; Hoggett JG et al.; The activation of transcription initiation from the P4 promoter of pBR322 by the Escherichia coli cyclic AMP receptor protein (CRP) has been investigated using a fluorescence abortive initiation assay . The effect of the cyclic-AMP/CRP complex on the linear P4 promoter was to increase the initial binding (KB) of RNA polymerase to the promoter by about a factor of 10, but the rate of isomerization of closed to open complex (kf) was unaffected . One molecule of CRP per promoter was required for activation, and the concentration of cyclic AMP producing half-maximal stimulation was about 7-8 microM . Supercoiling caused a 2-3-fold increase in the rate of isomerization of the CRP-activated promoter, but weakened the initial binding of polymerase by about one order of magnitude . The unactivated supercoiled promoter was too weak to allow reliable assessment of kinetic parameters against the high background rate originating from the rest of the plasmid.

Anal Biochem, 1992 Nov 1, 206(2), 363 - 8
Affinity electrophoretic detection of primary amino groups in nucleic acids: application to modified bases of tRNA and to aminoacylation; Igloi GL; Thiolation of primary amino groups in tRNA with the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate gives rise to species which are retarded during electrophoresis in organomercury-containing polyacrylamide gels . Since such amino groups occur, as far as is known, only as part of the modified bases 3-(3-amino-3-carboxypropyl)uridine and N-2-(5-amino-5-carboxypentyl)cytidine or as the alpha-amino group of aminoacylated tRNAs, this extension of the principle of affinity electrophoresis can be used for the detection and analysis of a specific functional group in both single tRNA species and in a mixed population . The strength of the interaction may be quantified and provides information on the chemical environment/conformation of the derivatized bases.

Anal Biochem, 1992 Nov 1, 206(2), 293 - 9
Preparation of DNA topoisomers by RP-18 high-performance liquid chromatography; Kapp U et al.; A method for the separation of superhelical DNA on the basis of superhelical density by reverse-phase HPLC on RP-18 columns is described . The technique can be used to prepare superhelical DNA in milligram amounts and narrow topoisomer distributions in 0.1 mg amounts . We show example separations of the plasmids pUC18 (2687 bp) and pi AN13 (895 bp) . While the best separation for pUC18 yields topoisomer distributions of two or three major components, the small plasmid can be separated into single topoisomer fractions . The basis of the separation is probably an interaction of partially opened bases with the hydrophobic column matrix . This hypothesis is supported by the elution behavior of DNA fragments on this column: DNA fragments with sticky ends, even at a length of several hundred base pairs, elute at much higher methanol concentrations than blunt-ended fragments.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10547 - 51
Functional interactions between putative intramembrane charged residues in the lactose permease of Escherichia coli; Sahin-Toth M et al.; Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys . Individual replacements for Asp-237, Asp-240, Glu-269, Arg-302, Lys-319, His-322, Glu-325, or Lys-358 abolish active lactose transport . When Asp-237 and Lys-358 are simultaneously replaced with Cys and/or Ala, however, high activity is observed . Therefore, when either Asp-237 or Lys-358 is replaced with a neutral residue, leaving an unpaired charge, the permease is inactivated, but neutral replacement of both residues yields active permease {King, S . C., Hansen, C . L . & Wilson, T . H . (1991) Biochim . Biophys . Acta 1062, 177-186} . Remarkably, moreover, when Asp-237 is interchanged with Lys-358, high activity is observed . The observations provide a strong indication that Asp-237 and Lys-358 interact to form a salt bridge . In addition, the data demonstrate that neither residue nor the salt bridge plays an important role in the transport mechanism . Thirteen additional double mutants were constructed in which a negative and a positively charged residue were replaced with Cys . Only Asp-240-->Cys/Lys-319-->Cys exhibits significant activity, accumulating lactose to 25-30% of the steady state observed with C-less permease . Replacing either Asp-240 or Lys-319 individually with Ala also inactivates the permease, but double mutants with neutral substitutions (Cys and/or Ala) at both positions exhibit essentially the same activity as Asp-240-->Cys/Lys-319-->Cys . In marked contrast to Asp-237 and Lys-358, interchanging Asp-240 and Lys-319 abolishes active lactose transport . The results demonstrate that Asp-240 and Lys-319, like Asp-237 and Lys-358, interact functionally and may form a salt bridge . However, the interaction between Asp-240 and Lys-319 is clearly more complex than the interaction between Asp-237 and Lys-358 . In any event, the findings suggest that putative transmembrane helix VII lies next to helices X and XI in the tertiary structure of lactose permease.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10454 - 8
ATPase activity of transcription-termination factor rho: functional dimer model; Seifried SE et al.; Transcription-termination factor rho of Escherichia coli functions as an RNA-dependent ATPase that causes transcript release at specific rho-dependent termination sites on the DNA template . Rho exists as a hexagon of identical subunits, physically organized as a trimer of dimers with D3 symmetry . The structural asymmetry of the dimer is reflected in the binding properties of rho; each dimer has a strong and a weak binding site for both the ATP substrate and the RNA cofactor . Here we use homopolynucleotides in competition and complementation experiments to characterize the ATPase activation properties of the cofactor binding sites of the functional rho dimer . We show that (i) no ATPase activity is observed unless both the high- and the low-affinity cofactor binding sites of the functional rho dimer are occupied; (ii) saturating levels of poly(rC), poly(rC) in combination with poly(rU), or poly(rU) alone can fully activate the ATPase of rho; and (iii) poly(dC) can serve as a fully competitive inhibitor of half of the ATPase activity of rho when one of the cofactor sites is filled with poly(rC) . These observations lead to a set of phenomenological rules that describe the cofactor dependence of the ATPase activation of the functional dimer of rho and help to define a mechanistic basis for interpreting rho function in termination.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10380 - 4
Identification of the gene for an Escherichia coli poly(A) polymerase; Cao GJ et al.; Many bacterial mRNAs, like those of eukaryotes, carry a polyadenylate sequence at their 3' termini, but neither the function of the bacterial poly(A) moieties nor their biosynthesis have been elucidated . To develop a genetic tool to approach the problem of bacterial poly(A) RNA, we have sought to identify the genes responsible for mRNA polyadenylylation . A poly(A) polymerase was purified to homogeneity from extracts of Escherichia coli and subjected to N-terminal sequence analysis . The 25-residue amino acid sequence obtained was used to design primers for the amplification of the corresponding coding region by the PCR from an E . coli DNA template . A 74-base-pair DNA segment was obtained that matched a region in the pcnB locus of E . coli, a gene that had originally been identified as controlling plasmid copy number {J . Lopilato, S . Bortner & J . Beckwith (1986) Mol . Gen . Genet . 205, 285-290} and was subsequently cloned and sequenced {J . Liu & J . S . Parkinson (1989) J . Bacteriol . 171, 1254-1261} . Direct evidence that the pcnB locus encodes poly(A) polymerase was provided by the observation that a bacterial strain transformed with an inducible expression vector carrying pcnB as a translational fusion produced 100-fold elevated levels of poly(A) polymerase upon induction . No increased poly(A) polymerase activity was observed in cells transformed with expression vectors carrying truncated forms of the pcnB gene . The identification of a gene encoding bacterial poly(A) polymerase opens the way for the study of the biosynthesis and function of bacterial polyadenylylated mRNA.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10345 - 9
DnaJ, DnaK, and GrpE heat shock proteins are required in oriP1 DNA replication solely at the RepA monomerization step; Wickner S et al.; We have found that three Escherichia coli heat shock proteins, DnaK (the hsp70 homolog), DnaJ, and GrpE, function in oriP1 DNA replication in vitro solely to activate DNA binding by the replication initiator protein RepA . Activation results from the conversion of P1 or P7 RepA dimers to monomers that bind with high affinity to the origin of replication of plasmid P1 . Thus, the essential role of these three heat shock proteins in this replication system is to change the quaternary structure of a single protein, RepA.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10139 - 43
Structure of the ColE1 DNA molecule before segregation to daughter molecules; Nakasu S et al.; The segregation of daughter DNA molecules at the end stage of replication of plasmid ColE1 was examined . When circular ColE1 DNA replicates in a cell extract at a high KCl concentration (140 mM), a unique class of molecules accumulates . When the molecule is cleaved by a restriction enzyme that cuts the ColE1 DNA at a single site, an X-shaped molecule in which two linear components are held together around the origin of DNA replication is made . For a large fraction of these molecules, the 5' end of the leading strand remains at the origin and the 3' end of the strand is about 30 nucleotides upstream of the origin . The 3' end of the lagging strand is located at the terH site (17 nucleotides upstream of the origin) and the 5' end of the strand is a few hundred nucleotides upstream of the terH site . Thus the parental strands of the molecule intertwine with each other only once . When the KCl concentration is lowered to 70 mM, practically all of these molecules are converted to daughter circular monomers or to catenanes consisting of two singly interlocked circular units.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10129 - 33
Immobilization of DNA for scanning probe microscopy; Allison DP et al.; Reproducible scanning tunneling microscope and atomic force microscope images of entire molecules of uncoated plasmid DNA chemically bound to surfaces are presented . The chemically mediated immobilization of DNA to surfaces and subsequent scanning tunneling microscope imaging of DNA molecules demonstrate that the problem of molecular instability to forces exerted by the probe tip, inherent with scanning probe microscopes, can be prevented.

Oncogene, 1992 Nov, 7(11), 2329 - 33
The human tpr gene encodes a protein of 2094 amino acids that has extensive coiled-coil regions and an acidic C-terminal domain; Mitchell PJ et al.; We recently provided evidence that the human tpr gene encodes a 726 amino acid protein (designated tpr-S) and identified an alternatively spliced transcript that encodes a larger tpr protein with an extended C-terminal domain . In this study, through isolation and sequencing of tpr cDNA clones, we have established that this alternatively spliced transcript encodes a protein of 2094 amino acids (designated tpr-L) . The larger tpr protein is predicted to have extensive regions of alpha-helix and several stretches of a heptad repeat that is characteristic of proteins adopting a coiled-coil conformation . Furthermore the carboxy domain of this protein is very rich in acidic residues and exhibits homology (58-80%) to the acidic regions of several nuclear proteins, including the Drosophila engrailed protein, Escherichia coli RNA polymerase sigma subunit and nucleolin . To gain additional insight into the function of tpr we examined the expression of tpr transcripts in tissues from adult rat . The highest levels of tpr transcripts were observed in testis, lung, thymus and spleen.

Mol Pharmacol, 1992 Nov, 42(5), 917 - 21
Mechanism of inhibition of aldose reductase by menadione (vitamin K3); Bhatnagar A et al.; Incubation of human placental aldose reductase (EC 1.1.1.21) with menadione (0.5-3.0 mM) resulted in time-dependent loss of the catalytic activity of the enzyme . Kinetic analysis of the data suggests that the inactivation process follows a single apparent rate constant that displays hyperbolic dependence on menadione concentration, indicating that menadione forms a kinetically significant, dissociable complex with the enzyme before the formation of an inactive enzyme-menadione complex . The inactivation of the enzyme with menadione was reversed upon dialysis of the inactivated enzyme against buffer containing 10 mM dithiothreitol suggesting that menadione reacts with enzyme sulfhydryl residue(s) . Inactivation of the enzyme was significantly prevented by dithiothreitol (5 mM), NADPH (0.1 mM), and DL-glyceraldehyde (10 mM) . Correlation of the fractional remaining activity with the extent of modification indicates that loss of catalytic activity corresponds to the modification of a single amino acid residue of the enzyme protein . Recombinant human aldose reductase, obtained by overexpression in Escherichia coli, and aldose reductase in which Cys-80 or Cys-303 was replaced by serine were also inactivated by menadione . However, enzyme in which Cys-298 was replaced by serine was insensitive to menadione . On the basis of these observations, it is suggested that menadione forms a thiodione-like adduct with Cys-298, leading to inactivation of the enzyme.

J Cataract Refract Surg, 1992 Nov, 18(6), 602 - 6
Cellular reaction following cataract surgery with implantation of the heparin-surface-modified intraocular lens in rabbits with experimental uveitis; Lundgren B et al.; The inflammatory response after cataract surgery and intraocular lens (IOL) implantation was studied in rabbits with endotoxin-induced uveitis . On days 1, 3, 7, 14, and 30 postoperatively the rabbits were sacrificed and the number of white blood cells in the aqueous humor and cellular deposits on the IOLs were estimated . On days 14 and 30 the rabbits also had slitlamp examination to study the clinical outcome of the surgery . At day 1 after lens extraction and IOL implantation, the number of white blood cells in the aqueous humor was significantly lower (P < .05) in eyes with heparin-surface-modified (HSM) IOLs (795.2 +/- 262.9; mean +/- SEM) than in eyes with poly(methyl methacrylate) (PMMA) lenses (1386.5 +/- 247.9) . No differences were seen at day 3, 7, 14, or 30 postoperatively . The choice of IOL material had no effect on the amount of cell deposits on the IOL surface or on clinical parameters such as anterior synechias, posterior synechias, fibrosis, and posterior capsular opacification . There was a trend toward a greater number of cellular deposits on the PMMA lenses, but this was not statistically significant . This study provides further evidence of improved biocompatibility of the HSM PMMA lens, as demonstrated by a decreased acute inflammatory response.

J Leukoc Biol, 1992 Nov, 52(5), 551 - 7
Augmented expression and release of nonspecific cross-reacting antigens (NCAs), members of the CEA family, by human neutrophils during cell activation; Kuroki M et al.; Nonspecific cross-reacting antigens (NCAs) are a group of human glycoproteins immunologically cross-reactive with carcinoembryonic antigen (CEA) . Our previous studies have shown that at least seven NCA glycoproteins different in molecular weight and antigenic reactivity, including a species corresponding to CD67, can be detected in neutrophil granulocytes . In the present paper, it is demonstrated that neutrophil activation induced with soluble stimulators, the calcium ionophore A23187, N-formylmethionyl-leucyl-phenylalanine, and phorbol myristate acetate, results in augmented release and cell surface expression of NCAs . The NCA release was correlated with the discharge of azurophil granules but not with that of specific granules and was attributable to the release of NCA species of 53 and 30 kd . The increased NCA expression on the cell surface was due to increments of the NCAs of 160, 100 (CD67), 95, 90, 30, and 26 kd . These results, together with the previous findings that the CEA family members can mediate intercellular adhesion and bind Escherichia coli in vitro, imply that the neutrophil NCAs participate in the functions of neutrophils such as phagocytosis, chemotaxis, and adherence.

J Comput Assist Tomogr, 1992 Nov-Dec, 16(6), 941 - 3
CT and MRI of mycotic aneurysms of the abdominal aorta; Moriarty JA et al.; In two of three patients with mycotic aneurysms of the abdominal aorta, diagnosis was delayed, with a fatal outcome in one patient . A combination of studies, such as indium white blood cell scanning, and anatomical imaging modalities, such as CT and MRI, may be necessary to arrive at the correct diagnosis.

J Bacteriol, 1992 Nov, 174(22), 7436 - 44
Physiological consequences of DnaK and DnaJ overproduction in Escherichia coli; Blum P et al.; The physiological consequences of molecular chaperone overproduction in Escherichia coli are presented . Constitutive overproduction of DnaK from a multicopy plasmid containing large chromosomal fragments spanning the dnaK region resulted in plasmid instability . Co-overproduction of DnaJ with DnaK stabilized plasmid levels . To examine the effects of altered levels of DnaK and DnaJ in a more specific manner, an inducible expression system for dnaK and dnaJ was constructed and characterized . Differential rates of DnaK synthesis were determined by quantitative Western blot (immunoblot) analysis . Moderate levels of DnaK overproduction resulted in a defect in cell septation and formation of cell filaments, but co-overproduction of DnaJ overcame this effect . Further increases in the level of DnaK terminated culture growth despite increased levels of DnaJ . DnaK overproduction was found to be bacteriocidal, and this effect was also partially suppressed by DnaJ . The bacteriocidal effect was apparent only with cultures which were allowed to enter stationary phase, indicating that DnaK toxicity is growth phase dependent.

J Bacteriol, 1992 Nov, 174(22), 7202 - 6
Characterization of the Escherichia coli membrane domain responsible for binding oriC DNA; Chakraborti A et al.; It has previously been shown that hemimethylated DNA from the Escherichia coli replication origin (oriC) binds with high specificity to membrane fractions isolated from disrupted cells . In this article, the membrane localization of oriC-binding activity was studied by subjecting crude membrane preparations to successive cycles of sedimentation and flotation gradient analysis . This revealed that approximately two-thirds of the membrane-associated oriC-binding activity of the cell was not associated with the outer membrane fraction as previously suggested but was recovered instead in a unique membrane fraction (OCB1) whose buoyant density and protein profile differed from those of both inner and outer membranes . The specific activity of oriC binding in OCB1 was approximately fivefold higher than the activity of the isolated outer membrane peak . It is likely that membrane fraction OCB1 includes the membrane domain responsible for the binding of hemimethylated oriC to the cell envelope in intact cells.

J Bacteriol, 1992 Nov, 174(22), 7174 - 9
An internal region of rpoB is required for autogenous translational regulation of the beta subunit of Escherichia coli RNA polymerase; Passador L et al.; In order to delineate the region involved in feedback regulation of the RNA polymerase beta subunit (encoded by rpoB), a collection of rpoB-lacZ translational fusions with different endpoints both upstream and downstream of the rpoB start site was assembled on lambda phage vectors . The extent of translational repression of beta was monitored by measuring beta-galactosidase levels in monolysogens of the fusions under conditions of increased intracellular concentrations of beta and beta' achieved via the induction of rpoBC expression from a multicopy plasmid . A construct containing as little as 29 bp upstream of the start of rpoB exhibited repression of beta-galactosidase activity to the same extent as a construct encoding the full upstream region . A construct which carried only 70 bp of the rpoB structural gene exhibited very little repression, while constructs which carried 126 or 221 bp of rpoB exhibited approximately the same degree of repression as a construct which carried 403 bp . These data suggest that the sequences important for feedback regulation of beta translation extend more than 70 bp into rpoB but are completely contained within a region which spans the sequences from 29 bp upstream to 126 bp downstream of the translational start site.

J Bacteriol, 1992 Nov, 174(22), 7121 - 7
Repression of Escherichia coli purB is by a transcriptional roadblock mechanism; He B et al.; Escherichia coli purB is regulated by a repressor-operator interaction . The purB operator is 242 bp downstream from the transcription start site and overlaps condons 62 to 67 in the protein-coding sequence (B . He, J . M . Smith, and H . Zalkin, J . Bacteriol . 174:130-136, 1992) . The mechanism by which the repressor-operator interaction functions to repress transcription was investigated by a combination of promoter replacement experiments and RNA analyses . By using a trp promoter replacement that deleted 5' flanking DNA to position -986, purB expression was increased sevenfold, yet normal two- to threefold regulation was maintained . This indicates that repressor-operator control is independent of the purB promoter and other 5' flanking sequences . Transcriptional regulation was likewise independent of coupled translation . An approximately 260-nucleotide truncated in vivo purB mRNA was identified which was dependent upon repressor-operator interaction . Thus, binding of purine repressor to the purB operator inhibits transcription elongation by a roadblock mechanism . The roadblock was not influenced by a sevenfold increase in promoter strength or by an operator mutation resulting in a 2.5-fold increase in repressor-operator affinity.

J Bacteriol, 1992 Nov, 174(22), 7104 - 11
Regulation of narK gene expression in Escherichia coli in response to anaerobiosis, nitrate, iron, and molybdenum; Kolesnikow T et al.; The regulation of the narK gene in Escherichia coli was studied by constructing narK-lacZ gene and operon fusions and analyzing their expression in various mutant strains in response to changes in cell growth conditions . Expression of narK-lacZ was induced 110-fold by a shift to anaerobic growth and a further 8-fold by the presence of nitrate . The fnr gene product mediates this anaerobic response, while nitrate control is mediated by the narL, narX, and narQ gene products . The narX and narQ gene products were shown to sense nitrate independently of one another and could each activate narK expression in a NarL-dependent manner . We provide the first evidence that NarL and FNR interact to ensure optimal expression of narK . IHF and Fis proteins are also required for full activation of narK expression, and their roles in DNA bending are discussed . Finally, the availability of molybdate and iron ions is necessary for optimal narK expression, whereas the availability of nitrite is not . Although the role of the narK gene product in cell metabolism remains uncertain, the pattern of narK gene expression is consistent with a proposed role of NarK in nitrate uptake by the cell for nitrate-linked electron transport.

Genes Dev, 1992 Nov, 6(11), 2214 - 20
Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration; Iwasaki H et al.; The Escherichia coli ruvA and ruvB genes are involved in DNA repair and in the late step of homologous genetic recombination . We have demonstrated previously that the RuvA-RuvB protein complex in the presence of ATP promotes reabsorption of cruciform structures extruded from a supercoiled plasmid with an inverted repeat sequence . Because the cruciform structure is topologically analogous to the Holiday structure, we have proposed that the role of the RuvA and RuvB proteins in recombination is to promote a strand exchange reaction at the Holliday junction . Here, we studied the specific interaction of the RuvA-RuvB complex with the Holliday structure using synthetic analogs prepared by annealing four oligonucleotides . The affinities of the RuvA protein for synthetic Holliday junctions are much higher (> 20-fold) than for duplex DNA, and the affinities of the RuvA protein for the junctions are further enhanced (> 4-fold) by the interaction with the RuvB protein . The RuvA-RuvB protein complex in the presence of ATP promotes dissociation of the synthetic Holliday junction with homology in the central core into two halves by catalyzing branch migration to the DNA ends, but it does not affect the structure of the synthetic Holliday junction without the homology . The separation of the synthetic Holliday junction is a result of the activity of the RuvA-RuvB complex that promotes strand exchange and DNA unwinding . Furthermore, RuvA and RuvB promote the strand exchange reaction at the Holliday junctions made by RecA . These results provide further evidence that the RuvA-RuvB complex recognizes the Holliday junction and promotes branch migration in homologous recombination.

Genes Dev, 1992 Nov, 6(11), 2066 - 76
Interaction of HTLV-1 Tax1 with p67SRF causes the aberrant induction of cellular immediate early genes through CArG boxes; Fujii M et al.; Tax1 of human T-cell leukemia virus type 1 (HTLV-1) is a transcriptional activator for viral gene expression and is also a transforming protein through inducing the expression of several cellular genes under the control of mitogenic signals . We identified the CArG boxes as a Tax1-responsive cis-acting element for the cellular immediate early genes c-fos, egr-1, and egr-2 . Using a chimeric protein consisting of the CArG-binding factor p67SRF and the heterologous DNA-binding domain of a yeast transcription factor GAL4, we demonstrated that Tax1 activates the transcriptional activity of p67SRF through the GAL4-binding site . The carboxy-terminal half of p67SRF, which lacks domains for DNA-binding, dimerization, and ternary complex formation with p62TCF, was sufficient for the activation by Tax1 . Tax1 produced in Escherichia coli bound p67SRF in vitro . The complex formation in vivo was also indicated by the finding that the acidic activation domain of VP16, by fusion to p67SRF, can complement the transcriptional activation function of a mutant Tax1 in trans . Thus, Tax1 activates CArG-mediated transcription without mitogenic signals through interaction with a CArG-binding factor, p67SRF . This must be one of the primary steps by which Tax1 causes aberration in growth control of the infected cells.

Exp Parasitol, 1992 Nov, 75(3), 308 - 22
Schistosoma mansoni: cloning of a complementary DNA encoding a cytosolic Cu/Zn superoxide dismutase and high-yield expression of the enzymatically active gene product in Escherichia coli; Hong Z et al.; We recently purified a 16-kDa cytosolic Cu/Zn superoxide dismutase (CT Cu/Zn-SOD) from Schistosoma mansoni, a human parasite . Three peptide sequences were obtained, one from the unblocked N-terminal and two from internal peptides which were generated by digestions with trypsin and cyanogen bromide . These sequences were aligned to the corresponding sequences of 19 cytosolic Cu/Zn-SODs from various species . Degenerate oligonucleotides were then designed according to the sequence and the position of each peptide . The oligonucleotides were used to amplify a complete cDNA using the polymerase chain reaction with either adult schistosome total RNA or a cercariae lambda gt11 phage cDNA library as the template . The protein encoded by the cDNA has 153 amino acids with a calculated molecular weight of 15,693 . It also has 60-65% homology to 19 cytosolic Cu/Zn-SOD from various species . All of the copper/zinc binding sites and SOD activity sites are conserved . Computer analysis predicts that the Cu/Zn-SOD has a pI value of 6.6, which is very close to the experimental results of IEF analysis (6.0 and 6.3) . The entire coding sequence from the cDNA was cloned into a bacterial alkaline phosphatase cytosolic expression vector and a large amount of soluble product was expressed and purified to homogeneity . We compared the bacterially expressed Cu/Zn-SOD with the native enzyme derived from schistosomes and found that they are identical by the following criteria: (1) They focus at the same positions on IEF gels; (2) they form dimers in solution as measured by gel filtration; (3) they have the same unblocked N-terminal sequence; (4) they both are enzymatically active with comparable specific activities . The specific activity of the bacterially derived enzyme was increased somewhat (approximately 10%) by incubation with copper and zinc ions.

Exp Cell Res, 1992 Nov, 203(1), 1 - 4
Addition of an ER retention signal to the ricin A chain increases the cytotoxicity of the holotoxin; Wales R et al.; With the exception of diphtheria toxin, which translocates from acidified endosomes, the intracellular organelle from which the catalytic moieties of several plant and bacterial toxins enter the target cell during endocytic uptake has not been identified . We have recently proposed that some toxins may travel the entire secretory pathway in reverse, moving from the cell surface to the lumen of the ER, before entering the cytosol . Several bacterial toxins have the ER retention sequence KDEL or a related analogue at their carboxyl termini, suggesting that the KDEL receptor may play a role in delivering these toxins to the ER . Here we provide further support for this possibility since the cytotoxicity of ricin, which lacks a KDEL sequence, can be significantly increased by adding KDEL to the C-terminus of its A chain.

Eur J Biochem, 1992 Nov 1, 209(3), 985 - 92
A substrate-like form of plasminogen-activator-inhibitor type 1 . Conversions between different forms by sodium dodecyl sulphate; Urano T et al.; Recombinant plasminogen-activator-inhibitor type 1 (PAI-1) purified in an active form from Escherichia coli and eucaryotic cells was found to contain a mixture of three functionally distinct forms: an active form that forms complexes with plasminogen activators (PAs), an inactive (latent) form that remains intact after incubation with PAs, and a substrate-like form which is easily cleaved by PAs . Since active PAI-1 purified from bacteria (rpPAI-1) contains only trace amounts of the inactive latent and the substrate-like forms, this material was used to study the effect of sodium dodecyl sulphate (SDS) on the structure and function of active PAI-1 . After treatment with 0.01% SDS, active rpPAI-1 was converted to an inactive form that did not form complexes with PAs, but exhibited characteristics similar to those of latent PAI-1 . After treatment with 0.1% SDS, PAI-1 lost its inhibitory activity and was cleaved as a substrate in the reactive center . Circular dichroism spectral analysis reveals that SDS changed the conformation of PAI-1 dramatically, mainly by increasing its alpha-helical content.

Eur J Biochem, 1992 Nov 1, 209(3), 945 - 50
Specific phosphorylation of the acidic central region of the N-myc protein by casein kinase II; Hagiwara T et al.; The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins . Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein . We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII) . Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII . For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells . Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins . Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc . However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low . Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII . Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263 . On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions . These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro . The functional significance of this acidic domain is discussed.

Eur J Biochem, 1992 Nov 1, 209(3), 933 - 7
Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin . Molecular cloning and expression studies; Abe M et al.; A full-length cDNA clone for a cysteine proteinase inhibitor (cystatin) was isolated from a lambda gt10 cDNA library of immature corn kernels by screening with a mixture of cDNA inserts for oryzacystatins I and II . The cDNA clone spans 960 base pairs, encoding a 135-amino-acid protein containing a signal peptide fragment . The protein, named corn cystatin I, is considered to be a member of the cystatin superfamily, since it contains the commonly conserved Gln-Val-Val-Ala-Gly region that exists in most known cystatins as a probable binding site and is significantly similar to other cystatins in its overall amino acid sequence . Corn cystatin I expressed in Escherichia coli showed a strong papain-inhibitory activity . Northern blot analysis showed that the amount of mRNA for corn cystatin I reaches a maximum 2 weeks after flowering and then decreases gradually.

Eur J Biochem, 1992 Nov 1, 209(3), 869 - 74
Mutational replacements in subtilisin 309 . Val104 has a modulating effect on the P4 substrate preference; Bech LM et al.; The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated . The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site-directed mutagenesis . It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue . With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates . The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change . However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket . The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.

Endocrinology, 1992 Nov, 131(5), 2419 - 29
Antipeptide polyclonal antibodies specifically recognize each human thyroid hormone receptor isoform; Falcone M et al.; We characterized four antipeptide polyclonal antibodies (abs) able to specifically recognize each thyroid hormone receptor (TR) isoform . The abs immunoprecipitated both the in vitro synthesized receptor and the receptor expressed in E . coli and their specificity was confirmed by competition studies and immunohistochemistry . Ab activity measured by enzyme-linked immunosorbent assay decreased after preabsorption of each ab with the immunizing peptide or the specific receptor protein expressed in E . coli . No specific activity was detectable in enzyme-linked immunosorbent assay, no nuclear staining was observed after affinity column immunoabsorption, and the specific bands obtained in Western blot analysis disappeared after preabsorption with the specific TR isoform expressed in E . coli . By immunohistochemical studies we detected coordinate expression of each receptor isoform in most tissues examined . However, in heart and muscle, the beta-isoform is expressed at a very low level compared to the alpha-isoform in spite of the significant TR beta mRNA levels previously demonstrated by Northern blot analysis . We also demonstrated a different pattern of distribution of alpha- and beta-isoforms in rat testis . In this tissue the TR alpha is significantly expressed in spermatogonia nuclei, but in spermatids the beta-isoform is predominant, and only the TR beta is detectable in mature spermatozoa.

Curr Genet, 1992 Nov, 22(5), 399 - 406
Transformation of Acremonium coenophialum, a protective fungal symbiont of the grass Festuca arundinacea; Tsai HF et al.; Acremonium coenophialum is a mutualistic mycosymbiont and natural agent of biological protection of the widely distributed grass Festuca arundinacea (tall fescue) . An electroporative transformation system was developed for A . coenophialum . Segments of DNA 5' to the beta-tubulin gene (tub2) of the closely related ascomycete Epichloe typhina, fused to the Escherichia coli hph gene encoding hygromycin B phosphotransferase, conferred hygromycin resistance when introduced into A . coenophialum by electroporation . The incorporation of the Emericella nidulans trpC terminator greatly increased protoplast germination on selective medium and improved transformation efficiencies 30-200% depending on the plasmid construct . Plasmid pCSN43, which incorporates the trpC controlling elements for hph expression, was also used to transform A . coenophialum . Southern blot analysis of ten pCSN43 transformants indicated the possibility of random integration of this vector into the genome.

Blood, 1992 Nov 1, 80(9), 2188 - 95
A controlled trial of recombinant human granulocyte-macrophage colony-stimulating factor after total body irradiation, high-dose chemotherapy, and autologous bone marrow transplantation for acute lymphoblastic leukemia or malignant lymphoma; Link H et al.; Infections during granulocytopenia are major complications of autologous bone marrow transplantation (ABMT) . Since recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) has proved to accelerate bone marrow recovery after cytostatic chemotherapy, we studied its effects on hematopoietic regeneration and on infectious complications after total body irradiation (TBI) and high-dose chemotherapy followed by ABMT . Eighty-one patients with acute lymphoblastic leukemia (ALL) in complete remission (CR) or with non-Hodgkin's lymphoma (NHL) in CR or partial remission were randomized in a double-blind, placebo-controlled trial . They received either rhuGM-CSF 250 micrograms/m2 (Escherichia coli-derived) daily by continuous infusion after ABMT, or placebo . Treatment was continued until the neutrophil counts reached greater than 500/microL for 1 week . The maximum treatment duration was 30 days . Thirty-nine patients in the rhuGM-CSF group and 40 patients in the placebo group were evaluable . The median time needed to reach a neutrophil count of 500/microL was 15 days with rhuGM-CSF and 28 days with placebo (P = .0001) . Bacterial infections occurred in 14 (35.9%) of the patients with rhuGM-CSF and in 25 (62.5%) of the patients given the placebo (P = .024) . Nine of the 14 bacterial infections in the rhuGM-CSF group and 20 of the 25 infections in the placebo group were diagnosed within the first 10 days after ABMT . Capillary leakage and a reversible fluid retention were seen in five of the rhuGM-CSF-treated patients . Patients treated with rhuGM-CSF had lower serum protein and albumin levels than patients in the placebo group . There was no statistically relevant difference in overall survival between the two groups (P = .47) . Relapse occurred in 14 (34%) patients with rhuGM-CSF and in 18 (45%) patients with placebo . We conclude that continuous infusion of rhuGM-CSF after ABMT accelerates the regeneration of granulocytes and reduces the number of bacterial infections.

Plant Mol Biol, 1992 Nov, 20(3), 459 - 65
Purification and characterization of seven chloroplast ribosomal proteins: evidence that organelle ribosomal protein genes are functional and that NH2-terminal processing occurs via multiple pathways in chloroplasts; Schmidt J et al.; Putative genes for 21 ribosomal proteins (RPs) have been identified in the chloroplast DNA of four plants by nucleotide sequencing and homology comparison but few of the gene products have been characterized . Here we report the purification and N-terminal sequencing of seven proteins from the spinach chloroplast ribosome . The data show them to be the homologues of Escherichia coli RPs L20, L32, L33, L36, S12, S16 and S19, and thus support the view that their genes identified in the chloroplast DNA represent functional genes . The initiating methionine residue was not detected in the mature protein in most cases but it was present in S16, indicating that only the formyl group is removed in this case . This result and the previously reported finding of N-methyl alanine at the N-terminus of chloroplast L2 indicate the existence of multiple N-terminal processing pathways in the chloroplast.

Biotechniques, 1992 Nov, 13(5), 780 - 9
A rapid and reliable method to create tandem arrays of short DNA sequences; Graham GJ et al.; Tandemly polymerized regulatory elements, antisense RNA segments or ribozymes are potentially useful in selective gene silencing . However, existing methods of tandemly polymerizing short DNA segments are laborious . We present a procedure that can create cloned arrays of 40-70 monomer units in two steps . We have created long arrays of regulatory elements and potential ribozyme sequences . Silencing of human immunodeficiency virus (HIV-1) activation by tandem arrays of a regulatory element in human immune system cells and in other human and monkey cells is discussed.

Arch Biochem Biophys, 1992 Nov 1, 298(2), 594 - 601
Characterization of the proto-oncogene pim-1: kinase activity and substrate recognition sequence; Friedmann M et al.; The human pim-1 proto-oncogene was expressed in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein and the enzymatic properties of its kinase activity were characterized . Likewise, a Pim-1 mutant lacking intrinsic kinase activity was constructed by site-directed mutagenesis (Lys67 to Met) and expressed in E . coli . In vitro assays with the mutant Pim-1 kinase showed no contaminating kinase activity . The wild-type Pim-1 kinase-GST fusion protein showed a pH optimum of 7 to 7.5 and optimal activity was observed at either 10 mM MgCl2 or 5 mM MnCl2 . Higher cation concentrations were inhibitory, as was the addition of NaCl to the assays . Previous work by this laboratory assaying several proteins and peptides showed histone H1 and the peptide Kemptide to be efficiently phosphorylated by recombinant Pim-1 kinase . Here we examine the substrate sequence specificity of Pim-1 kinase in detail . Comparison of different synthetic peptide substrates showed Pim-1 to have a strong substrate preference for the peptide Lys-Arg-Arg-Ala-Ser*-Gly-Pro with an almost sixfold higher specificity constant kcat/Km over that of the substrate Kemptide (Leu-Arg-Arg-Ala-Ser*-Leu-Gly) . The presence of basic amino acid residues on the amino terminal side of the target Ser/Thr was shown to be essential for peptide substrate recognition . Furthermore, phosphopeptide analysis of calf thymus histone H1 phosphorylated in vitro by Pim-1 kinase resulted in fragments containing sequences similar to that of the preferred synthetic substrate peptide shown above . Therefore, under optimized in vitro conditions, the substrate recognition sequence for Pim-1 kinase is (Arg/Lys)3-X-Ser/Thr*-X', where X' is likely neither a basic nor a large hydrophobic residue.

Arch Biochem Biophys, 1992 Nov 1, 298(2), 498 - 504
A reactive nucleophile proximal to vicinal thiols is an evolutionarily conserved feature in the mechanism of Arg aminoacyl-tRNA protein transferase; Berleth ES et al.; Aminoacyl-tRNA protein transferases post-translationally aminoacylate protein N-termini . At least in part, these enzymes function to allow a subset of cellular proteins to be targeted for protein degradation . A eukaryotic enzyme of this class, Arg aminoacyl-tRNA protein transferase, arginylates N-terminal Glu or Asp residues of proteins, allowing such proteins to be recognized by a specific ubiquitin-protein ligase . We showed previously that inorganic arsenite, a reagent expected to bind specifically to protein vicinal thiol groups, inhibited Arg aminoacyl-tRNA transferase activity in rabbit reticulocyte lysate (N . S . Klemperer and C . M . Pickart, 1989, J . Biol . Chem . 264, 19245-19252) . We now report that a bifunctional arsenoxide reagent, p-{(bromoacetyl)-amino}phenylarsenoxide, is a potent and irreversible inactivator of the same enzyme (K0.5 = 11.5 microM) . Bromoacetyl aniline, which lacks the arsenoxide moiety, has no effect . These results show that the transferase has a reactive nucleophile proximal to the site which binds arsenoxides . The related monofunctional arsenoxide reagent, p-aminophenylarsenoxide, is a reversible inhibitor whose potency (K0.5 = 7.7 microM) is 20-fold greater than that of inorganic arsenite . As expected for a mechanism in which p-aminophenylarsenoxide binds to vicinal thiol groups: (i) pretreatment of reticulocyte lysate with a thiol-blocking reagent prevents binding of the transferase to a phenylarsenoxide-Sepharose column; and (ii) inhibition by p-aminophenylarsenoxide is reversed by a competing chemical dithiol, but not by a monothiol reagent . Like the rabbit enzyme, Arg aminoacyl-tRNA protein transferase from the yeast Saccharomyces cerevisiae (expressed in Escherichia coli) is reversibly inhibited by the monofunctional phenylarsenoxide and irreversibly inactivated by the bifunctional phenylarsenoxide (but not by bromoacetylaniline) . Thus, a reactive nucleophile proximal to vicinal thiol groups is a conserved feature of the activity of the transferase . We speculate that these groups are catalytic elements in the transferase active site.

Arch Biochem Biophys, 1992 Nov 1, 298(2), 332 - 9
Slow-onset inhibition of ribosomal peptidyltransferase by lincomycin; Kallia-Raftopoulos S et al.; In a system derived from Escherichia coli, we carried out a detailed kinetic analysis of the inhibition of the puromycin reaction by lincomycin . N-Acetylphenylalanyl-tRNA (Ac-Phe-tRNA; the donor) reacts with excess puromycin (S) according to reaction {1}, C+S Ks <--> CS k3 --> C'+P, where C is the Ac-Phe-tRNA-poly(U)-ribosome ternary complex (complex C) . The entire course of reaction {1} appears as a straight line when the reaction is analyzed as pseudo-first-order and the data are plotted in a logarithmic form (logarithmic time plot) . The slope of this straight line gives the apparent ksobs = k3{S}/(Ks + {S}) . In the presence of lincomycin the logarithmic time plot is not a straight line, but becomes biphasic, giving an early slope (ke = k3{S}/(Ks(1 + {I}/Ki) + {S})) and a late slope (k1 = k3{S}/(Ks(1 + {I}/K'i + {S})) . Kinetic analysis of the early slopes at various concentrations of S and I shows competitive inhibition with Ki = 10.0 microM . The late slopes also give competitive inhibition with a distinct inhibition constant K'i = 2.0 microM . Excluding alternative models, the two phases of inhibition are compatible with a model in which reaction {1} is coupled with reaction {2}, C+I k4 <--> k5 CI k6 <--> k7 C*I, where the isomerization step CI <--> CI* is slower than the first step C+I <--> CI, Ki = k5/k4 and K'i = Ki {k7/(k6 + k7)} . Corroborative evidence for this model comes from the examination of reaction {2} alone in the absence of S . This reaction is analyzed as pseudo-first-order going toward equilibrium with kIeq = k7 + (k6 {I}/(Ki + {I})) . The plot of kIeq versus {I} is not linear . This plot supports the two-step mechanism of reaction {2} in which k6 = 5.2 min-1 and k7 = 1.3 min-1 . This is the first example of slow-onset inhibition of ribosomal peptidyltransferase which follows a simple model leading to the determination of the isomerization constants k6 and k7 . We suggest that lincomycin inhibits protein synthesis by binding initially to the ribosome in competition with aminoacyl-tRNA . Subsequently, as a result of a conformational change, an isomerization occurs (CI <--> C*I), after which lincomycin continues to interfere with the binding of aminoacyl-tRNA to the isomerized complex.

Mol Cell Biol, 1992 Nov, 12(11), 5059 - 68
Conformational activation of a basic helix-loop-helix protein (MyoD1) by the C-terminal region of murine HSP90 (HSP84); Shaknovich R et al.; A murine cardiac lambda gt11 expression library was screened with an amphipathic helix antibody, and a recombinant representing the C-terminal 194 residues of murine HSP90 (HSP84) was cloned . Both recombinant and native HSP90s were then found to rapidly convert a basic helix-loop-helix protein (MyoD1) from an inactive to an active conformation, as assayed by sequence-specific DNA binding . The conversion process involves a transient interaction between HSP90 and MyoD1 and does not result in the formation of a stable tertiary complex . Conversion does not require ATP and occurs stoichiometrically in a dose-dependent fashion . HSP90 is an abundant, ubiquitous, and highly conserved protein present in most eukaryotic cells . These results provide direct evidence that HSP90 can affect the conformational structure of a DNA-binding protein.

J Virol, 1992 Nov, 66(11), 6781 - 3
Human immunodeficiency virus type 1 gag-protease fusion proteins are enzymatically active; Kotler M et al.; We have introduced mutations into the region of the genome of human immunodeficiency virus type 1 (HIV-1) that encodes the cleavage sites between the viral protease (PR) and the adjacent upstream region of the polyprotein precursor . Segments containing these mutations were introduced into plasmids, and the retroviral proteins were expressed in Escherichia coli . The mutations prevented cleavage between the PR and the adjacent polypeptide; however, other PR cleavage sites in the polyprotein were cleaved normally, showing that the release of free PR is not a prerequisite for the appropriate processing of HIV-1 precursors.

J Virol, 1992 Nov, 66(11), 6714 - 20
A recombinant human Fab expressed in Escherichia coli neutralizes rabies virus; Cheung SC et al.; A recombinant human anti-rabies monoclonal antibody (MAb-57) Fab was prepared by cloning the heavy (Fd)- and light-chain domains into the same bacterial expression vector . To construct the recombinant Fab, mRNA was extracted from MAb-57-producing hybridoma cells, reverse transcribed, and then amplified by polymerase chain reaction (PCR) by using oligonucleotides specific for immunoglobulin heavy- and light-chain DNA sequences . PCR-amplified Fd-chain cDNA was fused, in frame, between a bacterial leader peptide (PelB) at the amino terminus and a 10-amino-acid peptide tag at the carboxy terminus . The PCR-amplified lambda-chain cDNA was also fused to the PelB leader peptide . The immunoglobulin Fab was then expressed as a dicistronic message in bacteria by using the isopropyl-beta-D-thiogalactopyranoside-inducible lactose promotor (lacZ) . DNA sequencing was used to define the gamma-chain isotype (immunoglobulin G1) and VH (VHI) chain and VL (V lambda II) chain gene usage . The recombinant Fab (rFab57) specifically bound the rabies virus coat glycoprotein, while the Fd and lambda chains, when expressed individually, did not . The binding specificity of rFab57 was indistinguishable from that of the intact MAb in direct enzyme-linked immunosorbent assays; however, the dissociation constant of rFab57 for rabies virus protein G was approximately 1 log10 U lower than that of complete MAb-57 in competition enzyme-linked immunosorbent assays . A fluorescent-focus inhibition assay showed that bacterially expressed rFab was capable of neutralizing rabies virus strain CVS-11 . We conclude that a human Fab expressed in bacteria maintains its specificity and biologic activity.

J Virol, 1992 Nov, 66(11), 6470 - 9
Transcription of viral late genes is dependent on expression of the viral intermediate gene G8R in cells infected with an inducible conditional-lethal mutant vaccinia virus; Zhang Y et al.; There are three temporal classes of vaccinia virus genes: early, intermediate, and late . The object of this study was to determine the effects on virus replication of regulating the expression of G8R, an intermediate gene that encodes a late transcription factor . We inserted the lac operator adjacent to the RNA start site of the G8R gene in a recombinant vaccinia virus that constitutively expresses the Escherichia coli lac repressor to make expression of the G8R gene dependent on the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) . In case repression would not be complete, we also weakened the promoter of the G8R gene by making a single-nucleotide substitution designed to reduce its basal level of transcription . The mutant virus replicated well in the presence of the inducer, although synthesis of the G8R-encoded 30,000-M(r) protein was only 10% of that of the wild-type virus . In the absence of IPTG, (i) synthesis of the G8R protein was inhibited by more than 99% relative to that of the wild-type virus, (ii) synthesis of early and intermediate mRNAs appeared to be unaffected, (iii) intermediate proteins accumulated to higher than normal levels, (iv) synthesis of late mRNA and protein was reduced by about 90%, (v) viral DNA was replicated but incompletely resolved concatemeric molecules accumulated, (vi) not even the earliest stages of virion assembly were detectable by transmission electron microscopy, and (vii) virus yield under one-step growth conditions and plaque formation were 10(-3) and 10(-4) times the wild-type values, respectively . The defect in late gene expression could be overcome by transfection of a G8R gene that was not under lac operator control, as well as by addition of IPTG, further demonstrating the specificity of the repression . The correlation between decreased expression of the G8R intermediate gene and inhibition of late mRNA synthesis is consistent with the notion that the G8R product serves as an essential late transcription factor and supports a cascade mechanism of vaccinia virus gene regulation . In addition, the inducer-dependent vaccinia virus mutant provided a tool for selective inhibition of late gene expression while allowing synthesis of early and intermediate mRNAs and proteins.

J Immunol, 1992 Nov 1, 149(9), 2879 - 86
C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including CD9, CD37, CD53, and CD63; Imai T et al.; C33 Ag was originally identified by mAb inhibitory to syncytium formation induced by human T cell leukemia virus type 1 . The Ag was shown to be a highly heterogeneous glycoprotein consisting of a 28-kDa protein and N-linked oligosaccharides ranging from 10 to 50 kDa . In the present study, cDNA clones were isolated from a human T cell cDNA expression library in Escherichia coli by using mAb C33 . The identity of cDNA was verified by immunostaining and immunoprecipitation of transfected NIH3T3 cells with mAb . The cDNA contained an open reading frame of a 267-amino acid sequence which was a type III integral membrane protein of 29.6 kDa with four putative transmembrane domains and three putative N-glycosylation sites . The C33 gene was found to belong to a newly defined family of genes for membrane proteins, such as CD9, CD37, CD53, CD63, and TAPA-1, and was identical to R2, a cDNA recently isolated because of its strong up-regulation after T cell activation . Availability of mAb for C33 Ag enabled us to define its distribution in human leukocytes . C33 Ag was expressed in CD4+ T cells, CD19+ B cells, CD14+ monocytes, and CD16+ granulocytes . Its expression was low in CD8+ T cells and mostly negative in CD16+ NK cells . PHA stimulation enhanced the expression of C33 Ag in CD4+ T cells by about 5-fold and in CD8+ T cells by about 20-fold . PHA stimulation also induced the dramatic size changes in the N-linked sugars previously shown to accompany human T cell leukemia virus type 1-induced transformation of CD4+ T cells.

J Bacteriol, 1992 Nov, 174(21), 7013 - 25
Molecular cloning, sequencing, and overexpression of the structural gene encoding the delta subunit of Escherichia coli DNA polymerase III holoenzyme; Carter JR et al.; Using an oligonucleotide hybridization probe, we have mapped the structural gene for the delta subunit of Escherichia coli DNA polymerase III holoenzyme to 14.6 centisomes of the chromosome . This gene, designated holA, was cloned and sequenced . The sequence of holA matches precisely four amino acid sequences obtained for the amino terminus of delta and three internal tryptic peptides . A holA-overproducing plasmid that directs the expression of delta up to 4% of the soluble protein was constructed . Sequence analysis of holA revealed a 1,029-bp open reading frame that encodes a protein with a predicted molecular mass of 38,703 Da . holA may reside downstream of rlpB in an operon, perhaps representing yet another link between structural genes for the DNA polymerase III holoenzyme and proteins involved in membrane biogenesis . These and other features are discussed in terms of genetic regulation of delta-subunit synthesis.

J Bacteriol, 1992 Nov, 174(21), 6904 - 10
Function of the N-terminal half of RepA in activation of Rts1 ori; Terawaki Y et al.; The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for replication . A mutant repA gene, repA delta C143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but at a low frequency, an Escherichia coli polA strain, JG112, when repA delta C143 was cloned into pBR322 with Rts1 ori in the natural configuration . The transformation was less efficient without the dyad DnaA box in the ori region, and no transformation occurred at 42 degrees C, characteristic of Rts1 replication . A fusion of the 3'-terminal half of repA of the P1 plasmid to repA delta C143 yielded a pBR322 chimeric plasmid that contained Rts1 ori through hybrid (Rts1-P1) repA . This plasmid was maintained much more stably in JG112 at 37 degrees C . At 42 degrees C, however, it was quite unstable . The overproduced hybrid RepA protein showed interference with mini-Rts1 replication in trans and also exhibited an autorepressor function, although both activities were decreased . These findings suggest that the N-terminal half of the RepA molecule of Rts1 is involved in the activation of the replication origin.

J Bacteriol, 1992 Nov, 174(21), 6862 - 71
Sequence elements in the Escherichia coli araFGH promoter; Hendrickson W et al.; The Escherichia coli araFGH operon codes for proteins involved in the L-arabinose high-affinity transport system . Transcriptional regulation of the operon was studied by creating point mutations and deletions in the control region cloned into a GalK expression vector . The transcription start site was confirmed by RNA sequencing of transcripts . The sequences essential for polymerase function were localized by deletions and point mutations . Surprisingly, only a weak -10 consensus sequence, and no -35 sequence is required . Mutation of a guanosine at position -12 greatly reduced promoter activity, which suggests important polymerase interactions with DNA between the usual -10 and -35 positions . A double mutation toward the consensus in the -10 region was required to create a promoter capable of significant AraC-independent transcription . These results show that the araFGH promoter structure is similar to that of the galP1 promoter and is substantially different from that of the araBAD promoter . The effects of 11 mutations within the DNA region thought to bind the cyclic AMP receptor protein correlate well with the CRP consensus binding sequence and confirm that this region is responsible for cyclic AMP regulation . Deletion of the AraC binding site nearest the promoter, araFG1, eliminates arabinose regulation, whereas deletion of the upstream AraC binding site, araFG2, has only a slight effect on promoter activity.

J Bacteriol, 1992 Nov, 174(21), 6844 - 51
The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein; Hauser J et al.; Inducible mutagenesis in Escherichia coli requires the direct action of the chromosomally encoded UmuDC proteins or functional homologs found on certain naturally occurring plasmids . Although structurally similar, the five umu-like operons that have been characterized at the molecular level vary in their ability to enhance cellular and phage mutagenesis; of these operons, the mucAB genes from the N-group plasmid pKM101 are the most efficient at promoting mutagenesis . During the mutagenic process, UmuD is posttranslationally processed to an active form, UmuD' . To explain the more potent mutagenic efficiency of mucAB compared with that of umuDC it has been suggested that unlike UmuD, intact MucA is functional for mutagenesis . To examine this possibility, we have overproduced and purified the MucA protein . Although functionally similar to UmuD, MucA was cleaved much more rapidly both in vitro and in vivo than UmuD . In vivo, restoration of mutagenesis functions to normally nonmutable recA430, recA433, recA435, or recA730 delta(umuDC)595::cat strains by either MucA+ or mutant MucA protein correlated with the appearance of the cleavage product, MucA' . These results suggest that most of the differences in mutagenic phenotype exhibited by MucAB and UmuDC correlate with the efficiency of posttranslational processing of MucA and UmuD rather than an inherent activity of the unprocessed proteins.

Infect Immun, 1992 Nov, 60(11), 4468 - 74
Some properties of purified Escherichia coli heat-stable enterotoxin II; Hitotsubashi S et al.; We examined the biological properties of purified Escherichia coli heat-stable enterotoxin II (STII) using mouse intestinal loop assays and compared these properties with those of heat-stable enterotoxin I (STI) and cholera toxin (CT) . The action of STII over time differed from those of STI and CT . STII did not alter cyclic GMP or cyclic AMP levels in intestinal mucosal cells . Our results supported the idea that the mechanism of action of STII in inducing fluid secretion is different from the mechanisms of action of STI and CT . This hypothesis was further supported by the fact that an anti-STII neutralizing serum did not neutralize the activities of STI and CT . Subsequently, we examined the involvement of prostaglandins in the action of STII . The level of prostaglandin E2 in the fluid accumulated as a result of the action of STII increased, and the prostaglandin synthesis inhibitors aspirin and indomethacin significantly reduced the response to STII . These results implicate prostaglandin E2 in the mechanism of action of STII.

Infect Immun, 1992 Nov, 60(11), 4460 - 7
Role of crl in avian pathogenic Escherichia coli: a knockout mutation of crl does not affect hemagglutination activity, fibronectin binding, or Curli production; Provence DL et al.; This study determined the role of crl in the production of curli by, the hemagglutination activity of, and fibronectin binding by avian pathogenic Escherichia coli chi 7122 . Curli, an extracellular structure that binds fibronectin, was recently described (A . Olsen, A . Jonsson, and S . Normark, Nature {London} 338:652-655, 1989) . The crl gene product was hypothesized to be the subunit monomer of curli and to bind fibronectin . E . coli HB101, which does not contain crl, binds fibronectin and produces curli when harboring a plasmid containing the crl gene . We show that HB101 hemagglutinates chicken erythrocytes when harboring the crl gene and that chi 7122 hemagglutinates chicken erythrocytes, binds fibronectin, and produces curli . Hemagglutination activity, fibronectin binding, and curli production are best expressed by chi 7122 and HB101 harboring the crl gene when the bacteria are grown on colonization factor antigen agar at 26 degrees C . The expression of hemagglutination activity, fibronectin binding, and curli production by both strains is decreased by growth on this agar at an increased temperature, of an increased osmolarity, or in an anaerobic atmosphere . This results indicates that the crl gene plays a role in the expression of the three phenotypes in HB101 and possibly in chi 7122 as well . We inactivated crl in chi 7122 by allele replacement in the expectation of abolishing hemagglutination activity, fibronectin binding, and curli production . The mutation was verified by Southern blot analysis and by a polymerase chain reaction, and there was no evidence of a second crl gene in chi 7122 . However, the mutant of chi 7122 lacking crl hemagglutinated chicken erythrocytes, bound fibronectin, and produced curli at wild-type levels . This result indicates that crl plays a nonessential role in the expression of these three phenotypes in chi 7122.

EMBO J, 1992 Nov, 11(11), 4219 - 25
DNA damage-inducible origins of DNA replication in Escherichia coli; Magee TR et al.; Upon induction of the SOS response in Escherichia coli, the mode of initiation of DNA replication is altered such that it can occur in the absence of normally required protein synthesis . This type of DNA replication has been termed induced stable DNA replication (iSDR) . We examined the origin usage during iSDR and found that the initiation of iSDR occurs primarily in the oriC and terC regions of the chromosome in a manner completely independent of transcription, translation and DnaA protein . Minichromosomes (oriC plasmids) pOC23 and pOC81 were induced to replicate in the absence of DnaA protein and transcription after SOS induction . The results localized one of the iSDR origin activities in a 596 bp region which includes the minimal oriC.

EMBO J, 1992 Nov, 11(11), 4167 - 73
The leaky UGA termination codon of tobacco rattle virus RNA is suppressed by tobacco chloroplast and cytoplasmic tRNAs(Trp) with CmCA anticodon; Zerfass K et al.; RNA-1 molecules from tobacco rattle virus (TRV) and pea early-browning virus (PEBV), two members of the tobravirus group, have recently been shown to contain internal, in-frame UGA termination codons which are suppressed in vitro . Our results suggest that a UGA stop codon also exists in RNA-1 of pepper ringspot virus (PRV), another tobravirus . UGA suppression may therefore be a universal feature of the expression of tobravirus genomes . We have isolated two natural suppressor tRNAs from uninfected tobacco plants on the basis of their ability to promote readthrough over the leaky UGA codon of TRV RNA-1 in a wheat germ extract depleted of endogenous mRNAs and tRNAs . Their amino acid acceptance and nucleotide sequences identify the two UGA-suppressor tRNAs as chloroplast (chl) and cytoplasmic (cyt) tryptophan-specific tRNAs with the anticodon CmCA . These are the first UGA suppressor tRNAs to be identified in plants . They have several interesting features . (i) Chl tRNA(Trp) suppresses the UGA stop codon more efficiently than cyt tRNA(Trp) . (ii) Chl tRNA(Trp) contains an A24:U11 pair in the D-stem as does the mutated Escherichia coli UGA-suppressor tRNA(Trp) which is a more active suppressor than wild-type tRNA(Trp) . (iii) The suppressor activity of chl tRNA(Trp) is dependent on the nucleotides surrounding the stop codon because it recognizes UGA in the TRV context but not the UGA in the beta-globin context.

EMBO J, 1992 Nov, 11(11), 4159 - 65
Recognition of bases in Escherichia coli tRNA(Gln) by glutaminyl-tRNA synthetase: a complete identity set; Hayase Y et al.; The fidelity of protein biosynthesis rests largely on the correct aminoacylation of transfer RNAs by their cognate aminoacyl-tRNA synthetases . Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl-tRNA synthetase (GlnRS) provides an excellent system for studying the basis of this highly specific recognition process . Correct aminoacylation depends on the set of nucleotides (identity elements) in tRNA(Gln) responsible for correct interaction with GlnRS . Specific contacts between tRNA(Gln) and GlnRS include the 2-amino group of guanosines . Therefore, we made a set of tRNA(Gln) variants in which specific guanosines were replaced by inosine using recombinant RNA technology . This resulted in a set of tRNAs that varied by single deletions of the amino group from guanine residues, thus allowing us to test the functional importance of these contacts . In addition, a number of mutants were made by transcription of mutated tRNA genes with base changes at position 10, 16 or 25 . In vitro aminoacylation of these mutants showed decreases in the specificity constant (kcat/KM) of up to 300-fold, with kcat being the parameter most affected . These experiments reveal G10 as a new element of glutamine identity . In addition, the interaction of G2, G3 and G10 with GlnRS via the 2-amino group is significant for tRNA discrimination . Based on these results, and on earlier data, we propose a complete set of bases as identity elements for tRNA(Gln).

EMBO J, 1992 Nov, 11(11), 3837 - 44
Only some members of a gene family in Trypanosoma cruzi encode proteins that express both trans-sialidase and neuraminidase activities; Uemura H et al.; Trypomastigotes, the blood stage form of the human parasite Trypanosoma cruzi, contain an enzyme on their surface, trans-sialidase, which catalyses the transfer of sialic acid from host glycoconjugates to acceptors on its own cell surface . At least a subset of the sialic acid-bearing acceptor molecules are involved in parasite invasion of host cells, an essential step in the life cycle of the parasite . Another trypomastigote surface enzyme that affects host cell invasion is neuraminidase and recent evidence suggests that both trans-sialidase and neuraminidase activities may be expressed by the same proteins on the parasite surface . We describe here the isolation and expression of several members of a trans-sialidase--neuraminidase gene family from T.cruzi . One of the isolated genes does indeed encode a protein with both trans-sialidase and neuraminidase activities, while other members of the gene family encode closely related proteins that express neither enzymatic activity . Chimeric protein constructs combining different portions of active and inactive genes identified a region of the gene necessary for enzymatic activity . Sequence analysis of this portion of the gene revealed a limited number of amino acid differences between the predicted active and inactive gene products.

J Bacteriol, 1992 Nov, 174(22), 7273 - 82
Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp . strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes; Brahamsha B et al.; The sigA gene of Anabaena sp . strain PCC 7120, encoding the principal RNA polymerase sigma factor, and the complement of the rpoD oligonucleotide (K . Tanaka, T . Shiina, and H . Takahashi, Science 242:1040-1042, 1988) were used as probes to isolate two genes, sigB and sigC, which encode two putative sigma factors exhibiting high degrees of similarity to SigA, to HrdA, -B, -C, and -D of Streptomyces coelicolor, and to KatF of Escherichia coli . sigB and sigC code for polypeptides of 332 and 416 amino acids with predicted molecular weights of 38,431 and 47,459, respectively . sigB and sigC mRNAs are detectable only under nitrogen-limiting conditions . Insertional inactivation of sigB and sigC indicates that neither gene alone is essential for nitrogen fixation or heterocyst differentiation.

J Bacteriol, 1992 Nov, 174(22), 7128 - 37
How a mutation in the gene encoding sigma 70 suppresses the defective heat shock response caused by a mutation in the gene encoding sigma 32; Zhou YN et al.; In Escherichia coli, transcription of the heat shock genes is regulated by sigma 32, the alternative sigma factor directing RNA polymerase to heat shock promoters . sigma 32, encoded by rpoH (htpR), is normally present in limiting amounts in cells . Upon temperature upshift, the amount of sigma 32 transiently increases, resulting in the transient increase in transcription of the heat shock genes known as the heat shock response . Strains carrying the rpoH165 nonsense mutation and supC(Ts), a temperature-sensitive suppressor tRNA, do not exhibit a heat shock response . This defect is suppressed by rpoD800, a mutation in the gene encoding sigma 70 . We have determined the mechanism of suppression . In contrast to wild-type strains, the level of sigma 32 and the level of transcription of heat shock genes remain relatively constant in an rpoH165 rpoD800 strain after a temperature upshift . Instead, the heat shock response in this strain results from an approximately fivefold decrease in the cellular transcription carried out by the RNA polymerase holoenzyme containing mutant RpoD800 sigma 70 coupled with an overall increase in the translational efficiency of all mRNA species.

Exp Hematol, 1992 Nov, 20(10), 1229 - 34
Neutrophil function in normal and Chediak-Higashi syndrome cats following administration of recombinant canine granulocyte colony-stimulating factor; Colgan SP et al.; The effects of recombinant canine granulocyte colony-stimulating factor (rcG-CSF) on leukocyte counts and neutrophil function in clinically normal cats and in cats heterozygotic and homozygotic for Chediak-Higashi syndrome (CHS) were examined . CHS is a genetic disease characterized by neutropenic episodes and defects in a variety of phagocyte functions . Short-term administration of rcG-CSF at 10 micrograms/kg body weight resulted in a five- to tenfold increase in circulating granulocytes by day 10 of administration and normalizes CHS neutrophil counts by day 3 . The drug was specific for neutrophils as determined by differential cell counts . Neutrophil chemotaxis under agarose and phagocytosis of Escherichia coli were characterized following administration of rcG-CSF in vivo . Granulocytes elicited by rcG-CSF show enhanced chemotactic abilities toward activated serum, increased spontaneous migration, and an enhanced ability to ingest opsonized E . coli . At a concentration of 1 nM rcG-CSF in vitro, chemotaxis and spontaneous migration were increased, with no effect on phagocytosis . CHS neutrophil function was improved by administration of rcG-CSF in all parameters studied, although the defect in chemotaxis was present throughout the treatment period . We conclude from this study that neutrophils elicited by rcG-CSF are functionally enhanced and that rcG-CSF may be a viable therapy for CHS and other related disorders.

Exp Cell Res, 1992 Nov, 203(1), 259 - 69
Nucleolin is an Ag-NOR protein; this property is determined by its amino-terminal domain independently of its phosphorylation state; Roussel P et al.; The Ag-NOR proteins are defined as markers of "active" ribosomal genes . They correspond to a set of proteins specifically located in the nucleolar organizer regions (NORs), but have not yet been clearly identified . We adapted the specific detection method of the Ag-NOR proteins to Western blots in order to identify these proteins . Using a purified protein, Western blots, and immunological characterization, the present study brings the first direct evidence leading to the identity of one Ag-NOR protein . We found that nucleolin is specifically revealed by Ag-NOR staining . Using different nucleolin fragments generated by CNBr cleavage and by overexpression in Escherichia coli, we demonstrate that the amino-terminal domain of nucleolin and not the carboxy-part of the protein is involved in silver staining . Moreover, as the pattern of staining does not vary using casein kinase II- and cdc2-phosphorylated nucleolin or dephosphorylated nucleolin, we conclude that the reduction of the silver ions is not linked to the phosphorylation state of the molecule . We propose that the concentration of acidic amino acids in the amino-terminal domain of nucleolin is responsible for Ag-NOR staining . This hypothesis is also supported by the finding that poly L-glutamic acid peptides are silver stained . These results provide data that can be used to explain the specificity of Ag-NOR staining . Furthermore, we clearly establish that proteolysis of the amino-terminal Ag-NOR-sensitive part of nucleolin occurs in vitro, leading to the accumulation of the carboxy-terminal Ag-NOR-negative part of the protein . We argue that this cleavage occurs in vivo as already proposed, bearing in mind that nucleolin is present in the fibrillar and in the granular component of the nucleolus, whereas no Ag-NOR staining is observed in the latter nucleolar component.

Am J Respir Cell Mol Biol, 1992 Nov, 7(5), 471 - 6
Cytokines and lipopolysaccharide induce nitric oxide synthase in cultured rat pulmonary artery smooth muscle; Nakayama DK et al.; In the current study, we describe cytokine and Escherichia coli lipopolysaccharide (LPS) induction of nitric oxide (NO) synthase mRNA levels in cultured smooth muscle from rat pulmonary artery (RPASM) . Exposure of RPASM to interleukin-1 beta, interferon-gamma, or LPS alone did not significantly affect NO synthesis, as determined by nitrite concentrations in media . Exposure to tumor necrosis factor-alpha caused a modest (2x) increase in nitrite production . In contrast, exposure to a combination of the above three cytokines and LPS caused a large increase in NO synthesis . Exposure of RPASM to this combination caused an increase in mRNA levels of NO synthase (as described by Northern blot analysis with 32P-cDNA probe to an inducible form of NO synthase present in murine macrophages) that was apparent as early as 4 h . Expression of the induced gene product after exposure to the cytokine and LPS mixture was evident by significant increases in nitrite production at 12 h . Production of nitrite was completely abolished in the presence of NG-monomethyl-L-arginine (NMA), and this inhibition was reversible by the addition of excess L-arginine . NO synthase mRNA levels were not affected by NMA . The nitrite production induced by the combination of cytokines and LPS was abolished by pretreating cells with cycloheximide . These data indicate that a combination of cytokines and LPS affect expression of the gene for the inducible form of NO synthase in cultured RPASM.

Arch Biochem Biophys, 1992 Nov 1, 298(2), 514 - 21
Rat cystathionine beta-synthase: expression of four alternatively spliced isoforms in transfected cultured cells; Roper MD et al.; The gene for rat cystathionine beta-synthase consists of 17 exons . Its transcripts are alternatively spliced, forming four distinct mRNA species . Type III consists of exons 1 through 12, 14, 15, and 17; type I also contains exon 16 . The open reading frame of type IV spans exons 1-->13; type II, 3-->13 . We cloned the corresponding cDNAs into appropriate expression vectors and inserted the constructs into Escherichia coli (I, III, and IV) and Chinese hamster (CH) cells (I through IV); all sequences were transcribed and translated . Catalytic activity was observed only for types I and III in lysates of transfected CH cells and transformed E . coli . The catalytic and kinetic properties of I and III were identical despite their structural difference (exon 16) . Both isoforms exhibited 6 mM Km constants for homocysteine which were reduced approximately eightfold by AdoMet; this elucidates the mechanism by which AdoMet regulates synthase activity . The four isoforms were differentially degraded by transfected cultured cells . Type III (t1/2 = 18 h) was degraded at 1/3 the rate of type I (t1/2 = 6 h); thus the 14 amino acid residues encoded by exon 16 appear to enhance degradation of CBS . The half-lives of both types II and IV were markedly shorter (ca . 1 h) . Western blots comparing rat liver to lysates from transfected CH cells revealed that hepatocytes express both isoforms . Type III was predominant, as predicted by its longer half-life and more abundant mRNA . PCR analysis of cDNA from various tissues revealed that type III mRNA was preferred in liver, kidney, and heart; equal amounts of I and III were found in brain.

Mutat Res, 1992 Nov, 293(1), 71 - 7
Inhibition of DNA polymerase activity by thymine hydrates; Ganguly T et al.; Ultraviolet irradiation of DNA results in various pyrimidine modifications . We have demonstrated formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in UV-irradiated poly(dA-dT):poly(dA-dT) . Both are released from DNA as free bases by bacterial and human glycosylases . Thymine hydrates are stable in DNA and can be detected in control, unirradiated substrates . We examined the effects of thymine hydrates in UV-irradiated substrate poly(dA-dT):poly(dA-dT) on E . coli DNA polymerase I activity . Enzymic incorporation of labeled thymidine-5'-monophosphate significantly decreased with increasing UV dose . Reversal of DNA thymine hydrates to thymines by mild heating of the substrate prior to enzymic reaction resulted in partial recovery of nucleotide incorporation . Cyclobutane thymine dimers are formed between non-adjacent thymines in UV-irradiated poly(dA-dT):poly(dA-dT) . These are responsible for the incomplete recovery of DNA polymerase activity following heating due to their heat stability . Analyses of the irradiated and hydrolyzed substrate also demonstrated formation of minor yields of photoproducts formed by covalent linkage of adjacent thymines and adenines by UV-irradiation . Therefore, the thymine hydrates formed in UV-irradiated DNA partially inhibit polymerase activity during DNA synthesis and thus could be potentially lethal if unrepaired.

Mutat Res, 1992 Nov, 293(1), 21 - 30
Relationship between the functional regions of the RecA protein and ATP hydrolysis in UV-irradiated Escherichia coli cells; Pueyo M et al.; The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutive (Cptc) mutants of E . coli has been studied . All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298-301) increased ATP after UV damage but without any subsequent decrease . Nevertheless, these mutants induced the SOS response after UV irradiation . Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells . On the other hand, mutants in region 1 (including amino acids 25-39) or 2 (amino acids 157-184) of the RecA protein showed an increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level . All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process . Furthermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found . Accordingly, both lexA71::Tn5 and null lexA mutants of E . coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA (Ind-) gene.

Mol Immunol, 1992 Nov, 29(11), 1383 - 9
Mapping of antibody binding epitopes of a recombinant Poa p IX allergen; Zhang L et al.; Antibody binding epitopes of a recombinant Poa p IX allergen were delineated using recombinant DNA and solid-phase peptide synthesis procedures . The full-length cDNA clone KBG60 and its four overlapping recombinant fragments, KBG60.1, KBG60.2, KBG8.3 and KBG10 which spanned the entire molecule were synthesized in E . coli with aid of the plasmid expression vector, pWR590.1 . The antigenic and allergenic sites of these recombinant proteins were analyzed by ELISA using human IgE and murine IgG antibodies . It was thus demonstrated that although the epitopes were found on all the fragments tested, the majority of these were located on a C-terminal fragment, rKBG8.3 . Furthermore, synthetic peptides were also employed to identify the epitopes of rKBG60 protein . The use of antisera raised against native KBG pollen extract and the recombinant KBG8.3 protein to scan a total of 56 overlapping deca-penta peptides, covering the entire rKBG60 protein, revealed that 10 positive peptides involved in the antibody-binding site(s) . Taken together, the results of these studies indicate that rKBG60 protein possesses at least 10 antibody binding epitopes.

J Virol, 1992 Nov, 66(11), 6806 - 12
In vitro enzymatic activity of human immunodeficiency virus type 1 reverse transcriptase mutants in the highly conserved YMDD amino acid motif correlates with the infectious potential of the proviral genome; Wakefield JK et al.; Reverse transcriptases contain a highly conserved YXDD amino acid motif believed to be important in enzyme function . The second amino acid is not strictly conserved, with a methionine, valine or alanine occupying the second position in reverse transcriptases from various retroviruses and retroelements . Recently, a 3.5-A (0.35-nm) resolution electron density map of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase positioned the YMDD motif within an antiparallel beta-hairpin structure which forms a portion of its catalytic site . To further explore the role of methionine of the conserved YMDD motif in HIV-1 reverse transcriptase function, we have substituted methionine with a valine, alanine, serine, glycine, or proline, reflecting in some cases sequence motifs of other related reverse transcriptases . Wild-type and mutant enzymes were expressed in Escherichia coli, partially purified by phosphocellulose chromatography, and assayed for the capacity to polymerize TTP by using a homopolymeric template {poly(rA)} with either a DNA {oligo(dT)} or an RNA {oligo(U)} primer . With a poly(rA).oligo(dT) template-primer, reverse transcriptases with the methionine replaced by valine (YVDD), serine (YSDD), or alanine (YADD) were 70 to 100% as active as the wild type, while those with the glycine substitution (YGDD) were approximately 5 to 10% as active . A proline substitution (YPDD) completely inactivated the enzyme . With a poly(rA).oligo(U) template-primer, only the activity of mutants with YVDD was similar to that of the wild type, while mutants with YADD and YSDD were approximately 5 to 10% as active as the wild-type enzyme . The reverse transcriptases with the YGDD and YPDD mutations demonstrated no activity above background . Proviruses containing the reverse transcriptase with the valine mutation (YVDD) produced viruses with infectivities similar to that of the wild type, as determined by measurement of p24 antigen in culture supernatants and visual inspection of syncytium formation . In contrast, proviruses with reverse transcriptases containing the YADD and YSDD mutations were less infectious than wild-type virus . These results point to the critical role of methionine of the YMDD motif in the activity of HIV-1 reverse transcriptase and subsequent replication potential of the virus.

J Virol, 1992 Nov, 66(11), 6370 - 8
Requirements for strand transfer between internal regions of heteropolymer templates by human immunodeficiency virus reverse transcriptase; DeStefano JJ et al.; We have examined the ability of the reverse transcriptase (RT) from human immunodeficiency virus (HIV) to carry out strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions of natural-sequence RNA . A 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide was used to initiate synthesis . DNA oligonucleotides with homology to internal regions of the donor were used as acceptors . In this system, HIV RT produced strand transfer products . An HIV RT having RNase H depleted to 3% of normal (HIV RTRD) catalyzed the transfer reaction inefficiently . An RNase H-minus deletion mutant of murine leukemia virus RT was unable to catalyze strand transfer . HIV RTRD, however, efficiently catalyzed transfer when Escherichia coli RNase H was included in the reactions, while the mutant murine leukemia virus RT was not efficiently complemented by the E . coli enzyme . Evidently, RNase H activity enhances, or is required for, internal strand transfer . Two acceptors homologous to 27-nucleotide regions of the donor, one offset from the other by 6 nucleotides, were tested . The offset eliminated a sequence homologous to a prevalent DNA synthesis pause site in the donor . Strand transfer to this acceptor was about 25% less efficient, suggesting that RT pausing can enhance strand transfer . When the deoxynucleoside triphosphates in the reactions were reduced from 50 to 0.2 microM, increasing RT pausing, the efficiency of strand transfer also increased . A model for RT-catalyzed strand transfer consistent with our results is presented.

Biosci Biotechnol Biochem, 1992 Nov, 56(11), 1849 - 53
Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae; Tsuchiya K et al.; The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose . The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) . To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A . oryzae met locus by homologous recombination . Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377 . Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene . The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.

Biosci Biotechnol Biochem, 1992 Nov, 56(11), 1780 - 5
Conversion of dethiobiotin to biotin in cell-free extracts of Escherichia coli; Ifuku O et al.; We constructed the plasmid pTTB151 in which the E . coli bioB gene was expressed under the control of the tac promoter . Conversion of dethiobiotin to biotin was demonstrated in cell-free extracts of E . coli carrying this plasmid . The requirements for this biotin-forming reaction included fructose-1,6-bisphosphate, Fe2+, S-adenosyl-L-methionine, NADPH, and KCl, as well as dethiobiotin as the substrate . The enzymes were partially purified from cell-free extracts by a procedure involving ammonium sulfate fractionation . Our results suggest that an unidentified enzyme(s) besides the bioB gene product is obligatory for the conversion of dethiobiotin to biotin.

Biosci Biotechnol Biochem, 1992 Nov, 56(11), 1773 - 5
Isolation and synthesis of a new bio-antimutagen, petasiphenol, from scapes of Petasites japonicum; Iriye R et al.; A new bio-antimutagen, petasiphenol {3-(3,4-dihydroxyphenyl)-2-oxopropyl caffeate} (1) was isolated from scapes of Petasites japonicum (AD50 = 95 micrograms/ml against UV-induced mutagenic E . coli WP2 B/r Trp- . Petasiphenol (1) and its isomer (2) were synthesized . The activity of 1 was observed in the presence of soybean oil (glyceride), although the isomer (2) did not show any activity in doses up to 300 micrograms/ml.

Res Microbiol, 1992 Nov-Dec, 143(9), 831 - 8
FimC, a chaperone-like periplasmic protein of Escherichia coli involved in biogenesis of type 1 fimbriae; Klemm P; The product of the fimC gene of Escherichia coli K12 is required for the biogenesis of type 1 fimbriae . Mutations within the fimC gene abolish fimbrial synthesis . The FimC protein was found to be processed and the mature version was located in the periplasm . Unlike similar fimbrial systems, the major type 1 fimbriae structural protein FimA was found to be significantly resistant to proteolytic degradation when present in the periplasm in a fimC- host background . The fimC gene was sequenced, and the deduced primary structure of the FimC protein was compared to other similar known proteins involved in the biogenesis of various fimbriae.

FEMS Microbiol Lett, 1992 Nov 1, 77(1-3), 149 - 53
Identification and characterization of GroEL and DnaK homologues in Thiobacillus ferrooxidans; Varela P et al.; The major heat shock proteins from Thiobacillus ferrooxidans were identified as DnaK and GroEL equivalents by Western blotting and analysis of the N-terminal amino acid sequence of spots isolated from dried 2-D polyacrylamide electrophoresis gels . The T . ferrooxidans chaperonins showed 70% and 80% identity with the Escherichia coli GroEL and DnaK, respectively . By using electrophoresis with a transverse pore gradient of cross-linked polyacrylamide and nondenaturing conditions followed by Western blotting, we found that the GroEL proteins from both bacteria formed a 14-mer, whereas E . coli DnaK protein existed partially as a dimer and the T . ferrooxidans DnaK-equivalent showed only a monomeric nature under our experimental conditions.

Biochem J, 1992 Nov 1, 287 ( Pt 3), 1007 - 10
Uracil-DNA glycosylases preferentially excise mispaired uracil; Verri A et al.; We have investigated the substrate specificity of human, viral and bacterial uracil-DNA glycosylases employing as substrate double-stranded oligonucleotides containing in the same position of the 5'-32P-labelled strand an uracil residue facing, on the complementary strand, guanine (mimicking cytosine deamination) or adenine (mimicking dUTP misincorporation) . The enzyme removal of uracil was monitored and quantified by the generation of alkali-sensitive apyrimidinic sites . All three uracil-DNA glycosylases excise uracil from mispaired oligonucleotides (U/G) more efficiently than from paired oligonucleotides (U/A) . The enzymes also remove uracil from single-stranded oligonucleotide with an efficiency similar to that observed with U/A paired oligonucleotide . The efficient recognition of U/G mispair by uracil-DNA glycosylase is important in minimizing miscoding transcripts and C/G-->T/A transitions in proliferating cells.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10341 - 4
Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli; Gragerov A et al.; Newly synthesized proteins aggregate extensively in Escherichia coli rpoH mutants, which are deficient in the heat shock proteins (hsp) . Overproduction of either GroEL and GroES or DnaK and DnaJ prevents aggregation . If expressed together, the four hsp are effective at physiological concentrations . Our data suggest that the GroEL and GroES proteins and the DnaK and DnaJ proteins have complementary functions in the folding and assembly of most proteins.

J Bacteriol, 1992 Nov, 174(22), 7407 - 18
The lethal phenotype caused by null mutations in the Escherichia coli htrB gene is suppressed by mutations in the accBC operon, encoding two subunits of acetyl coenzyme A carboxylase; Karow M et al.; Insertion mutations in the Escherichia coli htrB gene result in the unique phenotype of not affecting growth at temperatures below 32.5 degrees C but leading to a loss of viability at temperatures above this in rich media . When htrB bacteria growing in rich media were shifted to the nonpermissive temperature of 42 degrees C, they continued to grow at a rate similar to that at 30 degrees C but they produced phospholipids at the rate required for growth at 42 degrees C . This led to the accumulation of more than twice as much phospholipid per milligram of protein compared with that in wild-type bacteria . Consistent with HtrB playing a role in phospholipid biosynthesis, one complementation group of spontaneously arising mutations that suppressed htrB-induced lethality were mapped to the accBC operon . This operon codes for the biotin carboxyl carrier protein and biotin carboxylase subunits of the acetyl coenzyme A carboxylase enzyme complex, which catalyzes the first step in fatty acid biosynthesis . Four suppressor mutations mapped to this operon . Two alleles were identified as mutations in the accC gene, the third allele was identified as a mutation in the accB gene, and the fourth allele was shown to be an insertion of an IS1 transposable element in the promoter region of the operon, resulting in reduced transcription . The suppressor mutations caused a decrease in the rate of phospholipid biosynthesis, restoring the balance between the biosynthesis of phospholipids and growth rate, thus enabling htrB bacteria to grow at high temperatures.

Genes Dev, 1992 Nov, 6(11), 2177 - 89
Differential regulation of transcription preinitiation complex assembly by activator and repressor homeo domain proteins; Johnson FB et al.; Different eukaryotic transcription factors can act through the same upstream binding site to differentially regulate target gene expression, but little is known of the underlying mechanisms . Here, we show that Ultrabithorax and even-skipped homeo domain proteins (UBX and EVE) of Drosophila melanogaster exert active and opposite effects on in vitro transcription when bound to a common site upstream of a core promoter . Both the activator UBX and the repressor EVE affect the extent but not the rate constant of preinitiation complex (preIC) formation . Both regulators act early in preIC assembly and are dispensable later . Assembling complexes become resistant to regulation by the bound proteins, but activation by UBX is restored upon ATP or dATP addition, and regulation by both proteins is restored after the addition of all four nucleoside triphosphates and transcription initiation . The results establish that upstream activators and repressors can function by fundamentally similar mechanisms, by differentially regulating an early step in preIC assembly, leading to formation of functionally distinct transcription complexes . A subsequent step renders mature complexes transiently refractory to activation and repression . Implications for the mechanism of transcription complex assembly and turnover and its regulation are discussed, including a new role for ATP in turnover.

Genes Dev, 1992 Nov, 6(11), 2047 - 57
Recognition of the surface of a homeo domain protein; Pomerantz JL et al.; Homeo domain proteins exhibit distinct biological functions with specificities that cannot be predicted by their sequence specificities for binding DNA . Recognition of the surface of the Oct-1 POU homeo domain provides a general model for the contribution of selective protein-protein interactions to the functional specificity of the homeo domain family of factors . The assembly of Oct-1 into a multiprotein complex on the herpes simplex virus alpha/IE enhancer is specified by the interactions of its homeo domain with ancillary factors . This complex (C1 complex) is composed of the viral alpha TIF protein (VP16), Oct-1, and one additional cellular component, the C1 factor . Variants of the Oct-1 POU homeo domain were generated by site-directed mutagenesis, which altered the residues predicted to form the exposed surface of the domain-DNA complex . Proteins with single amino acid substitutions on the surface of either helix 1 or 2 of the Oct-1 POU homeo domain had decreased abilities to form the C1 complex . The behavior of these mutants in a cooperative DNA-binding assay with alpha TIF suggested that the Oct-1 POU homeo domain is principally recognized by alpha TIF in the C1 complex . The preferential recognition of Oct-1 over the closely related Oct-2 protein is critically influenced by a single residue on the surface of helix 1 because the introduction of this residue into the Oct-2 POU homeo domain significantly enhanced its ability to form a C1 complex.

J Bacteriol, 1992 Nov, 174(21), 7003 - 12
Thermoregulation of the pap operon: evidence for the involvement of RimJ, the N-terminal acetylase of ribosomal protein S5; White-Ziegler CA et al.; Our previous work showed that pap pilin gene transcription is subject to a thermoregulatory control mechanism under which pap pilin is not transcribed at a low temperature (23 degrees C) (L . B . Blyn, B . A . Braaten, C . A . White-Ziegler, D . H . Rolfson, and D . A . Low, EMBO J . 8:613-620, 1989) . In order to isolate genes involved in this temperature regulation of gene expression, chromosomal mini-Tn10 (mTn10) mutations that allowed transcription of the pap pilin gene at 23 degrees C were identified, and the locus was designated tcp, for "thermoregulatory control of pap" (C . A . White-Ziegler, L . B . Blyn, B . A . Braaten, and D . A . Low, J . Bacteriol . 172:1775-1782, 1990) . In the present study, quantitative analysis showed that the tcp mutations restore pap pilin transcription at 23 degrees C to levels similar to those measured at 37 degrees C . By in vivo recombination, the tcp mutations were mapped to phage E4H10S of the Kohara library of the Escherichia coli chromosome (Y . Kohara, K . Akiyama, and K . Isono, Cell 50:495-508, 1987) . The tcp locus was cloned by complementation, in which a 1.3-kb DNA fragment, derived from the Kohara phage, was shown to restore thermoregulation to the mTn10 mutants . DNA sequencing revealed two open reading frames (ORFs) encoding proteins with calculated molecular masses of 22.7 and 20.3 kDa . The sequence of the 22.7-kDa ORF was identical to that of rimJ, the N-terminal acetylase of the ribosomal protein S5 . The gene encoding the 20.3-kDa ORF, designated g20.3 here, did not display significant homology to any known DNA or protein sequence . On the basis of Northern (RNA) blot data, rimJ and g20.3 are located within the same operon . Two of the mTn10 transposons in the thermoregulatory mutants were inserted within the coding region of rimJ, indicating that the RimJ protein plays an important role in the temperature regulation of pap pilin gene transcription . However, rimJ itself is not thermoregulated, since rimJ transcripts were detected at both 23 and 37 degrees C . Disruption of the g20.3 gene by insertion and deletion mutagenesis did not affect thermoregulation of the pap operon, suggesting that, although g20.3 lies within the same operon as rimJ, it does not play a role in thermoregulation.

J Bacteriol, 1992 Nov, 174(21), 6938 - 47
Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli; Allen SP et al.; In Escherichia coli high-level production of some heterologous proteins (specifically, human prorenin, renin, and bovine insulin-like growth factor 2) resulted in the induction of two new E . coli heat shock proteins, both of which have molecular masses of 16 kDa and are tightly associated with inclusion bodies formed during heterologous protein production . We named these inclusion body-associated proteins IbpA and IbpB . The coding sequences for IbpA and IbpB were identified and isolated from the Kohara E . coli gene bank . The genes for these proteins (ibpA and ibpB) are located at 82.5 min on the chromosome . Nucleotide sequencing of the two genes revealed that they are transcribed in the same direction and are separated by 110 bp . Putative Shine-Dalgarno sequences are located upstream from the initiation codons of both genes . A putative heat shock promoter is located upstream from ibpA, and a putative transcription terminator is located downstream from ibpB . A temperature upshift experiment in which we used a wild-type E . coli strain and an isogenic rpoH mutant strain indicated that a sigma 32-containing RNA polymerase is involved in the regulation of expression of these genes . There is 57.5% identity between the genes at the nucleotide level and 52.2% identity at the amino acid level . A search of the protein data bases showed that both of these 16-kDa proteins exhibit low levels of homology to low-molecular-weight heat shock proteins from eukaryotic species.

EMBO J, 1992 Nov, 11(11), 4119 - 29
A POU-A related region dictates DNA binding specificity of LFB1/HNF1 by orienting the two XL-homeodomains in the dimer; Tomei L et al.; LFB1/HNF1 regulates the hepatocyte-specific transcription of several genes, binding as a dimer to cis-acting elements that match the inverted palindrome GTTAATNATTAAC . The DNA binding domain of LFB1/HNF1 is characterized by a unique tripartite structure that includes an unusually long homeodomain (domain C), a region related to the POU-specific A-box (domain B) and a short N-terminal dimerization domain (domain A) . We report that a recombinant peptide corresponding to the isolated homeodomain of LFB1/HNF1 binds as a monomer to a half-palindrome binding site, but shows diminished sequence specificity . Domain B, in addition to the homeodomain, is required and sufficient for proper recognition of LFB1/HNF1-responsive sites . A protein consisting of only these latter two domains is a monomer in solution, but forms dimers upon DNA binding . The protein-protein contacts established within the bound dimer restrain the orientation of the two homeodomains with respect to one another, thus contributing in a critical fashion to the recognition of the dyad symmetry-related LFB1/HNF1 sites . The DNA-independent dimerization domain (domain A) is required to increase the affinity of DNA binding, but does not influence the dimer geometry.

J Biochem (Tokyo), 1992 Nov, 112(5), 677 - 88
Gene structure and multiple mRNA species of Drosophila melanogaster aldolase generating three isozymes with different enzymatic properties; Kai T et al.; Genomic clones encoding the Drosophila aldolase gene were isolated and the organization of the gene was determined . The protein-coding region spanning nearly 3.5 kb consists of five coding exons (exon 2, 3, 4 alpha, 4 beta, and 4 gamma) . The insect exon 2 corresponds to exons 2 to 7 of vertebrate aldolase genes and thus appears to have been formed by the fusion of these 6 exons into a single exon during evolution . The Drosophila aldolase gene is predicted to generate mRNAs for three isozymes (alpha-, beta-, and gamma-types) from the primary transcripts by alternative usage of the final three exons . The reverse transcriptase-PCR assay revealed the occurrence of mRNAs for the three isozymic forms at different developmental stages, and tissue-specific expression was also found to occur in adult flies . In addition to the usual type mRNA species for the alpha-, beta-, and gamma-isozymes, two novel forms of mRNAs, alpha beta- and beta gamma-type mRNAs, were detected tissue-specifically in adult flies, although their functions are unpredictable . The alpha beta-mRNA is an alpha-type mRNA in which exon 4 beta remains unspliced, while the beta gamma-mRNA is a beta-type mRNA with the exon 4 gamma remaining unspliced . Recombinant enzymes expressed in Escherichia coli were all active and exhibited different enzymatic properties.

Biochem Int, 1992 Nov, 28(3), 451 - 60
Expression of human metallothionein-II fusion protein in Escherichia coli; Yamazaki S et al.; In order to obtain antibody against metallothionein-II (MT-II), a capsid protein metallothionein fusion protein was prepared . A gene encoding human MT-II was cloned into a plasmid for expression of MT fusion protein in Escherichia coli . MT cDNA was generated from human astrocytoma U373MG and amplified by polymerase chain reaction . The nucleotide sequence was identical to reported MT-II . The cDNA was inserted into plasmid pGEM EXTM-2 which carries the T7 promoter and T7 phage 10A major head protein . This expressed phage protein-MT fusion protein, has a molecular weight of 37kDa, forms inclusion bodies and constitutes about 20% of the total protein in transformed E . coli.

Mikrobiologiia, 1992 Nov-Dec, 61(6), 1030 - 7
{Effect of heavy water on the growth, glucose assimilation and stability of Escherichia coli to freezing-thawing}; Litvinenko LA et al.; Growth and resistance to freezing--thawing of Escherichia coli B-1640 were investigated during cultivation in synthetic media prepared with H2O and D2O . It is found that during cultivation in D2O the maximum specific growth rate decreases and the duration of the exponential growth phase increases . During the growth in D2O the glucose consumption rate drops in the exponential growth phase, the lactate content in the culture liquid is lower by two orders than that in H2O; the resistance to freezing--thawing is lower than that in H2O . After leaving the exponential phase the culture in D2O restores specific growth rate, glucose consumption rate and resistance to freezing--thawing up to the values obtained during the growth in H2O . The translation ability of ribosomes isolated from cells grown in D2O and H2O is the same . We conclude that the culture adapts to D2O during the exponential growth phase . It is suggested that during the adaptation the second carbon source is used which compensates the consequences of the disturbances of glucose metabolism and transport caused by deuteration of the cell content in the adaptation to D2O.

Cytokine, 1992 Nov, 4(6), 506 - 12
1,25-Dihydroxyvitamin D3 inhibits cytokine production by human blood monocytes at the post-transcriptional level; Muller K et al.; 1,25-Dihydroxyvitamin D3 {1,25-(OH)2D3} inhibits lymphocyte proliferation and production of antibodies and lymphokines such as interleukin (IL)-2 and interferon gamma . These lymphocyte functions are dependent upon cytokines, including IL-1 alpha, IL-1 beta, IL-6 and tumour necrosis factor alpha (TNF-alpha), produced by the antigen presenting cells . In the present study we examined the effect of 1,25-(OH)2D3 on the production of these cytokines, as well as superoxide generation by freshly isolated mononuclear cells and partially purified monocytes . The immediate precursor of 1,25(OH)2D3, 25-OH D3, and the synthetic analogue MC 903 ('Calcipotriol') were examined in parallel . 1,25-(OH)2D3 dose-dependently inhibited the production of IL-alpha, IL-6 and TNF-alpha by Escherichia coli lipopolysaccharide (LPS)-stimulated monocytes, without affecting superoxide production . MC 903 had comparable effects while 25-OH D3 was ineffective . The inhibition caused by 1,25-(OH)2D3 was not abolished by supraoptimal concentrations of LPS or indomethacin . 1,25-(OH)2D3 had similar effects on secreted and cell-associated IL-alpha . Nuclear run-off analysis indicated that inhibition of these cytokines was not caused by impaired production of mRNA . Taken together, the study demonstrates a vitamin D-induced inhibitory effect of LPS-driven monokine production, which is most likely a vitamin D-receptor mediated phenomenon exerted at a post-transcriptional, presecretory level . Impaired monokine production may be of importance in 1,25-(OH)2D3-mediated inhibition of lymphocyte functions in vitro.

J Exp Biol, 1992 Nov, 172, 451 - 9
The Fo complex of the proton-translocating F-type ATPase of Escherichia coli; Deckers-Hebestreit G et al.; The ATP synthase (F1Fo) of Escherichia coli consists of two structurally and functionally distinct entities . The F1 part is composed of five subunits alpha, beta, gamma, delta and epsilon (3:3:1:1:1) and carries the catalytic centres of the enzyme . The membrane-bound Fo complex functions as a proton channel and consists of the three subunits a, b and c (1:2:10 +/- 1) . Subunit c (8288 M(r)) exhibits a hairpin-like structure within the membrane . A conserved acidic residue (Asp-61) in the C-terminal hydrophobic segment is absolutely required for proton translocation through Fo, whereas the hydrophilic loop region is necessary for F1 binding . Expression of the chloroplast proteolipid together with subunits a and b of E . coli did not produce an active Fo hybrid complex . Therefore, the construction of hybrid c subunits consisting of parts of the proteolipid from both organisms is in progress to determine those parts of subunit c that are essential for a functional interplay with subunits a and b . Subunit a (30,276 M(r)), which is also involved in proton translocation, is an extremely hydrophobic protein with 5-8 membrane-spanning helices . Studies with alkaline phosphatase fusion proteins resulted in controversial conclusions about the localization of the N and C termini of the protein . A foreign epitope (13 amino acids) has been inserted into the N- or C-terminal region of subunit a without affecting the function of Fo . Binding studies with a monoclonal antibody against this epitope are now under investigation to determine the orientation of subunit a.(ABSTRACT TRUNCATED AT 250 WORDS)

J Exp Biol, 1992 Nov, 172, 443 - 9
Escherichia coli ATP synthase (F-ATPase): catalytic site and regulation of H+ translocation; Futai M et al.; We discuss our recent results on the Escherichia coli F-ATPase, in particular its catalytic site in the beta subunit and regulation of H+ transport by the gamma subunit . Affinity labelling experiments suggest that beta Lys-155 in the glycine-rich sequence is near the gamma-phosphate moiety of ATP bound at the catalytic site . The enzyme loses activity upon introduction of missense mutations in beta Lys-155 or beta Thr-156 and changes catalytic properties upon introduction of other mutations . By analysis of mutations and their pseudo revertants, residues beta Ser-174, beta Glu-192 and beta Val-198 were found to be located near the glycine-rich sequence . The combined approaches of chemical labelling and genetics have been fruitful in visualizing the structure of the catalytic site . Analysis of mutations in the gamma subunit suggests that this subunit has an essential role in coupling catalysis with proton translocation.

Mol Microbiol, 1992 Nov, 6(22), 3331 - 42
Expression of Escherichia coli beta-galactosidase in Mycobacterium bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular immune responses; Murray A et al.; A promoter sequence, PAN, was isolated from Mycobacterium paratuberculosis and characterized . This promoter lies adjacent to, and outside, the 3' end of an IS900 insertion element . IS900 contains an open reading frame, ORF2, on the complementary strand which codes for the putative transposase of this insertion sequence . A DNA fragment containing PAN and part of ORF2 was fused to the lacZ gene and inserted into the replicative shuttle vector pRR3 . Mycobacterium smegmatis and Mycobacterium bovis BCG (BCG) transformed with this plasmid exhibited beta-galactosidase activity . However, lacZ was only expressed in Escherichia coli under the control of PAN, when ORF2 was deleted . Immunization of mice with the recombinant M . bovis BCG expressing lacZ resulted in the induction of a high humoral and cellular response directed against beta-galactosidase . The PAN-ORF2 expression system may prove to be particularly useful for cloning and expression of heterologous genes in the BCG vaccine strain.

Mutagenesis, 1992 Nov, 7(6), 439 - 45
Spectrum of mutations produced by specific types of restriction enzyme-induced double-strand breaks; Winegar RA et al.; Rejoining of DNA double-strand breaks (DSB) plays a central role in the various processes leading to DNA rearrangements . We have analyzed DNA alterations induced by restriction enzymes that produce DSB with specific types of ends . Restriction enzymes were electroporated into a human lymphoblastoid cell line that stably maintains pHAZE, an EBV-based vector containing the lacZ gene . After allowing time for DSB repair, pHAZE DNA was rescued and screened in Escherichia coli . Mapping and sequence analysis of mutant copies of pHAZE indicated that restriction enzymes induced all classes of alterations except base substitutions (base deletions and insertions, large-scale deletions, inversions, and insertions) . The spectra of alterations were distinctive for each enzyme and appear to be the consequence of specific end-modification processes.

Br J Pharmacol, 1992 Nov, 107(3), 826 - 32
Endotoxin impairs the response of rabbit mesenteric artery to electrical stimulation via a prejunctional mechanism; Tomikawa S et al.; 1 . The effect of E . coli lipopolysaccharide (LPS) on sympathetic neuro-effector transmission was studied in the rabbit mesenteric artery . The experiments were performed on artery rings isolated 5 or 20 h after intravenous treatment with LPS or saline as well as on artery rings isolated from non-treated rabbits (for assessment of the effect of in vitro preincubation with LPS) . In most experiments, neural elements in the arteries were stimulated electrically (10 V, 2 ms, 1-32 Hz) . 2 . Preincubation with LPS (10 micrograms ml-1) for 5 or 20 h had no effect on the contraction responses of endothelium-intact artery rings to electrical stimulation . In contrast, in vivo intravenous pretreatment with LPS (10 micrograms) led to an inhibition of the contraction; LPS elicited this effect when injected 20 h, but not 5 h, before the experiment . The effect of LPS was eliminated in artery rings isolated from animals receiving an inhibitor of protein synthesis (actinomycin D or cycloheximide) before treatment with LPS . LPS (injected 20 h before the experiment) had no effect on the concentration-response curves for exogenous noradrenaline and tyramine in endothelium-intact artery rings . 3 . The inhibition of electrically induced contractions produced by LPS treatment in endothelium-intact artery rings was attenuated by atropine and yohimbine, but not by phentolamine . Yohimbine plus atropine restored the depressed contraction to the normal level . Clonidine and acetylcholine mimicked the effect of LPS in endothelium-intact artery rings isolated from saline-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1992 Nov, 235(2-3), 325 - 32
DNA sequences required for translational frameshifting in production of the transposase encoded by IS1; Sekine Y et al.; The transposase encoded by insertion sequence IS1 is produced from two out-of-phase reading frames (insA and B'-insB) by translational frameshifting, which occurs within a run of six adenines in the -1 direction . To determine the sequence essential for frameshifting, substitution mutations were introduced within the region containing the run of adenines and were examined for their effects on frameshifting . Substitutions at each of three (2nd, 3rd and 4th) adenine residues in the run, which are recognized by tRNA(Lys) reading insA, caused serious defects in frameshifting, showing that the three adenine residues are essential for frameshifting . The effects of substitution mutations introduced in the region flanking the run of adenines and in the secondary structures located downstream were, however, small, indicating that such a region and structures are not essential for frameshifting . Deletion of a region containing the termination codon of insA caused a decrease in beta-galactosidase activity specified by the lacZ fusion plasmid in frame with B'-insB . Exchange of the wild-type termination codon of insA for a different one or introduction of an additional termination codon in the region upstream of the native termination codon caused an increase in beta-galactosidase activity, indicating that the termination codon in insA affects the efficiency of frameshifting.

Mol Gen Genet, 1992 Nov, 235(2-3), 317 - 24
Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1; Sekine Y et al.; Previous genetic analyses indicated that translational frameshifting in the--1 direction occurs within the run of six adenines in the sequence 5'-TTAAAAAACTC-3' at nucleotide positions 305-315 in IS 1, where the two out-of-phase reading frames insA and B'-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84-87 . IS 1 mutants with a 1 bp insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84-87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-Lys-Lys-Leu . An IS 1 mutant with the DNA segment 5'-CTTAAAAACTC-3' at positions 305-315 carrying the termination codon TAA in the B'-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting . The beta-galactosidase activity specified by several IS 1-lacZ fusion plasmids, in which B'-insB is in-frame with lacZ, showed that the region 292-377 is sufficient for frameshifting . The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu-Lys-Lys-Leu encoded by the DNA segment 5'-TTAAAAAACTC-3', indicating that--1 frameshifting does occur within the run of adenines.

J Virol Methods, 1992 Nov, 40(2), 195 - 204
Plasmid-associated effects on test gene expression and Marek's disease virus plaque formation during recombination trials; Marshall DR et al.; Marek's disease virus (MDV), a herpesvirus of avian origin, is being examined for suitability as a vector for expressing foreign genes . We observed that plasmids encoding the LacZ gene of E . coli under the control of either the herpes simplex virus alpha 4 immediate-early promoter or the cytomegalovirus major immediate-early promoter inhibited MDV plaque formation . Plaque numbers were decreased by one-third, and transient expression of the beta-galactosidase reporter gene was increased by up to 6-fold, when the plasmids were linearized . Sequences associated with the heterologous promoter were identified as being responsible for inhibiting MDV replication.

Biochem J, 1992 Nov 1, 287 ( Pt 3), 1019 - 22
Protein phosphatase 2A is a specific protamine-kinase-inactivating phosphatase; Amick GD et al.; Purified preparations of a protamine protein kinase from bovine kidney cytosol {Damuni, Amick & Sneed (1989) J . Biol . Chem . 264, 6412-6416} were inactivated after incubation with near-homogeneous preparations of protein phosphatase 2A1 and protein phosphatase 2A2 . These protein phosphatase 2A-mediated inactivations of the protamine kinase were unaffected by highly purified preparations of inhibitor 2, but were prevented when the incubations were performed in the presence of 100 nM microcystin-LR, 100 nM okadaic acid or 0.2 mM-ATP . By contrast, highly purified preparations of protein phosphatase 2B, protein phosphatase 2C, the catalytic subunit of protein phosphatase 1, and two forms of a protein tyrosine phosphatase, designated PTPase 1B and T-cell PTPase, had little effect, if any, on protamine kinase activity . Purified preparations of the protamine kinase did not react with anti-phosphotyrosine antibodies, as determined by Western blotting and immunoprecipitation analysis . The results indicate that protein phosphatase 2A is a specific protamine-kinase-inactivating phosphatase.

Mol Pharmacol, 1992 Nov, 42(5), 802 - 7
Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions; Ling YH et al.; The incorporation of 6-thioguanine (S6G) in place of guanine proceeds readily in DNA synthesis reactions catalyzed by mammalian and bacterial polymerases . This report summarizes the consequences of such incorporation studied to date . S6G was incorporated into one strand of a defined M13mp18 phage sequence in a (+)reaction catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I . After denaturation of the newly synthesized strand (containing S6G) and annealing with a reverse (-) 32P-labeled primer, polymerization catalyzed by the Klenow enzyme as well as by human DNA polymerases alpha, gamma, and delta was slowed considerably, compared with that across the corresponding guanine-containing template . To evaluate S6G-containing DNA as a substrate for DNA ligases, two oligodeoxynucleotides (19- and 20-mers) antisense to a 40-mer were synthesized so that the 40-mer coded for guanine at the 3' terminus of the 19-mer . After annealing of the synthetic oligonucleotides to form a duplex DNA containing a one-nucleotide gap (opposite cytosine in the 40-mer), the 19-mer was extended with 2'-deoxythioguanosine 5'-triphosphate using DNA polymerase, forming a nicked duplex DNA . The abilities of T4 DNA ligase and HeLa and calf thymus DNA ligase I to join the 5'-phosphate with the 3'-S6G-OH were severely inhibited, compared with the 3'-guanine-extended control . This finding suggests that incorporation of S6G at the 3' terminus of Okazaki fragments would inhibit lagging strand DNA synthesis . In other experiments, cleavage of S6G-containing DNA by some but not all restriction endonucleases progressed poorly, compared with the control guanine-containing DNA, independently of the location of S6G at recognition or cleavage sites, as previously observed by Iwaniec et al . {Mol . Pharmacol . 39:299-306 (1991)} with a different spectrum of enzymes . These findings indicate altered DNA-protein interactions due to S6G incorporation . The poor template function of S6G-containing DNA is consistent with the known delayed cytotoxicity and DNA damage previously reported to occur in S6G-treated cells.

J Bacteriol, 1992 Nov, 174(22), 7482 - 5
Properties of recombinant cells capable of growing on serine without NhaB Na+/H+ antiporter in Escherichia coli; Kayahara T et al.; Escherichia coli HIT-1 has a mutation in the Na+/H+ antiporter gene, nhaB (P . Thelen, T . Tsuchiya, and E . B . Goldberg, J . Bacteriol . 173:6553-6557, 1991) . This strain is not able to utilize serine as a carbon source (T . Ishikawa, H . Hama, M . Tsuda, and T . Tsuchiya, J . Biol . Chem . 262:7443-7446, 1987), because an active NhaB is required to maintain the electrochemical potential of Na+, which drives serine transport via the Na+/serine carrier, the major transport system for serine . We isolated recombinant cells from a cross between strains HIT-1 and Hfr, and these cells were able to grow on serine even though the NhaB Na+/H+ antiporter of the recombinant cells was still defective . We found that the activity of the H+/serine cotransport system, one of the minor serine transport systems in E . coli, was elevated in the recombinant cells . H+/serine cotransport activity was induced by leucine in the recombinant cells more strongly than in strain HIT-1 . A kinetic analysis showed that the Vmax, but not the Km, of the transport system was much higher in the recombinant cells than in strain HIT-1 cells.

J Bacteriol, 1992 Nov, 174(22), 7262 - 72
Differential action and differential expression of DNA polymerase I during Escherichia coli colony development; Shapiro JA; A mini-Tn10 insertion in the polA cistron (polA2099) was isolated in a search for mutations that affect patterned Mudlac replication in colonies . The polA2099 mutation had a dramatic effect on cell morphogenesis during the first few hours of microcolony development . Abnormal microcolonies containing filamentous cells were produced as a result of SOS induction . Despite gross abnormalities in early microcolonies, mature polA2099 colonies after 2 to 4 days were morphologically indistinguishable from Pol+ colonies, and 44-h polA2099 colonies displayed a cell size distribution very similar to that of Pol+ colonies . These results suggested the involvement of a protective factor produced during colony growth that compensated for the polA deficiency . The action of a diffusible substance that stimulates growth of polA2099 microcolonies was shown by spotting dilute polA2099 cultures next to established colonies . Differential transcription of polA during colony development was visualized by growing colonies containing polA-lacZ fusions on beta-galactosidase indicator agar . When polA-lacZ colonies were inoculated next to established colonies, a diffusible factor was seen to inhibit polA transcription during the earliest stages of colony development . These results show that a basic housekeeping function, DNA polymerase I, is subject to multicellular control by the changing conditions which the bacteria create as they proliferate on agar.

Genes Dev, 1992 Nov, 6(11), 2221 - 32
DNA-promoted assembly of the active tetramer of the Mu transposase; Baker TA et al.; A stable tetramer of the Mu transposase (MuA) bound to the ends of the Mu DNA promotes recombination . Assembly of this active protein-DNA complex from monomers of MuA requires an intricate array of MuA protein-binding sites on supercoiled DNA, divalent metal ions, and the Escherichia coli HU protein . Under altered reaction conditions, many of these factors stimulate assembly of the MuA tetramer but are not essential, allowing their role in formation of the complex to be analyzed . End-type MuA-binding sites and divalent metal ions are most critical and probably promote a conformational change in MuA that is necessary for multimerization . Multiple MuA-binding sites on the DNA contribute synergistically to tetramer formation . DNA superhelicity assists cooperativity between the sites on the two Mu DNA ends if they are properly oriented . HU specifically promotes assembly involving the left end of the Mu DNA . In addition to dissecting the assembly pathway, these data demonstrate that the tetrameric conformation is intrinsic to MuA and constitutes the form of the protein active in catalysis.

Dig Dis Sci, 1992 Nov, 37(11), 1738 - 45
Systemic tumor necrosis factor-alpha production in experimental colitis; Mack DR et al.; Tumor necrosis factor-alpha (TNF) is a cytokine released by mononuclear cells in response to inflammation and sepsis . Since the biological effects of TNF are consistent with the systemic and intestinal features of ulcerative colitis, the role of TNF was examined in a rabbit model of chronic colitis . Peripheral blood mononuclear cells were isolated, stimulated with lipopolysaccharide, and cultured supernatants assayed for TNF levels using a cytotoxic assay on mouse fibrosarcoma L929 cells . Basal levels of TNF production by mononuclear cells from 13 normal rabbits (124.3 units/ml +/- 27.1 units/ml, mean +/- SE) were not different from nine rabbits with colitis (83.6 units/ml +/- 24.4 units/ml, P > 0.05) . Treatment with lipopolysaccharide (100 micrograms/ml) induced increased TNF production by mononuclear cells isolated from both normals (672.0 units/ml +/- 197.5 units/ml, P < 0.05) and rabbits with colitis (1114.0 units/ml +/- 489.6 units/ml, P < 0.05) . However, at all lipopolysaccharide concentrations stimulated TNF levels were comparable in experimental and control groups (P > 0.05) . In light of the role of leukotrienes in inflammation, a separate group of rabbits with colitis was investigated following treatment with an oral leukotriene B4 receptor antagonist . Serum TNF levels in 15 control rabbits (32.5 units/ml +/- 7.6 units/ml, mean +/- SE) were not significantly different from rabbits with colitis receiving either leukotriene B4 receptor antagonist (35.7 units/ml +/- 9.2 units/ml, N = 13) or vehicle alone (50.3 units/ml +/- 10.2 units/ml, N = 14) (ANOVA, P > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Virology, 1992 Nov, 191(1), 9 - 18
Serine protein kinase associated with varicella-zoster virus ORF 47; Ng TI et al.; Varicella-zoster virus (VZV) ORF 47 lies in the unique long region of the VZV genome . Sequence homology studies have demonstrated that gene 47 possessed conserved protein kinase motifs . In this study, we investigated the properties of the ORF 47 product . First, a rabbit antiserum was raised against a protein generated from the fusion of the most antigenic ORF 47 domain with Escherichia coli beta-galactosidase . The high-titer antiserum reacted specifically with ORF 47 polypeptides translated in vitro . When incubated with VZV-infected cell lysate, the antiserum immunoprecipitated a phosphoprotein of M(r) 54,000, a size comparable with the predicted molecular mass . The precipitated viral protein was phosphorylated in a protein kinase assay; subsequent phosphoamino acid analysis indicated that the phosphotransferase associated with the ORF 47 protein was a serine protein kinase . Synthesis of the ORF 47 product in VZV-infected cell culture increased in the first and second days and plateaued after the third day of infection . The protein kinase activity associated with VZV ORF 47 had several distinctive biochemical properties: (i) its phosphotransferase activity was enhanced more by manganese than by magnesium, (ii) it utilized both ATP and GTP as donors of phosphate, and (iii) it phosphorylated both acidic and basic substrates . In summary, this report lends support to the computer homology data which predicted that VZV ORF 47 would encode a serine protein kinase.

J Virol, 1992 Nov, 66(11), 6609 - 15
The UL16 gene of human cytomegalovirus encodes a glycoprotein that is dispensable for growth in vitro; Kaye J et al.; The UL16 gene of human cytomegalovirus (HCMV) encodes a predicted translation product with features characteristic of glycoproteins (signal and anchor sequences and eight potential N-linked glycosylation sites) . Antisera were raised against the UL16 gene product expressed in Escherichia coli as a beta-galactosidase fusion protein . The antisera detected a 50-kDa glycoprotein in HCMV-infected cells that was absent from purified virions . The UL16 glycoprotein was synthesized at early times after infection and accumulated to the highest levels at late times after infection . A recombinant HCMV in which UL16 coding sequences were interrupted by a lacZ expression cassette was constructed by insertional mutagenesis . Analysis of the phenotype of the recombinant virus indicated that the UL16 gene product is nonessential for virus infectivity and growth in tissue culture.

J Bacteriol, 1992 Nov, 174(21), 7059 - 62
Conditional growth of Escherichia coli caused by expression of vaccinia virus DNA topoisomerase I; Fernandez-Beros ME et al.; Active vaccinia virus topoisomerase I is expressed in Escherichia coli containing plasmid p1940 (S . Shuman, M . Golder, and B . Moss, J . Biol . Chem . 263:16401-16407, 1988) . Growth curves showed a decline of 2 to 3 logs in the number of viable cells at 42 degrees C after shift from 30 degrees C because of increased vaccinia virus topoisomerase I level . Mutations in the gyrA and gyrB genes allowed cells to grow equally well at 42 and 30 degrees C . The presence of gyrase inhibitor also improved growth at 42 degrees C.

J Bacteriol, 1992 Nov, 174(21), 6918 - 27
Catabolite gene activator protein and integration host factor act in concert to regulate tdc operon expression in Escherichia coli; Wu Y et al.; Anaerobic expression of the tdcABC operon of Escherichia coli requires cyclic AMP and the catabolite gene activator protein (CAP) . Purified CAP binds to a 30-bp sequence in the tdc promoter between positions -55 and -26, and a mutant CAP site with base substitutions at positions -48, -47, and -45 failed to bind CAP and also drastically reduced the beta-galactosidase expression from a tdcB'-'lacZ fusion plasmid . Recently, we showed that efficient expression of the tdc operon also requires a functional integration host factor (IHF) and an IHF-binding site in the tdc promoter between positions -118 and -88 . The levels of beta-galactosidase activity from the tdcB'-'lacZ fusion plasmids were also reduced in an IHF-deficient strain with the wild-type or mutant plasmid CAP sequence . In vitro footprinting experiments revealed that CAP and IHF occupy their specific binding sites on tdc DNA when they are present separately or together . These regulatory proteins also induced significant bending of the tdc promoter DNA . Our results suggest that CAP and IHF act in concert as positive transcription factors for tdc operon expression in vivo.

J Bacteriol, 1992 Nov, 174(21), 6872 - 7
Further evidence that transposition of Tn5 in Escherichia coli is strongly enhanced by constitutively activated RecA proteins; Kuan CT et al.; We have shown that excision and transposition of Tn5 in Escherichia coli are greatly increased by recA(Prtc) genes, which encode constitutively activated RecA proteins (C.-T . Kuan, S.-K . Liu, and I . Tessman, Genetics 128:45-57, 1991) . Contrary results, showing a significant decrease in Tn5 transposition under SOS conditions, were subsequently reported (M . D . Weinreich, J . C . Makris, and W . S . Reznikoff, J . Bacteriol . 173:6910-6918, 1991) . We have extended our studies to examine the following: (i) transposition of Tn5 from sites in the phoA, phoB, proC, trpD, and ilvD genes; (ii) the effect of gene transcription; (iii) the comparative effect of dinD+ and dinD(Def) alleles; (iv) the use of a mating-out assay of transposition; (v) the effect of a recA(Prtc) allele located at the normal chromosomal site; and (vi) the effect at 41.5 degrees C of the recA441(Prtc) allele . The new results fully confirm our previous conclusions, including the fact that the frequency of Tn5 transposition under constitutive SOS conditions is site dependent.

Infect Immun, 1992 Nov, 60(11), 4542 - 8
Limited T-cell receptor beta-chain diversity of a T-helper cell type 1-like response to Mycobacterium leprae; Uyemura K et al.; Delayed-type hypersensitivity (DTH) is the standard measure of T-cell responsiveness to infectious organisms . For leprosy, the Mitsuda reaction, a local immune response to cutaneous challenge with Mycobacterium leprae, is considered to represent a measure of DTH against the pathogen . We analyzed the diversity of the T-cell receptor beta-chain repertoire in Mitsuda reactions to determine the breadth of the mycobacterial antigens involved . The polymerase chain reaction was used to compare V beta usage in the Mitsuda reaction T-cell lines established and unstimulated peripheral blood . These molecular analyses revealed a skewed T-cell receptor V beta gene usage in the Mitsuda reaction and in T-cell lines from lesions . To examine the reactivity of T cells from these lesions, T-cell lines were tested against the available native and recombinant antigens of M . leprae . T-cell lines derived from Mitsuda reactions responded more strongly to the 10-kDa M . leprae antigen, a homolog of GroES in Escherichia coli, than to other M . leprae proteins . T-cell lines were also shown to proliferate strongly in response to the 17- and 3-kDa proteins . The pattern of the lymphokine mRNA of these cells was reminiscent of the pattern of murine TH1 cells, positive for interleukin-2 and gamma interferon and weakly positive for interleukin-4 . These data indicate that a limited array of T cells, perhaps recognizing stress proteins, secrete a type 1 lymphokine profile in the DTH response to mycobacteria.

Zhonghua Zhong Liu Za Zhi, 1992 Nov, 14(6), 414 - 7
{Minimum residual leukemic cells in genetically marked brown Norway rat myelocytic leukemia model}; Yan Y et al.; Different methods were used to detect minimal residual leukemic cells (LT 12 nl), which had been genetically marked with E . coli 1 acZ and neo-gene by retrovirus vector mediated gene transfer . The detection levels of flow cytometry based FDG staining and fluorophotometric method based MUG staining were 10(-3) to 10(-4) and 10(-2) to 10(-3), respectively . The method of G 418 selective agar culture was demonstrated as a 10(-4) to 10(-5) levels for the detection of LT 12 nl residual leukemic cells in bone marrow . The results indicated that the selective agar culture can be used as a sensitive method for the study of minimum residual disease in the BNML leukemia model . We have used the selective agar culture to study the distribution of clonal LT 12 nl cells in BNML during minimum residual disease (MRD) . A heterogenous distribution pattern of the clonal leukemic cells was found in the genetically marked BNML leukemia model during the MRD phase.

Protein Sci, 1992 Nov, 1(11), 1435 - 46
Arginine 54 in the active site of Escherichia coli aspartate transcarbamoylase is critical for catalysis: a site-specific mutagenesis, NMR, and X-ray crystallographic study; Stebbins JW et al.; The replacement of Arg-54 by Ala in the active site of Escherichia coli aspartate transcarbamoylase causes a 17,000-fold loss of activity but does not significantly influence the binding of substrates or substrate analogs (Stebbins, J.W., Xu, W., & Kantrowitz, E.R., 1989, Biochemistry 28, 2592-2600) . In the X-ray structure of the wild-type enzyme, Arg-54 interacts with both the anhydride oxygen and a phosphate oxygen of carbamoyl phosphate (CP) (Gouaux, J.E . & Lipscomb, W.N., 1988, Proc . Natl . Acad . Sci . USA 85, 4205-4208) . The Arg-54-->Ala enzyme was crystallized in the presence of the transition state analog N-phosphonacetyl-L-aspartate (PALA), data were collected to a resolution limit of 2.8 A, and the structure was solved by molecular replacement . The analysis of the refined structure (R factor = 0.18) indicates that the substitution did not cause any significant alterations to the active site, except that the side chain of the arginine was replaced by two water molecules . 31P-NMR studies indicate that the binding of CP to the wild-type catalytic subunit produces an upfield chemical shift that cannot reflect a significant change in the ionization state of the CP but rather indicates that there are perturbations in the electronic environment around the phosphate moiety when CP binds to the enzyme . The pH dependence of this upfield shift for bound CP indicates that the catalytic subunit undergoes a conformational change with a pKa approximately 7.7 upon CP binding . Furthermore, the linewidth of the 31P signal of CP bound to the Arg-54-->Ala enzyme is significantly narrower than that of CP bound to the wild-type catalytic subunit at any pH, although the change in chemical shift for the CP bound to the mutant enzyme is unaltered . 31P-NMR studies of PALA complexed to the wild-type catalytic subunit indicate that the phosphonate group of the bound PALA exists as the dianion at pH 7.0 and 8.8, whereas in the Arg-54-->Ala catalytic subunit the phosphonate group of the bound PALA exists as the monoanion at pH 7.0 and 8.8 . Thus, the side chain of Arg-54 is essential for the proper ionization of the phosphonate group of PALA and by analogy the phosphate group in the transition state . These data support the previously proposed proton transfer mechanism, in which a fully ionized phosphate group in the transition state accepts a proton during catalysis.

Protein Sci, 1992 Nov, 1(11), 1403 - 12
Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA-binding domain as studied by amide proton exchange; Mau T et al.; Backbone amide proton exchange rates in the DNA-binding domain of GAL4 have been determined using 1H-15N heteronuclear correlation NMR spectroscopy . Three forms of the protein were studied-the native Zn-containing protein, the Cd-substituted protein, and a Zn-GAL4/DNA complex . Exchange rates in the Zn-containing protein are significantly slower than in the Cd-substituted protein . This shows that Cd-substituted GAL4 is destabilized relative to the native Zn-containing protein . Upon DNA binding, global retardation of amide proton exchange with solvent was observed, indicating that internal fluctuations of the DNA-recognition module are significantly reduced by the presence of DNA . In all forms of the protein, the internal dyad symmetry of the DNA-recognition module of GAL4 is reflected by the backbone amide proton exchange rates.

Plant J, 1992 Nov, 2(6), 845 - 54
Molecular characterization of cDNA encoding for adenylate kinase of rice (Oryza sativa L.); Kawai M et al.; Two types of genes (Adk-a, and Adk-b) encoding for adenylate kinase (AK, EC 2.7.4.3.) were isolated from the cDNA library constructed from poly(A)+ RNA of rice (Oryza sativa L.) . Two cDNAs were heterogeneous at 5' and 3' ends of non-coding sequences and had possible polyadenylation signals . One of the genes, Adk-a, had 1154 bp sequences encoding 241 amino acid residues, while the other type, Adk-b, contained 1085 bp sequences encoding for 243 amino acid residues . Homology between Adk-a and Adk-b was 73.7% in nucleotide sequences, and 90.8% in amino acid level . Two genes showed about 53% homology to bovine mitochondrial adenylate kinase (AK2) at nucleotide and amino acid levels . Concerning the codon usage of rice AK genes, T was abundant at the third position of a codon in the reading frames . In order to examine the enzyme activity of the protein encoded by the rice cDNA, Adk-a was cloned into an expression vector, pUC119, which was introduced into Escherichia coli strain CV2, a temperature-sensitive mutant of adenylate kinase . We found that the transformant carrying the rice Adk-a gene in the sense orientation recovered cell growth at non-permissive high temperature (42 degrees C) and expressed enzyme activities higher than the untransformed CV2 and the transformant possessing Adk-a cDNA in the antisense orientation . These observations suggest that rice Adk-a codes a biologically active enzyme . Furthermore, sucrose was found to regulate the transcription of AK genes in rice cell cultures . Organ related accumulation of mRNA in whole plants was also found.

Mikrobiologiia, 1992 Nov-Dec, 61(6), 1087 - 95
{Survival of Escherichia coli cells during storage in suspensions of varying concentrations}; Vakhitov TIa et al.; The presence of positive correlative connection between death rate of Escherichia coli M-17 cells and the density of their suspension have been estimated . It has been shown, that the accumulation in extracellular medium (ECM) of death-stimulating (DS) metabolites, the concentration of which was higher in the suspensions of higher densities, was the immediate cause of the acceleration of death in suspensions with densities higher than 1 x 10(9) cells/ml . DS metabolites could be removed from ECM by adsorption or dialysis, and, thus, they had comparably low molecular mass . The presence of DS metabolites led to the acceleration of death of test-cultures E . coli M-17 . The filtrates of ECM of suspensions with density lower than 1 x 10(9) cells/ml did not accelerate the death rate of test-cultures, and, most probably, did not contain any DS-substances . It is supposed, that the role of DS-substances is the maintenance of optimal size of the population of bacteria . The low effective concentrations of these substances make it possible to consider that their functions are nothing but regulatory.

In Vivo, 1992 Nov-Dec, 6(6), 585 - 8
Induction of anti-Escherichia coli activity in mice by phenothiazines, benzo{a}phenothiazines and benz{c}acridines; Motohashi N et al.; The abilities of 14 phenothiazines, 8 benzo{a}phenothiazines and 12 benz{c}acridines to induce anti-Escherichia coli activity in mice were compared . Pretreatment with several benzo{a}phenothiazines or benz{c}acridines protected mice from lethal infection of Escherichia coli in a dose-dependent manner, whereas most of the phenothiazines induced much weaker anti-Escherichia coli activity . However, direct contact of Escherichia coli with these compounds or their administration just after bacterial inoculation were ineffective . These data suggest the immunopotentiation activity of benzo{a}phenothiazines and benz{c}acridines.

J Struct Biol, 1992 Nov-Dec, 109(3), 219 - 34
Assembly of 2-D membrane protein crystals: dynamics, crystal order, and fidelity of structure analysis by electron microscopy; Engel A et al.; Membrane protein reconstitution into two-dimensional (2-D) ordered arrays is described . The assembly of 2-D crystals may be modeled as a two-step process: the membrane protein is first integrated in the lipid bilayer and then crystallized by removal of excess detergent or lipid and/or by precipitating agents . Lipid-detergent, protein-detergent, and lipid-protein interactions are critical during the first step, while lipid-protein and protein-protein interactions dominate events in the second step . The evidence supporting this model results from quasielastic light scattering analyses and electron microscopy of different lipid-detergent systems and reconstitution experiments with Escherichia coli porin OmpF, Phormidium laminosum photosystem I reaction centers, and integral membrane proteins of mammalian lens fiber cells.

Kansenshogaku Zasshi, 1992 Nov, 66(11), 1538 - 42
{Enteroadherent Escherichia coli exhibiting localized pattern of adherence among infants with diarrhoea in Brazil--incidence and prevalence of serotypes}; Tsukamoto T et al.; The incidence of enteroadherent Escheridhia coli exhibiting localized adherence to HeLa cells was investigated using the EAF probe (Nataro et al., J . Infect . Dis., 152:560-563, 1985) among 126 infants below 3 years of age along with 126 age-matched healthy controls in Brazil . The EAF probe proved to be sensitive and specific in detection of enteroadherent E . coli . EAF-probe positive E . coli was isolated from 23.0% of the infants with acute diarrhoea while the corresponding rate of isolation from healthy controls was 11.9% . EAF-probe positive E . coli strains belonging to the classical enteropathogenic E . coli (EPEC) serogroups were more often associated with diarrheal cases (18.3%) than with strains isolated from control healthy infants (5.6%) . The predominant EAF-probe positive E . coli serotypes were O55:H-, O111:H2 and O119:H6 . These serotypes, especially O111:H2, were mainly isolated from cases with diarrhoea suggesting a strong causal association . Among the EAF positive non-EPEC serotypes, the most prevalent serotype was O88:H25 and this represents a, hitherto, unrecognized diarrheagenic E . coli serotype.

J Dermatol, 1992 Nov, 19(11), 797 - 801
A model to study the fate of genetically-marked keratinocytes in culture; Garlick JA et al.; In this study we demonstrate a method for analyzing the spatial distribution or fate of progeny keratinocytes derived from single progenitor cells . The method relies upon the use of retroviral vectors to introduce a reporter gene into replicating cells and to effect integration and expression of that new gene . All progeny cells from that initial cell inherit and express the transferred gene . The reporter gene is the E . coli beta-galactosidase gene (B-gal), which encodes a histochemically-detectable product in the cytoplasm . Using this method, we show that foci of genetically marked, B-gal positive cells can be readily identified in submerged cultures and we term this grouping of cells a "clonal proliferation unit" . Analysis of B-gal stained whole mounts and paraffin sections allows visualization of the proliferative potential and differentiating capacity of clonogenic cells . This model will allow exploration of how agents known to alter epidermal proliferation and differentiation affect lineage relationships.

Indian J Med Res, 1992 Nov, 95, 284 - 7
Loss of some virulence factors of enterotoxigenic Escherichia coli on repeated subcultures; Ram S et al.; Thirty enterotoxigenic Esch . coli (ETEC) strains of predominant serogroups, isolated from patients with diarrhoea in Ludhiana, Punjab were investigated for expression of heat labile (LT) enterotoxin and colonization factor antigens (CFAs) on repeated subculture . These belonged to serogroup 078 (10), 080 (2), 0114 (6), 020 (3), 0128 (3), 0153 (2) and 08 (4) respectively . The isolates exhibited a differential response for expression of LT and CFAs on repeated subculturing . All the strains were positive for both LT and CFA up to six subcultures . Three strains of serogroup 0114 and one of 080 failed to express CFA while one strain each of serogroups 080, 0114, 020 and 08 failed to elaborate LT in the 8th subculture . Only 25 and 19 isolates were detected as stable producer of LT and CFAs up to 10th subculture.

Indian J Med Res, 1992 Nov, 95, 278 - 83
Intestinal colonization & production of diarrhoea by enteroadherent-aggregative Escherichia coli; Tickoo SK et al.; The ability of HEp-2 cell adherent Esch . coli of aggregative phenotype (EA-Agg EC) to cause diarrhoea and to colonize the bowel of rabbits was studied . Thirty six rabbits were challenged with one of three EA-Agg EC strains (F23A; H766C and F17A-15, 3 and 3 rabbits respectively) or a control strain (K12-15 rabbits) in reversible ileal-tie in adult rabbit diarrhoea (RITARD) model . The animals were sacrificed 72 h post challenge . Severe diarrhoea occurred in greater number of F23A challenged rabbits than the controls (P < 0.05) . Mucosal cultures from proximal and distal small intestine and colon yielded about 1000 times more Esch . coli in the test than control rabbits (P < 0.001 in each case) . EA-Agg EC were consistently grown from mucosa in the test rabbits who commonly showed mild to moderate villous stunting and grade + to nuclear fragmentation (karyorrhexis) in the small and large bowel epithelium . The control animals had either normal villi or very mild villous stunting . Results comparable to F23A were obtained with the other two EA-Agg EC strains tested in a smaller number of animals.

Indian J Med Res, 1992 Nov, 95, 259 - 62
Childhood diarrhoea due to rotavirus in a community; Nath G et al.; The etiologic role of rotavirus in acute diarrhoeal illness in children under five years of age was studied over a period of one year in an urban slum community . Rotavirus was detected in 17.7 per cent of 376 children with diarrhoea and 4 per cent of 299 healthy children with maximum prevalence at 19-24 months of age with statistically comparable detection in 0-6 months of age . The overall prevalence was significantly higher in children below 2 yr as compared to those between 2-5 yr of age . Girls (24.1%) were more susceptible amongst the diarrhoeal group in comparison to boys (13.8%) . Diarrhoea due to rotavirus was more prevalent during the cooler months (November-February) of the year and no correlation was observed with rainfall and relative humidity . Rotavirus as the sole pathogen was observed in 9 per cent children with diarrhoea and in the rest, association of Cryptosporidium (3%) was observed for the first time with this virus along with other known enteropathogenic bacteria and parasites, enterotoxigenic Escherichia coli being the commonest organism.

Biull Eksp Biol Med, 1992 Nov, 114(11), 508 - 10
{The effect of exogenous whole-body hyperthermia on humoral immunity}; Solov'ev AS; We investigated the proliferative responses of spleen cells (SC) to polyclonal mitogens lipopolysaccharide (LPS) and pokeweed mitogen (PWM), immune responses to sheep red cells (SRC) in mice undergoing hyperthermia . There were increased proliferative responses of lymphocytes to PWM if we used mice having rectal temperature 42 degrees C . Thermal shock in mice was accompanied by suppression of immune response . If we used mice suffering from hyperthermia (43-44 degrees C) for 20 minutes; there were decreased proliferative responses of lymphocytes to PWM or LPS for 10-30 days . We observed low immune response to sheep red cells in mice for 5-20 days . The changes of immune response were not revealed on the 40th day after induction of hyperthermia in mice.

Development, 1992 Nov, 116(3), 573 - 83
Expression of Pax-3- and neuroectoderm-inducing activities during differentiation of P19 embryonal carcinoma cells; Pruitt SC; A P19 embryonal carcinoma stem cell line carrying an insertion of the E . coli LacZ gene in an endogenous copy of the Pax-3 gene was identified . Expression of the Pax-3/LacZ fusion gene in neuroectodermal and mesodermal lineages following induction of differentiation by chemical treatments (retinoic acid and dimethylsulfoxide) was characterized using this line and is consistent with the previous localization of Pax-3 expression in the embryo to mitotically active cells of the dorsal neuroectoderm and the adjacent segmented dermomyotome . Pax-3/LacZ marked stem cells were also utilized as target cells in mixing experiments with unmarked P19 cells that had been differentiated by pretreatment with chemical inducers . Induction of beta-galactosidase and neuroectodermal markers in the target cells demonstrates that: (1) some differentiated P19 cell derivatives transiently express endogenous Pax-3- and neuroectoderm-inducing activities, (2) undifferentiated target stem cells respond to these activities even in the presence of leukemia inhibitory factor and (3) the endogenous activities can be distinguished from, and are more potent than, retinoic acid treatment in inducing neuroectoderm . These observations demonstrate that P19 embryonal carcinoma cells provide a useful in vitro system for analysis of the cellular interactions responsible for neuroectoderm induction in mammals.

Somat Cell Mol Genet, 1992 Nov, 18(6), 529 - 41
Characterisation and correction of a mammalian cell mutant defective in late step of base excision repair; Ryan AJ et al.; An Indian muntjac cell line, SVM, is unusually sensitive to cell killing induced by a range of alkylating agents . Cells transfected with the Escherichia coli ada gene or human genomic DNA have allowed the response of SVM to alkylating agents to be dissociated into two distinct components . Thus, in SVM, which expresses very low levels of alkyltransferase (AT), O6-alkylguanine appears to be the major cytotoxic, clastogenic, and recombinogenic lesion following exposure to agents such as methylnitrosourea (MNU) . However, SVM is also very sensitive to agents such as dimethylsulfate (DMS), which produce only very low levels of O6-methylguanine damage . Sensitivity to DMS resides in an inability to complete base excision repair, with the appearance of persistent single-strand DNA breaks (SSBs), and does not appear to involve defects in glycosylase, apurinic/apyrimidinic endonuclease, or DNA ligase activities . Another, possibly related, phenotypic trait in SVM is its limited ability to ligate transfected linear plasmid DNA . Transfectants of SVM, harboring human DNA sequences, show a significant correction of DMS-induced cytotoxicity and clastogenicity and a reduction in the levels of DMS-induced DNA SSBs . The DMS-resistant transfectants have an increased ability to ligate linear plasmid DNA, and also express AT, making these lines resistant to alkylating agents such as MNU . These results suggest that cells possess a mechanism that regulates AT expression, plasmid break-joining ability, and certain aspects of base excision repair . Transfectants of SVM containing human DNA provide a means to isolate genes involved in a coordinate response to alkylation damage.

J Clin Immunol, 1992 Nov, 12(6), 440 - 50
Effects of in vivo endotoxin infusions on in vitro cellular immune responses in humans; Rodrick ML et al.; Studies of the immune response of patients following major injury have identified significant abnormalities, some of which may be due to the effects of endotoxin . To evaluate the effect of endotoxin on the immune system without conflicting variables, we studied 18 normal, healthy male volunteers each on two occasions . In one study, Escherichia coli endotoxin was administered intravenously at a dose of 4 ng/kg . In the other, saline was given . Blood for immune function studies was obtained at either 0, 4, or 24 hr (seven volunteers), 0, 1, and 4 hr (five volunteers), or 0, 4, and 6 hr (six volunteers) postinfusion . Peripheral blood mononuclear cells (PBMC) were isolated and adjusted to the same concentration . Measurements following endotoxin infusion were compared with those of the same volunteers following saline infusion and with those from normal ambulatory laboratory volunteers . Interleukin 1 (IL-1) production by adherent cells was significantly reduced at 1 hr post endotoxin infusion . Significant decreases in number of mononuclear cells, response to phytohemagglutinin (PHA), and production of IL-2 and IL-1 were observed by 4 hr after endotoxin infusion . No significant changes in percentages of monocytes, lymphocytes, or CD3, CD4, or CD8 lymphocytes were observed at any time . By 24 hr postinfusion all values had returned to normal or, in some cases, supranormal levels . Response to PHA by PBMC from volunteers 4 hr following endotoxin was completely restored by in vitro addition of recombinant human IL-2 but was only marginally improved by IL-1 . In vitro addition of indomethacin to PBMC cultures responding to PHA reduced the suppression observed after in vivo endotoxin but also was not as effective as IL-2 . In a fourth study, seven volunteers were treated as above either with two doses (800 mg each) of the cyclooxygenase inhibitor ibuprofen before endotoxin infusion or with ibuprofen alone . Ibuprofen pretreatment completely restored the PBMC response to PHA to normal and caused a significant decrease in the endotoxin-induced suppression of IL-2 production . However, the decrease in circulating PBMC number and adherent cell secretion of IL-1 was not affected by inhibition of the cyclooxygenase pathway . These results suggest that endotoxin has immunomodulatory effects on both adherent mononuclear-cell and T-lymphocyte function and that more than one mechanism is involved.

Mikrobiol Zh, 1992 Nov-Dec, 54(6), 41 - 5
{The isolation of ribosomes from Acholeplasma and the study of their physicochemical characteristics}; Korobkova ES et al.; Ribosomes are produced from Acholeplasma laidlawii PG-8 and A . laidlawii var . granulum str . 118 . It is proved as possible to use the standard method of differential centrifugation for isolation of mollicute ribosomes . The physico-chemical properties of acholesplasma ribosomes are studied . The protein content for A . laidlawii PG-8 amounted to 39.6%, rRNA content--60.4% and for A . laidlawii var . granulum--39.1 and 60.8%, respectively . The RNA: protein ratio equals 1.5:1, the ratio of optic density indicates at 260 and 280 nm--2, that is peculiar to treated preparations of ribosomes . Sedimentation coefficient of acholeplasma ribosomes is 70S . The produced preparations of acholeplasma ribosomes can be used subsequently for creating the system of translation in vitro to study the biosynthesis processes of mollicute protein.

Mol Microbiol, 1992 Nov, 6(22), 3385 - 93
High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II; Lin-Chao S et al.; The plasmids pUC18 and pUC19 are pBR322 derivatives that replicate at a copy number several fold higher than the parent during growth of Escherichia coli at 37 degrees C . We show here that the high copy number of pUC plasmids results from a single point mutation in the replication primer, RNA II, and that the phenotypic effects of this mutation can be suppressed by the Rom (RNA one modulator)/Rop protein or by lowering the growth temperature to 30 degrees C . The mutation's effects are enhanced by cell growth at 42 degrees C, at which copy number is further increased . During normal cell growth, the pUC mutation does not affect the length or function of RNA I, the antisense repressor of plasmid DNA replication, but may, as computer analysis suggests, alter the secondary structure of pUC RNA II . We suggest that the pUC mutation impedes interactions between the repressor and the primer by producing a temperature-dependent alteration of the RNA II conformation . The Rom/Rop protein may either promote normal folding of the mutated RNA II or, alternatively, may enable the interaction of sub-optimally folded RNA II with the repressor.

Nippon Hinyokika Gakkai Zasshi, 1992 Nov, 83(11), 1801 - 7
{Production of polyclonal antibody against human androgen receptor and immunohistochemical study of human androgen receptor in prostatic tissues}; Mizokami A et al.; We produced polyclonal antibody against human androgen receptor (hAR) by means of immunizing a rabbit with hAR fusion protein that was expressed in E . coli . In Western blot analysis, this antibody, NH27, recognized two protein bands at the site of 110 kDa and 107 kDa in androgen-independent human prostatic cancer cells (PC-3), transfected with full-length hAR expression plasmid DNA and at the site of 114 kDa and 108 kDa in androgen-dependent human prostatic cancer cells (LNCaP) . In immunohistochemical examination with NH27, the nuclei of epithelial and stromal cells in human benign prostatic hyperplasia were mainly stained as did with AN1-15, commercially available hAR monoclonal antibody . Titer of NH27, however, was about five times more high than that of AN1-15 . In prostatic cancer cells the nuclei were stained with NH27 as did with AN1-15 . Intensity of staining was various between the nuclei of cancer cells . The polyclonal antibody, NH27, produced in the present study is useful in investigating the characterization of AR in androgen-dependent and -independent prostatic cancers.

J Gen Microbiol, 1992 Nov, 138 ( Pt 11), 2337 - 45
Isolation and characterization of adhesin-defective TnphoA mutants of septicaemic porcine Escherichia coli of serotype O115:K-:F165; Harel J et al.; Non-enterotoxigenic porcine Escherichia coli strains belonging to the serogroup O115 have been associated with septicaemia and diarrhoea . Putative factors important in the pathogenicity of E . coli of serogroup O115 include fimbrial antigen F165, haemagglutination (MRHA), lipopolysaccharide, serum resistance, capsule and production of aerobactin . Using TnphoA transposon insertion mutagenesis, two classes of mutants were obtained from E . coli of serotype O115:F165 with respect to the phenotypic expression of fimbrial antigen F165 and MRHA of sheep erythrocytes: class I, F165-MRHA-, serum resistant; class II, F165+MRHA-, serum resistant . In a chicken lethality model, class I mutants were either virulent or of intermediate virulence, while class II mutants were of intermediate virulence . Alkaline phosphatase activity of class I and class II TnphoA mutants showed similar environmental regulation to that of fimbrial antigen F165 . Moreover, class I and class II mutants were mutated in the prs-like locus, and lacked a 18.5 kDa and/or a 17.5 kDa fimbrial band.

Immunology, 1992 Nov, 77(3), 322 - 9
A delayed-type hypersensitivity-inducing T-cell epitope of Semliki Forest virus mediates effective T-helper activity for antibody production; Snijders A et al.; The rational development of peptide vaccines requires the identification of both B- and T-cell epitopes . In this study, potential T-helper cell epitopes of Semliki Forest virus (SFV) were identified on the basis of their ability to induce delayed-type hypersensitivity (DTH) in mice using recombinant SFV fragments produced as hybrid proteins with beta-galactosidase in Escherichia coli and synthetic peptides coupled to beta-galactosidase . Although the tested fragments spanned almost the entire amino acid sequence of the structural proteins of SFV, only one DTH-inducing region (located between amino acid 137 and 151 of the SFV E2 membrane protein) was identified . Peptides containing this E2 region stimulated lymph node cells from SFV-primed mice in vitro . The ability of the identified T-cell epitope to induce a specific T-helper response in mice was evaluated using synthetic peptides that contained combinations of the DTH-inducing region and different previously identified linear B-cell epitopes of E2 . These peptides proved able to induce an antipeptide IgG response in mice in an H-2d-restricted fashion . One of the peptides was also able to induce high titres of IgG reactive with SFV-infected cells and protected 70-100% of the peptide-immunized mice after challenge with virulent SFV . Our findings suggest that DTH and T-helper activity are mediated by different doses of the same T-cell epitope.

Mol Gen Genet, 1992 Nov, 235(2-3), 422 - 31
Transcriptional analysis of the fix ABCXORF1 region of Azorhizobium caulinodans suggests post-transcriptional processing of the fix ABCXORF1 mRNA; Arigoni F et al.; We report here the transcriptional analysis of the fixABCXORF1 region of Azorhizobium caulinodans . This led to the identification of a 0.9 kb transcript covering fixX and ORF1, which was synthesized only under conditions of nitrogen fixation . The 5' end of this transcript was mapped by primer extension and S1 nuclease protection analyses and shown to be located 70 +/- 1 nucleotides upstream of the fixX start codon . By means of transcriptional fixX- and ORF1-lacZ fusions, it was shown that fixX and ORF1 were most probably transcribed from the fixA promoter and that expression of fixX and ORF1 was dependent on NifA activation . This suggests that the 0.9 kb mRNA results from post-transcriptional processing of a large mRNA covering fixA,B,C,X and ORF1 . In addition, ORF1 mutants were constructed and were shown not to be impaired in nitrogenase activity.

Plasmid, 1992 Nov, 28(3), 247 - 57
Convergent and overlapping transcripts of the Chlamydia trachomatis 7.5-kb plasmid; Fahr MJ et al.; Transcription of the 7.5-kb cryptic plasmid of Chlamydia trachomatis serovar L2 was investigated . Faint, diffuse transcripts of about 1.6 and 2.2 kb and intense short transcripts of about 250 and 430 bases were identified by Northern blot analysis . The short transcripts were found to have a common 5' end corresponding to bp 501 relative to the unique BamHI site of the plasmid and to terminate at different downstream sites . Putative promoter sequences of TTGCCA and TATATT, which closely resemble the consensus recognition site of Escherichia coli sigma 70, were identified at the -35 and -10 positions upstream from the 5' end of the short transcripts made in chlamydia . Transcripts of similar sizes were also expressed from this promoter in E . coli harboring a recombinant plasmid encoding the short transcripts . The short transcripts encode a common open reading frame (ORF) of 34 codons; however, a strong ribosome binding site was not found in the vicinity of the initiator codon, and it is not known whether the transcripts are translated in vivo . A large ORF of 330 codons, which has been shown to encode a hypothetical protein containing conserved domains of recombinase-like proteins, is antisense to the short transcripts . Transcripts encoding the large ORF could not be detected directly by Northern blot or primer extension analysis . However, transcripts were detected by polymerase chain reaction amplification of the large ORF cDNA and when Southern blots of single-stranded antisense DNA for the large ORF were probed with radiolabeled RNA synthesized by host-free chlamydial reticulate bodies . Thus, both strands of the chlamydial plasmid are transcribed in the region encoding the short transcripts . We propose that the short transcripts play a regulatory role as antisense RNAs.

Am J Physiol, 1992 Nov, 263(5 Pt 2), R1122 - 9
Modulation of glucose metabolic response to endotoxin by granulocyte colony-stimulating factor; Lang CH et al.; The present study examines whether in vivo administration of granulocyte colony-stimulating factor (G-CSF) and the resultant neutrophilia alters basal glucose metabolism or modulates the glucose metabolic response to a subsequent endotoxin {lipopolysaccharide (LPS)} challenge . Rats were injected with human recombinant G-CSF (50 micrograms/kg sc) twice daily for 2 days preceding an injection of LPS . Animals treated with G-CSF showed an eightfold increase in blood polymorphonuclear leukocytes (PMNs) but no detectable changes in hemodynamics or glucose metabolism . In control animals, LPS transiently decreased circulating PMN number, but by 4 h neutrophils had returned to control levels . LPS produced a greater reduction in circulating neutrophils in G-CSF-treated animals, which did not return to pretreatment levels by 4 h . G-CSF also produced marked changes in the glucose metabolic response to LPS . Rates of whole body glucose production and utilization in both control and G-CSF-treated rats were rapidly increased by LPS; however, the increment in glucose flux was 55-100% greater in the latter group . The enhanced rate of hepatic glucose production in this group occurred despite lower plasma levels of lactate and glucagon . The elevated rate of whole body glucose utilization was attributable to the G-CSF-enhanced LPS-induced increase in glucose uptake by the ileum, spleen, liver, and lung . Furthermore, LPS increased glucose uptake by skeletal muscle in G-CSF-treated rats but not in control animals . The enhanced glucose disposal in G-CSF-treated rats was not mediated by increases in plasma glucose or insulin concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Virol, 1992 Nov, 73 ( Pt 11), 2969 - 73
Human sera from varicella-zoster virus (VZV) infections cross-react with human T cell leukaemia virus type 1 (HTLV-1): common epitopes in VZV gene 22 protein and HTLV-1 p19 gag protein; Sato A et al.; Twenty-nine of 100 sera from patients recently infected with varicella-zoster virus (VZV) were found to cross-react with human T cell leukaemia virus type 1 (HTLV-1) antigen in the particle agglutination (PA) assay using HTLV-1 antigen-coated gelatin particles . Anti-VZV IgM antibodies were shown to be responsible for this cross-reactivity . Western blot analysis revealed that PA-positive anti-VZV sera reacted with the HTLV-1 gag p19 protein in HTLV-1-infected cells and recombinant p19 protein produced in Escherichia coli . By using a truncated p19, the cross-reactive region was located to the C-terminal 17 amino acids of p19 . One oligopeptide derived from the C terminus, PQIPPPYVEPT (amino acids 115 to 125), was capable of inhibiting PA, suggesting that this peptide carries the cross-reactive epitope . A homologous sequence was found in the VZV gene 22 protein by database analysis, and the oligopeptide TNIPPPLALLR (amino acids 1330 to 1340) had the ability to inhibit PA . These findings suggest that some IgM antibodies against the VZV gene 22 protein produced in the early phase of VZV infection are cross-reactive with the HTLV-1 gag p19 protein because they recognize an antigenic determinant containing an IPPP tetrapeptide.

Biochem Biophys Res Commun, 1992 Oct 30, 188(2), 754 - 9
Casein kinase II-catalysed phosphorylation of calmodulin is altered by amino acid deletions in the central helix of calmodulin; Sacks DB et al.; Calmodulin is phosphorylated by casein kinase II on Thr-79, Ser-81, Ser-101 and Thr-117 . To determine the consensus sequences for casein kinase II in intact calmodulin, we examined casein kinase II-mediated phosphorylation of engineered calmodulins with 1-4 deletions in the central helical region (positions 81-84) . Total casein kinase II-catalyzed phosphate incorporation into all deleted calmodulins was similar to control calmodulin . Neither CaM delta 84 (Glu-84 deleted) nor CaM delta 81-84 (Ser-81 to Glu-84 deleted) has phosphate incorporated into Thr-79 or Ser-81, but both exhibit increased phosphorylation of residues Ser-101 and Thr-117 . These data suggest that phosphoserine in the +2 position may be a specificity determinant for casein kinase II in intact proteins and/or secondary structures are important in substrate recognition by casein kinase II.

Biochem Biophys Res Commun, 1992 Oct 30, 188(2), 638 - 43
Heat stress of cultured GC cells enhances triiodothyronine-induced growth hormone production by action within the 5'-flanking region of the rat growth hormone gene; Mokshagundam S et al.; We studied the effect of incubation at 41 C on a clone of GC cells that had previously been stably transfected with a gene construct, pGHXGPT, containing -1800 to +8 of the rat growth hormone promoter fused to the structural gene for E . Coli xanthine guanine phosphoribosyl-transferase . The effect of incubation of the clone containing pGHXGPT at 41 C was to enhance triiodothyronine induction of growth hormone secretion (2-fold, p < 0.01) and of xanthine quanine phosphoribosyl-transferase activity (3-fold, p < 0.01) . We conclude that the increase in triiodothyronine-induced growth hormone production during heat stress occurs by stimulation of the growth hormone promoter.

Science, 1992 Oct 30, 258(5083), 812 - 5
A GDP dissociation inhibitor that serves as a GTPase inhibitor for the Ras-like protein CDC42Hs; Hart MJ et al.; Members of the family of Ras-related guanosine triphosphate (GTP) binding proteins appear to take part in the regulation of a number of biological processes, including cell growth and differentiation . Three different classes of proteins that regulate the GTP binding and GTP hydrolytic activities of the Ras family members have been identified . These different regulatory proteins inhibit guanosine diphosphate (GDP) dissociation (designated as GDIs), stimulate GDP dissociation and GDP-GTP exchange (designated as GDSs), or stimulate GTP hydrolysis (designated as GAPs) . In the case of the Ras-like protein CDC42Hs, which is the human homolog of a Saccharomyces cerevisiae cell division cycle protein, the GDI protein also inhibited both the intrinsic and GAP-stimulated hydrolysis of GTP . These findings establish an additional role for the GDI protein--namely, as a guanosine triphosphatase (GTPase) inhibitory protein for a Ras-like GTP binding protein.

Biochim Biophys Acta, 1992 Oct 30, 1128(2-3), 227 - 36
Black lipid membranes of tetraether lipids from Thermoplasma acidophilum; Stern J et al.; Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol . Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used . Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent . This finding corresponds to the results of Gliozzi et al . for the lipids of another archaebacterium, Sulfolobus solfataricus . Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration . In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin . At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2) . Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness . Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments . Poly(L-lysine) destroyed black lipid membranes . Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves . The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes . This influence was stronger than that of the polysaccharide dextran.

Biochem Biophys Res Commun, 1992 Oct 30, 188(2), 879 - 87
EPR stopped-flow studies of the reaction of the tyrosyl radical of protein R2 from ribonucleotide reductase with hydroxyurea; Lassmann G et al.; The reaction of the functional tyrosyl radical in protein R2 of ribonucleotide reductase from E . coli and mouse with the enzyme inhibitor hydroxyurea has been studied by EPR stopped-flow techniques at room temperature . The rate of disappearance of the tyrosyl radical in E . coli protein R2 is k2 = 0.43 M-1 s-1 at 25 degrees C . The reaction follows pseudo-first-order kinetics up to 450 mM hydroxyurea indicating that no saturation by hydroxyurea takes place even at this high concentration . Transient nitroxide-like radicals from hydroxyurea have been detected for the first time in the reaction of hydroxyurea with protein R2 from E . coli and mouse, indicating that 1-electron transfer from hydroxyurea to the tyrosyl radical is the dominating mechanism in the inhibitor reaction . The hydroxyurea radicals appear in low steady-state concentrations during 2-3 half-decay times of the tyrosyl radical and disappear thereafter.

Nature, 1992 Oct 29, 359(6398), 855 - 8
Structure of hisactophilin is similar to interleukin-1 beta and fibroblast growth factor; Habazettl J et al.; The fast reaction of the actin-based cytoskeleton in motile cells after stimulation with a chemoattractant requires a signal-transduction chain that creates a very specific environment at distinct regions beneath the plasma membrane . Dictyostelium hisactophilin, a unique actin-binding protein, is a submembranous pH sensor that signals slight changes of the H+ concentration to actin by inducing actin polymerization and binding to microfilaments only at pH values below seven . It has a relative molecular mass of 13.5K and its most unusual feature is the presence of 31 histidine residues among its total of 118 amino acids . The transduction of an external signal from the plasma membrane to the cytoskeleton is poorly understood . Here we report the protein's structure in solution determined by nuclear magnetic resonance spectroscopy . The nuclear Overhauser effect intensities of the three-dimensional nuclear Overhauser spectra were used directly in the calculations . The overall folding of histactophilin is similar to that of interleukin-1 beta and fibroblast growth factor, but the primary amino-acid sequence of hisactophilin is unrelated to these two proteins.

Nature, 1992 Oct 29, 359(6398), 851 - 5
Crystal structure of a Src-homology 3 (SH3) domain; Musacchio A et al.; The Src-homologous SH3 domain is a small domain present in a large number of proteins that are involved in signal transduction, such as the Src protein tyrosine kinase, or in membrane-cytoskeleton interactions, but the function of SH3 is still unknown (reviewed in refs 1-3) . Here we report the three-dimensional structure at 1.8 A resolution of the SH3 domain of the cytoskeletal protein spectrin expressed in Escherichia coli . The domain is a compact beta-barrel made of five antiparallel beta-strands . The amino acids that are conserved in the SH3 sequences are located close to each other on one side of the molecule . This surface is rich in aromatic and carboxylic amino acids, and is distal to the region of the molecule where the N and C termini reside and where SH3 inserts into the alpha-spectrin chain . We suggest that a protein ligand binds to this conserved surface of SH3.

Biochemistry, 1992 Oct 27, 31(42), 10380 - 9
Determination of recognition nucleotides for Escherichia coli phenylalanyl-tRNA synthetase; Peterson ET et al.; The nucleotides in Escherichia coli tRNA(Phe) required for recognition by its cognate synthetase have been determined in vitro by measuring the kinetic parameters for aminoacylation using mutant tRNA(Phe) transcripts with purified E . coli tRNA(Phe) synthetase . The substitution of 11 nucleotides in E . coli tRNA(Phe) is shown to decrease the kcat/KM by as much as 1000-fold relative to the wild type . The most important recognition elements are the three anticodon nucleotides G34, A35, and A36 . The recognition set also includes nucleotides in the variable pocket (U20 and U59), the acceptor end (A73), and the tRNA central core (G10, C25, A26, G44, and U45) . Many of the recognition nucleotides are also among the residues comprising the identity set determined in vivo using an amber suppressor tRNA(Phe) {McClain, W . H., & Foss, K . (1988) J . Mol . Biol . 202, 697-709} . As could be anticipated from the very different methods used, some nucleotides in the identity set determined by the suppressor method were not among the recognition nucleotides and vice versa . The E . coli tRNA(Phe) recognition data can also be compared to the recognition sets for yeast and human tRNA(Phe) determined previously . The results indicate that the mechanism by which phenylalanyl-tRNA synthetases recognize their substrates seems to have diverged somewhat among different species . For example, nucleotide 20 in the D-loop, the anticodon nucleotides and the discriminator base 73 are important for the recognition by all three enzymes . However, recognition of the tRNA central core nucleotides is unique to E . coli FRS.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Oct 27, 31(42), 10363 - 9
The low-spin heme site of cytochrome o from Escherichia coli is promiscuous with respect to heme type; Puustinen A et al.; Cytochrome o of Escherichia coli is able to incorporate two different structures of heme, either heme B (protoheme) or heme O, in its low-spin heme site . In contrast, the heme of the binuclear O2 reduction site is invariably heme O . Heme O is a newly discovered heme that is related to heme A, but with the formyl group of the latter replaced by methyl . Enzyme isolated from wild type E . coli has predominantly heme B in the low-spin site, whereas enzyme isolated from various overexpressing strains contains both types of enzyme in different proportions . In some strains, 70% of the enzyme has heme O in the low-spin site . Despite this variation in the structure of one of the prosthetic groups, the enzymatic activity and polypeptide composition of the enzyme remain virtually constant . EPR and activity data both indicate that heme B and heme O occupy the same low-spin heme site in the enzyme . With heme O in this site, the alpha-absorption band is narrower and further to the blue, and the Em,7 is lower, than when there is heme B in the site . In contrast to previous proposals, we show here that the enzyme does not exhibit significant spectral interactions between the hemes . The structural heterogeneity of the low-spin heme accounts for the variation in the optical spectra and redox properties of the enzyme as isolated from different strains of E . coli.

Biochemistry, 1992 Oct 27, 31(42), 10310 - 4
Functional compensation of a recognition-defective transfer RNA by a distal base pair substitution; Hou YM et al.; A single G3:U70 base pair in the acceptor helix is the major determinant of alanine acceptance in alanine transfer RNAs . Transfer of this base pair into other transfer RNAs confers alanine acceptance . A G3:C70 substitution eliminates alanine acceptance in vivo and in vitro . In this work, a population of mutagenized G3:C70 alanine tRNA amber suppressors was subjected to a selection for mutations that compensate for the inactivating G3:C70 substitution . No compensatory mutations located in the acceptor helix were obtained . Instead, a U27:U43 substitution that replaced the wild-type C27:G43 in the anticodon stem created a U27:U43/G3:C70 mutant alanine tRNA that inserts alanine at amber codons in vivo . The U27:U43 substitution is at a location where previous footprinting work established an RNA-protein contact . Thus, this mutation may act by functionally coupling a distal part of the tRNA structure to the active site.

Biochemistry, 1992 Oct 27, 31(42), 10303 - 9
Subunit complementation of thymidylate synthase; Pookanjanatavip M et al.; Each of the two active sites of thymidylate synthase contains amino acid residues contributed by the other subunit . For example, Arg-178 of one monomer binds the phosphate group of the substrate dUMP in the active site of the other monomer {Hardy et al . (1987) Science 235, 448-455} . Inactive mutants of such residues should combine with subunits of other inactive mutants to form heterodimeric hybrids with one functional active site . In vivo and in vitro approaches were used to test this hypothesis . In vivo complementation was accomplished by cotransforming plasmid mixtures encoding pools of inactive Arg-178 mutants and pools of inactive Cys-198 mutants into a host strain deficient in thymidylate synthase . Individual inactive mutants of Arg-178 were also cotransformed with the C198A mutant . Subunit complementation was detected by selection or screening for transformants which grew in the absence of thymidine, and hence produced active enzyme . Many mutants at each position representing a wide variety of size and charge supported subunit complementation . In vitro complementation was accomplished by reversible dissociation and unfolding of mixtures of purified individual inactive Arg-178 and Cys-198 mutant proteins . With the R178F + C198A heterodimer, the Km values for dUMP and CH2H4folate were similar to those of the wild-type enzyme . By titrating C198A with R178F under unfolding-refolding conditions, we were able to calculate the kcat value for the active heterodimer . The catalytic efficiency of the single wild-type active site of the C198A + R178F heterodimer approaches that of the wild-type enzyme.

Biochemistry, 1992 Oct 27, 31(42), 10185 - 93
Modular mutagenesis of exons 1, 2, and 8 of a glutathione S-transferase from the mu class . Mechanistic and structural consequences for chimeras of isoenzyme 3-3; Zhang P et al.; Exons 1 and 2 and exon 8 of the mu class GSH transferases from rat encode sequence-variable regions 1 and 4 of mu class isoenzymes, respectively . These two of four variable regions are located at the N- and C-termini of this isoenzyme class and impinge on the active site . In order to assess the influence of these variable regions on the catalytic diversity of the class mu isoenzymes, seven chimeric isoenzymes were constructed by transplantation of the variable regions of the sequence of the type 4 subunit into the corresponding regions of the type 3 subunit . The chimeric isoenzymes exhibit unique catalytic properties . Replacement of all, or part, of variable region 4 of the type 3 subunit with that of the type 4 subunit results in chimeric catalysts with higher turnover numbers in nucleophilic aromatic substitution reactions . Analysis of the crystal structure of isoenzyme 3-3 {Ji, X., Zhang, P., Armstrong, R . N., & Gilliland, G . L . (1992) Biochemistry (preceding paper in this issue)} suggests that interaction of the flexible C-terminal tail with the N-terminal domain helps limit the rate of product release from the active site of isoenzyme 3-3 in this type of reaction . Substitution of all, or part, of the sequence-variable region 1 of subunit 3 with that of subunit 4 results in chimeric isoenzymes that mimic the high stereoselectivity but not the catalytic efficiency of isoenzyme 4-4 toward alpha,beta-unsaturated ketones.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Oct 27, 31(42), 10331 - 7
Fumarase a from Escherichia coli: purification and characterization as an iron-sulfur cluster containing enzyme; Flint DH et al.; It has been shown previously that Escherichia coli contains three fumarase genes designated fumA, fumB, and fumC . The gene products fumarases A, B, and C have been divided into two classes . Class I contains fumarases A and B, which have amino acid sequences that are 90% identical to each other, but have almost no similarity to the sequence of porcine fumarase . Class II contains fumarase C and porcine fumarase, which have amino acid sequences 60% identical to each other {Woods, S.A., Schwartzbach, S.D., & Guest, J.R . (1988) Biochim . Biophys . Acta 954, 14-26} . In this work it is shown that purified fumarase A contains a {4Fe-4S} cluster . This conclusion is based on the following observations . Fumarase A contains 4 Fe and 4 S2- per mole of protein monomer . (The mobility of fumarase A in native polyacrylamide gel electrophoresis and the elution volume on a gel permeation column indicate that it is a homodimer.) Its visible and circular dichroism spectra are characteristic of proteins containing an Fe-S cluster . Fumarase A can be reduced to an EPR active-state exhibiting a spectrum consisting of a rhombic spectrum at high fields (g-values = 2.03, 1.94, and 1.88) and a broad peak at g = 5.4 . Upon addition of substrate, the high field signal shifts upfield (g-values = 2.035, 1.92, and 1.815) and increases in total spins by 8-fold, while the g = 5.4 signal disappears.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1992 Oct 26, 311(3), 311 - 4
Deletion of internal twenty-one amino acid residues of Escherichia coli prolipoprotein does not affect the formation of the murein-bound lipoprotein; Zhang WY et al.; Mutation pgsA affecting the phosphatidylglycerol phosphate synthesis is lethal for all but certain E . coli strains such as strains deleted for the lpp gene or strains containing unmodifiable prolipoprotein like lppD14 . Strain SD312 pgsA3 is tolerant to pgsA mutation, which suggests the lpp alleles in strain SD312 pgsA3 and its parental strain SD12 may be defective . DNA sequence analysis of the lpp genes in Escherichia coli strains SD12 and SD312 pgsA using asymmetric polymerase chain reaction showed that the lpp alleles in these two strains contained a 63 base pair deletion corresponding to the 37th to 57th codons of the wild-type lpp gene . {3H}Palmitate labeling of strains SD12 and SDS312 showed that the mutant lipoprotein in SD12 strain was modified with lipid, while the prolipoprotein in SD312 was not modified . The shortened mature lipoprotein in SD12 and the lipid-modified prolipoprotein in globomycin-treated SD12 were found to be covalently attached to the peptidoglycan, while the unmodified prolipoprotein in SD312 did not form significant amounts of murein-bound lipoprotein.

FEBS Lett, 1992 Oct 26, 311(3), 276 - 80
Sequence analysis of pigeon delta-crystallin gene and its deduced primary structure . Comparison of avian delta-crystallins with and without endogenous argininosuccinate lyase activity; Lin CW et al.; delta-Crystallin is a major lens protein present in the avian and reptilian lenses . To facilitate the cloning of the delta-crystallin gene, cDNA was constructed from the poly(A)+ RNA of pigeon lenses, amplified by the polymerase chain reaction (PCR) . The PCR product was then subcloned into pUC19 vector and transformed into E . coli strain JM109 . Plasmids purified from the positive clones were prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method . Sequencing two clones, containing 1.4 kb DNA inserts coding for delta-crystallin allowed the construction of a complete, full-length reading frame of 1,417 bp covering a deduced protein sequence of 466 amino acids, including the universal translation-initiating methionine . The pigeon delta-crystallin shows 88, 83 and 69% sequence identity to duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively . It is also shown that, in contrast to duck delta 2 crystallin which has a high argininosuccinate lyase activity, pigeon delta-crystallin appears to contain very low activity of this enzyme, despite the fact that they share a highly homologous structure . A structural comparison of delta-crystallins with or without enzymatic activity suggested several amino acid replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain avian species.

FEBS Lett, 1992 Oct 26, 311(3), 199 - 202
Ribosomal localization of the mRNA in the 30S initiation complex as revealed by UV crosslinking; Brandt R et al.; Translation initiation complexes consisting of 30S ribosomal subunits, 32P-labelled mRNA (002 mRNA), fMet-tRNA and the three initiation factors were subjected to UV-crosslinking to determine the protein and rRNA neighbors of the bound mRNA by immunochemical methods and by nucleic acid hybridization techniques . The mRNA was found to be crosslinked to a specific region of the 16S rRNA spanning from nucleotide 418 to 615 and to ribosomal proteins S1 and S21 (the main targets), S3, S10, S12 and S14; a low level of crosslinking was also detected with S2, S7, S13, S18 and S19.

Neurosci Lett, 1992 Oct 26, 146(1), 72 - 4
Involvement of opioid receptors in nucleus tractus solitarii in modulating endotoxic hypotension in rats; Xu T et al.; Opioid antagonists selective for the mu-receptor (naltrexone) and kappa-receptor (nalmefene), respectively, were unilaterally microinjected into the nucleus tractus solitarii (NTS) of the pentobarbital anaesthetized (50 mg/kg, i.p.) rat subjected to E . coli endotoxin (15 mg/kg, i.v.) . Naltrexone (0.5 microgram) ameliorated or totally abolished the endotoxic hypotension and tachycardia, and nalmefene at a dose of 1/25 (0.02 microgram) that of naltrexone showed a similar effect . The results suggest that the kappa- and mu-opioid receptors in NTS are actively involved in mediating the hypotensive and tachycardiac effects induced by systemic endotoxin in rats.

FEBS Lett, 1992 Oct 26, 311(3), 271 - 5
Role of leucine residues in the C-terminal region of human interleukin-6 in the biological activity; Nishimura C et al.; Site-directed mutagenesis of two sets of three periodic leucine residues which appear at every seventh position in the C-terminal region of human interleukin-6 (IL-6) was performed . Both receptor-binding and immunoglobulin (Ig)-induction activities of a triple mutant Leu168,175,182-->Val were only 1% compared with those of wild-type IL-6 . However, the mutant Leu152,159,166-->Val had 13% receptor-binding and 2% Ig-induction activities of those of wild-type IL-6 . In order to obtain more direct information on the receptor-binding region, we prepared two synthetic peptides . A significant binding activity was observed for the peptide Leu168-Met185, but not for the peptide Leu152-Arg169 . These results indicate that leucine residues in the C-terminal region, especially Leu168, Leu175, and Leu182, play an important role in the receptor-binding and Ig-induction activities.

Nucleic Acids Res, 1992 Oct 25, 20(20), 5407 - 12
DeoR repression at-a-distance only weakly responds to changes in interoperator separation and DNA topology; Dandanell G; The interoperator distance between a synthetic operator Os and the deoP2O2-galK fusion was varied between 46 and 176 bp . The repression of the deoP2 directed galK expression as a function of the interoperator distance (center-to-center) was measured in vivo in a single-copy system . The results show that the DeoR repressor efficiently can repress transcription at all the interoperator distances tested . The degree of repression depends very little on the spacing between the operators, however, a weak periodic dependency of 8-11 bp may exist.

Nucleic Acids Res, 1992 Oct 25, 20(20), 5383 - 7
Cytosine nucleoside inhibition of the ATPase of Escherichia coli termination factor rho: evidence for a base specific interaction between rho and RNA; Richardson LV et al.; The function of rho factor in transcription termination depends on interactions with nascent RNA molecules that contain unpaired cytidylate residues . We show that cytidine, as a free nucleoside, inhibits the binding of rho to lambda cro mRNA and is a competitive inhibitor of rho-ATPase activity with lambda cro mRNA as cofactor . The relative ability of various cytidine analogs and other nucleosides to inhibit the rho-RNA interaction was used to probe features responsible for the base specificity of rho action . The results suggest that rho has a specificity pocket in its polynucleotide-binding site that apparently can make H-bond interactions with the side of the cytosine ring that normally faces away from the sugar ring and that may involve a relatively close fit along the edge of the ribose ring at the C2' carbon . The nature of the complex of rho with cytidine nucleotides was analyzed further by determining whether incubation with BrCMP caused inactivation of rho ATPase . Although BrCMP could form Michaelis inhibition complexes, it did not activate rho . Rho thus lacks a diagnostic property of enzymes that make specific covalent addition complexes with pyrimidines.

Nucleic Acids Res, 1992 Oct 25, 20(20), 5351 - 6
Characterization of Chlorella virus PBCV-1 CviAII restriction and modification system; Zhang Y et al.; A second DNA site-specific (restriction) endonuclease (R.CviAII) and its cognate adenine DNA methyltransferase (M.CviAII) were isolated from virus PBCV-1 infected Chlorella strain NC64A cells . R.CviAII, a heteroschizomer of the bacterial restriction endonuclease NlaIII, recognizes the sequence CATG, and does not cleave CmATG sequences . However, unlike NlaIII, which cleaves after the G and does not cleave either CmATG or mCATG sequences, CviAII cleaves between the C and A and is unaffected by mCATG methylation . The M.CviAII and R.CviAII genes were cloned and their DNA sequences were determined . These genes are tandemly arranged head-to-tail such that the TAA termination codon of the M.CviAII methyltransferase gene overlaps the ATG translational start site of R.CviAII endonuclease . R.CviAII is the first chlorella virus site-specific endonuclease gene to be cloned and sequenced.

Nucleic Acids Res, 1992 Oct 25, 20(20), 5305 - 10
RFP is a DNA binding protein associated with the nuclear matrix; Isomura T et al.; We reported that the RFP gene encodes a protein with putative zinc finger domains and was involved in the activation of the ret proto-oncogene . To further characterize the RFP protein, we developed a polyclonal antibody against the product synthesized from a fragment of the RFP cDNA expressed in Escherichia coli . Western blot analysis showed that RFP was identified as a 58 kDa protein in cell lysates from four human and rodent cell lines and from mouse testis . In addition, a unique 68 kDa protein was detected in the testis . Using AH7974 (rat ascites hepatoma) and Raji (human Burkitt lymphoma) cells, we demonstrated strong association of RFP with the nuclear matrix . Furthermore, RFP solubilized from the nuclear matrix had DNA-binding activity although it appears to bind more preferentially to double-stranded DNA than to single-stranded DNA . These results thus suggest that RFP may play a role in molecular processes which occur in the nuclear matrix.

J Biol Chem, 1992 Oct 25, 267(30), 21873 - 8
Effects of site-directed mutagenesis of the highly conserved aspartate residues in domain II of farnesyl diphosphate synthase activity; Marrero PF et al.; Comparison of the farnesyl diphosphate (FPP) synthase amino acid sequences from four species with amino acid sequences from the related enzymes hexaprenyl diphosphate synthase and geranylgeranyl diphosphate synthase show the presence of two aspartate rich highly conserved domains . The aspartate motif ((I, L, or V)XDDXXD) of the second of those domains has homology with at least 9 prenyl transfer enzymes that utilize an allylic prenyl diphosphate as one substrate . In order to investigate the role of this second aspartate-rich domain in rat FPP synthase, we mutated the first or third aspartate to glutamate, expressed the wild-type and mutant enzymes in Escherichia coli, and purified them to apparent homogeneity using a single chromatographic step . Approximately 12 mg of homogeneous protein was isolated from 120 mg of crude bacterial extract . The kinetic parameters of the purified wild-type recombinant FPP synthase containing the DDYLD motif were as follows: Vmax = 0.84 mumol/min/mg; GPP Km = 1.0 microM; isopentenyl diphosphate (IPP) Km = 2.7 microM . Substitution of glutamate for the first aspartate (EDYLD) decreased the Vmax by over 90-fold . The Km for IPP increased, whereas the Km for GPP remained the same in this D243E mutant . Substitution of glutamate for the third aspartate (DDYLE) did not result in altered enzyme kinetics in the D247E mutant . These results suggest that the first aspartate in the second domain is involved in the catalysis by FPP synthase.

J Biol Chem, 1992 Oct 25, 267(30), 21577 - 83
Site-directed mutagenesis reveals the involvement of an additional thioredoxin-dependent regulatory site in the activation of recombinant sorghum leaf NADP-malate dehydrogenase; Issakidis E et al.; The chloroplastic enzyme NADP-malate dehydrogenase is activated by a reversible thiol/disulfide interchange with reduced thioredoxin . Its target disulfide bridge is considered to be located at the amino terminus . To further substantiate the regulatory role of this disulfide, site-directed mutagenesis has been used to replace each or both of the amino-terminal cysteines of the sorghum leaf NADP-malate dehydrogenase, expressed in Escherichia coli, by serines . A truncation mutant lacking the amino terminus has also been produced . Surprisingly, the mutant proteins still required activation by reduced thioredoxin . However, their activation was almost instantaneous, whereas the native enzyme reached full activity after a 10-20 min preincubation . The 8 1/2 for reduced thioredoxin was decreased 2-fold in the mutants, but their Km values for NADPH and oxaloacetate did not change significantly . The inhibition of activation by NADP and inhibition of activity by thiol-derivatizing agents were also retained . These results are interpreted as an indication that two thioredoxin-dependent reduction steps are involved in NADP-dependent malate dehydrogenase light activation, hence that two disulfides per monomer participate in the process . The overall activation rate would depend on a conformational change following the reduction of the amino-terminal disulfide bridge . The amino terminus also plays a role in the dimerization of the protein.

J Biol Chem, 1992 Oct 25, 267(30), 21512 - 7
Inhibition of eukaryotic DNA polymerase alpha with a novel lysophosphatidic acid (PHYLPA) isolated from myxoamoebae of Physarum polycephalum; Murakami-Murofushi K et al.; A specific inhibitor of DNA polymerase alpha was isolated from the lipid fraction prepared from myxoamoebae of a true slime mold, Physarum polycephalum . The purified substance was subjected to structural studies by fast atom bombardment mass spectroscopy, infrared spectroscopy, and two-dimensional nuclear magnetic resonance spectroscopy . The structure of this substance was thereby suggested to be a novel lysophosphatidic acid (LPA) composed of cyclic phosphate and cyclopropane-containing hexadecanoic acid . Then we named this substance PHYLPA (Physarum LPA) . PHYLPA inhibited more than 80% of the affinity-purified calf thymus DNA polymerase alpha activity at a concentration of 10 micrograms/ml (approximately 20 microM) . Inhibition was observed for DNA polymerase alpha but not for DNA polymerase beta or gamma from various eukaryotic species, nor did it inhibit DNA polymerase I from E . coli . From kinetic analyses, the inhibition was considered to be caused by the interaction of PHYLPA with the template DNA.

J Biol Chem, 1992 Oct 25, 267(30), 21471 - 8
Further examination of seventeen mutations in Escherichia coli F1-ATPase beta-subunit; Senior AE et al.; Seventeen mutations in beta-subunit of Escherichia coli F1-ATPase which had previously been characterized in strain AN1272 (Mu-induced mutant) were expressed in strain JP17 (beta-subunit gene deletion) . Six showed unchanged behavior, namely: C137Y; G142D; G146S; G207D; Y297F; and Y354F . Five failed to assemble F1F0 correctly, namely: G149I; G154I; G149I,G154I; G223D; and P403S,G415D . Six assembled F1F0 correctly, but with membrane ATPase lower than in AN1272, namely: K155Q; K155E; E181Q; E192Q; D242N; and D242V . AN1272 was shown to unexpectedly produce a small amount of wild-type beta-subunit; F1-ATPase activities reported previously in AN1272 were referable to hybrid enzymes containing both mutant and wild-type beta-subunits . Purified F1 was obtained from K155Q; K155E; E181Q; E192Q; and D242N mutants in JP17 . Vmax ATPase values were lower, and unisite catalysis rate and equilibrium constants were perturbed to greater extent, than in AN1272 . However, general patterns of perturbation revealed by difference energy diagrams were similar to those seen previously, and the new data correlated well in linear free energy relationships for reaction steps of unisite catalysis . Correlation between multisite and unisite ATPase activity was seen in the new enzymes . Overall, the data give strong support to previously proposed mechanisms of unisite catalysis, steady-state catalysis, and energy coupling in F1-ATPases (Al-Shawi, M . K., Parsonage, D . and Senior, A . E . (1990) J . Biol . Chem . 265, 4402-4410) . The K155Q, K155E, D242N, and E181Q mutations caused 5000-fold, 4000-fold, 1800-fold, and 700-fold decrease, respectively, in Vmax ATPase, implying possibly direct roles for these residues in catalysis . Experiments with the D242N mutant suggested a role for residue beta D242 in catalytic site Mg2+ binding.

J Biol Chem, 1992 Oct 25, 267(30), 21368 - 74
Molecular cloning, expression, and characterization of the cDNA for the rat hepatic squalene synthase; McKenzie TL et al.; Amino acid sequence information was obtained for the NH2 terminus, and for endogenous peptides generated by trypsin digestion, of a purified, truncated form of rat hepatic squalene synthase (RSS, EC 2.5.1.21) (Shechter, I., Klinger, E., Rucker, M . L., Engstrom, R . G., Spirito, J . A., Islam, M . A., Boettcher, B . R., and Weinstein, D . B . (1992) J . Biol . Chem . 267, 8628-8635) . Degenerate primers, based on the amino acid sequences, were synthesized and used for the amplification and sequencing of a 1708-base pair (bp) cDNA for RSS from the rat hepatoma cell line H35 . An open reading frame of 1248 bp encoding 416 amino acids (M(r) = 48,103) was detected for RSS . We have constructed a pRSS1327 expression vector by molecular cloning of a 1327-bp cDNA, which includes sequences of the entire coding region for RSS, into pBluescript . Expression in Escherichia coli of a functional, full-length RSS was confirmed by immunoblot analysis and enzymatic activity . We present and evaluate a model for the secondary structure of RSS and its possible membrane orientation . The model predicts a 315-residue domain at the center of the protein that contains the catalytic site and is released in a soluble form by partial proteolysis . The 33-residue NH2-terminal and 98-residue COOH-terminal sections are not involved in catalysis . Sequence analysis of the catalytic domain of RSS indicate three regions with high homology to sequences in a number of functionally distinct proteins that utilize polyprenyl diphosphate substrates.

J Biol Chem, 1992 Oct 25, 267(30), 21355 - 9
Cross-linking of the gamma subunit of the Escherichia coli ATPase (ECF1) via cysteines introduced by site-directed mutagenesis; Aggeler R et al.; The gamma subunit of the Escherichia coli F1 ATPase (ECF1) has been altered by site-directed mutagenesis to create five different mutants, gamma-S8C, gamma-S81C, gamma-T106C, gamma-S179C, and gamma-V286C, respectively . ECF1 isolated from four of these mutants had ATPase activities similar to that of a wild-type isogenic strain used as a control, the exception was enzyme isolated from mutant gamma-S81C, which had an ATPase activity of around 70-80% of the wild type . ECF1 isolated from each of the various mutants was reacted with N-(4-(7-(diethylamino)-4-methylcoumarin-3-yl))maleimide (CM) . The fluorescent reagent was incorporated into Cys residues placed at positions 8, 106, 179, and 286, but not at 81, indicating which of these Cys residues are on the surface of the gamma subunit in the enzyme complex . Modification of the Cys at position 106 with CM activated the enzyme, and modification of the Cys at position 8 inhibited ATPase activity a small amount; however, modification of Cys at 179 or 286 had no effect on enzyme activity . The four mutants with a reactive Cys were reacted with tetrafluorophenylazide maleimides (TFPAMs), novel photoactivatable cross-linkers . In the mutant gamma-S8C, cross-links were formed between the introduced Cys on the gamma subunit and sites on the beta subunit . This cross-linking between gamma and beta depended on nucleotide conditions under which the photolysis was carried out, with differently migrating cross-linked products being obtained in ATP + EDTA compared with ATP + Mg2+ or ATP + Mg2+ Pi . Cross-linking between beta and gamma inhibited ATPase activity in proportion to the yield of cross-linked product . In the mutant gamma-V286C, cross-links were formed between the introduced Cys on gamma and the alpha subunit which were the same in all nucleotide conditions and which led to inhibition of ATPase activity.

J Biol Chem, 1992 Oct 25, 267(30), 21844 - 9
Inhibitory effect of src homology (SH) 2/SH3 fragments of phospholipase C-gamma on the catalytic activity of phospholipase C isoforms . Identification of a novel phospholipase C inhibitor region; Homma Y et al.; In order to study the regulatory mechanisms of phospholipase C-gamma (PLC-gamma) via the intrinsic SH2/SH3 region (Z region), two recombinant Z proteins, rP45Z and rP38Z, derived from rat PLC-gamma 1 and PLC-gamma 2, respectively, were purified from the inclusion bodies of Escherichia coli . We examined their direct effects on phosphoinositide hydrolysis induced by four different PLC isoforms purified from bovine brain and thymus, and found that both of these Z proteins suppress the enzyme activity of all four PLC isoforms in a dose-dependent manner . This suppressive effect is very potent and stoichiometric . The kinetics studies indicate that the suppression is non-competitive . This suppression is eliminated by treatment with proteases but is not affected by heat treatment at 95 degrees C for 15 min, indicating that the primary structure might be important for the action of Z proteins . Comparative studies suggested that two Z proteins but not Src and phosphatidylinositol 3-kinase possess, adjacent to their SH2 and SH3 motifs, a phospholipase C inhibitor (PCI) region that strongly suppresses their phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity . A series of synthetic peptides identical with the sequence of the proposed PCI region, including an octamer, YRKMRLRY, inhibited PIP2 hydrolysis induced by four different phospholipase C isoforms . These results demonstrate that both types of phospholipase C-gamma contain the PCI sequence which is responsible for the inhibition of PIP2 hydrolysis, indicating that phospholipase C-gamma is a self-regulating enzyme.

J Biol Chem, 1992 Oct 25, 267(30), 21698 - 704
Molecular forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expressed in rat skeletal muscle; Crepin KM et al.; The rat cDNA for the muscle-type (M) isozyme of 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) contains two putative translation initiation sites . To determine whether the M isozyme expressed in rat skeletal muscle corresponds to the short (PFK2M-sf) or the long (PFK2M-lf) isoform, we have expressed them in Escherichia coli . A third construction was also expressed in which the second ATG codon was deleted (PFK2M-lf delta ATG) to ensure that initiation started at the first ATG . The properties of these recombinant proteins were compared with those of the PFK-2/FBPase-2 present in rat skeletal muscle and liver . The recombinant proteins displayed PFK-2 and FBPase-2 activities and the M(r) values of the subunits measured by SDS-polyacrylamide gel electrophoresis were compatible with the calculated ones . The purified recombinant lf form contained not only the expected lf band (54,500 M(r)) but also the sf band (52,000 M(r)), indicating that the expression system could synthesize the long and the short isoforms from the same mRNA . The kinetic properties of the recombinant sf form were not different from those of the rat muscle enzyme . By contrast, lf delta ATG PFK-2 displayed a higher Km for its substrates and a lower Vmax . Immunoblotting with an antibody directed against the long isoform revealed a 54,500 M(r) band both in the lf and the lf delta ATG recombinant, but no band in rat skeletal muscle extracts . In these extracts, one band of 52,000 and a minor one of 54,500 M(r) were detected by an anti PFK-2/FBPase-2 antibody . The 54,500 M(r) band was recognized by an antibody directed against the L isozyme, suggesting that a small amount of the latter is expressed in skeletal muscle . Thus, the M isozyme differs from the L isozyme by replacement of the first 32 amino acids of the L isozyme by an unrelated nonapeptide.

J Biol Chem, 1992 Oct 25, 267(30), 21588 - 94
Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase . The role of surface loop basic residues in substrate binding to the fructose-2,6-bisphosphatase domain; Li L et al.; Lys-356 has been implicated as a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Li, L., Lin, K., Correia, J., and Pilkis, S . J . (1992) J . Biol . Chem . 267, 16669-16675) . To ascertain whether the three other basic residues (Arg-352, Arg-358, and Arg-360), which are located in a surface loop (residues 331-362) which contains Lys-356, are important in substrate binding, these arginyl residues were mutated to Ala, and each arginyl mutant was expressed in Escherichia coli and purified to homogeneity . The far UV circular dichroism spectra of the mutants were identical to that of the wild-type enzyme . The kinetic parameters of 6-phosphofructo-2-kinase of the mutants revealed only small changes . However, the Km for fructose 2,6-bisphosphate, Ki for fructose 6-phosphate, and Ka for inorganic phosphate of fructose-2,6-bisphosphatase for Arg352Ala were, respectively, 2,800-, 4,500-, and 1,500-fold higher than those for the wild-type enzyme, whereas there was no change in the maximal velocity or the Ki for inorganic phosphate . The Km for fructose 2,6-bisphosphate and Ki for inorganic phosphate of Arg360Ala were 10- and 12-fold higher, respectively, than those of the wild-type enzyme, whereas the maximal velocity and Ki for fructose 6-phosphate were unchanged . In addition, substrate inhibition was not observed with Arg352Ala and greatly reduced with Arg360Ala . The properties of the Arg358Ala mutant were identical to those of the wild-type enzyme . The results demonstrate that in addition to Lys-356, Arg-352 is another critical residue in fructose-2,6-bisphosphatase for binding the C-6 phospho group of fructose 2,6-bisphosphate and that Arg-360 binds the C-2 phospho group of fructose 2,6-bisphosphate in the phosphoenzyme.fructose 2,6-bisphosphate complex . The results also provide support for Arg-352, Lys-356, and Arg-360 constituting a specificity pocket for fructose-2,6-bisphosphatase.

Nucleic Acids Res, 1992 Oct 25, 20(20), 5443 - 50
Temperature-dependent template switching during in vitro cDNA synthesis by the AMV-reverse transcriptase; Ouhammouch M et al.; Reverse transcriptase template switching has been invoked to explain several aspects of retroviral replication and recombination, and has been reported in vitro for the Moloney murine leukemia virus (M-MuLV) reverse transcriptase . During in vitro cDNA synthesis, the avian myeloblastosis virus (AMV) reverse transcriptase can switch from one template to another in a homology-dependent and temperature-dependent manner . Chimeric cDNA molecules are generated within 30 min at high incubation temperatures, with an increasing efficiency from 42 degrees C to 50 degrees C . Such products are detectable only after much longer incubation times when primer extension reactions are carried out at lower temperatures (90 min at 37 degrees C).

Biochemistry, 1992 Oct 13, 31(40), 9744 - 51
Interaction of C225SR1 mutant subunit of ribonucleotide reductase with R2 and nucleoside diphosphates: tales of a suicidal enzyme; Mao SS et al.; Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides . This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide . One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail . Incubation of C225SR1 and R2 with {3'-3H,U-14C}UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm . There is an isotope effect (kH/k3H) on uracil production of 3.2 . In addition, the tyrosyl radical on R2 is reduced . The observation of 3H2O, indicative of 3' carbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction . Incubation of {3'-2H}UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively . These studies provide the first direct evidence for protein radical involvement in catalysis . Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the R1 polypeptide into two new polypeptides of 26 and 61 kDa . The 26-kDa polypeptide is the N-terminus of R1, and hence cleavage of the polypeptide is occurring in the region of the mutation . The N-terminus of the 61-kDa polypeptide is blocked.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Oct 13, 31(40), 9733 - 43
A model for the role of multiple cysteine residues involved in ribonucleotide reduction: amazing and still confusing; Mao SS et al.; Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides . Multiple cysteins have been postulated to play a key role in this process . To test the role of various cysteines in nucleotide reduction, a variety of single and double mutants of the R1 subunit were prepared: C754S, C759S, C754-759S, C462S, C462A, C230S, and C292S . Due to the expression system, each mutant contains small amounts of contaminating wt-R1 (estimated to be 1.5-3% based on activity) . An epitope tagging method in conjunction with anion exchange chromatography was used to partially resolve the mutant R1 from the wt-R1 . The interaction of these mutants with the normal substrate was studied, which allowed a model to be proposed in which five cysteines of the R1 subunit of RDPR play a role in catalysis . C754S and C759S R1s catalyze CDP formation at rates similar to wt-R1 when DTT is used as a reductant . However, when thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH is used as reductant, the rates of dNDP production are similar to those expected for contaminating wt-R1 present as a heterodimer with the mutant . The impaired nature of these mutants with respect to reduction by TR suggests that their function is to transfer reducing equivalents from TR to the active site disulfide of R1 produced during NDP reduction . Single-turnover experiments, designed to avoid the problem of contaminating wt-R1, also support this role for C754 and C759 . The double serine mutant of 754 and 759 has catalytic activity with DTT that is one-third the rate of wt-R1 with thioredoxin . C225 and C462 are thought to be the active site cysteines oxidized concomitantly with NDP reduction . Conversion of these cysteines to serines results in R1 mutants which convert the normal substrate into a mechanism-based inhibitor . C462SR1 upon incubation with R2 and {3'-3H,U-14C}UDP results in uracil release, 3H2O production, 3H,14C-labeled protein which has an absorbance change at 320 nm, and slow loss of the tyrosyl radical on R2 . The isotope effect (kH/k3H) on 3' carbon-hydrogen bond cleavage is 1.7 . This sequence of events is independent of the reductant, consistent with the postulate that C462 is an active site thiol . The C462AR1 has properties similar to C462SR1 . Several additional mutant R1s, C230SR1, and C292SR1 were shown to have activities similar to wt-R1 with both TR/TRR/NADPH and DTT.

Proc R Soc Lond B Biol Sci, 1992 Oct 22, 250(1327), 51 - 5
Role of the phosphoenolpyruvate-dependent fructose phosphotransferase system in the utilization of mannose by Escherichia coli; Kornberg HL et al.; Mutants of Escherichia coli devoid of the membrane-spanning proteins PtsG and PtsMP, which are components of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and which normally effect the transport into the cells of glucose and mannose, do not grow upon or take up either sugar . Pseudorevertants are described that take up, and grow upon, mannose at rates strongly dependent on the mannose concentration in the medium (apparent Km > 5 mM); such mutants do not grow upon glucose but are derepressed for the components of the fructose operon . Evidence is presented that mannose is now taken up via the fructose-PTS to form mannose 6-phosphate, which is further utilized for growth via fructose 6-phosphate and fructose 1,6-bisphosphate.

Gene, 1992 Oct 21, 120(2), 315 - 6
Sequence of a cDNA encoding the alpha-subunit of wheat translation elongation factor 1; Metz AM et al.; A cDNA encoding the alpha-subunit of wheat protein synthesis elongation factor 1 (EF-1 alpha) was isolated from a wheat cDNA expression library and sequenced . The deduced amino acid sequence is compared to EF-1 alpha from other species and to elongation factor Tu (EF-Tu) from Escherichia coli . Putative GTP-binding sites are identified.

J Mol Biol, 1992 Oct 20, 227(4), 996 - 1008
Positive and negative effects of DNA bending on activation of transcription from a distant site; Claverie-Martin F et al.; Transcription of the Escherichia coli glnHPQ operon, which encodes components of the high-affinity glutamine transport system, is activated by nitrogen regulator I (NRI)-phosphate in response to nitrogen limitation . NRI-phosphate binds to sites upstream from the sigma 54-dependent glnHp2 promoter and activates transcription by catalyzing the isomerization of the closed sigma 54-RNA polymerase promoter complex to an open complex . On linear DNA, the initiation of glnHp2 transcription requires in addition to NRI-phosphate the presence of integration host factor (IHF), which binds to a site located between the NRI-binding sites and the promoter . On supercoiled DNA, IHF does not play an essential role, but enhances the activation of transcription by NRI-phosphate . We found that at a mutant glnHp2 promoter with increased affinity for sigma 54-RNA polymerase, the initiation of transcription can be activated equally well by NRI-phosphate in the presence or absence of IHF . Binding of IHF to its site does not increase the binding of sigma 54-RNA polymerase to the glnHp2 promoter; instead, our data suggest that IHF bends the DNA to align the activator with the closed sigma 54-RNA polymerase promoter complex to facilitate the interactions that result in open complex formation . In the absence of IHF, NRI-phosphate can activate transcription whether its binding sites are on the same face of the DNA helix as the sigma 54-RNA polymerase or on the opposite face . IHF enhances transcription when the three proteins are located on the same face of the helix, but strongly inhibits transcription when any one of the proteins is located on the opposite face.

Biochim Biophys Acta, 1992 Oct 20, 1132(3), 309 - 10
Improved plasmids containing the Escherichia coli dam gene under the control of the tac promoter; Guha S et al.; We report the construction of a series of plasmids containing the dam gene under the control of the tac promoter . Cells containing these plasmids produce about 8 to 10-fold more Dam methyltransferase (Mtase) than the previously used plasmid pTP166 and avoid the use of a high temperature step necessary for the expression of Dam Mtase in the plasmid pDOX1 and thus allow its use for the study of thermosensitive mutants.

Biochim Biophys Acta, 1992 Oct 20, 1132(3), 255 - 8
Expression of heme oxygenase and its RNA in mouse liver after injection of heme and splenectomy; Kurata S et al.; Heme is known to activate the HO (heme oxygenase) gene in cultured cells, but little is known about the effect of heme on the HO gene in intact organisms . The expressions of HO and its RNA in mouse liver were measured using mouse HO cDNA and HO antibody after injection of heme or splenectomy . The antibody was prepared against a beta-galactosidase-HO hybrid protein made in Escherichia coli . The HO mRNA level increased to a maximum 15 h after heme injection . In contrast, expression of HO was maximal about 45 h after heme injection . Essentially the same results were obtained in mice after splenectomy . These results suggest that the HO gene in mouse liver was activated by the injection of heme and splenectomy.

Biochemistry, 1992 Oct 20, 31(41), 9904 - 11
Oligomerization of the cytoplasmic fragment from the aspartate receptor of Escherichia coli; Long DG et al.; We have observed that a 31-kDa cloned fragment from the Escherichia coli aspartate receptor exhibits a reversible monomer-oligomer reaction . The fragment, derived from the cytoplasmic region of the receptor (c-fragment), contains the signaling functions of the receptor . The wild-type and nine missense mutant fragments were analyzed . The latter were selected by the effect of the mutations on the signaling properties of the intact receptor, which induced either persistent smooth swimming or tumbling in bacteria {Mutoh, N., Oosawa, K., & Simon, M . I . (1986) J . Bacteriol . 167, 992-998} . In pH 7.0 buffer, the mutations caused five out of the six smooth mutant c-fragments to form oligomers, while neither the three tumble mutant nor wild-type fragments exhibited significant oligomer formation . At a lower pH (5.5), all of the fragments displayed some tendency to form oligomers . The equilibria between the monomer and the oligomers were monitored by gel permeation chromatography (GPC) which resolved two to three forms with apparent molecular weights between 110,000 and 270,000 . The proportions of the different forms depended on concentration, indicating an association-dissociation reaction . Static light scattering (SLS) was used to demonstrate that the solution molecular mass of the wild-type c-fragment was 31 kDa and not 110 kDa as indicated by chromatography . One oligomer-forming c-fragment (S461L) eluted as the monomer and one other form, which was determined to be a dimer by SLS . The weight-average molecular weights, calculated from GPC data as a function of protein concentration, agreed well with the weight-average molecular weights obtained by SLS for this mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Oct 20, 31(41), 9883 - 90
Structural basis for recognition of polyglutamyl folates by thymidylate synthase; Kamb A et al.; Thymidylate synthase (TS) catalyzes the final step in the de novo synthesis of thymidine . In vivo TS binds a polyglutamyl cofactor, polyglutamyl methylenetetrahydrofolate (CH2-H4folate), which serves as a carbon donor . Glutamate residues on the cofactor contribute as much as 3.7 kcal to the interaction between the cofactor, substrate, and enzyme . Because many ligand/receptor interactions appear to be driven largely by hydrophobic forces, it is surprising that the addition of hydrophilic, soluble groups such as glutamates increases the affinity of the cofactor for TS . The structure of a polyglutamyl cofactor analog bound in ternary complex with deoxyuridine monophosphate (dUMP) and Escherichia coli TS reveals how the polyglutamyl moiety is positioned in TS and accounts in a qualitative way for the binding contributions of the different individual glutamate residues . The polyglutamyl moiety is not rigidly fixed by its interaction with the protein except for the first glutamate residue nearest the p-aminobenzoic acid ring of folate . Each additional glutamate is progressively more disordered than the previous one in the chain . The position of the second and third glutamate residues on the protein surface suggests that the polyglutamyl binding site could be utilized by a new family of inhibitors that might fill the binding area more effectively than polyglutamate.

Biochemistry, 1992 Oct 20, 31(41), 10099 - 107
Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch; Barak R et al.; CheY, a key protein in the mechanism of bacterial chemotaxis, is known to interact with the flagellar switch and thereby cause clockwise rotation . This activity of CheY was significantly increased by producing acetyladenylate (AcAMP) within cytoplasm-free bacterial envelopes containing purified CheY . This was achieved by including in the envelopes the enzyme acetyl-CoA synthetase (ACS) and ATP, and adding acetate externally . The fraction of clockwise-rotating envelopes, tethered to glass by their flagella, increased from 14% to 58% by the presence of AcAMP (or its derivative) . In parallel experiments carried out with {14C}acetate under similar conditions, CheY became acetylated: {1-14C}acetate was as effective as {2-14C}acetate in labeling CheY, and ACS-dependent labeling of CheY by {alpha-32P}ATP was not detected . The switch proteins, FliG, FliM, and FliN, isolated to purity, were not acetylated . The acetylation was specific for CheY and dependent on its native conformation . The acetylated form the CheY was estimated to be more active than its nonacetylated form by 4-5 orders of magnitude . Acetylated CheY was stable in the presence of the strong nucleophiles hydroxylamine or ethanolamine, indicative of N-acetylation . There was a correlation between the activity of CheY in vivo and its ability to be acetylated in vitro . Thus, proteins with a single substitution at their active site, CheY57DE and CheY109KR, are not active in vivo and accordingly were not acetylated in vitro; in contrast, the protein CheY13DK is active in vivo and was normally acetylated in vitro . The possibility that CheY acetylation plays a role in bacterial chemotaxis is discussed.

Biochemistry, 1992 Oct 20, 31(41), 9877 - 82
Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli; Seetharam R et al.; Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors . We have previously described methods to express {Sisk et al . (1990) Gene 96, 305-309} and purify {Reilly et al . (1990) J . Biol . Chem . 265, 9570-9574} a highly active form of the protein in substantial amounts, from Escherichia coli . Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1 . The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms . Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin . It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin . Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine . Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses . However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.

Biochemistry, 1992 Oct 20, 31(41), 9891 - 903
Folding topology of the disulfide-bonded dimeric DNA-binding domain of the myogenic determination factor MyoD; Starovasnik MA et al.; The myogenic determination factor MyoD is a member of the basic-helix-loop-helix (bHLH) protein family . A 68-residue fragment of MyoD encompassing the entire bHLH region (MyoD-bHLH) is sufficient for protein dimerization, sequence-specific DNA binding in vitro, and conversion of fibroblasts into muscle cells . The circular dichroism spectrum of MyoD-bHLH indicates the presence of significant alpha-helical secondary structure; however, the NMR spectrum lacks features of a well-defined tertiary structure . There is a naturally occurring cysteine at residue 135 in mouse MyoD that when oxidized to a disulfide induces MyoD-bHLH to form a symmetric homodimer with a defined tertiary structure as judged by sedimentation equilibrium ultracentrifugation and NMR spectroscopy . Oxidized MyoD-bHLH retains sequence-specific DNA-binding activity, albeit with an apparent 100-1000-fold decrease in affinity . Here, we report the structural characterization of the oxidized MyoD-bHLH homodimer by NMR spectroscopy . Our findings indicate that the basic region is unstructured and flexible, while the HLH region consists of two alpha-helices of unequal length connected by an as yet undetermined loop structure . Qualitative examination of interhelical NOEs suggests several potential arrangements for the two helix 1/helix 2 pairs in the symmetric oxidized dimer . These arrangements were evaluated for whether they could incorporate the disulfide bond, satisfy loop length constraints, and juxtapose the two basic regions . Only a model that aligns helix 1 parallel to helix 1' and antiparallel to helix 2 was consistent with all constraints . Thus, an antiparallel four-helix bundle topology is proposed for the symmetric dimer . This topology is hypothesized to serve as a general model for other bHLH protein domains.

J Mol Biol, 1992 Oct 20, 227(4), 1173 - 81
Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA; Chu WC et al.; To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified . Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed . FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine . FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12 . Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme . Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure . 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil . However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m . downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops . The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m . range, when fluorouracil was base-paired with adenine in helical stems . This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix . An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m . downfield of those from fluorouracil residues paired with A . In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.

J Mol Biol, 1992 Oct 20, 227(4), 1019 - 31
Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site; Svard SG et al.; The RNase P cleavage reaction was studied as a function of the number of base-pairs in the acceptor-stem and/or T-stem of a natural tRNA precursor, the tRNA(Tyr)Su3 precursor . Our data suggest that the location of the Escherichia coli RNase P cleavage site does not depend merely on the lengths of the acceptor-stem and T-stem as previously suggested . Surprisingly, we find that precursors with only four base-pairs in the acceptor-stem are cleaved by M1 RNA and by holoenzyme . Furthermore, we show that both disruption of base-pairing, and alteration of the nucleotide sequence (without disruption of base-pairing) proximal to the cleavage site result in aberrant cleavage . Thus, the identity of the nucleotides near the cleavage site is important for recognition of the cleavage site rather than base-pairing . The important nucleotides are those at positions -2, -1, +1, +72, +73 and +74 . We propose that the nucleotide at position +1 functions as a guiding nucleotide . These results raise the possibility that Mg2+ binding near the cleavage site is dependent on the identity of the nucleotides at these positions . In addition, we show that disruption of base-pairing in the acceptor-stem affects both Michaelis-Menten constants, Km and kcat.

FEBS Lett, 1992 Oct 19, 311(2), 119 - 23
Molecular cloning of a cDNA clone for tobacco lipid transfer protein and expression of the functional protein in Escherichia coli; Masuta C et al.; A cDNA clone encoding a lipid transfer protein (LTP) was isolated from tobacco by screening a library with a PCR-amplified spinach LTP gene . DNA sequence analysis showed a large open reading frame (344 bp) encoding a polypeptide of 114 amino acids . The first 23 amino acids of the deduced protein have the characteristics of a signal peptide for protein secretion or targeting into dense microbody-like vesicles . The cDNA clone was then inserted into an expression vector, pMAL, and expressed in E . coli as a fusion with the maltose binding protein (MBP) . The MBP-LTP fusion protein was purified to homogeneity and subjected to factor Xa cleavage to yield the LTP domain . A lipid transfer assay demonstrated that the resulting LTP was functional . The availability of the expression system in E . coli will facilitate the elucidation of in vivo function(s) of plant LTPs.

Cell, 1992 Oct 16, 71(2), 201 - 10
Protein splicing in the maturation of M . tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence; Davis EO et al.; The M . tuberculosis recA locus comprises an 85 kd open reading frame but produced 38 kd RecA and 47 kd products in E . coli . No RNA processing was detected; rather, an 85 kd precursor protein was spliced, releasing a 47 kd spacer protein, and joining its terminal fragments to form mature RecA protein . "Spacer" protein was also produced in M . tuberculosis and from a hybrid spacer-LacZ alpha fusion molecule . Mutagenesis at codon wobble positions at one splice junction showed that protein rather than nucleotide sequence determined splicing activity . Other mutants defined additional regions needed for splicing and allowed processing to be followed . Splicing was essential for RecA activity in E . coli . The possibility that splicing is a manifestation of a novel class of genetic element is discussed.

Biochem J, 1992 Oct 15, 287 ( Pt 2), 611 - 9
Overproduction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli and site-directed substitutions in the E1p and E2p subunits; Russell GC et al.; The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH complex) . An isopropyl beta-D-thiogalactopyranoside-inducible expression system was developed for amplifying fully lipoylated wild-type and mutant PDH complexes to over 30% of soluble protein . The extent of lipoylation was related to the degree of aeration during amplification . The specific activities of the isolated PDH complexes and the E1p component were 50-75% of the values normally observed for the unamplified complex . This could be due to altered stoichiometries of the overproduced complexes (higher E3 and lower E1p contents) or inactivation of E1p . The chaperonin, GroEL, was identified as a contaminant which copurifies with the complex . Site-directed substitutions of an invariant glycine residue (G231A, G231S and G231M) in the putative thiamine pyrophosphate-binding fold of the E1p component had no effect on the production of high-molecular-mass PDH complexes but their E1p and PDH complex activities were very low or undetectable, indicating that G231 is essential for the structural or catalytic integrity of E1p . A minor correction to the nucleotide sequence, which leads to the insertion of an isoleucine residue immediately after residue 273, was made . Substitution of the conserved histidine and arginine residues (H602 and R603) in the putative active-site motif of the E2p subunit confirmed that H602 of the E . coli E2p is essential, whereas R603 could be replaced without inactivating E2p . Deletions affecting putative secondary structural elements at the boundary of the E2p catalytic domain inhibited catalytic activity without affecting the assembly of the E2p core or its ability to bind E1p, indicating that the latter functions are determined elsewhere in the domain . The results further consolidate the view that chloramphenicol acetyltransferase serves as a useful structural and functional model for the catalytic domain of the lipoate acyltransferases.

Biochem J, 1992 Oct 15, 287 ( Pt 2), 521 - 31
Bivalent-metal binding to CheY protein . Effect on protein conformation; Kar L et al.; CheY is a 14 kDa cytoplasmic protein that is activated by the transfer of a phosphoryl moiety to Asp-57 from phosphoCheA during signal transduction in bacterial chemotaxis . It has been established that metal ions are necessary for the autophosphorylation of CheA, the transfer of phosphate from phosphoCheA to CheY and the autodephosphorylation of phosphoCheY . In this work, paramagnetic relaxation enhancement has been used in conjunction with one- and two-dimensional n.m.r . to study the interaction of CheY with bivalent metal ions . These studies have led to the discovery of two conformations of the protein in water, corresponding to the metal-free and the metal-bound states . Binding of bivalent cations like Mg2+, Ca2+, Sr2+, Zn2+ and Mn2+ results in a conformational change from the metal-free to the metal-bound state . Preliminary assignments of the aromatic proton resonances are reported . Comparison of phase-sensitive double-quantum-filtered COSY, homonuclear Hartmann-Hahn coherence transfer and nuclear Overhauser enhancement spectra from the metal-bound and metal-free protein indicates that Trp-58, Thr-87 and Tyr-106 are particularly affected by the conformational change involved, and that this change is limited to a small number of residues . In addition, homonuclear Hartmann-Hahn coherence transfer experiments with paramagnetic Mn2+ show significant suppression of cross-peaks associated with Trp-58 and several neighbouring residues . Comparison of the distances estimated using n.m.r . with the CheY crystal structure indicates that the n.m.r . results are consistent with bivalent metal binding at the cluster of aspartic acid residues that includes Asp-13 and Asp-57 . These studies also demonstrate the utility of paramagnetic metal-induced relaxation in conjunction with two-dimensional n.m.r . measurements for exploring ligand-binding sites.

Biochem J, 1992 Oct 15, 287 ( Pt 2), 501 - 8
Studies on the binding of the Escherichia coli MelR transcription activator protein to operator sequences at the MelAB promoter; Caswell R et al.; Escherichia coli MelR protein binds to two sites located upstream of the melAB transcription start site . Although both sites are required for optimal melibiose-dependent expression from the melAB promoter, some MelR-dependent expression is found if the upstream site is deleted or if the spacing between the two sites is altered . Gel retardation assays have been exploited to study MelR binding to a DNA fragment carrying just the upstream site . Methylation interference analysis was used to identify one guanine (at -104) which is important for MelR binding . Mutational analysis confirmed the importance of this base and revealed a second position (at -110) where mutations interfere with melAB promoter activity . Experiments using potassium permanganate as a probe suggested that the DNA sequence around -110 adopts a distorted conformation . We propose that the mutation at -104 alters MelR binding by interfering with a direct contact, whereas the mutation at -110 primarily affects DNA conformation . The binding of purified MelR protein to a melAB promoter fragment carrying both binding sites has also been studied: binding results in four retarded bands in gel assays . Methylation interference experiments have been exploited to identify the binding sites occupied in each complex . Although both binding sites share a common 18 bp sequence, MelR binding to the more upstream site is stronger . We could find no evidence for co-operative interactions between MelR and RNA polymerase and no major effects of melibiose . Some evidence for melibiose-dependent distortion in complexes between MelR and the melAB promoter is discussed.

Biochem J, 1992 Oct 15, 287 ( Pt 2), 493 - 9
Overexpression, purification and characterization of the Escherichia coli MelR transcription activator protein; Caswell R et al.; The gene encoding Escherichia coli MelR protein has been cloned in the expression vector pJLA502 . MelR has been overexpressed, substantially purified and shown to bind to DNA fragments carrying the melAB promoter . A truncated version of the melR gene, encoding the C-terminal half of MelR, was also cloned into pJLA502; the protein product of this truncated gene binds to the melAB promoter but was not overproduced . A number of amino acid substitutions were made in the recognition helices of two putative helix-turn-helix motifs in the C-terminal part of MelR, and the effects of these mutations on MelR-dependent transcription initiation at the melAB promoter have been measured.

Eur J Biochem, 1992 Oct 15, 209(2), 759 - 64
Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli; Cioni P et al.; The Trp phosphorescence spectrum, intensity and decay kinetics of apo-aspartate aminotransferase, pyridoxamine-5P-aspartate-aminotransferase and pyridoxal-5P-aspartate aminotransferase were measured over a temperature range 160-273 K . The fine structure of the phosphorescence spectra in low-temperature glasses, with 0-0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine-5P-enzyme reveals a marked heterogeneity of the chromophore environments . Only for the pyridoxal-5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0-0 vibrational band centered at 415 nm . Concomitant to quenching, there is Trp-sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer . All three forms of the enzyme are phosphorescent for temperatures up to 273 K . However, across the glass transition temperature the pyridoxal-5P enzyme shows a decrease in lifetime-normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature . In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites . For the pyridoxal-5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.

Eur J Biochem, 1992 Oct 15, 209(2), 549 - 53
Effect of heat shock on expression of proteins not involved in the heat-shock regulon; Fabisiewicz A et al.; The effect of heat shock on the expression of some genes of Escherichia coli was tested . To avoid side effects, promoters of the genes were fused to lacZ and their expression measured by the level of beta-galactosidase . The results show that expression of umuC, recA and polB, after induction of the SOS response, was somewhat higher in the heat-shocked than in the non-shocked cells, whereas expression of ada, alkB and alkA genes, after induction of the adaptive response, was about the same . Unexpectedly, it was found that expression of lacZ from its own promoter was drastically lowered in the heat-shocked cells . This effect, however, seems not to be dependent on the induction of heat-shock proteins.

Eur J Biochem, 1992 Oct 15, 209(2), 541 - 8
Concerted activities of the RNA recognition and the glycine-rich C-terminal domains of nucleolin are required for efficient complex formation with pre-ribosomal RNA; Ghisolfi L et al.; Nucleolin is an abundant nucleolar protein which is involved in the early stages of ribosome assembly . The central 40-kDa domain of nucleolin comprises four RNA recognition motifs (RRM) which are presumed to be involved in specific interactions with pre-rRNA . In order to examine in detail the role of this central domain and the contribution of the N-terminal and C-terminal domains of nucleolin to RNA binding, we have used an Escherichia coli expression system to synthezise polypeptides corresponding to various combinations of the three domains and their subdomains . By means of an in-vitro binding assay and a synthetic RNA corresponding to a specific recognition site in pre-rRNA we have been able to demonstrate conclusively that the central 40-kDa domain is indeed responsible for the specificity of RNA recognition and that the N-terminal domain can be removed without affecting RNA binding . Most interestingly, it appears that the C-terminal 10-kDa domain, which is rich in glycine and arginine residues, is essential for efficient binding of nucleolin to RNA, but does not itself contribute to the specificity of the interaction . Circular dichroic spectroscopic probing of the RNA component shows that the C-terminal domain significantly modifies the RNA-binding properties of the central RRM core . Finally, infrared spectroscopic studies reveal that the central 40-kDa domain is structured in alpha helices and beta sheets and that the interaction with the specific pre-rRNA site induces subtle changes in the beta sheet conformation.

Biochem Biophys Res Commun, 1992 Oct 15, 188(1), 424 - 32
Characterization of cysteine residues of glutathione S-transferase P: evidence for steric hindrance of substrate binding by a bulky adduct to cysteine 47; Nishihira J et al.; Glutathione S-transferase P (GST-P) lost the enzymatic activity by 7-fluoro-4-sulfamoyl-2, 1, 3-benzodiazole (ABD-F), a thiol-group chemical modifier, but did not by methylmethanethiol-sulfonate . Both ABD-F and methylmethanethiolsulfonate reacted with Cys47 and Cys101 . These two cysteine residues were site-directedly mutated with serine residues . Only the Cys101Ser lost the enzymatic activity by the treatment of ABD-F . On carbon 13 NMR experiments, a NMR signal of S-{13C}CH3 adduct to Cys47 did not show any change by the addition of S-hexylglutathione . These facts revealed that Cys47 did not locate at the active site, and a bulky adduct to Cys47 hindered the binding of substrates to the active site.

Biochem Biophys Res Commun, 1992 Oct 15, 188(1), 372 - 8
Deficiency of 8-hydroxyguanine DNA endonuclease activity and accumulation of the 8-hydroxyguanine in mutator mutant (mutM) of Escherichia coli; Bessho T et al.; In this report, we have characterized mutM mutant with respect to its ability to repair 8-hydroxyguanine (oh8Gua) in DNA . The oh8Gua DNA endonuclease activity in mutM strain was minimal as compared with that in the wild-type cells . The presence of oh8Gua in DNA of mutM was 6-fold that of the wild-type strain corresponding to a characteristically higher frequency of G.C-->T.A transversions in this mutant strain . These results suggest that mutator phenotype of mutM is at least partially due to a spontaneous accumulation of oh8Gua resulting from a greatly reduced oh8Gua DNA endonuclease activity.

Biochem Biophys Res Commun, 1992 Oct 15, 188(1), 228 - 34
Casein kinase-2 structure-function relationship: creation of a set of mutants of the beta subunit that variably surrogate the wildtype beta subunit function; Boldyreff B et al.; Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form of CK-2 exhibiting the expected sedimentation coefficient and 2) the enhancement of catalytic activity of CK-2 alpha . Extended deletions of 71 and 44 residues from the C-terminal end, but not a 7 residue deletion (including the cdc2 phosphorylation site) prevent both reconstitution of the holoenzyme and, consequently, stimulation of activity . This indicates that residue(s) located in the 171-209 sequence is essential for reconstitution . Also a four residue's N-terminal deletion (removing the autophosphorylation site) and single to quintuple substitutions of alanine for the acidic residues clustered in the 55-70 sequence give rise to mutants that still assemble with the alpha subunit to give a tetrameric holoenzyme . However, in the case of the mutants A57,59, A63,64, A59-61,63,64 in vitro assembly with the CK-2 alpha subunit was not complete . There were also intermediate complexes, free alpha-subunit and beta-mutants found to sediment at various positions in the sucrose density gradient . In comparison to CK-2 beta +, mutants A57,59, A59-61 and A59-61,63,64 show an increased stimulation of the catalytic activity supporting the view that these residues play a crucial role in determining the basal activity of reconstituted CK-2 holoenzyme.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9799 - 803
Second-site revertants of an arginine-210 to lysine mutation in the a subunit of the F0F1-ATPase from Escherichia coli: implications for structure; Howitt SM et al.; Arg-210 of the a subunit of the Escherichia coli F0F1-ATPase has been proposed previously as a component of the proton pore . A mutant in which lysine was substituted for Arg-210 was generated and was found to be unable to translocate protons . A plasmid carrying this mutation, along with wild-type genes encoding the c and b subunits, was unusual in that it failed to complement a chromosomal c-subunit mutation on succinate minimal medium . Three revertants on succinate minimal medium contained plasmids that showed complementation with chromosomal c-subunit but not with a-subunit mutations . One of these had a deletion in the a subunit . The other two were point mutations, resulting in the substitution of aspartic acid by Gly-53 and of arginine for Leu-211 . The Gly-53 to aspartic acid change implied that Gly-53 and Arg-210 are normally in close proximity . To test this idea further, a series of mutants in which aspartic acid was placed in helix I at positions ranging from 42 to 57 was generated . Full complementation was regained only when the aspartic acid residue was present on the same side of a putative helix as Gly-53 over a span of three turns of the alpha-helix . These results and others suggest modifications of a previously proposed model for the transmembrane helices of the F0 portion of the F0F1-ATPase . The implications of these modifications for the mechanism of proton translocation are discussed.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9754 - 8
Metal-binding chimeric antibodies expressed in Escherichia coli; Sawyer JR et al.; Metallothionein, a well-characterized biological chelator of metals, has been genetically fused to the binding domain of an antibody and expressed in the periplasm of Escherichia coli . Specific delivery of 109Cd to immobilized hapten or to haptenated cells was demonstrated directly in periplasmic extracts . This approach is potentially useful for targeted radiotherapy and diagnostic imaging . We find six to seven atoms of metal per active antigen-combining site . Absence of the Fc portion of the immunoglobulin along with low immunogenicity of metallothionein-metal complexes should reduce immunologic reactions.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9725 - 9
Asn177 in Escherichia coli thymidylate synthase is a major determinant of pyrimidine specificity; Hardy LW et al.; The substrate preference of recombinant Escherichia coli thymidylate synthase (TS) has been altered from 2'-deoxyuridylate (dUMP) to 2'-deoxycytidylate (dCMP) by site-directed mutagenesis of the codon for Asn177, which was changed to aspartic acid . The side-chain amide of Asn177 forms hydrogen bonds with O4 and N3 of dUMP bound to the crystalline enzyme {Montfort, W . R., Perry, K . M., Fauman, E . B., Finer-Moore, J . S., Maley, G . F., Hardy, L., Maley, F . & Stroud, R . M . (1990) Biochemistry 29, 6964-6977} . This Asn is invariant in all natural sequences for TS known . The values of kcat for the mutant enzyme, TS(N177D), with dCMP and dUMP are, respectively, 0.09 and 0.002 times the value of kcat of wild-type TS with dUMP as substrate . TS(N177D) turns over dCMP at 35 times its rate of dUMP turnover, whereas wild-type TS turns over dCMP at < 10(-5) of its rate of dUMP turnover . Thus Asn177 is a major determinant of the pyrimidine nucleotide specificity of TS . The mutant enzyme, like wild-type TS, forms a covalent complex with 5-fluoro-dUMP in the presence of 5,10-methylenetetrahydrofolate . TS(N177D) also has a newly acquired ability to be transiently inactivated by dUMP . This time-dependent inactivation requires the presence of methylenetetrahydrofolate and may be due to the accumulation of the enzyme in the form of a catalytic intermediate . The likely mechanistic basis for discrimination by TS between dUMP and dCMP is their differing requirements for charge stabilization during covalent catalysis.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9700 - 4
Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation; Kim J et al.; The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit . It requires binding of cAMP for a conformational change in order to function as a site-specific DNA-binding protein that regulates gene activity . The hinge region connecting the cAMP-binding domain to the DNA-binding domain is involved in the cAMP-induced allosteric change . We studied the structural changes in CRP that are required for gene regulation by making a large number of single and double amino acid substitutions at four different positions in or near the hinge . To achieve cAMP-independent transcription by CRP, amino acid residues 138 (located within the hinge region) and 141 (located in the D alpha-helix adjacent to the hinge) must be polar . This need for polar residues at positions 138 and 141 suggests an interaction that causes the C and D alpha-helices to come together . As a consequence, the F alpha-helix is released from the D alpha-helix and can interact with DNA . At position 144 in the D alpha-helix and within interacting distances of the F alpha-helix, replacement of alanine by an amino acid with a larger side chain, regardless of its nature, allows cAMP independence . This result indicates that pushing against the F alpha-helix may be a way of making the helix available for DNA binding . We believe that the cAMP-induced allosteric change involves similar hinge reorientation to adjust the C and D alpha-helices, allowing outward movement of the F alpha-helix.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9405 - 9
Grammatical model of the regulation of gene expression; Collado-Vides J; Based on a formal proof that justifies the search for generative grammars in the study of gene regulation, a linguistic formalization of an exhaustive data base of Escherichia coli sigma 70 promoters and their regulatory binding sites has been initiated . The grammar presented here generates all the arrays of the collection plus those that are predicted as consistent with the principles of regulation of sigma 70 promoters . "Systems of regulation," sets of regulatory sites that collaborate in a mechanism of regulation, are represented by means of syntactic categories . A small set of phrase structure rules restricted by an X-bar principle and by a hierarchical, c-command relation generates a representation of arrays of sites of regulation where the selection of the protein(s) identifying the system(s) of regulation occurs . Based on the features of the proteins, optional duplicated proximal and remote sites are generated by means of transformational rules . Consistency with the data, the predictions that the grammar generates, and important similarities and differences with some aspects of the generative theory of natural language are discussed.

J Biol Chem, 1992 Oct 15, 267(29), 21146 - 53
Specific binding sites for the activator protein, ALCR, in the alcA promoter of the ethanol regulon of Aspergillus nidulans; Kulmburg P et al.; ALCR is the specific activator of the Aspergillus nidulans ethanol-utilization pathway, mediating the induction of its own transcription and that of the structural genes alcA and aldA, encoding respectively, alcohol dehydrogenase I and aldehyde dehydrogenase . ALCR is a DNA binding protein in which 6 cysteines are coordinated in a zinc binuclear cluster . This domain was fused to glutathione-S-transferase (GST) and isolated as a GST-ALCR(7-58*) fusion protein from Escherichia coli . Mobility shift assays showed that the ALCR fusion protein binds at sites upstream of the alcA promoter . DNaseI protection footprinting experiments revealed three specific binding sites, two that are direct repeats and one that is an inverted repeat with the same half-site 5'-CCGCA-3' . The half-sites are separated by a variable number of nucleotides in both types of target . The interaction of the ALCR fusion protein with direct and inverted repeats were examined by using interference and protection footprinting assays . In both binding sites, modification of the guanines in the half-sites interfered with the formation of the DNA complex, but the adjacent ones did not . Our results suggest that the ALCR protein makes contact in the major groove of the DNA helix of the half-sites . The functionality of two out of three binding sites of the GST-ALCR protein was demonstrated after their deletion . Therefore, the region encompassing these binding sites is a cis-acting element involved in the full induction of the alcA gene.

J Biol Chem, 1992 Oct 15, 267(29), 21119 - 23
Expression and analysis of recombinant Amb a V and Amb t V allergens . Comparison with native proteins by immunological assays and NMR spectroscopy; Rafnar T et al.; The Amb V allergens are small, highly disulfide-bonded ragweed pollen allergens that serve as useful models for understanding the molecular basis of the human immune response . We have produced recombinant Amb a V and Amb t V (from short and giant ragweed pollens, respectively) in Escherichia coli and have compared their structural and functional characteristics to those of the native proteins . Recombinant Amb t V was indistinguishable from native Amb t V as determined by NMR spectroscopy and antibody-binding studies . Whereas inhibition analysis showed that recombinant Amb a V possessed only approximately 50% of the antibody-binding activity of native Amb a V, the two proteins were similarly effective in stimulating Amb a V-specific T-cells . Our results demonstrate that even highly homologous proteins exhibit different abilities to fold into their native three-dimensional conformations and establish the potential and limits of expressing the recombinant Amb V allergens intracellularly in E . coli.

J Biol Chem, 1992 Oct 15, 267(29), 20980 - 6
Functional analysis of nucleosome assembly protein, NAP-1 . The negatively charged COOH-terminal region is not necessary for the intrinsic assembly activity; Fujii-Nakata T et al.; A nucleosome assembly protein (NAP-1) of Saccharomyces cerevisiae facilitates the association of histones with DNA to form nucleosomes in vitro at physiological ionic conditions . The cloned gene was expressed in Escherichia coli using a T7 expression system, and the protein (417 amino acid residues) was purified by Mono Q column chromatography . Various deletion fragments of NAP-1 protein were also produced, and their nucleosome assembly activity was examined by supercoiling assay . The internal fragment containing the residues 43-365 was necessary and sufficient for the activity, and a long stretch of negatively charged region near the carboxyl terminus was dispensable . This minimal size fragment could form the 12 S NAP-1-histone complex as the whole protein could, whereas deleted fragments on either side could bind with core histones only to form aggregates.

J Biol Chem, 1992 Oct 15, 267(29), 20927 - 31
Regulation of Hsp70 function by a eukaryotic DnaJ homolog; Cyr DM et al.; We report that a purified cytoplasmic Hsp70 homolog from Saccharomyces cerevisiae, Hsp70SSA1, exhibits a weak ATPase activity, which is stimulated by a purified eukaryotic dnaJp homolog (YDJ1p) . Stable complex formation between Hsp70SSA1 and the permanently unfolded protein carboxymethylated alpha-lactalbumin (CMLA) was assayed by native gel electrophoresis . The affinity of Hsp70SSA1 for CMLA appeared to be regulated by YDJ1p . Significant reduction in both CMLA-Hsp70SSA1 complex formation and the release of CMLA pre-bound to Hsp70SSA1 was observed only in the presence of both YDJ1p and ATP . Thus, Hsp70SSA1 and YDJ1p interact functionally in the execution of Hsp70SSA1 chaperone activities in the eukaryotic cell.

J Biol Chem, 1992 Oct 15, 267(29), 20835 - 9
F0F1-ATPase gamma subunit mutations perturb the coupling between catalysis and transport; Shin K et al.; We introduced mutations to test the function of the conserved amino-terminal region of the gamma subunit from the Escherichia coli ATP synthase (F0F1-ATPase) . Plasmid-borne mutant genes were expressed in an uncG strain which is deficient for the gamma subunit (gamma Gln-14-->end) . Most of the changes, which were between gamma Ile-19 and gamma Lys-33, gamma Asp-83 and gamma Cys-87, or at gamma Asp-165, had little effect on growth by oxidative phosphorylation, membrane ATPase activity, or H+ pumping . Notable exceptions were gamma Met-23-->Arg or Lys mutations . Strains carrying these mutations grew only very slowly by oxidative phosphorylation . Membranes prepared from the strains had substantial levels of ATPase activity, 100% compared with wild type for gamma Arg-23 and 65% for gamma Lys-23, but formed only 32 and 17%, respectively, of the electrochemical gradient of protons . In contrast, other mutant enzymes with similar ATPase activities (including gamma Met-23-->Asp or Glu) formed H+ gradients like the wild type . Membranes from the gamma Arg-23 and gamma Lys-23 mutants were not passively leaky to protons and had functional F0 sectors . These results suggested that substitution by positively charged side chains at position 23 perturbed the energy coupling . The catalytic sites of the mutant enzymes were still regulated by the electrochemical H+ gradient but were inefficiently coupled to H+ translocation in both ATP-dependent H+ pumping and delta mu H+ driven ATP synthesis.

J Biol Chem, 1992 Oct 15, 267(29), 20758 - 64
Possible salt bridges between transmembrane alpha-helices of the lactose carrier of Escherichia coli; Lee JI et al.; Although it is energetically extremely unfavorable to have charged amino acid residues of a polypeptide in the hydrophobic environment of the membrane phospholipid bilayer, a few such charged residues are found in membrane-spanning regions of membrane proteins . Ion pairs (salt bridges) would be much more stable in low dielectric media than single ionized residues . This paper provides indirect evidence for a salt bridge between Asp-240 and Lys-319 in the lactose carrier of Escherichia coli . When Asp-240 was changed to alanine by site-directed mutagenesis, there was a loss of the ability to accumulate methyl-beta-D-thiogalactopyranoside (TMG), melibiose, or lactose . Fast-growing revertants were isolated on melibiose minimal agar plates . Two second-site revertants were isolated: Asp-240-->Ala plus Gly-268-->Val and Asp-240-->Ala plus Lys-319-->Gln . These revertants showed extremely poor accumulation of TMG, melibiose, and lactose, but showed significant "downhill" lactose entry into beta-galactosidase-containing cells with sugar concentrations of 2 and 5 mM . It is concluded that there is some important interaction between Asp-240 and Lys-319, possibly a salt bridge.

J Biol Chem, 1992 Oct 15, 267(29), 20648 - 58
Biochemical basis of the constitutive repressor cleavage activity of recA730 protein . A comparison to recA441 and recA803 proteins; Lavery PE et al.; The recA730 mutation results in constitutive SOS and prophage induction . We examined biochemical properties of recA730 protein in an effort to explain the constitutive activity observed in recA730 strains . We find that recA730 protein is more proficient than the wild-type recA protein in the competition with single-stranded DNA binding protein (SSB protein) for single-stranded DNA (ssDNA) binding sites . Because an increased aptitude in the competition with SSB protein has been previously reported for recA441 protein and recA803 protein, we directly compared their in vitro activities with those of recA730 protein . At low magnesium ion concentration, both ATP hydrolysis and lexA protein cleavage experiments demonstrate that these recA proteins displace SSB protein from ssDNA in a manner consistent with their in vivo repressor cleavage activity, i.e . recA730 protein > recA441 protein > recA803 protein > recAwt protein . Additionally, a correlation exists between the proficiency of the recA proteins in SSB protein displacement and their rate of association with ssDNA . We propose that an increased rate of association with ssDNA allows recA730 protein to displace SSB protein from the ssDNA that occurs naturally in Escherichia coli and thereby to become activated for the repressor cleavage that leads to SOS induction . RecA441 protein is similarly activated for repressor cleavage; however, in this case, significant SSB protein displacement occurs only at elevated temperature . At physiological magnesium ion concentration, we argue that recA803 protein and wild-type recA protein do not displace sufficient SSB protein from ssDNA to constitutively induce the SOS response.

J Biol Chem, 1992 Oct 15, 267(29), 20571 - 6
Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase beta subunit of Escherichia coli; Omote H et al.; beta Lys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M . (1991) J . Biol . Chem . 266, 5424-5429) . For examination of the roles of beta Lys-155 and beta Thr-156, mutants (beta Lys-155-->Ala, Ser, or Thr; beta Thr-156-->Ala, Cys, Asp, or Ser; beta Lys-155/beta Thr-156-->beta Thr-155/beta Lys-156; and beta Thr-156/beta Val-157-->beta Ala-156/beta Thr-157) were constructed, and their properties were studied extensively . The beta Ser-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type . Other mutants were defective in ATP synthesis, had < 0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient . The mutants had essentially the same amounts of F1 in their membranes as the wild type . Purified mutant enzymes (beta Ala-155, beta Ser-155, beta Ala-156, and beta Cys-156) showed low rates of multisite (< 0.02% of the wild type) and unisite (< 1.5% of the wild type) catalyses . The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the beta Ala-156 and beta Cys-156 enzymes and 10(2)-fold lower with the beta Ala-155 and beta Ser-155 enzymes . The beta Thr-156-->Ala or Cys enzyme showed an altered response to Mg2+, suggesting that beta Thr-156 may be closely related to Mg2+ binding . These results suggest that beta Lys-155 and beta Thr-156 are essential for catalysis and are possibly located in the catalytic site, although beta Thr-156 could be replaced by a serine residue.

Eur J Biochem, 1992 Oct 15, 209(2), 523 - 31
Characterization of the reverse transcriptase of 1731, a Drosophila melanogaster retrotransposon; Champion S et al.; The nucleotide sequence of 1731, a retrotransposon cloned from the genome of Drosophila melanogaster, reveals a structural similarity with the proviral form of the retroviruses including a pol-like gene containing a putative reverse-transcriptase(RT)-coding sequence . Diverse parts of that sequence were subcloned and expressed in Escherichia coli . It has been demonstrated that the expression of the RT-like sequence, when translated, gives rise to peptides displaying enzyme activity characteristic of a true RT enzyme . In addition, rabbit antisera directed against such recombinant proteins allowed us to detect an immunoreactive protein of around 110 kDa, which was only present in D . melanogaster cell lines, but not in cells derived from Drosophila virilis or Drosophila hydei, whose genomes do not bear the 1731 element . This protein is expected to correspond to a non-processed pol-gene translated product and cosediments with virus-like particles exhibiting RT activity.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9880 - 4
Antirepression function in Escherichia coli for the cAMP-cAMP receptor protein transcriptional activator; Forsman K et al.; The cAMP receptor protein (CRP) complex (cAMP-CRP) is a global regulator of gene expression . It influences transcription from a number of promoters in Escherichia coli, including two divergently oriented promoters in the pap pili-adhesin gene system . To further define the role of cAMP-CRP in pap regulation we monitored protein-DNA interactions in vitro and levels of pap transcription in vivo in wild-type and mutant pap-containing clones . The results showed that activation was mediated by a single cAMP-CRP-binding site centered at nucleotide positions -215.5 and -115.5 relative to the transcriptional start points . A target for the pap-specific regulatory protein PapB was localized adjacent to the cAMP-CRP-binding site . The long-range effects exerted from the protein-binding sites were consistent with the idea that cAMP-CRP caused a change in the local DNA conformation and that a nucleoprotein complex (involving cAMP-CRP and PapB) was formed in the region between the pap promoters . Moreover, transcription became independent of activation of cAMP-CRP and the PapB protein in a mutant lacking the nucleoid-associated protein H-NS . Our findings suggest that the cAMP-CRP complex mediates its positive regulatory function by alleviating transcriptional silencing and, as such, plays a role as antirepressor.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9652 - 6
Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay; Kohlstaedt LA et al.; Although the reverse transcriptase (RT) of human immunodeficiency virus (HIV) uses human tRNA(3Lys) as a primer of viral genome DNA synthesis in vivo, HIV RT binds Escherichia coli glutamine tRNA and in vitro-made human lysine tRNA with nearly equivalent affinities . We show that HIV RT can use either tRNA(3Lys) or tRNA(2Gln) as a primer for DNA synthesis in vitro without the addition of any other host or viral proteins . E . coli tRNA(2Gln) can serve as a primer for HIV RT if a primer-binding site sequence complementary to the 3' end of tRNA(2Gln) is at the 3' end of the template . With this reduced template, the specificity of binding the proper tRNA is due to base-pairing between a bound tRNA to the primer-binding site of the viral RNA template rather than sequence-specific recognition of tRNA(3Lys) by RT . If an 8-nucleotide viral sequence 3' to the primer-binding site is included in the template, then addition of Zn2+ or Co2+ is required for tRNA(3Lys)-primed synthesis, and tRNA(2Gln) now fails to prime synthesis . The latter result implies that a template sequence adjacent to the primer-binding site and containing 6 nucleotides complementary to the anticodon loop of human tRNA(3Lys) plays an active role in tRNA discrimination.

J Immunol, 1992 Oct 15, 149(8), 2576 - 84
Recombinant Escherichia coli express a defined, cytoplasmic epitope that is efficiently processed in macrophage phagolysosomes for class II MHC presentation to T lymphocytes; Pfeifer JD et al.; Although the processing of soluble Ag for presentation to T cells has been extensively studied in vitro, similar studies of phagocytic Ag processing have been limited . We have developed an in vitro model system to study the ability of macrophages to process recombinant Escherichia coli strain HB101 with cytoplasmic or surface expression of the well characterized T cell epitope of hen egg lysozyme (HEL) 52-61 . This epitope was expressed within full length HEL or within a fusion protein containing the HEL epitope . Phagocytosis of E . coli with cytoplasmic expression of HEL or the HEL fusion protein resulted in strong presentation of HEL(52-61) to T cells . Surface-conjugated HEL was processed with even greater efficiency . Processing required viable macrophages, was inhibited by cytochalasin D, and was achieved within 20 min of bacterial contact with the macrophages . Within this time span, phagosomes containing bacteria fused with lysosomes, and the bacteria were extensively degraded . Uptake of as few as four bacteria per macrophage produced an Ag-specific T cell response . We conclude that bacterial compartmentalization of the antigenic epitope (cytoplasmic vs surface) had some effect on its processing, but that phagocytic Ag processing organelles contain extensive capacity to degrade internalized bacteria and liberate intracellular Ag epitopes for recycling and presentation, consistent with a central role for phagolysosomes . Thus, future recombinant bacterial vaccines may be effectively designed with T cell epitopes expressed either on the surface or within the bacterial cytoplasm.

J Biol Chem, 1992 Oct 15, 267(29), 21004 - 10
Cloning and expression of the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase; Gillespie W et al.; Sequence information obtained by NH2-terminal sequence analysis of two molecular weight forms (45 and 48 kDa) of the porcine Gal beta 1,3GalNAc alpha 2,3-sialyltransferase was used to clone a full-length cDNA of the enzyme . The cDNA sequence revealed an open reading frame coding for 343 amino acids and a putative domain structure consisting of a short NH2-terminal cytoplasmic domain, a signal-anchor sequence, and a large COOH-terminal catalytic domain . This domain structure was confirmed by construction of a recombinant sialyltransferase in which the cytoplasmic domain and signal-anchor sequence of the enzyme was replaced with the cDNA of insulin signal sequence . Expression of the resulting construct in COS-1 cells produced an active sialyltransferase which was secreted into the medium in soluble form . Comparison of the cDNA sequence of the sialyltransferase with GenBank produced no significant homologies except with the previously described Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase . Although the cDNA sequences of these two enzymes were largely nonhomologous, there was a 45-amino acid sequence which exhibited 65% identity . This observation suggests that the two sialyltransferases were derived, in part, from a common gene.

J Biol Chem, 1992 Oct 15, 267(29), 20532 - 5
Escherichia coli topoisomerase III-catalyzed cleavage of RNA; DiGate RJ et al.; Relaxation of superhelical DNA by Escherichia coli topoisomerase III (Topo III) was inhibited by the inclusion of tRNA in the reaction mixture . Investigation of the basis of this inhibition revealed that Topo III could bind RNA and establish a cleavage-religation equilibrium . The addition of SDS to these reaction mixtures induced cleavage of the RNA by Topo III . The nucleotide sequences of RNA and DNA cleavage sites were identical, although cleavage site preference differed . Thus, the possibility that Topo III can pass strands of RNA as well as strands of DNA must be considered in accounting for the role of this enzyme in nucleic acid metabolism.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9593 - 7
Regulation of recombinant rat tyrosine hydroxylase by dopamine; Ribeiro P et al.; Recombinant rat PC12 tyrosine hydroxylase, also called tyrosine 3-monooxygenase {L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2}, purified from Escherichia coli is in an activated form with a low Km for the tetrahydrobiopterin cofactor and a pH optimum of 6.5 . Pretreatment with low levels of the derived product, dopamine, inhibits catalytic activity, increases the Km for the cofactor, and shifts the pH curve towards a more acidic pH optimum . Labeled dopamine binds to tyrosine hydroxylase with high affinity (Kd = 1 microM) but low stoichiometry (r = 0.08 mol/mol of enzyme subunit) . The binding of dopamine results in the appearance of a blue-green chromophore with lambda max at approximately 660 nm, which is consistent with the formation of a catecholamine-iron complex . In the absence of dopamine, the recombinant enzyme cannot be further activated by phosphorylation with cAMP-dependent protein kinase, although as much as 1 mol of phosphate is incorporated per mol of subunit . In contrast, the enzyme pretreated with dopamine is activated by phosphorylation in the same fashion and to the same extent as the native hydroxylase . The results suggest that the high-affinity binding of catecholamine products is a pivotal post-translational modification that determines the state of enzyme activation and the response to phosphorylation.

J Biol Chem, 1992 Oct 15, 267(29), 20844 - 9
GroE dependence of refolding and holoenzyme formation of 6-hydroxy-D-nicotine oxidase; Brandsch R et al.; In Escherichia coli cells expressing 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoprotein with covalently bound FAD, approximately 40% of the polypeptide is in its apoform . We investigated whether in vivo holoenzyme formation was influenced by the association of the apoenzyme with cellular chaperones . Immunoprecipitation of apoenzyme-containing cell extract with protein-A-Sepharose-bound 6-HDNO- or GroEL-specific antibodies failed to reveal the formation of complexes between these proteins . The limiting factor in holoenzyme formation in vivo appeared to be the intracellular supply of phosphorylated tricarbon compounds (e.g . glycerol-3-P) acting as allosteric effectors in the flavinylation reaction . When holoenzyme formation from purified apo6-HDNO was investigated in vitro, addition of GroEL and GroES to the reaction assays increased the yield of holoenzyme formation . The observed increase in apoenzyme to holoenzyme transition was ATP independent, and the effect of GroE could be simulated by high concentrations of glycerol (40%) . Apparently, a nonspecific protein-protein interaction between the GroE proteins and the apo6-HDNO favored holoenzyme formation . The refolding of guanidinium hydrochloride-unfolded holoenzyme, however, was catalyzed by GroEL and GroES in an ATP-dependent reaction . Recovery of the native, enzymatically active, conformation ranged from 30 to 40% . When apo6-HDNO was denatured and refolded, the same dependence on GroE and ATP was observed in the recovery of a conformation able to incorporate FAD and to holoenzyme . {14C} FAD in the refolding assay yielded radioactively labeled 6-HDNO demonstrating the autocatalytical covalent incorporation of FAD into the polypeptide during the folding process.

Biochem J, 1992 Oct 15, 287 ( Pt 2), 627 - 32
19F n.m.r . studies of conformational changes accompanying cyclic AMP binding to 3-fluorophenylalanine-containing cyclic AMP receptor protein from Escherichia coli; Hinds MG et al.; A fluorine-containing analogue of the cyclic AMP (cAMP) receptor protein (CRP) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorophenylalanine (3-F-Phe) . 19F n.m.r . studies on this protein have provided direct evidence for cAMP-induced conformational changes not only within the cAMP-binding domain but also within the hinge region connecting the cAMP-binding domain to the DNA-binding headpiece . At 313 K, the 19F n.m.r . spectrum of {3-F-Phe}CRP showed five signals corresponding to the five phenylalanine residues as expected for a symmetrical dimer . Proteolysis of {3-F-Phe}CRP with subtilisin produced a fragment (the alpha-fragment) containing the cAMP-binding domain . The alpha-fragment contains all the phenylalanines except for Phe-136, a residue located in the hinge region . By comparing the 19F spectra of {3-F-Phe}CRP and its alpha-fragment, the signal for Phe-136 was assigned . The chemical shifts of the corresponding signals in the two spectra are similar, indicating that the alpha-fragment retains the structure it has in the intact protein . The largest cAMP-induced shift was observed for the signal from Phe-136 providing direct evidence for a conformational change in the hinge region . However, whereas binding of a single cAMP molecule to a CRP dimer is known to be sufficient to activate the DNA binding, the n.m.r . data indicate that the hinge region does not have the same conformation in both subunits when only one cAMP molecule is bound.

Eur J Biochem, 1992 Oct 15, 209(2), 735 - 43
Cloning, overexpression and mechanistic studies of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus; Pollack SJ et al.; The enzyme carboxyphosphonoenolpyruvate mutase catalyses the formation of one of the two C-P bonds in bialaphos, a potent herbicide isolated from Streptomyces hygroscopicus . The gene encoding the enzyme has been cloned from a subgenomic library from S . hygroscopicus by colony hybridisation using an exact nucleotide probe . An open reading frame has been identified that encodes a protein of molecular mass 32700 Da, in good agreement with the subunit molecular mass of the carboxyphosphonoenolpyruvate mutase recently isolated from this source {Hidaka, T., Imai, S., Hara, O., Anzai, H., Murakami, T., Nagaoka, K . & Seto, H . (1990) J . Bacteriol . 172, 3066-3072} . The gene shares significant sequence similarity with that of phosphoenolpyruvate mutase, an enzyme that catalyses the related interconversion of phosphoenolpyruvate and phosphonopyruvate . When the carboxyphosphonoenolpyruvate-mutase gene was subcloned into the vector pET11a, the mutase was expressed as about 20% of the total soluble cellular protein in Escherichia coli . The mutase has been purified to homogeneity in three steps in 40% yield . With malate dehydrogenase/NADH, (hydroxyphosphinyl)pyruvate gives (hydroxyphosphinyl)lactate (kcat 164 s-1 and Km 680 microM) and this spectrophotometric assay for the product of the mutase reaction has been employed in the mechanistic studies . The kinetics for the mutase reaction have been evaluated for the substrate, carboxyphosphonoenolpyruvate, and for the putative reaction intermediate carboxyphosphinopyruvate, both of which have been prepared by chemical synthesis . Carboxyphosphonoenolpyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 0.020 s-1 and a Km of 270 microM, and carboxyphosphinopyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 7.6 x 10(-4) s-1 and a Km of 2.2 microM . Although the exogenously added intermediate is not kinetically competent, these results suggest that the mechanism for the mutase reaction involves an initial rearrangement to the intermediate carboxyphosphinopyruvate, followed by decarboxylation to yield the product (hydroxyphosphinyl)pyruvate.

Eur J Biochem, 1992 Oct 15, 209(2), 643 - 9
Biochemical characterization of thioredoxin 1 from Dictyostelium discoideum; Wetterauer B et al.; Multiple genes for thioredoxins (TRX) have been demonstrated in Dictyostelium discoideum . We expressed the cDNA for one of these genes (DdTrx1) in E . coli and purified the protein to homogeneity . The interaction with classic substrates as well as TRX reductases was analysed . It reacted with every tested substrate: insulin, NADP-dependent malate dehydrogenase and fructose-1,6-bisphosphatase . With a S0.5 of 20 microM, the reactivity with the fructose-1,6-bisphosphatase is the highest ever found for a heterologous TRX . DdTRX1 itself is accepted as a substrate by the chloroplast ferredoxin-dependent TRX reductase, as well as by the E . coli NADPH-dependent TRX reductase . Thus, the Dictyostelium TRX is functionally promiscuous . Its reactivity with insulin, chloroplast NADP-dependent malate dehydrogenase and ferredoxin-dependent TRX reductase resemble those of other TRX . It is, however, clearly different in its good interaction with chloroplast fructose-1,6-bisphosphatase and in its poor interaction with E . coli NADP-dependent TRX reductase.

Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9964 - 8
RNA binding determinant in some class I tRNA synthetases identified by alignment-guided mutagenesis; Shepard A et al.; The N-terminal nucleotide binding folds of all 10 class I tRNA synthetases (RSs) contain characteristic conserved sequence motifs that define this class of synthetases . Sequences of C-terminal domains, which in some cases are known to interact with anticodons, are divergent . In the 676-amino acid Escherichia coli methionyl-tRNA synthetase (MetRS), interactions with the methionine tRNA anticodon are sensitive to substitutions at a specific location on the surface of the C-terminal domain of this protein of known three-dimensional structure . Although four class I synthetases of heterogeneous lengths and unknown structures are believed to be historically related to MetRS, pair-wise sequence similarities in the region of this RNA binding determinant are obscure . A multiple alignment of all sequences of three of these synthetases with all MetRS sequences suggested a location for the functional analog of the anticodon-binding site in these enzymes . We chose a member of this set for alignment-guided mutagenesis, combined with a functional analysis of mutant proteins . Substitutions within two amino acids of the site fixed by the multiple sequence alignment severely affected interactions with tRNA but not with ATP or amino acid . Multiple individual replacements at this location do not disrupt enzyme stability, indicating this segment is on the surface, as in the MetRS structure . The results suggest the location of an RNA binding determinant in each of these three synthetases of unknown structure.

J Biol Chem, 1992 Oct 15, 267(29), 20992 - 8
Purification and characterization of 3-ketoacyl-acyl carrier protein synthase III from spinach . A condensing enzyme utilizing acetyl-coenzyme A to initiate fatty acid synthesis; Clough RC et al.; The 3-ketoacyl-acyl carrier protein (ACP) synthase III from spinach was purified to homogeneity by an eight-step procedure that included an ACP-affinity column . The size of the native enzyme was M(r) = 63,000 based on gel filtration, and its subunit size was M(r) = 40,500 based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that 3-ketoacyl-ACP synthase III may be a homodimer . The purified enzyme was highly specific for acetyl-CoA and malonyl-ACP . The Km for acetyl-CoA was 5 microM when assayed in the presence of 10 microM malonyl-CoA . Acetyl-, butyryl-, and hexanoyl-ACP would not substitute for acetyl-CoA as substrates . The specificity for acetyl-CoA suggested that the physiological function of 3-ketoacyl-ACP synthase is to catalyze the initial condensation reaction in fatty acid biosynthesis . The homogeneous 3-ketoacyl-ACP synthase was capable of catalyzing acetyl-CoA:ACP transacylation but at a rate about 90-fold slower than the condensation reaction with malonyl-ACP . The 3-ketoacyl-ACP synthase was inhibited 100% by 5 mM N-ethylmaleimide or 20 mM sodium arsenite.

J Biol Chem, 1992 Oct 15, 267(29), 20682 - 8
Substitution of manganese for iron in ribonucleotide reductase from Escherichia coli . Spectroscopic and crystallographic characterization; Atta M et al.; Each polypeptide chain of protein R2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a stable tyrosyl radical and two antiferromagnetically coupled oxo-bridged ferric ions . A refined structure of R2 has been recently obtained . R2 can be converted into apoR2 by chelating out the metal cofactor and scavenging the radical . This study shows that apoR2 has a very strong affinity for four stable Mn2+ ions . The manganese-containing form of R2, named Mn-R2, has been studied by EPR spectroscopy and x-ray crystallography . It contains two binuclear manganese clusters in which the two manganese ions occupy the natural iron-binding sites and are only bridged by carboxylates from glutamates 115 and 238 . This in turn explains why the spin-exchange interaction between the two ions is very weak and why Mn-R2 is EPR active . Mn-R2 could provide a model for the native diferrous form of protein R2, and a detailed molecular mechanism for the reduction of the iron center of protein R2 is proposed.

Biochim Biophys Acta, 1992 Oct 13, 1180(1), 33 - 43
The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys . Structural and in vitro biosynthetic studies; Holgersson J et al.; Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4) . The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A2 kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A2 kidneys . If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides . In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Gal alpha 1-4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of a Gal alpha 1-3(Fuc alpha 1-2)Gal beta-HexNAc-Gal alpha 1-4Gal beta-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide . In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant blood group A glycolipid . No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which might explain the low expression of type-4 chain blood group B heptaglycosylceramides in human blood group B kidneys.

Biochemistry, 1992 Oct 13, 31(40), 9752 - 9
Characterization of C439SR1, a mutant of Escherichia coli ribonucleotide diphosphate reductase: evidence that C439 is a residue essential for nucleotide reduction and C439SR1 is a protein possessing novel thioredoxin-like activity; Mao SS et al.; Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides . Cysteine 439 is proposed to be the protein radical on R1 which initiates the reduction reaction by cleavage of the 3' carbon-hydrogen bond of the nucleotide (Mao et al., 1992a,b) . C439 is thus proposed to be essential for catalysis . The C439S mutant of R1 (C439SR1) was prepared . The structure of this mutant was determined to be similar to wt-R1, based on identical CD spectra, isolation via an affinity column specific for the allosteric binding domain, binding of the substrate GDP, and competition with R1 for binding to R2 . Preparations of C439SR1 are contaminated with low levels of wt-R1 due to the expression system . The wt-R1 in these preparations can be specifically inactivated by the stoichiometric mechanism-based inhibitor, 2'-azido-2'-deoxyuridine 5'-diphosphate . The activity of the resulting C439SR1 was shown to be less than 0.03% that of the corresponding wt-R1 . This is the lower limit of detection with the present assay method . Thus C439 appears to be essential for catalysis . During these studies an unexpected activity of the C439SR1 was uncovered . Its additional cysteines, presumably C754 and C759, appear to function as a thioredoxin with the wt-R1, even though it is incapacitated with respect to nucleotide reduction.

Biochemistry, 1992 Oct 13, 31(40), 9725 - 32
Synergistic inhibition of Escherichia coli aspartate transcarbamylase by CTP and UTP: binding studies using continuous-flow dialysis; England P et al.; The allosteric control of Escherichia coli aspartate transcarbamylase (ATCase) involves feedback inhibition by both CTP and UTP, although it is only in the presence of CTP that UTP appears to inhibit the activity of the enzyme . In order to better understand the parts played by both pyrimidine nucleotides in this synergistic inhibition, binding studies were performed by continuous-flow dialysis and ultracentrifugation methods . The results obtained show that UTP binds to ATCase in the absence of CTP . Nevertheless, this binding does not induce any inhibition unless CTP is present . The mutual influence of CTP and UTP on their respective binding constants suggests that they bind to the same regulatory sites . However, the results obtained cannot be satisfactorily explained by a simple competition between the nucleotides, and it is shown that reciprocal affinity enhancements play a fundamental role . CTP enhances the affinity of UTP for the regulatory sites 80-fold, and conversely, UTP enhances the affinity of CTP 5-fold . Interestingly, the isolated regulatory subunits bind the two pyrimidine nucleotides following the same pattern as the entire enzyme . These observations indicate that the synergistic inhibition mechanism relies entirely on interactions between the two adjacent allosteric sites which belong to the same regulatory dimer.

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