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