Tokushima J Exp Med, 1993 Jun, 40(1-2), 13 - 7 Production of dihydrofolate reductase by an improved continuous flow cell-free translation system using wheat germ extract; Endo Y et al.; We have examined the characteristics of protein synthesis in an improved continuous flow cell-free translation system prepared from wheat germ extract with dihydrofolate reductase mRNA as the translated message . Continuous buffer flow and separation of the product from the reaction mixture were accomplished by the use of a modified Amicon ultrafiltration chamber as the reaction vessel . The system worked for 19 hours and produced 1.52 nmol (27.4 micrograms) of enzymatically active dihydrofolate reductase. Plant Physiol, 1993 Jun, 102(2), 529 - 36 Identification of the uridine-binding domain of sucrose-phosphate synthase . Expression of a region of the protein that photoaffinity labels with 5-azidouridine diphosphate-glucose; Salvucci ME et al.; The uridine diphosphate-glucose (UDP-Glc) binding domain of sucrose-phosphate synthase (SPS) was identified by overexpressing part of the gene from spinach (Spinacia oleracea) . Degenerate oligonucleotide primers corresponding to two tryptic peptides common to both the full-length 120-kD SPS subunit and an 82-kD form that photoaffinity labeled with 5-azidouridine diphosphate-glucose (5-N3UDP-Glc) were used in a polymerase chain reaction to isolate a partial cDNA clone . Comparison of the deduced amino acid sequence of spinach SPS with the sequences of potato sucrose synthase showed that the partial cDNA included one region that was highly conserved between the proteins . Expression of the partial cDNA clone of SPS in Escherichia coli produced a 26-kD fusion protein that photoaffinity labeled with 5-N3UDP-Glc . Photoaffinity labeling of the 26-kD fusion protein was specific, indicating that this portion of the SPS protein harbors the UDP-Glc-binding domain . Isolation of a modified peptide from the photolabeled protein provided tentative identification of amino acid residues that make up the uridine-binding domain of SPS. Plant Physiol, 1993 Jun, 102(2), 387 - 99 Isolating the Arabidopsis thaliana genes for de novo purine synthesis by suppression of Escherichia coli mutants . I . 5'-Phosphoribosyl-5-aminoimidazole synthetase; Senecoff JF et al.; We have initiated an investigation of the de novo purine nucleotide biosynthetic pathway in the plant Arabidopsis thaliana . Functional suppression of Escherichia coli auxotrophs allowed the direct isolation of expressed Arabidopsis leaf cDNAs . Using this approach we have successfully suppressed mutants in 4 of the 12 genes in this pathway . One of these cDNA clones, encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase (PUR5) has been characterized in detail . Analysis of genomic DNA suggests that the Arabidopsis genome contains a single AIR synthetase gene . Analysis of the cDNA sequence and mRNA size suggests that this enzyme activity is encoded by a monofunctional polypeptide, similar to that of bacteria and unlike other eukaryotes . The Arabidopsis AIR synthetase contains a basic hydrophobic transit peptide consistent with transport into chloroplasts . Comparison of both the predicted amino acid and nucleotide sequence from Arabidopsis to those of eight other distant organisms suggests that the plant sequence is more similar to the bacterial sequences than to other eukaryotic sequences . This study provides the groundwork for future investigations into the regulation of de novo purine biosynthesis in plants . Additionally, we have demonstrated that functional suppression of bacterial mutants may provide a useful method for cloning a variety of plant genes. Bioorg Khim, 1993 Jun, 19(6), 612 - 22 {Synthesis of somatostatin in Escherichia coli cells: isolation and characteristics}; Karpova SK et al.; A synthetic gene coding for somatostatin-14 (SST) was cloned in plasmid expression vectors in frame with the chloramphenicol acetyl transferase (CAT) gene, both genes being divided by a Met residue . The hybrid gene was expressed under the control of the CAT gene promoter (Pcat) or the tryptophan operon promoter (Ptrp) . Them fused genes gave insoluble polypeptide products amounting from 5% of the total cellular protein under constitutive biosynthesis conditions (Pcat) to 30% upon induction (Ptrp) . SST was liberated from the fused polypeptide by treatment with cyanogen bromide, purified to homogeneity by gel-filtration and reverse phase HPLC, and finally refolded by dilution and air oxidation . The renaturated recombinant SST showed the specific biological and immunological activities of the native peptide. Genome, 1993 Jun, 36(3), 459 - 66 Molecular cloning and expression of a cDNA encoding the proliferating cell nuclear antigen from Brassica napus (oilseed rape); Markley NA et al.; A cDNA clone encoding the proliferating cell nuclear antigen (PCNA) has been isolated from a Brassica napus apical meristem cDNA library . The putative full-length cDNA contains an open reading frame of 1004 nucleotides, which predicts a protein of 263 amino acids (M(r) = 29,231) . Sequence analysis has revealed that the plant PCNA exhibits 81.6% amino acid similarity with the human PCNA . Genomic Southern blot analysis indicates the presence of at least two copies of PCNA per genome . The B . napus PCNA mRNA (1.0 kb) was expressed in rapidly dividing tissues such as flower buds, apical meristems, and young leaves, while mature stem and fully expanded leaves showed significantly lower levels of PCNA transcript . The B . napus PCNA cDNA was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) in the bacterial expression vector pGEX-2T . A broad specificity monoclonal antibody raised against rabbit PCNA cross-reacted with the GST-PCNA fusion peptide but not with the GST moiety alone . This antibody also recognized the human PCNA (36 kDa) polypeptide, confirming the structural similarities between the human and plant PCNA . The high degree of structural conservation of PCNA from such diverse organisms as humans and higher plants suggests that the plant PCNA may function in a manner analogous to that found in mammals with respect to plant cell DNA replication . Such conservation suggests that PCNA is also a critical component of the plant cell DNA replication complex. J Crit Care, 1993 Jun, 8(2), 117 - 27 Organ blood flow and distribution of cardiac output in dopexamine- or dobutamine-treated endotoxemic rats; van Lambalgen AA et al.; Endotoxemia causes a decrease of blood flow to most organs . If this could be prevented, chances of survival might improve . In endotoxemic rats, we studied the effect of a therapeutic infusion of dopexamine (dopaminergic, beta 2-adrenergic) on blood flow and percentage of the cardiac output distributed to heart, brain, hepatic artery, stomach, intestines, spleen, pancreas, kidneys, adrenals, diaphragm, skeletal muscle, and skin . Dopexamine action was compared with that of dobutamine (beta 1-adrenergic) . Endotoxin shock was induced in 28 rats with infusion of 8 mg/kg Escherichia coli O127:B8 endotoxin from 0 to 60 minutes; the rats were then divided into 3 groups, which received from 60 to 135 minutes of an infusion of saline (ES; n = 10), dopexamine hydrochloride (DX, 3 x 10(-8) mol/kg.min; n = 10) or dobutamine (DB, 10(-7) mol/kg.min; n = 8) . A fourth group served as time-matched controls (C, saline from 0 to 135 minutes; n = 8) . In the untreated endotexemic rats, cardiac output decreased and organ blood flow decreased except in the diaphragm, heart, and brain; the percentage of the cardiac output to those organs increased . Dopexamine and dobutamine similarly improved cardiac output in endotoxemic rats . All organs benefitted to the same extent from the increased cardiac output . Therapeutic infusion of dopexamine during endotoxemia did not favor flow to any particular organ; redistribution of cardiac output changed little after administration of dopexamine, and its effects were not significantly different from those of dobutamine. Antimicrob Agents Chemother, 1993 Jun, 37(6), 1278 - 84 Functional studies of truncated soluble intercellular adhesion molecule 1 expressed in Escherichia coli; Martin S et al.; We have expressed in Escherichia coli the two N-terminal immunoglobulin (Ig)-like domains of the intercellular adhesion molecule 1 (ICAM-1) . The first 188 residues of ICAM-1 were expressed with an N-terminal methionine (MP188) or as a maltose-binding fusion protein which was cleaved with factor Xa (XP188) . After refolding, both MP188 and XP188 were active in binding to the leukocyte integrin lymphocyte function-associated antigen 1, which has previously been shown to bind to the N-terminal Ig domain of ICAM-1 . The major group of rhinoviruses and malaria-infected erythrocytes bind to distinct sites within the first Ig-like domain of ICAM-1 . Both MP188 and XP188 bound to malaria-infected erythrocytes; however, only XP188 inhibited human rhinovirus plaque formation . A product (MdQ1P188) with the initiation methionine fused to residue 2, i.e., with glutamine 1 deleted, inhibited plaque formation . MdQ1P188 was able to induce a conformational change of the virus capsid as shown by conversion of 149S particles to 85S particles, whereas MP188 had no effect . These results show that functionally active fragments of ICAM-1 can be produced in E . coli, that glycosylation is not required for ligand binding, and that the N-terminal residue of ICAM-1 is proximal to or part of the human rhinovirus-binding site. J Bacteriol, 1993 Jun, 175(11), 3679 - 84 Close linkage of genes encoding glutamine synthetases I and II in Frankia alni CpI1; Hosted TJ et al.; Frankia alni CpI1 has two glutamine synthetases (GSs), GSI and GSII . The GSI gene (glnA) was isolated from a cosmid library of F . alni CpI1 DNA by heterologous probing with glnA from Streptomyces coelicolor . The glnA gene was shown to be located upstream of the GSII gene (glnII) by DNA-DNA hybridization . The nucleotide sequences of the 1,422-bp CpI1 glnA gene and of the 449-bp intervening region between glnA and glnII were determined, and the glnA amino acid sequence was deduced . In common with GSIs from other organisms, CpI1 GSI contains five conserved regions near the active site and a conserved tyrosine at the adenylylation site . F . alni CpI1 glnA complemented the glutamine growth requirement of the Escherichia coli glnA deletion strain YMC11 but only when expressed from an E . coli lac promoter . While the functional significance of maintaining two GSs adjacent to one another remains unclear, this arrangement in F . alni provides support for the recently proposed origin of GSI and GSII as resulting from a gene duplication early in the evolution of life. Infect Immun, 1993 Jun, 61(6), 2526 - 31 Piglet ileal mucus contains protein and glycolipid (galactosylceramide) receptors specific for Escherichia coli K88 fimbriae; Blomberg L et al.; The aim of this study was to characterize the Escherichia coli K88-specific receptors in mucus from the small intestines of 35-day-old piglets with the isogenic strains E . coli K-12(pMK005) (K88+) and E . coli K-12(pMK002) (K88-) . These strains differed only in that the latter one cannot produce intact K88 fimbriae because of a deletion in the gene coding for the major fimbrial subunit . Adhesion was studied by incubating 3H-labeled bacteria with crude mucus, pronase-treated whole mucus, mucus fractionated by gel filtration, delipidated mucus, or extracted lipids immobilized in microtiter wells . In addition, E . coli strains were tested for adhesion to glycolipids extracted from mucus by overlaying glycolipid chromatograms with 125I-labeled bacteria . The recently reported finding that K88 fimbriae bind to glycoproteins in mucus from the piglet small intestine was confirmed in two ways . Pronase treatment of immobilized mucus reduced adhesion by 82%, and adhesion to delipidated mucus was 14 times greater for the K88+ than for the K88- strain . E . coli K88+ adhered to several of the fractions collected after gel filtration of crude mucus, including the void volume (M(r), > 250,000) . Receptor activity specific for the K88 fimbriae was demonstrated in the lipids extracted from mucus, as the neutral lipids contained six times as much receptor activity as the acidic lipid fraction . Specificity was confirmed by demonstrating that adhesion to the total lipids could be inhibited by pretreatment of the immobilized lipids with K88 fimbriae . Relative to K-12 (K88-), the K-12 (K88+) bacterial cells bound more avidly to galactosylceramide when the neutral lipids were separated on thin-layer chromatography plates . No adhesion to lipids in the acidic fraction separated on thin-layer plates was detected . Relative to adhesion of K-12 (K88-), adhesion of K-12 (K88+) to commercially available galactosylceramide immobilized in microtiter wells confirmed the results with the thin-layer plates . It can be concluded that 35-day-old piglet mucus contains both protein and glycolipid receptors specific for K88 fimbriae, the latter being galactosylceramide. Infect Immun, 1993 Jun, 61(6), 2453 - 61 Structure and copy number analyses of pap-, sfa-, and afa-related gene clusters in F165-positive bovine and porcine Escherichia coli isolates; Maiti SN et al.; Pathogenic F165-positive Escherichia coli isolates of porcine and bovine origin possess gene clusters related to extraintestinal E . coli fimbrial operons pap, sfa, and afaI . Probes from different segments of the pap, sfa, and afaI operons were used in Southern hybridization to analyze 18 F165-positive, mannose-resistant hemagglutinating E . coli isolates possessing pap- and sfa-, pap- and afa-, or pap-related sequences . Only single copies of the pap-, sfa-, or afa-related sequences were found among the isolates, and the position of each sequence with respect to those of adjacent sequences was variable . Expression of the P and F adhesins individually or concurrently was associated with the presence of a single copy of pap-related sequences . Gene clusters related to pap were structurally similar to the pap operon in certain isolates or the prs operon in others . sfa-related sequences showed few internal structural polymorphisms and were structurally similar to the foc rather than the sfa operon . afa-related sequences showed many internal structural polymorphisms compared with the afa-related sequences from prototype strain KS52 . These results demonstrate that the pap- and sfa-related sequences in F165-positive isolates are closely related to the prototype pap operon and foc operon of the P family and Sfa family, respectively . afa-related sequences, on the other hand, display heterogeneity and differ from the prototype afaI operon. J Virol, 1993 Jun, 67(6), 3534 - 43 Mutational analysis of the central domain of adenovirus virus-associated RNA mandates a revision of the proposed secondary structure; Pe'ery T et al.; Protein synthesis in adenovirus-infected cells is regulated during the late phase of infection . The rate of initiation is maintained by a small viral RNA, virus-associated (VA) RNAI, which prevents the phosphorylation of eukaryotic initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI . On the basis of nuclease sensitivity analysis, a secondary-structure model was proposed for VA RNA . The model predicts a complex stem-loop structure in the central part of the molecule, the central domain, joining two duplexed stems . The central domain is required for the inhibition of DAI activation and participates in the binding of VA RNA to DAI . To assess the significance of the postulated stem-loop structure in the central domain, we generated compensating, deletion, and substitution mutations . A substitution mutation which disrupts the structure in the central domain abolishes VA RNA function in vitro and in vivo . Base-compensating mutations failed to restore the function or structure of the mutant, implying that the stem-loop structure may not exist . To confirm this observation, we tested mutants with alterations in the hypothetical loop and short stem that constitute the main features of the wild-type model structure . The upper part of the hypothetical loop could be deleted without abolishing the ability of the RNA to block DAI activation in vitro, whereas other loop mutations were deleterious for function and caused major rearrangements in the molecule . Base-compensating mutations in the stem did not restore the expected base pairing, even though the mutant RNAs were still functional in vitro . Surprisingly, a mutant with a noncompensating substitution mutation in the stem was more effective than wild-type VA RNAI in DAI inhibition assays but was ineffective in vivo . The structural and functional consequences of these mutations do not support the proposed model structure for the central domain, and we therefore suggest an alternative structure in which tertiary interactions may play a significant role in shaping the specificity of VA RNA function in the infected cell . Discrepancies between the functionality of mutant forms of VA RNA in vivo and in vitro are consistent with the existence of additional roles for VA RNA in the cell. J Neurochem, 1993 Jun, 60(6), 2181 - 91 Development of polyclonal anti-D2 dopamine receptor antibodies to fusion proteins: inhibition of D2 receptor-G protein interaction; Boundy VA et al.; Portions of the cDNA encoding the third intracellular loop (i3 loop) of the long and short isoforms of the rat D2 dopamine receptor were subcloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins . The fusion proteins were gel-purified and used to immunize rabbits for the production of polyclonal anti-receptor antisera . The anti-fusion protein antisera recognized synthetic peptides corresponding to segments of the i3 loops of D2 dopamine receptors in a solid-phase radioimmunoassay . Antisera were tested in an immunoprecipitation assay using the reversible D2 antagonist {125I}NCQ 298 and digitonin-solubilized extracts of canine and rat caudate . {125I}-NCQ 298 bound reversibly and with high affinity (KD = 0.14 nM) to receptors in solubilized extracts enriched by chromatography on heparin-agarose . The anti-UBI-D2i3L and anti-UBI-D2i3s antisera were able to immunoprecipitate quantitatively D2 dopamine receptors labeled with {125I}NCQ 298 from solubilized rat caudate . The antibodies were tested for their ability to affect the coupling of D2 dopamine receptors to GTP-binding proteins in digitonin-solubilized rat caudate . Both anti-UBI-D2i3L and anti-UBI-Di3s antisera were able to inhibit the high-affinity binding of the agonist N-propylnorapomorphine to digitonin-solubilized rat caudate . These findings indicate that the i3 loop of the D2 dopamine receptor is an important determinant for coupling of the G protein. C R Acad Sci III, 1993 Jun, 316(6), 593 - 7 Identification of a Kin nuclear protein immunologically related to RecA protein in the rat CNS; Araneda S et al.; A polypeptide, called Kin, has been identified in cells of the central nervous system (CNS) by using antibodies raised against RecA protein of E . coli and by in situ hybridization with identified cDNA . RecA protein is a recombination enzyme associated with DNA repair . The RecA cross-reacting polypeptide was immunocytochemically demonstrated in the nuclei of various cells of adult rats . Following Western blot analysis using anti-RecA antibodies, the Kin protein showed a band with an apparent molecular weight of 41 kDa . Double labelling experiments, using in situ hybridization of Kin-17 mRNA with serotonin immunocytochemistry, demonstrated a cytoplasmic distribution of radiolabelling indicating the translation of this messenger RNA in serotonergic neurons . These data indicate the presence of a Kin nuclear protein in the CNS and suggest that neurons may possess some DNA-repair pathways analogous to those described in bacteria. Development, 1993 Jun, 118(2), 553 - 62 The chicken CdxA homeobox gene and axial positioning during gastrulation; Frumkin A et al.; The chicken homebox containing gene, CdxA (formerly CHox-cad), was previously shown to be expressed during gastrulation . Localization of CdxA transcripts by in situ hybridization to tissue sections revealed that, during gastrulation, expression of this gene exhibits a posterior localization along the primitive streak . The transcripts are localized to epiblast cells in the vicinity of the primitive streak, to cells of the primitive streak itself and in the definitive endoderm as it replaces the hypoblast . In order to study in greater detail the pattern of expression of the CdxA gene during gastrulation, we expressed the full-length CdxA protein as a fusion protein in E . coli and generated monoclonal antibodies against it . Chicken embryos at different stages of gastrulation were processed for whole-mount immunohistochemical localization of the protein using anti-CdxA antibodies . Once the pattern of expression in the whole embryo was determined, the same embryos were sectioned to determine the identity of the cells expressing the CdxA protein . Detailed analysis of the CdxA protein in embryos, from the onset of primitive streak formation to the beginning of the tail bud stage (stages 2 to 10), has shown different patterns of expression during primitive streak elongation and regression . The CdxA protein is initially detected at the posterior marginal zone and the expression moves rostrally into the primitive streak during mid-streak stages . As the primitive streak elongates, the CdxA stripe of expression moves anteriorly . By definitive streak stages, the CdxA stripe of expression delineates a position along the anterior-posterior axis in the primitive streak . CdxA, like its Drosophila homologue cad, is expressed during gastrulation in a stripe localized to the posterior region of the embryo . These observations suggest that CdxA as a homebox gene may be part of a regulatory network coupled to axial determination during gastrulation in the early chick embryo. Afr J Med Med Sci, 1993 Jun, 22(2), 39 - 41 Cloning and characterization of the tetracycline resistance determinant from enteropathogenic Escherichia coli p1479; Daini OA et al.; As part of an epidemiological study, R plasmids coding for tetracycline resistance were isolated from enteropathogenic Escherichia coli . A 1.4kb Pst 1 fragment of one of them p1479 (size 5.7kb) has been cloned into plasmid pGL101 . This recombinant plasmid containing the tetracycline gene was then transformed into E . coli DHI where the tetracycline resistant gene was fully expressed . Attempts to develop this clone to a diagnostic probe is now in progress. J Biotechnol, 1993 Jun, 29(3), 299 - 306 Enhanced production of pL-controlled recombinant proteins and plasmid stability in Escherichia coli RecA+ strains; Benito A et al.; Overexpression of pL-controlled foot-and-mouth disease virus recombinant proteins was studied in Escherichia coli RecA+ strains and in a recA mutant . Higher protein yield and extractable plasmid DNA amounts were found in wild type cells, in absence of detectable RecA proteolytic activity . Minor but still significant differences in pBR322 DNA amounts were also detected between RecA+ and its recA13 and lexA1 derivatives . These data should be seriously considered to select expression systems and to design production processes for recombinant proteins, specially if they are expected to be toxic for Escherichia coli cells. Appl Microbiol Biotechnol, 1993 Jun, 39(3), 324 - 8 Optimization of the synthesis of porcine somatotropin in Escherichia coli; Wang H et al.; We report on the influence of choice of promoter and RNA polymerase, 5'-untranslated regions and ribosome binding sites, codon usage, leader peptide coding sequences and poly A tail in the 3'-untranslated region on the synthesis of porcine somatotropin (PST) in Escherichia coli . A total of 12 different constructs were tested in this study for the production of porcine somatotropin (PST) in E . coli . Several factors have significant effects on PST synthesis . In the presence of a strong promoter and a strong ribosome binding site, the next most important factor seems to be the combination of sequences at the 5'-end of the mRNA including both the 5'-untranslated region and the start of the coding sequence . Codon usage in the 5'-coding sequence per se is not important in determining the level of PST synthesis where high level expression is achieved from a strong ribosome binding site . However, where low level synthesis of recombinant PST (rPST) is achieved, codon usage in the 5'-coding sequence is important in determining the level of PST synthesis . Leader sequences dramatically reduce the level of PST synthesis . The presence of a poly A tail in the 3'-untranslated region has no significant effect on PST synthesis. Appl Microbiol Biotechnol, 1993 Jun, 39(3), 309 - 17 Cloning and characterization of the Methylobacterium extorquens polyhydroxyalkanoic-acid-synthase structural gene; Valentin HE et al.; A cosmid gene bank of partially EcoRI-digested genomic DNA from Methylobacterium extorquens IBT no . 6 was screened for DNA fragments restoring polyhydroxyalkanoic-acid (PHA) accumulation in the PHA-negative mutant Alkaligenes eutrophus H16 PHB-4 . The M . extorquens PHA-synthase structural gene phaCMex was mapped on a 23-kbp EcoRI fragment by complementation studies, by hybridization experiments with heterologous DNA probes from A . eutrophus H16 encoding for phaA, phaB and phaC and by nucleic acid sequence analysis . Evidence for the presence of genes for a beta-ketothiolase or an acetoacetyl-coenzyme A reductase on this fragment was not obtained . The nucleotide sequence of a 3.7-kbp region was obtained . It contained the entire 1.815-kbp phaCMex plus approximately each 900-bp upstream and downstream of phaCMex.PhaCMex encoded a protein of 605 amino acids with a relative molecular mass (M(r)) of 66742, which exhibited 38.1% amino acid identity with the A . eutrophus PHA synthase . Determination of the N-terminal amino acid sequence of an M(r) 65,000 protein, which was enriched concomitantly with the purification of PHA granules in sucrose gradients, revealed a sequence that was identical with the amino acid sequence deduced from the most probable translation start codon except for a valine, which was obviously removed post-translationally . Enzyme analysis, which was done with the native gene and a phaCMex'-'lacZ fusion gene, gave no evidence for expression of phaCMex in Escherichia coli. J Membr Biol, 1993 Jun, 134(2), 85 - 92 A carboxy-terminal fragment of colicin Ia forms ion channels; Ghosh P et al.; A carboxy-terminal, 18 kD fragment of colicin Ia, a bacterial toxin, forms ion channels in artificial phospholipid bilayers . This fragment, which comprises a quarter of the intact 70 kD molecule, is resistant to extensive protease digestion and probably constitutes a structural domain of the protein . The ion channels formed by the 18 kD fragment are functionally heterogeneous, having conductances that range from 15 to 30 pS at positive voltages and from 70 to 250 pS at negative voltages, and open lifetimes that range from at least 25 msec to 5 sec . In contrast, ion channels formed by whole colicin Ia open only at negative voltages, at which their conductances range from 6 to 30 pS, and their open lifetimes range from 1 sec to 3 min . Additionally, the open state of the 18 kD fragment channel is characterized by noisy fluctuations in current, while the open state of the whole molecule ion channel is often marked by numerous, stable subconductance states . Since the properties of the fragment channel differ substantially from those of the whole molecule channel, we suggest that portions of the molecule outside of the 18 kD fragment are involved in forming the whole molecule ion channel. J Electron Microsc (Tokyo), 1993 Jun, 42(3), 202 - 4 Visualization of lipopolysaccharide aggregates by freeze-fracture and negative staining; Risco C et al.; The morphology of Escherichia coli 0111:B4 lipopolysaccharide (LPS) in aqueous medium was studied by freeze-fracture and negative staining . Samples processed by freeze-fracture showed LPS aggregates that were mainly spherical or elliptical and of rather homogeneous size . Negative staining, however, showed a more heterogeneous population, although globular structures revealed by both procedures had a similar size . Considering the mechanisms involved in the processing of the samples, we suggest that the genuine shape of LPS aggregates is more likely to be globular, thus being artefactual those forms visualized by negative staining, traditionally associated with the organization of LPS in aqueous suspension. Biometrics, 1993 Jun, 49(2), 543 - 55 Phylogenetic inference: linear invariants and maximum likelihood; Navidi WC et al.; We develop a new statistical method for inferring phylogenies, based on a likelihood ratio test . This method does not require parameter constraints but does require identical evolutionary processes in the sites considered . Another method of phylogenetic inference is the method of linear invariants, described by Cavender (1989, Molecular Biology and Evolution 6, 301-316), based on a notion of Lake (1987, Molecular Biology and Evolution 4, 167-191) . We describe a sound mathematical basis for the use of linear invariants . We show that the validity of the method requires parameter constraints, but does not require that the evolutionary processes in differing sites be identical . We show that the method of linear invariants is asymptotically equivalent to a less powerful version of our likelihood ratio test, and is thus essentially a maximum likelihood technique. J Cardiovasc Pharmacol, 1993 Jun, 21(6), 926 - 30 Effects of methylene blue on blood pressure and reactivity to norepinephrine in endotoxemic rats; Paya D et al.; The effects of methylene blue, an inhibitor of the activation of the soluble guanylyl cyclase by nitric oxide (NO), were studied on blood pressure (BP) and on hyporesponsiveness to norepinephrine (NE) induced by Escherichia coli lipopolysaccharide (LPS) in pentobarbital-anesthetized rats . Methylene blue intravenous (i.v.) injection (3 mg/kg) produced a transient increase in BP which, in LPS-treated rats, was followed by a more sustained increase in BP . Methylene blue restored the reactivity to NE in LPS-treated rats but did not change either BP or reactivity to NE in saline-infused control rats . Cyclic GMP level was significantly increased in small femoral resistance arteries removed from LPS-treated rats as compared with controls (125.2 +/- 19.5 and 83.5 +/- 18.8 fmol/mg DNA, respectively, n = 8) . In rats receiving methylene blue, there was no significant difference in cyclic GMP content of the arteries of LPS-treated rats as compared with controls (59.4 +/- 8.1 and 78.5 +/- 6.1 fmol/mg DNA, respectively, n = 8) . These results support the involvement of increased stimulation of arterial guanylyl cyclase in hyporeactivity to NE elicited by LPS . They show that in vivo administration of methylene blue is able to restore both vascular cyclic GMP level and pressor responses to NE to control levels in LPS-treated rats. Antimicrob Agents Chemother, 1993 Jun, 37(6), 1227 - 31 Inhibition of Pneumocystis carinii dihydropteroate synthetase by para-acetamidobenzoic acid: possible mechanism of action of isoprinosine in human immunodeficiency virus infection; Kovacs JA et al.; Isoprinosine has been reported to decrease progression to AIDS, primarily by preventing Pneumocystis carinii pneumonia (PCP), in human immunodeficiency virus-infected patients, but the mechanism of action is unknown . para-Acetamidobenzoic acid (PAcBA), one component of isoprinosine, is structurally related to para-aminobenzoic acid (PABA), a precursor of de novo folate synthesis . This pathway is known to be important for P . carinii because sulfonamides, which are effective anti-P . carinii agents, inhibit incorporation of PABA into folate precursors by the enzyme dihydropteroate synthetase (DHPS) . Inhibition of P . carinii DHPS by PAcBA was investigated by using two assays . In short-term cultures of P . carinii from rats, {3H}PABA incorporation into reduced folates was inhibited by both isoprinosine (mean +/- standard error 50% inhibitory concentration {IC50}, 20 +/- 8.4 microM) and PAcBA free acid (IC50, 240 +/- 100 microM); a soluble PAcBA salt was more potent than PAcBA free acid alone (IC50, 29 +/- 48 microM) . The activity of PAcBA free acid was confirmed in a cell-free DHPS inhibition assay (IC50, 120 +/- 120 microM) . Inosine and dimethylaminopropanol, two other components of isoprinosine, were poor inhibitors of PABA incorporation (IC50, > 1,000 microM) . PAcBA free acid also showed activity in inhibiting the DHPS of Toxoplasma gondii, but was a poor inhibitor of the DHPSs of Escherichia coli and Saccharomyces cerevisiae . In a rat model of PCP, the PAcBA salt administered intraperitoneally demonstrated no activity against established PCP either alone or when used in combination with trimethoprim; the lack of efficacy in this model may be due to the rapid metabolism of the drug . Prevention of PCP by PaCBA through inhibition of P . carinii DHPS may explain the activity of isoprinosine in decreasing the progression to AIDS in human immunodeficiency virus-infected patients. Genomics, 1993 Jun, 16(3), 551 - 61 DNA sequence and analysis of 136 kilobases of the Escherichia coli genome: organizational symmetry around the origin of replication; Burland V et al.; The DNA sequence of a 136-kb segment (81.5 to 84.5 min) from the Escherichia coli origin of replication region has been determined and analyzed . Of the 122 protein coding regions that were found, we could assign no gene name or function to half of them, even in this well-studied part of the genome . The newly sequenced region also includes five RNA genes . The arrangement of open reading frames and potential promoters suggests 63 transcription units . The sequence was also analyzed for bend sites and two types of repeated sequence elements . Together with our sequence of the 84.5 to 86.5 min region, this new determination forms a 227-kb contiguous region centered on oriC . A global analysis of this region reveals a remarkable symmetry: most genes are transcribed divergently from the replication origin, and Chi octanucleotide (5'GCTGGTGG3') recombinational hot spots are also strikingly oriented with respect to the directions of replication and translation. Mol Gen Genet, 1993 Jun, 239(3), 400 - 8 Nitrogen regulation in an Escherichia coli strain with a temperature sensitive glutamyl-tRNA synthetase; Osorio AV et al.; Escherichia coli cells carrying the gltX351 allele are unable to grow at 42 degrees C (Ts phenotype) due to an altered glutamyl-tRNA synthetase . We found that gltX351 cells display a new phenotype termed Gsd-, i.e . an inability to raise glutamine synthetase activity above low constitutive levels in minimal medium with 6.8 mM glutamine as sole nitrogen source . When 0.5 mM NH4+ or 12 mM glutamate replaced glutamine, the glutamine synthetase activities of gltX351 cells were raised to wild-type levels . Northern experiments showed that the Gsd- phenotype is the result of an impairment in transcription initiation from the Ntr-regulated promoter, glnAp2 . Intragenic and extragenic secondary mutations appeared frequently in gltX351 cells, which suppressed their Gsd- but not their Ts phenotype . Moreover, in heterozygous gltX+/gltX351 partial diploids, gltX351 was dominant for the Gsd- phenotype and recessive for the Tr phenotype . A slight increase in the glutamine pool and in the intracellular glutamine: 2-oxoglutarate ratio was also observed but this could not account for the Gsd- phenotype of gltX351 cells . In cells carrying gltX351 and a suppressor of the Gsd- phenotype, sup-1, tightly linked to gltX351, the glutamine pool and glutamine: 2-oxoglutarate intracellular ratio were even higher than in the gltX351 single mutant . These results indicate that the gltX351 mutant polypeptide may be the direct cause of the Gsd- phenotype . The possibility that it interacts with one or more components that trigger the Ntr response is discussed. Protein Expr Purif, 1993 Jun, 4(3), 187 - 99 Expression of polypeptides of human immunodeficiency virus-1 reverse transcriptase in Escherichia coli; Becerra SP et al.; We have prepared a plasmid, pRC-RT, for expression of HXB2 HIV-1 reverse transcriptase (RT) in Escherichia coli (Becerra et al., Biochemistry 30, 11707-11719, 1991) . Here we describe the optimization of RT overexpression and its purification . In pRC-RT, the precise RT coding region of HXB2 proviral DNA is flanked by start and stop codons, and expression is driven by the phage lambda pL promoter in a temperature-inducible system . The 64,484-Da RT polypeptide (termed p66) is expressed as approximately 10% of total cell protein after 2 h of induction, and the RT is readily solubilized and purified free of DNA Pol I and to near homogeneity as a homodimer of p66 or as a heterodimer of p66 and p51, resembling the natural enzyme . After achieving appropriate expression of the full-length p66 RT, we next created vectors to express multiple individual segments of the p66 polypeptide . These segments are: a 51,000-Da peptide, representing C-terminal truncation of p66, and several peptides representing consecutive N-terminal, central, and C-terminal segments of p66 . The latter peptide, corresponding to the RNase H domain of RT, has been purified in large quantities and is currently under study for solution of its structure by NMR . This peptide is devoid of enzyme activity and of substrate-binding capacity, but exists in solution as a folded globular protein with structure resembling that of E . coli ribonuclease H and that of a similar HIV-1 RT RNase H domain peptide examined by X-ray crystallography (Becerra et al., FEBS Lett . 270, 67-80, 1990) . Various other RT peptides described here should prove to be similarly useful for structural studies, as well as other approaches. Lab Invest, 1993 Jun, 68(6), 629 - 36 New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope; Key G et al.; BACKGROUND: The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all cells that are not in G0 . Recently, we could demonstrate that Ki-67 detects a double band in Western blots of proliferating cells with apparent molecular weights of 345 kilodaltons and 395 kilodaltons, respectively . Furthermore, initial molecular biologic data favored the view that the epitope detected by Ki-67 might be encoded by a repetitive 66 bp element . EXPERIMENTAL DESIGN: In order to verify this assumption, parts of the Ki-67 cDNA were bacterially expressed, and the fusion proteins obtained were used to elicit new monoclonal antibodies . The specificities of the new reagents were tested by immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay techniques . RESULTS: The somatic cell fusions revealed a number of antibodies with immunoreactivities comparable to Ki-67 . Three antibodies, designated MIB 1-3, were further characterized . Besides the fact that their immunostaining reactivity is identical with that of Ki-67, all new antibodies react in Western blots with native Ki-67 antigen . Furthermore, Western blot and competitive binding assays by enzyme-linked immunosorbent assay clearly demonstrate that MIB 1 and MIB 3, like the original Ki-67 antibody, react with an epitope that is encoded by the 66 bp repetitive element mentioned above . MIB 2, however, reacts with an epitope distinct from this latter structure . In addition, after antigen unmasking by microwave treatment, MIB 1 and MIB 3 detect the Ki-67 antigen in paraffin sections . CONCLUSIONS: Our results demonstrate that it is possible to use bacterially expressed parts of the Ki-67 antigen as immunogen to elicit antibodies that react with the native antigen . While MIB 1 and MIB 3 detect the same or a very similar epitope as the original antibody Ki-67, MIB 2 clearly differs in its fine specificity . Our results provide a circle of evidence that the cDNA sequence thus far determined encodes for the Ki-67 antigen . Furthermore, the new antibodies may become powerful tools for routine histopathology and for further functional characterization of the Ki-67 antigen. Exp Parasitol, 1993 Jun, 76(4), 358 - 70 Schistosoma mansoni: comparison of cloned tropomyosin antigens shared between adult parasites and Biomphalaria glabrata; Weston DS et al.; Rabbit antisera, raised against whole, homogenized hepatopancreas from uninfected Biomphalaria glabrata, were used to screen a cDNA library prepared from adult Schistosoma mansoni . Of 1.8 x 10(5) recombinants screened, 34 clones were specifically immunoreactive with the antisera . Twenty of the clones were subcloned for further analysis, and 13 of the 20 were demonstrated to hybridize to a cDNA probe encoding schistosome tropomyosin I (pSMTM) at high stringency (Xu et al . Experimental Parasitology 69, 373-392, 1989) . One partial cDNA clone (pSM4), which failed to hybridize to pSMTM, was used to rescreen the adult worm cDNA library and a 1381-bp cDNA clone, SmTMII, was identified and characterized . SmTMII contained a single 284-amino acid open reading frame and was shown to be 65.8% homologous to SMTM . Computer-assisted analysis of the predicted protein structure depicted a hydrophilic molecule with the extensive alpha helical secondary structure characteristic of tropomyosins . SmTMII was expressed in Escherichia coli and the resulting 45-kDa fusion protein was recognized by both a polyclonal antiserum specific for SMTM and the antiserum of mice chronically infected with S . mansoni . Northern analysis showed the SmTMII mRNA in the adult stage to be about 1.6 kb in size . Analysis of the SmTMII gene by Southern blot revealed a complex hybridization pattern suggesting the presence of introns or multiple gene copies . The 650-bp EcoRI fragment of SmTMII was used to screen a cDNA library prepared from uninfected Biomphalaria glabrata and two clones, each encoding a novel snail tropomyosin, BgTMII, were isolated and characterized . The two clones, consisting of 2816 and 2314 bp, respectively, differed from each other only in the polyadenylation signal used and each contained a single and identical 284-amino acid open reading frame having 94.7% homology with the previously reported snail tropomyosin, BG39 (Dissous et al . Molecular and Biochemical Parasitology 43, 245-256, 1990) . BgTMII was shown to have a higher amino acid homology with SMTM (68.3%) than with BG39 (65.1%) and a higher amino acid homology with SMTM than the two schistosome tropomyosins (SMTM and SmTMII) have with each other (65.8%) . Implications of these observations in relation to the concept of host-parasite antigen mimicry are discussed. J Bacteriol, 1993 Jun, 175(12), 3767 - 75 Methanol oxidation genes in the marine methanotroph Methylomonas sp . strain A4; Waechter-Brulla D et al.; Methanol dehydrogenase has been purified from the type I marine methanotroph Methylomonas sp . strain A4 and found to be similar to other methanol dehydrogenase enzymes in subunit composition, molecular mass, and N-terminal sequence of the two subunits . A heterologous gene probe and a homologous oligonucleotide have been used to identify a DNA fragment from Methylomonas sp . strain A4 which contains moxF, the gene encoding the large subunit of methanol dehydrogenase . Protein expression experiments with Escherichia coli, immunoblotting of expression extracts, and partial DNA sequence determination have confirmed the presence of moxF on this DNA fragment . In addition, expression and immunoblot experiments have shown the presence of the genes for the small subunit of methanol dehydrogenase (moxI) and for the methanol dehydrogenase-specific cytochrome c (moxG) . The moxG gene product has been shown to be cytochrome c552 . The expression experiments have also shown that two other genes are present on this DNA fragment, and our evidence suggests that these are the homologs of moxJ and moxR, whose functions are unknown . Our data suggest that the order of these genes in Methylomonas sp . strain A4 is moxFJGIR, the same as in the facultative methylotrophs . The transcriptional start site for moxF was mapped . The sequence 5' to the transcriptional start does not resemble other promoter sequences, including the putative moxF promoter sequence of facultative methylotrophs . These results suggest that although the order of these genes and the N-terminal amino acid sequence of MoxF and MoxI are conserved between distantly related methylotrophs, the promoters for this gene cluster differ substantially. EMBO J, 1993 Jun, 12(6), 2483 - 94 The Escherichia coli FIS protein is not required for the activation of tyrT transcription on entry into exponential growth; Lazarus LR et al.; The Escherichia coli DNA bending protein factor for inversion stimulation (FIS), is neither necessary nor responsible for the stimulation of transcription from the wild type promoter for the tyrT operon (encoding a species of tyrosine tRNA) that occurs upon resumption of exponential growth . This conclusion is unexpected given that the regulatory element required for optimal transcription of tyrT contains three binding sites for FIS protein . In addition, it is in apparent conflict with reports from other laboratories which have described FIS-dependent activation of the stable RNA promoters rrnB P1 and thrU(tufB) in vivo . However, tyrT transcription is stimulated in a FIS-dependent manner both in vivo and in vitro when promoter function is impaired by mutation of the promoter itself or by the addition of the polymerase effector guanosine 5'-diphosphate 3'-diphosphate . These conditions, which expose a requirement for activation of stable RNA synthesis by FIS, suggest that FIS serves an adaptive role permitting high levels of stable RNA transcription on nutritional shift-up when RNA polymerase levels are depleted . In principle such a mechanism could confer a significant selective advantage thus accounting for the conservation of FIS binding sites in the regulatory regions of stable RNA promoters. Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 4902 - 6 Kinetics of p56lck and p60src Src homology 2 domain binding to tyrosine-phosphorylated peptides determined by a competition assay or surface plasmon resonance; Payne G et al.; Src homology 2 (SH2) domains are phosphotyrosine-binding modules found within various signal-transducing proteins . We have determined by 125I competition assay and surface plasmon resonance that the SH2 domains of Src and Lck bind to a variety of phosphopeptides with similar affinity and specificity . Both bound with highest affinity {Kd(app) approximately 3.7 nM; ka = 2.4 x 10(5) M-1 x s-1; kd = 1.2 x 10(-3) s-1} a phosphopeptide having a Tyr(P)-Glu-Glu-Ile motif found in the hamster polyomavirus middle-sized tumor antigen . Intermediate affinity (5- to 40-fold lower) was observed with phosphopeptides corresponding to the regulatory domains of Src and Lck, containing Tyr527 and Tyr505, respectively . Lowest affinity (80- to 300-fold lower) was observed with phosphopeptides corresponding to phosphorylated tyrosines of GTPase-activating protein, insulin receptor substrate 1, and SH2 domain-containing protein-tyrosine-phosphatase 1. Mutat Res, 1993 Jun, 291(3), 181 - 92 A plasmid system to monitor gene conversion and reciprocal recombination in vitro; Oppliger T et al.; A plasmid system allowing for the detection of recombinagenic activities in cell-free extracts is described . Two truncated alleles of the bacterial neomycin resistance gene (neo), differing from each other at a polymorphic restriction site, were constructed . Recombinations involving both alleles mediated by Drosophila embryo nuclear protein extracts or Drosophila larva whole cell protein extracts were selected by their ability to confer kanamycin resistance to E . coli . Restriction analysis of plasmids recovered from E . coli transformants allowed the monitoring of the two molecular mechanisms which can lead to functional neo genes, gene conversion and reciprocal recombination . A dose dependent increase in the recombination frequency with increasing amounts of cell extract was observed . Recombination was further increased by linearizing one of the two substrate plasmids . The Drosophila cell extracts catalyzed recombination in vitro since after incubation a recombination product could be identified by polymerase chain reaction (PCR) technology . The recombination was absolutely dependent on the presence of an active cell extract, since no diagnostic PCR product was detected in a reaction where extract was omitted . Analysis of a representative number of recombinant plasmids by restriction analysis revealed that in the absence of an exogenous recombinational system less than 2% of kanamycin resistant recombinant plasmids occurred by gene conversion upon transformation into E . coli . In contrast, recombinants exhibiting restriction patterns diagnostic for gene conversion were observed at frequencies between 5.1% and 9.8% after incubation with Drosophila larva cell extracts . These results strongly argued that gene conversion is a prominent mechanism of recombination in Drosophila mitotic cells. Biochemistry, 1993 Jun 1, 32(21), 5508 - 17 A large deletion in the connection subdomain of murine leukemia virus reverse transcriptase or replacement of the RNase H domain with Escherichia coli RNase H results in altered polymerase and RNase H activities; Post K et al.; The functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT) was investigated by studying the activities of AKR murine leukemia virus (MuLV) enzymes . In addition to the wild type, an RNase H-minus RT missing the entire RNase H domain and two other mutants having abnormal polymerase:RNase H ratios were expressed . These mutants include (i) a chimeric protein in which the MuLV RNase H domain was replaced by the entire Escherichia coli RNase H sequence and (ii) an RT with a 126 amino acid deletion in a region analogous to the "connection" subdomain in the p66 subunit of human immunodeficiency virus type 1 RT (Kohlstaedt, L . A., Wang, J., Friedman, J . M., Rice, P . A., & Steitz, T . A . (1992) Science 256, 1783-1790) . With the wild-type RT, the major RNase H cleavage reaction was coordinated with DNA synthesis and occurred at a position corresponding to 15 nucleotides from the 3'-terminus of the DNA primer . Additional cleavages closer to the 5'-end of the RNA were explained in terms of a model relating binding of the RNA.DNA hybrid substrate and enzyme structure . The chimeric RT behaved like E . coli RNase H, exhibited 300-fold higher RNase H activity than wild-type RT, and was limited in its ability to synthesize DNA . Qualitative and quantitative changes in the polymerase and RNase H activities of the deletion mutant were also observed . The RNase H domain appeared to function independently of the polymerase domain, supporting the idea that the proper spatial relationship between the two active centers was disrupted by the mutation . Taken together, our results indicate that alteration of the normal polymerase:RNase H ratio can have profound effects on both polymerase and RNase H cleavage activities, as expected for an enzyme with two interdependent domains. Oncogene, 1993 Jun, 8(6), 1477 - 85 Identification of a human guanine nucleotide-releasing factor (H-GRF55) specific for Ras proteins; Schweighoffer F et al.; A critical step in the activation of cellular Ras is the release of bound GDP . Oligonucleotide primers derived from a mouse cDNA sequence homologous to the Saccharomyces cerevisiae CDC25 gene product were used to screen a human brain cDNA library . The cloning led to the isolation of a 2.8-kb cDNA predicted to encode a protein of 488 amino acids . This protein was produced in Escherichia coli as a glutathione S-transferase fusion protein and functioned in vitro as a specific guanine nucleotide-releasing factor . Polyclonal antibodies raised against the last 281 amino acids of the protein allowed a protein in the molecular weight range of 55 kDa to be identified in human cortex homogenates . Analysis by Northern blotting led to the identification of a 5.5-kb mRNA in brain poly(A)+ RNA . The functionality of the encoded protein was evaluated after expression in different cells: (i) in Saccharomyces cerevisiae the effects of the cdc25.5 and RAS2 Ala-22 mutations were reversed; (ii) in chinese hamster ovary cells, a RAS-responsive element was transactivated as demonstrated by the expression of a CAT reporter gene under the control of the polyomavirus enhancer . Finally, in situ hybridization on of human chromosomes revealed a localization on band 15q2.4. J Virol, 1993 Jun, 67(6), 3696 - 701 Hybrid hepatitis B virus nucleocapsid bearing an immunodominant region from hepatitis B virus surface antigen; Borisova G et al.; A hepatitis B core antigen (HBcAg) gene bearing the 39-amino-acid-long domain A of hepatitis B surface antigen (HBsAg) within the HBcAg immunodominant loop has been constructed and expressed in Escherichia coli . Chimeric capsids demonstrated HBs but not HBc antigenicity and elicited in mice B-cell and T-cell responses against native HBcAg and HBsAg. J Virol, 1993 Jun, 67(6), 3489 - 96 Mutational analysis of the envelope gene of Moloney murine leukemia virus; Gray KD et al.; The env gene products of Moloney murine leukemia virus are required for binding and entry of the virus into the target cell . Thirty-three linker insertion mutations were constructed throughout the env gene of Moloney murine leukemia virus . Twenty of the mutations were located in the surface protein (SU), and the remaining thirteen were located in the transmembrane protein (TM) . The viability of the viruses containing these env gene mutations was determined by performing transient transfections and screening for the release of reverse transcriptase . Eleven viable mutants were isolated, nine in SU and two in TM . Three of the viable mutants were temperature sensitive . Four of the viable mutants were clustered in the carboxy terminus of SU . The env gene products of transfected cell lines which produced viable virus were analyzed . Our results indicated two regions of SU important for the stability of the SU/TM heteropolymer and one region important for the interaction of the env gene products with the viral core. Mutat Res, 1993 Jun, 294(1), 43 - 50 Enzymatic recognition and biological effects of DNA damage induced by 3-carbethoxypsoralen plus UVA; Boiteux S et al.; The specific recognition of DNA modifications by repair endonucleases was used to characterize damage induced by 3-carbethoxypsoralen (3-CPs) plus UvA in M13mp8 replicative form I (RF-I) DNA . Under the conditions used, 3-CPs plus UVA generates DNA base modifications which are recognized by the UvrABC complex and the Fpg protein of E . coli . The rate of formation of UvrABC sensitive sites is 3-4-fold higher than that of Fpg sensitive sites . In addition a small number of sites of base loss (sensitive to Nfo protein) were observed . M13mp8 RF-I DNA treated with 3-CPs plus UVA was tested for transfection efficiency in E . coli mutants defective in either Fpg protein and/or UvrABC complex . The survival of 3-CPs plus UVA damaged M13mp8 RF-I DNA was significantly reduced when transfected into uvrA mutants compared to that in the wild-type strain . On the other hand, the survival of 3-CPs plus UVA damaged RF-I DNA was not altered in fpg-1 mutants . These results show that nucleotide excision repair mediated by the UvrABC complex is the major repair pathway involved in the elimination of lethal lesions induced in DNA by 3-CPs plus UVA . Our data suggest that in vitro exposure of M13mp8 RF-I DNA to 3-CPs plus UVA produces predominantly thymine photoaddition and to a lesser extent guanine photooxidation partially due to singlet oxygen generated during photoreaction . The photoaddition products are primarly responsible for the observed lethal effect. Mutat Res, 1993 Jun, 294(1), 1 - 8 The level of GC-->AT transitions induced by MMS is not affected by the adaptive response in Escherichia coli K12; Sledziewska-Gojska E; I have examined the effect of the adaptive response on the frequency of MMS-induced umuC-dependent AT-->TA transversions and umuC-independent, GC-->AT transitions . It was found that the induction of the adaptive response causes a moderate decrease (50-60%) in the frequency of AT-->TA transversions and surprisingly has no effect on the level of GC-->AT transitions . In contrast, a dramatic decrease in MNNG-induced mutations has been observed in adapted cultures . However, this effect was completely abolished by MMS pretreatment of adapted cells before MNNG challenge . A mechanism for the MMS interference with the repair of MNNG-induced mutations in adapted cells is proposed. Eur J Neurosci, 1993 Jun 1, 5(6), 624 - 32 The proximal region of the MBP gene promoter is sufficient to induce oligodendroglial-specific expression in transgenic mice; Goujet-Zalc C et al.; To characterize regulatory DNA sequences involved in oligodendroglial expression of myelin basic protein (MBP), transgenic mice carrying a 256 bp fragment of the mouse MBP promoter fused to an Escherichia coli lacZ gene were generated . Of four transgenic families, two (lines 2 and 4) expressed beta-galactosidase activity in the nervous system but not in most other tissues . Histochemical and immunohistochemical analysis of adult brain from these two lines showed oligodendroglial-specific expression of the transgene . In line 2, only a small proportion of oligodendrocytes expressed the transgene, and in labelled cells the product of the enzymatic reaction with beta-galactosidase was confined to a small round vesicle in the vicinity of the nucleus . In contrast, in tissue sections from line 4 adult brain and spinal cord beta-galactosidase activity was much more intense and at least 80-90% of oligodendrocytes expressed the transgene . Detection of the MBP-lacZ transcript by in situ hybridization showed that the transgene mRNA was confined to the oligodendrocyte cell body . These results suggest that cis-acting regulatory elements, specifying oligodendrocytes identity, are located within 256 bp upstream from the MBP gene. Braz J Med Biol Res, 1993 Jun, 26(6), 591 - 603 Expression of the VP3-VP1 sequence of foot-and-mouth disease virus in Escherichia coli; Rossetti ML et al.; 1 . cDNA recombinants containing the VP3 and VP1 sequences of foot-and-mouth disease virus were isolated and the VP3-VP1 sequence was reconstructed . 2 . The reconstructed VP3-VP1 sequence was subcloned into expression vector pEX31b and a fusion protein of about 62,000 Da was expressed . 3 . When injected into mice, the fusion protein was able to elicit the production of antibodies that recognized viral VP1 and VP3 . 4 . Antibodies present in sera from mice immunized with VP3-VP1 protein did not neutralize the foot-and-mouth disease virus in vitro. Gene, 1993 May 30, 127(2), 215 - 9 Cloning and expression of the gene encoding Acacia confusa trypsin inhibitor that is active without post-translational proteolysis; Hung CH et al.; A recombinant plasmid containing the coding regions for Acacia confusa trypsin inhibitor (ACTI) has been constructed and expressed in Escherichia coli cells, as a fusion protein between ACTI and glutathione S-transferase (GST) . The GST-fusion was produced as a soluble protein which did not require denaturing agents such as urea to solubilize it . The recombinant ACTI (reACTI) was obtained by treating the GST-fusion protein with thrombin . Both the reACTI and fusion protein have a strong inhibitory effect on trypsin activity without post-translational proteolysis. Gene, 1993 May 30, 127(2), 193 - 7 Functional starch-binding domain of Aspergillus glucoamylase I in Escherichia coli; Kusnadi AR et al.; We have fused three starch-binding domains (SBD) encoding gene fragments (residues 511-616, 495-616 and 481-616) of glucoamylase I (GAI) to the 3' end of the Escherichia coli malE gene encoding maltose-binding protein (MBP) . The fusion proteins were produced in E . coli and were purified by chromatography on cross-linked amylose . Factor Xa digestion of the fusion proteins resulted in the release of functional SBD fragments which were separated from MBP on the basis of their differential binding to cross-linked amylose . The amino acid (aa) composition of the purified SBD fragments agreed with the respective amino acid compositions of GAI . The sizes of the SBD fragments were 11.9, 13.8 and 15.6 kDa, respectively. Gene, 1993 May 30, 127(2), 221 - 5 Nucleotide sequence and expression in Escherichia coli of cDNAs encoding papaya proteinase omega from Carica papaya; Revell DF et al.; We have cloned and sequenced two similar, but distinct, cDNAs from both fruit and leaf tissues of Carica papaya . The C-terminal portion of the predicted amino acid (aa) sequence of one of the clones has complete identity with the mature enzyme sequence of the cysteine proteinase papaya proteinase omega (Pp omega) . The second clone contains ten individual bp changes compared with the first and encodes a protein with three single-aa substitutions, only one of which is located in the mature sequence, but most noticeably carries an additional 19-aa C-terminal extension . The clones encode pre-pro precursor isoforms of Pp omega . The former of these clones has been expressed in Escherichia coli using a T7 polymerase expression system to produce insoluble pro-enzyme which has been solubilized and refolded to yield auto-activable pro-Pp omega. Biochem Biophys Res Commun, 1993 May 28, 193(1), 453 - 9 Binding activity of the human transcription factor TFIID; Icard-Liepkalns C; In order to investigate the conformational state of the human TFIID, we studied the structure of the TATA-box binding protein (TBP) which is the DNA-binding subunit of the transcription factor TFIID required for transcriptional initiation by RNA polymerase II . We showed that TBP was able to form dimers and tetramers by chemical crosslinking, subunit exchange, ultracentrifugation and gel shift experiment . These findings indicate that the TBP homodimers could be the inactive binding form of TFIID and therefore could explain the lack of Gal4-activated transcriptional activity of the E . coli-expressed human TBP. Biochem Biophys Res Commun, 1993 May 28, 193(1), 228 - 34 Sec-Y protein is localized in both the cytoplasmic and thylakoid membranes in the cyanobacterium Synechococcus PCC7942; Nakai M et al.; Members of the SecY protein family mediate protein export in bacterial cells . Southern analyses showed that secY is likely a single copy gene in the cyanobacterium Synechococcus PCC7942 . Then the subcellular location of the cyanobacterial SecY protein was determined; i) antiserum raised against a fusion protein between the SecY fragment and maltose binding protein were used for immunoblotting of the membrane fractions, and ii) a modified SecY protein carrying the c-Myc peptide tag was expressed in the cyanobacterial cells, and the subcellular distribution of the SecY-c-Myc fusion protein was analyzed with the anti-c-Myc antibodies . The obtained results suggest that the SecY protein is localized in the thylakoid membrane as well as the cytoplasmic membrane; the SecY protein probably mediates protein translocation across both the cytoplasmic and thylakoid membranes in Synechococcus PCC7942. Biochem Biophys Res Commun, 1993 May 28, 193(1), 155 - 60 Expression of bovine seminal ribonuclease in Escherichia coli; de Nigris M et al.; Bovine seminal RNAase (BS-RNAase), an unusually dimeric member of the pancreatic-like ribonuclease superfamily, is also a multifunctional biological effector, with antitumor, immunosuppressive, and antispermatogenic activities . We report here the cloning of a semi-synthetic cDNA coding for the protein subunit chain, its expression with a T7 expression system in Escherichia coli inclusion bodies, the dimerization of correctly reoxidized monomeric protein, followed by the purification in high yields of the recombinant enzyme, and by its conversion to a protein undistinguishable from BS-RNAase as isolated from seminal vesicles, both in its catalytic activity and in the micro-heterogeneity of its quaternary structure. Brain Res, 1993 May 28, 612(1-2), 165 - 71 Interleukin-6 and tumor necrosis factor in cerebrospinal fluid: changes during pyrogen fever; Coceani F et al.; Several peptides (cytokines), viz., interleukin-1 (IL-1), interferon-alpha (IFN-alpha), interleukin-6 (IL-6), tumor necrosis factor (TNF), are formed in response to conditions causing tissue inflammation or damage and are implicated in reactive changes of the host, including fever, while IL-1 has been considered an important mediator of fever, the other cytokines, specifically IL-6 and TNF, have recently acquired prominence . The present study extends earlier research on IL-1 and addresses the question of the role of IL-6 and TNF in the genesis of fever . Experiments were conducted in the conscious cat, and IL-6 and TNF were assayed concomitantly in cerebrospinal fluid (CSF) from the third ventricle using specific bioassays . In the absence of fever, IL-6 was usually below the threshold of the assay (4-32 pg/ml), while TNF appeared measurable (424 +/- 57 pg/ml) in most experiments . A single intravenous injection of endotoxin (bolus) or continuous infusion of IL-1 at doses eliciting a sustained fever increased CSF levels of IL-6, but had no effect on concentrations of TNF . Intracerebroventricular injection of a pyrogenic dose of endotoxin led to an elevation of TNF and IL-6 and, in either case, the effect was manifest during the latent period before the fever . In addition, by the same route, IL-1 caused a rise in IL-6 . We conclude that brain is intrinsically capable of producing both IL-6 and TNF depending on the site of challenge . However, since IL-6 CSF levels are elevated regardless of the site of pyrogen injection, IL-6 lends itself better to a role in the pathogenesis of fever. Nucleic Acids Res, 1993 May 25, 21(10), 2459 - 64 The DNA binding affinity of HhaI methylase is increased by a single amino acid substitution in the catalytic center; Mi S et al.; The HhaI methyltransferase recognizes the sequence GCGC and transfers a methyl group to C5 of the first cytosine residue . All m5C-methyltransferases contain a highly conserved sequence motif called the P-C motif . The cysteine residue of this motif is involved in catalysis by forming a covalent bond with the 6-position of cytosine prior to methyl group transfer . For the EcoRII methyltransferase, it has been shown that substitution of this catalytic cysteine by glycine is cytotoxic to E.coli cells expressing the mutant methyltransferase (Wyszynski et al . Nucl . Acids Res . 20: 319, 1992) . We now show that this observation can be extended to the HhaI system and suggest that the cytotoxicity is due to abnormally tight DNA binding by the mutant methyltransferase, which probably interferes with replication or transcription. Nucleic Acids Res, 1993 May 25, 21(10), 2423 - 7 African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases; Yanez RJ et al.; A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli . After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced . The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases . The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit . Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV . ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication. Nucleic Acids Res, 1993 May 25, 21(10), 2363 - 7 DNA sequence determinants of LexA-induced DNA bending; Lloubes R et al.; The LexA repressor from Escherichia coli induces DNA bending upon interaction with the two overlapping operators which regulate the transcription of the colicin A encoding gene caa . Both caa operators harbor T-tracts adjacent to their recognition motifs . These tracts have been suggested to be especially favorable for the promotion of LexA-induced DNA bending . Here we show that this is indeed the case, since disruption of the TTTT-tract adjacent to operator O1 by the replacement of the two central thymine bases by AA, GA or CG markedly reduces LexA-induced DNA bending . Simple A.T-richness in this position is thus not sufficient to promote full LexA-induced bending, albeit a TAAT sequence is always more efficient to promote bending than those sequences containing one or two C/G base pairs. Nucleic Acids Res, 1993 May 25, 21(10), 2309 - 13 Gene structure and expression of the MboI restriction--modification system; Ueno T et al.; The genes from Moraxella bovis encoding the MboI restriction--modification system were cloned and expressed in Escherichia coli . Three open reading frames were found in the sequence containing the genes . These genes, which we named mboA, mboB, and mboC, had the same orientation in the genome . Genes mboA and mboC encoded MboI methyltransferases (named M.MboA and M.MboC) with 294 and 273 amino acid residues, respectively . The mboB gene coded for MboI restriction endonuclease (R.MboI) with 280 amino acid residues . Recombinant E.coli-MBOI, which contained the whole MboI system, overproduced R.MboI . R.MboI activity from E.coli-MBOI was 480-fold that of M.bovis . The amino acid sequences deduced from these genes were compared with those of other restriction--modification systems . The protein sequences of the MboI system had 38-49% homology with those of the DpnII system. Biochemistry, 1993 May 25, 32(20), 5455 - 65 Reversible electrochemistry of fumarate reductase immobilized on an electrode surface . Direct voltammetric observations of redox centers and their participation in rapid catalytic electron transport; Sucheta A et al.; Fumarate reductase (Escherichia coli) can be immobilized in an extremely electroactive state at an electrode, with retention of native catalytic properties . The membrane-extrinsic FrdAB component adsorbs to monolayer coverage at edge-oriented pyrolytic graphite and catalyzes reduction of fumarate or oxidation of succinate, depending upon the electrode potential . In the absence of substrates, reversible redox transformations of centers in the enzyme are observed by cyclic voltammetry . The major component of the voltammograms is a pair of narrow reduction and oxidation signals corresponding to a pH-sensitive couple with formal reduction potential E degree' = -48 mV vs SHE at pH 7.0 (25 degrees C) . This is assigned to two-electron reduction and oxidation of the active-site FAD . A redox couple with E degree' = -311 mV at pH 7 is assigned to center 2 ({4Fe-4S}2+/1+) . Voltammograms for fumarate reduction at 25 degrees C, measured with a rotating-disk electrode, show high catalytic activity without the low-potential switch-off that is observed for the related enzyme succinate dehydrogenase . The catalytic electrochemistry is interpreted in terms of a basic model incorporating mass transport of substrate, interfacial electron transfer, and intrinsic kinetic properties of the enzyme, each of these becoming a rate-determining factor under certain conditions . Electrochemical reversibility is approached under conditions of low turnover rate, for example, as the supply of substrate to the active site is limited . In this situation, electrocatalytic half-wave potentials, E1/2, are similar for oxidation of bulk succinate and reduction of bulk fumarate and coincide closely with the E degree' value assigned to the FAD . At 25 degrees C and pH 7, the apparent KM for fumarate reduction is 0.16 mM, and kcat is 840 s-1 . Accordingly the second-order rate constant, kcat/KM, is 5.3 x 10(6) M-1 s-1 . Under the same conditions, oxidation of succinate is much slower . As the supply of fumarate to the enzyme is raised to increase turnover, the electrochemical reaction eventually becomes limited by the rate of electron transfer from the electrode . Under these conditions a second catalytic wave becomes evident, the E1/2 value of which corresponds to the reduction potential of the redox couple suggested to be center 2 . This small boost to the catalytic current indicates that the low-potential {4Fe-4S} cluster can function as a second center for relaying electrons to the FAD. Biochemistry, 1993 May 25, 32(20), 5442 - 7 Flow-flash study of the reaction between cytochrome bo and oxygen; Svensson M et al.; The reaction between reduced cytochrome bo from Escherichia coli and oxygen has been studied using flash photolysis of the CO complex of the reduced protein after rapid mixing with oxygen . Absorbance changes were monitored in the alpha and Soret spectral regions . Two kinetic phases taking place at catalytically competent rates could be detected . The apparent rate constant obtained for both the first and second phase showed a hyperbolic dependence on the oxygen concentration . For the first phase, we obtained limiting first- and second-order rate constants at saturating and low oxygen concentrations of 4.5 x 10(4) s-1 and 1.6 x 10(8) M-1 s-1, respectively . The corresponding values for the second phase were 5 x 10(3) s-1 and 1.7 x 10(7) M-1 s-1 . The first phase accounted for 30% of the total absorbance change in the Soret band (430 nm) and 15% of the total absorbance change in the alpha band (555 nm) . These reactions are followed by a very slow phase with a lifetime of about 1 s . We have also studied the interaction between the fully oxidized enzyme and hydrogen peroxide, and we have found that peroxide binding induces an absorbance increase in the alpha band and a red shift of the Soret band . A consideration of the magnitude of the absorbance changes taking place during the first phase suggests that this reaction includes at least partial oxidation of the low-spin cytochrome b. Biochemistry, 1993 May 25, 32(20), 5419 - 24 Exchange, efflux, and substrate binding by cysteine mutants of the lactose permease of Escherichia coli; van Iwaarden PR et al.; In this study, wild-type lac permease and lac permease mutated at each of the eight cysteinyl residues in the molecule were solubilized from the membrane, purified, and reconstituted into proteoliposomes . Lactose equilibrium exchange and efflux activities of mutants with Ser in place of Cys117, Cys176, Cys234, Cys333, Cys353, or Cys355 are essentially the same as wild-type permease . In contrast, mutants in Cys148 and Cys154 exhibit diminished exchange and efflux activities . These mutants in Cys148 and Cys154, except for the C148S mutant, have previously been shown to slow down active transport as well {Van Iwaarden, P.R., Driessen, A . J . M., Menick, D . R., Kaback, H.R., & Konings, W . N . (1991) J . Biol . Chem . 266, 15688-15692} . C148S permease shows monophasic kinetics with a high apparent KM with respect to external lactose in the exchange reaction under nonequilibrium conditions, whereas wild-type permease exhibits biphasic kinetics with both a high and low KM component . Moreover, the absence of the low Km pathway in the C148S permease is correlated with the absence of a high-affinity binding site for p-nitrophenyl alpha-D-galactopyranoside (NPG) . Interestingly, the affinity of the permease for NPG appears to increase with the hydrophobicity of the side chain at position 154 (Ser < Cys < Gly < Val) . Finally, the presence of a high-affinity binding site for NPG in C154V is consistent with the biphasic exchange kinetics exhibited by this mutant . The results are discussed in the context of a model in which lac permease has two substrate binding sites, a catalytic site and a regulatory site. Biochemistry, 1993 May 25, 32(20), 5282 - 90 Structure and stability of an early folding intermediate of Escherichia coli trp aporepressor measured by far-UV stopped-flow circular dichroism and 8-anilino-1-naphthalene sulfonate binding; Mann CJ et al.; The refolding kinetics of Escherichia coli trp aporepressor were monitored using stopped-flow far-ultraviolet circular dichroism and 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy . Significant gains in secondary structure and the development of hydrophobic surface, respectively, were observed within the dead time of mixing (4-5 ms) . These initial increases, or burst phase amplitudes, plotted as a function of final urea concentration, exhibited sigmoidal, coincident unfolding transition curves . The transition curves were fit to a two-state model, and the resulting free energies of folding in the absence of denaturant were found to be similar (approximately 3.3 kcal/mol) . Three subsequent slow refolding phases exhibited relaxation times and amplitudes similar to those previously observed for tryptophan fluorescence {Gittelman, M . S., & Matthews, C . R . (1990) Biochemistry 29, 7011-7021} . These results support the proposals that a stable, monomeric intermediate is rapidly formed during the folding of trp aporepressor and that this species contains a significant amount of secondary structure and hydrophobic surface . This early intermediate is then processed through folding and association reactions that result in the formation of the remaining secondary, tertiary, and quaternary structure. Biochemistry, 1993 May 25, 32(20), 5273 - 81 Multiple magnesium ions in the ribonuclease P reaction mechanism; Smith D et al.; The ribozyme ribonuclease (RNase) P cleaves precursor transcripts to produce the mature 5'-end of tRNAs . This hydrolysis reaction has a divalent cation requirement that is primarily catalytic, rather than structural; RNase P can be considered a metalloenzyme . Kinetic analysis shows that the RNase P catalytic mechanism has a cooperative dependence upon Mg2+ concentration . At least three Mg2+ ions are required for optimal activity, suggesting a multiple metal ion mechanism . The 2'-OH at the site of substrate cleavage may act as a ligand for a catalytically important Mg2+: deoxyribose substitution reduces the apparent number of Mg2+ bound from three to two and increases the apparent dissociation constant for Mg2+ from the micromolar to the millimolar range . In addition to these cation effects, the deoxyribose substitution reduces the rate of catalysis by 3400-fold; substitution with 2'-O-methyl at the cleavage site reduces the catalytic rate 10(6)-fold . If we presume no significant conformational effects of the substitutions, these results suggest that the 2'-OH serves as hydrogen-bond donor . The kinetic analysis of the catalytic mechanism is based upon the characterization of the pH dependence of the reaction . There is a hyperbolic (saturable) dependence on hydroxide concentration, with the half-maximal rate achieved at pH 8.0-8.5 . The rate of the cleavage step is about 200 min-1 at pH 8.0, which is 500-fold faster than the steady-state parameter kcat. J Biol Chem, 1993 May 25, 268(15), 11143 - 51 Preparation and characterization of a set of deoxyoligonucleotide 49-mers containing site-specific cis-syn, trans-syn-I, (6-4), and Dewar photoproducts of thymidylyl(3'-->5')-thymidine; Smith CA et al.; Deoxyoligonucleotide 49-mers containing a central cis-syn, trans-syn-I, (6-4), or Dewar photoproduct of TpT were constructed for use in repair and replication studies by ligation of shorter photoproduct-containing oligonucleotides . A (6-4) product-containing 6-mer was prepared in 3.4% yield by 254 nm irradiation of d(AATTAA) and converted in nearly quantitative yield to the Dewar isomer by irradiation with Pyrex- and Mylar-filtered medium-pressure mercury arc light . d(CGAATTAAGC) containing a site-specific cis-syn or trans-syn-I dimer was prepared via automated solid-phase DNA synthesis utilizing photoproduct building blocks . The photoproduct-containing 49-mers were characterized by their susceptibility to base cleavage and a number of enzymes routinely used to map the sites of DNA photoproduct formation . 1 M piperidine at 90 degrees C cleaved the Dewar product faster than the (6-4) product, but did not cleave the cyclobutane dimers . The 3'-->5' exonuclease activity of T4 DNA polymerase was completely blocked by all the lesions except the (6-4) product, which it slowly bypassed . T4 endonuclease V did not cleave the (6-4) or Dewar photoproduct, but unexpectedly cleaved the trans-syn-I dimer at most 1% the rate of the cis-syn dimer in double-stranded DNA . The trans-syn-I dimer was cleaved at a 50-fold higher rate in double- than in single-stranded DNA . Escherichia coli photolyase was found to be specific for the cis-syn dimer at low concentrations . Implications of this work to methodology for mapping and quantifying DNA photoproducts are also discussed. J Biol Chem, 1993 May 25, 268(15), 11028 - 40 Mutational analysis of the human DNA polymerase alpha . The most conserved region in alpha-like DNA polymerases is involved in metal-specific catalysis; Copeland WC et al.; Five site-directed mutations were introduced at the most conserved amino acids in region I (YGDTDS) of the human DNA polymerase alpha catalytic subunit . Mutant proteins were expressed in the baculovirus system by an improved method and purified by a rapid one-step purification in high yield and high specific activity . The Asp1004 to Asn mutation produced a protein with no detectable polymerase activity while other mutations gave activities from 1 to 20% of the wild type polymerase activity . Steady state kinetic analysis of the active mutants indicates that none of the mutations caused a change in Km(dNTP) or KD(DNA), but all active mutants showed a decrease in kcat . Thus, the effect of these conserved mutations is manifest in altered rates of catalysis . Two mutations, Asp1002 to Asn and Thr1003 to Ser, caused the enzyme to utilize Mn2+ more effectively in catalysis than Mg2+, suggesting that these amino acids are involved in metal binding . Rates of catalysis by the D1002N and T1003S mutants, as well as Y1000F mutant were improved 80-, 30-, and 70-fold, respectively, on homopolymer templates when Mn2+ replaced Mg2+ as the activator metal . The results from these mutational studies suggest that this highly conserved region binds the metal which is essential for catalysis . The Asp1002 may participate directly in chelating the metal . Results from the T1003S mutant suggest that the beta-methyl group of the threonine side chain might be locked in a hydrophobic pocket preventing free rotation around the C alpha-C beta bond, thus positioning the Thr1003 hydroxyl group to form a crucial bond with the metal ion . In addition, D1002N and T1003S displayed a 20-fold resistance to aphidicolin compared to the wild type polymerase alpha, and all of the active mutants displayed altered sensitivity to butylphenyl-dGTP . Models of the involvement of region I in catalysis and aphidicolin interaction are proposed . The mutational studies presented in this report will serve as a prototype for the functional role of region I in catalysis for all alpha-like DNA polymerases. J Biol Chem, 1993 May 25, 268(15), 10914 - 9 Interaction of GTPase activating proteins (GAPs) with p21ras measured by a novel fluorescence anisotropy method . Essential role of Arg-903 of GAP in activation of GTP hydrolysis on p21ras; Brownbridge GG et al.; Ras GTPase activating proteins (GAPs) contain an invariant motif, -FLR-, within the most conserved region of their catalytic domains . Certain mutations in this motif have greatly reduced activity (Skinner, R . H., Bradley, S., Brown, A . L., Johnson, N . J., Rhodes, S., Stammers, D . K., and Lowe, P . N . (1991) J . Biol . Chem . 266, 14163-14166), but it was not determined whether the reduced activity was due to loss of binding or impaired catalysis . In order to address this question, we have developed a simple physical method to study formation of GAP.p21ras complexes . This utilizes the increase of fluorescence anisotropy upon binding of GAP to p21ras complexed with 2'(3')-O-(N-methylanthraniloyl) (mant) derivatives of guanine nucleotides . Dissociation constants obtained for the catalytic domains of either p120-GAP (GAP-344) or neurofibromin (NF1-GRD) with normal and Leu-61 p21ras proteins are comparable with those obtained by kinetic methods . In the course of these studies, we found, in contrast to previous observations, that both GAP and NF1-GRD can weakly activate the GTPase of Leu-61 mutant p21, showing that Gln-61 is not absolutely required for the stimulation of GTPase activity by GAPs . The fluorescence anisotropy method allowed us to show that mutation of Arg-903, within the FLR motif of GAP, can result in protein defective in catalysis but not in binding to p21ras . These data suggest a direct role for this residue in catalyzing GTP hydrolysis on p21ras, possibly by contributing a catalytic group to the p21 active site . This method is independent of the catalytic activity of the proteins, and so it could be extended generally to the measurement of binding of effector molecules, exchange factors, or other macromolecules to guanine nucleotide-binding proteins. J Biol Chem, 1993 May 25, 268(15), 10826 - 35 Differential usage of the carboxyl-terminal region among aldolase isozymes; Berthiaume L et al.; Sequence homology among nonconserved residues 357-362 of the COOH-terminal region in fructose-1,6-bisphosphate aldolases correlates with isozyme classification of aldolases . Recombinant chimers of human liver and maize aldolases were constructed by exchanging residues 357-362 with those from muscle, maize, and liver isozyme and by insertion in the maize sequence at position 349 rabbit muscle and liver residues 346-349 . Activity variation among the chimers relative to native controls ranged from less than 10% to greater than 300% of Vm . Exchange of residues 357-362 significantly affected both Vm and Km without modifying catalytic efficiency kcat/Km, whereas insertion of residues 346-349 modified Vm and Km and increased catalytic efficiency . Steady state carbanion oxidation rates varied inversely with activity and were differentially affected with respect to equilibrium oxidation rates . Sequence exchange of residues 357-362 appears to modulate carbanion proton exchange, whereas sequence insertion of residues 346-349 modifies substrate and aldehyde interaction with C6 phosphate binding locus . Low intrinsic susceptibility to carboxypeptidase A degradation of the COOH terminus in liver aldolase is consistent with tight association of this COOH terminus in a conformation unfavorable for promoting high catalytic activity . Efficient carbanion protonation promoted by specific sequences 357-362 represents a mechanistic feature which distinguishes catalytically active maize and muscle isozymes from less active liver isozyme . Conservation of active site residues among aldolases suggests that isozyme diversity among aldolases arose from divergent evolution of the COOH-terminal sequence. J Biol Chem, 1993 May 25, 268(15), 10760 - 5 Refolding of luciferase subunits from urea and assembly of the active heterodimer . Evidence for folding intermediates that precede and follow the dimerization step on the pathway to the active form of the enzyme; Ziegler MM et al.; Conditions have been established that allow reversible refolding of luciferase from 5 M urea . The kinetics of formation of the active enzyme showed a concentration-independent lag, suggesting the existence of intermediate structures on the pathway of refolding . The rate of approach to the final level of activity was strongly concentration-dependent at protein concentrations below 10 micrograms/ml, but at concentrations above about 20 micrograms/ml, the rate of approach to the final activity value did not change with concentration . The concentration dependence presumably reflects the second-order step yielding the heterodimeric structure . The finding that at concentrations above 20 micrograms/ml, the rate becomes insensitive to concentration suggests that under these conditions, some step subsequent to dimerization become rate-limiting . When the refolding reaction was initiated by dilution out of 5 M urea at 50 micrograms/ml followed at various times by a secondary dilution to a final concentration of 5 micrograms/ml, it was found that the increase in activity continued at the rate characteristic of the higher protein concentration for a period of about 1-2 min following the dilution before slowing to the rate expected for the lower protein concentration . These observations indicate that there are inactive heterodimeric species that form from assembly of the individual subunits and that these species must undergo further folding to yield the active heterodimeric species . At protein concentrations of 5-50 micrograms/ml, the final yield of active enzyme was about 65-85%, decreasing at higher and lower concentrations . At higher concentrations, aggregation probably accounts for the limit in recovery, whereas at lower concentrations, it appears that the reduced yield of activity is due to the competing process of the folding of one or both individual subunits into some form incompetent to interact with each other . These experiments demonstrate the existence of slow steps in the refolding of luciferase subunits from urea and the formation of the active heterodimeric structure, both preceding and following the dimerization . Furthermore, the failure of protein at low concentrations to efficiently reassemble into the active heterodimer is consistent with the prior finding that luciferase subunits produced independently in Escherichia coli fold into conformations that cannot interact to form the active heterodimer upin mixing (Waddle, J . J., Johnston, T . C., and Baldwin, T . O . (1987) Biochemistry 26, 4917-4921). Biochemistry, 1993 May 25, 32(20), 5431 - 5 Redesign of the interior hydrophilic region of mitochondrial cytochrome c by site-directed mutagenesis; Davies AM et al.; Heme propionate-7 in cytochrome c is an ionizable group located in a region of the protein that is inaccessible to bulk solvent . Electrostatic stabilization of this functional group appears to be achieved through interaction of heme propionate-7 with several amino acid residues that occur within hydrogen-bonding distance of it . To investigate the functional and spectroscopic roles of the amino acid residues that contribute to the immediate environment of heme propionate-7, the following variant forms of yeast (Saccharomyces cerevisiae) cytochrome c have been prepared and characterized by electrochemical and spectrochemical analyses: Arg38Ala, Tyr48Phe, Ala38Phe, Tyr48Phe/Trp59Phe, and Arg38Ala/Tyr48Phe/Trp59Phe . For each protein, the dependence of midpoint reduction potential and NMR spectrum on pH was determined, and the UV (250-450 nm) circular dichroic (CD) spectrum was measured . All of the variant proteins exhibited decreased reduction potentials with the greatest difference (-65 to -70 mV) exhibited by the multiply mutated proteins . The electrostatic properties of the variant proteins as reflected by the oxidation-state dependence of the His-39 pKa value were similar to those of the wild-type protein . Previous indirect assignments of minima in the CD spectrum of cytochrome c at 282 and 289 nm to Trp-59 are confirmed by spectra of the variant cytochromes in which this residue is replaced by Phe . The present results establish that the electrochemical effects of eliminating hydrogen-bonding interactions with heme propionate-7 are not additive and that the functional modulation of cytochrome c through regulation of the heme propionate-7 dielectric environment involves a complex combination of solvation effects and electrostatic or hydrogen-bonding interactions. J Biol Chem, 1993 May 25, 268(15), 11435 - 9 Cloning and characterization of two distinct human extracellular signal-regulated kinase activator kinases, MEK1 and MEK2; Zheng CF et al.; Mitogen-induced signal transduction is mediated by a cascade of protein phosphorylation and dephosphorylation . One of the immediate responses of mitogen stimulation is the activation of a family of protein kinases known as mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK) . MEK (MAP kinase or ERK kinase) is the immediate upstream activator kinase of ERK . Two cDNAs, MEK1 and MEK2, were cloned and sequenced . MEK1 and MEK2 encode 393 and 400 amino acid residues, respectively . The human MEK1 shares 99% amino acid sequence identity with the murine MEK1 and 80% with human MEK2 . Both MEK1 and MEK2 were expressed in Escherichia coli and shown to be able to activate recombinant human ERK1 in vitro . The purified MEK2 protein stimulated threonine and tyrosine phosphorylation on ERK1 and concomitantly activated ERK1 kinase activity more than 100-fold . The recombinant MEK2 showed lower activity as an ERK activator as compared with MEK purified from tissue . However, the recombinant MEK2 can be activated by serum-stimulated cell extract in vitro . MEKs, in a manner similar to ERKs, are likely to consist of a family of related proteins playing critical roles in signal transduction. J Biol Chem, 1993 May 25, 268(15), 10754 - 9 Phospholipase C-gamma 1 associates with viral and cellular src kinases; Nakanishi O et al.; A tyrosine kinase inhibitor, genistein, caused the subcellular translocation of phosphoinositide-specific phospholipase C (PLC) activity from membrane fractions to cytosolic fractions in rat 3Y1 fibroblasts and their transformants by Rous sarcoma virus, SR-3Y1 . The ratio of PLC activities associated with the membrane fractions to those of the homogenate fractions was greater in SR-3Y1 (32.6%) than in 3Y1 (20.8%) whereas membrane-associated PLC activities were strikingly reduced to the same levels in both cells by treatment with genistein . Moreover, it was found by immunoblotting analyses of membrane fractions that the amounts of PLC-gamma 1 isozyme were reduced to 20.4% of initial level in SR-3Y1 and to 30.2% of that in 3Y1 cells . While the levels of PLC-delta, another detectable PLC isozyme, were not altered by genistein suggesting that tyrosine kinase plays an important role in the association of PLC-gamma 1 with membranes . PLC-gamma 1 molecules were detected in anti-p60arc antibody immunoprecipitates of both 3Y1 and SR-3Y1 cells . The amounts of PLC-gamma 1 co-immunoprecipitating with src kinases were higher in SR-3Y1 than 3Y1 cells and were reduced in both cell types by treatment with genistein . In addition, it was confirmed that PLC-gamma 1 purified from rat liver was phosphorylated at a tyrosine residue and associated with viral src kinase and that src kinases associated with the recombinant SH2 region of PLC-gamma 1, expressed in Escherichia coli, depending upon phosphorylation of tyrosine residues . These findings suggest that both viral and cellular src kinases associate with PLC-gamma 1 and may mediate cellular signaling in normal and transformant cell growth. J Biol Chem, 1993 May 25, 268(15), 10946 - 52 Acetyl-CoA carboxylase from yeast is an essential enzyme and is regulated by factors that control phospholipid metabolism; Hasslacher M et al.; We have isolated a 1.2-kilobase pair cDNA fragment in a screening for yeast genes regulated at the level of transcription by soluble lipid precursors, inositol and choline . Sequence analysis and comparison of the deduced amino acid sequence to protein databases unveiled 68% similarity of a 374-amino acid peptide fragment to published C termini of chicken and rat acetyl-CoA carboxylase and almost 100% identity to the product of the FAS3 gene from yeast . Several lines of evidence confirm that the cloned gene represents the yeast structural gene ACC1 encoding acetyl-CoA carboxylase . Overexpression of the ACC1 gene from a high copy number plasmid resulted in overexpression of a 250-kDa biotin-enzyme and enzymatic activity of acetyl-CoA carboxylase . Disruption of one ACC1 allele in a diploid wild-type strain resulted in 50% reduction of ACC1-specific mRNA and acetyl-CoA carboxylase specific activity and a marked decrease of biotin associated with a 250-kDa protein, compared to wild-type . After sporulation of diploid disruptants, spores containing the disrupted acc1 allele failed to enter vegetative growth, despite fatty acid supplementation, suggesting that acetyl-CoA carboxylase activity is essential for a process other than de novo fatty acid synthesis and that only a single functional copy of the ACC1 gene exists . ACC1 transcription was repressed 3-fold by lipid precursors, inositol and choline, and was also controlled by regulatory factors Ino2p, Ino4p, and Opi1p, providing evidence that the key step of fatty acid synthesis is regulated in conjunction with phospholipid synthesis at the level of gene expression . The 5'-untranslated region of the ACC1 gene contains a sequence reminiscent of an inositol/choline-responsive element identified in genes encoding phospholipid biosynthetic enzymes. Nucleic Acids Res, 1993 May 25, 21(10), 2429 - 35 Domain I of 23S rRNA competes with a paused transcription complex for ribosomal protein L4 of Escherichia coli; Zengel JM et al.; Ribosomal protein L4 of Escherichia coli regulates expression of its own eleven gene S10 operon both by inhibiting translation and by stimulating premature termination of transcription . Both regulatory processes presumably involve L4 recognition of the S10 leader RNA . To help define L4's regulatory target, we have investigated the protein's cognate target on 23S rRNA . Binding of L4 to various fragments of the 23S rRNA was monitored by determining their ability to sequester L4 in an in vitro transcription system and thereby eliminate the protein's effect on transcription . Using this approach we identified a region of about 110 bases within domain I of 23S rRNA which binds L4 . A two base deletion within this region, close to the base to which L4 has been cross-linked in intact 50S subunits, eliminates L4 binding . These results also confirm the prediction of the autogenous control model, that L4 bound to its target on rRNA is not active in regulating transcription of the S10 operon. J Biol Chem, 1993 May 25, 268(15), 11449 - 55 Aldehyde dehydrogenase-derived omega-crystallins of squid and octopus . Specialization for lens expression; Zinovieva RD et al.; omega-Crystallin of the octopus lens is related to aldehyde dehydrogenases (ALDH) of vertebrates (Tomarev, S . I., Zinovieva, R . D., and Piatigorsky, J . (1991) J . Biol . Chem . 266, 24226-24231) and ALDH1/eta-crystallin of elephant shrews (Wistow, G., and Kim, H . (1991) J . Mol . Evol . 32, 262-269) . Only very low amounts of omega-crystallin are present in the squid lens . Here, we have cloned omega-crystallin cDNAs of the octopus (Octopus dofleini) and squid (Ommastrephes sloani pacificus) lenses . The deduced amino acid sequences of omega-crystallin from these species are 78% identical to each other, 56-58% identical to cytoplasmic ALDH1 and mitochondrial ALDH2 of vertebrates (which are 66-68% identical to each other), and 40% identical to Escherichia coli and spinach ALDHs . These data are consistent with the idea that the ALDH1/ALDH2 gene duplication in vertebrates occurred after divergence of cephalopods from the line giving rise to vertebrates, but before the separation of squid and octopus . Southern blot hybridization indicated that omega-crystallin is encoded by few genes (possibly just one) in octopus and squid . Northern blot hybridization revealed two bands (2.7 and 9.0 kilobases) of omega-crystallin RNA in the octopus lens and one band (4.2 kilobases) in the squid lens; omega-crystallin RNAs were undetectable in numerous non-lens tissues of octopus and squid, suggesting lens-specific expression of this gene(s) . Finally, extracts of the octopus lens had no detectable ALDH activity using different substrates, consistent with omega-crystallin having no enzymatic activity . Taken together, our results suggest that omega-crystallin evolved by duplication of an ancestral gene encoding ALDH and subsequently specialized for refraction in the transparent lens while losing ALDH activity and expression in other tissues. J Biol Chem, 1993 May 25, 268(15), 11409 - 16 Translational repression of endogenous thymidine kinase mRNA in differentiating and arresting mouse cells; Knofler M et al.; We observed that decline of thymidine kinase (TK) enzyme activity was severalfold faster than the decay of full length TK mRNA during growth arrest of 3T6 mouse fibroblasts or during differentiation of myoblasts (C2Cl12) or F9 embryonal carcinoma cells . In order to study the molecular mechanism of this disparate behavior, a polyclonal antiserum against mouse TK was raised in rabbit . High level expression of mouse TK polypeptide in Escherichia coli was achieved with a T7 RNA polymerase-directed expression system . Using the antiserum in immunoblotting, no indication for a pool of inactive enzyme was found during differentiation of F9 or growth arrest of 3T6 cells . Pulse labeling of these cells in vivo with {35S}methionine showed a more than 6-fold decrease in the rate of TK-protein synthesis of in F9 cells after 3 days of treatment with retinoic acid as well as in 3T6 cells after 16 h under low serum . This was not due to increased turnover of the protein as measured in pulse chase experiments . In addition, full length TK mRNA stayed associated with polysomes under these conditions in F9 as well as 3T6 cells . Taken together the results suggest that endogenous TK mRNA becomes translationally repressed under a variety of conditions when mouse cells cease to grow. J Biol Chem, 1993 May 25, 268(15), 11335 - 9 FK-506-binding protein: three-dimensional structure of the complex with the antagonist L-685,818; Becker JW et al.; L-685,818 differs only slightly in structure from the immunosuppressive drug FK-506, and both compounds bind with comparable affinity to the 12-kDa FK-506-binding protein (FKBP12), the major intracellular receptor for the drug . Despite these similarities, L-685,818 is a potent antagonist of both the immunosuppressive and toxic effects of the drug . Here, we present a structural analysis of this problem . Although FK-506 and L-685,818 differ greatly in pharmacology, we have found that the three-dimensional structures of their complexes with FKBP12 are essentially identical . Approximately half of each ligand is in contact with the receptor protein, and half is exposed to solvent; the exposed region includes the two sites where the compounds differ . These results indicate that the profound differences in the pharmacology of these two compounds are not caused by any difference in their interaction with FKBP12 . Rather, these effects arise because relatively minor changes in the exposed part of a bound ligand have a strong effect on how FKBP12-ligand complexes interact with calcineurin, their putative intracellular target . In addition, FK-506 complexes with FKBP12 proteins from several species all inhibit mammalian calcineurin . Analysis of the three-dimensional structure of the complex with respect to residues conserved among these proteins suggests a small number of surface residues near the bound ligands that may play a critical role in interactions between the protein-drug complex and calcineurin. J Biol Chem, 1993 May 25, 268(15), 10851 - 62 Characterization of RNA and DNA synthesis in Escherichia coli strains devoid of ppGpp; Hernandez VJ et al.; The synthesis rates of DNA, rRNA, bulk mRNA, protein, and RNA polymerase beta- and beta'-subunits were determined as functions of growth rate in a wild-type Escherichia coli strain, which produces guanosine tetraphosphate (ppGpp), and in a delta relA delta spoT mutant which does not produce ppGpp . The rate of stable RNA synthesis per amount of protein depends on three factors: RNA polymerase concentration, RNA polymerase activity, and the distribution of active RNA polymerase between stable and mRNA genes, measured as the stable RNA synthesis rate/total RNA synthesis rate, rs/rt . In the wild-type strain, all three factors increase with growth rate . In the ppGpp-deficient strains, only RNA polymerase synthesis and activity, but not rs/rt, increased with growth rate . Thus, adjustments of rs/rt require ppGpp . In the absence of ppGpp, the synthesis of rRNA and bulk mRNA both varied in direct proportion to the concentration of active RNA polymerase, in contrast to the wild-type strain, in which only rRNA synthesis increased with growth rate, while mRNA synthesis remained constant . Thus, a control specific for rRNA is absent in strains lacking ppGpp . In rich media, the ppGpp-deficient strain synthesized up to 4-fold more mRNA than wild-type bacteria, which was associated with a similarly increased RNA polymerase activity . We propose that RNA polymerase is rendered inactive in wild-type bacteria due to ppGpp-dependent transcriptional pausing during the synthesis of mRNA . Finally, the control of replication initiation was altered in ppGpp-less bacteria, apparently reflecting indirect changes in the cell physiology, rather than a direct effect of ppGpp on replication initiation. J Biol Chem, 1993 May 25, 268(15), 10802 - 7 Functional size analysis of F-ATPase from Escherichia coli by radiation inactivation; Ma JT et al.; A radiation inactivation technique was employed to determine the functional size of adenosine triphosphatase from Escherichia coli (EF0EF1-ATPase) . Functional units of the membrane-bound and the soluble ATPases were estimated to be 300 +/- 39 and 295 +/- 32 kDa, respectively . The presence of the free radical scavenger dithiothreitol was crucial in measuring the radiation inactivation size of ATPase . When gramicidin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were added, an increase in the functional mass of membrane-bound ATPase was observed . In contrast, valinomycin and KCl had hardly any effect on the functional size of ATPase . We also determined a functional unit of 355 +/- 33 kDa for proton translocation by a fluorescence quenching technique . A reconstitution study using irradiated coupling factor 1 (EF1)-depleted membrane revealed that the functional mass of the proton channel was 96 +/- 11 kDa . A similar functional size for ATP-Pi exchange and ATP hydrolysis implies that both reactions might utilize identical machinery . Furthermore, functional units of soluble EF1 for unisite (nonsteady state) and multisite (steady state) ATP hydrolysis were calculated as 200 +/- 32 and 298 +/- 32 kDa, respectively . A working hypothesis was proposed from radiation inactivation analysis to elucidate the structure and mechanism of F1-ATPase. J Biol Chem, 1993 May 25, 268(15), 10705 - 8 Evaluation of mutagenesis for epitope mapping . Structure of an antibody-protein antigen complex; Prasad L et al.; The location and description of epitopes on proteins describe the basis of immunological specificity . The 2.8-A structure of the phosphocarrier protein, HPr from Escherichia coli, complexed to the Fab fragment of the monoclonal antibody, Jel42, has been determined . This allows the first comparison of epitope predictions from extensive site-directed mutagenesis experiments, coupled with biological activity studies (Sharma, S., Georges, F., Klevit, R . E., Delbaere, L . T . J., Lee, J . S., and Waygood, E . B . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 4877-4881), with those from x-ray analysis . There are 14 amino acid residues of E . coli HPr that interact with the Jel42 antigen-binding site . Nine of these were correctly assigned by the mutagenesis studies . Of the 5 remaining residues, Met-1 could not be altered; two others appear to have critical roles in determining protein conformation; the other 2 residues have a minimal effect on antibody binding since they are located on the periphery of the epitope with one face of their side chains in van der Waals contact with the antibody and the other face in contact with solvent . Four residues were incorrectly assigned to the epitope . These residues were located adjacent to epitope residues that were likely perturbed by these mutations . This study demonstrates that mutations which caused greater than 10-fold changes in antibody binding affinity were correctly assigned to the epitope by the mutagenesis experiments . Guidelines are also presented in order to minimize incorrect assignments. J Chromatogr, 1993 May 21, 638(1), 9 - 19 Reversed-phase capillary high-performance liquid chromatography with on-line UV, fluorescence and electrospray ionization mass spectrometric detection in the analysis of peptides and proteins; Heath TG et al.; Analysis of peptide mixtures by reversed-phase capillary HPLC with gradient elution using three detectors in series: UV (214 nm), fluorescence (lambda exc . = 280 nm, lambda emiss . = 356 nm), and electrospray ionization mass spectrometry (ES-MS) is reported . The chromatographic integrity of the system and the detection limits were evaluated . The effect of the mass spectrometer's acquisition rate on the total ion current (TIC) profile was also examined . The utility of fluorescence monitoring with UV and ES-MS detection was demonstrated in the analysis of proteolytic digests of proteins . The native fluorescence character of tryptophan-containing peptides provides selectivity in peptide mapping, while monitoring UV absorption at 214 nm affords detection of the peptide bond . Three tryptophan-containing tryptic peptides of bovine serum albumin were immediately located by fluorescence among many UV peaks and ES-MS provided molecular masses allowing the peptides to be identified. J Theor Biol, 1993 May 21, 162(2), 153 - 86 Variation in concentrations of RNAs and proteins involved in gene expression of Escherichia coli; Mahaffy JM; A growing cell is not a steady-state situation . At some point in the cell cycle each gene doubles its concentration, which may result in a doubling of the production rate of its mRNA . The variations in concentration of mRNAs and proteins in an individual exponentially growing cell are studied for a gene which is constitutive, autorepressed, or autoactivated . Analysis of the mathematical model for a constitutive gene shows a fixed variation in concentration of a stable RNA between its minimum and maximum concentration of approximately 6%, independent of cell cycle time or gene location . The variation in the concentrations of the mRNA and protein for a constitutive gene are studied as gene position, molecular stability, and cell cycle time are varied . One result shows that doubling the growth rate can more than double the percentage of product from a gene near the origin of replication compared to one near the terminus . Results from these theoretical studies compare favorably to experimental results for rRNAs and the fully induced lac gene . Additional studies were performed to determine the effects of autorepression and autoactivation on the variation of concentration of mRNAs and proteins . The studies show that RNA and protein products of an autorepressed gene have almost the same variation in concentration as products of a constitutively expressed gene though the absolute concentrations are decreased . It is shown that the stability of an mRNA or protein affects variation in its concentration throughout the cell cycle, much more than the type of genetic control . The strength of repression has no effect on the variation in concentration of RNA and protein gene products through a cell cycle . Studies of an autoactivated gene show that it is significantly more responsive to a shift up or down, such as those caused by nutritional changes . An example is provided where the autoactivated gene is not expressed at one growth rate, but turns on at a higher growth rate . Furthermore, it is shown that gene position may determine whether or not the autoactivated gene is expressed. J Mol Biol, 1993 May 20, 231(2), 274 - 92 Solution structure of selenocysteine-inserting tRNA(Sec) from Escherichia coli . Comparison with canonical tRNA(Ser); Baron C et al.; Selenocysteine-inserting tRNAs (or tRNA(Sec)) are structurally untypical tRNAs that are charged by seryl-tRNA synthetase before being recognized by the selenocysteine synthase that converts serine into selenocysteine . tRNA(Sec) from Escherichia coli contains 95 nucleotides and is the longest tRNA known to