J Biol Chem, 1991 Jul 15, 266(20), 12964 - 70 Cloning and expression of a yeast protein tyrosine phosphatase; Guan KL et al.; To study the regulation of tyrosine phosphorylation/dephosphorylation in Saccharomyces cerevisiae, a protein tyrosine phosphatase (PTPase) was cloned by the polymerase chain reaction (PCR) . Conserved amino acid sequences within the mammalian PTPases were used to design primers which generated a yeast PCR fragment . The sequence of the PCR fragment encoded a protein with homology to the mammalian PTPases . The PCR fragment was used to identify the yeast PTP1 gene which has an open reading frame encoding a 335-amino acid residue protein . This yeast PTPase shows 26% sequence identity to the rat PTPase, although highly conserved residues within the mammalian enzymes are invariant in the yeast protein . The yeast PTP1 is physicallt linked to the 5'-end of a heat shock gene SSB1 . This yeast PTP1 gene was expressed in Escherichia coli and obtained in a highly purified form by a single affinity chromatography step . The recombinant yeast PTPase hydrolyzed phosphotyrosine containing substrates approximately 1000 times faster than a phosphoserine containing substrate . Gene disruption of yeast PTP1 has no visible effect on vegetative growth. Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6264 - 8 Isoprenoid modification of rab proteins terminating in CC or CXC motifs; Khosravi-Far R et al.; Mevalonate starvation of hamster fibroblasts resulted in a shift of rab1b from the membrane to the cytosolic fraction, suggesting that rab1b depends upon an isoprenoid modification for its membrane localization . rab1b and rab3a proteins expressed in insect cells incorporated a product of {3H}mevalonate, and gas chromatography analysis of material released by Raney nickel cleavage demonstrated that rab1b and rab3a are modified by geranylgeranyl groups . Additionally, in vitro prenylation analysis demonstrated farnesyl modification of H-ras but geranylgeranyl modification of five rab proteins (1a, 1b, 2, 3a, and 6) . Together, these results suggest that the carboxyl-terminal CC/CXC motifs (X = any amino acid) specifically signal for addition of geranylgeranyl, but not farnesyl, groups . A rab1b mutant protein lacking the two carboxyl-terminal cysteine residues was not prenylated in vitro . However, since a mutant H-ras protein that terminates with tandem cysteine residues was also not modified, the CC motif may be essential, but not sufficient, to signal prenylation of rab1b . Finally, rab1b and rab3a proteins were not efficient substrates for either farnesyl- or geranylgeranyltransferase activities that modify CAAX-containing proteins (A = any aliphatic amino acid) . Therefore, rab proteins may be modified by a prenyltransferase(s) distinct from the prenyltransferases that modify carboxyl-terminal CAAX proteins. Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6072 - 6 Trans-acting transposase mutant from Tn5; DeLong A et al.; Transposition of Tn5 and of its component insertion sequence IS50R is regulated through the action of two proteins it encodes: a cis-acting transposase, Tnp, and a trans-acting inhibitor of transposition, Inh . The mechanism of the cis-acting Tnp and the relevance of inhibition to cis action have been addressed in the current study . A specific colony morphology assay for transposition of Tn5 was shown to be sensitive to Inh produced in trans and was used to screen for mutants in Inh and/or Tnp with altered regulation . A dominant mutant in IS50R that promotes transposition in trans was isolated and characterized . The mutant (449F) carries a Leu----Phe mutation at position 449 in Tnp . This mutation reduces the frequency of Tn5 or IS50R transposition in cis but allows Tnp-449F to act as efficiently in trans as it does in cis . Tnp-449F is sensitive to inhibition and, furthermore, Inh-449F is a competent inhibitor in trans . These results show that Tnp-449F is a trans-acting transposase, unlike wild-type Tnp, which is cis-acting. Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 5979 - 83 Proteinase trapping: screening for viral proteinase mutants by alpha complementation; Liebig HD et al.; Many virally encoded proteinases cleave themselves out of a polyprotein, with cleavage occurring usually at their own N terminus . This property was used to develop an in vivo screening system using the lacZ gene fragment of M13mp18 . When a fusion protein of the alpha fragment of beta-galactosidase and an active 2A proteinase of human rhinovirus 2 was expressed, alpha complementation was not affected, as the 2A proteinase cleaved itself off the alpha fragment . However, fusion of an inactive 2A prevented alpha complementation, as the 2A polypeptide remained fused to the alpha fragment . After random mutation of the 2A gene by PCR amplification, mutants were screened; M13 phage defective in alpha complementation were obtained at an efficiency of 5% and were shown to contain mutated 2A genes . Intermolecular cleavage was then examined by expressing an alpha fragment-inactive proteinase fusion protein as substrate for an active 2A proteinase expressed from an M13 vector . alpha complementation indicated intermolecular processing of the 2A cleavage site on the alpha fragment-inactive proteinase fusion protein . This versatile system thus allows the high-density screening of both active and inactive proteinase mutants, cleaving either intramolecularly or intermolecularly, and should be applicable to other proteinases of high specificity. Cell, 1991 Jul 12, 66(1), 121 - 8 Three-dimensional structure of yeast RNA polymerase II at 16 A resolution; Darst SA et al.; The structure of yeast RNA polymerase II has been determined by three-dimensional reconstruction from electron micrographs of two-dimensional crystals at approximately 16 A resolution . The most prominent feature of the structure is an arm of protein density surrounding a channel about 25 A in diameter, similar to that found previously for E . coli RNA polymerase . The 25 A-diameter channel bifurcates on one face of the protein, connecting with a 25 A-wide groove and with a channel about half as wide . The 25 A channel and groove, and the narrow channel, may bind double- and single-stranded nucleic acids, respectively . A finger of protein density projecting from the molecule adjacent to the arm-like feature may represent the C-terminal domain of the largest subunit . These results provide a structural basis for analyses of the transcription process and its regulation. J Chromatogr, 1991 Jul 12, 548(1-2), 165 - 78 Characterization of synthetic macroporous ion-exchange resins in low-pressure cartridges and columns . Evaluation of the performance of Macro-Prep 50 S resin in the purification of anti-Klenow antibodies from goat serum; Dunn L et al.; Three ion-exchange materials and one hydrophobic-interaction chromatography packing, based on a rigid macroporous polymer with large, relatively uniform pores, have been evaluated for low-pressure liquid chromatography of antibodies . These sorbents have high capacities for both small and large proteins and are mechanically, chemically, and thermally stable . Macro-Prep 50 S . CM and Q ion-exchange materials are strongly acidic, weakly acidic, and strongly basic, respectively . Protein binding and recovery, pressure-flow properties, and chemical and thermal stability were determined for each sorbent . A rapid, two-step method for the purification of anti-Klenow antibodies from goat serum was developed, based on the Macro-Prep 50 S strong-acid cation-exchange material and the Econo-Pac HIC prepacked hydrophobic-interaction cartridge. Science, 1991 Jul 12, 253(5016), 164 - 70 A method to identify protein sequences that fold into a known three-dimensional structure; Bowie JU et al.; The inverse protein folding problem, the problem of finding which amino acid sequences fold into a known three-dimensional (3D) structure, can be effectively attacked by finding sequences that are most compatible with the environments of the residues in the 3D structure . The environments are described by: (i) the area of the residue buried in the protein and inaccessible to solvent; (ii) the fraction of side-chain area that is covered by polar atoms (O and N); and (iii) the local secondary structure . Examples of this 3D profile method are presented for four families of proteins: the globins, cyclic AMP (adenosine 3',5'-monophosphate) receptor-like proteins, the periplasmic binding proteins, and the actins . This method is able to detect the structural similarity of the actins and 70- kilodalton heat shock proteins, even though these protein families share no detectable sequence similarity. Mol Cell Endocrinol, 1991 Jul, 78(3), 163 - 70 A synthetic peptide corresponding to hFSH-beta-(81-95) has thioredoxin-like activity; Grasso P et al.; The thioredoxin-like activity of human follicle stimulating hormone (hFSH), hFSH-beta-(83-88) peptide amide (hFSH-beta-(83-88) which has a sequence similar to the thioredoxin active center (-His-Cys-Gly-Lys-Cys-Asp-)) and thioredoxin-(31-36)-peptide amide (TD-(31-36) which contains the redox-active dithiol of thioredoxin (-Trp-Cys-Gly-Pro-Cys-Lys-)) was characterized by their ability to reactivate reduced and denatured bovine pancreatic ribonuclease (RNase) . This assay reflects the recently recognized ability of thioredoxin to catalyze disulfide bond formation in proteins . Compared to uncatalyzed refolding of reduced, denatured substrate, hFSH was approximately 10-fold more active than thioredoxin on a molar basis . The catalytic activity of hFSH-beta-(83-88) and TD-(31-36) was equivalent to that of an equimolar concentration of thioredoxin . Screening of 11 overlapping peptide amides representing the entire primary structure of hFSH-beta-subunit indicated that hFSH-beta-(81-95), which contains the sequence similar to the thioredoxin active center within a receptor-binding region of the hFSH-beta-subunit, possesses strong thioredoxin-like activity and was more active than an equimolar concentration of thioredoxin . In contrast, hFSH-beta-(33-53), a thiol-containing peptide which corresponds to a second FSH receptor-binding domain but lacks the sequence similar to the thioredoxin active center, was inactive . Synthetic peptide amides corresponding to other regions of hFSH-beta-subunit were less effective than hFSH-beta-(81-95) in reactivating reduced and denatured RNase . Our data provide evidence that the recently reported thioredoxin-like catalytic activity of FSH may be due, at least in part, to the redox-active dithiol present within a receptor-binding domain of its beta-subunit, and thus may have a physiological role in receptor binding or signal transduction. Nature, 1991 Jul 11, 352(6331), 172 - 4 Convergent evolution of similar function in two structurally divergent enzymes; Kuriyan J et al.; An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase . Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin . Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site . Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other . We have determined the crystal structure of E . coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure . The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system . This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently. Nucleic Acids Res, 1991 Jul 11, 19(13), 3533 - 41 Viral RNA annealing activities of the nucleocapsid protein of Moloney murine leukemia virus are zinc independent; Prats AC et al.; The zinc fingers of retroviral gag nucleocapsid proteins (NC) are required for the specific packaging of the dimeric RNA genome into virions . In vitro, NC proteins activate both dimerization of viral RNA and annealing of the replication primer tRNA onto viral RNA, two reactions necessary for the production of infectious virions . In this study the role of the zinc finger of Moloney murine leukemia virus (MoMuLV) NCp10 in RNA binding and annealing activities was investigated through modification or replacement of residues involved in zinc coordination . These alterations did not affect the ability of NCp10 to bind RNA and promote RNA annealing in vitro, despite a complete loss of zinc affinity . However mutation of two conserved lysine residues adjacent to the finger motif reduced both RNA binding and annealing activities of NCp10 . These findings suggest that the complexed NC zinc finger is not directly involved in RNA-protein interactions but more probably in a zinc dependent conformation of NC protein modulating viral protein-protein interactions, essential to the process of viral RNA selection and virion assembly . Then the NC zinc finger may cooperate to select the viral RNA genome to be packaged into virions. Nucleic Acids Res, 1991 Jul 11, 19(13), 3499 - 506 Distinctive patterns of translational reinitiation in the lac repressor mRNA: bridging of long distances by out-of-frame translation and RNA secondary structure, effects of primary sequence; Matteson RJ et al.; In the early region of the Escherichia coli lac repressor mRNA, translational reinitiation events triggered by nonsense codons occur over long distances and in a distinctive pattern not explained by simple use of the next available initiator triplet . Defined fusions of the restart sites to the lacZ coding region have been used to explore the basis for these reinitiation patterns and to ask whether the sites can function in independent initiation at the 5' end of an mRNA . The results obtained confirm earlier indications that the restart sites may have little or no inherent capacity for binding free 30S ribosomes . The data also add to growing evidence that primary sequence elements are important determinants of reinitiation efficiency . On the basis of the reinitiation activities for nonsense sites throughout the early region of the mRNA, we suggest that out-of-frame restarts and RNA secondary structure bridge long distances between the point of termination and downstream restart codons . Such bridging mechanisms could serve more generally as a means of propagating translational activity across long polycistronic mRNAs. Nucleic Acids Res, 1991 Jul 11, 19(13), 3577 - 81 Characterization of a protein binding sequence in the promoter region of the 16S rRNA gene of the spinach chloroplast genome; Baeza L et al.; By means of mobility-shift assays and Exonuclease III mapping we have determined a 14 bp sequence (named CDF2 binding site) located in front of the 16S rRNA initiation start site which is protected by a spinach chloroplast extract . This region does not include neither one of the two '-35' nor of the two '-10' E . coli-like promoter elements which are recognised by E . coli RNA polymerase in vitro . The CDF2 binding site is specifically recognized by two small polypeptides which migrate corresponding to 35 and 33 kDa respectively as shown by UV cross-linking experiments . In vivo transcription initiation of the 16S rRNA gene occurs 13 nucleotides downstream of the 14 bp sequence and is different from the transcription start site which is used by E.coli polymerase in vitro. Nucleic Acids Res, 1991 Jul 11, 19(13), 3511 - 6 Amino acid misincorporation during high-level expression of mouse epidermal growth factor in Escherichia coli; Scorer CA et al.; To determine whether the high-level expression of foreign proteins in Escherichia coli can lead to frequent translational errors, we analyzed amino acid misincorporation in mouse epidermal growth factor (mEGF) produced as a TrpE fusion protein . The mEGF DNA does not encode phenylalanine and determining the phenylalanine content of the purified protein will measure missense errors . Using this approach, we found an error frequency of about 1 in 40 for codons differing by a single base from those for phenylalanine . This is at least ten times higher than the error rate found for normal E . coli protein synthesis and may be due to limiting supply of charged tRNAs and GTP, brought about by the high-level production of the heterologous protein . The unexpectedly high error rate has implications for the clinical use of E . coli-derived therapeutic proteins. Nucleic Acids Res, 1991 Jul 11, 19(13), 3653 - 60 Conformation and structural fluctuations of a 218 nucleotides long rRNA fragment: 4-thiouridine as an intrinsic photolabelling probe; Dubreuil YL et al.; The structure in solution of an RNA fragment (218 nucleotides long) containing part of E . coli 16S rRNA domain 2 has been studied using the intrinsic photoaffinity probe 4-thiouridine (s4U) . In vitro transcription with T7 polymerase, in the presence of s4U triphosphate yielded complete RNA molecules . An affinity electrophoresis system based on Phenylmercuric substituted polyacrylamide (APM) gels allows separation of the RNA chains as a function of their s4U content . Distribution of s4U within chains follows a binomial law indicating that (i) substitution is close to random, (ii) efficiency of s4U incorporation is 0.22 times that of U . The monothiolated RNA fraction isolated from APM gel was irradiated at 366 nm under native conditions and the intramolecularly crosslinked molecules, (34%), were separated on denaturing polyacrylamide gel according to loop size . The positions of the two partners of bridges were identified by mean of reverse transcription and RNA sequencing . 17 of the 41 possible s4U positions lead to detectable bridges . These crosslinks formed efficiently at the border of bihelical regions or when structural mobility is allowed . The pattern of crosslinks is in agreement with the previously proposed secondary structure but indicates that it is much more flexible than expected. Nucleic Acids Res, 1991 Jul 11, 19(13), 3517 - 24 The three-dimensional folding of ribosomal RNA; localization of a series of intra-RNA cross-links in 23S RNA induced by treatment of Escherichia coli 50S ribosomal subunits with bis-(2-chloroethyl)-methylamine; Doring T et al.; Intact 50S ribosomal subunits from E.coli were cross-linked with the symmetrical bifunctional reagent bis-(2-chloroethyl)-methylamine . After deproteinization, selected regions of the 23S RNA were excised by treatment with ribonuclease H in the presence of appropriate complementary decadeoxynucleotides, and screened for the presence of intra-RNA cross-links by two-dimensional gel electrophoresis . Individual isolated cross-linked RNA fragments were analysed by our established procedures . Sixteen intra-RNA cross-links were identified, three of which corresponded to those previously published . The thirteen 'new' cross-links were localized in the 23S RNA at positions 774-78 linked to 792-94, 876-79 linked to 899-900, 979-81 or 983-84 to 2029, 1715 to 1743-46, 1911-21 to 1964, 1933 to 1966, 2032 to 2054-55, 2112 to 2169-71, 2116-17 to 2163-67, 2128-32 to 2156-59, 2392-93 to 2422-23, 2737-38 to 2763-66, and 2791 to 2890 . These results are discussed in the context of three-dimensional model-building studies with the 23S RNA, with particular reference to the environment of the 'active centre' of the 50S subunit. Nucleic Acids Res, 1991 Jul 11, 19(13), 3629 - 32 MutM, a protein that prevents G.C----T.A transversions, is formamidopyrimidine-DNA glycosylase; Michaels ML et al.; We have cloned chromosomal DNA bordering an insert that inactivates mutM . Sequencing of this clone has revealed that the insertion element is located between the promoter and structural gene for formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase) . An overproducing clone of Fapy-DNA glycosylase complements the original mutM strain that had been isolated after EMS mutagenesis . Thus, we conclude that MutM is actually Fapy-DNA glycosylase . mutM has previously been characterized as a mutator strain that leads specifically to G.C----T.A transversions . This in vivo characterization correlates well with the mutagenic potential of one of the lesions Fapy-DNA glycosylase removes, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-OxodG). Nucleic Acids Res, 1991 Jul 11, 19(13), 3613 - 8 DNA helicase IV from HeLa cells; Tuteja N et al.; Human DNA helicase IV, a novel enzyme, was purified to homogeneity from HeLa cells and characterized . The activity was measured by assaying the unwinding of 32P labeled 17-mer annealed to M13 ss DNA . From 440g of HeLa cells we obtained 0.31 mg of pure protein . Helicase IV was free of DNA topoisomerases, DNA ligase and nuclease activities . The apparent molecular weight is 100 kDa . It requires a divalent cation for activity (Mg2+ = Mn2+ = Zn2+) and the hydrolysis of only ATP or dATP . The activity is destroyed by trypsin and is inhibited by 200 mM KCl or NaCl, 100 mM potassium phosphate, 45 mM ammonium sulfate, 5 mM EDTA, 20 microM ss M13 DNA or 20 microM poly {G} (as phosphate) . The enzyme unwinds DNA by moving in the 5' to 3' direction along the bound strand, a polarity opposite to that of the previously described human DNA helicase I (Tuteja et al Nucleic Acids Res . 18, 6785-6792, 1990) . It requires more than 84 bases of single-stranded DNA in order to exert its unwinding activity and does not require a replication fork-like structure . Like human DNA helicase I the enzyme can also unwind RNA-DNA hybrid. Biochim Biophys Acta, 1991 Jul 10, 1093(2-3), 135 - 43 The GTP-binding Sar1 protein is localized to the early compartment of the yeast secretory pathway; Nishikawa S et al.; SAR1, the yeast gene which encodes a novel type of small GTP-binding protein, has been shown to be required for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus . To further the understanding of the function of its product, a lacZ-SAR1 hybrid gene was constructed and a polyclonal antibody was raised against the hybrid protein . This antibody specifically recognizes the SAR1 gene product (Sar1p) as a 23-kDa protein in the yeast cell lysate . We examined the subcellular localization of Sar1p using this antibody . In wild-type cells, Sar1p was predominantly recovered in a rapidly sedimenting membrane fraction that includes the ER . The soluble form of Sar1p was also detected when the protein was overproduced . Immunofluorescence microscopy with the anti-Sar1p antibody showed perinuclear staining that was exaggerated in the ER-accumulating sec18 mutant . Membrane association of Sar1p was shown to be very light . Sar1p was not extracted from the membrane by treatment with alkaline sodium carbonate, and only 1% deoxycholic acid solubilized Sar1p completely . From these results, we suggest that Sar1p is firmly located on the ER membrane where it regulates the ER-Golgi traffic. Biochim Biophys Acta, 1991 Jul 10, 1093(2-3), 216 - 22 Hepatic phosphatidylinositol kinase activity in continuously endotoxemic rats; Rodriguez de Turco EB et al.; The activity of phosphatidylinositol (PI) kinase and the content and fatty acid composition of inositol phospholipids (IPLs) were analyzed in the livers of rats that had been continuously infused with Escherichia coli endotoxin (ET) or saline for 30 h . Maximal enzymatic activity in total liver membrane fractions was observed in the presence of 1 mM ATP, 20 mM MgCl2, exogenously added 0.3 mM PI and Triton X-100 (0.25%) . The activity of PI kinase for endogenous and exogenous PI was 43 and 79% higher respectively, in ET- as compared with saline-infused rats . The Km of the enzyme for ATP was not altered (0.175 mM), while the apparent Vmax was higher for ET- as compared with saline-infused rats (0.48 and 0.38 nmol of phosphatidylinositol 4-phosphate formed/mg protein per min, respectively) . The ET-induced higher activity of PI kinase was paralleled by a 68-78% increase in the content of polyphosphoinositides (PPI), while PI content was unchanged . All IPLs from livers of endotoxemic rats had a lower content of arachidonic acid . We demonstrate for the first time that ET can directly and/or indirectly stimulate the net synthesis of PPI in liver cells . This effect could serve to modulate the PPI derived signals by increasing the availability of the substrate phosphatidylinositol 4,5-bisphosphate. Biochemistry, 1991 Jul 9, 30(27), 6775 - 9 Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence; Loewenthal R et al.; Fluorescence spectra of wild-type barnase and mutants in which tryptophan and histidine residues have been substituted have been analyzed to give the individual contributions of the three tryptophan residues . The spectrum is dominated by the contribution of Trp-35 . The fluorescence intensity varies with pH according to an ionization of a pKa of 7.75 . This pKa is close to that previously determined by NMR titration of the C2-H resonances of His-18 as a function of pH (Sali et al., 1989) . This histidine residue is close to Trp-94 . The pH dependence of the spectrum is abolished when either His-18 or Trp-94 is mutated, and so appears to be caused by the His-18/Trp-94 interaction . The spectral response of this interaction can serve as a probe of the folding pathway and of electrostatic effects within the protein . Changes in the fluorescence spectra on substitution of Trp-94 and His-18 suggest that there is net energy transfer from Trp-71 to Trp-94. Biochemistry, 1991 Jul 9, 30(27), 6721 - 6 Interaction between the cytoplasmic and membrane-bound domains of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system; Lolkema JS et al.; Sulfhydryl reagents affected the binding properties of the translocator domain, NIII, of enzyme IImtl in two ways: (i) the affinity for mannitol was reduced, and (ii) the exchange rate of bound and free mannitol was increased . The effect on the affinity was very much reduced after solubilization of enzyme IImtl in the detergent decylPEG . The effects were caused exclusively by reaction of the sulfhydryl reagents with the cysteine residue at position 384 in the primary sequence . Interaction between two domains is involved, since Cys384 is located in the cytoplasmic domain, CII . When Cys384 was mutated to serine, the enzyme exhibited the same binding properties as the chemically modified enzyme . The data support our proposal that phosphorylation of enzyme IImtl drastically reduces the activation energy for the translocation step through interaction between domains CII and NIII {Lolkema J . S., ten Hoeve-Duurkens, R . H., Swaving Dijkstra, D., & Robillard, G . T . (1991) Biochemistry (preceding paper in this issue)} . Functional interaction between the translocator domain, NIII, and domain CI was investigated by phosphorylation of His554, located in domain CI, in the C384S mutant . No effect on the binding properties was observed . In addition, the binding properties were insensitive to the presence of the soluble phosphotransferase components enzyme I and HPr. Biochemistry, 1991 Jul 9, 30(27), 6716 - 21 Mechanistic coupling of transport and phosphorylation activity by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system; Lolkema JS et al.; Mannitol bound to enzyme IImtl could be trapped specifically by rapid phosphorylation with P-HPr . The assay was used to demonstrate transport of mannitol across the cytoplasmic membrane with and without phosphorylation of mannitol . The latter was 2-3 orders of magnitude slower . The fraction of bound mannitol molecules that was actually phosphorylated, the efficiency of the trap, was less than 50% . The efficiency was not very different for enzyme IImtl embedded in the membrane of vesicles with an inside-out orientation or solubilized in detergent . Subsequently, it is argued that the fraction of the bound mannitol molecules that was not phosphorylated dissociated into the cytoplasmic space . A model for the catalytic mechanism of enzyme IImtl is proposed on the basis of interpretations of the present experiments . The main features of the model are the following: (i) mechanistically, the coupling between transport and phosphorylation is less than 50%; (ii) in the physiological steady state of mannitol transport and metabolism, the coupling is 100%; (iii) phosphorylated enzyme IImtl catalyzes facilitated diffusion at a high rate; (iv) the state of phosphorylation of the cytoplasmic domain modulates the activity of the translocator domain; (v) the enzyme catalyzes phosphorylation of free cytoplasmic mannitol at least as fast as it catalyzes transport plus phosphorylation of free periplasmic mannitol. Biochemistry, 1991 Jul 9, 30(27), 6788 - 95 Nucleotide sequence of the promoter and fadB gene of the fadBA operon and primary structure of the multifunctional fatty acid oxidation protein from Escherichia coli; Yang XY et al.; The primary structure of a multifunctional protein, the large alpha-subunit of the Escherichia coli fatty acid oxidation complex, was determined by sequencing the fadB region of the fadBA operon . The amino-terminal sequence of this protein had been established by Edman degradation . The transcription start site of the fadBA operon was located 42 nucleotides upstream of the initiator codon of the fadB gene by primer extension analysis . Sequences of -10 and -35 regions of the promoter responsible for interaction with RNA polymerase were found to be CACACT and TTTGCA, respectively . The location of the promoter of the fadBA operon was defined, and the transcription direction of this operon, from fadB to fadA, as previously proposed {Yang, S.-Y., et al . (1990) J . Biol . Chem . 265, 10424-10429}, was corroborated . The multifunctional protein is composed of 729 amino acid residues and has a calculated Mr of 79,593 . A putative NAD-binding beta alpha beta-fold necessary for L-3-hydroxyacyl-CoA dehydrogenase function was found in the central region of the fadB gene product . Sequence analyses suggest that the functional domains of the multifunctional protein are arranged in the order enoyl-CoA hydratase:L-3-hydroxyacyl-CoA dehydrogenase: delta 3-cis-delta 2-trans-enoyl-CoA isomerase and suggest that the genes of the E . coli multifunctional protein and rat peroxisomal trifunctional beta-oxidation enzyme evolved from a common ancestral gene. FEBS Lett, 1991 Jul 8, 285(1), 31 - 4 Autoprocessing of Drosophila copia gag precursor to generate a unique laminate structure in Escherichia coli; Yoshioka K et al.; Drosophila copia protease is likely to be encoded in the gag gene . We have expressed copia gag polyprotein precursor in E . coli . The gag precursor was correctly processed to generate a unique laminate structure in E . coli . The processing was almost completely blocked by a mutation at the putative active site of copia protease, and resulted in accumulation of the precursor . Furthermore, the laminate structure was not found in E . coli expressing the mutant precursor . These results indicate that the protease is involved in cleaving the gag precursor itself . Also, the assembly of copia gag protein should correlate to the autoprocessing of copia gag polyprotein precursor. FEBS Lett, 1991 Jul 8, 285(1), 127 - 31 A chemically cross-linked nonlinear proOmpA molecule can be translocated into everted membrane vesicles of Escherichia coli in the presence of the proton motive force; Tani K et al.; The chemical cross-linking between the two cysteine residues at positions + 290 and + 302 of proOmpA was performed with N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine . In the absence of the proton motive force (delta muH+), the cross-linked proOmpA was only partially translocated into everted membrane vesicles, leading to accumulation of translocation intermediates . In the presence of delta mu H+, the cross-linked proOmpA was completely translocated . The translocated OmpA still possessed the cross-linked loop composed of 13 amino acid residues and the cross-linker . It is concluded that polypeptide chains need not be necessarily linear and fully expanded to be translocated. J Biol Chem, 1991 Jun 25, 266(18), 11610 - 3 The priB gene encoding the primosomal replication n protein of Escherichia coli; Allen GC Jr et al.; The gene encoding protein n of the Escherichia coli primosome has been discovered in the rpsF-rpsR-rplI ribosomal protein operon and designated priB . The low copy number of PriB protein and the distinctive codon usage of its gene argue against its being a ribosomal protein . A strain which overproduces PriB was constructed and has been used to purify the protein to homogeneity . The overproduced protein behaves like that purified from wild-type cells. J Mol Biol, 1991 Jul 5, 220(1), 19 - 33 Initiation of the UvrABC nuclease cleavage reaction . Efficiency of incision is not correlated with UvrA binding affinity; Snowden A et al.; The incision steps of Escherichia coli nucleotide excision repair are mediated by the UvrABC nuclease complex . We have previously shown that the UvrABC nuclease specifically incises apyrimidinic (AP) sites less efficiently than o-benzylhydroxylamine-modified apyrimidinic (BA) sites . To investigate these differences, quantitative DNase I footprinting titration studies were performed . The UvrA binding isotherms were similar for both the AP site (Kd = 6 x 10(-9) M) and the bulkier BA lesion (Kd = 14 x 10(-9) M), despite the fact that the extent of incision differs for these two lesions . It was also found that the relative binding affinity of the preincision UvrA2B complex to the AP and BA substrates differs significantly with estimated apparent equilibrium dissociation constants (Kd) of 4 x 10(-9) M and 80 x 10(-9) to 120 x 10(-9) M, respectively . These results indicate that incision efficiency does not correlate to UvrA binding affinity, but is a direct result of interactions between the UvrA2B complex and the site of the DNA damage . It is also shown that high UvrA concentrations are inhibitory to the UvrABC nuclease reaction. J Mol Biol, 1991 Jul 5, 220(1), 13 - 6 Use of chemical modification in the crystallization of isocitrate lyase from Escherichia coli; Abeysinghe SI et al.; Two different crystal forms of isocitrate lyase (ICL) from Escherichia coli have been grown following the chemical modification of the enzyme by either 3-bromopyruvate or ethyl mercuri thiosalicylate (EMTS), contrasting strongly with difficulties in obtaining ordered crystals of the native enzyme . Both crystal forms are obtained using the hanging drop method of vapour diffusion with ammonium sulphate as the precipitant . The crystals diffract well and X-ray photographs of the crystals have established that they are in space groups C222(1) and P3(1) (or its enantiomorph P3(2), respectively . Considerations of the values of Vm and measurements on the crystal density indicate that the asymmetric unit of both crystals contains four subunits. J Biol Chem, 1991 Jul 5, 266(19), 12759 - 65 Identification and characterization of the functional amino acids at the active center of pig liver thioltransferase by site-directed mutagenesis; Yang YF et al.; By using site-directed mutagenesis techniques, the essential amino acids at the catalytic center of porcine thioltransferase (glutaredoxin) were determined . Seven oligonucleotides were designed, synthesized, and used to construct mutants, ETT-C22S, ETT-C25S, ETT-C25A, ETT-R26V, ETT-K27Q, ETT-R26V: K27Q, and ETT-C78S:C82S, by altering their codons in pig liver thioltransferase cDNA/M13mp18 clones . Each of the thioltransferases was purified to homogeneity and its dithiol-disulfide exchange, and dehydroascorbate reductase activities were compared with those of the wild-type (ETT) . Evidence was obtained that Cys22 was essential for catalytic activity, and the extremely low pKa value of its sulfhydryl group was facilitated primarily by Arg26 . The role of Lys27 at the active center was different from that of Arg26 and may be important in stabilizing the E.S intermediate by electrostatic forces . The second pair of cysteines, Cys78 and Cys82, nearer the C terminus, were not directly involved in the active center, but may play a role in defining the native protein structure . The replacement of the original Cys with a Ser at position 25 increased rather than decreased the enzyme activity, suggesting that the proposed intramolecular disulfide bond between Cys22 and Cys25 is not necessary for the catalytic mechanism of the Ser25 mutant, but does not rule out such a mechanism for the wild-type enzyme. J Biol Chem, 1991 Jul 5, 266(19), 12469 - 73 Identification of a calcium-dependent calmodulin-binding domain in Xenopus membrane skeleton protein 4.1; Kelly GM et al.; Xenopus membrane skeleton protein 4.1 is expressed constitutively during embryonic development and accumulates to high levels within the retina during normal morphogenesis . There exists a high degree of amino acid identity between Xenopus protein 4.1 and human protein 4.1, suggesting that the mechanisms known to modulate the function(s) of human protein 4.1 may also serve to regulate Xenopus protein 4.1 . Calmodulin (CaM) is one regulatory protein known to affect membrane-cytoskeletal interactions . An in vitro binding assay was used to test the ability of Xenopus protein 4.1 to bind CaM . Two independent approaches, involving protein 4.1 synthesized in vitro from synthetic RNA or a partial length protein 4.1 fusion protein expressed in Escherichia coli, demonstrate calcium-dependent, CaM binding . Both approaches demonstrate that the CaM-binding site is within the amino-terminal region of Xenopus protein 4.1 . Results of this calmodulin binding activity suggest a possible regulatory mechanism by which calcium and calmodulin may affect the function of protein 4.1 during development. J Biol Chem, 1991 Jul 5, 266(19), 12439 - 41 Spermidine acetylation in response to a variety of stresses in Escherichia coli; Carper SW et al.; Heat shock, cold shock, ethanol, and alkaline shift, but not hydrogen peroxide, stimulate the accumulation of monoacetylspermidine in Escherichia coli . Acetylation occurs with nearly equal frequencies at both the N1 and N8 positions of this ubiquitous polycation . Spermidine acetylation does not appear to be associated with known stress regulons, such as htpR, oxyR, and SOS . E . coli, capable of acetylating spermidine, constitutively express a spermidine acetyltransferase activity during all phases of growth, and this activity is unaffected by cold shock . A mutant strain, incapable of acetylating spermidine, does not express this enzyme activity but grows at an identical rate as the parent strain at 37 degrees C . These results demonstrate that the monoacetylation of spermidine in E . coli is regulated by some mechanism other than a stress-inducible acetyltransferase and is not essential for growth of these cells . They suggest that polyamine acetylation is involved in the responses of these organisms to a variety of chemical and physical stresses. J Biol Chem, 1991 Jul 5, 266(19), 12228 - 33 Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis; Dong Q et al.; Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1 . The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification . The other substrate, aspartate, had no such effect even at a high concentration . In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed . Stoichiometry studies with {7-14C}phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP . Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification . This arginine has been changed to leucine by site-directed mutagenesis . The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme . In addition, kcat of the R147L mutant decreased by a factor of 1.3 x 10(4) . On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis. J Biol Chem, 1991 Jul 5, 266(19), 12495 - 501 Gabaculine-resistant glutamate 1-semialdehyde aminotransferase of Synechococcus . Deletion of a tripeptide close to the NH2 terminus and internal amino acid substitution; Grimm B et al.; Glutamate 1-semialdehyde aminotransferase (GSA-AT) is the last enzyme in the C5 pathway converting glutamate into the tetrapyrrole precursor delta-aminolevulinate in plants, algae, and several bacteria . Sequence analysis of the genes encoding GSA-AT in barley, Synechococcus, and Escherichia coli revealed 50-70% similarity in the primary structures of the proteins . The enzyme is inhibited rapidly by gabaculine when added in approximately stoichiometric amounts with the enzyme . A gabaculine-tolerant Synechococcus strain, GR6, was found to produce a GSA-AT less sensitive to the inhibitor . Accordingly, the mutant gene was isolated and sequenced . In comparison with the wild-type gene it contains a deletion of nine nucleotides (position 12-20) and a guanine to adenine substitution (position 743) . This resulted in the loss of the amino acids serine, proline, and phenylalanine (position 5-7) close to the NH2 terminus of the enzyme and an exchange of Met-248 for isoleucine in the middle of the polypeptide chain . Wild-type and mutant GSA-AT were expressed in E . coli and purified close to homogeneity . Although the specific activity of the mutant GSA-AT was only one-fifth of the wild type, it displayed a 100-fold increased resistance to gabaculine . Peaks in the absorption spectrum of the purified recombinant GSA-ATs at 335 and 417 nm are typical of a transaminase containing a B6 cofactor . Incubation with substrate and with inhibitor induced spectral changes characteristic of other gabaculine-sensitive, B6-requiring enzymes. J Biol Chem, 1991 Jul 5, 266(19), 12722 - 33 A serine/threonine kinase activity is closely associated with a 65-kDa phosphoprotein specifically recognized by the kappa B enhancer element; Ostrowski J et al.; The immunoglobulin kappa light chain enhancer, kappa B, is an important cis-acting transcriptional element . kappa B binds a number of proteins including the members of the ubiquitous NF-kappa B family of transcription factors . Agarose beads coupled to a double-stranded oligonucleotide containing the kappa B motif were used to isolate a 65-kDa predominantly nuclear phosphoprotein . Southwestern blot analysis demonstrated that this phosphoprotein can bind the kappa B element directly and specifically . This kappa B-associated protein was phosphorylated in vivo and in vitro by a nuclear serine/threonine kinase(s) which, in a number of different cell lines, appeared to be stimulated in response to interleukin-1 alpha and lipopolysaccharide treatment . In the B cell lines 70Z/3 and CH12 LX2B, and the T cell line EL-4 6.1 C10 the activity of the kappa B-associated kinase(s) correlated with the binding activity of nuclear NF-kappa B displayed in a gel shift assay . In vitro, the 65-kDa protein was phosphorylated in the absence of exogenously added kinase . The 65-kDa phosphoprotein and the kinase activity remained associated following sequential anion-exchange and hydrophobic interaction chromatography . These results suggest that the kappa B-associated phosphoprotein is either autophosphorylated or is phosphorylated by a closely associated kinase(s) . Stimulation of a nuclear protein kinase which is closely associated with a sequence-specific DNA may reflect a novel mechanism by which growth factors regulate gene expression. J Mol Biol, 1991 Jul 5, 220(1), 5 - 7 Crystallization and preliminary X-ray diffraction analysis of oucleoside diphosphate kinase from Myxococcus xanthus; Williams RL et al.; Nucleoside diphosphate (NDP) kinase catalyzes the transfer of the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate . Human and rodent forms of this enzyme have been shown to be suppressors of metastasis . Crystals that diffract X-rays to high resolution have been obtained for the recombinant Myxococcus xanthus NDP kinase expressed in and purified from Escherichia coli . Two crystal forms have been obtained . Both forms are orthorhombic, space group I222 (or I2(1)2(1)2(1)) with a = 267.1 A, b = 74.0 A and c = 75.1 A for form I and a = 53.5 A, b = 74.0 A and c = 75.1 A for form II . Form I appears to have five molecules in the asymmetric unit approximately related to each other by a translation of 0.2 along the a axis . Diffraction data have been recorded to 1.9 A for form I and to 2.2 A for form II. J Biol Chem, 1991 Jul 5, 266(19), 12289 - 93 Diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion toxin . Internal in-frame deletion from Thr387 to His485 results in the formation of a highly potent fusion toxin which is resistant to proteolytic degradation; Wen ZL et al.; We have previously reported the genetic construction and properties of a fusion protein which was composed of the enzymatically active and membrane translocation domains of the diphtheria toxin and the receptor-specific ligand alpha-melanocyte-stimulating hormone (alpha-MSH) (Murphy, J.R., Bishai, W., Borowski, M., Miyanohara, A., Boyd, J., and Nagle, S . (1986) Proc . Natl . Acad . Sci . U.S.A . 83, 8258-8262) . While this fusion toxin was found to be selectively toxic for MSH receptor-bearing cells in vitro, it was subject to profound proteolytic degradation in recombinant Escherichia coli making purification difficult . We now report that the deletion of diphtheria toxin fragment B sequences between Thr387 and His485 results in a protease-resistant form of the fusion toxin, DAB389-alpha-MSH . We show that DAB389-alpha-MSH is expressed in high yield in recombinant Escherichia coli, that it is readily purified from crude bacterial lysates by immunoaffinity and high performance liquid chromatography, and its cytotoxic activity toward both human and murine malignant melanoma cell lines is mediated through the MSH receptor. J Biol Chem, 1991 Jul 5, 266(19), 12140 - 5 Molybdenum cofactor biosynthesis in Escherichia coli . Requirement of the chlB gene product for the formation of molybdopterin guanine dinucleotide; Johnson JL et al.; The chlorate-resistant mutants of Escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor . The molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin . The molybdenum cofactor in a number of E . coli enzymes has been shown to contain GMP in addition to the metal-molybdopterin complex, with the GMP appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain . In this paper, we have examined the biochemistry of the chlB mutant and show that the gene product of the chlB locus is essential for the addition of the GMP moiety to form molybdopterin guanine dinucleotide, a step which occurs late in the cofactor biosynthetic pathway in E . coli . Sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of E . coli cells . Wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide, while cells of chlB mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide. Biochemistry, 1991 Jul 2, 30(26), 6454 - 64 Selectivity of Escherichia coli RNA polymerase for template conformation; Butzow JJ et al.; Escherichia coli RNA polymerase (RNAP) exhibits a strong selectivity for the secondary structure of its template DNA, as shown by the influence both of the DNA conformation on the transcription cycle and of the enzyme on the DNA conformation itself . Binding, chain initiation and elongation characteristics of RNAP, and DNA conformational characteristics were examined by use of the alternating copolymer poly(dGdm5C).poly(dGdm5C) as template . Transcription is impeded when the DNA is in the Z conformation as compared with the B; the initial conformation is determined by the concentration of the conformational effectors of Mg2+ and {Co(NH3)6}3+ . RNAP binds to both Z and B conformers; the total binding is moderately greater when the template is in the B conformation than when it is strongly stabilized in the Z, by {Co(NH3)6}3+ concentrations much higher than those required for B-Z transition . However, the Z conformer is much more easily displaced competitively from the bulk of its complexes with RNAP than is the B, indicating a specific binding preference for the B conformer . When the template is in the B conformation, or is moderately stabilized in the Z by Mg2+ concentrations such that the polynucleotide is just fully converted from B to Z, elongation is predicted well by chain initiation, indicating that on the Z conformer RNAP is effectively inhibited at the chain initiation or at an earlier stage . The average chain growth rates for polymeric product synthesized on B and on moderately stabilized Z are similar, even though overall RNA synthesis is considerably lowered on the Z form, again indicating that the limiting events precede elongation . When the Z conformer is strongly stabilized, chain initiation and elongation are further inhibited . Elongation is still roughly correlated with chain initiation, but some additional inhibition of elongation takes place independently . Circular dichroism analysis shows that RNAP-DNA binding affects the B-Z conformational equilibrium, leading to reformation of the B conformer from Z and interference with conversion of B to Z, under conditions that would otherwise favor the Z conformer . Thus, there is an RNAP concentration dependent shift of the B-Z transition to higher concentrations of Z-inducing cation, and there is an RNAP concentration dependent decrease in the rate of B to Z conversion . These effects were observed for poly(dGdm5C).poly(dGdm5C), with Z stabilized by {Co(NH3)6}3+ or Mg2+ . (They were observed as well for the unmethylated copolymer poly(dGdC).poly(dGdC), with Z stabilized by {Co(NH3)6}3+.) Perturbation of the Z conformer was detectable by circular dichroism at an RNAP:polynucleotide ratio down to a practical limit of approximately 1 RNAP:500 bp.(ABSTRACT TRUNCATED AT 400 WORDS) Biochemistry, 1991 Jul 2, 30(26), 6484 - 90 Open conformation of a substrate-binding cleft: 19F NMR studies of cleft angle in the D-galactose chemosensory receptor; Luck LA et al.; The Escherichia coli D-galactose and D-glucose receptor is a two-domain structure with a sugar-binding site at the interface between domains . The structure of the closed cleft containing bound D-glucose has been determined crystallographically, but the open cleft remains to be characterized . The present study illustrates a generalizable approach that is used to detect and analyze both the open- and closed-cleft conformations in solution . A 19F nucleus located inside the cleft is monitored by 19F NMR . When the cleft is occupied by D-glucose, the 19F nucleus is found to be inaccessible to the aqueous paramagnetic probe Gd-EDTA, verifying that the occupied cleft is closed in solution and inaccessible to bulk solvent . When the cleft is empty, the 19F nucleus becomes accessible to the paramagnet such that the distance of closest approach is r less than or equal to 10 A, indicating that the empty cleft opens at least transiently by an angle theta greater than or equal to 18 +/- 3 degrees. Biochemistry, 1991 Jul 2, 30(26), 6476 - 83 Human fibroblast stromelysin catalytic domain: expression, purification, and characterization of a C-terminally truncated form; Marcy AI et al.; Stromelysin-1 is a member of a tissue metalloproteinase family whose members are all capable of degrading extracellular matrix components . A truncated form of human fibroblast prostromelysin 1 lacking the C-terminal, hemopexin-like domain has been expressed in Escherichia coli and purified to homogeneity . Treatment of this short form of prostromelysin with (aminophenyl)mercuric acetate resulted in activation and loss of the propeptide in a manner identical with the wild-type, full-length protein . Kinetic comparisons using Nle11-substance P as a substrate showed that the wild-type stromelysin and the truncated form of the enzyme had similar kcat and Km values . Likewise, both enzymes displayed similar Ki values for a hydroxamate-containing peptide inhibitor . Taken together, these results indicate that the C-terminal portion of stromelysin is not required for proper folding of the catalytic domain, maintenance of the enzyme in a latent form, activation with an organomercurial, cleavage of a peptide substrate, or interaction with an inhibitor . Moreover, the active short form of stromelysin displayed a reduction in the C-terminal heterogeneity, a characteristic degradation of the full-length stromelysin, and thereby provides a more suitable protein for future structural studies. Biochemistry, 1991 Jul 2, 30(26), 6416 - 22 Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis; Thielking V et al.; Guided by the X-ray structure analysis of a crystalline EcoRV-d(GGGATATCCC) complex (Winkler, in preparation), we have begun to identify functionally important amino acid residues of EcoRV . We show here that Asn70, Asp74, Ser183, Asn185, Thr186, and Asn188 are most likely involved in the binding and/or cleavage of the DNA, because their conservative substitution leads to mutants of no or strongly reduced activity . In addition, C-terminal amino acid residues of EcoRV seem to be important for its activity, since their deletion inactivates the enzyme . Following the identification of three functionally important regions, we have inspected the sequences of other restriction and modification enzymes for homologous regions . It was found that two restriction enzymes that recognize similar sequences as EcoRV (DpnII and HincII), as well as two modification enzymes (M.DpnII and, in a less apparent form, M.EcoRV), have the sequence motif -SerGlyXXXAsnIleXSer- in common, which in EcoRV contains the essential Ser183 and Asn188 residues . Furthermore, the C-terminal region, shown to be essential for EcoRV, is highly homologous to a similar region in the restriction endonuclease SmaI . On the basis of these findings we propose that these restriction enzymes and to a certain extent also some of their corresponding modification enzymes interact with DNA in a similar manner. Biochemistry, 1991 Jul 2, 30(26), 6412 - 6 A novel alpha-proton exchange reaction catalyzed by Escherichia coli methionyl-tRNA synthetase; Williams JS et al.; The Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) was shown to catalyze alpha-carbon hydrogen-deuterium exchange of L-selenomethionine, L-methionine, L-ethionine, and L-norleucine in the presence of deuterium oxide . The rate of alpha-proton exchange for L-methionine was shown to be linear with respect to delta MTS concentration . The exchange reaction showed saturation kinetics with apparent Km values of 21 and 4 mM in the absence and presence of saturating adenosine concentrations, respectively . As expected, delta MTS did not catalyze alpha-proton exchange of D-methionine since the enzyme has been shown to be specific for L-amino acids . In the absence of enzyme or in the presence of an equivalent concentration of Zn2+, no hydrogen-deuterium exchange was detected . The exchange reaction was not observed with L-methioninol, an analogue of L-methionine lacking the carboxylate group . These results suggest that the alpha-carboxylate group is a requirement for the delta MTS-catalyzed exchange reaction . The E . coli methionyl-tRNA synthetase (MTS) has previously been shown to be a zinc metalloprotein {Posorske, L . H., Cohn, M., Yanagisawa, N., & Auld, D . S . (1979) Biochim . Biophys . Acta 576, 128} . On the basis of the structural and mechanistic information available on MTS, we propose that the enzyme-bound zinc coordinates the carboxylate of the amino acid, while a base on the enzyme is responsible for exchange of the alpha-proton . The role of the enzyme-bound metal is to render the alpha-proton more acidic through coordination of the carboxylate group.(ABSTRACT TRUNCATED AT 250 WORDS) Carcinogenesis, 1991 Jul, 12(7), 1241 - 6 The reaction of pristane (2,6,10,14-tetramethylpentadecane) with radiolytically generated reactive oxygen intermediates results in a stable genotoxic compound as assessed by the SOS chromotest; Janz S et al.; The most widely studied model of plasmacytomagenesis is the induction of plasmacytomas in BALB/c mice by i.p . injections of the isoalkane pristane (2,6,10,14-tetramethylpentadecane) . Employing a simple quantitative and well-established short-term bacterial genotoxicity assay, the SOS chromotest, as a model system, we have investigated whether pristane may potentially be involved in causing or modulating the genotoxic events thought to induce plasma cell tumorigenesis . We found that incorporation of pristane into the cell membranes enhance the SOS response in Escherichia coli PQ37 and PQ300 induced by gamma-radiation under hyperoxic conditions . Moreover, the oxidation of pristane by radiolytically generated reactive oxygen intermediates yielded a stable, genotoxic product active on E . coli PQ300, a SOS tester strain designed to detect oxidative genotoxins . We discuss these findings in relation to the tumor-promoting role of the chronic i.p . inflammation that accompanies plasmacytomagenesis and conclude that, under these specific conditions, pristane may possess a previously unrecognized genotoxic activity in its tumorigenic potential. Mutat Res, 1991 Jul, 255(1), 89 - 93 Theoretical and experimental measures of DNA helix stability and their relation to sequence specific repair of O6-ethylguanine lesions; Foley CK et al.; Recent work (Breslauer et al . (1986) Proc . Natl . Acad . Sci . (U.S.A.), 83, 3746) has provided a method for calculating empirical thermodynamic quantities for helix to coil transitions from the base sequence of any oligomer . It is shown in this work that the DNA helix binding energy, calculated with the AMBER force field, for 9-mers of the type 5'-GGGXGeYGGG-3', where X and Y are any base and the central Ge is O6-ethylguanine, correlates well with the empirical delta G for helix to strand transitions . The mutation spectrum of ethane methylsulfonate (EMS) in the lacI gene of Escherichia coli can be modeled using the calculated local binding energy but the empirical free energies, enthalpies and melting temperatures predict these levels of repair less well . The relation of the binding energy to the mutation spectrum can be somewhat improved by including entropic effects in a theoretical free energy of binding as given by delta G theoretical identical to delta E binding - T delta S. Mutat Res, 1991 Jul, 255(1), 39 - 48 Survival of M13mp18 gapped duplex DNA as a function of gap length; Hartke A et al.; Gaps of various lengths were generated in duplex M13mp18DNA by exonuclease III digestion of nicked DNA . The length of the gap increased essentially linearly with time of digestion . Survival in E . coli, however, was not a linear function of gap length . Similar results were obtained when gaps were produced by stopping the polymerization reaction . The survival (N/No) of the gapped DNA in SOS-induced E . coli cells transformed by electroporation and uninduced cells transformed by the calcium chloride method can be quantitatively accounted for by a kinetic model assuming a single-strand endonucleolytic activity (Pd) in the cell which increases linearly with gap length (L) and a repair activity by a polymerase (Pr) which is independent of gap length (formula 1) . With uninduced cells transformed by electroporation the results can be mathematically described if assumptions are made concerning the protection of single-stranded parts of the DNA by single-strand affinic proteins. Mutat Res, 1991 Jul, 249(1), 19 - 27 Genetic requirements for frameshift reversion induced by bulky DNA adducts in M13 DNA; Bennett CB et al.; In order to analyze the genetic requirements and mechanisms of frameshift mutagenesis by activated aflatoxin B1 (AFB1), in vitro-modified phage M13 replicative form (RF) DNA was transfected into appropriate Escherichia coli cells and +1 or -1 frameshift revertants in the lacZ(alpha) gene were isolated . This analysis shows that both +1 and -1 frameshift mutagenesis by AFB1 is significantly reduced in a umuC- background . On the other hand, in the absence of RecA, +1 frameshift mutagenesis is partially reduced, but -1 frameshift mutagenesis is unaffected . DNA sequence analysis of +1 frameshifts induced by AFB1 in recA- cells suggests that the mutations occur at the same sites as in recA+ cells, but that there are significant differences in the specificity of the observed base changes . A model consistent with the observed effects in the absence of RecA suggests that an appreciable fraction of AFB1-adducted guanines can correctly template for a cytosine. Mutat Res, 1991 Jul, 249(1), 177 - 87 Plasmid pGW16, a derivative of pKM101, increases post-UV DNA synthesis, but sensitises some strains of Escherichia coli to UV; Little CA et al.; Plasmid pKM101, which carries muc genes that are analogous in function to chromosomal umu genes, protected Escherichia coli strains AB1157 uvrB+ umuC+, JC3890 uvrB umuC+, TK702 uvrB+ umuC and TK501 uvrB umuC against ultraviolet irradiation (UV) . Plasmid pGW16, a derivative of pKM101 selected for its increased spontaneous mutator effect, also gave some protection to the UmuC-deficient strains, TK702 and TK501 . However, it sensitised the wild-type strain AB1157 to low, but protected against high doses of UV, whilst sensitising strain JC3890 to all UV doses tested . Even though its UV-protecting effects varied, pGW16 was shown to increase both spontaneous and UV-induced mutation in all strains . Another derivative of pKM101, plasmid pGW12, was shown to have lost all spontaneous and UV-induced mutator effects and did not affect post-UV survival . Plasmids pKM101 and pGW16 increased post-UV DNA synthesis in strains AB1157 and TK702, whereas pGW12 had no effect . Similarly, the wild-type UV-protecting plasmids R46, R446b and R124 increased post-UV DNA synthesis in strain TK501, but the non-UV-protecting plasmids R1, RP4 and R6K had no effect . These results accord with the model for error-prone DNA repair that requires umu or muc gene products for chain elongation after base insertion opposite non-coding lesions . They also suggest that the UV-sensitizing effects of pGW16 on umu+ strains can be explained in terms of overactive DNA repair resulting in lethal, rather than repaired UV-induced lesions. J Surg Res, 1991 Jul, 51(1), 46 - 53 Adaptive regulation of alanine transport in hepatic plasma membrane vesicles from the endotoxin-treated rat; Pacitti AJ et al.; The mechanisms by which hepatic alanine consumption is increased during endotoxemia were investigated to gain further insight into the altered amino acid metabolism which characterizes critical illness . Rats were studied 12 hr after receiving endotoxin (ENDO) or saline . Hepatic alanine delivery was determined in vivo and hepatic alanine content was measured . Hepatocyte transport activity was studied by evaluation of {3H}-alanine accumulation in hepatocyte plasma membrane vesicles (HPMVs) . Vesicle integrity was demonstrated by electron microscopy and a 14-fold enrichment in 5'-nucleotidase . Endotoxin treatment resulted in a state of hyperalaninemia and a threefold increase in hepatic alanine delivery (2.79 +/- 0.17 mu mole/100 g body weight/min in controls vs 8.13 +/- 0.98 in ENDO animals; P less than 0.001) . Data from HPMVs revealed the presence of a high- and low-affinity component of alanine transport . Endotoxin treatment resulted in a 30% decrease in the Vmax of the high-affinity transport component (3355 +/- 177 pmole/mg protein/10 sec in controls vs 2338 +/- 270 in the ENDO group; P less than 0.05) . Concomitant with the observed changes in alanine delivery and transport activity, endotoxin treatment resulted in a 56% rise in hepatic alanine content (2.53 +/- 0.29 mu mole/g liver in controls vs 3.95 +/- 0.23 in ENDO; P less than 0.005) . These data indicate that the accelerated hepatic alanine consumption which occurs during endotoxemia is primarily the result of increased hepatic substrate delivery . Despite the resultant repression of transport activity, delivery begins to outdistance the metabolic capacity of the hepatocyte to utilize alanine and intracellular alanine levels rise. J Med Chem, 1991 Jul, 34(7), 2094 - 101 Stereospecific synthesis of (R)- and (S)-S-adenosyl-1,8-diamino-3-thiooctane, a potent inhibitor of polyamine biosynthesis . Comparison of asymmetric induction vs enantiomeric synthesis; Liu C et al.; Two diastereomers of the potent spermidine synthase inhibitor S-adenosyl-1,8-diamino-3-thiooctane have been prepared in high (greater than 96% de) stereochemical purity . Two synthetic routes were investigated, one based on asymmetric induction and the other involving an enantiomeric synthesis . The latter route gave the desired products in greater than 96% de, whereas the synthesis based on asymmetric induction resulted in only 80% de in the final product . Evaluation of the two diastereomers as inhibitors of spermidine synthase showed that the R diastereomer is a more potent inhibitor than the S diastereomer. J Med Chem, 1991 Jul, 34(7), 1969 - 74 Lipopeptides containing 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid: synthesis, stereospecific stimulation of B-lymphocytes and macrophages, and adjuvanticity in vivo and in vitro; Metzger J et al.; Lipopeptides, carrying the N-terminal lipoamino acid 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid (Pam3Adh-OH, 1), were obtained by solid-phase synthesis and by synthesis in solution . 2-Amino-6,7-dihydroxyheptanoic acid (Adh) can be regarded as a methylene analogue of S-glycerylcysteine, the N-terminal amino acid of lipoprotein from the outer cell membrane of Escherichia coli (a methylene group substitutes for the sulfur atom) . The lipopeptides Pam3Adh-Ser-Ser-Asn-Ala 2a-d, in which the four possible stereoisomers of Pam3Adh-OH (2S,6S)-1 (1a), (2S,6R)-1 (1b), (2R,6S)-1 (1c), and (2R,6R)-1 (1d) are linked to the naturally occurring sequence Ser-Ser-Asn-Ala of the N-terminus of lipoprotein, and also Pam3Adh-Ser-(Lys)4 {2S,6S)-3), with a peptide part rendering the molecule water soluble, were capable of stimulating murine splenocytes polyclonally in vitro, as determined in a proliferation assay and in a hemolytic plaque assay against trinitrophenylated sheep erythrocytes . The diastereomers (2S,6S)-2 and (2R,6S)-2 with S-configurated C-6 were more active than the diastereomers (2S,6R)-2 and (2R,6R)-2 with R-configurated C-6; a change of the configuration at C-2 had less effect on the stimulatory activity . (2S,6S)-2 and (2S,6S)-3 are potent immunoadjuvants . A significantly enhanced primary immune response against trinitrophenylated sheep erythrocytes was obtained in vitro at lipopeptide concentrations of about 5 micrograms/mL and an immunization dose of 10(7) sheep erythrocytes/mL . Balb/c mice, which were immunized with a mixture of ovalbumin and (2S,6S)-2 or (2S,6S)-3, respectively, had a substantially higher antiovalbumin titer 28 days after immunization than mice which had received ovalbumin, (2S,6S)-2 or (2S,6S)-3 alone . Finally, the novel lipopeptides constitute potent macrophage activators: (2S,6S)-3 was able to induce tumor cytotoxicity against the tumor cell line L929 in bone marrow derived macrophages. J Bacteriol, 1991 Jul, 173(14), 4537 - 9 The hemimethylated replication origin of Escherichia coli can be initiated in vitro; Boye E; Unmethylated, fully methylated, and hemimethylated oriC-containing plasmids were assayed as substrates for DNA replication in vitro by using a system reconstituted with pure proteins . In contrast to the in vivo situation, all three substrates were initiated efficiently; the fully methylated plasmid was about twice as active as the other two. J Bacteriol, 1991 Jul, 173(14), 4424 - 32 Mutational analysis of nitrate regulatory gene narL in Escherichia coli K-12; Egan SM et al.; The narL gene product, NarL, is the nitrate-responsive regulator of anaerobic respiratory gene expression . We used genetic analysis of narL mutants to better understand the mechanism of NarL-mediated gene regulation . We selected and analyzed seven nitrate-independent narL mutants . Each of three independent, strongly constitutive mutants had changes of Val-88 to Ala . The other four mutants were weakly constitutive . The narL505(V88A) allele was largely dominant to narL+, while narX+ had a negative influence on its constitutive phenotype, suggesting that NarX may play a negative role in nitrate regulation . We also constructed two narL mutations that are analogous to previously characterized constitutive degU alleles . The first, narL503(H15L), was a recessive null allele . The second, narL504(D110K), functioned essentially as wild type but was dependent on narX+ for full activity . We changed Asp-59 of NarL, which corresponds to the site of phosphorylation of other response regulators, to Asn . This change, narL502(D59N), was a recessive null allele, which is consistent with the hypothesis that NarL requires phosphorylation for activation . Finally, we tested the requirement for molybdate on regulation in a narL505(V88A) strain . Although narL505(V88A) conferred some nitrate-independent expression of fdnGHI (encoding formate dehydrogenase-N) in limiting molybdate, it required excess molybdate for full induction both in the absence and in the presence of nitrate . This finding suggests that narL505(V88A) did not confer molybdate-independent expression of fdnGHI. J Bacteriol, 1991 Jul, 173(14), 4404 - 10 Role of heat shock protein DnaK in osmotic adaptation of Escherichia coli; Meury J et al.; Escherichia coli can adapt and recover growth at high osmolarity . Adaptation requires the deplasmolysis of cells previously plasmolyzed by the fast efflux of water promoted by osmotic upshift . Deplasmolysis is essentially ensured by a net osmo-dependent influx of K+ . The cellular content of the heat shock protein DnaK is increased in response to osmotic upshift and does not decrease as long as osmolarity is high . The dnaK756(Ts) mutant, which fails to deplasmolyze and recover growth, does not take up K+ at high osmolarity; DnaK protein is required directly or indirectly for the maintenance of K+ transport at high osmolarity . The temperature-sensitive mutations dnaJ259 and grpE280 do not affect the osmoadaptation of E . coli at 30 degrees C. J Bacteriol, 1991 Jul, 173(14), 4288 - 96 Subcellular localization and immunological detection of proteins encoded by the vir locus of Bordetella pertussis; Stibitz S et al.; The DNA sequence of the central regulatory locus vir of Bordetella pertussis predicts that three gene products, BvgA, BvgB, and BvgC, are encoded . Features of the predicted primary structures of these proteins and their homology to other two-component systems suggest that BvgA is located in the cytoplasm, BvgB is located in the periplasm, and BvgC spans the inner membrane . We have used gene fusions to the phoA and lacZ genes of Escherichia coli to investigate the subcellular localization and membrane topology of these proteins . PhoA fusion proteins were also purified and used to raise antibodies that allowed visualization of the vir-encoded polypeptides by Western immunoblotting . Our results have largely confirmed the predictions of the DNA sequence, with the exception that BvgB and BvgC were found to constitute one larger protein that was homologous to the sensor class of two-component systems . We propose that this protein be named BvgS (for sensor) and that its gene be named bvgS. J Bacteriol, 1991 Jul, 173(14), 4254 - 62 ClpB is the Escherichia coli heat shock protein F84.1; Squires CL et al.; ClpB is thought to be involved in proteolysis because of its sequence similarity to the ClpA subunit of the ClpA-ClpP protease . It has recently been shown that ClpP is a heat shock protein . Here we show that ClpB is the Escherichia coli heat shock protein F84.1 . The F84.1 protein was overproduced in strains containing the clpB gene on a plasmid and was absent from two-dimensional gels from a clpB null mutation . Besides possessing a slower growth rate at 44 degrees C, the null mutant strain had a higher rate of death at 50 degrees C . We used reverse transcription of in vivo mRNA to show that the clpB gene was expressed from a sigma 32-specific promoter consensus sequence at both 37 and 42 degrees C . We noted that the clpB+ gene also caused the appearance of a second protein spot, F68.5, on two-dimensional gels . This spot was approximately 147 amino acids smaller than F84.1 and most probably is the result of a second translational start on the clpB mRNA . F68.5 can be observed on many published two-dimensional gels of heat-induced E . coli proteins, but the original catalog of 17 heat shock proteins did not include this spot. Eur J Biochem, 1991 Jul 1, 199(1), 47 - 51 Properties of recombinant NADP-malate dehydrogenases from Sorghum vulgare leaves expressed in Escherichia coli cells; Jacquot JP et al.; In this study, a cDNA clone coding for sorghum leaf NADP-malate dehydrogenase {Cretin, C., Luchetta, P., Joly, C., Decottignies, P., Lepiniec, L., Gadal, P., Sallantin, M., Huet, J . C . & Pernollet, J . C . (1990) Eur . J . Biochem . 192, 299-303} was used either in the full-length form or in a shorter form deprived of the 5' end coding for the transit peptide . Both cDNA fragments were cloned into the expression vector pKK233-2 and the resulting constructions were used to transform E . coli cells . The bacterial cells which do not contain any NADP-dependent malate dehydrogenase before transformation were able to express the protein after transformation and induction, as detected both by activity measurements and by immunoblot . The recombinant proteins could be purified to homogeneity and their biochemical characteristics studied . They were identical to those of the enzyme isolated from corn or sorghum leaves, including the well known redox regulatory properties . The NADP-malate dehydrogenases derived from both constructions had a similar subunit size and the analysis of their N-terminal sequences revealed that E . coli cells were able to recognize the processing signal of the precursor polypeptide and to mature and assemble the protein in a manner similar to higher plants. Biochim Biophys Acta, 1991 Jul 1, 1066(1), 21 - 8 Escherichia coli membranes during electrotransformation: an electron microscopy study; Sabelnikov AG et al.; Structural changes undergone by Escherichia coli cell envelope membranes under the conditions of electrically induced gene (DNA) transfer (exponential pulse of about 13 kV/cm, tau = 5 ms) were studied by freeze-fracture electron microscopy . Special device similar to that of Stenger and Hui {1986) J . Membr . Biol . 93, 43-53), that allowed cryofixation of samples almost simultaneously with application of electric pulse, was employed to examine the cells within a short time (less than or equal to 1 s) after the pulse . Extensive blebbing of cells was observed immediately after the pulse . At later times (30-40 s after the pulse) blebbing was not detected, instead infrequent cellular membrane fusion and formation of large membrane 'opening' or pores were observed . An attempt to relate the observed membrane changes with cellular viability and permeability to exogenous DNA failed . Challenge of cells with a plasmid DNA 10 s after the pulse application resulted in a dramatic loss (at least four orders of magnitude) of the number of transformants compared to cells pulsed in the presence of DNA . On the other hand the results on additional pulsing of cell prior to the main electrotransformation procedure suggested that the life-time of membrane defects is at least no less than 2 min . Possible ways to reconcile the results are suggested. Am Rev Respir Dis, 1991 Jul, 144(1), 173 - 8 Lipopolysaccharide-induced pulmonary vascular sequestration of polymorphonuclear leukocytes is complement independent; Cardozo C et al.; Lipopolysaccharide (LPS) injected intravenously produces leukopenia and sequestration of polymorphonuclear leukocytes (PMN) in the pulmonary vascular bed . To evaluate the role of complement in this process, we used C5-sufficient (B10.D2/nSn) and C5-deficient (B10.D2/oSn) mice and Sprague-Dawley rats depleted of complement with Naja naja cobra venom factor (CVF) . We found a comparable increase in the number of PMN in lung tissue of C5-sufficient and C5-deficient mice given Escherichia coli LPS (0127:B8, 3 mg/kg), revealing that LPS acts independently of C5 and its biologically active fragments . Intravenous injection of LPS (3 mg/kg) into rats caused significant intravascular complement activation as assessed by serum CH50 and resulted in an almost 10-fold increase in numbers of PMN in lung tissue . Pretreatment of rats with CVF (50 U) did not reduce LPS-induced PMN sequestration, suggesting that the process is independent of C3 . As reported previously, we found large numbers of PMN in bronchoalveolar lavage samples of 24 h after injection of LPS (3 mg/kg) . Complement depletion did not prevent LPS-induced migration of PMN . No PMN migration occurred 2, 6, 12, 24, or 48 h after injection of CVF alone, indicating that complement activation is not sufficient to cause PMN migration . In contrast to our findings in rats, no PMN migrated into airspaces of C5-sufficient and C5-deficient mice 24 or 48 h after injection of LPS (3 to 20 mg/kg). Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5882 - 6 Adaptive evolution that requires multiple spontaneous mutations: mutations involving base substitutions; Hall BG; A previous study has demonstrated that adaptive missense mutations occur in the trp operon of Escherichia coli . In this study it is shown that, under conditions of intense selection, a strain carrying missense mutations in both trpA and trpB reverts to Trp+ 10(8) times more frequently than would be expected if the two mutations were the result of independent events . Comparison of the single mutation rates with the double mutation rate and information obtained by sequencing DNA from double revertants show that neither our classical understanding of spontaneous mutation processes nor extant models for adaptive mutations can account for all of the observations . Despite a current lack of mechanistic understanding, it is clear that adaptive mutations can permit advantageous phenotypes that require multiple mutations to arise and that they appear enormously more frequently than would be expected. Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5799 - 803 Conformational and membrane-binding properties of a signal sequence are largely unaltered by its adjacent mature region; McKnight CJ et al.; We have synthesized a peptide corresponding to the 25-residue signal sequence plus the first 28 residues of the Escherichia coli outer membrane protein LamB in order to explore the properties of a signal sequence in the presence of the N-terminal region of its passenger . In the last few years, there have been several observations of differing efficiencies of export when signal sequences are attached to different passenger proteins or when the first part of a passenger protein undergoes mutation . In the LamB case, gene fusions with lacZ have shown that the signal sequence plus the first 28 residues of mature LamB are necessary to direct beta-galactosidase into the export pathway {Rasmussen, B . A . & Silhavy, T . J . (1987) Genes Dev . 1, 185-196} . The origin of these observations and whether there is an influence of the mature region on the properties of the signal sequence have not been known . We find that the conformational and membrane-binding properties of the LamB signal sequence manifest in a 25-residue peptide are essentially unaltered in the context of the 53-residue peptide corresponding to this signal sequence plus the first 28 residues of the mature LamB protein . CD spectra show that the signal peptide and passenger domains are conformationally independent of each other in micelle or bilayer environments . Furthermore, the signal sequence leads to the spontaneous association of the 53-residue peptide with a lipid bilayer; alone, the mature domain does not interact with lipid bilayers . Fluorescence results show that the mode of interaction of the signal peptide with a bilayer is essentially unaltered by the presence of its mature region . This lack of influence of the mature domain on the behavior of the signal sequence is unexpected for juxtaposed polypeptides of comparable length and may be of physiological importance: N-terminal regions of secreted proteins may be selected to be passive, by comparison with their cognate signal sequences, which themselves must engage the export apparatus and actively interact with its components. Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5729 - 33 A phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of Caenorhabditis elegans; Maruyama IN et al.; Mutations in the unc-13 gene cause diverse defects in the nervous system of the nematode Caenorhabditis elegans . Molecular cloning of the gene and sequencing of the cDNA revealed that the product encodes a protein, 1734 amino acids in length, with a central domain with sequence similarity to the regulatory region of protein kinase C . The domain was expressed in Escherichia coli and shown to bind specifically to a phorbol ester in the presence of calcium; diacylglycerol inhibited the binding in a competitive manner . These findings confirm that the unc-13 gene product has binding sites similar to those of protein kinase C and may be a component of an alternative transduction pathway of the diacylglycerol signal to a different effector function in the nervous system. J Bacteriol, 1991 Jul, 173(13), 4188 - 94 The putative sigma factor KatF has a central role in development of starvation-mediated general resistance in Escherichia coli; McCann MP et al.; KatF is required for the expression of some 32 carbon starvation proteins in Escherichia coli including 6 previously identified as Pex . Mutants with the katF gene survive carbon and nitrogen starvation poorly . Many of the KatF-regulated starvation proteins are common to those induced by other stresses, and the mutant failed to develop starvation-mediated cross protection to osmotic, oxidative, and heat stresses . Furthermore, thermal resistance was not induced in the mutant by heat preadaptation, and it exhibited an altered pattern of protein synthesis at elevated temperature . Thus, KatF is a major switch that controls the starvation-mediated resistant state in E . coli. J Bacteriol, 1991 Jul, 173(13), 4133 - 43 Regulation of expression of the Escherichia coli K-12 mtr gene by TyrR protein and Trp repressor; Sarsero JP et al.; The Escherichia coli K-12 mtr gene, which encodes a tryptophan-specific permease, was cloned, and its nucleotide sequence was determined . The precise location of the mtr gene at 69 min on the E . coli chromosome was determined . The mtr gene product was identified as a 414-amino-acid residue protein with a calculated molecular weight of 44,332 . The protein is very hydrophobic, consistent with its presumed location spanning the cytoplasmic membrane . The initiation sites of transcription and translation were identified . Construction of an mtr-lacZ transcriptional fusion facilitated investigation of the molecular basis of mtr regulation . The TyrR protein in association with phenylalanine or tyrosine is responsible for the activation of mtr expression, whereas the Trp repressor in conjunction with tryptophan serves to repress expression of this gene . Site-directed mutagenesis confirmed that sequences in the mtr regulatory region homologous to TyrR protein and to Trp repressor-binding sites were involved in the activation and repression of mtr expression, respectively . Sequences homologous to sigma 70- and sigma 54-dependent promoters were identified upstream of the transcription start point of mtr . It was determined that transcription of mtr occurs only via a sigma 70-dependent promoter. J Bacteriol, 1991 Jul, 173(13), 4049 - 55 Mutant MotB proteins in Escherichia coli; Blair DF et al.; The MotB protein of Escherichia coli is an essential component in each of eight torque generators in the flagellar rotary motor . Based on its membrane topology, it has been suggested that MotB might be a linker that fastens the torque-generating machinery to the cell wall . Here, we report the isolation and characterization of a number of motB mutants . As found previously for motA, many alleles of motB were dominant, as expected if MotB is a component of the motor . In other respects, however, the motB mutants differed from the motA mutants . Most of the mutations mapped to a hydrophilic, periplasmic domain of the protein, and nothing comparable to the slow-swimming alleles of motA, which show normal torque when tethered, was found . Some motB mutants retained partial function, but when tethered they produced subnormal torque, indicating that their motors contained only one or two functional torque generators . These results support the hypothesis that MotB is a linker. J Leukoc Biol, 1991 Jul, 50(1), 77 - 85 Effect of chronic endotoxemia on concanavalin A-stimulated inositol lipid metabolism in rat splenocytes; Hagar AF et al.; The effect of a chronic, nonlethal infusion of endotoxin (ET) on the phosphatidylinositol (PI) cycle in rat splenocytes was evaluated . Rats were infused intravenously with sterile saline or Escherichia coli ET (0.1 mg/100 g body weight/24 hr) for 30 hr via subcutaneously implanted osmotic pumps . The splenocytes were then labeled in vitro for 90 min with {32P}PO4 or for 3 hr with {3H}myoinositol to assess the status of the resynthesis and degradative parts of the PI cycle, respectively . PI cycle activity was depressed in splenocytes of ET-infused rats as evidenced by a 25% reduction in the incorporation of {32P}PO4 into phosphatidic acid (PA), PI, and the polyphosphoinositides and by a similar decrease in the production of {3H}inositol phosphates . Stimulation of splenocytes with concanavalin A (Con A) resulted in dose-dependent increases in the incorporation of {32P}PO4 into PA and PI and in the production of {3H}inositol phosphates, indicating that Con A stimulates the PI cycle in these cells . The Con A-stimulated increase in inositol phosphate production was higher in splenocytes from ET-infused rats . We have previously shown that splenocytes from rats infused for 30 hr with ET exhibit a decreased blastogenic responsiveness to Con A and lipopolysaccharide {Spitzer et al., Proc . Soc . Exp . Biol . Med . 186,27, 1987} . The present data do not support the notion that inositol lipid-mediated signaling mechanisms are solely responsible for the expression of the appropriate functional response and suggest that in ET-infused rats there is an uncoupling of the initial response to Con A (i.e., the production of inositol lipid-derived second messengers) and the long-term (i.e., mitogenic) response. J Clin Invest, 1991 Jul, 88(1), 93 - 100 Mechanism of mammalian cell lysis mediated by peptide defensins . Evidence for an initial alteration of the plasma membrane; Lichtenstein A; Defensins induce ion channels in model lipid bilayers and permeabilize the membranes of Escherichia coli . We investigated whether similar membrane-active events occur during defensin-mediated cytolysis of tumor cells . Although defensin-treated K562 targets did not release chromium-labeled cytoplasmic components for 5-6 h, they experienced a rapid collapse (within minutes) of the membrane potential, efflux of rubidium, and influx of trypan blue . Defensin treatment also blunted the subsequent acidification response induced by nigericin, thereby further supporting the notion of enhanced transmembrane ion flow during exposure . These initial effects on the plasma membrane were not sufficient for subsequent lysis; a second phase of injury was required which involved the continued presence of defensin . The rapid membrane permeabilization phase was inhibited by azide/2-deoxyglucose, cytochalasin B, and increased concentrations of extracellular potassium and was unaffected by actinomycin-D, cycloheximide, and varying the calcium concentration . In contrast, the second phase was unaffected by cytochalasin B, inhibited by azide/2-deoxyglucose, enhanced by actinomycin D and cycloheximide, and varied with calcium concentration . These results indicate the initial adverse effect of defensins on mammalian cells occurs at the cell membrane . It is possible that the second phase of injury is mediated intracellularly by defensin that has been internalized through this leaky membrane. Crit Care Med, 1991 Jul, 19(7), 955 - 62 Lung fluid dynamics and supply dependency of oxygen uptake during experimental endotoxic shock and volume resuscitation; D'Orio V et al.; BACKGROUND AND METHODS: We studied the effect of volume resuscitation on lung fluid balance and systemic oxygen extraction during septic shock in eight anesthetized dogs . Sepsis was induced using a 2-hr continuous infusion of Escherichia coli endotoxin at 0.25 micrograms/min.kg . Relationships between oxygen uptake (VO2) and oxygen supply (DO2) were performed acutely during stepwise controlled decrements in cardiac output by progressive inflation of an intracardiac balloon . At each stage, DO2 and corresponding VO2 were measured independently and the individual critical DO2 level was referred to as the point below which the relationship held . The slope of such a constructed relationship was defined as the maximal oxygen extraction ratio . Lung fluid balance was assessed by measurements of extravascular lung water . All values were studied at baseline, after endotoxin insult, and after reversing hypotension by a 10% dextran infusion . RESULTS: Endotoxin infusion led to a shock state that associated hypotension (from 135 to 63 mm Hg) with increases in blood lactate (from 0.53 to 3.9 mmol/L) . The mean critical DO2 and maximal oxygen extraction ratio were significantly altered from 7.9 to 17.8 mL/min.kg and from 0.81 to 0.38, respectively . After reversing hypotension by 28 mL/kg colloid infusion, the critical DO2 (11.4 mL/min.kg) and maximal oxygen extraction ratio (0.48) were significantly improved . However, restoration of normal values required a state of fluid overload by further dextran infusion (8 mL/kg) . At the end of the fluid challenge, extravascular lung water significantly increased from 6.4 to 17.4 mL/kg . CONCLUSIONS: These data suggest that volume loading may reverse endotoxin-induced peripheral perfusion abnormalities . However, substantial pulmonary edema may occur, possibly jeopardizing the beneficial effects of fluid expansion. Virology, 1991 Jul, 183(1), 151 - 9 Biochemical and biological comparison of HIV-1 NEF and ras gene products; Nebreda AR et al.; Human immunodeficiency virus type 1 (HIV-1) NEF protein has been reported to share certain biochemical and structural properties with known oncoproteins like src or rats . To determine whether this is a general property of NEF from various HIV isolates, three different NEF proteins were expressed in Escherichia coli using a thermoinducible expression system previously exploited to overproduce functionally active p21 ras proteins . ras and NEF proteins expressed in this manner were evaluated in parallel to compare their biochemical and biological properties . In contrast to ras, our NEF protein preparations had no detectable GTP binding but showed autophosphorylation activity when incubated in the presence of either GTP or ATP . This putative autokinase activity was higher in NEF proteins containing threonine at position 15 than in those carrying alanine at that position . Two different NEF genes also failed to induce oncogenic transformation of permanently transfected NIH 3T3 cells under conditions that led to oncogenic transformation using activated ras genes . Also, unlike ras, the NEF gene products failed to induce meiotic maturation when injected into fully grown Xenopus oocytes. J Immunol, 1991 Jul 1, 147(1), 312 - 9 Sequence and immunogenicity of the 70-kDa heat shock protein of Mycobacterium leprae; McKenzie KR et al.; The gene encoding the Mycobacterium leprae 70-kDa heat shock protein has been isolated from a cosmid library using a fragment of the clone JKL2 . Southern blot analysis of a positive clone identified a 4.4-kb fragment containing the entire coding region of the gene plus 2.4 kb upstream . Sequencing revealed the gene to encode a 621-amino acid protein, bearing 56% identity with the Escherichia coli dnaK gene product and 47% and 46% identity with the human and Caenorhabditis elegans hsp70, respectively . Comparison with the C-terminal 203 amino acids of the Mycobacterium tuberculosis 71-kDa Ag yielded 70% identity . Recombinant M . leprae p70 was produced in E . coli as a fusion protein (rp70f) with a portion of the schistosomal glutathione-S-transferase, using the expression vector, pGEX-2T . Cleavage with thrombin resulted in the release of a 70.0-kDa protein (rp70c) from the glutathione-S-transferase . Examination of the proteins by immunoblotting demonstrated that anti-M . leprae mAb, L7, and sera from lepromatous leprosy patients bound to both the cleaved and fusion proteins . We compared the T cell reactivity of the M . leprae recombinant proteins with that of mAb affinity-purified bacille Calmette-Guerin (BCG) 70-kDa Ag using proliferation assays . PBMC of BCG vaccinees responded to both M . leprae cleaved and fusion p70, though more subjects responded to the rp70c (18 of 20) than to rp70f (13 of 20) . Responses were generally higher to rp70c than to rp70f, however all responses to the M . leprae recombinant proteins were lower than to mAb affinity-purified BCG p70 . Thus, the M . leprae 70-kDa heat shock protein elicits T and B cell responses in subjects exposed to mycobacteria, despite its homology with the human hsp70. J Cell Biol, 1991 Jul, 114(1), 83 - 99 Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a central alpha-helical coiled coil; Wiche G et al.; We have determined the complete cDNA sequence of rat plectin from a number of well-characterized overlapping lambda gt11 clones . The 4,140-residue predicted amino acid sequence (466,481 D) is consistent with a three-domain structural model in which a long central rod domain, having mainly an alpha-helical coiled coil conformation, is flanked by globular NH2- and COOH-terminal domains . The plectin sequence has a number of repeating motifs . The rod domain has five subregions approximately 200-residues long in which there is a strong repeat in the charged amino acids at 10.4 residues that may be involved in association between plectin molecules . The globular COOH-terminal domain has a prominent six-fold tandem repeat, with each repeat having a strongly conserved central region based on nine tandem repeats of a 19-residue motif . The plectin sequence has several marked similarities to that of desmoplakin (Green, K . J., D . A . D . Parry, P . M . Steinert, M . L . A . Virata, R . M . Wagner, B . D . Angst, and L.A . Nilles . 1990 . J . Biol . Chem . 265:2,603-2,612), which has a shorter coiled-coil rod domain with a similar 10.4 residue charge periodicity and a COOH-terminal globular domain with three tandem repeats homologous to the six found in plectin . The plectin sequence also has homologies to that of the bullous pemphigoid antigen . Northern blot analysis indicated that there is a significant degree of conservation of plectin genes between rat, human, and chicken and that, as shown previously at the protein level, plectin has a wide tissue distribution . There appeared to be a single rat plectin gene that gave rise to a 15-kb message . Expression of polypeptides encoded by defined fragments of plectin cDNA in E . coli has also been used to localize the epitopes of a range of monoclonal and serum antibodies . This enabled us to tentatively map a sequence involved in plectin-vimentin and plectin-lamin B interactions to a restricted region of the rod domain. EMBO J, 1991 Jul, 10(7), 1853 - 62 TFIID is required for in vitro transcription of the human U6 gene by RNA polymerase III; Simmen KA et al.; We present evidence that transcription factor TFIID, known for its central role in transcription by RNA polymerase II, is also involved in RNA polymerase III transcription of the human U6 snRNA gene . Recombinant human TFIID, expressed either via a vaccinia virus vector in HeLa cells or in Escherichia coli, affects U6 transcription in three different in vitro assays . First, TFIID-containing fractions stimulate U6 transcription in reactions containing rate-limiting amounts of HeLa nuclear extract . Second, TFIID addition relieves transcriptional exclusion between two competing U6 templates . Third, TFIID can replace one of two heat labile fractions essential for U6 transcription . Thus, at least one basal transcription factor is involved in transcription by two different RNA polymerases. J Virol, 1991 Jul, 65(7), 3715 - 20 A transcription factor for expression of vaccinia virus late genes is encoded by an intermediate gene; Wright CF et al.; A factor, designated VLTF-1, that is required in vitro for specific transcription of vaccinia virus late genes was previously isolated from vaccinia virus-infected cells . Subsequent genetic experiments identified three vaccinia virus genes, encoding proteins of 17, 26, and 30 kDa, that together trans activate late gene expression in vivo . The purpose of this study was to determine whether VLTF-1 corresponded to one of the three trans activators . Toward this end, VLTF-1 was further purified, the trans-activator genes were expressed in Escherichia coli, and antisera were made to the native and recombinant proteins . Antibody to the 30-kDa recombinant protein reacted on Western immunoblots with a protein of approximately Mr 30,000 that cochromatographed and cosedimented with VLTF-1 activity from virus-infected cells . Conversely, antibody to purified VLTF-1 bound to products produced by in vitro transcription and translation of the open reading frame encoding the 30-kDa trans-activator protein . Both antisera depleted VLTF-1 activity and blocked late gene transcription by partially purified extracts of vaccinia virus-infected cells . Taken together, these data demonstrate that the 30-kDa trans activator comprises part, if not all, of VLTF-1 activity. Res Microbiol, 1991 Jul-Aug, 142(6), 623 - 31 Isolation and characterization of intragenic suppressors of an Escherichia coli ftsA mutation; Robinson AC et al.; Mutagenesis of a strain of Escherichia coli carrying a temperature-sensitive (Ts) mutation in the cell division gene ftsA yielded a number of temperature-resistant variants . In certain cases, restoration of viability at the restrictive temperature could not be attributed to suppressor mutations occurring in other genes or to structural gene reversion . DNA sequencing of the variants demonstrated the continuing presence of the original Ts mutation (ftsA13) and revealed secondary mutations within the same gene . These secondary mutations are able to rescue the ftsA13 mutation in cis, but not in trans. J Gen Microbiol, 1991 Jul, 137 ( Pt 7), 1529 - 36 Molecular analysis of a Leptospira borgpetersenii gene encoding an endoflagellar subunit protein; Mitchison M et al.; A flagellin gene, flaB, from Leptospira borgpetersenii (formerly L . interrogans) serovar hardjo was cloned and expressed in Escherichia coli . Expression of the 32 kDa FlaB protein was dependent upon the lacZ promoter from pUC18 . Nucleotide sequence data showed an open reading frame encoding 283 amino acid residues, corresponding to a protein of molecular mass 31.3 kDa . The G + C content of the flaB gene was 54.7 mol% . Comparison of the deduced FlaB amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum FlaB proteins. Biotechniques, 1991 Jul, 11(1), 18, 20, 22 - 4 Purification of plasmid and high molecular mass DNA using PEG-salt two-phase extraction; Cole KD; A method for the rapid preparation of DNA is described . The method utilizes a polymer (polyethylene glycol) and salt solution to form a two-phase system . A crude source of DNA is added to a phase-forming mixture, it is mixed and phase separation occurs . Under the appropriate conditions, the nucleic acids remain in the lower (salt-rich) phase, while the proteins, cellular debris and other constituents are in the upper phase (polymer-rich) or are precipitated at the interphase region . Incorporation of protein denaturants (detergents and chaotropes) stop the action of liberated nucleases in the sample . The nucleic acids are obtained in an intact state and in a form suitable for further manipulation, as shown by gel electrophoresis and DNA restriction digestion . This method describes the conditions of the two-phase systems that are important for the separation of nucleic acids and proteins . The important phase-forming conditions shown in this paper are pH, polymer molecular weight and concentration, salt type and concentration and the addition of detergents and chaotropic agents . With the use of these extraction conditions, proteins can be moved selectively from the lower to the upper phase . The paper describes a method for DNA isolation that is rapid, simple and economical. Biotechniques, 1991 Jul, 11(1), 14 - 6 Chemiluminescent substrates increase sensitivity of antigen detection in western blots; Sandhu GS et al.; Replacement of colorimetric substrates in Western blots by chemiluminescent reagents enhances the sensitivity of detection by more than an order of magnitude . Protein levels beyond the detection limit of colorimetric substrates can be consistently detected without modifying pre-existing laboratory protocols. Zentralbl Veterinarmed A, 1991 Jul, 38(6), 445 - 59 Role of eicosanoids in abortion and its prevention by treatment with flunixin meglumine in cows during the first trimester of pregnancy; Giri SN et al.; Intravenous infusion of E . coli endotoxin at a rate of 4.16 ng/kg/min over 6 hr (total dose 1.5 micrograms/kg) in 5 cows in the first trimester of gestation induced abortion between 60 and 72 hr in three cows . Plasma PGF2 alpha levels in the aborting cows increased significantly to 289% of the zero time control (ZTC) at 1 hr and remained elevated for 9 hr . The PGF2 alpha level remained unaffected in the non-aborting cows except at 2 hr . The plasma TxB2 levels were increased by 6 to 18 fold for 6 hr in both the aborting and non-aborting cows relative to their ZTC controls . The 6-keto-PGF1 alpha levels were significantly increased to 2 to 3 fold only in the aborting cows . Plasma cortisol levels were increased maximally to 1,500% of ZTC at 5 hr in the aborting cows . Thereafter, the levels gradually declined but remained significantly elevated for 24 hr . The increases in the cortisol levels in the non-aborting cows were only 280% of ZTC at 5 hr and returned to ZTC value by 12 hr . Plasma progesterone levels in the aborting cows remained unaffected until 12 hr followed by a progressive decline through 18 hr to extremely low levels at 3, 4, and 5 days . Endotoxin-infusion caused hyperglycemia in both aborting and non-aborting cows and lactic acidemia in the aborting cows . Treatment with two doses of flunixin meglumine (FM, 1.1 mg/kg), an inhibitor of cyclooxygenase, 1 hr prior to endotoxin infusion and then 13 hr later, completely prevented the endotoxin-induced abortion and increases in the plasma PGF2 alpha, TxB2 and 6-keto-PGF1 alpha concentrations . The PGE level remained unaffected . Although FM treatment failed to abolish endotoxin-induced increases in the plasma cortisol and lactic acid levels, it effectively prevented marked decreases in the progesterone and increases in the glucose concentrations . It was concluded that the use of FM offers therapeutic promise in preventing bovine abortion caused by endotoxin resulting from bacterial infection during the 1st trimester of gestation. Vet Q, 1991 Jul, 13(3), 155 - 62 The role of endotoxin in the pathogenesis of acute bovine laminitis; Boosman R et al.; To study the possible role of endotoxin in the pathogenesis of bovine laminitis, local and systemic injections of endotoxin (E . coli 0111 B4) with different doses were given to three groups of four cows each . Clinical and haematologic parameters indicated an acute-phase response, including positive plasma ethanol gelation (soluble fibrin), the occurrence of fibrin degradation products and decreased thrombocyte counts . Local Shwartzman reactions were not evoked . Clinical examination of the claws and the gait of the animals revealed no signs of laminitis . However, on histopathological examination of the claw corium signs of laminitis such as vacuolisation of the Stratum basale, lymphocyte and leucocyte infiltration and thrombosis were found . These results indicate that endotoxin indeed may be involved in the pathogenesis of laminitis . For the development of a clinical acute laminitis model in cattle either another dosage, other toxins or factors in addition to the endotoxin used in this experiment are needed. Mutagenesis, 1991 Jul, 6(4), 271 - 8 Mutagenic activity of aziridinyl steroids and their mechanism of action in biological systems; Islam S et al.; The aziridinyl derivatives of steroids, structurally related to cholesterol, were tested for their mutagenic activity in the Ames tester strains . The test compounds were mutagenic without metabolic activation, although metabolic activation markedly enhanced their activity . A significant decrease in the survival of SOS defective mutants, recA and lexA of Escherichia coli was observed as compared with their wild-type counterpart in the presence of the steroids . The role of SOS repair genes gains further support from the lambda prophage induction in the lysogen and beta-galactosidase induction in the Mud strains as well as mutagenesis with Ames tester strains . Structural features which appear essential for mutagenic activity in these strains are (i) a reactive N-aminophthalamide group at the (5,6-b) position and (ii) an acetoxy/chlorine group at the third position of the steroidal nucleus . The individual moieties/groups were not mutagenic per se . These steroids appear to generate H2O2 as well as superoxide and hydroxyl radicals in the model biological system. Mol Microbiol, 1991 Jul, 5(7), 1801 - 10 Location of the RNA-processing enzymes RNase III, RNase E and RNase P in the Escherichia coli cell; Miczak A et al.; Cells overexpressing the RNA-processing enzymes RNase III, RNase E and RNase P were fractionated into membrane and cytoplasm . The RNA-processing enzymes were associated with the membrane fraction . The membrane was further separated to inner and outer membrane and the three RNA-processing enzymes were found in the inner membrane fraction . By assaying for these enzymatic activities we showed that even in a normal wild-type strain of Escherichia coli these enzymes fractionate primarily with the membrane . The RNA part of RNase P is found in the cytosolic fraction of cells overexpressing this RNA, while the overexpressed RNase P protein sediments with the membrane fraction; this suggests that the RNase P protein anchors the RNA catalytic moiety of the enzyme to a larger entity . The implications of these findings for the cellular organization of the RNA-processing enzymes in the cell are discussed. Mol Microbiol, 1991 Jul, 5(7), 1791 - 9 Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE-dependent endonucleolytic processing; Nilsson P et al.; Differential expression of the genes expressing Pap pili in Escherichia coli was suggested to involve mRNAs with different stabilities . As the result of a post-transcriptional processing event, a papA gene-specific mRNA product (mRNA-A) accumulates in large excess relative to the primary mRNA-BA transcript . Our results show that the processed product, mRNA-A, is a translationally active molecule and that it is generated from the mRNA-BA precursor by an RNaseE-dependent mechanism . The processing did not occur under non-permissive conditions in an E . coli rne mutant strain with a temperature-sensitive RNaseE . The endonuclease RNaseE was previously described as being chiefly involved in the processing of the 9S precursor of 5S rRNA . A comparison of nucleotide sequences of mRNA-BA and three other RNAs processed by RNAseE revealed a conserved motif around the cleavage sites . Mutations abolishing the activity of either of two other endoribonucleases, RNaseIII and RNaseP, did not affect the pap mRNA processing event . However, a conditional mutation in the ams locus, causing altered stability of bulk mRNA in E . coli, led to reduced pap mRNA processing in a manner similar to the effect caused by RNaseE deficiency . Our findings are consistent with the idea that ams is related/allelic to rne . Absence of the processing event in the RNaseE mutant (rne-3071) strain led to a four-fold stabilization of the mRNA-BA primary transcript . We conclude that the RNaseE-dependent processing event is the rate-limiting step in the decay of the papB-coding part of the primary transcript and in the production of the stable mRNA-A product.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Microbiol, 1991 Jul, 5(7), 1779 - 90 Characterization of the oriT region of the IncFV plasmid pED208; Di Laurenzio L et al.; DNA sequence analysis of a 2.2kb EcoRI-HindIII fragment from pED208, the derepressed form of the IncFV plasmid Folac, revealed sequences highly homologous to the oriT region, traM, and traJ genes of other IncF plasmids . The TraM protein was purified and immunoblots of fractionated cells containing pED208 or Folac showed that TraM was predominantly in the cytoplasm . Using DNA retardation assays and the DNase I footprinting technique, the TraM protein was found to bind to three large motifs in the oriT region: (I) an inverted repeat, (II) two direct repeats, and (III) the traM promoter region . These three footprint regions contained a Hinfl-like sequence (GANTC) that appeared 16 times, spaced 11-12 bp (or multiples thereof) apart, suggesting that TraM protein binds in a complex manner over this entire region. Mol Microbiol, 1991 Jul, 5(7), 1681 - 6 Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation; Pla J et al.; An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild-type copy of the gene in a thermosensitive plasmid . Under conditions in which the ftsZ+ allele is unable to be replicated at the same pace as the chromosome, the cells become non-viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival . Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell . Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm. Mol Microbiol, 1991 Jul, 5(7), 1639 - 48 The gef gene from Escherichia coli is regulated at the level of translation; Poulsen LK et al.; We describe post-transcriptional regulation of the chromosomal gene, gef, from Escherichia coli . The gef gene is a member of a gene family consisting of the chromosomal gef