Escherichia coli

J Virol, 1991 Mar, 65(3), 1149 - 59
The conserved DNA-binding domains encoded by the herpes simplex virus type 1 ICP4, pseudorabies virus IE180, and varicella-zoster virus ORF62 genes recognize similar sites in the corresponding promoters; Wu CL et al.; Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), pseudorabies virus (PRV), varicella-zoster virus (VZV), and equine herpesvirus 1 (EHV-1) are all classified as Alphaherpesvirinae . Each of these five viruses encodes an essential immediate-early (IE) regulatory protein referred to as HSV-1 ICP4, HSV-2 ICP4, PRV IE180, VZV ORF62 protein, and EHV-1 IE1, respectively . These five proteins share extensive homology with each other in domains referred to as regions 2 and 4 . The HSV-1 ICP4 region 2 domain contains residues that are required for the DNA-binding capability of ICP4 . In this report, we describe the expression of region 2 domains from the ICP4, IE180, and ORF62 genes as fusion proteins in Escherichia coli . DNA-binding assays revealed that each of these region 2 fusion proteins binds to a sequence that overlaps the transcription start site in the promoter for the gene encoding the corresponding protein . Each of the sites with high affinity for one or more of these fusion proteins contains the sequence 5'-ATCGT-3' . This sequence spans the mRNA cap site in the HSV-2 ICP4 gene promoter and is immediately upstream from the transcription start site in the EHV-1 IE1 gene . These results suggest that formation of a specific complex between an IE protein and its own gene promoter may be a common mechanism used by Alphaherpesvirinae to autoregulate transcription of an essential IE gene.

Virology, 1991 Mar, 181(1), 78 - 90
Simian virus 40 Vp2/3 small structural proteins harbor their own nuclear transport signal; Clever J et al.; We have used a microinjection approach to identify a domain of the simian virus 40 (SV40) structural proteins Vp2 and Vp3(Vp2/3) responsible for their nuclear transport . By using both synthetic peptides, containing small regions of Vp2/3 conjugated to bovine serum albumin (BSA), and beta-galactosidase-Vp3 fusion proteins, we have narrowed this nuclear transport signal (NTS) to 9 amino acids (198 to 206 of Vp3 or 316 to 324 of Vp2), Gly-Pro-Asn-Lys-Lys-Lys-Arg-Lys-Leu . The porter proteins carrying the NTS or mutant NTS were microinjected into the cytoplasm of TC7 cells and their subcellular localization following the subsequent incubation period was determined immunologically using anti-BSA IgG or anti-beta-galactosidase . The 9-residue NTS peptide localized BSA into the nucleus of injected cells, changing lysine-202 to threonine or valine abolished this accumulation while changing arginine-204 to lysine did not grossly affect transport . A peptide containing the carboxyl-terminal 13 residues of Vp3 failed to localize BSA to the nucleus . Several single or double point mutations at Vp3 residues 202 and 204 have been introduced by site-directed mutagenesis . Vp3 residues 194-234, containing either a wild-type or mutated sequence at 202 and/or 204, were expressed in Escherichia coli as Vp3-beta-galactosidase fusion proteins . Addition of the carboxyl-terminal 40 residues, but not an internal 150 residues, to otherwise cytoplasmic beta-galactosidase promoted entry of the fusion protein into the nucleus . Changing lysine-202 into threonine, valine, or methionine abolished this nuclear accumulation as did changing arginine-204 into lysine . A double mutant at both positions was also blocked . We have also observed that the lectin wheat germ agglutinin inhibits the nuclear accumulation of BSA carrying the Vp2/3 NTS while the lectin concanavalin A had no effect . These data indicate that even small nuclear proteins can contain NTS's which most likely utilize a mechanism for nuclear import similar to that described for other larger proteins.

Gig Sanit, 1991 Mar, (3), 26 - 8
{Standardization of miral levels in the soil}; Motuzinskii NF et al.; On the basis of results of studies on experimental substantiation of MAC for miral in soil it has been found out, that the sub-threshold miral amounts according to the water-migrational, air-migrational, translocational and general sanitary indices of harmfulness equal 0.03, 3, 0.03 and 0.15 mg/kg respectively . Therefore, water-migrational and translocational indices are the limiting indices of harmfulness for miral . 0.03 mg per 1 kg of absolutely dry soil is recommended for miral MAC according to the acting substance.

Khirurgiia (Mosk), 1991 Mar, (3), 103 - 6
{Causes of suppurative complications after appendectomy}; Deviatov VA et al.; The causes of purulent complications in 9,518 patients after appendectomy were studied . The total percentage of purulent complications was 8.0 (6.4 among males and 10.1 among females) . It is shown that the form of appendicitis (phlegmonous in 2.0% of males and in 2.0% of females and perforating in 17.7% and 44.9%, respectively) and the time from the onset of the disease to the operation (less than 6 hours in 4.0% of males and in 6.1% of females; more than 48 hours in 11.7% and 18.8%, respectively) have an effect on the frequency of purulent complications . The authors revealed a correlation between the frequency of purulent complications and some factors (the time spent by the patients in the clinic before the operation, surgical team with an incomplete staff, insufficient anesthesia, insufficient operative approach, irrational use of gauze tampons and antibiotics.

Gene, 1991 Mar 1, 99(1), 9 - 14
Genomic hsd-Mu(lac) operon fusion mutants of Escherichia coli K-12; Prakash A et al.; Genomic (chromosomal)hsd-Mu(lac) operon fusions have been constructed in two strains of Escherichia coli K-12 for the three hsd genes, hsdRK, hsdMK and hsdSK, using MudX and lambda placMu53 . Expression of hsdK mutants ranged from 16 to 74 units (u) (with a mean of 52 u) for fusions to promoter pres and ranged from 26-75 u (also with a mean of 52 u) for fusions to promoter pmod . The expression of the two hsdK promoters was measured in different stages of growth . The pres fusion mutant showed a lag in beta-galactosidase (beta Gal) production, as compared to the pmod fusion mutant . One r-Km-K mutant (JR205) showed more than ten times the beta Gal activity of other insertion mutants . The activity of this mutant decreased by 20-fold upon the transfer of F101-102, which includes the wild-type hsd region . Positive gene-dosage effect was observed using F' plasmids containing the hsd-lacZ region.

Mutat Res, 1991 Mar, 254(2), 107 - 17
Introduction of a UV-damaged replicon into a recipient cell is not a sufficient condition to produce an SOS-inducing signal; Sommer S et al.; Three models have been proposed for the nature of the SOS-inducing signal in E . coli . One model postulates that degradation products of damaged DNA generate an SOS-inducing signal; another model surmises that the very lesions produced by UV damage constitute the SOS-inducing signal in vivo; a third model proposes that DNA damage is processed upon DNA replication to form single-stranded DNA (the SOS signal) that activates RecA protein . We tested the models by measuring SOS induction produced by introducing into recipient cells the UV-damaged DNA of 2 constructed phagemids . We used phagemids since they transferred DNA to the recipients with 100% efficiency . The origin of replication of the phagemids was either oriC from the E . coli chromosome, or oriF from F plasmid . Replication of the oriC phagemid was dependent on methylation . A UV-damaged oriC phagemid failed to induce SOS functions in a recipient cell whereas an oriF phagemid did induce them . Our results disprove the first and the second model proposed for the nature of the SOS-inducing signal . The failure of a UV-damaged oriC replicon to induce SOS can be explained by the third model if one assumes that replication of a UV-damaged oriC plasmid does not generate single-stranded DNA as does the E . coli chromosome after UV damage.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1938 - 42
Mutational analysis of yeast vacuolar H(+)-ATPase; Noumi T et al.; Yeast mutants in which genes encoding subunits of the vacuolar H(+)-ATPase were interrupted were assayed for their vacuolar ATPase and proton-uptake activities . The vacuoles from the mutants lacking subunits A (72 kDa), B (57 kDa), or c (proteolipid, 16 kDa) were completely inactive in these reactions . Immunological studies revealed that in the absence of each one of those subunits the catalytic sector was not assembled . Labeling with N,N'-{14C}dicyclohexylcarbodiimide showed the presence of the proteolipid in vacuoles of mutants in which genes encoding subunits of the catalytic sectors were interrupted . No labeling was detected in the mutant in which the gene encoding the proteolipid was interrupted . We conclude that of all the ATPase subunits only the proteolipid is assembled independently and it serves as a template for the assembly of the other subunits . Site-specific mutations were generated in the gene encoding the proteolipid . All of the drastic changes and replacements gave inactive proteins . About half of the single amino acid replacements gave active proteins . Replacing glutamic acid-137 by any of several amino acids, except for aspartic acid, abolished the activity of the enzyme . Other amino acids that may function in proton conductance were changed . It was found that glycine residues may replace amino acids with exchangeable protons.

Glycobiology, 1991 Mar, 1(2), 187 - 91
CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N-acetylneuraminic acid and CMP-neuraminic acid derivatives; Shames SL et al.; The gene encoding CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) in Escherichia coli serotype O7 K1 was isolated and overexpressed in E.coli W3110 . Maximum expression of 8-10% of the soluble E.coli protein was achieved by placing the gene with an engineered 5'-terminus and Shine-Dalgarno sequence into a pKK223 vector derivative behind the tac promoter . The overexpressed synthetase was purified to greater than 95% homogeneity in a single step by chromatography on high titre Orange A Matrex dye resin . Enzyme purified by this method was used directly for the synthesis of CMP-NeuAc and derivatives . The enzymatic synthesis of CMP-NeuAc was carried out on a multigram scale using equimolar CTP and N-acetylneuraminic acid as substrates . The resultant CMP-NeuAc, isolated as its disodium salt by ethanol precipitation, was prepared in an overall yield of 94% and was judged to be greater than 95% pure by 1H NMR analysis . N-Carbomethoxyneuraminic acid and N-carbobenzyloxyneuraminic acid were also found to be substrates of the enzyme; 5-azidoneuraminic acid was not a substrate of the enzyme . N-Carbomethoxyneuraminic acid was coupled to CMP at a rate similar to that observed with NeuAc, whereas N-carbobenzyloxyneuraminic acid was coupled greater than 100-fold more slowly . The high level of expression achieved with the E.coli synthetase, together with the high degree of purity readily obtainable from crude cell extracts, make the recombinant bacterial enzyme the preferred catalyst for the enzymatic synthesis of CMP-N-acetylneuraminic acid.

J Mol Recognit, 1991 Mar-Jun, 4(2-3), 77 - 83
Immunological mapping of fine molecular surface structures of citrate synthase enzymes from different cell types; Nemeth P et al.; Citrate synthase (EC 4.1.3.7), which is present in all living organisms as a key enzyme in aerobic energy metabolism, is one of the most highly phylogenetically conserved enzymes known in terms of its primary and active site structure . However, in terms of other parameters such as in vitro stability, tolerance to changes in pH, degree of self-polymerization, etc., citrate synthases from different sources are markedly different . These divergences can be observed even between isoforms of the enzyme within the same species . Data documenting these diversities suggest that a high degree of difference in tertiary structures may occur . Therefore, the surface profiles of citrate synthase enzymes from yeast, pig, rat, tomato and Escherichia coli were investigated with immunological methods using monoclonal antibody families generated against either pig citrate synthase (alpha-PCS) or yeast citrate synthase-2 (alpha-YCS-2) . A high degree of homology of enzyme epitopes was detected on the mitochondrial citrate synthases originating from yeast, tomato, pig and rat cells . Major differences were found between the hexameric citrate synthase originating from E . coli compared with those dimeric forms prepared from eukaryotic cells . Only modest similarities were detected between the highly homologous peroxisomal and mitochondrial yeast citrate synthases . Furthermore, a point mutation of one of the catalytic residues (H274R on recombinant pig and H313R on yeast enzyme) of mitochondrial citrate synthase (CS-1) resulted in a significant increase in immunological similarity with the peroxisomal isoenzyme (CS-2) . These findings are discussed in terms of the possible mechanism of evolution of CS-2 in yeast.

Zhongguo Yao Li Xue Bao, 1991 Mar, 12(2), 135 - 40
Influences of a novel immunopotentiator polyactin A on interleukin 1 production and responsiveness in mice; Hu QL et al.; Effects of a novel immunopotentiator Polyactin A (PAA), developed in China, on production and responsiveness of murine interleukin 1(IL-1) were investigated . The results demonstrated that: (1) PAA 0.01-100 micrograms.ml-1 directly induced IL-1 synthesis and secretion from murine peritoneal macrophages (PMO) and markedly enhanced IL-1 production of the mouse PMO stimulated by lipopolysaccharides (LPS) of E coli; (2) IL-1 release from the PMO cultured in PAA 0.1 micrograms.ml-1 was detectable as early as 2 h after the incubation, peaked at 24 h, and then decreased gradually; (3) PAA stimulated and enhanced both IL-1 synthesis and release, but its effect on IL-1 release was stronger; (4) PMO from the mice given po PAA 200 mg.kg-1.d-1 for 7 d produced a higher level of IL-1 than those from control group, and the increase in extracellular IL-1 was more significant than that in intracellular one; (5) in vivo, PAA had no effect on IL-1 receptor expression and IL-1 responsiveness of murine lymphocytes, but eliminated the suppressing effects of cyclophosphamide (Cyc) on IL-1 receptor expression and IL-1 responsiveness of mouse lymphocytes . The above findings provide new explanation for action of PAA and new basis for wider clinical applications of PAA.

Mol Gen Mikrobiol Virusol, 1991 Mar, (3), 22 - 4
{Polymerase and immunologic activity of reverse transcriptase of the HIV-1 virus, isolated from Escherichia coli}; Shvetsov IuP et al.; The recombinant reverse transcriptase of HIV-1 virus has been isolated from Escherichia coli cells transformed by the plasmid pRT40 DNA . The 103 Kd protein produced by these cells is shown to be processed to proteins with lower molecular masses by the reverse transcriptases own protease activity as well as Escherichia coli proteases . The resulting 103-66 Kd proteins possess the polymerase activity while 51 Kd and smaller proteins are lacking the activity . The 66 and 51 Kd reverse transcriptase fragments demonstrate the positive immunological reaction with the human blood serum from the people possessing antibodies to HIV-1 virus . The recombinant reverse transcriptase of HIV-1 produced by Escherichia coli cells is shown to be useful in AIDS diagnosis in humans.

Anticancer Res, 1991 Mar-Apr, 11(2), 793 - 800
Establishment, characterization and determination of cell surface sugar receptor (lectin) expression by neoglycoenzymes of a human myeloid marker-expressing B lymphoblastoid cell line; Gabius S et al.; Mediation of cellular interactions by protein (lectin)-carbohydrate recognition presupposes the expression of respective surface determinants . Due to the importance of cellular contacts between bone marrow stromal cells, recently shown to express cell surface lectins, and tumor or normal progenitor cells for biosignaling and marrow egress, quantitation of cell surface sugar receptor expression by a panel of chemically glycosylated enzymes (tetrameric E . coli beta-galactosidase) for human leukemia/lymphoma cells was initiated . Cells of the new B lymphoblastoid line Croco II that are partially positive for the CD15-specific epitope expressed receptors for various sugar specificities on their surface, fulfilling an indispensable prerequisite for establishment of glycobiological interactions . Binding studies with increasing neoglycoenzyme concentrations up to saturation in four cases disclosed values for apparent affinity constants in the range of 25-200 nM with 0.25-3 x 10(5) bound probes per cell . The presence of receptors for constituents of carbohydrate chains of cellular glycoconjugates was also ascertained biochemically, namely for beta-galactosides, alpha-mannosides, alpha-fucosides and N-acetylgalactosaminides . Expression of this property was modulated by changes in the culture conditions, as revealed by binding studies with cells, derived from growth in medium containing different serum concentrations . These findings indicate that cell surface sugar receptors of tumor cells warrant further attention with respect to recognitive interactions.

Mol Biochem Parasitol, 1991 Mar, 45(1), 101 - 7
Molecular characterisation of a protective, 11-kDa excretory-secretory protein from the parasitic stages of Trichostrongylus colubriformis; Dopheide TA et al.; An 11-kDa protein occurring as a major component of the non-glycosylated fraction of 4th larval stage (L4) and adult Trichostrongylus colubriformis excretory-secretory (ES) fluid has been found to be highly protective in guinea pigs, an alternate host for T . colubriformis . The protein has been purified, characterised and partly sequenced . With a reverse-complement oligonucleotide based on the carboxy-terminal sequence of the protein, recombinant lambda gt11 clones were detected in an L4 cDNA library . The DNA sequence from one clone has a single extended open reading frame coding for a highly charged 11-kDa protein which lacks a leader sequence and contains a potential N-glycosylation site . Expression of the cloned DNA in Escherichia coli was detected with an antibody, raised in rabbits against gel-purified 11-kDa protein.

Biophys J, 1991 Mar, 59(3), 516 - 22
Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1; Rath P et al.; The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy . This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500 . The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures . The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins . Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal . Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange . These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.

Mol Microbiol, 1991 Mar, 5(3), 747 - 55
Osmotic induction of the periplasmic trehalase in Escherichia coli K12: characterization of the treA gene promoter; Repoila F et al.; The Escherichia coli treA gene encodes an osmotically inducible periplasmic trehalase . A strain carrying a treA-lacZ transcriptional fusion was constructed . The beta-galactosidase activity produced in this strain growing exponentially in a medium of high osmotic pressure was 10-fold higher than that produced in a medium of low osmotic pressure, demonstrating that treA transcription is osmotically inducible . treA transcriptional induction depends neither on the presence of trehalase itself nor on the synthesis of cytoplasmic trehalose which occurs in response to osmotic stress in wild-type E . coli strains . The treA promoter was identified by S1 nuclease protection . Deletion analysis demonstrated that sequences sufficient for the osmotic induction lie downstream from nucleotide -40 with respect to the transcription start . Transcription initiation at treAp required the presence of a functional sigma 70 subunit of RNA polymerase . treA expression was increased in the presence of a mutation in osmZ, which was previously identified as leading to a partially constitutive expression of the osmotically inducible proU operon.

Mol Microbiol, 1991 Mar, 5(3), 631 - 40
A copy-number mutant of plasmid pSC101; Xia GX et al.; Copy-number mutants of plasmid pSC101 were isolated by u.v . mutagenesis and selection for elevated expression of ampicillin resistance . Three independent mutations were identical and mapped in codon 93 of the initiation protein RepA . The mutated plasmids were maintained at a level four to five times higher than that of the wild type . For one of them, it was determined that: (i) the mRNA of the autoregulated repA gene, cloned onto a pUC19 plasmid under the control of its own promoter, was expressed at a level 1.7 times higher than that of the wild type; (ii) the RepA protein, under the same conditions, was expressed at a similarly higher level; (iii) the affinity of the mutated protein for three repeated sequences in the origin region of the plasmid was, on average, 3.4 times higher than that of the wild-type protein . We postulate that the copy-number effect is due to a combination of these two effects, i.e . higher protein concentration and increased affinity of the protein for the repeated sequences.

J Gen Microbiol, 1991 Mar, 137 ( Pt 3), 679 - 84
Role of ribosome release in the basal level of expression of the Escherichia coli gene pheA; Gavini N et al.; In Escherichia coli, the expression of the phenylalanine biosynthetic operon pheA is regulated by an attenuation mechanism . In the presence of excess phenylalanine, gene expression was decreased to 10% of the fully deattenuated level . To understand the factors that determine the basal level of pheA expression, we examined the role of ribosome release from the leader peptide stop codon UGA . The transcriptional readthrough from the pheA attenuator decreased by over 2-fold in the presence of the defective release factor 2 . However, a release factor 1 (UAG and UAA specific) mutation did not influence the basal level of pheA expression . These results support the proposal that the release of translating ribosomes from the leader peptide stop codon in stem 2 of the pheA attenuator plays a crucial role in determining the basal level of expression of this gene.

Sci China B, 1991 Mar, 34(3), 297 - 305
Arginyl-tRNA synthetase from Escherichia coli affinity labeling with 3'-oxidized tRNA(Arg); Cheng XD et al.; The covalent modification of E . coli arginyl-tRNA synthetase by the 2',3'-dialdehyde derivative of tRNA(Arg) (tRNA(oxArg)) resulted in the complete inactivation of the ATP-PPi exchange and aminoacylation activities of the enzyme . Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the ArgRS-tRNA(oxArg) covalent complexes indicated that two bands simultaneously appeared on the gel parallel with inactivation corresponding to different higher molecular weights . This result was different from that of the other aminoacyl-tRNA synthetase labeling systems as previously reported . Upon the ribonuclease treatment of the modified ArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities were recovered . During the whole process of labeling and RNase treatment, the two activities of the enzyme were closely associated.

Mol Gen Genet, 1991 Mar, 225(3), 435 - 42
Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli; Chiang CS et al.; The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA+ gene . In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift . This inhibition did not occur when the plasmid dnaA+ gene was expressed in the presence of the inducer isopropyl-1-thio-beta-D-galactopyranoside (IPTG) . Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol . Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30 degrees C, or after inactivation of DnaA by a shift to 42 degrees C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo . The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature . Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component . Rom- plasmids show only the late effect . After heat inactivation of DnaC, plasmid replication ceased immediately.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1991 Mar, 11(3), 1607 - 13
Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins; Matsuda M et al.; The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner . In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins . Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src . The deletion of SH2 resulted in the loss of binding activity . Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins . Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity . We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells . Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.

J Virol, 1991 Mar, 65(3), 1611 - 5
Epitope mapping and characterization of the infectious hematopoietic necrosis virus glycoprotein, using fusion proteins synthesized in Escherichia coli; Xu L et al.; A characterization of the antigenic determinants (epitopes) of the glycoprotein (G) of infectious hematopoietic necrosis virus was made by expressing different regions of the G gene in Escherichia coli . A cDNA copy of the G gene was divided into four fragments by TaqI digestion, and the fragments were subcloned into pATH vectors, placing the expression of each G gene fragment under control of the trpE promoter . The resulting plasmids, pXL2, pXL3, and pXL7, encoded trpE-G fusion proteins subsequently detected with anti-infectious hematopoietic necrosis virus sera by Western immunoblots . A comparison of reactivities of the fusion proteins encoded by these plasmids was made by Western immunoblot and radioimmunoassay with a number of anti-G specific monoclonal antibodies (MAbs) . The nonneutralizing MAb 136J reacted with the trpE-G fusion protein encoded by pXL3 and fusion proteins encoded by plasmids p52G and p618G, which were described in previous studies (R . D . Gilmore, Jr., H . M . Engelking, D . S . Manning, and J . C . Leong . Bio/Technology 6:295-300, 1988) . Another nonneutralizing MAb, 2F, bound to the pXL3 fusion protein, and the neutralizing MAb RB/B5 recognized the pXL7 fusion protein . All fusion proteins were tested as vaccines in rainbow trout fry . Although significant protection was induced by all fusion proteins, the pXL3 fusion protein was most effective as a vaccine.

J Virol, 1991 Mar, 65(3), 1507 - 15
Retrovirus promoter-trap vector to induce lacZ gene fusions in mammalian cells; Reddy S et al.; A retrovirus promoter-trap vector (U3LacZ) has been developed in which Escherichia coli lacZ coding sequences were inserted into the 3' long terminal repeat (LTR) of an enhancerless Moloney murine leukemia virus . The U3LacZ virus contains the longest reported LTR (3.4 kbp); nevertheless, lacZ sequences did not interfere with the ability of the virus to transduce a neomycin resistance gene expressed from an internal promoter . Duplication of the LTR placed lacZ sequences in the 5' LTR just 30 nucleotides from the flanking cellular DNA . Approximately 0.4% of integrated proviruses expressed beta-galactosidase as judged by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, and individual clones expressing lacZ were isolated by fluorescence-activated cell sorting . In all clones examined, beta-galactosidase expression resulted from the fusion of lacZ sequences to transcriptional promoters located in the flanking cellular DNA . Furthermore, by differential sorting of neomycin-resistant cell populations, clones were isolated in which lacZ expression was induced and repressed in growth-arrested and log phase cells, respectively.

Virology, 1991 Mar, 181(1), 14 - 21
Peptide mapping of neutralizing and nonneutralizing epitopes of duck hepatitis B virus pre-S polypeptide; Yuasa S et al.; Antibodies to the envelope proteins of duck hepatitis B virus neutralize viral infection in vitro . Using a library of murine monoclonal antibodies (Mabs) against the envelope proteins, we previously identified four neutralizing and two non-neutralizing epitopes on the pre-S region of the large envelope proteins . In this study we report the localization of all but one of these epitopes at the amino acid level . All but 28 nucleotides of the pre-S and S genes were cloned in pUC vectors and expressed in Escherichia coli . All Mabs in this study reacted with the expressed gene products in Western blots . Deletion mutants of the pre-S region were generated and their expressed products tested on Western blots for reactivity with the Mabs . Of the three epitopes involved in neutralization, the epitope found to be immunodominant in convalescent ducks was localized to nine amino acids of the middle portion of the pre-S gene product, while a second epitope was mapped to nine amino acids upstream of the immunodominant epitope and the third epitope to seven amino acids adjacent to the S gene . One of the two non-neutralizing epitopes was located between the two groups of neutralizing epitopes while the other mapped to the same region as one of the neutralizing epitopes . Our data indicate that several regions of the pre-S polypeptide may play a role in neutralization of hepadnaviruses.

Int J Radiat Biol, 1991 Mar, 59(3), 717 - 27
The relationship between the anoxic sensitivity and the extent of sensitization by nitrous oxide; Ewing D et al.; Nitrous oxide reacts during irradiation to increase the yield of .OH, a radical many believe to be a major cause of lethality . Logically, one would expect N2O to be a radiation sensitizer . In some instances it is, while in others it is not . In some cases we can explain why N2O fails to sensitize; factors such as dose rate, cell concentration, buffer composition and ionic strength all influence when N2O will sensitize and, if it sensitizes, by what magnitude . Based on the results presented here with multiple strains of procaryotic and eucaryotic cells, we believe the anoxic sensitivity is another critical factor that governs whether N2O will sensitize . Our data, with data from the literature, show a relationship between the anoxic sensitivity and the N2O enhancement ratio . N2O does not sensitize in vitro unless the anoxic sensitivity (inactivation constant, k) is less than approximately 0.2 daGy-1.

J Bacteriol, 1991 Mar, 173(6), 1894 - 901
Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli; Ray JM et al.; The aroH gene of Escherichia coli, which encodes the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzyme of the common aromatic biosynthetic pathway, was cloned behind the tac promoter in expression plasmid pKK223-3 . The enzyme was overexpressed, purified to homogeneity, and characterized . The native enzyme was found to be a dimeric metalloprotein containing 0.3 mol of iron per mol of subunit and variable amounts of zinc . The activity of the native enzyme was stimulated two- to threefold when assayed in the presence of Fe2+ ions . Pretreatment of the enzyme with Fe2+ also resulted in activation, accompanied by an equivalent increase in iron content . Treatment of the enzyme with chelating agents led to inactivation, which was fully reversed by the presence of Fe2+ in the assay mixture . The native enzyme exhibited a unique absorption profile, having a shoulder of absorbance on the aromatic band with a maximum around 350 nm and a broad, weak band with a maximum around 500 nm . Treatment of the enzyme with Fe2+ enhanced the absorbance at 350 nm and eliminated the band at 500 nm . Treatment with reducing agents caused the disappearance of both bands and destabilized the enzyme . Feedback regulation of the activity of the enzyme was specific for tryptophan, with maximum inhibition at about 70%.

J Bacteriol, 1991 Mar, 173(5), 1789 - 800
Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in Escherichia coli; Braaten BA et al.; Transcription of the pyelonephritis-associated pilus (pap) operon of Escherichia coli is subject to regulation by a phase variation control mechanism in which the pap pilin gene alternates between transcriptionally active (phase-on) and inactive (phase-off) states . Pap phase variation appears to involve differential inhibition of deoxyadenosine methylase (Dam) methylation of two pap GATC sites, GATC1028 and GATC1130, located in the regulatory region upstream of the papBA promoter . DNA from phase-on cells contains an unmethylated adenosine in the GATC1028 site, whereas DNA from phase-off cells contains an unmethylated adenosine in the GATC1130 site . papI and papB are two regulatory genes in the pap operon . Analysis of pap deletion mutants suggests that papI is required for methylation inhibition at the GATC1028 site; however, neither papI nor papB is required for inhibition of methylation at the GATC1130 site . We have identified a chromosomal locus, mbf (methylation-blocking factor), that is required for methylation protection of both the pap GATC1028 and GATC1130 sites . The mbf locus was identified after transposon mTn10 mutagenesis and mapped to 19.6 min on the E . coli chromosome . The effect of transposon mutations within mbf on pap pilin transcription was determined by using a papBAp-lac operon fusion which places lacZ under control of the papBA promoter . E . coli containing mbf::mTn10 and phase-off mbf+ E . coli cells both expressed beta-galactosidase levels about 30-fold lower than the beta-galactosidase level measured for phase-on mbf+ E . coli cells . These results indicated that mbf was necessary for pap pilin transcription and were supported by Northern (RNA) blotting and primer extension analyses . Moreover, transposon insertion within mbf greatly reduced Pap pilus expression . The mbf locus was isolated on a low-copy-number cosmid, pMBF1 . Complementation analysis indicated that each of seven mbf::mTn10 mutants isolated contained a transposon insertion within the same gene or operon . The identification of the mbf locus, required for pap transcription, supports the hypothesis that pap phase variation is controlled by a mechanism involving alternation between different methylation states.

Infect Immun, 1991 Mar, 59(3), 971 - 6
The msp1 beta multigene family of Anaplasma marginale: nucleotide sequence analysis of an expressed copy; Barbet AF et al.; A gene for the beta subunit of the immunoprotective surface antigen MSP-1 of Anaplasma marginale was previously cloned and expressed in Escherichia coli . A nucleic acid probe based on this gene detects A . marginale infection in carrier cattle and in the tick vector . We report here the sequence and structural features of the cloned msp1 beta gene and expressed polypeptide . The gene codes for a polypeptide of 756 amino acids that contains domains of tandemly repeated sequence and glutamine-rich regions at the N and C termini . The cloned copy is a member of a multigene family with multiple restriction fragment length polymorphisms in isolates of this rickettsia from different geographical regions . The availability of the sequence will allow use of the polymerase chain reaction in diagnostic assays and the preparation and testing of different vaccine constructs in cattle.

Infect Immun, 1991 Mar, 59(3), 1015 - 23
Mucin isolated from rabbit colon inhibits in vitro binding of Escherichia coli RDEC-1; Mack DR et al.; The rabbit enteric pathogen Escherichia coli RDEC-1 (serotype O15:H-) mediates attaching and effacing binding to colonic epithelium in a manner morphologically identical to that observed in both human enteropathogenic E . coli and enterohemorrhagic E . coli infections . The aim of this study was to determine if colonic mucus and its constituents, including mucin derived from goblet cells, inhibited RDEC-1 adherence in vitro . Crude mucus was prepared from mucosal scrapings of rabbit colon and separated by buoyant density into eight fractions . Purified mucin was characterized by gel electrophoresis, dot immunoblotting, indirect immunofluorescence, and amino acid composition . RDEC-1 bacteria were grown to promote and suppress the expression of mannose-resistant, hydrophobic pili . A nonpiliated mutant, strain M34, was also used as a negative control . Binding of radiolabeled RDEC-1 expressing pili was quantitated in the presence of crude mucus, purified mucin, and nonmucin fractions . Binding of piliated RDEC-1 to hydrophobic polystyrene wells was greater than for both nonpiliated RDEC-1 and strain M34 (P less than 0.05) . Both crude mucus and purified mucin mediated a concentration-dependent inhibition of piliated-RDEC-1 binding . Fractions of mucus without immunoreactive mucin did not inhibit the binding of RDEC-1 expressing hydrophobic pili . We conclude that colonic goblet cell-derived mucin mediates inhibition of piliated RDEC-1 attachment in vitro . Inhibition of bacterial adherence could prevent access of attaching and effacing E . coli enteric pathogens to the colonic mucosa in vivo.

Mol Biol (Mosk), 1991 Mar-Apr, 25(2), 515 - 24
{Construction and expression of hybrid genes based on a deletion mutant of the herpes virus thymidine kinase gene in Escherichia coli}; Zemskova MIu et al.; The plasmids with the deletion mutant of thymidine kinase gene (tk') of Herpes simplex virus were constructed . The promoter, transcription initiation site and first 35 codons of tk gene were removed and replaced with a series of DNA restriction sites . The DNA fragments, containing the gene regulatory sequences specific for bacteria, were cloned into these sites and shown to express enzymatically active proteins . The obtained ene fusions were able to complement in E . coli strain deficient in thymidine kinase function . Such plasmid vectors carrying tk' are useful for construction and selection of hybrid gene fusions.

Plasmid, 1991 Mar, 25(2), 145 - 8
A sequence at the inside end of IS50 down regulates transposition; Dodson KW et al.; Deletion analysis has shown that the segment at the IS50 inside (I) end that is needed for efficient transposition is approximately 19 bp long . Dam methylation at two 5' GATC sequences within this segment decreases I-end transposition activity . A third 5' GATC sequence is present at bp 21-24 of the I end . The comparisons presented here show that extension of the I end from 19 to 24 bp decreases its transposition activity in dam cells 5- to 50-fold, depending on the overall transposon structure.

Bioorg Khim, 1991 Mar, 17(3), 379 - 86
{Site-directed mutagenesis with the use of addressed fragmentation of single-stranded DNA for obtaining hybrids of homologous proteins}; Shekhter II et al.; Site-directed multiple mutagenesis to obtain hybrids of homologous proteins was carried out by means of the oligonucleotide-targeting digestion of ss DNA . The procedure is more convenient, rapid and simple than the step-by-step approach . To demonstrate different approaches to targeting digestion of ss DNA, NcoI, HinfI, FokI endonucleases were used for DNA cleavage . A hydride of the human and porcine IFN-alpha-2-genes has been constructed.

J Electron Microsc Tech, 1991 Mar, 17(3), 336 - 43
Suitability of different silver enhancement methods applied to 1 nm colloidal gold particles: an immunoelectron microscopic study; Stierhof YD et al.; In order to exploit the recently introduced 1 nm gold colloids in routine electron microscopic labeling experiments, an efficient enhancement step for a better visualization of this small marker is a prerequisite . Efficiency and reproducibility of enhancement as well as growth homogeneity of gold particles were evaluated for three different silver intensifying solutions: silver lactate/hydroquinone/gum arabic (Danscher, 1981), Ilford L4/Metol (Bienz et al., 1986), and the commercially available IntenSE M silver enhancement kit (Janssen Pharmaceutica) . The best results were obtained by using the silver lactate/hydroquinone/gum arabic mixture . The quality of enhancement of the IntenSE M kit was considerably increased by the addition of the protective colloid gum arabic.

Brain Res Mol Brain Res, 1991 Mar, 9(4), 347 - 51
A phospholipase A2-stimulating protein regulated by protein kinase C in Aplysia neurons; Calignano A et al.; We describe some properties on an Mr 30,000 thermolabile and trypsin-sensitive protein that activates phospholipase A2 (PLA2) and which was isolated from nervous tissue of the marine mollusk, Aplysia californica . A similar protein is present in rat cerebral cortex . This protein was partially purified from crude homogenates of nervous tissue by ion exchange chromatography on DEAE-Sephadex followed by size-exclusion high performance liquid chromatography (HPLC) . It is loosely associated with membrane fractions, and is extracted by 0.05% Tween 20 . Although similar in size to several previously described PLA2-stimulating proteins from non-neural mammalian cells and tissues, it differs from them in some aspects of biological activity . The protein promotes the release of eicosanoids from the membranes of intact Aplysia neurons prelabeled with {3H}arachidonic acid and appears to be an in vitro substrate for protein kinase C (PKC) . PLA2-stimulating activity is greatly enhanced after exposing isolated ganglia to phorbol dibutyrate (PDBu) and is reduced by treatment with immobilized E . coli alkaline phosphatase . These observations suggest that phosphorylation of this stimulatory protein by PKC regulates PLA2 in neurons.

Biotechnology (N Y), 1991 Mar, 9(3), 273 - 8
The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique; Skerra A et al.; We have previously demonstrated that the expression of fully functional Fv and Fab fragments in E . coli is possible by the simultaneous secretion of both chains to the periplasm . To increase production levels and facilitate engineering and random mutagenesis, we improved our previous vectors by introducing a resident repressor gene and a filamentous phage origin . We also developed a new purification strategy based on immobilized metal ion chromatography, with which a single-chain Fv fragment can be purified to homogeneity in a single step . We investigated the most efficient tail constructions and found that only a minimal structural change of three additional C-terminal amino acids is necessary . This modification has no deleterious effect on in vivo transport and folding or antigen affinity.

Biochem Biophys Res Commun, 1991 Feb 28, 175(1), 250 - 5
Zn+2 induces reversible cross-linking of human placental thyroid hormone nuclear receptor with no effect on hormone binding; Lin KH et al.; Zn+2 is required for specific binding of c-erbA proteins to the hormone response elements of target genes . It is unclear whether Zn+2 is important for the binding of ligand to c-erbA proteins . The present study evaluated the effect of Zn+2 and other divalent cations on the binding of 3,3',5-triiodo-L-thyronine(T3) to the purified human placental c-erbA protein (h-TR beta 1) . Zn+2 induced cross-linking of h-TR beta 1 to form aggregates in a dose-dependent manner with an apparent half-maximal concentration of approximately 200 microM at 22 degrees C . Cross-linking was reversible by the addition of 5 microM EDTA or 10 mM dithiothreitol . The cross-linked h-TR beta 1 bound T3 . These results indicated Zn+2 had no effect on T3 binding and suggested that the cysteines and histidines involved in cross-linking are not essential for T3 binding.

Eur J Biochem, 1991 Feb 26, 196(1), 87 - 93
Amino acid 55 plays a central role in tetramerization and function of Escherichia coli single-stranded DNA binding protein; Curth U et al.; The histidine at position 55 of the amino acid sequence of the Escherichia coli single-stranded DNA binding protein was replaced by tyrosine, glutamic acid, lysine, phenylalanine, and isoleucine . The properties of the mutant proteins were determined using analytical ultracentrifugation, NMR spectroscopy, gel filtration, and fluorimetric detection of their single-stranded DNA binding ability . While the phenylalanine and isoleucine substitutions did not change the properties of the protein measurably, tyrosine and lysine mutants dissociate into subunits and loose some of their binding affinity for poly(dT) . For the lysine mutant we show by electron microscopy that the protein, although fully dissociated and possibly denatured in the free state, binds to poly(dT) as a tetramer indistinguishable from the wild-type protein . The process of tetramerization as observed via single-stranded DNA binding ability is composed of a variety of steps ranging in time from some milliseconds to several hours; it probably involves several forms of dissociated and non-native protein.

Biochemistry, 1991 Feb 26, 30(8), 2273 - 80
Deduced amino acid sequence of Escherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function; Chang ZY et al.; The goal of the research reported here is to identify evolutionarily conserved amino acid residues associated with enzymatic deamination of adenosine . To do this, we isolated molecular clones of the Escherichia coli adenosine deaminase gene by functional complementation of adenosine deaminase deficient bacteria and deduced the amino acid sequence of the enzyme from the nucleotide sequence of the gene . Nucleotide sequence analysis revealed the presence of a 996-nucleotide open reading frame encoding a protein of 332 amino acids having a molecular weight of 36,345 . The deduced amino acid sequence of the E . coli enzyme has approximately 33% identity with those of the mammalian adenosine deaminases . With conservative amino acid substitutions the overall sequence homology approaches 50%, suggesting that the structures and functions of the mammalian and bacterial enzymes are similar . Additional amino acid sequence analysis revealed specific residues that are conserved among all three adenosine deaminases and four AMP deaminases for which sequence information is currently available . In view of previously published enzymological data and the conserved amino acid residues identified in this study, we propose a model to account for the enzyme-catalyzed hydrolytic deamination of adenosine . Potential catalytic roles are assigned to the conserved His 214, Cys 262, Asp 295, and Asp 296 residues of mammalian adenosine deaminases and the corresponding conserved amino acid residues in bacterial adenosine deaminase and the eukaryotic AMP deaminases.

Biochemistry, 1991 Feb 26, 30(8), 2227 - 39
Crystal structure of unliganded Escherichia coli dihydrofolate reductase . Ligand-induced conformational changes and cooperativity in binding; Bystroff C et al.; The crystal structure of unliganded dihydrofolate reductase (DHFR) from Escherichia coli has been solved and refined to an R factor of 19% at 2.3-A resolution in a crystal form that is nonisomorphous with each of the previously reported E . coli DHFR crystal structures {Bolin, J . T., Filman, D . J., Matthews, D . A., Hamlin, B . C., & Kraut, J . (1982) J . Biol . Chem . 257, 13650-13662; Bystroff, C., Oatley, S . J., & Kraut, J . (1990) Biochemistry 29, 3263-3277} . Significant conformational changes occur between the apoenzyme and each of the complexes: the NADP+ holoenzyme, the folate-NADP+ ternary complex, and the methotrexate (MTX) binary complex . The changes are small, with the largest about 3 A and most of them less than 1 A . For simplicity a two-domain description is adopted in which one domain contains the NADP+ 2'-phosphate binding site and the binding sites for the rest of the coenzyme and for the substrate lie between the two domains . Binding of either NADP+ or MTX induces a closing of the PABG-binding cleft and realignment of alpha-helices C and F which bind the pyrophosphate of the coenzyme . Formation of the ternary complex from the holoenzyme does not involve further relative domain shifts but does involve a shift of alpha-helix B and a floppy loop (the Met-20 loop) that precedes alpha B . These observations suggest a mechanism for cooperativity in binding between substrate and coenzyme wherein the greatest degree of cooperativity is expressed in the transition-state complex . We explore the idea that the MTX binary complex in some ways resembles the transition-state complex.

Biochemistry, 1991 Feb 26, 30(8), 2192 - 5
Kinetics of electron transfer from thioredoxin reductase to thioredoxin; Navarro JA et al.; The reduction of Escherichia coli thioredoxin by thioredoxin reductase was studied by stopped-flow spectrophotometry . The reaction showed no dependence on thioredoxin concentration, indicating that complex formation was rapid and occurred during the dead time of the instrument . The kobs for the reaction of approximately 20 s-1 probably reflects the rate of electron transfer from thioredoxin reductase to thioredoxin and agrees with the kcat observed by steady-state kinetics . The reaction rate was unaffected by increasing the ionic strength, suggesting a lack of electrostatic stabilization in the interaction of the two proteins . A mutant thioredoxin in which a positively charged lysine in the active-site region was changed to a glutamic acid residue resulted in an electrostatic destabilization . Thioredoxin K36E was still a substrate for the reductase, but binding was impaired so that the rate could be measured by stopped-flow techniques as reflected by a dependence on protein concentration . Raising the ionic strength in this reaction served to shield the negative charge and increased the rate of binding to the reductase.

Biochemistry, 1991 Feb 26, 30(8), 2184 - 91
Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli; Falzone CJ et al.; Two-dimensional 1H NMR methods and a knowledge of the X-ray crystal structure have been used to make resonance assignments for the amino acid side chains of dihydrofolate reductase from Escherichia coli complexed with methotrexate . The H7 proton on the pteridine ring of methotrexate was found to have NOEs to the methyl protons of Leu-28 which were assigned by using the L28F mutant . These NOEs indicated that the orientation of the methotrexate pteridine ring is similar in both solution and crystal structures . During the initial assignment process, it became evident that many of the resonances in this complex, unlike those of the folate complex, are severely broadened or doubled . The observation of two distinct sets of resonances in a ratio of approximately 2:1 was attributed to the presence of two protein isomers . At 303 K, NOESY spectra with mixing times of 100 ms did not show interconversion between these isomers . However, exchange cross-peaks were observed in a 700-ms NOESY spectrum at 323 K which demonstrated that these isomers are interconverting slowly on the NMR time scale . Many of the side chains with clearly doubled resonances were located in the beta-sheet and the active site . Preliminary studies on the apoprotein also revealed doubled resonances in the absence of the inhibitor, indicating the existence of the protein isomers prior to methotrexate binding . In contrast to the methotrexate complex, the binary complex with folate and the ternary MTX-NADPH-DHFR complex presented a single enzyme form . These results are proposed to reflect the ability of folate and NADPH to bind predominantly to one protein isomer.

Biochemistry, 1991 Feb 26, 30(8), 2156 - 65
1H NMR study of the solution molecular and electronic structure of Escherichia coli ferricytochrome b562: evidence for S = 1/2 in equilibrium S = 5/2 spin equilibrium for intact His/Met ligation; Wu JZ et al.; The solution 500-MHz 1H NMR spectral parameters for ferricytochrome b562, a soluble 12-kDa electron carrier from Escherichia coli with axial His/Met coordination, are shown to be strongly influenced by protein concentration and ionic strength at low pH and 25 degrees C in a manner consistent with significant aggregation at low ionic strength . At high ionic strength a well-resolved 1H NMR spectrum reveals over 40 hyperfine-shifted resonances which arise from two isomeric species in the ratio 2:1 . 2D COSY and NOESY maps at 25 degrees C for the hyperfine-shifted resonances allow the assignment of a number of axial His resonances and all heme peripheral substituent peaks . The resulting asymmetric heme contact shift patterns, together with the halving of the number of lines when reconstituting with 2-fold symmetric hemin, demonstrate the molecular basis of the solution heterogeneity to be heme orientational disorder . The strongly upfield-shifted axial Met-7 resonances, characteristic of low-spin ferricytochromes c with His/Met ligation, appear upfield only at very low temperatures . At elevated temperatures, all resonances, in particular those of the axial Met, move strongly downfield . Detailed analysis of the deviation from Curie behavior for different functional groups demonstrates the presence of a low spin in equilibrium high spin equilibrium with an intact His-Fe-Met coordination . The weaker axial field in ferricytochrome b562, relative to the purely low-spin ferricytochromes c, is attributed to a perturbed iron-Met bond . The contact shifts for a coordinated Met in the high-spin state are estimated . A link between equatorial hemin and axial ligand interactions is indicated by a differential population of the high-spin form for the two hemin orientations.

Biochemistry, 1991 Feb 26, 30(8), 2012 - 7
Toward a simplification of the protein folding problem: a stabilizing polyalanine alpha-helix engineered in T4 lysozyme; Zhang XJ et al.; In an attempt to simplify the protein folding problem, and also to further investigate the role of alanine as a helix-stabilizing residue, a series of alanines was introduced within the alpha-helix that includes residues 126-134 of T4 lysozyme . In wild-type lysozyme this alpha-helix contains alanine residues at positions 129, 130, and 134 . Mutant lysozymes with alanines substituted at positions 128, 131, 132, and 133, either as single substitutions or in selected combinations, were constructed by oligonucleotide-directed mutagenesis . With the exception of the replacement of Leu 133, which is buried within the hydrophobic core of the protein, all the variants were more stable than wild-type lysozyme . The variant with alanines substituted at positions 128, 131, and 132 (E128A/V131A/N132A), which incorporates the sequence Ala 128-Ala 129-Ala 130-Ala 131-Ala 132-Leu 133-Ala 134, has a melting temperature 3.3 degrees C above that of wild-type lysozyme . Determination of the crystal structure of this mutant lysozyme shows that the replacement of Glu 128, Val 131, and Asn 132 with alanine causes alpha-helix 126-134 to rotate 3.4 degrees about an axis parallel to its own axis . This rotation seems to be triggered primarily by the loss of a hydrogen bond between Asn 132 and Ser 117 and is associated with the repacking of several side chains at the interface between alpha-helix 126-134 and the adjacent alpha-helix 115-122.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1991 Feb 26, 196(1), 123 - 8
A characterization of copper/zinc superoxide dismutase mutants at position 124 . Zinc-deficient proteins; Banci L et al.; Substitution of the completely conserved aspartic acid residue at position 124 of Cu,Zn superoxide dismutase with asparagine and glycine has been performed through site-directed mutagenesis on the human enzyme . Asp124 is H-bonded to the NH of two histidines, one of which is bound to copper and the other to zinc . The mutant proteins, as expressed in Escherichia coli, result in an essential zinc-free enzyme which is similar to that obtained from the wild-type derivative through chemical manipulation . Only by extensive dialysis against 0.5 M ZnCl2 or CoCl2 at pH 5.4 was it possible to reconstitute approximately 50% of the molecules in the Cu2Zn2 or Cu2Co2 form . The new derivatives have been characterized through EPR, CD and nuclear magnetic relaxation dispersion techniques . The Cu2Cox derivatives (x approximately 1) were used to monitor, through electronic and 1H-NMR spectroscopies, the metal sites which are found to be similar to those of the wild type . In addition, a double substitution with asparagine has been made, replacing the invariant aspartate at position 124 and the highly conserved aspartate at position 125 . The behavior is similar to that of the other mutants in most respects . The Cu2E2 (E = empty) derivatives of the mutants are stable, even in the pH range 8-10, whereas in the case of the Cu2E2 derivative of the wild type, copper migration occurs at high pH, producing both Cu2Cu2 and apo derivatives . The activity measurements indicate that the various Cu2E2 derivatives have the same activity at low pH and similar to that of the holoenzyme . A full profile up to pH 10.5 was obtained for the mutants.

Biochemistry, 1991 Feb 26, 30(8), 2022 - 6
A novel dipyridodiazepinone inhibitor of HIV-1 reverse transcriptase acts through a nonsubstrate binding site; Wu JC et al.; A novel dipyridodiazepinone, 6,11-dihydro-11-cyclopropyl-4-methyldipyrido{2,3-b:2',3'-e}- {1,4}diazepin-6-one (BI-RG-587), is a selective noncompetitive inhibitor of HIV-1 reverse transcriptase (RT-1) . An azido photoaffinity analogue of BI-RG-587 was synthesized and found to irreversibly inhibit the enzyme upon UV irradiation . BI-RG-587 and close structural analogues competitively protected RT-1 from inactivation by the photoaffinity label . A thiobenzimidazolone (TIBO) derivative, a nonnucleoside inhibitor of RT-1, also protected the enzyme from photoinactivation, which suggests a common binding site for these compounds . Substrates dGTP, template-primer, and tRNA afforded no protection from enzyme inactivation . A tritiated photoaffinity probe was found to stoichiometrically and selectively label p66 such that 1 mol of probe inactivates 1 mol of RT-1.

Nucleic Acids Res, 1991 Feb 25, 19(4), 867 - 9
Suppression of a double missense mutation by a mutant tRNA(Phe) in Escherichia coli; Pages D et al.; We report here the isolation of a mutant tRNAPhe that suppresses a double missense auxotrophic mutation in trpA of Escherichia coli, trpA218 . The doubly mutant protein product differs from wild-type TrpA by the replacements of Phe22 by Leu and Gly211 by Ser . A partial revertant TrpA phenotype can be obtained from trpA218 by changing either Leu22 back to Phe or Ser211 back to Gly . Translational suppressors were previously obtained that act at codon 211, replacing the Ser211 in the TrpA218 protein, presumably with Gly . In the present study, we selected for trpA218 suppressors caused by mutation of a cloned tRNAPhe gene, pheV . DNA sequence analysis of the suppressor isolated reveals a singular structural alteration, changing the anticodon from 5'-GAA-3' to 5'-GAG-3' . Sequencing of trpA218 confirmed the likely identity of Leu22 as CUC . The new missense suppressor, designated pheV(SuCUC), is lethal to the cell when highly expressed, as from a high copy number plasmid . This may be due to efficient replacement of Leu by Phe at CUC (and, probably, CUU) codons throughout the genome . We anticipate that pheV(SuCUC) will prove, like other missense suppressors, to be extremely useful in studies on the specificity and accuracy of decoding.

Nucleic Acids Res, 1991 Feb 25, 19(4), 809 - 13
A novel DNA joining activity catalyzed by T4 DNA ligase; Western LM et al.; The use of T4 and E . coli DNA ligases in genetic engineering technology is usually associated with nick-closing activity in double stranded DNA or ligation of 'sticky-ends' to produce recombinant DNA molecules . We describe in this communication the ability of T4 DNA ligase to catalyze intramolecular loop formation between annealed oligodeoxyribonucleotides wherein Watson-Crick base pairing is absent on one side of the ligation site . Enzyme concentration, loop size, substrate specificity, and base composition were explored in an effort to maximize yield . Amounts of T4 DNA ligase in large molar excess to DNA template and ligated product are necessary to achieve high yields.

Nucleic Acids Res, 1991 Feb 25, 19(4), 727 - 31
Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation of integration vectors; Hillemann D et al.; For the isolation of single stranded plasmid DNA, various E . coli and E . coli-Streptomyces shuttle plasmids were equipped with the phage f1 replication origin . The transformation of some representative Streptomyces species with plasmid vectors occurred irrespective of whether single or double stranded DNA was used . In contrast, the transformation of Streptomyces was 10 to 100 times more efficient when an integration vector was in the single stranded form as opposed to the double stranded form . Streptomyces viridochromogenes was transformed by single stranded DNA integration vectors in order to replace the pat by the tsr gene and generate mutants unable to synthesize phosphinothricin-tripeptide (PTT).

FEBS Lett, 1991 Feb 25, 279(2), 233 - 6
Purification of SecE and reconstitution of SecE-dependent protein translocation activity; Tokuda H et al.; SecE was solubilized from SecE-overproducing E . coli cells and purified through ion exchange and size exclusion chromatographies . When the solubilized membrane containing overproduced amounts of SecY and SecE was fractionated by means of size exclusion chromatography, the two proteins were eluted in different fractions with slight overlapping . Proteoliposomes active in protein translocation were reconstituted from these fractions only when both SecE and SecY were present . When reconstitution was carried out with the purified SecE and fractions containing SecY but only a small amount of SecE, the resultant proteoliposomes exhibited appreciable translocation activity, indicating that SecE is essential for protein translocation . The translocation activity of proteoliposomes was proportional to the amount of purified SecE used for reconstitution . SecE-dependent protein translocation absolutely required ATP and SecA.

J Biol Chem, 1991 Feb 25, 266(6), 3752 - 9
The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita . Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis; Sato T et al.; The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14) . We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl . The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each . The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product . DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 6621-6625) . Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da . The data suggest that the enzyme is post-translationally processed during protein purification.

J Biol Chem, 1991 Feb 25, 266(6), 3744 - 51
Heteroduplex repair in extracts of human HeLa cells; Thomas DC et al.; A general repair process for DNA heteroduplexes has been detected in HeLa cell extracts . Using a variety of M13mp2 DNA substrates containing single-base mismatches and extra nucleotides, extensive repair is observed after incubation with HeLa cell cytoplasmic extracts and subsequent transfection of bacterial cells with the treated DNA . Most, but not all, mispairs as well as two frameshift heteroduplexes are repaired efficiently . Parallel measurements of repair in HeLa extracts and in Escherichia coli suggest that repair specificities are similar for the two systems . The presence of a nick in the molecule is required for efficient repair in HeLa cell extracts, and the strand containing the nick is the predominantly repaired strand . Mismatch-dependent DNA synthesis is observed when radiolabeled restriction fragments, produced by reaction of the extract with heteroduplex and homoduplex molecules, are compared . Specific labeling of fragments, representing a region of approximately 1,000 base pairs and containing the nick and the mismatch, is detected for the heteroduplex substrate but not the homoduplex . The repair reaction is complete after 20 min and requires added Mg2+, ATP, and an ATP-regenerating system, but not dNTPs, which are present at sufficient levels in the extract . An inhibitor of DNA polymerase beta, dideoxythimidine 5'-triphosphate, does not inhibit mismatch-specific DNA synthesis . Aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilom, inhibits both semiconservative replication and repair synthesis in the extract . Butylphenyl-dGTP also inhibits both replicative and repair synthesis but at a concentration known to inhibit DNA polymerase alpha preferentially rather than delta or epsilon . This suggests that DNA polymerase alpha may function in mismatch repair.

J Biol Chem, 1991 Feb 25, 266(6), 3408 - 10
A de novo designed signal peptide cleavage cassette functions in vivo; Nilsson I et al.; Leader peptidase (Lep) is a membrane-bound enzyme of the Escherichia coli inner membrane that serves to remove signal peptides from exported proteins . Statistical and experimental studies of known signal peptides have defined a short C-terminal region that seems to provide the information for correct cleavage by Lep . Based on the patterns of conserved amino acids found in this region, we have designed a signal peptide "cleavage cassette." This cassette is processed at the expected site when introduced after an uncleaved signal peptide . Furthermore, processing is blocked in the predicted manner when the (-3, -1)-rule for signal peptide cleavage is violated . This suggests that current understanding of the sequence requirements for signal peptide cleavage is sufficiently advanced to be used in, e.g . protein engineering applications.

Nucleic Acids Res, 1991 Feb 25, 19(4), 841 - 50
Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts; Brooks JE et al.; The BamHI restriction modification system was previously cloned into E . coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl . Acids Res . 17, 979-997) . The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined . The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570 . Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351 . The M . BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied . The BamHI methylase modifies the internal C within its recognition sequence at the N4 position . Comparisons of the deduced amino acid sequence of M . BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity . Finally, stability and expression of the BamHI system in both E . coli and B . subtilis have been studied . The results suggest R and M expression are carefully regulated in a 'natural' host like B . subtilis.

J Biol Chem, 1991 Feb 25, 266(6), 3760 - 7
Overexpression of the relA gene in Escherichia coli; Schreiber G et al.; Intracellular levels of guanosine 3',5'-bispyrophosphate (ppGpp) governed by the relA gene are normally regulated by aminoacyl-tRNA availability for protein synthesis . An experimental system is described in which cellular levels of ppGpp are controlled instead by induction of plasmid pKK223-3 derivatives with the relA structural gene, or portions thereof, under control of the Ptac promoter . In amino acid-rich media, isopropyl-1-thio-beta-D-galactopyranoside induction of transcription of the wild type relA gene in pSM10 yields about a 100-fold overexpression of a metabolically stable, full length (743 amino acid) RelA protein to levels approximating the number of cellular ribosomes . This overexpression is accompanied by a roughly parallel and relC-dependent elevation of ppGpp levels . Induction of a relA gene deletion mutant in pSM11 containing 455 amino-terminal amino acids results in much lower levels of expression of a metabolically unstable 55-kDa protein and elevated ppGpp levels that are almost equivalent to induced pSM10 and are relC-independent . Induction of a larger deletion in pSM12 containing 331 amino-terminal amino acids does not provoke ppGpp accumulation . We are able to elicit high levels of ppGpp without changing nutritional abundance and without massive overexpression of the RelA protein by inducing the metabolically unstable, truncated RelA protein . We find the effects of elevated ppGpp levels to include a slowing of growth, an inhibition of stable RNA accumulation, an inhibition of cellular rrn P1 promoter activities as measured by primer extension, and changes in the pattern of gene expression viewed by two-dimensional electrophoresis of cellular proteins.

Biochim Biophys Acta, 1991 Feb 25, 1062(2), 177 - 86
The interaction between aspartic acid 237 and lysine 358 in the lactose carrier of Escherichia coli; King SC et al.; The lacY from Escherichia coli strains 020 and AE43 have been cloned on plasmids which were designated p020-K358T and pAE43-D237N . These lacY mutants contain amino acid substitutions changing Lys-358 to Thr or Asp-237 to Asn, respectively . The charge neutralizing effect of each mutation is associated with a functional defect in melibiose transport which we exploited in order to isolate second site revertants to the melibiose-positive phenotype . Eleven melibiose-positive revertants of p020-K358T were isolated . All contained a second-site mutation converting Asp-237 to a neutral amino acid (8 to Asn, 1 to Gly, and 2 to Tyr) . Twelve melibiose-positive revertants of pAE43-D237N were isolated . Two were second-site revertants converting Lys-358 to a neutrally Gln residue, while the remainder directly reverted Asn-237 to the wild-type Asp-237 . We conclude that the functional intimate relationship between Asp-237 and Lys-358 suggests that these residues may be closely juxtaposed in three-dimensional space, possibly forming a 'charge-neutralizing' salt bridge.

FEBS Lett, 1991 Feb 25, 279(2), 285 - 8
Glycine-144 is required for efficient folding of outer membrane protein PhoE of Escherichia coli K12; de Cock H et al.; Short stretches of similar sequences have been detected in unrelated bacterial outer membrane proteins (Nikaido and Wu (1984) Proc . Natl . Acad . Sci . USA 81, 1048-1052) . In the most pronounced similarity region, only a glycine residue is absolutely conserved . To investigate whether this glycine residue is essential for outer membrane incorporation, oligonucleotide-directed mutagenesis was applied to replace this residue, i.e . Gly-144, as well as two other Gly-residues in pore protein PhoE . Substitution of Gly-52 and Gly-258 by Ala and Val, respectively, did not influence outer membrane incorporation . However, the substitution of Gly-144 by Leu affected the efficiency of outer membrane incorporation . After in vitro synthesis this mutant protein was less efficiently precipitated with monoclonal antibodies that recognize conformational epitopes than wild-type PhoE, showing that the mutation interferes with correct folding of the protein.

J Biol Chem, 1991 Feb 25, 266(6), 3863 - 9
Efficient purification of recombinant human tumor necrosis factor beta from Escherichia coli yields biologically active protein with a trimeric structure that binds to both tumor necrosis factor receptors; Schoenfeld HJ et al.; A fast and efficient method for medium scale purification of recombinant human tumor necrosis factor beta (rTNF-beta) from Escherichia coli cells is described . The purified rTNF-beta displayed biological activity similar to rTNF-alpha in a WEHI 164 cell cytotoxicity assay . The titration curve of rTNF-beta and elution profiles of rTNF-beta in gel filtration experiments were different from those of rTNF-alpha . However, light scattering and ultra-centrifugation studies showed that both cytokines have trimeric structures in solution at 0.5 mg/ml, with minor differences in the distribution of nontrimeric species . rTNF-beta bound to purified 55- and 75-kDa TNF receptors with high affinity . The binding of rTNF-beta to either receptor was analyzed on Scatchard plots and compared with that of rTNF-alpha.

J Biol Chem, 1991 Feb 25, 266(6), 3654 - 60
Analysis of an Escherichia coli dnaB temperature-sensitive insertion mutation and its cold-sensitive extragenic suppressor; Chang SF et al.; An Escherichia coli mutant, ts121, was isolated following random insertional mutagenesis using phage lambda Mu transposition . The mutant phenotype includes inability to form colonies at temperatures above 38 degrees C and inability to propagate phage lambda at all temperatures . A lambda i434 cI- (ts121)+ transducing phage was isolated on the basis of its ability to form plaques on ts121 mutant bacteria . Using this transducing phage, it was shown through complementation and protein analyses, that the ts121 mutation is located in the dnaB gene . The exact insertion event was identified by polymerase chain reaction amplification of the DNA sequences containing the insertion junction . The mutational insertion event in ts121 was mapped precisely between base pairs 1514 and 1515 of the dnaB gene . This result predicts that the mutant dnaB protein has lost its six terminal amino acids . The reading frame shifts into Mu-specific DNA sequences resulting in an additional 20 amino acid residues . The E . coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage lambda DNA replication . Our results demonstrate that the extreme carboxyl end of the dnaB protein is required for productive interaction with the lambda P replication protein at all temperatures, and is important for dnaB function at temperatures above 38 degrees C . Cold-sensitive extragenic suppressors of the ts121 mutation were isolated on the basis of their ability to restore colony formation at 42 degrees C . One of these extragenic suppressors was mapped at 54 min on the E . coli genetic map and localized to the suhB gene, whose product may affect the expression of a number of genes at the translational level.

J Biol Chem, 1991 Feb 25, 266(6), 3547 - 53
Mutations that alter the charge of type I regulatory subunit and modify activation properties of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells; Steinberg RA et al.; Mutations in regulatory (R) subunit of cAMP-dependent protein kinase were analyzed from cAMP-resistant mutants of S49 mouse lymphoma cells by direct sequencing of amplified regions of mutant R subunit cDNAs . Eight distinct single base-change lesions were identified in 24 independent mutants that were hemizygous for expression of mutant R subunits with altered protein charge . CG----TA transitions predominated, but AT----GC transitions and GC----TA transversions were also observed . Four of five spontaneous mutants had identical C----T transitions at CG causing substitution of Trp for Arg-334 . Sites mutated in isolates obtained after mutagenesis with ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine were more varied . Six of the lesions (two in binding site A and four in site B) were at amino acid residues that are highly conserved among cAMP-binding sites of R subunits and the Escherichia coli catabolite activator protein . These mutations all either prevented or strongly hindered binding of cyclic nucleotides to the mutated site . One of the remaining lesions (at Arg-242) also prevented cyclic nucleotide binding to the mutated binding site; the other (at Gly-170) had only minimal effects on binding of cyclic nucleotides but, nevertheless, increased the apparent constant for cAMP-dependent kinase activation . These results are discussed with reference to a model for the cAMP-binding sites of R subunit based on the crystal structure of the E . coli catabolite activator protein.

J Biol Chem, 1991 Feb 25, 266(6), 3540 - 6
Probing the limits of the DNA breakage-reunion domain of the Escherichia coli DNA gyrase A protein; Reece RJ et al.; In a previous report (Reece, R . J., and Maxwell, A . (1989) J . Biol . Chem . 264, 19648-19653) we showed that treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two stable fragments . The N-terminal 64-kDa fragment supports DNA supercoiling, while the C-terminal 33-kDa fragment shows no enzymic activity . We proposed that the 64-kDa fragment represents the DNA breakage-reunion domain of the A protein . We have now engineered the gyrA gene such that the 64-kDa protein is generated as a gene product . The properties of this protein confirm the findings of the experiments with the 64-kDa tryptic fragment . We have also generated a series of deletions of the gyrA gene such that C-terminal and N-terminal truncated versions of the A protein are produced . The smallest of the N-terminal fragments found to be able to carry out the DNA breakage-reunion reaction is GyrA(1-523) . The cleavage reaction mediated by this protein occurs with equal efficacy as that performed by the intact GyrA protein . Deletion of the N-terminal 6 amino acids from either the A protein or these deletion derivatives has no effect on enzymic activity, while deletion of the N-terminal 69 amino acids completely abolishes the DNA breakage-reunion reaction . Therefore the smallest GyrA protein we have found that will perform DNA breakage and reunion is GyrA(7-523) . A model is proposed for the domain organization of the gyrase A protein.

J Biol Chem, 1991 Feb 25, 266(6), 3361 - 4
Expression of hepatitis B virus surface antigen in adult rat liver . Co-introduction of DNA and nuclear protein by a simplified liposome method; Kato K et al.; We established a simple and efficient method for gene transfer in vitro (to cultured cells) and in vivo (to an adult organ) using liposomes . Plasmid DNA and proteins were efficiently co-encapsulated in liposomes by agitation and sonication, and were co-introduced into cells by hemagglutinating virus of Japan (HVJ)-mediated membrane fusion . Introduction of the Escherichia coli beta-galactosidase gene with non-histone chromosomal protein high mobility group 1 (HMG1) into LLCMK2 cells resulted in about 3 times higher beta-galactosidase activity than that on introduction of the gene alone . Two days after injection of HVJ-liposomes containing the beta-galactosidase gene and HMG1 under the perisplanchnic membrane of adult rat liver, hepatic cells near the injection site were found by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to have beta-galactosidase activity . After similar injection of HVJ-liposomes containing the hepatitis B virus surface antigen (HBsAg) gene and HMG1, HBsAg was detected in the serum for 9 days with a maximum of 25-45 ng/ml on day 2 after the injection.

FEBS Lett, 1991 Feb 25, 279(2), 193 - 7
A heptapeptide repeat contributes to the unusual length of chloroplast ribosomal protein S18 . Nucleotide sequence and map position of the rpl33-rps18 gene cluster in maize; Weglohner W et al.; The rpl33-rps18 gene cluster of the maize chloroplast genome has been mapped and sequenced . The derived amino acid sequence of the S18 protein shows a 7-fold repeat of a hydrophilic heptapeptide domain, S K Q P F R K, in the N-terminal region . Such a sequence is absent in the E . coli S18 and in the chloroplast S18 of the lower plant liverwort . In tobacco and rice chloroplast S18 it is present 2 and 6 times, respectively . Thus a long N-terminal repeat (resembling in composition the large C-terminal heptapeptide repeat in the eukaryotic pol II) appears to be characteristic of monocot cereal S18.

J Biol Chem, 1991 Feb 25, 266(6), 3630 - 5
Catalysis of protein folding by cyclophilins from different species; Schonbrunner ER et al.; Cyclophilins are a class of ubiquitous proteins with yet unknown function . They were originally discovered as the major binding proteins for the immunosuppressant cyclosporin A . The only known catalytic function of these proteins in vitro is the cis/trans isomerization of Xaa-Pro bonds in oligopeptides . This became clear after the discovery that bovine cyclophilin is identical with porcine prolyl isomerase . This enzyme accelerates slow, proline-limited steps in the refolding of several proteins . Here we demonstrate that the cyclophilins from man, pig, Neurospora crassa, Saccharomyces cerevisiae, and Escherichia coli are all active as prolyl isomerases and as catalysts of protein folding . This evolutionary conservation suggests that catalysis of prolyl peptide bond isomerization may be an important function of the cyclophilins . It could be related with de novo protein folding or be involved in regulatory processes . Catalysis of folding is very efficient in the presence of the high cellular concentrations of prolyl isomerase.

J Biol Chem, 1991 Feb 25, 266(6), 3387 - 95
The catalytic mechanism of the amidotransferase domain of the Syrian hamster multifunctional protein CAD . Evidence for a CAD-glutamyl covalent intermediate in the formation of carbamyl phosphate; Chaparian MG et al.; The multifunctional protein CAD catalyzes the first three steps in pyrimidine biosynthesis in mammalian cells, including the synthesis of carbamyl phosphate from bicarbonate, MgATP and glutamine . The Syrian hamster CAD glutaminase (GLNase) domain, a trpG-type amidotransferase, catalyzes glutamine hydrolysis in the absence of MgATP and bicarbonate (Km = 95 microM and kcat = 0.14 s-1) . Unlike E . coli carbamyl phosphate synthetase (Wellner, V.P., Anderson, P.M., and Meister, A . (1973) Biochemistry 12, 2061-2066), a stable thioester intermediate did not accumulate when the mammalian enzyme was incubated with glutamine . However, a covalent adduct could be isolated when the protein was denatured in acid . The steady state concentration of the intermediate increased with increasing glutamine concentration to nearly one mole per mole of enzyme with half saturation at 105 microM, close to the Km value for glutamine . The adduct formed at the active site of the glutaminase domain . The rate of breakdown of the intermediate (k4), determined directly, was 0.17 s-1 and the rate of formation (k3) was estimated as 0.52 s-1 . In the absence of MgATP and bicarbonate, k4 = kcat indicating that the decomposition of the intermediate is the rate-limiting step . The intermediate was chemically and kinetically competent, and the glutamine dissociation constant (330 microM) and rate constants were consistent with steady state kinetics and accurately predicted the steady state concentration of the intermediate . These studies suggest a mechanism similar to the cysteine proteases such as recently proposed by Mei and Zalkin (Mei, B., and Zalkin, H . (1989) J . Biol . Chem . 264, 16613-16619) who identified a catalytic triad in glutamine phosphoribosyl-5'-pyrophosphate amidotransferase, a purF-type enzyme . MgATP and bicarbonate increased kcat of the glutaminase reaction 14-fold by accelerating both the rate of formation and the rate of breakdown of the intermediate, and prevented the accumulation of the intermediate; however, the Km value for glutamine was not significantly altered . The instability of the thioester intermediate leads to appreciable hydrolysis of glutamine in the absence of the other substrates . However, bicarbonate alone spares glutamine by increasing the Km and Ks of glutamine to 600 and 8960 microM, respectively, thus reducing kcat/Km 3-fold when MgATP is limiting . In the absence of MgATP and bicarbonate, ammonia decreased the rate of hydrolysis and the accumulation of the thioester intermediate indicating that ammonia had direct access to the thioester at the GLNase domain active site.(ABSTRACT TRUNCATED AT 400 WORDS)

J Chromatogr, 1991 Feb 22, 539(2), 501 - 5
Purification of cloned trypanosomal calmodulin and preliminary NMR studies; Sweeney PJ et al.; Cloned trypanosomal calmodulin was expressed in Escherichia coli and purified to homogeneity using hydrophobic interaction chromatography on phenyl-Sepharose . The purified protein was subjected to NMR analysis which allows detailed changes to be observed when, firstly, calcium, and secondly, the drug calmidazolium bind . These spectral changes are the result of conformational changes in the protein and proximity effects due to the drug.

J Chromatogr, 1991 Feb 22, 539(2), 465 - 73
Purification and characterization of recombinant tropomyosins; Ferraz C et al.; The cloning of a cDNA coding for the skeletal human beta-tropomyosin in the bacterial expression vector pKK233-2 is reported . Deletion mutants were also constructed . pCF-T1088 was obtained by elimination of exon 9 and pCF-T1089 was built by deleting 2/3 of the first exon . The recombinant tropomyosins were synthesized in E . coli after induction by IPTG . The mutant proteins were characterized by western blot using antibodies raised against native tropomyosin . The amount of the human protein synthesized in E . coli varies with each mutant, suggesting the involvement of the structure of the protein or of the mRNA on the synthesis or the stability of the recombinant protein . After precipitation of most of the bacterial proteins at 100 degrees C, purification was achieved by high-performance liquid chromatography (HPLC) using TSK-DEAE, hydroxyapatite and reversed-phase columns . The chromatographic behaviour of the mutants were compared . Characterization of the mutated tropomyosins was achieved by tryptic digestion and analysis of the peptide composition by reversed-phase HPLC . A computer program for predicting the retention times of the peptides generated was written . It is shown that it is possible to identify the mutations solely by comparing the chromatogram of the tryptic digest with the profile obtained by computer simulation.

J Chromatogr, 1991 Feb 22, 539(2), 383 - 91
Unfolding of truncated and wild type aspartate aminotransferase studied by size-exclusion chromatography; Herold M et al.; The reversible unfolding of globular proteins with increasing concentration of guanidinium chloride (GuCl) can be analysed by size-exclusion chromatography, because the hydrodynamic volume of the proteins increases during unfolding . The dimeric enzyme aspartate aminotransferase (AAT) shows an uncoupled dissociation of the identical subunits followed by the unfolding of the monomers . During the monomer unfolding formation of an intermediate is observed . A monomeric mutant of AAT unfolds with a similar shape of the unfolding transition phase, but is less stable, as shown by a shift of the transition mid-point from 1.7 M GuCl for the wild type to 1.3 M GuCl for the mutant.

Proc R Soc Lond B Biol Sci, 1991 Feb 22, 243(1307), 155 - 60
Site-directed mutagenesis of the lipoate acetyltransferase of Escherichia coli; Russell GC et al.; Remote but significant similarities between the primary and predicted secondary structures of the chloramphenicol acetyltransferases (CAT) and lipoate acyltransferase subunits (LAT, E2) of the 2-oxo acid dehydrogenase complexes, have suggested that both types of enzyme may use similar catalytic mechanisms . Multiple sequence alignments for CAT and LAT have highlighted two conserved motifs that contain the active-site histidine and serine residues of CAT . Site-directed replacement of Ser550 in the E2p subunit (LAT) of the pyruvate dehydrogenase complex of Escherichia coli, deemed to be equivalent to the active-site Ser148 of CAT, supported the CAT-based model of LAT catalysis . The effects of other substitutions were also consistent with the predicted similarity in catalytic mechanism although specific details of active-site geometry may not be conserved.

Nature, 1991 Feb 21, 349(6311), 713 - 5
A dynamin-like protein encoded by the yeast sporulation gene SPO15; Yeh E et al.; The tightly centromere-linked gene SPO15 is essential for meiotic cell division in the yeast Saccharomyces cerevisiae . Diploid cells without the intact SPO15 gene product are able to complete premeiotic DNA synthesis and genetic recombination, but are unable to traverse the division cycles . Electron microscopy of blocked cells reveals a duplicated but unseparated spindle-pole body . Thus cells are unable to form a bipolar spindle . Sequence analysis of SPO15 DNA reveals an open reading frame that predicts a protein of 704 amino acids . This protein is identical to VPS1, a gene involved in vacuolar protein sorting in yeast which has significant sequence homology (45% overall, 66% over 300 amino acids) to the microtubule bundling-protein, dynamin . The SPO15 gene product expressed in Escherichia coli can be affinity-purified with microtubules . SPO15 encodes a protein that is likely to be involved in a microtubule-dependent process required for the timely separation of spindle-pole bodies in meiosis.

J Mol Biol, 1991 Feb 20, 217(4), 661 - 79
CAP and Nag repressor binding to the regulatory regions of the nagE-B and manX genes of Escherichia coli; Plumbridge J et al.; The divergent nagE-BACD operons located at 15.5 min on the Escherichia coli chromosome encode genes involved in the uptake and metabolism of N-acetylglucosamine . The start sites of the divergent transcripts are separated by 133 base-pairs (bp) . A repressor protein for the regulon is encoded by the gene nagC, one of the genes of the nagBACD operon . Strains overproducing the NagC protein have been used to investigate the binding of repressor to the intergenic nagE-B regulatory region . Two binding sites have been detected, overlapping the promoters of the nagE and nagB genes . NagC binding produces a series of DNase I hypersensitive sites separated by 9 to 11 bp in the region between the two NagC binding sites, supporting a model where the NagC proteins bind co-operatively to these two sites on the DNA and interact to form a DNA loop . A strong CAP binding site exists between the two operator sites . It is located at -61.5 and -71.5 relative to the nagE and nagB transcription start sites . CAP and NagC can bind simultaneously and produce a complex more stable than the binary NagC-DNA complex . In addition NagC and CAP binding sites have been found upstream from the manXYZ operon . Although the sites exhibit a similar organization there is no evidence for formation of a DNA loop in this operon.

Biochemistry, 1991 Feb 19, 30(7), 1980 - 5
Activity and structure of the active-site mutants R386Y and R386F of Escherichia coli aspartate aminotransferase; Danishefsky AT et al.; Arginine-386, the active-site residue of Escherichia coli aspartate aminotransferase (EC 2.6.1.1) that binds the substrate alpha-carboxylate, was replaced with tyrosine and phenylalanine by site-directed mutagenesis . This experiment was undertaken to elucidate the roles of particular enzyme-substrate interactions in triggering the substrate-induced conformational change in the enzyme . The activity and crystal structure of the resulting mutants were examined . The apparent second-order rate constants of both of these mutants are reduced by more than 5 orders of magnitude as compared to that of wild-type enzyme, though R386Y is slightly more active than R386F . The 2.5-A resolution structure of R386F in its native state was determined by using difference Fourier methods . The overall structure is very similar to that of the wild-type enzyme in the open conformation . The position of the Phe-386 side chain, however, appears to shift with respect to that of Arg-386 in the wild-type enzyme and to form new contacts with neighboring residues.

Biochemistry, 1991 Feb 19, 30(7), 1948 - 57
Construction, expression, and purification of recombinant kringle 1 of human plasminogen and analysis of its interaction with omega-amino acids; Menhart N et al.; An Escherichia coli expression vector, containing the alkaline phosphatase promoter and the stII heat-stable enterotoxin signal sequence, along with the cDNA of the kringle 1 (K1) region of human plasminogen (HPg), has been employed to express into the periplasmic space amino acid residues 82-163 (E163----D) of HPg . This region of the molecule contains the entire K1 domain (residues C84-C162) of HPg, as well as two non-kringle amino-terminal amino acids (S82-E83) that are present in their normal locations in HPg and a carboxyl-terminal amino acid, D163, that results from mutation of the E163, normally present at this location in the HPg amino acid sequence . After purification of r-K1 by chromatographic techniques, we have investigated its omega-amino acid binding properties by titration calorimetry, intrinsic fluorescence, and differential scanning microcalorimetry (DSC) . The antifibrinolytic agent, epsilon-aminocaproic acid (EACA), possesses a single binding site for r-K1 . The thermodynamic properties of this interaction, studied by calorimetric titrations of the heats of binding with this ligand, reveal a Kd of 12 +/- 2 microM at 25 degrees C and pH 7.4, a corresponding delta G of -6.7 +/- 0.1 kcal/mol, a delta H of -3.6 +/- 0.1 kcal/mol, and a delta S of 10.5 +/- 0.8 eu . The intrinsic fluorescence of r-K1 decreases by approximately 44% when its binding site is saturated with EACA, and titrations of this perturbation with EACA lead to calculation of a Kd of approximately 13 microM, a value in good agreement with that obtained from titration calorimetric analysis . EACA represents the strongest binding ligand of a variety of simple aliphatic omega-amino acids examined . A cyclic analogue of EACA, trans-4-(aminomethyl)cyclohexanecarboxylic acid, interacts with r-K1 with an approximate 12-fold tighter Kd (1.0 +/- 0.2 microM) . Investigations by DSC, at pH 7.4, demonstrate that a significant stabilization of the r-K1 structure occurs when EACA binds to this domain . The temperature of maximum heat capacity change (Tm) in the thermal denaturation of r-K1 increases from approximately 340.8 to 359.1 K as a consequence of EACA binding . These studies demonstrate that a fully functional EACA-binding kringle from HPg can be expressed and secreted in E . coli, purified by techniques that do not require refolding, and investigated as an independent structural unit.

Biochemistry, 1991 Feb 19, 30(7), 1845 - 51
Structural microheterogeneity of a tryptophan residue required for efficient biological electron transfer between putidaredoxin and cytochrome P-450cam; Stayton PS et al.; The carboxy-terminal tryptophan of putidaredoxin, the Fe2S2.Cys4 iron-sulfur physiological redox partner of cytochrome P-450cam, is essential for maximal biological activity {Davies, M . D., Qin, L., Beck, J . L., Suslick, K . S., Koga, H., Horiuchi, T., & Sligar, S . G . (1990) J . Am . Chem . Soc . 112, 7396-7398} . This single tryptophan-containing protein thus represents an excellent system for studying the solution dynamics of a residue directly implicated in an electron-transfer pathway . Steady-state and time-resolved measurements of the tryptophan fluorescence have been conducted across the emission spectrum as a function of redox state to probe potential structural changes which might be candidates for structural gating phenomena . The steady-state emission spectrum (lambda max = 358 nm) and anisotropy (alpha = 0.04) suggest that Trp-106 is very solvent-exposed and rotating partially free of global protein constraints . The time-resolved fluorescence kinetics for both oxidized and reduced putidaredoxin are fit best with three discrete components of approximately 5, 2, and 0.3 ns . The lifetime components were assigned to physical species with iodide ion quenching experiments, where differential quenching of the longer components was observed (k tau = 2 = 5.9 X 10(8) M-1 s-1, k tau = 5 = 1.3 X 10(8) M-1 s-1) . These findings suggest that the multiexponential fluorescence decay results from ground-state conformational microheterogeneity and thus demonstrate that the essential tryptophan exists in at least two distinguishable conformations . Small differences in the relative proportions of the components between redox states were observed but not cleanly resolved.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Feb 19, 30(7), 1788 - 95
Sites of contact of mRNA with 16S rRNA and 23S rRNA in the Escherichia coli ribosome; Wollenzien P et al.; The locations of close encounter between ribosomal RNA (rRNA) and messenger RNA (mRNA) were determined by photochemical cross-linking experiments that employ an artificial mRNA, 51 nucleotides long, containing 14 U residues that were randomly substituted by 1-4 4-thiouridine (s4U) residues . The mRNA was bound to 70S ribosomes or 30S subunits and then was irradiated at 366 nm to activate cross-linking between the s4U residues and rRNA . Cross-linking occurred to both 16S rRNA and 23S RNA . The rRNA was then analyzed by a series of reverse transcriptase experiments to determine the locations of cross-linking . Twelve sites in the 16S rRNA and two sites in the 23S rRNA have been detected . In the 16S rRNA, two of the sites (U1381, C1395) are in the middle part of the secondary structure close to position C1400, and the remaining sites (G413, U421, G424; A532; G693; U723; A845; G1131/C1132; G1300; G1338) are distributed between six regions that are peripheral in the secondary structure . In the 23S rRNA, one site (U1065) is located in the GTPase center close to A1067, the site of thiostrepton-resistance methylation in domain II, and the other site (U887) is located a short distance away also in domain II . The distribution of these rRNA sites in the ribosome specifies an mRNA track that is consistent with other information . In addition, some of the contact points represent new constraints for the three-dimensional folding of the rRNA.

Biochemistry, 1991 Feb 19, 30(7), 1768 - 73
Details of mannitol transport in Escherichia coli elucidated by site-specific mutagenesis and complementation of phosphorylation site mutants of the phosphoenolpyruvate-dependent mannitol-specific phosphotransferase system; van Weeghel RP et al.; The mannitol transport protein (EIImtl) carries out translocation with concomitant phosphorylation of mannitol from the periplasm to the cytoplasm, at the expense of phosphoenolpyruvate (PEP) . The phosphoryl group which is needed for this group translocation is sequentially transferred from PEP via two phosphorylation sites, located exclusively on the C-terminal cytoplasmic domain, to mannitol . Oligonucleotide-directed mutagenesis was used to investigate the precise role of these sites in phosphoryl group transfer, by producing specific amino acid substitutions . The first phosphorylation site, His-554 (P1), was replaced by Ala, which renders the EII-H554A completely inactive in PEP-dependent mannitol phosphorylation, but not in mannitol/mannitol 1-phosphate exchange . The P2 site mutant, EII-C384S, was inactive both in the mannitol phosphorylation reaction and in the exchange reaction, due to replacement of the essential Cys-384 by Ser . Although EII-H554A and EII-C384S were both catalytically inactive in the PEP-dependent phosphorylation, EII-C384S was able to restore up to 55% of the wild-type mannitol phosphorylation activity with the EII-H554A mutant, indicating a direct phosphotransfer between two subunits . These phosphorylation data together with the data obtained from mannitol/mannitol phosphate exchange kinetics, after mixing EII-H554A and EII-C384S, indicated the formation of functionally stable heterodimers, which consist of an EII-H554A and an EII-C384S monomer.

Biochemistry, 1991 Feb 19, 30(7), 1939 - 47
Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit; Mann GJ et al.; Overexpression of recombinant mouse and herpes simplex virus ribonucleotide reductase small subunit (protein R2) has been obtained by using the T7 RNA polymerase expression system . Both proteins, which constitute about 30% of the soluble Escherichia coli proteins, have been purified to homogeneity by a rapid and simple procedure . At this stage, few of the molecules contain the iron-tyrosyl free-radical center necessary for activity; however, addition of ferrous iron and oxygen under controlled conditions resulted in a mouse R2 protein containing 0.8 radical and 2 irons per polypeptide chain . In this reaction, one oxygen molecule was needed to generate each tyrosyl radical . Both proteins had full enzymatic activity . EPR spectroscopy showed that iron-center/radical interactions are considerably stronger in both mouse and viral proteins than in E . coli protein R2 . CD spectra showed that the bacterial protein contains 70% alpha-helical structure compared to only about 50% in the mouse and viral proteins . Light absorption spectra between 310 and 600 nm indicate close similarity of the mu-oxo-bridged binuclear iron centers in all three R2 proteins . Furthermore, the paramagnetically shifted iron ligand proton NMR resonances show that the antiferromagnetic coupling and ligand arrangement in the iron center are nearly identical in all three species.

Biochemistry, 1991 Feb 19, 30(7), 1801 - 7
Requirement for the beta,gamma-pyrophosphate bond of ATP in a stage between transcription initiation and elongation by Escherichia coli RNA polymerase; Fujioka M et al.; A linear fragment of DNA was fixed to acrylamide or agarose beads by its ends . When a fragment containing the lambda PR promoter is immobilized and transcribed, the RNA products are unchanged from those obtained on the unfixed DNA . Transcription from the immobilized fragment can be interrupted by diluting the reaction mixture into a large volume of the same buffer . Brief centrifugation allows isolation of the transcription complex with the immobilized DNA . If interruption occurs during elongation, the elongation can be resumed upon a second addition of substrates . If ATP is replaced by a beta, gamma-unhydrolyzable analogue in the second addition, the elongated products are similar to those obtained when the substrate contain ATP . When ATP is replaced by the analogue at the initiation step, however, the yield of elongated products is decreased to less than one-sixth and that of short abortive products is increased . Thus the ATP analogues are good substrates once elongation has been established in the presence of ATP, but not good enough to get past a stage just after initiation in the absence of ATP . We conclude that the beta, gamma-pyrophosphate bond of ATP is important for preparation of efficient elongation.

Biochim Biophys Acta, 1991 Feb 16, 1088(2), 323 - 4
Nucleotide sequence of the lipase gene lip3 from the antarctic psychotroph Moraxella TA144; Feller G et al.; A lipase gene (lip3) from the psychotrophic strain Moraxella TA144 has been cloned and sequenced . The deduced primary structure of the lipase preprotein is composed of 315 amino acids with a predicted Mr of 34,772 . This enzyme contains two consensus peptides showing cluster of glycine residues that may be involved in domain flexibility . The cloned gene product conserves the low temperature activity and the thermolability properties of the wild enzyme.

Biochim Biophys Acta, 1991 Feb 16, 1088(2), 292 - 300
Cloning and sequencing of the gene cluster encoding two subunits of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes; Tamaki T et al.; The membrane-bound alcohol dehydrogenase (ADH) from Acetobacter polyoxogenes NBI1028 is composed of a 72 kDa subunit and a 44 kDa cytochrome c subunit . The amino acid sequences of the two regions of the 72 kDa subunit were determined to prepare oligonucleotides for the purpose of amplification of a DNA fragment corresponding to the intermediate region by the polymerase chain reaction . A 0.5 kb DNA fragment thus amplified was used as the probe to clone a 7.0 kb PstI fragment coding for the whole 72 kDa subunit . Nucleotide sequencing and immunoblot analysis revealed that the cloned fragment contained the full structural genes for the 72 kDa and the 44 kDa subunits and they were clustered with the same transcription polarity . The predicted amino acid sequence of the gene for the 72 kDa subunit showed homology with that of the 72 kDa subunit from ADH of A . aceti and those of methanol dehydrogenase from methylotrophic bacteria . The 72 and 44 kDa subunits contained one and three typical haem binding sequences, respectively.

Biochim Biophys Acta, 1991 Feb 16, 1088(2), 197 - 207
Isolation and characterization of a human cDNA encoding uracil-DNA glycosylase; Muller SJ et al.; DNA repair of genetic information is an essential defense mechanism, which protects cells against mutation and transformation . The biochemistry of human DNA repair is in its beginning stages . Our research has concentrated on the enzymes involved in the removal of atypical bases from DNA . We present information on the identification and characterization of a cDNA isolate encoding uracil-DNA glycosylase . Uracil-DNA glycosylase was purified to homogeneity from HeLa S3 cells and used to generate polyclonal antibodies . These antibodies were in turn used to isolate a uracil-DNA glycosylase specific cDNA from a human T cell (Jurkat) lambda-gt11 library . The identity of this 1.25 kb cDNA was verified using in vitro transcription and translation systems to generate specific uracil-DNA glycosylase activity . Sequence data revealed a 327 amino acid open reading frame, which encodes a protein with a predicted molecular weight of 35351 . No significant amino acid homology was found between this human uracil-DNA glycosylase and the glycosylases of yeast, Escherichia coli, herpes simplex virus, or a recently identified 26,000 Da species of human uracil-DNA glycosylase . This apparent lack of homology prompted an investigation of uracil-DNA glycosylase in a variety of eukaryotic species . Western analysis demonstrated the presence of a 36 kDa uracil-DNA glycosylase protein in human fibroblast, human placental and Vero cell extracts . Interestingly, these antibodies did not detect glycosylase protein in Chinese hamster ovary (CHO) or mouse NIH3T3 fibroblast cells . Under conditions of reduced stringency, Southern blot analysis of BamHI digested DNA from human fibroblasts, human placental cells and Vero cells revealed common 12 kb and 3 kb fragments . In contrast, using the same reduced stringency protocol, 6 and 8 kb fragments for CHO and NIH3T3 DNA were seen, respectively, as well as a common 3 kb fragment . Under more stringent wash conditions, the common 3 kb band was absent in all samples analyzed, and no hybridization signal was detected from DNA of hamster or mouse origin . The lack of immunological reactivity between the human uracil-DNA glycosylase and the rodent forms is therefore reflected at the genetic level as well . This distinction in human and CHO hybridization patterns enabled us to localize this human uracil-DNA glycosylase cDNA to chromosome 5 by somatic cell hybridization.

Gene, 1991 Feb 15, 98(2), 289 - 93
Cloning and expression in Escherichia coli of a synthetic gene encoding the extracellular domain of the human muscle acetylcholine receptor alpha-subunit; Talib S et al.; To better define the antigenic sites on the human muscle acetylcholine receptor (AChR) that are involved in stimulating the production of pathogenic antibodies in myasthenia gravis (MG), the nucleotide sequence encoding the major extracellular domain of the AChR alpha subunit was chemically synthesized . The gene cassettes encoding amino acids (aa) 1-85 (AChR-I) and 86-210 (AChR-II), were cloned individually, and the coding sequence representing the complete major extracellular domain (aa 1-210; AChR-C) was obtained by subsequent fusion of cassettes encoding AChR-I and AChR-II . The genes were inserted into the inducible expression plasmid, pKK-223-3, and expressed in vitro and in vivo in Escherichia coli . Biological activity was demonstrated by immunoprecipitation of in vitro-synthesized AChR-C by sera from MG patients and by the alpha-bungarotoxin-binding activity of E . coli-synthesized AChR-II and AChR-C . The availability of the recombinant AChR polypeptides should facilitate studies on the molecular basis of the autoimmune response in MG.

Gene, 1991 Feb 15, 98(2), 259 - 63
The rodent B2 sequence can affect expression when present in the transcribed region of a reporter gene; Bladon TS et al.; The mouse B2 element is a moderately repetitive nt sequence of 180 bp transcribed by RNA polymerase III (Pol III) at high levels in embryonic and transformed cells . The B2 sequence is present in either orientation within the noncoding regions of a number of genes transcribed by RNA polymerase II (Pol II) . We sought to determine if the small B2 transcripts generated by Pol III are natural antisense RNA molecules which might hybridize to complementary sequences present within Pol II transcripts . Chimaeric reporter genes encoding Escherichia coli gpt were constructed containing a B2 repeat in either orientation within the 5'- or 3'-untranslated regions . These constructs were transfected into embryonal carcinoma (EC) cells and expression of the reporter gene was analysed in EC cells and retinoic acid-treated EC cells, which contain high and low levels of small B2 RNAs, respectively . Although the B2 sequences affected expression of the reporter gene, these effects did not appear to be due to hybridization of the small B2 RNA to the reporter transcripts . The presence of B2 sequences near a Pol II-transcribed gene can alter expression of that gene in a position- and orientation-dependent manner, suggesting these repetitive elements may be cis-acting regulators of gene expression.

Gene, 1991 Feb 15, 98(2), 243 - 8
Rapid isolation of a rice waxy sequence: a simple PCR method for the analysis of recombinant plasmids from intact Escherichia coli cells; Shimada H et al.; Polymerase chain reaction (PCR) is an efficient method for obtaining a desired nt sequence if both required primers can hybridize to the target DNA molecule specifically . A rapid and simple PCR-based method for analyzing plasmids using intact cells was established . An attempt to target a rice waxy sequence by PCR using homologous primers was also carried out . In three cases, specific fragments were amplified and their nucleotide sequences were determined . However, the cloned rice waxy gene contained base substitution mutations . The cumulative frequency of mutation after 30 polymerization cycles was estimated to be one in 500 bp.

Gene, 1991 Feb 15, 98(2), 217 - 23
Effects of second-codon mutations on expression of the insulin-like growth factor-II-encoding gene in Escherichia coli; Cantrell AS et al.; Expression plasmids encoding random sequence mutant proteins of insulin-like growth factor II (IGFII) were constructed by cassette mutagenesis, to improve the efficiency of IGFII synthesis in Escherichia coli . A pool of oligodeoxyribonucleotide linkers containing random trinucleotide sequences were used to introduce second-codon substitutions into the gene encoding Met-Xaa-Trp-IGFII in expression vectors . E . coli RV308 cells transformed with these vectors synthesized IGFII at levels varying from 0-22% of total cell protein . This variable synthesis is a function of the random second-codon sequence and its corresponding amino acid, Xaa . Our data showed that mRNA stability, protein stability and translational efficiency all contributed to variable expression levels of Met-Xaa-Trp-IGFII in E . coli . Furthermore, an efficiently synthesized IGFII mutant protein, Met-His-Trp-IGFII, was converted to natural sequence IGFII by a simple oxidative cleavage reaction.

Biochim Biophys Acta, 1991 Feb 15, 1076(3), 387 - 94
Specific modification of Escherichia coli RNA polymerase with monomercury derivative of fluorescein acetate; Ozoline ON et al.; The method of specific modification of RNA polymerase with a monomercuric fluorescein derivative, fluorescein-monomercuriacetate (FMMA), is proposed . Under an appropriate condition of modification, FMMA is capable of mercaptid bonding with one of the alpha-subunits . It is shown that covalent modification with FMMA does not affect the kinetic parameters (KB and k2) of RNA synthesis nor does it lead to the inhibition of the overall RNA synthesis . The spectral characteristics of FMMA covalently bound to RNA polymerase were found to be sensitive to some temperature-induced conformational alterations of RNA polymerase, indicating that the labeled enzyme allows study of conformational behaviour of RNA polymerase during its functioning.

Biochim Biophys Acta, 1991 Feb 15, 1076(3), 374 - 8
The importance of glycine-30 for enzymatic activity of phospholipase A2; Bekkers AC et al.; The nearly conserved glycine-30 in porcine pancreatic phospholipase A2 has been replaced by serine . The resulting mutant G30S was expressed in Escherichia coli, purified and characterized . The mutation caused a significant drop in enzymatic activity towards monomeric and aggregated substrates, but had a limited effect on substrate binding . In contrast the affinity for calcium ions, the essential cofactor, was reduced 10-fold . The reduced enzymatic activity is attributed to a reduced stabilization of the transition state . The results are discussed in view of naturally occurring inactive phospholipase A2 homologues from snake venom.

J Immunol Methods, 1991 Feb 15, 136(2), 211 - 9
Recombinant fusion proteins of protein A and protein G with glutathione S-transferase as reporter molecules; Lew AM et al.; The regions encoding the IgG-binding domains of protein A (PA) and protein G (PG) were cloned into the bacterial expression vector pGEX . Both proteins were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (PA-GST and PG-GST) and were found to be soluble, abundant and easily purified in one step from the bacterial lysate by affinity chromatography on immobilized glutathione . Yields of 50 mg/litre of cultures were obtained . Both purified fusion proteins were shown to be functional in a variety of immunochemical procedures . In radial diffusion tests, PA-GST precipitated IgG from human, squirrel monkey, rabbit, dog, cat and pig but not mouse, sheep, goat, cow, horse or chicken . PG-GST formed precipitin bands with IgG from human, rabbit, mouse, pig, sheep, goat, cow and horse but not squirrel monkey, dog, cat and chicken IgG . The fusion proteins were shown to function as effective detection reagents in ELISA and Western blotting . Glutathione agarose beads with bound fusion protein were shown to be useful for immunoprecipitation.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1531 - 5
Cloning and expression of human deoxycytidine kinase cDNA; Chottiner EG et al.; Deoxycytidine (dCyd) kinase is required for the phosphorylation of several deoxyribonucleosides and certain nucleoside analogs widely employed as antiviral and chemotherapeutic agents . Detailed analysis of this enzyme has been limited, however, by its low abundance and instability . Using oligonucleotides based on primary amino acid sequence derived from purified dCyd kinase, we have screened T-lymphoblast cDNA libraries and identified a cDNA sequence that encodes a 30.5-kDa protein corresponding to the subunit molecular mass of the purified protein . Expression of the cDNA in Escherichia coli results in a 40-fold increase in dCyd kinase activity over control levels . In dCyd kinase-deficient murine L cells, transfection with dCyd kinase cDNA in a mammalian expression vector produces a 400-fold increase over control in dCyd phosphorylating activity . The expressed enzyme has an apparent Km of 1.0 microM for dCyd and is also capable of phosphorylating dAdo and dGuo . Northern blot analysis reveals a single 2.8-kilobase mRNA expressed in T lymphoblasts at 5- to 10-fold higher levels than in B lymphoblasts, and decreased dCyd kinase mRNA levels are present in T-lymphoblast cell lines resistant to arabinofuranosylcytosine and dideoxycytidine . These findings document that this cDNA encodes the T-lymphoblast dCyd kinase responsible for the phosphorylation of dAdo and dGuo as well as dCyd and arabinofuranosylcytosine.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1344 - 8
Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein; Ramdas J et al.; To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones . We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed . In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange . The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing . The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing . The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.

J Biol Chem, 1991 Feb 15, 266(5), 3239 - 45
CD63 antigen . A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells; Metzelaar MJ et al.; To clone the CD63 antigen, originally described as a blood platelet activation marker, we adapted the expression cloning procedure of Seed and Aruffo (Seed, B., and Aruffo, A . (1987) Proc . Natl . Acad . Sci . U.S . A . 84, 3365-3369) to allow cloning of intracellular antigens . A megakaryocyte expression cDNA library was transiently transfected into MOP-8 mouse fibroblasts cultured on polyvinylidene difluoride membranes . Individual cells expressing intracellular CD63 were identified by autoradiography . cDNA was extracted from positive spots and reintroduced into Escherichia coli . After two screening rounds, a CD63 cDNA clone was isolated as assessed by immunofluorescence and Western blot analysis . The single long open reading frame of 238 amino acids contained four putative transmembrane regions and three N-glycosylation sites . The CD63 gene was expressed in a wide variety of cells . Surprisingly, CD63 was identical to ME491, an antigen reported as a melanoma-associated antigen (Hotta, H., Ross, A . H., Huebner, K., Isobe, M., Wendeborn, S., Chao, M . V., Ricciardi, R . P., Tsujimoto, Y., Croce, C . M., and Koprowski, H . (1988) Cancer Res . 48, 2955-2962) . By immunoelectron microscopy, co-localization with the lysosomal glycoproteins lamp-1 and -2 identified CD63 as a novel lysosomal membrane glycoprotein . CD63 was not related to the lysosomal glycoprotein family but contained the putative lysosomal targeting signal Gly-Tyr in its short cytoplasmic tail.

J Biol Chem, 1991 Feb 15, 266(5), 3101 - 6
DNA binding specificity of mutant glucocorticoid receptor DNA-binding domains; Zilliacus J et al.; Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P . (1989) Nature 338, 271-274) . We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays . We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements . Using binding sites of intermediate sequence we have further characterized the interaction with DNA . The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast . Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.

J Biol Chem, 1991 Feb 15, 266(5), 3045 - 51
Role of type III homology repeats in cell adhesive function within the cell-binding domain of fibronectin; Kimizuka F et al.; Recombinant fibronectin (FN) fragments and their mutant proteins were produced to elucidate the role of type III homology repeats in cell adhesive activity within the cell-binding domain of FN . Cell adhesive activity of the 11.5-kDa fragment, the cell attachment site of the cell-binding domain, was less than 0.1% that of native FN despite the presence of the Arg-Gly-Asp-Ser sequence . The activity increased as type III homology repeats were added to the N terminus of the 11.5-kDa fragment, and a 52-kDa fragment with four additional type III repeats had almost the same activity of native FN . Deletion of Arg-Gly-Asp from the fully active fragments completely abolished the cell adhesive activity . Deletion of one or two repeats from the 52-kDa fragment affected the extent of the cell adhesive activity, the degree of the effect being inversely correlated with the distance of the deletion from the type III repeat containing Arg-Gly-Asp-Ser . Rearrangement of type III repeats caused much loss of activity . These results suggest that the number and kinds of type III repeats and their correct alignment rather than the putative synergistic site decide the extent of the specific cell adhesive activity.

J Biol Chem, 1991 Feb 15, 266(5), 3022 - 30
Localization of a region of the S1 subunit of pertussis toxin required for efficient ADP-ribosyltransferase activity; Cortina G et al.; Purified recombinant S1 subunit of pertussis toxin (rS1) possessed similar NAD glycohydrolase and ADP-ribosyltransferase activities as S1 subunit purified from pertussis toxin . Purified rS1 and C180 peptide, a deletion peptide which contains amino acids 1-180 of rS1, had Km values for NAD of 24 and 13 microM and kcat values of 22 and 24 h-1, respectively, in the NAD glycohydrolase reaction . In contrast, under linear velocity conditions, the C180 peptide possessed less than 1% of the ADP-ribosyltransferase activity of rS1 using transducin as target . Radiolabeled tryptic peptides of transducin that had been ADP-ribosylated by either rS1 or C180 peptide were identical which suggested that both rS1 and C180 peptide ADP-ribosylated the same amino acid within transducin . To extend the functional primary amino acid map of the S1 subunit, two carboxyl-terminal deletions were constructed . One deletion, C195, removed the 40 carboxyl-terminal amino acids and the other, C219, removed the 16 carboxyl-terminal amino acids of the S1 subunit . Both C195 and C219 migrated in reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular masses of 22,000 and 27,500 Da, respectively . Relative to the C180 peptide C195 possessed 10-20-fold increase and C219 possessed 100-150-fold increase in ADP-ribosyltransferase activities . In addition, C219 appeared to have the same ADP-ribosyltransferase activity as rS1 . These studies indicate that (i) rS1, purified from Escherichia coli, possesses biochemical properties similar to S1 subunit purified from pertussis toxin, (ii) amino acids 1-180 of the S1 subunit contain residues required for NAD binding, N-glycosidic cleavage, and transfer of ADP-ribose to transducin, and (iii) residues between 181 and 219 of the S1 subunit are required for efficient ADP-ribosyltransferase activity.

J Biol Chem, 1991 Feb 15, 266(5), 3016 - 21
Human NAD(+)-dependent mitochondrial malic enzyme . cDNA cloning, primary structure, and expression in Escherichia coli; Loeber G et al.; Mitochondrial NAD(+)-dependent malic enzyme (EC 1.1.1.40) is expressed in rapidly proliferating cells and tumor cells, where it is probably linked to the conversion of amino acid carbon to pyruvate . In this paper, we report the cDNA cloning, amino acid sequence, and expression in Escherichia coli of functional human NAD(+)-dependent mitochondrial malic enzyme . The cDNA is 1,923 base pairs long and contains an open reading frame coding for a 584-amino acid protein . The molecular mass is 65.4 kDa for the unprocessed precursor protein . Comparison of the amino acid sequence of the human protein with the published NADP(+)-dependent mammalian cytosolic or plant chloroplast malic enzymes reveals highly conserved regions interrupted with long stretches of amino acids without significant homology . Expression of the processed protein in E . coli yielded an enzyme with the same kinetic and allosteric properties as malic enzyme purified from human cells.

J Biol Chem, 1991 Feb 15, 266(5), 2872 - 7
Purification and characterization of Tet(M), a protein that renders ribosomes resistant to tetracycline; Burdett V; The tet(M) tetracycline resistance gene has been found in a wide variety of clinically important bacteria . It has been shown previously (Burdett, V . (1986) J . Bacteriol . 165, 564-569) that the tet(M) gene product mediates resistance at the level of protein synthesis as judged by in vitro assay . Using this assay, large amounts of protein were purified from an Escherichia coli overproducer expressing the gene under control of a T7 promoter . The purified activity consists of a single polypeptide of molecular weight 68,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed to be the tet(M) gene product by amino-terminal sequence analysis . Purified Tet(M) has an associated ribosome-dependent GTPase with the specific activity being similar to that of the corresponding activity associated with elongation factor G . Since Tet(M) also displays substantial homology to elongation factor G throughout its length, Tet(M) may function as an analog of this elongation factor.

J Biol Chem, 1991 Feb 15, 266(5), 2831 - 5
Specific degenerate codons enhanced selective expression of human parathyroid hormone in Escherichia coli; Sung WL et al.; Specific degenerate codons in the amino-terminal region of a synthetic human parathyroid hormone (PTH) gene exerted dramatic effects on both products and yield of expression of this 84-amino acid polypeptide in Escherichia coli . With adenine-rich degenerate codons constituting the PTH-(1-5) region, intact PTH has been expressed as the only PTH product at 6.5 mg/liter . In contrast, with guanine-rich degenerate codons, the predominent product was analogue PTH-(8-84) . Use of cytosine- or thymine-rich degenerate codons generated only a small amount of immunoreactive product (0.2 mg/l) . With the amino terminal region reconstituted with adenine-rich degenerate codons, the mid and carboxyl regions of the synthetic gene were also reconstructed to imitate the E . coli-favored codon degeneracy . Expression yielded the intact PTH at 20 mg/liter . Gel electrophoresis and Western blots, with antibodies specific to the amino or carboxyl terminus of PTH, indicated only a single PTH-related polypeptide, with the same mobility as a synthetic intact PTH sample . Amino acid sequencing, composition analysis, mass spectrometry, and the adenylate cyclase bioassays confirmed the purified product as the processed intact PTH.

Gene, 1991 Feb 15, 98(2), 169 - 75
Ferredoxin and ribosomal protein S10 are encoded on the cyanelle genome of Cyanophora paradoxa; Bryant DA et al.; The petF and rsp10 genes of the cyanellar genome of the taxonomically ambiguous flagellate Cyanophora paradoxa have been cloned, mapped, and sequenced . In higher plants these genes are not encoded in the chloroplast DNA, but are encoded in the nucleus . The C . paradoxa petF gene predicts a protein of 99 amino acids (aa) which is more similar to type-I ferredoxins of diverse cyanobacteria than to those of green algae, dinoflagellates, and higher plants . The rsp10 gene (rspJ) predicts a protein of 105 aa which is about 50% identical and 71% homologous to the proteins of Escherichia coli and Mycoplasma capricolum . The results are discussed within the context of the endosymbiotic origins of chloroplasts from cyanobacteria.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1511 - 5
Inducer-dependent conditional-lethal mutant animal viruses; Zhang YF et al.; Regulatory elements of the Escherichia coli lac operon were used to construct an inducer-dependent conditional-lethal mutant animal virus . The gene encoding the repressor protein of the lac operon was integrated into the vaccinia virus genome so that it was expressed constitutively, and the lac operator was inserted next to the promoter of a gene that encodes an 11-kDa virion-associated protein of unknown function . The addition of inducer to the cell culture medium provided permissive conditions for isolation of a conditional-lethal mutant virus . Under nonpermissive conditions, the isolated virus did not form plaques, and the yield was decreased by at least 1000-fold under one-step growth conditions . Transcription of the operator-controlled gene was inducer-dependent and necessary for synthesis of the 11-kDa protein . Application of this mutagenesis strategy to other viruses is discussed.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1217 - 21
Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation; Ponder KP et al.; One approach to gene therapy for hepatic diseases is to remove hepatocytes from an affected individual, genetically alter them in vitro, and reimplant them into a receptive locus . Although returning hepatocytes to the liver itself would be advantageous, the feasibility of this approach has never been evaluated due to the inability to distinguish donor from host hepatocytes . To unambiguously identify transplanted hepatocytes after transplantation, and to better quantitate their number and degree of liver function, two transgenic mouse lines were generated in a C57BL/6 background . The first expresses the Escherichia coli beta-galactosidase gene from the relatively liver-specific human alpha 1-antitrypsin (hAAT) promoter and allows transgenic hepatocytes to be readily identified after 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining; the second produces the hAAT protein under control of the same promoter, which enables hepatocyte survival and maintenance of liver function to be quantitated by measuring the serum levels of hAAT . Hepatocytes isolated from transgenic donors were transplanted into nontransgenic C57BL/6 recipients by intrasplenic injection . Surprisingly, a large fraction of these cells were identified within the liver parenchyma but not the spleen at 2 months after transplantation . The high levels of serum hAAT detected in transplant recipients were stable for greater than 6 months, suggesting that established cells will survive indefinitely . These results have important implications for liver organogenesis and hepatic gene therapy.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1479 - 83
Transcribed sequences of the Escherichia coli btuB gene control its expression and regulation by vitamin B12; Lundrigan MD et al.; The Escherichia coli btuB gene product is an outer membrane protein required for the active transport of vitamin B12 and other cobalamins . Synthesis of BtuB is repressed when cells are grown in the presence of cobalamins . Mapping of the 5' end of the btuB transcript revealed that a 240-nucleotide transcribed leader precedes the coding sequence . Point mutations causing increased expression under repressing conditions were isolated by use of a btuB-lacZ gene fusion . Mutations at many sites within the leader region affected btuB-lacZ regulation, whereas some base changes upstream of the start of transcription affected the absolute level of expression but not its repressibility . Analysis of btuB-phoA gene fusions and btuB-lacZ operon and gene fusions of various lengths showed that sequences within the btuB coding region (between nucleotides +250 and +350) had to be present for proper expression and transcriptional regulation . Sequences within the leader region (up to +250) conferred regulation of translational fusions . These results indicate that btuB expression is controlled at both the transcriptional and translational levels and that different but possibly overlapping sequences in the transcribed region, including the coding region for the transport protein itself, mediate these two modes of regulation.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1247 - 50
Transposon-facilitated DNA sequencing; Strathmann M et al.; We describe here a transposon-based DNA sequencing strategy that allows the introduction of sequencing priming sites throughout a target sequence by bacterial mating . A miniplasmid was designed to select against transposon insertions into the vector . Sites of transposon insertion are mapped by the polymerase chain reaction with bacterial overnight cultures providing the templates . A small set of plasmids with transposons spaced several hundred base pairs apart can then be sequenced . Sequencing primers corresponding to the transposon ends allow sequencing in both directions . Thus, the entire sequence of both strands can be easily determined.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1212 - 6
ATP-dependent partitioning of the DNA template into supercoiled domains by Escherichia coli UvrAB; Koo HS et al.; The helicase action of the Escherichia coli UvrAB complex on a covalently closed circular DNA template was monitored using bacterial DNA topoisomerase I, which specifically removes negative supercoils . In the presence of E . coli DNA topoisomerase I and ATP, the UvrAB complex gradually introduced positive supercoils into the input relaxed plasmid DNA template . Positive supercoils were not produced when E . coli DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I or when both E . coli and eukaryotic DNA topoisomerases I were added simultaneously . These results suggest that like other DNA helix-tracking processes, the ATP-dependent action of the UvrAB complex on duplex DNA simultaneously generates both positive and negative supercoils, which are not constrained by protein binding but are torsionally strained . The supercoiling activity of UvrAB on UV-damaged DNA was also studied using UV-damaged plasmid DNA and a mutant UvrA protein that lacks the 40 C-terminal amino acids and is defective in preferential binding to UV-damaged DNA . UvrAB was found to preferentially supercoil the UV-damaged DNA template, whereas the mutant protein supercoiled UV-damaged and undamaged DNA with equal efficiency . Our results therefore suggest that the DNA helix-tracking activity of UvrAB may be involved in searching and/or prepriming the damaged DNA for UvrC incision . A possible role of supercoiled domains in the incision process is discussed.

J Biol Chem, 1991 Feb 15, 266(5), 3294 - 9
A new {2Fe-2S} ferredoxin from Rhodobacter capsulatus . Coexpression with a 2{4Fe-4S} ferredoxin in Escherichia coli; Grabau C et al.; A 285-base pair open reading frame was found immediately upstream of the fdxN gene (encoding ferredoxin I) of Rhodobacter capsulatus and coded for a 95-amino acid protein with a predicted molecular weight of 10,156 . The deduced amino acid sequence contained 5 cysteines, 4 of which exhibited spacing characteristic of {2Fe-2S} plant and cyanobacterial ferredoxins . The amino acid sequence was found to share approximately 25% amino acid similarity with plant-type ferredoxins . The gene was named fdxC . Expression of the fdxC and fdxN genes together in Escherichia coli was accomplished by subcloning the genes in the vector pUC18 downstream of the lac promoter . Cells containing this plasmid produced a red and a brown protein corresponding to the fdxC and fdxN gene products, respectively . EPR and UV-visible absorption spectroscopy confirmed that the FdxC protein contained a {2Fe-2S} cluster and the FdxN protein contained two {4Fe-4S} clusters and that the centers were correctly assembled and inserted in the ferredoxins expressed in E . coli . Transcription (Northern blot) analysis showed that the genes were transcribed only under nitrogen-limiting (nif-derepressing) growth conditions.

J Biol Chem, 1991 Feb 15, 266(5), 3154 - 61
Adenylate cyclase toxin from Bordetella pertussis . The relationship between induction of cAMP and hemolysis; Rogel A et al.; Bordetella pertussis produces a calmodulin-activated adenylate cyclase (AC) that exists in several forms . Only one form of AC, of apparent 200 kDa, is a toxin that penetrates eukaryotic cells and generates uncontrolled levels of intracellular cAMP . Recombination studies in transposon Tn5-insertion mutants of B . pertussis and amino acid sequence homology with alpha-hemolysin of Escherichia coli suggested that AC toxin may also have a hemolytic activity . Here, we demonstrate that only the toxic form of B . pertussis AC possesses hemolytic activity . Immunoblotting of membranes from sheep erythrocytes throughout the process of cell lysis detects the presence and accumulation of only the 200-kDa form of B . pertussis AC . cAMP generation induced by AC toxin in sheep erythrocytes is immediate whereas appearance of hemolysis is delayed by about 1 h and requires a higher level of AC toxin activity . Addition of exogenous calmodulin to sheep erythrocyte incubation medium potentiates the hemolytic activity of AC toxin but blocks cAMP generation . Extracellular Ca2+ at mM concentrations is absolutely required for cAMP generation but not for hemolysis . However, binding of AC toxin to sheep erythrocytes in the absence of exogenous Ca2+ followed by reincubation of cells in a toxin-free buffer containing Ca2+ leads to an immediate rise in intracellular cAMP . Human erythrocytes bind AC toxin and generate cAMP but are resistant to lysis . These results show that binding of AC toxin to erythrocytes can cause both cAMP generation and hemolysis or only one of these depending on conditions applied and cell type used.

J Biol Chem, 1991 Feb 15, 266(5), 3228 - 32
Alternative splicing of human synexin mRNA in brain, cardiac, and skeletal muscle alters the unique N-terminal domain; Magendzo K et al.; Several synexin (annexin VII) mRNAs have been identified by screening a human fibroblast cDNA library . One type of message contained an alternatively spliced cassette exon, predicting two isoforms of synexin differing in the N-terminal domain . Polymerase chain reaction analysis of synexin mRNA from various fetal and adult tissues, from human and monkey, revealed that the alternative splicing event is tissue-regulated; synexin mRNA containing the cassette exon is prevalent in brain, heart, and skeletal muscle . This is supported by Western blot analysis showing that muscle synexin (annexin VIIb) is larger than synexin from lung (annexin VIIa) . The muscle and lung isoforms have the same molecular mass as the recombinant synexins expressed in Escherichia coli using cDNAs containing or lacking the cassette exon, respectively . The difference in size is consistent with the molecular masses predicted from the proteins encoded by the alternatively spliced synexin mRNAs . Another type of synexin mRNA contained a longer 3'-noncoding region generated by the selection of an alternate poly(A) signal . Northern analysis of human fibroblast RNA showed the presence of two bands (2.0- and 2.4-kilobase) when hybridized to a cDNA fragment of the coding region of synexin, but only the 2.4-kilobase band hybridized to a probe made from the longer 3' end.

Gene, 1991 Feb 15, 98(2), 225 - 30
Synthesis and characterization of the Kunitz protease-inhibitor domain of the beta-amyloid precursor protein; Schilling J et al.; To understand the pathological process by which amyloid is deposited in Alzheimer's disease, it is important to characterize the proteolytic processing events of the beta-amyloid precursor protein (beta-APP) from which the amyloid-forming fragment is excised . A potentially important component in beta-APP processing is the 57-amino acid (aa) Kunitz serine protease inhibitor (KPI) located within the extracellular domain of both the 751- and 770-aa isoforms of beta-APP . We have synthesized DNA encoding the 57-aa KPI domain as a necessary step in identifying the role of the protease inhibitor in beta-APP processing and amyloid formation . A bacterial secretion system directed by the alkaline phosphatase signal peptide of Escherichia coli linked to a synthetic gene encoding KPI was used to produce soluble, extracellular recombinant KPI (reKPI) protein . The reKPI protein was purified to homogeneity from bacterial supernatants and was biochemically and biologically characterized . Complete aa sequence analysis confirmed the fidelity of the reKPI, and fast-atom bombardment mass-spectral analysis was used to document that reKPI was of the predicted Mr . The reKPI is as active on a molar basis as the inhibitor-containing beta-APP when assayed for inhibition of trypsin activity . Together these data suggest that reKPI protein is properly folded and lacking in modified aa . Hence, this reKPI will be an important reagent in gaining a better understanding of the role of the KPI domain in beta-APP function and metabolism, as well as in the proteolytic events involved in beta-amyloid formation.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1148 - 52
Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase; Hostomsky Z et al.; Two constituent protein domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase were expressed separately and purified to homogeneity . The N-terminal domain (p51) behaves as a monomeric protein exhibiting salt-sensitive DNA polymerase activity . The C-terminal domain (p15) on its own has no detectable RNase H activity . However, the combination of both isolated p51 and p15 in vitro leads to reconstitution of RNase H activity on a defined substrate . These results demonstrate that domains of HIV-1 reverse transcriptase are functionally interdependent to a much higher degree than in the case of reverse transcriptase from Moloney murine leukemia virus.

J Biol Chem, 1991 Feb 15, 266(5), 2843 - 51
Analysis of the altered mRNA stability (ams) gene from Escherichia coli . Nucleotide sequence, transcriptional analysis, and homology of its product to MRP3, a mitochondrial ribosomal protein from Neurospora crassa; Claverie-Martin F et al.; The product of the altered mRNA stability (ams) gene of Escherichia coli is involved in decay of mRNA . The complete nucleotide sequence of a 4-kilobase BamHI restriction fragment containing the ams coding sequence was determined . Transcription of the ams gene was analyzed by high resolution S1 mapping . A promoter was found with a homology score of 58% 361 nucleotides upstream from the start codon of ams . The ams structural gene consists of an open reading frame of 2,445 nucleotides . The protein predicted from this open reading frame has a molecular mass of 91,327 Da, which is significantly smaller than that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis . Confirmation of the ams coding sequence was obtained by comparing the predicted amino acid sequence with that derived by amino-terminal analysis of gel-purified Ams protein . The predicted protein sequence of the ams gene was screened against translations of the GenBank DNA sequence data base . A homology of 18% over a region of 315 amino acids of the carboxyl terminus of the Ams product was found to MRP3, a mitochondrial ribosomal protein from Neurospora crassa . A smaller region of homology (29% in 86 residues) was found to the human U1 small nuclear ribonucleoparticle 70,000-Da protein.

J Biol Chem, 1991 Feb 15, 266(5), 2705 - 8
Aminoacylation of alanine minihelices . "Discriminator" base modulates transition state of single turnover reaction; Shi JP et al.; RNA hairpin minihelices that recreate the acceptor-T psi C stem of Escherichia coli alanine tRNA are charged specifically with alanine, provided that they encode the critical G3:U70 base pair that is the major determinant for the identity of an alanine tRNA . These model substrates were used to investigate the role in charging of N73, the unpaired nucleotide that is just three positions removed from the amino acid attachment site and which is sometimes referred to as the "discriminator" base . Previous work showed that, while substrates which encode G3:U70 are all charged by alanine tRNA synthetase regardless of the base at position 73, catalytic efficiency is substantially higher with substrates that have the wild-type A73 . To identify a specific step in aminoacylation that is affected by substitutions of A73, we studied the single turnover charging of A73, U73, C73, and G73 minihelices, using preformed, enzyme-bound alanyl adenylate and saturating concentrations of the respective minihelices . Compared to the A73 substrate, the transfer of activated amino acid to bound RNA is sharply reduced for the substituted N73 minihelices . The low efficiency of transfer is not due to an abortive reaction in which the adenylate or a transiently charged RNA is hydrolyzed . Instead, under the conditions used, the active adenylate remains on the enzyme for extended periods and simply reacts slowly with bound N73 RNA . The results suggest that the nature of the discriminator base is a critical determinant of the transition state for the reaction of bound alanyl adenylate with RNA on the surface of the enzyme.

Gene, 1991 Feb 15, 98(2), 193 - 200
Cloning of murine SeGpx cDNA and synthesis of mutated GPx proteins in Escherichia coli; Rocher C et al.; Glutathione peroxidase (GPx) of mammalian cells and Escherichia coli formate dehydrogenase both contain a selenocysteine (SeCys) in their amino acid (aa) sequence . In these two enzymes, this aa is encoded by a UGA codon, which is usually a stop codon for protein synthesis . We constructed plasmids to test the synthesis of GPx in E . coli . These constructions permitted high-level production of GPx mutants, where the SeCys codon was replaced by cysteine (UGC, UGU) or serine (UCA) codons, but synthesis of selenoprotein could not be detected: our data suggest that signals used for the recognition of the UGA codon as a SeCys codon are not conserved between E . coli and mammalian cells.

Eur J Biochem, 1991 Feb 14, 195(3), 823 - 30
An acyl-carrier-protein-thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea . High-level production, purification and characterisation in Escherichia coli; Caffrey P et al.; The C-terminal region of a multifunctional polypeptide from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity {Cortes, J., Haydock, S . F., Roberts, G . A., Bevitt, D . J . & Leadlay, P . F . (1990) Nature 348, 176-178} . Site-directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7-based expression plasmid for this domain . The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post-translationally modified by attachment of a 4'-phosphopantetheine group . However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.

Biochem Biophys Res Commun, 1991 Feb 14, 174(3), 1248 - 54
Reduction in the Ca2(+)-induced Ca2+ release from canine cardiac sarcoplasmic reticulum following endotoxin administration; Liu MS et al.; Effects of endotoxin administration on the Ca2(+)-induced Ca2+ release from canine cardiac sarcoplasmic reticulum (SR) were studied . Results show that the Ca2(+)-induced Ca2+ release from either passively or actively loaded SR vesicles was decreased by 28 to 46% (p less than 0.05) 4 h after endotoxin administration . Kinetic analysis reveals that the Vmax for Ca2+ was decreased significantly without changing the S0.5 and the Hill coefficient values . The binding of {3H}ryanodine to cardiac SR was reduced by 25.3% (p less than 0.01) following endotoxin administration . These data demonstrate that the Ca2(+)-induced Ca2+ release via the ryanodine-sensitive Ca2+ channel in canine cardiac SR was reduced during endotoxin shock . A reduction in the SR Ca2(+)-induced Ca2+ release may have a pathophysiological significance in contributing to the development of myocardial depression during endotoxin shock.

Eur J Biochem, 1991 Feb 14, 195(3), 691 - 7
Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies; Orsini G et al.; Prourokinase is a plasminogen activator of 411 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction . The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain . In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (delta 125-prourokinase) in which 125 amino acids of the N-terminal region have been removed . Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-1 for delta 125-prourokinase and 0.25 h-1 for prourokinase) . Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain . When applied to pure, non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure . Lower yields (15% and 30%, respectively, for prourokinase and delta 125-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria . After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160,000 and 250,000 IU/mg protein, respectively, for prourokinase and delta 125-prourokinase . As with prourokinase, the deletion mutant delta 125-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.

Biochemistry, 1991 Feb 12, 30(6), 1515 - 23
Disulfide assignments in recombinant mouse and human interleukin 4; Carr C et al.; The disulfide pairings of mouse and human interleukin 4 (IL-4) proteins have been determined . The purified proteins, synthesized by recombinant DNA technology, are fully active as judged by their ability to stimulate an appropriate biological response in a variety of functional assays . Peptide maps were produced by digesting the proteins with pepsin and separating the resulting fragments by reverse-phase HPLC using linear acetonitrile-TFA gradients . Cystine-containing peptides were identified by determining which reverse-phase peaks showed an altered elution pattern after reduction . These peptides were purified further and defined by composition and sequence analysis . Three sets of disulfide-linked peptides were consistently identified for each protein . For mouse IL-4, the first and fifth, second and fourth, and third and sixth cysteines are joined . The disulfide bonds in human IL-4 are between the first and sixth, second and fourth, and third and fifth cysteines . A large double-loop region within the central three-fifths of each protein is stabilized by these bonds . Sequence analysis of the peptides containing the third and fifth cysteines of human IL-4 also demonstrated that only one of the potential N-glycosylation sites is used by C127 mammary tumor cells . Complete alkylation of mouse IL-4 under mild conditions completely destroyed its biological activity in a hematopoietic precursor cell proliferation assay.

Biochemistry, 1991 Feb 12, 30(6), 1497 - 503
Phosphorylation and formation of hybrid enzyme species test the "half of sites" reactivity of Escherichia coli succinyl-CoA synthetase; Mann CJ et al.; Recent sequencing experiments have identified alpha-His246 as the phosphorylation site of Escherichia coli succinyl-CoA synthetase {Buck, D., Spencer, M . E., & Guest, J . R . (1985) Biochemistry 24, 6245-6252} . We have replaced alpha-His246 with an asparagine residue using site-directed mutagenesis techniques . The resulting mutant enzyme (designated H246N) exhibited no enzyme activity, as expected, but was found as a structurally intact, stable tetramer . Small differences in the net charge of H246N and wild-type enzymes were first detected on native polyacrylamide gels . These charge differences were resolved by using native isoelectric focusing gels to further separate the wild-type enzyme into diphosphorylated, monophosphorylated, and unphosphorylated species . The enzyme species were found to be interconvertible upon incubation with the appropriate enzyme substrate(s) . Sample mixtures containing increasing molar ratios of H246N (alpha H246N beta)2 to wild-type enzyme (alpha beta)2 were unfolded and then refolded . The refolded enzyme mixtures were analyzed for enzymatic activity and separated on native isoelectric focusing gels . The hybrid enzyme (alpha beta alpha H246N beta) retained a significant amount of enzyme activity and also exhibited substrate synergism (stimulation of succinate in equilibrium succinyl-CoA exchange in the presence of ATP) . Substrate synergism with this enzyme has been interpreted as evidence for interaction between active sites in such a way that only a single phosphoryl group is covalently attached to the enzyme at a given time {Wolodko, W . T., Brownie, E.R., O'Connor, M . D., & Bridger, W . A . (1983) J . Biol . Chem . 258, 14116-14119} . On the contrary, we conclude that tetrameric succinyl-CoA synthetase from E . coli is comprised of two independently active dimer molecules associated together to form a "dimer of dimers" that displays substrate synergism within each dimer and not necessarily between dimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Feb 12, 30(6), 1461 - 9
Conformation of NADP+ bound to a type II dihydrofolate reductase; Brito RM et al.; Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are sequence and structurally different from known chromosomal DHFRs . These plasmid-derived DHFRs are responsible for confering trimethoprim resistance to the host strain . A derivative of R388 DHFR, RBG200, has been cloned and its physical properties have been characterized . This enzyme has been shown to transfer the pro-R hydrogen of NADPH to its substrate, dihydrofolate, making it a member of the A-stereospecific class of dehydrogenases {Brito, R . M . M., Reddick, R., Bennett, G . N., Rudolph, F . B., & Rosevear, P . R . (1990) Biochemistry 29,9825} . Two distinct binary RBG200.NADP+ complexes were detected . Addition of NADP+ to RBG200 DHFR results in formation of an initial binary complex, conformation I, which slowly interconverts to a second more stable binary complex, conformation II . The binding of NADP+ to RBG200 DHFR in the second binary complex was found to be weak, KD = 1.9 +/- 0.4 mM . Transferred NOEs were used to determine the conformation of NADP+ bound to RBG200 DHFR . The initial slope of the NOE buildup curves, measured from the intensity of the cross-peaks as a function of the mixing time in NOESY spectra, allowed interproton distances on enzyme-bound NADP+ to be estimated . The experimentally measured distances were used to define upper and lower bound distance constraints between proton pairs in distance geometry calculations . All NADP+ structures consistent with the experimental distance bounds were found to have a syn conformation about the nicotinamide-ribose (X = 94 +/- 26 degrees) and an anti conformation about the adenine-ribose (X = -92 +/- 32 degrees) glycosidic bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Feb 12, 30(6), 1484 - 90
Enhancement of the catalytic properties of human carbonic anhydrase III by site-directed mutagenesis; Jewell DA et al.; Among the seven known isozymes of carbonic anhydrase in higher vertebrates, isozyme III is the least efficient in catalytic hydration of CO2 and the least susceptible to inhibition by sulfonamides . We have investigated the role of two basic residues near the active site of human carbonic anhydrase III (HCA III), lysine 64 and arginine 67, to determine whether they can account for some of the unique properties of this isozyme . Site-directed mutagenesis was used to replace these residues with histidine 64 and asparagine 67, the amino acids present at the corresponding positions of HCA II, the most efficient of the carbonic anhydrase isozymes . Catalysis by wild-type HCA III and mutants was determined from the initial velocity of hydration of CO2 at steady state by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and water at chemical equilibrium by mass spectrometry . We have shown that histidine 64 functions as a proton shuttle in carbonic anhydrase by substituting histidine for lysine 64 in HCA III . The enhanced CO2 hydration activity and pH profile of the resulting mutant support this role for histidine 64 in the catalytic mechanism and suggest an approach that may be useful in investigating the mechanistic roles of active-site residues in other isozyme groups . Replacing arginine 67 in HCA III by asparagine enhanced catalysis of CO2 hydration 3-fold compared with that of wild-type HCA III, and the pH profile of the resulting mutant was consistent with a proton transfer role for lysine 64 . Neither replacement enhanced the weak inhibition of HCA III by acetazolamide or the catalytic hydrolysis of 4-nitrophenyl acetate.

Biochemistry, 1991 Feb 12, 30(6), 1655 - 63
Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study; Chu WC et al.; Interactions of 5-fluorouracil-substituted Escherichia coli tRNAVal with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance . Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E . coli, differs somewhat from VRS previously isolated from E . coli K12 . Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene {Heck, J.D., & Hatfield, G.W . (1988) J . Biol . Chem . 263, 868-877} . Apparent KM and Vmax values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNAVal . Binding of VRS to (FUra)tRNAVal induces structural perturbations that are reflected in selective changes in the 19F NMR spectrum of the tRNA . Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most . At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur . These results indicate that VRS interacts with tRNAVal along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon . Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNAVal, suggesting conformational changes in this part of the molecule . No 19F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNAVal that has been proposed as a common intermediate in the aminoacylation reaction.

Biochemistry, 1991 Feb 12, 30(6), 1642 - 50
Bis(pyrrolecarboxamide) linked to intercalating chromophore oxazolopyridocarbazole (OPC): selective binding to DNA and polynucleotides; Subra F et al.; We have investigated some properties related to interaction with DNA and recognition of AT-rich sequences of netropsin-oxazolopyridocarbazole (Net-OPC) (Mrani et al., 1990), which is a hybrid groove-binder-intercalator . The hybrid molecule Net-OPC binds to poly{d(A-T)} at two different sites with Kapp values close to 7 x 10(6) and 6 x 10(8) M-1 (100 mM NaCl, pH 7.0) . Data obtained from melting experiments are in agreement with these values and indicate that Net-OPC displays a higher binding constant to poly{d(A-T)} than does netropsin . On the basis of viscometric and energy transfer data, the binding of Net-OPC to poly{d(A-T)} is suggested to involve both intercalation and external binding of the OPC chromophore . In contrast, on poly{d(G-C)}, Net-OPC binds to a single type of site composed of two base pairs in which the OPC chromophore appears to be mainly intercalated . The binding constant of Net-OPC to poly{d(G-C)} was found to be about 350-fold lower than that of the high-affinity binding site in poly{d(A-T)} . As evidenced by footprinting data, Net-OPC selectively recognizes TTAA and CTT sequences and strongly protects the 10-bp AT-rich DNA region 3'-TTAAGAACTT-5' containing the EcoRI site . The binding of Net-OPC to this sequence results in a strong and selective inhibition of the activity of the restriction endonuclease EcoRI on the plasmid pBR322 as substrate . The extent of inhibition of the rate constant of the first strand break catalyzed by the enzyme is about 100-fold higher than the one observed in the presence of netropsin under similar experimental conditions.

Biochemistry, 1991 Feb 12, 30(6), 1478 - 84
Designing an allosterically locked phosphofructokinase; Kundrot CE et al.; Six site-directed mutants of Escherichia coli phosphofructokinase (PFK) were made in an attempt to produce an enzyme "locked" in the inactive or "T"-state . The kinetic properties of the mutants were examined as a function of the substrates fructose 6-phosphate (Fru6P) and ATP, the positive effector GDP, and the negative effector phosphoenolpyruvate (PEP) . All mutants exhibited lower activity than wild-type PFK . Three mutants (RS63, LV153, and VT246) had apparent dissociation constants for substrates and effectors similar to those of wild type . One mutant, HN160, had a 10-fold reduced affinity for Fru6P and reduced apparent affinity for the effectors . Two mutants, SN159 and T(GS)156, exhibited hyperbolic kinetics consistent with a "locked" T-state protein . Surprisingly, T(GS)156 showed hyperbolic activation in response to the physiological inhibitor PEP . The mutant PFK properties are discussed in terms of the PFK structure . These results suggest that the kinetic properties of PFK are sensitive to interactions in the homotropic interface; residues 156-160 in particular are critical in mediating the interactions between effector and active sites and in the T to R quaternary transition.

Biochemistry, 1991 Feb 12, 30(6), 1586 - 91
GroE facilitates refolding of citrate synthase by suppressing aggregation; Buchner J et al.; The molecular chaperone GroE facilitates correct protein folding in vivo and in vitro . The mode of action of GroE was investigated by using refolding of citrate synthase as a model system . In vitro denaturation of this dimeric protein is almost irreversible, since the refolding polypeptide chains aggregate rapidly, as shown directly by a strong, concentration-dependent increase in light scattering . The yields of reactivated citrate synthase were strongly increased upon addition of GroE and MgATP . GroE inhibits aggregation reactions that compete with correct protein folding, as indicated by specific suppression of light scattering . GroEL rapidly forms a complex with unfolded or partially folded citrate synthase molecules . In this complex the refolding protein is protected from aggregation . Addition of GroES and ATP hydrolysis is required to release the polypeptide chain bound to GroEL and to allow further folding to its final, active state.

J Bacteriol, 1991 Feb, 173(3), 968 - 77
Molecular characterization of the Escherichia coli K-12 zwf gene encoding glucose 6-phosphate dehydrogenase; Rowley DL et al.; In Escherichia coli K-12, expression of zwf, the gene for glucose 6-phosphate dehydrogenase, is coordinated with the cellular growth rate and induced by superoxide-generating agents . To initiate the study of the molecular mechanisms regulating its expression, the gene was cloned and its DNA sequence was determined . The 5' ends of zwf mRNA isolated from cells growing in glucose and acetate minimal media were mapped . The map was complex in that transcripts mapped to -45, -52, and -62, with respect to the beginning of the coding sequence . Three analytical methods were used to search the DNA sequence for putative promoters . Only one sequence for a promoter recognized by the sigma 70 form of RNA polymerase was found by all three search routines that could be aligned with a mapped transcript, indicating that the other transcripts arise by processing of the mRNA . A computer-assisted search did not reveal a thermodynamically stable long-range mRNA secondary structure that is capable of sequestering the translation initiation region, which suggests that growth-rate-dependent regulation of glucose 6-phosphate dehydrogenase level may not be carried out by a mechanism similar to the one for the gene (gnd) for 6-phosphogluconate dehydrogenase . The DNA segment between the -10 hexamer and the start point of transcription resembles the discriminator sequence of stable RNA genes, which has been implicated in stringent control and growth-rate-dependent regulation.

Nucleic Acids Res, 1991 Feb 11, 19(3), 657 - 63
RecA-dependent incision of psoralen-crosslinked DNA by (A)BC excinuclease; Cheng S et al.; Previous work to elucidate the mechanism of crosslink repair by (A)BC excinuclease has shown that a psoralen-crosslinked duplex is selectively incised in the furan-side strand, while a three-stranded structure is incised in the pyrone-side strand of the crosslink . These observations support a sequential incision and recombination model for the complete error-free repair of a psoralen crosslink . The work presented here extends these findings by demonstrating that in the presence of RecA protein and a homologous DNA oligonucleotide, (A)BC excinuclease is induced to incise the pyrone-side strand of a crosslinked double-stranded plasmid molecule . This finding adds further support to the current model for error-free crosslink repair.

Nucleic Acids Res, 1991 Feb 11, 19(3), 553 - 7
Isolation and stability of ternary complexes of elongation factor Tu, GTP and aminoacyl-tRNA; Abrahams JP et al.; Intact, native EF-Tu, isolated using previously described methods and fully active in binding GTP, was never found to be fully active in binding aminoacyl-tRNA as judged by high performance liquid chromatography (HPLC) gel filtration and zone-interference gel-electrophoresis . In the presence of kirromycin, however, all these EF-Tu.GTP molecules bind aminoacyl-tRNA, although with a drastically reduced affinity . For the first time, the purification of milligram quantities of ternary complexes of EF-Tu.GTP and aminoacyl-tRNA, free of deacylated tRNA and inactive EF-Tu, has become possible using HPLC gel filtration . We also describe an alternative new method for the isolation of the ternary complexes by means of fractional extraction in the presence of polyethylene glycol . In the latter procedure, the solubility characteristics of the ternary complexes are highly reminiscent to those of free tRNA . Concentrated samples of EF-Tu.GMPPNP.aminoacyl-tRNA complexes show a high stability.

FEBS Lett, 1991 Feb 11, 279(1), 5 - 8
Evidence for gene duplication forming similar binding folds for NAD(P)H and FAD in pyridine nucleotide-dependent flavoenzymes; McKie JH et al.; For pyridine nucleotide-dependent flavoenzymes, binding both FAD and NAD(P)H on a single amino-acid chain, we have found a high degree of internal sequence similarity for certain regions of the FAD and NAD(P)H binding portions of the chain for any given protein . This was the case for a range of enzyme classes, including disulphide oxidoreductases (such as glutathione reductase, trypanothione reductase, lipoamide dehydrogenase, mercuric reductase), mono- and dioxygenases, nitrite reductase, alkyl hydroperoxidase and NADH dehydrogenase from E . coli . This provides strong support for gene duplication as the origin of at least part of the FAD and NAD(P)H recognising domains of such enzymes.

Nature, 1991 Feb 7, 349(6309), 487 - 93
Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes; Company M et al.; The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome . The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing . The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases . A putative RNA-binding motif originally identified in bacterial ribosomal protein S1 and Escherichia coli polynucleotide phosphorylase has also been found in PRP22.

J Mol Biol, 1991 Feb 5, 217(3), 421 - 8
Construction, expression and release of hybrid colicins; Frenette M et al.; Colicins A and E1 are two pore-forming colicins sharing homology in their C-terminal domains but not in their N-terminal or central domains . Using site-directed mutagenesis, restriction sites were inserted at the proper locations to allow recombination of these domains . Six different constructs were obtained . All these proteins were expressed in Escherichia coli and properly recognized by monoclonal antibodies directed against epitopes located in different domains of colicin A . Out of the six hybrids, only two were released to the extracellular medium . Immunocytolocalization indicated that some of the hybrids aggregated within the cytoplasm . With some hybrids, the defect in release was related to a defect in synthesis of the lysis protein that normally promotes release.

Biochemistry, 1991 Feb 5, 30(5), 1291 - 8
Role of proline residues in the structure and function of a membrane transport protein; Consler TG et al.; By use of site-directed mutagenesis, each prolyl residue in the lac permease of Escherichia coli at positions 28 (putative helix I), 31 (helix I), 61 (helix II), 89 (helix III), 97 (helix III), 123 (helix IV), 192 (putative hydrophilic region 7), 220 (helix VII), 280 (helix VIII), and 327 {helix X; Lolkema, J . S., et al . (1988) Biochemistry 27, 8307} was systematically replaced with Gly, Ala, or Leu or deleted by truncation of the C-terminus {i.e., Pro403 and Pro405; Roepe, P.D., et al . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 3992} . Replacements were chosen on the basis of side-chain helical propensity: Gly, like Pro, is thought to be a "helix breaker", while Ala and Leu are "helix makers" . With the exception of Pro28, each prolyl residue can be replaced with Gly or Ala, and Pro403 and -405 can be deleted with the C-terminal tail, and significant lac permease activity is retained . In contrast, when Pro28 is replaced with Gly, Ala, or Ser, lactose transport is abolished, but permease with Ser28 binds p-nitrophenyl alpha-D-galactopyranoside and catalyzes active transport of beta-galactopyranosyl-1-thio-beta-D- galactopyranoside . Replacement of Pro28, -31, -123, -280, or -327 with Leu abolishes lactose transport, while replacement of Pro61, -89, -97, or -220 with Leu has relatively minor effects . None of the alterations in permease activity is due to inability of the mutant proteins to insert into the membrane or to diminished lifetimes after insertion, since the concentration of each mutant permease in the membrane is comparable to that of wild-type permease as judged by immunological analyses.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Feb 5, 30(5), 1284 - 90
Kinetic analysis of lactose exchange in proteoliposomes reconstituted with purified lac permease; Lolkema JS et al.; Lactose exchange catalyzed by purified lac permease reconstituted into proteoliposomes was analyzed with unequal concentrations of lactose on either side of the membrane and at low pH so as to prevent equilibration of the two pools . Exchange with external concentrations below 1.0 mM is a single-exponential process, and the apparent affinity constants for external and internal substrate are close to the apparent KMs reported for active transport and efflux, respectively {Viitanen, P.V., Garcia, M . L., & Kaback, H . R . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 1629} . At external lactose concentrations above 1.0 mM, a second kinetic pathway becomes evident with an apparent affinity constant of about 6 mM which is similar to the apparent KM for facilitated influx . A second pathway is not observed with respect to internal lactose even when the concentration is increased up to 80 mM . Furthermore, high internal or external lactose concentrations do not inhibit the exchange reaction . Biphasic kinetics with respect to external lactose are retained in a mutant permease that catalyzes exchange but is defective in H(+)-coupled lactose transport . It is suggested that lac permease has more than one binding site and that this may be the underlying reason for the biphasic kinetics observed for both exchange and H(+)-coupled lactose transport.

Biochemistry, 1991 Feb 5, 30(5), 1259 - 64
Analysis of the conformation and stability of Escherichia coli derived recombinant human interleukin 4 by circular dichroism; Windsor WT et al.; The conformation and stability of Escherichia coli derived recombinant human interleukin 4 (rhuIL-4) have been examined by circular dichroism (CD) . Protein unfolding was detected by ellipticity changes at 222 nm with increasing concentrations of guanidine hydrochloride (GdnHCl) . The unfolding midpoint ({GdnHCl}1/2) was 3.7 M, the free energy of unfolding, (delta GDH2O), was 5.9 kcal/mol and the dependence of delta GD on the GdnHCl concentration (m) was 1.6 (kcal/mol)/M . This unfolding was demonstrated to be reversible upon removal of the GdnHCl by dialysis . Analysis of the far-UV CD spectrum indicated the presence of a high percentage of alpha-helical structure (ca . 73%) . A small change in ellipticity was noted over the pH range 1.9-9.6, suggesting that the protein undergoes a minor conformational change with an apparent pKa of 4.17 . Virtually complete biological activity, measured in vitro in a T-cell proliferation assay, was recovered following exposure to extreme values of pH (i.e., pH 3 and 10) . An analysis of the near-UV CD spectrum indicated that the single tryptophan residue at position 91 was unconstrained and most likely exposed to the solvent . Titration with 4,4'-dithiodipyridine and 2-nitrothiosulfobenzoate established that the six cysteine residues in rhuIL-4 were involved in intramolecular disulfide linkages . These data support that rhuIL-4 has a highly stable three-dimensional structure.

Biochemistry, 1991 Feb 5, 30(5), 1188 - 92
Small-angle X-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly alpha-helical conformation; Kataoka M et al.; Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein . Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4) . The deletions change both the radii of gyration and the maximum dimensions of the molecules . In the presence of Ca2+, the observed radii of gyration are 22.4 A for wild-type bacterially expressed calmodulin, 19.5 A for Des2 calmodulin, and 20.3 A for Des4 calmodulin . A reduction in the radius of gyration by 1-2 A on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant . The large calcium-dependent conformational change in calmodulin induced by the binding of melittin {Kataoka, M., Head, J.F., Seaton, B.A., & Engelman, D.M . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 6944-6948} was observed in all the bacterially expressed proteins . Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2 . Modeling shows the central linker region of the molecule . Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide.

J Biol Chem, 1991 Feb 5, 266(4), 2480 - 5
Purification and cDNA-derived sequence of adenylosuccinate synthetase from Dictyostelium discoideum; Wiesmuller L et al.; Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP), EC 6.3.4.4) plays an important role in purine biosynthesis catalyzing the GTP-dependent conversion of IMP to AMP . The enzyme was purified from the cytosol of Dictyostelium discoideum using GTP-agarose chromatography as the critical step . It has an apparent molecular mass of 44 kDa . Monoclonal antibodies identified several forms of the enzyme with pI values between 8.1 and 9.0 . Michaelis-Menten constants (Km) were low for the nucleotide substrates IMP (Km = 30 microM) and GTP (Km = 35 microM) as compared with the value for aspartic acid (Km = 440 microM) . These values are in good agreement with constants reported from other organisms . Immunological studies indicated that the protein is predominantly localized in the cytosol and only partially associated with particulate fractions . The enzyme is present throughout the developmental cycle of D . discoideum . Using monoclonal antibodies, the gene was cloned from a lambda gt11 expression library . The complete sequence represents the first reported primary structure of an eucaryotic adenylosuccinate synthetase . Southern blots hybridized with a cDNA probe demonstrate that adenylosuccinate synthetase is encoded by a single gene and contains at least one intron . The deduced amino acid sequence shows 43% identity to adenylosuccinate synthetase from Escherichia coli . Homologous regions include short sequence motifs, such as the glycine-rich loop which is typical for GTP-binding proteins.

J Biol Chem, 1991 Feb 5, 266(4), 2474 - 9
Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin; Holzman TF et al.; We report the cloning of a neutral isoelectric form of the human peptidyl prolyl isomerase, cyclophilin, its expression in Escherichia coli, and its purification and comparison to bovine thymus cyclophilin . The cloned protein exhibited a pI of approximately 7.8 and formed a simple 1:1 complex with cyclosporin A . This cloned form had a pI similar to that observed for the neutral isoform (pI approximately 7.4) of human splenocyte cyclophilin . The bovine thymus proteins exhibited anomalous behavior on CM-cellulose chromatography but were resolved into alkaline (pI approximately 9.3) isoforms and a new neutral (pI approximately 7.8) isoform by isoelectric focusing gel electrophoresis and ultimately into at least four discrete isoforms by capillary electrophoresis . For cyclosporin A binding we observe a Kd of approximately 160 nM for an electrophoretically heterogeneous preparation of the natural bovine protein and approximately 360 nM for the more homogeneous preparation of the cloned human neutral isoform . Stopped-flow measurements of the activation energies for peptidyl-prolyl isomerase activity indicate the recombinant human protein has an activation enthalpy of 3.67 kcal/mol and an activation entropy of -47.3 cal/K-mol for cis----trans isomerization.

J Biol Chem, 1991 Feb 5, 266(4), 2313 - 9
Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase; Van Etten RL et al.; The pairing of the half-cysteine residues of human prostatic acid phosphatase was established by proteolytic digestion and analysis of the resulting peptide mixtures by fast atom bombardment mass spectrometry (FAB-MS) . An independently derived, full length cDNA clone was used as the basis for the interpretation of the FAB-MS data . The sequence of the native protein is that predicted from the present cDNA sequence, except for the carboxyl-terminal end and some possible post-translational deamidations . Isolated human prostatic acid phosphatase was found to have multiple carboxyl-terminal ends, terminating in Thr, Glu, and Asp, corresponding to residues 349-351 of the 354-residue protein that is predicted from the cDNA sequence after removal of a leader peptide . The protein contains no free sulfhydryl groups . The identical monomer chains of the dimeric native enzyme are found to contain three disulfide bonds, specifically Cys-129 to Cys-340, Cys-183 to Cys-281, and Cys-315 to Cys-319 . In view of the conserved positions of cysteines in the homologous human and rat liver lysosomal acid phosphatases, an identical disulfide bonding pattern may be predicted for those proteins . The location of a potential antigenic site was established by selective labeling of proximate tyrosine residues predicted to be on the surface . A conserved RHGXRXP sequence is present in the prostatic, lysosomal, Escherichia coli, and yeast acid phosphatases and is predicted to be of mechanistic significance . In addition, residue Arg-54 is shown to be an active site residue by reaction of the enzyme with phenylglyoxal . Interestingly, this residue is present in a sequence RXRY (R,H) that is also present in lysosomal phosphatase and in recently described protein tyrosine phosphatases.

J Biol Chem, 1991 Feb 5, 266(4), 2303 - 12
Interaction of a regulatory protein with a DNA target containing two overlapping binding sites; Lloubes R et al.; The LexA repressor from Escherichia coli regulates the transcription of about 20 different genes upon binding to single or multiple operators . In this work we study the interaction of LexA with the control region of the caa gene (coding for the bacterial toxin colicin A) that contains two operators (O1 and O2) which overlap by at least 2 base pairs relevant for sequence specific DNA recognition . This arrangement raises the question of how the LexA molecules which bind to the central overlapping part of the two operators avoid steric clashes and further, of whether the interaction of LexA with the two operators is cooperative or not . To address these questions we have constructed two mutant operators (O1+O2- and O1-O2+) for which the two most strongly conserved base pairs in each of the external operator half-sites have been mutated . Using methylation interference with the complex formation of LexA with the wild-type and these two mutant operators we could show: 1) that the two mutant operators behave symmetrically in that the methylation of one crucial guanine base in both operator half-sites interferes strongly with complex formation, 2) but that in the wild-type operator (containing four functional operator half-sites) only the two external half-operators give rise to interference if this crucial guanine base is methylated, whereas methylation of the two equivalent guanine bases within the two central (overlapping) operator half-sites does not lead to interference with the formation of a complex where both operators are occupied simultaneously . These data suggest that the centrally bound LexA molecules adopt a somewhat different binding mode than those bound to the external half-operators in order to avoid steric clashes and/or to optimize protein-protein contacts which are likely to be at the origin of the binding cooperativity that we could demonstrate by quantitative DNase I footprinting and gel retardation experiments . While the methylation interference experiments revealed a non-equivalence for the binding of externally and centrally bound LexA molecules, both methylation protection and hydroxyl radical footprinting were unable to reveal this difference, suggesting that the difference between the two binding modes should be fairly subtle.

J Biol Chem, 1991 Feb 5, 266(4), 2089 - 98
Nuclear Overhauser effect studies on the conformations of Mg(alpha,beta-methylene)ATP bound to Escherichia coli methionyl-tRNA synthetase; Williams JS et al.; Internuclear distances obtained from nuclear Overhauser effects were used in combination with a distance geometry algorithm to determine the conformation of Mg(alpha,beta-methylene)ATP bound to the Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) both in the absence and presence of cognate and noncognate amino acids . Mg(alpha,beta-methylene)ATP, a nonhydrolyzable analog of ATP, was used to prevent hydrolysis of the nucleotide in the presence of either cognate or noncognate amino acids . Kinetic analysis showed that Mg(alpha,beta-methylene)ATP was a linear competitive inhibitor with respect to ATP in the ATP-pyrophosphate exchange reaction with a Ki = 1.2 mM . The pattern of internuclear Overhauser effects on Mg(alpha,beta-methylene)ATP bound to delta MTS was qualitatively consistent only with an anti glycosidic torsional angle, suggesting that the adenosine portion of the nucleotide is uniquely oriented in the binary enzyme-nucleotide complex . Nearly identical patterns of nuclear Overhauser effects were also observed in ternary complexes containing either cognate L-methionine or noncognate L-homocysteine amino acids . Distance geometry calculations permitted the range and conformational space of the allowed adenine-ribose glycosidic torsional angles in each of the complexes to be better defined and compared . Average adenine-ribose glycosidic torsional angles for enzyme-bound Mg(alpha,beta-methylene)ATP of -106 +/- 9 degrees, -99 +/- 11 degrees, and -97 +/- 11 degrees were determined for the delta MTS.Mg(alpha,beta-methylene)ATP, delta MTS.Mg(alpha,beta-methylene)ATP.L-methionine, and delta MTS.Mg(alpha,beta-methylene)ATP.L-homocysteine complexes, respectively . Comparison of the three enzyme-bound conformations showed that a single nucleotide structure having an adenine-ribose glycosidic torsional angle of -98 degrees with a 3'-endo to O4'-exo ribose sugar pucker was, within error, consistent with the experimental internuclear distances obtained in all three complexes . The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrates that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or the nature of the amino acid bound at the aminoacyladenylate site . Therefore, conformational changes known to occur in the methionyl-tRNA synthetase upon ligand binding appear not to alter the bound conformation of the nucleotide . Information on the conformation and arrangement of substrates bound at the aminoacyladenylate site of delta MTS is necessary for understanding the molecular mechanisms involved in amino acid activation and discrimination.

J Biol Chem, 1991 Feb 5, 266(4), 2054 - 62
A human autoantibody specific for a unique conserved region of 28 S ribosomal RNA inhibits the interaction of elongation factors 1 alpha and 2 with ribosomes; Uchiumi T et al.; An autoantibody reactive with a conserved sequence of 28 S rRNA (anti-28 S) was identified in serum from a patient with systemic lupus erythematosus . Anti-28 S protected a unique 59-nucleotide fragment synthesized in vitro against RNase T1 digestion . RNA sequence analysis revealed that it corresponded to residues 1944-2002 in human 28 S rRNA and 1767-1825 in mouse 28 S rRNA . These sequences are identical and highly conserved throughout all known eukaryotic 28 S rRNAs . In addition, this fragment is homologous to residues 1052-1110 of Escherichia coli 23 S rRNA that lies within the GTP hydrolysis center of the 50 S ribosomal subunit . Anti-28 S and its Fab fragments strongly inhibited poly(U)-directed polyphenylalanine synthesis, but had no effect on ribosomal peptidyltransferase activity . This effect resulted from inhibition of the binding of elongation factors EF-1 alpha and EF-2 to ribosomes and of the associated GTP hydrolysis . The inhibitory effect was almost completely suppressed by preincubation of anti-28 S with 28 S rRNA or in vitro synthesized RNA fragments containing the immunoreactive region . These results show that the immunoreactive conserved region of 28 S rRNA participates in the interaction of ribosomes with the two elongation factors in protein synthesis.

J Biol Chem, 1991 Feb 5, 266(4), 2028 - 35
Molecular cloning and characterization of complementary DNA encoding for ferredoxin-dependent glutamate synthase in maize leaf; Sakakibara H et al.; The sequence of ferredoxin-dependent glutamate synthase (EC 1.4.7.1) mRNA from maize has been determined . Complementary DNAs were isolated from a cDNA library of light-induced leaf poly(A)+ RNA constructed in an expression vector . An open reading frame beginning at an ATG codon at nucleotide 328 of the longest cDNA (5617-bases long) encoded 1616 amino acid residues . The amino terminus of the purified mature enzyme coincided with the cysteine residue at position 98 of the predicted sequence . This enzyme is homologous with the large subunit of Escherichia coli NADPH-dependent glutamate synthase having about 42% identical residues between the two proteins . The enzyme also contains a short region similar to a potential FMN-binding region of yeast flavocytochrome b2 . The cDNA hybridizes to an RNA band about 5.5 kilobases whose steady-state level is markedly increased upon illumination of etiolated maize seedlings . Analysis of genomic DNA indicates the presence of a single-copy gene for ferredoxin glutamate synthase in maize.

J Biol Chem, 1991 Feb 5, 266(4), 2290 - 6
Identification of phosphorylation sites in the repetitive carboxyl-terminal domain of the mouse RNA polymerase II largest subunit; Zhang J et al.; The largest subunit of eukaryotic RNA polymerase II contains a carboxyl-terminal domain (CTD) which is comprised of repetitive heptapeptides with a consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 . We demonstrate here that the mouse CTD expressed in and purified from Escherichia coli can be phosphorylated in vitro by a p34cdc2/CDC28-containing CTD kinase from mouse ascites tumor cells . The product of this reaction, a phosphorylated form of the CTD, contains phosphoserine and phosphothreonine, but not phosphotyrosine . The same phosphoamino acid content is observed in the in vivo phosphorylated CTD from a mouse cell line . Synthetic peptides with naturally occurring non-consensus heptapeptide sequences can also be phosphorylated by CTD kinase in vitro . Phosphoamino acid analysis of these non-consensus heptapeptides together with direct sequencing of a phosphorylated heptapeptide reveals that serines (or threonines) at positions two and five are the sites phosphorylated by mouse CTD kinase . Thus, the -Ser(Thr)-Pro- motif common to p34cdc2/CDC28-containing protein kinases is the recognition site for mouse CTD kinase.

J Mol Biol, 1991 Feb 5, 217(3), 465 - 75
Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli; Mechulam Y et al.; The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f . Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA . Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase . To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis . Substitution of Lys61 slightly affected the enzyme activity . In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln . Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction . A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain . Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position . It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+ . We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre.

J Biol Chem, 1991 Feb 5, 266(4), 2041 - 7
Mechanism of Tn3 resolvase recombination in vivo; Bliska JB et al.; To determine the physiologically important features of site-specific recombination by Escherichia coli Tn3 resolvase we analyzed the salient properties of the reaction in vivo . A two-plasmid system in which one plasmid served as substrate while the other encoded both resolvase and a thermolabile repressor of resolvase transcription provided controlled, synchronous recombination . Recombination proceeded rapidly and was promoted by (-) DNA supercoiling . The structures of the in vivo recombination products were predominantly the same as those previously identified in vitro . By examination of the products of successive rounds of recombination of a four-site substrate, we ruled out a tracking mechanism for site alignment . Inversion and plasmid fusion occurred in vivo at a much lower rate than resolution but ultimately reached a higher extent than found in vitro . We propose that inversion and fusion exploit topologically interlinked substrates that occur at low levels in vivo . This proposal is supported by the unexpected specificity of fusion . Our data imply that supercoiled DNA, the resolvase synaptic complex, and the mechanism of strand exchange are fundamentally similar in vivo and in vitro, but that the repertoire of resolvase substrates and products is expanded in vivo by the action of other enzymes that alter DNA topology.

Biochemistry, 1991 Feb 5, 30(5), 1278 - 84
Catalytic site nucleotide and inorganic phosphate dependence of the conformation of the epsilon subunit in Escherichia coli adenosinetriphosphatase; Mendel-Hartvig J et al.; The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1 (ECF1) has been found to be ligand-dependent, as measured indirectly by the activation of the enzyme that occurs on protease digestion, or when followed directly by monitoring the cleavage of this subunit using monoclonal antibodies . The cleavage of the epsilon subunit was fast in the presence of ADP alone, ADP + MG2+, ATP + EDTA, or AMP-PNP, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site(s) . The half-maximal concentration of Pi required in the presence of ADP + Mg2+ to protect the epsilon subunit from cleavage by trypsin was 50 microM, which is in the range measured for the high-affinity binding of Pi to F1 . The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-{3-(dimethylamino)propyl}carbodiimide (EDC) . In the presence of ATP + Mg2+ or ADP + Mg2+ + Pi, the epsilon subunit cross-linked to beta in high yield . With ATP + EDTA or ADP + Mg2+ (no Pi), the yield of the beta-epsilon cross-linked product was much reduced . We conclude that the epsilon subunit undergoes a conformational change dependent on the presence of Pi . It has been found previously that binding of the epsilon subunit to ECF1 inhibits ATPase activity by decreasing the off rate of Pi {Dunn, S . D., Zadorozny, V . D., Tozer, R . G., & Orr, L . E . (1987) Biochemistry 26, 4488-4493} . This reciprocal relationship between Pi binding and epsilon-subunit conformation has important implications for energy transduction by the E . coli ATP synthase.

J Mol Biol, 1991 Feb 5, 217(3), 429 - 39
Individual domains of colicins confer specificity in colicin uptake, in pore-properties and in immunity requirement; Benedetti H et al.; Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and E1 . These hybrid colicins were purified and their properties were studied . All of them were active against sensitive cells, although to varying degrees . From the results, one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin E1 is located in the N-terminal domain of colicin E1; (4) the 183 N-terminal amino acid residues of colicin E1 are sufficient to promote E1AA uptake and thus probably colicin E1 uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C-terminal domains of colicin A and colicin E1 by their respective immunity proteins.

J Biol Chem, 1991 Feb 5, 266(4), 2632 - 8
Use of in vitro protein synthesis from polymerase chain reaction-generated templates to study interaction of Escherichia coli transcription factors with core RNA polymerase and for epitope mapping of monoclonal antibodies; Lesley SA et al.; The interaction of two Escherichia coli transcription factors (sigma 54 and sigma 32) with the core RNA polymerase was studied here . We examined the core binding ability of various fragments of these two transcription factors using a novel method for the in vitro synthesis of truncated proteins in a coupled transcription-translation (S-30) system . The method uses DNA templates generated by the polymerase chain reaction to direct synthesis of precisely truncated fragments of the encoded protein . Primers for the polymerase chain reaction contain transcription and translation signals so that the resulting product can be incubated in the S-30 directly . The synthesized proteins were used for mapping of both functional domains and epitopes of monoclonal antibodies to sigma 54 and sigma 32.

Biochemistry, 1991 Feb 5, 30(5), 1413 - 8
Effect of metal ions and adenylylation state on the internal thermodynamics of phosphoryl transfer in the Escherichia coli glutamine synthetase reaction; Abell LM et al.; Experiments were conducted to study the differences in catalytic behavior of various forms of Escherichia coli glutamine synthetase . The enzyme catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia via a gamma-glutamyl phosphate intermediate . The physiologically important metal ion for catalysis is Mg2+; however, Mn2+ supports in vitro activity, though at a reduced level . Additionally, the enzyme is regulated by a covalent adenylylation modification, and the metal ion specificity of the reaction depends on the adenylylation state of the enzyme . The kinetic investigations reported herein demonstrate differences in binding and catalytic behavior of the various forms of glutamine synthetase . Rapid quench kinetic experiments on the unadenylylated enzyme with either Mg2+ or Mn2+ as the activating metal revealed that product release is the rate-limiting step . However, in the case of the adenylylated enzyme, phosphoryl transfer is the rate-limiting step . The internal equilibrium constant for phosphoryl transfer is 2 and 5 for the unadenylylated enzyme with Mg2+ or Mn2+, respectively . For the Mn2(+)-activated adenylylated enzyme the internal equilibrium constant is 0.1, indicating that phosphoryl transfer is less energetically favorable for this form of the enzyme . The factors that make the unadenylylated enzyme most active with Mg2+ are discussed.

Kansenshogaku Zasshi, 1991 Feb, 65(2), 175 - 80
{Isolation of Vero toxin-producing Escherichia coli (VTEC) from imported meats}; Tanaka H et al.; One hundred and four samples of various imported meats obtained from the stores and supermarkets in Matsuyama-city and Iyo-city from November, 1987 to October, 1988 were examined and two strains of Vero toxin-producing Escherichia coli were isolated: one was from beef from Australia and the other was from pork from Canada . By bead-ELISA, it was confirmed that the strain from beef produced Vero Toxin 1 (VT1) and Vero Toxin 2 variant (VT2vh) and the strain from pork produced VT2vh . These results suggest that imported meats may be contaminated with VTEC and thus are possible sources of human VTEC infection, such as hemorrhagic colitis and hemolytic uremic syndrome.

Zentralbl Veterinarmed B, 1991 Feb, 38(1), 17 - 20
The diagnostic value of immunoperoxidase in detecting K99+ Escherichia coli on histological sections; Scanziani E et al.; The small intestine of 51 calves was examined for the presence of K99+ Escherichia coli by means of both an immunoperoxidase procedure performed on paraffin sections and by the slide agglutination test after isolation . Twelve cases resulted immunoperoxidase positive (23.5%) and 8 of them were also agglutination positive . Results of the 2 diagnostic tests agreed in 46 cases (90.2%) . In immunoperoxidase positive sections a thick layer of immunoreactive bacteria was seen on the luminal surface of the enterocytes . Post-mortem autolysis or prolonged fixation did not seem to affect the immunoperoxidase reactivity of the sample.

Zhong Xi Yi Jie He Za Zhi, 1991 Feb, 11(2), 110 - 1, 70
{Preventive effect of re du qing on hepatocytes and mitochondria damaged by lipid peroxidation in experimental rabbits with endotoxin-induced disseminated intravascular coagulation}; Deng ZM et al.; In this study, the general Shwartzman reaction of rabbits induced by Escherichia Coli endotoxin was made as DIC models . The experiments showed that the levels of lipid peroxide (LPO) in hepatic tissue and mitochondria in the model group were increased significantly compared with the control group (P less than 0.01), while superoxide dismutase (SOD) activity in hepatic tissue and glutathione peroxidase (GSH-Px) activity in hepatic tissue and mitochondria were decreased significantly (P less than 0.01) . The levels of LPO in hepatic tissue and mitochondria in Re Du Qing (RDQ) group and vitamin E (VE) group were decreased significantly (P less than 0.01 and P less than 0.05 respectively) compared with the model group . The levels of LPO in the RDQ group did not differ from the control group (P greater than 0.05), but the levels of LPO in the VE group were still higher than those in the control group significantly (P less than 0.05) . The SOD activity in hepatic tissue and GSH-Px activity in hepatic tissue and mitochondria in both RDQ group and VE group were also significantly higher than those in the model group (P less than 0.01) . These data suggest that the levels of oxygen free radicals were increased in hepatocytes and mitochondria . This is related to the decreased activities of SOD and GSH-Px in the course of pathogenesis of endotoxin-induced DIC . This study indicates that lipid peroxidation might be one of the important mechanisms resulting in hepatocellular and mitochondria from oxidative damage.

Can J Microbiol, 1991 Feb, 37(2), 116 - 21
{Implantation of Escherichia coli in pilot experiments and the influence of competition on the flora}; Le Guyader F et al.; Implantation in seawater and (or) sediment of bacterial flora and the influence of such flora upon the survival and growth of an Escherichia coli of human origin have been the object of experimental pilot studies . The selected pilot plant permitted work on large volumes of seawater and sediment, and maintenance of the structure of the latter . Diverse experiments were carried out in the presence or absence of seawater and (or) sediment bacterial flora during 13 days . Escherichia coli bacteria were introduced in the seawater experimental system at concentrations of 1 to 3 X 10(5) colony-forming units (cfu) per 100 mL . In sterile sediment, E . coli bacteria first went through a proliferative phase and then implanted themselves (3 X 10(4) cfu/100 g at 0 days and 4 X 10(5) cfu/100 g at 13 days) . Diffusion in the supernatant sterile seawater of organic matter released from sediment allowed the strain to proliferate (8 X 10(6) cfu/100 mL at 1 day) and survive for a few days (1 X 10(4) cfu/100 mL at 6 days), prior to an ultimate decreasing phase (1 cfu/100 mL at 13 days) . In the presence of the seawater indigenous flora, an immediate decrease (2 X 10(3) cfu/100 mL at 6 days), without a growth or even a survival phase, evidenced a selection pressure . In a nonsterile sediment, in the presence or absence of seawater indigenous flora, E . coli bacteria implanted themselves quickly (5 X 10(4) cfu/100 g at 1 day) and survived (1 X 10(4) cfu/100 g at 13 days) . In the supernatant seawater, a decrease was observed from the 1st day.(ABSTRACT TRUNCATED AT 250 WORDS)

J Membr Biol, 1991 Feb, 119(3), 267 - 75
A single amino acid substitution alters conductance and gating of OmpC porin of Escherichia coli; Delcour AH et al.; We have reconstituted into liposomes outer-membrane fractions from Escherichia coli strains which express OmpC porins with altered pore properties . Single-channel experiments were performed with the patch-clamp technique on blisters generated from the reconstituted liposomes . Our goal was to identify positively the activity pattern of OmpC in our reconstituted system . The properties of the parent strain were compared to those of a strain whose OmpC porin has a single amino acid substitution in a postulated transmembrane segment . The parent and the mutant strain each exhibit a cation-selective channel of high open probability and gating to closed levels of various amplitudes . However, the mutant channel appeared to be 9 to 30% larger in unit conductance . It tended to close and reopen most often in groups of three units, as opposed to two units in the parent channel . The results are discussed in terms of the observed phenotype and of their implication as to the structure-function relationship of the porin channels.

J Med Assoc Thai, 1991 Feb, 74(2), 108 - 11
Necrotizing fasciitis of previous surgical wound complicating intensive postremission chemotherapy; Intragumtornchai T et al.; Necrotizing fasciitis of previous surgical wound developed in a leukemic patient after receiving intense postremission chemotherapy . A unique feature was profound granulocytopenia and the recovery of Escherichia coli in the blood and necrotic tissue . We believe the hematogenous seeding of the surgical wound during the episode of E . coli septicemia was the cause of our patient's necrotizing fasciitis.

Fundam Appl Toxicol, 1991 Feb, 16(2), 249 - 56
Influence of the oral administration of excess copper on the immune response; Pocino M et al.; We have studied the influence of the oral administration of excess copper (Cu) on the immune response . With this aim, mice maintained on standard laboratory diet received 50, 100, 200, or 300 ppm of Cu as copper sulfate in the drinking water during 3 to 10 weeks . Inhibition of the proliferative response to concanavalin A was observed in mice exposed to 100 ppm of Cu for 8 weeks and to 200 ppm of Cu for either 3 or 8 weeks . Conversely, a significant increase in the proliferative response to Escherichia coli lipopolysaccharide (LPS) was observed in mice exposed to 50 or 100 ppm of Cu for 3 weeks . However, the response to LPS was also significantly inhibited following prolonged Cu administration . In contrast, mice exposed to low or high Cu doses during short or long periods showed increased production of autoantibodies directed to bromelain-treated mouse erythrocytes . The DTH response to sheep red blood cells was not modified following short-term administration of 100 ppm of Cu, but was depressed after prolonged exposure to this dose of the metal . Significant inhibition of the DTH response was observed in mice exposed to 300 ppm of Cu for 5 or 10 weeks . Thus, oral administration of excess Cu altered the immune response in a fashion related to the dose and duration of treatment.

Mol Biochem Parasitol, 1991 Feb, 44(2), 287 - 95
Paramyosin is the Schistosoma mansoni (Trematoda) homologue of antigen B from Taenia solium (Cestoda); Laclette JP et al.; Antigen B, a major antigen of the cestode parasite Taenia solium, has been purified and a portion of amino acid sequence obtained . Paramyosin of the trematode parasite Schistosoma mansoni, an immunogenic protein that has shown promise as a vaccine candidate, has several biochemical and immunological properties in common with antigen B . A full-length cDNA clone of S . mansoni paramyosin has been obtained and the predicted translation product contains a sequence that is highly homologous to the sequence obtained for antigen B . The predicted amino acid composition and isolectric point of paramyosin are nearly identical to those established for antigen B . Recombinant S . mansoni paramyosin, expressed in Escherichia coli as a fusion protein with beta-galactosidase, was recognized by antisera against T . solium antigen B . We conclude from these results that S . mansoni paramyosin and T . solium antigen B are homologous proteins . Since S . mansoni paramyosin is thought to be a muscle protein and T . solium antigen B a secreted glycoprotein with anti-complement activity, this conclusion raises some interesting questions regarding the role of this class of proteins in the host-parasite relationship.

Mol Biochem Parasitol, 1991 Feb, 44(2), 207 - 11
Large fragments of Plasmodium falciparum DNA can be stable when cloned in yeast artificial chromosomes; Triglia T et al.; A major problem in cloning large segments of Plasmodium falciparum DNA is the instability of this very A + T-rich DNA in Escherichia coli . We have therefore investigated whether P . falciparum DNA segments over 100 kb in size cloned in the yeast Saccharomyces cerevisiae as yeast artificial chromosomes (YACs) are stable . An analysis of EcoRI fragments adding up to 230 kb, or nearly 1% of the P . falciparum genome, showed that sequences cloned as YACs were indistinguishable from the DNA in the P . falciparum genome, showing neither deletions nor rearrangements . Over 400 YACs have been obtained so far with an average insert size between 110 and 130 kb . Screening the YACs with single-copy probes suggested that most sequences should be represented in a library of less than 10(3) YACs . Hence YACs provide an approach to cloning large segments of P . falciparum DNA.

Behring Inst Mitt, 1991 Feb, (88), 147 - 56
A recombinant hybrid protein as antigen for an anti-blood stage malaria vaccine; Knapp B et al.; Based on investigations on several blood stage antigens from Plasmodium falciparum we have expressed a hybrid protein in E . coli containing 262 amino acids of the serine-stretch protein SERP and 189 amino acids of the histidine alanine rich protein HRPII . Antibodies raised against the hybrid protein by immunization of rabbits and Aotus monkeys react with both corresponding schizont polypeptides . Two Aotus monkeys immunized with the SERP/HRPII hybrid protein showed only low parasitemias after challenge infection with P . falciparum, compared to the control group . The result suggests that hybrid proteins of this type may be the basis for the development of a malaria vaccine.

Behring Inst Mitt, 1991 Feb, (88), 112 - 9
Induction and modulation of interleukin 1 production by defined partial structures of lipopolysaccharide; Flad HD et al.; Bacterial lipopolysaccharides (LPS) consist of a hydrophilic heteropolysaccharide component and a covalently linked hydrophobic lipid portion termed lipid A . Lipid A is responsible for most of the toxic and immunoregulatory effects of LPS . In studying the structure-function relationship of lipid A partial structures we now report that certain synthetic analogues, with themselves being biologically "inactive", exert immunomodulating effects on LPS-induced Interleukin 1 production in vitro.

Mol Microbiol, 1991 Feb, 5(2), 467 - 76
The trpC gene of Phanerochaete chrysosporium is unique in containing an intron but nevertheless maintains the order of functional domains seen in other fungi; Schrank A et al.; The Phanerochaete chryososporium trpC gene has been isolated by complementation of an Escherichia coli trpC mutant . The full extent of the fungal gene, determined by sequence analysis, was found to be 2414bp . This includes a single intron of 50bp, the presence of which was confirmed by RNA-primed polymerase chain reaction analysis . This features makes the P . chrysosporium gene unique when compared to equivalent genes from other filamentous fungi . The P . chrysosporium trpC gene encodes a single protein containing three enzyme activities involved in tryptophan biosynthesis arranged in the order: NH2-GAT-IGPS-PRAI-COOH . This order is conserved in all filamentous fungi so far examined and, indeed, is the gene order within the E . coli trp operon.

Mol Microbiol, 1991 Feb, 5(2), 455 - 65
Two global regulators repress the anaerobic expression of MnSOD in Escherichia coli::Fur (ferric uptake regulation) and Arc (aerobic respiration control); Tardat B et al.; The expression of sodA, the Escherichia coli gene encoding manganese superoxide dismutase (MnSOD) is induced by aerobiosis and superoxide generators such as paraquat . Analysis of variants expressing sodA in the absence of oxygen has revealed that mutations in genes for two global regulatory systems, Fur (ferric uptake regulation) and Arc (aerobic respiration control), are simultaneously required for the expression of sodA in anaerobiosis . The Fur protein still represses sodA in an iron-dependent fashion in aerobiosis . Moreover, all mutants remain inducible by paraquat, indicating that the positive control of SoxR, which mediates the response to superoxide in E . coli, is still operative . Thus, in addition to the response to the superoxide-mediated oxidative stress which depends on SoxR, two global controls regulate MnSOD expression: ArcA couples MnSOD expression to respiration, and Fur couples it to the intracellular concentration of iron.

Mol Microbiol, 1991 Feb, 5(2), 439 - 46
The TraM protein of plasmid R1 is a DNA-binding protein; Schwab M et al.; The TraM protein of the resistance plasmid R1 was purified to homogeneity and used for DNA-binding studies . Both gel retardation- and footprint experiments showed that TraM specifically binds to DNA of plasmid R1 comprising the region between the origin of transfer and the traM gene . Several TraM molecules bind and, according to the footprint experiments, two distinct sites of specific binding exist . The two sites are separated from each other by 12 nucleotides and each contains an inverted repeat . DNase I protection assays showed that the initial TraM binding occurs at these palindromic sequences . At higher protein concentrations the lengths of the DNA segments protected by TraM were increased towards the traM gene . In one region this extension leads to binding of TraM protein at its own promoters.

Mol Microbiol, 1991 Feb, 5(2), 409 - 17
P.70 pertactin, an outer-membrane protein from Bordetella parapertussis: cloning, nucleotide sequence and surface expression in Escherichia coli; Li LJ et al.; The gene prn encoding the outer-membrane protein P.70 (pertactin) from Bordetella parapertussis has been cloned in Escherichia coli and its DNA sequence determined . Analysis of the DNA sequence reveals that the gene has an open reading frame comprising 922 amino acids capable of encoding a protein with a molecular weight of 95,177 (P.95) . In vivo processing of this precursor yields a protein with an estimated Mr of 70 kDa (P.70) which is located on the surface of B . parapertussis . Homology between the prn gene from B . parapertussis and that from Bordetella pertussis is 91.3% . The homology is 93% when the protein sequence of P.95 is aligned with that of P.93 from B . pertussis . The major differences between the P.70 pertactin from B . parapertussis and the P.69 pertactin from B . pertussis occur in the number of reiterated units within the repeat motifs found in both proteins; the sequence Gly-Gly-Xaa-Xaa-Pro is repeated four times in the P.70 pertactin, and five times in the P.69 pertactin, while the sequence Pro-Gln-Pro occurs nine times in P.70 pertactin and five times in P.69 pertactin . Cloning of the gene for P.95 in an E . coli expression vector results in the synthesis of a protein that mimics native gene expression in B . parapertussis, i.e . the P.95 protein is synthesized and subsequently processed to yield the P.70 form of the protein on the surface of the cell.

Mol Microbiol, 1991 Feb, 5(2), 393 - 9
Functioning of the stable signal peptide of the pCloDF13-encoded bacteriocin release protein; Luirink J et al.; The pCloDF13-encoded bacteriocin release protein (BRP) is a lipoprotein which is synthesized as a precursor with an amino-terminal signal peptide that appears to be stable after cleavage . The role of the stable signal peptide in the functioning of the BRP was studied with respect to the release of cloacin DF13, 'lysis' and leakage of periplasmic proteins . The BRP gene fragment encoding the stable signal peptide was replaced by a fragment encoding the unstable peptide of the murein lipoprotein (Lpp) . The resulting hybrid protein was normally acylated and processed by signal peptidase II, leaving no stable signal peptide in the cells . Expression of the hybrid protein did not result in the specific release of cloacin DF13, whereas 'lysis' and the release of periplasmic enzymes were unaffected . These results indicated a role for the stable BRP signal peptide in the translocation of cloacin DF13 across the cytoplasmic membrane.

Mol Microbiol, 1991 Feb, 5(2), 353 - 60
Promoter sequence requirements for Fnr-dependent activation of transcription of the narGHJI operon; Walker MS et al.; The sequence requirements for Fnr-dependent transcription of the narGHJI operon of Escherichia coli were studied in plasmids carrying a narG::lacZ protein fusion with the 5' end of the promoter deleted so that expression was controlled exclusively by Fnr . These plasmids were subjected to in vitro mutagenesis, and beta-galactosidase activities were determined in transformed strains after aerobic and anaerobic growth . A single base-pair change in the Fnr box, a sequence which is highly conserved in all Fnr-dependent promoters, essentially abolished anaerobic induction of expression . Primer extension analysis located the transcription start site 57 bp upstream from the narG translation start site and placed the Fnr box at a position centred between -41 and -42 bases from the transcription start site . The position of the Fnr box relative to the transcription start site was critical for anaerobic induction of expression . The deletion of 2 bp or addition of 4, 6, 10, 14, 20, 22, 28, 30, and 40 bp immediately downstream from the Fnr box abolished anaerobic induction . Sequence changes between the Fnr box and the transcription start site had different effects, depending upon the region mutagenized . Base changes immediately downstream from the Fnr box, including bases -20 to -29, did not lead to any decrease in anaerobic induction of expression, but in most instances resulted in increased expression . Base changes further downstream prevented anaerobic induction of expression and suggested the requirement for a -10 hexamer which is partially homologous to the -10 consensus sequence for sigma 70-specific promoters of E . coli.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1991 Feb, 62(1), 81 - 4
Evaluation of a small-scale method for the extraction of the K88 antigen from enterotoxigenic Escherichia coli; Payne DW et al.; A small-scale method for the extraction of the K88 major fimbrial subunit from enterotoxigenic Escherichia coli (ETEC) based on heat extraction has been developed . Variation in the buffer composition, time and temperature of extraction had negligible effect on the subsequent SDS-PAGE profile . There was, however, a correlation between the pH of the extraction buffer and the ensuing amount of K88 released . Reduction of the pH from 7 to 4 reduced by six-fold the amount of K88 released from the cells . We suggest that the relative stability of the K88 fimbriae at acid pH may influence the site of infection by ETEC in the intestine.

FEMS Microbiol Lett, 1991 Feb, 62(1), 37 - 41
Identification and subcellular localization of apolipoprotein N-acyltransferase in Escherichia coli; Gupta SD et al.; Apolipoprotein N-acyltransferase, the enzyme catalyzing the conversion of apolipoprotein to mature lipoprotein, was detected by an in vitro assay using {35S}methionine-labeled apolipoprotein as the substrate . Triton X-100 solubilized the enzyme, and was required for its activity . The enzyme showed a broad pH optimum (pH 6.5-7.5) . N-Acylation of apolipoprotein with ethanol-washed membranes was dependent on exogenous phospholipids, with phosphatidylethanolamine, phosphatidylglycerol and cardiolipin all showing about 10- to 20-times enhancement of the enzyme activity in the delipidated membranes . Incubation of apolipoprotein with {3H}palmitate-labeled membranes resulted in the incorporation of {3H}palmitate into lipoprotein . The enzyme was found to be enriched in the inner membrane and in the inner membrane/outer membrane mixed fractions of the E . coli cell envelope.

Immunol Lett, 1991 Feb, 27(2), 157 - 62
Serum levels of tumor necrosis factor determine the fatal or non-fatal course of endotoxic shock; Mozes T et al.; The role of tumor necrosis factor alpha (TNF alpha) in endotoxin-induced shock was investigated in pigs receiving 5 micrograms kg-1 of Escherichia coli endotoxin (LPS) during 60 min of continuous infusion into the superior mesenteric artery . LPS concentration in aortic plasma, as determined by a chromogenic Limulus amoebocyte lysate (LAL) test, reached a peak of approximately 1000 ng l-1 during LPS infusion, and declined rapidly after discontinuation of the infusion . Serum TNF levels were determined by a bioassay using the L929 murine transformed fibroblast line . Eight of the 17 animals infused with LPS died within 30 min after beginning LPS administration, while the other 9 pigs survived beyond the experimental observation period of 3 h, although they were in a state of shock . No difference in LPS concentration was found between the survivors and the non-survivors . However, the serum TNF levels in non-survivors were significantly higher than in survivors when measured at 30 min after beginning LPS administration . In survivors, the peak increase in serum TNF levels was measured at 60 min after the beginning of LPS injection and returned rapidly to the baseline values . Although the role of TNF inducing rapid death seems to be dominant, the hemodynamic, hematology and blood chemistry disturbances seen during shock continued in survivors long after the return of TNF to baseline levels . These findings indicate that besides TNF other mediators are also involved in the LPS infusion-induced shock.

Acta Anaesthesiol Scand, 1991 Feb, 35(2), 134 - 40
Adrenergic support during anesthesia in experimental endotoxin shock: norepinephrine versus dobutamine; Van der Linden P et al.; The effects of norepinephrine and dobutamine were compared during endotoxin shock in dogs anesthetized either with enflurane (E: 1.5%, N = 12) or with i.v . ketamine (K: 5 mg.kg-1 + 0.2 mg.kg-1.min-1, N = 12) . An i.v . bolus of 1.5 mg.kg-1 E . coli endotoxin was followed by saline infusion to restore left-sided filling pressures at baseline . With E, heart rate, mean arterial pressure and stroke index decreased (P less than 0.01) . The decrease in oxygen delivery (DO2) and in oxygen consumption (VO2) was associated with an increase in blood lactate . In contrast, K anesthesia was associated with remarkable hemodynamic stability . DO2 was well maintained, VO2 decreased (P less than 0.01) and blood lactate did not change . Under E anesthesia, mean arterial pressure increased more with norepinephrine and heart rate increased more with dobutamine . Under K anesthesia, cardiac index, stroke index and left ventricular stroke work index increased similarly with both agents . In both groups DO2 and VO2 increased markedly . The amount of fluid infused was higher with dobutamine than with norepinephrine . Thus, enflurane but not ketamine had depressant cardiovascular effects at the doses used in this model . With both anesthetics, norepinephrine and dobutamine could effectively improve cardiac function . Dobutamine could therefore represent a valuable alternative to norepinephrine for cardiovascular support during anesthesia in septic shock.

No Shinkei Geka, 1991 Feb, 19(2), 157 - 9
{A case report: abscess of the cavum septi pellucidi}; Oda S et al.; A rare case of an abscess in the cavum septi pellucidi (CSP) is described and previously reported cases are reviewed . A 60-year-old male was admitted to the hospital because a diagnosis of cerebellar hemisphere infarction was made on CT scan . Seven years earlier, the patient had undergone a craniotomy for aneurysm clipping, and a ventriculo-peritoneal shunt was installed for normal pressure hydrocephalus 14 days after the aneurysmal rupture . On his second hospitalization CT scan also demonstrated CSP but this was not associated with ventriculomegaly . He was placed on a rehabilitation regimen and his hospital course was uneventful . Two months later, however, he developed hyponatremia due to the syndrome of inappropriate secretion of antidiuretic hormone . After analysis of CSF obtained from the shunting device, a diagnosis of meningitis was made and CSF culture revealed E . coli infection . A part of the peritoneal tubing was torn and missing when the tube was removed from the peritoneal cavity and converted to outer drainage . Being treated with intrathecal and intravenous antibiotics administration, the meningitis subsided . However, CT scan taken twelve days after the onset of the infection showed an abscess in CSP which showed ring enhancement after contrast media . Therefore, the patient continued to receive intravenous antibiotics to counter the mass effect due to the abscess . The abscess had disappeared on follow-up CT scan obtained ten days later . The patient, however, eventually expired after iatrogenic hypernatremia associated with acute renal failure . The patient was submitted to an autopsy . The authors speculate that the abscess developed through a retrograde cisternal route after infection which had originated from bowel perforation by the peritoneal shunt tube.

Acta Chem Scand, 1991 Feb, 45(2), 186 - 92
A multivariate representation and analysis of DNA sequence data; Jonsson J et al.; A new way to represent and analyze DNA sequence data is described . This approach complements methods currently used, in that it allows the systematic part of the variation between different sequences to be modeled . This can prove as informative as absence of variation (homology), which is the most widely used criterion for comparing sequence data . A multivariate sequence-activity model (SAM), for DNA-promoter sequences is presented, by which the relative promoter strength is modeled in terms of the primary DNA-sequence . The model is shown to have a good predictive capability . The coefficients from the model are interpreted, and used to design new structures predicted to be strong promoters in the system investigated . The approach described is also applicable to other kinds of sequence data, e.g . RNAs, proteins or peptides.

J Appl Bacteriol, 1991 Feb, 70(2), 177 - 80
RecA-independent resistance to irradiation with u.v . light in acid-habituated Escherichia coli; Goodson M et al.; Growth of Escherichia coli 1829 ColV, I-K94 at pH 5.0 led to an increase in u.v . resistance compared with cells grown at pH 7.0 . This was due to a phenotypic change, since organisms grown at pH 7.0 showed increased resistance after only 2.5-5.0 min incubation at the mildly acid pH . Other E . coli K12 derivatives became more u.v.-resistant at pH 5.0 including uvrA, recA and polA1 mutants . Organisms grown at pH 5.0 also showed increased Weigle reactivation of u.v.-irradiated lambda phage and this applied to the repair-deficient mutants as well as the parent strains . Both the increased u.v . resistance of acid-habituated cells and their increased ability to bring about Weigle reactivation appear to involve RecA-independent processes and are presumably, therefore, independent of the SOS response.

J Appl Bacteriol, 1991 Feb, 70(2), 156 - 60
Inhibition of Escherichia coli K12 by short-chain organic acids: lack of evidence for induction of the SOS response; Cherrington CA et al.; Sublethal concentrations of formic acid (10 mmol/l) and propionic acid (5 mmol/l) at pH 5.0 preferentially inhibit DNA synthesis and stop cell multiplication in the absence of a corresponding cessation in the increase of culture turbidity . The possibility that the acids induce the SOS response by starving cells of thymine or by causing physical damage to the DNA molecule has now been investigated . Accumulation of thymine into the cytoplasm of whole cells was not inhibited by either acid . Mutants defective in excision repair (uvr A6), recombination repair (rec A56) and polymerase activity (pol A1) were not more sensitive to the acids than their isogenic parent . No significant increase in cell length was observed from measurements of transmission electron microscope images of acid-treated cells . It is concluded, therefore, that sublethal concentrations of formic and propionic acid inhibit DNA synthesis without physically damaging DNA molecule, or starving the cell of essential thymine or otherwise inducing an SOS response.

Prostaglandins, 1991 Feb, 41(2), 169 - 83
The effects of in vivo administration of endotoxin on the functions and interaction of hepatocytes and Kupffer cells; Ogle CK et al.; It was the purpose of this study to determine the effects of the in vivo administration of endotoxin on certain in vitro hepatocyte and Kupffer cell functions . An Alzet osmotic pump that contained endotoxin (LPS, 2.5 mg/100g) was implanted into the peritoneal cavity of 300g guinea pigs and delivered the endotoxin over a period of four days . In vivo administration of LPS did not cause a change in the in vitro release of albumin by isolated hepatocytes . However, when hepatocytes were co-cultured with Kupffer cells there was a significant decrease in albumin release for both control and LPS-treated animals . There was no difference between control and LPS-treated animals in the release of C3 by hepatocytes . However, there was a significant increase over the control group in C3 release by Kupffer cells from LPS-treated animals . When hepatocytes and Kupffer cells were cultured together, their release of C3 was not additive . Kupffer cells from LPS-treated animals released significantly greater amounts of PGE2 than control animals when stimulated in vitro with LPS . Thus, these Kupffer cells appeared to be primed to respond to an in vitro challenge of LPS . Kupffer cells from LPS-treated animals had significantly depressed antibody dependent cellular cytotoxicity (ADCC) . This endotoxin model is useful for determining the in vivo effects of endotoxin on cellular function and gives some indirect evidence for the detrimental effects of LPS on the immune system and host defense.

J Gen Microbiol, 1991 Feb, 137 ( Pt 2), 361 - 7
A molecular analysis of the 53.3 minute region of the Escherichia coli linkage map; Andrews SC et al.; The coding characteristics of four plasmids expressing a protein (BCP) which comigrates with bacterioferritin were examined and the nucleotide sequence of a common 1985 bp segment from the 53 min region of the Escherichia coli linkage map was determined . Three open reading-frames (orf1, orf2 and orf3) were detected, and orf2 (bcp, 156 amino acid codons) appeared to encode the bacterioferritin comigratory protein, BCP . The translation product of orf3 (205 amino acid codons) resembled the iron-sulphur protein component (DMS B subunit) of the anaerobic dimethylsulphoxide reductase complex of E . coli.

J Chromatogr, 1991 Feb 1, 538(2), 311 - 21
Affinity partitioning of restriction endonucleases . Application to the purification of EcoR I and EcoR V; Vlatakis G et al.; Partitioning of restriction endonucleases between two liquid aqueous phases can be strongly influenced by group-specific ligands included in the two-phase system . Three restriction endonucleases, namely EcoR I, EcoR V and BamH I, were partitioned within an aqueous dextran-polyethylene glycol (PEG) system . The enzymes could be extracted into the upper PEG phase by using either triazine dyes or herring DNA as affinity ligands . The influence of the endogenous bacterial nucleic acids, concentration of polymerbound dye and concentration of sodium chloride on the system were examined . A partial purification of EcoR I (up to 52-fold) and EcoR V (up to 37-fold) was achieved using a combination of affinity partitioning and ion-exchange chromatography, providing an extremely fast and economical method for the isolation of restriction endonucleases free from contaminating nuclease activities.

Appl Environ Microbiol, 1991 Feb, 57(2), 592 - 3
Assay for beta-glucuronidase in species of the genus Escherichia and its applications for drinking-water analysis; Rice EW et al.; Recently, Escherichia species other than Escherichia coli have been isolated from potable water . Environmental isolates as well as clinical isolates of E . adecarboxylata, E . blattae, E . fergusonii, E . hermannii, and E . vulneris were assayed for the enzyme beta-glucuronidase by using EC MUG medium and the Colilert system . None of the isolates were positive for the enzyme by either method.

Gene, 1991 Feb 1, 98(1), 89 - 93
Physical map and genetic early region of the T7-related coliphage, BA14; Michalewicz J et al.; The chromosome of the bacterial virus, BA14, a member of the T7 lytic coliphage group, was characterized by direct measurement of its length and construction of a restriction map . The chromosome (39.6 kb) is essentially the same size as T7 (39.9 kb), is devoid of a large number of restriction sites expected for a DNA of this size, and moreover, lacks modification sites for the Escherichia coli Dam and Dcm methyltransferases . The BA14 early region was assigned by testing the ability of specific chromosomal restriction fragments to direct RNA synthesis by E . coli RNA polymerase, and analysis of in vitro RNase III cleavage products of the transcripts . The data support and extend the previous assertion that BA14 is a representative of a distinct T7 subgroup, and limited nucleotide sequence analysis of the BA14 DNA ligase-encoding gene suggests a closer relationship of BA14 to T7 than to T3 phage, another member of the T7 group.

Gene, 1991 Feb 1, 98(1), 135 - 40
Acquisition of mitochondrial DNA by a transformation vector for Ustilago violacea; Bej AK et al.; Plasmid pUCH1 is a 5.2-kb pUC18 construct bearing the hygB gene fused to a promoter from Cochliobolus heterostrophus . Haploid cells of the basidiomycete, Ustilago violacea, were transformed with this plasmid . In addition to multiple integrations of plasmid sequences into U . violacea nuclear DNA, vector sequences independent of the nuclear genome were indicated by Southern-blot analysis using all or part of pUCH1 as a probe . Hybridization also revealed intact pUCH1 and several larger derivatives in satellite bands from CsCl-bis-benzamide gradients of whole cellular DNA and in DNA from purified mitochondria {mitochondrial (mt) DNA preparations} of transformed U . violacea; circular DNAs consistent with the sizes of DNAs in these satellite bands were seen in electron microscope analyses of the same mt DNA preparations as well . The plasmids could be detected in mt DNA preparations even after 30 generations of transformant growth under selective pressure . Transformation of Escherichia coli by these mt DNA preparations produced bacterial transformants bearing intact pUCH1, as well as several pUCH1 derivatives, including pUCH2, an approx . 8.0-kb plasmid . A 2.5-kb EcoRI fragment from pUCH2 showed only weak hybridization with pUCH1 . This unique fragment did hybridize strongly with mt DNA from untransformed U . violacea . This derivative thus appears to have acquired mt sequences from U . violacea.

Chromosoma, 1991 Feb, 100(2), 87 - 96
Two dispersed highly repeated DNA families of Triturus vulgaris meridionalis (Amphibia, Urodela) are widely conserved among Salamandridae; Vignali R et al.; Two BamHI families of repeated sequences were characterized from the genome of the Italian smooth newt, Triturus vulgaris meridionalis (Amphibia, Urodela) . The first family, which is divided into subfamilies, consists of tandemly arranged arrays whose basic repeat is around 398 bp long; these arrays are dispersed throughout the entire chromosome sets of the various species of Triturus tested . Moreover the family is widely conserved among Salamandridae, being detected by genomic DNA blotting of Notophthalmus viridescens, Taricha granulosa, Salamandrina terdigitata and Euproctus platycephalus . The second BamHI family is represented by a cloned sequence of 419 bp, which is dispersed in the chromosome set of several species of Triturus . The sequence is also conserved in S . terdigitata and in E . platycephalus but is not detectable in N . viridescens or T . granulosa . The cloned sequence is most probably only part of a longer unit interspersed within the Triturus genome.

Mol Gen Genet, 1991 Feb, 225(2), 342 - 5
A physiological role for DNA supercoiling in the anaerobic regulation of colicin gene expression; Malkhosyan SR et al.; The mechanism of anaerobic regulation of synthesis of colicins E1, E2, E3, K and D was studied . It was found that anaerobiosis significantly increases expression of the genes for colicins E1, E2, E3, K, and D . Experiments with novobiocin (a DNA gyrase inhibitor) showed that colicin synthesis in minicells and derepressed colicin synthesis in cells are dramatically reduced by relaxation of DNA supercoiling . A good correlation was observed between the levels of colicin synthesis and plasmid DNA supercoiling and the degree of aeration of the cultures . Thus, the regulation of colicin gene expression in response to a change in aeration appears to be mediated by environmentally induced variations in DNA supercoiling.

Mol Gen Genet, 1991 Feb, 225(2), 314 - 9
Characterisation of the gene cysH and of its product phospho-adenylylsulphate reductase from Escherichia coli; Krone FA et al.; The nucleotide sequence of the gene cysH from Escherichia coli K12 was determined . The open reading frame was 735 nucleotides in length; it was flanked by a repetitive palindromic sequence centred 36 nucleotides upstream of cysH and a terminator-like structure located 20 nucleotides downstream . CysH encoded a polypeptide of Mr 27927 consisting of 244 amino acids . The gene product was isolated as a homodimer exhibiting phospho-adenylylsulphate reductase (PAPS reductase) activity . The active enzyme was devoid of electron transferring cofactors and contained only one cysteine per subunit . Reduction of the enzyme by dithiols resulted in a shift of the apparent molecular weight from 44,000 to 62,000 without formation of an enzyme-thioredoxin complex.

Mol Gen Genet, 1991 Feb, 225(2), 297 - 304
Subcellular location and expression level of a chimeric protein consisting of the maize waxy transit peptide and the beta-glucuronidase of Escherichia coli in transgenic potato plants; Klosgen RB et al.; The transit peptide of the maize waxy protein (a nuclear-encoded amyloplast protein of the maize endosperm) was studied with respect to its role in subcellular protein targeting in transgenic potato plants . TP30, a chimeric precursor protein consisting of the waxy transit peptide and an additional 34 amino acids of the mature waxy protein fused to the beta-glucuronidase of Escherichia coli, was expressed in potato plants under the control of the 35S promoter of cauliflower mosaic virus . This fusion protein is imported not only into amyloplasts, the natural target organelles in the maize plant, but also into chloroplasts . In contrast, Gus, the beta-glucuronidase alone, which was also expressed in parallel experiments in transgenic potato plants is always found in the cytosol of the plant cells . As a consequence of the different subcellular locations of TP30 and Gus, we observed differences in the expression rates of the respective proteins in leaf cells, resulting in higher steady state levels of TP30 compared to Gus . In tuber cells, no correlation between intracellular location and expression of the proteins was found.

Mol Gen Genet, 1991 Feb, 225(2), 266 - 72
Evidence of abortive recombination in ruv mutants of Escherichia coli K12; Benson F et al.; Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids . Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold . Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants . Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec+ sbc+ strains, depending on the plasmid used . Recombinant plasmids carrying ruv+ were transferred efficiently . With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA . It is proposed that the failure to recover transconjugants in ruv recA+ strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.

Mol Gen Genet, 1991 Feb, 225(2), 234 - 40
recA gene dependence of replication of the Escherichia coli chromosome initiated by plasmid pUC13 integrated at predetermined sites; Mao YM et al.; Plasmid pUC13 was used to clone DNA fragments of known sites from the chromosome of Escherichia coli . Each chimeric plasmid was introduced individually into the same dnaA46 mutant strain LC381 and suppressive integration (Sin) strains were selected . By means of cotransduction the null mutation recA56 was then introduced into each Sin strain and growth of each recA56 derivative at 42 degrees C was scored . Strains that failed to grow at 42 degrees C depended upon the recA gene for replication . Three factors were shown to limit the viability of LC381 harboring different chimeric plasmids and affect the degree of recA gene dependence of chromosome replication in the Sin strains at 42 degrees C . It is suggested that these three constraints are the consequence of the organization of the E . coli chromosome, particularly the unique ability of terC to retard the progression of replication forks . Two classes of hypotheses concerning the function of the recA gene are considered.

Mol Gen Genet, 1991 Feb, 225(2), 193 - 8
Integrative transformation by homologous recombination in the zygomycete Mucor circinelloides; Arnau J et al.; Transformation of a Mucor circinelloides Leu- strain with the plasmid pAD45, harbouring the wild-type allele (leuA+) and a chymosin gene, led to the identification of mitotically stable transformants after one to three vegetative growth cycles on non-selective medium . Southern analysis of the stable transformed strains demonstrated that the vector is integrated, as an intact molecule, into the resident Mucor leuA locus . Retransformation of Escherichia coli with genomic DNA restricted with enzymes having no or only a single recognition site within the inserted sequence did not permit isolation of plasmids or fragments carrying the leuA or chymosin gene.

Oncogene, 1991 Feb, 6(2), 297 - 301
Identification of the ret proto-oncogene products in neuroblastoma and leukemia cells; Takahashi M et al.; Monoclonal and/or polyclonal antibodies were generated against the products synthesized from two portions of the ret proto-oncogene (c-ret) cDNA expressed in Escherichia coli . These antibodies were reactive in immunoblotting with 150 kd and 170 kd proteins in cell lysates from three human neuroblastoma cell lines expressing the ret proto-oncogene . When the neuroblastoma cells were treated with tunicamycin, a protein with an apparent molecular weight of 120 kd, which is consistent with that of the c-ret protein predicted from the cDNA sequence, appeared on immunoblots . These results indicated that the 150 kd and 170 kd proteins in neuroblastoma cells are produced from a single polypeptide of 120 kd by posttranslational glycosylation . Furthermore, the antibodies detected a unique 190 kd protein as well as 150 kd protein in a cell lysate from THP-1 human monocytic leukemia cell line, suggesting that glycosylated forms of the c-ret protein are different between neuroblastoma and leukemia cells.

Oncogene, 1991 Feb, 6(2), 265 - 73
Determinants of sequence-specific DNA-binding by p48v-myb; Garcia A et al.; The v-myb oncogene of the avian myeloblastosis virus encodes a nuclear protein, p48v-myb, which binds to DNA in a sequence-specific manner . We have used wild type and mutant forms of this protein expressed in E . coli to study the protein and DNA determinants for sequence-specific DNA-binding . We have shown that only the highly conserved domain at the amino terminus of p48v-myb is required for sequence-specific DNA-binding . However, neither of the tandem 50 amino acid repeats present in this domain is alone sufficient for such binding . We have also demonstrated that p48v-myb can recognize a single consensus myb binding site and appears to interact with DNA as a protein monomer . In addition, we have shown that sequence-specific binding by p48v-myb requires nucleotides which flank the previously reported PyAACT/GG consensus.

Mutat Res, 1991 Feb, 262(2), 145 - 50
Starvation as an inducer of error-free DNA repair in Escherichia coli; Fitt PS et al.; Previous work in this laboratory has shown that heat shock or vitamin B1 deprivation induces an error-free DNA-repair process in Escherichia coli . The system is absolutely dependent on excision repair, while its induction is delayed in lon- or recA- cells . We have now shown that starvation of E . coli for amino acids, glucose or phosphate, conditions known to induce the stringent response or the glu and pho regulons, respectively, leads to a similar uvrA-dependent increase in UV resistance and decrease in UV-induced mutation frequency . These results support the hypothesis that the effect is a general response to non-mutagenic stress that may play an important role in the survival of cells exposed to harsh environments.

Biochem J, 1991 Feb 1, 273 ( Pt 3), 725 - 31
Metalloendopeptidase QG . Isolation from Escherichia coli and characterization; Polgar L et al.; A new proteinase, which preferentially cleaves the Gln-Gly bond, was isolated from Escherichia coli . Because of this narrow specificity, the enzyme was called metalloendopeptidase QG . The proteinase is a monomer and consists of a single polypeptide chain of Mr 67,000, which is significantly smaller than the other known metalloendopeptidases of E . coli . It is found in the cytoplasm, but not in the periplasm . The enzyme cleaves the substrate benzyloxycarbonyl-Gln-Gly-Pro 2-naphthylamide between the glutamine and glycine residues, as well as its extended homologues including a nonapeptide, but it does not hydrolyse either the oxidized A and B chains of insulin or azo-casein . The pH-dependence of substrate hydrolysis gives a bell-shaped curve with pK1 = 6.6 and pK2 = 8.8 . The metallopeptidase is inhibited in Tris and imidazole buffers, the basic components of which are presumably liganded to the essential Zn2+ ion . 2-Aminobenzoyl-Gln-Gly-Pro 2-naphthylamide, designed as a fluorescent substrate for the metallopeptidase, proved to be a strong inhibitor . Bestatin, an inhibitor of aminopeptidases in the micromolar concentration range, inhibits the metalloendopeptidase only in the millimolar concentration range . Captopril, the widely used inhibitor of angiotensin-converting enzyme, is a fairly good inhibitor of the metalloendopeptidase . The simplest inhibitor that can be used to protect recombinant proteins from degradation by the metalloendopeptidase may be EDTA, which is effective at low millimolar concentration.

Biochem J, 1991 Feb 1, 273 ( Pt 3), 659 - 66
Cloning, sequencing and expression of cDNA for chick liver haem oxygenase . Comparison of avian and mammalian cDNAs and deduced proteins; Evans CO et al.; A cDNA from a chick liver library that encodes for haem oxygenase has been cloned and sequenced . Positive clones were identified with monospecific antibodies to the purified enzyme from chick liver and a cDNA of rat haem oxygenase-1 . The length of the cDNA is 1258 bases . An open reading frame of 888 bases was identified by comparison of nucleotide and amino acid sequences with those previously identified for haem oxygenase of mammalian or avian origin . The protein corresponding to this fragment of DNA is composed of 296 amino acid residues and has a molecular mass of 33,509 Da, which is similar to that previously estimated for haem oxygenase purified from chick liver . Unequivocal identification of this clone as that complementary to haem oxygenase was provided by (a) comparison of amino acid compositions and partial sequences with those previously established for the purified enzyme, (b) comparison with nucleotide and amino acid sequences for haem oxygenase from rat and human sources and (c) expression in Escherichia coli with production of high levels of mRNA, protein and haem oxygenase activity after exposure of the transfected bacteria to isopropyl beta-D-thiogalactopyranoside (IPTG) . Overall, the similarity of chick haem oxygenase to rat and human haem oxygenase (nucleotides 66% and amino acids 62%) is moderately high . The region between proline-129 and alanine-157 is identical in all three enzymes, including histidine-135, which is proposed to play a key role in binding the substrate haem at the active centre of the enzyme . Northern blots also show that treatment of chicks with CdCl2, a potent inducer of haem oxygenase, results in increases in 1.65-1.70 kb mRNA, which hybridizes selectively to the full-length cDNA or to a synthetic 24-base oligonucleotide with sequence identical to that of a portion of the haem oxygenase cDNA . These results suggest that Cd-dependent induction of haem oxygenase is due to increased transcription of the gene or stabilization of its message.

Biochem J, 1991 Feb 1, 273 ( Pt 3), 621 - 6
Human cystatin C . role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase; Abrahamson M et al.; Leucocyte elastase in catalytic amounts was observed to rapidly cleave the Val-10-Gly-11 bond of the human cysteine-proteinase inhibitor cystatin C at neutral pH . The resulting modified inhibitor had size and amino acid composition consistent with a cystatin C molecule devoid of the N-terminal Ser-1-Val-10 decapeptide . Leucocyte-elastase-modified cystatin C had more than 240-fold lower affinity than native cystatin C for papain . Removal of the N-terminal decapeptide of human cystatin C also decreased inhibition of human cathepsins B and L by three orders of magnitude, but decreased inhibition of cathepsin H by only 5-fold . A tripeptidyldiazomethane analogue of of the N-terminal portion of cystatin C was a good inhibitor of cathepsins B and L but a poor inhibitor of cathepsin H . It therefore appears that amino acid side chains of the N-terminal segment of cystatin C bind in the substrate-binding pockets of cathepsins B and L but not in those of cathepsin H . It is argued that the N-terminal cystatin C interaction with cathepsin B is physiologically important and hence that leucocyte elastase could have a function as a regulator of extracellular cysteine-proteinase inhibitory activity at sites of inflammation.

Radiat Res, 1991 Feb, 125(2), 197 - 205
Investigation of the neutral filter elution technique I . Effects of pore density and pore diameter on elution rate at pH 9.6; Mayer PJ et al.; To understand better the biophysical mechanism of neutral filter elution (pH 9.6), we eluted genomes of known size and shape: coliphage T4c (Mr 1.15 x 10(8), E . coli (Mr 2.7 x 10(9)), and Chinese hamster lung fibroblasts (V79, Mr 2-4 x 10(10)) . DNA eluted through 15% sucrose atop the filter in a biphasic pattern . The elution rate of the initial component correlated (r greater than 0.97) exponentially with 1/Mr for monodisperse samples of DNA eluted through pore sizes 0.1-3.0 microns . Using this relationship between elution rate and Mr, we estimated Mn of polydisperse, X-irradiated (253 Gy) samples of DNA from E . coli or V79 cells to be 3.15 +/- 1.46 and 1.42 +/- 0.33, respectively, compared to expected values of 2.93 and 3.52 (10(8) Da) . The best predictor of elution rate for DNA from T4c and intact and X-irradiated V79 cells was pore density, and pore diameter for DNA from X-irradiated E . coli . The rate of elution of DNA from unirradiated E . coli was unrelated to pore density or diameter . While the mechanism of neutral filter elution remains unknown, its use for linear DNAs with Mn ca . 10(8) Da appears to be valid quantitatively.

J Trop Med Hyg, 1991 Feb, 94(1), 57 - 60
Giardiasis and amoebiasis infections in three Saudi closed communities; Omar MS et al.; Two hundred and eighteen residents (131 healthy and 87 mentally or physically retarded) of a children's nursery, a foster house and a rehabilitation centre for the handicapped in Abha, southwestern Saudi Arabia, were examined for intestinal parasitism . About 30% of the population of the three communities were found to harbour asymptomatic infections with either Giardia lamblia and/or Entamoeba spp . Giardia cysts were identified in 19.3% of those examined . Entamoeba histolytica was found in 18.4% of the residents of the rehabilitation centre only . Entamoeba coli infections were found in residents of both the foster house and rehabilitation centre (6.0 and 25.3% respectively) . The prevalence of infection with the three parasites was significantly higher in the rehabilitation centre than in the children's nursery and foster house.

J Bacteriol, 1991 Feb, 173(4), 1367 - 75
A family of regulatory genes associated with type II restriction-modification systems; Tao T et al.; Restriction-modification systems must be regulated to avoid autorestriction and death of the host cell . An open reading frame (ORF) in the PvuII restriction-modification system appears to code for a regulatory protein from a previously unrecognized family . First, interruptions of this ORF result in a nonrestricting phenotype . Second, this ORF can restore restriction competence to such interrupted mutants in trans . Third, the predicted amino acid sequence of this ORF resembles those of known DNA-binding proteins and includes a probable helix-turn-helix motif . A survey of unattributed ORFs in 15 other type II restriction-modification systems revealed three that closely resemble the PvuII ORF . All four members of this putative regulatory gene family have a common position relative to the endonuclease genes, suggesting a common regulatory mechanism.

Carcinogenesis, 1991 Feb, 12(2), 221 - 4
Mutational specificities of environmental carcinogens in the lacI gene of Escherichia coli: IV . The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; Jiao JL et al.; We have determined the mutational specificity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by the characterization of 58 induced Escherichia coli lacI-d mutants at the DNA sequence level . Metabolic activation of NNK was carried out using the S9 fraction from Aroclor 1254-treated rats . G:C----A:T transitions dominated the spectrum, accounting for 55% of the mutations recovered . The other base substitutions recovered include three A:T----G:C transitions as well as two A:T----T:A, three A:T----C:G, five G:C----C:G and five G:C----T:A transversions . Other classes of mutational events included two deletions, three duplications and three frame-shifts . The complexity of the NNK mutational spectrum appears consistent with a model that this compound induces mutations by both the methylation and the pyridoloxobutylation of DNA.

Epidemiol Infect, 1991 Feb, 106(1), 63 - 70
Detection of novel trimethoprim resistance determinants in the United Kingdom using biotin-labelled DNA probes; Towner KJ et al.; Two collections of trimethoprim R plasmids, isolated from strains of Escherichia coli during 1978-83 and 1987-8 respectively, were retrospectively screened with specific biotinylated DNA probes for the presence of genes encoding particular DHFR enzymes . The results confirmed that the type I DHFR gene was the predominant plasmid-encoded gene conferring trimethoprim resistance in strains of E . coli from the Nottingham area of the UK, but indicated that genes encoding the more recently recognized types of DHFR enzymes had appeared in the bacterial gene pool and could be recognized with increased frequency in the latter plasmid collection . This was particularly true of the type IIIa and type VII enzymes which together accounted for 27% of the trimethoprim R plasmids examined in 1987-8.

Am J Pathol, 1991 Feb, 138(2), 395 - 402
In vivo biologic and immunohistochemical analysis of interleukin-1 alpha, beta and tumor necrosis factor during experimental endotoxemia . Kinetics, Kupffer cell expression, and glucocorticoid effects; Chensue SW et al.; Using a model of sepsis induced by parenteral challenge of mice with bacterial lipopolysaccharide (LPS), the authors analyzed the in vivo expression of interleukin-1 (IL-1) alpha,beta and tumor necrosis factor (TNF) . Both TNF and IL-1 alpha,beta were detected in hepatic sinusoidal macrophages (Kupffer cells), immunohistochemically . Kinetic analysis showed a clear sequence of synthesis . Tumor necrosis factor was produced first, reaching maximal expression at 1 hour after LPS challenge, then rapidly disappeared . IL-1 beta followed, reaching maximal expression at 2 to 3 hours, then dropped off by 6 hours . Interleukin-1 alpha expression reached a peak at 6 hours and had disappeared by 18 hours . Analysis of serum bioactivity also revealed sequential expression that correlated with immunohistochemical findings . Tumor necrosis factor was maximal at 1 hour and IL-1 at 6 hours . The IL-1 bioactivity was not due to interleukin-6 (IL-6), as this was depleted from specimens by immunoabsorption . Also IL-6 bioactivity reached maximal levels at 3 hours, earlier than IL-1 . Pretreatment with 4 mg/kg dexamethasone significantly decreased Kupffer cell expression of TNF and IL-1 alpha (about 80% and 60% suppression, respectively) but had less effect on IL-1 beta expression (about 30% suppression) . Accordingly, serum levels of TNF were suppressed by 75% while serum IL-1 was decreased by 39%, indicating differential sensitivity of these cytokines to glucocorticoids . Endogenous corticosteroid levels increased as TNF levels decreased, supporting the contention that glucocorticoids regulate TNF synthesis . In contrast, IL-1 levels rose concurrently with corticosterone . These data indicate a sequential activation of cytokine gene expression in vivo, which may be critical to the cascade of events leading to septic shock, and provide evidence that Kupffer cells are a major source of cytokines in endotoxemia . Finally, the differential sensitivity of cytokine expression to glucocorticoids may in part explain the inadequacy of the latter in the treatment of sepsis.

Surgery, 1991 Feb, 109(2), 154 - 9
Increased sensitivity to endotoxemia by tissue necrosis; Maessen JG et al.; In this study the interaction of endotoxemia and ischemic organ injury was investigated in a rat model . Animals received lipopolysaccharide to induce endotoxemia and were simultaneously subjected to renal ischemia . If only renal ischemia was induced, moderate azotemia occurred and all animals survived . Lipopolysaccharide treatment caused neither renal failure nor death . However, rats with both endotoxemia and renal ischemia showed severe azotemia, and 50% of the animals died within 48 hours . The observed mortality rate is unlikely related to renal failure since animals subjected to bilateral nephrectomy did not die within 48 hours after treatment with lipopolysaccharide . To further exclude the role of renal failure in the enhanced effect of endotoxemia, experiments were performed in which ischemic kidneys were excised from littermates and were placed in the abdomens of lipopolysaccharide-treated animals . A similar effect was observed: 50% of the animals died within 48 hours . Azotemia did not occur . Since tumor necrosis factor (TNF) is an important cytokine involved in endotoxemia-induced morbidity and death, we studied the role of TNF in our model . Plasma levels of TNF were increased during endotoxemia . Concomitant renal ischemic injury did not influence the concentration of TNF . When animals were treated with recombinant TNF and were subsequently subjected to renal ischemic injury, again a 50% mortality was observed, a rate similar to that in lipopolysaccharide-treated animals . We conclude that the sensitivity to endotoxemia is enhanced by tissue necrosis and may lead to death in the experimental model used in this study.

Proc Natl Acad Sci U S A, 1991 Feb 1, 88(3), 976 - 80
Sequence determination and modeling of structural motifs for the smallest monomeric aminoacyl-tRNA synthetase; Hou YM et al.; Polypeptide chains of 19 previously studied Escherichia coli aminoacyl-tRNA synthetases are as large as 951 amino acids and, depending on the enzyme, have quaternary structures of alpha, alpha 2, alpha 2 beta 2, and alpha 4 . These enzymes have been organized into two classes which are defined by sequence motifs that are associated with specific three-dimensional structures . We isolated, cloned, and sequenced the previously uncharacterized gene for E . coli cysteine-tRNA synthetase (EC 6.1.1.16) and showed that it encodes a protein of 461 amino acids . Biochemical analysis established that the protein is a monomer, thus establishing this enzyme as the smallest known monomeric synthetase . The sequence shows that cysteine-tRNA synthetase is a class I enzyme that is most closely related to a subgroup that includes the much larger methionine-, isoleucine-, leucine-, and valine-tRNA synthetases, which range in size from 677 to 951 amino acids . The amino-terminal 293 amino acids of the cysteine enzyme can be modeled as a nucleotide-binding fold that is more compact than that of its closest relatives by virtue of truncations of two insertions that split the fold . This smaller nucleotide-binding fold accounts for much of the reduced size of the cysteine enzyme and establishes the limit to which the structure of this domain is contracted in the five members of this subgroup of class I enzymes.

J Bacteriol, 1991 Feb, 173(3), 1360 - 2
Suppression of the defects in rdgB mutants of Escherichia coli K-12 by the cloned purA gene; Clyman J et al.; A gene suppressing the defects of Escherichia coli rdgB (recA-dependent growth) and recA(Ts) rdgB mutants was cloned . The cloned gene suppresses the hyper-Rec phenotype and the enhanced level of SOS functions in rdgB mutants at the nonpermissive temperature . We identified the cloned gene as purA.

J Bacteriol, 1991 Feb, 173(3), 1335 - 8
Map position and genomic organization of the kps cluster for polysialic acid synthesis in Escherichia coli K1; Vimr ER; The multigenic kps cluster in Escherichia coli K1 encodes functions for synthesis of a polysialic acid capsule . DNA probes flanking each side of the cluster were hybridized to lambda clones bearing overlapping E . coli W3110 genomic fragments . These fragments covered the region between 60 and 70 map units on the chromosome . The results located kps to an accretion domain near 64 map units and established the orientation of kps cluster genes . Acquisition of kps by the E . coli genome was apparently the result of an ancestral transpositionlike addition event.

J Bacteriol, 1991 Feb, 173(3), 1279 - 86
Integration host factor of Escherichia coli reverses the inhibition of R6K plasmid replication by pi initiator protein; Dellis S et al.; Integration host factor (IHF) protein is the only host-encoded protein known to bind and to affect replication of the gamma origin of Escherichia coli plasmid R6K . We examined the ability of R6K origins to replicate in cells lacking either of the two subunits of IHF . As shown previously, the gamma origin cannot replicate in IHF-deficient cells . However, this inability to replicate was relieved under the following conditions: underproduction of the wild-type pi replication protein of R6K or production of normal levels of mutant pi proteins which exhibit relaxed replication control . The copy number of plasmids containing the primary R6K origins (alpha and beta) is substantially reduced in IHF-deficient bacteria . Furthermore, replication of these plasmids is completely inhibited if the IHF-deficient strains contain a helper plasmid producing additional wild-type pi protein . IHF protein has previously been shown to bind to two sites within the gamma origin . These sites flank a central repeat segment which binds pi protein . We propose a model in which IHF binding to its sites reduces the replication inhibitor activity of pi protein at all three R6K origins.

J Bacteriol, 1991 Feb, 173(3), 1268 - 78
Mutations in the Escherichia coli dnaG gene suggest coupling between DNA replication and chromosome partitioning; Grompe M et al.; Eleven conditional lethal dnaG(Ts) mutations were located by chemical cleavage of heteroduplexes formed between polymerase chain reaction-amplified DNAs from wild-type and mutant dnaG genes . This entailed end labeling one DNA strand of the heteroduplex, chemically modifying the strands with hydroxylamine or osmium tetroxide (OsO4) at the site of mismatch, and cleaving them with piperidine . The cleavage products were electrophoresed, and the size corresponded to the position of the mutation with respect to the labeled primer . Exact base pair changes were then determined by DNA sequence analysis . The dnaG3, dnaG308, and dnaG399 mutations map within 135 nucleotides of one another near the middle of dnaG . The "parB" allele of dnaG is 36 bp from the 3' end of dnaG and 9 bp downstream of dnaG2903; both appear to result in abnormal chromosome partitioning and diffuse nucleoid staining . A suppressor of the dnaG2903 allele (sdgA5) maps within the terminator T1 just 5' to the dnaG gene . Isogenic strains that carried dnaG2903 and did or did not carry the sdgA5 suppressor were analyzed by a combination of phase-contrast and fluorescence microscopy with 4',6-diamidino-2-phenylindole to stain DNA and visualize the partitioning chromosome . Overexpression of the mutant dnaG allele corrected the abnormal diffuse-nucleoid-staining phenotype associated with normally expressed dnaG2903 . The mutations within the dnaG gene appear to cluster into two regions which may represent distinct functional domains within the primase protein.

J Bacteriol, 1991 Feb, 173(3), 1201 - 7
Mutagenic frequencies of site-specifically located O6-methylguanine in wild-type Escherichia coli and in a strain deficient in ada-methyltransferase; Rossi SC et al.; The adaptive response of Escherichia coli involves protection of the cells against the toxic and mutagenic consequences of exposure to high doses of a methylating agent by prior exposure to low doses of the agent . Ada protein, a major repair activity for O6-methylguanine, is activated to positively control the adaptive response; O6-methylguanine is one of the major mutagenic lesions produced by methylating agents . We investigated the mutation frequency of wild-type Escherichia coli and strains containing the ada-5 mutation in response to site-specifically synthesized O6-methylguanine under conditions in which the adaptive response was not induced . Site-directed mutagenesis and oligonucleotide self-selection techniques were used to isolate the progeny of M13mp18 DNAs constructed to contain O6-methylguanine at any of eight different positions . The progeny were isolated from E . coli strains isogeneic except for deficiency in Ada-methyltransferase repair, UvrABC excision repair, or both . The resulting O6-methylguanine mutation levels at each position were determined by using differential oligonucleotide hybridization . We found that the wild type had up to a 2.6-fold higher mutation frequency than ada-5 mutants . In addition, the mutation frequency varied with the position of the O6-methylguanine in the DNA in the wild type but not in ada-5 mutants; O6-methylguanine lesions at the 5' ends of runs of consecutive guanines gave the highest mutation frequencies . Determination of the mutation frequency of O6-methylguanine in wild-type and mutS cells showed that mismatch repair can affect O6-methylguanine mutation levels.

J Bacteriol, 1991 Feb, 173(3), 1193 - 200
A transcription terminator in the thymidylate synthase (thyA) structural gene of Escherichia coli and construction of a viable thyA::Kmr deletion; Bell-Pedersen D et al.; A transcription terminator has been identified within the coding sequence of the Escherichia coli thyA gene . Fusion of a relevant segment of the thyA structural gene to galK sequences showed that the terminator functions in vivo . Primer extension and Northern hybridization (RNA blot) analysis of thyA RNA suggested that the terminator acts as the transcription stop signal for an upstream gene and for thyA-specific transcripts . Results from antitermination studies utilizing a lambda PL-thyA fusion also offer evidence that the terminator is capable of attenuating thyA expression by reducing the amount of full-length thyA transcripts . This gene arrangement suggested that previous unsuccessful attempts to create a chromosomal thyA deletion in E . coli were attributable to the presence of the overlapping transcript . Introducing a deletion into the nonoverlapping portion of the cloned thyA gene and inserting a gene encoding kanamycin resistance produced a (delta thyA::Kmr) that was easily transferred to the chromosome of a recD host by marker replacement . This delta thyA::Kmr allele provides a useful and readily transducible chromosomal marker.

J Bacteriol, 1991 Feb, 173(3), 1120 - 4
Thermosensing ability of Trg and Tap chemoreceptors in Escherichia coli; Nara T et al.; The thermosensing ability of the Trg and Tap chemoreceptors in Escherichia coli was investigated after amplifying these receptors in a host strain lacking all four known chemoreceptors (Tar, Tsr, Trg, and Tap) . Cells with an increased amount of either Trg or Tap showed mostly smooth swimming and no response to thermal stimuli . However, when the smooth-swimming bias of the cells was reduced by adding Trg- or Tap-mediated repellents, the cells showed clear changes in the swimming pattern upon temperature changes; Trg-containing cells showed tumbling at 23 degrees C but mostly smooth swimming at 32 degrees C, while Tap-containing cells showed smooth swimming at 20 degrees C but tumbling at 32 degrees C . These results indicate that although both Trg and Tap have the ability to sense thermal stimuli, Trg functions as a warm receptor, as reported previously for Tar and Tsr, while Tap functions as a cold receptor.

J Bacteriol, 1991 Feb, 173(3), 1064 - 72
Mini-F plasmid mutants able to replicate in the absence of sigma 32: mutations in the repE coding region producing hyperactive initiator protein; Kawasaki Y et al.; Mini-F plasmids cannot replicate in Escherichia coli strains (delta rpoH) lacking sigma 32, presumably because transcription of the repE gene encoding the replication initiator protein (RepE protein) depends mostly on RNA polymerase containing sigma 32 . We have isolated and characterized mini-F mutants able to replicate in delta rpoH cells . Contrary to the initial expectation, five mutants with mutations in the repE coding region that produce altered RepE proteins were obtained . The mutations caused replacement of a single amino acid: the 92nd glutamic acid was replaced by lysine (repE10, repE16, and repE25) or glycine (repE22) or the 109th glutamic acid was replaced by lysine (repE26) . These plasmids overproduced RepE protein and exhibited very high copy numbers . Two major activities of mutated RepE proteins have been determined in vivo; the autogenous repressor activity was significantly reduced, whereas the initiator activity was much enhanced in all mutants . These results indicate the importance of a small central region of RepE protein for both initiator and repressor activities . Thus the decreased repE transcription in delta rpoH cells can be compensated for by an increased initiator activity and a decreased repressor activity of RepE, resulting in the increased synthesis of hyperactive RepE protein.

J Bacteriol, 1991 Feb, 173(3), 1012 - 20
Deletion analysis of the F plasmid oriT locus; Fu YH et al.; Functional domains of the Escherichia coli F plasmid oriT locus were identified by deletion analysis . DNA sequences required for nicking or transfer were revealed by cloning deleted segments of oriT into otherwise nonmobilizable pUC8 vectors and testing for their ability to promote transfer or to be nicked when tra operon functions were provided in trans . Removal of DNA sequences to the right of the central A + T-rich region (i.e., from the direction of traM) did not affect the susceptibility of oriT to nicking functions; however, transfer efficiency for oriT segments deleted from the right was progressively reduced over an 80- to 100-bp interval . Deletions extending toward the oriT nick site from the left did not affect the frequency of transfer if deletion endpoints lay at least 22 bp away from the nick site . Deletions or insertions in the central, A + T-rich region caused periodic variation in transfer efficiency, indicating that phase relationships between nicking and transfer domains of oriT must be preserved for full oriT function . These data show that the F oriT locus is extensive, with domains that individually contribute to transfer, nicking, and overall structure.

EMBO J, 1991 Feb, 10(2), 247 - 54
Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast; Schneider A et al.; Several precursors transported from the cytoplasm to the intermembrane space of yeast mitochondria are first cleaved by the MAS-encoded protease in the matrix space and then by additional proteases that have not been characterized . We have now developed a specific assay for one of these other proteases . The enzyme is an integral protein of the inner membrane; it requires divalent cations and acidic phospholipid for activity, and is defective in yeast mutant pet ts2858 which accumulates an incompletely processed cytochrome b2 precursor . The protease contains a 21.4 kd subunit whose C-terminal part is exposed on the outer face of the inner membrane . An antibody against this polypeptide inhibits the activity of the protease . As overproduction of the polypeptide does not increase the activity of the protease in mitochondria, the enzyme may be a hetero-oligomer . This 'inner membrane protease I' shares several key features with the leader peptidase of Escherichia coli and the signal peptidase of the endoplasmic reticulum.

Mol Cell Biol, 1991 Feb, 11(2), 872 - 85
Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily; Dascher C et al.; In Saccharomyces cerevisiae, the GTP-binding Ypt1 protein (Ypt1p) is essential for endoplasmic reticulum-to-Golgi protein transport . By exploiting a GAL10-YPT1 fusion to regulate YPT1 expression, three multicopy suppressors, SLY2, SLY12, and SLY41, and a single-copy suppressor, SLY1-20, that allowed YPT1-independent growth were isolated . Wild-type Sly1p is hydrophilic, is essential for cell viability, and differs from Sly1-20p by a single amino acid . SLY2 and SLY12 encode proteins with hydrophobic tails similar to synaptobrevins, integral membrane proteins of synaptic vesicles in higher eucaryotes . Sly41p is hydrophobic and exhibits sequence similarities with the chloroplast phosphate translocator . SLY12 but not SLY41 is an essential gene . The SLY2 null mutant is cold and heat sensitive . The SLY gene products may comprise elements of the protein transport machinery.

Mol Cell Biol, 1991 Feb, 11(2), 813 - 21
Yeast CBP1 mRNA 3' end formation is regulated during the induction of mitochondrial function; Mayer SA et al.; Alternative mRNA processing is one mechanism for generating two or more polypeptides from a single gene . While many mammalian genes contain multiple mRNA 3' cleavage and polyadenylation signals that change the coding sequence of the mature mRNA when used at different developmental stages or in different tissues, only one yeast gene has been identified with this capacity . The Saccharomyces cerevisiae nuclear gene CPB1 encodes a mitochondrial protein that is required for cytochrome b mRNA stability . This 66-kDa protein is encoded by a 2.2-kb mRNA transcribed from CPB1 . Previously we showed that a second 1.2-kb transcript is initiated at the CBP1 promoter but has a 3' end near the middle of the coding sequence . Furthermore, it was shown that the ratio of the steady-state level of 2.2-kb CBP1 message to 1.2-kb message decreases 10-fold during the induction of mitochondrial function, while the combined levels of both messages remain constant . Having proposed that regulation of 3' end formation dictates the amount of each CBP1 transcript, we now show that a 146-bp fragment from the middle of CBP1 is sufficient to direct carbon source-regulated production of two transcripts when inserted into the yeast URA3 gene . This fragment contains seven polyadenylation sites for the wild-type 1.2-kb mRNA, as mapped by sequence analysis of CBP1 cDNA clones . Deletion mutations upstream of the polyadenylation sites abolished formation of the 1.2-kb transcript, whereas deletion of three of the sites only led to a reduction in abundance of the 1.2-kb mRNA . Our results indicate that regulation of the abundance of both CBP1 transcripts is controlled by elements in a short segment of the gene that directs 3' end formation of the 1.2-kb transcript, a unique case in yeast cells.

Virology, 1991 Feb, 180(2), 763 - 9
The termini of the chlorella virus PBCV-1 genome are identical 2.2-kbp inverted repeats; Strasser P et al.; The Chlorella virus PBCV-1 genome is a linear nonpermuted 333-kbp dsDNA molecule with covalently closed hairpin termini . The termini (minus the hairpin) are identical inverted repeats of at least 2185 bases after which the sequence diverges . The inverted repeats contain two small potential open reading frames and several direct repeats . However, neither the open reading frames nor the remainder of the inverted repeats are transcribed during PBCV-1 replication . Twenty-nine other Chlorella virus DNAs, of 36 tested, hybridized to the PBCV-1 terminal fragments.

Virology, 1991 Feb, 180(2), 625 - 32
Vaccinia virus DNA ligase is nonessential for virus replication: recovery of plasmids from virus-infected cells; Kerr SM et al.; The essentiality of the vaccinia virus DNA ligase gene, SalF 15R, for virus growth was tested by insertional mutagenesis . A plasmid containing E . coli gpt inserted within a large deletion in the DNA ligase gene was transfected into vaccinia virus-infected cells and recombinant viruses selected by three cycles of plaque purification in the presence of mycophenolic acid (MPA) . Surprisingly, in some isolates, which replicated in a manner indistinguishable from wild type (WT) virus, the WT gene was replaced by the gpt allele, demonstrating that the DNA ligase gene is nonessential for growth in cultured cells . In other isolates the entire plasmid was integrated into the virus genome by a single crossover event and a functional copy of the DNA ligase was retained . Southern blot analyses of the latter, drug-resistant viruses indicated extra DNA fragments, of sizes inconsistent with predicted viral structures, which represent the plasmid products of homologous recombination . Hirt extracts from cells infected with such multiply plaque purified virus isolates yielded plasmids that produced ampicillin-resistant colonies after transformation of E . coli . These plasmids were of two structures, representing either the original plasmid used for transfection, or a plasmid containing the WT ligase gene rescued by recombination with the virus genome . Similarly, insertional mutagenesis of the vaccinia virus thymidine kinase (TK) gene with gpt yielded plasmids containing mutant or wild type TK alleles when recombinant viruses were selected in MPA . Such plasmids were not isolated when TK minus viruses were selected in 5-bromodeoxyuridine (BUdR).

Virology, 1991 Feb, 180(2), 527 - 34
Moloney murine leukemia virus IN protein from disrupted virions binds and specifically cleaves its target sequence in vitro; Ishimoto LK et al.; The integration of retroviral DNA plays an essential role in the viral life cycle . Previous studies of the Moloney murine leukemia virus (M-MuLV) have shown that viral integration is mediated by the integrase (IN) protein acting on the 13-bp inverted repeats that flank the linear viral DNA produced during reverse transcription . Prior studies have also shown that when the M-MuLV IN protein is produced in Escherichia coli it retains an ability to specifically associate with the viral inverted repeats (Krogstad and Champoux, 1990) . In this study we present evidence that the IN protein present in detergent-disrupted virions is capable of specifically interacting with double-stranded oligonucleotides that correspond to the viral inverted repeats, and that this interaction may change after integration-related processing of the viral att sites . We further present evidence that, in vitro, detergent-disrupted virions are capable of specifically cleaving ds-IR oligonucleotides in an IN-dependent reaction that mimics the trimming step that precedes integration.

J Neurochem, 1991 Feb, 56(2), 720 - 3
Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions; Kaufman DL et al.; Glutamate decarboxylase (GAD) catalyzes the production of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter . The mammalian brain contains two forms of GAD, with Mrs of 67,000 and 65,000 (GAD67 and GAD65) . Using a new antiserum specific for GAD67 and a monoclonal antibody specific for GAD65, we show that the two forms of GAD differ in their intraneuronal distributions: GAD67 is widely distributed throughout the neuron, whereas GAD65 lies primarily in axon terminals . In brain extracts, almost all GAD67 is in an active holoenzyme form, saturated with its cofactor, pyridoxal phosphate . In contrast, only about half of GAD65 (which is found in synaptic terminals) exists as active holoenzyme . We suggest that the relative levels of apo-GAD65 and holo-GAD65 in synaptic terminals may couple GABA production to neuronal activity.

J Immunol, 1991 Feb 1, 146(3), 793 - 8
TraT: a powerful carrier molecule for the stimulation of immune responses to protein and peptide antigens; Croft S et al.; A number of integral membrane proteins (Imps) isolated from Escherichia coli have been examined for their ability to generate serum antibody responses in the absence of adjuvant . These proteins were found to stimulate high titers of serum antibody when injected into rabbits or mice in saline . The antibody titers elicited were not significantly increased by the addition of a powerful adjuvant such as IFA . Covalent conjugation of BSA, of the DNP group, and of a peptide Ag from Plasmodium falciparum to these protein carriers resulted in a significant enhancement of the immune response to the conjugated material in comparison with the response elicited when the immunogen was injected without adjuvant or was not conjugated to Imps . The antibody response to these conjugates could not be significantly increased by the addition of IFA . Thus, the Imps of E . coli represent powerful carrier molecules which, when injected into mice and rabbits, are not only capable of generating high titers of antibody to themselves, but also to molecules conjugated to them . Immunization with immunogens coupled to these proteins results in the production of high titers of antibody without the need for oil-based adjuvants, thereby avoiding the unwanted side effects of such adjuvants.

J Virol, 1991 Feb, 65(2), 1046 - 52
Expression and characterization of the thymidine kinase gene of African swine fever virus; Martin Hernandez AM et al.; The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome by using two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TK genes . The TK gene was mapped within a 0.72-kbp BglII-XhoI fragment (0.242 to 0.246 map units) derived from a 23.9-kbp SalI-B fragment of the ASFV genome . Identification of this region as the ASFV TK gene was confirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free system programmed with RNA synthesized in vitro . The sequenced gene for TK includes an open reading frame of 588 nucleotides encoding a protein of 196 amino acids . The deduced amino acid sequence shows 32.4% identity with the TK of vaccinia virus.

Infect Immun, 1991 Feb, 59(2), 721 - 5
Sequence analysis of the gene encoding the Chlamydia pneumoniae DnaK protein homolog; Kornak JM et al.; The antigen-coding region of a 4.2-kb PstI fragment of Chlamydia pneumoniae (pLC3), which encodes a 75-kDa immunoreactive protein recognized during human C . pneumoniae infection, was localized to a 2.0-kb EcoRI fragment . This subclone expressed an immunoreactive fusion protein of ca . 82 kDa . Nucleotide sequence analysis of the C . pneumoniae gene revealed that it consisted of a 1,980-base open reading frame with an inferred 71,550-Da protein of 660 amino acids . Putative Escherichia coli-like promoters and a ribosomal binding site were located in the 5' upstream region, and an 11-base dyad forming a stable stem-loop structure following two in-frame stop codons was identified . The C . pneumoniae 75-kDa protein is a member of the hsp70 family of heat shock proteins and has 87% amino acid similarity with the Chlamydia trachomatis protein.

Arch Dis Child, 1991 Feb, 66(2), 181 - 4
Endotoxin induced damage to the cochlea in guinea pigs; Tarlow MJ et al.; An apparently unique form of cochlear damage was produced in guinea pigs by perfusing the cochlea or injecting the cerebrospinal fluid with bacterial endotoxin . This developed rapidly (within two hours) and was characterised by swelling of the tectorial membrane and damage to both inner and outer hair cells, with parallel functional damage demonstrable electrophysiologically . All these changes could be attenuated by pretreatment with dexamethasone . Such endotoxin mediated lesions may be the mechanism by which hearing loss occurs in bacterial meningitis.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1991 Feb, 24(1), 71 - 83
Protease-like sequence in hepatitis B virus core antigen is not involved in the cleavage processes of core protein in Escherichia coli; Hwang LH et al.; A DNA fragment, coding for hepatitis core antigen (HBcAg), was amplified by polymerase chain reaction and inserted into a lambda PL promoter-derived expression vector . The recombinant plasmid was transformed into Escherichia coli and proteins produced after heat induction were analyzed . In addition to the 21 kDa HBcAg protein, several smaller related polypeptides, particularly one of 17 kDa in size, were also detected with rabbit anti-HBcAg antiserum . Whether the protease-like sequence of core protein involved in the self-cleavage process to form the 17 kDa polypeptide was investigated by a deletion experiment . Our results with a mutant in which 7 amino acids of the conserved protease-like region in the core protein have been deleted suggest that the cleavage does not depend on the presence of these protease-like sequence . In addition, the core protein synthesized from in vitro translation reaction was not cleaved . Core particles from E . coli lysate were purified by sucrose and cesium chloride density gradient centrifugations and subsequently treated with 0.2% of SDS and 0.2% of beta-mecaptoethanol . Immunoblotting analysis, however, did not reveal any conversion of the 21 kDa protein to smaller ones . In conclusion, our results suggest that the protease-like domain at the N-terminus of the core protein does not contain intrinsic autocleavage activity, nor could the HBcAg be converted to smaller antigens by detergent treatment.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 321 - 4
Biogenesis of cell division sites in ftsA and ftsZ filaments; Cook WR et al.; The development of nascent cell division sites was studied in Escherichia coli strains containing ftsAts and ftsZts mutations in which septal development is arrested after shift to the restrictive temperature . Division sites were studied by measuring positions of plasmolysis bays, a visible marker for periseptal annuli . Annuli are circumferential zones of membrane adhesion which represent the earliest known structural differentiation at developing septal sites . Two patterns of annulus development were observed in the mutant strains . Normal numbers of new annuli were generated in filaments of both mutants, but in ftsZ filaments annuli failed to mature and to become properly localized, suggesting that ftsZ gene product is first required for maturation of annuli . In contrast, mature annuli accumulated at division sites in ftsA filaments, suggesting that the ftsA gene product is required at a stage after maturation and localization of nascent annuli.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 301 - 7
Localization of the membrane binding sites of Era in Escherichia coli; Gollop N et al.; Era is a GTP-binding protein that is essential for normal cell growth and division in Escherichia coli . In view of the fact that eukaryotic proteins similar to Era are membrane-associated and important in membrane signalling pathways, experiments were carried out to establish the intracellular location of Era . Immunoelectron microscopy was employed to demonstrate that Era resides at or very near the internal surface of the cytoplasmic membrane, which is a location expected for a membrane signalling protein . In addition, Era occurs in patches that may correspond to areas that are potential sites of septation.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 209 - 16
Unique sequence requirements for the P1 plasmid replication origin; Brendler TG et al.; We have carried out a detailed genetic analysis of the P1 plasmid replication origin and shown that it has four major structural requirements: the DnaA box, a series of five 7-base pair (bp) repeats, a GC-rich spacer and five 19-bp repeats that bind the P1 RepA protein . The origin requires the DnaA protein and its recognition sequence (the DnaA box) . However, although five boxes are present in two separate blocks in the wild type, just one, placed either to the left or right of the core origin sequences, is sufficient for function as long as the box conforms exactly to the proposed consensus . Each of the five 7-bp repeats that constitute the core of the origin is required; mutations within any of the first six base pairs can block origin function . The required bases include, but are not limited to, those constituting dam methylation sites . Also essential is a 39-bp GC-rich sequence . We show this to be a spacer of critical length that separates the 7-bp repeats from the last required region; a series of 19-bp repeats that bind the P1 RepA initiator protein.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 195 - 200
Characterization of Escherichia coli mutants with altered ploidy; Trun NJ et al.; We describe the isolation and characterization of new mutants in the cell cycle of Escherichia coli . The mutants were selected as gain of function mutants that are able to maintain more than the normal number of chromosomes . Our increased ploidy mutants were isolated as resistant to camphor vapours, which selects for cells with more chromosomes than normal . The mutants (called mbr for moth-ball-resistant) map to four chromosomal locations: mbrA at 68 min; mbrB at 88.5 min; mbrC at 89.5 min; and mbrD at 90 min . To investigate the nature of these cell cycle mutants, we have coupled them with defects in recA, to test for induction of the SOS response, and dam, to determine if methylation is required for mbr function . Based on the results of these and other tests, we have made a preliminary placement of the mbr mutants within the context of the cell cycle . mbrA mutations appear to be defective in the coupling of the DNA replication cycle to the cell division cycle, and as such, may define a new link between the two processes . mbrB does not seem to be able to coordinate the cell cycle and the growth rate of the cell . mbrC appears to be defective in partitioning of chromosomes . mbrD, which may be allelic to rpoB (a subunit of RNA polymerase), appears to be defective in either chromosomal partitioning or the later stages of DNA replication.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 177 - 80
In vivo effect of the tus mutation on cell division in an Escherichia coli strain where chromosome replication is under the control of plasmid R1; Dasgupta S et al.; The phenotypic effect of the tus::kan mutation in an Escherichia coli strain, where the chromosome is replicated unidirectionally by an integrated R1 miniplasmid, was examined by flow cytometry and phase fluorescence microscopy . The tus+ cells exhibited perturbed cell division, as indicated by the presence of many elongated cells and filaments . Inactivation of the tus gene led to a reduction in the frequency of such elongated cells, presumably by eliminating Tus-mediated polar arrests of replication forks at ter sites, thereby shortening the time required for completion of chromosome replication.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 161 - 7
The initiator titration model: computer simulation of chromosome and minichromosome control; Hansen FG et al.; The initiator titration model was formulated to explain the initiation control of the bacterial chromosome . In particular, features concerning the replication behaviour of minichromosomes, such as their high copy number and Escherichia coli's ability to coinitiate chromosome and many minichromosome origins, were considered during the formulation of the model . The model is based on the initiator protein DnaA and its binding sites, DnaA boxes, in oriC, in the dnaA promoter and at other positions on the chromosome . Another important factor in the model is the eclipse period created by the hemimethylation of a new oriC which makes it refractory to initiation . The model was analysed by computer simulations using a stochastic approach varying the different input parameters, and the resulting computer cells were compared with data on living E . coli cells . Here we present the outcome of a few of these simulations concerning the eclipse period, in silico-shift experiments blocking initiation or elongation of replication, and introduction of minichromosomes into the computer cells . We also discuss the synthesis of DnaA protein in the computer cells . From our simulations, we conclude that, whether true or not, the model can mimic the in vivo initiation control of E . coli.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 155 - 9
Only oriC and its flanking region are recovered from the complex formed at the time of initiation of chromosome replication in Escherichia coli; Kataoka T et al.; The oriC region of Escherichia coli constructs a specific complex to associate with the outer membrane fraction (oriC complex) . The oriC complex was periodically formed before as well as in the short period after initiation of DNA replication . Using the DNA extracted from outer membrane fractions of the cells just after initiation as a probe, the whole E . coli genomic library was assayed by plaque hybridization . DNA regions that hybridized with the probe corresponded to about a 100-kb length of chromosome DNA which included oriC . In addition, clones located in a counterclockwise direction from oriC were more preferentially hybridized among these positive clones . Thus, we conclude that the oriC region is a unique locus of the chromosome that binds to the outer membrane at the time of initiation of chromosome replication.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 141 - 9
Analysis of a copy number mutant of plasmid pSC101: co-maintenance of wild type and mutant plasmids; Goebel T et al.; We have isolated a high copy number mutant of plasmid pSC101 which is maintained at a level 4 times higher than that of the wild type . The mutation is a single base change that maps in codon 93 of the initiation protein RepA . We find that the mutation relaxes the autoregulation of the protein but increases its affinity for the repeated sequences in the origin . The wild type and the mutant repA genes are co-dominant and the mutated protein acts in trans even in the presence of the wild type protein . Co-maintenance of the two types of plasmids results in an intermediate copy number . Computer simulation indicates that simple models can explain the behaviour of the two plasmids.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 137 - 40
A calcium-binding protein that may be required for the initiation of chromosome replication in Escherichia coli; Guzman EC et al.; Starvation for isoleucine but not for other amino acids in an ilv- strain or the addition of valine in an ilv+ strain inhibits initiation of chromosome and minichromosome replication in stringent (Rel+) Escherichia coli, but it does not inhibit replication in relaxed (relA) mutants (Guzman et al, 1988) . From these results, we concluded that, (1) oriC initiation of replication is inhibited by ppGpp, and (2) isoleucine is not needed for the protein synthesis required at initiation . These results led us to find an isoleucine-free protein whose de novo synthesis is the sole protein synthesis requirement for oriC initiation . We also present evidence that this protein may be a calcium-binding protein located at 73 min in the genetic map.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 119 - 25
The complex of oriC DNA with the DnaA initiator protein; Messer W et al.; We describe several experimental approaches relating to the early steps in the initiation of DNA replication at oriC . 1) A matrix is given which enables calculatation of the relative affinity of DnaA boxes for DnaA protein; 2) base changes within single Dna A boxes in oriC have little effect on oriC function; 3) mutations which change the distance between DnaA boxes inactivate oriC, but changes by one helical turn (+ and -) result in near wild-type oriC activity; 4) a Fis binding site was located at oriC coordinates 206-220; 5) KMnO4 probing demonstrates Dna-A-dependent unwinding in the left part of oriC in vivo and in vitro.

Mol Microbiol, 1991 Feb, 5(2), 381 - 91
Genetic analysis of superoxide dismutase, the 23 kilodalton antigen of Mycobacterium tuberculosis; Zhang Y et al.; The gene encoding a 23 kilodalton protein antigen has been cloned from Mycobacterium tuberculosis by screening of a recombinant DNA library with monoclonal antibodies . The product of the gene has been identified as the superoxide dismutase (SOD) of M . tuberculosis on the basis of sequence comparison and by expression of the recombinant protein in a functionally active form . The derived amino acid sequence of M . tuberculosis SOD reveals a close similarity to manganese-containing SODs from other organisms, in spite of the fact that previous studies using the purified enzyme have identified iron as the preferred metal ion ligand . SOD is present in the extracellular fluid of logarithmic-phase cultures of M . tuberculosis, but the structural gene is not preceded by a signal peptide sequence . Insertion of the M . tuberculosis SOD gene into a novel shuttle vector demonstrated the mycobacteria but is ineffective in Escherichia coli.

J Microsc, 1991 Feb, 161 ( Pt 2), 183 - 203
The potential of cryofixation and freeze substitution: observations and theoretical considerations; Kellenberger E; The theoretical and experimental evidence in favour of cryofixation and freeze-substitution are critically reviewed . The solubility of macromolecules in water is due to the hydration shells . Their behaviour at different temperatures and the consequences of their removal during the processing for embedding are explained . Gelation prior to the transfer into solvents prevents macromolecules aggregating . During substitution at low temperatures, DNA is gelled, justifying the use of the term cryofixation . It is proposed that the preservation of hydration shells at the lowest temperature, and their transformation into minute gaps after a rise of temperature, facilitates the exhibition of epitopes.

AIDS, 1991 Feb, 5(2), 153 - 8
HIV-1 indicator cell lines; Akrigg A et al.; A simple quantitative bioassay for infectious HIV-1 has been developed . The assay is based on adherent CD4+ HeLa cell lines stably transfected with episomal vectors carrying the Escherichia coli beta-galactosidase gene under the control of the HIV long terminal repeat (LTR) promoter . HIV infection of these cell lines transactivates the LTR promoter inducing beta-galactosidase production . Infected cells and virus foci can be stained dark blue by the addition of the chromogenic substrate X-Gal . Alternatively, a readily automatable quantitative enzyme assay can be performed on the infected cultures . Because of its simplicity the bioassay may be useful for routine quantification of HIV-infected cultures, plaque purification, virus neutralization studies and for the screening of antiviral agents.

Mol Gen Genet, 1991 Feb, 225(2), 320 - 30
Identification and organization of carbon dioxide fixation genes in Xanthobacter flavus H4-14; Meijer WG et al.; The genes encoding the large (cfxL) and small (cfxS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisC/O) from Xanthobacter flavus H4-14 were identified and characterized . The RuBisC/O genes are separated by 11 bp and cotranscribed in Escherichia coli from the lac promoter in the order cfxLS . Primer extension and R-loop experiments with RNA isolated from autotrophically grown X . flavus H4-14 showed that transcription of cfxL and cfxS initiated 22 bp upstream from cfxL and resulted in a mRNA of at least 2.3 kb . DNA sequence analysis identified the start of an open reading frame transcribed divergently from cfxL, and displaying significant similarities with genes belonging to the lysR family of transcriptional activators . Downstream from cfxS an additional open reading frame was identified with unknown function . Expression studies showed that the genes encoding fructosebisphosphatase (cfxF) and phosphoribulokinase (cfxP) are located downstream from cfxLS . The cfxF and cfxP genes are cotranscribed in the same direction as cfxLS in the order cfxFP.

Biochem J, 1991 Feb 1, 273 ( Pt 3), 605 - 10
A proton-magnetic-resonance study of hydrogen-exchange reactions of yeast tryptophan synthase; Bailey CJ et al.; 1H n.m.r . was used to observe tryptophan formation from indole and L-serine, proton exchange at C-2 of L-tryptophan, and proton exchange at C-2 of L-serine, catalysed by yeast tryptophan synthase in the presence of 2H2O . Tryptophan synthesis took place with compulsory replacement of C-2 hydrogen by solvent hydrogen . The exponential decay rate (kobs) of the serine exchange reaction was insensitive to serine concentration in the range 2-20mM and was used to calculate kcat./Km values . However, kobs . was very sensitive to pH* values in the range 6.5-8.5 and the data require that the free enzyme is active in the base form resulting from two inseparable ionizations of pKa 7.3, and inactive after a third ionization controlled by a pKa of 7.5 . Initial rates measured by u.v . absorbance and colorimetric procedures were used to calculate kinetic parameters of the tryptophan synthesis reaction . From pH 6.5 to 7, kcat./Km values for L-serine in the tryptophan synthesis and hydrogen exchange reactions were indistinguishable and increased rapidly under the control of two acid-base groups of pKa 6.7 and 7.2 . Above pH 7, this equivalence breaks down because the exchange reaction alone is responsive to the third pKa value of the free enzyme . The pH dependence of the catalytic constant for tryptophan synthesis was qualitatively similar to that of the kobs . for serine exchange . A mechanism to explain the results is contrasted with recent proposals for the Escherichia coli system.

Mutat Res, 1991 Feb, 252(1), 51 - 60
Sources of variability of the Escherichia coli PQ37 genotoxicity assay (SOS chromotest); Mersch-Sundermann V et al.; To determine the variability in test results obtained with the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) when varying the test protocol, we examined the influences of sodium dodecylsulfate (SDS) concentrations, of buffer pH and composition on the enzyme assays, the effects of E . coli PQ37 density and culture conditions on the expression and/or determination of alkaline phosphatase (ap) and beta-galactosidase (beta-g) activities, the calculated induction factors (IF) and the SOS-inducing potentials (SOSIP) . Initially, we used 0-190 ng (0-1 nmole) 4-nitroquinoline-1-oxide (4-NQO) as a reference compound for the standard procedure in the absence of metabolic activation . Subsequently, to evaluate the results of protocol variations we examined several mutagenic compounds of differing chemical classes using both the standard and a modified assay procedure . We observed the highest enzyme activities using 1 mg SDS per tube and calibrating the ap buffer to pH 8.05 and the beta-g buffer to pH 7.75 . The longer the incubation period, the higher the enzyme activities . However, with respect to IF and SOSIP there is no reason to incubate in excess of 90 min . We found no significant differences in the IF and SOSIP values when varying substrate conversion times . There was, however, a definite decrease in beta-g activity when extended substrate incubation times were used . Higher enzyme activities are obtained when the bacterial count is increased . Using lower bacterial counts the enzyme activities decreased, but the sensitivity of E . coli towards genotoxic compounds increased.

Neuron, 1991 Feb, 6(2), 187 - 99
Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas; Zack DJ et al.; To define the cis-acting DNA elements required for rhodopsin expression, we generated lines of transgenic mice carrying sequences upstream of the bovine rhodopsin gene fused to the E . coli beta-galactosidase gene (lacZ) . Upstream sequences extending from -2174 to +70 bp, from -734 to +70 bp, and from -222 to +70 bp direct photoreceptor-specific expression . All three -2174 lines demonstrate a superior-temporal to inferior-nasal gradient of expression across the retina, whereas lines carrying the shorter constructs demonstrate either spatially continuous expression across the retina, discrete clusters of expression, or both . As a complementary approach to defining regulatory elements, we compared DNA sequences 5' of the murine, bovine, and human rhodopsin genes . Significant homology between all three species was found just upstream of the transcription start site and at approximately 1.5 kb upstream.

Arch Surg, 1991 Feb, 126(2), 231 - 5
Tumor necrosis factor alone does not explain the lethal effect of lipopolysaccharide; Sanchez-Cantu L et al.; Lethality and tumor necrosis factor production induced by different types of lipopolysaccharide were studied in naive (non-primed) rats during the late phase of endotoxin tolerance . The correlation with antilipopolysaccharide antibodies was also analyzed . No correlation was found between tumor necrosis factor levels and lipopolysaccharide-induced mortality in naive animals . Low-toxicity lipopolysaccharide preparations induced levels of tumor necrosis factor similar to those induced with more toxic types of lipopolysaccharide . Late tolerance was associated with progressively lower levels of lipopolysaccharide-induced tumor necrosis factor and increasing titers of antilipopolysaccharide antibodies after repeated injections of homologous lipopolysaccharide . During late endotonxin tolerance, a direct correlation between the lipopolysaccharide dose and peak tumor necrosis factor serum levels was found . We conclude that since tumor necrosis factor serum levels do not correlate with mortality, tumor necrosis factor alone cannot explain the lethal effect of lipopolysaccharide.

Arch Surg, 1991 Feb, 126(2), 211 - 8
Increased intestinal permeability in endotoxic pigs . Mesenteric hypoperfusion as an etiologic factor; Fink MP et al.; Infusing pigs with lipopolysaccharide (LPS) decreases superior mesenteric artery blood flow (Qsma), suggesting that mesenteric hypoperfusion may be responsible for LPS-induced alterations in gut mucosal permeability . To test this hypothesis, we studied four groups of anesthetized swine . Group 1 animals (N = 6) were infused with LPS (250 micrograms/kg over 1 hour beginning at 60 minutes) and continuously resuscitated with Ringer's lactate (48 mL/kg per hour) . In group 2 (N = 5), Qsma was decreased by 50% by means of a mechanical occluder to mimic the LPS-induced alterations in Qsma observed in group I . Group 3 (N = 5) was included to document our ability to detect ischemia/reperfusion-induced alterations in mucosal permeability; in these pigs, Qsma was decreased in steps to zero flow (at 150 to 210 minutes) and then perfusion was restored (at 210 to 270 minutes) . Pigs in group 4 (N = 6) served as normal controls; these animals were resuscitated with Ringer's lactate at the same rate as in group 1 but were not infused with LPS . To assess mucosal permeability, we measured plasma-to-lumen clearances for two markers, chromium 51-labeled edetic acid monohydrate (EDTA) and urea . Loading and maintenance infusions of the markers were given intravenously, and a 20-cm isolated segment of small intestine was continuously perfused at 2 mL/min with Ringer's lactate at 37 degrees C . Results were expressed as the ratio of the clearances for the two probes (CEDTA/CUREA) . In group 3, CEDTA/CUREA was 999% +/- 355% of baseline at 270 minutes . In group 1, CEDTA/CUREA was 572% +/- 235% of baseline at 270 minutes . In groups 2 and 4, however, CEDTA/CUREA did not change significantly from the baseline value over the duration of the study . These data suggest that increased mucosal permeability after LPS is due to factors other than (or in addition to) mesenteric hypoperfusion.

J Clin Invest, 1991 Feb, 87(2), 463 - 9
Enhanced production of neutrophil-activating peptide-1/interleukin-8 in rheumatoid arthritis; Seitz M et al.; Production of the neutrophil-activating peptide (NAP)-1/IL-8 by mononuclear phagocytes from patients with RA and from control subjects was studied under various conditions . Mononuclear cells from bone marrow (BMMC), PBMC, and synovial fluid (SFMC) were cultured for up to 48 h in the absence or presence of Escherichia coli LPS, different interleukins, interferon-gamma, zymosan, or immune complexes, and the neutrophil-stimulating activity released into the culture medium was determined . As shown by neutralization with an antiserum raised against human recombinant NAP-1/IL-8, over 90% of this activity could be attributed to NAP-1/IL-8 . In unstimulated mononuclear cells from control individuals and BMMC from RA patients, the production of NAP-1/IL-8 was very low and was enhanced moderately by stimulation with LPS . By contrast, the spontaneous production of NAP-1/IL-8 was 3- to 10-fold higher in PBMC and even much higher in SFMC from RA patients . In all instances, the yield of NAP-1/IL-8 could be enhanced by stimulation in culture . In addition to LPS, rheumatoid factor-containing immune complexes, zymosan, and IL-1 were highly effective in inducing NAP-1/IL-8 production, while IL-3, GM-CSF, tumor necrosis factor (TNF), and IL-2 were somewhat less potent . An inhibitory effect was obtained with IFN-gamma, which significantly decreased the spontaneous NAP-1/IL-8 release from SFMC and the IL-1- and LPS-induced NAP-1/IL-8 from RA and control PBMC . Inhibition was also observed with glucocorticoids . The production of NAP-1/IL-8 was markedly reduced by dexamethasone in phagocytosis-stimulated PBMC, and almost totally inhibited in SFMC obtained from joints after intraarticular administration of betamethasone . By contrast, the cyclooxygenase inhibitor, indomethacin, tended to increase the NAP-1/IL-8 yield from PBMC in culture.

J Bacteriol, 1991 Feb, 173(3), 1161 - 7
Effects of structural changes in the dsdA-dsdC intergenic region on D-serine deaminase synthesis; McFall E et al.; Single-base-pair changes well upstream of its transcription initiation site resulted in partially to fully constitutive expression of the D-serine deaminase structural gene, dsdA, independently of the cyclic AMP-cyclic AMP-binding protein complex and of the specific D-serine deaminase activator protein . These promoter mutations appear to define a consensus sequence that is repeated several times . Basal expression of dsdA+ was also strongly enhanced by subcloning on multicopy plasmids, by the DNA gyrase inhibitor novobiocin, and in dsdC(Con) mutants by increasing growth temperature . These results suggest that activation of dsdA+ expression by the dsdC-encoded protein involves distortion of promoter DNA . A dsdA translation start at bp -731 was verified by subcloning of dsdC+ . Plasmid-specified activator at a high concentration interfered with chromosomal dsdC(Con) expression, and the interference was enhanced by deletion of most of the intergenic region from the plasmid . Even at a high concentration, however, plasmid-specified activator did not activate expression of chromosomal dsdA+, and in one case it was actually repressive . These results confirm the strong cis tropism of plasmid-specified dsdC-encoded protein and suggest that it is mediated by multiple sites in the dsdA-dsdC intergenic region.

EMBO J, 1991 Feb, 10(2), 289 - 96
Regulatory region of human amyloid precursor protein (APP) gene promotes neuron-specific gene expression in the CNS of transgenic mice; Wirak DO et al.; The accumulation of beta-amyloid protein in specific brain regions is a central pathological feature of Alzheimer's disease (AD) . The 4 kd beta-amyloid protein derives from a larger amyloid precursor protein (APP) by as yet unknown mechanisms . In the absence of a laboratory animal model of AD, transgenic mice expressing various APP gene products may provide new insights into the relationship between APP and beta-amyloid formation and the pathogenesis of AD . beta-amyloid accumulation in AD brain may result from interactions between APP and other molecules . Such interactions are likely to be developmentally regulated and tissue-specific . A transgenic mouse model of AD, therefore, would aim for APP transgene expression that mimics the endogenous APP gene . As an initial step in developing an animal model, we have identified a 4.5 kb DNA fragment from the 5' end of the human APP gene, which mediates neuron-specific gene expression in the CNS of transgenic mice, using E . coli lacZ as a reporter gene . Detectable levels of transgene expression are found in most neurons but not in glial and vascular endothelial cells . The expression pattern of this reporter gene closely resembles the distribution of endogenous APP mRNA in both the human and mouse CNS.

Circulation, 1991 Feb, 83(2), 578 - 83
Introduction of vascular smooth muscle cells expressing recombinant genes in vivo; Plautz G et al.; Vascular smooth muscle cells contribute to the formation of atherosclerotic plaques by proliferating in response to vascular injury and releasing growth-promoting factors . Because their autocrine and paracrine effects are not fully understood, expression of such growth factor genes in specific cell types in vivo would help to determine their mechanism of action . We describe a method to transfer vascular smooth muscle cells expressing recombinant gene products to localized segments of the arterial wall . Vascular smooth muscle cells from the inbred Yucatan minipig were infected in vitro with an amphotropic, replication-defective retrovirus transducing the gene for Escherichia coli beta-galactosidase . Vascular smooth muscle cells expressing this recombinant gene were implanted, using a catheter, into denuded iliofemoral artery segments of pigs in vivo . These arteries subsequently demonstrated beta-galactosidase activity in cells of the intima and media . This method, which provides for the introduction of genetically modified smooth muscle cells, can be used to define the biological effects of recombinant genes in the vessel wall and potentially to provide alternative treatments of vascular diseases.

Arch Biochem Biophys, 1991 Feb 1, 284(2), 407 - 12
5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli--the substrate analogue bromopyruvate inactivates the enzyme by modifying Cys-408 and Lys-411; Huynh QK; In order to identify the essential reactive amino acid residues of 5-enolpyruvylshikimate-3-phosphate synthase, the reaction of the enzyme with its substrate analogue bromopyruvate was investigated . Incubation of the enzyme with bromopyruvate resulted in a time-dependent loss of enzyme activity . The inactivation followed pseudo-first-order and saturation kinetics with a Kinact of 28 microM and a maximum rate constant of 0.31 min-1 . The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvylshikimate 3-phosphate or by the combination of shikimate 3-phosphate plus glyphosate (N-phosphonomethylglycine), an inhibitor of the enzyme . Addition of sodium {3H}borohydride to the reaction mixture had no effect on the rate of inactivation but resulted in the incorporation of 3H label to the modified enzyme . Upon 90% inactivation, approximately 1 mol of bromo{14C}pyruvate was incorporated per mole of enzyme modified in the absence or presence of sodium borohydride . When the enzyme was incubated with bromopyruvate in the presence of sodium {3H}borohydride, approximately 1 mol of 3H label was found to be associated per mole of the modified enzyme . Tryptic digestion of these labeled proteins followed by reverse phase chromatographic separation resulted in the isolation of three radioactive peptides . Analyses of these three peptides indicated that bromopyruvate inactivated the enzyme by modifying Cys-408 and Lys-411, which are conserved in all enzyme sequences studied to date.

Biochimie, 1991 Feb-Mar, 73(2-3), 335 - 40
Cloning and preliminary characterization of srf-2020 and srf-801, the recF partial suppressor mutations which map in recA of Escherichia coli K-12; Wang TC et al.; We have located the single nucleotide changes suffered in recA sequence of 2 recF partial suppressor mutations: srf-2020 at codon 121 and srf-801 at codon 257 . srf-2020 changes codon 121 from threonine (ACC) to isoleucine (ATC) . srf-801 changes codon 257 from glutamine (CAG) to proline (CCG) . Consequently these mutations were renamed recA2020 and recA801 respectively . Preliminary characterization of recA2020 revealed that it is transdominant to recA+, like recA803, another recF partial suppressor . Interactions of recA2020 with recA803 were examined using genetic studies . Heterozygotes containing recA2020 and recA803 failed to produce a synergistic suppression effect in suppressing the recF deficiency . Presence of both recA2020 and recA803 mutations in the same recA gene also failed to produce any greater amount of UV resistance to a uvrA6recF143del(recA) strain indicating no interactions between these suppressors.

Biochimie, 1991 Feb-Mar, 73(2-3), 329 - 34
Positive and negative regulatory elements in the dnaA-dnaN-recF operon of Escherichia coli; Perez-Roger I et al.; The recF gene of E coli lies within a cluster of genes which play essential roles in DNA replication; the gene order is dnaA dnaN recF gyrB . Each of these genes has its own promoters which, with the exception of dnaA promoters, reside entirely within the translated region of the respective preceding gene . In this report, we analyze the effect of the dnaA and dnaN promoters on recF expression by translational fusions between recF and the lacZ reporter gene . Our results indicate that recF is a distal gene of the dnaA operon, and support the previous proposal that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters . They also suggest that dnaA, dnaN and recF are predominantly expressed from the same mRNA although transcriptional and/or post-transcriptional mechanisms should be specifically involved in lowering expression of the recF gene . Recently, we have localized 3 tandem transcription termination sites in the second half of the dnaN gene, downstream from the recF promoters . Neither of them shows the typical features of simple terminators and apparently they do not work in a minimal system of in vitro transcription . In this report, we present evidence that only one of them is dependent on the Rho protein . Although the operon structure allows coordinate expression of dnaA, dnaN and recF, the presence of internal promoters (the dnaN and recF promoters), which appear to be inducible by DNA damage, and intracistronic terminators, whose activity is inversely proportional to the efficiency of translation, permits expression of individual genes to be independently regulated in response to altered growth conditions.

Biochimie, 1991 Feb-Mar, 73(2-3), 313 - 20
Overlapping functions of recD, recJ and recN provide evidence of three epistatic groups of genes in Escherichia coli recombination and DNA repair; Lloyd RG et al.; The recD, recJ and recN genes of Escherichia coli K-12 have been shown to be involved in genetic recombination and DNA repair in this organism . Yet, mutation of any one of these genes does not seem to interfere much with the recovery of recombinants from conjugational crosses . Strains carrying all possible combinations of mutations inactivating these genes were constructed and examined for their recombination proficiency and sensitivity to UV light . The recD recJ and recJ recN double mutants are moderately sensitive to UV light and slightly deficient in recombination . A combination of mutations in all 3 genes produced strains that are very deficient in recombination (50- to 100-fold reduction) and strikingly sensitive to UV light . We conclude that these genes provide overlapping activities that compensate for one another in the single mutants . On the basis of these and other data, recombination genes are classified into 3 epistatic groups that define activities which function pre-synaptically or post-synaptically to promote genetic exchanges catalysed by RecA.

Biochimie, 1991 Feb-Mar, 73(2-3), 289 - 304
Biochemical and biological function of Escherichia coli RecA protein: behavior of mutant RecA proteins; Kowalczykowski SC; The recA protein of E coli participates in several diverse biological processes and promotes a variety of complex in vitro reactions . A careful comparison of the phenotypic behavior of E coli recA mutations to the biochemical properties of the corresponding mutant proteins reveals a close parallel both between recombination phenotype and DNA strand exchange and renaturation activities, and between inducible phenomena and repressor cleavage activity . The biochemical alterations manifest by the mutant recA proteins are reflected in the strength of their interaction with ssDNA . The defective mutant recA proteins fail to properly assume the high-affinity DNA-binding state that is characteristic of the wild-type protein and, consequently, form less stable complexes with DNA . The mutant proteins displaying an 'enhanced' activity bind ssDNA with approximately the same affinity as the wild-type protein but, due to altered protein-protein interactions, they associate more rapidly with ssDNA . These changes proportionately affect the ability of recA protein to compete with SSB protein, to interact with dsDNA, and, perhaps, to bind repressor proteins . In turn, the DNA strand exchange, DNA renaturation, and repressor cleavage activities mirror these modifications.

Biochimie, 1991 Feb-Mar, 73(2-3), 281 - 4
Site-directed mutagenesis in the Escherichia coli recA gene; Cazaux C et al.; Escherichia coli RecA protein plays a fundamental role in genetic recombination and in regulation and expression of the SOS response . We have constructed 6 mutants in the recA gene by site-directed mutagenesis, 5 of which were located in the vicinity of the recA430 mutation responsible for a coprotease deficient phenotype and one which was at the Tyr 264 site . We have analysed the capacity of these mutants to accomplish recombination and to express SOS functions . Our results suggest that the region including amino acid 204 and at least 7 amino acids downstream interacts not only with LexA protein but also with ATP . In addition, the mutation at Tyr 264 shows that this amino acid is essential for RecA activities in vivo, probably because of its involvement in an ATP binding site, as previously shown in vitro.

Biochimie, 1991 Feb-Mar, 73(2-3), 177 - 85
Head to head dimer model; an alternative model for the strand exchange reaction by RecA protein of Escherichia coli; Horii T; The RecA protein of E coli promotes a strand exchange reaction in vitro which appears to be similar to homologous genetic recombination in vivo . A model for the mechanism of strand transfer reaction by RecA protein has been proposed by Howard-Flanders et al based on the assumption that the RecA monomer has two distinctive DNA binding sites both of which can bind to ssDNA as well as dsDNA . Here, I propose an alternative model based on the assumption that RecA monomer has a single domain for binding to a polynucleotide chain with a unique polarity . In addition, the model is based on a few mechanical assumptions that, in the presence of ATP, two RecA molecules form a head to head dimer as the basic binding unit to DNA, and that the binding of RecA protein to a polynucleotide chain induces a structural change of RecA protein that causes a higher state of affinity for another RecA molecule that is expressed as cooperativy . The model explains many of the biochemical capabilities of RecA protein including the polar polymerization of RecA protein on single stranded DNA and polar strand transfer of DNA by the protein as well as the formation of a joint DNA molecule in a paranemic configuration . The model also presents the energetics in the strand transfer reaction.

Biochimie, 1991 Feb-Mar, 73(2-3), 143 - 55
Dynamic light scattering investigations of RecA self-assembly and interactions with single strand DNA; Benight AS et al.; Dynamic light scattering (DLS) measurements were performed on self-assembled solutions of RecA as a function of assembly time under strand exchange ionic strength conditions (10 mM MgCl2, 65 mM NaCl, 10 mM Tris-HCl, pH = 7.5, 1 mM DTT, 3-4 microM RecA) in the absence of ATP . These measurements yield distributions of the translational diffusion coefficients of the changing populations of assembling protein species . Interpretations of results of DLS measurements are made in terms of model hydrodynamic calculations that indicate, under the solution conditions employed, the smallest fundamental quaternary subunit of RecA is a hexamer in a toroidal or lock-washer configuration . Interactions of M13mp19 circular single strand DNA (ssDNA) with RecA assembled to different stages were also investigated . Additions of ssDNA to self-assembled solutions of RecA acts to dissociate the associated structures into hexamer subunits . However, the effect of ssDNA on assembled RecA is highly dependent on the RecA self-assembly state . The longer the assembly time, the less reversible the self-assembled structures of RecA become . Binding isotherms of titrated mixtures of ssDNA with RecA self-assembled to various stages were also determined . Evaluated dissociation constants of RecA/ssDNA complexes were found to increase with increases of the associated state of RecA . These results strongly suggest that, under the solvent conditions employed, the active ssDNA binding form of RecA is a hexamer.

Biochimie, 1991 Feb-Mar, 73(2-3), 133 - 41
RecA protein in the SOS response: milestones and mysteries; Witkin EM; The role of RecA protein in the SOS response of Escherichia coli is traced from the isolation of the first recA mutant to our current understanding of the scope and regulation of this DNA damage-inducible system . In addition, possible RecA protein activities that may be essential in the expression of several SOS phenotypes (stable DNA replication, DNA replication recovery, SOS mutagenesis and RecA association with the cell membrane) are discussed.

Mol Biol Rep, 1991 Feb, 15(1), 39 - 43
Isolation and partial characterization of a protein kinase NII from wheat germ chromatin; Angiolillo A et al.; A protein kinase, type NII, has been purified from wheat germ chromatin . The enzyme, which uses both ATP and GTP as phosphoryl donors, catalyzes the phosphorylation of casein, phosvitin and E . coli RNA polymerase, but not of histone proteins . Polypeptide bands at 46 kDa, 37 kDa and 25 kDa were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Autophosphorylation of the 25 kDa subunit was observed following incubation of the purified kinase with (gamma-32P)ATP and (gamma-32P)GTP.

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