Escherichia coli

EMBO J, 1986 Sep, 5(9), 2371 - 6
Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2; Jones IM et al.; In order to re-examine the sub-cellular location of the three influenza A/NT/60/68 polymerase proteins PB1, PB2 and PA in infected cells, specific antisera for each polymerase component have been prepared by immunizing rabbits with polymerase-beta-galactosidase fusion proteins synthesized in Escherichia coli . We show that polymerase PB1, PB2, and PA are predominantly associated with the nucleus of influenza-infected MDCK cells by immunocytochemical techniques . In the case of polymerase PB2 we investigate the possibility that nuclear accumulation is an intrinsic property of the PB2 protein . Using a vaccinia-PB2 recombinant virus, we show that PB2 accumulates intra-nuclearly in monkey CV-1 cells in the absence of any other influenza protein, suggesting it contains an intrinsic nuclear signal.

Bioorg Khim, 1986 Sep, 12(9), 1189 - 202
{Nucleotide sequence of the genome region of the tick-borne encephalitis virus coding for structural virion proteins}; Pletnev AG et al.; RNA of a flavivirus-tick-borne encephalitis virus (strain Sofjin) was subjected to reverse transcription and the DNA copy was transformed into double-stranded DNA by action of E . coli DNA-polymerase I (Klenow's fragment) . This DNA was annealed with pBR322 plasmid . The recombinant plasmids were cloned in E . coli K802 . The nucleotide sequence of the inserts of the clones coding for region of structural proteins C, pre-M, E and nonstructural protein ns1 was determined by the Maxam-Gilbert method . The nucleotide sequence of these regions is translatable into an amino acid sequence of proteins without interruption . The amino acid sequences of proteins and nucleotide sequence of genome of the tick-borne encephalitis virus are extensively homologous to that found for the flaviviruses Yellow Fever and West Nile.

Mol Gen Genet, 1986 Sep, 204(3), 435 - 42
Iron hydroxamate transport of Escherichia coli: nucleotide sequence of the fhuB gene and identification of the protein; Koster W et al.; The genes fhuA, fhuC and fhuD encode products involved in the transport of ferrichrome into cells of Escherichia coli . The nucleotide sequence of an additional locus contiguous to fhuD and formerly designated fhuB was determined . It contains an open reading frame for a polypeptide that consists of 659 amino acids . Expression of plasmids containing the fhuB region in an in vitro transcription/translation system and in E . coli minicells revealed a polypeptide with an electrophoretic mobility on SDS-polyacrylamide gels extremely dependent on the experimental conditions employed . This property could be explained by the very hydrophobic nature of the protein and it was concluded that the fhuB locus consists of a structural gene that encodes a membrane protein with a molecular weight of 82,181 . The fhuB gene is required for all iron transport systems which operate via hydroxamate compounds . The order of the genes in the fhu operon is fhuA fhuC fhuD fhuB, and transcription proceeds from fhuA to fhuB.

Mol Gen Genet, 1986 Sep, 204(3), 424 - 9
Reduced superhelicity of plasmid DNA produced by the rho-15 mutation in Escherichia coli; Fassler JS et al.; The plasmid pJSF6, a derivative of pBR327, could be maintained at 30 degrees C in strains of Escherichia coli containing the strong rho mutation, rho-15 . Plasmids extracted from rho-15 cells were always less negatively supercoiled than plasmids from rho+ cells . Transduction experiments designed to separate the rho gene from possible extragenic suppressors showed that the rho allele consistently determined the degree of plasmid superhelicity . Comparison of the superhelicity of plasmids extracted from the rho-15 and from a gyrB mutant showed that at 30 degrees C the negative supercoiling was reduced by the amounts delta Wrho = 4.0 +/- 0.3 and delta Wgyr = 6.0 +/- 0.3 turns; the effect of the rho-15 mutation on supercoiling was thus comparable to that of the gyrB mutation . A similar effect of the rho-15 mutation on the superhelicity of pBR329 was observed . The observation that the Rho protein has a role in determining DNA superhelicity (though not necessarily a direct role) provides a new point of view for studying the pleiotropic properties of rho mutants.

Mol Gen Genet, 1986 Sep, 204(3), 367 - 73
Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli; Yamagishi J et al.; DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants . The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis . Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid . This indicates tha mutations in the gyrB gene are responsible for nalidixic acid resistance.

Eur J Biochem, 1986 Sep 1, 159(2), 347 - 51
Cloning and sequencing of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough); Voordouw G et al.; The gene encoding the redox protein cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) has been cloned using two synthetic oligonucleotides (one 17-mer and one 18-mer), designed to recognize the structural gene . Plasmid pCYC3 was derived from the clone and contains a 7.5 X 10(3)-base EcoRI-HindIII insert of D . vulgaris DNA in pUC9 . A 674-base-pair fragment of this insert was sequenced with the dideoxy-chain-termination procedure and found to contain the entire structural gene encoding cytochrome c3 bracketed by apparent Escherichia coli consensus sequences for initiation and termination of transcription . The amino acid sequence of 107 residues, derived from protein sequencing {Trousil, E . B . and Campbell, L . L . (1974) J . Biol . Chem . 249, 386-393}, is confirmed by the nucleic acid sequence, which shows in addition that it is preceded by a hydrophobic, positively charged signal sequence of 21 residues . This amino-terminal extension functions in the export of cytochrome c3, which is thought to reside in the periplasm of D . vulgaris.

Eur J Biochem, 1986 Sep 1, 159(2), 263 - 6
Overproduction, isolation and determination of the amino-terminal sequence of the SecY protein, a membrane protein involved in protein export in Escherichia coli; Akiyama Y et al.; The gene product of secY (prlA) is an integral membrane protein with an essential role in protein export in Escherichia coli . When the protein was overproduced, using a plasmid, it was degraded rapidly in the cell . The lon or the htpR mutation did not slow down this degradation, but low-temperature growth conditions (30 degrees C) did so appreciably . On the other hand, the copy number of the pUC8-based plasmid was higher at higher temperatures . Thus, the plasmid was first amplified at 42 degrees C and the protein was then accumulated at 30 degrees C . The SecY protein was isolated in sodium dodecyl sulfate (SDS)-denatured form from the membranes of the overproducing cells, using SDS-SDS two-dimensional gel electrophoresis . Its NH2-terminal sequence confirmed the secY reading frame and the translation initiation site assigned previously . The SecY protein does not undergo NH2-terminal processing except for the removal of the initiator methionine.

Ann Surg, 1986 Sep, 204(3), 282 - 99
A systematic study of host defense processes in badly injured patients; Polk HC Jr et al.; A prospective study of factors predisposing to infection in badly injured patients has disclosed: the dominant roles of two specific parameters: monocyte antigen presenting capacity, and opsonic capacity of diluted serum; the potential value of further assessment of: the predictive value of plots of activated T-cells/total T-cells versus monocyte antigen presenting capacity, the apparent protective effect of the ability to sharply increase specific IgM in response to infection, and the apparent protective effect of cytomegalovirus (CMV) infection in the first 28 days after injury against major bacterial infection; the lack of value of analysis of other T- and B-cell subsets in such patients; and the need to clarify CMV and transfusion status with respect to interpretation of such data . The specific role of variable transfusion and of specific serum immunoglobulins will require further and more discriminating study.

Proc Natl Acad Sci U S A, 1986 Sep, 83(18), 6930 - 4
Analysis of mutations in the transmembrane region of the aspartate chemoreceptor in Escherichia coli; Oosawa K et al.; Site-specific mutagenesis was used to replace an alanine with a lysine residue and to create a deletion of seven amino acids into the first transmembrane region (TMI region) of the aspartate chemoreceptor in Escherichia coli . The mutations resulted in the loss of aspartate chemotaxis on tryptone motility plates . However, both mutant proteins were able to associate with the membrane and to bind aspartate . They were both refractory to methylation or to modification of the C-terminal region of the protein by the cheB gene product . These results suggested that the integrity of the TMI domain of the protein was required to maintain the function of the cytoplasmic portion of the receptor . The Lys-19 mutant retained the ability to generate a repellent response . Analysis of suppressor mutations of the Lys-19 mutation suggested that formation of an ion pair or specific changes in a 40 amino acid stretch in the cytoplasmic region of the protein (from amino acid 264 to amino acid 303) could suppress the effects of the Lys-19 mutation . The TMI region of the protein may be involved in transmembrane transmission of signals from the periplasmic portion of the cell to the cytoplasmic portion of the Tar protein.

Proc Natl Acad Sci U S A, 1986 Sep, 83(18), 6672 - 6
Synthesis of the complete trans-activation gene product of human T-lymphotropic virus type III in Escherichia coli: demonstration of immunogenicity in vivo and expression in vitro; Aldovini A et al.; Human T-lymphotropic virus type III (HTLV-III) contains a gene (tat-III) the product of which activates the expression of viral genes in trans . We have expressed in Escherichia coli the complete tat-III-encoded protein as well as a truncated form that lacks three amino acids from the amino terminus . These proteins are recognized by sera of many, but not all, infected individuals including patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex, as well as asymptomatic seropositive persons . Seropositivity for the tat-III protein does not appear to correlate with the clinical stage of HTLV-III-related disease . Antibodies raised in rabbits against the E . coli-produced protein detect the native protein (apparent molecular mass, 14.5 kDa) in a virus-producing cell line . A second protein (26 kDa), of unknown origin but viral related, is also specifically recognized by the immune serum.

J Med Microbiol, 1986 Sep, 22(2), 125 - 31
Distribution and genetic location of Tn7 in trimethoprim-resistant Escherichia coli; Kraft CA et al.; A series of 178 strains of Escherichia coli, highly resistant to trimethoprim, isolated from hospital patients and patients in the community between 1979 and 1983, was examined for the presence of Tn7 on a plasmid or on the chromosome only, by transposition to RP4 and restriction endonuclease digestion with Hind III . Of the isolates, 57% carried Tn7 . Comparison of hospital isolates from 1979 to 1980 and 1982 showed that although the proportions that carried Tn7 were similar (63% and 57%) there had been a significant change in the genetic location of the transposon . The proportion of plasmid-mediated Tn7 had fallen from 62% to 30% with a corresponding rise in Tn7 located exclusively on the chromosome from 38% to 70% . This change may be the result of continuing transposition of Tn7 from plasmids to the bacterial chromosome followed by plasmid loss . The consequent reduction in the mobility of trimethoprim-resistance genes may in turn lead to changes in the incidence of resistance.

Int J Lepr Other Mycobact Dis, 1986 Sep, 54(3), 416 - 22
Construction of genomic libraries of mycobacterial origin: identification of recombinants encoding mycobacterial-specific proteins; Khandekar PS et al.; A complete genomic library from Mycobacterium vaccae (2785 recombinants) and a partial genomic library of M . leprae and BCG (300 and 1750 clones, respectively) were constructed in the plasmid pBR322 . Bam HI was selected as the restriction endonuclease for obtaining DNA cleavage products . Evidence was obtained for limited expression of the cloned mycobacterial DNA inserts in Escherichia coli . A recombinant has been identified which codes for antigen immunoreactive with rabbit anti-M . leprae antibody but not with anti-H37Rv antibody.

J Bacteriol, 1986 Sep, 167(3), 992 - 8
Characterization of Escherichia coli chemotaxis receptor mutants with null phenotypes; Mutoh N et al.; Hydroxylamine mutagenesis was used to alter the tar gene that encodes the transmembrane Tar protein required for chemotaxis . Mutants defective in chemotaxis were selected, and the mutation was characterized by DNA sequencing . Two classes of mutations were found: nonsense and missense . The nonsense mutations were distributed throughout the gene, while the missense mutations were found to cluster in a region that includes 185 amino acids at the C-terminal end of the Tar protein . Partial characterization of mutant phenotypes suggested that some are completely defective in signaling while responding to attractants and repellents by differential methylation . Other mutants are undermethylated and constantly tumble, while yet another class of mutants is overmethylated and biased toward constant swimming with little or no tumbling . These mutants will be useful in experiments designed to understand the mechanism of chemotaxis.

J Bacteriol, 1986 Sep, 167(3), 1004 - 8
Interaction between membrane proteins PBP3 and rodA is required for normal cell shape and division in Escherichia coli; Begg KJ et al.; In Escherichia coli, the products of several genes are required for septation, and the products of several others are required for the maintenance of the rod shape of the cells . We show here that the combination of certain mutations in a division gene (ftsI) with a specific mutation in one of the shape genes (rodA) could produce cells with normal shape and division, although separately these mutations led to a loss of the capacity to divide (ftsI) or to form normal rod-shaped cells (rodA) . In contrast, combinations between other mutant alleles of these genes produced double mutants which had lost the capacity both to divide and to form rod-shaped cells . The mutual phenotypic correction observed within particular pairs of mutant genes suggests that the normal morphogenetic cycle of growth and division may require direct interaction between the two membrane proteins which are the products of these genes.

Infect Immun, 1986 Sep, 53(3), 464 - 73
Purification and characterization of type II heat-labile enterotoxin of Escherichia coli; Holmes RK et al.; Type II heat-labile enterotoxin (LT-II) of Escherichia coli has several biologic activities similar to cholera toxin (CT) and E . coli type I heat-labile enterotoxin (LT-I), but it is not neutralized by antiserum prepared against CT or LT-I . LT-II was purified from E . coli SA53 and from E . coli HB101(pCP3837), a strain that contains the cloned LT-II genes in a hybrid plasmid and produces up to 600 times more LT-II than does SA53 . Purification involved sonic disruption of bacterial cells, ammonium sulfate fractionation, chromatography on Affi-Gel Blue, chromatofocusing, and gel filtration on Sephadex G-100 . The LT-II purified to apparent homogeneity from HB101(pCP3837) had an isoelectric point of 6.8, induced increased vascular permeability in rabbit intracutaneous tests, caused rounding of cultured Y1 adrenal cells accompanied by increased intracellular cyclic AMP, and was 25 to 50 times more potent than CT or LT-I in the Y1 adrenal-cell assay . In contrast, purified LT-II did not cause secretion in ligated rabbit ileal segments at doses corresponding to CT controls that gave strongly positive reactions . LT-II was composed of two different polypeptides with MrS of 28,000 (A) and 11,800 (B); treatment of LT-II with trypsin cleaved the A polypeptide to fragments A1 (Mr, 21,000) and A2 (Mr, 7,000) . The activity of LT-II was not blocked by ganglioside GM1 at concentrations that inactivated LT-I or CT . Antiserum against the LT-II from E . coli HB101(pCP3837) completely neutralized purified LT-II and the LT-II in crude extracts of SA53, but it did not neutralize purified LT-I or CT.

J Urol, 1986 Sep, 136(3), 715 - 8
Polyglycolic acid mesh in experimental renal trauma; Lau JL et al.; We investigated the efficacy of kidney wrapping with polyglycolic acid (PGA) mesh for control of hemorrhage and preservation of renal function following extensive potentially lethal kidney lacerations in the dog . Wrapping of lacerated kidneys resulted in reapposition of the renal parenchyma and prompt, sustained hemostasis . At 21 days following injury the renal lacerations were well healed . Among five dogs with lacerations involving the entire surface of one kidney, the mean of the ratios of the creatinine clearance of the affected kidney divided by the creatinine clearance of the uninjured contralateral kidney was 0.83 +/- 0.14 . Among ten dogs with lacerations confined to the lower pole of one kidney, five were treated by mesh wrapping and five by partial nephrectomy . The mean of the ratios of the creatinine clearance of the affected kidney divided by the creatinine clearance of the uninjured contralateral kidney was 0.93 +/- 0.17 for the former group and 0.58 +/- 0.06 for the latter group . Perirenal infection following kidney wrapping developed in only one dog who had an E . coli bacteriuria at the time of injury . Blood pressure was monitored in eight dogs treated with mesh wrapping . None became hypertensive . These data suggest that PGA mesh may have clinical utility in the management of selected renal injuries in humans.

J Virol, 1986 Sep, 59(3), 635 - 45
Pseudorabies virus gene encoding glycoprotein gIII is not essential for growth in tissue culture; Robbins AK et al.; We have established that in the Becker strain of pseudorabies virus (PRV), the glycoprotein gIII gene is not essential for growth in cell culture . This was accomplished by construction and analysis of viral mutants containing two defined deletion mutations affecting the gIII gene . These mutations were first constructed in vitro and introduced into Escherichia coli expression plasmids to verify structure and protein production . Each mutation was then crossed onto PRV by cotransfection of plasmid DNA and parental viral DNA by using gIII-specific monoclonal antibodies as selective and screening reagents . One resultant virus strain, PRV-2, contained an in-frame deletion of a 402-base-pair (bp) SacI fragment contained within the gIII gene . Another virus strain, PRV-10, contained a deletion of a 1,480-bp XhoI fragment removing 230 bp of the upstream, putative transcriptional control sequences and 87% of the gIII coding sequence . The deletion mutants were compared with parental virus by analysis of virion DNA, gIII specific RNA, and proteins reacting with gIII specific antibodies . Upon infection of PK15 cells, the deletion mutants did not produce any proteins that reacted with two gIII specific monoclonal antibodies . However, two species of truncated glycosylated proteins were observed in PRV-2 infected cells that reacted with antiserum raised against bacterially produced gIII protein . PRV-10 produced no detectable gIII-specific RNA or protein . PRV-10 could be propagated without difficulty in tissue culture . Virus particles lacking gIII were indistinguishable from parental PRV virus particles by analysis of infected-cell thin sections in the electron microscope . We therefore conclude that expression of the gIII gene was not absolutely essential for PRV growth in tissue culture.

Plasmid, 1986 Sep, 16(2), 90 - 100
An F-derived conjugative cosmid: analysis of tra polypeptides in cosmid-infected cells; Ray A et al.; The genes involved in the conjugational transfer of F plasmid DNA are organized into three closely linked operons spanning an overall length of approximately 33 kilobase pairs of F . The entire transfer (tra) region comprising all three operons has been cloned into the cosmid vector pHC79 by in vitro recombination and packaging techniques . The transfer-proficient chimeric cosmid pRS2405 was packaged into lambda capsids, and uv-irradiated E . coli cells were infected with these DNA-filled particles . A number of polypeptides programmed by the infecting DNA were identified as tra-specified products; a traJ90 mutation on pRS2405 resulted in the significant reduction of synthesis of all detectable pRS2405-specified tra polypeptides, with the exception of TraTp.

J Gen Microbiol, 1986 Sep, 132 ( Pt 9), 2497 - 503
Effects of immune colostrum on the expression of a K88 plasmid encoded determinant: role of plasmid stability and influence of phenotypic expression of K88 fimbriae; Nagy LK et al.; Passaging of the K88-positive Escherichia coli strain CN6913 through synthetic medium containing immune colostrum gave rise to large numbers of K88-negative CN6913 variants . These K88-negative variants had all lost a single large plasmid known to encode the K88 genetic determinant . Four other large plasmids harboured by this strain were unaffected . Viable K88-positive and K88-negative variants of CN6913 accumulated at a similar rate in synthetic medium and in medium containing non-immune colostrum . In the presence of immune colostrum, viable cells of the K88-negative variant accumulated faster and to a greater extent in cultures than the K88-positive variant if incubated at 37 degrees C, which favours the phenotypic expression of K88 . However, when similar cultures were incubated at 18 degrees C, a temperature known to inhibit phenotypic expression of K88, the accumulation of viable cells of the two variants was strictly comparable in all media and no loss of plasmid or increase in K88-negative variants was observed . Cells containing a pBR322-based K88-encoding recombinant plasmid were also eliminated by immune colostrum whereas cells containing pBR322 were not . Plasmids encoding the K99 antigen were not readily eliminated from strains passaged through medium containing immune colostrum . K99-negative variants that were detected still harboured the K99-encoding plasmid.

Vet Microbiol, 1986 Sep, 12(3), 221 - 8
Isolation of piliated Escherichia coli from diarrheic foals; Ward AC et al.; Escherichia coli was isolated from the feces and intestines of foals with and without diarrhea . Piliation of isolates was demonstrated by electron microscopy and agglutination in antisera having specificity for K88, K99, P987 and F41 pili . Piliation was also demonstrated by electron microscopy on organisms which did not react with any of the antisera.

Acta Physiol Scand, 1986 Sep, 128(1), 57 - 63
The net fluid secretion caused by cyclic 3'5'-guanosine monophosphate in the rat jejunum in vivo is mediated by a local nervous reflex; Eklund S et al.; The tissue concentration of cyclic 3'5'-guanosine monophosphate (cGMP) has been shown to increase in the small intestine when net fluid secretion is evoked by the heat-stable enterotoxine of Escherichia coli . Lipophilic cGMP analogues are also known to elicit intestinal fluid secretion . It is therefore believed that an increase in intracellular cGMP concentration in enterocytes mediates this secretion . The present study reports that the fluid secretion, elicited by placing two different cGMP analogues, dibutyryl-cGMP and 8-Br-cGMP, in the intestinal lumen of anaesthetized rats in vivo, is significantly inhibited by atropine, hexamethonium and lidocaine . It is proposed that cGMP activates a reflex in the enteric nervous system which, in part, explains the observed fluid secretion.

J Bacteriol, 1986 Sep, 167(3), 968 - 74
Helical structure of Bordetella pertussis fimbriae; Steven AC et al.; The helical structures of Bordetella pertussis fimbriae of serotypes 2 and 6 were determined by optical diffraction analysis of electron micrographs of negatively stained paracrystalline bundles of purified fimbriae . The fimbrial structure is based on an axial repeat of 13 nm that contains five repeating units in two complete turns of a single-start helix . This structure was confirmed by direct measurements of mass per unit length for individual fimbriae performed by dark-field scanning transmission electron microscopy of unstained specimens . These data further established that the helically repeating unit is a monomer of fimbrial protein (Mr congruent to 22,000 for type 2 and Mr congruent to 21,500 for type 6) . Radial density profiles calculated from the scanning transmission electron micrographs showed that the fimbria has peak density at its center, i.e., no axial channel, consistent with the results of conventional negative-staining electron microscopy . The radial profile gives an outermost diameter of approximately 7.5 nm, although the peripheral density is, on average, diffuse, allowing sufficient intercalation between adjacent fimbriae to give a center-to-center spacing of approximately 5.5 nm in the paracrystals . Despite serological and biochemical differences between type 2 and type 6 fimbriae, the packing arrangements of their fimbrial subunits are identical . From this observation, we infer that the respective subunits may have in common conserved regions whose packing dictates the helical geometry of the fimbria . It is plausible that a similar mechanism may underlie the phenomenon of phase variations in other systems of bacterial fimbriae.

J Bacteriol, 1986 Sep, 167(3), 799 - 804
Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88; Moseley SL et al.; The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning . The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon . Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88 . Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E . coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain . This observation was extended to other F41-producing animal isolates . A large number of animal E . coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production . All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes . Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production . In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal . The K88 antigen-producing strains showed no homology with the K99 probe . A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens . This suggests that there are adhesins among animal isolates of E . coli which are genetically related to but antigenically distinct from K88 and F41.

J Bacteriol, 1986 Sep, 167(3), 759 - 65
Transglycosylase and endopeptidase participate in the degradation of murein during autolysis of Escherichia coli; Kitano K et al.; The cell wall degradation products released from Escherichia coli during autolysis triggered by cephaloridine or trichloroacetic acid were isolated and characterized . Murein was selectively lost from the disaccharide tetrapeptides and the bisdisaccharide tetrapeptide components . Two major autolytic products accounted for more than 85% of the released material . Compound 1 (60 to 80% of released material) was a disaccharide tetrapeptide monomer containing a 1,6-anhydromuramic acid residue . Compound 2 (15 to 30% of released material) was a mixture of a tritripeptide and a tritetrapeptide without hexosamines . Taken together the findings suggest that autolytic cell wall degradation in E . coli is selective and involves the activity of both the hydrolytic transglycosylase and an endopeptidase . Upon release, at least some of the wall components were also exposed to the activity of the N-acetylmuramic acid-L-alanine amidase.

Infect Immun, 1986 Sep, 53(3), 693 - 6
Receptor-binding function of type 1 pili effects bladder colonization by a clinical isolate of Escherichia coli; Keith BR et al.; The role of type 1 pili in promoting bladder colonization was examined by constructing two mutant strains of a clinical Escherichia coli isolate . One mutant was isogenic to the parental strain save for a lesion in a gene required for pilus receptor binding; the other mutant was isogenic save for a lesion in the gene encoding the pilus structural subunit . Using mixed infections of the parental and mutant strains in an ascending rat cystitis model, we found that type 1-piliated mutants that lacked the receptor-binding function were as ineffective in bladder colonization as were mutants lacking the entire organelle.

J Gen Microbiol, 1986 Sep, 132 ( Pt 9), 2571 - 6
Effect of DNA gyrase inhibitors and urea on the expression of cysB, the regulatory gene of the cysteine regulon; Bielinska A et al.; cysB, the regulatory gene of the cysteine regulon, is autoregulated . Inhibitors of both gyrase subunits, nalidixic acid and novobiocin, affect the expression of cysB, as monitored by beta-galactosidase activity in cysB::lac fusion strains . In gyrA mutants that are resistant to nalidixic acid, this drug does not affect cysB expression . The amount of mRNA transcribed from the cysB promoter isolated from cultures grown in the presence of gyrase inhibitors was significantly lower than that from the control culture without inhibitors . Urea also decreased cysB expression . These results suggest that DNA topology could play a role in cysB expression.

Mol Immunol, 1986 Sep, 23(9), 991 - 7
Characterization of monoclonal antibodies specific for the pre-S2 region of the hepatitis B virus envelope protein; Neurath AR et al.; Monoclonal antibodies (McAb) specific for the pre-S region of the hepatitis B virus (HBV) envelope protein were prepared using HBV particles of hepatitis B surface antigens (HBsAg) as immunogens . The antibodies reacted in Western blot analyses and in ELISA with pre-S2 sequences of the HBV envelope protein . Pepsin or protease V8 treatment of the antigen abolished reactivity . The fine specificity of one of the McAb (F376) was established by immunoassays using synthetic peptides and a pre-S2-beta-galactosidase fusion protein expressed in E . coli . The shortest peptide recognized by F376 is demarcated by residues pre-S(132) at the N-terminal and pre-S(140)-pre-S(145) at the C-terminal . The corresponding amino acid sequence (for HBV subtype adw2) is: QDPRVRGLY(LPAGG) . Additional amino acid residues at the N-terminal, and possibly at the C-terminal ends contribute to the binding of McAb, probably due to conformational influences . The McAb was applied to immunoassays of pre-S2 sequences in purified HBsAg and in human sera containing HBsAg.

Mol Biol (Mosk), 1986 Sep-Oct, 20(5), 1337 - 43
{Transcription of ribosomal protein genes rplKAJL and RNA-polymerase genes rpoBC in Escherichia coli cells: metabolic regulation of attenuation and the effect of rifampicin}; Kliachko EV et al.; The E . coli genes rplKAJL specifying ribosomal proteins L11, L1, L10, L7/L12 are co-transcribed with the genes rpoBC encoding the beta- and beta'-subunits of RNA polymerase, but are separated by the site of attenuation . The efficiency of attenuation within rplKAJL-rpoBC operon was determined as a ratio of rplKAJL transcription frequency to the same of rpoBC genes . The efficiency of attenuation was found to be a growth-rate dependent parameter of E . coli cells . At growth rate 1.2 doublings per hour the attenuation is rare and simultaneously increases with the increase in the growth rate (at mu = 1.2 doublings per hour the efficiency of attenuation is 4) . Rifampicin (10-30 micrograms/ml) inhibits the transcription of both rplKAJL and rpoBC genes in fast growing cells but paradoxically stimulates their transcription in slowly growing cells . The stimulatory effect of rifampicin on rplKAJL genes transcription is supposed to be based on its ability to repress the ppGpp synthesis . The possible role of ppGpp in the regulation of transcription attenuation in rplKAJL-rpoBC operon is discussed.

Biokhimiia, 1986 Sep, 51(9), 1541 - 8
{Threonyl-tRNA synthase from rabbit reticulocytes: a reversible transition from the RNA-non-binding to the RNA-binding form}; Fedorov AN et al.; A simple three-step procedure was used to isolate threonyl-tRNA synthetase of rabbit reticulocytes which is in a ribosome-free extract in the RNA-non-binding form . According to SDS electrophoresis, the enzyme has a molecular weight of 86 000 Da and is heterogeneous by isoelectric point; pI of the major component is near 6.2 . Threonyl-tRNA synthetase is capable of interacting with a high molecular weight RNA (E . coli rRNA) . Thus, in the course of purification threonyl-tRNA synthetase passes from the RNA-non-binding to the RNA-binding form . This transition was shown to be reversible.

Mutat Res, 1986 Sep, 166(2), 123 - 34
Characterization of Escherichia coli mutant strains deficient in AP DNA-repair synthesis; Weinberger S et al.; Deficiency of apurinic/apyrimidinic (AP) DNA-repair enzymes in crude extracts of E . coli mutants was determined by following general and specific AP DNA-repair synthesis via nick translation in the presence of either all four dNTPs, or only one dNTP . We have shown that mutations either in DNA polymerase I or in AP endonucleases or in both, inhibit to different degrees the ability to repair AP DNA . The polA mutation totally abolishes the ability to perform both general and specific AP DNA repair, while the polAex mutation affects only general AP DNA repair . The xthA tight mutants, including the deletion mutant BW9101, can cope with small amounts of AP sites but hardly with high amounts of these lesions . In addition we have found that crude extracts of the xthA mutants degrade AP DNA by two modes: a nonspecific, and an AP-specific mode . These phenomena are common to all xth mutants and enabled us to discover this mutation . In contrast to the xth mutants so far isolated, BW2001 exhibits marked sensitivity to MMS and to X-ray irradiation . We found that this strain has a proficient DNA polymerase I but is absolutely deficient in AP endonucleases . We attribute its sensitivities to a secondary mutation at the structural gene of endonuclease IV.

Proc Natl Acad Sci U S A, 1986 Sep, 83(17), 6538 - 42
Base substitutions in the tRNA anticodon arm do not degrade the accuracy of reading frame maintenance; Curran JF et al.; We have examined the activities of a set of 34 site-directed mutants of tRNA Su7 for their ability to shift reading frame during translation of amber codons in vivo . The set includes variants at every position in the distal three base pairs of the anticodon stem and saturates the anticodon loop, with the exception of the anticodon itself . Most anticodon-stem mutations were made pairwise to preserve the secondary structure of that region . Variants of the Hirsh (A24) coding alteration were also tested . The mutations have varied and often dramatic effects on the ability of Su7 to act in translation, which indicates that they cause distortions of the codon-anticodon complex . However, none of the tested mutations affects the intrinsic accuracy of translocation, which we show to be very high . These results suggest that translocation must be independent of the conformational detail of the codon-anticodon complex and stand in contrast to frameshifts that occur when tRNAs misread codons . We suggest that when the tRNA is properly paired to the codon, translocation proceeds normally . Thus, we conclude that selection of a cognate tRNA ensures highly accurate reading frame maintenance . As a corollary, inefficient amber suppressors are not inefficient because they frameshift . Instead, they are likely to fail because a release factor translates the amber codon.

J Bacteriol, 1986 Sep, 167(3), 1095 - 7
Transcription of ribosomal genes during a nutritional shift-up of Escherichia coli; Zengel JM et al.; We measured the differential transcription rates of individual ribosomal operons after a nutritional shift-up . All operons showed a transient increase in transcription . However, the response of the S10 ribosomal protein operon was much stronger than that of any other operon . We propose that only the S10 operon is autogenously regulated by a transcription attenuation mechanism.

J Virol, 1986 Sep, 59(3), 743 - 5
Expression of reverse transcriptase activity of human T-lymphotropic virus type III (HTLV-III/LAV) in Escherichia coli; Tanese N et al.; The pol gene from a biologically active clone of the human T-cell lymphotropic virus type III provirus was inserted into a bacterial expression vector . The resulting gene fusion induced the formation of active reverse transcriptase that could be readily detected in extracts of bacterial cells . The activity exhibited the template and divalent cation requirements of the authentic enzyme . These constructs will be useful for safe and rapid analysis of potential inhibitors of this important enzyme.

Biochem Biophys Res Commun, 1986 Aug 29, 139(1), 275 - 80
A human auto-immune antibody specifically recognizing initiator methionine tRNA from yeast and higher eucaryotes; Sri-Widada J et al.; Analysis of sera from 168 patients with autoimmune disorders revealed that one patient with Sjogren's syndrome produced antibodies against deproteinized initiator methionine tRNA in addition to those against La protein . This anti-tRNAimet recognizes also tRNAimet from yeast but not from Phaseolus vulgaris chloroplasts (bean) or E . coli . It appears therefore that the epitope could be located in the TF loop in which an A residue in position 60 and the AUCG sequence are the only common features in yeast and human tRNAimet.

Biochemistry, 1986 Aug 26, 25(17), 4784 - 90
Leaving group dependence in the phosphorylation of Escherichia coli alkaline phosphatase by monophosphate esters; Hall AD et al.; Values of kcat and Km have been measured for the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 18 aryl and 12 alkyl monophosphate esters at pH 8.00 and 25 degrees C . A Bronsted plot of log (kcat/Km) (M-1 s-1) vs . the pK of the leaving hydroxyl group exhibits two regression lines: log (kcat/Km) = -0.19 (+/- 0.02) pKArOH + 8.14 (+/- 0.15) log (kcat/Km) = -0.19 (+/- 0.01) pKROH + 5.89 (+/- 0.17) Alkyl phosphates with aryl or large lipophilic side chains are not correlated by the above equations and occupy positions intermediate between the two lines . The observed change in effective charge on the leaving oxygen of the ester (-0.2) is very small, consistent with substantial electrophilic participation of the enzyme with this atom . Cyclohexylammonium ion is a noncompetitive inhibitor against 4-nitrophenyl phosphate substrate at pH 8.00, and neutral phenol is a competitive inhibitor (Ki = 82.6 mM); these data and the 100-fold larger reactivity of aryl over alkyl esters are consistent with the existence of a lipophilic binding site for the leaving group of the substrate . The absence of a major steric effect in kcat/Km for substituted aryl esters confirms that the leaving group in the enzyme--substrate complex points away from the surface of the enzyme . Arguments are advanced to exclude a dissociative mechanism (involving a metaphosphate ion) for the enzyme-catalyzed substitution at phosphorus.

Nucleic Acids Res, 1986 Aug 26, 14(16), 6735 - 43
C4-methyldeoxythymidine replacing deoxythymidine in poly{d(A-T)} renders the polymer resistant to the 3'----5' exonuclease activity of the Klenow and T4 DNA polymerases; Singer B; We previously reported that O4-alkyl dTTPs could replace, for short times, dTTP in polymer synthesis {Singer et al., PNAS 83, 26-32, 1986} . The reasons for such early termination of synthesis could be either proofreading or the eventual formation of weakly paired primer termini . Utilizing the known 3'----5' exonucleolytic activity of polymerases, in the absence of dNTPs, enabled us to conclude that, in contrast to the digestibility of poly{d(A-T)} which yielded the expected 3'-mononucleotides, the polymerizing enzymes did not digest O4-methyl dT or its neighbors . The presence of the resistant alpha-phosphorothionate linkage did not prevent measurable digestion of poly{d(A-T)} by the Klenow fragment . This, together with evidence that polymerization of O4-methyl dTTP is favored at low temperatures, supports the model proposed by Ollis et al . {Nature 313, 762-766, 1985} showing independent domains for the two activities in the Klenow fragment.

Nucleic Acids Res, 1986 Aug 26, 14(16), 6621 - 31
Photoalkylated DNA and ultraviolet-irradiated DNA are incised at cytosines by endonuclease III; Weiss RB et al.; Photoalkylation, the ultraviolet irradiation of DNA with isopropanol and di-tert-butylperoxide, causes a variety of base alterations . These include 8-(2-hydroxy-2-propyl)guanines, 8-(2-hydroxy-2-propyl)adenines and thymine dimers . An E . coli endonuclease against photoalkylated DNA was assayed by conversion of superhelical PM2 phage DNA to the nicked form . Enzyme activities were compared between extracts of strain BW9109 (xth-), lacking exonuclease III activity, and strain BW434 (xth-,nth-), deficient in both exonuclease III and endonuclease III . The endonuclease level in the double mutant against substrate photoalkylated DNA was under 20% of the activity in the mutant lacking only exonuclease III . Irradiation of the DNA substrate in the absence of isopropanol did not affect the activity in either strain . Analysis by polyacrylamide gel electrophoresis identified the sites of DNA cleavage by purified E . coli endonuclease III as cytosines, both in DNA irradiated at biologically significant wavelengths and in photoalkylated DNA . Neither 8-(2-hydroxy-2-propyl)purines, pyrimidine dimers, uracils nor 6-4'-(pyrimidin-2'-one)pyrimidines were substrates for the enzyme.

Nucleic Acids Res, 1986 Aug 26, 14(16), 6613 - 20
Interaction of the tight-binding I12-X86 lac repressor with non operator DNA: salt dependence of complex formation; Grebert P et al.; The interaction of the wild-type lac repressor and its tight binding double mutant I12-X86 with a non operator-210 base pair-DNA fragment has been investigated using the nitrocellulose filter binding assay . While the affinity of the double mutant for this non specific DNA is increased as compared to that of the wild-type repressor, the number of ions released from the vicinity of the DNA upon complex formation is less important for the mutant than for the wild-type . These results demonstrate that the adaptation in the recognition surface of the repressor recently proposed by Mossing et al (J . Mol . Biol., 1985, 186, 295-305) in the case of an Oc mutant may be a more general phenomenon.

Biochemistry, 1986 Aug 26, 25(17), 4969 - 78
Structural and functional differences between the two intrinsic zinc ions of Escherichia coli RNA polymerase; Giedroc DP et al.; DNA-dependent RNA polymerase (RPase) from Escherichia coli contains 2 mol of intrinsic Zn(II)/mol of core enzyme (alpha 2 beta beta') . In techniques analogous to those employed with the Zn(II) metalloenzyme aspartate transcarbamoylase {Hunt, J . B., Neece, S . H., Schachman, H . K., & Ginsberg, A . (1984) J . Biol . Chem . 259, 14793-14803}, we show that titration of core or holoRPase with 10 or 16 equiv, respectively, of the sulfhydryl reagent p-(hydroxymercuri)benzenesulfonate (PMPS) results in the facile release of 1 mol of Zn(II) {B-site Zn(II)} in a reaction totally reversible with the addition of excess thiol provided no metal chelator is present . If ethylenediaminetetraacetic acid (EDTA) is present, reversal of the PMPS-enzyme complex results in formation of a Zn1 RPase {A-site Zn(II)} . This enzyme retains full transcriptional activity relative to Zn2 RPase on both calf thymus (nonspecific) and T7 (sigma-dependent, specific) DNA templates . If the core enzyme-PMPS complex is incubated with a large excess of another metal such as Cd(II) followed by thiol treatment, a hybrid ZnACdB RPase is formed . Direct treatment of the enzyme with excess Cd(II) also gives rise to a hybrid ZnACdB RPase . Transcription by these enzymes is also comparable to that of the starting Zn2 enzyme . Isolation of in vivo synthesized Co2 RPase and Cd2 RPase and treatment of either enzyme with PMPS/EDTA results in formation of a CoA and CdA enzyme, respectively . Co(II)A and Cd(II)A enzymes show 123 and 76%, respectively, of the elongation rates on T7 DNA observed for the Zn(II) enzyme . Visible absorption spectroscopy of the Co2 enzyme exhibits four d-d transition bands positioned at 760 (epsilon = 800), 710 (epsilon = 900), 602 (epsilon = 1500), and 484 (epsilon = 4000) nm . In addition, two charge-transfer bands are found at 350 (epsilon = 9600) and 370 (epsilon = 9500) nm . Only the Co(II) ion bound at site A is associated with this unique set of intense d-d transitions . The positions and intensities of both the visible and charge-transfer bands of Co(II)A RPase approximate those shown by Co(II)-substituted metalloenzyme sites where the ligands are four S rather than mixed S,N or S,O sites.

Nucleic Acids Res, 1986 Aug 26, 14(16), 6633 - 48
Drosophila ARSs contain the yeast ARS consensus sequence and a replication enhancer; Mills JS et al.; A number of restriction fragments that function as autonomously replicating sequences (ARSs) in yeast have been isolated from Drosophila melanogaster DNA . The behaviour in yeast of plasmids containing Drosophila ARS elements was studied and compared to that exhibited by the archetypal yeast ARS-1 plasmid . ARS functions were localised by subcloning and BAL-31 deletion analysis . These studies demonstrated the structural and functional complexity of Drosophila ARSs . Each Drosophila ARS element has at least two domains, one essential for replication (the replication sequence, RS) and a second (the replication enhancer, RE) which is essential for maximum function of the RS . The RS of three Drosophila ARSs was shown to contain a sequence identical to an 11 bp yeast ARS consensus sequence (5' A/T TTTATPuTTT A/T 3') . These observations lend support to the hypothesis that heterologous ARS elements may be of biological significance.

Biochemistry, 1986 Aug 26, 25(17), 4855 - 63
Nucleotide sequence of the cDNA coding for human complement C1r; Leytus SP et al.; C1r is a zymogen of a serine protease that is involved in the activation of the first component of the classical pathway of the complement system . cDNAs coding for human C1r have been isolated from libraries prepared from poly(A) RNA from human liver and Hep G2 cells . From DNA sequence analysis, the overlapping cDNA inserts were shown to span 2493 nucleotides of the C1r mRNA, not including the poly(A) tail . The cDNA sequence coding for C1r contained a 5' noncoding region, 2115 nucleotides coding for a polypeptide precursor of 705 amino acids, and a 3' noncoding region . Some variability in the length of the 3' noncoding sequence was observed with the cDNA inserts, although most contained a polyadenylation signal followed by a poly(A) tail . The A or noncatalytic chain of C-1r, which originates from the amino-terminal end of the precursor molecule, contains a potential growth factor domain and two different pairs of internal repeats . One pair of these internal repeats is closely related to the amino-terminal sequence of C1s, while the other pair of repeats is homologous to the tandem repeats present in beta 2-glycoprotein I, complement factor B, the b subunit of factor XIII, and a single region present in the alpha 1 chain of haptoglobin . The B chain of C-1r contains the catalytic portion of the enzyme and is homologous to the trypsin family of serine proteases.

Nucleic Acids Res, 1986 Aug 26, 14(16), 6541 - 9
Nucleotide sequence of the Escherichia coli replication gene dnaZX; Yin KC et al.; The Escherichia coli 2.2 kilobase dnaZX region contains one 1929 nucleotide reading frame which directs the synthesis of two protein products involved in DNA polymerization . The larger consists of 643 amino acids in a deduced 71,114 dalton chain which could be the tau subunit of DNA polymerase III . The smaller, the DNA polymerase III gamma subunit, is encoded by the same reading frame as the larger . The dnaZX sequence contains a region homologous to ATP binding sites, suggesting that these replication factors are adenine nucleotide binding proteins.

J Biol Chem, 1986 Aug 25, 261(24), 11320 - 7
Catalytic domains of carbamyl phosphate synthetase . Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase; Rubino SD et al.; We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine . When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor . Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine . The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate . The NH3-dependent activity of the mutant enzymes was equal to that of wild-type . Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate . The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH . The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme . Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate . Allosteric properties of the large subunit are also unaffected . However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively . The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.

J Biol Chem, 1986 Aug 25, 261(24), 11091 - 6
Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli; Saraswat LD et al.; An expression vector has been constructed for the type I regulatory subunit of cAMP-dependent protein kinase . A cDNA clone for the bovine RI-subunit has been inserted into pUC7 . When Escherichia coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 4 mg/liter . The expressed protein was visualized in total cell extracts by photolabeling with 8-azidoadenosine 3':5'-mono{32P}phosphate following transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose . Expression of R-subunit was independent of isopropyl-beta-D-thiogalactopyranoside . R-subunit accumulated in large amounts only in the stationary phase of growth, and the addition of isopropyl-beta-D-thiogalactopyranoside during the log phase of growth actually blocked the accumulation of R-subunit . Maximum expression (20 mg/liter) was achieved when E . coli 222 was transformed with the RI-containing plasmid . E . coli 222 is a strain that contains two mutations; it is cya- and also has a mutation in the catabolite gene activator protein (crp) that enables the protein to bind to DNA in the absence of cAMP . The expressed RI-subunit was a soluble, dimeric protein, and no significant proteolysis was apparent in the cell extract . The purified RI-subunit bound 2 mol of cAMP/mol of R monomer, reassociated with C-subunit to form holoenzyme, and migrated as a dimer on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agents . The expressed protein was also susceptible to limited proteolysis, yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000 . In all of these properties, the expressed protein was indistinguishable from RI purified from bovine tissue even though the R-subunit expressed in E . coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z' gene of the vector . This NH2-terminal sequence was confirmed by amino acid sequencing.

J Biol Chem, 1986 Aug 25, 261(24), 10990 - 5
Efficiency of reconstitution of the membrane-associated sn-glycerol 3-phosphate acyltransferase of Escherichia coli; Scheideler MA et al.; Membrane-associated enzymes are often solubilized with detergents, purified, and then reconstituted with phospholipid cofactors to regain function . Insofar as most purification and reconstitution procedures are not quantitative, the final reconstituted preparations could reflect a population of molecules ranging from fully functional to completely inactive . Quantitative studies on the efficiency of reconstitution of the Triton X-100-solubilized sn-glycerol 3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane were undertaken at each step of purification . Physical recovery of the 83,000 Mr polypeptide was quantitated in polyacrylamide gels using membranes from cells labeled with {3H}leucine . The 83,000 Mr polypeptide in such gels was demonstrated to consist exclusively of the glycerol-P acyltransferase peptide by V8 peptide mapping . Comparison between physical recovery of 83,000 Mr polypeptide and reconstituted activity allowed the efficiency of reconstitution to be determined . Unexpectedly, disproportionalities occurred during the purification . However, the final purification of reconstituted enzyme activity matched that of the 83,000 Mr polypeptide . This method also allowed measurement of the specific activities of the glycerol-P acyltransferase in membranes from a wild type E . coli strain and from plasmid-containing strains which express the plsB gene product to different extents . The physical amounts of the 83,000 Mr polypeptide and glycerol-P acyltransferase activity measured in membranes were not strictly proportional . In strains where the amount of 83,000 Mr polypeptide was enhanced, a larger proportion of latent activity was observed following solubilization and reconstitution . The results establish the suitability of the reconstituted preparations of glycerol-P acyltransferase for detailed kinetic analysis and permit inferences pertaining to regulation.

J Biol Chem, 1986 Aug 25, 261(24), 10970 - 5
Structural requirement at the cleavage site for efficient processing of the lipoprotein secretory precursor of Escherichia coli; Inouye S et al.; A phenotypically silent mutation in the signal peptide of the Escherichia coli outer membrane prolipoprotein was combined with other mutations in the mature lipoprotein structure . Under conditions where the individual mutations permit normal lipoprotein secretion, the prolipoprotein with both mutations was unable to be normally modified or processed . These results demonstrate that a given signal peptide is fully functional only if it is structurally compatible with the protein to be secreted . This structural compatibility between the signal peptide and the secretory protein is considered to be dependent on the secondary structure formed at or near the signal peptide cleavage site.

J Biol Chem, 1986 Aug 25, 261(24), 10963 - 5
The effect of Mg2+ on the guanine nucleotide exchange rate of p21N-ras; Hall A et al.; There is growing evidence that the protein products of the ras gene family, p21ras, can couple growth factor receptors to intracellular second messenger production and in particular to phosphoinositol lipid turnover . So far, however, there has been no direct proof that the ras proteins function as typical regulatory G proteins . We show here that the human p21N-ras protein, isolated from an Escherichia coli expression system, can exist as a stable GDP complex which exchanges very slowly with exogenous GTP, the half-life of the p21N-ras X GDP complex being around 20 min . However, in low Mg2+ (0.5 microM) the exchange rate is dramatically increased and the half-life of the p21N-ras X GDP complex is less than 30 s . Furthermore, in low Mg2+, the relative binding affinity of the protein for GTP as compared to GDP is increased 10-fold . The effect of low Mg2+ on the exchange rate of both normal and oncogenic mutant p21ras molecules is identical . We propose that removal of Mg2+ in vitro induces a similar conformational change to stimulation in vivo . The properties described here are consistent with a G protein-like activity for p21N-ras.

J Biol Chem, 1986 Aug 25, 261(24), 11374 - 7
3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate . A substrate and photoaffinity label for CMP-N-acetylneuraminic acid synthetase; Abeijon C et al.; A photoreactive, radiolabeled pyrimidine nucleotide, 3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate was synthesized from benzoylbenzoic acid and radiolabeled CTP . Benzoylbenzoyl-{5-3H}CTP could substitute for CTP, in an enzymatic reaction with N-acetylneuraminic acid catalyzed by Escherichia coli or rat liver CMP-NeuAc synthetase, to yield radiolabeled benzoyl-benzoyl-CMP-NeuAc . E . coli CMP-NeuAc synthetase could be specifically radiolabeled using benzoylbenzoyl-{alpha-32P}CTP as a photoaffinity label . This specific covalent binding occurred using enzyme preparations of different degrees of purity . These results suggest that benzoylbenzoic acid derivatives of pyrimidines should be of general use in the identification and active site mapping of pyrimidine-requiring proteins and enzymes . These include glycosyltransferases, sugar nucleotide synthetases, and transporters, and enzymes participating in the conjugation of bile acids and biosynthesis of nucleic acids and choline nucleotides.

J Biol Chem, 1986 Aug 25, 261(24), 11021 - 7
Regulation of diacylglycerol kinase biosynthesis in Escherichia coli . A trans-acting dgkR mutation increases transcription of the structural gene; Walsh JP et al.; The mechanism of a trans-acting mutation, dgkR1, which causes a 7-fold elevation of diacylglycerol kinase activity in membranes (Raetz, C . R . H., Kantor, G . D., Nishijima, M., and Jones, M . L . (1981) J . Biol . Chem . 256, 2109-2112) was investigated by direct measurement of diacylglycerol kinase polypeptide by high performance liquid chromatography and by construction of fusions of the dgkA promoter to beta-galactosidase and galactokinase . The dgkR1 mutation was demonstrated to act by increasing the transcription of the structural gene for diacylglycerol kinase, dgkA . Additionally, sn-glycerol-3-phosphate acyltransferase activities were shown to be decreased 30-50% in membranes from dgkR1 mutant strains . Increased diacylglycerol levels occurred when cells were grown on low osmolarity media . This did not affect dgkA expression . In a dgkR+ background, enhanced expression of sn-1,2-diacylglycerol kinase activity in cells containing a high copy number plasmid bearing dgkA decreased sn-1,2-diacylglycerol levels . However, overproduction of diacylglycerol kinase in a dgkR1 genetic background did not affect diacylglycerol levels, suggesting that the dgkR1 mutation affects diacylglycerol metabolism by mechanisms additional to enhancement of dgkA transcription.

J Biol Chem, 1986 Aug 25, 261(24), 11202 - 6
Promoter recognition by Escherichia coli RNA polymerase . Effects of substitutions in the spacer DNA separating the -10 and -35 regions; Auble DT et al.; A family of variants of the PRM promoter of lambda phage was constructed, bearing nine base pair substitutions in a stretch of the spacer DNA separating the contacted -10 and -35 regions . The substituted sequences were chosen for their potential to adopt structures different from those of average B-form DNA and thus to affect the interaction of RNA polymerase with the two contacted regions . Characterization of the promoters in vitro and in vivo provides additional support for the lack of specific contacts in the substituted spacer region and shows that a small change in the relative rotational orientation of the -10 and -35 regions is inconsequential to promoter function . However, a 2-3-fold reduction in promoter activity is observed with promoters bearing substitutions of nonalternating dG-dC base pairs in either orientation . This corroborates other studies indicating the anomalous behavior of such sequences and suggests that the structure of the spacer DNA can modulate promoter recognition.

J Biol Chem, 1986 Aug 25, 261(24), 11355 - 61
An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export; Freudl R et al.; Pulse-chase experiments were performed to follow the export of the Escherichia coli outer membrane protein OmpA . Besides the pro-OmpA protein, which carries a 21-residue signal sequence, three species of ompA gene products were distinguishable . One probably represented an incomplete nascent chain, another the mature protein in the outer membrane, and the third, designated imp-OmpA (immature processed), a protein which was already processed but apparently was still associated with the plasma membrane . The pro- and imp-OmpA proteins could be characterized more fully by using a strain overproducing the ompA gene products; pro- and imp-OmpA accumulated in large amounts . It could be shown that the imp- and pro-OmpA proteins differ markedly in conformation from the OmpA protein . The imp-OmpA, but not the pro-OmpA, underwent a conformational change and gained phage receptor activity upon addition of lipopolysaccharide . Utilizing a difference in detergent solubility between the two polypeptides and employing immunoelectron microscopy, it could be demonstrated that the pro-OmpA protein accumulated in the cytoplasm while the imp-OmpA was present in the periplasmic space . The results suggest that the pro-OmpA protein, bound to the plasma membrane, is processed, and the resulting imp-OmpA, still associated with the plasma membrane, recognizes the lipid A moiety of the lipopolysaccharide . The resulting conformational change may then force the protein into the outer membrane.

Biochim Biophys Acta, 1986 Aug 22, 867(4), 201 - 8
Inactivation of transforming activity of plasmid DNA by lipid peroxidation; Akasaka S; DNA damage due to NADPH-dependent lipid peroxidation of liposomes was examined using liposomes prepared from lipids, NADPH-cytochrome P-450 reductase and cytochrome P-450 isolated from rat liver microsomes . Plasmid pBR322 DNA was incubated in the reaction mixture for liposomal lipid peroxidation and introduced to Escherichia coli CSR603 (uvrArecA) . More of the transforming activity of the DNA was lost as the lipid peroxidation progressed, and this inactivation was dependent on the extent of lipid peroxidation . Single strand breaks occurred in the plasmid DNA . Hydroxyl radical scavengers could not prevent most of the strand breaks or the lipid peroxidation reaction . Chloroform extracts from the reaction mixture of peroxidized microsomes also inactivated the transforming activity of pBR322 DNA but did not cause strand breaks . The 105 000 X g supernatant of the reaction mixture, which contained more than 85% of the thiobarbituric acid-reactive substances, did not inactivate the plasmid DNA . The degradative products of {U-14C}arachidonic acid in the liposomes did not bind to DNA . These results led to the conclusion that at least two types of DNA damaging agent are produced during NADPH-dependent microsomal lipid peroxidation . One induces single strand breaks of DNA and another inactivates the plasmid-transforming activity without inducing strand breaks.

J Mol Biol, 1986 Aug 20, 190(4), 635 - 8
Recognition of B and Z forms of DNA by Escherichia coli DNA polymerase I; Ramesh N et al.; Since the substrate binding domain of the large proteolytic fragment of Escherichia coli DNA polymerase I has been shown to interact with the B forms of DNA, we have studied the ability of this enzyme to recognize structures other than the B form . The polymerase activity has been used to evaluate the degree of recognition of the B and Z forms of DNA . The Z form was found to promote less activity, indicating the probable inability of the polymerase to move along the conformationally rigid form of the template . The present study indicates that the Z-DNA found in vivo may have a role in the control of replication.

FEBS Lett, 1986 Aug 18, 204(2), 269 - 72
An infrequent generation of catenated network of pBR322 in Escherichia coli; Komiyama N et al.; It was demonstrated that Escherichia coli infrequently generates the catenated network of pBR322 . This complex pBR322 form was detected when DNA molecules could hardly enter the agarose gel during electrophoresis and was found to comprise monomers and dimers of the plasmid.

Carbohydr Res, 1986 Aug 15, 151, 349 - 58
Structural studies of the O-specific side-chains of the Escherichia coli O 10 lipopolysaccharide; Kenne L et al.; The structure of the O-specific side-chains of the Escherichia coli O 10 lipopolysaccharide has been investigated . Methylation analysis, n.m.r . spectroscopy, and various specific degradations were the principal methods used . It is concluded that the polysaccharide is composed of pentasaccharide repeating-units having the following structure, in which D-Fuc4NAcyl is 4-amino-4,6-dideoxy-D-galactose, acylated with acetic acid (60%) or D-3-hydroxybutyric acid (40%) . (Formula: see text).

Anal Biochem, 1986 Aug 15, 157(1), 144 - 53
Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies; Dunn SD; Two modifications to Western blots which enhance immunochemical recognition have been developed . The first is transfer in carbonate buffer at pH 9.9, rather than the more commonly used Tris-glycine buffer at pH 8.3 . This alteration improved the recognition of four of the five subunits of Escherichia coli F1-ATPase by monoclonal antibodies, the smaller subunits showing the greatest effects . Recognition of dinitrophenyl groups attached to the subunits by polyclonal antibodies was improved by the carbonate buffer only for the smallest ATPase subunit, epsilon . The second modification was incubation of the gel in mild buffers, designed to promote the renaturation of proteins, before the electrophoretic transfer step . The most effective buffer was 20% glycerol in 50 mM Tris-HCl, pH 7.4 . Improvements in the signal obtained with monoclonal antibodies to all the subunits of ATPase were obtained by this procedure . As the subunits vary markedly in size, isoelectric point, and other properties, this method should be useful for most proteins . The fate of the 15,000-Da epsilon subunit, labeled with 125I, was followed through a blotting experiment . As long as no sodium dodecyl sulfate was added to the transfer buffer, epsilon was bound to nitrocellulose efficiently in either Tris-glycine or carbonate buffer . However, the epsilon was retained much more strongly during the subsequent incubation steps if the transfer was done in the carbonate buffer . The binding of epsilon to the nitrocellulose was even more stable when the gel had been treated with the buffered glycerol solution before transfer . These results indicate that the conditions under which epsilon subunit first encounters the nitrocellulose markedly affect the stability of binding during subsequent steps . The F1-ATPase was partially fragmented by treatment with proteases and then run on a gel and either transferred immediately in Tris-glycine buffer or else treated with the buffered glycerol solution and transferred in the carbonate buffer . The second blot gave stronger recognition of residual alpha subunit and fragments by an anti-alpha monoclonal antibody, with the largest improvement for the smaller fragments . This result suggests that the modified procedure may be particularly useful in enhancing the detection of small proteins.

Arch Biochem Biophys, 1986 Aug 15, 249(1), 137 - 47
The role of magnesium and potassium ions in the molecular mechanism of ribosome assembly: hydrodynamic, conformational, and thermal stability studies of 16 S RNA from Escherichia coli ribosomes; Allen SH et al.; In an attempt to understand the role of magnesium ion in ribosome assembly in vitro, the hydrodynamic shape, conformation, and thermal stability of ribosomal 16 S RNA were studied systematically as a function of Mg2+ concentration by sedimentation velocity, intrinsic viscosity, circular dichroism, and difference ultraviolet absorption spectroscopy . These results were then compared with the corresponding parameters obtained for 16 S RNA under the optimal conditions of reconstitution, i.e., at 37 degrees C, 20 mM Mg2+, an ionic strength equal to 0.37, and pH 7.8 {S . H . Allen, and K.-P . Wong (1978) J . Biol . Chem . 253, 8759-8766} . When the 360 mM KCl required for reconstitution of 30 S ribosomes is added to the medium, only subtle conformational changes are observed, consistent with the destabilization of the conformation, thus making the RNA molecule more "open" and accessible to protein binding . However, when the concentration of Mg2+ is lowered from 20 to 1 mM, the hydrodynamic parameters indicate that the 16 S RNA is partially unfolded, while thermal denaturation studies suggest that the amount of base-stacking and base-pairing is not concomitantly altered . Further removal of the Mg2+ by dialysis against a pH 7.8 buffer containing no Mg2+ results in a drastic decrease of secondary structure and indicates that the Mg2+ is required for maintenance of the pairing, stacking, and stability of the nucleotide bases, in addition to the long range interactions which result in a compact structure . The results suggest that the 20 mM Mg2+ is required for the 16 S RNA molecules to assume the proper secondary and tertiary structure containing the protein-binding sites, while the high K+ concentration (360 mM KCl) is needed for "loosening up" the RNA, making the protein binding sites more accessible to the ribosomal proteins for molecular recognition and binding as well as for the conformational changes that occur during ribosome assembly.

J Biol Chem, 1986 Aug 15, 261(23), 10496 - 505
The translational efficiency of tRNA is a property of the anticodon arm; Yarus M et al.; We have reciprocally transplanted the anticodon arm sequences of a set of amber suppressor tRNA genes, using recombinant DNA techniques . By this means, a very efficient suppressor may be converted to a poor one, and the poorest tRNA to the efficiency of the best one . In tRNA molecules of normal 2 degrees and 3 degrees structure, the suppressor efficiencies of different composite tRNAs having the same anticodon arm sequence are approximately the same . Large numbers of simultaneous changes throughout the rest of the molecule do not affect the efficiency . Selective nucleotide modification as a result of varied anticodon arm sequences cannot explain these efficiencies . Efficiencies are also unlikely to differ because of selective aminoacylation . Measurement of in vivo tRNA shows, however, that tRNA levels do vary if the anticodon arm sequence is changed . If tRNA levels are normalized, the anticodon arm effect on the translational efficiency remains . Therefore, different anticodon arms, all of normal secondary structure, are not equivalent in translation . The most efficient sequences in this series resemble those found in natural tRNAs associated with similar anticodons, as is proposed in the extended anticodon theory (Yarus, M . (1982) Science 218, 646-652) . These molecules also provide some information on the specificity of nucleotide modification enzymes and on determinants of the steady-state tRNA level.

Cell, 1986 Aug 15, 46(4), 531 - 9
Host protein requirements for in vitro site-specific DNA inversion; Johnson RC et al.; Flagellar phase variation is mediated by a recombination event that occurs at specific sites leading to inversion of a chromosomal segment of DNA . The presence of a 60 bp recombinational enhancer sequence on the DNA substrate molecule results in a 150-fold stimulation in the initial rate of inversion . The protein components required for inversion have been purified . They include the 21,000 dalton recombinase (Hin), a 12,000 dalton host protein (Factor II), and one of the major histone-like proteins of E . coli HU . The dependence of the initial rate of recombination on HU varies with respect to the location of the recombinational enhancer . The role of HU, Factor II, and the enhancer in facilitating site-specific recombination is discussed.

Biochim Biophys Acta, 1986 Aug 15, 872(3), 243 - 52
Molybdenum cofactor: a compound in the in vitro activation of both nitrate reductase and trimethylamine-N-oxide reductase activities in Escherichia coli K12; Silvestro A et al.; Nitrate reductase (nitrite: (acceptor) oxidoreductase, EC 1.7.99.4) and trimethylamine N-oxide reductase (NADH : trimethylamine-N-oxide oxidoreductase, EC 1.6.6.9) activities were reconstituted by incubation of the association factor FA (the putative product of the chlB gene) with the soluble extract of the chlB mutant grown anaerobically in the presence of trimethylamine N-oxide . When soluble extracts of the chlB mutant grown on 10 mM sodium tungstate, a molybdenum competitor, were used in complementation systems, no enzymatic reactivation was observed . Heated extracts of the parental strain 541 were shown to contain a thermoresistant molybdenum cofactor by their ability to reactivate NADPH-nitrate reductase activity in the nit1 mutant of Neurospora crassa . By complementation of parental strain heated extract with association factor FA and soluble extract of the chlB mutant grown in the presence of sodium tungstate, we were able to show for the first time that the molybdenum cofactor is an activator common to the in vitro reconstitution of both nitrate reductase and trimethylamine-N-oxide reductase activities.

Carbohydr Res, 1986 Aug 15, 151, 329 - 35
Branch specificity of beta-D-galactosidase from Escherichia coli; van den Eijnden DH et al.; The "branch specificities" of the beta-D-galactosidases from Escherichia coli, jack bean, Aspergillus niger, and human liver were investigated with two branched oligosaccharide substrates, one which forms part of a complex-type biantennary N-linked glycan (compound 1) and a structure having blood group I activity (compound 2), respectively . Both substrates were available as radioactive compounds having a known distribution of 3H and 14C label in each of the terminal galactosyl groups, which allowed accurate estimation of the branch specificity of the enzymes from the ratio of 3H and 14C radioactivity in the galactose released by these hydrolases . It was found that the beta-D-galactosidase from E . coli preferentially released the galactosyl group at the 1----3 branch of compound 1 and that the 1----6 branch of compound 2 . By contrast, the other beta-D-galactosidases investigated showed little or no branch specificity . These results suggest that the branch specificity of the beta-D-galactosidase from E . coli has to be explained from a specific recognition of certain parts of the aglycon of the substrates by this enzyme rather than from a better accessibility of the galactose at one particular branch.

Eur J Biochem, 1986 Aug 15, 159(1), 175 - 80
Natural selection versus primitive gene structure as determinant of codon usage; Wong JT et al.; Different codons are not utilized equally in known gene sequences . One of the important biases of codon usage is observed in the form of an enrichment of RNY codons, especially within RNN codon families . Such biases could represent the residue of a primitive repeating-RNY gene structure, or the outcome of natural selection, or both . Analyses based on the rates of silent substitutions, the frequencies of base doublets, and synonymous codon ratios for Escherichia coli, yeast, Drosophila and Xenopus proteins have been performed . The results rule out any significant support for a primitive repeating-RNY or repeating-RRY gene structure, and establish the important role of natural selection in determining the choice of codons . With strong intervention by natural selection, the relationship between primitive gene structure and codon usage necessarily becomes minimal.

J Biol Chem, 1986 Aug 15, 261(23), 10936 - 40
Interactions of the Escherichia coli methionine repressor with the metF operator and with its corepressor, S-adenosylmethionine; Saint-Girons I et al.; The metJ gene encoding the methionine aporepressor was placed under the control of a strong and inducible promoter, ptac . Bacterial strains carrying the recombinant plasmid pIP35 overproduced the regulatory protein by a factor of 200 over the wild type strain as determined by the immunoblot technique . The purified metJ gene product negatively controls the expression of the metF gene, in a cell-free system as shown by repression of beta-galactosidase synthesis under the control of the metF promoter . The metJ protein binds to a DNA fragment containing the potential operator of the metF gene with an affinity which is 10 times greater in the presence of S-adenosylmethionine than in its absence . Equilibrium dialysis experiments showed that the met aporepressor binds 2 mol of S-adenosylmethionine per mol of dimer with a dissociation constant of 200 microM.

J Biol Chem, 1986 Aug 15, 261(23), 10885 - 90
The binding of catabolite activator protein and RNA polymerase to the Escherichia coli galactose and lactose promoters probed by alkylation interference studies; Shanblatt SH et al.; The Escherichia coli galactose and lactose promoter regions have been studied by alkylation interference experiments . The data reveal those bases or phosphate groups which, when modified, prevent the binding of the catabolite activator protein (CAP) or RNA polymerase and hence are presumably in contact with the proteins . Interference contacts made by CAP at its primary binding sites at gal and lac are quite similar, indicating that CAP-cAMP uses the same mode of binding at these two operons . RNA polymerase, when bound in the presence of CAP-cAMP, exhibits contacts at the gal and lac P1-10 regions very much like those of the lac UV5 and T7 A3 promoters (Siebenlist, U., Simpson, R . B., and Gilbert, W . (1980) Cell 20, 269-281) . CAP, therefore, does not detectably alter the structure of the open complex . The binding sites for CAP and RNA polymerase at lac, as deduced from interference experiments, do not overlap . However, at gal a CAP molecule is found much closer to the enzyme, and there is competition for a set of mutual contacts . These experiments thus reveal both similarities and differences in the mechanisms whereby CAP activates transcription at catabolite-sensitive operons.

J Biol Chem, 1986 Aug 15, 261(23), 10797 - 801
Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli . Homology among outer membrane receptors that interact with TonB; Lundrigan MD et al.; We have determined the nucleotide sequence of the Escherichia coli fepA gene, which codes for the outer membrane receptor for ferrienterochelin and colicins B and D . The predicted FepA polypeptide has a molecular weight of 79,908 and consists of 723 amino acids . A 22-amino acid leader or signal peptide preceded the mature protein . With respect to overall composition, hydropathy, net charge and distribution of nonpolar segments, the FepA polypeptide was typical of other E . coli outer membrane proteins, except that FepA contained 2 cysteine residues . Comparison of the deduced amino acid sequence of FepA with that of three other TonB-dependent receptors (BtuB, FhuA, and IutA) revealed only a few regions of sequence homology; one of these included the amino-termini . An amino acid substitution within the conserved amino-terminal region of BtuB resulted in production of a receptor that had normal binding functions but was incapable of energy-dependent transport of vitamin B12 . This result suggests that the amino-terminal end of these four polypeptides is involved in interaction with the TonB protein or another step of energy transduction . Three other regions of homology were shared among the four proteins: one near residues 50 to 70, another at about residue 100 to 140, and the last between 20 and 40 amino acid residues from the carboxyl terminus . The function of these three regions remains speculative.

J Biol Chem, 1986 Aug 15, 261(23), 10632 - 6
Nucleotide sequence of the purM gene encoding 5'-phosphoribosyl-5-aminoimidazole synthetase of Escherichia coli K12; Smith JM et al.; 5'-Phosphoribosyl-5-aminoimidazole synthetase (EC 6.3.3.1), encoded by the purM gene of Escherichia coli, catalyzes the synthesis of 5'-phosphoribosyl-5-aminoimidazole from 5'-phosphoribosylformylglycinamidine . The purM gene was subcloned from the Clarke and Carbon (Clarke, L., and Carbon, J . (1976) Cell 9, 91-99) plasmid pLC1-41 and the nucleotide sequence determined . The mature protein, as deduced from the purM structural gene sequence, contains 344 amino acid residues and has a calculated Mr of 36,726 . The 5' end of the purM mRNA was determined by mung bean nuclease mapping to be 44-45 nucleotides up-stream of the proposed GTG translation initiation codon . A C-G-rich region characteristic of stringently controlled promoters is located immediately in front of the proposed purM promoter region . Comparison of the upstream sequences of the purM and the coregulated purF loci revealed a highly conserved (33 of 39 base pairs are identical) sequence . The presumptive purM promoter is located in this region, thus suggesting that both purine loci share a common mechanism of regulation mediated through this sequence.

J Biol Chem, 1986 Aug 15, 261(23), 10610 - 7
Glutamyl-tRNA synthetase of Escherichia coli . Isolation and primary structure of the gltX gene and homology with other aminoacyl-tRNA synthetases; Breton R et al.; The gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli and adjacent regulatory regions was isolated and sequenced . The structural gene encodes a protein of 471 amino acids whose molecular weight is 53,810 . The codon usage is that of genes highly expressed in E . coli . The amino acid sequence deduced from the nucleotide sequence of the gltX gene was confirmed by mass spectrometry of large peptides derived from the glutamyl-tRNA synthetase . The observed peptides confirm 73% of the predicted sequence, including the NH2-terminal and the COOH-terminal segments . Sequence homology between the glutamyl-tRNA synthetase and other aminoacyl-tRNA synthetases of E . coli was found in four segments . Three of them are aligned in the same order in all the synthetases where they are present, but the intersegment spacings are not constant; these ordered segments may come from a progenitor to which other domains were added . Starting from the NH2-end, the first two segments are part of a longer region of homology with the glutaminyl-tRNA synthetase, without need for gaps; its size, about 100 amino acids, is typical of a single folding domain . In the first segment, containing sequences homologous to the HIGH consensus, the homology is consistent with the following evolutionary linkage: gltX----glnS----metS----ileS and tyrS.

Cell, 1986 Aug 15, 46(4), 513 - 9
Germ line transmission of autonomous genetic elements in transgenic mouse strains; Rassoulzadegan M et al.; Upon microinjection into fertilized mouse eggs of circular molecules of plasmid pPyLT1 carrying the gene encoding the large T protein of polyoma virus within bacterial vector sequences, autonomous circular plasmids were stably maintained in low copy numbers in transgenic strains . These plasmids could be rescued in E . coli by transfection . Integrated forms could be detected neither in somatic tissues, nor in spermatozoa . Efficiency of paternal or maternal transmission was close to 100% . The plasmids had lost or had extensively rearranged the polyoma sequences . In addition, they had acquired defined segments of genomic mouse DNA, which might be responsible for correct segregation of daughter copies at both mitosis and meiosis (centromeric function).

Eur J Pharmacol, 1986 Aug 15, 127(3), 205 - 10
Vascular beta-adrenoceptor blocking activity of endotoxin and pertussis toxin from Bordetella pertussis in rats; De Wildt DJ et al.; Isolated and purified leucocytosis promoting factor (LPF), alternatively described as pertussis toxin, reduced the hypotension after beta 2-adrenoceptor stimulation with salbutamol as well as the negative chronotropic activity induced by the muscarinic receptor stimulant arecoline 4 days after its injection into rats . These inhibitory effects of LPF were accompanied by a reduction in basal blood pressure . No effect on autonomic responsiveness or blood pressure was observed 5 h after injection of LPF . Sublethal doses of purified B . pertussis endotoxin (LPS) elicited neither vascular beta 2-adrenergic nor cardiac cholinergic blockade 4 days following injection . Only a distinct vascular beta 2-adrenolytic effect was measured 5 h after pretreatment with the same doses of LPS . This beta 2-adrenoceptor hyporesponsiveness was accompanied by neither an anticholinergic nor a hypotensive effect, but rather by a slight but significant elevation of the blood pressure . In conclusion, both components of B . pertussis (LPS and LPF) give rise to vascular beta 2-adrenergic hyporesponsiveness irrespective of blood pressure effects . There is an important difference between both components with respect to their various kinetic profiles for this phenomenon: an early occurring and short-lasting beta 2-adrenergic blockade for LPS and a late occurring LPF-mediated beta 2-adrenergic blockade.

J Biol Chem, 1986 Aug 15, 261(23), 10653 - 8
Topology and function of "stalk" proteins in the bovine mitochondrial H+-ATPase; Joshi S et al.; Proton translocating ATPases comprise a hydrophilic sector F1, a membrane sector F0, and, in the case of bovine mitochondria, a connecting "stalk" which is believed to contain the oligomycin sensitivity-conferring protein (OSCP) and coupling factor 6 (F6) . The present study was undertaken to verify the accessibility of F6 and OSCP to trypsin and to examine the functional consequences of such treatment . Our data show that F1 binds equally to trypsin-treated F0 and untreated F0, but the former complexes exhibit cold lability and only partial sensitivity to oligomycin . Furthermore, these complexes fail to exhibit ATP-driven proton translocation or ATP-32Pi exchange activity . Trypsinization of F0 does not, however, inhibit passive proton conductance through the membrane sector but actually enhances it . Immunological data indicate extensive degradation of OSCP under conditions where F6 proteolysis is insignificant . Intact H+-ATPase complexes are relatively resistant to both the structural and functional effects of trypsin . We conclude that OSCP is predominantly an extrinsic protein which is shielded by F1 in the native membrane . F6 may also be an extrinsic protein but is shielded from trypsinization by OSCP and/or other F0 polypeptides . The exposed, trypsin-sensitive segments of OSCP are not required for passive proton conductance through F0 but may be required for ATP-driven reactions . We propose that bovine mitochondrial OSCP is a functional analogue of subunit b in the Escherichia coli H+-ATPase.

J Biol Chem, 1986 Aug 15, 261(23), 10587 - 91
Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence; Colombo G et al.; Glutamine synthetase is encoded by the glnA gene of Escherichia coli and catalyzes the formation of glutamine from ATP, glutamate, and ammonia . A 1922-base pair fragment from a cDNA containing the glnA structural gene for E . coli glutamine synthetase has been sequenced . An open reading frame of 1404 base pairs encodes a protein of 468 amino acid residues with a calculated molecular weight of 51,814 . With few exceptions, the amino acid sequence deduced from the DNA sequence agreed very well with the amino acid sequences of several peptides reported previously . The secondary structure predicted for the E . coli enzyme has approximately 36% of the residues in alpha-helices which is in agreement with calculations of approximately 39% based on optical rotatory dispersion data . Comparison of the amino acid sequences of glutamine synthetase from E . coli (468 amino acids) and Anabaena (473 amino acids) (Turner, N . E., Robinson, S . T., and Haselkorn, R . (1983) Nature 306, 337-342) indicates that 260 amino acids are identical and 80 are of the same type (polar or nonpolar) when aligned for maximum homology . Several homologous regions of these two enzymes exist, including the sites of adenylylation and oxidative modification, but the regulation of each enzyme is different.

Eur J Biochem, 1986 Aug 15, 159(1), 95 - 101
Induction of polynucleotide-protein cross-linkages by ultraviolet irradiation . Peculiarities of the high-intensity laser pulse irradiation; Budowsky EI et al.; The efficiency and specificity of RNA-protein cross-linking in the 30S subunit of Escherichia coli ribosomes, induced by low-intensity (10(15) photons cm-2 s-1, 254 nm) and high-intensity {(1.6-6.8) X 10(24) photons cm-2 s-1, 266 nm, pulse duration 10(-8) s} ultraviolet radiation, are studied . Under the former conditions proteins S4, S7 and S9/S11, and under the latter conditions these proteins together with S3, S18 and S20, are cross-linked to 16S RNA . Biphotonic processes operate in the latter case . In the presence of 2-mercaptoethanol cross-linking occurs either directly, via a higher excited state or via activated intermediates with life-times less than 25 ns . Cross-links thus formed are specific, i.e . they are formed between regions of macromolecules which are in contact in the native (non-disturbed) complex prior to excitation . The efficiency of cross-linking (per photon absorbed) is 20-100 times higher upon two-step excitation as compared with single-step excitation and an analysable number of cross-links are produced in a single pulse . Only base U-1239 of 16S RNA is cross-linked to protein S7 by low-intensity radiation, whereas the adjacent base, G-1240 is also involved in laser-induced cross-linking . A transition from the former to the latter conditions allows one to reduce the duration of irradiation from several minutes to several nanoseconds.

Eur J Biochem, 1986 Aug 15, 159(1), 103 - 9
Structural characteristics and classification of some tRNA-binding sites of elongating Escherichia coli ribosome; Abdurashidova GG et al.; Ultraviolet(254 nm)-irradiation-induced cross-linkages in ribosomal complexes allowed identification of proteins in contact with tRNA at different elongation steps . Both the set and the ratio of cross-linked proteins, i.e . the structural characteristics of the tRNA-binding sites of the ribosome, were shown to depend strongly not only on the position of the mRNA codon with which tRNA interacts as a component of a ribosomal complex, but also on its functional state, i.e . on the elongation step . A new classification of tRNA-binding sites of ribosome is suggested.

Biochemistry, 1986 Aug 12, 25(16), 4504 - 7
Formaldehyde metabolism by Escherichia coli . Detection by in vivo 13C NMR spectroscopy of S-(hydroxymethyl)glutathione as a transient intracellular intermediate; Mason RP et al.; In vivo 13C NMR has been used to detect the transient formation of S-(hydroxymethyl)glutathione (GSCH2OH) from glutathione and {13C}formaldehyde in Escherichia coli . Two-dimensional 1H-13C shift correlation was used to locate the chemical shift of the formaldehyde-derived protons of the adduct . The adduct GSCH2OH is formed by chemical reaction in the first few minutes after cells are challenged with formaldehyde and remains within the cell until consumed by metabolism.

Biochemistry, 1986 Aug 12, 25(16), 4483 - 5
lac permease of Escherichia coli: histidine-205 and histidine-322 play different roles in lactose/H+ symport; Puttner IB et al.; The lac permease of Escherichia coli was modified by site-directed mutagenesis such that His-205 or His-322 is replaced with either Asn or Gln . Permease with Asn or Gln in place of His-205 exhibits normal activity, while permease with Asn or Gln in place of His-322 exhibits no activity . The results are consistent with the interpretation that His-205 and His-322 play different roles in lactose/H+ symport, the former involving hydrogen bonding of the imidazole nitrogens and the latter requiring positive charge in the imidazole ring . In addition, it is demonstrated that permease with Arg in place of His-322 does not catalyze efflux, exchange, or counterflow . The observations, in conjunction with those in the accompanying paper {Carrasco, N., Antes, L . M., Poonian, M . S., & Kaback, H . R . (1986) Biochemistry (following paper in this issue)}, suggest that His-322 plays an important role in H+ translocation, possibly as a component of a charge-relay system with Glu-325, a neighboring residue in helix 10.

Biochemistry, 1986 Aug 12, 25(16), 4555 - 61
Identification of peptide sequences at the tRNA binding site of Escherichia coli methionyl-tRNA synthetase; Valenzuela D et al.; Four different structural regions of Escherichia coli tRNAfMet have been covalently coupled to E . coli methionyl-tRNA synthetase (MetRS) by using a tRNA derivative carrying a lysine-reactive cross-linker . We have previously shown that this cross-linking occurs at the tRNA binding site of the enzyme and involves reaction of only a small number of the potentially available lysine residues in the protein {Schulman, L . H., Valenzuela, D., & Pelka, H . (1981) Biochemistry 20, 6018-6023; Valenzuela, D., Leon, O., & Schulman, L . H . (1984) Biochem . Biophys . Res . Commun . 119, 677-684} . In this work, four of the cross-linked peptides have been identified . The tRNA-protein cross-linked complex was digested with trypsin, and the peptides attached to the tRNA were separated from the bulk of the tryptic peptides by anion-exchange chromatography . The tRNA-bound peptides were released by cleavage of the disulfide bond of the cross-linker and separated by reverse-phase high-pressure liquid chromatography, yielding five major peaks . Amino acid analysis indicated that four of these peaks contained single peptides . Sequence analysis showed that the peptides were cross-linked to tRNAfMet through lysine residues 402, 439, 465, and 640 in the primary sequence of MetRS . Binding of the tRNA therefore involves interactions with the carboxyl-terminal half of MetRS, while X-ray crystallographic data have shown the ATP binding site to be located in the N-terminal domain of the protein {Zelwer, C., Risler, J . L., & Brunie, S . (1982) J . Mol . Biol . 155, 63-81}.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Aug 12, 25(16), 4711 - 8
Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites; Pettigrew DW; Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C . The inactivation by DTNB is reversed by dithiothreitol . In the cases of both reagents, the kinetics of activity loss are pseudo first order . The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration . Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit . However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly . The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol . The protection afforded by ATP or AMP is not dependent on Mg2+ . The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Aug 12, 25(16), 4647 - 54
57Fe and 1H electron-nuclear double resonance of three doubly reduced states Escherichia coli sulfite reductase; Cline JF et al.; We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the 57Fe hyperfine interactions in the bridged-siroheme {4Fe-4S} cluster that forms the catalytically active center of the two-electron-reduced hemoprotein subunit of Escherichia coli NADPH-sulfite reductase (SiR2-) . Previous electron paramagnetic resonance (EPR) and Mossbauer studies have shown that this enzyme oxidation state can exist in three distinct spectroscopic forms: (1) a "g = 2.29" EPR species that predominates in unligated SiR2-, in which the siroheme Fe2+ is believed to be in an S = 1 state; (2) a "g = 4.88" type of EPR species that predominates in SiR2- in the presence of small amounts of guanidinium sulfate, in which the siroheme Fe2+ is in an S = 2 state; and (3) a classical "g = 1.94" type of EPR species that is seen in SiR2- ligated with CO, in which the siroheme Fe2+ is in an S = 0 state . In all three species, the cluster is in the {4Fe-4S}1+ state, and two distinct types of Fe site are seen in Mossbauer spectroscopy . ENDOR studies confirm the Mossbauer assignments for the cluster 57Fe in the g = 1.94 state, with A values of 37, 37, and 32 MHz for site I and ca . 19 MHz for site II . The hyperfine interactions are not too different on the g = 2.29 state, with site I Fe showing more anisotropic A values of 32, 24, and 20 MHz (site II was not detected).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Aug 12, 25(16), 4486 - 8
lac permease of Escherichia coli: histidine-322 and glutamic acid-325 may be components of a charge-relay system; Carrasco N et al.; When Glu-325 in the lac permease of Escherichia coli is replaced with Ala, lactose/H+ symport is abolished . Thus, the altered permease catalyzes neither uphill lactose accumulation nor efflux . Remarkably, however, permease with Ala-325 catalyzes exchange and counterflow at completely normal rates . Taken together with the results presented in the accompanying paper {Puttner, I . B., Sarkar, H . K., Poonian, M . S., & Kaback, H . R . (1986) Biochemistry (preceding paper in this issue)}, the findings suggest that the His-322 and Glu-325 may be components of a charge-relay system that plays an important role in the coupled translocation of lactose and H+.

Nucleic Acids Res, 1986 Aug 11, 14(15), 6159 - 68
Simultaneous synthesis of human-, mouse- and chimeric epidermal growth factor genes via 'hybrid gene synthesis' approach; Sung WL et al.; Simultaneous synthesis of two DNA duplexes encoding human and mouse epidermal growth factors (EGF) was accomplished in a single step . A 174 b.p . DNA heteroduplex, with 16 single and double base pair mismatches, was designed . One strand encoded the human EGF, and the opposite strand indirectly encoded the mouse EGF . The heteroduplex DNA was synthesized by ligation of seven overlapping oligodeoxyribonucleotides with a linearized plasmid . After transformation in E . coli HB101 (recA 13), the resulting heteroduplex plasmid served as the template in plasmid replication . Two different plasmid progenies bearing either the human or mouse EGF-coding sequence were identified by colony hybridization using the appropriate probes . However, in E . coli JM103, the same process yielded plasmid progenies encoding different chimeric EGF molecules, presumably due to crossover of human and mouse EGF gene sequences.

FEBS Lett, 1986 Aug 11, 204(1), 97 - 9
Adjacent codon-anticodon interactions of both tRNAs present at the ribosomal A and P or P and E sites; Rheinberger HJ et al.; A labeled tRNA present at the A, P or E site can be partially chased from the ribosome, a cognate nonlabeled tRNA as chasing substrate being 3-12-times more efficient than non-cognate tRNA at a molar ratio tRNA: 70 S = 10:1 . These findings indicate that a tRNA bound to a programmed ribosome undergoes codon-anticodon interaction at all three sites (A, P and E site) . Furthermore, both labeled tRNA present on the ribosome can be chased more effectively with cognate than with non-cognate substrate at the same time . This finding provides strong evidence that both tRNAs present on the ribosome exhibit simultaneous codon-anticodon interaction . This is valid for both the pretranslocational state (Ac{3H}Lys-tRNALys in the A and {14C}tRNALys in the P site) as well as the posttranslocational state (Ac{3H}Lys-tRNALys in the P and {14C}tRNALys in the E site).

FEBS Lett, 1986 Aug 11, 204(1), 145 - 50
Expression, secretion and folding of human growth hormone in Escherichia coli . Purification and characterization; Becker GW et al.; An efficient secretion vector containing a gene coding for an E . coli signal peptide fused to human growth hormone (hGH) was cloned into E . coli . The recombinant fusion protein was expressed and correctly processed hGH was secreted into the periplasmic space at a yield of 10-15 micrograms hGH/A600 . Purification of hGH from the periplasmic fraction by anion exchange and size exclusion gave hGH of greater than 90% purity . Characterization by SDS-PAGE, amino terminal analysis, trypsin mapping, and circular dichroism demonstrated that the fusion protein was correctly processed to authentic hGH and that the E . coli periplasm provided an appropriate environment for proper folding of hGH and disulfide bond formation.

Biochim Biophys Acta, 1986 Aug 7, 860(1), 51 - 6
Membrane action of colicin E1: detection by the release of carboxyfluorescein and calcein from liposomes; Kayalar C et al.; Colicin E1 induces the efflux of carboxyfluorescein and calcein from liposomes whose phospholipid composition is similar to that of Escherichia coli . This colicin action takes place at protein-to-liposome ratios and within pH ranges that are physiologically meaningful . Colicin-induced permeability of carboxyfluorescein is not limited to the initial phase of colicin membrane interaction but is sustained thereafter . Colicin E1 requires negatively charged phospholipids in the liposomal membrane in order to bind and induce efflux.

Biochim Biophys Acta, 1986 Aug 7, 860(1), 44 - 50
Substrate specificity of the Escherichia coli maltodextrin transport system and its component proteins; Ferenci T et al.; Maltooligosaccharides up to maltoheptaose are transported by the maltodextrin transport system of Escherichia coli . The overall substrate specificity of the transport system was investigated by using 15 maltodextrin analogues with various modifications at the reducing end of the oligosaccharides as competing substrates . The binding interaction of the analogues with maltoporin in the outer membrane and the periplasmic maltose-binding protein, the two protein components of the transport system with known specificity for maltodextrins, was also investigated . All analogues containing several alpha, 1----4-glucosyl linkages were bound with high affinity by maltoporin and maltose-binding protein, regardless of O-methyl, O-nitrophenyl, beta-glucosyl or beta-fructosyl substitutions at the reducing end of the dextrins . Introduction of a negative charge or lack of a ring structure at the reducing end were also ineffective in abolishing binding by these two proteins . These results suggest that the structure of the reducing glucose is not important in the binding specificity of maltoporin or maltose-binding protein . However, the high affinity of these proteins for analogues was not in itself sufficient for recognition by the transport system overall . Maltohexaitol, 4-nitrophenyl alpha-maltotetraoside and 4-beta-D-maltopentaosyl-D-glucopyranose were bound with the same affinity as comparable maltodextrins by both maltoporin and maltose-binding protein but were poorly recognized by the transport system . These results suggest that another, yet uninvestigated component of the transport system has a more restricted specificity towards changes at the reducing end of the maltodextrin molecule.

J Mol Biol, 1986 Aug 5, 190(3), 499 - 507
Specificity of N-acetoxy-N-2-acetylaminofluorene-induced frameshift mutation spectrum in mismatch repair deficient Escherichia coli strains mutH, L, S and U; Granger-Schnarr M et al.; The mismatch repair system of Escherichia coli is known to contribute to the fidelity of the replicational process . This system involves the functions of mutH, mutL, mutS and mutU (uvrD) loci which recognize mispaired bases as a consequence of errors due to the polymerase itself . Chemical modifications of DNA have also been suspected to create mispaired bases which, if the mispaired bases are removed, will lead to mutations by frameshift . Using the pBR322 plasmid DNA modified by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) we have investigated this possibility in a forward mutational assay (tetracycline sensitivity) . This fluorene derivative has been shown to induce predominantly frameshift mutations . Our results show that: The sensitivity of the deficient strains mutH, mutL and mutS to the AAF adducts is similar to that of the corresponding wild-type strain . However, the mutU strain appears much more sensitive to those adducts although less than a uvrA, B or C-deficient strain . This suggests that the mutU gene product is involved in the repair of AAF adducts . For the four mut deficient strains, and as it was shown with the wild-type strain, AAF adducts induced mutations to tetracycline sensitivity are only observed when the SOS system of the host bacteria is induced by irradiation of the cells prior to transformation with the modified plasmid . The mutation frequencies depend upon the ultraviolet light doses and similar maxima were found for the four mut strains and the corresponding wild-type strain . In agreement with the results obtained with wild-type or uvrA strains we observe that AAF adducts induce mostly frameshift mutations in the mut strains . Two types of hot spots of mutagenesis were described in wild-type and uvrA strains occurring either at repetitive sequences or at sequences of the type 5' G-G-C-G-C-C 3' (NarI restriction enzyme recognition sequence) . While the second type of mutational hot spot does exist in the mismatch repair-deficient strains, we observe that the repetitive sequences are no longer hot spots of mutations in these strains, suggesting that the mismatch repair protein complex is involved in the establishment of AAF-induced frameshift mutations at repetitive sequences.

J Biol Chem, 1986 Aug 5, 261(22), 10340 - 7
Yeast mitochondrial RNA polymerase . Purification and properties of the catalytic subunit; Kelly JL et al.; An RNA polymerase was purified 6500-fold to near homogeneity from a whole cell extract of Saccharomyces cerevisiae . The purified enzyme consists of a single 145,000-dalton polypeptide . By subcellular fractionation, the enzyme was localized to the mitochondria . The RNA polymerase activity is alpha-amanitin- and rifampicin-resistant . With single-stranded DNA templates, the enzyme catalyzes the synthesis of polyribonucleotide chains with lengths ranging from less than 10 to greater than 100 residues . It is inactive with double-stranded DNA . Specific antisera inhibit the RNA polymerase activity and recognize the 145,000-dalton polypeptide . The antisera relate this enzyme to previously described yeast mitochondrial RNA polymerase preparations capable of initiation of transcription at mitochondrial promoter sequences (Winkley, C . S., Keller, M . J., and Jaehning, J . A . (1985) J . Biol . Chem . 260, 14214-14223) . It therefore appears that the enzyme is a subunit of the yeast mitochondrial RNA polymerase.

J Biol Chem, 1986 Aug 5, 261(22), 10277 - 81
The heme and Fe4S4 cluster in the crystallographic structure of Escherichia coli sulfite reductase; McRee DE et al.; Isolated hemoprotein subunits of Escherichia coli NADPH:sulfite reductase catalyze the 6-electron reduction of SO2-3 to S2- . The prosthetic groups of the hemoprotein, a siroheme and a Fe4S4 cluster, have been shown by spectroscopy to be tightly coupled . We have crystallized the isolated hemoprotein subunits and produced a 3-A electron density map by x-ray crystallography . A single heavy atom derivative and the native anomalous scattering (from the protein's 5 Fe and several S) were used to calculate the phases . In the electron density map, the cluster has a geometry similar to other Fe4S4 clusters . Both the cluster and the siroheme are found near the surface of the protein . The siroheme and the Fe4S4 cluster pack next to each other in the structure, apparently with a common ligand, consistent with a cysteine S gamma, shared by the siroheme Fe and one of the cluster Fe . The distance from the siroheme Fe to the center of the cluster is 5.5 A and the distance from the siroheme Fe to the nearest cluster Fe is 4.4 A . The edge of the siroheme macrocycle appears to be in Van der Waals contact with a cubane S atom of the cluster . The sixth coordination position of the siroheme Fe appears unoccupied and is quite exposed to the solvent . Some possible implications of the proposed structure on the role of the bridged siroheme-Fe4S4 cluster in catalysis are discussed.

J Biol Chem, 1986 Aug 5, 261(22), 10087 - 92
Nonessentiality of histidine 291 of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase as determined by site-directed mutagenesis; Niyogi SK et al.; Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B . A., and El-Gul, T . (1985) Biochemistry 24, 3957-3962) . From the pH dependence of inactivation, the pKa of His-298 was observed to be approximately 6.8, and it was suggested that this histidine might be the essential base that initiates catalysis (Paech, C . (1985) Biochemistry 24, 3194-3199) . To explore further the possible function of His-298, we have used site-directed mutagenesis to replace the corresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine . Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is approximately 40% as active catalytically as the normal carboxylase . After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue . The purified mutant carboxylase had a kcat of 1.5 s-1 and a kcat/Km of 1.7 X 10(4) M-1 s-1 in contrast to values of 3.6 s-1 and 6 X 10(5) M-1 s-1 for the normal enzyme . The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R . rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.

J Mol Biol, 1986 Aug 5, 190(3), 269 - 79
Partitioning of plasmid R1 . Structural and functional analysis of the parA locus; Gerdes K et al.; The stability locus, parA+, of plasmid R1 is shown to be localized within a 1500 base-pair region of DNA on the largest EcoRI restriction fragment of plasmid R1 . The nucleotide sequence of the region revealed the presence of two open reading frames, one of 320 codons, and another of 60 codons . The larger open reading frame encodes a polypeptide of 36,000 Mr . Deletions covering the promoter distal end of the 36,000 Mr reading frame give rise to synthesis of large amounts of truncated protein . Construction of promoter fusions between the parA+ promoter and the lacZ gene showed that the parA+ region encodes a factor that negatively regulates the expression of the 36,000 Mr protein . The locus exerting parA+-associated incompatibility, denoted incA+, was mapped to a 60 base-pair region covering the parA+ promoter . Most likely, this region is involved both in the negative regulation of the parA+ operon and in the parA+-associated incompatibility . Two explanations are suggested to explain this possible dual function of the parA+ promoter region . The parA+ region was cloned into an unstably inherited (par-) derivative of a mini-F derivative . The low copy number plasmid mini-F devoid of its own partition genes was stabilized more than 100-fold by carrying the parA+ genes . This observation is in accordance with the proposal that the parA+ locus specifies the true partition function of plasmid R1.

J Biol Chem, 1986 Aug 5, 261(22), 10450 - 5
Isolation and characterization of Limulus C-reactive protein genes; Nguyen NY et al.; Three homologous genes coding for Limulus C-reactive protein (CRP) have been isolated and characterized from a lambda phage EMBL-3 library containing genomic DNA sequences from Limulus amebocytes . The genes have a typical promoter region with a CAAT (nucleotides 50-53) and a TATAA (nucleotides 77-81) box located, respectively, 178 and 149 base pairs 5' upstream from the initiation codon ATG . The polyadenylation site AATAAA is situated within 300 base pairs downstream from the stop codon TAG . Nucleotide sequence analysis reveals a 24-residue signal peptide preceding a coding region of 218 amino acids . Significant differences were found between the genes coding for human and Limulus CRPs . In the human CRP gene there is an intron separating the signal peptide and the coding region . In Limulus this intervening sequence is missing . The Drosophila heat shock consensus sequence CTnGAAnnTTnAG (Simon, J . A., Sutton, C . A., Lobell, R . B., Glaser, R . L., and Lis, J . T . (1985) Cell 40, 805-817), found in the genes of human (Woo, P., Korenberg, J . R., and Whitehead, A . S . (1985) J . Biol . Chem . 260, 13384-13388) and rabbit (Syin, C., Gotschlich, E . C., and Liu, T.-Y . (1986) J . Biol . Chem . 261, 5473-5479) CRP at the 5' end, is not found in the Limulus CRP genes . Whereas a single CRP gene was found in the human, multiple genes were found for the Limulus CRPs . All CRPs exhibit calcium-dependent phosphorylcholine ligand binding properties . The coding regions of the Limulus and human CRP genes share approximately 25% identity and two stretches of highly conserved regions, one of which falls in the region proposed as the phosphorylcholine binding site, while the other site is very similar to the consensus sequence required for calcium binding in calmodulin and related proteins . The nucleotide sequence analysis provides convincing evidence to support the evolutionary relatedness of the human and Limulus CRPs.

J Biol Chem, 1986 Aug 5, 261(22), 10218 - 27
The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates; Lindenbaum JO et al.; The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro . Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis . In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails . DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails . Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed . Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule . This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II) . During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred . This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.

J Biol Chem, 1986 Aug 5, 261(22), 10169 - 75
Escherichia coli helicase II (urvD gene product) translocates unidirectionally in a 3' to 5' direction; Matson SW; Escherichia coli helicase II, product of the uvrD gene, is a single-stranded DNA-dependent nucleoside 5'-triphosphatase with helicase activity . As a DNA-dependent ATPase, helicase II translocates processively along single-stranded DNA (S . W . Matson, unpublished results) . The direction of translocation has been determined using a helicase assay that directly measures the ability of helicase II to catalyze the displacement of a labeled DNA fragment from one end of a single-stranded linear DNA molecule . The translocation of helicase II along single-stranded DNA is unidirectional and in the 3' to 5' direction with respect to the DNA strand on which the enzyme is bound . A kinetic analysis of the displacement of a labeled DNA fragment annealed to a linear single-stranded DNA molecule is also consistent with unidirectional translocation in the 3' to 5' direction . These results are contrary to results previously obtained using an indirect helicase assay (Kuhn, B., Abdel-Monem, M., Krell, H., and Hoffmann-Berling, H . (1979) J . Biol . Chem . 254, 11343-11350).

J Biol Chem, 1986 Aug 5, 261(22), 10043 - 50
Impaired proton conductivity resulting from mutations in the a subunit of F1F0 ATPase in Escherichia coli; Cain BD et al.; Mutations in the uncB gene which encodes the a subunit of F1F0-ATPase in Escherichia coli were isolated and characterized . Eight mutations caused premature polypeptide chain termination . Two mutations were single amino acid substitutions resulting in the replacements of serine 206 with leucine (ser-206----leu) and histidine 245 with tyrosine (his-245----tyr) . The ser-206----leu mutation does not alter F1 binding and allows ATP driven membrane energization at a low level . Stripping of F1 from membranes containing the ser-206----leu mutation does not render the membranes permeable to protons indicating impaired proton conductivity . The his-245----tyr mutation also blocks Fo-mediated proton conduction but has normal F1 binding properties . F1 bound to membranes with both ser-206----leu and his-245----tyr mutant a subunits is sensitive to dicyclohexylcarbodiimide . Apparently, both missense mutations impair proton conduction without altering assembly of the F1F0-ATPase complex . The direct involvement of the a subunit in proton translocation is discussed.

J Biol Chem, 1986 Aug 5, 261(22), 10037 - 42
Genetic evidence for interaction between the a and b subunits of the F0 portion of the Escherichia coli proton translocating ATPase; Kumamoto CA et al.; A mutation of the b subunit of the Escherichia coli proton translocating ATPase was previously described (Porter, A . C . G., Kumamoto, C., Aldape, K., and Simoni, R . D . (1985) J . Biol . Chem . 260, 8182-8187) . This mutation, which causes substitution of aspartic acid for glycine at position 9 (basp9), results in loss of function of the ATPase complex . In this paper we describe the isolation and characterization of two mutations that partially suppress the effects of the basp9 alteration . The suppressor mutations cause amino acid substitutions at position 240 of the a subunit . Membranes derived from strains carrying a suppressor mutation and the basp9 mutation exhibited ATP-dependent proton translocating activity.

J Mol Biol, 1986 Aug 5, 190(3), 513 - 7
Mutation in the D arm enables a suppressor with a CUA anticodon to read both amber and ochre codons in Escherichia coli; Raftery LA et al.; Su9 of Escherichia coli differs from tRNATrp by only a G to A transition in the D arm, yet has an enhanced ability to translate UGA by an unusual C X A wobble pairing . In order to examine the effects of this mutation on translation of the complementary and wobble codons in vivo, we constructed the gene for an amber (UAG) suppressing variant of Su9, trpT179, by making the additional nucleotide change required for an amber suppressor anticodon . The resultant suppressor tRNA, Su79, is a very strong amber suppressor . Furthermore, the D arm mutation enables Su79 to suppress ochre (UAA) codons by C X A wobble pairing . These data demonstrate that the effect of the D arm mutation on wobble pairing is not restricted to a CCA anticodon . The effect extends to the CUA anticodon of Su79, thereby creating a new type of ochre suppressor . The new coding activity of Su79 cannot be explained by alterations in the level of aminoacylation, steady-state tRNA concentration, or nucleotide modification . The A24 mutation could permit unorthodox wobble pairings by generally enhancing tRNA efficiency at all codons or by altering codon specificity.

J Biol Chem, 1986 Aug 5, 261(22), 10112 - 8
Effect of replacing uridine 33 in yeast tRNAPhe on the reaction with ribosomes; Dix DB et al.; We have determined several kinetic parameters for the reaction of poly(U)-programmed ribosomes with ternary complexes of elongation factor Tu, GTP, and yeast Phe-tRNA analogs with different bases substituted for uridine in position 33 . These analogs test whether disruption of the hydrogen bonds normally formed by uridine 33 and steric crowding in the anticodon loop are detrimental to tRNA function on the ribosome . Single-turnover kinetic studies of the reaction of these ternary complexes with ribosomes show that these Phe-tRNA analogs decrease the apparent rate of GTP hydrolysis (kGTP) and the ratio of peptide formed to GTP hydrolyzed . Thus, the substitution of uridine 33 affects not only the selection of a ternary complex by the ribosome but also the selection of an aminoacyl-tRNA in the proofreading reaction . The effects become greater as first one, and then the other, H-bond is disrupted . Steric crowding in the anticodon loop is also important, but does not have as great an effect on the rate constants . An analysis of the elementary rate constants which comprise the rate constant, kGTP, demonstrates that the reduction in kGTP results from a decreased rate of ternary complex association with the ribosome (k1) and that there is little or no effect on the rate of GTP cleavage (k2) . An analysis of the rate constants involved in proofreading shows that all the modified (tRNAs have increased rates of aminoacyl-tRNA rejection (k4) but that the rate of peptide bond formation (k3) is unaffected.

Bioorg Khim, 1986 Aug, 12(8), 1088 - 100
{Site-localized mutagenesis directed by phosphotriester analogs of oligonucleotides}; Petrenko VA et al.; 17- and 20-mer oligodeoxyribonucleotides and their analogues, containing one to four phosphate groups esterified with ethyl alcohol in different positions of oligonucleotide chain, were synthesized by modified triester method . Ethylated di- and trinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives . The structures of the oligonucleotides were confirmed by Maxam-Gilbert sequencing method . Oligonucleotides were not totally complementary to the N-terminal region of lac Z'gene (coding for N-terminal fragment of beta-galactosidase) of phage M13mpB DNA and induced the formation of the proposed deletion mutant DNA M13mp1 delta T . Phosphotriester analogues were more effective mutagens as compared to phosphodiester oligonucleotides due to their stability to nucleases . The use of E . coli DNA-polymerase I provided the increase in the mutant yields in case of the phosphotriester analogues . The stability of the analogues to 5'----3'----5'-endonuclease action, the specificity of oligonucleotide: DNA binding and the structure of mutant DNA were studied by the Sanger sequencing method.

Acta Anaesthesiol Scand, 1986 Aug, 30(6), 463 - 9
Pulmonary function, extravascular lung water and chest radiography in a porcine model of adult respiratory distress syndrome; Forsgren P et al.; To study the pathophysiology and the value of chest radiography in the diagnosis of early adult respiratory distress syndrome spontaneously air-breathing pigs under ketamine anaesthesia were investigated . Five control animals received physiological saline and showed no notable changes in physiological or radiological data . Eleven animals were infused i.v . with E . coli endotoxin over 6 h . The pulmonary dysfunction in the endotoxin animals was characterized by an early increase in venous admixture with hypoxaemia and a peak increase in pulmonary vascular resistance at 0.5 h after start of endotoxin infusion . Subsequently there was a tendency towards a restitution to baseline physiology, but from 3 h onwards a "second wave" of pulmonary dysfunction developed in addition to an increase in extravascular lung water . No significant correlation (r = 0.44) existed between the increase in extravascular lung water and venous admixture . The increase in calculated pulmonary microvascular pressure correlated significantly (r = 0.77) with the increase in extravascular lung water . Radiographic signs of pulmonary oedema were sparse . Thus, only three of 11 animals displayed increased density on chest radiography indicative of pulmonary oedema.

J Appl Toxicol, 1986 Aug, 6(4), 291 - 5
The effect of topically applied n-butylester of 2,4-dichlorophenoxyacetic acid on the immune response in mice; Blakley BR et al.; Six-week-old female CD-1 mice were administered the n-butylester of 2,4-dichlorophenoxyacetic acid (2,4-D) . The 2,4-D ester was applied dermally at dosages ranging from 0 to 500 mg/kg (2,4-D content) in the acute studies and 0 to 300 mg/kg in the 3 week subacute studies . Following acute exposure, antibody production against sheep red blood cells was suppressed at higher exposure levels . Evidence of clinical toxicity, myotonia and depression, and histopathological alterations in the central nervous system including perivascular edema and ganglial cell necrosis, was also seen in the mice . No alterations were observed in the T- and B-lymphocyte proliferative responses induced by concanavalin A, a T-lymphocyte mitogen, or Escherichia coli lipopolysaccharide, a B-lymphocyte mitogen . Subacute 2,4-D ester exposure which produced minimal clinical or pathological alterations, had no effect on antibody production, but did enhance the B- and T-lymphocyte proliferative responses . The immunosuppressive effects of acute 2,4-D ester exposure which were observed in this study, were unlikely a direct immunological alteration, but rather a secondary manifestation of the clinical syndrome . Since the subacute exposure levels may be more comparable to occupational or environmental exposure, it is unlikely that 2,4-D esters will have any major immunotoxicological significance in agricultural communities.

J Protozool, 1986 Aug, 33(3), 397 - 404
A light and electron microscopical study of a new, polymorphic free-living amoeba, Phreatamoeba balamuthi n . g., n . sp; Chavez LA et al.; A new amoeba, isolated from well water in Gambia, West Africa, is described and named Phreatamoeba balamuthi n . g., n . sp . Requiring anaerobic conditions for growth, it is easily cultured monoxenically with Escherichia coli or axenically in complex, undefined organic media . Three phenotypes have been observed in the life cycle: an amoeba, a flagellate, and a cyst . The amoeba moves by monopodia, is predominantly multinucleate, and varies from 11 to 160 microns in length . The flagellate has a single flagellum and is from 6 to 50 microns long . The cyst is surrounded by a resistant wall that lacks pores and ranges from 9 to 18 microns in diameter . The transformation from amoeba to flagellate can be induced nutritionally, the exact inducing factor(s) being unknown . Sexual reproduction has not been observed.

Laryngoscope, 1986 Aug, 96(8), 899 - 903
Head and neck war injuries: 10-year experience at the American University of Beirut Medical Center; Zaytoun GM et al.; Lebanon has witnessed over the past 10 years fierce outbreaks of violence resulting in heavy casualties . Head and neck injuries secondary to bullets, shrapnel, and/or glass were quite frequent: 1,357 injuries in 1,021 patients were taken care of by members of the Department of Otolaryngology between 1975 and 1984 . They were distributed as follows: (Formula: see text) . Fractures of the mandible were treated by closed reduction in 54% of cases and by open reduction in 46%; 74% healed well and 26% required secondary surgery . Primary repair of oral cavity injuries resulted in healing in 68% of cases; 32% had dehiscences or fistulae . In around one-third of the orbital injuries, the orbital contents herniated into the maxillary sinus, so orbital floor repairs had to be done with good results in 82% of cases . The nasal fractures were treated by closed reduction in 75% of cases and open reduction when the wound was open in the rest . The overall infection rate was 12% . The most common offending organisms were, in order of frequency, S . aureus, P . aeruginosa, and E . coli.

Carcinogenesis, 1986 Aug, 7(8), 1383 - 6
Repair of oligodeoxynucleotides containing O6-methylguanine by O6-alkylguanine-DNA-alkyltransferase; Scicchitano D et al.; O6-Alkylguanine-DNA-alkyltransferase is a DNA repair protein known to carry out the transfer of alkyl groups from the O6-position of guanine in alkylated DNA to a cysteine acceptor site contained within its own protein sequence . We have examined the ability of this protein isolated from either E . coli or mammalian cells to perform this repair reaction in short oligodeoxynucleotides . Dodecadeoxynucleotides of the sequence 5'-dCGNGAATTCm6GCG-3' where N is any one of the normal four bases were all repaired very rapidly by the protein with 50% repair in less than 15 s at 0 degree C . The hexadeoxynucleotide 5'-dCGCm6GCG-3' was repaired slightly more slowly with 50% removal taking 7 min at 0 degree C and 1.5 min at 37 degrees C . The tetradeoxynucleotide 5'-dTm6GCA-3' was also a substrate but was repaired much more slowly requiring 45 min for 50% repair at 37 degrees C . These results indicate that (a) the AGT has a strong but not absolute preference for double-stranded DNA substrates; (b) the repair of O6-methylguanine is independent of the base opposite the lesion; and (c) that oligodeoxynucleotides as short as tetramers are substrates for repair by this protein.

Am Surg, 1986 Aug, 52(8), 413 - 7
The effect of imidazole in endotoxin shock in dogs; VanWylen SJ et al.; The effect of imidazole, a thromboxane A2 inhibitor, in endotoxin shock in dogs was studied . Thromboxane A2 is a vasoconstrictor and enhances platelet and neutrophil aggregation . The purpose of this study was to examine the hemodynamic modifications of endotoxin shock with imidazole . Thirty dogs were studied, all receiving 1 X 10(8) live Escherichia coli/kg over a 30-minute period . The dogs were divided into three groups of ten dogs each . The first group received no treatment . The second group received a concomitant infusion of imidazole 25 mg/kg per hour . In a third group the imidazole infusion was delayed for 1 hour after the E . coli infusion . Mean arterial pressure (MAP), pulmonary wedge pressure (PWP), central venous pressure (CVP), cardiac output (CO), and systemic vascular resistance (SVR) were measured at 30-minute intervals . Hemoglobin (HGB), white blood count (WBC), a-A gradient (a-A grad), A-V 02 difference (AV02 diff), and fluid requirements were measured at 60-minute intervals . The endotoxin shock response in untreated control dogs was characterized by significant changes over time in MAP and SVR (decreased), and CO and a-A grad (increased) . The imidazole group was significantly different, as the MAP and SVR did not decrease and the CO did not increase as in the control group . The control group required significantly more fluids than the imidazole group, while the HGB remained stable suggesting more capillary permeability in the control group . The delayed treatment group was statistically similar to the control animals . These data suggest that imidazole significantly modifies the hemodynamic response to endotoxin shock and may reduce the associated capillary permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Microbiol, 1986 Aug, 132 ( Pt 8), 2337 - 43
Cloning and expression of the pepD gene of Escherichia coli; Klein J et al.; Peptidase D of Escherichia coli, cleaving the unusual dipeptide carnosine, was found to be encoded by the ColE1 hybrid plasmid pLC44-11 . From this plasmid the pepD gene was subcloned into small vectors . As shown by successive reduction of the flanking sequences of genomic DNA, the order of genes in the region at 6 min of the E . coli K12 map is phoE, pepD, in the clockwise orientation . Insertional inactivation of the pepD gene and expression of recombinant plasmids in maxicells allowed the identification of the pepD product as a 52 kDa protein . Comparison with the 100 kDa protein molecular mass determined by gel filtration suggests that active peptidase D is probably a dimer.

J Gen Microbiol, 1986 Aug, 132 ( Pt 8), 2287 - 95
Suppression by the ColV,I-K94 plasmid of a growth lesion in ompA mutants of Escherichia coli; Deeney CM et al.; Organisms of three independently isolated ompA mutants of Escherichia coli failed to form colonies on glucose minimal agar (glucose MA) at 44 degrees C after growth in glucose minimal salts medium at 37 degrees C, although all three strains formed colonies on nutrient agar at 44 degrees C . Supplementation of the glucose MA with individual amino acids including L-methionine and/or L-cysteine did not allow colony formation at 44 degrees C, although addition of 0.1% Casamino acids was effective; replacement of glucose with other energy sources or ammonium ions with glutamate also did not allow growth at 44 degrees C . The failure to form colonies at 44 degrees C was not due to killing of the organisms, because colonies were formed if plates of the ompA mutant initially incubated at 44 degrees C were shifted to 30 degrees C after 16 h . Introduction of the ColV, I-K94 plasmid into P678-54 ompA, 1131 ompA or an ompC ompA mutant suppressed the 44 degrees C growth lesion, but other plasmids (F lac, R483ColIa, RI, ColB-K98, R124) tested in P678-54 ompA did not . Growth of the ColV, I-K94+ derivative at 44 degrees C was due to a suppressing effect of the plasmid rather than to introduction of the plasmid into a variant with normal or altered OmpA protein . An attempt was made to ascertain which component(s) encoded by ColV, I-K94 was (were) responsible for allowing growth at 44 degrees C . Transfer components appeared unlikely to be involved and plasmids which conferred individual colicins (plus the corresponding immunity component) did not suppress.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1986 Aug, 100(2), 425 - 32
Synthesis and characterization of human prorenin in Escherichia coli; Imai T et al.; DNA sequences encoding Ile-Glu-Gly-Arg and human prorenin were joined and placed under the transcriptional control of the Escherichia coli trp promoter-operator in the expression vector pTR501 . E . coli cells transformed with pTR501 expressed high levels (30% of total cell protein) of prorenin as part of a hybrid protein with the trp E gene product . The chimeric protein, accumulated in a sedimentable form, was dissolved in 6 M guanidine X HCl, purified to near homogeneity, and renatured by dialysis . The complete prorenin sequence was then excised from the renatured hybrid protein using blood coagulation factor Xa, a proteinase which is highly specific for the tetrapeptide insert Ile-Glu-Gly-Arg introduced between the 9 amino-terminal residues of the trp E gene product and the first amino acid (Thr 1) of prorenin . Human prorenin thus obtained was readily activatable with trypsin and showed close similarities to naturally occurring prorenin in its biochemical and immunochemical properties.

Mol Biol Med, 1986 Aug, 3(4), 351 - 68
Sorting large numbers of clones expressing Plasmodium falciparum antigens in Escherichia coli by differential antibody screening; Stahl HD et al.; We describe an approach to classifying a large number of clones expressing Plasmodium falciparum antigens in Escherichia coli by virtue of their differing reactivities with 100 human anti-malarial sera . Individual sera exhibited marked differences in the patterns of reactivity with these clones . These patterns led to the identification of sets of clones, here termed "serological families", which were shown to encode distinct P . falciparum antigens . A serological family was found to be composed of non-identical clones derived from portions of the same antigen . Using this approach six new P . falciparum antigens were identified . One of these is described in detail and is a 102 X 10(3) Mr antigen, predominantly of schizonts . Sequencing studies on four cDNA clones encoding parts of this antigen revealed blocks of hydrophilic dipeptide and tripeptide repeats and so the antigen has been termed the acidic basic repeat antigen (ABRA).

Lab Anim Sci, 1986 Aug, 36(4), 370 - 4
Endotoxemia in Yucatan miniature pigs: metabolic derangements and experimental therapies; Fettman MJ; Endotoxemia is a frequent complication of many health disorders . It is characterized by systemic release of a variety of endogenous inflammatory mediators which effect cardiovascular depression, reductions in organ blood flow, tissue ischemia and derangements in cellular metabolism leading to death . During a continuous intravenous infusion of Escherichia coli lipopolysaccharide, the chronology of alterations in hepatosplanchnic blood flow, hepatic carbohydrate metabolism and pancreatic insulin secretion has been studied in awake Yucatan miniature pigs (Sus scrofa) . Endotoxic shock in this model is characterized by reductions in portal venous and hepatic arterial blood flow, early transient increases in pancreatic insulin secretion, increases in the 3H-glucose-derived rates of glucose appearance and disappearance, profound hypoglycemia, hyperlactatemia and metabolic acidosis . Reductions in hepatic oxygen delivery are compensated for by enhanced oxygen extraction efficiency, but hepatic gluconeogenesis continues at an inadequate rate to compensate for increased glucose utilization . Experimental therapies including lidocaine, naloxone, captopril, dichloroacetate and glucagon each effect specific improvements in cardiovascular or metabolic function, but none significantly alter the composite derangements responsible for lethality in this model.

J Appl Bacteriol, 1986 Aug, 61(2), 157 - 61
The effect of water activity on Legionella spp . growth and survival; Danielson RE et al.; Growth and survival of Legionella spp . at various water activity (aw) levels were determined . Compared with Escherichia coli, the growth of Legionella spp . was limited to a high aw environment (greater than or equal to 0.98).

Biol Chem Hoppe Seyler, 1986 Aug, 367(8), 731 - 40
Chemical synthesis and expression of a gene coding for hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis; Bergmann C et al.; A DNA containing the coding sequence for the proteinase inhibitor protein hirudin from the leech Hirudo medicinalis has been obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides . The 226 bp synthetic gene carries signals for the translation initiation and termination . Fragment synthesis was performed by the Khorana ligation method as well as by the fill-in method . Efficiencies of these two methods are compared . The synthetic gene was expressed in E . coli as a fusion protein with beta-galactosidase under the control of the lac-promoter as well as a non-hybrid protein under the control of the lambda PL-promoter . The non-hybrid expression product was shown to have similar biological properties as the authentic protein isolated from the leech.

Biochem Int, 1986 Aug, 13(2), 343 - 50
Partial purification and characterization of fatty acid binding protein(s) in Escherichia coli membranes and reconstitution of fatty acid transport system; Kameda K; Fatty acid binding protein(s) in E . coli membranes was solubilized and partially purified by oleate-AH Sepharose 4B column chromatography . The binding of palmitate to the protein was saturable . The protein bound all fatty acids with chain lengths of 10-18 tested, and its maximum activity was observed with palmitate . The incorporation of the protein into liposomes which contained a system for acyl-CoA synthesis significantly increased the uptake of the extracellular {14C} palmitate by the liposomes in comparison with the liposomes without the protein . The uptake of {14C} palmitate was also saturable, and the accumulated radioactive compound was found to be palmitoyl-CoA.

EMBO J, 1986 Aug, 5(8), 1903 - 11
Identification and characterization of the protein encoded by the human c-myb proto-oncogene; Klempnauer KH et al.; We have identified the product of the human c-myb proto-oncogene as a 80,000-Mr protein, p80c-myb, by using polyclonal and monoclonal antibodies raised against a bacterially synthesized polypeptide from the amino terminus of the viral myb protein . p80c-myb shares at least two distinct antigenic sites with the amino terminal region of the v-myb protein . p80c-myb is found only in hematopoietic cells or in cells that contain amplified c-myb genes . Like the chicken myb proteins, p80c-myb is a nuclear DNA-binding protein that is predominantly associated with chromatin and exhibits a short half-life of approximately 1 hour.

Genetics, 1986 Aug, 113(4), 811 - 9
DNA sequence analysis of mutagenicity and site specificity of ethyl methanesulfonate in Uvr+ and UvrB- strains of Escherichia coli; Burns PA et al.; EMS-induced mutations within a 180 base pair region of the lacI gene of E . coli were cloned and sequenced . In total, 105 and 79 EMS-induced mutations from a Uvr+ and a UvrB- strain, respectively, were sequenced . The specificity of EMS-induced mutagenesis was very similar in the two strains; G:C----A:T transitions accounted for all but three of the mutants . The overall frequency of induced mutation was fivefold higher in the UvrB- strain compared to the Uvr+ strain . This demonstrates, at the DNA sequence level, that the presumed premutagenic lesion, O6-ethylguanine, is subject to repair by the uvrABC excision repair system of E . coli . An analysis of mutation frequencies with respect to neighboring base sequence, in the two strains, shows that O6-ethylguanine lesions adjacent to A:T base pairs present better targets for the excision repair machinery than those not adjacent to A:T base pairs.

DNA, 1986 Aug, 5(4), 325 - 32
Oligodeoxynucleotide-directed mutagenesis of Escherichia coli and yeast by simple cotransformation of the primer and template; Burke DT et al.; A method of oligodeoxynucleotide-directed mutagenesis is presented which requires no in vitro DNA synthesis . Cotransformation of the synthetic primer and single-stranded template into competent spheroplasts of Escherichia coli or Saccharomyces cerevisiae generates the directed mutation . The desired event is detected genetically or by hybridization screening using the mutagenic oligodeoxynucleotide as a probe . The targeted mutation arises at a frequency of approximately 0.1% . In one extensively studied case in E . coli, involving creation of a 99-bp deletion, this procedure produced many fewer untargeted mutation events than did conventional protocols.

Transplant Proc, 1986 Aug, 18(4), 959 - 62
Staghorn calculus in a renal allograft; Jain AB et al.; A case of staghorn calculus in a transplanted kidney associated with tertiary hyperparathyroidism in the presence of infection has been described . Possible mechanisms of pathophysiology have been discussed.

Surgery, 1986 Aug, 100(2), 349 - 55
Impaired metabolic response to endotoxin in obstructive jaundice; Warren RS et al.; Surgical management of extrahepatic cholestasis is frequently complicated by sepsis, which can be explained in part by diminished function of the reticuloendothelial system . We have explored the possibility that the metabolic response to infection may also be abnormal . Fischer 344 rats underwent either bile duct ligation (BDL) or sham operation and were studied 3 days after operation . Hepatic amino acid uptake measured in vivo by the accumulation of 14C-alpha-aminoisobutyric acid or in vitro by the rate of transport of 14C-alanine by isolated hepatocytes was unaltered in the BDL animals, while gluconeogenesis from alanine by viable hepatocytes from BDL rats was actually enhanced . However, the expected increase in hepatic amino acid uptake in response to endotoxin was diminished in the BDL animals . In addition, we observed impaired responses of the jaundiced animals to glucagon and interleukin-1, two mediators of the hepatic acute phase response to endotoxin . These data suggest that while hepatic amino acid transport is normal in the basal state, the rat with extrahepatic biliary obstruction does not respond appropriately to stress and that this defect cannot be explained solely on the basis of altered handling of endotoxin by the reticuloendothelial system.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 6040 - 4
Spermidine synthase of Escherichia coli: localization of the speE gene; Tabor CW et al.; We have obtained Escherichia coli mutants lacking spermidine synthase (putrescine aminopropyltransferase) and have found that the mutated gene (speE) is located immediately upstream from the gene coding for S-adenosylmethionine decarboxylase (speD); these genes are located at 2.7 minutes on the E . coli chromosome . Both genes are present in a 1795-base-pair fragment of E . coli DNA that was cloned into pBR322 . Deletion of 105 bases upstream of speE caused a coordinate loss of both activities, indicating that speE and speD constitute a single operon . speE and speD have also been cloned separately in a high-expression vector; strains carrying these plasmids overproduce the respective enzymes.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5934 - 8
Effect of amino acid substitutions on the catalytic and regulatory properties of aspartate transcarbamoylase; Robey EA et al.; Although intensive investigations have been conducted on the allosteric enzyme, aspartate transcarbamoylase, which catalyzes the first committed reaction in the biosynthesis of pyrimidines in Escherichia coli, little is known about the role of individual amino acid residues in catalysis or regulation . Two inactive enzymes produced by random mutagenesis have been characterized previously but the loss of activity is probably attributable to changes in the folding of the chains stemming from the introduction of charged and bulky residues (Asp for Gly-128 and Phe for Ser-52) . Site-directed mutagenesis of pyrB, which encodes the catalytic chains of the enzyme, was used to probe the functional roles of several amino acids by making more conservative substitutions . Replacement of Lys-84 by either Gln or Arg leads to virtually inactive enzymes, confirming chemical studies indicating that Lys-84 is essential for catalysis . In contrast, substitution of Gln for Lys-83 has only a slight effect on enzyme activity, whereas chemical modification causes considerable inactivation . Gln-133, which has been shown by x-ray crystallography to reside near the contact region between the catalytic and regulatory chains, was replaced by Ala . This substitution has little effect on catalytic activity but leads to a marked increase in cooperativity . The Gln-83 mutant, in contrast, exhibits much less cooperativity . Since a histidine residue may be involved in catalysis and His-134 has been shown by x-ray diffraction studies to be in close proximity to the site of binding of a bisubstrate analog, His-134 was replaced by Ala, yielding a mutant with only 5% wild-type activity, considerable cooperativity, and lower affinity for aspartate and carbamoylphosphate . All of the mutants, unlike those in which charged or bulky residues replaced small side chains, bind the bisubstrate analog, which promotes the characteristic "swelling" of the enzymes indicative of the allosteric transition.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5904 - 8
Affinity chromatography with an immobilized RNA enzyme; Vioque A et al.; M1 RNA, the catalytic subunit of Escherichia coli RNase P, has been covalently linked at its 3' terminus to agarose beads . Unlike M1 RNA, which is active in solution in the absence of the protein component (C5) of RNase P, the RNA linked to the beads is active only in the presence of C5 protein . Affinity chromatography of crude extracts of E . coli on a column prepared from the beads to which the RNA has been crosslinked results in the purification of C5 protein in a single step . The protein has been purified in this manner from cells that contain a plasmid, pINIIIR20, which includes the gene that codes for C5 protein . A 6-fold amplification of the expression of C5 protein is found in these cells after induction as compared to cells that do not harbor the plasmid.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5866 - 70
Importance of the loop at residues 230-245 in the allosteric interactions of Escherichia coli aspartate carbamoyltransferase; Middleton SA et al.; Site-directed mutagenesis has been used to replace tyrosine-240 with phenylalanine in each of the catalytic chains of aspartate carbamoyltransferase . Tyrosine-240 is part of a loop in the structure of the enzyme, between residues 230 and 245, which undergoes a substantial conformation change as the enzyme becomes ligated {Krause, K . L., Volz, K . W . & Lipscomb, W . N . (1985) Proc . Natl . Acad . Sci . USA 82, 1643-1647} . The mutant enzyme with phenylalanine at position 240 has substantially reduced homotropic interactions and an increased affinity for the substrate aspartate but displays no alteration in maximal observed specific activity . The Hill coefficient decreases from 2.4 for the wild-type enzyme to 1.8 for the mutant, and the aspartate concentration at half the maximal observed velocity decreases from 11.9 mM to 4.7 mM at pH 8.3 . Heterotropic interactions of the mutant enzyme are altered to a lesser extent . The catalytic subunit derived from the mutant enzyme exhibits kinetics identical to that of the wild-type catalytic subunit . Reactivity of the mutant enzyme with p-hydroxymercuribenzoate suggests that the unligated enzyme exists in an altered conformation . The properties of the mutant enzyme are explained in terms of the structure of the wild-type enzyme, and a model is proposed to account for the allosteric interactions of the wild-type enzyme in terms of specific interactions involving the 230-245 loop of the enzyme.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5854 - 7
Autogenous regulation of splicing of the transcript of a yeast ribosomal protein gene; Dabeva MD et al.; The gene for a yeast ribosomal protein, RPL32, contains a single intron . The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript . This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5723 - 6
An inhibitor of SOS induction, specified by a plasmid locus in Escherichia coli; Bagdasarian M et al.; Plasmid R6-5 contains a locus whose product inhibits induction of sfiA and prophage lambda in a recA441 mutant at 42 degrees C and in a recA+ host after treatment with nalidixic acid . This plasmidic SOS-inhibition locus (psi) is situated on an 8.1-kilobase DNA fragment near oriT, the origin of plasmid R6-5 conjugational transfer . Loss of the Psi function, resulting from the insertion of Tn3 into psi+, greatly reduced the synthesis of two proteins, designated PsiA (Mr 24,500) and PsiB (Mr 12,500) . Using host cells in which there was an inactive LexA repressor, we found that Psi function does not act by interfering with the expression of the SOS pathway . The Psi function may affect the generation of an SOS signal . We postulate that during the course of evolution, the Psi function has been selected in some conjugative plasmids so as to permit them to transfer single-stranded DNA without generating an SOS signal.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5607 - 10
Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes; Samson L et al.; The ada-alkB operon protects Escherichia coli against the effects of many alkylating agents . We have subcloned it into the pSV2 mammalian expression vector to yield pSV2ada-alkB, and this plasmid has been introduced into Mer- HeLa S3 cells, which are extremely sensitive to killing and induction of sister chromatid exchange by alkylating agents . One transformant (the S3-9 cell line) has several integrated copies of pSV2ada-alkB and was found to express a very high level of the ada gene product, the 39-kDa O6-methylguanine-DNA methyltransferase . S3-9 cells were found to have become resistant to killing and induction of sister chromatid exchange by two alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine and N,N'-bis(2-chloroethyl)-N-nitro-sourea . This shows that bacterial DNA alkylation-repair genes are able to suppress the alkylation-repair defects in human Mer- cells.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5558 - 62
recD: the gene for an essential third subunit of exonuclease V; Amundsen SK et al.; Exonuclease V (EC 3.1.11.5) of Escherichia coli, an enzyme with multiple activities promoting genetic recombination, has previously been shown to contain two polypeptides, the products of the recB and recC genes . We report here that the enzyme contains in addition a third polypeptide (alpha) with a molecular mass of about 58 kDa . The alpha polypeptide is not synthesized by a class of mutants (previously designated recB) lacking the nuclease activity of exonuclease V but retaining recombination proficiency . The gene, recD, coding for the alpha polypeptide is located near recB in the order thyA-recC-ptr-recB-recD-argA on the E . coli chromosome . The recB and recD genes appear to be governed by a common promoter to the left of recB; a weaker promoter appears to govern recD alone . In the light of these results we discuss the relation between the structure and function of the three polypeptides of exonuclease V, hereby alternatively designated RecBCD enzyme.

Mutat Res, 1986 Aug, 174(4), 265 - 9
Escherichia coli xthA mutant is not hypersensitive to ascorbic acid/copper treatment--an H2O2 generating reaction; Van Sluys MA et al.; Ascorbate (vitamin C) in the presence of copper yields H2O2, which seems to be responsible for its toxic effects in bacteria . However, we found that the Escherichia coli xthA mutant strain, which is hypersensitive to H2O2, has almost the same sensitivity as the wild-type strain to ascorbate and copper treatment . Our results suggest that the DNA damage induced in E . coli by H2O2 generated in oxidized ascorbate solutions is different from that induced by direct H2O2 treatment.

Lab Invest, 1986 Aug, 55(2), 164 - 76
Repeated Escherichia coli endotoxin-induced pulmonary inflammation causes chronic pulmonary hypertension in sheep . Structural and functional changes; Meyrick B et al.; Chronic pulmonary hypertension occurs in several human diseases in which there is evidence of chronic or repeated bouts of pulmonary inflammation . To determine whether prolonged lung inflammation causes persistent pulmonary hypertension Escherichia coli endotoxin was given to seven chronically instrumented awake sheep three times a week for 10 to 14 weeks . Pulmonary artery, left atrial and systemic arterial pressures, cardiac output, arterial blood gases and pH were monitored before starting endotoxin treatment and twice weekly, immediately before endotoxin infusion . Three sheep receiving saline over a similar time period served as controls . Pulmonary vasoreactivity to breathing 12% oxygen and a bolus infusion of an analog of prostaglandin H2 was also assessed . Peripheral lung biopsy tissue was taken at baseline and at periods throughout the experiment to assess pulmonary inflammation . Repeated endotoxin infusions resulted in a significant increase in mean pulmonary artery pressure from the 8th week of treatment and more than a 50% increase from week 10 (baseline = 18.4 cm H2O +/- 1.0 (mean +/- SE); 10 weeks endotoxin = 27.8 +/- 4.3; p less than 0.05) . Pulmonary vasoreactivity to both an analog of prostaglandin H2 and 12% oxygen decreased in the period from 4 to 8 weeks of endotoxin treatment . Light microscopic assessment of lung biopsy tissue showed a persistent four-fold increase above baseline in number of peripheral lung granulocytes . Electron microscopy revealed that granulocytes, lymphocytes, and monocytes sequestered in the lungs of these animals, and that structural damage to the endothelium was minimal . Morphometry of lungs obtained at autopsy in which the pulmonary arteries had been distended with barium-gelatin showed extension of muscle into the walls of smaller intra-acinar arteries (than normal) and a reduction in number of filled peripheral arteries . We conclude that repeated infusions of endotoxin into sheep cause persistent lung inflammation, altered pulmonary vasoreactivity, sustained pulmonary hypertension, and some of the structural changes characteristic of this disease . Chronic inflammation may play a role in the pathogenesis of chronic pulmonary hypertension.

J Med Chem, 1986 Aug, 29(8), 1329 - 40
Streptonigrin . 1 . Structure-activity relationships among simple bicyclic analogues . Rate dependence of DNA degradation on quinone reduction potential; Shaikh IA et al.; A series of simple aza and diaza bicyclic quinones related to the AB ring system of streptonigrin (1) have been synthesized and tested in vitro for their ability to degrade DNA under conditions similar to those used with the parent drug . The results obtained from a study of 22 quinones indicate that there is a quantitative linear relationship between their reduction potentials and the rate at which they degrade DNA under identical conditions in vitro . Almost all of the synthetic substances were superior to 1 in their DNA-degrading ability.

J Bacteriol, 1986 Aug, 167(2), 703 - 10
Genetic organization of plasmid R1162 DNA involved in conjugative mobilization; Brasch MA et al.; DNA involved in the mobilization of broad-host-range plasmid R1162 was localized to a region of 2.7 kilobases within coordinates 3.4 to 6.1 kilobases on the R1162 map . By examining the transfer properties of plasmids containing cloned fragments of DNA from within this region, we showed that at least four trans-active products and a cis-active site (oriT) were involved in mobilization . A cloned DNA fragment of 155 base pairs was capable of providing full oriT activity . This fragment was located within 600 base pairs of DNA containing the origin of replication of R1162, and its nucleotide sequence and that of neighboring DNA were determined . Activation of oriT required R1162-encoded, trans-acting products . Deletions which resulted in the loss of one or more of these had a variable effect on transfer efficiency and indicated the presence of both essential and nonessential Mob products . Regions encoding these products flanked oriT and in one case appeared to overlap a gene essential for plasmid replication . The implications of these findings with respect to the broad host range of R1162 are discussed.

J Bacteriol, 1986 Aug, 167(2), 556 - 61
Molecular analysis of the promoter operator region of the Escherichia coli K-12 tyrP gene; Kasian PA et al.; The nucleotide sequence of the tyrP promoter region from Escherichia coli has been determined . Two TYR R boxes have been identified, and one of these was shown to overlap the -35 region of a major tyrP promoter (p1) . S1 nuclease mapping of in vivo transcripts revealed that transcription from p1 is stimulated by phenylalanine and to a lesser extent by leucine . The demonstration that mutants in which TyrR-tyrosine-mediated repression of tyrP has been abolished have single base changes in the TYR R box which overlaps p1 suggests that TyrR-tyrosine-mediated repression of tyrP also involves p1 . TyrR-independent stimulation of tyrP expression by Casamino Acids involves a second promoter 140 bases upstream of p1 . There are no TYR R boxes in this region . The sequences of 10 TYR R boxes preceding the genes tyrP, tyrR, and aroG and the operons aroF tyrA and aroL aroM are compared and discussed.

Int J Radiat Biol Relat Stud Phys Chem Med, 1986 Aug, 50(2), 191 - 203
UV-type damage associated with ionizing radiation: a review; Redpath JL; The induction of UV-type damage by ionizing radiation in repair deficient strains of E . coli is reviewed . Both photoreactivable and non-photoreactivable types of damage can be observed . The induction of UV-type damage is largely independent of the presence of free-radical reactive agents (e.g . oxygen and thiols), but is dependent upon the energy of the photon--or electron--beam used, the radiation geometry and the optical absorbance of the extracellular medium . On the basis of calculations and experimental evidence, it is clear that one mechanism whereby such damage arises is through the generation of Cerenkov emission . However, small yields of UV-type damage can be produced using X-rays whose energy is below the threshold for production of Cerenkov emission . In this instance, the damage induction mechanism is thought to involve a direct excitation process.

Infect Immun, 1986 Aug, 53(2), 445 - 7
Characterization of heat-stable enterotoxin from a hypertoxigenic Escherichia coli strain that is pathogenic for cattle; Saeed AM et al.; An enterotoxigenic Escherichia coli (ETEC) strain isolated from a calf with clinical scours was found to produce over 17- to 60-fold more heat-stable enterotoxin (STa) than four laboratory-adapted bovine ETEC strains . The purified STa of this strain was identical to those produced by other ETEC strains . A severe form of scours was induced in 5- to 15-day-old colostrum-fed calves and in 1- to 2-week-old piglets by oral administration of the purified STa . This study demonstrates that STa is a mediator of diarrhea in newborn calves and piglets and that under identical growth conditions diverse strains of bovine ETEC may produce variable amounts of homologous STa's.

Infect Immun, 1986 Aug, 53(2), 435 - 7
Composition of affinity-purified alpha-hemolysin of Escherichia coli; Bohach GA et al.; Escherichia coli alpha-hemolysin was purified from culture supernatants by affinity chromatography, using a hemolysis-neutralizing monoclonal antibody ligand . Purified hemolysin contains several proteins and lipopolysaccharides . Thus, alpha-hemolysin exists as a macromolecular complex and may be exported from E . coli cells by outer membrane fragmentation.

Infect Immun, 1986 Aug, 53(2), 339 - 46
Natural and experimental infection with an attaching and effacing strain of Escherichia coli in calves; Moxley RA et al.; Gnotobiotic calves were inoculated with an O5:K4:H-, urease-positive strain of Escherichia coli isolated from a 2-day-old calf with diarrhea . The calves developed elevated temperatures and passed loose mucoid feces, with or without blood . The E . coli strain was negative for heat-stable and heat-labile enterotoxins but produced high levels of Shiga-like toxin . Bacteria attached diffusely to the epithelium of the large intestine and multifocally to the epithelium of the ileum . The duodenum and jejunum were not affected . At the sites of bacterial attachment, microvilli were effaced, enterocytes were degenerate, and necrosis and exfoliation had occurred . These results confirm a previous report from England that calves may naturally contract infections similar to those caused by enteropathogenic E . coli strains pathogenic to humans or rabbits . This suggests that the calf bacterial strains, like some enteropathogenic E . coli strains, produce high levels of Shiga-like toxin and cause attachment and effacement lesions in the colonic epithelium of the infected host.

Eur J Biochem, 1986 Aug 1, 158(3), 647 - 53
Nucleotide sequence of the pntA and pntB genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli; Clarke DM et al.; A 3240-base-pair DNA fragment spanning the pyridine nucleotide transhydrogenase (pnt) genes of Escherichia coli has been sequenced . The sequence contains two open-reading frames, pntA and pntB of 1506 and 1386 base pairs, coding for the transhydrogenase alpha and beta subunits, respectively . The coding sequences are preceded by a promoter-like structure and are most likely co-transcribed . Each coding sequence is preceded by a Shine-Dalgarno sequence . The amino-terminal amino acid sequences were determined from the purified alpha and beta subunits of the transhydrogenase . These sequences agree with those predicted from the nucleotide sequences of the pntA and pntB genes . The predicted relative molecular masses of 53906 (alpha) and 48667 (beta) are close to the values obtained by analysis of the subunits by sodium dodecyl sulfate/polyacrylamide gel electrophoresis . Several hydrophobic regions large enough to span the cytoplasmic membrane were observed in each subunit . These results indicate that transhydrogenase is an intrinsic membrane protein.

Eur J Biochem, 1986 Aug 1, 158(3), 575 - 9
Visualization of intermediary transcription states in the complex between Escherichia coli DNA-dependent RNA polymerases and a promoter-carrying DNA fragment using the gel retardation method; Heumann H et al.; DNA-dependent RNA polymerase in complex with a DNA fragment was analyzed by electrophoresis in non-denaturing gels as core enzyme, holoenzyme, during initiation and elongation . The DNA fragment carried the promoter A1 of the phage T7 . The stoichiometry between holoenzyme and promoter and between sigma and core enzyme in complex with DNA was determined . Holoenzyme bound as a monomer to the DNA, whereas core enzyme formed aggregates before binding to the DNA . If the molar ratio of holoenzyme to DNA exceeded 0.5:1 a second holoenzyme molecule interacted with the DNA fragment with diminished affinity . A large difference in the frictional coefficient of the holoenzyme-promoter and the core enzyme-DNA complex indicated a drastic conformational difference between the two types of complexes . The stability of the holoenzyme-promoter complex decreased with decreasing temperature, accompanied by at least partial dissociation of holoenzyme into core enzyme and sigma factor . Addition of nucleoside triphosphates did not change the electrophoretic mobility of the complex if abortive transcription only was allowed, but increased it after addition of all four nucleoside triphosphates owing to release of the sigma factor.

Eur J Biochem, 1986 Aug 1, 158(3), 483 - 90
Activation in vitro of respiratory nitrate reductase of Escherichia coli K12 grown in the presence of tungstate . Involvement of molybdenum cofactor; Saracino L et al.; The chlorate-resistant (chlR) mutants are pleiotropically defective in molybdoenzyme activity . The inactive derivative of the molybdoenzyme, respiratory nitrate reductase, present in the cell-free extract of a chlB mutant, can be activated by the addition of protein FA, the probable active product of the chlB locus . Protein FA addition, however, cannot bring about the activation if 10 mM sodium tungstate is included in the culture medium for the chlB strain . The inclusion of a heat-treated preparation of a wild-type or chlB strain prepared after growth in the absence of tungstate, restores the protein-FA-dependent activation of nitrate reductase . All attempts to activate nitrate reductase in extracts prepared from tungstate-grown wild-type Escherichia coli strains failed . It appears that during growth with tungstate, the possession of the active chlB gene product leads to the synthesis of a nitrate reductase derivative which is distinct from that present in the tungstate-grown chlB mutant . Heat-treated preparations from chlA and chlE mutants which do not possess molybdenum cofactor activity fail to restore the activation . Fractionation by gel filtration of the heat-treated preparation from a wild-type strain produced two active peaks in the eluate of approximate Mr 12000 and less than or equal to 1500 . The active material in the heat-treated extract was resistant to exposure to proteinases, but after such treatment the active component, previously of approximate Mr 12000, eluted from the gel filtration column with the material of Mr less than or equal to 1500 . The active material is therefore of low molecular mass and can exist either in a protein-bound form or in an apparently free state . Molybdenum cofactor activity, assayed by the complementation of the apoprotein of NADPH:nitrate oxidoreductase in an extract of the nit-1 mutant of Neurospora crassa, gave a profile following gel filtration similar to that of the ability to restore respiratory nitrate reductase activity to the tungstate-grown chlB mutant soluble fraction . This was the case even after proteinase treatment of the heat-stable fraction . Analysis of the chlC (narC) mutant, defective in the structural gene for nitrate reductase, revealed that heat treatment is not necessary for the expression of the active component . Furthermore both the active component and molybdenum cofactor activity are present in corresponding bound and free fractions in the non-heat-treated soluble subcellular fraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood, 1986 Aug, 68(2), 351 - 4
Increased infection mortality and decreased neutrophil migration due to a component of an artificial blood substitute; Lane TA et al.; We previously showed that an artificial blood substitute containing perfluorocarbons, Fluosol-DA, inhibited both neutrophil migration and adherence, due to its detergent component, Pluronic F-68 . The purpose of the studies we report here was to determine if Fluosol or Pluronic might also reduce in vivo neutrophil migration and impair host resistance to bacterial infection . We studied in vivo PMN migration by injecting mice intraperitoneally (IP) with glycogen, followed by intravenous (IV) infusion of saline, Fluosol, or Pluronic . Peritoneal lavage after eight hours showed a significant decrease in the accumulation of PMN in lavage fluids of animals given either Fluosol or Pluronic (control--.19 +/- .03 X 10(6) PMN/mL, glycogen--1.35 +/- .14; glycogen/Fluosol--0.63 +/- .12; glycogen/Pluronic--0.69 +/- .07) . We ascertained the effect of Fluosol and Pluronic on infection mortality by injecting mice IV with saline, Fluosol, or Pluronic, followed by a quantity of E coli (0.6 X 10(7} IP shown in preliminary studies to kill 20% to 50% of the mice in 24 hours . The 24-hour mortality was 14/45-saline, 24/32-Fluosol (chi 2 = 17.1; P less than .001) and 17/23 - Pluronic (chi = 11.2; P less than .001) . Neither Fluosol nor Pluronic caused mortality without E coli . The increase in infection mortality occurred when Fluosol was given either two hours before, or simultaneously with E coli, but only with the simultaneous administration of bacteria and Pluronic . Pluronic did not alter reticuloendothelial system (RES) clearance function . These studies indicate that, in an animal model, Fluosol-DA, due to its detergent component Pluronic F-68, impaired neutrophil delivery to an inflammatory locus, and resulted in an increased rate of infection mortality . Since Pluronic did not result in RES blockade, but did impair the delivery of PMN to an inflammatory locus, our results suggest that the latter effect is responsible for the increase in infection mortality.

Science, 1986 Aug 1, 233(4763), 561 - 3
Self-assembling cytotoxins; Rideout D; Decanal and N-amino-N'-1-octylguanidine (AOG), combined at 28 microM each, mediated erythrocyte lysis within 80 minutes under physiological conditions . By contrast, no lysis was observed after 20 hours with either decanal (56 microM) or AOG (100 microM) alone . The pronounced synergism observed for these chemicals and similar reactive pairs of chemicals is due to the self-assembly of more cytotoxic hydrazones in situ . Decanal and AOG also exhibit synergistic activity against cultured human cells (HeLa) and bacteria (Escherichia coli J96) . This synergism may be useful in the design of cytotoxins that would self-assemble selectively from nontoxic precursors within tumors, while sparing normal tissue.

J Immunol, 1986 Aug 1, 137(3), 1054 - 9
Cyclophilin: distribution and variant properties in normal and neoplastic tissues; Koletsky AJ et al.; The cytosolic concentration, Mr, and isoforms of cyclophilin (CyP), a specific cytosolic binding protein for cyclosporin A (CsA), were determined in normal and neoplastic human tissues as well as tissues from species of diverse phylogeny . CyP was present in all tissues examined; however, concentrations varied significantly among different tissue types . The CyP concentration was highest in lymphoblasts from a patient with T cell acute lymphocytic leukemia (1.15 micrograms/mg protein) and Hodgkin's and non-Hodgkin's lymphomas . CyP concentration in colon adenocarcinomas was twofold to threefold greater than that found in adjacent normal tissue . CyP from all normal and neoplastic human tissues examined had an apparent Mr of 17,000 determined by gel filtration HPLC . Major (pI 8.6 to 8.7) and minor (pI 6.7 to 6.9) CyP isoforms were identified in all human and murine tissue extracts by column sucrose gradient isoelectrofocusing; however, the ratio of the major to minor isoform varied widely . Among other species examined, significant concentrations of CyP were detected in cytosol extracts from sponges (Microciona prolifera), yeast (Saccharomyces cerevisiae), mushrooms, the giant cockroach (Blaberus discoidalis), and a trematode (Schistosoma mansoni) . By contrast, CyP was not detectable in extracts of Escherichia coli . A twofold to threefold elevation in the CyP content of murine splenocytes was detected 72 hr after Con A stimulation . A survey of a variety of natural products, synthetic compounds, and immunoregulating agents has failed thus far to identify compounds capable of competing with CsA for binding to CyP . The broad tissue and phylogenetic distribution of CyP, its highly conserved structure, and its increased content after mitogenic stimulation suggest a fundamental role in cellular metabolism.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5534 - 8
Isolation of cDNA clones for differentially expressed genes of the human parasite Schistosoma mansoni; Davis AH et al.; Little is known about the mechanisms that control transformations during the life cycle of Schistosoma mansoni . To enable isolation of DNA sequences encoding developmentally regulated antigens a cDNA expression library in the vector lambda gt11 amp3 was constructed from adult mRNA and immunologically screened with sera from infected individuals . We report here on the properties of three recombinant clones that derive from developmentally regulated genes . Clone 10-3 encoded a beta-galactosidase fusion protein present in high abundance in infected Escherichia coli . Clones 7-2 and 8-2 also produced immunologically recognized proteins; however, the peptides did not appear to be beta-galactosidase fusion proteins . The expression of mRNAs hybridizing to these cDNAs was examined in the different stages of the parasite life cycle . Messenger RNA corresponding to clone 10-3, approximately equal to 1000 bases in length, was present in higher abundance in male worms than in females but was not detected in schistosome eggs . A 900-base mRNA hybridizing to clone 7-2 was observed in adult worms and eggs . Both clone 10-3 and clone 7-2 hybridized to smaller mRNAs in cercariae and freshly transformed schistosomula than in adult worms . Clone 8-2 contained tandem cDNA inserts . One cDNA hybridized to a 1700-base mRNA present in all stages, while the second hybridized to an 800-base mRNA specific to adult female worms.

Biochim Biophys Acta, 1986 Aug 1, 887(3), 283 - 90
Cathepsin B synthesis by the HL60 promyelocytic cell line: effects of stimulating agents and anti-inflammatory compounds; Burnett D et al.; Cathepsin B synthesis by the human HL60 promyelocyte cell line was investigated by immunohistochemistry and by the assay of the enzyme in cell lysates using a fluorimetric substrate . HL60 cells were shown to produce cathepsin B in response to treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) . Intracellular levels of cathepsin B and immunohistochemical staining of the enzyme were related to time in culture with increasing concentrations of TPA from 1 nmol/1 to 8.0 nmol/1 . Synthesis of cathepsin B was associated with TPA-induced phagocytic activity of cells in culture, expression of alpha-naphthyl acetate esterase and reduced cell division . Cathepsin B production was, therefore, related to differentiation of the HL60 promyelocytes into mature macrophage-like cells . Cathepsin B activity in HL60 cell lysates was significantly increased by incubation of the cells with 10 micrograms/ml endotoxin (lipopolysaccharide) from Escherichia coli, but not carrageenan . The production of cathepsin B by TPA-induced HL60 cells was significantly reduced by 0.25 mumol/1 dexamethasone and the non-steroidal anti-inflammatory compound 4-(6-methoxy-2-naphthyl)-butan-2-one but not by indomethacin . The HL60 promyelocytic cell line is a useful model for the study of factors affecting proteinase synthesis by human mononuclear phagocytes.

J Gen Microbiol, 1986 Aug, 132 ( Pt 8), 2297 - 307
beta-Galactosidase and alkaline phosphatase do not become extracellular when fused to the amino-terminal part of colicin N; Pugsley AP et al.; A series of plasmids encoding hybrid proteins comprising various lengths of the NH2-terminal region of colicin N coupled to almost complete beta-galactosidase or alkaline phosphatase polypeptides was constructed by transposon mutagenesis of ColN plasmid derivatives . Synthesis of the hybrid proteins, like that of colicin N itself, was regulated by the SOS response . Large quantities of the hybrid proteins accumulated in the cytoplasm (beta-galactosidase) or particulate fractions (alkaline phosphatase) . When the gene fusions were expressed in cells that were producing colicin E2 and expressing the ColE2 lysis gene, only very low levels of the hybrid proteins were found in the medium . The results suggest that the amino-terminal part of colicin N does not contain sufficient biochemical information to promote the release of the hybrid proteins into the medium.

Genetika, 1986 Aug, 22(8), 2055 - 65
{Genetic analysis of Escherichia coli K-12 mutants defective for the structural and regulatory genes for second purine nucleoside phosphorylase}; Kocharian ShM et al.; Two types of mutants lacking the second purine nucleoside phosphorylase (PNPase 2) activity were isolated using the Escherichia coli K-12 pndR strains with constitutive or inosine-inducible synthesis of the PNPase 2 . The mutations of the first type are recessive to the pndR+ allele on the F' episome . They are closely linked to the original pndR+ mutations and therefore affect the pndR gene encoding the activator protein . The mutations of the second type affect the PNPase 2 structural gene (pndA) and are recessive to the pndA+ allele on the F' episome . The nupC-pndR-pndA-ptsH-cysA gene order was established by means of four- and five-factorial transductional crosses.

Mol Gen Genet, 1986 Aug, 204(2), 243 - 8
Characterization of DNA uptake by the cyanobacterium Anacystis nidulans; Daniell H et al.; The binding and uptake of nick-translated 32P-labeled pBR322 by Anacystis nidulans 6301 have been characterized . Both processes were considerably enhanced in permeaplasts compared to cells . The breakdown of labeled DNA was not correlated with binding or uptake by permeaplasts or cells . Uptake of DNA by permeaplasts was unaffected by: Mg2+ or Ca2+, light, or inhibitors of photophosphorylation such as valinomycin or gramicidin D in the presence or absence of NH4Cl . ATP at 2.5-10 mM inhibited both binding and uptake of labeled DNA by permeaplasts of A . nidulans whereas the ATP analog adenyl-5-yl imido-diphosphate was non-inhibitory in the same concentration range . In contrast to transformation of A . nidulans 6301 cells to ampicillin-resistance by pBR322, transformation to kanamycin-resistance by the plasmid pHUB4 was considerably enhanced in the dark . The transformation efficiency for permeaplasts by the plasmid pCH1 was 59% and 8% in the dark and light, respectively, whereas transformation of permeaplasts by pBR322 at an efficiency of 16% was absolutely light-dependent.

J Pathol, 1986 Aug, 149(4), 307 - 13
An experimental study into the cause of acute haemorrhagic gastritis in cirrhosis; Shibayama Y; To clarify the role of endotoxaemia and congestion of the stomach in the development of acute haemorrhagic gastritis in cirrhotic patients and to investigate the mechanisms of gastric mucosal haemorrhage, the present study was undertaken using rats . Congestion of the stomach was produced by the ligation of gastric veins . Congestion of the stomach or endotoxaemia could not produce gastric mucosal haemorrhage by itself . However, petechial haemorrhage was induced when endotoxin was given to the rats with congestion of the stomach, and the gastric mucosal haemorrhage was largely prevented by administration of gabexate mesilate, an anti-kallikrein drug . Administration of bromelain, which releases prekallikrein and high molecular weight kininogen, instead of endotoxin, also induced gastric mucosal haemorrhage . These findings suggest that the cause of acute haemorrhagic gastritis may be the coexistence of endotoxaemia and congestion of the stomach due to liver cirrhosis and portal hypertension . The mechanisms of the haemorrhage may be as follows: Endotoxin-induced bradykinin acts on the dilated capillaries and small veins in the mucosa and markedly increases their permeability.

Carbohydr Res, 1986 Aug 1, 150, 233 - 40
Structure of the serine-containing capsular polysaccharide K40 antigen from Escherichia coli O8:K40:H9; Dengler T et al.; The structure of the K40 antigenic capsular polysaccharide (K40 antigen) of E . coli O8:K40:H9 was elucidated by determination of the composition, 1H- and 13C-n.m.r . spectroscopy, periodate oxidation and Smith degradation, and methylation analysis . The K40 polysaccharide consists of {(O-beta-D-glucopyranosyluronic acid)-(1----4)-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1----6)-O -(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)} repeating units . All of the glucuronic acid residues are substituted amidically with L-serine.

Am Rev Respir Dis, 1986 Aug, 134(2), 205 - 9
Antibody-based enzyme-linked immunosorbent assay for determination of immune complexes in clinical tuberculosis; Bhattacharya A et al.; Immune complexes were isolated from sera of tuberculosis patients by precipitation with 2.5% polyethylene glycol . The precipitates were characterized by quantitative determination of different immunoglobulin classes by single radioimmunodiffusion, sodium dodecyl sulfate polyacrylamide gel electrophoresis for presence of serum components, Ouchterlony's double diffusion method for detection of complement components C3 and C4, and immuno-dot assay for detection of Mycobacterium tuberculosis antigens . The results showed that polyethylene glycol precipitates of patients' sera were indeed immune complexes, as they contained immunoglobulins, albumin, complement components, and mycobacterial antigens, whereas precipitates from control sera contained mainly albumin . The antibodies present in immune complexes were specific to M . tuberculosis antigen and showed no binding to Escherichia coli antigens . Immune complex levels, as determined by the ability to bind M . tuberculosis antigens in an enzyme-linked immunosorbent assay, were significantly higher in tuberculosis patients (n = 22) than in healthy control subjects (n = 18) . Thus, immune complex level could be a useful parameter in the diagnosis of active tuberculosis.

J Cell Physiol, 1986 Aug, 128(2), 180 - 8
Binding of iodinated multipotential colony-stimulating factor (interleukin-3) to murine bone marrow cells; Nicola NA et al.; Multipotential colony-stimulating factor (Multi-CSF or interleukin-3) was radioiodinated to high specific radioactivity (1-4 X 10(5) cpm/ng) with no detectable loss of biological activity and its binding to murine bone marrow cells and factor-dependent cell lines studied . Both the native glycosylated molecule purified from a cloned T-cell line (LB-3) and the purified non-glycosylated recombinant molecule produced by E . coli could be radioiodinated . Comparative binding studies with these derivatives demonstrated equal binding affinities and equal numbers of binding sites on various cell types indicating that carbohydrate moieties are not involved in the binding interactions . Binding of 125I-Multi-CSF to several factor-dependent continuous hemopoietic cell lines showed the presence of specific receptors on all cell lines, the receptor number per cell varying from 700 to 13,000 and the apparent dissociation constant from 400 pM to 1 nM . Specific binding of 125I-Multi-CSF was also observed to normal murine hemopoietic cells and the binding to murine bone marrow cells was studied in detail . Bone marrow cells showed 117-130 receptors per cell on average and an apparent dissociation constant of 126-233 pM . However, quantitative autoradiographic analysis indicated that receptors for 125I-Multi-CSF were not distributed randomly on bone marrow cells--nucleated erythroid and lymphoid cells were not labeled while essentially all neutrophilic granulocyte, eosinophilic granulocyte and monocytic cells were labeled . Moreover, in each of the labeled cell lineages grain counts (reflecting receptor number) decreased with increasing maturation and a small subpopulation of marrow cells (0.4-1.5% and including blast cells, monocytes, promyelocytes, and myelocytes) exhibited very high grain counts . The existence of such a subset of marrow cells raises the possibility of functional heterogeneity among marrow cells in their response to Multi-CSF.

Mutat Res, 1986 Aug, 162(1), 1 - 5
Induction of SOS functions by nitrogen dioxide in Escherichia coli with different DNA-repair capacities; Kosaka H et al.; The effect of gaseous nitrogen dioxide (NO2) on cytotoxicity, induction of synthesis of UmuC and RecA proteins, and mutagenesis was studied in Escherichia coli strains with different capacities of DNA repair . Gaseous NO2 (90, 180 microliter/l) killed Escherichia coli . The recA mutant was most sensitive, the lexA mutant moderately sensitive, and the uvrA mutant and the wild-type the least sensitive . When 90 microliter/l NO2 gas was bubbled into bacterial suspensions for 30 min at a flow rate of 100 ml/min, the induction of umuC gene expression increased in the wild-type strain . NO2 also induced the recA gene expression in the wild-type strain . The synthesis of neither RecA nor UmuC proteins was induced in the recA and lexA mutants . We further investigated the NO2 mutagenesis in the cells treated with bubbling of NO2 gas . NO2 caused mutation to Trp+ of WP2.

J Immunol, 1986 Aug 1, 137(3), 876 - 9
Lymphokine stimulation of human macrophage C2 production is partially due to interferon-gamma; Sanders KM et al.; Monocyte complement stimulator (MCS), a product of T lymphocytes, is defined by its ability to stimulate the synthesis and secretion of the second complement component (C2) by monocytes . Most macrophage-activating factor (MAF) activity present in lymphokine-rich culture supernatants has recently been found to be due to interferon-gamma (IFN-gamma) . We therefore hypothesized that IFN-gamma may have MCS activity as well . We tested recombinant, E . coli-derived, human IFN-gamma (rIFN-gamma) for its effects on C2 production by adherent peripheral blood monocytes and U937 cells, a human monocytic cell line . Recombinant IFN-gamma in concentrations ranging from 0.1 to 300 U/ml (0.003 to 8.8 ng/ml) stimulates C2 production by both cell populations . Exposure of responding cells for at least 24 hr is required for maximal stimulation . To determine the contribution of IFN-gamma toward total MCS activity in crude lymphokine-rich supernatants, we employed a solid-phase immunoabsorption technique with the use of a monoclonal anti-IFN-gamma antibody . This technique removed all IFN-gamma detectable by a sensitive ELISA, but MCS activity was decreased by only 40 to 50% . Additionally, MCS activity of these supernatants did not correlate with IFN-gamma content as determined by ELISA . By using another method to eliminate IFN-gamma activity, acid dialysis destroyed all rIFN-gamma activity, as measured by stimulation of U937 C2 synthesis, but eliminated only 30 to 67% of MCS activity from crude lymphokine preparations . Thus IFN-gamma stimulates C2 production by monocytes and U937 cells and apparently accounts for some, but not all, MCS activity present in lymphokine-rich supernatants . Other lymphokines are present in such supernatants that also possess this activity.

J Biomol Struct Dyn, 1986 Aug, 4(1), 11 - 21
Linguistics of nucleotide sequences: morphology and comparison of vocabularies; Brendel V et al.; The concept of "words" in continuous languages devoid of blanks is introduced and an operational definition of words given . With this novel concept nucleotide sequences become object for linguistic analysis . The typical word size of the nucleotide language is found to be 3 to 5 (tri- to pentamers) . Different genomes have distinct vocabularies . Comparison of these vocabularies can serve as a basis for revealing functional and evolutionary relatedness of sequences.

Sci Sin {B}, 1986 Aug, 29(8), 856 - 63
The expression of HBsAg gene in yeast under GAL-10 promoter control; Shen LP et al.; The surface antigen gene of HBV (adr subtype) is inserted into the YEP 1 which is replicated and selected in E . coli and baker yeast Saccharomyces cerevisiae . The HBsAg gene can be expressed in yeast under GAL-10 promoter control . The protein synthesized in yeast is assembled into particles that have the same size and shape as particles isolated from plasma . One microgram surface antigen can be yielded in 100 ml culture.

J Gen Microbiol, 1986 Aug, 132 ( Pt 8), 2071 - 8
Properties of in vitro recombinant derivatives of pJV1, a multi-copy plasmid from Streptomyces phaeochromogenes; Bailey CR et al.; The 10.8 kb plasmid pJV1, isolated from Streptomyces phaeochromogenes, has a high copy number (about 150) and a broad host range among Streptomyces spp . Several pJV1 derivatives carrying the thiostrepton resistance gene (tsr) of S . azureus were made . One derivative, pWOR191, was shown to promote its own transfer and to mobilize chromosomal markers in S . lividans . Another derivative, pWOR109, was non-transmissible . Deletion in vitro of a segment of pWOR109 gave pWOR120 (5.6 kb), which has single BamHI and Bg/II sites shown to be capable of accepting 'foreign' DNA such as a previously cloned S . antibioticus DNA fragment encoding tyrosinase, giving vectors (pWOR125, pWOR126) with properties resembling the well-established multicopy vector pIJ702 . Shuttle vectors capable of functioning in both S . lividans and Escherichia coli were also constructed . The region of pJV1 essential for replication and maintenance was localized to a 2.5 kb segment . Stable maintenance of pWOR109 and pWOR120 was observed in the presence of derivatives of pIJ101, the progenitor of pIJ702.

Genetika, 1986 Aug, 22(8), 2042 - 7
{Phasmids . Properties and the use in genetic engineering . II . Packing in vitro . Lysogeny of Escherichia coli K-12}; Fonshtein MIu et al.; Circular monomeric lambda DNA molecules were used as a substrate for packaging reaction in vitro . For obtaining lambda DNA in circular monomeric form only, Escherichia coli recA plasmid bearing cells were used . This hybrid DNA molecule which we designated phasmid lambda pMYF11, is the pBR322 plasmid in which lambda 47.1 DNA was introduced in vitro . The phasmid can exist in the plasmid form or as a non-defective phage . The efficiency of packaging reaction in vitro proved to be similar for monomeric circular and linear form of phasmid DNA molecules . The cI- variant of the phasmid is not able to exist as a plasmid even in the cells containing homoimmune prophage . Still, cI+ phasmid variants capable of lysogenizing arise with low frequency, as a result of recombination between the resident cI+ prophage and infecting cI- phasmid.

Genetika, 1986 Aug, 22(8), 2035 - 41
{Phasmids . Properties and the use in genetic engineering . I . Construction of the phasmid vector}; Fonshtein MIu et al.; A phasmid vector molecule designated pMYF11 has been constructed . The vector combines some useful features of plasmid and phage vector molecules . lambda pMYF11 is a hybrid of lambda 47.1 vector and pBR322 plasmid . CI- marker of pMYF11 is replaced with cI+ marker by recombination between the plasmid and prophage 434 . The phasmid molecule can be used as a replacement vector for BamHI, HindIII, SalGI endonucleases . The maximum size of fragments to be cloned is 21 kilobase pairs . Positive selection for hybrid molecules is possible because of the Spi phenotype expression after replacement of the central HindIII or BamHI DNA fragment with foreign DNA . A library of Escherichia coli genes is constructed with the help of lambda pMYF11 as a vector molecule . A hybrid phage harboring genes of the proline operon is detected by means of complementation.

Genetika, 1986 Aug, 22(8), 2025 - 34
{Molecular cloning and functional analysis of DNA regions of plasmid RP4 determining the incompatibility properties}; Riabchenko LE et al.; Sau3A-generated DNA fragments determining incompatibility functions of the plasmid RP4 were cloned on the vectors pTK16 and pBR322 . Inc+ recombinant plasmids were divided into two types: 1) expressing incompatibility only towards the homologous RP4 replicon, 2) expressing incompatibility - both towards the homologous RP4 replicon and towards the heterologous replicons of plasmids R906 and R751 . For one member of the first type plasmids it was shown that the cloned Inc+-specific insertion derived from the region of location of the EcoRI restriction site . The majority of the Inc+ recombinant plasmids showed asymmetric expression of incompatibility, predominantly eliminating the resident IncP plasmid.

Mol Gen Genet, 1986 Aug, 204(2), 355 - 8
Single amino acid replacements affecting the thermostability of kanamycin nucleotidyltransferase; Matsumura M et al.; Amino acid residues of the carboxyl-terminal region of kanamycin nucleotidyltransferase were modified using segment-directed mutagenesis . Six different mutant enzymes with single amino acid replacements were selected out of 59 clones by DNA sequence analyses . The mutant enzymes were purified and it was found that the thermostability of one mutant enzyme was identical to the wild type, whereas the other five were less thermostable at varying degrees . The data suggested that changes in the enzyme thermostability depend not only on the position but also on the species of amino acid residue replaced.

Mol Gen Genet, 1986 Aug, 204(2), 328 - 33
Rescue of a tk-plasmid from transgenic mice reveals its episomal transmission; Kiessling U et al.; This communication demonstrates the usefulness of the plasmid rescue procedure for recovery of plasmids from transgenic mice . We have microinjected the plasmid pSK1 harbouring the Herpes simplex virus thymidine kinase gene into fertilized mouse oocytes and succeeded in recovering plasmids from newborns by transformation of E . coli either with HindIII cut cellular DNA or with uncut DNA . The majority of the rescued plasmids were indistinguishable from pSK1 by restriction analysis . The rescued plasmids proved to be functionally active in a transient expression assay in mouse Ltk- cells . The pSK1 DNA sequences were inherited by up to 90% of the second generation progeny mice, which is not in agreement with a Mendelian transmission of heterozygous markers integrated into a single site of the chromosome . These data support the assumption that germ line transmission of non-integrated episomal plasmids can occur.

Mol Gen Genet, 1986 Aug, 204(2), 285 - 8
Cloning of the lkyB (tolB) gene of Escherichia coli K12 and characterization of its product; Lazzaroni JC et al.; The lkyB gene of Escherichia coli K12 has been cloned from the Clarke and Carbon colony bank by selecting a ColE1 plasmid conferring cholic acid resistance to lkyB mutants . The lkyB gene was localized on hybrid plasmid pJC778 by analysis of mutated plasmids generated by Tn5 insertions . Restriction analysis and complementation studies indicated that plasmid pJC778 carried genes nadA, lkyB and sucA which mapped at min 16.5; the lkyB+ allele was dominant over the lkyB207 mutant allele . Analysis of cell envelope proteins from strains carrying plasmids pJC778 (lkyB+), pJC2578 or pJC2579 (lkyB::Tn5), as well as plasmid-coded proteins in a maxicell system, made it likely that the ikyB gene product was a membrane protein of molecular weight 42,000.

Mol Gen Genet, 1986 Aug, 204(2), 214 - 20
Asymmetric replication of an oriC plasmid in Escherichia coli; Yoshimoto M et al.; Plasmid pTSO118 containing the Escherichia coli origin of replication, oriC, initiated replication simultaneously with the chromosome when temperature-sensitive host cells were synchronized by temperature shifts . Replicating intermediates of the plasmid as well as of the chromosome were isolated from the outer membrane fraction of the cell . Plasmid DNA with eye structures was enriched when cytosine-1-beta-arabinofuranoside was introduced into the culture during replication . Electron microscopy of the replicating molecules, after digestion with restriction endonucleases, showed that the replication fork proceeds exclusively counter-clockwise towards the unc operon . We conclude that the replication of the oriC plasmid is unidirectional or, if bidirectional, is highly asymmetric.

EMBO J, 1986 Aug, 5(8), 2037 - 40
Identification of the stable free radical tyrosine residue in ribonucleotide reductase; Larsson A et al.; The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue . Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine . The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing . Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum . The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity . These results identify Tyr122 of E . coli protein B2 as the tyrosyl radical residue . An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits . It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322 . Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.

EMBO J, 1986 Aug, 5(8), 2015 - 21
DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor; Valentin-Hansen P et al.; The sequences required for full repression of the Escherichia coli deoP1 and P2 promoters by the deoR repressor (DeoR) have been analyzed in vivo . Using recombinant techniques, we have constructed a set of deo-lacZ fusions which contain different parts of the sequences involved in the regulation of deo expression on low copy number fusion vectors . Since these vectors are present in only one copy per chromosome at temperatures below 37 degrees C, this vector system allows very accurate studies of gene control signals . Our results show that three DeoR operator sites exist in the deoP1-P2 regulatory region . Two of these loci overlap the initiation sites for deoP1 (O1) and deoP2 (O2) transcription located 599 bp apart, whereas the third site (OE) is present approximately 270 bp upstream of P1 . DeoR repression of both P1 and P2 transcription is weak on promoter fragments which only contain one operator site (O1 or O2) . Enhanced repression by deoR is observed on promoter fragments containing two operator sites . However, all three sites are needed for full repression . These findings are discussed with respect to upstream and downstream control regions of eukaryotic genes.

EMBO J, 1986 Aug, 5(8), 2009 - 13
GATC sequence and mismatch repair in Escherichia coli; Laengle-Rouault F et al.; The Escherichia coli mismatch repair system greatly improves DNA replication fidelity by repairing single mispaired and unpaired bases in newly synthesized DNA strands . Transient undermethylation of the GATC sequences makes the newly synthesized strands susceptible to mismatch repair enzymes . The role of unmethylated GATC sequences in mismatch repair was tested in transfection experiments with heteroduplex DNA of phage phi 174 without any GATC sequence or with two GATC sequences, containing in addition either a G:T mismatch (Eam+/Eam3) or a G:A mismatch (Bam+/Bam16) . It appears that only DNA containing GATC sequences is subject to efficient mismatch repair dependent on E . coli mutH, mutL, mutS and mutU genes; however, also in the absence of GATC sequence some mut-dependent mismatch repair can be observed . These observations suggest that the mismatch repair enzymes recognize both the mismatch and the unmethylated GATC sequence in DNA over long distances . The presence of GATC sequence(s) in the substrate appears to be required for full mismatch repair activity and not only for its strand specificity according to the GATC methylation state.

EMBO J, 1986 Aug, 5(8), 1959 - 66
Identification of an Epstein-Barr virus-coded thymidine kinase; Littler E et al.; We have demonstrated the presence of an Epstein-Barr virus (EBV)-coded thymidine kinase (TK) by producing biochemically transformed, TK-positive mammalian cell lines using either microinjection of whole EBV virions or calcium phosphate-mediated transfection of the SalI-B restriction endonuclease fragment of EBV DNA . Analysis of these cell lines showed that: (i) EBV DNA was present in the cell lines, (ii) sequences from the SalI-B restriction endonuclease fragment of EBV were expressed, (iii) a TK activity was present and (iv) a protein with antigenic cross-reactivity with the herpes simplex virus (HSV) TK was produced . The identity of the EBV TK gene was determined by demonstrating that a recombinant plasmid, which expressed the protein product of the BXLF1 open reading frame as a fusion protein, could complement TK- strains of E . coli . A comparison of the predicted amino acid sequences of the TK proteins of EBV and HSV-1 revealed significant regions of homology.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 6058 - 62
Introduction of plasmid DNA into the trypanosomatid protozoan Crithidia fasciculata; Hughes DE et al.; Crithidia fasciculata cells were treated with a plasmid (pDK96) containing pBR322 sequences, a Leishmania tarentolae maxicircle autonomously replicating sequence, and the bacterial gene for aminoglycoside 3' phosphotransferase I inserted between the yeast alcohol dehydrogenase 1 promotor and terminator sequences . Resistant colonies were selected on agar plates containing paromomycin and screened for vector DNA by hybridization . Approximately 1% of the resistant colonies contained detectable vector DNA, which was present as extrachromosomal closed circular molecules ranging in copy number from 1 to 160 per cell . The plasmids could be recovered from Escherichia coli transformed to ampicillin resistance with Crithidia total cell DNA . Most of the recovered plasmids were a deleted product of pDK96, which lacked the maxicircle autonomously replicating sequence and contained a unique fragment of Crithidia nuclear DNA present at a low copy number in the wild-type genome . The plasmid DNA in resistant Crithidia was unstable even under selective conditions and was lost within 30 cell divisions.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 6030 - 4
Genes for the eight ribosomal proteins are clustered on the chloroplast genome of tobacco (Nicotiana tabacum): similarity to the S10 and spc operons of Escherichia coli; Tanaka M et al.; The nucleotide sequence of a tobacco (Nicotiana tabacum) chloroplast gene cluster that encodes eight proteins homologous to Escherichia coli ribosomal proteins L23, L2, S19, L22, S3, L16, L14, and S8 has been determined . RNA gel blot hybridization revealed that all eight coding regions are expressed in the chloroplasts . The arrangement of the eight genes resembles that found in the E . coli S10 and spc operons . Among the eight genes, the L2 and L16 genes contain 666- and 1020-base-pair introns, respectively . These intron boundary sequences are consistent with the conserved boundary sequences of the chloroplast group III introns {Shinozaki, K., Deno, H., Sugita, M., Kuramitsu, S . & Sugiura, M . (1986) Mol . Gen . Genet . 202, 1-5}.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5798 - 802
Substitution of a serine residue for proline-87 reduces catalytic activity and increases susceptibility to proteolysis of Escherichia coli adenylate kinase; Gilles AM et al.; Amino acid analysis, HPLC separation of trypsin digests, and sequence analysis showed that the thermosensitivity of the adenylate kinase (EC 2.7.4.3) from Escherichia coli K-12 strain CR341 T28 results from substitution of a serine residue for proline-87 in the wild-type enzyme . This mutation is accompanied by decreased affinity for nucleotide substrates and decreased catalysis . Circular dichroism spectroscopy showed a significant change of the secondary structure . This mainly corresponds to a reduction in alpha-helix content (39%) of mutant protein as compared to wild-type adenylate kinase (50%) . Altered conformation of thermosensitive adenylate kinase was also manifested by an increase in susceptibility to proteolysis by trypsin . Ap5A and ATP, known to induce important conformational changes in eukaryotic adenylate kinase(s), protected the mutant enzyme against inactivation by trypsin . This seems to indicate that the "loosening" of the three-dimensional structure of E . coli adenylate kinase by proline----serine substitution is largely compensated for when an enzyme X ATP or enzyme X Ap5A complex is formed.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5769 - 73
Mechanism of DNA polymerase I: exonuclease/polymerase activity switch and DNA sequence dependence of pyrophosphorolysis and misincorporation reactions; Mizrahi V et al.; Mechanistic features of several processes involved in the idling-turnover reaction catalyzed by the large (Klenow) fragment of Escherichia coli DNA polymerase I have been established . The exonuclease----polymerase activity switch involved in the excision/incorporation mode of idling-turnover occurs without an intervening dissociation of the enzyme from its DNA substrate . Comparative studies on the pyrophosphorolysis kinetics of related DNA substrates indicate a significant dependence of the reaction rate upon the DNA sequence within the duplex region upstream of the primer-template junction . Finally, a gel electrophoretic analysis of the products of the idling-turnover reaction has provided direct evidence for an alternative DNA sequence-dependent misincorporation/excision pathway.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5693 - 7
Definitive identification of a member of the Epstein-Barr virus nuclear protein 3 family; Hennessy K et al.; Some Epstein-Barr virus (EBV) immune human antisera are known to react with a 142-kDa protein, EBV-encoded nuclear antigen 3 (EBNA3), which, like EBNA1 and EBNA2, is likely to be involved in the establishment of latent infection or growth transformation . We have now constructed gene fusions between Escherichia coli lacZ and an EBV DNA open reading frame (BERF1; BamHI E fragment rightward open reading frame 1), which is transcribed into an mRNA in latently infected cells . Purified hybrid protein from one of these constructs, chosen because of its reactivity with EBNA3-positive human antisera, was used to affinity purify the specific antibody from human antiserum . This specific antibody was used to prove that EBNA3 is encoded, at least in part, by BERF1, and that EBNA3 is in the nucleus of each latently infected cell . In rodent cells, BERF1 encodes a 120- to 130-kDa protein, which translocates to the nucleus and is recognized by EBNA3-positive human antisera . Two other proteins similar in size to EBNA3 are detected in latently infected cells by EBV immune human antisera . Two EBV open reading frames related to BERF1 may encode these proteins.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5392 - 6
Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli; Ivanoff LA et al.; We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector . The 3C region is a virus-specific protease, which, when expressed in E . coli, is shown to be active and autocatalytic . In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C) . Mutants in the 3C region have been constructed by oligonucleotide-directed mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease . The mutation of highly conserved residues (cysteine-47 or histidine-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.

J Virol, 1986 Aug, 59(2), 249 - 59
Replication and resolution of cloned poxvirus telomeres in vivo generates linear minichromosomes with intact viral hairpin termini; DeLange AM et al.; The covalently closed terminal hairpins of the linear duplex-DNA genomes of the orthopoxvirus vaccinia and the leporipoxvirus Shope fibroma virus (SFV) have been cloned as imperfect palindromes within circular plasmids in yeast cells and recombination-deficient Escherichia coli . The viral telomeres inserted within these recombinant plasmids are equivalent to the inverted-repeat structures detected as telomeric replicative intermediates during poxvirus replication in vivo . Although the telomeres of vaccinia and SFV show little sequence homology, the termini from both viral genomes exist as AT-rich terminal hairpins with extrahelical bases and alternate "flip-flop" configurations . Using an in vivo replication assay in which circular plasmid DNA was transfected into poxvirus-infected cells, we demonstrated the efficient replication and resolution of the cloned imperfect palindromes to bona fide hairpin termini . The resulting linear minichromosomes, which were readily purified from transfected cells, were shown by restriction enzyme mapping and by electron microscopy to have intact covalently closed hairpin termini at both ends . In addition, staggered unidirectional deletion derivatives of both the cloned vaccinia and SFV telomeric palindromes localized an approximately 200-base-pair DNA region in which the sequence organization was highly conserved and which was necessary for the resolution event . These data suggest a conserved mechanism of the resolution of poxvirus telomeres.

J Gen Virol, 1986 Aug, 67 ( Pt 8), 1633 - 43
Biological activities and receptor binding of two human recombinant interferons and their hybrids; Meister A et al.; Two human recombinant lymphoblastoid interferon-alpha subtypes, LyIFN-B (alpha 8) and LyIFN-D (alpha 1), and 10 hybrids generated therefrom were produced in Escherichia coli and purified . The antiviral and antiproliferative activities and the induction of (2'-5')oligoadenylate synthetase were compared to their receptor binding affinities . The IFN subtypes and their hybrids had similar specific antiviral activities on bovine cells . On human cells both the specific antiviral and antiproliferative activities of LyIFN-B were about 30-fold higher than those of LyIFN-D . This difference in activity could be attributed partly to the N-terminal amino acids 1 to 60 and partly to amino acids 61 to 92 . A third domain affecting the biological activities was found within the carboxy-proximal segment from amino acids 93 to 150 . The differences in these activities were found to correlate with their ability to bind the receptor, suggesting that the differences in activity might be due to altered binding of the IFNs to the cellular receptors . In contrast, the induction of (2'-5')oligoadenylate synthetase did not follow the same activity profile . On mouse cells, the efficiency of the hybrids was affected by at least four sites on the IFN protein . A hybrid with the N-terminal segment 1 to 60 from IFN-B and amino acids 61 to 166 from IFN-D had a specific antiviral activity on mouse cells as high as on human cells corresponding to a 500- and 5000-fold increase in specific activity compared to IFN-D and IFN-B, respectively . We suggest that on mouse cells the IFN activity may be more dependent on conformational differences than on human cells, which in turn might reflect a less precise fit to the mouse receptor than to the human receptor.

J Bacteriol, 1986 Aug, 167(2), 749 - 53
Cloning of the aroP gene and identification of its product in Escherichia coli K-12; Chye ML et al.; The aroP gene of Escherichia coli K-12 was located in a ca . 1.2-kilobase region of DNA . The aroP gene product was identified as a membrane-bound protein with an apparent molecular weight of approximately 37,000.

J Bacteriol, 1986 Aug, 167(2), 647 - 54
DNA environment of the aerobactin iron uptake system genes in prototypic ColV plasmids; Waters VL et al.; The aerobactin iron uptake system genes in the prototypic plasmid pColV-K30 are flanked by inverted copies of insertion sequence IS1 and by two distinct replication regions . To address the question of how these flanking regions may facilitate the maintenance and spread of the aerobactin system among the plasmids and chromosomes of enteric species, we investigated the DNA environment of 12 ColV plasmids . We found that the aerobactin system-specific genes are conserved in every plasmid phenotypically positive for the aerobactin system . The upstream IS1 and its overlapping replication region (REPI) are also conserved . This replication region was cloned from several ColV plasmids and found to be functional by transforming these cloned derivatives into a polA bacterial host . In contrast, the downstream flanking region is variable . This includes the downstream copy of IS1 and the downstream replication region (REPII) . We infer from these results that sequences in addition to the two flanking copies of IS1, in particular the upstream region including REPI, have been instrumental in the preservation and possible spread of aerobactin genes among ColV plasmids and other members of the FI incompatibility group.

J Bacteriol, 1986 Aug, 167(2), 594 - 603
Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli; Biek DP et al.; We isolated mutations that reduce plasmid stability in dividing cell populations and mapped these mutations to a previously undescribed gene, recD, that affects recombination frequency and consequently the formation of plasmid concatemers . Insertions of the transposable element Tn10 into recD resulted in increased concatemerization and loss of pSC101 and ColE1-like replicons during nonselective growth . Both concatemer formation and plasmid instability in recD mutants require a functional recA gene . Mutations in recD are recessive to recD+ and map to a small region of the Escherichia coli chromosome located between recB and argA . Although the recD locus is distinct from loci encoding the two previously identified subunits of the RecBC enzyme, mutations in recD appear to affect the exonuclease activity of this enzyme.

J Bacteriol, 1986 Aug, 167(2), 462 - 6
Translational control of exported proteins in Escherichia coli; Hengge-Aronis R et al.; We recently described the suppression of export of a class of periplasmic proteins of Escherichia coli caused by overproduction of a C-terminal truncated periplasmic enzyme (GlpQ') . This truncated protein was not released into the periplasm but remained attached to the inner membrane and was accessible from the periplasm . The presence of GlpQ' in the membrane strongly reduced the appearance in the periplasm of some periplasmic proteins, including the maltose-binding protein (MBP), but did not affect outer membrane proteins, including the lambda receptor (LamB) (R . Hengge and W . Boos, J . Bacteriol., 162:972-978, 1985) . To investigate this phenomenon further we examined the fate of MBP in comparison with the outer membrane protein LamB . We found that not only localization but also synthesis of MBP was impaired, indicating a coupling of translation and export . Synthesis and secretion of LamB were not affected . The possibility that this influence was exerted via the level of cyclic AMP could be excluded . Synthesis of MBP with altered signal sequences was also reduced, demonstrating that export-defective MBP which ultimately remains in the cytoplasm abortively enters the export pathway . When GlpQ' was expressed in a secA51(Ts) strain, the inhibition of MBP synthesis caused by GlpQ' was dominant over the precursor accumulation usually caused by secA51(Ts) at 41 degrees C . Therefore, GlpQ' acts before or at the level of recognition by SecA . For LamB the usual secA51(Ts) phenotype was observed . We propose a mechanism by which GlpQ' blocks an yet unknown membrane protein, the function of which is to couple translation and export of a subclass of periplasmic proteins.

J Bacteriol, 1986 Aug, 167(2), 455 - 61
Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product; Harayama S et al.; TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes . The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase . The synthesis of these enzymes is positively regulated by the product of xylR . Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene{s})-xylA (xylene oxygenase gene{s})-xylB (benzyl alcohol dehydrogenase gene) . Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order . Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde . The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.

J Bacteriol, 1986 Aug, 167(2), 433 - 8
Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli; Barron A et al.; Adaptation to osmotic stress alters the amounts of several specific proteins in the Escherichia coli K-12 envelope . The most striking feature of the response to elevated osmolarity was the strong induction of a periplasmic protein with an Mr of 31,000 . This protein was absent in mutants with lambda plac Mu insertions in an osmotically inducible locus mapping near 58 min . The insertions are likely to be in proU, a locus encoding a transport activity for the osmoprotectants glycine betaine and proline . Factors affecting the extent of proU induction were identified by direct examination of periplasmic proteins on sodium dodecyl sulfate gels and by measuring beta-galactosidase activity from proU-lac fusions . Expression was stimulated by increasing additions of salt or sucrose to minimal medium, up to a maximum at 0.5 M NaCl . Exogenous glycine betaine acted as an osmoregulatory signal; its addition to the high-osmolarity medium substantially repressed the expression of the 31,000-dalton periplasmic protein and the proU-lac+ fusions . Elevated osmolarity also caused the appearance of a second periplasmic protein (Mr = 16,000), and severe reduction in the amounts of two others . In the outer membrane, the well-characterized repression of OmpF by high osmolarity was observed and was reversed by glycine betaine . Additional changes in membrane composition were also responsive to glycine betaine regulation.

Infect Immun, 1986 Aug, 53(2), 267 - 71
Augmentation of host defense by a unicellular green alga, Chlorella vulgaris, to Escherichia coli infection; Tanaka K et al.; Protection against Escherichia coli inoculated intraperitoneally into mice was enhanced by intraperitoneal, intravenous, or subcutaneous administration of a water-soluble, high-molecular-weight fraction extracted from a dialyzed hot-water extract from a strain of Chlorella vulgaris (CVE-A) . The enhancing effect was detected with doses over 2.0 mg/kg and when doses were administered 1, 4, or 7 days before the infection . The elimination of bacteria from the spleen of CVE-A-treated mice was increased, and this enhanced elimination may have been related to the acceleration of superoxide generation and chemokinesis in polymorphonuclear leucocytes by CVE-A treatment . A cyclophosphamide-induced decrease in protection against E . coli could be prevented by subcutaneous administration of CVE-A.

J Interferon Res, 1986 Aug, 6(4), 437 - 43
Efficient expression of unfused human alpha D-interferon in Escherichia coli using overlapping termination and initiation codons (TGATG) in its signal sequence; Hou YT et al.; A plasmid carrying the lambda PL promoter was constructed to express efficiently unfused human alpha D-interferon (HuIFN-alpha D) in Escherichia coli using a TGATG site in its signal sequence, which occurs also in the lambda DNA sequence . The unfused nature of HuIFN-alpha D expressed by pBV867 in E . coli (BMH 71-18) was confirmed by the following evidence: first, the purified IFN showed a single band of 19.5K in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); a 27K band, representing lambda N-HuIFN-alpha D fusion protein, was not detected . Second, the peak of IFN activity coincided with the 19.5K protein . Third, the peak of absorbent material for human leukocyte IFN antibody coincided with that of IFN activity . Finally, amino-terminal sequencing of purified IFN demonstrated an unfused HuIFN-alpha D . This suggests that E . coli is able to process the signal sequence of HuIFN-alpha D . Studies on the mechanism of "translational coupling" initiation of gene expression were carried out by the construction of two hybrid plasmids and titration of the IFN activities produced by them . The level of expression by the ATG-TGATG initiation mode was found to be six times higher than that of the single ATG mode.

J Interferon Res, 1986 Aug, 6(4), 349 - 60
Molecular cloning and sequencing of a gene encoding biologically active porcine alpha-interferon; Lefevre F et al.; Nine distinct genomic clones containing human alpha 1-interferon (IFN-alpha 1) related sequences were isolated from a porcine genomic library constructed in phage lambda . Restriction mapping and Southern blot analysis revealed that these clones contained a total of 10 potential porcine IFN-alpha genes or pseudogenes belonging to a multigene family of at least 12 members . One of these genes was subcloned in plasmid pUC8 and the recombinant plasmid obtained was shown to direct the synthesis of a low but detectable IFN-alpha activity in Escherichia coli JM103 . The sequence of this porcine IFN-alpha gene (Po IFN-alpha 1) was determined . As expected, it contained no introns and encoded a 189-amino-acid-long preprotein with a putative signal peptide of 23 residues . The homology to human (Hu)IFN-alpha 1 was 78.5% at the nucleotide level and 64% at the amino acid level.

J Virol, 1986 Aug, 59(2), 420 - 7
Adenovirus type 12 E1A protein expressed in Escherichia coli is functional upon transfer by microinjection or protoplast fusion into mammalian cells; Krippl B et al.; We efficiently expressed, in Escherichia coli, and purified the protein product encoded by the human adenovirus type 12 (Ad12) 13S mRNA . The functional properties of the E1A protein were analyzed by introducing the protein by microinjection or protoplast fusion into living mammalian cells . We showed that the E . coli-expressed E1A protein induces gene expression of the adenovirus type 5 (Ad5) E1A deletion mutant Ad5dl312 . The purified E1A protein rapidly and quantitatively localized to the cell nucleus after microinjection into the cytoplasm . In addition, we raised high-titered monospecific antibodies to the purified Ad12 E1A protein . Using deleted forms of an adenovirus type 2 and Ad5 hybrid (Ad2/5) E1A protein, we showed that all of the epitopes conserved between Ad2/5 E1A and Ad12 E1A protein that are recognized by the Ad12 E1A-specific antiserum map to within the first exon-encoded amino-terminal half of the protein.

J Bacteriol, 1986 Aug, 167(2), 674 - 80
Escherichia coli iron enterobactin uptake monitored by Mössbauer spectroscopy; Matzanke BF et al.; Iron uptake by Escherichia coli under aerobic conditions of iron deficiency is mediated by a highly stable ferric enterobactin {Fe(ent)3-} siderophore complex . Mossbauer spectroscopy has been used to monitor the fate of the iron as 57Fe(ent) was taken up by the cells . Osmotic shock experiments were used to distinguish between the iron present in the periplasmic space and that in the cytoplasm of the cell . Iron delivery by a synthetic analog of enterobactin, 1,3,5-N,N',N''- tris-(2,3-dihydroxybenzoyl)triaminomethylbenzene (MECAM), was also studied . Although Fe-MECAM was transported at the same rate as was Fe(ent) across the outer membrane and was apparently accumulated in the periplasmic space, the subsequent behaviors of Fe(ent) and Fe-MECAM were very different . After more than 30 min, a major fraction of the iron originally absorbed as ferric enterobactin appeared as Fe(II), apparently in the cytoplasm of the cell . However, little iron was delivered to the cytoplasm by the MECAM complex . The differences in specificity of these two stages of iron uptake by E . coli are discussed.

Carcinogenesis, 1986 Aug, 7(8), 1359 - 64
The biological activity of single-stranded phi X174 DNA, modified by N-hydroxy-2-aminofluorene, is inhibited by guanine imidazole ring-opening of the major, non-lethal aminofluorene-DNA adduct; Lutgerink JT et al.; The major aminofluorene-DNA derivative, found in the liver of rats after administration of the hepatocarcinogen N-acetyl-2-aminofluorene and identified as N-(deoxyguanosin-8-yl)-2-aminofluorene (dGuo-C8-AF), was introduced in different amounts in single-stranded phi X174 DNA by reacting the DNA with tritium labeled N-hydroxy-2-aminofluorene . The modified DNA was subsequently incubated in 0.1 M NaOH at 37 degrees C for increasing periods of time to convert the dGuo-C8-AF residues into their guanine imidazole ring-opened forms . The degree of conversion was determined by measuring the amount of residual N-(guanin-8-yl)-2-aminofluorene in trifluoroacetic acid hydrolyzates of the alkali-treated DNA by h.p.l.c . In addition, the effect of ring opening on the biological activity of the DNA was monitored by transfecting the DNA to Escherichia coli wild-type spheroplasts . The results indicate that the major aminofluorene-DNA adduct formed initially, which contributes little to inactivation, becomes lethal when its guanine imidazole ring is opened.

Mutat Res, 1986 Aug, 162(1), 7 - 20
Deoxyuridine misincorporation causes site-specific mutational lesions in the lacI gene of Escherichia coli; Sedwick WD et al.; Spontaneous forward mutation in lacI was analyzed by DNA sequencing in a Dut- strain of E . coli . Hyperuracil incorporation into DNA due to the defect in deoxyuridinetriphosphatase caused a 5-fold increase in mutation frequency . Deletion, duplication and base-substitution frequencies were all enhanced in the Dut- strain . However, the analysis of the specificity of mutation revealed a remarkable site- and class-specificity . For example, base substitutions at a single site, a G:C = greater than A:T transition (Ochre 34) accounted for 55% of the base substitutions recovered . The spontaneous A:T = greater than G:C hotspot at position +6 at the lac operator was also recovered at an enhanced frequency in the Dut- strain where it accounted for 25% of the base substitutions . Many of the deletion and duplication events were recovered more than once; most had endpoints in A/T rich regions . The spontaneous frameshift hotspot involving the gain or loss of 5'-CTGG-3' in a region where this tetramer is tandemly repeated 3 times, was also greatly enhanced . No frameshifts involving a single base pair nor IS1 insertions were identified among the 86 lacI mutants sequenced . The analysis of these events reveals them to be generally consistent with a mechanism involving AP sites generated by the removal of misincorporated uracil by uracil-N-glycosylase . Considering the number of potential AP sites (approximately 1 per 170 base pairs) E . coli is remarkably refractory to mutational consequences of deoxyuridine misincorporation in place of thymidine.

EMBO J, 1986 Aug, 5(8), 2003 - 8
Two overlapping genes in bovine mitochondrial DNA encode membrane components of ATP synthase; Fearnley IM et al.; Two hydrophobic proteins have been purified to homogeneity from a mixture of about 13 proteins that are extracted from bovine mitochondria with a chloroform:methanol mixture . Sequence analysis shows that the smaller is a protein of 66 amino acids and is the product of a mitochondrial gene, A6L . The larger, a protein of 226 amino acids, is ATPase-6, a membrane component of ATP synthase, also encoded in mitochondrial DNA . The protein sequences determined establish that the genes for the two proteins overlap by 40 bases and indicate that translation of the second gene, ATPase-6, is initiated within the coding region of A6L . The A6L and the ATPase-6 proteins have also been isolated from the ATP synthase complex and so appear to be bona fide components of the enzyme . The function of A6L is unknown . However, weak structural homology suggests a functional similarity to the yeast mitochondrial protein, aapI, which is required for assembly of the fungal ATP synthase complex . Homologies between ATPase-6 and subunit a of the Escherichia coli ATP synthase complex indicate that the ATPase-6 protein has a similar role in the mitochondrial complex to its bacterial counterpart, being essential for the formation of an active proton channel.

Arch Biochem Biophys, 1986 Aug 1, 248(2), 540 - 50
Biosynthesis of 5-methylaminomethyl-2-selenouridine, a naturally occurring nucleoside in Escherichia coli tRNA; Wittwer AJ et al.; A selenium-containing nucleoside, 5-methylaminomethyl-2-selenouridine (mnm5se2U), is present in lysine- and glutamate-isoaccepting tRNA species of Escherichia coli . The synthesis of mnm5se2U is optimum (4 mol/100 mol tRNA) when selenium is present at about 1 microM concentration and is neither decreased by a high (8 mM) level of sulfur in the medium nor increased by excessive (10 or 100 microM) levels of selenium . Lysine- and glutamate-isoaccepting tRNA species that contain 5-methylaminomethyl-2-thiouridine (mnm5s2U) coexist with the seleno-tRNAs in E . coli cells and a reciprocal relationship between the mnm5se2U- and the mnm5s2U-containing species is maintained under a variety of growth conditions . The complete 5-methylaminomethyl side chain is not a prerequisite for introduction of selenium at the 2-position . In E . coli mutants deficient in the ability to synthesize the 5-methylaminomethyl substituent, both the 2-thiouridine and the corresponding 2-selenouridine derivatives of intermediate forms are accumulated . Broken cell preparations of E . coli synthesize mnm5se2U in tRNAs by an ATP-dependent process that appears to involve the replacement of sulfur in mnm5s2U with selenium.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5909 - 13
Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli; Ninfa AJ et al.; Transcription from nitrogen-regulated promoters, such as glnAp2, requires the glnG gene product, NRI, as well as the rpoN(glnF) gene product, sigma60, and is regulated by the glnL gene product, NRII . We find that in a reaction mixture containing NRI, NRII, and ATP, NRII catalyzes the transfer of the gamma phosphate of ATP to NRI . This covalent modification of NRI occurs concurrently with the acquisition of the ability by the reaction mixture to activate transcription from glnAp2 . In the presence of PII, the product of glnB, NRII catalyzes the removal of the phosphate from NRI-phosphate . This reaction occurs concurrently with the loss by the reaction mixture of the ability to activate transcription from glnAp2 . On the basis of this evidence, we propose that NRI-phosphate activates transcription from nitrogen-regulated promoters and that the role of NRII is control of the formation and breakdown of NRI-phosphate in response to cellular signals of nitrogen availability.

J Biomol Struct Dyn, 1986 Aug, 4(1), 1 - 9
RNA secondary structure formation during transcription; Mironov A et al.; A new approach has been proposed for predicting the kinetic ensemble of the RNA secondary structures during chain growth . It is based on an analysis of time intervals in structural reconstruction . The Markov chain employed for describing structural reconstruction was modelled on the Monte Carlo method . A calculation was made of possible secondary structures formed during transcription . An algorithm has also been suggested for the search of a helix with a bulge type defect in which a cooperative effect is retained . Kinetic ensembles of the SD-sites and initiation regions of the polycistronic mRNA transcribed from ATP operon E . coli were calculated . A correlation between the secondary structures of these mRNA regions and the relative cistronic expression was established.

Mol Cell Biol, 1986 Aug, 6(8), 2910 - 5
In vitro methylation of bovine papillomavirus alters its ability to transform mouse cells; Christy BA et al.; Bovine papillomavirus (BPV) was methylated in vitro at either the 29 HpaII sites, the 27 HhaI sites, or both . Methylation of the HpaII sites reduced transformation by the virus two- to sixfold, while methylation at HhaI sites increased transformation two- to fourfold . DNA methylated at both HpaII and HhaI sites did not differ detectably from unmethylated DNA in its efficiency of transformation . These results indicate that specific methylation sites, rather than the absolute level of methylated cytosine residues, are important in determining the effects on transformation and that the negative effects of methylation at some sites can be compensated for by methylation at other sites . BPV molecules in cells transformed by methylated BPV DNA contained little or no methylation, indicating that the pattern of methylation was not faithfully retained in these extrachromosomally replicating molecules . Methylation at the HpaII sites (but not the HhaI sites) in the cloned BPV plasmid or in pBR322 also inhibited transformation of the plasmids into Escherichia coli HB101 cells.

Biol Chem Hoppe Seyler, 1986 Aug, 367(8), 769 - 80
Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites; Goringer HU et al.; The ribosomal 5S RNA gene from E . coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103 . The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E . coli strain using a modified maxicell system . The mutant 5S RNA genes were found to be transcribed and processed normally . The 5S RNA molecules were assembled into 50S ribosomal subunits . Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type . The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25 . The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably . The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered . In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity . Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay . Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.

Zh Mikrobiol Epidemiol Immunobiol, 1986 Aug, (8), 72 - 5
{Escherichia-erythrocyte diagnostic agents}; Shamardin VA et al.; The conditions of a simple and practicable method for the preparation of effective antigenic nonprotein diagnosticums on the basis of water-phenol extracts of 23 Escherichia species have been developed . The method consists in heating the mixture of erythrocytes and the antigen in a boiling water bath for 60 minutes . The diagnosticums thus obtained are 16-30 times more sensitive in the passive hemagglutination test and 4-6 times more sensitive in the passive hemagglutination inhibition test than diagnosticums prepared with the use of tannin, rivanol, as well as by the common method for the preparation of nonprotein antigens . The minimum concentration of Escherichia cells detected in the passive hemagglutination inhibition test is 0.8-1.2 million cells/ml.

DNA, 1986 Aug, 5(4), 271 - 9
Influence on stability in Escherichia coli of the carboxy-terminal structure of cloned Moloney murine leukemia virus reverse transcriptase; Gerard GF et al.; We have cloned and expressed in Escherichia coli a section of the Moloney murine leukemia virus (Mo-MLV) pol gene which includes the entire coding region of mature reverse transcriptase (RT) plus 284 additional base pairs 3' to the coding region (Kotewicz et al., 1985) . To prepare cloned Mo-MLV RT as close as possible to authentic RT in structure and activity, the universal terminator sequence GC(TTAA)3GC was introduced at a number of positions inside and outside the RT coding region within 200 nucleotides of its 3' end . The level of RT activity expressed from these constructs varied sevenfold . This variation was found to be directly related to the stability of the RT protein products in the E . coli K-12 strain K802; half-lives varied from 2 to 35 min . The stability of most of the RT proteins was not increased in E . coli K802 lon- cells, with the exception of two, whose half-lives were increased by a factor of two.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5929 - 33
Identification and characterization of the NMYC gene product in human neuroblastoma cells by monoclonal antibodies with defined specificities; Ikegaki N et al.; Increased N-myc (now designated NMYC in human gene nomenclature) gene expression has been detected at the transcriptional level in certain types of neoplasms . As yet, the N-myc gene product has not been identified . To detect and characterize the N-myc gene product, we have developed monoclonal antibodies against the putative N-myc gene product made in Escherichia coli as a fusion protein . The antibodies that recognize the N-myc-specific regions were selected on the basis of their reactivities to different portions of the fusion protein . These monoclonal antibodies detect a pair of closely migrating polypeptides of 60 and 63 kDa in nuclear fractions of human neuroblastoma cells . The relative levels of the polypeptides are roughly proportional to the level of N-myc transcripts present in a panel of neuroblastoma lines . These two polypeptides have a half-life of approximately equal to 35 min, and they are indistinguishable from each other by their epitopic profiles.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5400 - 4
ras p21 deletion mutants and monoclonal antibodies as tools for localization of regions relevant to p21 function; Lacal JC et al.; Deletion mutants of the viral Harvey ras oncogene were generated by removing different lengths of the gene from either the amino or the carboxyl terminus . The deletion mutants, ras p21 expressed in Escherichia coli, yielded proteins of approximately 8, 10, 12, 14, 17, 18, 19, and 20 kDa . These proteins were utilized to identify epitopes recognized by a series of recently generated monoclonal antibodies as well as some previously reported monoclonal antibodies . Monoclonal antibodies that inhibited GTP binding, a major biochemical activity of the p21 protein, recognized two major regions of the protein . These regions were localized from amino acids 5 to 69 and 107 to 164, respectively, and were separated by another stretch from residues 70 to 106, whose antigenic determinants were not directly involved in GTP binding . Thus, the mapping of epitopes within the p21 molecule recognized by monoclonal antibodies has made it possible to localize important functional regions within the ras p21 molecule.

J Virol, 1986 Aug, 59(2), 328 - 40
Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli; Tanese N et al.; Portions of the pol gene of Moloney murine leukemia virus (MuLV) were expressed as fusion proteins in Escherichia coli, and the purified proteins were used to elicit antibodies in Escherichia coli, and the purified proteins were used to elicit antibodies in rabbits . The sera were used to examine the mature pol gene products contained in virion particles and identified the reverse transcriptase and a second protein, P46pol, encoded by the 3' portion of the gene . The P46 protein was not phosphorylated and was present at the same molar abundance as the reverse transcriptase . The sera were also used to detect the Pr200gag-pol intracellular precursor protein and to analyze its processing to the mature forms . The proteins formed by several Moloney MuLV mutants were analyzed . Further tests revealed cross-reactivity with Friend MuLV and feline leukemia virus proteins, but not with avian retrovirus proteins.

Infect Immun, 1986 Aug, 53(2), 257 - 63
A reexamination of the O1 lipopolysaccharide antigen group of Escherichia coli; Moll A et al.; A total of 64 Escherichia coli strains of the O1 serogroup were tested for the migration pattern of their lipopolysaccharides (LPS) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . O1:K1 and O1:K51 strains of the OMP5 outer membrane protein pattern possessed LPS with a doublet pattern (O1A1) or the lowermost band of the O1A1 doublet (O1A2) . O1:K1 strains of the OMP9 pattern possessed LPS referred to as O1A, which corresponded to the uppermost band of the O1A1 doublet pattern . A few O1:K? strains possessed LPS of different migration patterns (O1B and O1C) . O1A and O1A1 LPS were indistinguishable by chemical techniques, and both reacted with each of 10 different monoclonal antibodies tested . However, O1A1 had an additional epitope within the additional band in each doublet, as demonstrated by adsorption experiments with hyperimmune rabbit sera followed by Western blotting . Furthermore, purified polysaccharide from O1A bacteria was incapable of inhibition in enzyme-linked immunosorbent assays performed with O1A1 LPS as antigen and adsorbed, specific anti-O1A1 antibodies, whereas O1A1 polysaccharide inhibited this reaction . O1B and O1C LPS differed in all respects tested, including chemical composition, from O1A and O1A1 LPS.

Biochem Biophys Res Commun, 1986 Jul 31, 138(2), 826 - 34
Sequence and expression of a novel murine interferon alpha gene--homology with enhancer elements in the regulatory region of the gene; Dion M et al.; A murine interferon gene (MuIFN alpha) has been isolated from a cosmid library . The sequence of a 1.2-kb HindIII-PstI fragment revealed a new MuIFN alpha gene which has not yet been described and which was termed MuIFN alpha 7 . The coding sequence produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter . A comparison of the MuIFN alpha 7 gene with the known interferon genes in their coding and flanking sequences shows homologies between enhancer elements found in the 5' upstream region of the coding gene . The core element common to all known viral enhancers, GTGG(AAA/TTT)G is repeated four times in the MuIFN alpha 7 5'-flanking region, as in all known MuIFN alpha genes.

Biochem Biophys Res Commun, 1986 Jul 31, 138(2), 512 - 8
Multiple sequences in the Drosophila melanogaster U3 RNA gene are homologous to vertebrate U3 RNA; Akao M et al.; We have cloned and sequenced a DNA fragment from the genome of Drosophila melanogaster which is homologous to Novikoff hepatoma (rat) small nucleolar U3 RNA . DNA sequence analysis shows that the regions of homology between the cloned DNA and rat U3 RNA are distributed over the entire length of the molecule and the total homology of linear sequence is 53% . The present finding supports the generalization that U3 RNA has been evolutionally conserved but diverges between mammals and invertebrates . This clone contains the upstream and downstream sequences required for the efficient and correct transcription of small nuclear RNAs . The secondary structure proposed for the U3 RNA sequence deduced from the cloned DNA is similar in general topography to that reported for rat U3 RNA (Bernstein et al., ref . 23).

Biochem Biophys Res Commun, 1986 Jul 31, 138(2), 789 - 94
Modification of Glu 58, an amino acid of the active center of ribonuclease T1, to Gln and Asp; Nishikawa S et al.; Glu 58 is one of the amino acids which participates in its catalytic action of ribonuclease T1 . We mutated this residue to Gln 58 or Asp 58 by genetic engineering using chemically synthesized genes . The mutant enzymes were expressed in E . coli as fused proteins and purified to homogeniety on SDS-PAGE after cleavage with cyanogen bromide . Their activities in hydrolyzing pGpC were reduced to 10% in the Asp 58 mutant and about 1% in the Gln 58 mutant compared to that of the wild-type enzyme . These results suggest that Glu 58 is important but not essential for catalysis of ribonuclease T1.

Biochemistry, 1986 Jul 29, 25(15), 4366 - 71
Purification and characterization of aminoimidazole ribonucleotide synthetase from Escherichia coli; Schrimsher JL et al.; Aminoimidazole ribonucleotide (AIR) synthetase has been purified 15-fold to apparent homogeneity from Escherichia coli which contains a multicopy plasmid containing the purM, AIR synthetase, gene . The protein is a dimer composed of two identical subunits of Mr 38,500 . The N-terminal sequence, amino acid composition, and steady-state kinetics of the protein have been determined . AIR synthetase has been shown to catalyze the transfer of the formyl oxygen of {18O}formylglycinamide ribonucleotide to Pi.

Biochemistry, 1986 Jul 29, 25(15), 4240 - 9
Isomerization of (3S)-2,3-dihydro-5-fluoro-L-tryptophan and of 5-fluoro-L-tryptophan catalyzed by tryptophan synthase: studies using fluorine-19 nuclear magnetic resonance and difference spectroscopy; Miles EW et al.; We are exploring the active site and the mechanism of the pyridoxal phosphate dependent reactions of the bacterial tryptophan synthase alpha 2 beta 2 complex by use of substrate analogues and of reaction intermediate analogues . Fluorine-19 nuclear magnetic resonance studies and absorption spectroscopy are used to study the binding and reactions of the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)- and (3R)-2,3-dihydro-5-fluorotryptophan . Tryptophan synthase specifically and tightly binds the 3S diastereoisomer of both 2,3-dihydro-5-fluoro-D-tryptophan and 2,3-dihydro-5-fluoro-L-tryptophan, whereas it binds 5-fluoro-D-tryptophan more tightly than 5-fluoro-L-tryptophan . Unexpectedly, we find that the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)-2,3-dihydro-5-fluorotryptophan are slowly interconverted by isomerization reactions . Since these isomerization reactions are 10(3)-10(5) times slower than the beta-replacement and beta-elimination reactions catalyzed by tryptophan synthase, they have no biochemical significance in vivo . However, the occurrence of these slow reactions does throw some light on the nature of the active site of tryptophan synthase and its requirements for substrate binding . Our results raise the interesting question of whether tryptophan synthase itself serves a catalytic role in these slow reactions or whether the enzyme simply binds the substrate and pyridoxal phosphate stereospecifically and thus promotes the intrinsic catalytic activity of pyridoxal phosphate.

Biochemistry, 1986 Jul 29, 25(15), 4194 - 204
Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67; Matthews DA et al.; Crystalline R67 dihydrofolate reductase (DHFR) is a dimeric molecule with two identical 78 amino acid subunits, each folded into a beta-barrel conformation . The outer surfaces of the three longest beta strands in each protomer together form a third beta barrel having six strands at the subunit interface . A unique feature of the enzyme structure is that while the intersubunit beta barrel is quite regular over most of its surface, an 8-A "gap" runs the full length of the barrel, disrupting potential hydrogen bonds between beta-strand D in subunit I and the adjacent corresponding strand of subunit II . It is proposed that this deep groove is the NADPH binding site and that the association between protein and cofactor is modulated by hydrogen-bonding interactions along one face of this antiparallel beta-barrel structure . A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase . Geometrical comparison of this model with an experimentally determined structure for chicken DHFR suggests that chromosomal and type II R-plasmid specified enzymes may have independently evolved similar catalytic machinery for substrate reduction.

Biochemistry, 1986 Jul 29, 25(15), 4450 - 6
Study of the interaction of Escherichia coli methionyl-tRNA synthetase with tRNAfMet using chemical and enzymatic probes; Pelka H et al.; The accessibility of nucleotides in Escherichia coli tRNAfMet to chemical and enzymatic probes in the presence and absence of methionyl-tRNA synthetase has been investigated . Dimethyl sulfate was used to probe the reactivity of cytosine and guanosine residues . The N-3 position of the wobble anticodon base, C34, was strongly protected from methylation in the tRNA-synthetase complex . A synthetase-induced conformational change in the anticodon loop was suggested by the enhanced reactivity of C32 in the presence of enzyme . Cytosine residues in the dihydrouridine loop and in the 3'-terminal CCA sequence showed little or no change in reactivity . Methylation of the N-7 position of guanosine residues G42, G52, and G70 was partially inhibited by the synthetase . Nuclease digestion of tRNAfMet with alpha-sarcin in the presence of 1-2 mM Mg2+ resulted in cleavage mainly at C71 in the acceptor stem and was strongly inhibited by synthetase . Other nuclease digestion experiments using the single strand specific nucleases RNase A and RNase T1 revealed weak protection of nucleotides in the D loop and strong protection of nucleotides in the anticodon on complex formation . The present data, together with previous structure-function studies on this system, indicate strong binding of methionyl-tRNA synthetase to the anticodon of tRNAfMet, leading to a change in the conformation of the anticodon loop and stem . We propose that this, in turn, produces more distant, and possibly relatively subtle, conformational changes in other parts of the tRNA structure that ultimately lead to proper orientation of the 3' terminus of the tRNA with respect to the active site of the enzyme.

Biochemistry, 1986 Jul 29, 25(15), 4371 - 6
Covalent modification of the inhibitor binding site(s) of Escherichia coli ADP-glucose synthetase: specific incorporation of the photoaffinity analogue 8-azidoadenosine 5'-monophosphate; Larsen CE et al.; The photoaffinity agent 8-azidoadenosine 5'-monophosphate (8-N3AMP) is an inhibitor site specific probe of the Escherichia coli ADP-glucose synthetase (ADPG synthetase) . In the absence of light, 8-N3AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP . In the presence of light (254 nm), the analogue specifically and covalently modifies the enzyme, and photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation . The substrate ADPG provides nearly 100% protection from 8-N3AMP photoinactivation, while the substrate ATP provides approximately 50% protection and the inhibitor AMP, approximately 30% protection . These three adenylate allosteric effectors of E . coli ADPG synthetase also protect it from photoincorporation of 8-N3AMP . A structural overlap of the inhibitor and substrate binding sites is proposed which explains the protection data in light of the known binding and kinetic properties of this tetrameric enzyme.

Nucleic Acids Res, 1986 Jul 25, 14(14), 5575 - 89
Mutant Escherichia coli Ada proteins simultaneously defective in the repair of O6-methylguanine and in gene activation; Demple B; The activated Ada protein triggers expression of DNA repair genes in Escherichia coli in response to alkylation damage . Ada also possesses two distinct suicide alkyltransferase activities, for O6-alkylguanines and for alkyl phosphotriesters in DNA . The mutant Ada3 and Ada5 transferases repair O6-methylguanine in DNA 20 and 3000 times more slowly, respectively, than the wild-type Ada protein, but both exhibit normal DNA phosphotriester repair . These same proteins also exhibit delayed and sluggish induction of the ada and alkA genes . Since the C-terminal O6-methylguanine methyltransferase domain of Ada is not implicated in the direct binding of specific DNA sequences, this part of the Ada protein is likely to play an alternative mechanistic role in gene activation, either by promoting Ada dimerization, or via direct contacts with RNA polymerase.

J Biol Chem, 1986 Jul 25, 261(21), 9966 - 71
Structural analysis of the ileR locus of Escherichia coli K12; Weiss DL et al.; The Ile repressor protein negatively controls expression from the ilv and thr promoters of Escherichia coli K12 . Its existence was inferred from an analysis of the phenotypes of the ileR mutant avr-16 (Johnson, D . I., and Somerville, R . L . (1983) J . Bacteriol . 155, 49-55) . The nucleotide sequence of ileR, the structural gene for Ile repressor, has been determined . A DNA segment of 300 base pairs constitutes the ileR gene . The predicted gene product, a protein of 100 amino acids (molecular weight 11,823) has primary structural features reminiscent of other double-stranded DNA-binding regulatory proteins . S1 nuclease mapping of the 5' terminus of ileR mRNA revealed two discrete species whose startpoints differed by approximately 47 nucleotides . The ileR gene, like other repressors for amino acid biosynthetic systems, is autogenously regulated at the transcriptional level . Within the ileR promoter region lie two 18-base pair segments of DNA bearing significant homology to putative operator targets also found within the thr and ilv promoters . A second open reading frame capable of specifying a protein of 83 amino acids, designated orf83, is transcribed divergently from the ileR gene . There are 202 base pairs separating the first codons of the two genes . S1 nuclease mapping of the 5' terminus of orf83 mRNA revealed two discrete species whose startpoints differed by approximately 27 nucleotides . The upstream promoters for ileR and orf83 overlap in their -35 regions.

J Biol Chem, 1986 Jul 25, 261(21), 9951 - 8
Crystalline arrays of the Escherichia coli sn-glycerol-3-phosphate acyltransferase, an integral membrane protein; Wilkison WO et al.; The gene encoding the Escherichia coli sn-glycerol-3-phosphate acyltransferase, plsB, was inserted into hybrid plasmids under transcriptional control of the lambda PL and tac promoters . Enzymatic activities 35-50-fold above wild type and a large increase in glycerol-P acyltransferase polypeptide were obtained . Thin section electron microscopy of the cells overproducing the glycerol-P acyltransferase revealed 235-245-A diameter tubular structures associated with the cytoplasmic membrane . These structures were released from the cell by osmotic lysis and purified on Matrex Gel Green A . Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the dominant protein constituent of the tubules was the glycerol-P acyltransferase . Analysis of tubule-enriched fractions isolated by differential centrifugation revealed a decreased phospholipid to protein ratio as compared to total and cytoplasmic membrane fractions . At high magnification, negative stained tubules displayed ordered arrays of stain-excluding components projecting 50-60 A from the cytoplasmic surface . Optical diffraction patterns from the micrographs contained intense layer lines at (1/78 A) and (1/39 A) along the tubule axis and a prominent spot at (1/62 A) near the equator . From compositional and structural data, 18-37% of the polypeptide volume is estimated to lie within the hydrophobic domain of the tubule membrane.

J Biol Chem, 1986 Jul 25, 261(21), 9607 - 13
The DNA scanning mechanism of T4 endonuclease V . Effect of NaCl concentration on processive nicking activity; Gruskin EA et al.; T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism . A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers . The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction . Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl . The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining . Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased . At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism . The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM . The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction . In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed.

Biochim Biophys Acta, 1986 Jul 25, 872(1-2), 92 - 7
Localization of lysine residues in the binding domain of the K99 fibrillar subunit of enterotoxigenic Escherichia coli; Jacobs AA et al.; Modification of lysine residues with 4-chloro-3,5-dinitrobenzoate results in the loss of the binding capacity of K99 fibrillae to horse erythrocytes (Jacobs, A.A.C., van Mechelen, J.R . and de Graaf, F.K . (1985) Biochim . Biophys . Acta 832, 148-155) . In the present study we used dinitrobenzoate as a spectral probe to map the modified residues . After the incorporation of 0.7 mol CDNB per mol subunit, 90% of the binding activity disappeared and the lysine residues at positions 87, 132 and 133 incorporated 20%, 27.5% and 52.2% of the totally incorporated label, respectively . In the presence of the glycolipid receptor, Lys-132 and Lys-133 were partially protected against modification, while Lys-87 was not protected . The results suggest that Lys-132 and Lys-133 are part of the receptor-binding domain of the K99 fibrillar subunit and that the positive charges on these residues are important for the interaction of the fibrillae with the negatively charged sialic acid residue of the glycolipid receptor . A striking homology was found between a six-amino-acid residue segment of K99, containing Lys-132 and Lys-133, and segments of three other sialic-acid-specific lectins; cholera toxin B subunit, heat-labile toxin B subunit of Escherichia coli and CFA1 fimbrial subunit, suggesting that these segments might also be part of the receptor-binding domain in these three proteins.

Nucleic Acids Res, 1986 Jul 25, 14(14), 5693 - 711
Mechanism of autonomous control of the Escherichia coli F plasmid: different complexes of the initiator/repressor protein are bound to its operator and to an F plasmid replication origin; Masson L et al.; E protein, the 29 kd product of the F plasmid repE gene, plays both positive and negative roles in the autoregulation of F replication . We have cloned and expressed the repE gene in an inducible ATG-fusion vector and have detected specific binding of E protein to the repE operator and to four 19-base pair direct repeats (incB) within the F plasmid replication origin ori2 . Binding of E protein at the repE operator occurs with higher affinity than at ori2(incB) and gives almost complete protection to at least 30 base pairs, whereas binding of E protein to the direct repeats in the ori2 region shows an alternating pattern of enhanced and reduced sensitivity to DNAase cleavage consistent with a protein-induced folding of the DNA . These results provide direct biochemical support for a model of F plasmid replication in which the E protein serves both as an initiator of replication and as an autorepressor of its own synthesis.

Nucleic Acids Res, 1986 Jul 25, 14(14), 5615 - 27
Gene activation properties of a mouse DNA sequence isolated by expression selection; von Hoyningen-Huene V et al.; The MES-1 element was previously isolated from restricted total mouse cellular DNA by "expression selection"--the ability to reactivate expression of a test gene devoid of its 5' enhancer sequences . Mes-1 has been tested in long-term transformation and short-term CAT expression assays . In both assays MES-1 is active independent of orientation and at a distance when placed 5' to the test gene . The element is active with heterologous promoters and functions efficiently in both rat and mouse cells . MES-1 activates expression by increasing transcription from the test gene's own start (cap) site . Thus the expression selection technique can be used for the isolation of DNA sequences with enhancer-like properties from total cellular DNA.

Nature, 1986 Jul 24-30, 322(6077), 335 - 9
The cytoplasmic carboxy terminus of M13 procoat is required for the membrane insertion of its central domain; Kuhn A et al.; The M13 coat protein spans the Escherichia coli plasma membrane with its amino-terminus facing the periplasm . It is made as a precursor--the procoat--with a typical leader peptide . Mutations which destroy the basic character of the carboxy-terminal domain of procoat, a domain which is oriented towards the cytoplasm, block membrane assembly, while insertion of three lysyl residues near the carboxy terminus partially restores assembly . Thus the information specifying membrane insertion of M13 procoat protein is found in its mature region as well as the leader and is not simply decoded in an amino to carboxy direction.

J Mol Biol, 1986 Jul 20, 190(2), 191 - 9
Models for the structure of outer-membrane proteins of Escherichia coli derived from raman spectroscopy and prediction methods; Vogel H et al.; The secondary structure of porin, maltoporin and OmpA protein reconstituted in lipid membranes is determined by Raman spectroscopy . The three proteins have similar structures consisting of 50 to 60% beta-strand, about 20% beta-turn, and less than 15% alpha-helix . Employing a method for structural prediction that accounts for amphipathic beta-strands, folding models are developed for porin and for the segment of OmpA protein incorporated into the membrane . In the model, the OmpA fragment consists of eight amphipathic membrane-spanning beta-strands that form a beta-barrel . Similarly, porin is folded into ten amphipathic membrane-spanning beta-strands that are located at the surface of the trimer towards the lipids and eight predominantly hydrophilic strands in the interior.

Science, 1986 Jul 18, 233(4761), 347 - 51
Studies of the human c-myb gene and its product in human acute leukemias; Slamon DJ et al.; The myb gene is the transforming oncogene of the avian myeloblastosis virus (AMV); its normal cellular homolog, c-myb, is conserved across a broad span of evolution . In humans, c-myb is expressed in malignant hematopoietic cell lines and in primary hematopoietic tumors . Partial complementary DNA clones were generated from blast cells of patients with acute myelogenous leukemia . The sequences of the clones were compared to the c-myb of other species, as well as the v-myb of AMV . In addition, the carboxyl terminal region of human c-myb was placed in an expression vector to obtain protein for the generation of antiserum, which was used to identify the human c-myb gene product . Like v-myb, this protein was found within the nucleus of leukemic cells where it was associated with the nuclear matrix . These studies provide further evidence that c-myb might be involved in human leukemia.

Nature, 1986 Jul 17-23, 322(6076), 273 - 5
Expression of peptide chain release factor 2 requires high-efficiency frameshift; Craigen WJ et al.; Peptide chain release factors are soluble proteins that participate in the stop codon-dependent termination of polypeptide biosynthesis . In Escherichia coli, two release factors are necessary for peptide chain termination: release factor 1 (RF1) specifies UAG- and UAA-dependent termination whereas release factor 2 (RF2) specifies UGA- and UAA-dependent termination . Release factors are found in low concentrations relative to other translation factors, suggesting that their expression is tightly regulated and, accordingly, making the study of their structure-function relationship difficult . RF1 and RF2 exhibit significant sequence homology, probably reflecting their similar functions and perhaps a common evolutionary origin . DNA and peptide sequencing have suggested the existence of a unique mechanism for the autogenous regulation of RF2 in which an in-frame UGA stop codon requires an obligatory +1 frameshift within the coding region of the RF2 gene . In this report we present in vitro experimental results consistent with the autogenous regulation of RF2 . Additionally, we used RF2-lacZ gene fusions to demonstrate that autogenous regulation occurs, at least in part, by premature termination at the in-frame stop codon, since deletion of this stop codon leads to overproduction of the RF2-LacZ fusion protein . Frameshifting at this premature termination codon occurs at the remarkably high rate of 50%.

Nature, 1986 Jul 17-23, 322(6076), 228 - 32
Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria; Eilers M et al.; Methotrexate, a folate antagonist, blocks import into mitochondria of mouse dihydrofolate reductase fused to a mitochondrial presequence . Methotrexate does not mask the presequence, but stabilizes the dihydrofolate reductase moiety . It does not inhibit import of the authentic precursor from which the presequence is derived . This suggests that dihydrofolate reductase must at least partly unfold in order to be transported across mitochondrial membranes.

Biochem Biophys Res Commun, 1986 Jul 16, 138(1), 72 - 7
An Escherichia coli mutant exhibiting temperature-sensitive ATP synthesis; Ito M et al.; A mutant strain SM434 (ttr-3) of Escherichia coli that exhibits a temperature-sensitive Unc(succinate-nonutilizing) phenotype was characterized . The mutant allele ttr-3 was not linked to the ilvA gene, but was complemented by Fill carrying 81 min-91 min of the E . coli chromosome . The mutant strain SM434 exhibited resistance to N,N'-dicyclohexylcarbodiimide (DCCD) and a temperature-sensitive phenotype at the level of ATP synthesis, compatible with that of cell growth . These findings indicate that the mutant strain SM434 could carry a mutation (ttr-3) in an unknown gene responsible for the energy-transduction system.

Biochem Biophys Res Commun, 1986 Jul 16, 138(1), 110 - 7
Preferential nucleosome placement on pBR322 restriction fragments; McNamara PT et al.; Two restriction fragments of DNA containing the regulatory feature GTG/CAC were experimentally associated with core histones . The reconstituted DNA-histone complexes consisted of different forms of mononucleosomes . Lambda exonuclease and Fnu4HI were used to probe the structure of each distinct nucleoprotein complex . For each of the DNA fragments, one form of particle was produced that showed preferred placement of the core octamer on the DNA . The GTG/CAC base triplets may play some role in determining the final histone core positions in these reconstitutes.

Clin Chim Acta, 1986 Jul 15, 158(1), 99 - 108
Highly sensitive enzyme immunoassay for human creatine kinase MM and MB isozymes; Kato K et al.; A sensitive enzyme immunoassay system for measurement of MM and MB forms of human creatine kinase (CK) was developed using purified monospecific antibodies to the M subunit and to the B subunit of CK . The CK-MM assay system consisted of polystyrene balls with immobilized F(ab')2 fragments of anti-M and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli . The CK-MB was assayed with the polystyrene balls with either antibody (anti-M or anti-B) F(ab')2 fragments and the other antibody (anti-B and anti-M, respectively) Fab' fragments labeled with galactosidase . The assays were highly sensitive and 3 pg of CK-MM and CK-MB were measurable . The CK-MM assay was specific to the M subunit of creatine kinase, and it cross-reacted about 15% with CK-MB, but not with CK-BB . The CK-MB assay did not cross-react with CK-MM nor CK-BB . Therefore, concentrations of CK-MM could be estimated by subtracting the cross-reacting values of CK-MB . Coefficients of variation in within-run and between-run precision studies for serum CK-MM and CK-MB were less than 9% . Serum levels in healthy adults of various ages (16-59 yr old) were ranged from 35.2-132 ng/ml for CK-MM and from 0.40-1.77 ng/ml for CK-MB . There was apparently no statistical significance among the sex- and age-related differences . Concentrations of CK-MM and CK-MB in various human tissues were also determined . The CK-MM was present abundantly in the heart and the tissues composed of striated muscles (skeletal muscle, diaphragm and proximal esophagus) . The CK-MB was distributed not only in the heart but also in the striated muscle tissues at a relatively high level.

Biochemistry, 1986 Jul 15, 25(14), 4085 - 90
Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system has two sites of phosphorylation per dimer; Waygood EB; Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli has been reported to contain one phosphorylation site per dimer and thus operates by either a half of the sites or a flip-flop mechanism {Misset, O., & Robillard, G . T . (1982) Biochemistry 21, 3136-3142; Hoving, T., ten Hoeve-Duurkens, R., & Robillard, G . T . (1984) Biochemistry 23, 4335-4340} . In this paper, the determination of two phosphorylation sites per dimer of enzyme I was made by using a number of different methods . In some experiments, less than two sites per dimer were found, but a concomitant loss in enzyme I activity was also found . The phosphorylated residue in enzyme I was shown to have the properties expected for a N3-phosphohistidinyl residue.

Biochemistry, 1986 Jul 15, 25(14), 4046 - 51
Subunit interactions of the Escherichia coli mannitol permease: correlation with enzymic activities; Stephan MM et al.; A fraction of the phosphorylated form of the Escherichia coli mannitol permease (enzyme IIMtl) of the sugar phosphotransferase system can be extracted from the membrane in a dimeric form {Roossien, F.F., & Robillard, G.T . (1984) Biochemistry 23, 5682-5685} . Using E . coli minicells in which this protein can be specifically labeled with {35S}methionine, we show in this paper that part of the unphosphorylated form of enzyme IIMtl can also be extracted from the membrane as a dimer . We further demonstrate that both phosphoenolpyruvate-dependent phosphorylation of the permease and conditions promoting turnover of the enzyme decrease the amount of extractable dimer . Thus, the dimer of these forms of the enzyme appears to be less stable than that of the unmodified form, at least in detergent solution . In contrast, inorganic phosphate, which activates the permease-catalyzed phospho exchange between mannitol 1-phosphate and mannitol ("transphosphorylation"), stabilizes the dimer . These results support the hypothesis that the mannitol permease dimer is more active in transphosphorylation than the monomer . Treatment of minicell membranes with oxidizing agents produced heat-stable, high molecular weight aggregates of the permease on dodecyl sulfate gels, but no heat-stable dimer could be detected . The nonionic detergent Lubrol PX decreased the amount of dimer extractable at 30 degrees C with a concomitant increase in the monomeric form . These results suggest that the dimer depends predominantly on hydrophobic interactions for its stability and is not covalently cross-linked in that form by oxidizing agents.

Biochemistry, 1986 Jul 15, 25(14), 4041 - 5
Purification and characterization of recombinant single-chain urokinase produced in Escherichia coli; Winkler ME et al.; Recombinant single-chain urokinase (rUK1) has been purified from Escherichia coli . The purification utilizes a refractile body purification, followed by batch DE-52 cellulose extraction, hydroxylapatite chromatography, and S-200 chromatography . Two-chain rUK (rUK2) is separated from rUK1 on benzamidine--Sepharose . The purification eliminates proteases early in the procedure so the rUK1 will not be cleaved to rUK2 . The rUK1 has been characterized by amino-terminal analysis as well as carboxy-terminal analysis after cleavage by plasmin.

Eur J Biochem, 1986 Jul 15, 158(2), 423 - 8
Limited proteolysis of lactose permease from Escherichia coli; Stochaj U et al.; Escherichia coli lactose permease (also referred to as lactose carrier) is an integral protein of the cytoplasmic membrane . Using lactose permease either radiolabeled biosynthetically in plasmid-bearing E . coli minicells or radioalkylated post-synthetically by chemical modification, we have determined sites on the membrane-bound protein accessible to proteolytic attack and we have characterized several high-molecular-mass products . The most prominent polypeptide obtained from lactose permease radiolabeled biosynthetically is observed after digestion with different proteases . The fragment produced by thermolysin was shown to contain the intact N-terminus and to extend into the region around amino acid residue 140 which, according to secondary structure models, is presumed to be less tightly folded than the rest of the molecule . Evidence is presented that the corresponding fragments obtained after digestion with several other proteases also originate from the N-terminal part of the protein . This N-terminal segment of the lactose carrier is resistant to proteolytic digestion even in the presence of non-ionic detergents and it may represent a tightly folded domain . Additional proteolytic cleavage sites located C-terminal of the Cys148 residue can be inferred.

J Biol Chem, 1986 Jul 15, 261(20), 9405 - 11
Molecular cloning and sequencing of the sppA gene and characterization of the encoded protease IV, a signal peptide peptidase, of Escherichia coli; Ichihara S et al.; Clones carrying a gene causing overproduction of protease IV, a signal peptide peptidase of Escherichia coli, were isolated from the Clarke and Carbon's collection . Restriction mapping analysis revealed that pLC7-10 and pLC40-13, thus isolated, shared the same chromosomal DNA region . The 2.3-kilobase RsaI-SalI fragment in this region, which was found to carry the gene, was subjected to nucleotide sequence determination . Only one long open reading frame was found . The hypothetical polypeptide sequence deduced from the DNA sequence has a molecular mass of 67,241 daltons . The putative gene was named sppA . Protease IV was purified to homogeneity from the cytoplasmic membrane of an overproducing strain harboring a sppA gene-carrying plasmid . The purified enzyme gave a single polypeptide band of 67,000-dalton molecular mass on sodium dodecyl sulfate-polyacrylamide gel . This molecular mass and the amino acid composition of the purified enzyme were consistent with the deduced primary structure of the sppA gene product . The molecular mass thus determined was almost twice as large as that previously reported by Pacaud (Pacaud, M . (1982) J . Biol . Chem . 257, 4333-4339) . A cross-linking study revealed that protease IV is a tetramer of the polypeptide . From these results, we conclude that protease IV is a tetramer of the sppA gene product.

J Biol Chem, 1986 Jul 15, 261(20), 9083 - 6
Reconstitution of sugar phosphate transport systems of Escherichia coli; Ambudkar SV et al.; Studies with Escherichia coli cells showed that the transport systems encoded by glpT (sn-glycerol 3-phosphate transport) and uhpT (hexose phosphate transport) catalyze a reversible 32Pi:Pi exchange . This reaction could be used to monitor the glpT or uhpT activities during reconstitution . Membranes from suitably constructed strains were extracted with octylglucoside in the presence of lipid and glycerol, and proteoliposomes were formed by dilution in 0.1 M KPi (pH 7) . Both reconstituted systems mediated a 32Pi:Pi exchange which was blocked by the appropriate heterologous substrate, sn-glycerol 3-phosphate (G3P) or 2-deoxyglucose 6-phosphate (2DG6P), with an apparent Ki near 50 microM . In the absence of an imposed cation-motive gradient, Pi-loaded proteoliposomes also transported the expected physiological substrate; Michaelis constants for the transport of G3P or 2DG6P were near 20 microM . The heterologous exchange showed a maximal velocity of 130 nmol/min/mg protein via the glpT system and 11 nmol/min/mg protein for the uhpT system . This difference was expected because the G3P transport activity had been reconstituted from a strain carrying multiple copies of the glpT gene . Taken together, these results suggest that anion exchange may be the molecular basis for transport by the glpT and uhpT proteins.

Biochemistry, 1986 Jul 15, 25(14), 4091 - 7
Effects of certain 5'-substituted adenosines on polyamine synthesis: selective inhibitors of spermine synthase; Pegg AE et al.; A number of nucleosides related to S-adenosylmethionine were tested for their inhibitory action on three enzymes involved in the biosynthesis of polyamines . The particular objective of the experiments was to determine whether any of the compounds could be used as selective inhibitors of the synthesis of spermine by spermine synthase . None of the nucleosides examined were potent inhibitors of S-adenosylmethionine decarboxylase . 5'-{(3-Aminopropyl)amino}-5'-deoxyadenosine dihydrochloride was quite a strong inhibitor of spermidine synthase (I50 of 7 microM) but was more than an order of magnitude less active than S-adenosyl-1,8-diamino-3-thiooctane, which is a mechanism-based inhibitor of this enzyme . 5'-{(3-Aminopropyl)amino}-5'-deoxyadenosine also inhibited spermine synthase with an I50 of 17 microM, but more selective inhibition of spermine synthase was produced by 9-{6(RS),8-diamino-5,6,7,8-tetradeoxy-beta-D-ribo-octofuranosyl}-9 H-purin-6- amine (I50 of 12 microM) and by dimethyl(5'-adenosyl)sulfonium perchlorate (I50 of 8 microM) since these compounds were much less active against spermidine synthase . Both 9-{6(RS),8-diamino-5,6,7,8-tetradeoxy-beta-D-ribo-octofuranosyl}-9 H-purin-6- amine and dimethyl(5'-adenosyl)sulfonium perchlorate were able to reduce the synthesis of spermine in SV-3T3 cells, but there was a compensatory increase in the concentration of spermidine, and there was no effect on cell growth . These results and those from experiments in which these spermine synthesis inhibitors were combined with inhibitors of spermidine synthase and ornithine decarboxylase indicated that the cells compensated for the inhibition of the aminopropyltransferases by increasing the production of decarboxylated S-adenosylmethionine and putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem J, 1986 Jul 15, 237(2), 427 - 37
The cloning and expression of the aroL gene from Escherichia coli K12 . Purification and complete amino acid sequence of shikimate kinase II, the aroL-gene product; Millar G et al.; The aroL gene encoding the enzyme shikimate kinase II was cloned from Escherichia coli K12 . Construction of over-expressing strains permitted for the first time the purification to homogeneity of a monofunctional shikimate kinase . The complete amino acid sequence of shikimate kinase II was determined by a combined nucleotide and direct amino acid sequencing strategy . E . coli shikimate kinase II is a monomeric enzyme containing 173 amino acid residues with a calculated Mr 18,937 . The amino acid sequence contains a region homologous with other kinases and ATP-requiring enzymes . Evidence is presented suggesting that the transcriptional start site of the aroL gene is located within a potential operator site.

J Biol Chem, 1986 Jul 15, 261(20), 9534 - 9
Replication from one of the three origins of the plasmid R6K requires coupled expression of two plasmid-encoded proteins; Mukhopadhyay P et al.; The minimal beta-replicon of plasmid R6K contains an open reading frame for a 151-amino acid protein in addition to the seven 22-base pair direct nucleotide sequence repeats, the structural gene (pir) for the pi initiation protein, and the beta-origin sequence that have been shown to be required for replication activity . In this work, a site-specific mutation by linker insertion in this putative coding sequence, designated bis, resulted in a nonfunctional beta-replicon . The nonfunctional beta-replicon was complementable in trans and the protein coded by the bis sequence was detected in an immunoblot assay as a hybrid product from a bis-lac z fused gene . The bis gene is not required for a functional alpha or gamma origin replication origin of R6K . A site-specific mutation in the upstream pir gene was shown to lead to a loss of synthesis of the bis product and inactivation of the beta-replicon . Trans-complementation of this mutation for beta-replicon activity required the wild-type sequence of the pir gene joined to the intact bis sequence . These results indicate that the bis product is required for activity specifically of the beta-origin, and its synthesis is coupled in cis to the expression of pi protein from an unaltered pir gene.

J Biol Chem, 1986 Jul 15, 261(20), 9316 - 20
Regulation of the ompC gene of Escherichia coli . Involvement of three tandem promoters; Ikenaka K et al.; ompC expression in Escherichia coli K-12 is known to be regulated by the ompB locus, comprising the ompR and envZ genes, and the OmpR protein is believed to act as a positive transcriptional factor . We examined the transcriptional capability of the ompC gene in vitro and found that RNA polymerase could transcribe ompC without a requirement for other transcriptional factors . Furthermore, transcripts from three tandem promoters in ompC were identified in vitro . We employed oligonucleotide-directed site-specific mutagenesis to dissect the promoter region of the gene and assayed the promoters separately for transcriptional ability using fusions to the lacZ gene . The levels of beta-galactosidase indicate that ompC expression in vivo is dependent on the function of at least one of the upstream promoters . The function of OmpR appears to be the enhancement of a basal level of ompC expression . From the results of our experiments, the site of action of OmpR was deduced to be in the vicinity of the upstream promoters of ompC.

J Biol Chem, 1986 Jul 15, 261(20), 9526 - 33
Mechanism of replication of ultraviolet-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli . Implications for SOS mutagenesis; Livneh Z; Replication of UV-irradiated oligodeoxynucleotide-primed single-stranded phi X174 DNA with Escherichia coli DNA polymerase III holoenzyme in the presence of single-stranded DNA-binding protein was investigated . The extent of initiation of replication on the primed single-stranded DNA was not altered by the presence of UV-induced lesions in the DNA . The elongation step exhibited similar kinetics when either unirradiated or UV-irradiated templates were used . Inhibition of the 3'----5' proofreading exonucleolytic activity of the polymerase by dGMP or by a mutD mutation did not increase bypass of pyrimidine photodimers, and neither did purified RecA protein influence the extent of photodimer bypass as judged by the fraction of full length DNA synthesized . Single-stranded DNA-binding protein stimulated bypass since in its absence the fraction of full length DNA decreased 5-fold . Termination of replication at putative pyrimidine dimers involved dissociation of the polymerase from the DNA, which could then reinitiate replication at other available primer templates . Based on these observations a model for SOS-induced UV mutagenesis is proposed.

Virology, 1986 Jul 15, 152(1), 1 - 10
Induction of cellular DNA synthesis by purified adenovirus E1A proteins; Kaczmarek L et al.; The purified Escherichia coli-expressed products of the human subgroup-C adenovirus E1A 13 S and 12 S mRNAs are shown to induce cellular DNA synthesis when introduced by microinjection into quiescent, G0-arrested mammalian cells from immortalized cell lines . The E1A proteins stimulated cellular DNA synthesis in mouse Swiss 3T3 cells, when microinjected either individually or in combination . A truncated E1A protein, in which 169 carboxyl terminal residues of the 289-amino acid E1A 13 S mRNA product are deleted, was unable to induce cellular DNA synthesis in these cells . Our results provide evidence that E1A proteins can function, independent of other viral functions, in the stimulation of cellular DNA synthesis in certain cell types . The present results are consistent with the E1A gene products acting to modulate the expression of the cellular genes which control cell cycle progression into S phase.

J Biol Chem, 1986 Jul 15, 261(20), 9196 - 201
Replacement of arginine 246 by histidine in the beta subunit of Escherichia coli H+-ATPase resulted in loss of multi-site ATPase activity; Noumi T et al.; A mutant strain KF43 of Escherichia coli defective in the beta subunit of H+-translocating ATPase (F0F1) was examined . In this mutant, replacement of Arg246 by His was identified by DNA sequencing of the mutant gene and confirmed by tryptic peptide mapping . The mutant F1-ATPase was defective in multi-site hydrolysis of ATP but was active in uni-site hydrolysis . Studies on the kinetics of uni-site hydrolysis indicated that the k1 (rate of ATP binding) was similar to that of the wild-type, but the k-1 (rate of release of ATP) could not be measured . The mutant enzyme had a k3 (rate of release of inorganic phosphate) about 15-fold higher than that of the wild-type and showed 3 orders of magnitude lower promotion from uni- to multi-site catalysis . These results suggest that Arg246 or the region in its vicinity is important in multi-site hydrolysis of ATP and is also related to the binding of inorganic phosphate . Reconstitution experiments using isolated subunits suggested that hybrid enzymes (alpha beta gamma complexes) carrying both the mutant and wild-type beta subunits were inactive in multi-site hydrolysis of ATP, supporting the notion that three intact beta subunits are required for activity of the F1 molecule.

J Biol Chem, 1986 Jul 15, 261(20), 9140 - 3
Codon-anticodon interaction at the ribosomal E site; Rheinberger HJ et al.; The question of whether or not the tRNA at the third ribosomal binding site specific for deacylated tRNA (E site) undergoes codon-anticodon interaction was analyzed as follows . Poly(U)-programmed ribosomes each carrying two {14C}tRNAPhe molecules were subjected to a chasing experiment using various tRNA species . At 0 degree C Ac{3H}Phe-tRNAPhe did not trigger any chasing whereas deacylated cognate tRNAPhe provoked a strong effect; non-cognate tRNALys was totally ineffective . This indicates that the second {14C}tRNAPhe cannot be present at the A site but rather at the E site (confirming previous observations) . In the presence of poly(U) or poly(A) ribosomes bound the cognate tRNA practically exclusively as second deacylated tRNA, i.e . {14C}tRNAPhe and {14C}tRNALys, respectively . Thus, the second deacylated tRNA binds in a codon-dependent manner . {14C}tRNALys at the P site and Ac{3H}Lys-tRNALys at the A site of poly(A)-primed ribosomes were translocated to the E and P sites, respectively, by means of elongation factor G . The E site-bound {14C}tRNALys could be significantly chased by cognate tRNALys but not by non-cognate tRNAPhe, indicating the coded nature of the E site binding . Additional evidence is presented that the ribosome accommodates two adjacent codon-anticodon interactions at either A and P or P and E sites.

J Biol Chem, 1986 Jul 15, 261(20), 9133 - 9
Allosteric interactions between the ribosomal transfer RNA-binding sites A and E; Rheinberger HJ et al.; We have previously proposed a three-site model for the elongation cycle . The model is characterized by the presence of two tRNAs on the ribosome before and after translocation . We have already shown a first consequence of the model, namely that the translocation reaction is not coupled with a release of deacylated tRNA . Here we demonstrate the following conclusions . Occupation of the A site triggers the tRNA release from the E site, i.e . the A site occupation induces a drastic decrease in the affinity of the E site for deacylated tRNA . In the concentration range of deacylated tRNA in which a ribosome binds a second tRNA in addition to that one already present at the P site the deacylated tRNA does not compete for one and the same binding site with an A site ligand (AcPhe-tRNA) at 37 degrees C . It follows that the second deacylated tRNA binds to a site, the E site, which is physically distinct from the A site . When the ribosome binds a deacylated tRNA at the E site (in addition to a tRNA at the P site), the A site cannot be occupied by AcPhe-tRNA at 0 degree C and only poorly by the ternary complex elongation factor Tu . Phe-tRNA . guanyl-5'-yl imidodiphosphate . At 37 degrees C a significant A site binding is observed, with a corresponding tRNA release from the E site . In contrast, if the E site is free and only the P site occupied, the A site can bind significant amounts of charged tRNA already at 0 degree C . It follows that an occupied E site induces a low-affinity state of the A site . Thus, the ribosome always contains two high-affinity binding sites, which are A and P sites before and P and E sites after translocation . A and E sites are allosterically linked in a bidirectional manner.

Vet Rec, 1986 Jul 12, 119(2), 39 - 42
Evaluation of a combined rotavirus and enterotoxigenic Escherichia coli vaccine in cattle; Snodgrass DR; A vaccine of rotavirus and K99 antigen from enterotoxigenic Escherichia coli was emulsified in oil adjuvant and administered intramuscularly to pregnant cows . Calves born to and reared on vaccinated dams were protected against experimental rotavirus infection at five days old when compared with calves from unvaccinated control cows . Field trials of the vaccine were carried out in 40 commercial herds, in which half the cows in each herd were selected at random for vaccination and half were left unvaccinated . In 31 herds (2641 cows) there was no significant diarrhoea problem (less than 10 per cent morbidity); these herds were excluded from further analysis . The nine remaining herds did experience a calf diarrhoea problem of greater than 10 per cent morbidity, but on four farms the disease was associated with cryptosporidiosis and on a fifth no enteropathogens were detected; these five farms (461 cows) were also excluded from further analysis . Of the remaining four herds, two beef suckler herds (105 cows) had concurrent rotavirus and cryptosporidial infections, and vaccination was associated with a decreased excretion of rotavirus but not with a decreased incidence of diarrhoea . In the other two dairy herds (68 cows) with prevaccination rotavirus problems, there was a significantly decreased incidence of diarrhoea in calves born to vaccinated cows . No natural field challenge of enterotoxigenic E coli was encountered on any of the trial farms.

Nucleic Acids Res, 1986 Jul 11, 14(13), 5125 - 43
Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes; Sharp PM et al.; Codon usage data has been compiled for 110 yeast genes . Cluster analysis on relative synonymous codon usage revealed two distinct groups of genes . One group corresponds to highly expressed genes, and has much more extreme synonymous codon preference . The pattern of codon usage observed is consistent with that expected if a need to match abundant tRNAs, and intermediacy of tRNA-mRNA interaction energies are important selective constraints . Thus codon usage in the highly expressed group shows a higher correlation with tRNA abundance, a greater degree of third base pyrimidine bias, and a lesser tendency to the A+T richness which is characteristic of the yeast genome . The cluster analysis can be used to predict the likely level of gene expression of any gene, and identifies the pattern of codon usage likely to yield optimal gene expression in yeast.

Nucleic Acids Res, 1986 Jul 11, 14(13), 5449 - 60
Nucleotide sequence and deduced amino acid sequence of Escherichia coli pyruvate oxidase, a lipid-activated flavoprotein; Grabau C et al.; The entire nucleotide sequence of the poxB (pyruvate oxidase) gene of Escherichia coli K-12 has been determined by the dideoxynucleotide (Sanger) sequencing of fragments of the gene cloned into a phage M13 vector . The gene is 1716 nucleotides in length and has an open reading frame which encodes a protein of Mr 62,018 . This open reading frame was shown to encode pyruvate oxidase by alignment of the amino acid sequences deduced for the amino and carboxy termini and several internal segments of the mature protein with sequences obtained by amino acid sequence analysis . The deduced amino acid sequence of the oxidase was not unusually rich in hydrophobic sequences despite the peripheral membrane location and lipid binding properties of the protein . The codon usage of the oxidase gene was typical of a moderately expressed protein . The deduced amino acid sequence shares homology with the large subunits of the acetohydroxy acid synthase isozymes I, II, and III, encoded by the ilvB, ilvG, and ilvI genes of E . coli.

Science, 1986 Jul 11, 233(4760), 206 - 8
Conformations of signal peptides induced by lipids suggest initial steps in protein export; Briggs MS et al.; Despite the requirement for a functional signal sequence in protein export, little is known of the conformational properties and membrane interactions of these highly hydrophobic amino terminal extensions on nearly all exported proteins . The Escherichia coli lambda phage receptor signal sequence was studied in phospholipid monolayers by circular dichroism and Fourier transform infrared spectroscopy; the signal peptide was shown to prefer an alpha-helical conformation when inserted into the lipid phase . However, interaction with the lipid surface without insertion induced the signal sequence, which is unstructured in bulk aqueous solution, to adopt a beta structure . These observations are combined in a model for the initial steps in signal sequence-membrane interaction in vivo.

FEBS Lett, 1986 Jul 7, 202(2), 323 - 6
The effects of 1-aminooxy-3-aminopropane and S-(5'-deoxy-5'-adenosyl)methylthioethylhydroxylamine on ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase from Escherichia coli; Paulin L; 1-Aminooxy-3-aminopropane (APA) was shown to be a potent competitive inhibitor (Ki = 1.0 nM) of partially purified Escherichia coli ornithine decarboxylase . APA did not inhibit S-adenosyl-L-methionine decarboxylase and spermidine from E . coli . S-(5'-Deoxy-5'-adenosyl)methylthioethylhydroxylamine (AMA), which is a structural analogue of decarboxylated S-adenosyl-L-methionine, was for the first time shown to be an irreversible inhibitor of bacterial S-adenosyl-L-methionine decarboxylase and a competitive inhibitor (Ki = 47 microM) of bacterial ornithine decarboxylase . AMA had no effect on spermidine synthase.

FEBS Lett, 1986 Jul 7, 202(2), 295 - 7
On the sequence homology of the ribosomal proteins, Escherichia coli S11, yeast rp59 and Chinese hamster S14; Tanaka T et al.; A strong sequence homology was found among the ribosomal proteins of the different species, S11 of Escherichia coli, rp59 of Saccharomyces cerevisiae and S14 of Chinese hamster ovary cell . The significance of this series of highly conserved proteins is discussed.

FEBS Lett, 1986 Jul 7, 202(2), 340 - 4
Affinity labelling of Escherichia coli ribosomes with a benzylidene derivative of AUGU6 within initiation and pretranslocational complexes; Babkina GT et al.; Affinity labelling of E . coli ribosomes with the 2',3'-O-{4-(N-2-chloroethyl)-N-methylamino}benzylidene derivative of AUGU6 was studied within the initiation complex (complex I) obtained by using fMet-tRNAMetf and initiation factors and within the pretranslocational complex (complex II) obtained by treatment of complex I with the ternary complex Phe-tRNAPhe.GTP.EF-Tu . Both proteins and rRNA of 30 S as well as 50 S subunits were found to be labelled . Sets of proteins labelled within complexes I and II differ considerably . Within complex II, proteins S13 and L10 were labelled preferentially . On the other hand, within complex I, multiple modification is observed (proteins S4, S12, S13, S14, S15, S18, S19, S20/L26 were found to be alkylated) despite the single fixation of a template in the ribosome by interaction of the AUG codon with fMet-tRNAMetf.

J Mol Biol, 1986 Jul 5, 190(1), 133 - 4
Crystallization of the C-terminal domain of colicin A carrying the voltage-dependent pore activity of the protein; Tucker AD et al.; The C-terminal fragment (Mr, 21,800) of colicin A (a bacterial toxin that kills sensitive Escherichia coli cells) has been crystallized . This fragment, which possesses the pore-forming activity of the toxin, resulted from thermolysin digestion of the entire molecule . The crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2) with a = b = 72.8 A, c = 170.4 A . They contain a dimer in the asymmetric unit and diffract to 2.7 A.

J Mol Biol, 1986 Jul 5, 190(1), 37 - 44
Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12; Makino K et al.; phoB encodes a positive regulator for a number of the genes belonging to the phosphate regulon of Escherichia coli, including phoA, phoS, phoE and ugpAB . We have determined the nucleotide sequence of the chromosomal segment containing the promoter and the coding region of the phoB gene . The sequence data combined with the known amino-terminal amino acid sequence of a PhoB-LacZ hybrid protein suggest that the PhoB protein consists of 228 amino acid residues with a Mr of 26,302 . In the regulatory region of the gene, a consensus nucleotide sequence shared by the regulatory regions of the phoA, phoS and phoE genes, which we name the "phosphate box", was found . Since these genes are positively regulated by the product of phoB, this suggests that transcription of the phoB gene is also regulated positively by its own product . Extensive homology was found in the amino acid sequences of the products of phoB, ompR and dye, all of which are positive regulatory genes for a number of genes coding for envelope proteins . This implies that these genes were originally duplications of a protogene that evolved to have divergent but related functions.

J Biol Chem, 1986 Jul 5, 261(19), 8608 - 16
Different interactions used by Cro repressor in specific and nonspecific DNA binding; Takeda Y et al.; The mode of interaction of Cro repressor with specific and nonspecific sites on DNA was explored by chemical modification and protection of lysine and tyrosine residues . Cro has 8 lysines . In the presence of DNA, lysines 32 and 56 are fully protected and lysines 21, 62, and 63 are partially protected from alkylation . However, the terminal amino group and lysines 8, 18, and 39 are not protected . Location of the protected and unprotected lysines on the three-dimensional Cro structure defines a DNA-binding region . The results provide direct experimental support for a mode of interaction between Cro and DNA, in which Cro buries its 2-fold related alpha-helices in consecutive DNA major grooves (Anderson, W . F., Ohlendorf, D . H., Takeda, Y., and Matthews, B . W . (1981) Nature 290, 754-758; Ohlendorf, D . H., Anderson, W . F., Fisher, R . G., Takeda, Y., and Matthews, B . W . (1982) Nature 298, 718-723) . In the model, the carboxyl-terminal part of Cro was tentatively presumed to interact with the DNA minor groove . Protection of lysines 62 and 63 confirms the involvement of the carboxyl terminus in DNA binding . Although nonspecific and specific DNA protect the same lysine residues, there are differences in the nature of the interaction of Cro with nonspecific and specific DNA . Cro-nonspecific DNA interaction is salt-sensitive, suggesting that the interaction is predominantly electrostatic . On the other hand, Cro-specific DNA interaction is salt-resistant, suggesting that the interaction may include nonelectrostatic components (hydrogen bonds and hydrophobic interactions) as well . Protection experiments of tyrosine residues (against iodination) suggest that the conformation of Cro repressor changes in two stages: first, when Cro binds at nonspecific sites, and, second, when Cro binds to specific sites on DNA.

J Biol Chem, 1986 Jul 5, 261(19), 8914 - 8
Site-specific mutagenesis of cysteine 148 to serine in the lac permease of Escherichia coli; Sarkar HK et al.; Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the lac Y gene of Escherichia coli such that Cys148 in the lac permease is converted to Ser . A mutagenesis protocol is used that significantly improves the efficiency of mutant recovery by in vitro methylation of closed-circular heteroduplex DNA containing the mutation, followed by nicking with HindIII in the presence of ethidium bromide and heat denaturation prior to transfection . In contrast to Gly148 permease (Trumble, W.R., Viitanen, P.V., Sarkar, H.K., Poonian, M.S., and Kaback, H . R . (1984) Biochem . Biophys . Res . Commun . 119, 860-867), permease containing Ser at position 148 catalyzes active lactose transport at a rate comparable to wild-type permease . Like Gly148 permease, however, transport activity is less sensitive to inactivation by N-ethylmaleimide, and galactosyl-1-thio-beta-D-galactopyranoside affords no protection against inactivation . The observations provide strong support for the contention that Cys148 is obligatory for substrate protection against inactivation by sulfhydryl reagents, but does not play an essential role in lactose:H+ symport.

J Mol Biol, 1986 Jul 5, 190(1), 113 - 7
DNA sequence and coding properties of mutD(dnaQ) a dominant Escherichia coli mutator gene; Cox EC et al.; The mutD(dnaQ) gene in Escherichia coli codes for the epsilon subunit of the DNA polymerase pol III holoenzyme . Previous work has shown that this gene lies adjacent to the gene coding for RNase H (rnh) . The two products are translated from diverging promoters . Here we report on the 1.6 kb (1 kb = 10(3) bases or base-pairs) sequence of the region coding for both genes, and the transcripts encoded by them . mutD codes for two transcripts, one of whose origins lies within the rnh structural gene . Both transcripts overlap and are complementary to a region of the rnh transcript . Thus, they can potentially form double-stranded helices with rnh . Of the two possible double-stranded structures, the shorter does not interfere with a likely rnh ribosome binding site, while the longer one does . We suggest that this unique organization may regulate rnh translation rates.

J Biol Chem, 1986 Jul 5, 261(19), 8965 - 76
Structure and complete nucleotide sequence of the chicken alpha-smooth muscle (aortic) actin gene . An actin gene which produces multiple messenger RNAs; Carroll SL et al.; The alpha-smooth muscle (aortic) actin gene is a distinct member of the actin multigene family which is expressed in vascular smooth muscle cells . We have determined the complete nucleotide sequence of 11 kilobase pairs of genomic DNA encoding the chicken alpha-smooth muscle actin gene . This single copy gene specifies a protein identical in sequence to the major alpha-actin from bovine aorta . The protein-coding sequences are interrupted by seven introns which are at codons specifying amino acid residues 41/42, 84/85, 121/122, 150, 204, 267, and 327/328 . An eighth intron was found in the mRNA 5' untranslated region . The 5' flanking sequences contain elements which are conserved in other chicken muscle actin genes . Additional sequences at the 5' end of the gene may be conserved in at least one human actin gene . We have identified at least four messenger RNAs ranging in size from approximately 1370 to 2700 nucleotides (excluding poly(A) tails) which are transcribed from the alpha-smooth muscle actin gene . These RNAs differ in the length of their 3' untranslated regions, probably as a result of the utilization of alternative polyadenylation signals . This is the first report of an actin gene with multiple mRNA transcripts.

J Biol Chem, 1986 Jul 5, 261(19), 8953 - 7
Trimeric structure and localization of the major lipoprotein in the cell surface of Escherichia coli; Choi DS et al.; A hybrid gene consisting of the ompF promoter, the coding regions for the signal peptide, and the Ala-Glu residue of the OmpF NH2 terminus and the coding region for the major outer membrane lipoprotein devoid of the NH2-terminal cysteine residue was constructed . Escherichia coli carrying the cloned gene produced the predicted hybrid protein that is the same as the major lipoprotein except that the diacyl glycerylcysteine residue at the NH2 terminus is replaced by the Ala-Glu residue . The hybrid protein was localized in the periplasmic space as a trimer with a noncovalent interaction in addition to the previously known covalent interaction with the peptidoglycan . These results strongly indicate that the major lipoprotein exists as a trimer in the periplasmic space with covalent and noncovalent interactions with the peptidoglycan layer through the protein domain on one side and with the hydrophobic interaction with the outer membrane through the lipid domain on the other side . The trimeric structure of the lipoprotein was directly demonstrated by the chemical cross-linking of the native lipoprotein with both cleavable and uncleavable reagents . The cross-linking study also revealed interaction between the lipoprotein and the OmpA protein, a major outer membrane protein.

J Biol Chem, 1986 Jul 5, 261(19), 8597 - 600
Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells; Preiss J et al.; A simple enzymatic method for the quantitation of the mass of sn-1,2-diacylglycerol (DAG) present in crude lipid extracts was developed to assess the function of DAGs as intracellular "second messengers" of extracellular agents and of oncogene products . The assay employed Escherichia coli DAG kinase which constituted approximately 15% of the membrane protein of a plasmid-bearing strain and defined mixed micellar conditions to solubilize the DAG present and allow its quantitative conversion to {32P}phosphatidic acid . The assay was proportional with the amount of DAG added over the range of 25 pmol to 25 nmol . The rapid rise of DAG in platelets stimulated with thrombin (210% over basal) and in hepatocytes stimulated with vasopressin (230% over basal) was quantitated and the values agreed with previous measurements . The amounts of DAG in normal rat kidney (NRK) cells grown at 34 and 38 degrees C, respectively, were 0.47 and 0.61 nmol/100 nmol of phospholipid . In K-ras-transformed NRK cells grown at 34 or 38 degrees C, DAG levels were elevated 168 or 138%, respectively . When a temperature-sensitive K-ras NRK cell line was investigated, the amount of DAG present was elevated at the permissive but not at the restrictive temperature . These data are consistent with the K-ras protein functioning in transmembrane signalling by activating phospholipase C . Protein kinase C (Ca2+/phospholipid-dependent enzyme) activation by DAG may play an important role in cellular transformation.

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