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