J Bacteriol, 1986 Jan, 165(1), 244 - 51 Synthesis of a functional F0 sector of the Escherichia coli H+-ATPase does not require synthesis of the alpha or beta subunits of F1; Fillingame RH et al.; The uncB, E, F, and H genes of the Escherichia coli unc operon were cloned behind the lac promoter of plasmid pUC9, generating plasmid pBP101 . These unc loci code, respectively, for the chi, omega, and psi subunits of the F0 sector and the delta subunit of the F1 sector of the H+-ATP synthase complex . Induction of expression of the four unc genes by the addition of isopropyl-beta-D-thiogalactoside resulted in inhibition of growth . During isopropyl-beta-D-thiogalactoside induction, the three subunits of F0 were integrated into the cytoplasmic membrane with a resultant increase in H+ permeability . A functional F0 was formed from plasmid pBP101 in a genetic background lacking all eight of the unc structural genes coding the F1F0 complex . In the unc deletion background, a reasonable correlation was observed between the amount of F0 incorporated into the membrane and the function measured, i.e., high-affinity binding of F1 and rate of F0-mediated H+ translocation . This correlation indicates that most or all of the F0 assembled in the membrane is active . Although the F0 assembled under these conditions binds F1, only partial restoration of NADH-dependent or ATP-dependent quenching of quinacrine fluorescence was observed with these membranes . Proteolysis of a fraction of the psi subunit may account for this partial deficiency . The experiments described demonstrate that a functional F0 can be assembled in vivo in E . coli strains lacking genes for the alpha, beta, gamma, and epsilon subunits of F1. Infect Immun, 1986 Jan, 51(1), 320 - 6 Intestinal receptor for heat-stable enterotoxin of Escherichia coli is tightly coupled to a novel form of particulate guanylate cyclase; Waldman SA et al.; A novel form of particulate guanylate cyclase tightly coupled by cytoskeletal components to receptors for heat-stable enterotoxin (ST) produced by Escherichia coli can be found in membranes from rat intestinal mucosa . Intestinal particulate guanylate cyclase was resistant to solubilization with detergent alone, with only 30% of the total enzyme activity being extracted with Lubrol-PX . Under similar conditions, 70% of this enzyme was solubilized from rat lung membranes . The addition of high concentrations of sodium chloride to the extraction buffer resulted in greater solubilization of particulate guanylate cyclase from intestinal membranes . Although extraction of intestinal membranes with detergent and salt resulted in greater solubilization of guanylate cyclase, a small fraction of the enzyme activity remained associated with the particulate fraction . This activity was completely resistant to solubilization with a variety of detergents and chaotropes . Particulate guanylate cyclase and the ST receptor solubilized by detergent retained their abilities to produce cyclic GMP and bind ST, respectively . However, ST failed to activate particulate guanylate cyclase in detergent extracts . In contrast, guanylate cyclase resistant to solubilization remained functional and coupled to the ST receptor since enzyme activation by ST was unaffected by various extraction procedures . The possibility that the ST receptor and particulate guanylate cyclase were the same molecule was explored . ST binding and cyclic GMP production were separated by affinity chromatography on GTP-agarose . Similarly, guanylate cyclase migrated as a 300,000-dalton protein, while the ST receptor migrated as a 240,000-dalton protein on gel filtration chromatography . Also, thiol-reactive agents such as cystamine and N-ethylmaleimide inhibited guanylate cyclase activation by ST, with no effect on receptor binding of ST . These data suggest that guanylate cyclase and the ST receptor are independent proteins coupled by cytoskeletal components in membranes of intestinal mucosa. Infect Immun, 1986 Jan, 51(1), 24 - 30 Reversal of the biological activity of Escherichia coli heat-stable enterotoxin by disulfide-reducing agents; ElDeib MM et al.; Various disulfide-reducing agents, mostly thiols and thiol precursors, were examined for their ability to reduce the disulfide bonds in the Escherichia coli heat-stable enterotoxin STa; reduction of the bonds results in loss of biological activity . The biological activity measured was the stimulation of guanylate cyclase in pig intestinal brush border membranes by STa . Nearly all of the compounds inactivated STa, although at different rates; a smaller number appreciably decreased guanylate cyclase activity when they were introduced into the reaction mixture after STa bound to its receptor . With dithiothreitol, the decrease in reaction rate was both time and concentration dependent and resulted in a reversal to basal activity . The anionic thiols were relatively ineffective in reversing activation, the neutral monothiols were moderately effective, and the aminothiols and neutral dithiols were the most effective . The order of effectiveness of the compounds was S-2-(3-aminopropylamino)ethanethiol greater than 2,3-dimercaptopropanol = 2-aminoethylisothiuronium bromide greater than dithiothreitol greater than 2-mercaptoethylamine greater than alpha-thioglycerol . These compounds were used in weanling pig ligated-intestinal-loop bioassays to determine if STa-induced secretion was reduced when they were injected 20 min after the STa . Instead of S-2-(3-aminopropylamino)ethanethiol we used the phosphorylated derivative S-2-(3-aminopropylamino)ethylphosphorothioic acid; this compound and 2,3-dimercaptopropanol were the only compounds that reduced STa-induced secretion and had no direct secretory or pathological effects. Infect Immun, 1986 Jan, 51(1), 10 - 5 New fimbrial antigen F165 from Escherichia coli serogroup O115 strains isolated from piglets with diarrhea; Fairbrother JM et al.; Sixteen strains of Escherichia coli serogroup O115 isolated from piglets with diarrhea were examined for mannose-sensitive or mannose-resistant hemagglutination (MSHA or MRHA, respectively) for the presence of fimbriae by electron microscopy and for enterotoxigenicity by the ligated gut loop technique in 10-day-old piglets . Four strains demonstrated MRHA of sheep, goat, pig, dog, cat, chicken, and human erythrocytes but no MRHA of calf, horse, guinea pig, and rabbit erythrocytes . They were divided into pattern I (MSHA negative) and pattern II (MSHA positive) . The remaining 12 strains were classified as pattern III (MRHA negative, MSHA positive) and pattern IV (hemagglutination negative) . An antiserum produced against the MRHA-positive, MSHA-negative strain 4787 and absorbed by the same strain grown at 15 degrees C agglutinated all of the MRHA-positive strains but none of the MRHA-negative strains and completely inhibited the MRHA of these strains . The surface antigen against which this absorbed antiserum was directed was designated "F165." Fimbriae (pili) purified from strain 4787 hemagglutinated erythrocytes in the same mannose-resistant pattern as the strain itself and reacted with the anti-F165 antiserum in an enzyme-linked immunosorbent assay, thus demonstrating the fimbrial nature of the hemagglutinating F165 antigen . The F165 antigen showed no serological relationship with the fimbrial antigens F4, F5, F6, and "F41" . A positive correlation between the presence of F165 and the lack of enterotoxigenicity was demonstrated . Thus, we found a new mannose-resistant, hemagglutinating fimbrial antigen, F165, which is produced only by nonenterotoxigenic strains of E . coli serogroup O115 . The possible role of F165 as a virulence attribute of E . coli strains causing extraintestinal disease is discussed. Methods Enzymol, 1986, 126, 94 - 113 Purification and properties of two terminal oxidase complexes of Escherichia coli aerobic respiratory chain; Kita K et al.; Two terminal oxidase complexes, cytochrome b-562-o complex and cytochrome b-558-d complex, are isolated in highly purified forms which show ubiquinol oxidase activities . From the result of steady-state kinetics of cytochromes in the membrane and E'm values of purified cytochromes, we propose a branched arrangement of the late exponential phase of aerobic growth, as shown in Fig . 10 . Cytochrome b-556 is reduced by several dehydrogenases and the gene for this cytochrome (cybA) is located in the sdh gene cluster . Recently, we found another low-potential b-type cytochrome, cytochrome b-561 (Em' = 20 mV), which is also reduced by dehydrogenases . The position of this new cytochrome in the aerobic respiratory chain is under investigation . Two terminal oxidase complexes branch at the site of ubiquinone-8, and the Km value for oxygen of the purified cytochrome b-558-d complex is about 8-fold lower than that of the purified cytochrome b-562-o complex when ubiquinol-1 is used as substrate . This result is consistent with the idea that the cytochrome b-558-d complex is synthesized as an alternative oxidase for more efficient utilization of oxygen at low oxygen concentration . Thus, E . coli cells can maintain efficient oxidative energy conservation over a wide range of oxygen pressures by simply changing the contents of the two terminal oxidases, each of which functions as a coupling site. Princess Takamatsu Symp, 1986, 17, 85 - 91 Structure of the activated c-raf-1 gene from human stomach cancer; Shimizu K et al.; We previously isolated a novel human transforming gene from a primary stomach cancer and identified it as an activated version of the c-raf-1 gene which is the human homologue of v-raf, a viral oncogene encoding a serine/threonine-specific protein kinase . Analyses of cDNA and genomic clones of this gene revealed that it was generated by substitution of 5'-sequence (exons 1-5) of the normal c-raf-1 gene with an unrelated human sequence . We identified the region in the genomic clone where the rearrangement had occurred . The rearranged EcoRI fragment was detected in all the primary transformants obtained from two independent transfections, suggesting that the recombination had occurred in the primary cancer . By sequence analysis of cDNA, the putative product of the transforming gene was inferred to have a hydrophobic stretch ahead of the ser/thr-protein kinase domain of the c-raf-1 gene product . We introduced one of the cDNA which contains the 1.6-kb open reading frame into the pUC9 vector . An autophosphorylating, 58 kd protein was induced in Escherichia coli cells bearing the plasmid upon induction . Since ser/thr-protein kinase activity of the normal c-raf protein has not been evidenced, these results suggest that the truncation/replacement of the amino-terminal domain of the c-raf-1 protein leads to constitutive activation of the protein kinase probably residing on the downstream domain. Am J Physiol Imaging, 1986, 1(2), 77 - 82 A radionuclide model for studying changes in the regional blood volumes during early endotoxic shock: an experimental model; Christenson JT et al.; Septic shock constitutes a great threat to patients undergoing major abdominal surgery and also to trauma patients . The current state of knowledge on pathophysiological mechanisms in septic shock is not fully known . The aim of this study was to develop an experimental model that resembled the clinical situation and allowed the exploration of central and peripheral vascular mechanisms in endotoxic shock . Seven anesthetized sheep (weighing 30-45 kg) were provoked to lethal endotoxic shock by intravenous injection of 3 mg/kg bodyweight Escherichia coli endotoxin . The arterial pressure was monitored . Serial radionuclide images of the chest and abdomen were recorded after injecting technetium-99m-labeled RBC's . The volumetric (blood) alterations of liver, spleen, kidney, heart, and lungs, as well as peripheral muscle were measured . Prior to injection of endotoxin base-line data were obtained . Two to 4 minutes after injection, spleen volume decreased by -36% (mean) followed by a slow restoration; the left ventricular volume and kidneys increased by a maximum of 10-15%, while the liver volume increased by 38% of initial volume. Curr Genet, 1986, 11(1), 25 - 34 The genes encoding chloroplast ribosomal proteins S7 and S12 are located in the inverted repeat of Spirodela oligorhiza chloroplast DNA; Posno M et al.; We have used a variety of methods to localize the genes for ribosomal proteins S7 and S12 on Spirodela chloroplast DNA . Heterologous hybridization with a rps12 gene specific probe from Euglena has revealed the presence of rps12 homologous sequences within the inverted repeat of Spirodela chloroplast DNA on the fragment BamHI-V . In the partial nucleotide sequence of this fragment, two regions of amino acid sequence homology to Euglena S12 can be identified, separated from each other by a 542 bp intron with conserved boundary sequences . As was found for Nicotiana S12, the Spirodela S12 coding regions are for 85 amino acids homologous (79%) to E . coli S12 (starting from residue 38 to the C-terminus) . Likewise, we are unable to identify the 37 5' terminal codons of rps12 in Spirodela . The functionality of the Spirodela rps12 sequence is discussed . The rps7 gene is located adjacent to rps12 . Chloroplast ribosomal protein C-S11 (homologous to S7) has been detected by immunoprecipitation with both a polyspecific anti 30S serum and an anti C-S11 serum, among the in vitro translation products of mRNAs selected by Spirodela chloroplast DNA fragments BamHI-V and BamHI-P . Since in a DNA dependent E . coli cell free system, only BamHI-V appears to be capable of synthesis of C-S11, it is concluded that rps7 is located entirely within BamHI-V and is transcribed into a mRNA which extends into BamHI-P . As determined by Northern hybridization experiments, rps7 is cotranscribed with rps12; a stable transcript of approx . 1100 b is detected in total cellular Spirodela RNA with either rps12 and rps7 gene specific probes . The rps12 probe additionally detects an approx . 600 b transcript, which presumably corresponds to the excised rps12 intron RNA . Finally we have examined the expression of both rps7 and rps12 during light induced chloroplast development by Northern blotting and by immunoblotting . It is shown, that the steady-state levels of neither chloroplast ribosomal protein transcripts, nor those of the chloroplast ribosomal proteins itself, change significantly during the greening process. Curr Genet, 1986, 10(7), 557 - 60 Isolation of mutants sensitive to 2-aminopurine and alkylating agents and evidence for the role of DNA methylation in Penicillium chrysogenum; Rogers SD et al.; Using high performance liquid chromatography, the presence of N6-methyladenine has been found at a level of 0.1 mol percent in DNA extracted from Penicillium chrysogenum . No 5-methylcytosine was detected . A mutant strain HP547, which is sensitive to the lethal effects of N-methyl-N'-nitro-N-nitrosoguanidine, methylmethane sulphonate and the base analogue 2-aminopurine shows an increased spontaneous mutation rate and no detectable DNA methylation . Comparison of restriction enzyme digests of wild type and undermethylated strains indicated that methylation was occurring at a different sequence to that of the Dam methylase system of E . coli. J Hepatol, 1986, 3 Suppl 2, S157 - 60 The classification and biological functions of the interferons . A review; Finter NB; There are three main types of interferon, now designated alpha, beta and gamma . Human interferons-alpha exist as many subtypes, probably more than 26 . These are chemically quite closely related, but each has a unique chemical composition and biological properties . Preparations of interferon-alpha derived from stimulated peripheral blood leukocytes or lymphoblastoid cell lines contain mixtures of these sub-types; individual sub-types can be obtained by expression of the human gene concerned in bacteria . Human interferon-beta shows about 30% chemical homology with the interferons-alpha . The natural product is glycosylated, and there is only one molecular species . Natural interferon-gamma, also a glycoprotein, has the same non-specific antiviral properties as other types of interferon, but shows little, if any, chemical homology . Recombinant interferons-beta and -gamma have also been prepared by application of gene cloning techniques and expression in E . coli . These proteins are not glycosylated, and differ in their pharmacokinetic behaviour from the natural human proteins . In nature, interferons are normally formed and act locally . If relatively large amounts are administered in order to produce systemic effects, there are dose-related side effects which resemble an attack of influenza. Gene, 1986, 50(1-3), 361 - 9 Avian retrovirus nucleocapsid protein, pp12, produced in Escherichia coli has biochemical properties identical to unphosphorylated viral protein; Katz RA et al.; The avian sarcoma and leukosis viruses contain an RNA binding nucleocapsid protein, pp12, in the phosphorylated form . An Escherichia coli expression vector carrying the Rous sarcoma virus Prague C strain gene coding for this protein has been constructed using a site-directed deletion mutagenesis method to place start and stop codons into translational reading frame with the N- and C-terminal coding sequences of the protein, respectively . The protein is produced efficiently in bacteria (rp12), is soluble and readily purified . It is also indistinguishable from the unphosphorylated viral pp12 protein in its migration on SDS-polyacrylamide gels, reactivity with rabbit antisera directed against purified viral pp12, low RNA-binding affinity, and ability to serve as a substrate for in vitro phosphorylation at serine-40 by protease-activated kinase I . The ability to analyze the biochemical activities of the normal, modified, and mutant forms of this protein is an essential step in elucidating its role in the retroviral life cycle . This expression clone will be especially useful in testing for the effects of mutations before they are reconstructed into retroviral genomes for analyses. Microbiol Immunol, 1986, 30(12), 1271 - 9 Two monoclonal antibodies distinguish between human recombinant interferon-alpha 5s produced by Escherichia coli and by mouse cells; Tsukui K et al.; Two monoclonal antibodies against human IFN-alpha--one against natural leukocyte IFN-alpha and the other against recombinant human IFN-alpha 2 produced in E . coli--were prepared, and designated as HT-1, and 104-5-f, respectively . These monoclonal antibodies were used to examine the antigenicities of recombinant human IFN-alpha 5s produced by E . coli and by mouse cells . The HT-1 antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in mouse cells, but not recombinant human IFN-alpha 5 synthesized in E . coli . On the other hand, the 104-5-f antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in E . coli but not recombinant human IFN-alpha 5 synthesized in mouse cells . Then these monoclonal antibodies or sheep polyclonal antibody against human IFN-alpha were used to immunoprecipitate the radioactively labeled recombinant human IFN-alpha 5 synthesized either in E . coli or mouse cells, and analysed on polyacrylamide gel electrophoresis in the presence of NaDodSO4 . The labeled recombinant human IFN-alpha 5 produced by mouse cells could be immunoprecipitated with the HT-1 monoclonal antibody or sheep anti-(human IFN-alpha) polyclonal antibody but not with the 104-5-f monoclonal antibody and showed a band of Mr . 17,500 on polyacrylamide gel electrophoresis in the presence of NaDodSO4 . On the other hand, the labeled recombinant human IFN-alpha 5 produced by E . coli could be immunoprecipitated with the 104-5-f monoclonal antibody but not with the HT-1 monoclonal antibody and showed a band of similar Mr . on polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS) Gene, 1986, 49(3), 303 - 9 Nucleotide sequence of the pepN gene encoding aminopeptidase N of Escherichia coli; Foglino M et al.; We have sequenced a 3.3-kb fragment of the Escherichia coli chromosome that contains pepN gene encoding aminopeptidase N . This gene codes for a protein of 870 amino acid residues . From the size of the pepN transcript and the presence of inverted repeats in the nucleotide (nt) sequence, a putative transcription terminator has been identified . The N-terminal amino acid sequence deduced from the pepN nt sequence corresponds to the N-terminal sequence of the purified protein; the amino acid composition of the protein is also in good agreement with that deduced from the gene sequence . No obvious homology with previously sequenced peptidases has been detected. Gene, 1986, 49(2), 189 - 97 Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus; Broekhuijsen MP et al.; A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease virus (FMDV) sequences was constructed . The fusion proteins contain a large part of beta-galactosidase from Escherichia coli preceded (N-terminal) by 1, 2, 4 or 8 repeats of the antigenic determinant of FMDV consisting of amino acids 137-162 of the capsid polypeptide VP1 . All four fusion proteins were efficiently produced in E . coli host bacteria . Immunization of rabbits resulted in FMDV-specific, neutralizing antibodies, the response being dependent on the number of repeats . With enzyme-linked immunosorbent-assay techniques it was shown that the FMDV antigenic determinants are exposed on the surface of the fusion proteins under non-denaturing conditions. Gene, 1986, 49(1), 81 - 91 New pUC-derived expression vectors for rapid construction of cDNA libraries; Oberbaumer I; We have constructed a new series of the pUC-derived plasmids with an extended polylinker obtained from M13tg131 {Kieny et al., Gene 26 (1983) 91-99} . These vectors allowed us to design a simplified version of the method of Okayama and Berg {Mol . Cell . Biol . 2 (1982) 161-170} for establishing complete cDNA libraries . Improvements included easy recovery of the inserted cDNA due to the extended polylinkers; use of these vectors for gene expression in Escherichia coli, and amenability to supercoil sequencing with the universal primers of the M13 system {Chen and Seeburg, DNA 4 (1985) 165-170}, which speeds up the identification of positive clones . Moreover, there is no need for an additional linker fragment, as was required by the Okayama and Berg {Mol . Cell . Biol . 2 (1982) 161-170} method . We have successfully used this system to obtain cDNA clones coding for the different chains of the large basement membrane proteins type IV collagen and laminin. Acta Microbiol Pol, 1986, 35(3-4), 267 - 79 Production of enterotoxins by strains of Escherichia coli isolated from pigs; Niemialtowski M et al.; The production of enterotoxins by 237 hemolytic strains of Escherichia coli isolated from pigs was determined with the use of CTE in CHO . Vero and Hela cells and ILT . More frequent (p less than 0.01) production of enterotoxins, determined by ILT, was found for the serotypes being pathogenic for the animals (63.8% of the strains) . No correlation between intensity of ILT and particular serotype was observed . Both the serotypes pathogenic for pigs and other serotypes produced LT enterotoxins and ST toxin . The frequency of LT enterotoxin production was statistically insignificant compared to the frequency of ST enterotoxin production by strains with serotypes pathogenic for the pigs . Strains of E . coli producing only enterotoxin ST belonged both to the pathogenic serotypes as well as to other hemolytic serotypes . The cytotoxic activity of supernatants of E . coli strains with different serotypes isolated from pigs in Vero and Hela cells and simultaneous CTE in CHO cells was observed . This suggests the production by the strains of enterotoxin LT and cytotoxin VT . Seven out of the 96 isolates showing CTE in CHO cells gave no reaction in the ILT in pigs . This suggests the production by these isolates of a toxin (toxins) differing from the E . coli enterotoxins. Acta Microbiol Pol, 1986, 35(3-4), 241 - 9 Penetration of nalidixic acid in the cell cycle of Escherichia coli K-12; Hrebenda J et al.; Diffusion of nalidixic acid (NAL), DNA synthesis and filamentation of Escherichia coli K-12 cells induced by the drug were investigated in the division cycle . It was found that in nonsynchronous culture and within the range of sublethal NAL concentrations the length of the filaments depends on the dose or the time of action of the drug . In synchronous culture differences are observed in the values of measured parametres between samples from the successive culture phases . Maximum {3H} NAL penetration and DNA synthesis inhibition caused by the drug were noted in the period proceeding directly division . The extent of NAL penetration into the cell in the sample is correlated with the change in length of the NAL-induced filamentous cells. Acta Microbiol Pol, 1986, 35(3-4), 191 - 7 Purification of Escherichia coli DNA helicase I from plasmid-transformed cells; Benz I et al.; DNA helicase I was purified in large quantity from Escherichia coli cells harboring a plasmid that carries the gene encoding helicase I--the traI gene of the F sex factor--cloned in a high copy number vector . Electron microscopic studies on the purified material reveal new properties of the enzyme protein. Nauchnye Doki Vyss Shkoly Biol Nauki, 1986, (12), 78 - 81 {Macromolecular synthesis and cell division in Escherichia coli in the lag-phase of diauxie}; Kuz'min SM et al.; The pulse incorporation of radiolabelled mannitol, glycerol, thymidine, uridine and protein hydrolysate has been studied during the Escherichia coli growth . The incorporation of carbohydrates and predecessors is sharply changed before and after lag-phase, that is due evidently to the partial synchronization of cell population . Three cell divisions have been found: before lag-phase (determined by the curve of optical density), at the end of it and after it termination . Cell division is accompanied by removal of catabolic repression from glycerol utilization, by increase of protein, RNA and DNA synthesis rates . The rates of biopolymer synthesis decrease sharply at the beginning of lag-phase, but DNA synthesis is going on at a rather high level. J Cardiovasc Pharmacol, 1986, 8 Suppl 8, S57 - 60 Regulation of cyclic GMP synthesis and the interactions with calcium; Murad F et al.; The formation of cyclic GMP (cGMP) by guanylate cyclase, and the properties of the soluble and particulate isoenzymes, are reviewed . Regulation of guanylate cyclase and cGMP accumulation in intestinal mucosa and vascular smooth muscle with and without endothelium are summarized . The effects of E . coli heat-stable enterotoxin, nitrovasodilators, endothelium-dependent vasodilators, and atrial natriuretic factors in these systems are discussed . A potential role for free radicals, Ca, and unsaturated fatty acids in cGMP synthesis is reviewed . These tissue systems (intestinal mucosa and vascular smooth muscle with or without intact endothelium) are presented as model systems and as examples of the regulation of cGMP accumulation in a single cell type by peptides, drugs, and cell-cell interactions that are required to generate cGMP with a hormone or drug. J Basic Microbiol, 1986, 26(6), 317 - 22 Synthesis of poly(A)+-containing RNA during growth in Escherichia coli relA+ and relA- strains; Hanschke R et al.; There are very different results on the synthesis of poly(A)+-containing RNA in bacteria . We, therefore, studied the influence of growth and amino acid starvation on the synthesis of poly(A)+-RNA in a relA+ and relA- strains of E . coli . Only the relA+ strains is able to respond to an amino acid limitation by production of ppGpp which causes a strong reduction of stable RNA transcription . During growth we observed significant alterations of the percentage of {3H}-uridine labelled total RNA which bound to poly(U)-sepharose (% poly(A)-RNA) . It was mainly influenced by drastic changes of the synthesis of non-polyadenylated stable RNA (rRNA, tRNA) during growth and, therefore, it did not reflect the actual synthesis of polyadenylated mRNA . An amino acid starvation induced in the relA+ strain a stronger and more rapid reduction of the transcription of non-polyadenylated RNA as well as of poly(A)+-RNA than in the relA- strain which did not produce ppGpp under these conditions . Therefore, we conclude that ppGpp inhibited not only the synthesis of the non-polyadenylated stable RNA but also that of poly(A)+-containing mRNA, although the latter was apparently less affected. Gene, 1986, 46(2-3), 287 - 90 RNA colony hybridization method; Ivanov I et al.; A method for rapid screening of specific RNA sequences in recombinant colonies by hybridization in situ is presented . The method includes two consecutive steps of lytic treatment of the nitrocellulose-filter-supported colonies (10% sodium dodecyl sulfate and 3 X SSC at 65 degrees C) and hybridization with 32P-labelled specific oligodeoxynucleotides. C R Acad Sci III, 1986, 303(15), 633 - 6 {Preparation and characterization of specific antisera directed against different polypeptide domains encoded by the c-myc oncogene for studying the expression of this gene introduced into quail or rat cells}; Ferre F et al.; By using bacterial expression vectors, we have prepared antisera directed against two polypeptidic domains encoded by exons 2 and 3 of the human c-myc oncogene . These antisera which detect specifically the human c-myc proteins allow us to analyse the expression of human c-myc gene activated by retroviral sequences and introduced in quail embryo cells (QEC) or in established rat embryo fibroblastic cell line (208 F) . Although human myc mRNA are expressed in the two cell types, the p64/p67 human c-myc proteins are only detected in the QEC. Histochemistry, 1986, 86(1), 35 - 41 Silver-enhanced colloidal gold probes as markers for scanning electron microscopy; Scopsi L et al.; Silver enlargement of small colloidal gold particles has been extensively used for the light microscopical visualization of gold probes . Very recently, a few investigators have employed physical developers in electron microscopy (both pre-embedding and on-grid staining methods) . We now demonstrate that physical development of small colloidal gold particles advantageously can be exploited for labelling biological surfaces in scanning electron microscopy . This novel application of silver enhancement of colloidal gold particles is characterized by a high detection efficiency . Thus, specimens are labelled with small gold probes affording high immunocytochemical efficiency but being impossible to detect with the present scanning microscopes . These particles are subsequently scanning electronmicroscopically visualized by silver enhancement. IARC Sci Publ, 1986, (70), 359 - 71 Replication and transcription of polynucleotides containing ethenocytosine, ethenoadenine and their hydrated intermediates; Singer B et al.; Replication with Escherichia coli DNA polymerase I (Pol I) and transcription with DNA-dependent RNA polymerase from Escherichia coli or calf thymus, using as templates synthesized ribo- or deoxyribopolynucleotides containing 1,N6-ethenoadenine (epsilon A) or 3,N4-ethenocytosine (epsilon C), showed that only epsilon C could direct significant misincorporation . The hydrated intermediate of epsilon C caused errors only upon transcription, but not upon replication . epsilon A was a very poor mutagen as assessed by replication with Pol I . Transcription of polynucleotides containing epsilon A under error-prone conditions caused frequent A misincorporation which could not be detected in replication assays . It is concluded that epsilon C may lead to point mutations, specifically directing the misincorporation of thymine . The analogous derivative, epsilon A, is bulky and is likely to be bypassed rather than read . This mechanism could cause frameshift mutation, as generally found for other bulky adducts. J Membr Biol, 1986, 92(3), 237 - 45 Chemical modification of the two histidine and single cysteine residues in the channel-forming domain of colicin E1; Bishop LJ et al.; The two histidine residues of COOH-terminal channel-forming peptides of colicin E1 were modified by addition of a carbethoxy group through pretreatment with diethylpyrocarbonate . The consequences of the modification were examined by the action of the altered product on both phospholipid vesicles and planar membranes . At pH 6, where activity is low, histidine modification resulted in a decrease of the single channel conductance from 20 pS to approximately 9 pS and a decrease in the selectivity for sodium relative to chloride, showing that histidine modification affected the permeability properties of the channel . At pH 4, where activity is high, the single channel conductance and ion selectivity were not significantly altered by histidine modification . The histidine modification assayed at pH 4 resulted in a threefold increase in the rate of Cl- efflux from asolectin vesicles, and a similar increase in conductance assayed with planar membranes . This conductance increase was inferred to arise from an increase in the fraction of bound histidine-modified colicin molecules forming channels at pH 4, since the increase in activity was not due to an increase in binding of the modified peptide, a change in ion selectivity, a change of single channel conductance, or a change in the pH dependence of binding . The sole cysteine in the colicin molecule was modified in 6 M urea with 5,5'-dithiobis(2-nitrobenzoic acid) . The activities of the colicin and its COOH-terminal tryptic peptide were found to be unaffected by cysteine modification, arguing against a role of (-SH) groups in protein insertion and/or channel formation. Int J Immunopharmacol, 1986, 8(7), 819 - 24 In vitro and in vivo suppressive effects of delta-9-tetrahydrocannabinol on interferon production by murine spleen cells; Blanchard DK et al.; Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, has been reported to be suppressive on some immune functions . Since interferons (IFNs) are important immunomodulatory proteins, the effect of in vivo or in vitro administration of THC on induction of IFN by various mitogens was examined . Splenocytes from normal mice in the presence of THC produced significantly less IFN when stimulated by phytohemagglutinin (PHA), concanavalin A (Con A), or Escherichia coli lipopolysaccharide (LPS) . Induction of IFN by a bacterial antigen, Legionella pneumophila bacterial cells, was also suppressed by THC . Also, splenocytes which were incubated up to 24 h in the presence of THC partially recovered responses to mitogens when cells were washed before stimulation . This suggested that THC must be present in order to mitigate IFN induction . Splenocyte cultures from mice which were chronically injected with THC for 6-8 weeks were also less responsive to induction of IFN by the various mitogens . These results suggest that at least part of the immunosuppressive effects of THC may be related to depressed IFN production by stimulated lymphocytes . Since Con A and PHA are T cell mitogens and LPS is considered to be a macrophage and B cell stimulator, suppression of IFN production by these classes of cells indicate a wide range of effects of THC. Circ Shock, 1986, 20(2), 127 - 39 Reduced RBC versus plasma microvascular flow due to endotoxin; Baker CH et al.; The microvascular circuits traversed by red blood cells (RBCs) and plasma from first-order arterioles to first-order venules are complicated by variations in hemodynamic, rheologic, and dimensional parameters . Escherichia coli endotoxin causes microcirculatory derangements expected to alter RBC and plasma transport through these circuits . Wistar male rats were anesthetized with pentobarbital; the left cremaster muscle was spread over an optical port in a Krebs solution bath and administered endotoxin (6 mg/kg) iv over a 1-h period . The right femoral artery was cannulated for measurement of aortic pressure (Pm) and ia bolus injections of fluorescent DTAF-RBC and FITC-dextran . Fluorescence epi-illumination videomicroscopy and densitometry were used to obtained time-concentration curves (TCCs) in arterioles and venules . Control Pm averaged 106 +/- 8 mm Hg and progressively decreased during the 150-min observation period following endotoxin infusion . Arteriolar and venular diameters decreased approximately 50% during the 150-min observation period . At control DTAF-RBC flow velocity exceeded FITC-dextran velocities but by 90 min postendotoxin, even though both velocities were greatly reduced, plasma velocity, significantly exceeded red cell velocity . The control mean transit times for FITC-dextran exceeded the DTAF-RBC times in all vessels; 90 min postendotoxin the DTAF-RBC mean transit times significantly exceeded the FITC times and cell aggregates were in venous blood . The data suggest that cell aggregation, vasoconstriction and use of longer alternate parallel vascular circuits occur in endotoxin shock, restricting red cell flow . Plasma bypasses RBCs, flowing more rapidly than red cells in terminal shock. J Basic Microbiol, 1986, 26(4), 211 - 8 Proteins tightly bound to DNA and DNA-synthesizing activity of nucleoids from Escherichia coli; Gaziev AI et al.; Membrane-attached nucleoids were isolated from E . coli and separated from proteins by 2 M NaCl . Disintegration of such nucleoids by ultrasound and subsequent centrifugation resulted in the formation of two fractions: a sediment (fraction I) and a supernatant (fraction II) . The protein:DNA ratio of fraction I was equal to 27 and was different from that to fraction II (2.6) . More than 70% of the proteins not dissociating at 2 M NaCl and bound to DNA of both fractions were polypeptides with molecular weights (Mw) of 31,000-23,000 daltons (31-23 Kdal) . After pulse labelling of the cells with {3H}-thymidine, the specific radioactivity of newly synthesized DNA associated with fraction I was shown to be considerably higher than that of fraction II . The analysis of DNA-synthesizing activities in fractions I and II showed that both nucleoid fractions contained DNA polymerase I . After dissolving the two fractions in 8 M urea - 0.15% sodium dodecylsulphate (SDS) they were chromatographed on hydroxyapatite . DNA-protein complexes were obtained that did not dissociate at 4 M guanidine X HCl - 5 M urea and 1% SDS . The main protein of the complexes was a 31 Kdal polypeptide tightly bound to DNA. J Neurosci Res, 1986, 16(1), 127 - 39 pp60c-src encoded by the proto-oncogene c-src is a product of sensory neurons; Maness PF; The advent of recombinant DNA technology has led to the identification in the DNA of normal animal cells of over 30 proto-oncogenes that are homologous to retroviral transforming genes . One of these encodes a protein kinase (pp60c-src) of unknown function, that is preferentially synthesized in brain and neural retina . Here the expression of pp60c-src in the peripheral nervous system was examined in sensory neurons from chick dorsal root ganglia with antisera raised against the transforming protein of Rous sarcoma virus (pp60v-src) expressed in Escherichia coli carrying the cloned v-src gene . This antiserum recognizes pp60c-src specifically in normal chicken cells . Western immunoblotting showed that dorsal root ganglia of stage 30 (day 6.5) chick embryos contained elevated levels of pp60c-src . Immunoperoxidase staining of neuron-enriched cultures prepared from chick dorsal root ganglia showed pp60c-src immunoreactivity in cells with neuronal morphology; flat, fibroblastic cells contained no detectable immunoreactivity . Indirect double immunofluorescence with pp60src antibodies and monoclonal antibodies against the 200-kD subunit of neurofilament protein confirmed that the cells expressing pp60c-src were neurons . Ninety-six percent of the neurofilament-positive cells were immunoreactive with pp60src antibodies, and conversely, all pp60c-src-positive cells were immunoreactive with neurofilament antibodies . pp60c-src immunofluorescence appeared to be distributed over the cell body, processes, and growth cones . These results clearly demonstrate that pp60c-src is a product of neurons and is expressed in sensory neurons in culture. Gene, 1986, 43(3), 295 - 300 Expression of a Streptomyces plasmid promoter in Escherichia coli; Deng Z et al.; A 166-bp DNA fragment from the Streptomyces multicopy plasmid pIJ101 with in vivo promoter activity both in Streptomyces lividans and in Escherichia coli was isolated . The start point of the RNA transcribed from this fragment, determined by high resolution S1 nuclease mapping, was the same in S . lividans and in E . coli . This suggests that the E . coli RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding S . lividans enzyme . The putative promoter sequence shows good homology to the E . coli promoter consensus sequence in the '-35' region but poor homology in the '-10' region. Dev Biol Stand, 1986, 63, 41 - 51 The relationships between Ts-inducing and Th and Tp-inducing determinants on a large protein antigen; Krzych U et al.; The immunogenicity of several small monomeric protein antigens - lysozyme, myoglobin, cytochrome c and insulin - has been intensely studied during the past decade to try to learn the rules of the game . It is likely that before the stage is reached at which we can predict the nature of the determinants responded against by each lymphoid subpopulation, other types of molecules such as fibrous proteins, multimeric large proteins and viral capsid proteins will have to be examined, and such studies have commenced recently . In an effort to generalize our studies with lysozyme to one of these systems, we have been exploring the response of H-2k, CBA/J mice to E . coli B-galactosidase, GZ, a tetramer with a monomer molecular weight of 116,250 . We feel that aspects of this system have important implications for the vaccine problem, and these will be summarized at the end of the article. Acta Microbiol Pol, 1986, 35(1-2), 29 - 41 Free RNA-dependent ATPase activity of transcription termination factor Rho: a model of cyclic dissociation and reassembly of Rho protein; Wolska KI; RNA-dependent ATPase activity of Rho + and two mutant proteins Rho15 and Rho301 was studied . It was shown that monomeric Rho forms oligomers in the presence of ATP . This ATP-induced structural change of Rho allows protection of the protein from heat inactivation . Poly(C), which highly activates Rho ATPase, was found to potentiate heat inactivation of Rho301, but no Rho + and Rho15, only under optimal conditions of ATP hydrolysis . It was also shown that Rho301 is defective in interaction with RNA . The molecular model postulating that Rho-catalysed ATP hydrolysis with free RNA involves the cyclic process of protein dissociation and reassembly is postulated. Gene, 1986, 42(3), 303 - 12 Expression of the Epstein-Barr virus 138-kDa early protein in Escherichia coli for the use as antigen in diagnostic tests; Motz M et al.; We have attempted to produce the 138-kDa early protein (ep 138) of Epstein-Barr virus (EBV) in Escherichia coli . This protein was found, by immunoprecipitation, to be a clinically relevant antigen, especially for the determination of the IgA-titer in patients with nasopharyngeal carcinoma (NPC) . Since the expression of the entire ep 138 coding region was unsuccessful, we synthesized only the antigenic parts of this protein . Potential antigenic sites were predicted from the amino acid sequence by combining values for hydrophilicity with calculated estimates of the secondary structure . The two predicted fragments were found to be antigenic, but only one of them was stably expressed in E . coli as a non-fusion protein . This stable protein fragment was, in turn, able to stabilize the second antigenic fragment forming an autologous fusion protein, consisting exclusively of EBV-derived sequences . The resulting product reacts particularly well with IgA antibodies of NPC patients indicating its diagnostic value for NPC. Gene, 1986, 42(2), 175 - 83 Use of a portable ribosome-binding site for maximizing expression of a eukaryotic gene in Escherichia coli; Marquis DM et al.; To maximize expression of a eukaryotic gene in Escherichia coli, a series of plasmids were constructed containing various synthetic ribosome-binding sites (RBS) . These sites consist of a Shine-Dalgarno (SD) region (with translation stop codons in all three reading frames) positioned at distances 5-9 nucleotides (nt) from the AUG initiator codon of the gene coding for human T-cell growth factor (TCGF or IL-2) . The region encompassing the RBS through the TCGF structural gene from each of these plasmids was inserted as a 'cassette' into seven different E . coli expression vectors, and TCGF production was measured . Our results demonstrate a greater than 2000-fold range of TCGF synthesis dependent upon the promoter and the synthetic RBS used . The translational efficiency of the TCGF gene was found to be influenced by the quality of the RBS, which is in part determined by the external sequence context of this site . The synthetic RBS, containing the necessary information for the translation initiation process, readily accessible by restriction sites, should be of general usefulness in obtaining maximum expression of eukaryotic genes in E . coli. Antonie Van Leeuwenhoek, 1986, 52(1), 63 - 74 Expression of the SOS system in Escherichia coli growing under nitrate respiration conditions; Barbe J et al.; Induction of several SOS functions by mitomycin C, bleomycin or thermal treatment of a recA441 mutant growing under nitrate respiration conditions was studied in Escherichia coli . Mitomycin C caused inhibition of cell division, induction of prophages and expression of umuC gene but like in aerobically growing cells, it did not trigger the cessation of cell respiration . On the contrary, both recA+ and recA441 cultures either treated with bleomycin or incubated at 42 degrees C failed to induce any of the different SOS functions cited above . Furthermore, after bleomycin addition or thermal treatment both recA+ and recA441 cultures did not present any variation in the cellular ATP level, contrary to what happens under aerobic growth . The blocking of the expression of some SOS functions under nitrate respiration conditions is not an irreversible process because cells incubated under these anaerobic conditions were able to induce the SOS system when changed to an aerobic medium 30 min after the SOS-inducing treatment had been applied. Int J Vitam Nutr Res, 1986, 56(1), 11 - 20 Vitamin A status affects chromatin structure; Porter SB et al.; We have examined RNA synthesis by nuclei isolated from testes of rats of varying vitamin A status . Nuclei from retinol-deficient animals showed substantially decreased RNA synthesis by polymerase II when compared to nuclei from normal animals . Within 4 hours after oral administration of retinyl acetate (as the source of retinol) to deficient animals, RNA synthesis by polymerase II had significantly increased . Administration of retinoic acid had a similar but lesser effect . Nucleoside analysis after alkaline hydrolysis of the RNA synthesized by the endogenous polymerase II suggested that the increased activity was due to a greater number of actively transcribing polymerase II molecules on the DNA . Further, when the template capacity of testicular chromatin isolated from deficient and retinyl acetate refed animals was compared, the number of sites recognized by E . coli RNA polymerase was increased twofold after retinyl acetate administration . We conclude that these retinol-induced changes in transcription are due at least in part to changes in chromatin structure. Gene, 1986, 41(2-3), 281 - 8 Construction and characterization of pBR322-derived plasmids with deletions of the RNA I region; Gayle RB 3rd et al.; A region upstream from the origin of replication in ColE1-type plasmids has been shown to be necessary for replication . Two RNA transcripts are produced from this area, RNA II, which yields the primer for DNA polymerase initiation at the origin and RNA I, which is complementary to the 5' end of RNA II and acts to inhibit primer formation . We have constructed plasmids which do not possess the nucleotide sequence for RNA I, or the normal 5' terminus and promoter of RNA II . The RNA II analog, in these plasmids, is believed to be synthesized by the readthrough transcription of the upstream trimethoprim-resistant dihydrofolate reductase (DHFR) gene at a level comparable to that produced by the tryptophan promoter . These plasmids have a copy number of about tenfold higher than that of pBR322 during logarithmic growth and are compatible with other ColE1-type plasmids . These plasmids are stably maintained in several strains when selective pressure is present and the plasmids are stably maintained during exponential growth in W3110 strains without selective pressure . In all strains examined, the dimeric form of the plasmid was lost from cells much more rapidly than those containing the monomeric form. Mutat Res, 1986 Jan, 165(1), 21 - 30 Genotoxicity of formaldehyde and an evaluation of its effects on the DNA repair process in human diploid fibroblasts; Snyder RD et al.; Formaldehyde treatment of human fibroblasts gave rise to DNA damage detected by a nick translation assay . This damage was not repaired by typical 'long-patch'-type excision repair as evidenced by the failure of DNA repair inhibitor post-treatment to elevate the amount of DNA strand breakage . In addition, the effects of formaldehyde on DNA repair were examined in light of a recent report suggesting that formaldehyde inhibited the repair of X-ray-induced strand breaks and UV- and benzo {a}pyrene diol epoxide-induced unscheduled DNA synthesis in human bronchial cells . We report that formaldehyde (1) was ineffective at inhibiting the sealing of X-ray- or bleomycin-induced DNA strand breaks, (2) did not inhibit the removal of pyrimidine dimers from cellular DNA at short treatment times, and (3) that the previously observed inhibition of unscheduled DNA synthesis was most likely due to the inhibition of uptake of labeled precursor into formaldehyde-treated cells . Thus, our findings are not consistent with the notion that formaldehyde inhibits the repair process in human fibroblasts . Finally, formaldehyde was shown to elevate the level of misincorporation of bases into synthetic polynucleotides catalyzed by E . coli DNA polymerase I, indicating that the mutagenicity of formaldehyde may be due to covalent alteration of DNA bases. J Med Chem, 1986 Jan, 29(1), 69 - 74 Potential antitumor agents . 45 . Synthesis, DNA-binding interaction, and biological activity of triacridine derivatives; Atwell GJ et al.; A series of amide-linked triacridines of varying interchromophore separation were synthesized as potential DNA trisintercalating agents . The corresponding diamide diacridines (lacking the central chromophore) were also prepared, and the DNA-binding and biological activities of both series of compounds were evaluated . Although one of the triacridines shows evidence of a trisintercalative binding mode, most of the triacridines (and all the diacridines) bound by bisintercalation . The diacridines showed great cytotoxicity and higher DNA association constants than the corresponding 9-{{3-(dimethylamino)propyl}amino}acridine monomer, but addition of a third chromophore did not provide corresponding increases in either DNA affinity, inhibition of RNA synthesis, or cytotoxicity . Some members of both series show minimal in vivo antileukemic activity . The results suggest that further development of trimeric molecules as potential antitumor agents should focus on smaller chromophores with lower capacity for nonspecific binding and/or the employment of rigid linker chains to provide true molecular "staples" for DNA. J Bacteriol, 1986 Jan, 165(1), 116 - 22 Physical properties of short- and long-O-antigen-containing fractions of lipopolysaccharide from Escherichia coli 0111:B4; Peterson AA et al.; Aggregates of short- and long-chain O-antigen-containing fractions of lipopolysaccharide were analyzed by electron spin resonance probing to reveal differences in their physical properties . The fluidities of the lipid regions of the two fractions were quite similar, although the long-chain lipopolysaccharide aggregates appeared to be more hydrated as reflected by the polarity determined with a lipid probe . In contrast, the head-group region of the long-chain fraction was dramatically more mobile than that of the short-chain sample . The binding of polycations (e.g., polymyxin B, spermine) to lipopolysaccharide aggregates was measured by the partitioning of a cationic spin probe . Less probe was displaced from the long-chain fraction and unseparated lipopolysaccharide than from the short-chain fraction by the addition of cations, suggesting that the long O-antigen masks anionic sites on lipopolysaccharide . These results indicate that the aggregate shape and reactivity of lipopolysaccharide are affected by O-antigen length . Thus, the biological activity of lipopolysaccharide may be modulated directly by the presence of O-antigen and indirectly by the effects of O-antigen on the lipopolysaccharide aggregate structure. Infect Immun, 1986 Jan, 51(1), 54 - 9 Monoclonal antibodies against the nonhemagglutinating fimbrial antigen 1C (pseudotype 1) of Escherichia coli; Schmitz S et al.; Hybridoma-derived monoclonal antibodies were produced with fimbrial preparations from Escherichia coli 20025 (04:K12:H-) with fimbrial (F) antigens 1C, 13, one related to 12, and one preliminarily termed y and from E . coli 2980 (018ac:K5:H-) with F antigens 1C and 8 . Two clones of subclonal hybrid cells were studied which produced monoclonal antibodies (mc-20025-F2b, immunoglobulin G2b {IgG2b}; mc-2980-F2, IgG1) that were reactive with E . coli 20025, 2980, and a number of additional strains which exhibited the F1C antigen . Results of enzyme-linked immunosorbent assay and Western blot analysis indicated that the antibodies had F1C specificity, and competitive enzyme-linked immunosorbent assay with 125I-labeled antibodies showed that they recognized different epitopes on the fimbrial subunit . Neither of the antibodies agglutinated F1C-fimbriated E . coli but bound to the bacteria . There was no binding to E . coli without F1C fimbriae. Infect Immun, 1986 Jan, 51(1), 110 - 4 Immunogenicity and antigenicity of synthetic Escherichia coli lipid A; Brade L et al.; The immunogenicity and antigenicity of synthetic Escherichia coli lipid A (compound 506) and its 1- and 4'-monophosphorylated derivatives (compounds 505 and 504, respectively) and nonphosphorylated derivative (compound 503) were compared with those of bis- and 4'-monophosphorylated natural free lipid A from E . coli . The synthetic compounds under study were either coated onto sheep erythrocytes (except for the water-insoluble preparation 503) or incorporated into liposomes and used for the immunization of rabbits . Both types of immunogens (the latter representing fully synthetic immunogens) resulted in high-titered polyclonal antisera which were characterized before or after absorption in a passive hemolysis assay as well as in a passive hemolysis inhibition assay with the synthetic compounds as test antigens . All antisera were found to react with their corresponding homologous antigens coated onto sheep erythrocytes, with titers of up to 2,048, and were comparable to those antisera obtained after immunization with natural lipid A exposed on the bacterial surface after acid hydrolysis . Antisera against bisphosphorylated compound 506 were highly specific for the homologous antigens, showing no interaction with compounds 504 and 505 in the passive hemolysis test . The same held true for the absorption experiments in which glutaraldehyde-fixed sheep erythrocytes were sensitized with the respective antigens . Antisera against monophosphorylated compounds 504 and 505 exhibited, besides their expected homologous reactivity, complete cross-reactivity with compound 506, but they did not cross-react with each other . Thus, anti-504 and anti-505 antibodies recognized distinct antigenic determinants, being related to the ester linked 4'-phosphate or the glycosidically linked 1-phosphate, respectively . Both antigenic determinants were also expressed by bisphosphorylated compound 506 used as an antigen; however, upon immunization, only antibodies against compound 506 were elicited. Cancer Res, 1986 Jan, 46(1), 417 - 25 Immunological variables as predictors of prognosis in patients with Kaposi's sarcoma and the acquired immunodeficiency syndrome; Vadhan-Raj S et al.; Multivariate analysis was used to identify which of a large number of pretreatment immunological parameters correlated with therapeutic response, subsequent development of opportunistic infection, and survival from the time of diagnosis in a group of 70 patients with Kaposi's sarcoma and acquired immunodeficiency syndrome treated with recombinant leukocyte A interferon . In a logistic regression model, delayed type hypersensitivity response to one or more recall antigens and high proliferative response to Escherichia coli were significant predictors for response to recombinant leukocyte A interferon (for the model, P = 0.01) . For prediction of the development of opportunistic infection, the model selected low proliferative responses to phytohemagglutinin and E . coli (P less than 0.001) . Favorable factors predicting survival in the Cox regression model were the absence of endogenous serum interferon activity and a high proliferative response to E . coli (P less than 0.001) . The estimated median survival for the group with endogenous serum interferon activity and low E . coli response was 12 months; the median has not yet been reached for the group with no serum interferon and a high E . coli response . We conclude that immunological parameters may be useful in predicting prognosis in patients with Kaposi's sarcoma and acquired immunodeficiency syndrome. Biochemistry, 1985 Dec 31, 24(27), 7942 - 7 Electron-nuclear double resonance studies of oxidized Escherichia coli sulfite reductase: 1H, 14N, and 57Fe measurements; Cline JF et al.; We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the bridged siroheme--{Fe4S4} cluster that forms the catalytically active center of the oxidized hemoprotein subunit (SiRo) of Escherichia coli NADPH-sulfite reductase . The siroheme 57Fe hyperfine coupling (Az = 27.6 MHz, Ay = 26.8 MHz) is similar to that of other high-spin heme systems (A approximately equal to 27 MHz) . Bonding parameters obtained from the 14N hyperfine coupling constants of the siroheme pyrrole nitrogens are consistent with a model of a nonplanar pi system of reduced aromaticity . The absence of hyperfine coupling to the 14N of an axial ligand, such as is observed for the histidine 14N of metmyoglobin (Az = 11.55 MHz), rules out the possibility that imidazolate acts as the bridge between the siroheme and the {Fe4S4} cluster . Proton ENDOR of the deuterium-exchanged protein indicates that H2O does not function as a sixth axial ligand and suggests that the ferrisiroheme is five-coordinate . 57Fe ENDOR measurements confirm the results of Mossbauer spectroscopy for the {Fe4S4} cluster . They also disclose a slight anisotropy of the cluster 57Fe coupling that may be associated with the mechanism by which the siroheme and cluster spins are coupled. Biochemistry, 1985 Dec 31, 24(27), 7860 - 5 Specific interaction between ribosomal protein S4 and the alpha operon messenger RNA; Deckman IC et al.; The Escherichia coli ribosomal protein S4 is known to repress translation of its own gene and several other ribosomal protein (r-protein) genes in the alpha operon as part of a general mechanism coordinating the levels of rRNA and r-protein synthesis . Using a filter binding assay and RNA transcripts prepared in vitro, we have detected and quantitated specific interactions between S4 and alpha mRNA fragments . The main results are the following: Only the alpha mRNA leader is required for specific recognition, with a small fraction of the binding free energy derived from sequences at the ribosome initiation site . 16S rRNA and alpha mRNA compete for binding to S4 with about the same affinity (approximately equal to 2 X 10(7) M-1), suggesting that S4 utilizes the same recognition features in each RNA . Nonspecific binding of S4 to tRNA or other mRNA sequences is strongly salt dependent, while the specific S4-alpha mRNA affinity is nearly independent of salt . At physiological salt concentrations the nonspecific S4-RNA affinity (10(5)-10(6) M-1) is large enough to strongly buffer the free S4 concentration in vivo. Biochemistry, 1985 Dec 31, 24(27), 8043 - 9 DNA filter retention assay for exonuclease activities . Application to the analysis of processivity of phage T5 induced 5'-exonuclease; Joannes M et al.; The 5'-exonuclease of phage T5 has been purified nearly to homogeneity by using a simple and fast procedure . The kinetic properties of the purified enzyme have been studied by using a new sensitive assay based upon retention by nitrocellulose filters of DNA with short protruding single-stranded ends . The enzyme is specifically stimulated by KCl . Its Km is 2.2 X 10(-7) M at 30 degrees C, and its turnover number is 0.33 DNA molecule transformed per minute . The filter retention assay shows that the T5 exonuclease acts by a semiprocessive mechanism, removing from DNA ends about 30 nucleotides on the average per cycle . The degree of enzyme processivity increases with increasing magnesium concentrations. EMBO J, 1985 Dec 30, 4(13B), 3887 - 93 Transcriptional activation of a pap pilus virulence operon from uropathogenic Escherichia coli; Baga M et al.; A gene cluster mediating production of pili in uropathogenic Escherichia coli was analysed with respect to regulation of pili synthesis . Two cistrons, papB and papI, were localized upstream of the major pilus subunit gene, papA . The papI-papB-papA region was characterized by nucleotide sequencing and by transcriptional analysis . The papA gene was primarily represented by an 800 nucleotide long transcript but was also co-transcribed with papB as a less abundant 1300 nucleotide long mRNA . Both transcripts presumably terminated at the same site downstream of the papA coding sequence . The weakly expressed papI gene was transcribed in the opposite direction to that of papB and papA . Studies with lacZ operon fusions showed that the papB gene encoded a trans-active effector required for papA transcription . Similarly, the papI gene stimulated papB transcription in trans . Furthermore, full expression of papA was cis dependent upon the papI-papB region . Transcription of the papB gene was shown to be dependent upon cAMP and its receptor protein . A binding site for the cAMP-CRP complex was postulated in the DNA sequence upstream of the papB promoter. J Biol Chem, 1985 Dec 25, 260(30), 16310 - 5 RNA polymerase . Direct evidence for a unique topographical site for initiation; Ruetsch N et al.; The compound 9-(3'-azido-3'-deoxy-beta-D-xylofuranosyl)adenine 5'-monophosphate is an inhibitor (Ki = 330 microM) of the initiation binding site of the DNA-dependent RNA polymerase derived from Escherichia coli . The alpha-32P derivative of this photo-labile compound is used to derivatize a site on the sigma subunit of the holoenzyme (E sigma) using either T7 delta D111 or poly{d(A-T)} as a DNA template . The incorporation of the 32P label into the sigma subunit could be prevented by the addition of either 5'-AMP or 5'-ATP . The results are suggested to support the existence of a unique initiation binding site, topographically distinct from the sites employed during the elongation phase. J Biol Chem, 1985 Dec 25, 260(30), 16237 - 41 The rate of cleavage of GTP on the binding of Phe-tRNA.elongation factor Tu.GTP to poly(U)-programmed ribosomes of Escherichia coli; Eccleston JF et al.; Interaction of Phe-tRNA.elongation factor Tu.GTP with poly(U)-programmed ribosomes containing an occupied P site can be described by a three-step kinetic mechanism . Initial binding is followed by the cleavage of GTP, and then a new peptide bond is formed . Rate constants controlling the first and third of these reactions are known, but only a lower limit for the rate constant of the cleavage step has been reported . We have determined this rate constant to be 20 s-1 at 5 degrees C, 30 s-1 at 15 degrees C, and 50 s-1 at 25 degrees C . This is much faster than the reverse step of the initial binding process and implies that the intrinsic accuracy of the ribosome in the initial selection step is sacrificed in favor of speed . The similarity of the kinetic and chemical mechanism of this GTP cleavage step with other nucleoside 5'-triphosphatases is discussed. J Biol Chem, 1985 Dec 25, 260(30), 16181 - 6 Characterization of lactose carrier mutants which transport maltose; Brooker RJ et al.; Brooker and Wilson (Brooker, R . J., and Wilson, T . H . (1985) Proc . Natl . Acad . Sci . U . S . A . 82, 3959-3963) previously isolated lactose carrier mutants which were able to transport maltose . All of the mutants were found to be single amino acid substitutions for alanine 177 or for tyrosine 236 . In the present study, we have examined the ability of these mutants to transport maltose, lactose, o-nitrophenyl-beta-D-galactopyranoside, methyl-beta-D-thiogalactopyranoside, and H+ . Both the position 177 and 236 mutants have enhanced rates of maltose transport and exhibit apparent Km values for maltose which are substantially less than that of the wild-type strain . The position 177 mutants transport lactose and other galactosides at a normal rate and with normal affinity during downhill transport and show counterflow transport rates which are faster than the wild-type strain . Interestingly, these mutants are markedly defective in accumulating substrates against a concentration gradient, yet retain a normal H+:galactoside stoichiometry . The position 236 mutants appear to be defective in the downhill, uphill, and counterflow transport of galactosides but exhibit a normal H+:galactoside stoichiometry. J Biol Chem, 1985 Dec 25, 260(30), 16148 - 55 Kinetic studies of the lipid-activated pyruvate oxidase flavoprotein of Escherichia coli; Mather MW et al.; Pyruvate oxidase is a flavoprotein dehydrogenase isolated from Escherichia coli which catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 . In vivo, the enzyme can bind to the bacterial membrane and reduce ubiquinone-8, feeding electrons into the respiratory chain . The purified enzyme has been shown previously to bind to phospholipids and detergents and, upon doing so, is activated . The turnover with ferricyanide as an electron acceptor increases 20- to 30-fold upon lipid binding . In this work, initial velocity and stop-flow kinetics are used to investigate the activation of this enzyme . It is shown that the unactivated form of the enzyme is markedly hysteretic . Progress curves at low substrate concentrations show an initial acceleration in enzyme turnover . This is consistent with the results of stop-flow experiments . Rates obtained for either the reduction of the unactivated flavoprotein by pyruvate or its reoxidation by ferricyanide in single turnover experiments are much slower than the rates predicted by observed turnover in initial velocity studies, in some cases by more than 2 orders of magnitude . The data are best explained by the slow interconversion between two forms of the enzyme, one with low turnover and one which rapidly turns over . As isolated, the enzyme is highly unreactive, as revealed by the stop-flow experiments . During turnover, even in the absence of lipid activators, some of the enzyme converts to the rapid-turnover form . This slow interconversion is shown by kinetic simulation to preclude a steady state from being established . Lipid activators appear to shift the equilibrium to favor the rapid-turnover form of the enzyme . Once the enzyme is "locked" into an activated conformation, the hysteresis is no longer observed, and the stop-flow results are in agreement with data obtained from initial velocity experiments . Activation appears to result in both increased rates of electron transfer into and out of the flavin. J Biol Chem, 1985 Dec 25, 260(30), 16049 - 51 Quaternary structure of pyruvate dehydrogenase complex from Escherichia coli; Yang HC et al.; The pyruvate dehydrogenase complex of Escherichia coli and subcomplexes derived from it by selective removal of component enzymes have been subjected to quaternary structural analysis by scanning transmission electron microscopy . Scanning transmission electron microscopic images of the intact complex (E1E2E3), the dihydrolipoyl transacetylase-dihydrolipoyl dehydrogenase (E2E3) subcomplex, and the E2 core enzyme appear as cubic particles in various orientations . Mass distributions within this complex and its subcomplexes have been determined by radial mass analysis of similarly oriented scanning transmission electron microscopic images of each type . The data show that mass attributable to dihydrolipoyl dehydrogenase (E3) is well integrated into the structural framework of the E2 core, dihydrolipoyl transacetylase, whereas mass attributable to pyruvate dehydrogenase (E1) is located about the periphery of the core enzyme . The mass distributions are fully consistent with a structural model in which 6 E3 dimers are integrated into the six faces of the cubic E2 core, and 12 E1 dimers are associated along the 12 edges of the core enzyme. J Biol Chem, 1985 Dec 25, 260(30), 16433 - 8 Molecular cloning of cDNAs cognate to genes sensitive to hormonal control in rat liver; Lee KL et al.; Poly(A)-RNAs were prepared from livers of rats treated with hydrocortisone and cycloheximide, then enriched for large mRNAs by successive sucrose gradients and gel electrophoresis . The size-selected RNAs were used as templates for synthesis of double-stranded cDNAs that were cloned in Escherichia coli using the pBR322 plasmid vector . Recombinant plasmids characterized as carrying inserts of potential interest were further analyzed by differential hybridization to mRNAs from untreated and hydrocortisone-treated rats . Seven of the cloned cDNAs were identified as complementary to mRNAs whose content in liver is sensitive to modulation by the steroid . Further screening for hormonal responsiveness revealed that two of the cloned cDNAs hybridize to a 3.4-kilobase mRNA that is rapidly induced in liver by treatment with insulin or cAMP as well as by hydrocortisone; restriction enzyme analysis demonstrated that the cDNAs from these two clones are derived from the same mRNA . In isolated nuclei, the rate of transcription of this mRNA is increased by each of the inducing hormones . This unusually regulated mRNA codes for a protein of 53 kDa on denaturing gels, undergoes rapid intracellular degradation that is prevented by cycloheximide, and appears to be the product of a single copy gene. J Biol Chem, 1985 Dec 25, 260(30), 16383 - 94 Ligand-mediated conformational changes in Trp repressor protein of Escherichia coli probed through limited proteolysis and the use of specific antibodies; Tsapakos MJ et al.; Trp repressor of Escherichia coli K-12 is a dimeric protein (monomer size, 108 amino acids) that acquires high affinity for certain operator targets in double-stranded DNA upon interaction with L-tryptophan . High titer antiserum directed against E . coli Trp repressor protein, elicited in rabbits, was monospecific toward native or denatured Trp repressor . Using an enzyme-linked immunosorbent assay to measure antigen-antibody reaction, we found that the binding of L-tryptophan to Trp repressor was associated with a marked decrease in antibody reactivity that presumably accompanied a conformational change in this protein to a state with strong affinity for trp operator-bearing DNA . We analyzed the pattern of cleavage of Trp repressor by chymotrypsin and trypsin and the effect of L-tryptophan on such hydrolytic cleavages . Chymotrypsin cleaved Trp repressor mainly between residues 71 and 72 . In the presence of L-tryptophan this cleavage was slowed . The first-order rate constants for chymotryptic digestion of Trp repressor were 7.6 X 10(-2) and 4.6 X 10(-2) min-1 in the absence and presence of L-tryptophan, respectively . Tryptic digestion was more complex . Initial cleavage of Trp repressor occurred with approximately equal facility between residues 69-70 or 84-85 . Subsequent tryptic hydrolyses led eventually to a major core fragment containing the first 54 amino acids of Trp repressor plus four other fragments from the carboxyl-terminal half of the protein . In the presence of L-tryptophan, cleavage by trypsin between residues 54-55 and 84-85 was retarded, even when a previous hydrolytic event elsewhere in the protein had occurred . Tryptophan had essentially no effect on the tryptic hydrolysis of peptide bond 97-98, but accelerated cleavage at peptide bond 69-70 . The first-order rate constants for the first tryptic cleavage of Trp receptor were 1.55 X 10(-1) and 1.33 X 10(-1) min-1 in the absence and presence of ligand, respectively . Our results are compatible with a structural model wherein certain amino acid side chains and peptide bonds of Trp repressor (specifically, those of residues 69-85) lie on or near the surface of the protein . This region of Trp repressor has been predicted to contain the operator recognition site . The susceptibility to proteolytic attack of at least four peptide bonds in this area changes when the protein interacts with L-tryptophan. J Mol Biol, 1985 Dec 20, 186(4), 773 - 90 DNA bending and its relation to nucleosome positioning; Drew HR et al.; X-ray and solution studies have shown that the conformation of a DNA double helix depends strongly on its base sequence . Here we show that certain sequence-dependent modulations in structure appear to determine the rotational positioning of DNA about the nucleosome . Three different experiments are described . First, a piece of DNA of defined sequence (169 base-pairs long) is closed into a circle, and its structure examined by digestion with DNAase I: the helix adopts a highly preferred configuration, with short runs of (A, T) facing in and runs of (G, C) facing out . Secondly, the same sequence is reconstituted with a histone octamer: the angular orientation around the histone core remains conserved, apart from a small uniform increase in helix twist . Finally, it is shown that the average sequence content of DNA molecules isolated from chicken nucleosome cores is non-random, as in a reconstituted nucleosome: short runs of (A, T) are preferentially positioned with minor grooves facing in, while runs of (G, C) tend to have their minor grooves facing out . The periodicity of this modulation in sequence content (10.17 base-pairs) corresponds to the helix twist in a local frame of reference (a result that bears on the change in linking number upon nucleosome formation) . The determinants of translational positioning have not been identified, but one possibility is that long runs of homopolymer (dA) X (dT) or (dG) X (dC) will be excluded from the central region of the supercoil on account of their resistance to curvature. J Mol Biol, 1985 Dec 20, 186(4), 715 - 24 Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli . II . Involvement of the C-terminal region of the regulatory chain in homotropic and heterotropic interactions; Ladjimi MM et al.; The modified aspartate transcarbamylase (ATCase) encoded by the transducing phage described by Cunin et al . has been purified to homogeneity . In this altered form of enzyme (pAR5-ATCase) the last eight amino acids of the C-terminal end of the regulatory chains are replaced by a sequence of six amino acids coded for by the lambda DNA . This modification has very informative consequences on the allosteric properties of ATCase . pAR5-ATCase lacks the homotropic co-operative interactions between the catalytic sites for aspartate binding and is "frozen" in the R state . In addition, this altered form of enzyme is insensitive to the physiological feedback inhibitor CTP, in spite of the fact that this nucleotide binds normally to the regulatory sites . Conversely, pAR5-ATCase is fully sensitive to the activator ATP . However, this activation is limited to the extent of the previously described "primary effect" as expected from an ATCase form "frozen" in the R state . These results emphasize the importance of the three-dimensional structure of the C-terminal region of the regulatory chains for both homotropic and heterotropic interactions . In addition, they indicate that the primary effects of CTP and ATP involve different features of the regulatory chain-catalytic chain interaction area. Nucleic Acids Res, 1985 Dec 20, 13(24), 8893 - 9 Direct-acting mutagenicity of N4-aminocytidine derivatives bearing alkyl groups at the hydrazino nitrogens; Nomura A et al.; To investigate the mechanism of N4-aminocytidine-induced mutagenesis, N'-alkyl-N4-aminocytidines and N4-alkyl-N4-aminocytidines were prepared and their mutagenicity on bacteria were assayed . N'-Methyl-N4-aminocytidine, N'-(2-hydroxyethyl)-N4-aminocytidine and N',N'-dimethyl-N4-aminocytidine showed direct-acting mutagenicity on S . typhimurium TA100 and E . coli WP2 uvrA, tester strains that are sensitive to base-pair substitutions . In contrast, N4-methyl-N4-aminocytidine, N4-(2-hydroxyethyl)-N4-aminocytidine and N4,N'-dimethyl-N4-aminocytidine were not mutagenic on these bacteria . Since N'-methyl-N4-aminocytidine does not form hydrazones, the possibility that N4-aminocytidine causes mutation due to its reactivity with carbonyl compounds has been excluded . Furthermore, the fact that only those alkyl N4-aminocytidines having a hydrogen on the nitrogen at position 4 are mutagenic is consistent with the previously proposed mechanism in which the tautomerization between the amino and the imino forms of N4-aminocytosine allowing an ambiguous base pairing is the cause of the mutagenesis. Biochim Biophys Acta, 1985 Dec 20, 832(3), 343 - 50 Isolation and characterization of the multiple charge isoforms of acyl-CoA synthetase from Escherichia coli; Kameda K et al.; The three fractions of acyl-CoA synthetase differing in isoelectric pH were isolated from Escherichia coli by isoelectric focusing and characterized . They had the same molecular weight and identical immunochemical properties . The three fractions differed appreciably in pH-velocity profiles . These fractions had distinguishable thermal stabilities and peptide patterns obtained after limited proteolysis . Apparent Km and Vmax values for fatty acids were also significantly different in these fractions, although the specificity ranged from C8 to C18 fatty acids with maximum activity for lauric acid in all fractions. Science, 1985 Dec 20, 230(4732), 1383 - 5 Systematic variation in DNA length yields highly ordered repressor-operator cocrystals; Jordan SR et al.; Crystals have been grown that contain the operator-binding domain of the lambda repressor and the lambda operator site OL1 . Crystallization conditions were tested with a set of DNA fragments, ranging in length from 17 to 23 base pairs . The best crystals were grown with a 20-base pair DNA fragment . These crystals have space-group symmetry P2I, with unit cell dimensions a = 37.1 A, b = 68.8 A, c = 56.8 A, and a beta angle of 91.5 degrees . They diffracted to at least 2.5 A resolution . High resolution data from these crystals should allow the direct determination of how a repressor recognizes its operator site. Science, 1985 Dec 20, 230(4732), 1350 - 4 Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia; Saiki RK et al.; Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia . The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies . In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences . The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA. J Mol Biol, 1985 Dec 20, 186(4), 733 - 42 Functional analysis of lac repressor restart sites in translational initiation and reinitiation; Cone KC et al.; To define some of the features that influence ribosomal recognition of translational restart sites in the lac repressor mRNA, recombinant DNA methods have been used to construct lacI-Z fusions in which lacZ gene expression is dependent upon initiation or reinitiation within lacI mRNA sequences . Reinitiation efficiencies, as assessed by beta-galactosidase levels in strains bearing such plasmids, appear to be determined by at least three features of the RNA between the termination codon and reinitiation codon: the presence of competing out-of-frame AUG or GUG triplets, the distance between termination and reinitiation points, and the extent to which restart sequences remain accessible to ribosomes . While some of the restart sites are used with substantial efficiency for reinitiation, they do not function detectably as primary initiators if placed at the 5' end of the lacZ mRNA . This finding concurs with our observation that relative to the wild-type initiator region, which is recovered in quantitative yield from in vitro initiation reactions, ribosome protection of the four restart sites occurs at more than 100-fold lower efficiencies . In part, the lack of initiation activity is rationalized by the striking potential these sequences have for forming stable secondary structures that sequester elements essential for ribosome binding . However, the differential functioning of the restart sites in primary initiation versus reinitiation must also reflect real differences in the mechanisms operative in the two events. J Mol Biol, 1985 Dec 20, 186(4), 725 - 32 Messenger RNA conformation and ribosome selection of translational reinitiation sites in the lac repressor mRNA; Cone KC et al.; In the early region of the Escherichia coli lac repressor mRNA, the pattern of cleavage by nucleases specific for single or double-stranded RNA confirms the presence of secondary structures previously proposed to influence the pattern of translational reinitiation . These are positioned so as to mask a potential restart site centered on an in-phase GUG triplet corresponding to repressor amino acid position Val38 . Our finding that a restart polypeptide initiated at the Val38 GUG codon is observed only in situations that that preclude base-pairing of adjacent mRNA sequences establishes a functional role for these structures in vivo . This evidence for structure, considered with the overall pattern of reinitiation events, suggests that local mRNA conformation is the major determinant that dictates ribosomal selection of restart sites within the early region of the repressor cistron. Nucleic Acids Res, 1985 Dec 20, 13(24), 9031 - 42 Correlation between the rate of productive transcription initiation and the strand-melting property of Escherichia coli promoters; Tachibana H et al.; "Opening potential"s of DNA segments of about ten base pairs in length were calculated for eleven promoters of Escherichia coli using the thermodynamic parameter values {Gotoh and Tagashira (1981) Biopolymers 20, 1043-1058} for the stabilities of ten kinds of nearest neighbor base pair doublets in a melting reaction . They were compared with the strength of each promoter determined experimentally with a "mixed transcription" system {Kajitani and Ishihama (1983) Nucleic Acids Res . 11, 3873-3888} . A positive correlation was found between the calculated opening potential in the -9 to +3 region (+1 denotes the nucleotide position at which the transcription starts) and the rate of open complex formation. Nature, 1985 Dec 19-1986 Jan 1, 318(6047), 672 - 5 Cloning of the breakpoint of an X;21 translocation associated with Duchenne muscular dystrophy; Ray PN et al.; Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder which affects approximately 1 in 3,300 males, making it the most common of the neuromuscular dystrophies . The biochemical basis of the disease is unknown and as yet no effective treatment is available . A small number of females are also affected with the disease, and these have been found to carry X; autosome translocations involving variable autosomal sites but always with a breakpoint within band Xp21 of the X chromosome (implicated by other kinds of genetic evidence as the site of the DMD lesion) . In these female patients the normal X chromosome is preferentially inactivated, which it is assumed silences their one normal DMD gene, leading to expression of the disease . In one such affected female the autosomal breakpoint lies in the middle of the short arm of chromosome 21, within a cluster of ribosomal RNA genes . Here we have used rRNA sequences as probes to clone the region spanning the translocation breakpoint . A sequence derived from the X-chromosomal portion of the clone detects a restriction fragment length polymorphism (RFLP) which is closely linked to the DMD gene and uncovers chromosomal deletions in some male DMD patients. Biochim Biophys Acta, 1985 Dec 19, 821(3), 404 - 12 A pH titration study on the ionic bridging within lipopolysaccharide aggregates; Coughlin RT et al.; The packing of lipopolysaccharide aggregates from rough strains of Escherichia coli was examined at different pH values . Lipopolysaccharide head-group motion, measured with an electron spin resonance probe, was found to be dependent on pH, and indicated the existence of multiple ionizable groups . Lipopolysaccharide from a rough (Ra) and a heptose-less (Re) mutant were more rigid at pH 5 than at pH 10.5 . In addition, head-group mobility of the magnesium salt of Ra lipopolysaccharide was substantially less than that of the sodium salt at pH 7.0, whereas at high pH (pH 12) the two salts were equally fluid . Changes in head-group packing were also reflected in pH-dependent changes in the phase transition measured with differential scanning calorimetry . The enthalpy of the transition, delta Ht, for the sodium salt of Re lipopolysaccharide was greatest at pH 7.5 and approached zero in both the acidic and the basic pH ranges . We propose that fixed charges in the core and lipid A regions significantly influence lipopolysaccharide head-group motion and the lipopolysaccharide aggregation state . Furthermore, ionic bridging among phosphate groups dramatically rigidifies head group interactions in the neutral to acidic pH ranges. Biochim Biophys Acta, 1985 Dec 18, 826(4), 208 - 12 A new restriction endonuclease Eco31I recognizing a non-palindromic sequence; Butkus V et al.; A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31 . The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of lambda DNA have been compared . A common 5'GAGACC 3'CTCTGG sequence occurs near each cleavage site . Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence . This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5' GGTCTCN decreases 3' CCAGAGN NNNN increases. Biochemistry, 1985 Dec 17, 24(26), 7662 - 8 Equilibrium denaturation of pituitary- and recombinant-derived bovine growth hormone; Brems DN et al.; Holladay and co-workers {Holladay, L . A., Hammonds, R . G., & Puett, D . (1974) Biochemistry 13, 1653-1661} reported the presence of an equilibrium intermediate in the guanidine hydrochloride (GdnHCl) induced denaturation of pituitary-derived bovine growth hormone (p-bGH) . Since then, numerous reports have appeared demonstrating the inherent heterogeneity in p-bGH . In this report we show that a standard preparation of p-bGH can be separated into two components of almost equal abundance differing in molecular weight by approximately 1000 . Each of these two components could give rise to different denaturation transitions which would be interpreted as evidence for equilibrium intermediates . We report here the equilibrium denaturation of bGH produced by Escherichia coli through recombinant DNA technology . The recombinant-derived bGH (r-bGH) is more homogeneous than that derived from pituitary sources and is greater than 95% a single polypeptide entity . Nevertheless, the GdnHCl-induced denaturation profiles of both recombinant bGH and pituitary bGH are very similar . The presence of equilibrium intermediates is verified by the asymmetry of the denaturation transition as measured by size-exclusion high-performance liquid chromatography and by noncoincidence of the denaturation transitions as observed by ultraviolet absorbance, fluorescence intensity, and circular dichroism . These findings conclusively show that the secondary structure of bovine growth hormone is more stable than the tertiary structure and is consistent with a framework model of protein folding. Biochem Biophys Res Commun, 1985 Dec 17, 133(2), 436 - 41 Comparison of amino acid sequences between phosphoenolpyruvate carboxylases from Escherichia coli (allosteric) and Anacystis nidulans (non-allosteric): identification of conserved and variable regions; Ishijima S et al.; Amino acid sequences of phosphoenolpyruvate carboxylases of Escherichia coli (allosteric) and a cyanobacterium Anacystis nidulans (non-allosteric) were aligned . The pattern of homology suggests that the enzyme molecule is comprised of two distinct regions, namely, a conserved region (C-terminal half) and a variable region (N-terminal half) . Among the amino acid residues which have previously been presumed essential for the catalytic activity, three histidine residues were found to be conserved, but cysteine residues were not . Furthermore, the conserved sequence unique to the enzyme was identified by comparison of the enzyme sequence with amino acid sequences in our data bank. Biochemistry, 1985 Dec 17, 24(26), 7834 - 8 Acyl carrier protein from Escherichia coli . Structural characterization of short-chain acylated acyl carrier proteins by NMR; Mayo KH et al.; Acylated acyl carrier proteins (ACPs) with acyl chain lengths of 2, 4, 6, 8, and 10 carbons were investigated by NMR and nuclear Overhauser methods at 500 MHz . Chemical shift changes of downfield aromatic and upfield, ring-current-shifted, isoleucine proton resonances monotonically vary as a function of acyl chain length with the most prominent shifts occurring with chain lengths between four and six carbons . Chemical shifts are largest for one of the two phenylalanines; however, substantial shifts do exist for Tyr-71, His-75, and two isoleucines . Since these residues are distributed throughout the molecule, their associated resonance chemical shifts are most probably explained by an induced conformational change . Comparative NOE measurements on reduced ACP (ACP-SH) and ACP-S-C8 suggest, however, that these induced conformational changes are small except for around one of the phenylalanines . A tertiary structural model for acyl-ACP consistent with our previous model for ACP-SH {Mayo, K . H., Tyrell, P . M., & Prestegard, J . H . (1983) Biochemistry 22, 4485-4493} is presented. Biochemistry, 1985 Dec 17, 24(26), 7628 - 35 Site-directed mutagenesis of cysteine-148 in the lac permease of Escherichia coli: effect on transport, binding, and sulfhydryl inactivation; Viitanen PV et al.; By subjecting the lac y gene of Escherichia coli to oligonucleotide-directed, site-specific mutagenesis, Cys148 in the lac permease has been replaced with a Gly residue {Trumble, W . R., Viitanen, P . V., Sarkar, H . K., Poonian, M . S., & Kaback, H . R . (1984) Biochem . Biophys . Res . Commun . 119, 860} . Recombinant plasmids bearing wild-type or mutated lac y were constructed and used to transform E . coli T184 . Steady-state levels of lactose accumulation, the apparent Km for lactose under energized conditions, and the KD for p-nitrophenyl alpha-D-galactopyranoside are comparable in right-side-out vesicles containing wild-type or mutant permease . In contrast, the Vmax for lactose transport in vesicles containing mutant permease is significantly decreased . Although antibody binding studies reveal that vesicles from the mutant contain almost as much permease as wild-type vesicles, surprisingly only about one-fourth of the altered molecules bind p-nitrophenyl alpha-D-galactopyranoside with high affinity . Mutant permease is less sensitive to inactivation by N-ethylmaleimide, although the alkylating agent is still capable of completely inhibiting transport activity . Importantly, beta-galactosyl 1-thio-beta-D-galactopyranoside affords complete protection of wild-type permease against N-ethylmaleimide but has no protective effect whatsoever in the mutant . The rate of inactivation of wild-type and mutant permeases by N-ethylmaleimide is increased at alkaline pH and by the presence of a proton electrochemical gradient (interior negative and alkaline), and these phenomena are exaggerated in vesicles containing mutant permease . Finally, p-(chloromercuri)benzenesulfonate, which completely displaces bound p-nitrophenyl alpha-D-galactopyranoside from wild-type permease, does not affect binding in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1985 Dec 17, 24(26), 7617 - 21 Reaction of both active site thiols of reduced thioredoxin reductase with N-ethylmaleimide; O'Donnell ME et al.; Thioredoxin reductase from Escherichia coli, only in its reduced state, reacts rapidly with 2 mol of N-ethylmaleimide, which specifically alkylates both active site cysteine residues . This dual modification supports previous studies indicating that a base lowers the pK of both active site cysteine residues . The dual modification also indicates that the region around the active site dithiol is more open than is the case with the related enzymes lipoamide dehydrogenase and glutathione reductase, both of which can be alkylated only on one nascent thiol . Enhanced nucleophilicity of the active site thiols is consistent with the proposed chemical mechanism of thioredoxin reductase . The sequence of the amino-terminal 16 residues is presented. Biochem Biophys Res Commun, 1985 Dec 17, 133(2), 731 - 9 Regulation of methionine synthesis in Escherichia coli: effect of metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL and metJ genes; Shoeman R et al.; The regulation of the expression of three Escherichia coli met genes, metB, which codes for cystathionine gamma-synthetase (EC 4.2.99.9), metL, which codes for aspartokinase II-homoserine dehydrogenase II (EC 2.7.2.4-EC 1.1.1.3) and metJ, which codes for the methionine regulon aporepressor, has been studied using highly purified DNA-directed in vitro protein synthesis systems . In a system where the entire gene product is synthesized, the expression of the metB and metL genes is specifically inhibited by MetJ protein (repressor protein) and S-adenosylmethionine (AdoMet) . In a simplified system that measures the formation of the first dipeptide of the gene product (fMet-Ala for the metJ gene), MetJ protein and AdoMet partially repress (approximately 40-60%) metJ gene expression . Thus, the metJ gene can be partially autoregulated by its gene product. Biochem Biophys Res Commun, 1985 Dec 17, 133(2), 589 - 97 Repair of DNA O-alkylation damage by various human organs; Wani AA et al.; Protein extracts from human adult liver, fetal liver, intestine, brain, kidney, lung and skin were tested against poly(dT)methylated X poly(dA), poly(dA)methylated X poly(dT) and methylated DNA . The suitability of various substrates was established in assays using E . coli extracts that removed O4-methylthymidine (O4-MedT), O2-MedT, and O6-methylguanine (O6-MeG) . The human extracts efficiently removed O6-MeG and N3-methyladenine from methylated substrates . The adult liver exhibited low and fetal tissues negligible removal of O4-MedT . Only the liver showed limited removal of O2-MedT . The poor removal of the miscoding base O4-MedT by human organs could be an important factor in carcinogen induced mutagenesis, carcinogenesis and teratogenesis. Biochem Biophys Res Commun, 1985 Dec 17, 133(2), 511 - 9 Cloning and expression of human interleukin 2 in Streptomyces lividans using the Escherichia coli consensus promoter; Munoz A et al.; Streptomyces lividans was transformed with a plasmid containing the structural gene that specifies human interleukin 2 . The expression of interleukin 2 in this plasmid is controlled by both the consensus promoter and a consensus ribosome binding site characteristic of Escherichia coli . We have detected production of active human interleukin 2 in liquid cultures of the transformed Streptomyces by both, biological assay and immuno-blotting analysis. EMBO J, 1985 Dec 16, 4(13A), 3633 - 8 The size of the lactose permease derived from rotational diffusion measurements; Dornmair K et al.; The lactose permease of Escherichia coli was labeled with eosinyl-maleimide, reconstituted into vesicles of dimyristoylphosphatidylcholine and subjected to time-dependent phosphorescence anisotropy measurements in order to determine the rotational diffusion coefficient . By comparison with bacteriorhodopsin, the diffusion coefficient is evaluated in terms of an effective radius of the lactose permease in the plane of the membrane . This radius amounts to 20 +/- 2 A which implies that the lactose permease is a monomer . The monomeric state is maintained in the presence of a membrane potential. EMBO J, 1985 Dec 16, 4(13A), 3625 - 31 The structure of the lactose permease derived from Raman spectroscopy and prediction methods; Vogel H et al.; The secondary structure of the lactose permease of Escherichia coli reconstituted in lipid membranes was determined by Raman spectroscopy . The alpha-helix content is approximately 70%, the beta-strand content below 10% and beta-turns contribute 15% . About 1/3 of the residues in alpha-helices and most other residues are exposed to water . Employing a method for structural prediction which accounts for amphipathic helices, 10 membrane-spanning helices are predicted which are either hydrophobic or amphipathic . They are expected to form an outer ring of helices in the membrane . The interior of the ring would be made of residues which are predominantly hydrophilic and, evoking the analogy to sugar-binding proteins, suited to provide the sugar binding site. EMBO J, 1985 Dec 16, 4(13A), 3599 - 604 Control of gene expression in P2-related coliphages: the in vitro transcription pattern of coliphage 186; Pritchard M et al.; Transcription in vitro of coliphage 186 DNA generated four transcripts . The most abundant transcript was that of the late control gene B and an equivalent transcript was identified for the closely related phage P2 . A second transcript was from the rightward promoter at 75% and predicted to be under CI repressor control . The remaining two transcripts initiated from the one promoter located at 95% and are apparently under LexA control in vivo . The significance of these transcripts is discussed in relation to coliphage 186. EMBO J, 1985 Dec 16, 4(13A), 3593 - 8 The nature of information, required for export and sorting, present within the outer membrane protein OmpA of Escherichia coli K-12; Freudl R et al.; Information, in addition to that provided by signal sequences, for translocation across the plasma membrane is thought to be present in exported proteins of Escherichia coli . Such information must also exist for the localization of such proteins . To determine the nature of this information, overlapping inframe deletions have been constructed in the ompA gene which codes for a 325-residue major outer membrane protein . In addition, one deletion, encoding only the NH2-terminal part of the protein up to residue 160, was prepared . The location of each product was determined by immunoelectron microscopy . Proteins missing residues 4-45, 43-84, 46-227, 86-227 or 160-325 of the mature protein were all efficiently translocated across the plasma membrane . The first two proteins were found in the outer membrane, the others in the periplasmic space . It has been proposed that export and sorting signals consist of relatively small amino acid sequences near the NH2 terminus of an outer membrane protein . On the basis of sequence homologies it has also been suggested that such proteins possess a common sorting signal . The locations of the partially deleted proteins described here show that a unique export signal does not exist in the OmpA protein . The proposed common sorting signal spans residues 1-14 of OmpA . Since this region is not essential for routing the protein, the existence of a common sorting signal is doubtful . It is suggested that information both for export (if existent) and localization lies within protein conformation which for the former process should be present repeatedly in the polypeptide. Eur J Biochem, 1985 Dec 16, 153(3), 541 - 51 D-Cysteine desulfhydrase of Escherichia coli . Purification and characterization; Nagasawa T et al.; D-Cysteine-specific desulfhydrase is found in some intestinal bacteria . Escherichia coli W3110 delta trpED102/F' delta trpED102 was found to have the highest enzyme activity . The enzyme was purified from E . coli W3110 delta trpED102/F' delta trpED102 in six steps . After the last step the enzyme appeared to be homogeneous by the criteria of polyacrylamide gel electrophoresis, analytical ultracentrifugation and double diffusion in agarose . The enzyme has a molecular mass of about 67 000 Da and consists of two subunits identical in molecular mass . The enzyme exhibits absorption maxima at 278 nm and 418 nm, which are independent of pH (6.5-10.5), and contains 2 mol pyridoxal phosphate/mol enzyme . The holoenzyme is resolved to the apoenzyme by incubation with phenylhydrazine, and reconstituted on the addition of pyridoxal phosphate . D-Cysteine desulfhydrase also catalyzes the beta-replacement reaction of the chlorine of 3-chloro-D-alanine with thioglycolic acid to yield S-carboxymethyl-D-cysteine . Its catalytic and immunological properties are compared with those of 3-chloro-D-alanine dehydrochlorinase. Eur J Biochem, 1985 Dec 16, 153(3), 445 - 9 Synthesis and expression of a human growth hormone (somatotropin) gene mutated to change cysteine-165 to alanine; Tokunaga T et al.; We have mutated a synthetic human growth hormone (hGH) gene specifically at the codon for Cys-165 to a codon for Ala by replacement of synthetic deoxyoligonucleotides corresponding to this site . This modification prevented the formation of a disulfide bond between Cys-53 and Cys-165 in the hGH molecule . This mutated protein, {Ala165}hGH was expressed at the same level as the intact hGH, 4 X 10(5) molecules per cell under the control of the tryptophan promoter in Escherichia coli, and retained similar immunological activity to intact hGH . The limited digestion pattern of the mutated protein with human plasmin suggests that the tertiary structure of {Ala165}hGH resembles to that of the intact hGH molecule . {Ala165}hGH revealed full biological activity as examined by the body weight increase of hypophysectomized rats. J Biol Chem, 1985 Dec 15, 260(29), 15526 - 9 Biphasic recombination of photodissociated CO compound of cytochrome o(s) from Vitreoscilla; Webster DA et al.; The soluble cytochrome o from Vitreoscilla contains two identical subunits and two hemes . The reduced form binds 2 mol of CO in a cooperative manner with a Hill coefficient near 2 (Tyree, B., and Webster, D . A . (1978) J . Biol . Chem . 253, 6988-6991) . This carbonyl compound was photolysed with a dye laser and recombination followed at 437 or 420 nm where maximal absorbance changes were registered . Recombination kinetics were biphasic, and the fast phase was approximately 10 times the rate of the slow phase . Apparent rate constants of both phases showed a nonlinear dependence on CO concentration, respectively, in conformity with a reaction scheme which assumes the transient formation of an intermediate species in both slow and fast reactions . A study of temperature dependence of the reactions gave EA = 2.7 kcal/mol for the slow reaction and EA = 3.2 kcal/mol for the fast reaction below 23 degrees C; above this temperature the slope of the Arrhenius plot for the fast reaction became positive . Maximal rates for both phases were around pH 6.5 and fell to approximately 40% of maximal at pH 12 . The binding reaction was affected by even a low concentration of sodium dodecyl sulfate (0.0025%), which changed both the kinetic constant of each phase and the relative contribution of each phase to the reaction . A model which assumes the existence of fast and slow reaction conformers in equilibrium is proposed. J Biol Chem, 1985 Dec 15, 260(29), 16037 - 44 Heat-labile enterotoxin in Escherichia coli . Kinetics of association of subunits into periplasmic holotoxin; Hofstra H et al.; We have investigated the assembly of the heat-labile enterotoxin (LT) subunits after their processing and segregation into the periplasmic space as mature LT A and LT B polypeptides . LT B starts associating into oligomers during or immediately after translocation through the cytoplasmic membrane . Binding to LT A occurs immediately after oligomerization . Over 80% of the LT B subunits have oligomerized, and over 50% have associated with LT A into holotoxin within 1 min after synthesis . The fate of newly synthesized LT A is totally different . There is an extensive overproduction of LT A relative to LT B and after membrane translocation it becomes part of a periplasmic pool of free LT A . It is then bound by LT B oligomers or degraded at such a rate that the free periplasmic LT A disappears from the pool with a half-time of 20-25 min . About half of the LT A is incorporated into holotoxin, while the other half is degraded . We conclude that LT subunits are translocated and processed in a ratio of about 2 A to 5 B . Since free LT A is either degraded slowly or bound to newly synthesized LT B oligomers, the net result is a steady state of 1.4 to 1.7 A subunits to 5 B subunits in the periplasm . About 60% of this LT A is bound by LT B to form periplasmic holotoxin with a subunit ratio of about 1 A to 5 B . The remaining 40% of periplasmic LT A occurs free. J Biol Chem, 1985 Dec 15, 260(29), 15571 - 6 Cytoplasmic methionyl-tRNA synthetase from Bakers' yeast . A monomer with a post-translationally modified N terminus; Fasiolo F et al.; Methionyl-tRNA synthetase has been purified from a yeast strain carrying the MES1 structural gene on a high copy number plasmid (pFL1) . The purified enzyme is a monomer of Mr = 85,000 in contrast to its counterpart from Escherichia coli which is a dimer made up of identical subunits (Mr = 76,000; Dardel, F., Fayat, G., and Blanquet, S . (1984) J . Bacteriol . 160, 1115-1122) . The yeast enzyme was not amenable to Edman's degradation indicating a blocked NH2 terminus . Its primary structure as derived from the DNA sequence (Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P., and Fasiolo, F . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 2437-2441) has been confirmed using the fast atom bombardment-mass spectrometric method . This method was applied to tryptic digests of the carboxymethylated enzyme and the corresponding data provided extensive coverage of the translated DNA sequence, thus confirming its correctness . The ambiguity concerning which of the three NH2-terminally located methionine codons is the initiation codon was easily resolved from peptides identified in this region . It was possible to show that the first methionine had been removed and that the new NH2 terminus, serine, had been acetylated . A comparison between the yeast and E . coli sequences shows that the former has an N-terminal extension of about 200 residues as compared to the latter . It also lacks the C-terminal domain which is responsible for the dimerization of the E . coli methionyl-tRNA synthetase. J Biol Chem, 1985 Dec 15, 260(29), 15925 - 31 Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane; Dalbey RE et al.; Leader peptidase cleaves the amino-terminal leader sequences of many secreted and membrane proteins . We have examined the function of leader peptidase by constructing an Escherichia coli strain where its synthesis is controlled by the arabinose B promoter . This strain requires arabinose for growth . When the synthesis of leader peptidase is repressed, protein precursors accumulate, including the precursors of M13 coat protein (an inner membrane protein), maltose binding protein (a periplasmic protein), and OmpA protein (an outer membrane protein) . These precursors are translocated across the plasma membrane, as judged by their sensitivity to added proteinase K . However, pro-OmpA and pre-maltose binding protein are retained at the outer surface of the inner membrane . Thus, leader peptides anchor translocated pre-proteins to the outer surface of the plasma membrane and must be removed to allow their subsequent release into the periplasm or transit to the outer membrane. J Biol Chem, 1985 Dec 15, 260(29), 15402 - 5 Monoclonal antibodies with specific effects on partial activities of recA protein of Escherichia coli; Makino O et al.; recA protein of Escherichia coli promotes a wide variety of DNA reactions in vitro . Specific effectors of recA protein should be very useful in elucidating the mechanisms of these complex reactions . Six mouse hybridoma clones that secreted class G immunoglobulins specific to recA protein were obtained in three cell-fusion experiments . Five IgGs were purified by affinity chromatography . These monoclonal antibodies were characterized by examining their effects on the single-stranded DNA-dependent ATPase activity, negatively superhelical double-stranded DNA-dependent ATPase activity, and an activity in pairing negatively superhelical closed circular double-stranded DNA and homologous single-stranded DNA-fragments to form D-loops . These IgGs inhibited all, some, or one of these three activities, and from the spectra of their inhibitory effects they were classified into four groups . This classification suggests that each of the monoclonal antibodies binds to one of at least four antigenic determinants on recA protein and specifically inactivates one or more of the active centers on the protein . These monoclonal antibodies will be useful in analyzing the complex reactions promoted by recA protein. J Biol Chem, 1985 Dec 15, 260(29), 15458 - 65 Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex . Primary sequence analysis of ATP2 encoding the F1-ATPase beta-subunit precursor; Takeda M et al.; The yeast nuclear gene ATP2 encodes a F1-ATPase beta-subunit protein of 509 amino acids with a predicted mass of 54,575 daltons . In contrast to the ATPase beta-subunit proteins determined previously from Escherichia coli and various plant sources, the yeast mitochondrial precursor peptide contains a unique cysteine residue within its immediate amino terminus . Expression of an in-frame deletion in ATP2 between residues 28 and 34 to eliminate this single cysteine residue located near the processing site of the matrix protease does not prevent the in vivo delivery of the subunit to mitochondria or its assembly into a functional ATPase complex . Thus, the import F1 beta-subunit into mitochondria does not require a covalent modification of the type utilized for the secretion of the major lipoprotein from E . coli . In addition, analysis of the level of the major F1-ATPase subunits in mitochondria prepared f