Escherichia coli

Cell, 1984 Apr, 36(4), 1073 - 80
Yeast DNA topoisomerase II is encoded by a single-copy, essential gene; Goto T et al.; The gene TOP2 encoding yeast topoisomerase II has been cloned by immunological screening of a yeast genomic library constructed in the phage lambda expression vector, lambda gt11 . The ends of the message encoded by the cloned DNA fragment were delimited by the Berk and Sharp procedure (S1 nuclease mapping) for the 5' end and mapping of the polyA tail portion of a cDNA fragment for the 3' end . The predicted size of the message agrees with the length of the message as determined by Northern blot hybridization analysis . The identity of the gene was confirmed by expressing the gene in E . coli from the E . coli promoter lac UV5 to give catalytically active yeast DNA topoisomerase II . Disruption of one copy of the gene in a diploid yeast creates a recessive lethal mutation, indicating that the single DNA topoisomerase II gene of yeast has an essential function.

Gene, 1984 Apr, 28(1), 93 - 102
The rep mutation . VII . Cloning and analysis of the functional rep gene of Escherichia coli K-12; Bialkowska-Hobrzanska H et al.; The rep gene of Escherichia coli was isolated on a 6-kb PvuII fragment of plasmid pLC44-7 DNA from the Clarke-Carbon collection and cloned into pSC101 (to form pHBH8) and pBR322 (to form pHBH30) . The plasmids pHBH8 and pHBH30 were found to complement all rep mutations tested . The functional rep gene and its promoter were mapped to a 3.2-kb XhoI-BalI fragment on the basis of complementation data with deletion and insertion derivatives of the two plasmids; subcloning of various restriction fragments confirmed the assignment . EcoRI, HindIII, and HpaI restriction sites were found to reside within that region of the DNA required for expression of the rep function . A coupled in vitro transcription-translation system was used to show that only those plasmids containing a functional rep gene encoded a protein of about Mr 67 000 (the Mr of the rep protein) . No plasmids were found that complemented only the A or B classes of rep mutants (which differ in their ability to support the growth of P2 and M13 phages) . This result suggests that rep-A and rep-B are alleles of the same structural gene.

Gene, 1984 Apr, 28(1), 29 - 35
Lambda phagemids and their transducing properties; Melnikov AA et al.; Two recombinant lambda DNAs, lambda gt::pMB9 and lambda NM::pBR322, containing, respectively, the pMB9 and pBR322 replicon were constructed and characterized . Both constructs (phagemid DNAs) transfect Escherichia coli cells, producing mature infectious phage progenies . Alternatively, drug-resistant colonies of transductants can be selected upon infection with these phages (phagemid particles) that maintain phagemid DNA in the cell in the form of covalently closed circular plasmids . The efficiency of transduction for nonlysogenic E . coli strains with lambda gt::pMB9 phage producing lambda repressor cIts ranges from 10(-7) to 10(-2) transductant colonies per input phage, depending on the temperature and strain used, while lambda NM::pBR322 phage carrying imm21 transduces with a frequency of up to 1 . This means that each lambda NM::pBR322 phagemid particle is capable of establishing itself in the cell as a nonlethal plasmid, permitting formation of a resistant bacterial colony . The maximal level of transduction with lambda gt::pMB9 was obtained when E . coli cells lysogenic for lambda were used . Thus, we believe that the efficiency of transduction is determined by the turn-on of the phage repressor in the transductant . In addition, we have found that all lambda gt::pMB9-containing transductants under certain conditions harbor precisely excised pMB9; excision of pBR322 from lambda NM::pBR322 has not been observed.

Int J Radiat Biol Relat Stud Phys Chem Med, 1984 Apr, 45(4), 379 - 88
Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals; Nabben FJ et al.; Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E . coli wild-type spheroplasts . On the other hand, using spheroplasts prepared from E . coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found . Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage . The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals . Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair . Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions . We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.

Can J Microbiol, 1984 Apr, 30(4), 451 - 60
Morphological examination of cell surface structures of enterotoxigenic strains of Escherichia coli; Chan R et al.; The very fine sinuous K99 pili of enterotoxigenic strains of Escherichia coli can be visualized in shadowed and in negatively stained preparations, especially if the amorphous K30 glycocalyx is not produced, but these very delicate structures cannot be directly resolved in sectioned material . The K99 pili can, however, be thickened by the nonspecific accretion of K30 glycocalyx material, during its condensation as a result of dehydration, to the point where it can be resolved in sectioned material . This visualization is enhanced if the accreted and condensed glycocalyx is stained with ruthenium red . Alternatively and additionally, the K99 pilus can be thickened by the specific accretion of monoclonal antibodies so that it is made visible in sectioned material . The condensation of the hydrated K30 antigen glycocalyx of enterotoxigenic strains of Escherichia coli during dehydration can be prevented by stabilization using specific antibodies so that this capsular glycocalyx structure is identified in sectioned material and is seen in its correct distribution and dimensions . These methods allow the identification and visualization of bacterial surface structures, both in vitro and in vivo, and they provide a useful means of assessing the presence and distribution of these structures at all stages of the bacterial disease and a possible means of assessing their roles in the pathogenic process.

Anal Biochem, 1984 Apr, 138(1), 66 - 7
An improved method for increasing the resolution and sensitivity of silver staining of nucleic acid bands in polyacrylamide gels; Kolodny GM; Staining of polyacrylamide gels with methylene blue prior to silver staining increases band resolution and sensitivity . This method permits resolution of multiple bands less than 1 mm apart, and is able to detect bands containing only 100 pg of RNA.

Biochem J, 1984 Apr 1, 219(1), 205 - 10
Polyamine regulation of stringent control in a polyamine-auxotrophic strain of Escherichia coli; Goldemberg SH; Escherichia coli BGA8 , a mutant unable to synthesize putrescine, behaves as stringent or relaxed according to the presence or absence of polyamine, respectively, in the culture medium . The relaxed synthesis of RNA can be reverted back to stringent by addition of putrescine or spermidine . The stringent response depends on the concentration of the polyamine in the culture medium . The formation of guanosine 3'-diphosphate 5'-diphosphate elicited by amino acid starvation is stimulated at least 40-fold in putrescine-supplemented bacteria and only about 2-fold in putrescine-depleted cells.

Proc Natl Acad Sci U S A, 1984 Apr, 81(8), 2295 - 7
Cloning and sequence analysis of cDNA for rat spleen thymosin beta 4; Wodnar-Filipowicz A et al.; Molecular cloning of a cDNA has established the sequence of the translated portion of the mRNA for rat spleen thymosin beta 4 . The presence of a methionyl initiator codon immediately preceding the codon for the first seryl residue of mature thymosin beta 4 is consistent with previous results indicating the absence of a signal peptide in the product translated in vitro from rat spleen mRNA . The cDNA sequence analysis also established the presence of two terminator codons immediately following the codon for the COOH-terminal seryl residue . Thymosin beta 4 is thus synthesized as a 5100-dalton peptide containing 44 amino acid residues . Removal of the initiator methionyl residue and acetylation of the NH2-terminal serine residue would yield mature thymosin beta 4 containing 43 amino acids . The absence of a signal peptide makes it unlikely that thymosin beta 4 is a secreted peptide.

Proc Natl Acad Sci U S A, 1984 Apr, 81(7), 1966 - 70
A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA); Mizuno T et al.; The expression of the genes for the major outer membrane proteins OmpF and OmpC are osmoregulated . The ompC locus was found to be transcribed bidirectionally under conditions of high osmolarity and a 174-base transcript encoded upstream of ompC was found to inhibit the OmpF production and to substantially reduce the amount of the ompF mRNA . This RNA {mRNA-interfering complementary RNA (micRNA)} has a long sequence that is complementary to the 5' end region of the ompF mRNA . We propose that the micRNA inhibits the translation of the ompF mRNA by hybridizing with it . This RNA interaction may cause premature termination of the transcription of the ompF gene or destabilization of the ompF mRNA or both.

J Bacteriol, 1984 Apr, 158(1), 69 - 72
Conversion of D-mannitol to D-ribose: a newly discovered pathway in Escherichia coli; Rosenberg H et al.; A mutant (mtlD) strain of Escherichia coli unable to oxidize mannitol-1-phosphate to fructose-6-phosphate was used to study the fate of mannitol-1-phosphate . D-{1-14C}mannitol entered the cells via the phosphotransferase system and was phosphorylated equally at carbon 1 or 6 . The label disappeared gradually from the mannitol-1-phosphate pool, and some 60% of the 14C was recovered in nucleic acids . Ribose was isolated from the purified RNA . The 14C label distribution in the isolated ribose precluded a simple hexose-to-pentose conversion by elimination of one terminal carbon from mannitol-1-phosphate . The 14C from mannitol-1-phosphate that did not enter macromolecules was found in CO2 and in some organic, non-phosphorylated compounds that were not identified . We suggest that the de novo synthesis of mannitol-1-phosphate in E . coli may be a reaction specifically dedicated to the biosynthesis of ribose.

J Bacteriol, 1984 Apr, 158(1), 376 - 8
Mycoplasmas (Mollicutes) have a low number of rRNA genes; Amikam D et al.; DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction endonucleases was hybridized with probes consisting of portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of Mycoplasma capricolum . The results indicate the presence of only one or two sets of rRNA genes in the genome of Mollicutes linked in the procaryotic fashion, 16S-23S-5S.

J Bacteriol, 1984 Apr, 158(1), 362 - 4
Genetic screen for cloned release factor genes; Weiss RB et al.; Nonsense suppression reflects competition between a nonsense suppressor tRNA and a translational release factor . This provides a simple way to screen for release factor genes cloned into a multicopy plasmid . We have confirmed that the expected competition occurs with the gene for release factor 2, cloned by C.T . Caskey et al . (C.T . Caskey, W.C . Forrester, W . Tate, and C.D . Ward, J . Bacteriol . 158:365-368, 1984), and used it to clone the gene for release factor 1.

Arch Biochem Biophys, 1984 Apr, 230(1), 178 - 93
Relaxation time, interthiol distance, and mechanism of action of ribosomal protein S1; Odom OW et al.; The two sulfhydryl groups of ribosomal protein S1 from Escherichia coli have been labeled with fluorescent maleimides and the distance between them has been determined by nonradiative energy transfer . This distance was found to be approximately 27 A for both free S1 and S1 bound to 30 S subunits . This value probably represents an upper limit . The position of the fluorescence emission maximum indicates that both sulfhydryl groups are in a relatively hydrophobic environment . When poly(U) is added to labeled S1, either free or in 30 S subunits, the emission maximum shifts to the red by about 3 nm but without a detectable change in the interthiol distance . S1 labeled at one or both of its sulfhydryl groups retains most of its ability to enhance poly(U)-directed polyphenylalanine synthesis . About the same concentration of poly(U) is required to give the maximum shift in fluorescence as is required to give maximum polyphenylalanine synthesis, indicating that S1 binds poly(U) during translation . The peptide initiation inhibitor aurintricarboxylic acid almost completely quenches the fluorescence from either labeled sulfhydryl groups in S1 bound to ribosomes or free in solution . This quenching probably is due to energy transfer from the labeled sulfhydryls to bound aurintricarboxylic acid . Fluorescence anisotropy measurements indicated that the C-terminal domain of S1 is relatively rigid, but retains some independent movement when attached to ribosomes . The overall data are consistent with a model in which a region near the two sulfhydryl groups in the elongated C-terminal domain functions to sequester and bind mRNA to the ribosome during peptide synthesis.

Proc Soc Exp Biol Med, 1984 Apr, 175(4), 458 - 67
Surface phenotype of LPS-binding murine lymphocytes; Jacobs DM et al.; We have characterized LPS+ murine lymphocytes by determining their surface phenotype using double-labeling immunofluorescence . In the spleen, 40% of cells were LPS+mu+ (B cells), 5% were LPS+Thy 1.2+ (T cells), and 5% were LPS+ cells bearing neither mu nor Thy 1.2 (null cells) . Peripheral lymph nodes contained 11% LPS+ B cells and 2% LPS+ T cells, and mesenteric lymph nodes contained 15% LPS+ B cells and 3% LPS+ T cells . In contrast, the 4% LPS+ cells in Peyer's patches were all B cells, and the thymus contained 4% LPS+ T cells . Only the spleen contained LPS+ null cells . Within each lymphoid organ examined, the fraction of total lymphocytes identified as LPS+mu+, LPS+Ia+, or LPS+ delta + was similar, indicating that LPS+ B cells possessed the surface phenotype mu+Ia+ delta +, characteristic of a mature B cell . This conclusion was supported by the absence of LPS+mu+Ia+ delta + cells in newborn spleens . The fraction of mu+Ia+ delta + cells which also binds LPS was highest in spleen and lowest in Peyer's patches . Assuming that cells are, on the average, more mature in the progression from spleen to lymph nodes to Peyer's patches, it would appear that LPS+ cells are a less mature fraction of the mu+Ia+ delta + pool, distinguished by the presence of an LPS binding site or receptor . These data illustrate selective binding of LPS predominantly to mature B cells, but also to small numbers of null cells and T cells . The relationship of this binding to cell activation is discussed by considering the characteristics of cells which can be activated by LPS to clonal growth or differentiation under appropriate conditions.

J Pediatr, 1984 Apr, 104(4), 564 - 8
Appearance of secretory IgM and IgA antibodies to Escherichia coli in saliva during early infancy and childhood; Mellander L et al.; Unstimulated whole saliva was collected from 203 uninfected individuals at various ages from birth until adulthood . Levels of specific antibodies against Escherichia coli O antigens of secretory IgA, secretory IgM and IgG, as well as total amounts of SIgA, were determined using ELISA . Levels of SIgA antibodies found in adults were approached by the age of 12 months, but high levels could be attained earlier, presumably in response to antigenic exposure at the mucosal level . During the first few months of life, secretory IgM antibodies appeared in the saliva, possibly compensating for the relative lack of IgA.

Exp Cell Res, 1984 Apr, 151(2), 314 - 21
Cyclosporin A is a differential inhibitor of eukaryotic RNA polymerases; Brack C et al.; In order to investigate the molecular mechanism of biological action of the drug cyclosporin A (CsA), we have studied its effect on eukaryotic RNA polymerase activities in two systems: (a) in vitro transcription; (b) injection into frog oocytes . The electron microscopic transcription R-loop method {1} has been used to assay for specific transcription of an immunoglobulin gene clone in HeLa cell lysates . In this system, CsA inhibits efficiently the transcription of immunoglobulin genes . Dilution experiments show that RNA polymerase II activity is inhibited by nanomolar concentrations (i.e . amounts comparable to the ones needed for the inhibition of lymphocyte stimulation), whereas polymerase III is inhibited at micromolar concentrations . In vitro transcription with E . coli RNA polymerase is not inhibited . Parallel experiments with the mushroom toxin alpha-amanitin have shown that the eukaryotic RNA polymerase II displays similar sensitivity towards the two cyclic peptides CsA and alpha-amanitin in vitro . When CsA is injected into frog oocytes, together with a cloned Xenopus laevis U1 small nuclear RNA gene, transcription of this gene by endogenous RNA polymerase II is inhibited by 60% . RNA polymerase III and RNA polymerase I transcription is not inhibited in the oocyte . Various possibilities for the selective action of CsA on stimulated lymphocytes are discussed.

Cell, 1984 Apr, 36(4), 1089 - 95
Dispensable pieces of an aminoacyl tRNA synthetase which activate the catalytic site; Jasin M et al.; Recent data suggest that size polymorphism of aminoacyl tRNA synthetase is due to variable fusions of additional functional domains to a catalytic core so that, in a large synthetase, a substantial part of the polypeptide is dispensable for catalytic activity . We demonstrate here that a dispensable domain, joined to the catalytic core of a large synthetase, can activate the catalytic sites . This is shown by complementation of an activity-deficient mutant enzyme by protein fragments that contain internal deletions within the catalytic domain and are themselves devoid of activity . The complementation is dependent upon the presence of a defined segment of polypeptide that is remote in the sequence from the catalytic core . Substantial coupling has been established between dispensable and indispensable component pieces . This could be a mechanism to build efficiently large enzymes which integrate the catalytic sites with other previously shown functional roles.

J Gen Microbiol, 1984 Apr, 130 ( Pt 4), 951 - 7
F and type 1 piliation of Escherichia coli; Biebricher CK et al.; F and type 1 piliation of various Escherichia coli K12 Hfr and F+ strains was re-examined by using a new visualization assay . In anaerobic cultures in rich media, bacteria were well F piliated throughout all growth phases . In aerobic cultures in rich media, F piliation reached a maximum in the mid-exponential phase . The yield of pili was up to 15 mg 1(-1) . In aerated cultures, F pili disappeared in the late exponential phase . In rich media F pili were on average 10-20 micron long; in synthetic media they were an order of magnitude shorter, and less numerous . Addition of metabolic poisons (NaCN, NaN3, Na3AsO4, phenylethyl alcohol) and starving the bacteria caused rapid disappearance of F pili under aerobic conditions, but had no influence on F piliation under anaerobic conditions . Type 1 piliation was not influenced by these drugs or by an alteration of growth conditions.

J Gen Microbiol, 1984 Apr, 130 ( Pt 4), 941 - 9
Light-microscopic visualization of F and type 1 pili; Biebricher CK et al.; Methods for the direct visualization of F and type 1 pili of Escherichia coli in the light microscope are described . The method for visualizing F pili is based on the specific adsorption of fluorescent dye-labelled RNA phages to F pili . The best results were obtained with MS2 phages labelled with rhodamine B . Semi-quantitative determination of the amount of F pili is possible . Type 1 pili can be visualized rapidly and specifically by indirect immunofluorescence . Other structures on the cell surface are neither detected by, nor interfere with these assays . By using different fluorescent dyes the two methods can be combined and both F and type 1 pili can be determined in the same sample.

Proc Natl Acad Sci U S A, 1984 Apr, 81(7), 2011 - 5
Oxidative inactivation of glutamine synthetase subunits; Nakamura K et al.; Escherichia coli glutamine synthetase (GS) was inactivated by a nonenzymic mixed-function oxidation system composed of ascorbate, O2, and Fe(III) . Partial inactivation of GS by this system leads to the formation of hybrid GS molecules (dodecamers) composed of both active and inactive subunits . Subunit interactions in these hybrid molecules are weaker than in the native enzyme, as indicated by the kinetics of subunit dissociation in the presence of 4 M urea . Heterologous subunit interactions in these hybrid molecules do not affect the affinity of active subunits for glutamate . Incubation of partially adenylylated GS preparations (n = 6.7) with the ascorbate system in the absence of substrates leads to preferential oxidative inactivation of unadenylylated subunits, whereas incubation in the presence of ATP and glutamate leads to preferential inactivation of adenylylated subunits.

J Clin Microbiol, 1984 Apr, 19(4), 498 - 501
Monoclonal antibody enzyme-linked immunosorbent assay for identification of K99-positive Escherichia coli isolates from calves; Mills KW et al.; For Escherichia coli to produce diarrhea in animals it must possess the ability to attach to the epithelial cells of the intestine and to produce enterotoxins . Tests developed to differentiate pathogenic from nonpathogenic E . coli have relied on detection of adherence structures called pili or detection of the toxins . We utilized a monoclonal antibody to K99 pili in an enzyme-linked immunosorbent assay to detect the presence of K99 pili in E . coli isolated from calves . Twenty-three E . coli isolates that were known to be stable toxin positive were all shown to produce K99 pili . A 100% correlation also was shown between the presence of K99 antigen and production of stable toxin by E . coli isolates . Of the 251 isolates, 245 were negative by K99 enzyme-linked immunosorbent assay and stable toxin assay . The other six were positive on both tests . The enzyme-linked immunosorbent assay also was shown to be specific for K99 pili by antibody-blocking assays . The number of E . coli necessary for detecting K99 pili by enzyme-linked immunosorbent assay was determined to be 3.5 X 10(5) bacteria per ml.

J Med Microbiol, 1984 Apr, 17(2), 141 - 50
The role of different K antigens of Escherichia coli in phagocytosis by polymorphonuclear leukocytes; Verweij-Van Vught AM et al.; The importance of K antigens from six strains of Escherichia coli for the interaction with polymorphonuclear leukocytes (PMNL) was studied . The major factor influencing this interaction was the ability of strains to activate complement by the classical route during opsonisation, this process being reduced for most K-positive strains . Interference of K antigens with the functioning of common pili as adhesions of eukaryotic cells was not observed nor a toxic effect of K antigens on PMNL.

J Med Chem, 1984 Apr, 27(4), 429 - 32
Inhibitors of inosinic acid dehydrogenase . 2-Substituted inosinic acids; Wong CG et al.; A series of 2-substituted inosine monophosphate (IMP) and inosine derivatives were synthesized and tested for inhibitory activity against IMP dehydrogenase from Escherichia coli . All of the IMP analogues that possessed electron-withdrawing substituents on the phenyl ring of a benzylthio group placed at the 2-position of IMP showed strong inhibition, which was competitive with IMP . No evidence of hydrophobic interactions of the 2-substituent with the enzyme was observed.

Bioorg Khim, 1984 Apr, 10(4), 567 - 9
{The nature of a covalent bond between the relaxation protein and ColE1-DNA in the relaxation complex}; Zhuklis KL et al.; O-{32P}phosphoserine was found to be the only phosphoamino acid in the acid hydrolysate of the {32P}ColE1 DNA-peptide produced by action of proteases on the ColE1 DNA relaxation complex . This finding suggests that the relaxation protein is bound to ColE1 DNA in the relaxation complex via a phosphodiester linkage between a serine hydroxyl of the protein and the 5'-phosphate of the terminal deoxycytidine residue of the DNA.

J Biochem (Tokyo), 1984 Apr, 95(4), 909 - 16
The primary structure of phosphoenolpyruvate carboxylase of Escherichia coli . Nucleotide sequence of the ppc gene and deduced amino acid sequence; Fujita N et al.; The nucleotide sequence of the ppc gene, the structural gene for phosphoenolpyruvate carboxylase {EC 4.1.1.31}, of Escherichia coli K-12 was determined . The gene codes for a polypeptide comprising 883 amino acid residues with a calculated molecular weight of 99,061 . The amino acid sequence deduced from the nucleotide sequence was entirely consistent with the protein chemical data obtained with the purified enzyme, including the NH2- and COOH-terminal sequences and amino acid composition . The coding region is preceded by two putative ribosome binding sites, and is followed closely by a good representative of rho-independent terminator . The codon usage in the ppc gene suggests a moderate expression of the gene . The secondary structure of the enzyme was predicted from the deduced amino acid sequence.

Lancet, 1984 Mar 31, 1(8379), 698 - 702
Human recombinant interleukin-2 partly reconstitutes deficient in-vitro immune responses of lymphocytes from patients with AIDS; Lifson JD et al.; The lymphokine interleukin-2 is required for the development of various cell-mediated immune functions that are known to be deficient in patients with acquired immunodeficiency syndrome (AIDS) . The effects of pure human recombinant interleukin-2 (rIL-2), produced by Escherichia coli containing the cloned human gene, on in-vitro immune responses were studied in 16 patients with AIDS and 10 age-matched healthy heterosexual men . Exposure of lymphocytes from most AIDS patients to 1-100 U/ml rIL-2, increased mitogen and alloantigen induced proliferation and augmented natural killer (NK) cell function in a dose-dependent manner . NK activity was the function most consistently improved, with deficient patient responses uniformly restored to normal after incubation of effector cells with rIL-2 . Patient responsiveness to rIL-2 did not appear to depend upon the primary manifestation of disease (opportunistic infection, Kaposi's sarcoma, or both) or other clinical variables . rIL-2 also augmented the responses of lymphocytes from health subjects, but to a lesser degree . Pure rIL-2 seems capable of at least partly reconstituting some in-vitro immunological defects characteristic of AIDS . The availability of highly purified rIL-2 makes in-vivo testing feasible.

Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 926 - 32
Trp holorepressor-trp operator interaction studied by protein distribution analysis; Haydock PV et al.; Trp repressor protein of Escherichia coli (Mr 24,700) undergoes a conformational change upon interaction with L-tryptophan that enables the resulting binary complex to bind with high specificity to several operator targets in double-stranded DNA . By protein distribution analysis it was shown that a significant fraction of Trp repressor is inert in operator binding . The equilibrium dissociation constant for Trp holorepressor-Trp operator interaction is 6.7 nM at 20 degrees in 0.05M NaCl, pH 7.4 . The Trp holorepressor-trp operator complex consists of one molecule of each of the participating species, even at high molar ratios of protein to DNA.

Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 860 - 7
Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli; Trumble WR et al.; The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys148 is converted to a glycine residue . Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid . Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-beta-D-galactopyranoside affords no protection against inactivation . The findings suggest that although cys148 is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose: proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.

Science, 1984 Mar 30, 223(4643), 1412 - 4
Biological activity of recombinant human interleukin-2 produced in Escherichia coli; Rosenberg SA et al.; The gene for interleukin-2 was isolated from the Jurkat cell line and from normal peripheral blood lymphocytes and, when inserted in Escherichia coli, was expressed at high concentrations . This interleukin-2 was purified to apparent homogeneity and tested for biological activity in a variety of assays in vitro and in vivo . The recombinant lymphokine supports the growth of murine and human interleukin-2 dependent cell lines, enhances the generation of murine and human cytolytic cells in vitro, and generates lymphokine activated killer cells from murine and human lymphocytes . It has a serum half-life of 2 to 3 minutes in the mouse and significantly enhances the generation of cytolytic cells in vivo after alloimmunization . No functional differences between native and the recombinant interleukin-2 molecules have been detected.

Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 1103 - 8
Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate; Marcus F et al.; Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme . The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations . At a substrate concentration of 20 microM, 50% inhibition was observed at 4.8 microM fructose 2,6-bisphosphate . Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (Ki = 16 microM) and phosphoenolpyruvate caused release of AMP inhibition . However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate . The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate - fructose-1,6-bisphosphatase active site interaction.

Biochemistry, 1984 Mar 27, 23(7), 1468 - 73
T4 RNA ligase mediated preparation of novel "chemically misacylated" tRNAPheS; Heckler TG et al.; T4 RNA ligase was employed for the condensation of Escherichia coli tRNAPhe missing cytidine-75 and adenosine-76 (tRNAPhe-COH; the acceptor "oligomer") with each of several chemically acylated derivatives of pCpA (the donor "oligomer") . The resulting "chemically misacylated " tRNAPheS were obtained in 20-65% yields following chromatographic workup on DEAE-cellulose and benzoylated DEAE-cellulose . Characterization of the chemically misacylated tRNAs was accomplished by (i) enzymatic reaminoacylation of chemically misacylated tRNAPhe with phenylalanine by E . coli phenylalanyl-tRNA synthetase following chemical deacylation of the "incorrect" amino acid, (ii) comparison of the hydrolytic effects of Cu2+ solutions on chemically and enzymatically prepared samples of N-acetyl-L-phenylalanyl- tRNAPheS , and (iii) measurement of the chromatographic behavior of the tRNA species derived from chemical misacylation .

Biochemistry, 1984 Mar 27, 23(7), 1426 - 32
Defective proton ATPase of uncA mutants of Escherichia coli . 5'-Adenylyl imidodiphosphate binding and ATP hydrolysis; Wise JG et al.; The Escherichia coli uncA gene codes for the alpha-subunit of the F1 sector of the membrane proton ATPase . In this work purified soluble F1 enzymes from three mutant strains ( uncA401 , uncA447 , and uncA453 ) have been compared to F1 from a normal strain in respect to (a) binding of 5'-adenylyl imidodiphosphate (AMPPNP) to native enzyme in both the presence and absence of Mg, (b) high-affinity binding of MgATP to native enzyme, (c) total reloading of MgAMPPNP to nucleotide-depleted F1 preparations, (d, e) ability to hydrolyze MgATP at both high MgATP concentrations (d) (steady-state conditions) and low MgATP concentrations (e) where substrate hydrolysis occurs under nonsteady-state (" unisite ") conditions, and (f) sensitivity of steady-state ATPase activities to inhibitors of normal F1-ATPase activity . uncA mutant F1 showed normal stoichiometry of MgAMPPNP binding to both native (three sites per F1) and nucleotide-depleted preparations (six sites per F1) . Native uncA F1 preparations showed lower-than-normal affinity for MgAMPPNP and MgATP at the first site filled . Binding of AMPPNP in the absence of Mg was similar to normal, except that no increase in affinity for AMPPNP was induced by aurovertin . The uncA F1-ATPases had low but real steady-state rates of ATP hydrolysis, which were inhibited by aurovertin but relatively insensitive to inhibition by AMPPNP, efrapeptin, and sodium azide . Non-steady-state ( unisite ) ATP hydrolysis rates catalyzed at low substrate concentrations by uncA F1-ATPases were similar to normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1984 Mar 26, 12(6), 2745 - 58
Binding of Xenopus transcription factor A to 5S RNA and to single stranded DNA; Hanas JS et al.; Footprint competition assays are utilized to study the binding of Xenopus transcription factor A to a variety of single-stranded nucleic acids . The addition of Xenopus oocyte, yeast, or wheat germ 5S RNA as footprint competitors reveals that factor A binds these 5S RNAs with similar affinity . In contrast, factor A does not bind to E.coli 5S RNA or wheat germ tRNA in this assay . Factor A binding to single stranded DNA is also examined using footprint competition . Factor A binds preferentially to non-specific single stranded (M13) DNA versus double stranded (pBR322) DNA . Factor A binds equally well to single stranded DNA fragments containing either the coding or non-coding strands of the 5S RNA gene . Using single stranded M13 DNA as a competitor, the factor A-5S RNA gene complex is found to dissociate with a half-life of 5-6 min.

Nucleic Acids Res, 1984 Mar 26, 12(6), 2641 - 8
DNA replication regulated by the priming promoter; Panayotatos N; ColE1 derivative plasmids were constructed in which the natural promoter that primes replication or, in addition, the region coding for the RNA I control element had been deleted . In all of these molecules priming of the origin was effected by read-through transcription from constitutive or inducible (lacUV5) promoters inserted farther upstream . In the latter case, regulation of lac repressor activity with IPTG resulted in controlled plasmid levels in vivo . These results indicated that, at least in the absence of other control elements, regulation of the priming promoter was sufficient to control DNA replication and determine plasmid copy number.

Nucleic Acids Res, 1984 Mar 26, 12(6), 2969 - 85
The nucleotide and deduced amino acid sequences of the encephalomyocarditis viral polyprotein coding region; Palmenberg AC et al.; The nucleotide sequence of 7200 bases of encephalomyocarditis (EMC) viral RNA, including the complete polyprotein-coding region, was determined . The polyprotein is encoded within a unique translational reading frame, 6870 bases in length . Protein synthesis begins with the sequence Met-Ala-Thr, and ends with the sequence Leu-Phe-Trp, 126 bases from the 3' end of the RNA . Viral capsid and noncapsid proteins were aligned with the deduced amino acid sequence of the polyprotein . The proteolytic processing map follows the standard 4-3-4 picornaviral pattern except for a short leader peptide (8 kd), which precedes the capsid proteins . Identification of the proteolytic cleavage sites showed that EMC viral protease, p22, has cleavage specificity for gln-gly or gln-ser sequences with adjacent proline residues . The cleavage specificity of the host-coded protease(s) includes both tyr-pro and gln-gly sequences.

Nucleic Acids Res, 1984 Mar 26, 12(6), 2901 - 16
An enhancer sequence from bovine papilloma virus DNA consists of two essential regions; Weiher H et al.; A comprehensive structural analysis of an enhancer sequence from bovine papilloma virus DNA is presented based on the construction and functional analysis of 20 mutant derivatives . The results, obtained in CV-1 tissue culture cells, show that this enhancer is a small genetic element--only 40 bp in length--that contains two essential regions . The two regions exhibit homology to each other and to DNA fragments from other viral genomes that also act as enhancers in the assay used . However, there is a certain latitude in the sequences that have enhancer activity in CV-1 cells, even within the critical regions . The results are discussed with respect to the model that enhancers are binding sites for tissue specific transcription factors . The formation of Z-DNA might be involved in the enhancement process . However, single base pair transitions in an 8 base pair stretch of alternating purines and pyrimidines within the BPV enhancer which conserve this pattern destroy enhancer function.

Nucleic Acids Res, 1984 Mar 26, 12(6), 2851 - 9
The genes for the ribosomal proteins S12 and S7 are clustered with the gene for the EF-Tu protein on the chloroplast genome of Euglena gracilis; Montandon PE et al.; We characterize a DNA segment of the Euglena gracilis chloroplast DNA fragment Eco . N by nucleotide sequencing and S1 nuclease analysis . We show that this region, which is upstream of the previously sequenced tuf A gene, contains the genes for the ribosomal proteins S12 and S7 . The gene arrangement is 5'-rps 12-80 bp spacer-rps 7-174 bp spacer-tuf A, somewhat similar to the str operon of E . coli . The chloroplast S12 and S7 proteins contain 124 and 155 aminoacids, respectively, and are to 68% and 38% homologous with the corresponding E . coli proteins . The region is transcribed into a distronic mRNA of about 1.1 to 1.2 kb . The rps 12 and rps 7 genes, contrary to the tuf A gene, are not split.

J Mol Biol, 1984 Mar 25, 174(1), 113 - 20
Identification of a development-specific promoter of Myxococcus xanthus; Inouye S; In the chromosome of Myxococcus xanthus, two homologous genes for protein S, a development-specific protein, are tandemly repeated with a 1.4 X 10(3) base-pair sequence between the two genes . Two synthetic oligodeoxyribonucleotides were used as specific probes for individual transcripts from the upstream gene 1 and the downstream gene 2, respectively . The gene 2 transcript was detected only during developmental growth, while the gene 1 transcript was not detected during developmental or vegetative growth . The major initiation site for the gene 2 transcription was determined to be 51 bases upstream of the initiation codon for gene 2 . The development-specific promoter of gene 2 was identified; it shows some homologies to the Escherichia coli promoter structures.

J Biol Chem, 1984 Mar 25, 259(6), 3825 - 30
Prolipoprotein modification and processing enzymes in Escherichia coli; Tokunaga M et al.; Prolipoprotein signal peptidase, a unique endopeptidase which recognizes glycyl glyceride cysteine as a cleavage site, was characterized in an in vitro assay system using purified prolipoprotein as the substrate . This enzyme did not require phospholipids for its catalytic activity and was found to be localized in the inner cytoplasmic membrane of the Escherichia coli cell envelope . Globomycin inhibited this enzyme activity in vitro with a half-maximal inhibiting concentration of 0.76 nM . Nonionic detergent, such as Nikkol or Triton X-100, was required for the in vitro activity . The optimum pH and reaction temperature of prolipoprotein signal peptidase were pH 7.9 and 37-45 degrees C, respectively . Phosphatidylglycerol:prolipoprotein glyceryl transferase (glyceryl transferase) activity was measured using {2-3H}glycerol-labeled JE5505 cell envelope and {35S}cysteine-labeled MM18 cell envelope as the donor and acceptor of glyceryl moiety, respectively . 3H and 35S dual-labeled glyceryl cysteine was identified in the product of this enzymatic reaction . The optimal pH and reaction temperature for glyceryl transferase were pH 7.8 and 37 degrees C, respectively.

J Biol Chem, 1984 Mar 25, 259(6), 3729 - 33
Effects of mutations at glycine residues in the hydrophobic region of the Escherichia coli prolipoprotein signal peptide on the secretion across the membrane; Inouye S et al.; Each of the 2 glycine residues in the hydrophobic region of the prolipoprotein signal peptide of Escherichia coli was systematically deleted or substituted with a valine residue by oligonucleotide-directed site-specific mutagenesis . Functional analysis of four such mutants as well as four double mutants, resulting from combinations of any two of the single mutations, revealed that (a) glycine residues at positions 9 and 14 could be replaced individually or at the same time with a valine residue without affecting the secretion of prolipoprotein; (b) the deletion of glycine at position 9 had no effect on the secretion of prolipoprotein whereas, when glycine at position 14 was deleted, the glyceride modification and the processing of the mutant prolipoprotein occurred at a much slower rate at 42 degrees C than those of the wild type prolipoprotein; and (c) the effects of deleting glycine at position 14 could be suppressed by the deletion of glycine at position 9, which resulted in shortening the hydrophobic region of the prolipoprotein signal peptide by 2 amino acid residues . These results indicate that the hydrophobic region of the prolipoprotein signal peptide has remarkable flexibility in terms of the relationship between its primary structure and function in protein secretion.

J Biol Chem, 1984 Mar 25, 259(6), 3625 - 32
Differential scanning calorimetry of Cd(II) alkaline phosphatases; Roberts CH et al.; Differential scanning calorimetry has been employed to monitor structural alterations induced in the dimeric enzyme alkaline phosphatase on binding of Cd(II) (to the metal-free apoenzyme) and phosphate (Pi) (to the Cd(II) enzyme) . Cd(II) addition to the apoenzyme at pH 6.5 results in an increased transition temperature, suggesting a stabilizing effect of the bound metal ion . Two distinct structural forms of the protein are detected as discrete calorimetric transitions (Tm = 69-84 degrees C; 87-94 degrees C, respectively) . Distribution of the enzyme between these forms is found to depend on the exogenous Cd(II) concentration and the protocol of Cd(II) addition . These results indicate that conversion between the conformational forms is a slow process which appears to require specific levels of metal ion site occupancy . These studies, in which the exogenous Cd(II) concentration was varied from 10(-5) M to 10(-3) M suggest a structural basis for previously observed hysteretic phenomena observed on Cd(II) binding to the enzyme . Even at a minimum stoichiometry of Cd(II) (2 eq/mol of dimer) a single equivalent of Pi is sufficient to accelerate assumption of a stabilized form of the protein (Tm = 90 degrees C) . This is followed by a slow structural change paralleling the time course of formation of the functional 2 Cd(II) phosphoryl enzyme which displays two calorimetric transitions (Tm = 65 degrees C, 88 degrees C) . The low temperature transition does not appear if Pi is initially present at millimolar concentrations and is abolished on addition of Pi at concentrations in excess of 0.1 mM . These observations suggest the presence of a second, distinct Pi binding site on the 2 Cd(II) phosphoryl enzyme . This is supported by the changes observed in the 31P NMR chemical shift of Pi added to comparable enzyme samples . These data, including assessment of the effect of the presence of Mg(II), are discussed in terms of the mechanism of metal ion association to the enzyme and rearrangement of bound metal ions induced by Pi binding.

Science, 1984 Mar 23, 223(4642), 1299 - 301
Total synthesis and cloning of a gene coding for the ribonuclease S protein; Nambiar KP et al.; A gene for ribonuclease S protein, has been chemically synthesized and cloned . The gene is designed to have 25 specific restriction endonuclease sites spaced at short intervals, permitting its structure to be rapidly modified . This flexibility facilitates tests of hypotheses relating the primary structure of the enzyme to its physical and catalytic behavior.

Med J Aust, 1984 Mar 17, 140(6), 332 - 6
Acute obstructive suppurative cholangitis; Pillay SP et al.; Acute obstructive suppurative cholangitis is an uncommon condition which, if unrecognized, carries a high rate of mortality . This study of 12 patients highlights the clinical features as well as the poor outcome of delayed surgical decompression . Endoscopic and percutaneous drainage of the obstructed biliary tree in acutely ill patients may improve the results of subsequent surgery.

Carbohydr Res, 1984 Mar 15, 126(2), 249 - 59
Structural studies of the O-specific side chain of the lipopolysaccharide from Escherichia coli O:7; L'vov VL et al.; The structure of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O:7 has been investigated, using n.m.r . spectroscopy, methylation analysis, partial hydrolysis, and Smith degradation as the principal methods . It is concluded that the polysaccharide is constructed of repeating pentasaccharide units having the structure (formula; see text) where D-QuipNAc stands for 4-acetamido-4,6-dideoxy-D-glucopyranose . The 13C-n.m.r . spectrum of the polysaccharide has been interpreted completely.

J Mol Biol, 1984 Mar 15, 173(4), 437 - 61
Multiple IS10 rearrangements in Escherichia coli; Raleigh EA et al.; We have investigated the occurrence of multiple transposon-promoted chromosomal rearrangements in Escherichia coli K12 strains containing transposon Tn10 . We show that a single Tn10 element, with its two closely spaced insertion sequence (IS10) elements, frequently gives rise to complex rearrangements that can be accounted for as the sum of two "classical" IS10 events . Using a strain containing differentially marked Tn10 elements at widely separated locations, we have investigated the possibility that IS10-promoted rearrangements occur in cell-wide "bursts", as expected if cells could occasionally undergo brief periods when all IS10 transposition events were activated, interspersed with longer periods of relative quiescence . We find no evidence for strong (greater than 60-fold), periodic cell-wide activation under our experimental conditions . The sensitivity of this experiment has been evaluated using an expression for the accumulation of double mutations in populations with heterogeneous, fluctuating mutation rates (see Appendix) . We discuss several mechanisms by which two closely linked IS10 elements could undergo coupled double events without cell-wide activation: local activation of small chromosomal regions, periodic bursts of synthesis of cis-acting transposase protein, and/or a propensity for elements that have actually engaged in one rearrangement event to initiate a second successive event immediately thereafter . We favor the last possibility.

Eur J Biochem, 1984 Mar 15, 139(3), 541 - 5
Involvement of the 3' side of the anticodon loop of yeast tRNATyr in messenger-free binding to ribosomes . An electron-spin resonance study; Weygand-Durasevic I et al.; Electron-spin resonance (ESR) spectra of a nitroxide spin-label attached to residue i6A-37 of yeast tRNATyr were measured in complexes of deacylated tRNATyr with Escherichia coli ribosomes . A Scatchard plot, obtained in the absence of mRNA, indicated strong binding with an association constant of 1 X 10(7) l X mol-1, suggesting the P-site binding . The ESR spectrum of free tRNATyr, characteristic for a rapidly tumbling nitroxide, changes to a spectrum with extensively broadened lines in the ribosome-tRNA complex . The original spectrum can be restored upon long incubations of the complex with an excess of extraneous tRNA . ESR spectra suggest that the spin-label motion is drastically perturbed though not completely blocked in the ribosome-tRNATyr complex . Since ESR spectra of a spin-label attached to the opposite, i.e . 5', side of the anticodon loop are only slightly perturbed by the messenger-free binding to ribosomes {Rodriguez et al . (1980) J . Biol . Chem . 255, 8116-8120}, it is concluded that the two sides of the anticodon loop face entirely different environments when bound to the P site, the 3' side being oriented towards the surface of the ribosome, and the other side towards its environment or a large cavity.

Eur J Biochem, 1984 Mar 15, 139(3), 547 - 52
Metabolism of tRNA in near-ultraviolet-illuminated Escherichia coli . The tRNA repair hypothesis; Blanchetot A et al.; The relA+-dependent stringent response is an important component of the mechanism of the near-ultraviolet-induced growth delay . However, the behaviour of the intracellular level of ppGpp is unexpected {Thomas et al . (1981) Eur . J . Biochem . 118, 381-387} and this led us to examine the metabolism of tRNAs during the illumination period and the growth lag that follows . Analysis of the gel electrophoresis migration profiles of tRNA molecules, synthesized prior to the illumination period, provides no evidence for tRNA degradation . Rather, it is suggestive of the rearrangement of some cross-linked tRNA species during the growth lag . By the same technique the neosynthesis of one or several tRNA species escaping the stringent response could be ruled out at the beginning of the growth lag . The behaviour of the cross-linked tRNAs was followed by a chromatographic procedure allowing the quantitative evaluation of the 8-13 link present in vivo . Upon illumination of growing cells, one observes an initial linear increase of the 8-13 link content . Unexpectedly this is followed during the illumination period by an abrupt decrease . The 8-13 link content then remains stable . The data above suggest that part of the 8-13 link (25-40%) is eliminated from tRNA without degradation of the molecules involved . A tRNA repair hypothesis is proposed: elimination of the 8-13 link would occur by scission of the N1-C1' glycosidic bonds at positions 8 and 13 of tRNA . It would be followed by reinsertion of uracil and cytosine in their respective positions.

Biochemistry, 1984 Mar 13, 23(6), 1269 - 74
Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a radiolabeled bifunctional arsenoxide: evidence for an essential histidine residue at the active site of lipoamide dehydrogenase; Adamson SR et al.; Incubation of pyruvate dehydrogenase multienzyme complex (PD complex) from Escherichia coli with thiamin pyrophosphate, pyruvate, coenzyme A, Mg2+, and the radiolabeled bifunctional arsenoxide p-{(bromoacetyl)-amino}phenyl arsenoxide (BrCH214CONHPhAsO) led to the irreversible loss of lipoamide dehydrogenase (E3) activity . The mode of inactivation occurred by initial "anchoring" of the reagent via its -AsO group to reduced lipoyl residues on lipoate acetyltransferase (E2) (generated by substrates) followed by the delivery of the BrCH214CO- moiety into the active site of E3 where an irreversible alkylation ensued {Stevenson, K . J., Hale, G., & Perham, R . N . (1978) Biochemistry 17, 2189} . To account for nonspecific alkylations, not mediated by this delivery process, control experiments were conducted in which the radiolabeled bifunctional reagent was incubated with PD complex in the absence of substrates . E3 subunits were isolated from inhibited and control PD complexes by chromatography on hydroxylapatite in the presence of 8 M urea . Acid hydrolysis of the alkylated E3 and control E3 samples produced radiolabeled carboxymethylated amino acids that were identified and quantitated by high-voltage electrophoresis and amino acid/radiochemical analysis . The inhibited sample contained N3-(carboxymethyl)histidine and a small amount of S-(carboxymethyl)cysteine . These residues were not present in significant amounts in the controls . The loss of 81% of E3 activity correlated with the alkylation of about 0.7 residue of histidine and 0.1 residue of cysteine per mol of E3.

Biochemistry, 1984 Mar 13, 23(6), 1047 - 51
Function of the repeating homologous sequences in nucleic acid binding domain of ribosomal protein S1; Suryanarayana T et al.; Ribosomal protein S1 contains in its RNA binding domain four repeating, homologous stretches of sequences . Its functionally active mutant form m1-S1 {Subramanian, A.R., & Mizushima, S . (1979) J . Biol . Chem . 254, 4309} contains only three repeating stretches . In order to assess the functional importance of this repeating sequence, we cleaved S1 at its reactive SH group on Cys-349 and isolated a fragment (S1-F4) that has lost two of the homologous stretches but retains all other essential elements . We find that ribosomes reconstituted with S1-F4 instead of S1 are functionally active in translating poly(U) and poly(A) but totally inactive in translating phage MS2 RNA . The significance of this result is discussed vis-a-vis the initiation step in translating natural mRNA, and a functional role for the tetrarepeat of S1 is suggested.

Nucleic Acids Res, 1984 Mar 12, 12(5), 2243 - 58
The effect of nucleotide analogs on cell-free gene expression; Zimmer M et al.; The effects of the following pyrimidine nucleoside 5'-triphosphates: f5 UTP, br5 UTP, rTTP, s2 UTP, s4 UTP and s2 CTP on cell-free expression of the beta-galactosidase gene in lambda h80dlac DNA as well as the galactokinase gene in plasmid 01-14 were investigated . Only rTTP could substitute UTP in cell-free gene expression without restriction . Combinations of the other analogs with their respective natural congeners led to inhibition of gene expression . All analogs were found to inhibit transcription . Whereas br5 UTP and s4 UTP did not affect translation, mRNA containing s2 UMP or s2 CMP residues respectively was found to function poorly in translation . Only in the case of f5 UTP could ambiguitive behaviour be demonstrated . Whether mispairing of f5 UMP residues, responsible for this ambiguity takes place in transcription or in translation, could not be decided.

Nucleic Acids Res, 1984 Mar 12, 12(5), 2499 - 508
The modelling of the decoding site of the Escherichia coli ribosome; Sarapuu T et al.; Individual ribosomal proteins S4, S9 and S13 were tested for their ability to interact with tRNA and synthetic polynucleotides . All three proteins bind to immobilized to Sepharose poly(A) and poly(U), while S4 and S13 form stoichiometric (1:1) complexes with tRNA in solution . We show that only the polynucleotide X S13 complexes are able to select their cognate tRNAs . In particular, the affinity of tRNAPhe to the binary poly(U) X S13 complex is about three orders of magnitude higher than that for poly(U) alone.

FEBS Lett, 1984 Mar 12, 168(1), 166 - 8
Does the DNA methylase Eco dam pair nucleotide sequences to form site-specific duplexes?
Buryanov YaI, Zinoviev VV, Vienozhinskis MT, Malygin EG, Nesterenko VF, Popov SG, Gorbunov YuA.
The Eco dam methylase is active on denatured DNA and single-stranded synthetic oligonucleotides containing GATC sites . The results suggest that on interaction with single-stranded oligonucleotides the Eco dam methylase is able to form a duplex structure within the GATC site, and that this duplex site is a substrate for enzyme.

Nucleic Acids Res, 1984 Mar 12, 12(5), 2461 - 72
The complete nucleotide sequence of the RNA coding for the primary translation product of foot and mouth disease virus; Carroll AR et al.; The complete nucleotide sequence of the coding region of foot and mouth disease virus RNA (strain A1061) is presented . The sequence extends from the primary initiation site, approximately 1200 nucleotide from the 5' end of the genome, in an open translational reading frame of 6,999 nucleotides to a termination codon 93 nucleotides from the 3' terminal poly (A) . Available amino acid sequence data correlates with that predicted from the nucleotide sequence . The amino acid sequence around cleavage sites in the polyprotein shows no consistency, although a number of the virus-coded protease cleavage sites are between glutamate and glycine residues.

Nucleic Acids Res, 1984 Mar 12, 12(5), 2421 - 6
Physical mapping of the ribosomal RNA genes of Mycoplasma capricolum; Glaser G et al.; Physical mapping of the rRNA genes of Mycoplasma capricolum was done by digestion of the mycoplasmal DNA with EcoRI, PstI and BglII and hybridization with nick-translated probes consisting of defined portions of the rrnB ribosomal RNA operon of Escherichia coli . The results indicate that the rRNA genes in the chromosome of M . capricolum are arranged in two clusters, each organized in the order 5'-16S-23S-5S-3', resembling the order of the genes in the rrnB operon, with no large spacer regions separating the genes in each cluster.

J Biol Chem, 1984 Mar 10, 259(5), 3368 - 74
Terminal oxidases of Escherichia coli aerobic respiratory chain . I . Purification and properties of cytochrome b562-o complex from cells in the early exponential phase of aerobic growth; Kita K et al.; Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli K12, was isolated in a highly purified form . The purified oxidase is composed of equimolar amounts of two polypeptides, with Mr = 33,000 and 55,000, determined by gel electrophoresis in the presence of sodium dodecyl sulfate . It contains 19.5 nmol of heme and 16.8 nmol of copper/mg of protein, but no detectable nonheme iron, phospholipid, ubiquinone, or menaquinone . In the difference spectrum at room temperature, the oxidase shows a single alpha absorption peak at 560 nm and at 77 K it shows two alpha absorption peaks at 555 and 562 nm . This oxidase combines with CO and the CO difference spectrum at room temperature has a peak at 416 nm and a trough at 430 nm in the Soret region . Its oxidation-reduction potential is estimated to be 125 mV (pH 7.4) and it is pH-dependent (-60 mV/pH) in medium of pH 6.0 to 7.4 . It catalyzes electron transport to oxygen via ubiquinol and ascorbate in the presence of phenazine methosulfate or N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride . This oxidase activity depends on phospholipids and is sensitive to respiratory inhibitors, such as 2-heptyl-4-hydroxyquinoline N-oxide, piericidin A, KCN and NaN3 . The divalent cations Zn2+, Cd2+, and Co2+ inhibit the oxidase activity extensively . The oxidase activity of the cytochrome b562-o complex was inhibited by photoinactivation with rose bengal, suggesting that the inhibition by zinc ion results from modification of a histidine residue of cytochrome o.

J Biol Chem, 1984 Mar 10, 259(5), 2905 - 9
Escherichia coli pyruvate dehydrogenase complex . Thiamin pyrophosphate-dependent inactivation by 3-bromopyruvate; Apfel MA et al.; Inactivation of the pyruvate dehydrogenase complex by 3-bromopyruvate is thiamin pyrophosphate (TPP)-dependent . Inactivation with 2-14C- or 3-14C-labeled 3-bromopyruvate results in TPP-dependent covalent labeling of more than 60 sites in the complex, all of which are associated with the dihydrolipoyl transacetylase component . Inactivation by 3-bromo{1-14C}pyruvate labels up to 20 sites associated with dihydrolipoyl transacetylase, also with TPP dependence . Systemic chemical degradation of the complex inactivated by 3-bromo{2-14C}pyruvate under conditions that would convert lipoyl groups to S,S,-biscarboxymethyl dihydrolipoic acid produces S,S,-bis{14C}carboxymethyl dihydrolipoic acid . It is concluded that 3-bromopyruvate inactivates this complex by initially undergoing the first two steps of the usual catalytic pathway, TPP-dependent decarboxylation followed by reductive bromoacetylation of lipoyl moieties . The sulfhydryl groups of S-bromoacetyl dihydrolipoyl moieties generated by reductive bromoacetylation are then alkylated by 3-bromopyruvate as well as by bromoacetyl thioester groups associated with the complex.

J Biol Chem, 1984 Mar 10, 259(5), 2798 - 802
The conserved 5 S rRNA complement to tRNA is not required for translation of natural mRNA; Zagorska L et al.; We have tested a putative base-paired interaction between the conserved GT psi C sequence of tRNA and the conserved GAAC47 sequence of 5 S ribosomal RNA by in vitro protein synthesis using ribosomes containing deletions in this region of 5 S rRNA . Ribosomes reconstituted with 5 S rRNA possessing a single break between residues 41 and 42, deletion of residues 42-46, or deletion of residues 42-52 were tested for their ability to translate phage MS2 RNA . Initiator tRNA binding, aminoacyl-tRNA binding, ppGpp synthesis, and miscoding were also tested . All of the measured functions could be carried out by ribosomes carrying the deleted 5 S rRNAs . The sizes and relative amounts of the polypeptides synthesized by MS2 RNA-programmed ribosomes were identical whether or not the 5 S RNA contained deletions . Aminoacyl-tRNA binding and miscoding were essentially unaffected . Significant reduction in ApUpG (but not poly(A,U,G) or MS2 RNA)-directed fMet-tRNA binding and ppGpp synthesis were observed, particularly in the case of the larger (residues 42-52) deletion . We conclude that if tRNA and 5 S rRNA interact in this fashion, it is not an obligatory step in protein synthesis.

J Biol Chem, 1984 Mar 10, 259(5), 2734 - 41
Purification and characterization of succinyl-CoA: tetrahydrodipicolinate N-succinyltransferase from Escherichia coli; Simms SA et al.; Tetrahydrodipicolinate succinylase, an enzyme involved in the diaminopimelate-lysine pathway, was purified 1900-fold from crude extracts of Escherichia coli . The enzyme catalyzes the formation of CoA and N-succinyl-2-amino-6-keto-L-pimelate from succinyl-CoA and tetrahydrodipicolinate . The purified enzyme was shown to be homogeneous by polyacrylamide gel electrophoresis . The Stokes radius of the enzyme was determined from its elution volume on a Sephacryl S300 column and its sedimentation constant from sucrose density gradient centrifugation . These were 35 A and 4.7 (S20,w), respectively . The enzyme consists of two subunits each with a mass of 31,000 daltons, as determined using sodium dodecyl sulfate/polyacrylamide gel electrophoresis . Tetrahydrodipicolinate succinylase was shown to be a sulfhydryl enzyme . It has a pH optimum of 8.2 . The equilibrium lies predominantly in favor of product formation but the reverse reaction can be demonstrated in vitro.

J Biol Chem, 1984 Mar 10, 259(5), 3375 - 81
Terminal oxidases of Escherichia coli aerobic respiratory chain . II . Purification and properties of cytochrome b558-d complex from cells grown with limited oxygen and evidence of branched electron-carrying systems; Kita K et al.; Cytochrome b558-d complex, a terminal oxidase in the respiratory chain of Escherichia coli K12 grown under the condition of limited oxygen, was purified to near homogeneity . The purified oxidase complex is composed of equimolar amounts of two polypeptides, with Mr = 26,000 and 51,000, determined with gel electrophoresis in the presence of sodium dodecyl sulfate . It contains 12.3 nmol of protoheme, 9.54 nmol of cytochrome d, and 26.6 nmol of iron/mg of protein . The enzyme is a "cytochrome bd-type oxidase," which shows absorption peaks at 558 and 624 nm in the difference spectrum at 77 K . This oxidase combines with CO, and the Soret region of the CO difference spectrum at room temperature has a peak at 420 nm and troughs at 430 and 442 nm . The oxidation-reduction potentials of cytochrome b558 and cytochrome d at pH 7.4 were estimated to be +10 mV and +240 mV, respectively . The cytochrome b558-d complex catalyzes the oxidation of ubiquinol-1, menadiol, and ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride . This oxidase activity was inhibited by the respiratory inhibitors piericidin A, KCN, and NaN3, but the cytochrome b558-d complex was less sensitive to the inhibitors than the cytochrome b562-o complex . The Km values for oxygen of purified cytochrome b558-d complex and cytochrome b562-o complex were determined to be 0.38 and 2.9 microM, respectively . Formation of a membrane potential by the reconstituted cytochrome b558-d complex in liposomes was observed with the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide on addition of ubiquinol-1 . This is the first indication that there is a coupling site in the terminal oxidase, which contains cytochrome d . Steady state kinetics of cytochromes in the membrane showed that these oxidase complexes branch at the site of ubiquinone-8 in the respiratory chain . From these and previous results, branched electron-carrying systems of the E . coli respiratory chain are proposed.

J Biol Chem, 1984 Mar 10, 259(5), 3202 - 9
Site specific deletions of regulatory sequences in a ribosomal protein-RNA polymerase operon in Escherichia coli . Effects on beta and beta' gene expression; Dennis PP; The 319 nucleotide long intergenic region between the rplL (L12) and the rpoB (beta) genes of the L10 operon contains a transcription attenuation sequence and a RNase III mRNA processing sequence . Four site specific deletions located within this intergenic space which remove either the transcription attenuation sequence or the RNase III mRNA processing sequence or both sequences have been isolated on recombinant DNA plasmids carrying this operon . Deletions of sequences surrounding the RNase III processing site result in a uniform 80-90% reduction in the translational efficiency of beta subunit mRNA . This reduction in translation efficiency appears not to be related to processing per se; transcription of the rpoB and rpoC genes and the translation efficiency of the respective mRNA sequences were indistinguishable in an RNase III processing defective mutant (rnc) and its isogenic parent (rnc+) . Deletions of the attenuator sequence result in a substantial increase in the downstream transcription of the beta subunit gene . The translational efficiency of RNase III processed beta subunit mRNA was found to be related in an inverse manner to the level of beta subunit synthesis . These result suggest that sequences on the mRNA in the vicinity of the RNase III processing site (i) are essential for efficient translation of beta subunit mRNA and (ii) are utilized for reducing the translational efficiency of the beta subunit mRNA when the beta subunit protein is produced in excess of that required for RNA polymerase assembly.

J Biol Chem, 1984 Mar 10, 259(5), 2803 - 9
Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation; Tanaka K et al.; Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation . Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts . At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of {3H}methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP . This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate . Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates . When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate . In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation . In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs . This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells . Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes . However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes.

J Biol Chem, 1984 Mar 10, 259(5), 3293 - 8
Dihydroorotase from Escherichia coli . Purification and characterization; Washabaugh MW et al.; Dihydroorotase (4,5-L-dihydroorotate amidohydrolase (EC 3.5.2.3}, which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to dihydro-L-orotate, has been purified to homogeneity from an over-producing strain of Escherichia coli . Treatment of 70 g of frozen cell paste produces about 7 mg of pure enzyme, a yield of about 35% . The native molecular weight, determined by equilibrium sedimentation, is 80,900 +/- 4,300 . The subunit molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 38,400 +/- 2,600, and by amino acid analysis is 41,000 . The enzyme is thus a dimer and contains 0.95 +/- 0.08 tightly bound zinc atoms per subunit when isolated by the described procedure, which would remove any loosely bound metal ions . Isoelectric focusing under native conditions yields a major species at isoelectric point 4.97 +/- 0.27 and a minor species at 5.26 +/- 0.27; dihydroorotase activity is proportionately associated with both bands . The enzyme has a partial specific volume of 0.737 ml/g calculated from the amino acid composition and a specific absorption at 278 nm of 0.638 for a 1 mg/ml solution . At 30 degrees C, the Michaelis constant and kcat for dihydro-DL-orotate (at pH 8.0) are 0.0756 mM and 127 s-1, respectively; for N-carbamyl-DL-aspartate (at pH 5.80), they are 1.07 mM and 195 s-1.

Nature, 1984 Mar 8-14, 308(5955), 201 - 3
Mechanism of mutagenesis by O6-methylguanine; Eadie JS et al.; O6-methylguanine (O6meG) lesions of double-stranded DNA have been associated with mutation and neoplastic transformation . These lesions can, in principle, be produced by at least three different mechanisms: direct alkylation of G X C base pairs in double-stranded DNA; alkylation of guanine residues in single-stranded regions of DNA associated with replication forks; and alkylation of the DNA precursor pool followed by incorporation of O6-methyl deoxyguanosine triphosphate (O6-medGTP) during DNA replication . DNA biosynthesis subsequent to all three events will generate predominantly O6-meG X T base pairs as O6meG preferentially pairs with T . We show here that O6meG X T base pairs are mutagenic; that transalkylase repair has a direct role in the generation of mutations induced by alkylated pool nucleotides; and that the Escherichia coli mismatch repair system is capable of repairing mutagenic G X T intermediates.

J Mol Biol, 1984 Mar 5, 173(3), 293 - 305
Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light; Wood RD et al.; Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells . Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA . Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions . For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst . From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication . A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells . In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced . We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication . The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.

Plasmid, 1984 Mar, 11(2), 166 - 77
A mathematical model for lambda dv plasmid replication: analysis of copy number mutants; Lee SB et al.; A mathematical model based on the molecular control mechanisms for lambda dv plasmid replication in a single Escherichia coli cell has been applied to simulate replication of mutant lambda dv plasmids . Model simulations of changes in repressor level and copy number resulting from mutations in the promoter-operator PROR region are consistent with experimental data . Calculated effects on lambda dv plasmid copy number of oligomer formation and of alternations in termination efficiency at tR1 also agree with experiment . The model has been employed to simulate the influence of cro mutants and of cro and tR1 double mutants on copy number and stable maintenance of lambda dv plasmid copy number . The genetic structure included in formulation of the replicon model provides a framework for relating changes in specific genetic loci on the plasmid with resulting alterations in host-plasmid system function.

Plasmid, 1984 Mar, 11(2), 151 - 65
A mathematical model for lambda dv plasmid replication: analysis of wild-type plasmid; Lee SB et al.; A mathematical model for lambda dv plasmid replication in a growing single cell of Escherichia coli has been formulated and solved numerically . Quantitative description of the molecular control mechanism for initiation of lambda dv replication presumes regulatory functions of repressor and initiator proteins and transcriptional activation of the origin region . Random selection of a single plasmid for activation and replication is assumed, as is regular plasmid segregation to daughter cells . The model is capable of simulating the periodic changes in each regulatory element and the plasmid copy number during the cell cycle . The calculated average copy number, repressor concentration, and timing of plasmid replication agree well with experimental data . The simulated lambda dv plasmid replication rate is controlled primarily by transcription frequency . Initiation of plasmid replication is not related to variations in the levels of repressor or initiator proteins during the cell cycle . Simulation studies of perturbations in plasmid and repressor segregation indicate that replication regulation of the lambda dv plasmid compensates to readjust copy number to normal values in a few generations . Implications of these studies relative to the molecular mechanisms of replication control are discussed.

Plasmid, 1984 Mar, 11(2), 141 - 50
Sequence analysis of the maize mitochondrial 26 S rRNA gene and flanking regions; Dale RM et al.; A single copy of the large ribosomal 26 S rRNA gene is found in the maize mitochondrial genome . The sequence of this gene and the flanking regions has been determined using the M13 dideoxy sequencing method . The maize mt 26 S rDNA shares a high degree of homology with the Escherichia coli 23 S rDNA, and the approximate 5' and 3' ends of the maize 26 S rDNA have been located by comparison with the E . coli sequence . The maize mt 26 S rDNA has also been compared with the sequences of the maize chloroplast 23 S rDNA, the human mitochondrial 16 S rDNA, part of the yeast mitochondrial 21 S rDNA, and the yeast cytoplasmic 25 S rDNA . In all cases, there are numerous regions of 70% or higher homology.

Hoppe Seylers Z Physiol Chem, 1984 Mar, 365(3), 289 - 96
Purification and properties of phenylalanyl-tRNA synthetase from a higher plant (Phaseolus vulgaris); Rauhut R et al.; Phenylalanyl-tRNA synthetase from beans (Phaseolus vulgaris) was purified 2 800-fold to homogeneity with a 16% overall yield by salting-out chromatography, salting-out affinity chromatography, gel filtration and chromatography on DEAE-cellulose and hydroxylapatite . This combination minimizes potentially harmful effects of proteinases and products of the secondary metabolism of a green plant during the early steps . The molecular mass is 260 000 Da with a subunit structure of alpha 2 beta 2 (alpha = 59 000, beta = 70 000 Da) . Enzymatic activity was optimal with 20mM Mg2+ and 10mM KCl at pH 6.5 and pH 8.5, depending on the buffer substance . Kinetic measurements at low temperature and steady-state kinetics indicate that the esterification of tRNA or a step preceding it, but not the activation, are rate-determining at pH 7.65 . The cognate tRNAPhe is exclusively aminoacylated at the 2'-OH group . tRNAs from Escherichia coli and bean chloroplasts are not aminoacylated . No immunological relationship of the plant enzyme to other phenylalanyl-tRNA synthetases was revealed by immuno-diffusion and immunotitration with polyclonal antibodies raised against the enzymes from E . coli, yeast and hen liver . ATP analogs revealed a unique pattern of substrate properties with indication of conservation of ATP binding in the form of an ATP-Mg2+ complex in the anti-conformation with a coordination of the cation to the nitrogen in position 7 of the purine moiety.

Cancer Res, 1984 Mar, 44(3), 1044 - 7
Augmentation of natural killer cell activity by lipopolysaccharide through separable effects on the binding of nonadherent lymphocytes to tumor targets and tumor killing; Salata RA et al.; A single-cell assay was utilized to study the augmentation by Escherichia coli lipopolysaccharide (LPS) of the cytotoxicity of human lymphocytes for the human myeloid tumor K562 . Preincubation with LPS at 20 micrograms/ml for 30 min at 37 degrees increased the binding of all nonadherent (NA) lymphocyte populations to K562 tumors {unseparated NA lymphocytes from 13.1 to 25.1%, immunoglobulin G Fc receptor-enriched lymphocytes from 27.6 to 42.9%, and immunoglobulin G Fc receptor-depleted lymphocytes from 14.0 to 23.7%, at p less than 0.001} . In contrast, interferon (IFN) at 10 units/ml had no effect on the overall binding of lymphocytes to K562 tumors . When lymphocyte-tumor conjugates were dispersed in agarose, cytotoxic activity of unseparated NA lymphocytes at 1 to 3 hr was markedly increased by preincubation with LPS (p less than 0.001) . However, LPS did not enhance cytotoxicity if conjugates were formed in its absence . IFN, likewise, increased cytotoxic activity in unseparated NA lymphocytes at 1 to 3 hr (p less than 0.001) . No synergistic cytotoxicity was seen with concurrent exposure to LPS and IFN . LPS increased cytotoxicity in the Fc receptor-enriched:tumor conjugates at 1 to 3 hr (p less than 0.001) and appeared to promote more efficient killing in individual conjugates over time . Cytotoxicity in the Fc receptor-depleted:tumor conjugates was not enhanced by LPS . Thus, LPS may enhance natural killer cell-like activity by increasing the binding of human lymphocytes to K562 tumors and by rearranging the population of binding cells to include more efficient killer cells . While the effects of LPS on binding appear independent of IFN, selective recruitment of more efficient killer cells could be through an IFN mechanism.

Mikrobiologiia, 1984 Mar-Apr, 53(2), 285 - 9
{Expression of the resistance genes of plasmid RP4 in Escherichia coli cells grown by continuous cultivation}; Filonov AE et al.; The resistance to tetracycline decreased in Escherichia coli C600 cells containing plasmid RP4 and grown under the conditions of continuous cultivation . The population of cells containing plasmid RP4 is heterogeneous in the trait of tetracycline resistance, and most cells cannot grow in a selective medium with tetracycline at a concentration of 20 micrograms/ml . The decreased resistance to tetracycline was most pronounced for a glucose-limited chemostat culture and also in the presence of two plasmids, RP4 and pBS94 , in the cells . No decrease was found in the resistance to other drugs determined by plasmid genes.

J Gen Microbiol, 1984 Mar, 130 ( Pt 3), 687 - 92
Genetic and structural evidence for the presence of propanediol oxidoreductase isoenzymes in Escherichia coli; Ros J et al.; The synthesis of propanediol oxidoreductase, an enzyme permitting the anaerobic metabolism of fucose and rhamnose, has been described as being controlled by the prd locus closely linked to the fuc locus in wild-type cells of Escherichia coli . However, strain AA-787, deleted in the fuc and prd loci, grew anaerobically on rhamnose, displaying propanediol oxidoreductase activity . From the deleted strain we derived a constitutive producer of propanediol oxidoreductase able to grow on 1,2-propanediol by oxidizing the diol to lactaldehyde which was further metabolized to lactate . Transduction experiments showed that this ability to use propanediol was closely linked to the rha locus . Peptide mapping of fucose- and rhamnose-induced propanediol oxidoreductase of wild-type cells established structural differences between the two enzymes, indicating two structural genes, one for each sugar metabolizing system.

Can J Microbiol, 1984 Mar, 30(3), 411 - 5
Escherichia coli growth on lactose requires cycling of beta-galactosidase products into the medium; Huber RE et al.; Growth on lactose was found to be restricted in an Escherichia coli strain deficient in its ability to transport glucose and galactose . If the latter sugars were removed from the medium as they were being produced, a wild-type strain grew only poorly, while the transport-deficient strain did not grow at all . These results suggested that all of the products of beta-galactosidase action on lactose are released into the medium before being metabolized . This contention was strongly supported by the finding that the appearance of products in the medium was equal to lactose disappearance at three limiting lactose concentrations and by an experiment which showed that essentially all of the label from added lactose ( {1-14C}glucose) was found in the medium as glucose when chased with unlabelled lactose.

Can J Microbiol, 1984 Mar, 30(3), 339 - 44
Involvement of outer membrane proteins in freeze--thaw resistance of Escherichia coli; Calcott PH et al.; Two families of Escherichia coli mutants with altered outer membrane protein components were examined for sensitivity to freezing and thawing and other stresses . A mutant unable to make the lipoprotein (lpo) was extremely sensitive to freezing and thawing in water or saline and to challenge with detergent, while the mutant unable to make the porin proteins (ompB) was more resistant than the isogenic wild type; strains unable to make the tsx and ompA proteins were slightly more sensitive to the stresses . Similarly, the lpo deficient strain exhibited more and the ompB less wall and membrane damage than the wild-type strains . Little difference in the extent of wall damage, but more membrane damage, was seen for the two tsx and the ompA strains when compared with the wild-type strain . The roles of the specific proteins in determining sensitivity to freeze-thaw are discussed.

Vestn Khir Im I I Grek, 1984 Mar, 132(3), 95 - 6
{Use of CO2 laser for the prevention of postoperative wound suppuration}; Skobelkin OK et al.; The experiments with laboratory animals have convincingly shown that CO2-laser can be successfully used for prophylactics of suppurations of postoperative wounds in clinics.

EMBO J, 1984 Mar, 3(3), 623 - 9
Mutagenesis of the three bases preceding the start codon of the beta-galactosidase mRNA and its effect on translation in Escherichia coli; Hui A et al.; The effect on the translation efficiency of various mutations in the three bases (the -1 triplet) that precede the AUG start codon of the beta-galactosidase mRNA in Escherichia coli was studied . Of the 39 mutants examined, the level of expression varies over a 20-fold range . The most favorable combinations of bases in the -1 triplet are UAU and CUU . The expression levels in the mutants with UUC, UCA or AGG as the -1 triplet are 20-fold lower than those with UAU or CUU . In general, a U residue immediately preceding the start codon is more favorable for expression than any other base; furthermore, an A residue at the -2 position enhances the translation efficiency in most instances . In both cases, however, the degree of enhancement depends on its context, i.e . the neighboring bases . Although the rules derived from this study are complex, the results show that mutations in any of the three bases preceding the start codon can strongly affect the translational efficiency of the beta-galactosidase mRNA.

J Exp Med, 1984 Mar 1, 159(3), 828 - 43
Interrelationships of human interferon-gamma with lymphotoxin and monocyte cytotoxin; Stone-Wolff DS et al.; Crude preparations of interferon (IFN)-gamma derived from human peripheral blood leukocyte (PBL) cultures induced with 12-O-tetra-decanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) were more cytotoxic to HeLa cells than partially purified nautral or highly purified recombinant human IFN-gamma preparations . Conditioned media from PBL cultures contained, in addition to IFN-gamma, a mixture of cytotoxins, including classic lymphocyte-derived lymphotoxin (LT), and a TPA-induced cytotoxic activity produced by the adherent cell population (presumably monocytes) . These two types of cytotoxins, indistinguishable in the mouse L929 cell LT assay, could be differentiated by an antiserum prepared against LT derived from the B lymphoblastoid cell line RPMI 1788 . This antiserum neutralized lymphocyte-derived classic LT but failed to neutralize the activity of the monocyte-derived cytotoxin . Processing of conditioned media by sequential chromatography on silicic acid, Con A-Sepharose, and DEAE-Sephacel failed to separate IFN-gamma from the LT activity . However, this procedure did remove the monocyte-derived cytotoxic activity present in the original starting material, leaving predominantly classic LT . This LT showed a slightly basic isoelectric point (pI 7.6) which partially overlapped the more basic pI range of IFN-gamma . The two lymphokine activities also could not be completely separated by fast protein liquid chromatography or molecular sieve chromatography . LT in these partially purified preparations was associated with a protein having an apparent molecular weight of 58,000 on gel filtration . This form dissociated partially into a 20,000 mol wt species after denaturation with 0.1% NaDodSO4 . IFN-gamma could be selectively removed from preparations containing both IFN-gamma and LT with the aid of monoclonal antibody to IFN-gamma . The addition of purified LT to purified E . coli-derived recombinant human IFN-gamma resulted in a marked synergistic enhancement of cytotoxicity for HeLa cells.

J Bacteriol, 1984 Mar, 157(3), 828 - 32
Dual control of a common L-1,2-propanediol oxidoreductase by L-fucose and L-rhamnose in Escherichia coli; Chen YM et al.; Anaerobic growth of Escherichia coli on L-fucose or L-rhamnose as the sole source of carbon and energy depends on the regeneration of NAD from NADH by disposing the intermediate L-lactaldehyde as L-1,2-propanediol . The two parallel pathways, with their own permeases and enzymes encoded by two widely separated gene clusters, appear to share a single enzyme that catalyzes the formation of L-1,2-propanediol . Although this oxidoreductase is encoded by a gene at the fuc locus, the enzyme is inducible by both L-fucose and L-rhamnose . The inducibility by L-rhamnose is controlled by a gene at the rha locus with no other known functions, since the aerobic growth rate on L-rhamnose remains normal . L-1,2-Propanediol oxidoreductase activity is inducible only anaerobically, and the effect of the two methylpentoses operates at different levels: L-fucose exerts its influence post-transcriptionally; L-rhamnose exerts its influence transcriptionally.

J Immunol, 1984 Mar, 132(3), 1300 - 4
Natural and recombinant Escherichia coli-derived interferon-gamma differ in their reactivity with monoclonal antibody; Le J et al.; Monoclonal antibody GIF-1 was found to neutralize human natural immune interferon (IFN-gamma), but not Escherichia coli-derived recombinant IFN-gamma . In addition, GIF-1 antibody failed to immunoprecipitate 125I-labeled recombinant IFN-gamma, whereas it precipitated natural IFN-gamma in a concentration-dependent manner . The lack of recognition of recombinant IFN-gamma by antibody GIF-1 may not be due to the absence of the oligosaccharide moiety in the molecules of recombinant IFN-gamma alone, because removal of carbohydrate from natural IFN-gamma by treatment with a mixture of glycosidases did not alter the selective binding of antibody, i.e., deglycosylated and untreated natural IFN-gamma were equally neutralized and immunoprecipitated by GIF-1 antibody . In addition, a minor monomeric component of natural IFN-gamma with the m.w . of 15,500, which apparently lacks carbohydrate, was also recognized by antibody GIF-1 . These results suggest that the discriminative recognition of natural and recombinant IFN-gamma by monoclonal antibody GIF-1 may be due to a conformational difference at or near the active regions of natural and recombinant human IFN-gamma molecules.

Vaccine, 1984 Mar, 2(1), 93 - 9
Immunological activity of chitin and its derivatives; Nishimura K et al.; The effect of chitin and its derivatives on the activation of peritoneal macrophages in vivo, on the suppression of tumour growth in syngeneic mice and on the protection of the host against bacterial infection was examined . Thirty percent deacetylated chitin (30% DA-chitin), 70% DA-chitin and carboxymethyl-chitin (CM-chitin) induced cytotoxic macrophages most effectively . Chitosan, hydroxyethyl-chitin, dihydroxypropyl-chitin (DHP-chitin) and DHP-chitosan had moderate activities . Phosphorylated-, sulphonated- or acetyl-chitin, however, were less effective . Both 70% DA-chitin and DHP-chitosan were most active on the suppression of Meth-A tumour growth in BALB/c mice, and 30% DA-chitin had a moderate effect . For the stimulation of non-specific host resistance against Escherichia coli infection, 30% and 70% DA-chitin were effective.

Aust Vet J, 1984 Mar, 61(3), 77 - 82
The effect of Escherichia coli endotoxin and culture filtrate on the lactating bovine mammary gland; Frost AJ et al.; The pathogenesis of coliform mastitis was studied by observing pathological changes in lactating glands after infusion of either endotoxin or the sterile culture filtrate (CCF) of the medium in which Escherichia coli strain B117 had been grown . Both infusions produced a rapid and intense inflammatory response by 4 h with a marked increase of serum proteins in the milk . Before dispersing into the milk, neutrophils were attached to the ductular epithelium; highest cell counts in the milk were recorded when the tissue reaction had waned . Oedema of the ductular epithelium occurred, particularly where neutrophils were actively migrating . The infusion of CCF produced, in addition to inflammation, degeneration and necrosis of ductular cells . The smallest lesions healed very rapidly . There was evidence of differing cell susceptibility to the necrotising toxin as well as uneven distribution over the epithelial surface . All changes observed were confined to the regions of the teat and lactiferous sinuses with little effect on the secreting tissue . The role of the necrotising toxin in the natural disease remains undetermined.

Anal Biochem, 1984 Mar, 137(2), 351 - 9
Glycerol, sucrose, and other diol-containing reagents are not inert components in in vitro incubations containing aminoacyl-tRNA; Johnson AE et al.; The addition of glycerol, sucrose, or other diol-containing reagents to solutions of aminoacyl-tRNA (aa-tRNA) substantially increased the rate of hydrolysis of the aminoacyl ester bond . Glycerol at 4.9% (v/v) doubled the rate of deacylation for several aa-tRNAs and peptidyl-tRNAs, including fMet-tRNAMetf, while 1% (v/v) glycerol increased the deacylation rate by 20% . This effect was not caused by a nuclease contamination, and tRNA deacylated in the presence of glycerol could be fully recharged . The deacylation of aa-tRNA was accelerated by glycerol and sucrose even in the presence of EF-Tu X GTP . In addition, the extent of tRNA aminoacylation was reduced when glycerol was present at concentrations above 2% (v/v) . Thus, glycerol and sucrose are not necessarily inert or neutral additions to an in vitro incubation.

Plasmid, 1984 Mar, 11(2), 178 - 81
Location of a function on RP1 that fertility inhibits Inc W plasmids; Yusoff K et al.; Two fertility inhibition (Fi+) functions which reduce R388 (Inc W) transfer were detected on RP1 (Inc P) . Neither function affected R388 -mediated surface exclusion but they could be distinguished by their effect on pilus production . One of the functions was located in the 6.5-kb Pst1 -C region of RP1, part or all of which also occurs on six Fi+ but not two Fi- Inc P plasmids studied.

Med Tekh, 1984 Mar-Apr, (2), 12 - 6
{Automated biochemical analysis based on the use of electrosorption systems}; Andreev VS; An engineering method to separate biocomponents that is suitable for discrete and flow-type autoanalyzers is proposed . The principle is based on using electrosorption systems; it provides, to a high reproducibility, the separation of cells and biomacromolecules from another components of samples to be analysed and makes it possible to solve analytical problems which require the application of several currently available separation systems.

J Biochem (Tokyo), 1984 Mar, 95(3), 729 - 36
Two autonomously replicating sequences near oli-1 gene of yeast mitochondrial DNA; Mabuchi T et al.; We cloned two autonomously replicating sequences from a short segment of mtDNA of an oligomycin-resistant petite yeast, O-111, into a vector pYleu 12 constructed from yeast LEU 2 gene and pBR 322 . These plasmids, pYmit 4 and pYmit 1, had frequencies of transformation of yeast as high as that of YEp 13, having a replicator of 2 mu DNA . They were maintained as plasmids in yeast under selective conditions and shuttled from yeast to E . coli . No evidence was obtained that these plasmids were incompatible with the wild-type mitochondrial genome . These sequences were located in intergenic regions.

J Biochem (Tokyo), 1984 Mar, 95(3), 637 - 42
Phosphoenolpyruvate carboxylase of Escherichia coli . Specificity of some compounds as activators at the site for fructose 1,6-bisphosphate, one of the allosteric effectors; Kodaki T et al.; An investigation was performed to elucidate some unusual phenomena which had been observed with phosphoenolpyruvate (PEP) carboxylase {EC 4.1.1.31} of Escherichia coli . (i) Fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP--the allosteric activators--were competitive with each other in the activation . (ii) Some analogs of PEP such as DL-2-phospholactate and 2-phosphoglycolate, which behaved as inhibitors in the presence of the activator (acetyl-CoA or dioxane), activated the enzyme to some extent in the absence of the activator . (iii) Ammonium sulfate deprived the enzyme of sensitivity to Fru-1,6-P2 or GTP but had no effect on the sensitivity to other effectors . It was found that the activation by the analogs was lost upon desensitization of the enzyme to Fru-1,6-P2 by reaction with 2,4,6-trinitrobenzene sulfonate . The activation by the analogs was not observed in the presence of 200 mM ammonium sulfate . In the presence of lower concentrations (0.1 mM) of PEP, ammonium sulfate activated the enzyme at concentrations less than 700 mM but had an inhibitory effect on the desensitized enzyme . These findings suggest that the unusual phenomena described above are a result of binding of the phosphate esters and sulfate ions with the Fru-1,6-P2 site of the enzyme or the active site depending on the reaction conditions.

Res Vet Sci, 1984 Mar, 36(2), 187 - 93
Influence of diet on postweaning malabsorption and diarrhoea in the pig; Miller BG et al.; Five-day-old pigs challenged with 10(3) pathogenic Escherichia coli (nalidixic acid resistant) showed no clinical signs of disease until subsequently weaned at three weeks . Dietary manipulation was shown to influence xylose malabsorption, diarrhoea and bacterial proliferation after weaning . Brief, but not continuous, contact with the diet before weaning markedly increased the severity of subsequent disease after weaning . Immunogenicity of the weaning diet was critical for the development of the disease . Two diets, identical except that in one the protein source (casein) had previously been enzymatically hydrolysed, were compared . Pigs fed the predigested diet showed no clinical signs of post weaning diarrhoea whereas those fed the untreated casein all developed diarrhoea.

Prikl Biokhim Mikrobiol, 1984 Mar-Apr, 20(2), 208 - 16
{Purification and properties of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600}; Ankilova VN et al.; A preparative scale method for isolation of highly purified phenylalanyl-tRNA synthetase from E . coli MRE-600 was developed . It consists of cell destroying, nucleic acid precipitation with streptomycine sulfate, fractionation with ammonium sulfate followed by chromatography on different carriers (Sephadex G-200, DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite) . The mode of cell destroying was found to affect the process of the further enzyme purification . The phenylalanyl-tRNA synthetase was purified 540-fold, with recovery being 20.6% and the specific activity - 540 units per mg protein . The enzyme content in the purified preparation was 80-90% judging by electrophoresis in PAAG . The molecular weights of the subunits determined by electrophoresis under denaturative conditions were found to be 102,000 +/- 4000 (beta) and 42,000 +/- 2000 (alpha) . The molecular weight of the native enzyme determined by gel filtration through Sephadex G-200 and electrophoresis at varied concentrations of polyacrylamide was found to be 340,000 +/- 20,000 . The Km values for tRNA, ATP and phenylalanine in the aminoacylation reaction are equal to 5.4 X 10(-7) M, 1,9 X 10(-4) M, and 3.7 X 10(-6) M, respectively.

Mutat Res, 1984 Mar-Apr, 131(3-4), 97 - 100
UV irradiation alters deoxynucleoside triphosphate pools in Escherichia coli; Das SK et al.; UV irradiation of exponentially growing Escherichia coli increased intracellular concentration of dATP and dTTP without significantly changing the concentrations of dGTP and dCTP . These selective increases in dATP and dTTP pools are seen in wild-type E . coli K12 and AB1157, as well as in recA and umuC strains, and are proportional to UV dose . The possible significance of these findings with respect to induction of the SOS response and nontargeted mutagenesis are discussed.

Mol Biol (Mosk), 1984 Mar-Apr, 18(2), 362 - 9
{Changes in the state of tryptophan residues in T4 phage lysozyme during binding to a competitive inhibitor}; Vedenkina NS et al.; The analysis of the intrinsic fluorescence parameters of T4 phage lysozyme in free state and in complex with inhibitor--disaccharide-tetrapeptide from the E . coli cell wall has been carried out . A comparison of the fluorescence changes with the results obtained by difference spectrophotometry and with the data of Elwell and Schellman on the intrinsic fluorescence of wild type WT and mutant eRI T4 phage lysozymes and a consideration of the three dimensional structure of the protein allows to represent the protein fluorescence parameters as a sum of contributions of the individual tryptophan residues . According to the proposed scheme Trp-126 does not emit neither in the free protein nor in the complex; the fluorescence parameters of Trp-158 (lambda m 332 nm, q = 0.27) are not affected by binding of the inhibitor, but all the fluorescence changes are due to the rise of the quantum yield (from 0.135 to 0.315) and the blue shift (from 332 to 328 nm) of the fluorescence of Trp-138.

J Clin Microbiol, 1984 Mar, 19(3), 408 - 11
Colony incompatibility studies of enterotoxigenic Escherichia coli O126 isolated during one outbreak; Bettelheim KA; Strains of heat-stable enterotoxin-producing Escherichia coli belonging to O group O126 isolated during an outbreak of gastroenteritis were studied by the colony incompatibility phenomenon . They were compared with other isolates of O126 . All of the outbreak strains except one appeared related in these studies and were different from all other strains studied . The one different outbreak strain was isolated 6 weeks after all of the others and may represent some new factor . The study demonstrated the potential usefulness of monitoring strains during an outbreak for relatedness.

Genetika, 1984 Mar, 20(3), 373 - 81
{Deletions of plasmid pRP3.1ts12 derived from RP1 leading to the suppression of the thermosensitive ts12 mutation and mucoid phenotype induction in Escherichia coli K-12}; Danilevich VN et al.; Properties of a temperature-sensitive in replication mutant pRP3.1ts12 derived from the broad host range RP1 plasmid have been studied . pRP3.1ts12 is a shortened variant of the temperature-sensitive RP1ts12 mutant carrying a deletion in a region from 2.3 to 7.6 MD . In contrast to RP1ts12, the plasmid pRP3.1ts12 is a leaky ts mutant and is characterized by an elevated frequency of reversions to the temperature-independent phenotype . Temperature-independent derivatives of pRP3.1ts12 were studied . Approx . 15% of these were found to induce mucoid growth of the host cells . As revealed from restriction endonuclease analysis, most of the latter derivatives contain deletions of small DNA segments in the region 0.56 to 2.3 MD of the RP1 map . The possible nature of the gene(s), whose deletions suppress the temperature-sensitive ts12 mutation and results in superproduction of Escherichia coli capsular poly-saccharide is discussed.

EMBO J, 1984 Mar, 3(3), 631 - 5
A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant; Shiba K et al.; We describe the properties of a temperature-sensitive mutant, ts24, of Escherichia coli . The mutant has a conditional defect in export of periplasmic and outer membrane proteins . At 42 degrees C, precursor forms of these proteins accumulate within the cell where they are protected from digestion by externally added trypsin . The accumulated precursors are secreted and processed very slowly at 42 degrees C . The mutation is complemented by expression of the wild-type secY (or prlA) gene, which has been cloned into a plasmid vector from the promoter-distal part of the spc ribosomal protein operon . The mutant has a single base change in the middle of the secY gene, which would result in the replacement of a glycine residue by aspartic acid in the protein product . These results demonstrate that the gene secY (prlA) is essential for protein translocation across the E . coli cytoplasmic membrane.

EMBO J, 1984 Mar, 3(3), 545 - 50
Inducible repair of O-alkylated DNA pyrimidines in Escherichia coli; McCarthy TV et al.; The three miscoding alkylated pyrimidines O2-methylcytosine, O2-methylthymine and O4-methylthymine are specifically recognized by Escherichia coli DNA repair enzymes . The activities are induced as part of the adaptive response to alkylating agents . O2-Methylcytosine and O2-methylthymine are removed by a DNA glycosylase, the alkA+ gene product, which also acts on N3-methylated purines . O4-Methylthymine is repaired by a methyltransferase, previously known to correct O6-methylguanine by transfer of the methyl group to one of its own cysteine residues . It is proposed that certain common structural features of the various methylated bases allow each of the two inducible repair enzymes to recognize and remove several different kinds of lesions from alkylated DNA.

Ann Immunol (Paris), 1984 Mar-Apr, 135C(2), 251 - 60
{Assay of rabbit anti-thermolabile enterotoxin antibodies from Escherichia coli by an immunoenzyme technic}; Germani Y et al.; The titration of rabbit anti-Escherichia coli heat-labile enterotoxin antibodies by an immunosorbent assay (ELISA) generally uses a GM1 coating step to lend specificity to the ELISA reaction at a time when the method is tempered by a lack of purified E . coli toxin . We have developed an adsorption method of the toxin to the polystyrene in order to simplify the technique . Three different immunosorbent preparations were tested to determine which of them yielded the most sensitive results.

Am J Vet Res, 1984 Mar, 45(3), 586 - 91
Role of prostaglandins in pathogenesis of bovine mastitis induced by Escherichia coli endotoxin; Giri SN et al.; Four doses (5 to 100 micrograms, 1 dose/quarter) of Escherichia coli endotoxin were introduced into lactating mammary glands of 2 cows . There was no effect on milk prostaglandin (PG) E2 concentration, except that the concentration was increased from 200 pg/ml of milk to 1,060 pg/ml at post-treatment hour (PTH) 8 in cow 1 and from 75 to 420 pg/ml at PTH 4 in cow 2 after the highest dose 100 micrograms . Endotoxin caused a dose-dependent increase in milk PGF2 alpha concentrations in both cows . After the highest dose, PGF2 alpha was maximally increased from 200 to 3,500 pg/ml at PTH 4 in cow 1 and from 250 to 2,000 pg/ml in cow 2 at PTH 8 . The instillation of 50 micrograms of endotoxin in all 8 quarters of 2 more lactating cows caused no significant (P greater than 0.05) changes in milk PGE2 and thromboxane B2 concentrations, whereas milk PGF2 alpha was significantly increased from the base-line value of 642 to 2,683, 1,189, and 2,281 pg/ml at PTH 4, 8, and 12, respectively . The 6-keto-PGF1 alpha was also significantly increased from the base-line value of 305 to 871, 631, and 600 pg/ml at the corresponding times, respectively . A marked increase in vascular permeability, as judged by high concentrations of serum albumin in the whey, was observed as early as PTH 4 and peaked at PTH 12 followed by a gradual decline, although it remained significantly increased over the control for 48 hours after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Trop Med Hyg, 1984 Mar, 33(2), 281 - 4
Outbreak of invasive Escherichia coli gastroenteritis on a cruise ship; Snyder JD et al.; An invasive strain of Escherichia coli (ONT:NM) was isolated from stool specimens from 7 of 10 ill passengers who developed diarrhea during a 5-day ocean cruise . The ill passengers had shared no common exposures off the ship before or during the cruise . Three of the persons whose stools were cultured were part of a tour group of 219 persons, and a food consumption and health history questionnaire was completed by 190 members (87%) of this tour group . Forty-seven (25%) had had diarrhea during the cruise; other symptoms among those with diarrhea included nausea (72%), abdominal cramps (68%), headache (68%), chills (60%), dizziness (53%), myalgias (43%), subjective fever (36%), and vomiting (26%) . The median duration of symptoms was 3 days . Eating at cold buffets on ship and eating potato salad, a buffet food item, were significantly associated with illness . No evidence of secondary spread of illness in household contacts of the ill person was found.

Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1844 - 8
Molecular model for elongation of the murein sacculus of Escherichia coli; Burman LG et al.; Labeling experiments are presented that suggest that new (radioactive) strands of murein are initially inserted adjacent to old strands . After 8 min, new strands start to be inserted adjacent to the previously inserted radioactive strands . Analysis of these data suggests that, for Escherichia coli to double the length of the sacculus in each generation, about 90 separate membrane-bound enzyme complexes travel unidirectionally around the circumference of the cell . They travel at a constant rate, six times each generation, synthesizing, inserting, and crosslinking two strands of murein at a time, thereby doubling the length of the sacculus.

Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1624 - 8
Possible ideal lac operator: Escherichia coli lac operator-like sequences from eukaryotic genomes lack the central G X C pair; Simons A et al.; Five DNA fragments have been cloned from yeast, chicken, and mouse DNA that titrate lac repressor in an Escherichia coli lac+ I+Z+ wild-type strain when on a multi-copy plasmid . The five repressor-binding sequences have been identified by DNA sequence determinations and DNase cleavage-inhibition patterns . They share the 14-base-pair symmetrical consensus sequence 5' T-G-T-G-A-G-C:G-C-T-C-A-C-A 3' (the colon represents the center of symmetry), which is an inverted repeat of 7 base pairs of the left half of the E . coli lac operator . A similar perfect palindromic DNA fragment--an 11-base-pair inverted repeat of the left half of the lac operator--was synthesized . The cloned synthetic DNA 5' G-A-A-T-T-G-T-G-A-G-C:G-C-T-C-A-C-A-A-T-T-C 3' binds lac repressor 8-fold more tightly than does wild-type E . coli lac operator DNA.

Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1394 - 7
Contact points between transcription machinery and the fibroin gene promoter deduced by functional tests of single-base substitution mutants; Hirose S et al.; An efficient method for oligonucleotide-directed mutagenesis was developed to construct a set of site-specific mutations by using a mixture of oligodeoxyribonucleotides . With this method, as high as 40% of the tested clones turned out to be desired mutants . Seven single-point mutants were isolated in the "TATA" box region of the fibroin gene . In vitro transcription experiments showed that single-base transversions at the TATA box (A----T at position -29, T----A or G at -28, A----T at -27, and A----T at -26) resulted in decreased promoter activities, whereas A----G transitions at positions -24 and -23 had no effect . The initiation site of transcription was normal in three down-promoter mutants at positions -28 and -26, but the A----T transversions at positions -29 and -27 induced an additional transcription start from position +4 . Using 10 single-point mutants obtained as above or by nitrous acid-induced mutagenesis, we have prepared a pair of heteroduplex DNAs consisting of a mutant strand and the wild-type one . The molecules heterozygous at positions -30, -21, and -20 showed reduced transcription activities when the noncoding strand bears the mutation, whereas that at position -26 gave a low activity when the coding strand carries the mutation . Both types of heteroduplex at positions -29, -28, -27, and -17 exhibited decreased activities . These results suggest a transcription machinery contact to a major groove of the DNA helix at the TATA box and region of position -20.

Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1332 - 5
Post-translational activation introduces a free radical into pyruvate formate-lyase; Knappe J et al.; Pyruvate formate-lyase (formate acetyltransferase; EC 2.3.1.54) of Escherichia coli cells is post-translationally interconverted between inactive and active forms . Conversion of the inactive to the active form is catalyzed by an Fe2+-dependent activating enzyme and requires adenosylmethionine and dihydroflavodoxin . This process is shown here to introduce a paramagnetic moiety into the structure of pyruvate formate-lyase . It displays an EPR signal at g = 2 with a doublet splitting of 1.5 mT and could comprise an organic free radical located on an amino acid residue of the polypeptide chain . Hypophosphite was discovered as a specific reagent that destroys both the enzyme radical and the enzyme activity; it becomes covalently bound to the protein . The enzymatic generation of the radical, which is linked to adenosylmethionine cleavage into 5'-deoxyadenosine and methionine, possibly occurs through an Fe-adenosyl complex . These results suggest a radical mechanism for the catalytic cycle of pyruvate formate-lyase.

Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1312 - 6
The free energy of DNA supercoiling is enthalpy-determined; Seidl A et al.; The thermodynamics of superhelix formation was determined by combining superhelix density data with enthalpy values obtained from microcalorimetric measurements of the relaxation of supercoiled ColE1 amp plasmid DNA in the presence of topoisomerase I from Escherichia coli (omega protein) . The thermodynamic quantities for superhelix formation at 37 degrees C in 10 mM Tris/2 mM MgCl2/1 mM EDTA pH 8, are: delta G = 921 kJ X (mol of plasmid)-1; delta H 2260 kJ X (mol of plasmid)-1; deltaS = 4.3 kJ X (mol of plasmid X K)-1 . These data clearly demonstrate that the unfavorable Gibbs free energy associated with supercoiling of DNA results exclusively from the positive enthalpy involved in formation of superhelical turns . A positive overall entropy change accompanies superhelix formation, which overcompensates the expected decrease of configurational entropy . By neglecting contributions from bending, an estimate of the torsional rigidity C = 1.79 X 10(-19) erg X cm (1 erg = 0.1 microJ) of the supercoiled ColE1 amp plasmid DNA was made on the basis of the enthalpy value . This value is in excellent agreement with values of C derived from subnanosecond time-resolved fluorescence depolarization measurements for pBR322 DNA {Millar, D . P., Robbins, R . J . & Zewai, A.H . (1982) J . Chem . Phys . 76, 2080-2094} . The magnitude of C is larger than for linear DNAs, indicating that supercoiled DNA is more rigid than linear DNA.

Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1307 - 11
Cocrystals of the DNA-binding domain of phage 434 repressor and a synthetic phage 434 operator; Anderson J et al.; The amino-terminal domain of the phage 434 repressor forms cocrystals with a synthetic phage 434 operator . The cocrystals diffract to at least 4 A, and x-ray crystallographic analysis of them is in progress . An analysis of the packing in the cocrystals shows that complexes consisting of dimers of amino-terminal domain bound specifically to operators are stacked end to end in longer protein-DNA rods parallel to the unit cell body diagonals . The DNA in the complexes has 10.5 base pairs per turn and a rise per base of 3.26 A--values consistent with B-form DNA--indicating that DNA is neither unwound nor overwound by bound repressor . The packing analysis suggests an approach that might facilitate the cocrystallization of other DNA-binding proteins with the DNA they recognize.

Genetics, 1984 Mar, 106(3), 347 - 64
The role of pyrimidine dimers as premutagenic lesions: a study of targeted vs . untargeted mutagenesis in the lacI gene of Escherichia coli; Kunz BA et al.; We have employed conjugal transfer of an F' lac episome to examine targeted and untargeted mutagenesis in the lacI gene of Escherichia coli and to determine the relative importance of pyrimidine dimers as premutational UV lesions compared to (6-4) photoproducts that also may have a mutational role . This conjugal system allowed us to assess the premutagenic role of UV lesions independently from any role as inducers of SOS functions . F' DNA was transferred to an SOS-induced recipient strain from: unirradiated donor cells, UV-treated donor cells or donor cells that were irradiated and then exposed to photoreactivating light . The results indicate that SOS-related, untargeted events may account for as much as one-third of the nonsense mutations (i.e., base substitutions) recovered after undamaged F' DNA is transferred to UV-irradiated recipients . When the donor strain also is irradiated, in excess of 90% of the mutations detected following conjugation appear to be targeted . Photoreactivation of the UV-treated donors cells, prior to F' transfer to the SOS-induced recipient strain, demonstrated that in this experimental system virtually all recovered UV-induced mutations are targeted by photoreactivable lesions . We presume that these lesions are pyrimidine dimers because (6-4) photoproducts are not photoreactivable.

Genetics, 1984 Mar, 106(3), 335 - 45
Mutagenic specificity of a novel T4 DNA polymerase mutant; Reha-Krantz LJ et al.; The in vivo mutational specificity of a novel T4 DNA polymerase mutator mutant, tsM19, was determined . Two genetic tester systems were used to characterize the mutant . Results of our studies indicate that tsM19 promotes transition and transversion mutagenesis and, possibly, frameshift mutagenesis . Central G:C base pairs in runs of three or more consecutive G:C base pairs may be target sites for tsM19-induced transitions.

Am J Epidemiol, 1984 Mar, 119(3), 350 - 5
Seasonal and racial incidence of infantile gastroenteritis in South Africa; Robins-Browne RM; The etiology of summer diarrhea, which formerly caused extensive mortality in children living in industrialized countries, was never discovered . This condition no longer occurs in developed countries, having been replaced by winter diarrhea, which is associated with a low mortality . Summer epidemics of diarrhea still take place in black South African infants, whereas, in white South African children, the pattern of diarrhea is similar to that seen in children in industrialized countries today . In 1977-1980, the author studied the records of patients less than two years of age admitted for treatment of dehydrating diarrhea to two teaching hospitals in Johannesburg, the Johannesburg General and Baragwanath Hospitals, which serve the needs of white and black patients, respectively . The incidence of severe diarrhea (i.e., diarrhea necessitating hospitalization) showed highly significant seasonality (p less than 0.001) and distinctive patterns by race . Dehydrating diarrhea in black children was strongly associated with warm weather, while diarrhea in white children occurred more regularly throughout the year, with a peak incidence in late fall . Laboratory studies have shown that bacteria, in particular "classical" enteropathogenic Escherichia coli, are the leading cause of diarrhea in black South African children, and that diarrhea in white children is largely attributable to rotaviruses . The association of enteropathogenic E . coli with diarrhea in black children suggests that these bacteria were responsible for earlier outbreaks of summer diarrhea . The finding that the etiology of diarrhea varies according to socioeconomic class has important implications for diarrhea control programs.

Mutat Res, 1984 Mar, 139(3), 101 - 5
Comparison of the Escherichia coli umu+-encoded function with plasmid R46-mediated error-prone repair in DNA-damaged cells; Attfield PV et al.; Escherichia coli strain TK701 umu+ was more resistant than strain TK702 umu when tested against bleomycin (BLM), cis-platinum(II) diamminodichloride (PDD), ultraviolet light and methyl methanesulphonate (MMS), which produce single-strand DNA damage . However, the umu mutant was no more sensitive to mitomycin C (MTC) or proflavine (PF), which cause double-strand DNA binding . Strain TK702 umu was nonmutable by any of the agents, whereas mutations were induced in the wild-type strain by PDD, UV, MMS and MTC . The E . coli umu+ function therefore mimics plasmid R46-mediated error-prone repair in protecting only against single-strand DNA damage, whilst enhancing mutagenesis by both single- and double-strand damaging agents . Comparison of plasmid R46-mediated protection and mutagenesis in umu+ and umu strains indicated that the plasmid confers a greater error-prone DNA-repair activity in the mutant . Results are discussed in terms of analogy between host umu+ and plasmid muc+ functions.

Mutat Res, 1984 Mar, 126(1), 9 - 18
Metal-induced mutagenesis in the lacI gene of Escherichia coli; Zakour RA et al.; Mutagenesis in the lacI gene of Escherichia coli has been examined in cells grown in the presence of beryllium, manganese or chromium compounds, metals with suspected mutagenic or carcinogenic potential . 2--3-fold increases in mutation frequency were produced by BeCl2, MnCl2 and K2Cr2O7 . Among the cells grown in the presence of Be2+, the frequency of amber and ochre mutants was 3-fold higher than the spontaneous background, suggesting that at least part of the increased mutagenicity was due to base-substitution mutations . The specificity of base-substitution mutations induced by Be2+ and Mn2+ in the lacI gene was analyzed . Among the amber mutations induced in cells grown in the presence of Be2+, an increase in G:C----A:T transitions was detected . In contrast, following growth in Mn2+, no increase in amber and ochre mutation frequencies was observed, and the mutational spectrum resembled that obtained spontaneously indicating that mutations induced by Mn2+ in the lacI gene involve changes that do not yield nonsense mutations . These results suggest that metals may exert a number of different mutagenic effects and that these effects vary for each metal.

J Bacteriol, 1984 Mar, 157(3), 956 - 7
Mapping of a locus (mdoA) that affects the biosynthesis of membrane-derived oligosaccharides in Escherichia coli; Bohin JP et al.; Mutants of Escherichia coli defective in the newly discovered mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides . The mutation has now been mapped and found to be located near 23 min on the E . coli chromosome between putA and pyrC . The mdoA mutants are defective in the membrane-localized component of the glucosyl transferase system described by Weissborn and Kennedy (A . C . Weissborn and E . P . Kennedy, Fed . Proc . 42:2122, 1983).

J Bacteriol, 1984 Mar, 157(3), 909 - 17
Nucleotide sequence of the phoS gene, the structural gene for the phosphate-binding protein of Escherichia coli; Magota K et al.; phoS is the structural gene for the phosphate-binding protein, which is localized in periplasm and involved in active transport of phosphate in Escherichia coli . It is also a negative regulatory gene for the pho regulon, and the gene expression is inducible by phosphate starvation . The complete nucleotide sequence of the phoS gene was determined by the method of Maxam and Gilbert (A . M . Maxam and W . Gilbert, Methods Enzymol . 65:499-560, 1980) . The amino acid sequences at the amino termini of the pre-PhoS and PhoS proteins and at the carboxy terminus of the PhoS protein were determined by using the purified proteins . Furthermore, the amino acid sequence of enzymatically digested peptide fragments of the PhoS protein was determined . The combined data established the nucleotide sequence of the coding region and the amino acid sequence of the pre-PhoS and the PhoS proteins . The pre-PhoS protein contains an extension of peptide composed of 25 amino acid residues at the amino terminus of the PhoS protein, which has the general characteristics of a signal peptide . The mature PhoS protein is composed of 321 amino acid residues, with a calculated molecular weight of 34,422, and lacks the disulfide bond and methionine . The regulatory region of phoS contains a characteristic Shine-Dalgarno sequence at an appropriate position preceding the translational initiation site, as well as three possible Pribnow boxes and one -35 sequence . the nucleotide sequence of the regulatory region of phoS was compared with those of phoA and phoE, the genes constituting the pho regulon.

J Bacteriol, 1984 Mar, 157(3), 891 - 8
Assembly of the aspartate transcarbamoylase holoenzyme from transcriptionally independent catalytic and regulatory cistrons; Foltermann KF et al.; The cistrons encoding the regulatory and catalytic polypeptides of aspartate transcarbamoylase (EC 2.1.3.2) from Escherichia coli K-12 have been cloned separately on plasmids from different incompatability groups . The catalytic cistron (pyrB) was carried by pACYC184 and expressed from its own promoter, whereas the regulatory cistron was expressed from the lac po of pBH20 . The catalytic polypeptide chains assembled into enzymatically active trimers (c3) in vivo when expressed in the absence of regulatory subunits . Similarly, the regulatory polypeptide chains assembled into regulatory dimers (r2) in vivo in the absence of catalytic subunits . When cellular extracts containing regulatory dimers and catalytic trimers synthesized in separate cells were combined in vitro, partial spontaneous holoenzyme assembly occurred . When pyrB and pyrI were expressed from transcriptionally independent cistrons in the same cell, all detectable catalytic polypeptides were incorporated into the functional aspartate transcarbamoylase holoenzyme, 2(c3):3(r2) . Thus, it is clear that the in vivo assembly of ATCase holoenzyme is a direct, spontaneous process involving the association of preformed regulatory subunits (r2) and catalytic subunits (c3) . This procedure provides a general method for the construction of hybrid aspartate transcarbamoylase in vivo and may be applicable to other oligomeric enzymes constructed from different polypeptides.

J Bacteriol, 1984 Mar, 157(3), 779 - 84
The ftsA gene product participates in formation of the Escherichia coli septum structure; Tormo A et al.; The patterns of septation in filaments of Escherichia coli, formed as a consequence of the lack of an active ftsA gene product and then returned to permissive conditions, were analyzed in isogenic strains containing three different mutated alleles of ftsA . Septation was blocked for at least one doubling time at the potential septation sites that presumably contained inactive FtsA protein but not at those sites containing either the active gene product or no gene product at all . These results suggested a possible structural role for the ftsA gene product in the construction of the E . coli septum.

Infect Immun, 1984 Mar, 43(3), 1027 - 32
Characterization of monoclonal antibodies to heat-labile enterotoxin encoded by a plasmid from a clinical isolate of Escherichia coli; Belisle BW et al.; Eight selected hybridoma cell lines that produced monoclonal antibodies against heat-labile enterotoxin from an Escherichia coli strain of human origin (LTh) were characterized . Antibodies produced by these cell lines were tested for binding specificity in a series of solid-phase radioimmunoassays and Western blots by using as test antigens LTh, the A, A1, A2, and B polypeptides of LTh, the heat-labile enterotoxin from an E . coli strain of porcine origin, and cholera toxin . The monoclonal antibodies were also tested for isotype and ability to neutralize LTh . Two of the anti-LTh monoclonal antibodies cross-reacted with cholera toxin, and six were specific for determinants of LTh that were not present on cholera toxin . One was specific for a unique epitope of LTh that was not shared by the heat-labile enterotoxin from an E . coli strain of porcine origin or cholera toxin . Four antibodies specific for epitopes on the B subunit of LTh (LTh-B) reacted with pentameric LTh-B but did not react in Western blots with monomeric LTh-B . The remaining four antibodies were specific for epitopes on LTh-A; two of these antibodies bound to A1, one reacted with A2, and one recognized only intact LTh-A . Only one monoclonal antibody had detectable neutralizing activity, and it was specific for LTh-A.

Gene, 1984 Mar, 27(3), 331 - 3
Restriction endonuclease mapping and cloning of Mycobacterium intracellulare plasmid pLR7; Crawford JT et al.; A restriction map of Mycobacterium intracellulare plasmid pLR7 was developed . This 15.3-kb plasmid had unique sites for BamHI, HindIII, and XbaI . Various large fragments of pLR7 were cloned into pBR322 or pHP34 and propagated in Escherichia coli . A hybrid pLR7 ::pBR322 plasmid carrying the complete pLR7 sequence was constructed by joining the plasmids at their HindIII sites . The construction of these hybrids will facilitate the analysis and manipulation of pLR7 and may allow the development of this plasmid as a model system for genetic analysis in mycobacteria.

Gene, 1984 Mar, 27(3), 327 - 9
The cleavage site for the restriction endonuclease EcoRV is 5'-GAT/ATC-3'; Schildkraut I et al.; The cleavage site for the restriction endonuclease EcoRV has been found to be 5'-GAT/ATC-3', rather than 5'- GATAT /C-3' as reported earlier by Kholmina et al . { Dokl . Akad . Nauk . 253 (1980) 495-497}.

Gene, 1984 Mar, 27(3), 279 - 88
A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates; Polack A et al.; DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79 . The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage lambda L47 . The large overlapping clones were used to prepare a library of subclones with inserts of 1-15 kb . A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources . The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA- Escherichia coli hosts.

Gene, 1984 Mar, 27(3), 253 - 64
DNA sequence of the Escherichia coli gene, gnd, for 6-phosphogluconate dehydrogenase; Nasoff MS et al.; Expression of gnd of Escherichia coli, which encodes 6-phosphogluconate dehydrogenase, an enzyme of the hexose monophosphate shunt, is subject to growth rate-dependent regulation and is gene dosage-dependent: the level of the enzyme increases in direct proportion to the cellular growth rate at both low and high gene copy numbers . We have determined the nucleotide sequence of gnd and flanking control regions, the 5'-end of in vivo gnd mRNA, and the start codon of the structural gene . Analysis of the sequence indicated that: (i) the gnd promoter is typical of other E . coli promoters and the structural gene is followed by a rho-independent transcription termination signal; (ii) the 56-nucleotide leader of gnd mRNA does not contain a rho-independent transcription termination signal, so growth rate-dependent regulation of 6-phosphogluconate dehydrogenase level is not carried out by an attenuation mechanism analogous to the one that controls expression of the E . coli ampC gene; (iii) the codon composition of the structural gene resembles that of other highly expressed E . coli genes and thus is not responsible for the regulation either; (iv) the structural gene is preceded at an optimal distance by a strong Shine-Dalgarno (SD) sequence, AGGAG ; (v) the leader region of the mRNA contains regions of dyad symmetry that have the potential to sequester the SD sequence and the start codon . This latter feature of the gene suggests that growth rate-dependent regulation may involve regulation of translation initiation frequency.

Gene, 1984 Mar, 27(3), 239 - 51
The yeast cloning vector YEp13 contains a tRNALeu3 gene that can mutate to an amber suppressor; Fischhoff DA et al.; We have shown that the yeast-Escherichia coli shuttle vector YEp13 contains, as part of its yeast chromosomal segment, a tRNALeu3 gene . We have also isolated and characterized a variant of YEp13 , namely YEp13 -a, which is capable of suppressing a variety of yeast amber-suppressible alleles in vivo . YEp13 -a differs from YEp13 by a single point mutation, which changes the three-nucleotide, plus-strand sequence corresponding to the tRNALeu3 anticodon from the normal C-A-A to C-T-A . This nucleotide change creates a site for the restriction enzyme XbaI in the suppressor tRNALeu3 gene . We have taken advantage of the correlation between the suppressor mutation and the XbaI site formation, to show that the tRNALeu3 gene on YEp13 corresponds to the genetically characterized yeast chromosomal amber suppressor SUP53 . We have also shown that SUP53 is located just centromere-distal to LEU2 on chromosome III . Finally, comparison of the DNA sequence of SUP53 and its flanking regions with the sequences of other cloned yeast tRNALeu3 genes has revealed considerable sequence homology in the immediate 5'-flanking regions of these genes.

Recomb DNA Tech Bull, 1984 Mar, 7(1), 1 - 7
Assessing physical containment in recombinant DNA facilities; Fisher E et al.; This paper has presented a computer model that has been developed to explore the effectiveness of various physical containment strategies . The specific applications that are considered here concern recombinant DNA operations, a technology where technical constraints inhibit the direct monitoring of physical containment effectiveness . Simulations have been used to assess several aspects of recombinant DNA containment, including the effects of using different protocols and host organisms, establishing tradeoffs between physical and biological containment levels, and recognizing the variability introduced by human error and equipment failures . In considering these questions, we have focused on the operator's exposure, although the model that has been developed can also be used to estimate the total release of viable recombinant organisms to the environment . Comparing benchtop protocol simulation results with scale-up protocol results indicates that operator exposure in the former situation may be two or three orders of magnitude lower . A similar difference is apparent between the operator's exposure during a standard scale-up protocol and a protocol that replaces manual sampling with automated sampling . Because the specific activities in a facility influence organism release dramatically, physical containment effectiveness cannot be represented with a single estimate of organisms released per facility per time for each containment level . Results from simulations using E . coli chi 1776 and simulations using hardier organisms indicate that, in a well ventilated facility, the operator's inhalation of viable organisms is essentially unaffected by using the disabled host organism . It should be noted, however, that these simulation results only reflect the differences in the survival of the aerosolized organisms; the relative survival and establishment of organisms after inhalation is not considered.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Microbiol, 1984 Mar, 130 ( Pt 3), 701 - 10
Localization and characterization of a gene on the ColE3-CA38 plasmid that confers immunity to colicin E8; Chak KF et al.; Escherichia coli W3110 cells carrying the ColE3-CA38 plasmid are immune to externally added colicin E8, a newly described member of the E group colicins . By molecular cloning and transposon mutagenesis we localized the colicin E8 immunity gene between the EcoRI site (4.0 kb on the restriction map) and the PvuII site (3.68 kb) of the ColE3-CA38 plasmid . This placed the colicin E8 immunity gene between the colicin E3 immunity gene and lys, the region which determined mitomycin C sensitivity . Insertion of a transposon into the colicin E3 structural gene prevented the synthesis of active colicin and completely abolished mitomycin C sensitivity, but had no effect on the two immunity genes . In contrast, insertion of a transposon into the colicin E8 immunity gene had no effect upon colicin E3 production or colicin E3 immunity but did abolish mitomycin C sensitivity . The phenotype conferred by plasmids with a transposon inserted into the lys region of ColE3-CA38 was dependent upon the site of insertion.

Mol Cell Biol, 1984 Mar, 4(3), 399 - 406
Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector; Pennock GD et al.; The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase . The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA . Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside . Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell . By 48 h postinfection, a remarkably high level of expression is achieved . On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background . A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.

Mol Biol (Mosk), 1984 Mar-Apr, 18(2), 397 - 403
{Isolation and characterization of a phage T7 DNA fragment containing promoter B active in vivo and in vitro}; Kravchenko VV et al.; The fragment of 124 base pairs (Alu-124) has been isolated from the phage T7 DNA and cloned in the plasmid pSK . The sequence of the fragment was determined and position of this fragment on the physical map of phage DNA was found . It has been found that Alu-124 fragment contained an active in vivo and in vitro promoter for E . coli RNA-polymerase . In vitro transcription from this promoter was initiated with GTP . The start of the transcription was localised at about 104 or 105 nucleotide . The data obtained indicate that the Alu-124 fragment of the phage T7 promoter B, may be involved in the control of the transcription of the phage T7 early stage development.

Mol Biol (Mosk), 1984 Mar-Apr, 18(2), 370 - 81
{DNA-like duplexes containing repetitive sequences . VIII . Synthesis and properties of DNA fragments--substrates of restriction endonuclease EcoRII}; Gromova ES et al.; The optimal way of constructing a family of restriction endonuclease EcoRII substrates has been developed . The substrates are DNA-like duplexes containing regularly repeated native or modified sites of this enzyme as well as those of EcoRI and AluI . Synthesis of substrates was performed by water-soluble carbodiimide-induced polycondensation of two nonanucleotides, d(C-C-T-G-G-A-A-T-Tp) and d(C-C-A-G-G-A-G-C-Tp), as constituents of different complementary complexes . The products of reaction (degree of polymerization, 2-20) were isolated by G-200 gel-filtration . The yield of polymers was about 70% . The main products of reaction were dimers when dephosphorylated nonanucleotides (terminators of polycondensation) were used . The thermal stability of DNA-like duplexes is very high . The structure of the polymers obtained has been confirmed by UV-spectroscopy and by CD data as well as by the results of cleavage by EcoRI and AluI restriction endonucleases.

Genetika, 1984 Mar, 20(3), 382 - 8
{Characteristics of the expression of the Ap- and Tc-genes of plasmid origin in lambda vectors}; Chernykh SI et al.; Expression of Ap and Tc genes of the plasmid origin in lambda vectors where the effect of lac promoter and PL phage promoter can be traced has been studied . The different orientation of Ap gene relative to strong promoters (lac and PL) permitted the beneficial arrangement of Ap gene for its optimum expression to be established . The interfering interaction of the two closely arranged strong promoters, Ap gene and lac promoter, has been detected . This repressed considerably the expression of Ap gene . The enhanced expression of this gene has been observed under the effect of the removed PL phage promoter . The active expression of Tc gene has been only observed in clones with the recombinant lambda-pcv-20 molecule (without lac promoter), irrespective of the plasmid pcv-20 orientation . This, presumably, can be accounted for by the favourable secondary structure of the m-RNA of Tc gene providing the efficient translation.

EMBO J, 1984 Mar, 3(3), 575 - 9
The EcoA restriction and modification system of Escherichia coli 15T-: enzyme structure and DNA recognition sequence; Suri B et al.; The EcoA restriction enzyme from Escherichia coli 15T- has been isolated . It proves to be an unusual enzyme, clearly related functionally to the classical type I restriction enzymes . The basic enzyme is a two subunit modification methylase . Another protein species can be purified which by itself has no enzymatic activities but which converts the modification methylase to an ATP and S-adenosylmethionine-dependent restriction endonuclease . The DNA recognition sequence of EcoA has an overall structure that is very similar to previously determined type I sequences . It is: 5'-GAGNNNNNNNGTCA-3' 3'-CTCNNNNNNNCAGT-5' where N can be any nucleotide . Modification methylates the adenosyl residue in the specific trinucleotide and the adenosyl residue in the lower strand of the specific tetranucleotide.

Somat Cell Mol Genet, 1984 Mar, 10(2), 129 - 38
Herpes simplex virus thymidine kinase gene is stably maintained and expressed in cells transformed by protoplast fusion; Sandri-Goldin RM et al.; We examined a series of transformed cell lines resulting from transfer of the herpes simplex virus type 1 thymidine kinase gene to Ltk- cells by protoplast fusion gene transfer . We show that multiple copies of the transforming plasmid DNA, ranging from a minimum of two to greater than 20, were present in one or at most a few integration sites in each cell line . The TK+ phenotype was stable in five independent transformed cell lines after growth in nonselective medium for over a year . Transforming plasmid DNA was stable in one cell line containing from two to five copies after a year of growth in nonselective medium . In another cell line initially containing about 20 copies, the transforming DNA became rearranged soon after growth to mass culture, resulting in a decrease to two to five copies which then remained stably maintained . This suggests that TK+ transformants resulting from protoplast fusion are stable when the input DNA has integrated in a relatively low copy number.

Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1629 - 33
Purified reconstituted lac carrier protein from Escherichia coli is fully functional; Viitanen P et al.; Proteoliposomes reconstituted with lac carrier protein purified from the plasma membrane of Escherichia coli catalyze each of the translocation reactions typical of the beta-galactoside transport system (i.e., active transport, counterflow, facilitated influx and efflux) with turnover numbers and apparent Km values comparable to those observed in right-side-out membrane vesicles . Furthermore, detailed kinetic studies show that the reconstituted system exhibits properties analogous to those observed in membrane vesicles . Imposition of a membrane potential (delta psi, interior negative) causes a marked decrease in apparent Km (by a factor of 7 to 10) with a smaller increase in Vmax (approximately equal to 3-fold) . At submaximal values of delta psi, the reconstituted carrier exhibits biphasic kinetics, with one component manifesting the kinetic parameters of active transport and the other exhibiting the characteristics of facilitated diffusion . Finally, at low lactose concentrations, the initial velocity of influx varies linearly with the square of the proton electro-chemical gradient . The results provide quantitative support for the contention that a single polypeptide species, the product of the lac y gene, is responsible for each of the transport reactions typical of the beta-galactoside transport system.

Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1499 - 503
groEL and dnaK genes of Escherichia coli are induced by UV irradiation and nalidixic acid in an htpR+-dependent fashion; Krueger JH et al.; Two proteins with molecular weights of 61,000 and 73,000 were found to be induced by UV light in Escherichia coli mutants in which the SOS responses are constitutively expressed . The induction of these proteins by UV light and nalidixic acid was shown to be independent of the recA+ lexA+ regulatory system . Analysis of these proteins by two-dimensional gel electrophoresis and comparison with the "heat-shock" proteins of E . coli revealed that the Mr 61,000 protein comigrated with the groEL gene product, that the Mr 73,000 protein comigrated with the dnaK gene product, and that other heat-shock proteins were also induced . The induction of groEL and dnaK by UV light and nalidixic acid is controlled by the htpR locus . The results suggest that the regulatory response of E . coli to agents such as UV light and nalidixic acid is more complex than previously thought.

Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1470 - 4
Transformation of Aspergillus nidulans by using a trpC plasmid; Yelton MM et al.; We constructed a chimeric plasmid carrying a complete copy of the trifunctional trpC gene from the Ascomycete fungus Aspergillus nidulans . This plasmid, designated pHY201, replicates in Escherichia coli, where it confers resistance to ampicillin and chloramphenicol and complements trpC mutants lacking phosphoribosylanthranilate isomerase activity . We used pHY201 to transform an A . nidulans trpC- strain to trpC+ at frequencies of greater than 20 stable transformants per microgram of DNA . Southern blot analysis of DNA from transformants showed that pHY201 DNA had integrated into the A . nidulans chromosomes in a majority of cases . Most of the integration events appeared to occur at the site of the trpC- allele of the recipient strain . In several instances, we succeeded in recovering pHY201, or derivatives thereof, from A . nidulans transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of E . coli.

Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1336 - 40
Increased intracellular concentration of an initiator protein markedly reduces the minimal sequence required for initiation of DNA synthesis; Dotto GP et al.; One of the most common sites used for cloning in the filamentous phages f1, fd, and M13 lies within the phage "functional origin," a sequence of 140 nucleotides that is required for phage replication . Even small insertions (four nucleotides) at this location severely reduce origin function . Secondary trans-acting mutations in the phage genome are necessary to restore efficient replication . One of these mutations, present in one of our cloning vectors, R218, has been fully characterized . It consists of a regulatory mutation within gene V that leads to a marked increase in the intracellular level of the phage gene II protein, the "initiator" of viral replication . Increased gene II protein production is sufficient to reduce the minimal sequence required for a functional origin to only 40 nucleotides, while the remaining 100 (containing the cloning site) become entirely dispensable . The general implications of these findings are discussed.

Biull Eksp Biol Med, 1984 Mar, 97(3), 332 - 4
{Effect of transposons Tn1 and Tn9 on the genetic regulation system of transfer and incompatibility functions of Col-plasmid pAP19-1}; Buianova NI et al.; F-like pAP19-1 Col-plasmid was labeled with transposons Tn1 and Tn9 and transfer functions of its derepressed mutants were investigated . The plasmid indicated was compatible with reference plasmids of 9 F-like incompatibility groups . Thus it belongs to the new incompatibility group FX . The ability of Tn9 to change the incompatibility of the plasmid investigated was discovered.

Nature, 1984 Mar 1-7, 308(5954), 32 - 6
Cryo-electron microscopy of viruses; Adrian M et al.; Thin vitrified layers of unfixed, unstained and unsupported virus suspensions can be prepared for observation by cryo-electron microscopy in easily controlled conditions . The viral particles appear free from the kind of damage caused by dehydration, freezing or adsorption to a support that is encountered in preparing biological samples for conventional electron microscopy . Cryo-electron microscopy of vitrified specimens offers possibilities for high resolution observations that compare favourably with any other electron microscopical method.

J Virol, 1984 Mar, 49(3), 724 - 30
Organization of the left-hand end of the herpes simplex virus type 2 BglII N fragment; Galloway DA et al.; We have determined the complete nucleotide sequence surrounding the region coding for a 38,000-dalton protein of herpes simplex virus type 2 strain 333 that is encoded between map coordinates 0.58 to 0.595 on the viral genome . The sequence data have revealed an open translational reading frame of 1,011 nucleotides encoding a protein of 337 amino acids . Upstream of those sequences, transcriptional regulatory signals could be identified that overlap another open reading frame of 489 nucleotides, which presumably encodes the carboxy-terminal 163 amino acids of a 140,000-dalton protein . Downstream of the gene encoding the 38,000-dalton protein a polyadenylation signal was found as well as one further along on the other strand near a termination codon that presumably punctuates the gene specifying a 61,000-dalton protein . The DNA sequence data were compared with the amended sequence of the herpes simplex virus type 1 strain KOS 40,000-dalton protein (K . G . Draper, R . F . Frink, and E . K . Wagner, J . Virol . 43:1123-1128, 1982) and with the 5' end of the same gene from herpes simplex virus type 1 strain 17 (J . McLaughlan and J . B . Clements, J . Gen . Virol . 64:997-1006, 1983) to assess the degree of inter- and intratypic variation . The comparison of the sequences has provided a basis for the type specifity of this viral protein and for different mechanisms giving rise to the diversity between herpes simplex virus types 1 and 2.

J Virol, 1984 Mar, 49(3), 674 - 9
DNA methylation inhibits the transfecting activity of replicative- form phi X174 DNA; Wang RY et al.; Replacement of virtually all the cytosine residues with 5-methylcytosine residues in the complementary strand of the replicative form (RF) of phi X174 DNA caused a 300- to 500-fold loss in its transfecting activity . Similar results were obtained with analogously methylated M13 RF . Transfection experiments with phi X RF hemimethylated in only part of the molecule, as assessed by analysis with restriction endonucleases, indicated that gene A of phi X, which needs to be nicked at a specific site by the gene A protein for RF replication, was not the main target for this inhibition by DNA methylation . We propose that the loss of transfecting activity was due to hemimethylation of the phi X RF interfering with the processively catalyzed movement of the replication fork.

J Bacteriol, 1984 Mar, 157(3), 984 - 6
Clustering of genes for L-fucose dissimilation by Escherichia coli; Chakrabarti T et al.; Aerobic and anaerobic L-fucose utilization by Escherichia coli involves an inducible trunk pathway mediated by a permease, an isomerase, a kinase, and an aldolase . Tn5 insertion mutants of a parental strain expressing this pathway constitutively were used to map the positions of the structural genes by transduction . Results from this and previous studies show that all of the structural genes of the L-fucose trunk pathway map between eno and argA at minute 60.2 of the chromosome.

J Bacteriol, 1984 Mar, 157(3), 968 - 70
Affinity of two different regions of the chromosome to the outer membrane of Escherichia coli; Wolf-Watz H; It was found that DNA associated with the outer membrane of Escherichia coli K-12 is enriched for two different regions of the chromosome, which are both on the 5.9-megadalton EcoRI fragment containing the replication origin, oriC . One region overlaps oriC, whereas the other region was found to be associated with a 1-megadalton EcoRI-BamHI fragment located within the atp operon.

J Bacteriol, 1984 Mar, 157(3), 962 - 4
Effects of deletions in transposon Tn7 on its frequency of transposition; Smith GM et al.; Deletions in transposon Tn7 either abolished transposition or reduced transposition frequency . Except for a deletion in the right-hand terminus, these deletions could be complemented in trans . A 2.1-kilobase fragment of Tn7 encodes a diffusible gene product which stimulates transposition above the wild-type frequency . No cointegrate formation was detected.

J Bacteriol, 1984 Mar, 157(3), 940 - 1
Enzyme III stimulation of cyclic AMP synthesis in an Escherichia coli crp mutant; Daniel J; Cyclic AMP (cAMP) synthesis in Escherichia coli is altered in cAMP receptor protein mutants and in phosphoenolpyruvate:sugar phosphotransferase transport system mutants . The stimulation of cAMP synthesis observed in cAMP receptor protein-deficient mutants is largely dependent upon enzyme III of the phosphoenolpyruvate:sugar phosphotransferase transport system . The phosphoenolpyruvate:sugar phosphotransferase transport system enzyme I is not required for elevated cAMP synthesis . These results suggest that enzyme III plays an important role in regulating adenylate cyclase activity.

J Bacteriol, 1984 Mar, 157(3), 881 - 90
Reconstitution of maltose chemotaxis in Escherichia coli by addition of maltose-binding protein to calcium-treated cells of maltose regulon mutants; Brass JM et al.; Maltose chemotaxis was reconstituted in delta malE cells lacking maltose-binding protein (MBP) . Purified MBP was introduced into intact cells during incubation with 250 mM CaCl2 in Tris-hydrochloride buffer at 0 degrees C . After removal of extracellular CaCl2 and MBP, chemotaxis was measured with tethered bacteria in a flow chamber or with free-swimming cells in a capillary assay . About 20% of tethered cells responded to 10(-4) M maltose; the mean response times were about half those of CaCl2-treated wild-type cells (100 s as opposed to 190 s) . In capillary tests, the maltose response of reconstituted cells was between 15 and 40% of the aspartate response, about the same percentage as in wild-type cells . The best reconstitution was seen with 0.5 to 1 mM MBP in the reconstitution mixture, which is similar to the periplasmic MBP concentration estimated for maltose-induced wild-type cells . Strains containing large deletions of the malB region and malT mutants lacking the positive regulator gene of the mal regulon also could be reconstituted for maltose chemotaxis, showing that no product of the mal regulon other than MBP is essential for maltose chemotaxis.

J Bacteriol, 1984 Mar, 157(3), 857 - 62
Isolation of uvrA mutation on a multicopy plasmid: preliminary characterization of the mutant protein; Lorensen E et al.; A new uvrA mutation (uvrA276) has been isolated on a multicopy plasmid and shown to reside within the region of the uvrA gene defined by the KpnI to SalI endonuclease sites . The protein produced by the uvrA276 mutant gene is identical in size to the wild-type protein and binds to single-stranded DNA under the same conditions as the wild-type protein . However, extracts prepared from strains containing this mutant are deficient at incision of DNA that has been irradiated with UV light.

J Bacteriol, 1984 Mar, 157(3), 846 - 56
Correlation of 3,4-dihydroxybutyl 1-phosphonate resistance with a defect in cardiolipin synthesis in Escherichia coli; Hwang YW et al.; Escherichia coli treated for 1 h with 100 microM rac-3,4-dihydroxybutyl 1-phosphonate (DBP), a glycerol-3-phosphate analog, die when sorted at 5 degrees C, whereas the viability of untreated cells is relatively unaffected . This observation formed the basis of a selection procedure that was used to isolate mutants that are partially resistant to DBP . One such mutant, strain 6204, is constitutive for DBP transport, exhibits a particularly high degree of cold resistance, has the same doubling time as the parent, and is similar to the parent strain in terms of incorporation of DBP into the lipid fraction . Glycerol-3-phosphate and phosphatidylglycerol phosphate synthetases obtained from strain 6204 and its parent were identical in terms of DBP recognition . The parent strain is killed when incubated in the presence of a combination of 70 microM rac-DBP and 0.25% deoxycholate, whereas strain 6204 continues to grow, albeit more slowly, in the presence of this combination . Strain 6204 can be distinguished from the parent strain on agar plates (low phosphate minimal medium with glucuronate as the sole carbon source) containing 15 microM rac-DBP . The insertion of Tn10 near the 6204 mutation has facilitated genetic manipulations . All phenotypic effects attributed to strain 6204 appear to be due to a single mutation . Genetic analysis indicates that Tn10, inserted near the gene responsible for DBP resistance, maps in the vicinity of 27 min . Three-factor crosses reveal a gene order of hemA-Dbpr-Tn10(zch)-trp . The only gene for phosphoglyceride metabolism known to map in this region is the gene associated with cardiolipin synthetase, cls . Genetic results suggest that the mutation responsible for DBP resistance maps in or very near cls . Analysis of the lipids isolated from untreated strain 6204 (and from each of the transductants prepared by P1 vir-mediated transfer of DBP resistance of wild-type strains) reveals that cardiolipin synthesis is defective . These results strongly suggest that the mutation responsible for DBP resistance has its primary effect on cardiolipin synthesis . To further test this hypothesis, strains with an authentic cls mutation were constructed and examined for resistance to DBP . These strains had growth properties that were identical with those of strain 6204 . Wild-type strains and mutants defective in cardiolipin synthesis were treated with DBP and 20 mM magnesium or calcium chloride . Simultaneous treatment of either cell type with DBP and divalent cation not only failed to stimulate growth but, quite the contrary, had a marked synergistic growth inhibitory effect.

J Bacteriol, 1984 Mar, 157(3), 772 - 8
Structural gene for the phosphate-repressible phosphate-binding protein of Escherichia coli has its own promoter: complete nucleotide sequence of the phoS gene; Surin BP et al.; The complete nucleotide sequence of the phoS gene, the structural gene for the phosphate-repressible, periplasmic phosphate-binding protein Escherichia coli K-12, was determined . The phosphate-binding protein is synthesized in a precursor form which includes an additional N-terminal segment containing 25 amino acid residues, with the general characteristics of a signal sequence . The amino acid sequence derived from the nucleotide sequence shows the mature protein to be composed of 321 amino acids with a calculated molecular weight of 34,427 . The phoS gene is not part of an operon and is transcribed counterclockwise with respect to the E . coli genetic map . A promoter region has been identified on the basis of homology with the consensus sequence of other E . coli promoter regions . However, an alternative promoter region has been identified on the basis of homology with the promoter regions of the phoA and phoE genes, the structural genes for alkaline phosphatase and outer-membrane pore protein e, respectively.

Z Naturforsch {C}, 1984 Mar-Apr, 39(3-4), 293 - 9
Isolation and characterization of tributyltin resistant mutants of Escherichia coli; Singh AP et al.; Two classes of tributyltin (TBT) resistant, spontaneous mutants of Escherichia coli K-12 were isolated, using a cytochrome containing (W 1485) and a cytochrome deficient ( SASX76 ) strain . In contrast to the cytochrome sufficient strain, the cytochrome deficient strain was found to be fifty times more sensitive to TBT . The class I mutants, isolated from strain W 1485, also showed cross-resistance to triphenyltin (TPT) . As compared to its wild type parent, the TBT-resistant mutants exhibited mucoid colony type, aberrant cell morphology and reduced uptake of TPT . Based on these results, it was suggested that the resistance of class I mutants to TBT may be associated with above mentioned alterations . The class II TBT-resistant mutants were isolated from the cytochrome deficient strain, SASX76 . In comparison to class I mutants, these class II mutants were found to have TBT-resistant membrane bound adenosine triphosphatase (ATPase) which may account for their resistance to TBT.

Ann Microbiol (Paris), 1984 Mar-Apr, 135A(2), 181 - 90
Screening for lamB missense mutations which alter all lambda receptor activities in Escherichia coli K12; Braun-Breton C; Previously described missense mutations in gene lamB, the structural gene for the lambda receptor in Escherichia coli K12, affected only some of the activities of this multifunctional protein . We isolated lamB mutations, some of which could be of the missense type, and which affected all of the activities of the LamB protein . Among 8 of these mutations, 5 affected the stability of the LamB protein and 3 did not markedly decrease the amount of LamB protein in the mutant strains . In these 3 cases, the mutated LamB proteins were recovered with the envelope of the mutants . We briefly discuss the nature of these mutations and their possible effects on LamB protein structure and location.

Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1375 - 9
Autodigestion of lexA and phage lambda repressors; Little JW; Proteolytic cleavage of lexA repressor is an early step in derepression of the SOS regulatory system of Escherichia coli . In vivo and in vitro data have indicated a role for recA protein in this specific proteolytic reaction . I show here that, under certain conditions, specific in vitro cleavage of highly-purified lexA protein can take place in the absence of recA protein . This autodigestion reaction cleaved the same alanine-glycine bond as did the recA-dependent cleavage reaction . Several lines of evidence argued that it was not due to a contaminating protease activity . Autodigestion was stimulated by alkaline pH . It occurred in the presence of EDTA but was stimulated several fold by the presence of Ca2+, Co2+, or Mg2+ . The reaction appeared to be first-order, and its rate was independent of protein concentration over a wide range, strongly suggesting that it is intramolecular . Purified phage lambda repressor also broke down under similar conditions to yield products like those resulting from recA protein action . Phage lambda repressor broke down at a far slower rate than did lexA, as previously observed in the recA-catalyzed in vitro reaction and in vivo . This correlation between the two types of cleavage also extended to the reactions with mutant repressor proteins; taken together with the site specificity, it suggests that autodigestion and recA-dependent cleavage follow, at least in part, a similar reaction pathway . These findings indicate that specific cleavage of lexA protein can be catalyzed by the protein itself and suggest that recA protein plays an indirect stimulatory role, perhaps as an allosteric effector, in the recA-dependent reaction, rather than acting directly as a protease . The protease active site and the recA-recognition site lie in the central or COOH-terminal portion of the lexA protein, since a tryptic fragment containing these portions of lexA protein could take part in both reactions.

J Bacteriol, 1984 Mar, 157(3), 839 - 45
Isolation and analysis of two Escherichia coli K-12 ilv attenuator deletion mutants with high-level constitutive expression of an ilv-lac fusion operon; Bennett DC et al.; A lysogenizing lambda phage, lambda dilv-lac11, was constructed to carry an ilvD-lac operon fusion . Expression from the phage of the ilvE and lacZ genes is controlled by an intact ilv control region also carried by this phage . Two spontaneous mutants of lambda dilv-lac11 that have high-level constitutive expression of the ilv-lac fusion operon were isolated by growth on a beta-chloroalanine selective medium . The mutants were shown by nucleotide sequence determination to contain large deletions (delta 2216, approximately 1.6 kilobases; delta 2219, approximately 1.9 kilobases), which in both cases remove the proposed ilv attenuator terminator . The rest of the ilv leader and promoter region DNA remains intact in these mutants . Deletion 2216 also removed part of the downstream ilvG gene, whereas delta 2219 extended through the entire ilvG gene into the ilvGE intercistronic region . A possible mechanism of deletion formation is discussed.

J Bacteriol, 1984 Mar, 157(3), 815 - 20
A lacZ-ftsZ gene fusion is an analog of the cell division inhibitor sulA; Ward JE Jr et al.; An in-frame lacZ-ftsZ gene fusion under lac control was fortuitously constructed by subcloning an EcoRI fragment that contains approximately 90% of the ftsZ gene . The identity of the gene fusion was confirmed by isolating an amber mutation in the hybrid gene and then using it to reconstruct the ftsZ gene, which now contained an amber mutation . The hybrid protein (ZZ), which does not possess ftsZ activity, contains seven amino acids of lacZ at its amino terminal end, followed by 35,000 daltons of the carboxyl end of the ftsZ protein . Induction of the hybrid protein resulted in a rapid cessation of cell division which could be reversed by removing the lac inducer . This inhibition of division could be prevented by an increased gene dosage of ftsZ or the presence of the sulB allele of ftsZ, which is known to code for an altered but functional ftsZ protein . An increased gene dosage of ftsZ or the presence of the sulB allele of ftsZ is known to overcome sulA-mediated inhibition of division during the SOS response . Thus, our results suggest that ZZ is an analog of sulA and may aid in determining how sulA inhibits cell division.

Biochimie, 1984 Mar, 66(3), 179 - 201
Crystallization of transfer ribonucleic acids; Dock AC et al.; A compilation of crystallization experiments of tRNAs published in literature as well as original results are given and discussed in this paper . Up to now 17 different tRNA species originating from Escherichia coli and from the yeast Saccharomyces cerevisiae have been crystallized . All structural tRNA families are represented, namely the tRNAs with large or small extra-loops and among them the initiator tRNAs . The tRNAs with small variable loops (4 to 5 nucleotides), e.g . tRNAAsp and tRNAPhe, yield the best diffracting crystals . Crystalline polymorphism is a common feature; about 100 different crystal forms have been observed, but only 6 among them enabled structure determination studies by X-ray diffraction . Crystallization strongly depends upon experimental parameters such as the presence of polyamines and magnesium as well as upon the purity and the molecular integrity of the tRNAs . Crystals are usually obtained by vapour diffusion methods using salts (e.g . ammonium sulfate), organic solvents (e.g . isopropanol, dioxane or 2-methyl-2,4-pentane diol) or polyethylene glycol as precipitants . A methodological strategy for crystallyzing new tRNA species is described.

Biophys Chem, 1984 Mar, 19(2), 147 - 61
Mg2+-induced proton release from Escherichia coli ribosome and ribosomal RNA; Hagihara H et al.; Escherichia coli ribosome released protons upon addition of Mg2+ . The Mg2+-induced proton release was studied by means of the pH-stat technique . The number of protons released from a 70 S ribosome in the Mg2+ concentration range 1-20 mM was about 30 at pH 7 and 7.6, and increased to about 40 at pH 6.5 . The rRNA mixture extracted from 70 S ribosome showed proton release of amount and of pH dependence similar to those of the 70 S ribosome but the ribosomal protein mixture released few . This indicates that rRNA is the main source of the protons released from ribosome . The pH titration of rRNA showed that the pKa values of nucleotide bases were downward shifted upon Mg2+ binding . This pKa shift can account for the proton release . The Scatchard plots of proton release from rRNA and ribosome were concave upward, showing that the Mg2+-binding sites leading to proton release were either heterogeneous or had a negative cooperativity . A model assuming heterogeneous Mg2+-binding sites is shown to be unable to explain the proton release . Electrostatic field effect models are proposed in which Mg2+ modulates the electrostatic field of phosphate groups and the potential change induces a shift of the pKa values of bases that leads to the proton release . These models can explain the main features of the proton release.

Mol Biol (Mosk), 1984 Mar-Apr, 18(2), 350 - 7
{Structural studies of translating ribosomes . II . Comparative sedimentation analysis of pre- and post-translocation states}; Baranov VI et al.; Using the solid-phase translation system technique where template poly(U) is covalently coupled to Sepharose through cleavable disulfide bridges translating monoribosomes carrying a polypeptide (polyPhe) of 10 to 20 amino acids long have been isolated . Both pre-translocation state and post-translocation state ribosomes have been obtained . It has been shown that the sedimentation coefficient of the pre-translocation state ribosomes exceeds that of the post-translocation state ribosomes by a magnitude of about 1S . This difference is independent on the sedimentation rate (hydrostatic pressure) in the range of 20 000 to 40 000 rev/min and, most likely, is not a direct contribution of the increase of the particle mass at the expense of an additional tRNA in the pre-translocation state ribosomes . Together with other data, this result suggests that translating ribosomes in the pre-translocation state are more compact than post-translocation state ribosomes.

J Bacteriol, 1984 Mar, 157(3), 971 - 4
Unusual rRNA-linked complex of 50S ribosomal subunits isolated from an Escherichia coli RNase III mutant; Clark MW et al.; We have isolated and characterized complexes of ribosomal subunits from Escherichia coli mutant AB301-105 connected by strands of unprocessed RNA . By electron microscopy of these complexes, the location of the 5' end of 5S RNA was established and the location of the 3' end of 23S RNA was confirmed . We also note that in these complexes insertion of 5S rRNA can proceed without the 23S-5S spacer having been processed.

Plasmid, 1984 Mar, 11(2), 116 - 29
Synthesis of F-pilin polypeptide in the absence of F traJ product; Ippen-Ihler K et al.; The products of a lambda transducing phage (ED lambda 101) which carries a segment of the F tra operon expressing F traA , traL , and traE activity from the lambda leftward promoter were examined using a uv-irradiated host system . After infection of an F- host, products of traE (19,500 Da) and traA (14,000 Da) were detectable among the lambda early proteins synthesized . Infection of an Flac host altered the pattern of polypeptides synthesized by the phage in that the 14,000-Da traA product became barely detectable and was replaced by a polypeptide which migrated at 7000 Da . A derivative of ED lambda 101 carrying the traA1 amber mutation was unable to synthesize either the 14,000-Da polypeptide in F- cells or the 7000-Da polypeptide in Flac cells . The 7000-Da polypeptide derived from ED lambda 101 was synthesized in the absence of traJ product in F- cells coinfected with a second transducing phage which carried a tra operon segment including traQ . It was also a product of ED lambda 134 which expresses genes traA through traH . The 7000-Da polypeptide, like F-pilin, associated primarily with the inner membrane, and could be immunoprecipitated with antiserum prepared against purified F-pili . Analysis of membranes from F- cells infected with ED lambda 101 indicated that the 14,000-Da traA product synthesized under these conditions accumulated in the inner membrane . These results show that both the 14,000-Da traA product might be processed to F-pilin in a traQ -dependent reaction which occurs in or on the inner membrane of the Escherichia coli host . However, the possibility that traQ encodes a regulatory product which affects expression of the traA sequence has not been excluded.

J Bacteriol, 1984 Mar, 157(3), 802 - 8
Molecular cloning of a gene abundantly expressed during fruiting body initiation in Schizophyllum commune; Dons JJ et al.; Complementary DNA was synthesized on polyadenylated RNA from a dikaryotic mycelium of the basidiomycete Schizophyllum commune bearing fruiting body initials . The complementary DNA was cloned into the PstI site of pBR327 by the deoxyguanidylate-deoxycytidylate tailing approach . After transformation into Escherichia coli cells, a differential screening was performed by colony hybridization with complementary {32P}DNA made on the RNAs of the monokaryon and dikaryon strains . Two clones were selected for further analysis by Northern blotting and hybrid release translation . Clone 1D10 hybridized with an mRNA of 775 nucleotides, coding for a polypeptide with an Mr of 15,000 . Although this RNA was present in both monokaryotic and dikaryotic mycelia, its concentration appeared to change considerably over time and with different cultivation conditions . This mRNA is probably the most abundantly expressed sequence in S . commune . Clone 1G2 and its homologs hybridized with an mRNA of 650 nucleotides, coding for a polypeptide with an Mr of 13,000 . This gene was exclusively expressed in the dikaryon strain . In liquid-grown cultures, the concentration of this mRNA was low but increased ca . 20-fold during the establishment of fruiting body primordia . A chromosomal fragment of 9 kilobase pairs which contained the 1G2 gene was cloned into pBR327 and used as a probe in Northern blot hybridization . It was found that surrounding sequences were not expressed at the same time or to the same extent as the 1G2 gene.

Boll Ist Sieroter Milan, 1984 Mar, 63(1), 77 - 82
Flavonoids and hepatic cyclic monophosphates in liver injury; Scevola D et al.; Among the large spectrum of pharmacological activities of flavonoids, play an important role the recently investigated properties involving the arachidonic acid metabolism . In order to clarify the mechanisms of "cytoprotection" of the 3-palmitoyl-(+)-catechin (Palm-cat), a new flavonoid compound (C31 H44 O7) we have studied in experimental hepatitis of the rat, induced by Galactosamine (Ga1N) and E . coli 055:B 5 endotoxin (LPS), hepatic cAMP and cGMP, transaminases, bilirubin and endotoxemia . The Palm-cat significantly increases cyclic-GMP levels in the liver, whereas reduces or slightly modifies the cAMP . Transaminases and bilirubin values increase both in controls and flavonoid treated rats . The flavonoid significantly decreases the frequency of endotoxemia . These effects suggest that RES and hepatocytes functions, immune and inflammatory response can be affected in liver disease by flavonoids via cyclic nucleotides regulation.

Biochemistry, 1984 Feb 28, 23(5), 922 - 7
Conformation of double-stranded DNA during agarose gel electrophoresis: fractionation of linear and circular molecules with molecular weights between 3 X 10(6) and 26 X 10(6); Serwer P et al.; To answer several questions concerning the mechanisms of DNA fractionation during agarose gel electrophoresis, the electrophoretic mobility (mu) of double-stranded DNA has been measured as a function of (1) DNA topological conformation (linear, open circular, closed circular) and molecular weight (Mr) (molecular weights were between 2.9 X 10(6) and 26.4 X 10(6)), (2) gel concentration (A) and temperature, and (3) voltage gradient . It was found that mu extrapolated to an A of 0 (mu 0') was independent of DNA conformation . The effect of temperature was to raise values of mu 0' in inverse proportion to buffer viscosity . Semilogarithmic mu vs . A plots for linear DNAs had curvature that was opposite to the curvature for spherical particles (plots for linear DNA were concave) . As A approached 0, the plots became increasingly linear . For the larger DNAs, the negative slope (KR) in the region of linearity was decreased as voltage gradient increased . These and other data indicate deformation of linear DNA random coils during agarose gel electrophoresis . The data suggest both an asymmetric and a symmetric collapse of linear DNA random coils during agarose gel electrophoresis . However, end-first migration of linear DNA, previously suggested by others, does not explain the data . The semilogarithmic mu vs . A plots were more linear for closed and open circular DNAs than they were for linear DNAs . Closed circular DNAs had KR's lower than KR's of either open circular or linear DNAs of the same molecular weight . At the lower voltage gradients, open circular DNA had the same KR as linear DNA of the same molecular weight . However, as voltage gradient and molecular weight increased, the KR of open circular DNA became smaller than the KR of linear DNA (of the same molecular weight) . This and the concave curvature of semilogarithmic mu vs . A plots for linear DNA resulted in a previously unreported reversal of the relative migration of linear and open circular DNAs as A increased.

Biochemistry, 1984 Feb 28, 23(5), 928 - 36
Oxidative destruction of DNA by the adriamycin-iron complex; Eliot H et al.; The 2:1 adriamycin-Fe(III) complex is able to bind to DNA and to catalyze its oxidative destruction . The binding of the drug-metal complex to DNA is indicated by characteristic spectral changes which are different from those seen with adriamycin intercalation and by the propensity of the drug-metal complex to precipitate DNA . Furthermore, intercalated adriamycin appears not to be available for iron binding . The resulting ternary complex is quite stable: it is not disrupted by incubation in the presence of EDTA and can be isolated by using Sephadex G-50 column chromatography . Disruption of the ternary complex requires vigorous conditions (extraction with phenol at 60 degrees C) . The adriamycin-iron complex in free solution has the capacity to catalyze the reduction of oxygen by thiols . The DNA-bound drug-metal complex preserves this capacity over a wide range of complex/DNA ratios . As a consequence of this thiol-dependent oxygen reduction, DNA is cleaved . This thiol-dependent DNA cleavage has been shown to require hydrogen peroxide as an intermediate product . These results have led us to propose that the thiol-dependent DNA cleavage reaction has two stages involving (1) reduction of oxygen leading to hydrogen peroxide and then (2) peroxide-dependent DNA cleavage . An unusual property of this reaction is that the cleavage is not random but gives rise to a defined 2300 base pair fragment.

Biochemistry, 1984 Feb 28, 23(5), 1015 - 22
Relationships between the Na+-H+ antiport activity and the components of the electrochemical proton gradient in Escherichia coli membrane vesicles; Bassilana M et al.; The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient . An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents . The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline) . The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate . At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value . The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1984 Feb 25, 173(2), 177 - 209
Transcription of a gene cluster coding for two aminoacyl-tRNA synthetases and an initiation factor in Escherichia coli; Wu TH et al.; The alpha and beta subunits of phenylalanyl-tRNA synthetase are encoded by the pheS and pheT genes, respectively . These genes are clustered closely together with the genes for threonyl-tRNA synthetase (thrS) and translation initiation factor IF3 (infC); the gene order is thrS infC pheS pheT . We have used two methods to study the transcription pattern within this cluster . The first was the in vitro transcription of DNA restriction fragments with purified RNA polymerase, followed by fractionation of the RNA products by polyacrylamide gel electrophoresis . The second method was the mapping of promoters by means of the "abortive initiation" reaction of McClure and co-workers . This procedure consists of the incubation of RNA polymerase with DNA restriction fragments plus one nucleoside monophosphate and one {alpha-32P}nucleoside triphosphate; the polymerase synthesizes dinucleotide products of known sequence at promoter sites in the DNA . We found that transcription initiated at an internal site within infC (designated P1), and at two promoter sites between infC and pheS (designated P2 and P3) . Transcription terminated at two sites about 200 nucleotides apart, located just before pheS . The initiation and termination signals were arranged so as to yield a nested set of overlapping transcripts . At the P1 promoter, transcription initiated with G-C, at P2 with A-C and sometimes A-G, and at P3 with G-U . Promoter activity was also found in a 3000-base interval that includes the start of the thrS gene; eight or nine transcripts (not mapped in detail) were observed, which started with at least four different dinucleotides . All major initiation sites in the gene cluster represented purine starts, although some pyrimidine initiation was observed in trace amounts . No promoter activity was found between pheS and pheT with either of the two techniques; this observation supports the conclusion that these genes are co-transcribed . No evidence was found for any promoter between the termination sites and the beginning of the pheS gene . It is suggested that one of the terminators is an attenuation site controlling the extension of transcription into pheS and pheT . Attenuation may explain the observed regulation of phenylalanyl-tRNA synthetase by the amino acid supply.

J Biol Chem, 1984 Feb 25, 259(4), 2457 - 65
Cooperativity in highly aggregated enzyme systems . A slow transition model for the pyruvate dehydrogenase complex from Escherichia coli; Bisswanger H; Three models are compared describing cooperative phenomena in enzymatic reactions in order to explain sigmoidal saturation curves found with the pyruvate dehydrogenase complex from Escherichia coli: the concerted model, the sequential model, and the slow transition model . Both the concerted and the sequential model were considered especially with regard to the increasing number of identical interaction subunits (protomers) in order to get close to the situation found with the pyruvate dehydrogenase complex which consists of 24 protomers . Applying the sequential model to a great number of protomers results in a weak increase of the Hill coefficient, while, in addition to this effect, the concerted model drastically shifts the sigmoidal range of the saturation function to very low ligand concentrations . Such shift is seen with saturation curves of pyruvate and thiamine disphosphate with the pyruvate dehydrogenase complex and a good fit with theoretical curves derived from the concerted model is obtained . However, subcomplexes with a reduced number of protomers exhibited no change in saturation behavior, thus providing evidence against concerted conformational changes of all subunits of the enzyme complex . A scheme for the initial reaction of the pyruvate dehydrogenase complex based on slow transitions is presented and a rate equation has been derived . Ordered binding of thiamine diphosphate and pyruvate and a ligand-induced slow transition between a less active and a fully active enzyme form has been assumed . The curves simulated with this model are in agreement with all essential kinetic data, which are observed with the pyruvate dehydrogenase complex: the atypical shape of the saturation curves of pyruvate and thiamine diphosphate, the respective Hill coefficients and Michaelis constants, the hyperbolic binding behavior of thiamine diphosphate, and the inhibition pattern found for acetyl coenzyme A.

J Biol Chem, 1984 Feb 25, 259(4), 2407 - 10
Inosine biosynthesis in transfer RNA by an enzymatic insertion of hypoxanthine; Elliott MS et al.; An enzyme was discovered which incorporates hypoxanthine into mature tRNA macromolecules . This enzyme is postulated to be similar to tRNA-guanine ribosyltransferase which inserts 7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl )-7-deazaguanine into the first position of the anticodon of four tRNAs . The hypoxanthine-incorporating enzyme has been assayed in extracts of rat liver and cultured human leukemia cells and it has been resolved from tRNA-guanine ribosyltransferase by DEAE-cellulose column chromatography . The enzyme assay is based on the incorporation of radiolabeled hypoxanthine into unfractionated heterologous tRNA and the reaction rate is proportional to the amount of added enzyme extract . Hydrolysis of the radiolabeled tRNA and analysis of the nucleoside composition yields inosine (the nucleoside of hypoxanthine) as the only radiolabeled product . It is proposed that the enzyme, a tRNA-hypoxanthine ribosyltransferase, is responsible for the biosynthesis of inosine in the anticodon wobble position of specific tRNAs, resulting in greatly expanded codon recognition by these tRNAs.

J Biol Chem, 1984 Feb 25, 259(4), 2252 - 6
The interaction between Escherichia coli aspartokinase-homoserine dehydrogenase and 3-acetylpyridine-adenine dinucleotide phosphate (reduced), an analog of NADPH; Muller K et al.; The interaction of 3-acetylpyridine-adenine dinucleotide phosphate, a structural analog of NADPH, with aspartokinase-homoserine dehydrogenase has been studied by fluorescence and activity measurements . This analog binds to the same site and with the same affinity as does the natural coenzyme . Also, the binding of homoserine to the dehydrogenase site or that of threonine to the regulatory site is the same whether NADPH or its analog is bound to the enzyme . So NADPH and its analog appear as equivalent in the formation of various stable enzyme-ligand(s) complexes . The analog resembles NADPH enough so that it is a substrate that the enzyme can use to reduce aspartate semialdehyde; the maximum velocity of this dehydrogenase reaction is however reduced by 90% as compared to that with NADPH . It seems as if one of the catalytic steps is affected by the replacement of a--CONH2 group by--COCH3 . Another difference between the two coenzymes is that the reaction with the analog is insensitive to threonine, whereas that with NADPH is inhibited . The lack of inhibition is not due to a lack of binding, but rather to a difference in the ternary complexes composed of enzyme, coenzyme, and substrate . A possible relationship between the inhibition by threonine and the mechanism of the dehydrogenase reaction is thus suggested by this comparison between NADPH and its analog.

J Biol Chem, 1984 Feb 25, 259(4), 2243 - 51
Reconstitution of Escherichia coli thioredoxin reductase with 1-deazaFAD . Evidence for 1-deazaFAD C-4a adduct formation linked to the ionization of an active site base; O'Donnell ME et al.; The flavin prosthetic group (FAD) of thioredoxin reductase has been replaced by 1-deazaFAD (carbon substituted for nitrogen at position 1) . Reduction of 1-deazaFAD-thioredoxin reductase by four electrons proceeds in two stages having midpoint potentials that are separated by 0.063 V . Two-electron reduced 1-deazaFAD-thioredoxin reductase (EH2) has spectral characteristics that are different from both the fully oxidized and fully reduced enzyme . The fluorescence of the 2-electron reduced enzyme shows a mixture of two EH2 species . The spectrum of one EH2 species has a single absorption peak (lambda max, 414 nm; epsilon 414, 8750 M-1 cm-1) which is similar to the spectrum of 1-deazaFAD-C-4a adducts (referred to as the 414-nm absorbing species) . In the other EH2 species the electrons are in the dithiol, and it has an oxidized 1-deazaFAD spectrum (referred to as the 550-nm EH2 species) . The equilibrium between the two EH2 species of 1-deazaFAD-thioredoxin reductase is pH dependent, forming more of the 414-nm absorbing species as the pH is lowered . The pH dependence suggests the presence of an active center base having a pK of 7.41 on the 414-nm EH2 species and a thiol of pK 6.73 on the 550-nm EH2 species . These pK values are similar to the pK values determined for native enzyme having a disulfide or a dithiol (7.59 and 6.98, respectively) . Thus, the pH dependence of the equilibrium between the two EH2 species of 1-deazaFAD-thioredoxin reductase is further evidence for an active site base with an ionization behavior that is linked to the chemical state of the active site disulfide moiety . The nature of the linked ionization is consistent with a thiol base ion pair formed upon disulfide reduction.

J Biol Chem, 1984 Feb 25, 259(4), 2144 - 8
Escherichia coli succinyl coenzyme A synthetase . Inhibition of ATP-stimulated succinate----succinyl coenzyme A exchange at low succinyl coenzyme A concentrations by an ADP trap; Nishimura JS et al.; The hypothesis that Escherichia coli succinyl-CoA synthetase functions by a cooperative alternating sites mechanism is based largely on the results of {18O}phosphate exchange experiments (Bild, G . S., Janson, C . A., and Boyer, P . D . (1980) J . Biol . Chem . 255, 8109-8115) . In those experiments, {18O}Pi----succinate (predominantly) exchange appeared to proceed at greater rates (relative to the apparent amount of succinyl-CoA released from the enzyme) at low ATP in incubations containing ATP, CoA, succinate, {18O}Pi, 0.48 M hydroxylamine (as a succinyl-CoA trap), and a pyruvate kinase-lactate dehydrogenase ADP trap . The conclusion arrived at was that succinyl-CoA binding at one site was inversely related to ATP binding at the second site . Thus, the residence time of succinyl-CoA binding at a site would be longer at lower ATP concentrations . Our experiments show that, under the incubation conditions described by Bild et al . (Bild, G . S., Janson, C . A., and Boyer, P . D . (1980) J . Biol . Chem . 255, 8109-8115), succinyl-CoA is not efficiently trapped . Thus, at ATP concentrations from 3.6 to 150 microM, concentrations of succinyl-CoA from 13 to 78 microM were observed . Succinate----succinyl-CoA exchange reactions carried out in this range of ATP and subsaturating succinyl-CoA concentrations were found to be markedly inhibited by the addition of the ADP trap . This inhibition was more pronounced at higher ATP levels . At a saturating succinyl-CoA concentration (1.5 mM), addition of the ADP trap actually stimulated succinate----succinyl-CoA exchange . Under these conditions, ATP----Pi exchange was greatly depressed . These results are interpreted as follows . ADP is required for optimal binding of succinyl-CoA, but only when the latter is present at subsaturating concentrations; thus, the ADP trap inhibits the reaction . ATP exerts its stimulatory action on succinate---- succinyl-CoA exchange through an "other site" effect, i.e . in binding to the noncatalytic site of succinyl-CoA synthetase, it facilitates binding and release of succinyl-CoA at the catalytic site . ATP may also exert negative effects by inhibiting other site binding of ATP or by interfering with same site succinyl-CoA binding at subsaturating concentrations of the latter . These data support the notion that a half-sites mechanism applies to succinyl-CoA synthetase, but suggest that the {18O}Pi----succinate exchange data which have been instrumental in development of the cooperative alternating sites hypothesis should be re-evaluated.

J Biol Chem, 1984 Feb 25, 259(4), 2124 - 9
D-1-amino-2-propanol:NAD+ oxidoreductase . Purification and general properties of the large molecular form of the enzyme from Escherichia coli K12; Kelley JJ et al.; Growth of Escherichia coli K12 under relatively anaerobic conditions in a medium containing casein hydrolysate, 0.8% glycerol, and 0.8% hydroxyacetone has been found to induce the level of D-1-amino-2-propanol oxidoreductase activity 50- to 100-fold over that in cells grown in casein hydrolysate alone or with 0.8% glycerol added . A large molecular weight form of this oxidoreductase (designated Form L) has been purified to apparent homogeneity in good yield by three simple steps designed to obviate its conversion to a smaller species . The molecular weight of native Form L and its basic subunit are 417,000 +/- 20,700 and 50,500 +/- 2,770, respectively; hence Form L would appear to consist of eight identical subunits . The pH activity profile for Form L shows one optimum in the range of 8.3 to 8.6 and another at pH 10.0 to 10.2 . This form of the oxidoreductase has no apparent requirement for added metal ions (rather, numerous divalent transition metal ions are strongly inhibitory) or thiol compounds; it catalyzes the oxidation of several vic-glycols but is completely stereospecific for the D-isomer of 1-amino-2-propanol, utilizes only NAD+ as cosubstrate in the oxidation reaction (Km for NAD+ with DL-1-amino-2-propanol = 1.23 mM), but both NADH and NADPH serve as cosubstrate in the reduction of hydroxyacetone . Oxidoreductase activity of Form L is highly sensitive to inhibition by Hg2+, p-mercuribenzoate, or dithiodipyridine; inhibition by the latter two compounds is completely reversed by adding a thiol in excess.

J Biol Chem, 1984 Feb 25, 259(4), 2656 - 61
Proline dehydrogenase from Escherichia coli K12 . Reconstitution of a functional membrane association; Graham SB et al.; Soluble and membrane associated proline dehydrogenase differ in catalytic properties . The soluble enzyme transfers electrons from L-proline to exogenous electron acceptors . It has a high Km for L-proline (105 mM) and is insensitive to the respiratory chain inhibitors 5-ethyl-5-isopentyl-barbituric acid and cyanide . The membrane-associated enzyme transfers electrons from L-proline to O2 via the respiratory chain, with coupled transmembrane proton translocation . It has a low Km for L-proline (3 mM) and is inhibited by 5-ethyl-5-isopentyl-barbituric acid and cyanide . Proline:O2 oxidoreductase activity identical to that of native membranes can be reconstituted using enzyme purified in the absence of detergent and enzyme deficient membranes from a putA mutant strain . Reassociation of the enzyme with the membrane is an autocatalytic process that requires the simultaneous presence of L-proline, MgCl2, enzyme, and membranes . It can be monitored by observing the chromogenic reaction of delta 1-pyrroline carboxylic acid with o-aminobenzaldehyde . Reduction of membrane components or generation of a protonmotive force is apparently required to promote enzyme-membrane association or to activate electron transfer . The reconstituted activity is a saturable function of enzyme concentration at constant membrane concentration and the activity approached is 20-fold higher than that of native membranes isolated from bacteria that have been induced for proline utilization . It is therefore unlikely that saturation of the available membrane binding sites is achieved during induction of the put genes in vivo.

J Biol Chem, 1984 Feb 25, 259(4), 2268 - 73
Rho-dependent termination and concomitant NTPase activity requires a specific, intact RNA region; Sharp JA et al.; We have investigated the specific DNA and RNA requirements for rho-dependent transcription termination in vitro . As a model, we have used templates containing the rho-dependent terminator of the Escherichia coli trp operon, trp t' . Templates containing the trp t' region direct specific rho-dependent termination in vitro, with concomitant stimulation of the rho NTPase activity, and deletion of the trp t' region results in templates that do not induce rho-dependent termination or rho NTPase activity . Addition of ribonuclease T1 to transcription reactions specifically eliminated transcription termination and rho NTPase activity . These results demonstrate the requirement for a specific RNA component within the trp t' transcript necessary for NTPase activation and rho-dependent transcription termination . Active transcription is not a prerequisite for rho NTPase activation; trp t' RNA (rho-terminated transcripts) and read-through transcripts, which contain the trp t' region, activated the rho NTPase when rho was added after inhibition of transcription . As is true for synthetic polynucleotides known to activate the rho NTPase, the trp t' region has few G residues . This reduces the potential for the formation of stable secondary structures in the RNA transcript, and may be one determinant of sites specifying rho-dependent termination of transcription . The implications of this are discussed in the light of the lack of significant sequence homologies between rho-dependent transcription termination sites.

J Biol Chem, 1984 Feb 25, 259(4), 2031 - 4
Molecular cloning of cDNA for rat acyl-CoA oxidase; Osumi T et al.; Poly(A+) RNA was prepared from hepatic free polysomes of rats which had been fed di(2-ethylhexyl) phthalate for the induction of peroxisomal beta-oxidation enzymes . This preparation was enriched for the mRNAs of these enzymes by sucrose density gradient centrifugation, and used for the synthesis of double-stranded cDNA . Recombinant plasmids were constructed from the cDNA and pBR322 by dG X dC-tailing method and used for the transformation of an Escherichia coli strain, chi 1776 . By differential colony hybridization using {32P}cDNA of partially purified liver poly(A+) RNA from induced and noninduced rats as probes, and then by hybridization-selected translation, we obtained two clones with cDNA inserts which specifically selected acyl-CoA oxidase mRNA . On Northern blotting, both cDNA inserts hybridized to 3.8-kilobase RNA which was increased about 10-fold by di(2-ethylhexyl) phthalate treatment of the rats . The cleavage maps of the cDNA inserts showed they overlap with each other . We conclude that the above two recombinant plasmid clones contain cDNA sequences for rat acyl-CoA oxidase.

J Biol Chem, 1984 Feb 25, 259(4), 2594 - 601
The interaction of Escherichia coli replication factor Y with complementary strand origins of DNA replication . Contact points revealed by DNase footprinting and protection from methylation; Greenbaum JH et al.; A defined region of the viral (+) strand of phi X174 and of each strand of pBR322 DNA serves as an effector for the ATPase activity of replication factor Y from Escherichia coli . These loci can also function as complementary strand origins of DNA replication in a single-stranded circular leads to replicative form pathway whose protein requirements are characteristic of phi X174 DNA . Despite this functional similarity, these three sites possess no extensive sequence homology . To uncover a possible common structural determinant, factor Y recognition sequences were treated with pancreatic DNase or dimethyl sulfate in the presence and absence of this replication protein . When factor Y was present, the action of the nuclease was altered in a similar manner on each of the three templates, indicating that factor Y was bound to the entire length of its effector site . Factor Y-mediated modification of the dimethyl sulfate methylation patterns gave evidence of specific, tight protein-DNA contacts . Protection maps, devised by plotting the results of the methylation and footprinting experiments on duplex structures, suggest that tertiary interactions are either involved in the formation of a factor Y effector site or are induced by the binding of the protein.

Biochim Biophys Acta, 1984 Feb 24, 781(1-2), 64 - 75
Substrate and inhibitor specificity of tRNA-guanine ribosyltransferase; Farkas WR et al.; We have tested as inhibitors or substrates of tRNA-guanine ribosyltransferase (EC 2.4.2.29) a number of compounds, including derivatives of 7-deazaguanine, pteridines, purines, pyrimidines and antimalarials . Virtually all purines and pteridines that are inhibitors or substrates of the rabbit reticulocyte enzyme have an amino nitrogen at the 2 position . In addition the 9 position and the oxygen at the 6 position may be important for recognition by the enzyme . Saturation of the double bond in the cyclopentenediol moiety of queuine reduces substrate activity and queuine analogs that lack the cyclopentenediol moiety, such as 7-deazaguanine and 7-aminomethyl-7-deazaguanine, are relatively poor substrates for the enzyme . While adenosine is not an inhibitor, neplanocin A (an adenosine analog in which a cyclopentenediol replaces the ribose moiety) is a poor inhibitor . The incorporation of 7-aminomethyl-7-deazaguanine into the tRNA of L-M cells results in a novel chromatographic form of tRNAAsp, indicating that L-M cells cannot modify this Q precursor (in Escherichia coli) to queuosine . The specific incorporation of 7-deazaguanine and 8-azaguanine into tRNA by L-M cells also results in novel chromatographic forms of tRNAAsp . With intact L-M cells, the enzyme-catalyzed insertion into tRNA of queuine, dihydroqueuine, 7-aminomethyl-7-deazaguanine, or 7-deazaguanine is irreversible, while guanine or 8-azaguanine incorporation is reversible; suggesting that it is the substitution of C-7 for N-7 which prevents the reversible incorporation of queuine into tRNA.

Nucleic Acids Res, 1984 Feb 24, 12(4), 2171 - 80
TET repressor.tet operator complex formation induces conformational changes in the tet operator DNA; Altschmied L et al.; The structural changes of the tet operator DNA upon binding of the TET repressor protein are examined by circular dichroism . For this purpose a 70 bp DNA fragment was prepared which contains both tet operators . About 67% of the base pairs of this DNA are involved in specific interaction with the TET repressor . A rather large change in the CD of the DNA is induced by binding of the TET repressor . The shape of the CD difference spectrum is similar to the respective difference found for the lac operator DNA upon complex formation with the lac repressor . However, the effect induced by the TET repressor on tet operator DNA seems to comprise both the specific and non-specific effect of the lac repressor on the structure of DNA {Culard, F . and Maurizot, J.C . (1981) Nucl . Acids Res . 9, 5157-5184} . Specificity of binding is confirmed by the lack of any effect of the TET repressor on the CD of a 95 bp lac operator containing DNA fragment, by the reduced mobility of TET repressor.tet operator complexes on polyacrylamide gels under CD conditions, and by a titration experiment of tet operator DNA with TET repressor employing the CD change . The latter experiment reveals a stoichiometry of four TET repressors per tet operon control region.

Nucleic Acids Res, 1984 Feb 24, 12(4), 1977 - 89
Structural alterations of pathologically or physiologically modified DNA; Ciomei M et al.; We have studied the alterations of DNA conformation in in vitro depurinated or methylated topological isomers of the plasmid pAT 153 . Depurination by heat/acid treatment or alkylation by methyl methanesulfonate (pathological modifications) result in DNA unwinding detected as a reduction in the degree of supercoiling of DNA topoisomers as measured by the alteration of electrophoretic mobility on agarose gel . On the contrary, in vitro enzymic methylation at the C-5 position of cytosine (physiological modification) does not measurably alter the tertiary structure of the circular substrates . From the average number of modified sites needed to remove one superhelical twist from each single topoisomer of a population of partially relaxed DNA molecules, we have calculated an unwinding angle smaller than -3.4 degree per methylated purine and of approximately -12.0 degree per apurinic site . These results, together with previously reported values of unwinding by pyrimidine dimers, suggest a possible mechanism of recognition of damaged sites by repair mechanisms that are not single-damage specific.

Nucleic Acids Res, 1984 Feb 24, 12(4), 1791 - 810
Fluorescent labelling of tRNA and oligodeoxynucleotides using T4 RNA ligase; Cosstick R et al.; 3'-O-(5'-phosphoryldeoxycytidyl) phosphorothioate and fluorescent 3'-O-(5'-phosphoryldeoxycytidyl) S-bimane phosphorothioate can be ligated to tRNA by T4 RNA ligase . They are also efficient donors for the enzymatic ligation to oligodeoxynucleotides bearing a 3'-cytidine terminus . Cytidine 3',5'-bisphosphate is also a substrate for the ligation reaction with DNA restriction fragments with a 3'-terminate cytidylic acid residue . Oligo- and polynucleotides with a 3'-phosphorothioate group react readily with electrophiles as exemplified by the reaction with monobromobimane.

Nucleic Acids Res, 1984 Feb 24, 12(4), 2181 - 92
The nucleotide sequence of the Escherichia coli fus gene, coding for elongation factor G; Zengel JM et al.; We have determined the nucleotide sequence of the Escherichia coli fus gene, which codes for elongation factor G . The protein product of the sequenced gene contains 703 amino acids, with a predicted molecular weight of 77,444 . The fus gene shows the nonrandom pattern of codon usage typical of ribosomal proteins and other proteins synthesized at a high level . We have identified several potential promoter sequences within the gene . One of these sequences may correspond to the secondary promoter for expression of the downstream tufA gene (encoding elongation factor Tu) whose activity has been described previously (1,2) . A comparison of the nucleotide and amino acid sequences of elongation factors G and Tu reveals a limited but significant homology between the two proteins within the 150 amino acid residues at their amino-terminal ends.

Nucleic Acids Res, 1984 Feb 24, 12(4), 1875 - 87
Cloning and nucleotide sequence of the simian rotavirus gene 6 that codes for the major inner capsid protein; Estes MK et al.; The nucleotide sequence of the gene that codes for the major inner capsid protein of the simian rotavirus SA11 has been determined . A DNA copy of mRNA from gene 6 was cloned in the E . coli plasmid pBR322 . The full-length gene is 1357 nucleotides long with a 5'-noncoding region of 23 nucleotides and a 3'-noncoding region of 140 nucleotides . The gene contains a single, long, open reading-frame of 1194 nucleotides capable of coding for a protein of 397 amino acids with a molecular weight of 44,816 . The predicted protein product is relatively proline-rich with a net charge at neutral pH of -3.5 . One stretch of 53 amino acids (encoded by nucleotides 327-485) is basic.

Biochim Biophys Acta, 1984 Feb 24, 781(1-2), 45 - 55
Resistance of DNA from filamentous and unicellular cyanobacteria to restriction endonuclease cleavage; Lambert GR et al.; Chromosomal DNA from nine species of filamentous cyanobacteria as diverse as Nostoc, Gloeotrichia and Plectonema is suggested to be extensively modified (methylated) by its resistance to cleavage by a number of restriction endonucleases . A remarkably similar pattern of DNA modification in these species contrasts with the known heterogeneity of their type II restriction endonuclease content . In particular, Nostoc PCC 73102, which lacks detectable sequence-specific endonucleases, is shown to possess extensive DNA modification . The use of isoschizomers demonstrates the presence of a methylase in the filamentous strains analogous to the dam enzyme of Escherichia coli . As a preliminary to assessing the significance of the DNA modification, a study of susceptibility to restriction endonuclease cleavage of the genomes of five unicellular cyanobacteria revealed considerable variation between the different strains . The significance of the DNA modification patterns elucidated is discussed in terms of the restriction endonuclease content and cellular differentiation of the relevant cyanobacterial strains.

Biochim Biophys Acta, 1984 Feb 24, 781(1-2), 81 - 91
Effects of adduct formation on the biological activity of single- and double-stranded øX174 DNA, modified by N-acetoxy-N-acetyl-2-aminofluorene; Lutgerink JT et al.; In order to establish a good quantitative relationship between the number of acetylaminofluorene adducts and the extent of inactivation of DNA, single-stranded (ss) oX174 DNA and oX174 RF DNA were modified to various extents with 3H-labelled N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) and subsequently transfected to Escherichia coli spheroplasts having different repair capabilities . Exponential survival curves were obtained . In the case of ssDNA about one adduct per molecule appears to be lethal . On the other hand only 1 out of 10.2 adducts is found to inactivate RF DNA if tested on wild-type E . coli . However, when assayed on strains deficient in excision repair 1 out of 2.3 adducts leads to inactivation of RF DNA . RecA-dependent postreplication repair only has little influence on these figures . Product analysis of the modified DNAs shows that in RF DNA at least 76% of the interaction products is N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (dGuo-C8-AAF) and at least 6% and at most 12% is 3-(deoxyguanosin-N2-yl)-N-acetyl-2-aminofluorene (dGuo-N2-AAF) . In ssDNA only dGuo-C8-AAF is formed . No apurinic sites could be detected in the modified DNAs . From these results it can be concluded that in RF DNA most of the dGuo-C8-AAF is removed by excision repair . The remaining damage, consisting probably both of dGuo-N2-AAF and unexcised dGuo-C8-AAF, inactivates RF DNA . Inactivation can be explained by a model which shows that only damage in the minus strand of RF DNA inhibits replication and/or transcription.

Nucleic Acids Res, 1984 Feb 24, 12(4), 1863 - 74
Different restriction enzyme-generated sticky DNA ends can be joined in vitro; Hung MC et al.; We describe a simple method for joining the 5'-protruding, single-stranded DNA ends generated by restriction enzymes . The method allows ends with different sequences to be joined and prevents identical ends from being joined . This is accomplished by partially filling the single strands in a controlled reverse transcriptase reaction . Partial filling can create new single-stranded ends that can be ligated to different, partially filled ends . In almost all useful cases, partial filling simultaneously eliminates the self-complementarity of identical ends and thus prevents them from being joined by DNA ligase . Although all possible combinations of partially filled ends were not tested, the tests performed indicate that the method is fairly general . We demonstrate that ends of the same length can be ligated with useful efficiency if they are: 1) one nucleotide long and complementary; 2) two nucleotides long and complementary or have a mismatch (dA:dC) at one position; or 3) three nucleotides long and, in our test, have a dT:dC mismatch at the middle position.

Experientia, 1984 Feb 15, 40(2), 200 - 1
Monoclonal antibodies to L-asparaginase; Kitao T et al.; Five hybridomas secreting monoclonal antibody to E . coli L-asparaginase were isolated . These monoclonal antibodies were classified into 3 different subclasses; Ig G1 (1 clone), Ig G2 (2 clones) and Ig G3 (2 clones) . One of them possessed anti-L-asparaginase neutralizing activity . Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (Kd) ranging between 2.5 X 10(-9) M and 6.3 X 10(-10) M . The monoclonal antibodies should be useful probes for investigation of the enzyme activity.

Eur J Biochem, 1984 Feb 15, 139(1), 47 - 50
Fluorescence-quenching studies on a conformational transition within a domain of the beta 2 subunit of Escherichia coli tryptophan synthase; Chaffotte AF et al.; The fluorescence quenching by acrylamide of the single tryptophan residue in the beta 2 subunit of tryptophan synthase from Escherichia coli K12 is studied for different states of the protein: the native apo-enzyme and holo-enzyme, the nicked apo-protein and holo-protein and the isolated proteolytic fragment F1 corresponding to the N-terminal two thirds of beta 2 . The quenching constants measured are used to estimate the accessibility of the tryptophan residue in these different forms . The results are discussed in terms of conformational transition within the F1 domain, occurring in the presence of the cofactor, pyridoxal 5'-phosphate, in the native enzyme . The proteolytic cleavage of the native enzyme is shown to render the nicked protein unable to undergo this conformational change.

Arch Biochem Biophys, 1984 Feb 15, 229(1), 98 - 103
Energetics of sodium efflux from Escherichia coli; Borbolla MG et al.; When energy-starved cells of Escherichia coli were passively loaded with 22Na+, efflux of sodium could be initiated by addition of a source of metabolic energy . Conditions were established where the source of energy was phosphate bond energy, an electrochemical proton gradient, or both . Only an electrochemical proton gradient was required for efflux from intact cells . These results are consistent with secondary exchange of Na+ for H+ catalyzed by a sodium/proton antiporter.

J Mol Biol, 1984 Feb 15, 173(1), 75 - 91
Torsional rigidity of DNA and length dependence of the free energy of DNA supercoiling; Horowitz DS et al.; By analyzing the Boltzmann populations of DNA topoisomers that differ only in their linking numbers, the dependence of the free energy delta G tau of DNA supercoiling on the linking number alpha has been determined for DNA rings as small as 200 base-pairs (bp) in length . All experimental data can be fitted by the relation delta G tau = K (alpha-alpha)2, where alpha is a constant for a given DNA at a given set of conditions and K is a DNA length-dependent proportionality constant . For DNA rings with length N larger than 2000 bp, K is inversely proportional to N and the product NK is nearly a constant around 1150 RT X bp . For rings smaller than 2000 bp NK increases steadily with decreasing N; for a 200 bp ring NK is 3900 RT X bp . The increase in NK when N decreases can be interpreted as a result of the decrease in the contribution of the fluctuation in the writhing number to the equilibrium distribution in alpha . Assuming that the writhing contribution approaches zero for DNA rings 200 bp in size, the torsional rigidity of the DNA double helix is calculated to be 2.9 X 10(-19) erg cm . In addition, the large value of K for the small circles allows precise calculation of the helical repeat of DNA . For the 210 bp rings, the repeat is measured to be 10.54 bp.

Arch Biochem Biophys, 1984 Feb 15, 229(1), 320 - 8
Sulfhydryl groups of the F1 adenosine triphosphatase of Escherichia coli and the stoichiometry of the subunits; Stan-Lotter H et al.; The distribution and total number of sulfhydryl groups present in the F1 adenosine triphosphatase of Escherichia coli were used to calculate the stoichiometry of the alpha-delta subunits . Titration with 5,5'-dithiobis (2-nitrobenzoate) gave 19.1 +/- 2.2 sulfhydryl groups/mol ATPase . Labeling with {14C}iodoacetamide and {14C}N-ethylmaleimide showed that 11.9, 3.1, 1.9, and 1.8 sulfhydryl groups per molecule of ATPase were associated with the alpha, beta, gamma, and delta subunits, respectively . The epsilon subunit was not labeled . Application of the method of Creighton {Nature (London) (1980) 284, 487-489} showed that 4, 1, and 2 sulfhydryl groups were present in the alpha, beta, and gamma subunits, respectively . This, together with published data for the delta subunit, allowed a subunit stoichiometry of alpha 3 beta 3 gamma delta to be calculated . The presence of four cysteinyl residues in the alpha subunit, as shown by several different methods, does not agree with the results of DNA sequencing of the ATPase genes {H . Kanazawa, T . Kayano, K . Mabuchi, and M . Futai (1981) Biochem . Biophys . Res . Commun . 103, 604-612; N . J . Gay and J . E . Walker (1981) Nucl . Acids Res . 9, 2187-2194} where three cysteinyl residues/alpha subunit have been found . It is suggested that post-translational modification of the alpha subunit to add a fourth cysteinyl residue might occur.

Arch Biochem Biophys, 1984 Feb 15, 229(1), 212 - 9
The epsilon subunit as an ATPase inhibitor of the F1-ATPase in Escherichia coli; Dreyfus G et al.; The isolation of protein ATPase inhibitor was attempted directly from Escherichia coli membrane extracts to examine the possible presence of a Pullman-Monroy-type inhibitor {M . E . Pullman and G . C . Monroy (1963) J . Biol . Chem . 238, 3762-3769} distinct from the epsilon subunit of E . coli ATPase . Purification to homogeneity was achieved in a sequence of steps involving trichloracetic acid precipitation, DEAE-cellulose, Sephadex G75 chromatography, and a terminal isoelectric focusing step . An inhibitory protein was obtained and was identified by its physicochemical and inhibitory properties as the epsilon subunit of E . coli ATPase . The other inhibitory fraction observed in the purification procedure consisted of aggregated epsilon subunits.

J Mol Biol, 1984 Feb 15, 173(1), 131 - 6
Use of monoclonal antibodies in immuno-electron microscopy for the determination of subunit stoichiometry in oligomeric enzymes . There are three alpha-subunits in the F1-ATPase of Escherichia coli; Lunsdorf H et al.; The subunit stoichiometry of oligomeric enzymes can be determined by immuno-electron microscopy using monoclonal antibodies against the individual subunits . Monoclonal antibodies against native F1-ATPase of Escherichia coli were prepared that were specific for the alpha-subunit . The immune complexes of F1 and monoclonal antibodies were isolated . Electron microscopy revealed the presence of three immunoglobulins per molecule of F1-ATPase . This unequivocally demonstrates an alpha 3 stoichiometry for the F1-ATPase of E . coli.

Biochemistry, 1984 Feb 14, 23(4), 655 - 60
Folding of aspartokinase-homoserine dehydrogenase I is dominated by tertiary interactions; Muller K et al.; In the presence of guanidine hydrochloride concentrations above 2 M, aspartokinase-homoserine dehydrogenase I remains sufficiently soluble so that the fluorescence and circular dichroism of the protein can be measured . Both parameters show that, up to 3 M guanidine hydrochloride, the protein exists in a stable folded state which possesses a large amount of secondary structure and buried tryptophan residues . This intermediate species is probably monomeric; it is reversibly unfolded by guanidine hydrochloride concentrations between 3 and 4 M . This folded species is formed rapidly from unfolded protein when the denaturant is diluted out, and this rapid folding step precedes all the reactivation steps described previously . The existence of a stable monomeric and folded intermediate indicates that the tertiary interactions have a major contribution to the stability of the native structure of aspartokinase-homoserine dehydrogenase I . Similar measurements were performed on two complementary nonoverlapping fragments: a kinase fragment corresponding to the N-terminal third and a dehydrogenase fragment corresponding to the C-terminal two-thirds of the polypeptide chain . Both fragments exist in a stable folded state up to 2.5 M guanidine hydrochloride . Both fragments show cooperative unfolding transitions between 2.5 and 4 M denaturant . The stability of the folded state of a given region is about the same in an isolated fragment and in the entire chain of aspartokinase-homoserine dehydrogenase I: indeed, an equimolar mixture of these two fragments and the intact chain would give about the same results . This indicates that folding of the kinase and dehydrogenase regions occurs independent ly with a single subunit of the entire protein.

Biochemistry, 1984 Feb 14, 23(4), 651 - 4
Stepwise inactivation of Escherichia coli aspartokinase-homoserine dehydrogenase I; Muller K et al.; In the range of guanidine hydrochloride concentrations from 0.2 to 1.2 M, aspartokinase-homoserine dehydrogenase I loses its enzymatic properties, both kinase and dehydrogenase activities and their allosteric inhibition by L-threonine . Ligands which stabilize the tetrameric native structure protect the enzyme against inactivation . Under some conditions, all the functional properties do not disappear at the same rate: an intermediate species possessing only the kinase activity can be detected . Several arguments suggest that this partly active intermediate has a monomeric structure . These results show that deactivation of aspartokinase-homoserine dehydrogenase I is a stepwise process, compatible with the reverse of the previously described reactivation {Garel, J.-R., & Dautry-Varsat, A . (1980) Proc . Natl . Acad . Sci . U.S.A . 77, 3379-3383} . The same measurements performed with a monofunctional fragment carrying the dehydrogenase activity show that the loss of dehydrogenase activity is the same whether or not the polypeptide chain is intact or lacks the kinase region; this finding suggests that the protein is composed of independent regions . The influence of protein aggregation in studying unfolding-refolding of oligomeric enzymes is also discussed.

Biochem Biophys Res Commun, 1984 Feb 14, 118(3), 902 - 9
Construction of a cDNA clone for a nuclear-coded subunit of cytochrome c oxidase from rat liver; Parimoo S et al.; A cDNA library for 6S-9S poly(A)-containing RNA from rat liver was constructed in E . coli . Initial screening of the clones was carried out using single stranded 32P-labeled cDNA prepared against poly(A)-containing RNA isolated from immunoadsorbed polyribosomes enriched for the nuclear-coded subunit messenger RNAs of cytochrome c oxidase . One of the clones, pCO89, was found to hybridize with the messenger RNA for subunit VIC . The DNA sequence of the insert in pCO89 was carried out and it has got extensive homology with the C-terminal 33 amino acids of subunit VIC from beef heart cytochrome c oxidase . In addition, the insert contained 146 bp, corresponding to a portion of the 3'-non-coding region . Northern blot analysis of rat liver RNA with the nick-translated insert of pCO89 revealed that the messenger RNA for subunit VI would contain around 510 bases.

FEBS Lett, 1984 Feb 13, 167(1), 93 - 7
The complete sequence of human preprocalcitonin; Le Moullec JM et al.; DNA complementary to mRNA extracted from the thyroid glands of patients suffering from medullary carcinoma of the thyroid (MCT), a calcitonin-producing tumour, was inserted in the Pst site of pBR 322 by G-C tailing . The recombinant plasmids were used to transform Escherichia coli DP 50 . Ampicillin-resistant clones were screened using a 32P-labelled cDNA to mRNA extracted from a case of MCT particularly rich in calcitonin (CT) mRNA . Positive clones were subsequently rescreened using a 32P poly(T) probe . Eighty clones were thus purified, and the inserts obtained by digestion with PstI were subjected to positive hybridization selection with subsequent translation in vitro . An insert stimulating synthesis of the protein and containing restriction sites compatible with the previously published complete sequence of calcitonin mRNA from rat was sequenced . This cDNA insert contained the entire coding region of 426 bp, 70 bp at the 5'-end, and 295 bp upstream from the poly(A) tail . The complete amino acid sequence of human preprocalcitonin could thus be deduced.

FEBS Lett, 1984 Feb 13, 167(1), 59 - 63
Purification of Escherichia coli 50 S ribosomal proteins by high performance liquid chromatography; Kamp RM et al.; The 50 S subunit proteins from the Escherichia coli ribosome were purified by size-exclusion, ion-exchange or reversed phase high performance liquid chromatography (HPLC) avoiding any precipitation or desalting procedures during isolation . Best resolution of this complex protein mixture was achieved by reversed phase chromatography on supports with short alkyl chains and C18 hydrocarbon-bonded phases; 23 out of the 32 proteins from the 50 S subunit were purified as shown by two-dimensional gel electrophoresis, amino acid analysis and direct micro-sequencing . Protein recoveries varied between 25 and 84% as determined by amino acid analysis . Ribosomal proteins of other organisms can be separated under similar conditions.

Nucleic Acids Res, 1984 Feb 10, 12(3), 1697 - 706
A new experimental approach for studying the association between RNA polymerase and the tet promoter of pBR322; Bertrand-Burggraf E et al.; In order to follow the kinetics of the initiation of transcription by the E . coli RNA polymerase, we have used the procedure of abortive initiation as described by Mc Clure (1980) (7) . In place of radioactive labeling we have taken advantage of a fluorescent probe (UTP gamma ANS) to obtain fast and accurate determinations of the rate of transcription and to deduce from kinetic equations both the binding constant (KB) and the rate of isomerization (k2) which characterize the classical two-step model . This analysis was applied to the tet promoter of pBR322 in a linearized plasmid DNA and was studied in function of temperature (from 25 degrees C to 37 degrees C) and of pH (from 6 to 8.3) . The association is entropy driven (delta H degrees = 29 Kcal/mole and delta S degrees = 130 e.u.) . The activation energy of isomerization is 13 Kcal/mole . Both k2 and k-2 are increasing with pH . The insensitivity to pH of the KBK2 product could be tentatively explained in terms of the processive aspect of the polymerase binding to its specific site.

Nucleic Acids Res, 1984 Feb 10, 12(3), 1447 - 61
The sequence of the tms transcript 2 locus of the A . tumefaciens plasmid pTiA6 and characterization of the mutation in pTiA66 that is responsible for auxin attenuation; Sciaky D et al.; The incorporation of Ti plasmid sequences, the T-DNA, into the genomes of dicotyledenous plants causes the formation of tumors . Here we report the nucleotide sequence of one of the T-DNA "oncogenes", the transcript 2 gene of pTiA6 and we further characterize the 2.7 Kb element that has spontaneously inserted into this gene in plasmid pTiA66 . The results indicate that the transcript 2 portion of the T-DNA has an open reading frame that could encode a polypeptide of 49.8 Kd . The open reading frame is surrounded by sequences that typically have roles in eucaryotic gene expression . Nucleotide sequence and Southern blot analysis also indicates that the 2.7 Kb insert in the transcript 2 gene of pTiA66 is located within the coding sequence of the gene and suggests that the element is an insertion sequence . We designate this element, IS66.

J Biol Chem, 1984 Feb 10, 259(3), 1951 - 7
Promoter selectivity of Escherichia coli RNA polymerase . Differential stringent control of the multiple promoters from ribosomal RNA and protein operons; Kajitani M et al.; Using the in vitro mixed transcription system (Kajitani, M., and Ishihama, A . (1983) Nucleic Acids Res . 11, 671-686; Kajitani, M., and Ishihama, A . (1983) Nucleic Acids Res., 11, 3873-3889) we examined the effect of guanosine 3'-diphosphate, 5'-diphosphate (ppGpp), the chemical mediator of stringent control, on transcription of various Escherichia coli DNA fragments, each carrying a single specific promoter . We found that ppGpp inhibits transcription of stringently controlled genes, rrnE, rpsA, and rplJ, coding for ribosomal RNA, ribosomal protein S1 and L10, respectively, but not that of trp (tryptophan) and lacUV5 (lactose) genes . Among the multiple promoters of the rrnE and rpsA operons, the upstream promoters, rrnEp1 and rpsAp1, are subject to repression by ppGpp but the downstream promoters, rrnEp2 and rpsAp3, are insensitive . Taking these facts and the intrinsic strength of the respective promoters together, we suggest that the multiple promoters within the single and same operons play different physiological roles and are regulated by independent mechanisms . The inhibition by ppGpp takes place even after formation of open complexes, suggesting that the RNA polymerase bound to the sensitive promoters is accessible for interaction with ppGpp leading to rapid decay of the open complexes . During this study, we noticed that some promoters including recAp are activated in the presence of ppGpp, raising a possibility that ppGpp has dual effects on the promoter function.

J Biol Chem, 1984 Feb 10, 259(3), 1891 - 5
Turnover of the 4'-phosphopantetheine prosthetic group of acyl carrier protein; Jackowski S et al.; Acyl carrier protein is an essential cofactor in fatty acid biosynthesis, and in contrast to the stability of the protein moiety during growth, its 4'-phosphopantetheine prosthetic group is metabolically active . The biosynthetic incorporation of deuterium into nonexchangeable positions of acyl carrier protein was found to enhance the sensitivity of the protein to pH-induced hydrodynamic expansion . This constitutional isotope effect was exploited to separate deuterated from normal acyl carrier protein by conformationally sensitive gel electrophoresis, thus providing the analytical framework for separating pre-existing (deuterated) from newly synthesized acyl carrier protein in pulse-chase experiments . The rate of acyl carrier protein prosthetic group turnover was found to depend on the intracellular concentration of coenzyme A . At low coenzyme A levels, prosthetic group turnover was four times faster than the rate of new acyl carrier protein biosynthesis but at the higher coenzyme A concentrations characteristic of logarithmic growth, turnover was an order of magnitude slower, amounting to approximately 25% of the acyl carrier protein pool per generation . These observations suggest that the acyl carrier protein prosthetic group turnover cycle may be related to coenzyme A metabolism rather than to lipid biosynthesis.

J Biol Chem, 1984 Feb 10, 259(3), 1781 - 8
2,4-Dienoyl coenzyme A reductases from bovine liver and Escherichia coli . Comparison of properties; Dommes V et al.; 2,4-Dienoyl-CoA reductases, enzymes of the beta-oxidation of unsaturated fatty acids which were purified from bovine liver and oleate-induced cells of Escherichia coli, revealed very similar substrate specificities but distinctly different molecular properties . The subunit molecular weights, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 32,000 and 73,000 for the mammalian and the bacterial enzyme, respectively . The native molecular weights, calculated from sedimentation coefficients and Stokes radii yielded 124,000 for the bovine liver and 70,000 for the bacterial enzyme . Thus, bovine liver 2,4-dienoyl-CoA reductase is a tetramer consisting of four identical subunits . The E . coli 2,4-dienoyl-CoA reductase, however, possesses a monomeric structure . The latter enzyme contains 1 mol of FAD/mol of enzyme, whereas the former reductase is not a flavoprotein . The bovine liver reductase reduced 2-trans, 4-cis- and 2-trans,4-trans-decadienoyl-CoA to 3-trans-decenoyl-CoA . The E . coli reductase catalyzed the reduction of the same two substrates but in contrast yielded 2-trans-decenoyl-CoA as reaction product . Certain other properties of the two 2,4-dienoyl-CoA reductases are also presented . The localization of the reductase step within the degradation pathway of 4-cis-decenoyl-CoA, a metabolite of linoleic acid, is discussed.

J Biol Chem, 1984 Feb 10, 259(3), 1520 - 5
Location of a structural gene for xylose-H+ symport at 91 min on the linkage map of Escherichia coli K12; Davis EO et al.; Mutations in the xylose-H+ transport activity of Escherichia coli K12 were isolated using Mud(ApRlac) . The initial selection was for simultaneous acquisition of ampicillin and xylose resistance in an fda background . Colonies were then screened for xylose-inducible beta-galactosidase and for growth on xylose of their fda+ derivatives . Two of the xylose-positive derivatives were shown to be impaired in xylose-H+ symport in whole cells and in xylose transport into subcellular vesicles . Their xylose transport in whole cells showed increased sensitivity to arsenate . The site of prophage insertion was mapped to 91.4 min on the E . coli genome between pgi and malB . It is proposed that the gene for the xylose-H+ symport system be called xylE.

J Immunol Methods, 1984 Feb 10, 66(2), 245 - 51
Cascade immunization: a method of obtaining polyspecific antisera against crude fractions of antigens; Thalhamer J et al.; Between 20 and 30 precipitation lines are usually obtained by crossed immune electrophoresis of an Escherichia coli cytoplasmic extract against antisera produced against that extract in individual rabbits . With a combination of several such antisera, the number of precipitation lines increases to 30-40 . Nevertheless, extracts as used in this work contain many antigens in addition to those thus detected . Intermolecular immune competition may be avoided by removing strong immunogens from the extracts . The remaining antigens which give no immune response in the primary immunization are used for further immunization . New antibody production against 8-14 additional antigens occurs after one such 'cascade immunization' step . Separation of strong from weak immunogens is performed by preparative CIE and the use of immuno-affinity columns . The procedure is called cascade immunization because it involves repeated removal of antigens and production of further antisera directed against antigens in the remainder.

Nucleic Acids Res, 1984 Feb 10, 12(3), 1671 - 86
2',3'-Dideoxy-3' aminonucleoside 5'-triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases; Chidgeavadze ZG et al.; It is shown that 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates with adenine, guanine, cytosine and thymine bases are effective inhibitors of DNA polymerase I, calf thymus DNA polymerase alpha and rat liver DNA polymerase beta . The effect of the above-mentioned compounds is markedly higher than corresponding action of the well-known DNA synthesis inhibitors arabinonucleoside 5'-triphosphates and 2',3'-dideoxynucleoside 5'-triphosphates . 2',3'-dideoxy-3'-aminonucleoside 5'-monophosphate residues incorporate into the 3'-terminus of the primer and terminate the DNA chain elongation . The possibility of using 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates as terminators for DNA sequencing by the polymerization method is demonstrated.

Nucleic Acids Res, 1984 Feb 10, 12(3), 1657 - 70
Genes encoding alpha-heavy chains of rabbit IgA: characterization of cDNA encoding IgA-g subclass alpha-chains; Knight KL et al.; cDNA molecules encoding rabbit IgA alpha-heavy chains have been synthesized and six of these have been characterized . The complete nucleotide sequence of one cDNA, p 19 (942bp), showed that it encoded all but the N-terminal 57 amino acid residues of the constant region of alpha-chains . The cDNA molecules were subcloned into the expression vector pUC8 and E . coli were transformed . Radioimmunoassay of the molecules synthesized by these clones showed that all six cDNA molecules encoded alpha-chains of the IgA-g subclass . Comparison of the amino acids encoded by the alpha-cDNA with the amino acid sequence of mouse and human alpha-chains showed that although all of the intradomain disulfide bonds appear to be conserved, some positions, probably involved in interchain disulfide bonds, are not conserved . We propose that secretory component is covalently bound to cysteine 299 and/or cysteine 301 of the CH2 domain of mouse and human alpha-chains . The results from Southern blot analysis of genomic DNA with 32P-cDNA suggests that the rabbit genome has multiple C alpha genes.

J Biol Chem, 1984 Feb 10, 259(3), 1807 - 12
Physical and genetic characterization of the melibiose operon and identification of the gene products in Escherichia coli; Hanatani M et al.; We have identified hybrid plasmids carrying the melibiose operon of Escherichia coli in a colony bank of Clarke and Carbon (Tsuchiya, T., Ottina, K., Moriyama, Y., Newman, M., and Wilson, T . H . (1982) J . Biol . Chem . 257, 5125-5128) . Using one of the plasmids as a starting material, the DNA fragments containing the melibiose operon were recloned in a vector pBR322 . Restriction maps were prepared, and several DNA segments were subcloned into pBR322 . Genetic complementation tests and recombination analyses using those plasmids and melA- and melB- mutants as well as biochemical analyses of mel mutants transformed with those plasmids enabled us to determine the physical location of promoter, melA, and melB on the DNA segment . The size of the melAB region was about 3,000 base pairs . Gene products were identified using maxicells harboring plasmids carrying the melibiose operon . The apparent molecular weight of the alpha-galactosidase (coded by melA) was about 50,000 and that of the melibiose carrier (coded by melB) was about 31,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The melibiose carrier was also identified as a 30,000-dalton protein in reconstituted proteoliposomes which possessed melibiose transport activity.

J Biol Chem, 1984 Feb 10, 259(3), 1560 - 5
Identification of the uvrD gene product of Escherichia coli as DNA helicase II and its induction by DNA-damaging agents; Kumura K et al.; Taking advantage of overproduction of the uvrD protein in cells that harbor multicopy plasmids carrying the uvrD gene, we have purified protein to physical homogeneity . The purified protein possesses single-stranded DNA-dependent ATPase and ATP-dependent DNA unwinding activities, and both activities are equally inactivated by antibodies raised against DNA helicase II . Molecular weight (75,000) and chromatographic properties of the uvrD protein are also similar to those of DNA helicase II and DNA-dependent ATPase I (Richet, E., and Kohiyama, M . (1976) J . Biol . Chem . 251, 808-812; Abdel-Monem, M., Durwald, H., and Hoffmann-Berling, H . (1977) Eur . J . Biochem . 79, 39-45) . Thus, it is concluded that the uvrD protein is identical with DNA helicase II and DNA-dependent ATPase I . Expression of the uvrD gene, as assayed by DNA-dependent ATPase activity, was stimulated by exposure of bacteria to nalidixic acid or mitomycin C . No increase in ATPase activity was observed with recA mutant cells, although basic levels of DNA-dependent ATPase activity in recA+ and recA- strains were almost the same . Thus, the uvrD gene is constitutively expressed but also regulated in a recA-dependent fashion.

Nucleic Acids Res, 1984 Feb 10, 12(3), 1563 - 72
Lysogenization of ultraviolet-irradiated Escherichia coli with lambda: dependence on the recA and recB gene products; Salaj-Smic E et al.; The lysogenization of ultraviolet-irradiated Escherichia coli cells by the lambda phage was studied . Genetic analysis indicates that changes in the number of the lysogenized cell during post-UV growth is primarily due to the change in the proteolytic activity of RecA protein . Full proteolytic activity is achieved only in the presence of the functional recB gene product.

J Biol Chem, 1984 Feb 10, 259(3), 1539 - 45
On the fidelity of DNA replication . The accuracy of T4 DNA polymerases in copying phi X174 DNA in vitro; Kunkel TA et al.; The fidelity with which wild type T4 DNA polymerase copies phi X174 amber 3 plus strand DNA at position 587 in vitro has been measured . Synthesis is initiated by hybridizing to the template a HaeIII restriction fragment whose 3'-OH terminus is 83 nucleotides from the amber 3 site . Based on gel electrophoresis of product DNA molecules and genetic marker rescue data, T4 DNA polymerase copies significantly beyond the mutant site . Transfection analysis shows that the A X T leads to G X C mutation at position 587 occurs 10- to 100-fold less frequently with T4 DNA polymerase than with E . coli DNA polymerase I . The aberrant incorporation of cytosine opposite adenine at position 587 by the T4 polymerase alone is occurring at a frequency not greater than about 10(-7) which, for this particular locus, may be similar to the fidelity exhibited by the T4 accessory proteins plus the polymerase comprising the replication complex . A comparison of the accuracy of mutator L56 and antimutator L141 T4 DNA polymerases relative to wild type shows at most a 2- to 4-fold decrease and increase, respectively, in fidelity . When compared to 10- to 1000-fold effects on mutation frequencies that these same mutant alleles have in vivo, these results suggest that the wide range in expression of mutator and antimutator phenotypes in vivo may be dependent on an abnormal interaction of the aberrant DNA polymerases with other protein components of the replication complex.

Biochim Biophys Acta, 1984 Feb 9, 792(2), 192 - 8
The effects of triiodothyronine, hydrocortisone and insulin on lipid synthesis by cultured fibroblasts preincubated in a serum-free medium; Amorosa LF et al.; Studies of lipid metabolism in cell cultures are usually carried out after preincubation of cells in media containing lipoprotein-deficient or delipidated serum . The artifacts produced during delipidation prevent the standardization of assays and the study of the role of hormones on lipid metabolism . We studied the effects of triiodothyronine, hydrocortisone, insulin and their combination on cholesterol and fatty acid synthesis in cultured human skin fibroblasts preincubated for 24 h in an artificial medium (medium A) consisting of equal volumes of Dulbecco's modified Eagle's and Ham's F-12 media enriched with transferrin, biotin and calcium pantothenate . In cells preincubated in medium A the incorporation of acetate to cholesterol and the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase were much lower than in cells preincubated in standard medium containing lipoprotein-deficient serum . Addition of the three hormones caused a marked stimulation of the incorporation of acetate to cholesterol (from 3.1 to 17.7 pmol/min per mg protein), an activity similar to that in cells preincubated in lipoprotein-deficient serum plus hormones . The stimulatory effect of the hormones on HMG-CoA reductase activity was smaller, from 11 to 26 pmol/min per mg protein compared to 83 pmol/min per mg protein in cells preincubated in lipoprotein-deficient serum plus hormones . Most of the stimulatory effect was due to insulin . The lack of coordinate response between these two parameters in cells preincubated in artificial medium could not be explained by (a) stimulation of a post-mevalonate step as measured by the incorporation of mevalonate to cholesterol; (b) the in vitro inactivation of HMG-CoA reductase by phosphorylation: incubation of fibroblast microsomes with Escherichia coli alkaline phosphatase resulted in a decrease in HMG-CoA reductase activity, in contrast to an increase in hepatic microsomes; (c) the presence of inhibitors of HMG-CoA reductase in the microsomal extract . In cells preincubated in medium A the incorporation of acetate to fatty acids and the activities of acetyl-CoA carboxylase and fatty acid synthetase were approximately equal to that of cells preincubated in standard medium containing lipoprotein-deficient serum . Hormones added to medium A caused a stimulation of incorporation of acetate to fatty acids (from 5.1 to 19.8 pmol/min per mg protein), the activity of acetyl-CoA carboxylase (from 494 to 820 pmol/min per mg protein) and of fatty acid synthetase (from 300 to 678 pmol/mg protein) . These values were significantly higher than those obtained in cells preincubated with lipoprotein-deficient serum with or without hormones.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochim Biophys Acta, 1984 Feb 9, 792(2), 123 - 9
Formation of age pigment-like fluorescent substances during peroxidation of lipids in model membranes; Shimasaki H et al.; The formation of age pigment-like fluorescent substances during the lipid peroxidation of model membranes has been studied . Ferrous ion and ascorbate-induced lipid peroxidation of liposomal membranes containing phosphatidylethanolamine led to the formation of fluorescent substances which have characteristics similar to those of compounds derived from the reaction of phosphatidylethanolamine with purified fatty acid hydroperoxides . The fluorescent substances were accumulated in liposomal membranes, whereas thiobarbituric acid-reactive substances formed during lipid preoxidation were immediately released from the liposomal membranes . The thiobarbituric acid-reactive substances free from the membranes were not reactive with amino compounds such as phosphatidylethanolamine in liposomes or glycine in aqueous phase . It was suggested that the products reacting with amino compounds are short-lived, and may be rapidly inactivated after released into aqueous phase . The formation of fluorescent products was inefficient when phosphatidylethanolamine incorporated into the liposomes insensitive to lipid preoxidation was incubated with ferrous ion and ascorbate in the presence of liposomes sensitive to the peroxidation . The results suggest that some products generated from peroxidation-sensitive lipids react with the amino group of phosphatidylethanolamine molecules which are located on the same membranes, forming fluorescent substances . The presence of phosphatidylethanolamine in the membrane suppressed the formation of thiobarbituric acid-reactive substances, suggesting that phosphatidylethanolamine may react with radicals formed and terminate the propagation.

J Mol Biol, 1984 Feb 5, 172(4), 573 - 9
Reduced expression of the isoleucine and valine enzymes in integration host factor mutants of Escherichia coli; Friden P et al.; The level of the isoleucine and valine (Ilv) enzymes specified by the ilvB and ilvGEDA operons is reduced in integration host factor mutants (himA and himD) of Escherichia coli K-12 . Growth inhibition of these strains in minimal medium can be explained by the decreased amounts of one of the Ilv enzymes, acetohydroxy acid synthase I (AHASI) . No growth inhibition, or reduction in AHASI activity, was found in a himA derivative of a mutant strain containing high constitutive levels of AHASI . A strong correlation was observed in himA strains between the reduced amount of the Ilv enzymes and of Ilv-specific messenger RNA . These data suggest that integration host factor may be a positive effector for transcription of the ilvB and ilvGEDA operons.

J Mol Biol, 1984 Feb 5, 172(4), 405 - 16
Interaction of tight binding repressors with lac operators . An analysis by DNA-footprinting; Pfahl M et al.; To increase our understanding of protein-DNA interaction in general, and in particular that of lac repressor with lac operator, we have investigated the interaction of tight binding (Itb) repressors with wild type (WT) operator and Oc operators . Nine Oc and a WT operator were cloned and sequenced . Three different Oc and an O+ were then chosen for the footprint analysis of six Itb repressors and WT repressor . Distinct protection patterns for the various repressor-operator pairs were observed at low repressor concentrations whereas, at high repressor concentrations, a stretch of 24 bases of the lower strand of the four different operators was protected in most cases . This protection pattern at high repressor concentration was almost completely redundant for all repressor-operator pairs, in spite of the fact that the affinities of the various pairs differed by more than three orders of magnitude . Two exceptions to this general observation were the two tight binding repressors R67 and R78a . These had been mapped in a region that codes for amino acid residues involved in subunit interaction . The two repressors showed reduced protection of O+ and of some Oc operators at the 3' (right) end of the lower strand . Dimethylsulfoxide, which is known to increase the affinity of O+ for repressor, did not increase the number of bases protected by WT repressor on the lower strand of O+ . The footprinting results presented here clearly demonstrate that lac repressor can maximally protect about 24 bases of the lower strand of the operator and that the number and kind of interactions occurring in this region determine the strength of the repressor-operator interaction.

Biochimie, 1984 Feb, 66(2), 121 - 6
FFT method to compute solution X-ray scattering curves; Rey FA et al.; We present an efficient algorithm to compute X-ray intensities scattered by macromolecules in solution, from atomic positions found in crystal structures . The algorithm applies the Fast Fourier Transform to an electron density map created from the atomic coordinates and corrected for solvent density . We compute scattering curves for both allosteric forms of E . coli aspartate carbamoyltransferase . Calculated intensities are in agreement with the ones measured by Moody et al . which shows that the structures observed in solution in the presence or in the absence of a substrate analogue do correspond to those of two crystal forms analyzed by Lipscomb and collaborators .

Mod Vet Pract, 1984 Feb, 65(2), 136 - 8
Use of selenium in problem cattle herds; Sanders DE; A 150-cow beef herd had problems with diarrhea in all neonatal calves, with 50% mortality . Necropsy revealed changes consistent with colibacillosis . Vaccination of cows in late pregnancy with E coli and BVD vaccines reduced morbidity but did not eliminate the problem . Assays revealed low serum Se levels . Limited supplementation of Se in premix at 90 mg/lb premix, with 17 lb premix/ton feed, resolved the problem . A 70-cow dairy herd had long-term problems with increased numbers of cystic ovaries and retained placentas, and low conception rates . Serum Se levels were low; results of various other diagnostic tests were inconclusive . Dietary supplementation of Se greatly improved reproductive efficiency . Gross and microscopic lesions may not be inconclusive for a diagnosis of Se deficiency . Dietary supplementation of Se is recommended over injection.

Mol Cell Biol, 1984 Feb, 4(2), 310 - 6
Structure and distribution of Alu family sequences or their analogs within heterogeneous nuclear RNA of HeLa, KB, and L cells; Robertson HD et al.; We studied the distribution of repetitive sequence elements capable of forming double-stranded regions in nuclear RNA of HeLa, KB, and L cells . In human RNA populations, we called these regions duplex Alu family RNA (dAfRNA) because they represent transcripts of the highly reiterated family of DNA regions known as "Alu family DNA" (Rubin et al., Nature (London) 284:372-374, 1980) . Although the dAfRNA populations of both human cell lines (HeLa and KB) have low sequence complexity, they represent 5% of the total heterogeneous nuclear RNA and have identical fingerprints; mouse L-cell dAf-like RNA (which has a similar complexity) represents only 2% of the total heterogeneous nuclear RNA and has an entirely different fingerprint . We utilized Escherichia coli RNase III as a highly specific reagent for the recognition of RNA:RNA duplex structure . This enzyme cleaves within the six characteristic RNase T1-resistant oligonucleotides of HeLa- and KB-cell dAfRNA (Robertson et al., J . Mol . Biol . 115:571-589, 1977) . In addition, the size of heterogeneous nuclear RNA from all three cell types is reduced from greater than 32S to about 15S after RNase III treatment . We conclude that this size shift is a result of cleavage within dAfRNA regions and that such regions are present in most or all of the large RNA transcripts of these cells.

Infect Immun, 1984 Feb, 43(2), 775 - 8
Chemotactic and phagocytic responses of human alveolar macrophages to activated complement components; Richards SW et al.; Human alveolar macrophages (AMs) migrated toward and aggregated to C5a but not toward C5a-deficient serum . Human AMs from cigarette smokers migrated significantly farther than did AMs from nonsmokers . Human AMs phagocytized Escherichia coli opsonized with activated C3 . Immunoglobulin G was not required for phagocytosis . Human AMs demonstrate chemotaxis and aggregation to C5a . Further, human AMs can phagocytize bacteria coated with activated C3.

Infect Immun, 1984 Feb, 43(2), 622 - 30
Binding of Escherichia coli heat-stable enterotoxin to rat intestinal cells and brush border membranes; Frantz JC et al.; The association of heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli 431 with isolated rat intestinal epithelial cells and brush border membranes was characterized . Specific binding of strain 431 125I-STa to a single class of specific high-affinity receptors was saturable and temperature dependent and reached a maximum between 5 and 10 min . A 1,000-fold excess of unlabeled 431 STa competitively displaced 90 to 95% of radiolabeled enterotoxin bound to brush border membranes . In contrast, specific binding of 431 125I-STa to intestinal cells ranged from 40 to 65% . The number of STa-specific receptors on rat intestinal cells determined by Scatchard analysis was 47,520 +/- 14,352 (mean +/- standard error of the mean) per cell, with affinity constants (KaS) of 2.55 X 10(11)and 4.32 x 10(11) liters/mol determined for intestinal cells and brush border membranes, respectively . Villus intestinal cells appeared to possess about twice as many STa receptors as did crypt cells . Dissociation of specifically bound 431 125I-STa from intestinal cells and brush border membranes was minimal (2 to 5%) . In addition, neither the rate nor the extent of dissociation was increased by a 1,000-fold excess of unlabeled homologous 431 Sta . Binding experiments with 431 125I-STa and brush border membranes showed that purified unlabeled STas from enterotoxigenic E . coli strains 667 (class 1 porcine enteropathogen), B-41 (bovine enteropathogen), and human strains 213C2 (Mexico) and 153961-2 (Dacca, Bangledesh) exhibited patterns of competitive inhibition similar to those of homologous unlabeled 431 STa (class 2 enteropathogen) . A lipid extract which contained gangliosides and glycolipids exhibited dose-dependent competitive inhibition of heat-labile enterotoxin binding to brush border membranes but did not inhibit binding of 431 125I-STa . Purified heat-labile enterotoxin from strain 286C2 did not inhibit binding of 431 STa to brush border membranes . Pronase treatment of brush border membranes reduced binding of 431 125I-STa by about 30%, suggesting that the STa receptor was a protein or a glycoprotein . The putative STa receptor was radiolabeled with 431 125I-STa and solubilized with sodium deoxycholate . One major radioactive band was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography . These data suggested that STas bind essentially irreversibly to a specific receptor on the cell surface of intestinal cells before activation of guanylate cyclase.

Jpn J Pharmacol, 1984 Feb, 34(2), 241 - 8
Effect of lipopolysaccharide (from Escherichia coli) on the hepatic drug-metabolizing activities in successively LPS-treated mice; Sasaki K et al.; The effect of an acute or a successive administration of endotoxin (lipopolysaccharide obtained from Escherichia coli, LPS) on the hepatic drug-metabolizing system in vivo and in vitro was examined in mice . An acute LPS (5 mg/kg, i.v.) administration or a successive LPS (5-20 mg/kg, i.p., a day for 6 days) administration prolonged the duration of pentobarbital sleeping time and reduced the rate of hepatic microsomal metabolism of pentobarbital, aminopyrine, aniline and cyclophosphamide and reduced cytochrome P-450 content as compared with those in the control mice . No change of these parameters, however, was observed by an acute treatment with LPS to the successively LPS-treated mice . In addition, the LD50's of aminopyrine and pentobarbital and the ED50 of aminopyrine were reduced by an acute administration of LPS in control mice . No change of both parameters, however, was observed in the successively LPS-treated mice with or without an acute administration of LPS.

EMBO J, 1984 Feb, 3(2), 279 - 87
The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli; Lane AN et al.; The alpha subunit is bound with negative cooperativity to the holo beta 2 subunit of tryptophan synthase in phosphate buffer . Thus it is feasible to measure separately the rates of formation both of the stable alpha beta 2 subcomplex from beta 2, and of the mature alpha 2 beta 2 complex from alpha beta 2, using stopped-flow techniques . Addition of each alpha subunit proceeds in two steps; an initial alpha beta protomer is formed rapidly, which subsequently isomerizes slowly to the equilibrium state . The rates of dissociation of both the alpha beta 2 and alpha 2 beta 2 complexes were measured by trapping released alpha subunit with enzymically inactive reduced beta 2 subunit . The reversal of the slow isomerization both determines the rate of dissociation, and accounts for the high overall affinity of the beta protomer for the alpha subunit . The data fit to a sequential assembly mechanism consisting of seven protein species and yields values for most of the rate constants and all of the microscopic equilibrium constants . Negative cooperativity arises from a weaker initial binding of the second alpha subunit, as expressed by its larger off-constant, possibly due to steric hindrance . The kinetics of binding of L-serine and indolepropanol phosphate during the assembly process shows that the beta protomer is already partially activated in the initial alpha beta complex . Full activation is achieved in the slow isomerization reaction . In contrast, the alpha subunit gains high affinity for indolepropanol phosphate only in the isomerization reaction . These observations indicate that the isomerization involves synchronous conformation changes of both alpha and beta protomers.

J Appl Bacteriol, 1984 Feb, 56(1), 165 - 72
Cryosensitivity of Escherichia coli and the involvement of cyclopropane fatty acids; Calcott PH et al.; Strains of Escherichia coli proficient and deficient in cylopropane fatty acid synthesis were compared for fatty acid content, cryosensitivity, presence of freeze-thaw-induced wall and membrane damage, resistance to detergent-stimulated lysis and tolerance to salt and detergents during growth . The mutant populations synthesized much less cyclopropane fatty acids and were more resistant than the wild type to freezing and thawing in saline only, exhibiting less viability loss and less wall and membrane damage . While the resistance of the mutants to NaCl was unaltered, their detergent resistance was decreased under both growth and non-growth conditions . Although these physiological changes were associated with a lower cyclopropane fatty acid content in the mutant strains, it is proposed that the responses were due to the altered membrane fluidity of the mutants due to changes in their unsaturated fatty acid content.

Proc Natl Acad Sci U S A, 1984 Feb, 81(3), 767 - 70
Receptor-mediated activation of a phospholipase A2 in rabbit neutrophil plasma membrane; Bormann BJ et al.; Using the exogenous substrate {1-14C}oleate-labeled autoclaved Escherichia coli, we have demonstrated that the chemotactic factors fMet-Leu-Phe, complement component C5a, and leukotriene B4 {(5S,12R)-dihydroxy-6-cis,8-trans,11-trans,14-cis-icosatetraenoic acid} stimulate a phospholipase A2 of isolated plasma membranes of rabbit peritoneal neutrophils . Each of the chemotactic factors shows a biphasic concentration dependence with the optimal concentrations occurring at 1, 10, and 0.1 nM, respectively . The specific antagonists of fMet-Leu-Phe binding, carbobenzoxy-Phe-Met and t-butoxycarbonyl-Phe-Leu-Phe, effectively block the stimulation by fMet-Leu-Phe, indicating that the activation is receptor mediated . delta 6-trans-leukotriene {(5S-12R)-dihydroxy-all-trans-6,8,10,14-icosatetraenoic acid}, a biologically inactive stereoisomer of leukotriene B4, does not stimulate phospholipase activity, suggesting that the enhancement by leukotriene B4 is also receptor mediated . The unstimulated and activated phospholipase exhibit a broad range of maximal activity between pH 7.0 and pH 8.5, both with an optimal pH of 8.5 . The activation of the phospholipase by fMet-Leu-Phe is completely calcium dependent; no increase in activity is demonstrable if fMet-Leu-Phe is added in the absence of exogenous calcium or in the presence of EGTA . In contrast, the unstimulated plasma membrane activity of the phospholipase, as well as the activity arising after stimulation, is relatively insensitive to the concentration of calcium, being inhibited by less than 50% in the presence of 10 mM EGTA . The phospholipase hydrolyzes 1-{1-14C}palmitoyl-2-acyl-sn-glycerophosphoethanolamine to form only radioactive lysophosphatidylethanolamine as the product, indicating that the enzyme has an A2 specificity.

Infect Immun, 1984 Feb, 43(2), 549 - 54
F41 antigen among porcine enterotoxigenic Escherichia coli strains lacking K88, K99, and 987P pili; To SC; Colonial morphological variations among enterotoxigenic Escherichia coli which lack K88, K99, and 987P (3P-) were shown to be correlated with expression of several surface antigens, i.e., type 1 pili, F41 pili, and somatic and capsular antigens . Such correlations were established by electron microscopy, serology, and hemagglutination of cells derived from these specific colonial types . Identification of F41 produced by the 3P- enterotoxigenic E . coli strains was made by immunodiffusion in agarose gel, immunofluorescence microscopy, subunit molecular weight determination in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and hemagglutination of F41-piliated 3P- cells and purified F41 pili . Two of the 3P- enterotoxigenic E . coli strains were also shown to be virulent in conventional neonatal pigs and calves . Intense immunofluorescence staining by reference F41 serum of ileal villi of 3P- -infected animals indicated that F41 was expressed during the disease process.

Eur J Biochem, 1984 Feb 1, 138(3), 527 - 31
Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli; Herrchen M et al.; {3H} Conduritol C cis-epoxide (1,2-anhydro-epi-inositol, I) was synthesized as an active-site-directed inhibitor for lacZ beta-galactosidase from Escherichia coli . A considerable kinetic isotope effect was noted in the reduction by {3H}NaBH4 of the p-benzoquinone-derived precursor for I . Complete loss of beta-galactosidase activity occurred on incorporation of 4 mol I/mol beta-galactosidase tetramer . The inhibitor was very labile in the denatured enzyme at pH greater than 8, implying the formation of an ester bond between I and a carboxylate at the active site . The radioactive material released from the labeled enzyme was identified as allo-inositol . The stereochemistry of the expoxide reaction (trans-diaxial ring opening) is thus the same as for beta-glucosidases with the corresponding epoxides . The binding site for I was identified as Glu-461 by the isolation and partial sequence analysis of a radioactive octapeptide from the cyanogen bromide and pepsin fragments of the labeled enzyme . A failure to determine the N-terminal amino acid of the labeled peptide is ascribed to the great reactivity of the esterified gamma-carboxyl group of its N-terminal Glu-461 which causes rapid cyclisation of this residue to pyroglutamate, even under weakly basic conditions . The participation of the carboxylate of Glu-461 in catalysis is discussed.

Mol Biol Evol, 1984 Feb, 1(2), 171 - 82
Directed evolution of cellobiose utilization in Escherichia coli K12; Kricker M et al.; The cellobiose catabolic system of Escherichia coli K12 is being used to study the role of cryptic genes in evolution of new functions . Escherichia coli does not use beta-glucoside sugars; however, mutations in several loci can activate the cryptic bgl operon and permit growth on the beta-glucoside sugars arbutin and salicin . Such Bgl+ mutants do not use cellobiose, which is the most common beta-glucoside in nature . We have isolated a Cel+ (cellobiose-utilizing) mutant from a Bgl+ mutant of E . coli K12 . The Cel+ mutant grows well on cellobiose, arbutin, and salicin . Genes for utilization of these beta-glucosides are located at 37.8 min on the E . coli map . The genes of the bgl operon are not involved in cellobiose utilization . Introduction of a deletion covering bgl does not affect the ability to utilize cellobiose, arbutin, or salicin, indicating that the new Cel+ genes provide all three functions . Spontaneous cellobiose negative mutants also become arbutin and salicin negative . Analysis of beta-glucoside positive revertants of these mutants indicates that there are separate loci for utilization of each of the beta-glucoside sugars . The genes are closely linked and may be activated from a single locus . A fourth gene at an unknown location increases the growth rate on cellobiose . The cel genes constitute a second cryptic system for beta-glucoside utilization in E . coli K12.

Mol Biol Evol, 1984 Feb, 1(2), 162 - 70
Selective neutrality of glucose-6-phosphate dehydrogenase allozymes in Escherichia coli; Dykhuizen DE et al.; Six naturally occurring alleles representing four electromorphs of the enzyme glucose-6-phosphate dehydrogenase were transferred by P1-mediated transduction from natural isolates of Escherichia coli into the genetic background of E . coli K12 and were studied in pairwise competition in chemostats limited for glucose in order to estimate differences in growth rate associated with the alleles . Although the level of resolution of such experiments is a growth rate differential of approximately 0.002 h-1, no significant differences among the strains were found . Studies of apparent Km and Vmax in crude enzyme extracts of the strains also failed to reveal any significant differences among the electromorphs . These results support the view that the alleles are selectively neutral or nearly neutral under these conditions.

Biochem Int, 1984 Feb, 8(2), 209 - 15
Fractionation of DNA with Sephacryl S-1000; Suominen AI et al.; In this study the application of gel filtration for purification of heterogeneous DNA is described . The fractionation of partial restriction enzyme digests of bacterial chromosomal DNA on a Sephacryl S-1000 -column is easy and rapid . Simultaneously intact chromosomal DNA and low molecular weight substances are eliminated in the run . The method is also applicable to the purification of plasmid DNA, as has been previously reported (3) . Thus we are able to get pure DNA with yields over 80%.

Biochimie, 1984 Feb, 66(2), 115 - 9
A memory effect in DNA replication; Papanicolaou C et al.; A study of the polymerization/excision ratio in the replication of poly(dA), primed with oligo(dT), was carried out with E . coli DNA polymerase I, at various primer and enzyme concentrations . The variations in this ratio suggest that 1) the DNA polymerase is able to switch between two states of low and high exonuclease activities and 2) after dissociating from the template, the DNA polymerase drifts towards the low exonuclease state . The recovery of the high exonuclease state would require several successive incorporations.

Anal Biochem, 1984 Feb, 137(1), 80 - 7
A highly sensitive photometric method for proton release or uptake: difference protometry; Horie K et al.; A highly sensitive quantitative method was developed to detect protons released or taken up upon ligand binding . A small change in pH due to proton release or uptake was detected by measuring the difference in the absorbance of a pH indicator upon ligand addition . Owing to the difference detection of protons, the uncertainty of pH due to CO2 dissolution and unknown buffering capacities of sample solutes could be compensated with easy manipulations . Precise calibration of the absolute amount of protons could also be made very easily . The amount of protons measurable by the method is as small as 0.5 nmol that is 10 to 30 times more sensitive than the pH-stat method . We measured the Mg2+ ion-induced proton releases of ADP to confirm the accuracy and reliability of the method and of Escherichia coli ribosomes to show the improvement in sensitivity . The method is useful for protometric studies of biomolecules that are difficult to obtain in large amount.

Anal Biochem, 1984 Feb, 137(1), 143 - 5
Purification of oligo dG-tailed Okayama-Berg linker DNA fragments by oligo dC-cellulose chromatography; Forsdyke DR; A simple procedure for preparation of oligo dG-tailed DNA fragments is presented . The fragments are first purified by ultracentrifugation through sucrose gradients at low salt concentration . Appropriate gradient fractions are then adjusted to 1 M NaCl and immediately applied to a column of oligo dC-cellulose equilibrated in buffered 1 M NaCl at 4 degrees C . Fragments are eluted with water at room temperature . Passage through the column achieves, in one step, the concentration and purification of oligo dG-tailed DNA fragments free from sucrose.

Mod Vet Pract . 1984 Feb;65(2):118, 120.
Treatment of calf scours with colostrum; Gregory DW et al.; The calf of a cow vaccinated with an E coli strain 1751 bacterin developed scours 14 hours after oral challenge with E coli strain B44 . The calf became almost moribund but fully recovered in several days after repeated oral administration of the dam's colostrum.

J Gen Microbiol, 1984 Feb, 130 ( Pt 2), 299 - 308
Interrelationships between the enzymes of ethanolamine metabolism in Escherichia coli; Jones PW et al.; The activities of the enzymes ethanolamine ammonia-lyase, CoA-dependent and CoA-independent aldehyde dehydrogenases, and isocitrate lyase were assayed in Escherichia coli which had been grown on various sources of carbon and nitrogen . Induction of ethanolamine ammonia-lyase and of maximal levels of both aldehyde dehydrogenases required the concerted effects of ethanolamine and vitamin (or coenzyme) B12 . Molecular exclusion chromatography revealed that, in the absence of one or both co-inducers, two repressible isoenzymes of CoA-dependent aldehyde dehydrogenase (mol . wts 900000 and 120000) were produced, these being replaced by two inducible isoenzymes (mol . wts 520000 and 370000) in the presence of both co-inducers . A similar inducible repressible series of isoenzymes was also observed for CoA-independent aldehyde dehydrogenase . No evidence was found for structural relationships between ethanolamine ammonia-lyase, CoA-dependent aldehyde dehydrogenase and CoA-independent aldehyde dehydrogenase, but mutant and physiological studies demonstrated that the induction of the first two enzymes is under common control . Evidence is presented for the operation of a previously unreported pathway of ethanolamine metabolism in E . coli.

Gene, 1984 Feb, 27(2), 213 - 22
Mutagenesis and mutation transfer induced by ultraviolet light in plasmid-cloned DNA; Chattoraj DK et al.; We describe here simple techniques for increasing the frequency of UV-induced mutations in a DNA fragment cloned in plasmid pBR322 . Irradiation of both the host and the plasmid DNA before transformation is necessary to produce new mutations in the plasmid DNA, presumably because the UV-damaged pBR322 replicon cannot efficiently induce the error-prone repair pathway of Escherichia coli . In contrast, UV irradiation of the plasmid DNA alone before transformation primarily causes the transfer of preexisting mutations from the host chromosome to homologous DNA present in the plasmid . The only other kind of mutants obtained were large deletions of the plasmid DNA . Two chromosomal mutations from the host galK gene and one from the lacZ gene have been transferred to the plasmid by UV irradiation of the plasmid DNA alone . The technique can thus be of general use.

Gene, 1984 Feb, 27(2), 193 - 9
Complete nucleotide sequence of the glutamate dehydrogenase gene from Escherichia coli K-12; Valle F et al.; A 2.3-kb PstI- ClaI chromosomal DNA segment, carrying the complete coding region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, has been sequenced . The complete amino acid sequence (447 residues) of the GDH monomer has been deduced, and comparisons are made with reported amino acid sequences of GDH from other organisms.

Anal Biochem, 1984 Feb, 136(2), 497 - 502
A novel, general radioimmunoassay for acyl carrier proteins; Kuo TM et al.; A radioimmunoassay (RIA) for acyl carrier proteins (ACP) is described that is based on the competitive binding between {3H}acyl-ACP and unlabeled ACP of the same species . The radiolabeled antigen, {3H}palmitoyl-ACP, is enzymatically synthesized by Escherichia coli acyl-ACP synthetase . Because acyl-ACP synthetase can specifically radiolabel ACP in crude extracts from several plant sources, the use of this enzyme to prepare {3H}acyl-ACP obviates the need for pure preparations of each ACP . Preparation of {3H}acyl-ACP with a specific activity of 15 Ci/mmol allows RIA detection of total ACP in crude plant extracts at the nanogram level . Because antibodies against spinach ACP partially crossreact with ACP from many plant sources, RIAs for other plant species can be constructed using only one preparation of antibody . ACP preparations from safflower, soybean, avocado, corn, and E . coli show a decreasing order of partial immunocross-reactivity with spinach ACP-specific antiserum, as examined by RIA using spinach {3H}palmitoyl-ACP.

Anal Biochem, 1984 Feb, 136(2), 493 - 6
Purification of thiogalactoside transacetylase by affinity chromatography; Zabin I et al.; Thiogalactoside transacetylase, the product of the lacA gene of the lactose operon of Escherichia coli, has been purified by an improved procedure . The enzyme binds tightly to immobilized Cibacron Blue F3GA columns and can be eluted by potassium chloride in high concentrations . Final purification was obtained by affinity chromatography on an agarose-coenzyme A column followed by gel filtration.

J Biochem (Tokyo), 1984 Feb, 95(2), 311 - 7
Studies on the metabolism of unsaturated fatty acids . XIII . Properties of 2,4-dienoyl-CoA reductase from beef liver; Mizugaki M et al.; It is demonstrated that 3-alkenoyl-CoA is the product of the enzymic reduction catalyzed by 2,4-dienoyl-CoA reductase with the highly purified enzyme preparation from beef liver mitochondria . Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase from beef liver was investigated . When trans-2, trans-4-decadienoyl-CoA was incubated with the reductase in the presence of 4R-{4-2H1}NADPH and H2O, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to pyrrolidine amides of fatty acids . When the substrate was incubated with the enzyme in the presence of 4S-{4-2H1}NADPH and H2O, one deuterium atom was incorporated into the product, 3-decenoate, at the C-5 position . In the case of 3-decenoate enzymatically prepared in the presence of NADPH and 2H2O, two deuterium atoms were incorporated into the product at the C-2 position . These results indicate that the reaction catalyzed by 2,4-dienoyl-CoA reductase from beef liver is a unique example of 1,4-addition of hydrogen to a 1,3-diene system conjugated with a carbonyl group of a thioester in biochemical fields . Chain length specificity of the reductase, isotope effects on reaction rates and competitive inhibitors are also discussed.

Am J Vet Res, 1984 Feb, 45(2), 314 - 8
Cloning and comparison of heat-stable enterotoxin genes from Escherichia coli strains of bovine, porcine, and avian origins; Sekizaki T et al.; The Ent plasmid encoding Escherichia coli heat-stable enterotoxin (ST) isolated from bovine, porcine, and avian strains were used for the cloning of ST genes . The ST+ DNA regions were finally cloned into pBR322 as the 1.75 kilobases PstI fragment . The electron microscopic analysis of self-annealed molecules indicated that ST+ recombinant plasmid had a stem-loop structure of a size the same as that observed in their wild type Ent plasmids . The stem-loop structures and the restriction enzyme cleavage mappings indicated that 4 kinds of ST genes cloned in this experiment may be identical to Tn1681 . The ST production levels of the recombinant plasmids were higher than those of the original plasmids.

Am J Vet Res, 1984 Feb, 45(2), 255 - 9
Relationship of iron administration to susceptibility of newborn pigs to enterotoxic colibacillosis; Kadis S et al.; To determine whether supplemental iron (Fe) administration to newborn pigs reared in concrete pens not only prevents anemia, but renders the pigs more susceptible to Escherichia coli-induced diarrheal disease, pigs were given a large or a small dose of Fe IM or orally before or after challenge exposure with E coli . The controls were challenge-exposed pigs not given Fe and pigs not challenge exposed (Fe-treated and nontreated groups) . Although the mortality of the pigs challenge exposed with E coli and administered a large oral dose of Fe shortly after birth was greater than that of the challenge-exposed pigs given no Fe, differences in mortality were not noted between any of the groups tested when the Fe was injected IM . The Fe-treated survivors had severe diarrhea (oral Fe administration) or mild diarrhea (IM Fe administration) for longer periods than did the nontreated survivors . All challenge-exposed pigs treated with a large dose of Fe gained less weight than the nontreated pigs during the diarrheal period and for several days thereafter . Beyond this time period, the weight gain of the Fe-treated pigs was substantially greater than that of their nontreated littermates; the weight gain of the pigs given a small dose of Fe was intermediate . Hemoglobin and hematocrit values of the pigs shortly after birth and weekly thereafter revealed that within 2 weeks, both sets of values from the pigs treated with a large dose of Fe were within acceptable laboratory limits and substantially greater than the values obtained for the nontreated pigs, which were severely anemic.

J Pharm Sci, 1984 Feb, 73(2), 275 - 7
New compounds: peptide derivatives of the antitumor agent N-phosphonoacetyl-L-aspartic acid; Gigot D et al.; Two peptide forms of the antitumor transition state analogue N-phosphonoacetyl-L-aspartic acid (N2-phosphonoacetyl-N4-glycylglycinamidoethyl-L-asparagine and N1-glycylglycinamidoethyl-N2-phosphonoacetyl-L-isoasparagine ) have been synthesized to obtain potential medicinal agents useful as prodrugs or in a lysosomotropic carrier approach . The bridging unit, ethylenediamine, used for synthetic purposes might be of general interest.

J Appl Physiol, 1984 Feb, 56(2), 489 - 94
Effect of endotoxin on lung fluid balance in unanesthetized sheep; Gabel JC et al.; We used a gravimetric technique to test for increased pulmonary capillary permeability after Escherichia coli endotoxin infusion in unanesthetized sheep . The sheep were chronically prepared with cannulas placed into the left atrium and pulmonary artery 1-2 wk before the experiments . We estimated pulmonary capillary pressure (Pc) as the average of pulmonary arterial and left atrial pressures, and used the modified method of Pierce to estimate the ratio of extravascular fluid weight (EVF) to blood-free dry weight . In 15 sheep we inflated a left atrial balloon to raise Pc to -10.7, 5, 10, or 15 mmHg above plasma oncotic pressure (IIc) for 3 h, then measured EVF . EVF averaged 4.0 +/- 0.2 (base line), 4.3 +/- 0.1, 4.5 +/- 0.1, and 5.1 +/- 0.5 (SD), respectively, for the four levels of Pc - IIc . We gave seven additional sheep 1 microgram/kg of E . coli endotoxin (0127:B8) and measured EVF after 3 h of stable Pc . Endotoxin increased Pc in each sheep . EVF was higher than control for the endotoxin sheep with Pc - IIc greater than -1 . This finding is consistent with an increase in pulmonary capillary permeability caused by endotoxin . However, EVF was not elevated in the endotoxin sheep with Pc - IIc less than 1 mmHg . This shows that the increased permeability was insufficient to cause edema unless Pc was elevated . Thus endotoxin may cause edema by two mechanisms, 1) an increase in capillary permeability, and 2) an increase in Pc.

Genetika, 1984 Feb, 20(2), 219 - 23
{Dependence of Escherichia coli K-12 viability and mutability on the balance of DNA and protein syntheses . VI . Molecular mechanisms underlying the phenomena of imbalance death and metabolic mutagenesis}; Fradkin GE et al.; In this report the results of further studies of the molecular mechanisms of the disbalance death and metabolic (disbalance) mutagenesis are presented . As reported previously, the relationship of viability and mutability of Escherichia coli cells from the extent disturbance of DNA-protein synthesis balance is determined by the processes of stabilization and repair of metabolic disbalance gaps in DNA strands . It is established that polB, recB, SSB gene products are involved in the processes of stabilization and repair of metabolic disbalance gaps.

Eur J Clin Microbiol, 1984 Feb, 3(1), 30 - 4
Use of hepatitis B core antigen produced in Escherichia coli to detect immunoglobulin M specific antibodies in an enzyme-linked immunosorbent assay; Hasche G et al.; The antigenic activity of HBcAg produced in Escherichia coli and HBcAg from human liver was compared in a mu-specific solid-phase antibody-capture assay for detection of anti-HBc-IgM . HBcAg from liver could be detected in dilutions up to 1:3, HBcAg from Escherichia coli in dilutions up to 1:10,000 . Using HBcAg from Escherichia coli, sera from five patients with acute resolving hepatitis B and sera from four patients with acute hepatitis B who had developed chronic liver disease were tested for anti-HBc-IgM in ELISA . IgM fractions separated out of the same sera by immunoaffinity chromatography were tested for anti-HBc-IgM using a commercially available test . The results were in good agreement with those obtained by ELISA . Anti-HBc-IgM could be detected up to 900 days after onset of disease . Different groups of patients were tested for presence of anti-HBc-IgM in ELISA . Fifty-nine of 60 patients with acute hepatitis B were positive for anti-HBc-IgM at onset of illness . Ten of 16 patients with chronic aggressive hepatitis and seven of 23 HBsAg positive dialysis patients were also positive for anti-HBc-IgM, whereas only two of 12 patients with chronic persistent hepatitis and one of 15 HBsAg positive blood donors ("healthy" carriers of HBsAg) had detectable anti-HBc-IgM.

Proc Natl Acad Sci U S A, 1984 Feb, 81(4), 1048 - 52
Amino acid sequence homology among the major outer membrane proteins of Escherichia coli; Nikaido H et al.; Analysis of amino acid sequences reported for the major outer membrane proteins of Escherichia coli, including the porins (OmpF, OmpC, and PhoE), the phage lambda receptor (LamB), and another protein (OmpA), revealed several regions of local homology that is statistically significant . The implications of this observation are discussed in relation to the evolutionary origins of these proteins, as well as to the mechanism of export of these proteins to the outer membrane.

Proc Natl Acad Sci U S A, 1984 Feb, 81(3), 731 - 5
Androgen induction of ornithine decarboxylase mRNA in mouse kidney as studied by complementary DNA; Kontula KK et al.; To investigate the mechanisms by which androgens regulate ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) in mouse kidney, a cDNA clone encoding OrnDCase mRNA was prepared . Purification of OrnDCase mRNA from kidneys of androgen-treated mice was accomplished by immunoadsorption of renal polysomes to a protein A-Sepharose column and enrichment for poly(A)-containing RNA by oligo(dT)-cellulose . Double-stranded cDNA synthesized from this mRNA was inserted into the Pst I site of plasmid pBR322 by using oligo(dG . dC)-tailing and was propagated in Escherichia coli . Plasmids containing cDNA sequences coding for OrnDCase were identified by differential colony hybridization, by radioimmunological detection of OrnDCase-like antigens in bacterial cultures, and by cell-free translation of hybrid-selected mRNA followed by immunoprecipitation with monospecific OrnDCase antiserum . A restriction endonuclease fragment of the selected plasmid DNA (pODC54) was labeled by nick-translation and used to study changes in OrnDCase mRNA concentration . After a single dose of testosterone, renal OrnDCase mRNA concentration increased as soon as 6 hr and peaked 24 hr after steroid injection, as measured by RNA blot hybridization . Continuous androgen treatment for 4 days resulted in a 10- to 20-fold increase in OrnDCase mRNA concentration in normal animals, but no induction of this mRNA was detected in mice that have an inherent defect of the androgen receptor (testicular feminization) . These results indicate that androgens regulate OrnD-Case synthesis in mouse kidney, at least in part, by increasing OrnDCase mRNA accumulation.

Proc Natl Acad Sci U S A, 1984 Feb, 81(3), 722 - 5
Aminomalonic acid: identification in Escherichia coli and atherosclerotic plaque; Van Buskirk JJ et al.; Aminomalonic acid (Ama) has been isolated from proteins of Escherichia coli and human atherosclerotic plaque . The presence of Ama has important biological implications because the malonic acid moiety potentially imparts calcium binding properties to protein . Ama was obtained by anaerobic alkaline hydrolysis and identified by chromatographic behavior, quantitative acid-mediated decarboxylation to glycine, and unambiguous gas chromatographic/mass spectral detection . The chromatographic, chemical, and mass spectral properties of naturally occurring Ama were identical to those of the synthetic compound . Amino acid analysis and GC/mass spectrometry also revealed the presence of beta-carboxyaspartic acid and gamma-carboxyglutamic acid in the base hydrolysate of human atherosclerotic plaque . The ratio of Ama to beta-carboxyaspartic acid to gamma-carboxyglutamic acid was 20:1:10, and the quantity of Ama per 1,000 glycine residues was 0.2 . Ama is a relatively unstable, minor amino acid in complex structures such as bacteria or tissues . This may explain why it has escaped detection previously, despite intensive investigation.

Mol Biochem Parasitol, 1984 Feb, 10(2), 207 - 16
Expression of a Trypanosoma brucei brucei variant antigen in Escherichia coli; Parsons M et al.; A cDNA library derived from antigenically homogeneous bloodstream stage Trypanosoma brucei brucei was screened with an antiserum directed against the variant surface antigen (VSA) using an enzyme-linked filter immunoassay . Several recombinant clones were detected and the clone giving the most intense reaction was further analyzed . It contained a VSA-specific cDNA insert and synthesized a protein of the expected molecular weight bearing VSA determinants . The nucleotide sequence of the insert was determined and shown to have the unusual codon bias characteristic of T . brucei VSAs, frequently employing codons specifying tRNAs rare in Escherichia coli . These results indicate that a codon bias very different from that of E . coli does not preclude the expression of a cloned sequence to detectable levels in this heterologous host.

J Clin Microbiol, 1984 Feb, 19(2), 286 - 90
Whole-cell peroxidase test for identification of Legionella pneumophila; Pine L et al.; A simple combined peroxidase-catalase test has been developed which is applicable to live bacterial cells . Known strains of Legionella pneumophila were differentiated from other species of Legionella by being peroxidase positive and catalase negative.

Int J Radiat Biol Relat Stud Phys Chem Med, 1984 Feb, 45(2), 193 - 6
Degradation of Escherichia coli DNA synthesized after ultraviolet irradiation in the absence of repair; Trgovcevic Z et al.; DNA degradation in Escherichia coli uvrA recA bacteria exposed to a low dose (0.07 J/m2) of ultraviolet radiation was studied . A considerable amount of the newly-synthesized DNA, which contains gaps opposite pyrimidine dimers, is broken down . In contrast, parental, dimer-containing DNA is resistant to radiation-induced degradation.

Biophys J, 1984 Feb, 45(2), 463 - 7
Distinction between changes in membrane potential and surface charge upon chemotactic stimulation of Escherichia coli; Eisenbach M et al.; Galactose and other chemotactic attractants have been shown to trigger an apparent hyperpolarization in Escherichia coli (Eisenbach, M., 1982, Biochemistry, 21:6818-6825) . The probe used to measure membrane potential in that study, tetraphenylphosphonium (TPP+), may respond also to surface-charge changes in the membrane . The distinction between true changes in membrane potential and changes in the surface charge of the membrane is crucial for the study of this phenomenon in bacterial chemotaxis . To distinguish between these parameters, we compared the response to galactose with different techniques: K+ distribution in the presence of valinomycin (measured with a K+-selective electrode), TPP+ distribution (measured with a TPP+-selective electrode) at different ionic strengths, absorbance changes of bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol (oxonol V), and fluorescence changes of three probes with different mechanisms of response . All the techniques revealed stimulation by galactose of transient hyperpolarization, of comparable magnitude . This indicates the involvement of ion currents rather than alterations of local surface properties.

Arch Surg, 1984 Feb, 119(2), 173 - 9
Comparative pulmonary effects of intraperitoneal inoculation of live v dead Escherichia coli; Barke RA et al.; We studied the effect of 2.5 X 10(9) live Escherichia coli per kilogram v 2.7 X 10(9) dead E coli per kilogram injected into the peritoneal cavity of sheep with chronic pulmonary lymph fistulas . The effects of dead E coli were compared with those of live E coli, with respect to (1) pulmonary hypertension, (2) hemodynamic failure, (3) damage to the pulmonary microvasculature, (4) systemic arterial hypoxemia, (5) neutropenia and lymphopenia, (6) thrombocytopenia and platelet aggregation, (7) plasma fibrinogen concentration, and (8) classic- and alternative-pathway hemolytic complement . The time after injection of the bacteria was divided into an early period (zero to two hours) and a late period (two to seven hours) . We made two conclusions: (1) The early period effects, with the exception of the absolute neutrophil count and Pao2, were independent of bacterial viability, whereas the late period effects were strongly dependent on bacterial viability . (2) The early notable difference between the live v dead groups, with respect to the absolute neutrophil count and Pao2, could not be explained on the basis of an increase in bacterial numbers alone.

J Med Microbiol, 1984 Feb, 17(1), 53 - 8
Studies on Escherichia coli as a cause of acute diarrhoea in Calcutta; Sen D et al.; The prevalence of different types of diarrhoea-producing Escherichia coli among 240 patients with acute diarrhoea in hospital was investigated . The 25 patients (10.4% of the total) from whose faeces we isolated enteropathogenic E . coli (EPEC) were all less than 5 years old but the 29 (12.1%) from whom we isolated enterotoxigenic E . coli (ETEC) were of various ages, most of them greater than 12 years old . No enteroinvasive E . coli (EIEC) strains were isolated . ETEC strains that produced heat-labile toxin (LT) were encountered more often than those that produced either heat-stable toxin (ST) alone or both LT and ST . The ETEC isolates were distributed among eight different serotypes, the commonest being O148:H28 (38%) . Correlations between enterotoxin production, serotype pattern and possession of colonisation factor antigens I and II were observed.

J Bacteriol, 1984 Feb, 157(2), 690 - 3
Standard reference strains of Escherichia coli from natural populations; Ochman H et al.; A set of 72 reference strains of Escherichia coli isolated from a variety of hosts and geographical locations has been established for use in studies of variation and genetic structure in natural populations . The strains, which have been characterized by multilocus enzyme electrophoresis, are representative of the range of genotypic variation in the species as a whole.

J Bacteriol, 1984 Feb, 157(2), 661 - 4
Exonucleases I, III, and V are required for stability of ColE1-related plasmids in Escherichia coli; Bassett CL et al.; The stability of two ColE1-related plasmids (pRSF2124 and pMB9) was examined in strains of Escherichia coli multiply deficient in exonucleases I (sbcB), III (xthA), or V (recB recC) . Any combination of exonuclease I, III, and V deficiency resulted in dramatically decreased stability of both pRSF2124 and pMB9 . Inactivation of the RecF pathway by introducing either recF or recJ mutations to the recB recC subcB background resulted in nearly wild-type levels of stability for both plasmids . In contrast, the introduction of uvrD3 uvr-257, uvrE100, or recL152 into the recB21 recC22 sbcB15 strain did not affect plasmid stability . Furthermore, the amount of plasmid DNA recovered from pRSF2124 or pMB9 transformants of a xthA1 sbcB15 strain was strikingly reduced relative to that of a wild-type control . Taken together, these results suggest that some aspect of DNA repair is required for stable maintenance of ColE1-related plasmids in E . coli.

J Bacteriol, 1984 Feb, 157(2), 637 - 42
Cellular location of heat-labile enterotoxin in Escherichia coli; Hirst TR et al.; We demonstrated that both the A and B subunits of heat-labile enterotoxin from Escherichia coli are located in the periplasm . The toxin was shown to form aggregates in Tris-EDTA buffers which are routinely used for isolating membranes . The aggregates pellet upon centrifugation, and this may explain why several previous investigators have concluded that enterotoxin is associated with membranes.

J Bacteriol, 1984 Feb, 157(2), 591 - 8
Use of phi(glp-lac) in studies of respiratory regulation of the Escherichia coli anaerobic sn-glycerol-3-phosphate dehydrogenase genes (glpAB); Kuritzkes DR et al.; Expression of the glpA operon encoding the extrinsic membrane anaerobic sn-glycerol-3-phosphate dehydrogenase complex of Escherichia coli K-12 was studied in five strains carrying independent glpA-lac operon fusions . The location of the fusions was confirmed by transduction . Two of the strains produced an enzymatically active anaerobic sn-glycerol-3-phosphate dehydrogenase that accumulated in the cytoplasmic fraction of the cells . This suggests the loss of a specific membrane anchor subunit encoded by a distal gene, glpB, which was disrupted by the insertion . beta-Galactosidase in all five strains carrying phi(glpA-lac) was highly inducible by glycerol only anaerobically . A mutation in fnr, a pleiotropic activator gene, prevented full induction of the phi(glpA-lac), demonstrating that the Fnr protein is a positive regulator of the primary dehydrogenase as well as of the terminal reductases of anaerobic respiratory chains . Low concentrations of the respiratory poison KCN had a permissive effect on aerobic expression of phi(glpA-lac) . Aerobic expression of the hybrid operon was also enhanced in isogenic derivatives of the fusion strains deficient in protoporphyrin biosynthesis (hemA) . Thus, heme proteins may play a role in mediating aerobic repression of the anaerobic respiratory chain.

J Bacteriol, 1984 Feb, 157(2), 576 - 81
Demethylation of methyl-accepting chemotaxis proteins in Escherichia coli induced by the repellents glycerol and ethylene glycol; Oosawa K et al.; The addition of glycerol or ethylene glycol caused not only severe tumbling but also a drastic decrease in the methylation level of methyl-accepting chemotaxis proteins (MCPs) in Escherichia coli . Experiments with various mutants having defects in their MCPs showed that the demethylation occurred in all three kinds of MCPs, MCPI, II, and III . The addition of an attractant to the glycerol- or ethylene glycol-treated cells resulted in a distinct increase in the methylation level of the relevant MCP, indicating that glycerol and ethylene glycol do not directly damage the methylation-demethylation system in the cell . The time courses of adaptation and MCP demethylation upon addition of these repellents were consistent with each other . Furthermore, both the response time and the extent of MCP demethylation were increased in parallel with increasing concentrations of glycerol or ethylene glycol . These results indicate that the adaptation to these repellents is performed by the demethylation of MCPs . Thus, glycerol and ethylene glycol are novel repellents, which utilize not just one but all three kinds of MCPs for both information processing and adaptation.

J Bacteriol, 1984 Feb, 157(2), 498 - 506
Suppression of Escherichia coli recF mutations by recA-linked srfA mutations; Volkert MR et al.; Suppressors of recF (srfA) were found by selection for resistance to mitomycin C and UV irradiation in a recB21 recC22 sbcB15 recF143 strain . srfA mutations map in recA and are dominant to srfA+ . They suppress both the DNA repair and the recombination deficiencies due to recF mutations . Therefore, RecA protein which is altered by the srfA mutation can allow genetic recombination to proceed in the absence of recB, recC, and recF functions . recF is also required for induction of the SOS response after UV damage . We propose that recF+ normally functions to allow the expression of two recA activities, one that is required for the RecF pathway of recombination and another that is required for SOS induction . The two RecA activities are different and are separable by mutation since srfA mutations permit recombination to proceed but have not caused a dramatic increase in SOS induction in recF mutants . According to this hypothesis, one role for recF in DNA repair and recombination is to modulate RecA activities to allow RecA to participate in these recF-dependent processes.

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