Escherichia coli

Vopr Med Khim, 1976 May-Jun, 22(3), 343 - 6
{Enzyme splitting S-adenosylmethionine in the cells of Escherichia coli CK infected with phage CD}; Nikol'skaia II et al.; Extract of normal Escherichia coli CK cells methylated DNA of Cd phage in vitro in presence of 3H-S-adenosyl methionine, although in vivo methylation of viral DNA did not occur . Extract of cells, infected with Cd phage, also was unable to methylate the DNA in vitro due to de novo synthesis of the enzyme splitting S-adenosyl methionine to 5'-methyl thioadenosine and homoserine . The enzyme was not found in cells, infected with the phage in presence of chloramphenicol . The activity of the S-adenosyl methionine splitting enzyme was shown to be maximal between the fourth and fifth min of infection . This suggests that the enzyme is synthesized under control of the virus genome, it belongs to the early virus-specific proteins and inhibits the methylation of the phage DNA in vivo . In unpurified extracts of Escherichia coli CK the methylating activity was completely inhibited by 10(-5) M S-adenosyl homocysteine.

Microsc Acta, 1976 May, 78(2), 131 - 48
Preparation and counts of particles in electron microscopy: application of negative stain in the agarfiltration method; Kellenberger E et al.; The agarfiltration method, published by Kellenberger and Arber in 1957, has been adapted to negative stain with sodium phosphotungstate and uranyl acetate . The technique is described in detail including all recent improvements . The precision obtainable in particle counts is discussed for agarfiltration and sprayed droplets.

J Antibiot (Tokyo), 1976 May, 29(5), 579 - 83
The in vitro activity of ceftezol (demethylcefazolin) against dense populations of Escherichia coli; Greenwood D et al.; The activity of ceftezol was examined by continuous turbidimetric monitoring of dense populations of Escherichia coli exposed to the drug . Although ceftezol was found to be very active against strains of E . coli, its activity was consistently less than that of the closely related antibiotics, cefazolin . This difference was also found when strains of E . coli were examined in a dynamic system which simulates some of the conditions in which bacteria and drug interact in the treatment of bacterial cystitis . Evidence is presented that the difference in activity between the two cephalosporins resides in a differential ability to induce certain morphological changes in E . coli and in a differential rate of destruction by escherichial beta-lactamases.

Cell, 1976 May, 8(1), 115 - 22
Effect of the RelA gene on the synthesis of individual proteins in vivo; Furano AV et al.; We have shown that the relA gene can affect the rates of synthesis of many nonribosomal proteins in E . coli . We used rel+ and relA strains that contain a temperature-sensitive valyl tRNA synthetase . Upon transfer from a permissive temperature (30degreesC) to a semi-resitrictive one (36.5degreesC), these strains continue to grow, although undergoing a partial deprivation of valyl tRNA, In the rel+ strain, the concentration of ppGpp increases immediately, and the accumulation of RNA ceases abruptly but temporarily . In contrast, in the relA strain, the concentration of ppGpp falls, whereas the rate of accumulation of RNA increases . As judged by gel electrophoresis, the rates at which individual polypeptides are synthesized by the strains after their transfer to 36.5degreesC depend to a large extent upon the allelic state of the relA gene . In both strains, the concentration of ppGpp changes soon enough to have altered the synthesis of some of the proteins by affecting the transcription of their genes.

Zh Mikrobiol Epidemiol Immunobiol, 1976 May, (5), 68 - 71
{A new variant, for the USSR, of enteropathogenic escherichia 0151:K--, isolated from acute intestinal diseases under the conditional name "Crimea"}; Golubeva IV et al.; A study was made of enteropathogenic escherichia "Krym" isolated in the USSR from patients with acute enteric diseases; their antigenic structure was unknown . Also cultures of new serological groups officially recorded as standard test-strains were investigated . By its antigenic structure "Krym" escherichia corresponded to the standard strain of the international set 0151:K-- . Along with the H10 antigen described earlier "Krym" escherichia proved to possess H11 antigen; these two serological types of escherichia were revealed in the USSR, i.e . 0151:K--: H10 and 0151:K--: H11 . The statement on the presence of a new H50 antigen described in escherichia 880-67 with an antigenic formula of 0151:K--: H50 should be revised due to its complex identity with the 0151: K--: H10 strain . For proper serological identification the conditioned name "Krym" should be replaced by "escherichia of serological group 0151:K--".

Nucleic Acids Res, 1976 May, 3(5), 1351 - 71
Cleavage of T4 species I ribonucleic acid by Escherichia coli ribonuclease III; Paddock GV et al.; T4 Species I RNA, a molecule 140 nucleotides in length with some structural features very much like a tRNA, is specifically cleaved by an enzymatic activity in Escherichia coli extracts to give three segments with 19, 48 and 73 nucleotides . We report the purification and characterization of the E . coli RNase which cleaves two 3' phosphodiester bonds of T4 Species I RNA . This reaction has many properties in common with those catalyzed by E . coli RNase III, although the optimal salt conditions for T4 Species I RNA cleavage differ significantly from those for other RNase III-catalyzed reactions . The reaction is not catalyzed by extracts from an E . coli strain lacking RNase III activity . Furthermore, T4 Species I RNA is cleaved by highly purified E . coli RNase III to yield the same three specific fragments . We conclude that this specific cleavage is due to the action of RNase III, and that the requirement for lower ionic strength may reveal further important properties about this RNA processing enzyme.

Nucleic Acids Res, 1976 May, 3(5), 1307 - 22
Specific release of ribosomal proteins by nucleic acid-intercalating agents; Ballesta JP et al.; Increasing concentrations of ethidium bromide cause progressive inactivation of ribosomes, apparently by binding to double-stranded regions of the rRNA . At low drug concentrations (10(-4)M) the partial inhibition detected is due to specific release of proteins L7 and L12; activity can be restored by addition of an excess of these two proteins . At higher concentrations the inactivation is not reversed by supplementation with released proteins . The presence of ethanol affects the extent of ethidium binding and also the release of ribosomal proteins . In all tests the proteins most sensitive to the presence of the drug are L7 and L12, followed by L8/9, L11, L27, L28, L29 and L30 . Despite the fact that L7 and L12 are the first two proteins released by ethidium they are never totally missing from drug-treated ribosomes, though the other proteins can be displaced completely . About 50% of proteins L7 and L12 remain on the ribosomes at the highest drug concentrations tested, possibly indicating heterogeneity in the binding sites for the several copies present in the ribosome.

Nucleic Acids Res, 1976 May, 3(5), 1215 - 24
Fluorescent 2'(3')-O-aminoacylnucleosides-acceptor substrates for ribosomal peptidyltransferase+; Chladek S et al.; 2'(3')-O-L-Phenylalanylderivatives of fluorescent 1,N6-ethenoadenosine and 3,N4-ethenocytidine were prepared by chemical synthesis . Both compounds are good acceptor substrates in ribosomal peptidyltransferase reactions . Since these compounds cannot form Watson-Crick base pairs, the results indicate that the terminal aminoacyladenosine unit of AA-tRNA is bound to ribosomal protein on the acceptor site of peptidyltransferase and not to rRNA.

Nucleic Acids Res, 1976 May, 3(5), 1203 - 13
Stimulation of transcription on chromatin by polar organic compounds; Stratling WH; Polar organic compounds, including DMSO, increase RNA synthesis on isolated chromatin by E . coli RNA polymerase and RNA polymerase II from calf thymus . Transcription is stimulated on chromatin from Friend-virus-infected erythroleukemia cells and from various other sources . Using procedures which inhibit specifically the formation of a stable initiation complex, it is shown that the stimulation does not result from an increase in initiation of both E . coli and the eukaryotic RNA polymerase . After separation of chromatin into template active and inactive fractions, DMSO increases RNA synthesis by a factor of about 1.5 using the template inactive fraction, while stimulation of transcription on the template active portion is lower (factor of 1.2) . It is suggested that the effect on RNA synthesis is mediated by a weakening of the apolar interactions between histones in chromatin subunits, releasing transcription partially from the constraints imposed by histones.

Biochem J, 1976 May 1, 155(2), 429 - 32
Spin-label study of the mobility of enzyme-bound lipoic acid in the pyruvate dehydrogenase multienzyme complex of Escherichia coli; Ambrose MC et al.; The lipoic acid residues covalently bound to the transacetylase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl . The electron-spin-resonance spectrum of the spin-labelled enzyme indicates that the bound nitroxide groups have high mobilities relative to the protein molecule . This physicochemical evidence is consistent with the view that the dithiolane ring of a lipoyl residue is capable of rapid migration between the active sites of the component enzymes in the catalytic mechanism.

Biochem J, 1976 May 1, 155(2), 225 - 9
The concerted inactivation of Escherichia coli uridine diphosphate galactose 4-epimerase by sugar nucleotide together with a free sugar; Blackburn P et al.; 1 . The combined effect of the sugar nucleotides UDP-D-fucose or UDP-D-glucuronic acid together with the free sugars D-fucose or L-arabinose is the inactivation of the Escherichia coli enzyme UDP-galactose 4-epimerase (EC 5.1.3.2) . The sugar nucleotide or the free sugar alone or the sugar nucleotide plus 5'-Ump do not inactivate the enzyme . 2 . The inactivation of the enzyme by its substrate UDP-D-glucose was not affected by the presence of free sugar . 3 . In all cases the inactivation observed follows pseudo-first-order kinetics . 4 . A comparison of various sugar nucleotides indicates that the hydroxymethyl group at position 6 of the sugar moiety of the natural substrates is important for substrate binding.

Biochem J, 1976 May 1, 155(2), 209 - 16
A cyanogen bromide fragment of beta-galactosidase from Escherichia coli with alpha-donor activity in complementation of the enzyme from mutant M15; Marinkovic DV et al.; Aminoethylated beta-galactosidase from Escherichia coli was cleaved by CNBr . The fragment C4a was purified by gel filtration and ion-exchange chromatography . The molecular weight of the fragment C4a was determined to be 9000 +/- 600 . The N-terminal amino acid was found to be isoleucine . Qualitative examination of homogeneity was carried out by disc-gel electrophoresis . The fragment C4a was shown to be active as an alpha donor in complementation of beta-galactosidase activity in vitro with E . coli mutant M15, which has a deletion in the alpha region of the z gene . The molecular weights of complementable fractions from mutant M15 were found to be 123 000 +/- 2500 and 507 000 +/- 11 000, and of the complemented enzyme 522 500 +/- 11 400.

Res Vet Sci, 1976 May, 20(3), 233 - 6
Observations on the association of pathogenic Escherichia coli with small intestinal villi of pigs; Arbuckle JB; Pathogenic Escherichia coli were observed in association with microvilli of piglets experimentally infected with E coli O149:K91,K88a,c, but the bacteria were only rarely observed actually in contact with microvilli . The association of E coli with villi occurred most commonly in the ileum and least commonly in the duodenum: in some pigs, it was only observed in the ileum . The proliferation of pathogenic E coli in the small intestine and their association with villi commonly coexisted, although each phenomenon was also observed independently . It is suggested that the villous association of pathogenic E coli is not a prerequisite for their proliferation.

J Gen Virol, 1976 May, 31(2), 277 - 80
Genes 46 and 47 of phage T4: possible compensation for loss of their function; Minner CA et al.; The gene 46 and 47 functions of phage T4 are required for normal DNA replication, recombination, u.v . repair and host DNA breakdown, and yet am mutants defective in these genes characteristically form tiny plaques on Escherichia coli strains lacking an am suppressor . Our results imply that this limited growth is not due to misreading of am codons or partial function of nearly complete poly-peptides terminated at the am mutation . Thus it appears that genes 46 and 47 are not entirely essential, perhaps because other phage or host products can partially compensate for their loss.

J Gen Microbiol, 1976 May, 94(1), 75 - 89
Uptake of galactose into Escherichia coli by facilitated diffusion; Kornberg HL et al.; Strains of Escherichia coli devoid of systems for the active transport of galactose (galP mgl) still grow on galactose but at rates that are a function of the galactose concentration of the medium: half-maximal growth rates require more than 2 mM-galactose to be present . Evidence is presented that galactose is taken up by such strains by facilitated diffusion on a carrier specified by the umg gene (or by a gene highly co-transducible with it) which is thus a part of, or closely associated with, an enzyme II for glucose of the phosphoenolpyruvate-phosphotransferase system . However, the entry of galactose does not require phosphotransferase activity, and the sugar taken up appears in the cells as free galactose.

Eur J Biochem, 1976 May 1, 64(2), 597 - 606
The subcellular distribution and state of the elongation factor Tu in extracts of Escherichia coli B; Furano AV; The concentration of elongation factor Tu (EF-Tu) is about 8 times that of ribosomes in extracts of Escherichia coli B grown in glucose-minimal medium . 90% of the EF-Tu is found in the ribosome-free fraction of the cell and 75% of this is free of other macromolecules as judged by chromatography on Sephadex G-100; about 10% is bound to the elongation factor EF-Ts and the remaining 10-15% is eluted from Sephadex G-100 earlier than expected for its molecular weight . Tryptic peptide maps of the three forms of EF-Tu are essentially indistinguishable.

Eur J Biochem, 1976 May 1, 64(2), 511 - 8
The specific role of ribosomal protein S1 in the recognition of native phage RNA; van Dieijen G et al.; The previously reported requirement of ribosomal protein S1 for translation of phage RNA is now shown to be related to the involvement of the protein in initiation complex formation . The structure of the messenger RNA appears to be uniquely related to S1 function, since translation and initiation and midly unfolded phage RNA (by modification with formaldehyde) are independent of S1 . It is proposed that S1 functions in conjunction with initiation factor IF-3 by recognizing and unfolding elements of the tertiary structure of phage RNA . A model is suggested for S1 function in both initiation of protein synthesis and initiation of phage RNA replication.

Clin Exp Immunol, 1976 May, 24(2), 336 - 45
Immunological enhancement in the pathogenesis of pyelonephritis; Miller TE et al.; The role of the immune response in pyelonephritis was investigated by manipulation of the host's immune capacity using the immunosuppressive drugs 6-mercaptopurine, cyclophosphamide and thiamphenicol . Treatment with 6-mercaptopurine depressed the humoral immune response but did not have an adverse effect on the course of renal infection . Thiamphenicol administration prevented the development of pathological lesions but this was due to the anti-bacterial activity of thiamphenicol and not to its immunosuppressive activity . Pyelonephritic animals treated with cyclophosphamide did not produce anti-bacterial antibody . Despite this, cyclophosphamide-treated animals were able to eliminate organisms more readily from the infected kidney than untreated animals with a normal humoral immune response . We believe that blocking of the phenomenon of immunological enhancement explains these unexpected results and that the immune response to renal infection may have an immunoenhancing role protecting the bacterial cell from otherwise effective host defence mechanisms.

Can J Microbiol, 1976 May, 22(5), 745 - 51
Role of lipopolysaccharide in adsorption of coliphage T4D to Escherichia coli B; Watanabe T; Coliphage T4D was strongly adsorbed to intact lipopolysaccharides and alkaline and lipase-treated lipopolysaccharides from cells of Escherichia coli B, but was not so adsorbed to heat-treated cells . In contrast, coliphage T2h was not adsorbed to lipopolysaccharides and the heat-treated cells . Acid hydrolysate of lipopolysaccharides strongly inhibited the adsorption of phage T4D to acetone and ether-treated cells . The adsorption of phage T4D to the acetone and ether-treated cells was markedly inhibited by authentic D-glucosamine, N-acetyl-D-glucosamine, alpha-methyl-N-acetyl-D-glucosaminide, alpha-methyl-D-glucoside, and D-maltose . Authentic D-glucose and E,L-2,6-diaminopimelic acid also showed similar activity . These compounds did not affect the adsorption of phage T2h to the acetone- and ether-treated cells . Concanavalin A and wheat-germ agglutinin inhibited phage T4D adsorption to the acetone and ether-treated cells probably by blocking the phage-receptor sites on the cell wall . The blocking by concanavalin A and by wheat-germ agglutinin was reversed by alpha-methyl-D-glucoside and by alpha-methyl-N-acetyl-D-glucosaminide, respectively . Results suggested the possibility that coliphage T4D requires N-acetyl-D-glucosaminyl-glucose or glucosyl-D-glucosamine residues of the core of lipopolysaccharides for the initial attachment to the cell wall of Escherichia coli B.

Can J Biochem, 1976 May, 54(5), 470 - 6
Protein binding and subunit association activity in particles reconstituted from Escherichia coli MRE600 50S ribosomal components; Chu FK et al.; Reconstituted 37S and 48S ribonucleoprotein particles were constructed by incubating Escherichia coli ribosomal RNA with total 50S ribosomal proteins by a sequential incubation method . By comparing the protein compositions of the two types of particles, the proteins that bind to 37S complexes to form 48S particles have been determined . Although only 48S particles could associate with 30S subunits, isolated 37S reconstituted particles could do so if incubated with exogenous 50S proteins . The proteins that bind under these conditions and confer upon particles the ability to associate are L2, L11, L15, L18, and L25 . The involvement of these proteins in 5S RNA binding is discussed.

Am J Vet Res, 1976 May, 37(5), 531 - 4
Sensitivity of Escherichia coli after exposure to lincomycin in vitro and in vivo; DeGeeter MJ et al.; Exposure of 10 Escherichia coli isolates in vitro to a concentration of lincomycin found in the intestine of swine fed the maximun concentration recommended in feed did not significantly affect sensitivity to 8 antibiotics, 1 nitrofuran, and 1 sulfonamide when compared with sensitivity of E coli isolates not exposed to lincomycin . Changes in sensitivity, on the basis of Kirby-Bauer interpretation, did occasionally occur; however, these alterations were in zonal sizes, which were marginal for designation as sensitive, intermediate, or resistant . These same fluctuations were observed in E coli not exposed to lincomycin . Exposure of E coli to lincomycin in the intestinal tract of swine for 34 days did not alter sensitivity of E coli to tetracycline, dihydrostreptomycin, spectinomycin, lincomycin, or triple sulfa . The results indicated that addition of lincomycin to the feed did not appear to promote resistance transfer in E coli.

Proc Natl Acad Sci U S A, 1976 May, 73(5), 1698 - 701
A mutant of Escherichia coli with an altered elongation factor Tu; Pedersen S et al.; A previously isolated mutant of E . coli K12 HAK 88 {Kuwano, M., Endo, h & yamamoto, M . (1972) J . Bacteriol, 112, 1150-1156}, contains a new protein that in two-dimensional gel electropherorgrams has the same molecular weight as normal elongation factor Tu, but whose isoelectric point is altered approximately 0.1 pH unit in the acidic direction . Peptide mapping, purification properties and the ratio of leucyl plus isoleucyl to methionyl plus cysteinyl residues of the normal elongation factor Tu protein and the new protein show a close similarity between the two . The mutation causing the altered electrophoretic mobility is located between argH and rif (79 min on the E . coli genetic map) . These biochemical and genetic data indicate that strain HAK 88 has a mutationally altered tufB gene.

Proc Natl Acad Sci U S A, 1976 May, 73(5), 1518 - 22
Electron microscopy of gene regulation: the L-arabinose operon; Hirsh J et al.; Unlike normal cells, malignant rat and two simian virus 40-transformed human cell lines can neither grow nor survive in B12- and folate-supplemented media in which methionine is replaced by homocysteine . Yet three lines of evidence indicate that the malignant and transformed cells synthesize large amounts of methionine endogenously through the reaction catalyzed by 5-methyltetrahydropterolyl-L-glutamate: L-homocysteine S-methyltransferase (EC 2.1.1.13) . (1) The activities of this methyltransferase were comparable in extracts of malignant and normal cells . (2) The uptake of radioactive label from {5-14C}methyltetrahydropteroyl-L-glutamic acid (5-Me-H4PteGlu) was at least as great in the malignant cells as in the normals and was nearly totally dependent on the addition of homocysteine, the methyl acceptor; furthermore, 59-84% of the label incorporated by cells was recovered as methionine.

Proc Natl Acad Sci U S A, 1976 May, 73(5), 1509 - 12
Synthesis of an R plasmid protein associated with tetracycline resistance is negatively regulated; Yang HL et al.; Synthesis of proteins encoded by the R222 plasmid was observed in a DNA-directed cell-free system and the products were compared to those plasmid proteins synthesized in Escherichia coli minicells . A greater number of plasmid-specified proteins was detected in the in vitro system than in the minicell, suggesting the presence of control factors for plasmid gene expression in the minicell . Synthesis of a newly detected plasmid protein (TET protein) is induced by tetracycline in minicells containing tetracycline-resistant plasmids, including R222, and this induced synthesis correlates with induced host resistance to the drug . This TET protein was synthesized in vitro from R222 DNA in the absence of tetracycline, indicating that no positive regulatory role for tetracycline is required for the protein's synthesis . TET proteon synthesis was inhibited in vitro when cell-free extracts prepared from cells containing the R222 plasmid were used.

Klin Wochenschr, 1976 May 1, 54(9), 449 - 50
{Template activity of chromatin and DNA-dependent RNA polymerase III during postnatal development (author's transl)}; van der Meulen N et al.; The template-activity of chromatin isolated from liver nuclei of developing rats increases sharply at birth and decreases remarkable until the 10th day . Between the 10th and the 40th day a slow increase was shown in template activity of chromatin tested with purified E.-coli RNA-polymerase (EC 2.7.7.6) . We correlated these findings with changes of RNA-polymerase III-activity in rat liver during development.

JACEP, 1976 May, 5(5), 332 - 3
Infection rate of simple suturing; Galvin JR et al.; Four hundred consecutive patients with superficial lacerations participated in a study of infection rate of lacerations sutured in an emergency department . Of these, 322 (85%) returned for suture removal . An infection rate of 5% was noted . Reported incidence of infection among the 68 (17%) non-returnees was 5.8%.

Surgery, 1976 May, 79(5), 564 - 8
Direct effect of endotoxin on the gastric mucosal microcirculation and electrical gradient; Cheung LY et al.; The effects of intra-arterial infusion of E . coli endotoxin at 1.0 mg . per minute on the gastric total and mucosal blood flows, electrical potential difference, and ionic fluxes across the gastric mucosa were studied in an exteriorized, chambered preparation of canine fundic stomach . Gamma-labelled microsphere technique was used in addition to venous drainage and plasma aminopyrine clearance for the measurement of total and mucosal blood flow, respectively . In spite of normal systemic blood pressure throughout the experiment, E . coli endotoxin infusion caused a significant decrease in total gastric blood flow and in the fractional distribution of flow to the mucosae . There was no significant arteriovenous shunting of microspheres . Significant reduction in potential difference and hydrogen-ion back diffusion also was noted after endotoxin infusion, possibly as a consequence of reduced mucosal blood flow . The results indicate that significant gastric mucosal ischemia can occur and may represent a mechanism in the development of gastric erosions in endotoxemia, even in the absence of systemic hypotension.

J Bacteriol, 1976 May, 126(2), 990 - 6
In vivo methylation by Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase protects against in vitro cleavage by the RII restriction endonuclease (R . Eco RII); Schlagman S et al.; We have analyzed the susceptibility of the deoxyribonucleic acid (DNA) of phage fd replicative form (RF) and of Escherichia coli to in vitro cleavage by purified RII restriction endonuclease (R . Eco RII) . The results are summarized as follows: (i) fd, mec- RFI, isolated from infected E . coli K-12 mec- bacteria (a mutant strain lacking DNA-cytosine methylase activity), is cleaved into at least two fragments, whereas fd . mec+ RFI, isolated from the parental mec+ strain, is not cleaved . (ii) E . coli mec- DNA is extensively degraded, whereas mec+ DNA-cytosine methylase acts as an RII modification enzyme.

J Bacteriol, 1976 May, 126(2), 985 - 9
Pyrimidine dimer excision in surviving and nonsurviving cells of ultraviolet-irradiated cultures of Escherichia coli; Schenley RL et al.; We compared dimer excision in viable and nonviable cells fractions separated from Escherichia coli B/r cultures exposed to ultraviolet (UV) irradiation . For cells grown on minimal medium with glycerol as a carbon source, both fractions from the irradiated (20 J/m2, 5% survival) culture excised 60 to 70% of the thymine dimers from prelabeled DNA within 120 min . This percentage was, within experimental error, the same as that obtained from unseparated cultures . When isolated viable and nonviable populations were given a second UV exposure (20 J/m2) both types of cells were again able to excise dimers . The UV survival curve for the isolated viable population indicates that these cells are no more sensitive to radiation than exponentially growing cells not previously exposed to UV . The extent of dimer excision after UV irradiation was also the same in viable and nonviable cells separated from cultures grown on a glucose minimal medium in which both populations excised about 85% of the dimers within 120 min . These results show that the extent of removal of pyrimidine dimer from deoxyribonucleic acid is not precisely correlated with survival of repair-competent bacterial cells after exposure to UV light.

J Bacteriol, 1976 May, 126(2), 977 - 84
Centrifugal separation of irradiated cultures of Escherichia coli cells into viable and nonviable populations; Schenley RL et al.; Incubation of ultraviolet-irradiated Escherichia coli B/r cultures with 0.7% Triton X-100 resulted in a large decrease in turbidity . Under phase-contrast optics, most of the irradiated detergent-treated cells were smaller than normal and of low phase density; only a small percentage were normal or larger than normal and of normal phase density . Irradiated cells not treated with detergent showed fewer pronounced morphological changes . Irradiated cells treated with detergent lost large amounts of proteins and ribonucleic acid, but not of deoxyribonucleic acid . Such cultures could be separated by centrifugation into populations of (i) slowly sedimenting cells consisting of small, phase-light cells of low viability and (ii) large cells of normal phase density and high viability (100%) . A similar separation was effected in gamma-irradiated cultures.

J Bacteriol, 1976 May, 126(2), 951 - 8
Periplasmic protein related to the sn-glycerol-3-phosphate transport system of Escherichia coli; Silhavy TJ et al.; Two-dimensional gel electrophoresis of shock fluids of Escherichia coli K-12 revealed the presence of a periplasmic protein related to sn-glycerol-3-phosphate transport (GLPT) that is under the regulation of glpR, the regulatory gene of the glp regulon . Mutants selected for their resistance to phosphonomycin and found to be defective in sn-glycerol-3-phosphate transport either did not produce GLPT or produced it in reduced amounts . Other mutations exhibited no apparent effect of GLPT . Transductions of glpT+ nalA phage P1 into these mutants and selection for growth on sn-glycerol-3-phosphate revealed a 50% cotransduction frequency to nalA . Reversion of mutants taht did not produce GLPT to growth on sn-glycerol-3-phosphate resulted in strains that produce GLPT . This suggests a close relationship of GLPT to the glpT gene and to sn-glycerol-3-phosphate transport . Attempts to demonstrate binding activity of GLPT in crude shock fluid towards sn-glycerol-3-phosphate have failed so far . However, all shock fluids, independent of their GLPT content, exhibited an enzymatic activity that hydrolyzes under the conditions of the binding assay, 30 to 60% of the sn-glycerol-3-phosphate to glycerol and inorganic orthophosphate.

J Bacteriol, 1976 May, 126(2), 861 - 8
Clo DF13 plasmid deoxyribonucleic acid-directed in vitro synthesis of biologically active cloacin DF13 and clo DF13 immunity protein; Konings RN et al.; Clo DF13 plasmid deoxyribonucleic acid (DNA) was used as a template to direct transcription and translation in a DNA-dependent cell-free system prepared from Escherichia coli . Analysis of the invitro products on sodium dodecylsulfate-polyacrylamide gels revealed that Clo DF13 DNA directs the synthesis of at least 10 polypeptides ranging in molecular weight from approximately 7,000 to 70,000 . Two of these polypeptides could be identified, with respect to their physiochemical and biological characteristics, as the products of the Clo DF13 genes coding for cloacin DF13 and Clo DF13 immunity protein . These results confirm previous findings, obtained which Clo DF13-harbouring minicells of E . coli, that the structural tenes for the latter proteins residue on the Clo DF13 genome.

J Bacteriol, 1976 May, 126(2), 814 - 22
Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport; McKeown M et al.; A commonly used strain of Escherichia coli K-12 was shown to be deficient in the transport of a number of nucleosides, including thymidine . Thymidine incorporation was unaffected . Strain AB2497 exhibited a strikingly lower thymidine pulse-label incorporation at low (less than 1 mug/ml) thymidine concentrations than do many other strains . The deficiency appeared to be due to mutation in a single gene . This gene, which we designated nup (for nucleoside uptake), is located at 10 to 13 min on the E . coli linkage map . In nup+ strains, the transport of a given nucleoside was relatively insensitive to large excesses of other nucleosides but was competitively inhibited by the same nucleoside . Mutants deficient inthymidine kinase are deficient in thymidine uptake but normal in deoxyadenosine uptake . A two-step model for nucleoside transport is presented in which the first step, utilizing the nup gene product, is a nonspecific translocation of nucleoside to the interior of the cell . In the second step, the individual nucleosides are modified by cellular enzymes (e.g., nucleosides kinases) facilitate accumulation.

J Bacteriol, 1976 May, 126(2), 593 - 600
Characterization of an Escherichia coli K-12 F-Con-mutant; Havekes LM et al.; An Escherichia coli K-12 F-mutant defective in conjugation was isolated by means of a zygotic induction enrichment procedure . The recipient ability of the mutant was reduced about 50 times owing to a block in one of the first steps of the conjugation process . In the mutant, cell envelope alterations could not be observed . Sensitivity toward detergents, antibiotics, and phages was unaltered . The mutation appeared to be co-transducible with pyrD . The linkage order in the region of the mutation is origin KL 99-con-pyrD-aroA.

J Bacteriol, 1976 May, 126(2), 563 - 7
Extragenic suppression of two ribosomal protein cistrons lying near the rif locus in Escherichia coli; Molholt B; The experiments reported here involve temperature-sensitive mutations in two of five cistrons encoding 50S ribosomal proteins that lie near the rif locus in Escherichia coli . I selected spontaneous TS+ mutants able to grow at elevated temperatures in which the TS+ event takes place outside this tract of cistrons near rif . Six distinct classes of extragenic suppressors were found, five of which have been mapped . Two of these suppressors lie near 64 min, a region known to be rich in cistrons ribosomal proteins (Dennis and Nomura, 1975) . The remaining three extragenic suppressors were located near 16.5, 47, and 86 min.

Biochem J, 1976 May 1, 155(2), 419 - 27
Selective inactivation of the transacylase components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli; Brown JP et al.; 1 . The reaction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli with maleimides was examined . In the absence of substrates, the complex showed little or no reaction with N-ethylmaleimide . However, in the presence of pyruvate and N-ethylmaleimide, inhibition of the pyruvate dehydrogenase complex was rapid . Modification of the enzyme was restricted to the transacetylase component and the inactivation was proportional to the extent of modification . The lipoamide dehydrogenase activity of the complex was unaffected by the treatment . The simplest explanation is that the lipoyl groups on the transacetylase are reductively acetylated by following the initial stages of the normal catalytic cycle, but are thereby made susceptible to modification . Attempts to characterize the reaction product strongly support this conclusion . 2 . Similarly, in the presence of N-ethylmaleimide and NADH, much of the pyruvate dehydrogenase activity was lost within seconds, whereas the lipoamide dehydrogenase activity of the complex disappeared more slowly: the initial site of the reaction with the complex was found to be in the lipoyl transacetylase component . The simplest interpretation of these experiments is that NADH reduces the covalently bound lipoyl groups on the transacetylase by means of the associated lipoamide dehydrogenase component, thereby rendering them susceptible to modification . However, the dependence of the rate and extent of inactivation on NADH concentration was complex and it proved impossible to inhibit the pyruvate dehydrogenase activity completely without unacceptable modification of the other component enzymes . 3 . The catalytic reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by NADH in the presence of the pyruvate dehydrogenase complex was demonstrated . A new mechanism for this reaction is proposed in which NADH causes reduction of the enzyme-bound lipoic acid by means of the associated lipoamide dehydrogenase component and the dihydrolipoamide is then oxidized back to the disulphide form by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) . 4 . A maleimide with a relatively bulky N-substituent, N-(4-diemthylamino-3,5-dinitrophenyl)maleimide, was an effective replacement for N-ethylmaleimide in these reactions with the pyruvate dehydrogenase complex . 5 . The 2-oxoglutarate dehydrogenase complex of E . coli behaved very similarly to the pyruvate dehydrogenase complex, in accord with the generally accepted mechanisms of the two enzymes . 6 . The treatment of the 2-oxo acid dehydrogenase complexes with maleimides in the presence of the appropriate 2-oxo acid substrate provides a simple method for selectively inhibiting the transacylase components and for introducing reporter groups on to the lipoyl groups covalently bound to those components.

Can J Microbiol, 1976 May, 22(5), 712 - 8
Further studies on the relative antiviral efficacies of interferons induced by poly I:C and Mengo virus; Campbell JB; Mouse serum interferons induced by polyI:C, vesicular stomatitis virus (VSV), reovirus, and Mengo virus were assayed in monolayers of mouse L-929 cells by the plaque-reduction method using both VSV and Mengo as challenge viruses . Titers obtained with Mengo virus as challenge were all lower than with VSV . With the interferons induced by VSV, reovirus, and ployI:C, the reductions were of the order of two- to three-fold . With Mengo virus-induced interferon the reduction was much greater (about 17-fold) . This offers an explanation for the observation that, unit for unit (measured by the plaque reduction of VSV), Mengo virus-induced interferon is only about 1/10 as effective as polyI:C-induced interferon in protecting mice against lethal infection with Mengo virus . The data are consistent with the hypothesis that an interferon antagonist is produced in the serum of mice infected with Mengo virus . This antagonist, which is not produced in mice inoculated with polyI:C, or reovirus, effectively blocks the antiviral action of interferon during Mengo virus infections, both in vivo and in vitro.

Bol Med Hosp Infant Mex, 1976 May-Jun, 33(3), 595 - 606
{New concepts on the etiopathogenesis of diarrhea}; Olarte J; The author discusses new knowledge concerning E . coli enterotoxins and Duovirus in the production of diarrheal disease.

J Med Chem, 1976 May, 19(5), 643 - 7
Synthesis and biological studies of 3-(beta-D-ribofuranosyl)-2,3,-dihydro-6H-1,3-oxazine-2,6-dione, a new pyrimidine nucleoside analog related to uridine; Chwang TL et al.; Reaction of the trimethylsilyl derivative of 2,3-dihydro-6H-1,3-oxazine-2,6-dione (2, "uracil anhydride") with protected 1-O-acetylribofuranoses in the presence of stannic chloride gave the corresponding block nucleosides . 3-(2,3-5-Tri-O-2',2',2'-trichloroethoxycarbonyl-beta-d-ribofuranosyl)-2,3-dihydro-6H-1,3-oxazine-2,6-dione (4c) thus prepared from the protected sugar 3c, 1-O-acetyl-2,3,5-tri-O-(2,2,2-trichloroethoxycarbonyl)ribofuranose, gave, on removal of the protecting groups with zinc dust,3-(beta-d-ribofuranosyl)-2,3-dihydro-6H-1,3-oxazine-2,6-dione (1) . The structure of 1 was confirmed by uv, ir, NMR, and CD spectral data and was shown to be an N nucleoside . Uracil anhydride, 2, and, to a lesser extent, its ribonucleoside 1 exert a moderate growth inhibition of mouse leukemia L5178Y, HeLa, and Novikoff hepatoma cells i- culture . Both compounds produce weak inhibition of vaccinia viral replication in HeLa cells.

Cancer Res, 1976 May, 36(5), 1761 - 70
Inhibition of rat liver RNA polymerases by action of the methylating agents dimethylnitrosamine in vivo and methyl methanesulfonate in vitro; Herzog J et al.; Dimethylnitrosamine maximally inhibits rat liver nuclear RNA synthesis by 50% at a dose of 40 mg/kg of body weight . The inhibition develops during the first 4 hr and persists through the 12th hr . All parenchymal cells of the lever lobule seem to be affected . The decreased RNA synthesis can be accounted for entirely by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified . A similar inhibition of the polymerase activities was demonstrated in the intact nuclei by inactivating the endogenous template with actinomycin D and assaying the polymerases with an added exogenous template, poly(deoxy-adenylate-deoxythymidylate) . Chromatin was prepared by two methods differing in the extent to which they remove the endogenous polymerase activity . Each preparation was transcribed with either added Escherichia coli or partially purified rat liver nucleoplasmic RNA polymerase . With either polymerase or chromatin preparation, no inhibition of the template activity of liver nuclear chromatin isolated from the DMN-treated animals was detected . A similar mechanism of inhibition of RNA synthesis was produced by the action of the methylating agent methyl methanesulfonate on whole nuclei in vitro . The dose-dependent inhibition of RNA synthesis could be accounted for by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified from the affected nuclei . Chromatin prepared from the methyl methanesulfonate-treated nuclei had a normal template capacity with either E . coli or rat liver nucleoplasmic RNA polymerase . No preferential methylation of the RNA polymerases by {14C}methyl methanesulfonate could be demonstrated . It is concluded that the action of the two methylating agents on RNA metabolism is similar and that the inhibition of liver nuclear RNA synthesis results from inactivation of the RNA polymerases . At the same time, dimethylnitrosamine and methyl methanesulfonate leave the chromatin template intact, at least quantitatively, for the synthesis of RNA . The implications of such an effect on RNA synthesis are discussed.

J Bacteriol, 1976 May, 126(2), 999 - 1001
Altered deoxyribonucleotide pools in P2 eductants of Escherichia coli K-12 due to deletion of the dcd gene; Neuhard J et al.; Deletion of the Escherichia coli K-12 chromosome associated with P2 mediated education extend through the structural gene for uridine kinase, udk, and the dcd gene encoding 2'-deoxycytidine 5'-triphosphate deaminase . The lack of uridine kinase makes a positive selection possible for these strains . Due to the dcd mutation, P2 eductants show large alterations in their deoxyribonucleoside triphosphate pools.

J Bacteriol, 1976 May, 126(2), 852 - 60
Lack of glucose phosphotransferase function in phosphofructokinase mutants of Escherichia coli; Roehl RA et al.; Phosphofructokinase (pfkA) mutants of Escherichia coli are impaired in growth on all carbon sources entering glycolysis at or above the level of fructose 6-phosphate (nonpermissive carbon sources), but growth is particularly slow on sugars, such as glucose, which are normally transported and phosphorylated by the phosphoenolpyruvate, (PEP)-dependent phosphotransferase system (PTS).

J Bacteriol, 1976 May, 126(2), 785 - 93
Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12; Billen D et al.; Toluene-treated Escherichia coli mutants have been used to study the roles of deoxyribonucleic acid (DNA) polymerases I, II, and III, and of DNA ligase in repair synthesis and strand rejoining following X-irradiation . In cells possessing all three DNA polymerases, both a greater amount of repair synthesis ("exaggerated" repair synthesis) and failure of ligation are observed when DNA ligase activity is inhibited . In a mutant lacking the polymerizing activity of DNA polymerase I, exaggerated repair synthesis is not observed, and strand rejoining does not occur even if DNA ligase is fully activated . In a mutant possessing the polymerizing activity of DNA polymerase I but lacking its 5'leads to 3' exonuclease activity, exaggerated repair synthesis is minimal . After irradiation, DNA polymerases II and III are capable of carrying out an adenosine 5'-triphosphate-dependent repair synthesis,but rejoining of strand breaks does not occur and exaggerated synthesis is not seen whether DNA ligase is active or not . These results suggest that DNA polymerase I and DNA ligase act together to limit repair synthesis after X irradiation and that both are necessary in toluene-treated cells for strand rejoining . DNA polymerases II and III apparently cannot complete chain elongation and gap filling, and therefore repair carried out by these enzymes does not respond to ligase action.

J Bacteriol, 1976 May, 126(2), 646 - 53
Methyl methane sulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease specific for apurinic sites in deoxyribonucleic acid; Ljungquist S et al.; A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment . The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays . Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity . These endonuclease-deficient strains are more MMS-sensitive than wild-type strains . Revertants of these deficients strains to normal MMS resistance were isolated . They had increased levels of the endonuclease activity but did not attain wild-type levels . The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment . Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12 . A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts . The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition . The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity.

Z Naturforsch {C}, 1976 May-Jun, 31(5-6), 255 - 62
{Coalpha/Cobeta-isomerism of corrinoids . Partial synthesis and Escherichia coli activity of further isomer pairs of the (Co-methyl)-corrinoids (author's transl)}; Moskophidis M et al.; In 2-methyladenyl-(Cobeta-methyl)-cobamide and in adenyl-(cobeta-methyl)-cobamide the nucleotide base is coordinated to the cobalt atom in neutral and weak acidic aqueous solutions (in the corresponding adenosylcorrinoids the nucleotide base is not coordinated) . 2-Methylthioadenyl-(Cobeta-methyl)-cobamide resembles, with regard to the coordination of the nucleotide base, the benzimidazole-corrinoids . The partial synthesis via cobalt (I) corrinoids results in a variable proportion: (Coalpha-methyl) isomer/(Cobeta-methyl) isomer, e . g . 7/93 (cobalamin) and 50/50 (p-cresylcobamide) . This proportion is generally low, if in the corresponding cyanocorrinoid the nucleotide base is firmly coordinated; it is high, if the coordination in the corresponding cyanocorrinoid is weak or absent . These results are compatible with the assumption that in the cobalt (I) corrinoids the nucleotide base is to a certain extent coordinated . In Escherichia coli 113-3 the examined (Coalpha-methyl)-corrinoids show a weaker bioactivity than the corresponding (Cobeta-methyl)-corrinoids.

J Biochem (Tokyo), 1976 May, 79(5), 927 - 37
A novel adenosine triphosphatase isolated from RNA polymerase preparations of Escherichia coli . II . Enzymatic properties and molecular structure; Ishihama A et al.; An adenosinetriphosphatase (ATPase) {EC 3.6.1.3} copurified with the DNA-dependent RNA polymerase {EC 2.7.7.6} from Escherichia coli was isolated to apparent homogeneity and some of its functional as well as structural properties were examined . Although the novel ATPase exhibited metal requirements similar to those of Mg2+, Ca2+-ATPase, its response to NaN3 and antisera appeared completely different from that of the Mg2+, Ca2+-ATPase . The purified ATPase was found to be a large protein with a molecular weight of 9.3X10(5) daltons, composed of identical subunits of 7X10(4) daltons . When viewed under an electron microscope, the ATPase appeared to be very similar to material previously misidentified as the RNA polymerase . The physiological role of the novel ATPase, however, remains unclear.

J Biochem (Tokyo), 1976 May, 79(5), 917 - 25
A novel adenosine triphosphatase isolated from RNA polymerase preparations of Escherichia coli . I . Copurification and separation; Ishihama A et al.; Adenosinetriphosphatase (ATPase) {EC 3.6.1,3} activity has been found to exist in most preparations of DNA-dependent RNA polymerase {EC 2.7.7.6} obtained from Escherichia coli by a number of purification procedures so far established . Electrophoretic analysis on polyacrylamide gels demonstrated that ATP hydrolysis and RNA synthesis were catalyzed by two distinct enzyme proteins . It appears that the two enzymes are associated or have similar molecular properties . Separation of the two enzymes, the object of the present work, was achieved by three independent methods: ion exchange chromatography on a phosphocellulose column, electrophoresis in glycerol gradients, or high-salt glycerol gradient centrifugation.

J Virol, 1976 May, 18(2), 709 - 18
Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA; Davis J et al.; A sensitive nitrocellulose filter assay that measures the retention of 125I single-stranded calf thymus DNA has been used to detect and purify DNA-binding proteins that retain a biological function from Rauscher murine leukemia virus . By consecutive purification on oligo (dT)- cellulose and DEAE-Bio-Gel columns and centrifugation in 10 to 30% glycerol gradients, RNA-dependent DNA polymerase has been separated from a second virion DNA-binding protein . The binding of this protein to DNA was strongly affected by NaCl concentration but showed little change in activity over a wide range of temperature or pH . After glycerol gradient purification, polyacrylamide gel electrophoresis of this protein showed one major band with a molecular weight of approximately 9,800 . This protein binds about as well as to single-stranded Escherichia coli or calf thymus DNA or 70S type C viral RNA . The binding to 125I single-stranded calf thymus DNA is very efficiently inhibited by unlabeled single-stranded DNA from either E . coli or calf thymus and by 70S murine or feline viral RNA . Much larger amounts of double-stranded DNA are required to produce an equivalent percentage of inhibition . This protein, therefore, shows preferential binding to single-stranded DNA or viral RNA.

Lancet, 1976 May 1, 1(7966), 930 - 4
Enhanced antibody responses in active chronic hepatitis: relation to HLA-B8 and HLA-B12 and porto-systemic shunting; Galbraith RM et al.; Titres of antibodies to rubella, measles, smooth muscle, nuclei, and Escherichia coli were examined in relation to the presence of particular histocompatibility antigens in 57 patients with active chronic hepatitis, 8 of whom were HBsAg positive . With the exception of antibodies to E . Coli, the HBsAg-negative patients with HLA-B8 or HLA-B12 had higher titres than those with neither, and antibody titres were highest in the 7 cases with both these histocompatibility antigens . In contrast, E . coli antibody titres were not related to the presence of particular histocompatibility antigens but correlated closely with the degree of portosystemic shunting . None of the HBsAg-positive patients possessed HLA-B8, and titres of all the antibodies were significantly lower than in the HBsAg-negative cases . The increased antibody response in HBsAg-negative patients is likely to be due to a genetically determined increase in immunological responsiveness for which HLA-B8 and HLA-B12 are markers . The results obtained in healthy family members also suggest that this defect in immunoregulation is under polygenic control.

Cell, 1976 May, 8(1), 123 - 8
The mechanism of action of ppGpp on rRNA synthesis in vitro; van Ooyen AJ et al.; We have studied the mechanism of the specific inhibition of ribosomal RNA synthesis by ppGpp in a purified system using as templates E . coli DNA and DNA from lambdad5ilv, which carries a rRNA cistron from E . coli . Ribosomal RNA synthesis, as well as its inhibition by ppGpp, are critically salt-dependent . Of a number of guanosine phosphates tested, only pppGpp (MS II) mimicked the action of ppGpp, establishing the specificity of ppGpp . The two templates gave similar results for rRNA synthesis in all experiments . By using the initiation inhibitor rifampicin, we could show that the specific inhibition of rRNA synthesis by ppGpp is due to its effect on rRNA initiation . The somewhat variable inhibition of RNA synthesis in general by ppGpp is mainly or wholly a consequence of premature chain termination . We propose that ppGpp specifically inhibits rRNA synthesis by acting on the formation of the so-called "closed-promoter complex".

Can J Biochem, 1976 May, 54(5), 413 - 22
Modified 5'-nucleotides resistant to 5'-nucleotidase: isolation of 3-(3-amino-3-carboxypropyl) uridine 5'-phosphate and N2, N2-dimethylguanosine 5'-phosphate from snake venom hydrolysates of transfer RNA; Gray MW; A procedure for the quantitative measurement of the O2'-methylnucleoside constitutents of RNA has recently been developed in this laboratory (Gray, M.W . Can . J . Biochem . 53, 735-746 (1975)) . This assay method is based on the resistance of O2'-methylnucleoside 5'-phosphates (pNm) (generated by phosphodiesterase hydrolysis of RNA) to subsequent dephosphorylation by venom 5'-nucleotidase (EC 3.1.3.5) . In the present investigation, two base-modified 5'-nucleotides, each displaying an unusual resistance to 5'-nucleotidase, have been identified . These compounds have been characterized by a variety of techniques as N2, N2-dimethylguanosine 5'-phosphate (pm2/2G) and 3-(3-amino-3-carboxypropyl)uridine 5'-phosphate (p4abu3U) . Because of their resistance to 5'-nucleotidase, pm2/2G and p4abu3U are isolated along with the pNm in the mononucleotide fraction of venom hydrolysates of transfer RNA . Under hydrolysis conditions, the stability of p4abu3U is comparable to that of a pNm, allowing quantitative assay of the nucleotide . The proportion (mean +/- SD) of p4abu3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA has been determined to be 0.35 +/- 0.03 (n=5) and 0.14 +/- 0.02 (n=4) mol%, respectively . The absence of p4abu3U in venom hydrolysates of yeast tRNA implies the absence of the corresponding nucleoside in yeast tRNA, in agreement with existing data . The variable recovery of pm2/2G from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistent to 5'-nucleotidase . The complete absence of pm2/2G in venom hydrolysates of E . coli tRNA is consistent with the known absence of N2, N2-dimethylguanosine in this RNA . These observations demonstrate that resistance to 5'-nucleotidase is a necessary but not sufficient criterion for concluding that a 5'-nucleotide is O2'-methylated . When applied to wheat embryo ribosomal RNA, the analytical methods described in this report failed to reveal any compound having the distinctive charge properties of p4abu3U . It therefore appears that 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, recently characterized as a constituent of the 18 S rRNA of Chinese hamster cells (Saponara, A.G . & Enger, M.D . Biochim . Biophys . Acta 349, 61-77 (1974)), may not be present in wheat embryo ribosomal RNA.

J Bacteriol, 1976 May, 126(2), 601 - 8
Effect of colicin K on a membrane-associated, energy-linked function; Sabet SF; The purpose of this work was in investigate the capability of cell extracts of Escherichia coli and E . coli treated with colicin K to catalyze the following energy-dependent reverse transhydrogenase reaction: NADP + NADH + ATP in equilibrium NADPH + NAD +ADP + Pi . Under anaerobic conditions this reaction requires the presence of a specific portion of the electron transport chain, a functional energy coupling system, including an adenosine triphosphatase, enzyme, and ATP as energy source . The ATP-linked reaction was partially inhibited in French press extracts of E . coli K-12 C600 cells that had been pretreated with colicin K but not in extracts from similarly treated cells of a colicin-tolerant mutant . Ultracentrifugation of extracts yielded particulate fractions competent in catalyzing the reaction; this reaction is substantially inhibited in fractions from colicin-treated cells . The extent of inhibition increased with increasing concentration of colicin . Supernatants also supported ATP-linked formation of NADPH, but this reaction was insensitive to the colicin effect . A comparison between the requirement of the reaction in supernatant and particulate fractions suggests that the reaction in the supernatant is different from the one inhibited by colicin . The ATP-hydrolyzing ability of particulate fractions from the control or treated bacteria was identical . Likewise, the electron transport chain was not affected by colicin treatment, as evidenced from lack of effect on NADH oxidase, succinic dehydrogenase, and NADPH-NAD transhydrogenase . It is concluded that colicin K interferes with the coupling of ATP the utilization of the intermediate for the ATP-linked transdehydrogenase reaction.

N Engl J Med, 1976 Apr 29, 294(18), 965 - 72
Human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children; Kapikian AZ et al.; We found a human reovirus-like agent in the stools of 42 per cent of 143 infants and young children hospitalized with acute gastroenteritis between January, 1974, and June, 1975 . Half the patients studied by electron microscopy and serologic technics had evidence of infection with the agent . The infection had a seasonal pattern: 59 per cent of those admitted during the cooler months (November to April) shed the agent, with a peak of 78 per cent in December, 1974, and January, 1975, combined . None of the patients admitted during the warmer months (May to October) shed the agent . None of 275 Escherichia coli isolates from 32 patients with diarrhea produced heat-labile enterotoxin, whereas 17 of the 32 had evidence of infection with the reovirus-like agent . In addition, 14 of 40 parents of 37 patients with diarrhea associated with the reovirus-like agent were also infected, but most infectious were inapparent . This agent appears to be the major cause of diarrheal illness in the young during the cooler months.

Virchows Arch B Cell Pathol, 1976 Apr 29, 20(3), 229 - 38
Evaluation of a short term in vitro test for granulocyte chalone activity; Maurer HR et al.; A short term in vitro test for granulocyte chalone activity eas examined for its specificity and reliability . The test used the inhibition, by granulocyte extracts, of 3H-thymidine (3H-Tdr) uptake in to the acid-insoluble material by rat bone marrow cells in vitro to measure possible chalone activity . Among the many possible 3H-Tdr artifacts pool size dilution by Tdr contained in the extracts was excluded using an E . coli mutant requiring thymine . Several amino acids and biogenic amines do not affect the test . However, continuous and pulse labelling of bone marrow cells with 3H-Tdr, viability tests and micro flow fluorometric measurements of the cell cycle distribution following colcemid treatment strongly suggests that the cells do not proliferate in vitro during short term incubation, since practically no cells enter the S-phase, cells in the S-phase die and few if any cells proceed through G2 and mitosis . Moreover, the test cannot exclude cytotoxicity . Thus, the in vitro test may only sceem for an unspecific S-phase inhibitor and must hence be supplemented by another assay to prove the chalone nature of an extract or fraction . The test per se fails to meet most of the requirements of a valid granulocyte chalone assay.

J Biol Chem, 1976 Apr 25, 251(8), 2493 - 8
Different mechanisms of energy coupling for transport of various amino acids in cells of Mycobacterium phlei; Prasad R et al.; Whole cells of Mycobacterium phlei were shown to actively accumulate proline, leucine, lysine, tryptophan, histidine, glutamine, and glutamic acid to different steady state levels . The transport of proline, in contrast to that of other amino acids, was found to be insensitive to various respiratory inhibitors, e.g . cyanide, arsenate, azide, and sulfhydryl reagents . However, oxygen was an obligatory requirement for the uptake of proline, as well as for the other amino acids . The results indicate that the energy requirements for proline uptake are different from those of other amino acids . In contrast to the system from Escherichia coli, the mode of energy transduction for the uptake of proline, glutamine, and glutamic acid is different even though these amino acids are shock resistant in the M . phlei system.

J Biol Chem, 1976 Apr 25, 251(8), 2399 - 404
Terminal deoxynucleotidyltransferase . Serological studies and radioimmunoassay; Kung PC et al.; Mouse antisera against calf terminal deoxynucleotidyltransferase (terminal transferase) have been prepared . The sera have been used to characterize terminal transferase both by studying inhibition of enzyme activity and by developing a competition radioimmunoassay using highly purified 125I-labeled terminal transferase . By either assay, anti-terminal transferase serum did not cross-react significantly with calf DNA polymerases alpha and beta, Escherichia coli DNA polymerase I, or the reverse transcriptase of Moloney mouse leukemia virus . The calf terminal transferase did, however, share cross-reactive but not identical determinants with human and murine terminal transferase . The radioimmunoassay could detect as little as 2 ng of terminal transferase/mg of soluble protein in a tissue extract . Thymocytes were found to contain 280 ng of terminal transferase/mg of cell protein or about 1 X 10(5) molecules/cell; bone marrow had about 1% of the level of enzyme found in thymus . Extracts of spleen, peripheral white blood cells, lymph nodes, liver, muscle, and kidney all lacked detectable antigenicity of terminal transferase . These data indicate that terminal transferase is a tissue-specific enzyme and is not related to other DNA polymerases.

J Biol Chem, 1976 Apr 25, 251(8), 2487 - 92
Factors affecting the acyl selectivities of acyltransferases in Escherichia coli; Okuyama H et al.; In Escherichia coli the synthesis of phosphatidic acid from glycerophosphate involves first the intermediate formation of 1-acyl glycerophosphate . This reaction is catalyzed by the membrane-bound acyl-CoA(acyl-ACP):glycerophosphate acyltransferase . The 1-acylglycerophosphate is then converted to phosphatidic acid by acyl-CoA(acyl-ACP):1-acylglycerophosphate acyltransferase (Okuyama, H., and Wakil, S.J . (1973) J . Biol . Chem . 248, 5197-5205) . In vitro both acyltransferases utilize various saturated and unsaturated acyl-CoAs at comparable rates, resulting in the incorporation of both saturated and unsaturated fatty acids into position 1 as well as position 2 of the glycerophosphate moiety . Thus, the specificities of acyltransferase systems as compared with regard to the maximal velocities for various acyl-AoAs, do not explain the positional distribution of the individual fatty acid in phospholipid molecules . The selectivities of the acyltransferases for acyl-CoAs are variable depending upon the acceptor concentration . In the presence of both palmitoyl-CoA and oleoyl-CoA and at low concentrations of the acceptors comparable to those found in vivo, the acylation at position 1 of glycerophosphate by acyl-CoA:glycerophosphate acyltransferase showed higher preference for palmitate, whereas the acylation at position 2 by acyl-CoA:1-acylglycerophosphate acyltransferase showed higher selectivity for oleate . In the presence of saturating amounts of the acceptors, the acylation at position 1 or position 2 was less selective for the acyl-CoAs . The ratios of saturated acyl-CoA to unsaturated acyl-CoA also affect the ratios of the fatty acids incorporated in vitro into positions 1 and 2 of phosphatidic acid; relatively more palmitate was incorporated when the proportion of palmitoyl-CoA among the acyl donors was higher and vice versa . Thus, highly selective positioning of various acyl-CoAs observed at lower concentrations of the acceptors in phosphatidic acid synthesis in vitro helps to explain the selective distribution of saturated and unsaturated fatty acids at positions 1 and 2 of glycerophospholipids in the membranes . Another factor, the availability of acyl donors, affects the proportions of different molecular species of phosphatidic acid, which at least partly explains the variability of molecular species of phospholipids found in vivo.

J Biol Chem, 1976 Apr 25, 251(8), 2475 - 9
An antimutator deoxyribonucleic acid polymerase . I . Purification and properties of the enzyme; Lo KY et al.; The DNA polymerase induced by an antimutator T4 phage has been purified to apparent homogeneity and has been compared to the wild type polymerase . The mutant enzyme resembles the wild type in thermal stability, pH optimum, salt activation, divalent metal ion requirement, inhibition by a sulfhydryl reagent, and apparent affinity for DNA . However, the mutant enzyme differs from the wild type in its 8-fold higher 3'-exonuclease activity and in its decreased apparent affinity for deoxyribonucleoside triphosphates . Inhibition studies indicate that the exonuclease of the mutant enzyme is more vulnerable to physical and chemical modification than its wild type counterpart.

J Biol Chem, 1976 Apr 25, 251(8), 2299 - 306
Mode of action of bottromycin A2 . Release of aminoacyl- or peptidyl-tRNA from ribosomes; Otaka T et al.; Bottromycin A2 inhibited MS2 phage RNA-dependent protein synthesis as well as polyuridylic acid-(poly(U))- or polyadenylic acid (poly(A))-dependent polypeptide synthesis . When the ribosomal complex with N-acetyl-{14C}phenylalanyl-tRNA (N-acetyl-{14C}Phe-tRNA) at the A site was subjected to bottromycin A2, the release of N-acetyl-{14C}Phe-tRNA was observed while no release of N-acetyl-{14C}Phe-tRNA from the donor site was observed, indicating that the action of bottromycin A2 is specific to the A site of ribosomes . Due to bottromycin's capacity to release {14C}Phe-tRNA or N-acetyl-{14C}Phe-tRNA from the ribosomal acceptor site (A site), bottromycin A2 inhibited the nonenzymatic binding of N-acetyl-{14C}Phe-tRNA and elongation factor T (EF-T)-dependent binding if the concentration of EF-Tu-GTP-{14C}Phe-tRNA ternary complex was low . Our data are consistent with the possibility that the inhibition of overall polypeptide synthesis by bottromycin A2 is at least partly due to bottromycin A2's activity to release aminoacyl- or oligopeptidyl-tRNA from ribosomes . Among 10 antibiotics tested, bottromycin A2 and lincomycin released aminoacyl-tRNA from ribosomes.

J Biol Chem, 1976 Apr 25, 251(8), 2207 - 16
Association-dissociation behavior and subunit structure of heat-released nitrate reductase from Escherichia coli; Lund K et al.; Nitrate reductase, released from the membrane fraction of Escherichia coli by a neutral heat treatment, was purified to homogeneity by gel filtration chromatography . The purified enzyme behaved as an associating-dissociating system, exhibiting concentration-dependent sedimentation constants which ranged from 24 S at high concentrations in the ultracentrifuge down to 10 S at low concentrations in sucrose gradients . The molecular weight determined at high concentrations by sedimentation equilibrium was 880,000 +/- 30,000 . Large and small enzyme species were detected on polyacrylamide disc gels run with diluted samples of enzyme . The ratio of the two species was concentration-dependent and the dissociation was reversible . The purified enzyme appeared to be homogeneous and monodisperse in the ultracentrifuge, on sucrose gradients, during gel filtration on Bio-Gel and on polyacrylamide gels, but it had a heterogeneous subunit composition as determined by sodium dodecyl sulfate gel electrophoresis . Enzyme species with different subunit compositions were partially resolved by gel filtration . The fractions with the highest specific activity contained subunits of 150,000 and 55,000 daltons in a ratio of approximately 1:1 . Other fractions contained reduced amounts of the 55,000-dalton subunit and correspondingly increased amounts of 51,000-, 45,000-, and 10,000-dalton subunits, suggesting that the heterogeneity was the result of proteolytic degradation of the 55,000-dalton subunit . The enzyme contained approximately 12 non-heme irons, 12 acid-labile sulfides, 24 cysteine residues, and 1 molybdenum per 200,000 daltons.

J Biol Chem, 1976 Apr 25, 251(8), 2462 - 8
Involvement of the glucose enzymes II of the sugar phosphotransferase system in the regulation of adenylate cyclase by glucose in Escherichia coli; Harwood JP et al.; The nature of the interaction of glucose with toluene-treated cells of Escherichia coli leading to inhibition of adenylate cyclase was examined by the use of analogues . Those analogues with variations of the substituents about carbon atoms 1 or 2 (e.g . alpha-methylglucoside or 2-deoxyglucose) are inhibitory, and they are also substrates of the phosphoenolpyruvate-dependent sugar phosphotransferase system . Analogues with changes in other parts of the molecule (e.g . 3-O-methylglucose or galactose), L-glucose and several disaccharides and pentoses, do not inhibit adenylate cyclase and are not substrates of the phosphotransferase system . This correlation suggests some functional relationship between the adenylate cyclase and phosphotransferase systems . Further studies were done with mutants defective in glucose enzymes II of the phosphotransferase system (designated GPT and MPT); these two activities are measured by phosphorylation of alpha-methyl-glucoside and 2-deoxyglucose, respectively . The wild-type parent phosphorylates both analogues, and both inhibit adenylate cyclase . In the GPT- mutant, alpha-methylglucoside does not inhibit adenylate cyclase and is not phosphorylated, while 2-deoxyglucose is inhibitory and phosphorylated . In the GPT- MPT- double mutant, adenylate cyclase activity is present, but neither alpha-methylglucoside nor 2-deoxyglucose inhibits adenylate cyclase, and neither sugar is phosphorylated . These studies demonstrate that glucose inhibition of adenylate cyclase in toluene-treated cells requires an interaction of this sugar with either the GPT or mpt enzyme II of the phosphotransferase system.

J Biol Chem, 1976 Apr 25, 251(8), 2388 - 94
Increased intestinal chromatin template activity . Influence of 1alpha,25-dihydroxyvitamin D3 and hormone-receptor complexes; Zerwekh JE et al.; 1alpha,25-Dihydroxyvitamin D3 administration to rachitic chicks results in an increase in the chromatin template activity of intestinal target tissue assayed in vitro using Escherichia coli RNA polymerase . The maximum stimulation of template capacity was 12 to 20% over control values and occurred 2 hours after administration of the sterol . This rapid effect preceded the biologic response to 1alpha,25-dihydroxyvitamin D3 in the intestine and was not observed in other tissues such as liver or kidney . The in vivo enhancement of intestinal chromatin template activity was specific for the 1alpha,25-dihydroxyvitamin D3 hormone in that equivalent doses of 25-hydroxyvitamin D3 or vitamin D3 did not elicit a response in 2 to 3 hours . Only 1alpha-hydroxyvitamin D3, a synthetic sterol which is very rapidly metabolized to the 1alpha,25-dihydroxyvitamin D3 form, was able to minic the natural hormone in vivo . To further elucidate the nuclear mechanism of action of 1alpha,25-dihydroxyvitamin D3, the hormone was preincubated at 0 degrees with intestinal cytosol to form hormone-receptor complexes . After addition of the hormone-receptor complexes to purified intestinal mucosa nuclei and incubation for 1 hour at 25 degrees, chromatin isolated from this reconstituted system displayed a significant increase in template activity as compared to chromatin prepared from similar in vitro incubations not containing hormone . This stimulation was 12 to 24% over control values and exhibited an absolute requirement for intestinal cell cytosol . The response was specific for physiologic levels of 1alpha,25-dihydroxyvitamin D3, but occurred with pharmacologic doses of 25-hydroxyvitamin D3 . It is concluded that a stimulation of the chromatin template activity of intestinal target tissue by 1alpha,25-dihydroxyvitamin D3 may be an integral part of the ultimate physiologic response of enhanced calcium transport.

J Biol Chem, 1976 Apr 25, 251(8), 2520 - 4
ATPase activity required for termination of transcription by the Escherichia coli protein factor rho; Howard BH et al.; The relationship between the RNA-dependent beta-gamma ATPase in purified rho preparations and rho-mediated termination of transcription has been investigated . In a purified in vitro system, transcription from lambdagal DNA has been carried out using either ribonucleoside triphosphates (NTPs) or four ribonucleoside 5'-(beta-gamma-imino)triphosphates (NMP-P(NH)Ps) as RNA precursors . In the presence of NTPs, rho termination activity results in (a) the synthesis of rho-dependent transcripts which are of discrete size by polyacrylamide gel analysis and (b) a marked reduction by hybridization assay in RNA transcribed distal to the rho-sensitive termination site tR . In the presence of four NMP-P(NH)Ps, which are not substrates for the beta-gamma ATPase, termination by rho is completely abolished, whereas rho-independent termination occurs normally . Addition of ATP to transcription reactions containing four NMP-P(NH)Ps restores termination, ruling out the possibility that the termination activity of rho is nonspecifically inhibited by the analog preparations . We interpret our data as strongly suggesting that the RNA-dependent beta-gamma ATPase activity of rho is required for rho-mediated termination of transcription.

Mol Gen Genet, 1976 Apr 23, 145(1), 97 - 100
Stabilization and size of araC protein; Wilcox G et al.; AraC protein from Escherichia coli has been further stabilized and characterized . pH is a critical variable in conferring stability . araC protein has a sedimentation coefficient of 4.0 +/- 0.2s on standardized 5%-20% glycerol gradients . Its isoelectric point is at a pH of 7.1.

Mol Gen Genet, 1976 Apr 23, 145(1), 65 - 71
Glyceraldehyde 3-p dehydrogenase, glycerate 3-P kinase and enolase mutants of Escherichia coli: genetic studies; Irani MH et al.; The genetic loci for two enzymes of glycolysis have been established by transductional crosses . The eno locus, likely to be the structural gene for enolase maps in the 52-minute region of the Escherichia coli chromosome . A structural determinant for glycerate 3-P kinase (pgk) is located near serA . The map order is speB-pgk-serA-lysA-argA-eno-cysC . In the 35-minute region maps a locus affecting the structure of glyceraldehyde 3-P dehydrogenase.

Biochim Biophys Acta, 1976 Apr 22, 431(1), 54 - 61
Temperature-sensitive formation of the phospholipid molecular species in Escherichia coli membranes; Nishihara M et al.; Phospholipids in the membranes of Escherichia coli grown at 37 degrees C are composed of different proportions of molecular species than are those at 17 degrees C . The 1,2-disaturated and 1-saturated-2-unsaturated molecular species increased at 37 degrees C, but the 1,2-diunsaturated species increased at 17 degrees C . When the growth temperature was lowered from 37 to 17 degrees C during the middle exponential growth phase, phosphatidylethanolamine and phosphatidylglycerol composed of proportions of molecular species similar to those found at 17 degrees C were immediately synthesized . By using various membranes composed of different compositions of the phospholipid molecular species, the temperature-sensitive formation of the phospholipid molecular species was found to be independent of the composition of the membrane phospholipids and to be dependent on changes in the specificities of membrane-bound sn-glycerol 3-phosphate acyltransferase against the acyl-CoAs, due to temperature changes.

Biochemistry, 1976 Apr 20, 15(8), 1610 - 4
Production of high levels of phosphorylated F1 histone by zinc chloride; Tanphaichitr N et al.; Methods have been sought to perturb the level of phosphohistones . ZnCl2 (10 mM) exhibits histone phosphate phosphatase in vivo in HTC cells and leads to hyperphysiological levels of F1 phosphohistone . Treatment of tissue culture cells with this concentration of ZnCl2 leads to a reduction in medium pH to 6.4 . Control experiments have indicated that HTC cells grow efficiently at this pH and that the reduction of pH does not produce the hyperphosphorylated state per se . The optimum conditions for the ZnCl2 effect are described . That the effect of ZnCl2 on the heterogeneity of F1 histone is due to an effect on phosphorylation was demonstrated by the observation that the entire effect is abolished by treatment with alkaline phosphatase . The site of phosphorylation is in the carboxy-terminal end of the F1 molecule . The inhibitory effect of ZnCl2 on F3 phosphorylation in metaphase cells is also described.

Biochemistry, 1976 Apr 20, 15(8), 1755 - 60
Mechanistic studies of glutamine synthetase from Escherichia coli: kinetics of ADP and orthophosphate binding to the unadenylylated enzyme; Rhee SG et al.; The kinetics of protein fluorescence change exhibited by ADP or orthophosphate addition to the Mg2+-or Mn2+-activated unadenylylated glutamine synthetase from Escherichia coli were studied . The kinetic patterns of these reactions are incompatible with a simple bimolecular binding process and a mechanism which required protein isomerization prior to substrate binding . They are consistent with a mechanism in which direct substrate binding is followed by a substrate-induced conformational change step, ES in equilibrium ES . At pH 7.0 and 15 degrees C, the association constants for the direct binding (K1) of ADP to MnE1.0 and of Pi to MnE1.0ADP are 3.9 X 10(4) and 2.28 X 10(2) M(-1), respectively . The association constant for the direct binding of ADP to MnE1.0Pi is 2.3 X 10(4) M(-1) at pH 7.0 and 19 degrees C . The deltaG degrees for the substrate-induced conformational step are -3.5 and -1.3 kcal mol(-1) due to ADP binding to MnE1.0Pi and MnE1.0, respectively, and -1.4 kcal mol(-1) due to Pi binding to MnE1.0ADP . Rate constants, k2 and k(-2), for the isomerization step are: 90 and 9.5 s(-1) for ADP binding to MnE1.0, 440 and 0.36 s(-1) for ADP binding to MnE1.0Pi, and 216 and 1.8 s(-1) for Pi binding to MnE1.0ADP . Due to low substrate affinity, the association constant for direct Pi binding to MnE1.0 was roughly estimated to be 230 M(-1) and k2 = 750 s(-1), k(-2) = 250 s(-1) . At 9 degrees C and pH 7.0, the estimated association constants for the direct ADP binding to MgE1.0 and MgE1.0 Pi are 1.8 X 10(4) and 1.6 X 10(4) M(-1), respectively; and the rate constants for the isomerization step associated with the corresponding reaction are k2 = 550 s(-1), k(-2) = 500 s(-1), and k2 = 210 s(-1), k(-2) = 100 s(-1) . From the kinetic analysis it is evident that the inability of Mn2+ to support biosynthetic activity of the unadenylylated enzyme is due to the slow rate of ADP release from the MnE1.0PiADP complex . In contrast the large k(-2) obtained for ADP release from the MgE1.0ADP or MgE1.0PiADP complex indicates that this step is not rate limiting in the biosynthesis of glutamine since the k catalysis obtained under the same conditions is 7.2 s(-1).

Biochim Biophys Acta, 1976 Apr 15, 432(1), 49 - 59
Purification and properties of DNA-dependent RNA polymerase from Mycobacterium tuberculosis H37RV; Harshey RM et al.; RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase DNA-dependent), EC 2.7.7.6) was purified approximately 200 fold from Mycobacterium tuberculosis H37RV cells . The purified enzyme has a molecular weight of about 330 000-350 000 and is composed of four subunits . The subunits beta', beta and sigma have molecular weights different from those of Escherichia coli polymerase; the fourth, alpha subunit has a similar weight . The purified enzyme is a thousand-fold more sensitive to rifampicin, a potent antitubercular drug than the E . coli RNA polymerase, probably because of the difference in the beta subunits . This, with other data presented in this paper, indicate that the RNA polymerase of M . tuberculosis differs in its properties from that of E . coli.

Eur J Biochem, 1976 Apr 15, 64(1), 77 - 89
Studies on the environment of protein S7 within the 30-S subunit Escherichia coli ribosomes; Rinke J et al.; Analyses are described of three types of ribosomal fragment, all derived from the well-characterized ribonucleoprotein species consisting of proteins S7, S9, S10, S14 AND S19, together with RNA from sections O'-D-E'-K-P-P'-E-A of the 16-S sequence . 1 . When 30-S subunits were hydrolysed with ribonuclease T1 in presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S7 and S19, together with minor amounts of S13 or S14 . Oligonucleotide analysis of this fragment showed that it contained RNA from sections O'-E' and P-A of the 16-S RNA, but that section K (from the middle of this area) was missing . 2 . It has previously been shown that when 30-S subunits are irradiated with ultraviolet light, protein S7 is the primary target of cross-linking of protein to RNA . By making use of this reaction, ribonucleoprotein fragments were isolated from irradiated 30-S subunits the unbound proteins were removed, and RNA fragments containing covalently linked protein S7 were identified . It was possible to demonstrate that the site of cross-linking lies within the O'-A region of the 16-S RNA, and more precise experiments showed that this site almost certainly is in the P-A region . 3 . When the parent five-protein ribonucleoprotein fragment (above) was deproteinized under very mild conditions, RNA complexes could be isolated which consisted of non-contiguous sequences, but which migrated as a single species into a polyacrylamide gel . Analysis of one of these complexes showed that it contained sequences from sections O'-E' and P-A, but that section K was missing (cf . first paragraph above) . This demonstrated that these two separate regions of RNA interact within the 30-S subunit, independently of the presence of protein.

Eur J Biochem, 1976 Apr 15, 64(1), 313 - 20
The binding of indole to the alpha-subunit and beta2-subunit and to the alpha2beta2-complex of tryptophan synthase from Escherichia coli . Identification of a second indole-binding site on the alpha-subunit; Weischet WO et al.; The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual alpha and beta2-subunits and to the alpha2beta2-complex of tryptophan synthase was studied by equilibrium dialysis . The use of {14C}indole and indolepropanol {32P}phosphate permitted simultaneous binding studies to be carried out . Competition between indole and indolepropanol phosphate in binding to a particular site was taken as evidence for that site being part of the active site of the alpha-subunit . The binding of indole to the active site of the alpha-subunit is weak (Kd = 18mM) . A second distinct site binds indole more strongly (Kd = 1.5 mM) and interacts with the active site indirectly . It is therefore designated an effector site . Furthermore, the binding of indole and/or indolepropanol phosphate appears to stabilize different conformations of the alpha-subunit . The beta2-subunit binds indole only weakly (Kd = 12 mM) to many (n = 10) sites per polypeptide chain . The alpha2beta2-complex retains one or two sites per alphabeta-equivalent of relatively high affinity (Kd = 1.2 mM) . The active sites of the component alpha and beta-subunits probably belong to the second class of many (n = 40) sites of low (Kd = 30 mM) affinity for indole . These findings support conclusions from the literature that both bi-substrate reactions involving indole catalyzed by tryptophan synthase and its subunits must follow strictly ordered addition mechanisms with the respective other substrate adding first.

Eur J Biochem, 1976 Apr 15, 64(1), 295 - 300
Small-angle x-ray and light-scattering study of native and trypsin-modified methionyl-tRNA synthetase from Escherichia coli; Gulik A et al.; Small-angle X-ray scattering experiments were performed on an absolute scale on solutions of methionyl-tRNA synthetase from Escherichia coli in its native and trypsin-modified forms . A light-scattering study was performed on the same solutions to verify monodispersity . The structural parameters for the trypsin-modified enzyme, radius of gyration (2.48 nm), volume (90 nm3), surface/volume (1.5 nm-1) and the distribution of chords can account for an equivalent prolate ellipsoid of revolution having an axial ratio 2.3 and a maximum length of 9 nm, with a creviced surface . The rsults obtained for the native enzyme {i.e . radius of gyration (4.3 nm), volume (244 nm3), distribution of the scattering intensity and distribution of chords} exclude the possibility of a very compact quaternary structure and suggest that the enzyme consists of at least two globular parts, probably the two protomers, linked together by interactions involving a limited region of the structure.

Eur J Biochem, 1976 Apr 15, 64(1), 199 - 204
Purification of protease II from Escherichia coli by affinity chromatography and separation of two enzyme species from cells harvested at late log phase; Pacaud M; N-Acetyl-D-arginine linked to an agarose matrix has been used to purify protease II from Escherichia coli by affinity chromatography . The specific adsorption of protease II to this absorbent was achieved in 220 mM potassium phosphate buffer pH 7.6, and the enzyme was eluted with L-arginine . Enzyme preparations from cells harvested at late log phase have been resolved into two molecular species which differ in specific activity, kinetic constants and carbohydrate content . Both species appeared homogeneous by electrophoresis in conventional buffers and also in the presence of sodium dodecyl sulfate . Only one enzyme species was obtained by the same procedure using bacteria harvested at the middle of exponential growth.

Eur J Biochem, 1976 Apr 15, 64(1), 177 - 88
Structural properties of Escherichia coli RNA polymerase Subunits; Lowe PA et al.; 1 . The surface of the RNA-polymerase-DNA complex possesses an exposed polypeptide loop . 2 . Proteinases with differing specificities (trypsin, chymotrypsin, subtilisin and clostripain) preferentially cleave the exposed region . 3 . The cleaved polypeptide is reassembled into RNA polymerase by renaturation from a solvent which promotes a random coil conformation . 4 . Isolated beta subunit has a proteolytically resistant nucleus of approximately 70000 molecular weight . This resistant polypeptide may be generated by trypsin, chymotrypsin, subiilisin or clostripain . 5 . Isolated alpha subunits are comparatively resistant to proteolysis . 6 . Although of similar molecular weights beta and beta' appear to have unrelated primary sequences and markedly different conformations in free solution . 7 . Digestion of the beta subunit may be blocked by formation of the alpha2beta subassembly . 8 . Evidence is presented suggesting that beta' in the intact enzyme (alpha2beta beta') possesses the exposed polypeptide loop.

Eur J Biochem, 1976 Apr 15, 64(1), 115 - 21
Globin mRNA species containing poly(A) segments of different lengths . Their functional stability in Xenopus oocytes; Nudel U et al.; Rabbit globin mRNA species containing poly(A) segments of different lengths were prepared by partial phosphorolysis of mRNA with Escherichia coli polynucleotide phosphorylase . By varying the salt concentration and the time of incubation of the phosphorolysis mixture, as well as performing oligo(dT)-cellulose chromatography at 22 degrees C and at 4 degrees C, globin mRNA preparations containing poly(A) segments of approximately 122, 95, 68, 39, 32, 21, and 16 adenylate residues were obtained . It was found that the functional stability of the mRNA species containing 32 or more adenylate residues after injection into Xenopus laevis oocytes equaled that of the native globin mRNA . On the other hand, the functional stability of mRNA containing an average number of 21 adenylate residues was about 30% of the native mRNA, while that of mRNA containing 16 adenylate residues was as low as poly(A)-free globin MRNA.

Experientia, 1976 Apr 15, 32(4), 520 - 1
Induction of immunological memory in mice by RNA extract; Ziska P et al.; RNA extract isolated from spleens of mice immunized with lipopolysaccharide from E . coli induced an immunological memory in normal mice . Application of small amounts of corresponding antigen provoked a specific secondary immune response in RNA primed mice.

Biochim Biophys Acta, 1976 Apr 15, 432(1), 98 - 103
Release of certain ribosomal proteins from 70-S Escherichia coli ribosomes by mild ribonuclease digestion; Gast WH et al.; A method for the release of some proteins from Escherichia coli (MRE 600)ribosomes is described, avoiding extraction with denaturing reagents . High-salt-washed, 70-S ribosomes were treated with pancreatic ribonuclease which led to the release of 12 proteins of the larger ribosomal subunit . Separation of released protein was first attempted by gel filtration and by fractionation with (NH4)2SO4 . The number and type of the released proteins were identified by sodium dodecyl sulphate-polyacrylamide gels and two-dimensional gel electrophoresis . The method should prove of use in the large scale purification of proteins L1, L7, and L25.

Eur J Biochem, 1976 Apr 15, 64(1), 27 - 34
Conformation of Escherichia coli glutamic acid tRNA II as studied by hydrogen-tritium exchange catalyzed by cysteine methyl ester; Effect of estrogen on gene expression in the chick oviduct; Hen oviduct RNA polymerase II and Escherichia coli RNA polymerase holoenzyme and core enzyme were used to study the initiation of RNA synthesis on chromatin . In either the presence or absence of estrogenic stimulation, changes in the level of oviduct chromatin initiation sites as measured in the presence of either homologous or heterologous polymerases followed a similar pattern . Comparison of the initiation sited utilized by these enzymes on chick oviduct chromatin indicated that these enzymes compete with each other for the same initiation regions . In contrast to chromatin, however, the majority of the initiation sites on DNA which are utilized by the oviduct RNA polymerase II are different from those utilized by E . coli holoenzyem . These results suggest that chromatin proteins are involved in the selection of initiation sites on chromatin for RNA polymerases . The in vitro transcripts of these RNA polymerases on stimulated chick oviduct chromatin were analyzed by hybridization to a cDNA probe transcribed from ovalbumin mRNA . The relative concentration of ovalbumin sequences transcribed by these three polymerases was 4:1.5:1 for oviduct RNA polymerase II, E . coli core enzyme, and holoenzyme respectively . Therefore, the efficiency of transcribing a specific gene appears to depend on the interaction between RNA polymerase and chromosomal elements in the initiation region.

J Biol Chem, 1976 Apr 10, 251(7), 1896 - 901
Endonuclease II of Escherichia coli is exonuclease III; Weiss B; Exonuclease III, a phosphatase-exonuclease specific for bihelical DNA, wn the preparation was endonuclease II, an activity specific for DNA that has been partially depurinated by treatment with methyl methanesulfonate . The two activities, which could not be separated by electrophoresis, by sedimentation, or by gel filtration, were associated with a single monomeric protein of 28,000 daltons . To explain how a relatively small protein could have such diverse activities, it is proposed that one site on the enzyme can recognize interstrand spaces created either by depurination or by spontaneous terminal unwinding of a DNA duplex.

J Biol Chem, 1976 Apr 10, 251(7), 2063 - 9
Acetyl coenzyme A carbosylase . Circular dichroism studies of Escherichia coli biotin carboxyl carrier protein; Fall RR et al.; The biotin carboxyl carrier protein (BCCP) component of Escherichia coli acetyl coenzyme A carboxylase and three peptides derived from BCCP by proteolytic digestion have been examined by circular dichroism spectroscopy . BCCP, which has a peptide molecular weight of 22,500, has a spectrum typical of globular proteins with negative extrema at 222 nm and 208 nm . The two smallest peptides, BCCP(SC) and BCCP(9,100), with molecular weights of 8,900 and 9,100, respectively, exhibit unusual positive CD bands centered at 237 nm and 220 nm . BCCP(10,400), with a molecular weight of 10,400, has a CD spectrum intermediate between BCCP and that of the smallest peptides . Since d-biotin exhibits a positive CD band at 233 nm, it was suspected that the biotin prosthetic group might be the chromophore responsible for the 237 nm CD band seen in BCCP(SC) and BCCP(9,100) . Enzymatic carboxylation of BCCP(SC) to form CO2-BCCP(SC) caused the CD spectrum to change with a shift of the 237 nm band to 232 nm . The positive CD band at 220 nm was unaffected by carboxylation of the biotin prosthetic group . These date suggest that the 237 nm signal may be due either to the biotin which acts as a chromophore directly or to a chromophore that is perturbed by the carboxylation of biotin . A spectropolarimetric titration was carried out to investigate the possible contribution of the single tyrosine residue of BCCP(SC) to the CD spectrum of this peptide . At pH values over 9 the CD spetrum changed with the disappearance of the 237 nm band, suggesting that tyrosine might contribute to this CD band . Denaturation of BCCP(SC) or BCCP(9,100) with 8 M urea of 6 M guanidine HCl abolished the positive CD bands and resulted in spectra typical of a random coil, whereas treatment of BCCP(SC) with 1% sodium dodecyl sulfate abolished the positive bands and left a spectrum exhibiting a shoulder at 222 nm and a negative band at 205 nm, suggestive of a high degree of ordered structure . It is concluded that the CD band at 237 nm in BCCP(SC) and BCCP(9,100) is prabably due to a noncovalent interaction of biotin with an amino acid residue(s) of the protein . It is suggested that the biotin prosthetic group is partially buried in the surface of the protein, rather than swinging free at the end of the lysine side chain through which it is covalently linked to the protein, to permit this interaction to occur.

Biochim Biophys Acta, 1976 Apr 9, 430(1), 182 - 8
The accumulation of superoxide radical during the aerobic action of xanthine oxidase . A requiem for H2O4; Hodgson EK et al.; The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O2- during the first few minutes of the reaction . H2O2 decreases this accumulation of O2- presumably because of the Haber-Weiss reaction (H2O2+O2- leads to OH- +OH+O2) and very small amounts of superoxide dismutase eliminate it . This accumulation of O2- was demonstrated in terms of a burst of reduction of cytochrome c, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate . The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H2O4-, can now be satisfactorily explained entirely on the basis of known radical intermediates.

Biochim Biophys Acta, 1976 Apr 8, 429(2), 352 - 8
Uridine phosphorylase from Escherichia coli . Kinetic properties and mechanism; Krenitsky TA; Type I and Type II uridine phosphorylases (uridine: orthophosphate ribosyltransferase EC 2.4.2.3) are distinguished by their pH optima (Krenitsky et al . (1965) J . Biol . Chem . 240, 1281-1286) . A Type I enzyme was partially purified from Escherichia coli . The crossing pattern of the initial velocity analysis indicated that the catalytic mechanism involved the sequential addition of substrates to the enzyme . Product inhibition by uracil or by ribose 1-phosphate was linear competitive with uridine or with concentrations of phosphate below 3 mM . This indicated that the sequence of substrate addition was random rather than ordered . At concentrations of phosphate above 3 mM, product inhibition by uracil was complex . The random mechanism of this Type I enzyme contrasts with the ordered mechanism of a Type II enzyme from rat liver (Kraut, A . and Yamada, E.W . (1971) J . Biol . Chem . 246, 2021-2030).

Biochemistry, 1976 Apr 6, 15(7), 1510 - 6
Incorporation of noncomplementary nucleotides at high frequencies by ribodeoxyvirus DNA polymerases and Escherichia coli DNA polymerase I; Mizutani S et al.; The fidelity of DNA synthesis with synthetic homopolymer templates by two ribodeoxyvirus DNA polymerases and E . coli DNA polymerase I was examined by nearest neighbor frequency analyses . The experiments were designed to favor the incorporation of noncomplementary nucleotides, and the reactions were carried out only for short periods of time . All incorporations were template dependent . The frequency of incorporation of noncomplementary nucleotides next to complementary nucleotides by all three of these DNA polymerases significantly varied, depending on the conditions, from almost none (less than 0.2%) to 100% of the incorporation of the complementary nucleotide . In general, there was more incorporation of noncomplementary nucleotides in the presence of Mn2+ than in the presence of Mg2+ and with ribohomopolymer templates than with deoxyribohomopolymer templates . With a heteropolymer RNA template, the infidelity of DNA synthesis by one of the ribodeoxyvirus DNA polymerases was also increased by the presence of Mn2+ in the DNA synthesizing reaction . These findings indicate that, under appropriate conditions, any DNA polymerase has the potential to make a significatn number of errors in DNA synthesis and that altered conditions for DNA polymerase action might play a role in spontaneous mutation.

Biochemistry, 1976 Apr 6, 15(7), 1569 - 80
Glutamine synthetase adenylyltransferase from Escherichia coli: purification and physical and chemical properties; Caban CE et al.; The glutamine synthetase adenylyltransferase (EC 2.7.7.42), WHIch catalyzes the adenylylation and deadenylylation of glutamine synthetase in E . coli, has been stabilized and purified 2200-fold to apparent homogeneity . Sedimentation and electrophoresis studies show that the native enzyme is a single polypeptide chain of 115,000 +/- 5000 molecular weight with an isoelectric pH (PL) OF 4.98, a sedimentation coefficient (S20.w0) of 5.6S, and a molar frictional coefficient (f/f0) of 1.52 . An alpha-helical content of approximately equal to 25% and approximately equal to 28% beta-pleated sheet and approximately equal to 47% random coil structures were estimated from circular dichroism measurements . The amino acid composition of the protein has been determined . The intrinsic tryptophanyl residue flourescence of adenylyltransferase is two fold greater than that of L-tryptophan; this property has been used to monitor ligand-induced conformational changes in the enzyme . Activators of the adenylylation reaction (ATP, L-glutamine, or the E . coli PII regulatory protein) produced an enhancement of fluorescence; alpha-ketoglutarate, an inhibitor of adenylylation and an activator of deadenulylation, caused a net decrease in fluorescence . The adenylytransferase has separate interaction sites for L-glutamine and the regulatory PII protein.

Biochemistry, 1976 Apr 6, 15(7), 1547 - 61
31P nuclear magnetic resonance study of alkaline phosphatase: the role of inorganic phosphate in limiting the enzyme turnover rate at alkaline pH; Hull WE et al.; 31P nuclear magnetic resonance (NMR) was used to directly observe the binding of inorganic phosphate to alkaline phosphatase . Evidencq for the tight binding of 1.5-2.0 mol of inorganic phosphate per dimer of alkaline phosphatase is presented . Two distinct forms of bound phosphate are observed, one predominating above pH 7 and representing the non-covalent E-P1 complex and the other predominating below pH 5 and representing the covalent E-P1 complex . The 31P NMR line width of the E-P1 complex indicates that the dissociation of noncovalent phosphate is the rate-limiting step in the turnover of the enzyme at high pH.

Biochemistry, 1976 Apr 6, 15(7), 1535 - 46
Fluorine-19 nuclear magnetic resonance study of fluorotyrosine alkaline phosphatase: the influence of zinc on protein structure and a conformational change induced by phosphate binding; Hull WE et al.; 19F nuclear magnetic resonance (NMR) spectroscopy has been used to study a fully active E . coli fluorotyrosine alkaline phosphatase . The fluorotyrosine resonances provide sensitive probes of the conformational states of the protein . They were used to follow the addition of zinc or cobalt to the apoprotein, and the titration of the protein with inorganic phosphate or the inhibitor 2-hydroxy-5-nitrobenzylphosphonate . The results indicate that 2 molecules of inorganic phosphate per dimer of alkaline phosphatase are required to complete a general conformational change in the protein involving perturbations to the environment of several tyrosines . Spectra of the cobalt enzyme indicate that on specific tyrosine per subunit may be near the metal site . The 19F NMR results, combined with the 31P NMR results in the accompanying paper, lead directly to the conclusion that dissociation of noncovalently bound inorganic phosphate from the enzyme is the rate-limiting process in enzyme catalysis at high pH . The local environment of the individual fluorotyrosines is also discussed.

Biochemistry, 1976 Apr 6, 15(7), 1387 - 92
The state of energization of the membrane of Escherichia coli as affected by physiological conditions and colicin K; Brewer GJ; The bacterial protein colicin K, when added to sensitive Escherichia coli in the presence of 3,3'-dihexyloxacarbocyanine, cuases a doubling in fluorescence of the probe . Glucose and oxygen cause a decreased fluorescence while anoxia and cyanide cause a rise in fluorescence . These results in conjunction with the work of other laboratories suggest that colicin K causes a depolarization of the transmembrane electrical potential . Fluorescence in the absence of colicin K was relatively independent of KCl, NaCl, and MgCl2 concentrations below 0.1 M . Although colicin K caused rapid efflux of the K+ analogue 86Rb+, the fluorescence rise was only partially blocked by 0.13 M KCl . The level of fluorescence caused by the action of colcin K was inversely proportional to the logarithm of the concentration of MgCl2 over the range of 2 muM to 4 mM . This suggests that a Nernst electrochemical potential for an anion can counteract a membrane depolarization caused by colcin . After colcin K action, the fluorescence of the carbocyanine could be further increased by anoxia or cyanide . The distribution of the weak base dimethyloxazolidinedione indicated that the pH in the interior of aerobic E . coli supplied with lactate was alkaline by 0.1 unit and unaffected by colicin . These results suggest that colicin K does not completely depolarize the membrane potential and does not interfere with the component of membrane energization generated by electron transport . Colicin K does not act as a cationophore . The partial depolarization of the membrane may account for the inhibition of active solute transport caused by colicin K.

Biochim Biophys Acta, 1976 Apr 5, 426(4), 711 - 22
Kinetic processes in Escherichia coli membranes and cells . A laser photolysis study using derivatives of pyrene; Wong M et al.; Pyrene and several derivatives of pyrene are used to investigate photo-induced kinetic processes in whole cells and membranes extracted from Escherichia coli . A mutant of E . coli was used which, under appropriate growth conditions, produced a complete or incomplete lipopolysaccharide in the outer membrane . The pyrene derivatives used were: pyrene sulfonic acid, pyrene butyric acid and the ester of pyrene butyric acid and 10-hydroxydecanoic acid . The pyrene chromophore was excited by the ultraviolet pulse from a Q switch, frequency-doubled, ruby laser . The lifetimes of the pyrene fluorescence in the presence of the quenchers O2, thallous ion (T1+), I-and CH3NO2 were measured and tabulated as second order rate constants . For the most part the quenching rate constants were much lower than the corresponding values observed in simple nonviscous solution, e.g . ethanol . This is interpreted as being due to the location of the probe within the membrane . The membrane inhibits the movement of the quenchers to the excited state . Cell membranes containing complete lipopolysaccharide showed significantly lower quenching rates for the probes pyrene and pyrene sulfonic acid than cell membranes with incomplete lipopolysaccharide . From an amalysis of the kinetic data it is suggested that pyrene and pyrene sulfonic acid are located near and under lipopolysaccharide and close to membrane proteins . On the other hand, no effect of lipopolysaccharide composition was observed for the probes pyrene butyric acid and pyrene butyroyl decanoic acid . This may suggest that these probes are located primarily in the lipid part of the membrane . A simple model for the outer membrane of E . coli is suggested that accounts for the observed laser-induced kinetic processes.

Biochim Biophys Acta, 1976 Apr 2, 425(4), 396 - 400
Effects of irradiation on RNA synthesis primed by calf thymus deoxyribonucleoprotein treated with salt and salt-urea; Kobayashi T et al.; 1 . The effects of ionizing radiation on the activity of calf thymus templates were examined in a Escherichia coli RNA polymerase system . 2 . The template activity of native and 2 M NaCl-5M urea-treated deoxyribonucleoproteins was enhanced by relatively low doses of irradiation, while that of 2 M NaCl-treated deoxyribonucleoprotein was not enhanced by irradiation . 3 . The template activity of purified DNA was markedly decreased by irradiation, while that of native deoxyribonucleoprotein, 2 M NaCl-treated, and 2 M NaCl-5 M urea-treated ones were slightly decreased at a higher dose range . The doses for 50% inactivation of these templates were 1.3, 210, 140, and approximately 200 krad, respectively.

Biochim Biophys Acta, 1976 Apr 2, 425(4), 492 - 501
Regulation of nitrate reductase at the transcriptional and translational levels in Escherichia coli; Ruiz-Herrera J et al.; Nitrate reductase from Escherichia coli is induced by nitrate and derepressed by oxygen removal after a lag phase . Elimination of inducer, shift to aerobic conditions and addition of actinomycin D causes the decline in the rate of its synthesis, which eventually may stop . Kinetic analysis of the sensitivity of the biosynthetic process to oxygen, chloramphenicol, actinomycin D and rifampicin gave results which we interprete as evidence that oxygen (and possibly nitrate) affect simultaneously both the transcriptional and translational processes.

Biochim Biophys Acta, 1976 Apr 2, 425(4), 451 - 62
The effect of silver ion binding and pH on the buoyant density of DNA and its use in fractionating heterogeneous DNA; Rinehart FP et al.; 1 . The effect of pH on the buoyant density of the complexes of Ag+ with DNA has been studied using 3H-labeled human DNA and several bacterial DNAs to determine the conditions necessary for the maximum resolution of compositional heterogeneity . In neutral CS2SO4 density gradients, Ag+ complexes with (G - C)-rich components are always denser than those with (A - T)-rich components, since (G - C)rich DNAs have a larger affinity for Ag+ than (A - T)-rich DNAs and their complexes are denser than (A - T)-rich complexes . In alkaline (pH greater than 9) CS2SO4 gradients, the buoyant density of the Ag+ - DNA complex is not a simple function of base composition . The Ag+ affinity of (A - T)-rich DNA is larger than that of (G - C)-rich DNA but the density of a (G - C)-rich complex is larger . Thus the ordering of the buoyant density changes depends on the amount of added Ag+ . 2 . The problem of resolving the density heterogeneity within a tracer DNA, and minor components of DNA, is explored and useful fractionation techniques are developed.

Br J Haematol, 1976 Apr, 32(4), 573 - 7
Fine structural studies of peripheral blood leucocytes from children with kwashiorkor: morphological and functional properties; Schopfer K et al.; This is the first report of electron microscopic observation on circulating leucocytes from children with kwashiorkor, Neutrophils showed evidence of immaturity, prominent Golgi zones, granule pleomorphism; strands of endoplasmic reticulum and Dohle bodies were frequent . Although an in vitro microbicidal defect occurs with S . aureus, E . coli and C . albicans, the postphagocytic morphological events, including vacuole formation and degranulation, were normal . In mononuclear cell pellets plasmacytoid cells were frequently observed, and rare lymphoblasts occurred . The findings suggest activation of the phagocytic system and stimulation of humoral immunity in children with kwashiorkor . These fine structural observations are comparable to those which occur in trauma or thermal injury, both conditions associated with tissue breakdown and extensive antigenic stimulation.

Immunology, 1976 Apr, 30(4), 529 - 35
Changes in thymocyte reactivity to lectins induced by B-cell mitogens of the type of sulphated polyanions; Blitstein-Willinger E et al.; Dextran sulphate, polyvinyl sulphate and carrageenan (but not the other polyanions tested, such as poly I:poly C, poly-L-glutamic acid or E . coli lipopolysaccharide) act synergisticaly with PHA on mouse thymocyte stimulation, as measured by thymidine uptake and blast cell transformation . Furthermore the active compounds shift the dose response of thymocytes to Con A . The effect could be observed in four different strains of mice tested by using normal or cortisone-resistant thymocytes . Two possibilities are envisaged for explanation of these effects: (a) attachment of polyanions by their anionic groups to cell surface constituents and interaction of the sulphate groups of the polymers with plant lectins; and (b) modification of the cell membrane induced by sulphated polymers changing either the binding capacity or the effectiveness of the membrane-associated events leading to thymocyte stimulation by the lectins.

Proc Natl Acad Sci U S A, 1976 Apr, 73(4), 1131 - 5
Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex; Brown S et al.; Escherichia coli phage Qbeta RNA replicase, an RNA-dependent RNA polymerase (RNA-dependent RNA nucleotidyltransferase), is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host . Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors . Using a previously developed reconstitution system we have examined the effects of crosslinking the EF-Tu-Ts complex on reconstituted replicase activity . Renaturation is significantly more efficient when exogenously added native EF-Tu-Ts is crosslinked than when it is not . Crosslinked EF-Tu-Ts can be purified from a crude crosslinked postribosomal supernatant by its ability to replace EF-Tu and EF-Ts in the renaturation of denatured Qbeta replicase . A sample of Qbeta replicase with crosslinked EF-Tu-Ts replacing the individual elongation factors was prepared . Although it lacked EF-Tu and EF-Ts activities, it could initiate transcription of both poly(C) and Qbeta RNA normally and had approximately the same specific activity as control enzyme . Denatured Qbeta replicase formed with crosslinked EF-Tu-Ts was found to renature much more rapidly than untreated enzyme and, in contrast to normal replicase, its renaturation was not inhibited by GDP . The results demonstrate that EF-Tu and EF-Ts function as complex in Qbeta replicase and do not perform their known protein biosynthetic function in the RNA synthetic reaction.

Nucleic Acids Res, 1976 Apr, 3(4), 931 - 51
Cleavage patterns of Drosophila melanogaster satellite DNA by restriction enzymes; Shen CJ et al.; The five satellite DNAs of Drosophila melanogaster have been isolated by the combined use of different equilibrium density gradients and hydrolyzed by seven different restriction enzymes; Hae III, Hind II + Hind III, Hinf, Hpa II, EcoR I and EcoR II . The 1.705 satellite is not hydrolyzed by any of the enzymes tested . Hae III is the only restriction enzyme that cuts the 1.672 and 1.686 satellites . The cleavage products from either of these reactions has a heterogeneous size distribution . Part of the 1.688 satellite is cut by Hae III and by Hinf into three discrete fragments with M.W . that are multiples of 2.3 X 10(5) daltons (approximately 350 base pairs) . In addition, two minor bands are detected in the 1.688-Hinf products . The mole ratios of the trimer, dimer and monomer are: 1:6.30 : 63.6 for 1.688-Hae III and 1 : 22.0 : 403 for 1.688-Hinf . Circular mitochondrial DNA (rho = 1.680) is cut into discrete fragments by all of the enzymes tested and molecular weights of these fragments have been determined.

Can J Biochem, 1976 Apr, 54(4), 301 - 6
The purification from Escherichia coli of a protein relaxing superhelical DNA; Burrington MG et al.; The Escherichia coli omega protein was first described by Wang (Wang J.C.: J . Mol . Biol . 55, 523-533 (1971)) as having the ability to relax supercoiled covalently-closed circular DNA by changing the topological winding number, alpha . We have developed a rapid assay for omega activity which has allowed us to purify the protein to homogeneity . It appears to be an alphabeta-type subunit protein with a molecular weight of the intact protein of about 80,000 (determined by gel filtration) and of the individual subunits of 56000 and 31000 (sodium dodecyl sulfate polyacrylamide gels) . We have confirmed Wang's observation that it only partly relaxes negative supercoils, and is not active on a positive supercoils . Its characteristics with respect to pH, salts, temperature and chromatography are described . A method for rapid screening of E . coli for omega mutants is described.

J Bacteriol, 1976 Apr, 126(1), 183 - 91
Puromycin-resistant biosynthesis of a specific outer-membrane lipoprotein of Escherichia coli; Halegoua S et al.; The reported puromycin resistance of the in vivo biosynthesis of a specific outer-membrane lipoprotein of Escherichia coli was further investigated . The biosynthetic machinery making the lipoprotein was made more accessible to puromycin by disruption of the cell structure using ethylenediaminetetracetate or toluene, and finally in an in vitro protein biosynthesis system using polyribosomes . Puromycin sensitivity of overall protein synthesis increased by about 10-fold for each method of disruption of the cell structure; 50% inhibitions were obtained at 330, 35, 2.7, and 0.22 mug of puromycin per ml for intact cells, ethylenediaminetetraacetate-treated cells, toluene-treated cells, and the polyribosome system, respectively . However, the lipoprotein biosynthesis remained more resistant to puromycin than the biosynthesis of other proteins in all systems tested . These results strongly suggest that puromycin resistance of the lipoprotein biosynthesis is due to an intrinsic property of the lipoprotein biosynthetic machinery.

Eur J Biochem, 1976 Apr 1, 63(2), 563 - 8
Purification by immunoadsorbtion chromatography of the normal and a mutant form of the B2 subunit of Escherichia coli tryptophan synthase; Shannon LM et al.; Columns containing immobilized immunoglogulin G fractions from normal and immunized animals were used to purify the wild-type beta component of Escherichia coli tryptophan synthase, formula alpha2 beta2, and a mutant form of the beta component . The procedure yielded proteins with no detectable contaminants as measured by analytical acrylamide disc gel electrophoresis and immunodiffusion against proteins subject to sodium dodecylsulfate acrylamide electrophoresis . After elution from the antibody column at pH 11 the normal beta component, by dialysis against pyridoxal phosphate at pH 7.5, could be restored to the enzymatically-active beta2 dimer with a specific activity of 1700 enzyme units/mg protein . This compares with reported values of 2500-3000 enzyme units/mg when the beta2 dimer is purified by conventional means.

Eur J Immunol, 1976 Apr, 6(4), 269 - 73
Control mechanism of lymphocyte traffic . Altered distribution of 51Cr-labeled mouse lymph node cells pretreated in vitro with lipopolysaccharide; Freitas AA et al.; The effect of in vitro treatment of mouse lymph node cell populations with lipopolysaccharide (LPS) on their migration in syngeneic recipients was studied . LPS-exposed cells had a decreased lymph node localization and an increased spleen distribution when compared to control untreated cells, after intravenous injection into syngeneic recipients . This effect of LPS on cell traffic was maximal at 24 h and could not be detected at either 1 or 72 h after cell transfer . These changes induced on cell distribution by LPS treatment are interpreted as the consequence of a modified interaction between the circulating lymphocytes and the resident cells within the spleen of the recipients.

Eur J Immunol, 1976 Apr, 6(4), 250 - 6
Dual pathway of B lymphocyte differentiation in vitro; Askonas BA et al.; Direct visualization of the events resulting from LPS stimulation of mouse spleen cells in vitro was achieved by characterizing the cells during four days of culture for morphology, Ig and theta surface markers and autoradiography after {3H} thymidine uptake . The changes observed were related to biochemical parameters such as incorporation of {3H} thymidine into DNA, Ig biosynthesis and secretion . Two pathways of B lymphocyte differentiation were observed: a) the generation of a large number of small B lymphocytes with high density of surface Ig but no internal pool detectable by immunofluorescence, and b) the maturation of a very small proportion of cells with a large intracellular pool and the ability to secrete Ig . Both cell types arise from dividing blast cells, either physically separated or traced by pulse chase experiments with {3H} thymidine . We discuss whether this duality is caused by the triggering of different B cell subpopulations at different developmental stages, preprogramed to one or the other pathway or whether the final direction of development depends on the microenvironment of individual dividing cells.

Hoppe Seylers Z Physiol Chem, 1976 Apr, 357(4), 543 - 51
Multiple forms of lysyl-tRNA synthetase from Escherichia coli; Dittgen RM et al.; Lysyl-tRNA synthetase has been isolated from E . coli . The enzymatic activity elutes as three or four bands from a hydroxyl-apatite column . Polyacrylamide gel analysis shows that each of these bands contain more than one enzymatically active protein species . The molecular weights of the subunits of these species provides an explanation for the variation in the molecular weights previously reported for this enzyme.

Zh Mikrobiol Epidemiol Immunobiol, 1976 Apr, (3), 84 - 7
{Mechanism of the stimulating effect of protamines on the infectivity of DNA phages}; Moiseeva TF et al.; The interaction of styrin (protamine from sturgeon milt) with E . coli spheroplasts was investigated with the aid of the fluorescent probe--5-dimethylamino-1-naphthalenesulfonyl-chloride (DNS) . Not less than 40% of the total (15 mug ml-1) DNA-protamine concentration was bound to spheroplasts . A part of the protein was found in the isolated membranes and inside the cell . There was a correlation between the binding of protein with spheroplasts and its biological action . The dansylation somehow decreased the protein stimulating activity in transfection.

Cell, 1976 Apr, 7(4), 477 - 86
Adjacent repeating units of Xenopus laevis 5S DNA can be heterogeneous in length; Carroll D et al.; The distribution of length heterogeneity in adjacent repeating units of X . laevis 5S DNA has been examined by "cloning" 5S DNA in bacteria . Fragments of 5S DNA produced by partial digestion with Hind III and containing 1, 4, and 5 repeating units have been inserted at the single Hind III site of the tetracycline-resistance plasmid, pSC101, and the hybrid plasmids cloned in E . coli . Adjacent 5S DNA repeats in the cloned multi-repeat fragments can differ in length . This finding rules out some mechanisms which have been proposed to account for the parallel evolution of tandem repeated DNAs . The results are consistent with an unequal crossing-over mechanism and place some constraints on the molecular processes in this recombinatory event.

Jpn J Microbiol, 1976 Apr, 20(2), 141 - 6
Strain differences in the immunogenicity of aggregated human IgG and the adjuvant action of lipopolysaccharide on the low-responder strain of mice; Fujiwara M et al.; Strain differences in the antibody response to human IgG (HGG) were observed when aggregated HGG was injected intravenously . Lipopolysaccharide (LPS) administered subsequently markedly enhanced the antibody response to HGG in low responder C57BL/6 mice as compared with that in high responder DDD, C3H/He or (C57BL/6 X DDD)F1 mice . Aggregate-free preparation of HGG at a dose of 0.5 mg induced immunological tolerance in all strains of mice tested . LPS injected subsequently converted tolerogenic, aggregate-free HGG into immunogen in DDD mice but not in C57BL/6 mice . To determine the correlation between adjuvanticity and mitogenicity of LPS, spleen cells from normal mice were cultured in the presence of LPS and 3H-thymidine uptake was measured . Spleen cells of DDD mice incorporated three times as much 3H-thymidine as those of C57BL/6 mice . There seems no strong correlation between both activities of LPS . The data obtained are discussed in terms of strain differences in the macrophage function for processing the antigen.

Infect Immun, 1976 Apr, 13(4), 1214 - 20
Colonization of porcine small intestine by Escherichia coli: ileal colonization and adhesion by pig enteropathogens that lack K88 antigen and by some acapsular mutants; Nagy B et al.; Seven K88-negative porcine enteropathogenic Escherichia coli, representing three different serogroups, caused severe diarrhea and characteristically colonized the ileum, but not the jejunum, of intragastrically exposed newborn pigs . Bacterial counts of intestinal contents and wall, fluorescence, and scanning electron microscopy all suggested that these strains colonized the ileum by adhesion to the villous epithelium . However, in ligated intestinal loops, these enteropathogenic E . coli strains adhered to jejunal epithelium as well as to ileal epithelium . Acapsular (K-) mutants, derived from one of the principal strains, retained their colonizing and adhesive abilities, whereas K- mutants from three other enteropathogenic E . coli strains did not . It is suggested that: (i) these K88-negative enteropathogenic E . coli colonize the ileum by adhesion, and (ii) the adhesion of some K-88-negative strains is mediated by surface factors other than, or in addition to, the polysaccharide K antigen.

Infect Immun, 1976 Apr, 13(4), 1117 - 9
Occurrence and characteristics of enterotoxigenic Escherichia coli isolated from calves with diarrhea; Myers LL et al.; Of 1,004 isolates of Escherichia coli obtained during the spring of 1975 in seven different states from calves with diarrhea, 124 isolates were enterotoxigenic based upon ability to cause distention of the calf ligated intestinal segment . Isolates of enterotoxigenic E . coli (ETEC) were obtained from calves in six of the seven states . ETEC were detected in calves in 118 of 355 herds in Montana during the 1974 and 1975 spring beef calving seasons . The occurrence and serotypes of ETEC isolated from calves in states outside Montana were similar to ETEC isolated from calves in Montana . One hundred and fourteen of the 124 isolates of ETEC were placed in one of six different groups upon agglutination in OK antiserum . Serotyping of 35 of the 124 isolates of ETEC indicated the following serotype for isolates in each group: group 1, O9:K35; group 2, O101:K30; group 3, O8:K85; group 4, O20:K?; group 5, O8:K25; and group 6, O101:K28 . Determination of the presence of K99 antigen indicated that 28 of 35 isolates of ETEC had K99 antigen, whereas the antigen was not detected in any of the 10 isolates of non-ETEC studied.

Am J Pathol, 1976 Apr, 83(1), 213 - 36
Interstitial nephritis . A brief review; Heptinstall RH; Interstitial nephritis is a common condition, which in spite of a relatively constant pathologic picture has different etiologic agents and pathogenetic mechanisms . Failure to appreciate this, particularly in the chronic group, has led to considerable confusion and has been largely responsible for the overdiagnosis of chronic pyelonephritis . Although we are still largely ignorant of the causes of interstitial nephritis, it is now possible to define many of them . While experimental studies have not made spectacular contributions to our understanding, an attempt is now being made to develop appropriate models, and we hope these will enable us to still further clarify our understanding of other entities.

Nucleic Acids Res, 1976 Apr, 3(4), 863 - 77
Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase; Roychoudhury R et al.; Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion . The priming efficiency in the presence of Co+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fold higher than that observed with Mg+2 ion . In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3'-ends regardless of whether such ends are staggered or even . Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages . Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA . Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.

Nucleic Acids Res, 1976 Apr, 3(4), 1053 - 63
Enzymatic synthesis of duplex circular phiX174 DNA containing phosphoramidate bonds in the (-) strand; Letsinger RL et al.; Duplex circular phiX174 DNA (RF I) containing some phosphoramidate links in the backbone chain of the (-) strand was synthesized by reaction of 5'-amino-5'-deoxythymidine 5'-triphosphate, dCTP, dGTP, and 3H-dATP with DNA polymerase I and DNA ligase (T4) on a (+) strand phiX174 amber 3 DNA template . The yield of duplex DNA was higher when dTTP was included along with the amino analog in the initial reaction system or was added late in the synthesis . RF I DNA was observed as a rapidly sedimenting species in an alkaline sucrose gradient, and the presence of phosphoramidate linkages was demonstrated by the unusual lability of the duplex DNA in a weakly acidic solution.

Mutat Res, 1976 Apr, 35(1), 1 - 6
A study of possible mechanisms of the RNA-polymerase involement in mutagenesis in phage T4; Piruzian ES et al.; Spontaneous and induced mutation frequencies of phage T4 have been measured in Escherichia coli strains containing altered RNA-polymerase . In the strain E . coli rif-r stl-r, with double RNA-polymerase mutation, spontaneous reversion rates were increased in different mutants of phage T4 . The study of base analogues mutagenesis in rUV mutants of phage T4 has shown that the introduction of RNA-polymerase mutations did not increase reversion rates in a mutant of frame-shift type but enhanced the rate of mutagenesis in phage mutants with lesions of transition type.

Mol Biol Rep, 1976 Apr, 2(5), 401 - 6
A form of elongation factor G insensitive to N-ethyl-maleimide; Girbes T et al.; Treatment of elongation factor G (EF-G) with the thiol reagent N-ethylmaleimide only partially inhibits (10 to 70%) the activity of the factor in (a) guanosine nucleotide-EF-G-ribosome complex formation, (b) uncoupled ribosome-dependent GTP hydrolysis, and (c) polypeptide synthesis . Moreover, a similar treatment of the factor with N-{3H}ethylmaleimide does not lead to 3H-label being associated with a GDP-EF-G-ribosome-fusidic acid complex . Thus, the results indicate the presence in EF-G preparations of a form of the factor that does not react with N-ethylmaleimide.

Biotechnol Bioeng, 1976 Apr, 18(4), 465 - 71
Cell inactivation by ultrasound; Dakubu S; Cell survival curves have been obtained for Escherichia coli B (E . coli B) after the sonication of suspensions of the bacteria with continuous wave ultrasound at a fixed frequency of 2 MHZ between peak intensities of 8.7 and 2.25 W cm-2 . It was found that under suitable conditions the survival curves were reproducible and it also was found that there was a clear relationship between the rate of inactivation and the peak acoustic intensity of the ultrasound . There appeared to be a lower threshold of peak intensity below which no inactivation was observed.

Proc Natl Acad Sci U S A, 1976 Apr, 73(4), 1159 - 63
Asymmetric orientation of phage M13 coat protein in Escherichia coli cytoplasmic membranes and in synthetic lipid vesicles; Wickner W; At each stage of infection, the major coat protein of coliphage M13 binds to the E . coli cytoplasmic membrane with its antigenic site exposed to the cell exterior {Wickner, W . (1975) Proc . Nat . Acad . Sci . USA 72, 4749-4753} . This antigenic site is now shown to be at the amino-terminus of the protein . The amino-terminus of M13 coat protein is also found exclusively on the outside of dilauroyl or dimyristoyl lecithin vesicles, formed with coat protein by the cholate dilution technique {Racker, E., et al . (1975) FEBS Lett . 57, 14-18} near the lipid phase transition temperature . The basic carboxyterminus of the coat protein is exclusively on the inside of these vesicles . Vesicles of M13 coat protein and dimyristoyl lecithin when formed below the lipid phase transition temperature have both ends of the coat protein exposed to the vesicle exterior . The asymmetry of a membrane protein can, therefore, be established in the absence of other proteins and of lipid asymmetry; it depends on the physical state of the lipid phase . The factors which cause asymmetry in this model system may affect the distribution of proteins in biological membranes.

Proc Natl Acad Sci U S A, 1976 Apr, 73(4), 1121 - 5
Double-stranded RNA in chromatin transcripts formed by exogenous RNA polymerase; Pays E; RNA transcribed in vitro at low ionic strength, from either rat liver chromatin or DNA, contains a significant amount of structure resistant to RNase in high salt buffer . This is observed with rat liver (form B polymerase) as well as with Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleoside triphosphate: RNA nucleotidyltransferase; EC 2.7.7.6) . Treatment with RNases specific for either double-stranded or hybrid RNA indicates that resistance to RNase is due to the presence of double-stranded RNA sequences . Denaturation kinetics in the presence or absence of RNase suggest that these sequences are formed by intramolecular base pairing . Their mean length is about 20 to 30 nucleotides, but 15-20% are more than 100 nucleotides long . They contain 60-65% G-C base pairs . The proportion of double-stranded segments is higher in chromatin transcripts than in DNA-templated RNA, and is higher with homologous RNA polymerase than with the bacterial enzyme . On the other hand, chromatin endogenous RNA polymerase, which is unable to initiate transcription, does not synthesize double-stranded RNA . The problem of the location of these sequences is discussed; preliminary results suggest that the 5' end of the RNA transcripts could be enriched in complementary sequences.

Proc Natl Acad Sci U S A, 1976 Apr, 73(4), 1053 - 7
Involvement of escherichia coli dnaZ gene product in DNA elongation in vitro; Wickner S et al.; E . coli dnaZ gene product is required for conversion of phiX174, fd, and ST-1 single-stranded phage DNAs to duplex DNAs in vitro . This protein has been purified about 5000-fold . It functions in the elongation of RNA- or DNA-primed single-stranded DNA that is catalyzed by DNA polymerase III(DNA nucleotidyltransferase; deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) in conjunctions with two other E . coli protein preparations referred to as DNA elongation factors I and III . It also functions in similar reactions catalyzed by DNA polymerase II in combination with E . coli DNA binding protein and DNA elongation factors I and III.

J Gen Virol, 1976 Apr, 31(1), 9 - 20
Effects of the phage P1 restriction system on coliphage phi W: degradation and complex formation of phage phi W DNA; Skogman GS et al.; Growth of phages phi W and T7 was restricted in Escherichia coli lysogenic for phage P1 . Only a fraction of the infected cells gave burst of phages . Cells permitting phage growth gave normal burst size . Host strains carrying P1 mutants with defective endonuclease gave no restriction of phages T7 and phi 3, the latter a host-range mutant of phi W . Degradation but not modification of parental phage DNA could be demonstrated . Although no DNA, RNA or protein was synthesized in phi W infected P1 lysogenic cells, the parental phage DNA was found in increasingly larger complexes during the course of infection . At early times after infection, parental phage DNA was found to sediment about twice as fast as mature phage DNA . At later times during the infection the parental phage DNA was recovered as a very rapidly sedimenting material . Such material was also found in alkaline sucrose gradient centrifugation after treatment of the cell extract with sodium dodecyl sulphate, pronase digestion and phenol extractions.

J Gen Virol, 1976 Apr, 31(1), 21 - 33
Studies on phage internal proteins: formation of internal protein - T2 DNA complexes in vivo; Bachrach U et al.; Internal proteins, synthesized in T2-infected Escherichia coli B cells were recovered from bacterial membranes during the early stages of infection . Approx . 15 min after the onset of infection, T2 and T4 internal proteins were released from the bacterial membranes and sedimented along with newly synthesized phage DNA . Internal protein-DNA complexes were also obtained by chromatography on hydroxylapatite columns . Internal proteins were not released from bacterial membranes after infection with amber mutants defective in genes 21, 22 and 23 . It has been suggested that these complexes are intermediates in T-even phage morphogenesis.

J Bacteriol, 1976 Apr, 126(1), 80 - 90
Mapping of two loci affecting the regulation of branched-chain amino acid transport in Escherichia coli K-12; Anderson JJ et al.; Two mutant loci resulting in derepression of, respectively, the L-leucine-specific transport system (lstR) and both the leucine-specific and the general branched-chain amino acid transport LIV-I systems (livR) were mapped by conjugation and transduction . Both livR and lstR were found to be closely linked to aroA at min 20 on the Escherichia coli genetic map . The merodiploid livR+/livR displayed wild-type regulation of L-leucine transport, indicating that the livR product is a diffusible, negative controlling element for high-affinity leucine transport systems . Isogenic strains carrying lstR, livR, and wild-type transport alleles were compared for leucine uptake kinetic parameters and leucine-binding protein levels . The higher levels of leucine transport in the mutant strains under repressing conditions were generally due to increased high-affinity systems, which were accompanied by striking increases in the level of leucine-binding proteins.

J Bacteriol, 1976 Apr, 126(1), 64 - 71
Comparative studies on membrane-associated, folded chromosomes from Escherichia coli; Dworsky P; Nuclear bodies were isolated from Escherichia coli spheroplasts by two different lysis procedures . Their lipid and protein content and the superhelix density was measured . The preparations differed mainly in respect to the amount of the attached membrane material, which seems to be an essential factor in maintaining the stability of the nuclear bodies . The superhelix density was found to be higher than that of naturally occurring, covalently closed, circular deoxyribonucleic acids . The influence of the preparative method on the superhelicity was comparatively small.

J Bacteriol, 1976 Apr, 126(1), 56 - 63
Threonine deaminase from Escherichia coli: feedback-hypersensitive enzyme from a genetic regulatory mutant; Calhoun DH; A mutation, ilvA538, in the gene coding for the biosynthetic L-threonine deaminase of Escherichia coli K-12 has previously been demonstrated to have pleiotropic regulatory effects leading to low and invariant expression of some of the isoleucine-valine biosynthetic enzyme, and altered expression of the branched-chain aminoacyl-tRNA synthetases . Strain PS187, which carries the ilvA538 allele, has a partial growth requirement for L-isoleucine and is characterized by a sensitivity to growth inhibition by L-leucine . The experiments reported here demonstrate that the L-threonine deaminase produced by strain PS187 is hypersensitive to inhibition by the pathway end product L-isoleucine . In addition, L-leucine, which acts at relatively high concentrations in vitro as an inhibitor of L-threonine deaminase from the wild type, is a more potent inhibitor of the activity of the mutant enzyme . Forty-six derivatives of strain PS187 were isolated as spontaneous mutants resistant to the growth-inhibitory effects of L-leucine . Two of these, strains MSR14 and MSR16, produce an L-threonine deaminase that is more resistant than the wild type to L-isoleucine inhibition, and intermediate between the wild type and strain PS187 with respect to L-leucine inhibition . Strains MSR14 and MSR16 produce L-threonine deaminase and dihydroxyacid dehydrase, the ilvD gene product, at the low levels characteristic of the parent strain . Other L-leucine-resistant derivatives of strain PS187 produce higher levels of the feedback-hypersensitive L-threonine deaminase . Thus, the sensitivity to growth inhibition by L-leucine observed with strain PS187 appears to be related both to the hypersensitivity of L-threonine deaminase to inhibition of catalytic activity and to the low level of ilv gene expression . The results reported here indicated that L-threonine deaminase is structurally altered in strain PS187, and thus provide further support for the proposal that L-threonine deaminase participates as a genetic regulatory element for the expression of the branched-chain amino acid biosynthetic enzymes.

J Bacteriol, 1976 Apr, 126(1), 536 - 8
Methyl galactosidase activity: an alternative evolutionary destination for the ebgA0 gene; Hall BG; Previous studies (Campbell et al., 1973; Hall and Hartl, 1974; Hall and Hartl, 1975) have shown that the ebgA0 gene, whose product does not hydrolyze lactose may evolve so that its product does hydrolyze lactose; i.e., lactase activity is one evolutionary destination of the ebgA0 gene . Beginning with a strain that synthesizes ebgA0 gene product constitutively and grows extremely slowly (doubling time, 30 to 50 h) on methyl-beta-D-galactopyranoside (MG), a derivative was selected capable of growth on MG at a moderate rate (doubling time, 5.9 h) . Genetic evidence is presented showing that the gene that permits growth on MG is an allele of ebgA . A comparison among strains bearing several alleles of ebgA shows that the new allele, termed ebgAmg, synthesizes a product specific for MG and thus represents a true alternative evolutionary destination for the ebgA0 gene.

J Bacteriol, 1976 Apr, 126(1), 48 - 55
Essential genes in the metB-malB region of Escherichia coli K-12; Armstrong KA et al.; We isolated 25 independent mutants that are deficient in essential genes located in the metB-malB region of the Escherichia coli chromosome . The mutations were mapped within this region by using several F' factors and were also classified into 11 cistrons by complementation testing . There is clustering of mutations in essential genes within the metB-malB region.

J Bacteriol, 1976 Apr, 126(1), 478 - 86
Formation of the formate-nitrate electron transport pathway from inactive components in Escherichia coli; Scott RH et al.; When Escherichia coli was grown on medium containing 10 mM tungstate the formation of active formate dehydrogenase, nitrate reductase, and the complete formate-nitrate electron transport pathway was inhibited . Incubation of the tungstate-grown cells with 1 mM molybdate in the presence of chloramphenicol led to the rapid activation of both formate dehydrogenase and nitrate reductase, and, after a considerable lag, the complete electron transport pathway . Protein bands which corresponded to formate dehydrogenase and nitrate reductase were identified on polyacrylamide gels containing Triton X-100 after the activities were released from the membrane fraction and partially purified Cytochrome b1 was associated with the protein band corresponding to formate dehydrogenase but was not found elsewhere on the gels . When a similar fraction was prepared from cells grown on 10 mM tungstate, an inactive band corresponding to formate dehydrogenase was not observed on polyacrylamide gels; rather, a new faster migrating band was present . Cytochrome b1 was not associated with this band nor was it found anywhere else on the gels . This new band disappeared when the tungstate-grown cells were incubated with molybdate in the presence of chloramphenicol . The formate dehydrogenase activity which was formed, as well as a corresponding protein band, appeared at the original position on the gels . Cytochrome b1 was again associated with this band . The protein band which corresponded to nitrate reductase also was severely depressed in the tungstate-grown cells and a new faster migrating band appeared on the polyacrylamide gels . Upon activation of the nitrate reductase by incubation of the cells with molybdate, the new band diminished and protein reappeared at the original position . Most of the nitrate reductase activity which was formed appeared at the original position of nitrate reductase on gels although some was present at the position of the inactive band formed by tungstate-grown cells . Apparently, inactive forms of both formate dehydrogenase and nitrate reductase accumulate during growth on tungstate which are electrophoretically distinct from the active enzymes . Activation by molybdate results in molecular changes which include the reassociation of cytochrome b1 with formate dehydrogenase and restoration of both enzymes to their original electrophoretic mobilities.

J Bacteriol, 1976 Apr, 126(1), 467 - 77
Mode of insertion of lipopolysaccharide into the outer membrane of escherichia coli; Kulpa CF Jr et al.; A mutant of Escherichia coli that lacks uridine 5'-diphosphate galactose-4-epimerase makes lipopolysaccharide with less carbohydrate than the parent, unless galactose is present during growth . Carbohydrate is dense, and the outer membrane, which contains lipopolysaccharide, was found to be denser when isolated from cells grown with galactose then when galactose was omitted . Cells given galactose after growth in its absence rapidly formed dense regions within the outer membrane that disappeared when galactose was removed . These results indicate that lipopolysaccharide enters the outer membrane nonrandomly at a minimum of 10 to 22 discrete "insertion points." Isopycnic centrifugation provides a method for isolating these regions.

J Bacteriol, 1976 Apr, 126(1), 454 - 66
Mode of replication of the conjugative R-plasmid RSF1040 in Escherichia coli; Crosa JH et al.; Replicating deoxyribonucleic acid (DNA) molecules of plasmid RSF1040, a deletion mutant of the conjugative R plasmid R6K, appear in the electron microscope as partially supercoiled structures with two open circular branches of equal size, although open structures with three branches, two branching points and no supercoiled regions (theta structures) were also found at a lower frequency . The partially supercoiled molecules sediment more rapidly than native covalently closed circular DNA in neutral sucrose gradients and band at a position intermediate between covalently closed circular and open circular DNA in CsClethidium bromide gradients . Electron microscope measurements of the linear EcoRI-treated replicative intermediates indicate that replication can be initiated at two sites (origins) on the plasmid DNA molecule located at about 23% (alpha) and 39% (beta) of the total genome length from an EcoRI end designated arbitrarily as the "left-hand" end of the molecule . The overall replication of RSF1040 is asymmetrically bidirectional . Replication from the alpha origin proceeds first to the "right" to a unique termination site located some 55% of the total genome length from the left-hand end of the molecule . At this point replication proceeds from the alpha origin to the "left" (i.e., opposite to the original direction of replication) until replication of the molecule is completed . Replication also proceeds from the beta origin asymmetrically to the unique terminus site.

J Bacteriol, 1976 Apr, 126(1), 38 - 47
Method for the isolation of Escherichia coli K-12 mutants deficient in essential genes; Armstrong KA et al.; We developed a general procedure for the induction and identification of mutations in chromosomal essential genes that are located in a diploid region of Escherichia coli K-12 . The partial diploidy is conferred by an episome that is temperature sensitive for replication so that a mutant strain will form microcolonies at 42 C on complete media if an essential chromosomal gene in the diploid region is defective . Mutations identified by this procedure can be classified into cistrons by a complementation method devised for the purpose . To verify that the procedure works in practice, we fused an episome covering the rif region with an Ftslac+ and used the resulting temperature-sensitive episome to identify chromosomal mutations in essential functions near rif . As expected, a certain proportion of the mutations were in the rif gene, an essential gene that codes for the beta subunit of ribonucleic acid polymerase.

J Bacteriol, 1976 Apr, 126(1), 365 - 76
Effect of arginine on the stability and size of argECBH messenger ribonucleic acid in Escherichia coli; Krzyzek RA et al.; The chemical stability of argECBH messenger ribonucleic acid (mRNA) produced by Escherichia coli was found to be unaltered during steady-state repression by arginine . During extreme arginine deprivation, the increase in argECBH mRNA stability was related to general effects of amino acid starvation on mRNA stability . Thus a mechanism whereby argECBH gene expression is regulated by altering the decay rate of this mRNA is not consistent with our data . Sucrose gradient analysis followed by hybridization revealed that both heavy (14S) and light (8S) components of argECBH mRNA were produced by cells of E . coli K-12 grown without arginine, whereas predominantly light (8S) mRNA was produced by cells grown with arginine . A functional argR gene and the EC portion of the argECBH cluster were found essential for the arginine restriction of heavy-mRNA production . Experiments suggest that light argECBH mRNA did not arise from heavy message, and 8u% of both light and heavy mRNA was found bound to ribosomes . The data appear most consistent with the notion that a second site of control by arginine regulates the amounts of light and heavy arginine mRNA in the cell either by early termination of transcription or by endonucleolytic processing . Consideration of these data in conjunction with those of the accompanying report (Krzyzek and Rogers, 1976) permits the tentative conclusion that light argECBH mRNA is not translated into active enzymes and is thus responsible for the discrepancy between the high content of hybridizable mRNA and low rates of enzyme synthesis found during arginine repression.

J Bacteriol, 1976 Apr, 126(1), 348 - 64
Dual regulation by arginine of the expression of the Escherichia coli argECBH operon; Kryzek RA et al.; The correlation between the level of messenger ribonucleic acid (mRNA) specific for the argECBH gene cluster (argECBH mRNA) measured by ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization and the rates of synthesis of N-acetylornithine deacetylase (argE enzyme) and of argininosuccinate lyase (argH enzyme) of Escherichia coli strain K-12 were determined for steady-state growth with and without added L-arginine and during the transition periods between these two states . During the transient period after arginine removal (transient derepression), the synthesis of enzymes argE and argH was initially three to five times greater than the steady-state derepressed rate finally reached 50 min later . The level of argECHB mRNA correlated well both quantitatively and temporally with the rates of enzyme synthesis during this transition . The level of in vivo charged arginyl-transfer RNA (tRNAarg), monitored simultaneously, was initially only 5 to 10% and gradually increased to a final level of 80% after 45 min . During the transient period after arginine addition (transient repression), the rates of synthesis of enzymes argE and argH decreased to almost zero and gradually reached steady-state repressed rates after about 180 min . The argECBH mRNA level remained constant at the steady-state repressed level throughout transient repression, revealing a discontinuity between the level of this mRNA and rates of enzyme synthesis . A similar discrepancy was noted during the transition after ornithine addition . In vivo charged tRNAarg remained constant at 80% during this transition . After removal of arginine, the zero-level transient enzyme synthesis developed after only 7.5 min of arginine deprivation and was maximum after 30 min . The results suggest an accumulation of a molecule regulated by arginine that plays a role in transient repression . Our data indicate that arginyl-tRNA synthetase is not this molecule since its synthesis was unaffected by arginine . The ratios of steady-state argE and argH enzyme synthesis without arginine to that with arginine were 12 and 20, respectively, whereas the similar ratio for argECBH mRNA was 2 to 3 . The repressed level of argECBH mRNA was not affected by attempts to repress or derepress the ppc+ gene (carried on the DNA used for hybridization), and the repressed level of argECBH mRNA was lowered about 50% in cells carrying an internal argBH deletion . These data taken together indicate the presence of an excess of untranslated argECBH mRNA during both transient and steady-state repression by arginine . Thus, a second regulatory mechanism, not yet defined, appears to play an important role in arginine regulation of enzyme synthesis.

J Bacteriol, 1976 Apr, 126(1), 251 - 6
Effects of growth temperature on yield and maintenance during glucose-limited continuous culture of Escherichia coli; Mainzer SE et al.; The effects of growth temperature on the aerobic growth yield with respect to oxygen consumption (Y0-grams {dry weight} per gram-atom of O) and the rate of maintenance respiration (m0-milligram-atoms of O/gram {dry weight} per hour) are reported for Escherichia coli B cultivated continuously in the presence of oxygen with limiting glucose . During anaerobic continuous culture, YATP(max) (grams {dry weight} per mole of ATP corrected for maintenance) increases from 10.3 to 12.7 as the growth temperature is lowered from 37 to 25 C . Over this same range, Y0(max) (Y0 corrected for maintenance respiration) rises from 12.5 to 28.8 and remains at the higher value down to 17.5 C . From 37 to 32 C, m0 increases from 0.9 to 4.4 but then falls to 1.5 as the temperature is lowered to 17.5 C . The value of m0 sharply rises some 13-fold as the temperature is raised to 42 C without a significant change in the value of Y0(max) . Changes of Y0(max) are consistent with a temperature-sensitive doubling of the efficiency of oxidative phosphorylation, but the reasons for the changes of the rate of maintenance respiration are not known.

J Bacteriol, 1976 Apr, 126(1), 222 - 4
Temperature dependence of mating-pair formation in Escherichia coli; Walmsley RH; The temperature dependence of mating-pair formation by Escherichia coli has been studied from 2 to 45 C by means of a physical assay . No mating-pair formation was found below 24 C . Between 30 and 41 C pair formation increased very rapidly, followed by an equally sharp decline between 41 and 45 C . The results are interpreted in terms of the pilus adsorption process of recipient cells and pili production by donor cells.

J Bacteriol, 1976 Apr, 126(1), 147 - 56
Escherichia coli mutants altered in murein lipoprotein; Wu HC et al.; Mutants with alterations in the structure, biosynthesis, or assembly of murein lipoprotein were selected by a procedure based on radiation suicide of wild-type organisms by {3H}arginine under conditions where the radioactive arginine was preferentially incorporated into lipoprotein . Further screening for the potential mutants among the survivors of {3H}arginine suicide was carried out by using a sensitive immunodiffusion test, followed by radioactive double-labeling experiments . Three mutants were obtained and partially characterized.

J Bacteriol, 1976 Apr, 126(1), 140 - 6
Inactivation of membrane transport in Escherichia coli by near-ultraviolet light; Koch AL et al.; Evidence is presented that near-ultraviolet (near-UV) light can alter galactoside transport in Escherichia coli in several independent ways . It can inactivate the permease system per se, it can interfere with metabolic energy production or transfer, and it can cause an increase in the generalized permeability of the membrane . Earlier publications suggested that near-UV destroys cofactors needed for electron transport and thus places a limitation on energy reserves . In agreement, we found that the active accumulation of {14C}thiomethyl-beta-D-galactopyranoside is decreased after irradiation by a larger factor than that due to action directly on the permease system . The effect on the latter was measured by the decrease in the rate of o-nitrophenyl-beta-D-galactopyranoside (ONPG) transport . As evidence that energy supplies for this "downhill" process did not become rate limiting after irradiation, we found that carbonylcyanide-m-chlorophenyl-hydrazone did not stimulate ONPG transport of irradiated cells . Cells genetically deficient in functional permease or cells treated with formaldehyde still transport ONPG passively, although at much lower rates . With the use of such cells, it was found that high fluences (doses) made the cells leaky . Further evidence that the permease system and the metabolic energy system can be inactivated independently is also presented . It is shown that a photoproduct from the irradiation of chloramphenicol inactivates the permease system much more efficiently than the energy system . In addition, it is shown that thio-beta-D-digalactopyranoside protects the permease system, but not the energy system, both against direct inactivation by near-UV and against photosensitized inactivation in the presence of chloramphenicol.

J Bacteriol, 1976 Apr, 126(1), 132 - 9
Specific in vivo cleavage of D-serine deaminase and properties of tetrameric polypeptide aggregates of the fragments; Heincz MC et al.; The primary D-serine deaminase (D-serine dehydratase, EC 4.2.1.14) of Escherichia coli K-12 is unstable within the cell . The protein, a single polypeptide chain, is cleaved at a lysine residue by a cellular proteolytic activity . Fragments containing the active site then aggregate into tetramers, which retain substrate affinity and show very low catalytic activity . Such degradations may represent an evolutionary mechanism for the generation of new enzymes.

J Bacteriol, 1976 Apr, 126(1), 122 - 31
Biosynthesis of membrane-bound nitrate reductase in Escherichia coli: evidence for a soluble precursor; MacGregor CH; Membrane-bound nitrate reductase of Escherichia coli consists of three subunits designated as A, B, and C, with subunit C being the apoprotein of cytochrome b, A hemA mutant that cannot synthesize delta-aminolevulinic acid (ALA) produces a normal, stable, membrane-bound enzyme when grown with ALA . When grown without ALA, this mutant makes a reduced amount of membrane-bound enzyme that is unstable and contains no C subunit . Under the same growth conditions, this mutant accumulates a large amount of a soluble form of the enzyme in the cytoplasm . Accumulation of this cytoplasmic form begins immediately upon induction of the enzyme with nitrate . The cytoplasmic form is very similar to the soluble form of the enzyme obtained by alkaline heat extraction . It is a high-molecular-weight complex with a Strokes radius of 8.0 nm and consists of intact A and B subunits . When ALA is added to a culture growing without ALA, the cytoplasmic form of the enzyme is incorporated into the membrane in a stable form, coincident with the formation of functional cytochrome b . Reconstitution experiments indicate that subunit C is present in cultures grown without ALA but is reduced in amount or unstable . These results indicate that membrane-bound nitrate reductase is synthesized via a soluble precursor containing subunits A and B, which then binds to the membrane upon interaction with the third subunit, cytochrome b.

J Bacteriol, 1976 Apr, 126(1), 1 - 6
Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicons in Eschericha coli; Bennett PM et al.; A number of well-characterized R plasmids (R1drd 19 . K1.R100-1, R46, R55-1, R64-11, R388, and R751) will mobilize an integrated amp gene from the chromosome of Escherichia coli K-12 . R391 will not act in this way . The process involves recombination, and in each case the mobilizing plasmid acquires an additional piece of deoxyribonucleic acid of molecular weight 4 X 10(6) . Acquisition of this deoxyribonucleic acid may impair the transfer properties of the recombinant plasmid in some cases . The process will occur in a recA background.

Eur J Biochem, 1976 Apr 1, 63(2), 607 - 15
The study of DNA-RNA-polymerase complexes by kinetic formaldehyde method; Zarudnaya MI et al.; A modification of the kinetic formaldehyde method has been proposed providing a possibility for locally denatured regions (defects) formed in DNA preincubated with RNA polymerase (in the absence of nucleoside triphosphates) to be detected . This modification consists in a previous fixation of DNA-enzyme complex with small concentrations of formaldehyde, which do not induce formation of defects in DNA alone . The method has been calibrated under the conditions favourable to RNA synthesis . Studies of the effect of the fixation conditions on the number of defects in DNA interacting with RNA polymerase have shown that the number of defects is constant with formaldehyde fixation concentration between 0.05% and 0.3-0.5% and with fixation time between 2 min and 100 min . The dependence of the number of defects in DNA on RNA polymerase concentration at low ionic strength (0.05 M KCl) is presented by a curve with a plateau . From the initial linear part of the curve it has been found that the enzyme bound to DNA as a monomer . At the excess of the enzyme the mean number of nucleotide pairs between defects is 400-500 . Increase of ionic strength results in decrease of the number of defects in DNA . The number of defects depends on temperature of preincubation of the complex . There were no defects in DNA at temperatures below 20 degrees C . At temperatures above 30 degrees C the number of defects reaches saturation . A sharp transition occurs in the range of temperatures between 20 degrees C and 30 degrees C . Analysis of the experimental and literature data, concerning the interaction of formaldehyde and amino acid methylol derivatives with DNA bases, leads to the conclusion that the mechanism of the formation of defects in helical DNA most likely consists in its unwinding or sharp weakening upon binding of RNA polymerase, prior to addition of formaldehyde.

Eur J Biochem, 1976 Apr 1, 63(2), 469 - 75
Factors affecting the release of folded chromosomes from Escherichia coli; Meyer M et al.; 1 . The envelope-bound folded chromosome prepared by gentle lysis of Escherichia coli K 12 DG 75 cells in the presence of 1.0 M sodium chloride was found to be a non-specific complex of DNA strands entangled in vesicle-like envelope structures . This envelope remnant is still more or less rod-shaped . 2 . Both the envelope-bound and the envelope-free folded chromosomes arise directly from intact cells . Even under extreme conditions of lysis, envelope-bound chromosomes cannot be converted into envelope-free nucleoids.

Eur J Biochem, 1976 Apr 1, 63(2), 459 - 67
Membrane hybridization by centrifugation analysed by lipid phase transitions and reconstitution of NADH-oxidase-activity; Devor KA et al.; A procedure is described to hybridize cytoplasmic membranes of Escherichia coli . The method involves high-speed contrifugation of two different vesicle preparations at 37 degrees C in the presence of cations such as spermine or Mg2+ . The occurrence of hybridization is shown by the following experiments . Firstly, formation of a mixed lipis phase starting from two membranes having a different hydrocarbon chain composition in their phospholipids . Secondly, formation of a hybrid membrane having an intermediate lipid to protein ratio from two membrane fractions having different lipid to protein ratios . Thirdly, reconstitution of NADH oxidase activity by hybridization of two membrane fractions, one lacking active cytochromes and the other being deficient in quinones . It is proposed that hybridization occurs by fusion of vesicles during the tight association of collapsed vesicles under high centrifugal forces . This interpretation is supported by electron microscopy of the membrane pellets after centrifugation . However, lipid transfer as the mechanism of hybridization cannot be excluded and attempts to reconstitute active galactoside transport by complementation of beta-galactoside transport-deficient membranes and cytochrome-deficient membranes have been unsuccessful.

Eur J Biochem, 1976 Apr 1, 63(2), 431 - 40
Interactions of guanosine triphosphate analogues with elongation factor G of Escherichia coli; Hamel E; Previous studies from this laboratory have shown that the GTP analogue guanosine 3'-diphosphate 5'-triphosphate (pppGpp) was almost without activity in the translocation reaction catalyzed by elongation factor G (EF-G), while it was fully active with elongation factor T (EF-T) and initiation factor 2 (IF-2) . To assess the importance of the 3'-ribose hydroxyl itself in the translocation reaction, we examined polyphenylalamine synthesis and EF-G-dependent formation of N-acetylphenylalanylphenylalanylpuromycin (Ac-Phe2-puromycin) supported by 3'-deoxyguanosine 5'-triphosphate (3'dGTP) and 3'-deoxy-3'-aminoguanosine 5'-triphosphate (3'dNH2GTP) . Like pppGpp, these nucleotides were similar to GTP in EF-T and IF-2dependent reactions . We also examined the ability of the dialcohol derived from GTP by periodate oxidation and borohydride reduction (rroGTP) and of ITP to support translocation . These compounds had shown significant, although reduced, activity with EF-T and IF-2 . A spectrum of activity was found with these compounds in both poly(Phe) synthesis and Ac-Phe2-puromycin formation . All had significantly reduced activity relative to GTP, but all were significantly more active than pppGpp . A surprising finding was that the activities of all analogues relative to GTP were dependent on the reaction temperature in both poly(Phe) synthesis and Ac-Phe2-puromycin formation; these relative activities were significantly lower at 8 degrees C after than 37 degrees C . Furthermore, the extent of poly(Phe) synthesis with the analogues relative to GTP could be significantly affected by the amounts of EF-G and EF-T in the reaction mixture . Differences were minimized in the presence of rate-limiting EF-T and saturating EF-G and maximized in the presence of rate-limiting EF-G and saturating EF-T . This effect of reducing the level of EF-G in the reaction mixture thus mimicked the effect of lowering the incubation temperature, and a similar observation was made for Ac-Phe2-puromycin formation . GTP-supported formation of Ac-Phe2-puromycin at 8 degress C could be inhibited by 3'dNH2GTP, 3'dGTP, pppGpp, and ITP: these compounds were better inhibitors than they were substrates . Inhibition by other nucleotides was also noted . The GTP analogues were compared to GTP as substrates in EF-G-dependent reactions uncoupled from protein synthesis . Fusidic-acid-dependent binding of nucleotides to ribosomes and ribosome-dependent catalytic nucleotide hydrolysis were both examined, and the activity of the nucleotides as substrates in these ractions showed little correlation with their ability to support translocation.

Eur J Biochem, 1976 Apr 1, 63(2), 419 - 26
Labelling of L-isoleucine tRNA ligase from Escherichia coli with L-isoleucyl-bromomethyl ketone; Rainey P et al.; L-Isoleucine: tRNA ligase from Escherichia coli could be irreversibly inactivated by L-isoleucyl-bromomethyl ketone but not by L-isoleucyl-chromethyl ketone . The inactivation rate exhibited a saturation concentration dependence typical for an affinity reagent . L-Isoleucine provided 100% protection against inactivation at saturating concentration, whereas ATP, AMP, and pyrophosphate offered partial protection and tRNAIle, no protection . The ligase was labelled in preparative scale with L-{14C}isoleucyl-bromethyl ketone . The molar ration of label incorporated to enzyme inactivated was close to unity . The protein was subsequently subjected to tryptic digestion and the radioactive peptide isolated and identified . The labelled amino acid proved to be the same cysteine previously reported as being labelled with N-{14C}ethylmaleimide {Kula, M.-R . (1974) FEBS Lett . 46, 130-133}.

Chest, 1976 Apr, 69(4), 474 - 8
Phagocytosis and cutaneous delayed hypersensitivity in patients with chronic obstructive pulmonary disease; Ritts RE et al.; The objective of this study was to determine if the phagocytic and intracellular killing capacity of peripheral granulocytes or an expression of cellular-mediated immunity, delayed cutaneous reactivity, as measurements of native and acquired immunity, might be risk factors associated with chronic obstructive pulmonary disease (COPD) . Over 100 patients with a value for their forced expiratory volume in one second (FEV1) less than or equal to 70 percent of normal were carefully matched with healthy participants having an FEV1 greater than or equal to 86 percent of normal, and together they served as the study group . Phagocytosis and intracellular killing were normal in patients with COPD; however, these patients demonstrated a significant impairment in the ability of their peripheral leukocytes to reduce nitroblue tetrazolium . The delayed-hypertensitivity response rate and the degree of reactivity were similar in the two groups, except for the patients with COPD having a significantly greater degree of reactivity to Monilia albicans extract ("canadin.") This finding is thought to be a consequence of reduced mucociliary clearance rather than a risk factor . The significance of decreased resting and stimulated cells' reduction of nitroblue tetrazolium in patients with COPD is not clear.

Obstet Gynecol, 1976 Apr, 47(4), 423 - 6
Cephacetrile in the treatment of female pelvic inflammatory disease; Zehnder JL et al.; The efficacy and tolerability of cephacetrile administered by the intravenous or intramuscular route was evaluated in an open-label study of 55 hospitalized females diagnosed as having mild to severe pelvic inflammatory disease . Rapid improvement was observed in most patients . All patients were classified as clinically cured or significantly improved . Pain was absent or mild in 86% of the patients who received cephacetrile by intramuscular injection . Seventy-two percent of the patients given the drug intravenously experienced no localized pain . Phlebitis occurred in 6 of the 53 patients (11%) who received intravenous cephacetrile and thrombosis was present in 3 (6%) . Rash and puritus was reported in 1 patient . Results of this study suggest that cephacetrile is an important and useful drug in the treatment of pelvic inflammatory disease.

Nature, 1976 Apr 1, 260(5550), 406 - 10
Mechanism localisation and control of restriction cleavage of phage T4 and lambda chromosomes in vivo; Dharmalingam K et al.; The primary action of restriction endonuclease, cleaving infecting DNA, has been demonstrated in vivo . This primary cleavage is followed rapidly by hydrolysis of the cleaved DNA at its newly exposed termini . Infecting viruses can inactivate cytoplasmic and membrane restriction endonucleases to prevent cleavage of unmodified DNA replicas.

Biochem J, 1976 Apr 1, 155(1), 201 - 3
Electron-paramagnetic-resonance studies on the molybdenum of nitrate reductase from Escherichia coli K12; Bray RC et al.; Studies on the respiratory nitrate reductase (EC 1.7.99.4) from Escherichia coli K12 by electron-paramagnetic-resonance spectroscopy indicate that its molybdenum centre is comparable with that in other molybdenum-containing enzymes . Two Mo(V) signals may be observed; one shows interaction of Mo(V) with a proton exchangeable with the solvent and has: A (1H) 0.9-1.2mT; g1 = 1.999; g2=1.985; g3 = 1.964; gav . = 1.983 . Molybdenum of both signal-giving species may be reduced with dithionite and reoxidized with nitrate.

J Antibiot (Tokyo), 1976 Apr, 29(4), 433 - 7
Synthesis of (beta-methyl-3H-)-benzylpenicillin and (beta-methyl-3H)-6-aminopenicillanic acid; Meesschaert B et al.; The synthesis of benzylpenicillin and 6-aminopenicillanic acid, labeled with tritium in the beta-methyl group, is described . Benzylpenicillin S-sulfoxide benzyl ester is refluxed in benzene and tritiated water and is successively debenzylated and deoxygenated to (beta-methyl-3H)-benzylpenicillin . Removal of the side chain with a bacterial acylase gives (beta-methyl-3H)-6-aminopenicillanic acid.

Nucleic Acids Res, 1976 Apr, 3(4), 847 - 61
Spin labeled nucleic acids; Caspary WJ et al.; Homopolyribonucleotides and E . coli DNA wer spin labeled with an iodoacetamide-nitroxide compound . The extent of labeling is highly dependent upon the nature of the base and the secondary structure of the nucleic acid . This spin label-polymer linkage is unstable at high temperatures and in phosphate buffers . In order to determine the effect of changes in the environment of nucleic acids on the esr signals of their attached spin labels, the polynucleotides were subjected to temperature and viscosity perturbations . An increase in temperature (T) affects a linear decrease in the anisotropy factor of the esr signal . The log tau (tau = correlation time) versus (1/T) profile is linear with a positive slope when the spin label is attached to single stranded polynucleotides but exhibits discontinuities at certain critical temperatures when attached to the duplexes poly (As-U) and poly (I-Cs) . These critical temperatures are lower than the optical Tm . Logarithmic increase in viscosity was found to produce a linear increase in tau in aqueous sucrose solutions.

Proc Natl Acad Sci U S A, 1976 Apr, 73(4), 1126 - 30
Transcription-translation and translation-messenger RNA decay coupling: separate mechanisms for different messengers; Walker AC et al.; Antibiotics were used to inhibit protein synthesis at specific steps in the biosynthetic pathway . In this way, it was possible to study the coupling of protein synthesis to the accumulation of biologically active mRNA in T4-infected Escherichia coli . Functional mRNA for the phage enzymes deoxynucleotide kinase (EC 2.7.4.4; ATP: nucleoside monophosphate phosphotransferase or nucleosidemonophosphate kinase) and alpha-glucosyltransferase (EC 2.4.1.5; 1, 4-alpha-D-glucan: 1, 6-alpha-D-glucan 6-alpha-glucosyltransferase or dextrin dextranase) accumulated during inhibition of protein synthesis irrespective of the step in the synthesis of protein that was blocked . Under these conditions, however, the rate of mRNA synthesis for both enzymes was significantly inhibited . In contrast, the rate of degradation of these mRNAs was markedly dependent on the step in protein synthesis that was inhibited . That is, the site for mRNase action was different for each message . The most important step in protein synthesis required for the stability of deoxynucleotide kinase mRNA is the initiation step . A single ribosome bound to the 5' end of the deoxynucleotide kinase mRNA can stabilize the molecule . On the other hand, the initiation event does not seem to be important for stabilizing the alpha-glucosyltransferase mRNA . Instead, a high ribosome denisty on the alpha-glucosyltransferase messenger is required to achieve significant stability . Therefore, in studying messenger metabolism, it is important to focus on the functional stability of specific mRNAs instead of on total messenger since each mRNA can be metabolized differently.

J Bacteriol, 1976 Apr, 126(1), 7 - 12
Siderophore protection against colicins M, B, V, and Ia in Escherichia coli; Wayne R et al.; A variety of natural and synthetic siderophores capable of supporting the growth of Escherichia coli K-12 on iron-limited media also protect strain RW193+ (tonA+ ent-) from the killing action of colicins B, V, and Ia . Protective activity falls into two categories . The first, characteristic of enterobactin protection against colicin B and ferrichrome protection against colicin M, has properties of a specific receptor competition between the siderophore and the colicin . Thus, enterobactin specifically protects against colicin B in fes- mutants (able to accumulate but unable to utilize enterobactin) as predicted by our proposal that the colicin B receptor functions in the specific binding for uptake of enterobactin (Wayne and Neilands, 1975) . Similarly ferrichrome specifically protects against colicin M in SidA mutants (defective in hydroxamate siderophore utilization) . The second category of protective response, characteristic of the more general siderophore inhibition of colicins B, V, and Ia, requires the availability or metabolism of siderophore iron . Thus, enterobactin protects against colicins V and Ia, but only when the colicin indicator strain is fes+, and hydroxamate siderophores inhibit colicins B, V, and Ia, but only when the colicin indicator strain is SidA+ . Moreover, ferrichrome inhibits colicins B, V, and Ia, yet chromium (III) deferriferrichrome is inactive, and ferrichrome itself does not prevent adsorption of colicin Ia receptor material in vitro . Although the nonspecific protection against colicins B, V, and Ia requires iron, the availability of siderophore iron for cell growth is not sufficient to bring about protection . None of the siderophores tested protect cells against the killing action of colicin E1 or K, or against the energy poisons azide, 2, 4-dinitrophenol, and carbonylcyanide m-chlorophenylhydrazone . We suggest that nonspecific siderophore protection against colicins B, V, and Ia may be due either to an induction of membrane alterations in response to siderophore iron metabolism or to a direct interference by siderophore iron with some unknown step in colicin action subsequent to adsorption.

Zh Mikrobiol Epidemiol Immunobiol, 1976 Apr, (3), 44 - 8
{Protective properties of IgM in experiments on animals}; Kiseleva IA et al.; On a model of intraperitoneal infection of albino mice the authors demonstrated a protective action of the fraction enriched with IgM and of gamma-globulin isolated from the normal human blood serum, against E . coli O111 . The intensity of the protective action depended on the method, duration of administration of the preparation and also on the infective dose . Protective properties of the fraction enriched with IgM were more pronounced in comparison with the gamma-globulin preparation.

J Bacteriol, 1976 Apr, 126(1), 327 - 37
Protonmotive force as the source of energy for adenosine 5'-triphosphate synthesis in Escherichia coli; Wilson DM et al.; Net synthesis of adenosine 5'-triphosphate (ATP) in energy-depleted cells of Escherichia coli was observed when an inwardly directed protonmotive force was artificially imposed . In wild-type cells, ATP synthesis occurred whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside) . Formation of ATP did not occur unless the protonmotive force exceeded a value of 200 mV . Under these conditions, no ATP synthesis was found when cells were exposed to an inhibitor of the membrane-bound Ca2+- and Mg2+- stimulated adenosine triphosphatase (EC 3.6.1.3), dicyclohexylcarbodiimide, or to a proton conductor, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone . Adenosine triphosphatase-negative mutants failed to show ATP synthesis in response to either a membrane potential or a pH gradient . ATP synthesis driven by a protonmotive force was observed in a cytochrome-deficient mutant . These observations are consistent with the chemiosmotic hypothesis of Mitchell (1961, 1966, 1974).

Chromosoma, 1976 Mar 31, 55(1), 13 - 25
Electron microscopy of membrane-associated folded chromosomes of Escherichia coli; Kavenoff R et al.; Membrane-associated folded chromosomes were purified from log-phase cultures of Escherichia coli 15 TAU-bar and prepared for electron microscopy by aqueous spreading techniques . A spectrum of structures was observed, ranging from condensed structures with no DNA fibers visible, to extended structures with DNA fibers . In the extended structures, loops of DNA radiated from residual envelope, the loops sometimes appeared supercoiled, and both their number and apparent contour length approximated previous estimates from physical and biochemical data . It is proposed that the structures with free DNA arose from the condensed structures.

J Mol Evol, 1976 Mar 29, 7(2), 133 - 49
Frequency of insertion-deletion, transversion, and transition in the evolution of 5S ribosomal RNA; Sankoff D et al.; The problem of choosing an alignment of two or more nucleotide sequences is particularly difficult for nucleic acids, such as 5S ribosomal RNA, which do not code for protein and for which secondary structure is unknown . Given a set of 'costs' for the various types of replacement mutations and for base insertion or deletion, we present a dynamic programming algorithm which finds the optimal (least costly) alignment for a set of N sequences simultaneously, where each sequence is associated with one of the N tips of a given evolutionary tree . Concurrently, protosequences are constructed corresponding to the ancestral nodes of the tree . A version of this algorithm, modified to be computationally feasible, is implemented to align the sequences of 5S RNA from nine organisms . Complete sets of alignments and protosequence reconstructions are done for a large number of different configurations of mutation costs . Examination of the family of curbes of total replacements inferred versus the ratio of transitions/transversions inferred, each curve corresponding to a given number of insertions-deletions inferred, provides a method for estimating relative costs and relative frequencies for these different types of mutations.

Lancet, 1976 Mar 27, 1(7961), 659 - 63
Enterotoxigenic Escherichia coli and Reovirus-like agent in rural Bangladesh; Ryder RW et al.; PIP: 48 patients admitted to a rural Bangladesh hospital with dehydration secondary to diarrhea were examined for infection caused by (R.L.A.) reovirus-like agent or (E.T.E.C.) enterotoxigenic Escherichia coli . The diagnosis of R.L.A . infection was established by electron microscopy of stool filtrates and by a 4-fold or greater rise in serum complement-fixing antibodies to the Nebraska calf diarrhea virus . Evidence of infection by heat-labile-toxin producing E.T.E.C . was sought by stool culture and serological testing using the adrenal cell tissue-culture system . Infection by heat-stable-toxin producing E.T.E.C . was sought by stool culture using the infant mouse test . 12 patients, all less than 2 years old, had evidence of R.L.A . infection, accounting for illness in 5% of the 22 patients under 2 . None of these 22 had evidence of E.E.T.E.C . infection . R.L.A . diarrhea lasted 5-6 days, often led to serious dehydration, and was associated with vomiting and fever . 11 cases of E.T.E.C . diarrhea were detected, accounting for 56% of the cases of diarrhea in the 18 patients who were more than 10 years old . Diarrhea caused by E.T.E.C . was sudden in onset, shorter in duration, and caused pronounced dehydration . In a community survey, E.T.E.C . was isolated with equal frequency in the stools of control and case family members . Data suggest that E.T.E.C . is a common cause of adult diarrhea in Bangladesh while R.L.A . is a common cause of diarrhea in children .

Biochim Biophys Acta, 1976 Mar 25, 428(1), 146 - 56
Membrane-associated reactions in ubiquinone biosynthesis . 2-Octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase; Leppik RA et al.; The O-methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, which has been previously postulated to be the final reaction in the biosynthesis of ubiquinone was demonstrated in vitro using cell extracts of Escherichia coli . S-Adenosyl-L-methionine was active as the methyl donor for the reaction . The enzyme concerned, S-adenosyl-L-methionine: 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone-O-methyltransferase, was partially purified and shown to have a molecular weight of about 50 000 and to require a divalent metal and dithiothreitol for optimal activity in vitro . The methyltransferase was absent from extracts from ubiG- mutants suggesting that the ubiG gene is the structural gene coding for the methyltransferase . The enzyme, although not firmly membrane-bound, showed some affinity for the cell membrane in broken cell preparations and could utilize the benzoquinone substrate when the latter was free or bound to the cell membrane, with about equal efficiency . It is concluded that in vivo, the methyltransferase reaction probably occurs at the internal surface of the cytoplasmic membrane.

J Biol Chem, 1976 Mar 25, 251(6), 1718 - 22
Stoichiometry of polypeptide chain elongation; Cabrer B et al.; To quantitate the amount of GTP hydrolyzed during polypeptide chain elongation, an in vitro system containing purified endogenous Escherichia coli polysomes has been developed . The polysomes are washed with 1 M NH4Cl to eliminate endogenous GTPase activities and are depleted of subunits and free ribosomes to diminish the uncoupled elongation factor G-dependent GTP hydrolysis . These polysomes, supplemented with elongation factors, aminoacyl-tRNA, and low concentrations of GTP, incorporate amino acids in their nascent peptide chains . After correcting for a background of uncoupled GTP hydrolysis, it has been found that the incorporation of each molecule of amino acid is associated with the hydrolysis of 2 molecules of GTP.

J Biol Chem, 1976 Mar 25, 251(6), 1712 - 7
Structural studies on yeast RNA polymerases . Existence of common subunits in RNA polymerases A(I) and B(II); Buhler JM et al.; The subunits of yeast RNA polymerases A(I) and B(II) were characterized using several techniques . The present studies demonstrate that the A and B enzymes possess three subunits, which are indistinguishable on the basis of molecular weight, isoelectric point, and fingerprint pattern . The three common subunits belong to the small molecular weight components of the enzymes . By polyacrylamide gel electrophoresis with sodium dodecyl sulfate they migrate with apparent molecular weights of 27,000, 23,000, and 14,500, respectively . A two-dimensional subunit mapping technique on polyacrylamide gel was used to separate the subunits according to isoelectric point and molecular weight . The common polypeptides co-migrated on three spots corresponding to isoelectric points of 9.2 (27,000), 4.5 (23,000), and 4.6 (14,500) . The fingerprints of the 35S-labeled tryptic peptides of the presumptive common subunits were found to be essentially identical . Finally, the presence of common subunits was supported by the fact that antibodies against pure RNA polymerase A cross-react with and inhibit RNA polymerase B . Except for the common subunits, it is likely that RNA polymerases A and B are primarily made of distinct gene products for the following reasons . A total of 13 polypeptide chains are present in enzyme A, whereas 10 polypeptides are found in enzyme B . The molecular weight, isoelectric point, and sulfur content of the majority of these polypeptide chains are different in the two enzymes . No similarity was found in the 35S-peptide fingerprint from a number of A and B subunits of slightly different molecular weight . Finally, antibodies against the largest subunit from RNA polymerase A do not cross-react with or inhibit RNA polymerase B . The data are discussed in terms of structural organization of eukaryotic RNA polymerases.

Biochemistry, 1976 Mar 23, 15(6), 1324 - 30
Concerted allosteric transition in hybrids of aspartate transcarbamoylase containing different arrangements of active and inactive sites; Gibbons I et al.; Various hybrids of aspartate transcarbamoylase of Escherichia coli were constructed from native regulatory subunits and mixtures of active and inactive (pyridoxylated) catalytic chains in specific arrangements within the two catalytic subunits . The kinetic and physical properties of these well-defined hybrids were studied in order to determine the effects of reducing the number of substrate binding sites and distributing the active and inactive chains in different ways . Experiments on enzyme-like molecules containing six, four, three, two, and one active sites showed that the Hill coefficient decreased and the apparent Km increased as the number of active chains in the hybrids was reduced . The maximum inhibition and activation by the nucleotide effectors, CTP and ATP, were independent of the composition of the enzyme-like molecules . Two hybrids were of particular interest since one contained two active sites in one catalytic subunit and none in the other, and the second hybrid had one active site in each catalytic subunit . These two hybrids exhibited identical kinetic behavior despite the markedly different structural arrangements . The ligand-promoted conformational changes of the hybrids monitored both by sedimentation velocity measurements and the reactivity toward p-hydroxymercuribenzoate were similar to those of the native enzyme . These results indicate that there are no discrete "cooperative units" within the enzyme molecules but rather that the allosteric transition promoted by ligands is fully concerted . The various kinetic and physical properties can be accounted for satisfactorily in terms of the two-state model of Monod et al.

Biochemistry, 1976 Mar 23, 15(6), 1316 - 23
Alteration of the allosteric properties of aspartate transcarbamoylase by pyridoxylation of the catalytic and regulatory subunits; Blackburn MN et al.; Extension modification of aspartate transcarbamoylase from Escherichia coli with pyridoxal 5'-phosphate followed by reduction of the Schiff base with sodium borohydride caused only partial inactivation of the enzyme . Under comparable conditions, virtually complete loss of enzyme activity is obtained with the free catalytic subunits . The pyridoxylated, intact enzyme containing more than 60% of the bound pyridoxamine phosphate on the regulatory subunits exhibited considerable cooperativity, inhibition by CTP, and activation by ATP . When the modification was performed in the presence of the ligands which bind to the catalytic sites, the resulting product had virtually the same activity as the native enzyme, but it exhibited significantly reduced cooperativity and virtually no inhibition by CTP . The pyridoxylation of the regulatory subunits within the intact enzyme was enhanced markedly in the presence of ligands as compared with the reactivity of these subunits when the modificaiton was performed in the absence of the active site ligands . Both types of pyridoxylated derivatives exhibited the ligand-promoted conformational changes characteristic of the native enzyme . Spectrophotometric studies of inactive pyridoxylated catalytic subunits and intact enzyme showed that the substrate (carbamoyl phosphate) bound strongly but that the substrate analogue (succinate) did not bind . Both the pyridoxylation experiments in the presence and absence of ligands and the spectral behavior of a hybrid containing one native and one pyridoxylated catalytic subunit indicated that ligand binding was accompanied by a conformational change in the intact enzyme molecules.

Mol Gen Genet, 1976 Mar 22, 144(2), 151 - 8
An approach to genetic transformation in the Xiphophorine fish; Schwab M et al.; The particular suitability of the Xiphophorine fish system for achieving genetic transformation is presented, and it was analyzed whether information carrying donor DNA might be available to the propigment cells of embryos of Xiphosphorus helleri, which are the target cells for the transformation . Heterologous 2H3H-labelled donor DNA from E . coli, which was taken for technical reasons instead of homologous fish DNA, undergoes degradation both after injection into the neural crest region and after injection into the yolk sac (molecular weight at O h: 50 X10(6); at 2 h: 1 X 10(6); at 5 h: less than 3 X 10(5); at 10 h: less than 1 X 10(4)) . It is concluded therefore, that informative donor DNA is present for about 2 to 3 hours after injection . The DNA of the recipient embryo is labelled radioactively during that time at which informative DNA is present only, if the donor DNA is injected into the neural crest region . The probability that a foreign gene might become available to the propigment cells and might induce transformation is discussed.

Mol Gen Genet, 1976 Mar 22, 144(2), 217 - 21
The maintenance affinities of KLF-1 proximal F merogenotes in Escherichia coli; Habacek J et al.; In conjugation with donor strains carrying proximal F merogenotes of KLF-1 type about 100-fold lower frequency of Leu+ or Lac+ recombinants was found . The determination of the level of beta-galactosidase synthesis during the initial period of mating indicated that the transfer process of plasmid DNA was not impaired . Among the recombinants selected a large fraction have not expressed the plasmic fertility functions . This phenomenon was found to be replicon specific and was observed only with proximal F merogenotes but not with classical F'lac and F'ORF-1 elements or R1-19 plasmid . The expression of KLF-1 plasmid functions in the cell seems to be affected by a chromosomal gene of the proximal F merogenote closely linked to leu marker.

Mol Gen Genet, 1976 Mar 22, 144(2), 185 - 90
Consequences of losing ribonuclease III on the Escherichia coli cell; Apirion D et al.; An isogenic pair of Escherichia coli strains, one carrying an rnc+ and the other an rnc- allele (a mutation which reduces the level of ribonuclease III), was compared . The rnc- strain fails to grow at very elevated temperatures (for E . coli) while the rnc+ strain does grow exponentially . Assaying the residual RNase III like activity in extracts of the rnc- strain at different pHs and at different temperatures suggested that this residual RNase III like activity is not due to RNase III . This raised the possibility that the rnc- strain is devoid of any RNase III activity in the cell . Comparing the decay of newly synthesized RNA and functional decay of beta-galactosidase mRNA in such strains revealed that in both strains these parameters proceed in similar rates, which suggests that RNase III is not involved in the metabolism of mRNA . During carbon starvation preexisting total RNA, as well as 23S and 16S rRNA, decay faster in the rnc- strain, thus eliminating the possibility that RNase III is the endoribonuclease which initiates the decay of rRNA during starvation (Kaplan and Apirion, 1975a).

Mol Gen Genet, 1976 Mar 22, 144(2), 177 - 83
Plasmids controlling synthesis of hemolysin in Escherichia coli . II . Polynucleotide sequence relationship among hemolytic plasmids; Royer-Pokora B et al.; Plasmids of three different sizes, designated as plasmid A (mw: 65 X 10(6), plasmid B (mw: 41 X 10(6) and plasmid C (mw: 32 X 10(6) respectively, have been isolated from various hemolytic wild-type strains of E . coli . DNA-DNA hybridization was performed to determine their relationship . The wild-type strain, PM167a, harbours plasmids of all three sizes . Hybridization studies indicate that all three plasmids share extented sequence homologies but that plasmid A is not composed of plasmids B and C . Hybridization between plasmids of the donor strain and those of appropriate transconjugants demonstrates that in some cases plasmids with identical size are not longer completely homologous in their nucleotide sequences . This indicates that despite their defined sizes these plasmids are not stable genetic entities, but rather they undergo frequently recombination and dissociation during conjugation . In one particular transconjugant strain, K12-PM152/1, a plasmid D was found which is a stable recombined molecule of plasmids B and C of the original strain . Plasmids of size B found as the only extrachromosomal elements in a hemolytic wild-type strain (P224) and two transconjugant strains (e.g . K12-CM20 and K12-PM167/1) share extended nucleotide sequence homologies but are not identical . Little sequence homology was observed between two different hemolytic plasmids and the F and the Col Ib plasmids suggesting that the former do not belong to either the F-like or the I-like group of plasmids . Another hemolytic plasmid is F-like based on its sequence homologies with the F factor.

Mol Gen Genet, 1976 Mar 22, 144(2), 159 - 70
Mutants of the Clo DF13 plasmid in Escherichia coli with a decreased bacteriocinogenic activity; Andreoli PM et al.; Three Clo DF13 mutant plasmids (designated as clp03, clp05 and clp21) that show a decreased cloacin activity were isolated . The decreased cloacin activity was not due to a reduced number of Clo DF13 copies per cell . The cloacins produced by the clp03 and the clp21 mutant plasmids have a strongly decreased killing activity in vivo in comparison with the wild type cloacin and the cloacin of the clp05 mutant plasmid . Furthermore no lacunae could be observed from clp03 or clp21 harbouring strains, while strains harbouring the clp05 plasmid showed a 50-100 times decreased frequency of lacunae . In addition the clp05 mutant showed a decreased rate of RNA synthesis in clp05 harbouring Escherichia coli minicells . No complementation between the three mutant plasmids was observed . We suggest that the clp03 and clp21 mutations are located in the gene coding for the cloacin . Since the cloacin produced by the clp05 mutant plasmid has retained all the known wild type cloacin activities, the reduced inhibition zone in the stab test is probably caused by a mutation affecting the expression of the cloacin gene . The nature of this mutation is discussed.

Lancet, 1976 Mar 20, 1(7960), 629 - 31
Enterotoxin testing of Escherichia coli causing epidemic infantile enteritis in the U.K; Gross RJ et al.; Three test systems were used to study enterotoxin production by epidemic strains of Escherichia coli from cases of infantile enteritis in well-documented outbreaks in the U.K . The tests used were the Y1-mouse-adrenal-cell test and the Chinese-hamster-ovary-cell (C.H.O.) test for the detection of heat-labile enterotoxin and the infant-mouse test for the detection of heat-stable enterotoxin . All 6 outbreaks had been studied using full serotyping techniques and the results had been published . In each outbreak the epidemiological studies clearly implicated a particular serotype of E . Coli as the epidemic strain and cultures of that serotype were tested for enterotoxin production . Although a control strain validated by other workers was positive in all three systems, the epidemic strains from infantile enteritis were negative . It seems that the three enterotoxin tests used in this study are of little value in recognising strains of E . coli causing epidemics of infantile enteritis in the U.K.

Biochim Biophys Acta, 1976 Mar 19, 426(3), 581 - 6
Freeze etch morphology of outer membrane mutants of Escherichia coli K12; Verkleij AJ et al.; Freeze etching showed that the loss of each of the major outer membrane proteins b, c or d in mutants of Escherichia coki K12 does not influence the morphology of fracture faces of the outer membrane . Mutants that possess a heptose-deficient lipopolysaccharide and which in addition are deficient in one or more major outer membrane proteins exhibit a reduction in the number of intramembranous particles of the outer membrane . Moreover it was shown that lipid phase transitions induce a lateral lipid protein separation in the outer membrane, similar to that found in the cytoplasmic membrane.

Arch Microbiol, 1976 Mar 19, 107(2), 199 - 203
Induction and stabilization of synchrony in the cell division of Escherichia coli; Anagnostopoulos GD; Escherichia coli strains B5 and B/r/l were grown under conditions of periodic glucose starvation in a minimal medium . Such conditions of growth give rise to two synchronous populations that are out of phase regarding their time of division, one dividing shortly after a new supply of fresh medium and the other at a later stage of the feeding cycle . Preferential selection of one of the two populations using heat treatment resulted in a homogeneous synchronized culture that exhibited in a non-limiting medium of a high degree of synchrony that was long lasting . Synchrony and its persistence could survive preservation of such a synchronized culture by freeze drying . An explanation of the synchrony persistence was put forward and the practical implications of these findings were discussed.

Science, 1976 Mar 19, 191(4232), 1183 - 5
Phylogeny of DNA polymerase-beta; Chang LM; Analyses of various organisms for DNA polymerase-beta activity show that the enzyme is widely distributed in cells from multicellular animals but absent in bacteria, plants, and protozoa . These results suggest that DNA polymerase-beta may have evolved with the development of metazoan forms . Further evolutionary changes of the enzyme protein may account for some of the minor differences in properties of the enzyme in various organisms.

Biochim Biophys Acta, 1976 Mar 19, 426(3), 451 - 63
Regulation of uracil uptake in Escherichia coli by adenosine 3',5'-monophosphate; De Robertis EM Jr et al.; Culture of a wild-type strain of Escherichia coli in the presence of cyclic AMP leads to an impairment of uracil uptake . Half maximum inhibition of uracil uptake was observed at 1.5 mM cyclic AMP . The effect seems to be specific since no inhibition was found in cultures supplemented with ATP, ADP or 5'-AMP . Similarly the inhibition was not observed in cultures of a mutant deficient in the cyclic AMP receptor protein . The inhibition in uracil uptake, found in bacteria cultured in the presence of cyclic AMP, is not a consequence of a reduction in the growth rate . On the other hand, this inhibition was observed only in cultures containing glucose or pyruvate as carbon source.

Biochim Biophys Acta, 1976 Mar 17, 425(3), 356 - 67
Characterization of a replication mutant of the bacteriocinogenic plasmid Clo DF13; Veltkamp E et al.; In a previous paper (Kool, A.J . and Nijkamp, H.J.J . (1974) J . Bacteriol . 120, 569--578) the isolation of a mutant of the bacteriocinogenic plasmid Clo DF13-Rep3, has been described . It was observed that cells harbouring the wild type plasmid synthesize more plasmid DNA cells harbouring the wild type plasmid . This paper deals with the characterization of the nature of this plasmid-specific mutation . The following properties of the Clo DF13-Rep3 mutant plasmid could be observed: 1 . The plasmid-specific mutation did not lead to a significant change in the sedimentation value of Clo DF13 DNA . 2 . The specific rate of Clo DF13-Rep3 DNA synthesis (expressed as the number of plasmid DNA molecules synthesized per min) is on average seven times the specific rate of wild type Clo DF13 DNA synthesis . 3 . Also chromosomeless minicells, harbouring the Clo DF 13-Rep 3 plasmid, contain about seven times more plasmid DNA as wild type Clo DF13 harbouring minicells . 4 . The replication time of the Clo DF13-Rep3 plasmid is approx . 90 s at 30 degrees C and does not differ significantly from the replication time of the wild type plasmid . 5 . The Rep3 mutation did not alter the dependence of Clo DF13 plasmid replication on the dnaA and dnaC gene products . 6 . The plasmid-specific mutation is cis-dominant over wild type . The data presented in this paper indicate that this mutant plasmid is not affected in the elongation but in the initiation of plasmid DNA replication.

Biochim Biophys Acta, 1976 Mar 17, 425(3), 334 - 41
Transfer RNA methyltransferase activity in paramecium aurelia; Maki RA et al.; The tRNA methyltransferases from Paramecium aurelia were investigated . The effects of varying the Mg2+ and NH4+ concentrations, pH, and temperature on the methylation of Escherichia coli B tRNA using extracts from P . aurelia were determined . Optimum tRNA methyltransferase activity was observed at pH 7.8 and 37 degrees C . The Mg2+ optimum occurred at 0.66 mM in the absence of NH4+ while the NH4+ optimum occurred at 100 mM in the absence of Mg2+ . Analysis of the bases methylated in (E . coli B) tRNA by extracts of P . aurelia showed the presence of 1-methyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine and methylated pyrimidine nucleotides . In comparison, an analysis of the in vivo methylation of tRNA from P . aurelia showed the presence of 1-methyladenine, 6-methyladenine, 6,6-dimethyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine, 7-methylguanine, and methylated pyrimidine nucleotides . The pattern of methylation of tRNA in P . aurelia is similar to that observed in other eukaryotes.

Biochim Biophys Acta, 1976 Mar 17, 425(3), 296 - 304
Enzymatic properties of cloacin DF13 and kinetics of ribosome inactivation; Oudega B et al.; 1 . The cloacin DF13-induced inactivation of ribosomes in vitro can be described as an enzyme-catalyzed reaction according to the Michaelis-Menten equation . Most probably the cloacin acts as a unique endoribonuclease . 2 . At pH 7.8 and 37 degrees C the Km value for the reaction of cloacin DF13 with ribosomes is 13.2 - 10(-6) M . If under these conditions the reaction mixture is supplemented with all components necessary for protein synthesis, the Km changes to 17.7 - 10(-6) M . 3 . The in vitro activity of cloacin DF13 has a temperature optimum of 43 degrees C at pH 7.8 and a pH optimum of 8.4 at 37 degtees C . 4 . Experiments with cloacin DF13-immunity protein as an inhibitor of the cloacin activity in vitro have indicated that the immunity protein might be considered as a non-competitive and virtually "irreversible" inhibitor.

Eur J Biochem, 1976 Mar 16, 63(1), 83 - 92
The use of a cleavable crosslinking reagent to identify neighboring proteins in the 30-S ribosomal subunit of Escherichia coli; Peretz H et al.; A cleavable bifunctional reagent, dimethyl 3,3'-dithiobispropionimidate, has been used to crosslink proteins that occupy neighboring positions in the 30-S ribosomal subunit of Escherichia coli . The crosslinked proteins were identified, fully or partly, by their positions in two two-dimensional gel electrophoretic systems, one diagonal and the other quasi-diagonal, in which the complexes were cleaved after the first-dimensional run . It was found to be necessary to block the protein sulfhydryl groups in order to prevent artifactual disulfide crosslinking after extraction of the protein from ribosome . Eleven crosslinked complexes were detected . Four were fully identified: the triplet S4-S5-S8, and the pairs S2-S3, S4-S5, and S5-S8 . In five others one component was identified unambiguously . No additional complexes were seen when the longer homologous butyro and capro reagents were used.

Eur J Biochem, 1976 Mar 16, 63(1), 47 - 52
The major proteins of the Escherichia coli outer cell-envelope membrane . Heterogeneity of protein I; Schmitges CJ et al.; One of the major proteins of the Escherichia coli outer cell envelope membrane, protein I, can be separated electrophoretically into protein components Ia and Ib . Strain differences exist regarding presence or absence of component Ib and this component can selectively be lost by mutation to resistance against a phage . Both components Ia and Ib are further heterogeneous isoelectrically, and both together may contain at lease six separable isoelectric species . As judged by analysis of their cyanogen bromide fragments, Ia and Ib are almost identical concerning their primary structure; the difference (charge only or size and charge) was located in a part of the protein that does not correspond to the C-terminal or N-terminal regions . Components Ia and Ib thus represent essentially the same polypeptide and they may arise by a modification process in vitro or the existence of two almost identical genes.

Eur J Biochem, 1976 Mar 16, 63(1), 313 - 20
{Study of the lipopolysaccharide from Escherichia coli K12 CR34}; Benedetto JP et al.; The structure of the lipopolysaccharide from Escherichia coli K12, strain CR34 has been investigated . The lipopolysaccharide contains D-galactose, D-glucose, D-glucosamine, L-glycero D-mannoheptose, 2-keto-3-deoxyoctonate and lipid A . The core region does not contain D-glucosamine but contains galactose, glucose, and heptose in the molar ratios 1:3:6 . Methylations were performed on the lipopolysaccharide and on the degraded polysaccharide obtained after acetic acid hydrolysis of the lipopolysaccharide . It was found that galactose is in terminal position partly in the form of galactopyranose, partly in the form of galactofuranose . A part of the heptose was found as unsubstituted heptofuranose . A mole of glucose was 1 leads to 2 linked and another mole of glucose was 1 leads to 6 linked . Periodate oxidation of the lipopolysaccharide followed by borohydride reduction eliminates galactose and only a part of glucose and of heptose . A mole of heptose gave mannose and thus it is unsubstituted in C6 and C7 . One mole of glucose and one mole of heptose were not degraded by periodate oxidation.

Eur J Biochem, 1976 Mar 16, 63(1), 289 - 301
Ornithine carbamoyltransferase from Escherichia coli W . Purification, structure and steady-state kinetic analysis; Legrain C et al.; Ornithine carbamoyltransferase from Escherichia coli W was purified to homogeneity . The enzyme has a molecular weight of 105000 . It is composed of three apparently identical subunits with molecular weights of 35000 . The mechanism of the ornithine carbamoyltransferase enzyme system from E . coli W was investigated kinetically by using the approach of product inhibition and dead-end inhibition of both forward and reverse reactions . On the basis of the kinetic data and binding studies it appears that the mechanism of the reaction involves a compulsory sequence of substrate binding to the enzyme, in which carbamoylphosphate is the first substrate to bind to the enzyme and phosphate the last product to be released . The same studies also indicate that the mechanism involves dead-end complexes . The reaction mechanism appears consistent with that proposed by Theorell and Chance . Values have been determined for the Michaelis and dissociation constants involved in the combination of each reactant with the enzyme . Comparison of the values for the kinetic constants which are common to both forward and reverse reaction have shown that they are always of a comparable magnitude.

Experientia, 1976 Mar 15, 32(3), 301 - 2
A possibility to achieve genetic transformation in the platyfish-swordtail system (Platypoecilus maculatus - Xiphophorus helleri); Schwab M et al.; In order to determine how informative homologous donor DNA might be made available to propigment cells of the recipient Xiphophorus helleri for transformation, labelled heterologous DNA from E . coli was injected into the neural crest region or the yolk sac of embryos of the recipient . On the basis of the degradation rate of the donor DNA and the incorporation rate of radioactivity into the recipient DNA, it is concluded that injection into the neural crest region may be a suitable method to make available informative homologous donor DNA for transformation.

Biochem J, 1976 Mar 15, 154(3), 731 - 4
Energy coupling to active transport in anaerobically grown mutants of Escherichia Coli K12; Gutowski SJ et al.; 1 . Anaerobic uptake of proline requires either the presence of a coupled Mg2+-stimulated adenosine triphosphatase or anaerobic electron transport . 2 . Anaerobic uptake of glutamine does not require anaerobic electron transport even in the absence of a coupled Mg+2-stimulated adenosine triphosphatase . 3 . These results support previous suggestions {Berger (1973) Proc . Natl . Acad . Sci . U.S.A . 70, 1514--1518; Berger & Heppel (1974) J . Biol . Chem . 249, 7747-7755; Kobayashi, Kin & Anraku (1974) J . Biochem . (Tokyo) 76, 251-261} that two distinct mechanisms of energy coupling to active transport exist in Escherichia coli in that energization of anaerobic proline uptake requires the 'high-energy membrane state', whereas the energization of anaerobic glutamine uptake does not.

Biochim Biophys Acta, 1976 Mar 12, 423(3), 450 - 61
Anaerobic transport of amino acids coupled to the glycerol-3-phosphate-fumarate oxidoreductase system in a cytochrome-deficient mutant of Escherichia coli; Singh AP et al.; The uptake of proline and glutamine by cytochrome-deficient cells of Escherichia coli SASX76 grown aerobically on glucose or anaerobically on pyruvate was stimulated by these two substrates . Pyruvate could not stimulate transport in the glucose-grown cells . Uptake of these amino acids energized by glucose was inhibited by inhibitors of the Ca2+, Mg2+-stimulated ATPase such as DCCD, pyrophosphate, and azide, and by the uncouplers CCCP and 2,4-dinitrophenol . Glycerol (or glycerol 3-phosphate) in the presence of fumarate stimulated the transport of proline and glutamine under anaerobic conditions in cytochrome-deficient cells but not in membrane vesicles prepared from these cells although glycerol 3-phosphate-fumarate oxidoreductase activity could be demonstrated in the vesicle preparation . In contrast, in vesicles prepared from cytochrome-containing cells of E . coli SASX76 amino acid transport was energized under anaerobic conditions by this system . Inhibitors of the Ca2+, Mg2+-activated ATPase and uncoupling agents inhibited the uptake of proline and glutamine in cytochrome-deficient cells dependent on the glycerol-fumarate oxidoreductase system . Ferricyanide could replace fumarate as an electron acceptor to permit transport of phenylalanine in cytochrome-deficient or cytochrome-containing cells under anaerobic conditions . It is concluded that in cytochrome-deficient cells using glucose, pyruvate, or glycerol in the presence of fumarate, transport of both proline and glutamine under under anaerobic conditions is energized by ATP through the Ca2+, Mg2+-activated ATPase . In cytochrome-containing cells under anaerobic conditions electron transfer between glycerol and fumarate can also drive transport of these amino acids.

Chromosoma, 1976 Mar 10, 54(4), 349 - 62
Comparison of in vivo and in vitro RNA synthesis on polytene chromosomes of Drosophila; Leibovitch BA et al.; A comparative radioautographic study of the RNA precursors incorporation on polytene chromosomes of Drosophila in vivo in the cells of salivary glands, and in vitro during incubation of E.coli RNA polymerase on slides with fixed chromosomes was performed.--The pattern of in vivo 3H-uridine incorporation on different sections of the chromosomes drastically differed from the in vitro 3H-UTP incorporation which seems to be much more related to DNA content of the individual small sections . In both cases puffing of the loci resulted in the increase of RNA synthesis but in vitro only 2-3 fold and in vivo much more . Hence, RNA synthesis in vitro was unspecific and did not reflect the in vivo RNA synthesis.--On the other hand, E.coli RNA polymerase completely mimics in vitro the dosage compensation phenomenon making twice as much RNA on one X-chromosome of males (1X2A) as on each of X-chromosomes of diploid (2X2A) and triploid (3X3A) females and super-females (3X2A), and the intermediate amount of RNA on each of X-chromosomes of intersexes (2X3A) . It is suggested that the differences in the in vitro template activity of X-chromosomes of cells with different X:A ratio are due to different extent of condensation of their deoxyribonucleoprotein (DNP) . Yet, both male and each of female X-chromosomes bind the same amount of thymus histone FI labelled with fluorochrome which indicates that they contain the same amount of "open" regions with exposed chromosomal DNA accessible to external proteins.--On the basis of these observations a hypothesis is put forward which suggests that RNA transcription in animal chromosomes is regulated at two levels by different mechanisms; the first one controls the extent of condensation of DNP of genetic loci and determines their competence to the second mechanism which involves the action of gene-specific activator proteins . According to this hypothesis the phenomenon of dosage compensation of sex-linked genes is due to decondensation of DNP of male X-chromosome which renders its loci twice as responsive to activators as compared to the same loci in females.

J Biol Chem, 1976 Mar 10, 251(5), 1529 - 30
Evidence for a conformational change in tRNAPhe upon aminoacylation; Caron M et al.; A conformational difference between the structure of tRNAPhe and Cbz-Phe-tRNAPhe from Escherichia coli was detected using the spin label method . A comparison of the respective rotational correlation time (tau c) values of three differently located spin labels, indicates that upon aminoacylation of tRNAPhe, the 4-thiouridine residue region and the miniloop region become more flexible, while the environment of the anticodon loop is not affected . These observations suggest that the main difference in structure between charged and uncharged tRNA resides in the release and exposure of the TpsiC loop for eventual binding to the ribosomes.

J Biol Chem, 1976 Mar 10, 251(5), 1438 - 45
An endonuclease from Escherichia coli that introduces single polynucleotide chain scissions in ultraviolet-irradiated DNA; Radman M; An endonuclease that makes single polynucleotide chain scissions in ultraviolet-irradiated DNA has been purified from Escherichia coli . The activity has the following properties: (a) unirradiated DNA is attacked very little if at all; (b) single strand DNA is not attacked, whether irradiated or not; (c) there is no requirement for divalent cations and the activity is not affected by the addition of EDTA; (d) the pH optimum is approximately 7; (e) the activity is inhibited by 1 M NaCl, single strand DNA, transfer RNA and double strand DNA; (f) the sedimentation coefficient, S20,w, is approximately 2.6; (g) it is a basic protein . The enzyme is tentatively named E . coli endonuclease III . The physiological function of the endonuclease has not yet been established.

Virchows Arch B Cell Pathol, 1976 Mar 9, 20(2), 155 - 67
Ultrastructure of experimental xanthogranulomatous pyelonephritis; Povysil C; The ultrastructure of xanthogranulomas developing during obstructive suppurative pyelonephritis was studied in rats with a permanent ligature of one ureter and an intravenous injection of E . coli suspension . The xanthogranulomas consisted of macrophages with numberous phagolysosomes containing rests of phagocytosed polymorphonuclear leucocytes and bacteria . Lipids identifiable as triglycerides were found in such cells particularly as isolated intracytoplasmic droplets and, less frequently, within the phagolysosomes . The source of isolated intracytoplasmic lipid droplets within the macrophages has remained obscure . Their origin in the macrophages by synthesis could not be excluded, similarly as in the polymorphonuclear leucocytes . PAS positive cells occurring as late as 2 months after the beginning of the experiment at the periphery of xanthogranulomas contained multiple intracytoplasmic vacuoles, most probably phagolysosomes . These cells, with high probability the oldes ones of the macrophagic population in the xanthogranulomas were virtually devoid of lipid droplets.

Biochim Biophys Acta, 1976 Mar 5, 426(2), 317 - 24
Action of phospholipases on the cytoplasmic membrane of Escherichia coli . Stimulation by melittin; Mollay C et al.; The emission maximum of the single tryptophan residue of melittin was measured in the presence of phosphatidylethanolamine liposomes and Escherichia coli cytoplasmic membranes . In both cases, the fluorescence maximum was shifted to shorter wavelengths indicating a transfer of the indole ring to an apolar environment . E . coli membranes were labelled in position 2 of their phospholids with {14C}oleic acid . These membranes were used for measuring the activity of an endogenous phospholipase A2 . A slow hydrolysis is observed, which can be accelerated by adding melittin . The extent of the stimulation depends on the molar ratio of melittin to membrane phospholipid . Under suitable conditions, the initial rate of hydrolysis is six to seven times higher in the presence than in the absence of melittin . The action of the phospholipase A2 from bee venom is also stimulated by melittin . An identical stimulation was observed with either E . coli membranes or pure phosphatidylethanolamine liposomes as substrate.

Biochim Biophys Acta, 1976 Mar 4, 425(2), 175 - 84
Intraction of aminoacyl-tRNA synthetases with ribosomes and ribosomal subunits; Graf H; Salt-washed ribosomes from rabbit reticulocytes stimulate seven partially purified aminoacyl-tRNA synthetases up to threefold: arginyl-, alanyl-, isoleucyl-, lysyl-, methionyl-, phenylalanyl- and valyl-tRNA synthetase . Both isolated subunits but not total ribosomal RNA show the stimulation . The increase in synthetase activity is also found in a highly purified Escherichia coli system employing homogenous (E . coli)phenyl-alanyl-tRNA synthetase and (E . coli)- tRNAPhe . The biological significance of the stimulation is discussed.

Biochim Biophys Acta, 1976 Mar 4, 425(2), 157 - 67
Random cleavage of superhelical SV 40 DNA S1 nuclease; Waldeck W et al.; SV40 DNA FO I is randomly cleaved by S1 nuclease both at moderate (50 mM) and higher salt concentrations (250 mM NaC1) . Full length linear S1 cleavage products of SV40 DNA when digested with various restriction endonucleases revealed fragments that were electrophoretically indistinguishable from the products found after digestion of superhelical SV40 DNA FO I with the corresponding enzyme . Concordingly, when the linear S1 generated duplexes were melted and renatured, circular duplexes were formed in addition to complex larger structures . This indicated that cleavage must have occurred at different sites . The double-strand-cleaving activity present in S1 nuclease preparations requires circular DNA as a substrate, as linear SV40 DNA is not cleaved . With regard to these properties S1 nuclease resembles some of the complex type I restriction nucleases from Escherichia coli which also cleave SV40 DNA only once, and, completely at random.

Biokhimiia, 1976 Mar, 41(3), 469 - 75
{A proteolytic enzyme bound to the aspartate transaminase of swine heart cytosol}; Denisova GF et al.; Purified preparations of aspartate transaminase from pig heart cytosol contain a tightly bound proteolytic enzyme (approximately 2, 5%) . The enzyme was separated from aspartate transaminase by gel-filtration on Sephadex G-100 in the presence of sodium dodecyl sulfate and by affinity chromatography on the column with Sepharose, containing covalently bound denaturated aspartate transaminase . Protease has a pH optimum of 9.0 and molecular weight of about 23.000-25.000 . The proteolysis rates of different subforms of aspartate transaminase depend on their denaturation lability . A more stable choloenzyme is split at a slower rate than the apoenzyme . An enriched preparation of protease was also shown to split glutamate decarboxylase from E . coli and had no effect on cysteinlyase from hen egg, as well as on lactate dehydrogenase and albumin.

J Bacteriol, 1976 Mar, 125(3), 855 - 63
Partial purification and characterization of cytidine 5'-diphosphate-diglyceride hydrolase from membranes of Escherichia coli; Raetz CR et al.; Cytidine 5'-diphosphate (CDP)-diglyceride is hydrolyzed to phosphatidic acid and cytidine 5'-monophosphate by a specific membrane-bound enzyme in cell-free extracts of Escherichia coli . The hydrolase can be extracted from the particulate fraction with Triton X-100 and purified 1,000-fold in the presence of this detergent . Several nucleoside disphosphate diglycerides were synthesized to determine the substrate specificity of the hydrolase . CDP-diglyceride was hydrolyzed preferentially, although uridine 5'-diphosphate-diglyceride, guanosine 5'-diphosphate-diglyceride, and adenosine 5'-diphosphate (ADP)-diglyceride were also slowly hydrolyzed . Surprisingly, the purified enzyme did not catalyze detectable cleavage of deoxy-CDP (dCDP)-diglyceride . The liponucleotide pool of E . coli contains dCDP-diglyceride and CDP-diglyceride in approximately equal amounts (Raetz and Kennedy, 1973) . Water-soluble nucleoside pyrophosphates, such as CDP-choline, nicotinamide adenine dinucleotide, or adenosine 5'-triphosphate are not attacked by this specific hydrolase . Hydrolysis of CDP-diglyceride is strongly inhibited by adenosine 5'-monophosphate and by ADP-diglyceride.

J Bacteriol, 1976 Mar, 125(3), 1223 - 5
Lowered levels of colicin Ia membrane receptors in an Escherichia coli mutant defective in heme biosynthesis; Gilchrist MJ et al.; Cells of an Escherichia coli mutant defective in heme biosynthesis (hemA) grown under conditions for respiration deficiency (grown in the absence of aminolevulinic acid) have 20% of the number of specific colicin Ia receptors found in cells of the same strain grown under conditions for respiration competency (grown in the presence of aminolevulinic acid).

J Gen Microbiol, 1976 Mar, 93(1), 82 - 8
Lability of RNA from the large cytoplasmic ribosomal subunit of the protozoon Crithidia oncopelti; Spencer R et al.; Cytoplasmic ribosomalRNA extracted from Crithidia oncopelti and analysed by gel electrophoresis at 4 degrees C consisted of two components, with molecular weights (relative to E . coli rRNA) of 1-30 X 10(6) and 0-83 X 10(6) daltons, present in equimolar amounts . On heating briefly at 51 degrees C followed by rapid cooling, the 1-30 X 10(6) RNA completely dissociated into two components of molecular weights 0-70 X 10(6) and 0-56 X 10(6) (present in equimolar amounts) . Fifty per cent dissociation of the molecule occurred at 28 degrees C . That the integrity of the RNA molecule at low temperatures is maintained by its secondary structure was confirmed by electrophoresis under denaturing conditions (98%, v/v, formamide) . To account for these phenomena, latent cleavage of the molecule in vivo is proposed.

Nucleic Acids Res, 1976 Mar, 3(3), 789 - 808
The conformation of 16S RNA in the 30S ribosomal subunit from Escherichia coli; Milner JJ et al.; The digestion of E . coli 16S RNA with a single-strand-specific nuclease produced two fractions separable by gel filtration . One fraction was small oligonucleotides, the other, comprising 67.5% of the total RNA, was highly structured double helical fragments of mol . wt . 7,600 . There are thus about 44 helical loops of average size corresponding to 12 base pairs in each 16S RNA . 10% of the RNA could be digested from native 30S subunits . Nuclease attack was primarily in the intraloop single-stranded region but two major sites of attack were located in the interloop single-stranded regions . Nuclease digestion of unfolded subunits produced three classes of fragments, two of which, comprising 80% of the total RNA, were identical to fragments from 16S RNA . The third, consisting of 20% RNA, together with an equal weight of peotein, was a resistant core (sedimentation coefficient 7S).

Infect Immun, 1976 Mar, 13(3), 763 - 7
Antiviral activity of Brucella abortus preparations; separation of active components; Feingold DS et al.; Injection into mice of heat-killed Brucella abortus or aqueous ether-extracted B . abortus (Bru-pel) induced a "virus-type" interferon response, with peak titers at 6.5 h . The animals also were protected against challenge with otherwise lethal doses of Semliki forest virus . Extraction of either heated B . abortus or BRU-PEL with a mixture of chloroform-methanol (2:1, vol/vol) (C-M( yielded an insoluble residue (extracted cells) and a C-M extract . Neither extracted cells nor C-M extract alone induced interferon or afforded protection against Semliki forest virus infection in mice . Full interferon-inducing and protective activity was restored when extracted cells were recombined with C-M extract . C-M extract from heat-killed Escherichia coli also was effective in restoring activity to extracted Brucella cells . Neither heat-killed E . coli nor its C-M extract was active, nor was C-M estracted E . coli recombined with the C-M extract from B . abortus . These results suggest that the interferon-inducing and antiviral protective properties of B . abortus are constituted of a C-M-extractable component that is common to B . abortus and E . coli and an unextractable component that is unique to B . abortus.

J Gen Microbiol, 1976 Mar, 93(1), 63 - 8
Extrachromosomal DNA in chloramphenicol resistant myxococcus strains; Brown NL et al.; The presence of extrachromosomal DNA in strains of Myxococcus xanthus and M fulvus was examined by rate-zonal centrifugation of radioactively-labelled DNA in 'cleared lysates' . All the strains examined contained extrachromosomal DNA, with the exception of M . xanthus FBt . Chloramphenicol resistance is inducible in M . xanthus FBt . A peak of extrachromosomal DNA, containing covalently closed molecules, was found in one of the induced strains, implying that induction of chloramphenicol resistance is associated with the production of a plasmid . By incubating R+ strains of Escherichia coli with myxococci, R factor-mediated chloramphenicol resistance can be introduced into the latter . Evidence of extra chromosomal DNA in a derivative of M . xanthus with chloramphenicol resistance from R factor RI . 19 unique to the chloramphenicol strain, was obtained . By using a double-labelling technique, several chloramphenicol-resistant strains of M . fulvus M were examined . Evidence for a peak, unique for the chloramphenicol-resistant strain, was found in a strain with resistance derived from the R factor, S-a, but not from comparable strains with resistance derived from R factors R57b, R1 . 19 and R478.

Proc Natl Acad Sci U S A, 1976 Mar, 73(3), 775 - 9
Transcription in vitro of immunoglobulin kappa light chain genes in isolated mouse myeloma nuclei and chromatin; Smith MM et al.; Messenger RNA sequences for immunoglobulin kappa light chain were synthesized in vitro in isolated mouse myeloma nuclei using bound endogenous RNA polymerase (RNA nucleotidyltransferase; nucleoside triphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) and from isolated myeloma chromatin using exogenous Escherichia coli RNA polymerase . The in vitro RNA was transcribed using 5-mercuriuridine triphosphate and separated from in vivo RNA by chromatography on an agarose sulfhydryl affinity column . Template restriction is retained in vitro since synthesis of kappa chain messenger RNA, As determined by hybridization with complementary DNA, was much more efficient in nuclei and chromatin isolated from myeloma 66.2 tissue culture cells, a kappa-chain-producing cell line, than from MOPC 315 tissue culture cells, a lambda-chain-producing cell line . Transcription of kappa chain messenger RNA was 25 times more efficient in myeloma 66.2 nuclei than in myeloma 66.2 chromatin.

Basic Life Sci, 1976 Mar 1-7, 8, 431 - 8
Biophysical and genetic evidence for transformation in plants; Ledoux L; In thiamine mutants of Arabiodopsis, genetic corrections have been obtained by treatment with DNA bearing a thiamine information . When correction is attempted under selective conditions, about 0.7% of the treated plants grow and set fruit . Their progeny and the following ones, obtained by selfing, behave as homozygotes . Segregation of characters is found only when correction is attempted under nonselective conditions or when the correcting genes were of plasmidian origin . The correction is hereditary; results of backcrosses and test crosses indicate that it is dominant, nuclear, and strongly bound to the genome . The corrective factor appears to be added to the mutated genome and not substituted for the mutation, as it can be suppressed by outcrossing with the wild type or with a plant corrected by another DNA.

Biochem J, 1976 Mar 1, 153(3), 533 - 41
Purification and some properties of nitrate reductase (EC 1.7.99.4) from Escherichia coli K12; Clegg RA; 1 . Nitrate reductase was purified 134-fold from Escherichia coli K12 . The purification procedure involves the release by Triton X-100 of the enzyme from the cell envelope . i . The purified enzyme exists in aqueous solution either as a monomer (mol . wt . about 220 000) or as an associated form (probably a tetramer; mol.wt . about 880 000) . 3 . The purified enzyme has three subunits with apparent mol.wts . of 150 000, 67000 and 65000 . An additional subunit of apparent mol.wt . 20000 is present in a haem-containing fraction that is also produced by the preparative procedure described . 4 . None of the enzyme subunits is present in the cell envelope of cells grown in the absence of nitrate . 5 . Reversible changes in the activity of nitrate reductase in vitro with FMNH2 as reductant can be induced under circumstances which are without effect on the reduced Benzyl Viologen-NO3-activity.

Prim Care, 1976 Mar, 3(1), 83 - 90
The infectious diarrheas; Franklin JE Jr; In most cases, the physical examination will not adequately differentiate between the various infectious entities, though a careful history may be helpful . A stool guaiac test can help differentiate between invasive organisms and toxigenic organisms . All the organisms described usually cause a self-limited illness, and therapy is directed toward the control of symptoms.

J Biochem (Tokyo), 1976 Mar, 79(3), 673 - 8
Pantothenate synthetase of Escherichia coli B . I . Physicochemical properties; Miyatake K et al.; Using a new apparatus for preparative polyacrylamide gel electrophoresis, pantothenate synthetase (D-pantoate: beta-alanine ligase (AMP-forming), {EC 6.3.2.1} was purified about 500-fold from Escherichia coli B . It was found to be homogeneous in analytical disc gel electrophoresis and sedimentation ultracentrifugation (so20, w=4.9) . From sedimentation equilibrium ultracentrifugation, a molecular weight of 70,100 was obtained, which is in good agreement with the value obtained by the Sephadex G-150 gel filtration method (69,000); the diffusion constant was calculated to be 5.88X10(-7) cm2/sec . The minimum molecular weight calculated from the amino acid composition of this enzyme protein was 19,700, a value in reasonable accord with molecular weight of the enzyme subunit, 18,000, obtained by gel electrophoresis in the presence of sodium dodecylsulfate . The partial specific volume, v, was calculated to be 0.71 cm3/g . The enzyme had an amino-terminal glycyl residue and a Leu-Ala-Ser-OH sequence at the carboxyl end . Electrophoresis of the enzyme with carrier ampholine gave an isoelectric point of pH 4.6.

J Biochem (Tokyo), 1976 Mar, 79(3), 505 - 11
Fluorometric investigation of the local conformation of 16S rRNA in the regions of the 3'- and 5'-ends; Kumagai JI et al.; The conformational changes of ribonucleic acid induced by heat treatment were studied by measurements of fluorescence polarization, circular dichroism, and ultraviolet absorption . The fluorescence polarization of proflavine covalently bound to the 3'-end of 16S ribosomal RNA of Escherichia coli and that of ethenoadenosine conjugated to the 5'-end of the RNA indicated that a conformational difference existed between heat-treated and untreated samples of the RNA in the regions near the 3'- and 5'-ends . Comparison of the fluorescence polarization data with other optical measurements indicated that the local conformation around the 3'- and 5'-ends of the RNA was converted to a more rigid and stable form by the heat treatment, while the gross conformation did not appear to change.

Nucleic Acids Res, 1976 Mar, 3(3), 767 - 88
Studies of DNA bound RNA molecules isolated from nucleoids of Escherichia coli; Hecht RM et al.; Methods are developed for studying RNA molecules bound directly to DNA in bacterial nucleoids . It is found that among the 1000-3000 nascent RNA chains that normally are attached to the DNA via their associated RNA polymerase molecules, 74 +/- 14 chains per nucleoid can be bound differently . These chains unlike the other nascent RNAs remained bound to the DNA after the chromosome was deproteinized and sheared . Sensitive assays using radioactive labels detected no RNA polymerase involved in the RNA-DNA linkage . The linkage was stable at low temperatures, but the RNA separated from the DNA at high temperature . The bound RNA molecules were heterodisperse (weight average length 1200 bases) . Pulse-chase experiments and studies of the fate of these RNA molecules in rifampicin treated cells demonstrated that they are nascent RNAs, degraded or released from the DNA in vivo with kinetics similar to that of the total nascent RNA . Hybridization analyses showed that the chains are composed at least in part of nascent rRNA and known mRNA molecules . Some, but not more than 5% of the bound chains, contained sequences of about 300 nucleotides in length, bound to the DNA in an RNase resistant form.

Int J Radiat Biol Relat Stud Phys Chem Med, 1976 Mar, 29(3), 249 - 54
Lethality and double-strand scissions from 14C decay in the DNA of micro-organisms; Krisch RE; 14C-2-thymidine was incorporated into the DNA of E . coli B/r and of coliphage T4 . The labelled organisms were stored for several years at -196 degrees C . Both were periodically assayed for loss of viability, and the coliphage also for the appearance of double-strand breaks (DSBs) in DNA . E . coli B/r exhibited a survival curve with a substantial initial shoulder, extrapolation number 5-2 +/- 2-3, and a final exponential portion corresponding to a lethal efficiency per 14C decay per 2-5 X 10(9) daltons of DNA, of 0-009 +/- 0-002 . For coliphage T4, our best estimate for the lethal efficiency per 14C decay is 0-03 +/- 0-04, and that for the DNA breakage efficiency is -0-002 +/- 0-004 . The large standard errors result from the very small number of 14C decays occurring in each phage . These results suggest that 14C decay in the DNA of micro-organisms does not cause DSBs but does cause potentially lethal damage to the thymine bases in which decay occurs, and that wild-type E . coli can repair a large number of such DNA lesions.

Int J Radiat Biol Relat Stud Phys Chem Med, 1976 Mar, 29(3), 235 - 40
Production and rejoining of single-strand breaks in DNA of Escherichia coli cells after exposure to gamma-rays in the presence of iodoacetic acid under oxic and anoxic conditions; Nair CK et al.; The presence of iodoacetic acid (IAA, 10(4) M) during exposure of Escherichia coli cells to 25 krad gamma-radiation under anoxia resulted in a greater number of radiation-induced single-strand scissions in DNA . The single-strand breaks produced under oxic conditions of irradiation, however, were only marginally increased when IAA was present during irradiation . Although cells irradiated under anoxic or oxic condition and those irradiated under anoxic condition in the presence of IAA were capable of rejoining single-strand breaks produced in DNA, those irradiated under oxic condition in the presence of IAA had lost this capacity.

Infect Immun, 1976 Mar, 13(3), 741 - 9
Serum chemotactic inhibitory activity: heat activation of chemotactic inhibition; Epps DE et al.; Serum chemotactic inhibitory activity (CIA) was studied in 46 patients with various systemic diseases, using a system consisting of normal human leukocytes as indicator cells and 10% fresh normal serum as a control chemotactic attractant . It was shown, as previously reported, that an association exists between CIA and skin test anergy . Heat treatment of sera at 56 C for 30 min increased both the incidence and the degree of chemotactic inhibition observed in these patients . The effects of heat treatment of sera containing CIA on other chemotactic attractants (C3a, bacteria-derived chemotactic factor (BF), and casein) are shown . Before heat treatment, some sera suppressed chemotaxis mediated by BF in the absence of suppression of normal serum-mediated chemotaxis, indicating the possible involvement of more than one system of inhibition . Multiple systems were further supported by data indicating that room temperature incubation resulted in a loss of CIA as measured by normal serum-mediated chemotoxis with no apparent decrease in the inhibition of BF -mediated chemotaxis . Separation of sera containing CIA by Sephadex G-200 showed chemotactic inhibitory activity to be increased in both the void volume region . Experiments showed that heat treating before separation resulted in similar increases in both peaks, implying the presence of an antagonist to CIA . Experiments demonstrating that sera containing CIA do not suppress casein-mediated chemotaxis by means of an irreversible inactivation of chemotactic factor are included along with experiments demonstrating a cellular mode of action . The possible presence of two systems of chemotactic inhibition, one acting directly upon chemotactic factors and one interacting with the responding cell, are discussed.

Infect Immun, 1976 Mar, 13(3), 1005 - 7
Increased survival in calves of Escherichia coli K-12 carrying an Ent plasmid; Falkow S et al.; Escherichia coli K-12 strains with and without an Ent plasmid were fed to calves, and the survival of each was monitored by viable bacterial counts of the feces . The E . coli K-12 strain carrying the Ent plasmid survived in the calves at significantly higher levels and for a longer period of time than the E . coli F(-) stain.

Br J Cancer, 1976 Mar, 33(3), 241 - 59
A critique of the evidence for active host defence against cancer, based on personal studies of 27 murine tumours of spontaneous origin; Hewitt HB et al.; Extensive experience with isotransplants of 27 different tumours (leukaemias, sarcomata, carcinomata), all of strictly spontaneous origin in laboratory bred mice of low cancer strains CBA/Ht and WHT/Ht, has revealed no evidence of tumour immunogenicity . Of approximately 20,000 maintenance transplants, none failed and none regressed; of almost 10,000 carefully observed tumours arising from small or minimal inocula of tumour cells, none spontaneously regressed . The number of injected viable tumour cells required to give a 50% probability of successful transplantation (the TD50) ranged from approximately 1 cell to greater than 10,000 cells among the 27 tumours; high TD50 values, which were dramatically reduced by various procedures having no immunological significance, did not signify active "resistance" of the hosts . In the case of all of 7 randomly selected tumours, prior "immunization" of recipients with homologous lethally irradiated cells increased their tumour receptivity . Several experiments using various tumours failed to give evidence that immunity could be non-specifically induced or that a massive preponderance of lymphocytes from specifically sensitized mice could inhibit tumour transplantation or growth in vivo; no trace of "resistance" to tumour was adopted by isogeneic recipients of lymphocytes from regional nodes of tumour bearers . A limited review of the recent literature on tumour immunity shows that practically all the animal data presented in support of a general theory of tumour immunogenicity or to provide a basis for active clinical immunotherapy have been obtained from transplanted tumour systems which entail artefactual immunity associated with viral or chemical induction of the tumours or their allogeneic transplantation . It is suggested that isotransplants of spontaneously arising tumours are the only appropriate models of human cancer and that any genuine rapport between the animal laboratory and the clinic requires their exclusive use.

Res Vet Sci, 1976 Mar, 20(2), 131 - 8
Laboratory trials with inactivated vaccines against Escherichia coli (O78 K80) infection in fowls; Deb JR et al.; Laboratory trials made with five inactivated (O78 K80) Escherichia coli vaccines showed that the greatest degree of protection against an intramuscular challenge with the homologous strain was obtained with an alum precipitated vaccine . This vaccine was equally effective when administered by the subcutaneous, intramuscular or intraperitoneal route . Maximal protection, persisting for at least six weeks, was obtained by inoculating chicks at three weeks of age with a dose of approximately 1-5 X 10(9) cells in 0.5 ml . Some degree of protection was obtained, however, in chicks vaccinated earlier . There was no evidence that this vaccine provided any cross protection against other pathogenic serotypes of E coli . No adverse effect was noted on growth rate or carcase quality in the chicks vaccinated . Immunological studies showed that protection was not related to the production of O and K serum agglutinins.

J Gen Microbiol, 1976 Mar, 93(1), 133 - 40
Genetic analysis of deletions of R100-1 that are both transfer-deficient and tetracycline-sensitive; Foster TJ et al.; The extent of the deletions of five Tets Tra- mutants of R100-1 was determined by complementation experiments with wild-type and tra mutants of Flac . The presence or absence of the origin of transfer on the mutants was also investigated . Using the results, a tentative map of this region of the R factor was drawn: it was essentially similar to the analogous region of the E . coli K12 F factor, except that tet was located between traJ and traA . Some of the deletions had removed the promoter for the transfer operon . This allowed detection of the transcription of traC and distal genes from a weak, traJ-independent promoter . This is probably the Is2 promoter, since R100-1 carries and Is2 insertion sequence located immediately to the left of traC in the correct orientation . Since neither the transfer operon promoter nor the Is2 promoter seemed to be required for transcription of traI, it was concluded that, unlike the F factor, this was located in a separate operon.

J Gen Microbiol, 1976 Mar, 93(1), 103 - 10
R factor-mediated resistance to ultraviolet light in strains of Escherichia coli deficient in known repair functions; Tweats DJ et al.; The expression of resistance to u.v . irradiation mediated by R factor R46 has been studied in strains deficient in excision repair and recombination repair . The R factor protected wild-type bacteria and also wild-type cells in which repair had been inhibited by the substitution of bromouracil for chromosomal thymine . It increased the survival of strains defective in the endonucleolytic (uvr), repolymerizing (pol) and joining (lig) stages of the excision repair process . Recombination deficient bacteria mutant at the recB or recC loci were protected by R46, but the R factor had little effect on the survival of a recA strain or a recA recB double mutant . R46 increased the survival of cells that had been treated with chloramphenicol before u.v . irradiation, but did not protect cultures treated with chloramphenciol after irradiation . It is concluded that R46 confers resistance to the lethal effects of u.v . irradiation by a mechanism that is independent of excision repair . Resistance appears to be mediated by an inducible gene product, which is possibly a nuclease and dependent on a functional host recA gene for expression.

Eur J Biochem, 1976 Mar 1, 62(3), 551 - 4
A simple method for measuring specific radioactivities of ribonucleoside triphosphates using RNA polymerase; Maxson RE et al.; We describe a method for the rapid, one-step determination of the specific radioactivity and pool size of ATP, UTP, CTP or GTP . Escherichia coli RNA polymerase and poly{d(A-T)} or poly{d(G-C)} are used to synthesize an alternating copolymer from a {3H}nucleoside triphosphate of unknown specific activity and a {14C}nucleoside triphosphate of known specific activity . The fact that {3H}nucleotide and {14C}nucleotide are incorporated into poly{r(A-U)} or poly{r(G-C)} in equimolar amounts, coupled with a knowledge of the {14C}nucleotide specific activity, permits calculation of the {3H}nucleotide specific activity . The requirement for direct knowledge of the {14C}nucleotide specific activity may be bypassed by an isotope dilution procedure . The pool size of a nucleoside triphosphate can be estimated either from isotope dilution data or by determining the fraction of {3H}nucleotide polymerized, dividing the number of counts 3H/min in the polymer by this fraction and by the {3H}nucleotide specific activity . The method was successfully applied to acid extracts made from sea urchin embryos labeled with a {3H}RNA precursor.

Can J Biochem, 1976 Mar, 54(3), 291 - 5
Relationship between ppGpp levels and rates of protein and RNA synthesis in Escherichia coli; Chaloner-Larsson G et al.; When amino acid starvation is ended in stringent (relA+) strains of escherichia coli, the rates of RNA and protein synthesis as well as their accumulation return to normal more slowly in spoT- PSTRAIns than in the spoT+ strains . The level of ppGpp accumulated declines more slowly in the spoT- strains than in the spoT+ strains . Thus, there is an inverse relationship between ppGpp levels and the rates of RNA and protein synthesis . The slow resumption of protein synthesis in the spoT- relA+ strains could therefore be explained in terms of the limited synthesis of mRNA species coding for the bulk of cellular proteins.

Biophys Chem, 1976 Mar, 4(2), 113 - 21
Sustained oscillations and threshold phenomena in an operon control circuit; Sanglier M et al.; A genetic regulatory model involving a positive feedback (via induction) and a negative feedback (via catabolite repression) is analyzed and applied to the problem of the lac operon regulation in E . coli . Damped and sustained oscillations of the limit cycle type are found along with threshold phenomena corresponding to multiple limit cycles or to multiple steady states, for values of the parameters compatible with experimental data . A comparison with the observations of Knorre and Goodwin is outlined.

Am J Surg, 1976 Mar, 131(3), 270 - 4
Unusual abscesses in perforating colorectal cancer; Welch JP; Perforating colorectal carcinomas may be complicated by abscess formation . If unchecked, the abscess may subsequently extend to a number of sites thus requiring emergency medical attention . The cases of five such patients treated at Massachusetts General Hospital are reported . Sepsis may spread in a short period of time over a considerable area in anatomic spaces such as the retroperitoneal space . Recognition of the source of abscess may be difficult . At times, the abscess may be accompanied by subcutaneous emphysema . Prompt diagnosis and elimination of the source of sepsis may be lifesaving.

Acta Paediatr Scand, 1976 Mar, 65(2), 216 - 24
Studies of Escherichia coli O antigen specific antibodies in human milk, maternal serum and cord blood; Carlsson B et al.; Studies of Escherichia coli O antigen specific antibodies in human milk, maternal serum and cord blood . Acta Paediatr Scand, 65:216, 1976.--The quantity and class specificity of E . coli O antibodies in human milk, maternal serum and cord blood was determined by the enzyme-linked immunosorbent assay . The predominant Ig-class of these antibodies in milk was IgA . The initially high levels of antibodies decreased 10-fold during the first days, but there seemed to be a fairly constant daily production of IgA antibodies during the first two months of the nursing period . There was no obvious decline in antibody content from morning to night or during a meal . The ratio milk antibody/serum antibody was very high for IgA, suggesting a local production . The ratios for the IgG were all less than 1, suggesting a restricted transfer from the serum . Ratios around 1 for IgM did not exclude some local production . In umbilical cord serum the amount of IgG E . coli O antibodies was higher than that in maternal serum . Small amounts of IgM and IgA antibodies was also demonstrated in some cases . In milk as well as in serum there were antibodies against a wide variety of E . coli O groups, not only to the actual E . coli strain dominating the mother's gut flora.

Proc Natl Acad Sci U S A, 1976 Mar, 73(3), 707 - 11
Stringent factor from Escherichia coli directs ribosomal binding and release of uncharged tRNA; Richter D; Uncharged tRNA is preferentially bound to the peptidyl site of the ribosome in the absence of stringent factor ,but in its presence is directed to the acceptor site . The synthesis of pppGpp and ppGpp is initiated by tRNA bound in the acceptor but not in the peptidyl site . In this reaction, tRNA is not permanently attached to the acceptor site . Uncharged {32P} tRNA but not 3H-labeled stringent factor is released from the ribosome after each round of stringent factor-dependent hydrolysis of ATP . ATP-32PPi-exchange experiments reveal that exchange is independent of the presence of GTP but strongly enhanced by the addition of stringent factor and tRNA . The tRNA release is suppressed when ATP is replaced by beta, gamma-imido adenosine 5'-triphosphate, 5'-AMP, or GTP.

Proc Natl Acad Sci U S A, 1976 Mar, 73(3), 702 - 6
Specific in situ cleavage of 16S ribosomal RNA of Escherichia coli interferes with the function of initiation factor IF-1; Baan RA et al.; Specific in situ cleavage of 16S rRNA of E . coli has been accomplished by in vitro treatment of 70S ribosomes ("tight couples") with the bacteriocin cloacin DF13 . The defective ribosomes, which have fully lost their ability to sustain polypeptide synthesis, are still able to form initiation on complexes with MS2 RNA, but the kinetics are altered . This is apparently due to an improper functioning of initiation factor IF-1, for the defective ribosomal couples respond normally to dissociation by IF-3 but the dissociation is not stimulated by IF-1 . The initiation complexes formed with defective ribosomes are fully reactive with puromycin . Their ability to bind alanyl-tRNA is reduced by about 50% at all concentrations of elongation factor Tu studied . Cleavage of the 16S rRNA, not the release of the terminal fragment from the ribosome, causes the block of protein synthesis and the aberrations observed during initiation and elongation.

J Infect Dis, 1976 Mar, 133 Suppl, 41 - 54
Activation of Heat-labile Escherichia coli enterotoxin by trypsin; Rappaport RS et al.; Trypsin-treated, cell-free filtrates derived from enterotoxigenic Escherichia coli, strain H197 (O78:H11), exhibited a fourfold or greater increase in heat-labile vascular permeability factor activity and a 10-fold or greater increase in the ability to stimulate secretion of growth hormone by cultured rat pituitary cells . In contrast, trypsin-treated filtrates were not different from untreated filtrates in their ability to elicit a secretory response in ligated rabbit intestinal loops . However, incubation of culture filtrate in ligated intestinal loops, or with rabbit intestinal fluid (in vitro), resulted in at least a twofold increase in permeability factor that did not occur in the presence of trypsin inhibitor or with heat-inactivated intestinal fluid . Moreover, trypsin inhibitor could reduce the secretory response to culture filtrate . These findings suggest that activation of heat-labile E . coli enterotoxin by host enzymes may play a role in the development of a full pathogenic effect.

J Bacteriol, 1976 Mar, 125(3), 770 - 5
Glutamate transport in membrane vesicles of the wild-type strain and glutamate-utilizing mutants of Escherichia coli; Kahane S et al.; A highly specific energy-dependent glutamate transport system was demonstrated in membrane vesicles of glutamate-utilizing Escherichia coli K-12 mutants . The glutamate transport activity of membranes from the parent strain, unable to grow on glutamate, was very low . With ascorbate-phenazine methosulfate as the electron donor, mutant preparations displayed 17 to 20 times higher activity than did the wild type . However, the affinity of the mutant carrier for L-glutamate remained the same as in the parent strain . Comparative inhibition analysis of glutamate transport in whole cells and membrane vesicles and of in vitro binding of glutamate to a specific periplasmic-binding protein suggests that under certain conditions the latter may be a component of the E . coli K-12 glutamate transport system.

J Bacteriol, 1976 Mar, 125(3), 1232 - 4
Kinetics of thymineless death in Escherichia coli 15 as measured by single-cell observations; Mennigmann HD et al.; Kinetics of thymineless death for Escherichia coli 15 TAU-bar from plating on solid medium were compared with those from direct observations of single cells under a microscope . The latter method did not involve any physical change of the medium . The kinetics obtained for the two methods were identical . This rules out the assumption that in E . coli 15 TAU-bar death from the thymine deprivation is directly associated with the plating procedure.

J Bacteriol, 1976 Mar, 125(3), 1057 - 73
Escherichia coli mutants with altered ribosomal ribonucleic acid metabolism; Jackson JM 3rd et al.; This paper reports the partial characterization of two temperature-sensitive mutants of Escherichia coli with alterations in ribosomal ribonucleic acid (rRNA) metabolism at the restrictive temperature . Both mutants continue to synthesize deoxyribonucleic acid and protein at 42 C but showed little or no accumulation of RNA . Both strains are inducible for beta-galactosidase at the restrictive temperature, showing that messenger RNA synthesis continues and that the messenger RNA is translated into functional protein . One of the strains, 2S474, shows a rather severe depression (sevenfold) in the synthesis of all classes of RNA at 42 C . In addition, the synthesis of rRNA is selectively depressed, with the percentage of rRNA synthesis . However, there appears to be a selective depression in the rate of rRNA synthesis and, possibly, in the conversion of p16S rRNA to m16S rRNA.

J Bacteriol, 1976 Mar, 125(3), 1024 - 31
Changes in active transport, intracellular adenosine 5'-triphosphate levels, macromolecular syntheses, and glycolysis in an energy-uncoupled mutant of Escherichia coli; Lieberman MA et al.; The temperature-sensitive Escherichia coli mutant ecfts metC (Lieberman and Hong, 1974), previously shown to be defective in the coupling of metabolic energy to active transport, is also altered in a wide variety of cellular activities at the nonpermissive temperature . These alterations include a lowering of intracellular adenosine 5'-triphosphate levels, an alteration of glucose metabolism such that large quantities of pyruvate and dihydroxyacetone phosphate are excreted into the medium, excretion of accumulated potassium ions, and a cessation of deoxyribonucleic acid, ribonucleic acid, and phospholipid synthesis . Since these effects closely mimic the action of colicins E1 and K on E . coli cells, the possibility that the ecf gene product is the primary biochemical target for these colicins is discussed.

J Bacteriol, 1976 Mar, 125(3), 1013 - 7
Discontinuous synthesis of lac messenger ribonucleic acid in a mutant of Escherichia coli with a new lac promoter; Bruenn J et al.; A mutant of Escherichia coli with a new promoter for the lac operon exhibits dramatic discontinuities in the synthesis of lac messenger ribonucleic acid after induction . These discontinuities immediately precede similar discontinuities in the synthesis of beta-galactosidase . The discontinuous synthesis of beta-galactosidase persists after addition of rifampin.

Fertil Steril, 1976 Mar, 27(3), 275 - 81
Effect of prostaglandins, nitrofurantoin, and Escherichia coli on response of human vas deferens to norepinephrine; Hepperlen TW et al.; Surgical specimens of human vas deferens, mounted isometrically in vitro, were tested for their reactivity to norepinephrine, the major neurohumoral control mechanism in this tissue, under a variety of conditions . There was no significant difference in reactivity (measured as amplitude and frequency of contraction) between vasa obtained under either spinal or local anesthesia . Similarly, the age of the donor (range, 20 to 79 years) had no effect on either measure of reactivity . Prostaglandins A1 (10(-7) gm/ml) and E2 (10(-9) gm/ml), Escherichia coli (10(5) organisms/ml), and E . coli endotoxin (10(-7) gm/ml) did not affect norepinephrine responses, suggesting that the role of these compounds in problems of fertility is not related to an alternation in sperm transport through the vas . Nitrofurantoin (10(-5) gm/ml) also had no effect on reactivity to norepinephrine, providing further evidence that low sperm counts in patients taking this drug are more appropriately attributed to a direct effect on spermatogenesis than to an effect on sperm transport.

Cancer Res, 1976 Mar, 36(3), 927 - 9
Inhibitory effect of endotoxin on the growth of plasma cell tumor; Bober LA et al.; Small amounts (0.1 ng to 5.0 mug) of Escherichia coli endotoxin protect normal female BALB/c mice against challenge with low doses of syngeneic mineral oil-induced 315 plasma cell tumor . Significant protection was most evident when mice were treated with endotoxin 11 days and 5 days before inoculation with 50 to 100 tumor cells i.p., and endotoxin treatment continued twice a week for the entire experiment . Tumors induced by 10,000 cells s.c . were similarly affected by this treatment . The antitumor action of endotoxin was obliterated when higher challenges of tumor cells or solid tumor pieces were used . Omission of endotoxin pretreatment resulted in a loss of the effect against i.p.-induced tumors but not against s.c.-induced tumor.

J Exp Med, 1976 Mar 1, 143(3), 678 - 83
Amyloid-related serum protein SAA in endotoxin-induced amyloidosis of the mink; Anders RF et al.; The concentration of the amyloid AA-related serum protein (SAA) was markedly increased after endotoxin injections in mink, mouse, rabbit, and man . It was particularly studied during the development of endotoxin-induced amyloidosis of the mink . Protein SAA was markedly elevated in all mink 24 h after the first endotoxin injection but had fallen to relatively low levels before the onset of amyloidosis at about 4 wk . These results are consistent with SAA being a circulating precursor of the amyloid fibril protein, AA . However, both proteins may be derived by proteolysis from a common precursor.

J Cell Biol, 1976 Mar, 68(3), 642 - 53
Characterization of surface glycoproteins of mouse lymphoid cells; Gahmberg CG et al.; We have labeled exposed surface glycoproteins of mouse lymphoid cells by the galactose oxidase-tritated sodium borohydride technique . The labeled glyco-proteins were separated by polyacrylamide slab gel electrophoresis and visualized by autoradiography (fluorography) . The major thymocyte surface proteins have molecular weights of 170,000 and 125,000 . Thymocytes from TL antigen-positive mouse strains showed an additional band with a molecular weight of 27,000 . Highly purified T lymphocytes contain two major surface glycoproteins with molecular weights of 180,000 and 125,000 . Purified B lymphocytes have one major surface glycoprotein with a molecular weight of 210,000 . When T lymphocytes are stimulated in vitro by concanavalin A or phytohemag-glutinin, the major proteins characteristic of T cells are relatively weakly labeled, but new components of lower molecular weights appear on the cell surface . A similar change is seen in B lymphocytes stimulated by Escherichia coli lipopolysaccharide . T lymphoblasts isolated from mixed lymphocyte cultures show a slightly different surface glycoprotein pattern . A polypeptide with a molecular weight of 57,000, which was labeled without enzymatic treatment by tritiated sodium borohydride alone, is strongly labeled in proliferating cells.

Eur J Biochem, 1976 Mar 1, 62(3), 519 - 26
Homoserine kinase from Escherichia coli K12; Burr B et al.; Homoserine kinase was purified to apparent homogeneity from a derepressed strain of Escherichia coli K12, using standard fractionation techniques . It is a dimer (Mr = 60000) composed of apparently identical polypeptide chains (Mr = 29000) . Its amino acid composition and N-terminal sequence have been determined . L-Threonine is a competitive inhibitor of the substrate L-homoserine; this inhibition is straighforward and shows no sign of co-operativity . Evidence is presented that homoserine and threonine bind to the same site of this non-allosteric enzyme . The binding of homoserine and threonine can also be studied by difference spectroscopy; the latter studies reveal an unexpected effect of magnesium ions, which might be the basis for the unusual high Mg2+ requirement for optimal enzyme reaction.

J Bacteriol, 1976 Mar, 125(3), 1220 - 2
Transcriptional and post-transcriptional control of beta-galactosidase synthesis; Ennis HL et al.; lac messenger ribonucleic acid synthesis is dissociated from its translation in certain lac operon mutants . More beta-galactosidase messenger ribonucleic acid (as determined by hybridization) is made in fully induced lacOc mutants and in trp-lac fusions than is eventually translated into enzyme . Consequently, beta-galactosidase synthesis may be post-transcriptionally controlled under these conditions.

Z Naturforsch {C}, 1976 Mar-Apr, 31(3-4), 201 - 2
Kinetic properties of Mg, Ca ATPase from various Escherichia coli mutants; Ahlers J et al.; The ATPase is not lacking in the ATPase mutants DL 54 and AN 120 but has very different kinetic properties including a higher Cl- optimum and higher Km values for MgATP . In AN 120, the ATPase activity also has a higher Mg2+ optimum.

Nucleic Acids Res, 1976 Mar, 3(3), 677 - 95
DNA methylase: purification from ascites cells and the effect of various DNA substrates on its activity; Turnbull JF et al.; DNA methylase has been purified 405-fold from Krebs II ascites cells . The purified enzyme is homogeneous on SDS-poly acrylamide gel electrophoresis (molecular weight about 80,000) and the only product of the reaction with DNA is 5-methyl cytosine . Both native and denatured DNA are methylated by the enzyme; with calf thymus DNA the double stranded form is the better substrate but the enzyme preferentially methylates single stranded E.coli DNA even in "native" preparations . Our results do not support a mechanism whereby the enzyme methylates DNA by binding irreversibly and "walking" along it . By measuring maximum levels of methylation of DNAs from different sources we have estimated the proportion of unmethylated sites present in them . Homologous ascites DNA can be methylated, but only to about 5% of the level of the best substrate, undermethylated mouse L929 cell DNA . DNA isolated from growing cells or tissues is a better substrate than DNA from normal liver or pancreas, or from stationary cells.

J Bacteriol, 1976 Mar, 125(3), 811 - 7
Hfr formation by I pilus-determining plasmids in Escherichia coli K-12; Datta N et al.; R483, an atypical, I pilus-determining plasmid, and also R144, a typical one, were shown to suppress the DnaA phenotype by integration into the Escherichia coli chromosome.

J Bacteriol, 1976 Mar, 125(3), 796 - 9
Compatibility properties of R483, a member of the I plasmid complex; Datta N et al.; R483, an I pilus-determining plasmid previously reported as belonging to a distinct incompatibility group Ibeta, proved to be an atypical Ialpha plasmid; in a growing culture, the degree of inhibition of replication of one Ialpha plasmid by the presence of another was not uniform within the Ialpha group.

J Immunol Methods, 1976 Mar, 10(2-3), 127 - 32
Histochemical staining in lymphocyte suspensions complexed with beta-galactosidase as an antigen . Unspecific staining of spleen cells by indigo; Dankwarth L et al.; Spleen cells from mice immunized with E . coli beta-galactosidase were incubated with the enzyme and with 5-bromo-4-chloroindolyl-beta-galactosidase as a histochemical substrate . Some cells are stainable in this reaction, however, not all of them carry specific receptors for beta-galactosidases . Some spleen cells can take up dye and thereby become unspecifically stained . This system is therefore not suitable for the detection of antigen binding to cells.

J Exp Med, 1976 Mar 1, 143(3), 497 - 510
Modulation of regulatory mechanisms operative in the cyclical production of antibody; Romball CG et al.; Modulation of the cyclical response in rabbits to aggregated human gamma globulin (AHuIgG) was investigated in order to study some of the parameters involved in self-regulation of the immune response . Several mitogens (lipopolysaccharide {LPS}, phytohemagglutinin {PHA}, and concanavalin A {Con A}), when injected simultaneously with antigen, have been shown to modulate the normal splenic plaque-forming cell (PFC) response in rabbits to a single intravenous injection of AHuIgG . This response to AHuIgG has previously been characterized by the initial appearance of PFC in the spleen 3 days later, with a peak of PFC at 5 days after injection . The number of PFC in the spleen then decreases and remains at a low level until a second increase begins on day 10, peaking on day 13 . The 8-day cycle between peak PFC repeats, with a third peak appearing on day 21 . In the present studies, injection of LPS with AHuIgG was shown to affect the PFC response by enhancing only the initial peak of PFC, PHA was shown to enhance both the initial and secondary peaks of PFC, while injection of Con A with AHuIgG resulted in a prolonged increase in PFC with no apparent cycling . Irradiation 24 h after injection of antigen resulted in PFC kinetics similar to those observed with PHA, although the increase in PFC was more marked with irradiation . Thus, although LPS, PHA, Con A, and irradiation markedly affected the immune response to AHuIgG, Con A was the only substance which altered the cyclical appearance of PFC to HuIgG . The cyclical nature of the PFC kinetics was shown to occur with either intravenous or intraperitoneal injection of antigen and in both primary and secondary responses, provided that the rabbits were primed with a low dose of antigen . Data were obtained that suggest that the response in distal lymph nodes may be regulated by immunological events occurring in the spleen . Cycling of PFC was not observed in the draining node after subcutaneous injection of AHuIgG in the hind foot . However, if the antigen was also injected intravenously at the same time as the subcutaneous injection, the response in the node became cyclical.

Nucleic Acids Res, 1976 Mar, 3(3), 817 - 24
Preparation and properties of poly 2'-O-ethylcytidylic acid; Kielanowska M et al.; Poly 2'0-ethylcytidylic acid (poly (Ce)) was prepared by polymerization of 2'-0-ethylcytidine-5'-pyrophosphate with Escherichia coli polynucleotide phosphorylase in the presence of Mn++, and its properties compared with those of poly (rC), poly (Cm) and poly (dC) . The neutral form of pOLY (Ce) exhibits properties similar to those of poly (rC) and poly (Cm) . It also forms an acid twin-stranded helix with a transition pH of 5.9 in 0.1 M NaCl . The neutral form readily forms a double-stranded helical complex with poly (rI) . Relative to poly (Cm), replacement of the 2'-0-methyl by 2-0-ethyl leads to increased enhancement of the thermal stabilities of both the acid helical form of poly (Ce) and its complex with poly (rI).

J Gen Physiol, 1976 Mar, 67(3), 325 - 41
Cation transport in Escherichia coli . VIII . Potassium transport mutants; Rhoads DB et al.; Analysis of K transport mutants indicates the existence of four separate K uptake systems in Escherichia coli K-12 . A high affinity system called Kdp has a Km of 2 muM, and Vmax at 37 degrees C of 150 mumol/g min . This system is repressed by growth in high concentrations of K . Two constitutive systems, TrkA and TrkD, have Km's of 1.5 and 0.5 mM and Vmax's of 550 and 40 at 37 and 30 degrees C, respectively . Mutants lacking all three of these saturable systems take up K slowly by a process, called TrkF, whose rate of transport is linearly dependent on K concentration up to 105 mM . On the whole, each of these systems appears to function as an independent path for K uptake since the kinetics of uptake when two are present is the sum of each operating alone . This is not true for strains having both the TrkD and Kdp systems, where presence of the latter results in K uptake which saturates at a K concentration well below 0.1 mM . This result indicates some interaction between these systems so that uptake now has the affinity characteristic of the Kdp system . All transport systems are able to extrude Na during K uptake . The measurements of cell Na suggest that growing cells of E . coli have very low concentrations of Na, considerably lower than indicated by earlier studies.

Eur J Biochem, 1976 Mar 1, 62(3), 485 - 90
The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K-12 . Incubation of the enzyme in alkaline conditions: dissociation and disulfide-bridge formation; Jacques Y et al.; Aspartokinase I - homoserine dehydrogenase I from Escherichia coli K-12, a homotetrameric enzyme, dissociates into dimers upon alkaline treatment . Both aspartokinase and homoserine dehydrogenase inactivation, as well as desensitazion towards L-threonine, occur in a multi-step process . Dithiothreitol stabilizes a dimeric form retaining full activity and sensitivity; L-homoserine stabilizing another dimeric form devoid of aspartokinase activity and retaining a substantial dehydrogenase activity insensitive toward L-threonine . A model is proposed showing that dissociation into dimers occurs in a first step, the resulting dimer losing both aspartokinase and homoserine dehydrogenase sensitivity in two subsequent steps involving the formation of intrachain disulfide bonds.

J Biomed Mater Res, 1976 Mar, 10(2), 249 - 58
A Dacron wool packed-bed extracorporeal reactor: a kinetic study of immobilized Escherichia coli II L-asparaginase; KO RY et al.; An extracorporeal reactor containing a packed bed of Dacron fibers has been developed . Escherichia coli II L-asparaginase was coupled to the Dacron using gamma-aminopropyltriethoxysilane and glutaraldehyde . The preparation had an activity of 37 IU per gram of Dacron (37 degrees C) . The apparent Km was studied as a function of the flow rate . The data indicated that the apparent Km approached the Km of the native enzyme at flow rates of about 300 mg/min . In vivo use of L-asparaginase immobilized on the Dacron indicated effective lowering of plasmatic L-asparagine levels.

J Bacteriol, 1976 Mar, 125(3), 923 - 33
Sulfate-reducing pathway in Escherichia coli involving bound intermediates; Tsang ML et al.; Although a sulfate-reducing pathway in Escherichia coli involving free sulfite and sulfide has been suggested, it is shown that, as in Chlorella, a pathway involving bound intermediates is also present . E . coli extracts contained a sulfotransferase that transferred the sulfonyl group from a nucleosidephosphosulfate to an acceptor to form an organic thiosulfate . This enzyme was specific for adenosine 3'-phosphate 5'-phosphosulfate, did not utilize adenine 5'-phosphosulfate, and transferred to a carrier molecule that was identical with thioredoxin in molecular weight and amino acid composition . In the absence of thioredoxin, only very low levels of the transfer of the sulfo group to thiols was observed . As in Chlorella, thiosulfonate reductase activity that reduced glutathione-S-SO3- to bound sulfide could be detected . In E . coli, this enzyme used reduced nicotinamide adenine dinucleotide phosphate and Mg2+, but did not require the addition of ferredoxin or ferredoxin nicotinamide adenine dinucleotide phosphate reductase . Although in Chlorella the thiosulfonate reductase appears to be a different enzyme from the sulfite reductase, the E . coli thiosulfonate reductase and sulfite reductase may be activities of the same enzyme.

J Bacteriol, 1976 Mar, 125(3), 1096 - 1104
Role of D-tryptophan oxidase in D-tryptophan utilization by Escherichia coli; Hadar R et al.; Mutants of Escherichia coli K-12 that require L-tryptophan (trp) are normally unable to utilize D-tryptophan to fulfill their requirement . However, secondary mutations (dadR) that confer this ability can be isolated . In such strains two distinct enzymes are found to be produced at high levels: D-amino acid oxidase (EC 1.4.3.3) and D-tryptophan oxidase . A convenient assay procedure for D-tryptophan oxidase is described . The two enzymes could be distinguished on the basis of their sensitivity to inhibition by L-phenylalanine and L-tyrosine . Strains that were trp dadR could not grow with D-tryptophan in the presence of L-phenylalanine, but further mutations, Fyo, could be isolated that allowed growth under these conditions . Some of them were characterized by further increases in the level of D-tryptophan oxidase activity and a sharp decrease in D-amino acid oxidase . These kinds of Fyo mutations lay in or near the dadR gene . The substrate specificity of the two enzymes toward a large number of compounds was examined . The transamination of aromatic keto acids was investigated . In the wild-type strain only a single enzyme, transaminase A (EC 2.6.1.5), was found, and it was irreversibly activated when subjected to elevated temperatures . The present state of our knowledge on D-amino acid utilization in E . coli is summarized.

Mol Gen Genet, 1976 Feb 27, 144(1), 53 - 8
Mutagenic DNA repair in Escherichia coli . III . Requirement for a function of DNA polymerase III in ultraviolet-light mutagenesis; Bridges BA et al.; The polC (= dnaE) temperature-sensitive DNA polymerase III mutation from Escherichia coli BT1026 has been transduced into E . coli WP2 (to give CM731) and WP2 uvr A (to give CM741) . In excision-deficient CM741 UV-induced Trp+ mutations progressively lost their photoreversibility during post-irradiation incubation at 34 degrees . Immediately after transfer to 43 degrees, however, there was no further loss of reversibility although post-replication strand joining still occurred and uptake of 3H-thymidine into DNA continued for 20 to 30 min . In excision-proficient CM731, UV lesions capable of leading to Strr mutations disappeared during post-irradiation incubation at restrictive temperature and there was no increase in the number remaining after exposure to photoreversing light . In contrast, at permissive temperature, premutational lesions were not lost and became progressively converted into non-photoreverisble mutations . It is concluded that a function of the polC gene is necessary for error-prone repair to occur and that this function is defective at 43 degrees in the enzyme specified by the polC allele from BT1026 . This function seems not to be essential for most post-replication or excision repair or for normal DNA replication and may be particularly involved in the insertion of incorrect bases during error-prone repair.

Mol Gen Genet, 1976 Feb 27, 144(1), 17 - 20
In vitro synthesis and repression of argininosuccinase in Escherichia coli K12; partial purification of the arginine repressor; Kelker NE et al.; Phi80dargECBH DNA has been used to direct cell-free synthesis of argininosuccinase, the argH gene product in Escherichia coli K12 . In vitro enzyme synthesis is sensitive to repression by partially purified preparations from an argR+ strain but not by corresponding preparations from an argR- strain . Using DNA-cellulose chromatography, approximately seventyfold purification of repressor has been obtained . The partially purified preparation represses argininosuccinase synthesis but has no effect on beta-galactosidase synthesis.

Mol Gen Genet, 1976 Feb 27, 144(1), 115 - 8
Isolation of transducing phage carrying rps T, the structural gene for ribosomal protein S20; Friesen JD et al.; Lambda transducing phages carrying segments of the Escherichia coli chromosome in the dapB region have been isolated in their in vivo gene products analyzed by two-dimensional gel electrophoresis . One of these phages, lambdaddapB-2, carries the structural genes for ribosomal protein S20 (rps T) and isoleucyltransfer RNA synthetase (ileS.) The most likely gene order is thr-rpsT-ileS-dapB-pyrA.

Mol Cell Biochem, 1976 Feb 25, 10(3), 137 - 43
The role of thiol groups in nucleoside transport; Doskocil J; (1) The inactivation of various forms of nucleoside transport with reagents blocking thiol groups was studied in whole cells of E . coli B . No positive correlation between the efficiency of active transport and the extent or rate of inactivation could be demonstrated . (2) The most efficient constitutive nucleoside-transporting system was found to comprise a specific thiol component characterized by low rate of inactivation with N-ethylmaleimide; the less efficient inducible transport and the facilitated diffusion of guanosine require the integrity of another thiol component which is rapidly inactivated with N-ethylmaleimide . (3) The constitutive nucleoside-transporting system is completely inactivated with T4 phage, while other modes of nucleoside transport are much less affected . (4) Inactivation of constitutive transporting system in cells exposed to N-ethylmaleimide for a limited period of time continues long after the inhibitor has been removed, indicating storage of the inhibitor in some cellular compartment . Addition of dithiothreitol stops the inactivation immediately.

J Biol Chem, 1976 Feb 25, 251(4), 930 - 3
Selective chemical modification of Escherichia coli elongation factor G . N-Ethylmaleimide modification of a cysteine essential for nucleotide binding; Rohrbach MS et al.; Escherichia coli Elongation Factor G is inhibited ireversibly by the chemical modification of 1 cysteine residue with N-ethylmaleimide . At pH 5.2, this cysteine is approximately 130 times more reactive than beta-mercaptoethanol toward N-ethylmaleimide . Inhibition is not prevented by either the ribosome or GTP alone at concentrations approximately equal to that of Elongation Factor G, but in combination they reduce the inhibition by 50% . Increasing the stability of the Elongation Factor G-ribosome-GDP complex by the addition of fusidec acid, completely protects against N-ethylmaleimide inhibition . The modified protein cannot form either the Elongation Factor G-ribosome-GMP-P(CH2)P or the Elongation Factor G-ribosome-GDP-fusidic acidcomplex . However, the modification had no effect on its ability to form the Elongation Factor G-ribosome complex . These results suggest that the cysteine residue modified by N-ethylmaleimide is at or near the nucleotide binding site.

J Biol Chem, 1976 Feb 25, 251(4), 922 - 9
Isolation and characterization of large transfer ribonucleic acid precursors from Escherichia coli; Ilgen C et al.; Several transfer RNA precursors which accumulate in a strain of Escherichia coli temperature-sensitive for RNase P have been described . These precursors range from 135 to 690 nucleotides in length . Their tRNA content has been determined by digestion of the precursors to 4 S RNA, followed by Sanger fingerprint analysis of the purified 4 S material . Identification of some of these tRNAs, as well as an estimate of the number of copies of tRNA in each precursor has been achieved . Many of these precursor RNA molecules contain multiple copies of the same tRNA sequence, indicating a tandem arrangement of the corresponding tRNA genes in the E . coli genome.

J Biol Chem, 1976 Feb 25, 251(4), 1217 - 8
Superoxide dismutase . Reversible removal of manganese and its substitution by cobalt, nickel or zinc; Ose DE et al.; The maganese-containing superoxide dismutase, from Escherichia coli, lost metal and activity when dialyzed against 20 mM-8-hydroxyquinoline and 2.5 M guanidinium chloride in 5 mM Tris-chloride buffer at pH 7.8 . Subsequent dialysis against 0.01 m M McCl2, in this buffer, caused a restoration of manganese and activity . Reconstituted enzyme appeared identical with native enzyme, and removal and restoration of manganese could be repeated . Co (II), Zn(II), Ni(II), Mg(II), Cr (II), Cu(II), Fe(II), In(II), and Mo(VI), were tested for their abiltiy to replace manganese . None of these restored activity to apoenzyme . When present at a 100-fold molar excess over manganese only Co(II), Ni(II), and Zn(II) were effective as competitors of manganese . Co(II) was demonstrated to be tightly bound to the apoenzyme in place of the magnasese, with a stoichiometry of 1 Co(II) per molecule . In the cases of Co(II), Zn(II), or Ni(II), reconstituted enzyme; the metals could be removed and subsequently replaced by manganese, with restoration of catalytic activity . None of these metal cations exhibited superoxide dismutase activity . We conclude that manganese is essential for the catalytic activity of superoxide dismutase, and that the site which normally binds manganese can accommodate Co(II), Ni(II), and Zn(II).

J Biol Chem, 1976 Feb 25, 251(4), 1171 - 4
Kinetic studies of inducer binding to lactose repressor protein; Friedman BE et al.; The kinetics of binding of the inducer, isopropyl-beta, D-thiogalactoside to lactose repressor from Escherichia coli was studied by stopped flow rapid mixing techniques . Three different spectral probes for measuring changes in the conformation of the repressor were used: ultraviolet absorbance, fluorescence, and a reporter group, 2-mercuri-4-nitrophenol, in the visible region . Repressor can be reacted with this mercurial to modify two of the three free sulfhydryl groups per monomer without loss of inducer or operator binding activities . The observed first order rate constant for the reaction of repressor with 2-chloromercuri-4-nitrophenol at pH 7.5 and 20 degrees was found to be on the order of 0.1 s-1, an unexpectedly slow rate for this type of reaction . Once bound to repressor, the nitrophenol serves as a chromophoric probe to monitor changes in the surrounding environment . The binding of inducer to repressor causes a change in the absorbance of the bound 2-mercuri-4-nitrophenol moiety and exhibits a second order rate of 3.2 x 10(4) M-1 s-1 . Similar rates were obtained when binding of inducer is monitored by changes in either the ultraviolet absorbance of fluorescence of tryptophan residues . Since the same rate of spectral perturbation is observed for different regions of the primary structure of the protein, the conformational change produced in response to inducer binding appears to be translated rapidly throughout the protein molecule.

J Biol Chem, 1976 Feb 25, 251(4), 1147 - 53
The site of ribosome degradation in starved Escherichia coli cells; Kaplan R et al.; An attempt was made, in starved Escherichia coli cells, to locate the site at which the process of ribosome degradation is initiated . Supernatant and rapidly sedimenting pellet fractions from exponentially growing and from carbon-starved cells were prepared, and the ribonucleic acids from these fractions were seperated by polyacrylamide electrophoresis and quantitated . The data indicated that 23 S, 16 S, and 5 S RNA are lost only from the pellet; and also that the low molecular weight RNA degradation products are confined to this fraction . Ribosomes from supernatant and pellet fractions were seperated on sucrose density gradients . The sedimentation profiles obtained indicated that pellet fractions of starved cells contain for the most part 50 S and 30 S subunits, whereas 70 S monosomes were most abundant in the supernatant fraction . In vitro measurements on RNA degradation in supernatant and rapidly sedimenting pellet fractions confirmed the in vivo data on the exclusive degradation of ribosomal RNA in the pellet . Based on these data and on previous observations, we suggest that the endoribonucleolytic attack which triggers ribosome degradation, occurs in free subunits attached to the rapidly sedimenting membrane fraction . Subsequently, the ribosome falls apart, and the small RNA pieces generated remain attached to the pellet fraction until their final degradation by the exonuclease.

J Biol Chem, 1976 Feb 25, 251(4), 1137 - 46
Effect of estrogen on gene expression in the chick oviduct . Kinetics of initiation of in vitro transcription on chromatin; Hirose M et al.; The kinetics of initiation of in vitro RNA synthesis by Escherichia coli RNA polymerase on the chromatin template of chick oviduct was examined and compared to initiation on the template of deproteinized chick DNA . The formation of rapidly starting, highly stable binary complexes (RS complexes) between RNA polymerase and chromatin was an apparent first order process with a half-time (t1/2) of 9.4 min . By contrast, the t1/2 of formation of RS complex on chick DNA was 1.3 min . The apparent first order rate constants of RNA chain initiation from performed RS complexes were 0.57 s-1 for chromatin and 0.98 s-1 for DNA . Thus, although the formation of RS complex on chromatin was much slower than on DNA, the actual rate of RNA chain initiation on chromatin was similar to that on DNA . The effects of temperature on the formation of RS complex were examined . On chick DNA, both the rate of formation and the amount of RS complex formed decreased as the temperature of preincubation was lowered from 37 degrees to 0 degrees . On chromatin, the t1/2 of formation of RS complex was independent of temperature, and the amount of complex formed was only slightly affected at changing, temperatures . The kinetics of initiation on chromatin is consistent with the hypothesis that the rate-limiting step in the formation of RS complex is dissociation of RNA polymerase from nonspecific interaction with chromosomal proteins . Furthermore, chromosomal proteins seem to interact with initiation sites to facilitate the opening of the DNA strands required for RS complex formation.

J Biol Chem, 1976 Feb 25, 251(4), 950 - 61
A major polypeptide component of rat liver mitochondria: carbamyl phosphate synthetase; Clarke S; One of the major components of rat liver mitochondria detected by gel electrophoresis in sodium dodecyl sulfate is a 165,000 molecular weight polypeptide that makes up 15 to 20% of the total mitochondrial protein . This component appears to be a single molecular species . Evidence is presented here for the identification of this protein with the polypeptide chain of a urea cycle enzyme, carbamoylphosphate synthetase I (EC 2.7.2.5) . The 165,000 molecular weight polypeptide was solubilized from mitochondria with Triton X-100 and purified to 90% homogeneity by DEAE-cellulose chromatography . This component co-migrated with carbamyl phosphate synthetase activity when mitochondrial proteins were separated by gel filtration or sucrose gradient centifugation . The identification of the 165,000 molecular weight polypeptide with this activity was also supported by the presence or absence of this protein in a variety of rat tissue mitochondria, in liver and kidney mitochondria from various ureotelic and nonureotelic species, and in fetal rat liver mitochondria.

J Biol Chem, 1976 Feb 25, 251(4), 982 - 6
On the fidelity of DNA replication . Lack of exodeoxyribonuclease activity and error-correcting function in avian myeloblastosis virus DNA polymerase; Battula N et al.; Homogeneous DNA polymerase ("reverse transcriptase") from avian myeoblastosis virus was assayed for exodeoxyribonuclease activity . The substrates were defined template-initiator complexes in which different radioactive nucleotides were present at the 3'-OH termini of the initiator . Even when the number of molecules of enzyme was equal to the number of initiator termini there was no significant release of radioactivity with any of the template-initiator combinations tested . Under similar conditions, the nuclease activity associated with either Escherichia coli or T4DNA polymerases rendered more than 90% of the initiator termini acid-soluble . The ratio of exodeoxyribonuclease activity to protein with avian myeoblastosis DNA polymerase is less than 0.003% of that obtained with E . coli DNA polymerase I . Furthermore, avian myeloblastosis virus DNA polymerase failed to excise mispaired terminal nucleotides in both the presence and absence of polymerization.

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