Escherichia coli

Transfusion, 1982 Nov-Dec, 22(6), 504 - 6
Use of whole blood exchange transfusion to supply neutrophils to septic, neutropenic neonates; Christensen RD et al.; When neutropenia due to exhaustion of the marrow neutrophil reserve, develops in a neonate with bacterial sepsis the likelihood of survival is very small . We report such a case who was treated with a double-volume exchange transfusion using fresh unstored whole blood . We were able to determine a net gain of 5 x 10(8) neutrophils per kg . Then, in neutropenic neonatal animals, neutrophil transfusion by double-volume exchange transfusion with unstored blood was investigated.

Infect Immun, 1982 Nov, 38(2), 798 - 801
Failure to detect conventional enterotoxins in classical enteropathogenic (serotyped) Escherichia coli strains of proven pathogenicity; Robins-Browne RM et al.; Ammonium sulfate-precipitated supernatants of classical enteropathogenic Escherichia coli strains were negative when investigated for enterotoxin production in rabbit ligated ileal loops, rabbit skin vascular permeability factor tests, suckling mice, and Y-1 adrenal cells . They also failed to stimulate guanylate cyclase activity in homogenates of rabbit, rat, and infant mouse intestines . Furthermore, DNA from enteropathogenic E . coli lacked sequences that encode heat-labile and heat-stable enterotoxins . These studies fail to show conventional enterotoxin synthesis by classical enteropathogenic E . coli.

J Bacteriol, 1982 Nov, 152(2), 935 - 8
Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase; Lorowitz W et al.; Mutants of Escherichia coli resistant to allyl alcohol were selected . Such mutants were found to lack alcohol dehydrogenase . In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained . These mutations, designated adhE, are all located at the previously described adh regulatory locus . Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.

J Bacteriol, 1982 Nov, 152(2), 919 - 23
Escherichia coli polypeptide controlled by the lon (capR) ATP hydrolysis-dependent protease and possibly involved in cell division; Schoemaker JM et al.; Mutation in the gene lon (capR) of Escherichia coli K-12 causes conditional inhibition of cell division . Two-dimensional gel electrophoresis was used to compare polypeptides from isogenic capR+ and capR strains . One polypeptide was present in the capR strain but absent in the wild-type strain, and it was proteolyzed when the pure capR+ protease was added to the capR extract . This polypeptide could only be detected in the capR strain when cell division was inhibited, and its synthesis was independent of the SOS response.

J Bacteriol, 1982 Nov, 152(2), 901 - 3
Detection of primase specified by IncB plasmid R864a; Dalrymple BP et al.; Plasmid R864a (IncB) contains nucleotide sequences homologous with the sog primase determinant of IncI alpha plasmid ColIb-P9 . Extracts of Escherichia coli carrying a mutant R864a derepressed for transfer functions showed enhanced primase activity, and contained a large polypeptide identical in size (apparent Mr = 220,000) to the IncI alpha sog gene product.

J Bacteriol, 1982 Nov, 152(2), 897 - 900
Gene ompR and regulation of microcin 17 and colicin e2 syntheses; Hernandez-Chico C et al.; The production of microcin 17 is controlled by plasmid pRYC17 . Chromosomal mutants unable to produce a normal amount of microcin were isolated in Escherichia coli . One of the mutations maps in the ompR locus, indicating that an active OmpR product is required for the synthesis of microcin 17 . The same conclusion was obtained for the synthesis of colicin E2 . Therefore, two new functions of the regulatory gene ompR have been revealed.

J Bacteriol, 1982 Nov, 152(2), 572 - 83
Suppression of the ssb-1 and ssb-113 mutations of Escherichia coli by a wild-type rep gene, NaCl, and glucose; Tessman ES et al.; The ssb-1 mutation confers severe temperature sensitivity and UV sensitivity on many strains of Escherichia coli K-12 and C, including strain C1412 . However, ssb-1 confers only slight temperature sensitivity and slight UV sensitivity on strain C1a, suggesting that strain C1a contains extragenic suppressors of ssb-1 . We found that introduction of the wild-type rep gene from C1a into strain C1412 ssb-1 gave strong suppression of temperature sensitivity and moderate suppression of UV sensitivity . Also, the C1a rep+ gene mildly suppressed the temperature sensitivity conferred by the ssb-113 mutation, formerly called lexC113 . Suppression of the C1412 ssb-1 growth defect by C1a rep+ rendered the cells Gro- for phi X174 . In contrast to the positive suppression of ssb-1 and ssb-113 by a wild-type rep gene, mutant rep alleles enhanced the severity of the ssb-1 defect, with several C1a ssb-1 double mutants being either more temperature sensitive or more UV sensitive than C1a ssb-1, depending on which mutant rep allele was used . As a control, the same rep alleles in combination with a dnaB mutation gave an allele-independent increase in temperature sensitivity . Our results on suppression of ssb-1 by rep and on the role of the genetic background in this suppression suggested that the rep and ssb proteins interact to form a subcomplex of the total DNA replication complex and that this subcomplex has some function in repair . The effects of NaCl and glucose on suppression of both the temperature sensitivity and the UV sensitivity conferred by ssb-1 and ssb-113 are described . The degree of suppression of temperature sensitivity by salt or glucose was dependent on the source of the wild-type rep allele, as well as on the genetic background.

Gut, 1982 Nov, 23(11), 974 - 9
Antidiarrhoeal activity of loperamide: studies of its influence on ion transport across rabbit ileal mucosa in vitro; Hughes S et al.; Loperamide is a well-established antidiarrhoeal agent with effects on gastrointestinal motility . We have now shown that the drug influences ion transport . In isolated rabbit ileal mucosa loperamide caused a dose-related fall in potential difference and short-circuit current and reduced the serosa to mucosa flux of chloride . The electrical effects were inhibited by naloxone (10(-6)M) suggesting that they were mediated by opiate receptors . Loperamide (10(-6)M) inhibited secretion provoked by heat stable and heat labile E . coli toxins and by prostaglandin E2 . We conclude that loperamide is able to inhibit secretion mediated by cAMP or cGNP, and that this may be relevant to its antidiarrhoeal properties.

Gene, 1982 Nov, 20(1), 111 - 9
A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant; Tandeau de Marsac N et al.; A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described . This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis . A met::Tn901 mutant was isolated . Chromosomal DNA fragments were cloned in the Escherichia coli plasmid vector pACYC184 . A recombinant plasmid carrying the inactivated met::Tn901 gene was selected after transformation to E . coli . The cloned met::Tn901 DNA fragment was used as a probe to select the corresponding A . nidulans R-2 wild-type met gene from a gene library prepared in E . coli, using the newly constructed shuttle cosmid vector pPUC29 . When transformed into A . nidulans Met- mutants, this cloned gene allowed the mutants to grow prototrophically.

Cell, 1982 Nov, 31(1), 43 - 51
Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes; DiNardo S et al.; Escherichia coli deletion mutants lacking DNA topoisomerase I have been identified previously and shown to grow at a normal rate . We show that such strains grow normally only because of spontaneously arising mutations that compensate for the topoisomerase I defect . Several of these compensatory mutations have been found to map at or near the genes encoding DNA gyrase, gyrA and gyrB . DNA gyrase assays of crude extracts show that strains carrying the mutations have lower gyrase activity . Thus the mutations are in the gyrase structural genes or in nearby regulatory sequences . These results, in conjunction with DNA supercoiling measurements of others, indicate that in vivo DNA superhelicity is a result of a balance between topoisomerase I and gyrase activities . An excess of negative supercoils due to an absence of topoisomerase I is deleterious to the cell, but a moderate gyrase deficiency is not harmful.

Cell, 1982 Nov, 31(1), 35 - 42
Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes; Pruss GJ et al.; Bacterial chromosomes and plasmid (pBR322) DNA from topoisomerase I-defective Escherichia coli strains have been characterized with respect to superhelical density . The topoisomerase I defect results in increased negative superhelical density of both the bacterial chromosome and pBR322 . Thus topoisomerase I is involved in determining the level of supercoiling in bacteria . Three of the topoisomerase I-defective strains were studied carry secondary mutations that decrease superhelical density; these additional mutations are closely linked to the gyrB locus in two of the strains and to the gyrA locus in the third strain.

Cell, 1982 Nov, 31(1), 227 - 35
A previously unidentified gene in the spc operon of Escherichia coli K12 specifies a component of the protein export machinery; Shultz J et al.; The gene prlA codes for a factor that appears to function in the export of proteins in Escherichia coli . This conclusion is based on the finding that mutations altering the prlA gene product restore export of envelope proteins with defective signal sequences . Previous results showed that the prlA gene lies in an operon (spc) known to code for ten different ribosomal proteins . Our studies show that the prlA gene lies promoter-distal to the last known ribosomal protein gene in this operon . Evidence from gene fusions constructed in vitro suggests that prlA codes for a protein containing at least 300 amino acids . Thus a heretofore unidentified protein specified by a gene within the spc operon appears to be a component of the cellular protein export machinery.

Biochem J, 1982 Nov 1, 207(2), 333 - 9
Electron-spin-resonance studies of the NADH-dependent nitrite reductase from Escherichia coli K12; Cammack R et al.; The NADH-dependent nitrite reductase of Escherichia coli, which contains sirohaem, flavin, non-haem iron and labile sulphide, was examined by low-temperature e.s.r . spectroscopy . The enzyme, stored in the presence of nitrite and ascorbate, gave the spectrum of a nitrosyl derivative, with hyperfine splitting due to the nitrosyl nitrogen . On removal of these reagents, a series of signals centred around g = 6 was observed, typical of high-spin ferric haem . Cyanide converted this into a low-spin form . On reduction of the enzyme with NADH, an axial spectrum at g = 1.92, 2.01 was observed . The temperature-dependence of this signal is indicative of a {2Fe-2S} iron-sulphur cluster . The midpoint potential of this cluster was estimated to be -230 +/- 15 mV by two independent methods . Reduction of the enzyme with dithionite yielded further signals, which are at present unidentified, at g = 2.1-2.28 . No signals were observed that could be assigned to a {4Fe-4S} cluster, such as is found in other sulphite reductases and nitrite reductases that contain sirohaem.

Proc Natl Acad Sci U S A, 1982 Nov, 79(22), 6971 - 5
Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon; Johnston SA et al.; GAL4 is a classically defined positive regulatory gene controlling the five inducible structural genes of galactose/melibiose utilization in yeast . The positive regulatory function of the GAL4 gene product in turn is controlled by the product of another gene, the negative regulator GAL80 . We have cloned a 3.1-kilobase fragment containing GAL4 by homologous complementation using the multicopy chimeric vector YEp24 and demonstrated that multiple copies of GAL4 in yeast have pronounced dosage effects on the expression of the structural genes . Yeast transformed with GAL4-bearing plasmid become constitutive for expression of the galactose/melibiose genes, even in normally repressing (glucose) medium . Multiple copies of the GAL4 plasmid also increase expression of the structural genes in inducing (galactose) medium and can partially overcome the effects of a dominant super-repressor mutant, GAL80S . Using an internal deletion in GAL4, we have demonstrated that these dosage effects are due to overproduction of GAL4 positive regulatory product rather than an effect of the flanking sequences titrating out a negative regulator . These results point to the importance of competitive interplay between the positive and negative regulatory proteins in the control of this system . We have also used the dosage effect of GAL4 plasmid in combination with different GAL4 and GAL80 alleles to create new phenotypes . We interpret these phenotypes as indicating that (i) the repressing effects of glucose, at least in part, are mediated by the product of the negative regulatory gene, GAL80, and (ii) the GAL80 protein may have specific interactions with the control regions of the structural genes.

J Gen Virol, 1982 Nov, 63 (Pt 1), 25 - 36
Molecular cloning of the endogenous rat C-type helper virus DNA sequence: structural organization and functional analysis of some restricted DNA fragments; Yang SS et al.; Recently, we have identified and purified the integrated and proviral DNA sequences specific for two endogenous rat type C leukaemia helper viruses: WR-RaLV which originated from a fibrosarcoma induced in a feral rat and RHHV from the cell line HTC-H1 which originated from a Buffalo rat hepatoma . The rat leukaemia helper virus DNA sequences have previously been shown to be 8.4 to 8.8 kilobases (kb) in size . In this communication, we report the molecular cloning of the 8.8 kb DNA of RHHV by ligation at the BamHI site of the vector pBR322, cultured in an Escherichia coli RR1 host . After screening 5750 clones for ampicillin resistance and tetracycline sensitivity and testing by colony hybridization using 32P-labelled RHHV cDNA, four clones were isolated, two of which carried the total 8.8 kb DNA . A detailed restriction endonuclease map of the cloned RHHV DNA was deduced by sequential digestions of either 3'- or 5'-labelled DNA . Of the 14 restriction enzymes tested, EcoRI, BamHI, PstI, KpnI, TaqI, PvuII and SmaI gave informative cleavage patterns . At least two copies of long terminal repeated sequences (LTR) flanking the 3' and 5' termini of the proviral DNA were identified by TaqI and PstI cleavages . LTR in the rat endogenous leukaemia helper virus DNA measured 780 +/- 20 nucleotides in length . The genetic information encoded by the cloned DNA was also analysed by hybridization selection of RHHV mRNA, which was then used in cell-free protein synthesis in a rabbit reticulocyte lysate system . Essentially all major RaLV-specific proteins precipitable by anti-RaLV serum were synthesized in vitro, confirming that the RHHV genomic DNA was successfully cloned with little fidelity loss or scrambling of the genetic information.

J Bacteriol, 1982 Nov, 152(2), 954 - 8
Cloning of the trp-1 gene from neurospora crassa by complementation of a trpC mutation in Escherichia coli; Keesey JK Jr et al.; Studies with a hybrid plasmid containing 4.0 kilobase pairs of Neurospora crassa DNA cloned into plasmid pBR322 indicated that the plasmid restored to prototrophy a trpC mutant of Escherichia coli which lacked phosphoribosyl anthranilate isomerase but not a trpC mutant which lacked indole glycerol phosphate synthetase, that the relevant transcription was initiated at a promoter within the N . crassa DNA, and that the phosphoribosyl anthranilate isomerase could be specified by a subcloned segment of the original DNA.

J Bacteriol, 1982 Nov, 152(2), 829 - 39
The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region; Easton AM et al.; The incompatibility properties of IncFII R plasmid NR1 were compared with those of two of its copy number mutants, pRR12 and pRR21 . pRR12 produced an altered incompatibility product and also had an altered incompatibility target site . The target site appeared to be located within the incompatibility gene, which is located more than 1,200 base pairs from the plasmid origin of replication . The incompatibility properties of pRR21 were indistinguishable from those of NR1 . Lambda phages have been constructed which contain the incompatibility region of NR1 or of one of its copy mutants fused to the lacZ gene . In lysogens constructed with these phages, beta-galactosidase was produced under the control of a promoter located within the plasmid incompatibility region . Lysogens containing prophages with the incompatibility regions from pRR12 and pRR21 produced higher levels of beta-galactosidase than did lysogens containing prophages with the incompatibility region from the wild-type NR1 . The introduction into these inc-lac lysogens of pBR322 plasmids carrying the incompatibility regions of the wild-type or mutant plasmids resulted in decreased levels of beta-galactosidase production . For a given lysogen, the decrease was greater when the pBR322 derivative expressed a stronger incompatibility toward the plasmid from which the fragment in the prophage was derived . This suggested that the incompatibility product acts on its target to repress gene expression in the plasmid replication region.

J Bacteriol, 1982 Nov, 152(2), 773 - 9
Molecular mapping of glyW, a duplicate gene for tRNA3Gly of Escherichia coli; Tucker SD et al.; By the use of {5'-32P}tRNA3Gly from Escherichia coli as a hybridization probe, glyW was located on cloned fragments of the uvrC pgsA region of the bacterial chromosome . After determination of the sites of action of several restriction enzymes, glyW was found to be within approximately 300 base pairs of pgsA . The order of genes in this region is uvrC, pgsA, glyW, flaI . Comparison of the order of determined restriction sites with the sites predicted from the nucleotide sequence of tRNA3Gly indicates that the direction of transcription of glyW is counterclockwise on the circular E . coli map.

J Bacteriol, 1982 Nov, 152(2), 736 - 46
An amber mutation in the gene encoding the beta' subunit of Escherichia coli RNA polymerase; Ridley SP et al.; An Escherichia coli strain carrying an amber mutation (UAG) in rpoC, the gene encoding the beta prime subunit of RNA polymerase, was isolated after mutagenesis with nitrosoguanidine . The mutation was moved into an unmutagenized strain carrying the supD43,74 allele, which encodes a temperature-sensitive su1 amber suppressor, and sue alleles, which enhance the efficiency of the suppressor . In this background, beta prime is not synthesized at high temperature . Suppression of the mutation by the non-temperature-sensitive amber suppressor su1+ yields a protein which is functional at all temperatures examined (30, 37, and 42 degrees C).

J Bacteriol, 1982 Nov, 152(2), 692 - 701
Cloning of and complementation tests with alkaline phosphatase regulatory genes (phoS and phoT) of Escherichia coli; Amemura M et al.; The regulatory genes of alkaline phosphatase, phoS and phoT, of Escherichia coli were cloned on pBR322, initially as an 11.8-kilobase EcoRI fragment . A restriction map of the hybrid plasmid was established . Deletion plasmids of various sizes were constructed in vitro, and the presence of phoS and phoT genes on the cloned DNA fragments was tested by introducing the plasmids into phoS64 and phoT9 strains for complementation tests . One set complemented only phoS64 but not phoT9; the other set complemented only phoT9 but not phoS64 . We conclude that phoS64 and phoT9 mutations belong to different complementation groups and probably to different cistrons . The hybrid plasmid with the 11.8-kilobase chromosomal fragment also complemented the phoT35 mutation . A smaller derivative of the hybrid plasmid was constructed in vitro which complemented phoT35 but did not complement phoS64, phoT9, or pst-2 . Our results agree with the suggestion that phoT35 lies in a different complementation group from phoS, phoT, or pst-2 (Zuckier and Torriani, J . Bacteriol . 145:1249--1256, 1981) . Therefore, we propose to designate phoT35 as phoU . The effect of amplification of phoS or phoT on alkaline phosphatase production was examined . It was found that multiple copies of the phoS gene borne on pBR322 repressed enzyme production even in low-phosphate medium, whether it was introduced into wild-type strains (partially repressed) or phoR (phoR68 or phoR17) strains (fully repressed), whereas the introduction of multicopy plasmids bearing the phoT gene did not affect the inducibility of the enzyme.

J Bacteriol, 1982 Nov, 152(2), 650 - 60
Escherichia coli phenylalanyl-tRNA synthetase operon: characterization of mutations isolated on multicopy plasmids; Plumbridge JA et al.; Plasmid pB1 carries the genes for threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase, and translation initiation factor IF3 . Strains carrying this plasmid overproduce phenylalanyl-tRNA synthetase about 100-fold . Spontaneous mutant plasmids were obtained which no longer caused the overproduction of the enzyme . Three classes of mutations were found . (i) Deletion mutations were found, some of which had the interesting property of fusing different genes together, e.g., putting phenylalanyl-tRNA synthetase under the control of the threonyl-tRNA synthetase promoter . (ii) Insertion mutations were found; one insertion in particular was studied . This insertion is located in front of the structural gene for phenylalanyl-tRNA synthetase and is shown to interrupt a cis-acting regulatory region . (iii) Mutations that showed no major change in DNA structure were found . One of these mutations is apparently purely structural, as it produces a small subunit of phenylalanyl-tRNA synthetase with a reduced molecular weight . This protein is less stable than the wild-type enzyme . These mutations represent useful tools to investigate how the phenylalanyl-tRNA synthetase operon is regulated.

Mutat Res, 1982 Nov, 106(1), 11 - 26
Effects of a recA operator mutation on mutant phenotypes conferred by lexA and recF mutations; Clark AJ et al.; Derepression of recA by an operator mutation (recAo281) produces effects opposite to those obtained from its derepression following DNA damage . Inducible reactivation of lambda vir and S13 phages is decreased and inducible UV mutagenesis of a phi X174 amber mutant is lessened in a recAo281 strain compared to a recAo+ strain . The decreases could not be accounted for by increases in constitutive levels of these processes . Consistent with these results the UV resistance of a recAo281 strain is less than that of a recAo+ strain . This may indicate that too much recA protein immediately after irradiation interferes with derepression of the lexA regulon or functioning of its products . Effects of increasing the recAo+ and recA+ copy number on a Co1E1 plasmid are compared with the effects of recAo281 . recAo281 partially suppresses UV sensitivity due to lexA102 and lexA3 in E . coli K-12 . This increase in resistance is not correlated with an increase in constitutive or inducible reactivation of UV-irradiated lambda vir or S13 . This is consistent with the previous suggestion that the UV resistance stems from a decrease in DNA degradation allowing an increase in DNA repair . lexA3 blocks UV mutagenesis of phi X174 as measured by reversion of amber mutations and this was not suppressed by recAo281 . recF143 blocks UV mutagenesis of phi X174 . recAo281 suppresses neither this effect nor the decrease in bacterial UV resistance caused by recF143.

Gene, 1982 Nov, 20(1), 39 - 49
Isolation of the origin of replication of the IncW-group plasmid pSa; Tait RC et al.; The origin of replication of the IncW plasmid pSa has been cloned and the function of this origin in Escherichia coli examined . A 1.9-kb region of DNA is required for efficient autonomous replication, and a 0.47-kb fragment within this region can initiate replication only in the presence of an autonomously replicating derivative of pSa . An Mr 35,000 protein (repA) is encoded adjacent to the origin and is required for efficient initiation of replication . The derivatives examined provide information suggesting a direct role of partition factors in plasmid replication and incompatibility.

Cell, 1982 Nov, 31(1), 61 - 70
Analysis of nutR: a region of phage lambda required for antitermination of transcription; Olson ER et al.; The N gene product of coliphage lambda acts with host factors (Nus) through sites (nut) to render subsequent downstream transcription resistant to a variety of termination signals . These sites, nutR and nutL, are downstream, respectively, from the early promoters PR and PL . Thus a complicated set of molecular interactions are likely to occur at the nut sites . We have selected mutations in the nutR region that reduce the effectiveness of pN in altering transcription initiating at the PR promoter . DNA sequence analysis of three independently selected mutations revealed, in each case, a deletion of a single base pair in the cro gene . Consideration of the effect of such mutations on the extension of translation of cro message into the adjacent downstream nut region led to the identification of a consensus sequence CGCTCT(T)TAA that appears to play a role in the recognition of a host factor, possibly the NusA protein.

Cell, 1982 Nov, 31(1), 53 - 60
Transient generation of displaced single-stranded DNA during nick translation; Lundquist RC et al.; We show that displaced single-stranded overhangs are transiently generated and destroyed during nick translation by E . coli DNA polymerase I . Evidence that hyper-rec mutants have an increased frequency of such overhang structures is discussed . The transient generation of overhangs may be significant for general recombination . The 5' leads to 3' exonuclease activity of polymerase I specifically hydrolyzes such overhangs to yield a nick . Overhangs are generated by polymerization, but after every polymerization step, either polymerase or exonuclease can act--55% of the time, polymerization occurred first . At this frequency overhangs of greater than or equal to 12 nucleotides are generated every 1300 nucleotides polymerized . We suggest that many DNA strand discontinuities are displaced single-stranded overhangs, rather than gaps or simple nicks.

J Dairy Res, 1982 Nov, 49(4), 587 - 96
Qualitative and quantitative determination of proteolysis in mastitic milks; de Rham O et al.; Proteolytic activity in mastitic skim-milk was often 5-10 fold higher than in normal milk, its level being related to somatic cell count but not precisely correlated with it . In milks with the highest levels of activity plasmin accounted for about one third of the total proteinase . A further third was sedimented with the micellar fraction together with the plasmin, but unlike plasmin, was not inhibited by addition of soyabean trypsin inhibitor (SBTI) . The final third remained in the serum phase . Polyacrylamide gel electrophoresis (PAGE) showed that alpha-sl- and beta-caseins were degraded at about the same overall rate . The plasmin produced the usual readily identified fragments from beta-casein, but incubation of mastitic milk also produced changes in patterns in the gamma-casein region differing from plasmin-induced changes, which were also apparent when the micellar fraction was incubated . As they were inhibited by SBTI, a second trypsin-like enzyme in addition to plasmin may also have been present . Other proteinase(s) not inhibited by SBTI was also associated with casein micelles and produced at least 3 characteristic protein fragments seen on PAGE . The serum phase proteinase(s) was likewise not inhibited by SBTI, and did not produce any well-defined electrophoretic bands, suggesting a rather non-specific breakdown of caseins . After separation of mastitic whole milk, a considerable proportion of the proteolytic activity was found in the cream phase . The proportion was enhanced by freezing and thawing, and the enzyme appeared to be identical to the SBTI-resistant micellar proteinase . Because of the considerable proteolysis likely to occur under the time and temperature conditions involved, our results may provide some explanation for the problems encountered in cheesemaking with mastitic milks (e.g . yield losses, poor curd strength and off-flavour development).

J Bacteriol, 1982 Nov, 152(2), 963 - 7
Analysis of hemolytic determinants of plasmid pHly185 by Tn5 mutagenesis; Stark JM et al.; A nonconjugative hemolysin plasmid, pHly185, was isolated from Escherichia coli 185 . Tn5 mutagenesis produced nonhemolytic derivatives that either expressed no hemolytic activity or did not secrete activity . These Tn5 insertions were located in three contiguous EcoRI fragments . Insertions inactivating hemolysin structural gene(s) were identified on all three EcoRI fragments . Mutations affecting the secretory function were clustered in one portion of a single EcoRI fragment.

J Bacteriol, 1982 Nov, 152(2), 815 - 21
In vitro membrane association of the F0 polypeptides of the Escherichia coli proton translocating ATPase; Decker KP et al.; The F0 polypeptides a, b, and c of the H+-translocating ATPase associated with membranes when synthesized in vitro . This association occurred when the membranes were present either cotranslationally or post-translationally . In addition, the F0 polypeptides associated with liposomes . The membrane association seemed to be an insertion process since there was protection of polypeptides a and c from proteolysis . The in vitro insertion of the F0 polypeptides a, b, and c was independent of the synthesis of each polypeptide and of the F1 polypeptides.

Z Naturforsch {C}, 1982 Nov-Dec, 37(11-12), 1228 - 33
{Induction of single- and double-strand breaks in linear and superhelical DNA by phleomycin}; Schmulling F et al.; Phleomycin induced DNA breakage was investigated with superhelical Col . E1 DNA and linear T2 DNA as well . In both DNA-forms besides single-strand breaks direct double-strand breaks were produced by the drug . The double-strand breakage rate obtained after treatment with phleomycin, however, was considerably smaller than that found after bleomycin treatment . Whereas the single- and double-strand breakage rate in Col . E1 DNA showed a linear and nearly quadratic dependence on the phleomycin concentration, respectively, in T2 DNA the breakage rates increased faster than the first or second power of the concentration . This indicates various modes of drug-DNA interaction . Under nondegrading conditions a strong retardation of electrophoretic mobility was observed for all three topological isomers of Col . E1 DNA whereas the sedimentation behaviour remained unchanged . The in vitro effects (strand breakage) of phleomycin and bleomycin are compared with induction of chromosomal aberrations in human lymphocytes and oocytes of Drosophila melanogaster.

Vopr Virusol, 1982 Nov-Dec, 27(6), 677 - 81
{Electron microscopic analysis of the individual genes of the influenza virus}; Makhov AM et al.; The lengths of fragments of influenza virus strain A/USSR/90/77 (H1N1) genome RNA separated by electrophoresis and treatment with 0.25 M glyoxal solution were measured . The number of nucleotides of these fragments was estimated in regard to marker ribosomal 16 S and 23 S E . coli RNA . The results were compared with analogous data obtained by other methods.

Mol Cell Endocrinol, 1982 Nov-Dec, 28(3), 411 - 24
Nucleotide sequence of bovine parathyroid hormone messenger RNA; Weaver CA et al.; The sequence of bovine parathyroid hormone mRNA has been determined by sequence analysis of near full-length cloned DNA complementary to the mRNA . Restriction fragments hybridized to the mRNA and extended toward the 5' terminus with reverse transcriptase were analyzed to derive the sequence not present in cDNA . The reverse transcripts were heterogeneous in length with three major stopping points within 8 nucleotides of each other and a minor stop about 30 bases further toward the 5' terminus of the mRNA . The sequence of the gene corresponding to the minor reverse transcript begins with the sequence 5' XXXATATATAAAA which contains the consensus sequence for a TATA box, a putative eukaryotic promoter sequence . Assuming that the major reverse transcriptase stop nearest the 5' terminus of the mRNA, which is 24 bases downstream from the TATA box, represents the beginning of bovine PTH mRNA, the mRNA contains 672 nucleotides, 100 in the 5' noncoding region, 348 in the coding region and 224 in the 3' noncoding region . Bovine PTH mRNA contains 38% G and C bases . The 3' noncoding region is particularly rich in A and U bases with the last 100 nucleotides of the molecules containing 46% U and 32% A . As with other mRNAs, the sequences CG and UAG occur much less than expected . The 5' noncoding region does not contain an AUG before the initiator codon and contains two potential regions that could base-pair with sequences near the 3' terminus of 18S ribosomal RNA . The sequence AAUAAA is present 14 nucleotides from the polyadenylic acid at the 3' terminus . Bovine PTH mRNA exhibits extensive homology with human PTH mRNA.

Antimicrob Agents Chemother, 1982 Nov, 22(5), 824 - 31
7-Hydroxytropolone: an inhibitor of aminoglycoside-2"-O-adenylyltransferase; Allen NE et al.; Aminoglycoside-2"-O-adenylyltransferase was inhibited by 7-hydroxytropolone . Inhibition was competitive with respect to the cosubstrate ATP and appeared to require the unique vicinal arrangement of oxygens found in 7-hydroxytropolone . Combinations of 7-hydroxytropolone plus the appropriate aminoglycoside substrates were active against resistant bacteria possessing the adenylyltransferase . No potentiation was observed against other aminoglycoside-resistant or -susceptible strains . The fact that the inhibition of an aminoglycoside-modifying enzyme overcomes the poor uptake of aminoglycosides in resistant strains points to the singular importance of the inactivating enzyme as a determinant of resistance.

Eur J Biochem, 1982 Nov, 128(1), 47 - 52
70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding; Grajevskaja RA et al.; Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites . This conclusion is based on several findings . Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac{14C}Phe-tRNAPhe, ribosomes are capable of binding additionally deacylated non-cognate {3H}tRNA . If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes . In the absence of poly(U), when the A site can not bind, the P site and the 'additional' site can be filled simultaneously with Ac{14C}Phe-tRNAPhe and deacylated {3H}tRNA, or with {3H}tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes . The binding at the 'additional' site is not sensitive to the template . {3H}tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution . Deacylated tRNA is preferred to the aminoacylated one . The binding of AcPhe-tRNAPhe was not observed there at all . The 3'-end adenosine is essential for the affinity . The function of the 'additional' site is not known, but its existence has to be considered when tRNA . ribosome complexes are studied.

J Bacteriol, 1982 Nov, 152(2), 584 - 94
Different control circuits for growth rate-dependent regulation of 6-phosphogluconate dehydrogenase and protein components of the translational machinery in Escherichia coli; Farrish EE et al.; Previous studies showed that the level of 6-phosphogluconate (6PG) dehydrogenase increases about fourfold with increasing growth rate when the growth rate is varied by varying the carbon source . When the growth rate was reduced by anaerobic growth or by using mutations to divert metabolism to less efficient pathways, the level of 6PG dehydrogenase was the same as in a wild-type strain growing aerobically on other carbon sources that yielded the same growth rate . Thus, expression of gnd, which encodes 6PG dehydrogenase, is regulated by the cellular growth rate and not by specific nutrients in the medium . Growth rate-dependent regulation was independent of temperature . After a nutritional shift-up, 6PG dehydrogenase and total protein did not attain the postshift rate of accumulation for 30 min, whereas RNA accumulation increased immediately . The kinetics of accumulation of 6PG dehydrogenase and RNA were coincident after a nutritional shift-down . Partial amino acid starvation of a strain that controls RNA synthesis stringently (rel+) had no effect on the differential rate of accumulation of the enzyme . The level of 6PG dehydrogenase in cells harboring a gnd+ multicopy plasmid was in approximate proportion to gene dosage and somewhat higher at faster growth rates . Growth rate control of chromosomal gnd was normal in strains carrying multiple copies of the promoter-proximal and promoter-distal portions of gnd . These results show that gnd is not part of the same regulatory network as components of the translational apparatus since gnd shows a delayed response to a nutritional shift-up, is not autoregulated, and is not subject to stringent control . Models to account for growth rate-dependent regulation of gnd are discussed.

J Immunol, 1982 Nov, 129(5), 1952 - 9
Rapid induction of seven proteins in human lymphocytes by interferon; correlation to natural killer cell activity; Gustafsson A et al.; The early effects of interferon (IFN) on the synthesis of protein in human nylon wool-nonadherent lymphocytes have been stimulated by use of two-dimensional electrophoresis . IFN-alpha or -beta as well as Escherichia coli-produced IFN-alpha 2 induced the rapid formation of seven proteins (Mr 80, 75, 62, 53, 38, 36, and 33 kD) . At least five proteins were expressed within 2 hr of incubation with IFN . The synthesis of the seven proteins seemed to require rapid transcription of new RNA, because actinomycin D markedly inhibited their formation only when added less than 30 min after IFN . A good correlation was found between the ability of actinomycin D to inhibit both the formation of new proteins and the augmentation of natural killer (NK) cell activity . Screening of a panel of 10 hematopoietic and two anchorage-dependent cell lines revealed that p62 and p38 were induced in most cell lines, whereas p80 and p33 were preferentially induced in lymphoid cell lines . Three proteins could not be induced by IFN in any of the 12 cell lines, and thus could represent molecules mediating differentiated functions, possibly involved in NK cell function.

Infect Immun, 1982 Nov, 38(2), 764 - 73
Antigenic quantitation of type 1 fimbriae on the surface of Escherichia coli cells by an enzyme-linked immunosorbent inhibition assay; Dodd DC et al.; Type 1 fimbriae from two strains of Escherichia coli, K-12-derived CSH50 and a clinical isolate VL-2, were purified by a simplified procedure, which should be applicable to a variety of bacterial strains . After mechanical removal from the cells, the fimbriae were sedimented in the ultracentrifuge and resuspended in 5 M urea to disaggregate cell membranes and flagella, leaving the urea-resistant fimbriae intact . After several hours at 37 degrees C, this crude fimbrial suspension was diluted to 1 M urea, and the intact fimbriae were sedimented through a 1 M urea-1 M sucrose cushion . The pellet was found to be pure fimbriae by sodium docecyl sulfate-polyacrylamide gel electrophoresis, with apparent subunit molecular weights of 17,000 for the fimbriae from K-12 strain CSH50 and 19,000 for those from the clinical isolate VL-2 . High-titer rabbit antiserum raised against CSH50 fimbriae was specific for fimbriae by indirect ferritin labeling and immunoprecipitation and was used to develop an enzyme-linked immunosorbent assay . Competitive inhibition of antifimbrial antiserum in the enzyme-linked immunosorbent assay by a known amount of either purified fimbriae or fimbriae-bearing bacteria permitted precise quantitation of fimbrial antigen in cultures of strain CSH50, thereby providing a simple means of determining the effects of environmental conditions on the synthesis of type 1 fimbriae.

Infect Immun, 1982 Nov, 38(2), 739 - 44
Cloning of genes determining the production of mannose-resistant fimbriae in a uropathogenic strain of Escherichia coli belonging to serogroup O6; Clegg S; Chromosomal DNA from a uropathogenic strain of Escherichia coli was partially digested with the restriction enzyme EcoRI . The partial digests were ligated into a cosmid containing an ampicillin-resistant determinant and packaged into lambda phage particles . An ampicillin-resistant transductant of E . coli HB101 was found to possess mannose-resistant hemagglutinating activity associated with a 50-kilobase-pair plasmid . Subcloning of the mannose-resistant fimbrial genes revealed that the genetic determinants were encoded by a 6.9-kilobase-pair DNA fragment of a recombinant plasmid . Chimeric plasmids smaller in size were unable to transform E . coli to fimbrial production . Physical maps of the recombinant plasmids were prepared showing restriction endonuclease sites within the inserted DNA fragments . The hemagglutinating activities of the wild-type strain and of the recombinant derivative were compared . Both strains agglutinated human erythrocytes in the presence of D-mannose to the same degree and also failed to produce fimbriae after incubation at 18 degrees C . Also, both strains were agglutinated by antifimbrial serum at a high titer, whereas no such activity was observed when a strain of E . coli which did not possess a plasmid was used.

Biochim Biophys Acta, 1982 Oct 29, 699(1), 53 - 9
Cytosol induces apparent selectivity of glucocorticoid receptor binding to nucleic acids of different secondary structure; Romanov GA et al.; Unpurified rat liver glucocorticoid-receptor complexes within cytosol show a distinct binding preference for double-stranded DNA over single-stranded DNA; the binding to Escherichia coli rRNA is negligible . Extensive purification of the receptor abolishes its ability to distinguish among DNAs of different secondary structure and the affinity of the purified receptor toward RNA is greatly enhanced, reaching 30-50% of that of DNA . The purification effect is reversible: after cytosol addition to purified receptor preparation the binding preference restores . NaCl does not mimic the effect of cytosol . The flow-through fraction of a phosphocellulose column retains the ability of crude cytosol to produce selective decrease in the receptor binding to single-stranded DNA . This effect may also be observed by using two types of DNA-cellulose bearing double-stranded or denatured DNA, pretreated with crude cytosol . Additionally, pretreatment of immobilized DNA with even low cytosol concentrations has been shown to markedly enhance receptor binding, although this enhancement was lacking specificity with respect to DNA secondary structure . The nature of cytosolic active principle and some possible regulatory implications are discussed.

Biochim Biophys Acta, 1982 Oct 29, 699(1), 67 - 73
Inhibition of calf thymus DNA polymerase alpha and of normal and cancer cell growth by butylanilinouracil and butylphenylguanine; Rochowska M et al.; 6-(p-n-Butylanilino)uracil and N2-(p-butylphenyl)guanine inhibited the activity of DNA polymerase alpha from calf thymus but had no effect on other eukaryotic polymerases (DNA polymerases beta and gamma) or Escherichia coli DNA polymerase I . Inhibition was competitive with deoxyguanosine 5'-triphosphate and did not occur in the reaction of DNA polymerase alpha with a template that did not contain cytosine residues . The results support a mechanism which involves hydrogen bonding of inhibitors with cytosines in the DNA template and binding with an inhibitor specific site on the enzyme . A screen of inhibitor effects on normal and cancer cell growth in culture showed that cells were not uniformly sensitive to these compounds, a mouse lymphoma line being least sensitive and a human lung cancer line being most sensitive . It is suggested that these inhibitors may be useful to probe possible structural differences among DNA polymerases alpha.

Biochim Biophys Acta, 1982 Oct 29, 699(1), 28 - 39
In vitro transcription of phage T4 late genes on purified DNA by partially purified RNA polymerase from T4-infected Escherichia coli b cells; Westin G et al.; RNA polymerase was purified from 'late' phage T4-infected Escherichia coli B cells by DNA-cellulose affinity chromatography and high salt agarose filtration . The DNA-cellulose-purified RNA polymerase preparation contained T4-coded DNA endonuclease activity and several proteins, some with sizes comparable with the known T4 maturation factors, essential for late RNA synthesis . Some of these proteins, and the DNA endonuclease utilizing native, parental T4 DNA and supercoiled phi X 174 DNA as substrates, were partially separated from the RNA polymerase as a complex during agarose filtration . In vitro RNA was made by the DNA-cellulose-purified RNA polymerase using native, parental T4 DNA as template . About 26% of the in vitro RNA was transcribed from the DNA r-strand; 75% from the same r-strand region as in vivo late after infection . Both the abundancy and specificity of the in vitro r-strand transcription were markedly reduced after agarose filtration of the enzyme . Addition of the proteins separated from the RNA polymerase during agarose filtration caused a restoration of in vitro r-strand transcription abundance, but not its specificity . These results show that partially purified RNA polymerase from T4-infected E . coli B cells was able to transcribe late T4 genes in vitro with some abundancy and specificity on purified, parental T4 DNA, but further purification of the enzyme caused an irreversible reduction of this ability.

Biochim Biophys Acta, 1982 Oct 28, 719(1), 165 - 7
Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli; Alonso JM et al.; 5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway has been purified to 96% homogeneity . The native enzyme, which appears to be a tetramer, has an apparent molecular weight of 210000 . The purified enzyme shows a narrow pH optimum at pH 7.8 and does not require ions for its catalytic activity . Under standard assay conditions the enzyme acts preferentially with NAD but reduces NADP at 11% of the rate observed for NAD, primarily because of a difference in Km . Apparent Km values are 6.4 micro M for 5-carboxymethyl-2-hydroxymuconic semialdehyde and 52.2 micro M for NAD.

Biochemistry, 1982 Oct 26, 21(22), 5468 - 74
Synthesis and template properties of an ethyl phosphotriester modified decadeoxyribonucleotide; Miller PS et al.; The phosphate groups of nucleic acids are often the targets of mutagenic and carcinogenic alkylating agents . In order to study the effects of alkyl phosphotriester modification on the physical and biochemical properties of DNA, two diastereomeric ethyl phosphotriester modified decadeoxyribonucleotides, d-CpCpApApGp(Et)ApTpTpGpG isomer I and isomer II, were prepared . A phosphotriester synthetic procedure was used to specifically place ethyl triester groups with either an R or S configuration in the central dimer region of the decamer . Terminal deoxynucleotidyl transferase was used to add oligodeoxyadenylate tails to the 3' end of the decamers . The resulting oligomers were tested as templates for Escherichia coli DNA polymerase I with d-(pT)8pCpC as a primer . The rates and extents of polymerization directed by the modified templates were 25% (isomer I) and 50% (isomer II) less than those of an unmodified control template . Thus the presence of an ethyl triester group inhibits polymerization, the effectiveness of which is determined by the orientation of the ethyl group relative to the rest of the template backbone . These results suggest ethyl phosphotriester lesions could inhibit replication rates of cellular DNA.

Biochemistry, 1982 Oct 26, 21(22), 5539 - 51
Aggregation equilibria of Escherichia coli RNA polymerase: evidence for anion-linked conformational transitions in the protomers of core and holoenzyme; Shaner SL et al.; The aggregation equilibria of Escherichia coli RNA polymerase core and holoenzyme have been studied by velocity sedimentation as a function of {NaCl} both in the presence and in the absence of MgCl2 . Effects of other anions (F- and I-), pH, and temperature have also been examined . Diffusion coefficients obtained by quasi-elastic light scattering (QLS) at high and low salt concentrations were used in conjunction with sedimentation coefficients under these conditions to obtain molecular weights of the protomer and aggregates of the core enzyme . At low salt concentration, core aggregates to a tetramer in the absence of MgCl2 and to an octamer in the presence of MgCl2 . Some ambiguity exists in the interpretation of the sedimentation and QLS data for holoenzyme . The sedimentation results are consistent with the formation of dimers at low salt, both in the presence and in the absence of MgCl2 . In all cases, equilibrium constants were calculated assuming a simple monomer--j-mer stoichiometry . These equilibrium constants are extremely sensitive functions of the concentration and type of monovalent anion . In Cl-, aggregation of both core and holoenzyme begins abruptly when the salt concentration is reduced below approximately 0.2 M (at a protein concentration of approximately 0.30 mg/mL); for core, substitution of I- for Cl- suppresses aggregation while F- enhances aggregation at a fixed anion concentration . No specific effect of monovalent cations (Na+, NH4+) is observed; Mg2+ has no effect on holoenzyme dimerization and has little effect on the salt range of core aggregation, though the stoichiometries of the core aggregates in the presence and absence of Mg2+ differ . Anion effects on these equilibria were modeled by assuming that a class of anion-binding sites on the protomer is not present in the aggregate, so that anion release accompanies aggregation . Analytical expressions for several models of the effect of anions on the aggregation equilibria were derived by using the method of binding polynomials . The salt dependence of the aggregation equilibria in the absence of Mg2+ appears inconsistent with a model in which the anion-binding sites on the protomer are independent (noncooperative), but it is well described by a model in which anion binding to the protomers occurs in a completely cooperative manner . The molecular basis of this apparent cooperative effect of anions on the aggregation equilibria is proposed to be an allosteric effect of anions on conformational equilibria of the protomers of core polymerase and the holoenzyme . Implications of such a salt-dependent conformational transition for the DNA-binding interactions of the enzyme are considered.

Biochemistry, 1982 Oct 26, 21(22), 5634 - 8
Lactose-proton symport by purified lac carrier protein; Foster DL et al.; The lac carrier protein of Escherichia coli was purified by an improved procedure and its activity assayed by a rapid filter method . Following reconstitution of the carrier by octyl glucoside dilution, proteoliposomes were concentrated by filtration on a microporous filter . Lactose accumulation by adsorbed or entrapped proteoliposomes is driven by an artificially imposed pH gradient (interior alkaline), by a membrane potential (interior negative), or by a combination of both forces . Activity is almost completely abolished by the protonophore carbonyl cyanide m-chlorophenylhydrazone or by the competitive inhibitor thiodigalactoside . Addition of lactose to proteoliposomes under appropriate conditions results in alkalinization of the external medium . This effect is not observed with liposomes devoid of lac carrier or in the presence of proton conducting agents . The results provide a strong indication that the lac gamma gene product is the only protein in the cytoplasmic membrane of Escherichia coli required for lactose-proton symport.

Biochemistry, 1982 Oct 26, 21(22), 5534 - 8
Stoichiometry of the H+-ATPase of growing and resting, aerobic Escherichia coli; Kashket ER; The H+/ATP stoichiometry of the proton-translocating ATPase was investigated in growing and nongrowing, respiring cells of Escherichia coli . The protonmotive force, delta p, was determined by measuring the transmembrane chemical gradient of protons, delta pH, from the cellular accumulation of benzoate anions, and the electrical gradient, delta psi, from the accumulation of the lipophilic cation tetraphenylphosphonium (TPP+) . The accumulation of lactose was also used to calculate the delta p in this lactose operon constitutive beta-galactosidase negative mutant . The phosphorylation potential, delta GP', was determined by measuring the cellular concentration of ATP, ADP, and inorganic phosphate . According to chemiosmotic principles, at steady state the phosphorylation potential is in thermodynamic equilibrium with the protonmotive force, and thus the ratio delta p/delta GP' can be used to determine the H+/ATP ratio . Respiring E . coli cells, in mid-exponential phase of growth or incubated in buffer, at external pHs from 6.25 to 8.25 had a constant delta GP' of about 500 mV . The H+/ATP ratio was found to be 3 when the delta p value derived from lactose accumulation levels was used . However, when the delta p values derived from delta pH and delta psi were used in the calculations, the H+/ATP ratio varied from about 2.5 at external pH 6.25 to about 4 at pH 8.25 . Arguments are presented for the hypothesis that the delta psi values obtained from the TPP+ measurements are likely to be inaccurate and that a value of 3 H+/ATP, independent of the external pH, is likely to be the valid stoichiometry.

Nucleic Acids Res, 1982 Oct 25, 10(20), 6487 - 500
Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA; Zoller MJ et al.; This paper presents a versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA fragments cloned into M13 derived vectors . As an example, production of a transition mutation in a clone of the yeast MATa1 gene is described . The oligonucleotide is hybridized to the template DNA and covalently closed closed double stranded molecules are generated by extension of the oligonucleotide primer with E . coli DNA polymerase (large fragment) and ligation with T4 DNA ligase . The resulting double stranded closed circular DNA (CC-DNA) is separated from unligated and incompletely extended molecules by alkaline sucrose gradient centrifugation . This purification is essential for production of mutants at high efficiency . Competent E . coli JM101 cells are transformed with the CC-DNA fraction and single stranded DNA is isolated from individual plaques . The recombinants are screened for mutant molecules by 1) restriction endonuclease screening for the loss of the Hinf I site in the target region, and 2) by dot blot hybridization using the mutagenic oligonucleotide as probe . Double stranded DNA is isolated from the sequencing . Efficiency of mutant production is in the range of 10-45% and no precautions to prevent mismatch repair are required.

Nucleic Acids Res, 1982 Oct 25, 10(20), 6401 - 10
Sequence analysis of heteropolymeric DNA synthesized in vitro by the enzyme terminal deoxynucleotidyl transferase and cloned in Escherichia coli; Damiani G et al.; We have synthesized in vitro single strands of DNA in reaction mixtures containing the terminal deoxynucleotidyl transferase (Bollum's enzyme), an oligo-dG as primer, the four common deoxynucleoside triphosphates and both Mg and Co ions . The resulting heteropolymers have been converted into double strands, tailed with oligo-dC sequences, annealed with an oligo-dG tailed plasmid vector and cloned in E . coli . Six recombinant plasmids have been isolated and characterized . Two of them have been sequenced . The heteropolymeric chains produced by the terminal transferase, ranging in size between 200 and 400 nucleotides, are richer in purines than in pyrimidines, except in the last portions . Open reading frames for 7-20 amino acids with repeated, in phase translational stop codons are present in these sequences and in their complements.

J Biol Chem, 1982 Oct 25, 257(20), 12063 - 8
S-adenosylmethionine decarboxylase of Escherichia coli . Studies on the covalently linked pyruvate required for activity; Markham GD et al.; A covalently linked pyruvoyl group is essential for the enzymatic activity of S-adenosylmethionine decarboxylase from Escherichia coli . A rapid purification method based on affinity chromatography is described for the isolation of this enzyme from an E . coli K12 strain which contains a plasmid containing the structural gene for S-adenosylmethionine decarboxylase, and which overproduces this enzyme . The purified enzyme contains one pyruvate moiety on each of six subunits . The enzyme is inactivated by incubation with carbonyl group reagents such as NaBH4 and phenylhydrazine; after inactivation, 1 mol of lactate or 1 mol of phenylhydrazone is found/mol of enzyme subunit . The enzyme is also inactivated by NaCNBH3 but only in the presence of either substrate or product and the divalent metal ion activator Mg2+; inactivation is accompanied by incorporation of 1 mol of the product, decarboxylated adenosylmethionine, per mol of enzyme subunit, suggesting that the pyruvoyl group participates in catalysis by formation of a Schiff base with the substrate . Equilibrium dialysis studies indicated a single substrate (or product) binding site/enzyme subunit.

J Biol Chem, 1982 Oct 25, 257(20), 12060 - 2
Copies of protein S6 in Escherichia coli ribosomes; Bartsch M et al.; A reinvestigation of the copy number of protein S6 in Escherichia coli ribosomes shows that there is only one copy of S6 in 30 S subunits and in 70 S ribosomes . The previously reported higher value (Subramanian, A . R . (1980) J . Biol . Chem . 255, 6941-6946) is shown to arise from the presence in the usual ribosome preparations of a protein which co-electrophoreses with S6 but does not react with S6 antiserum . This protein is removed when ribosomes are purified by passage through a sucrose gradient.

J Biol Chem, 1982 Oct 25, 257(20), 11876 - 8
Studies on identifying the allosteric binding sites of deoxycytidylate deaminase; Maley F et al.; Thymidine triphosphate, a negative regulator of deoxycytidylate deaminase, was found to bind covalently to this enzyme on exposure to UV light at 254 nM . The rate of half-maximal fixation was extremely rapid, occurring within 30 s and probably attaining a maximum of about 1 mol of dTTP fixed/mol of enzyme subunit . In contrast to the case of ribonucleotide reductase (Ericksson, S., Caras, I . W., and Martin, D . W., Jr . (1982) Proc . Natl . Acad . Sci . U . S . A . 79, 81-85) where the fixation of dTTP inactivated this enzyme, the activity of the deaminase was unaffected . The bound nucleotide could be released on exposure to UV 254 nm light in the presence of dCTP or dTTP but not dATP or dGTP . The enzyme-fixed nucleotide was found to remain with the larger of the two peptides released as a result of CNBr treatment of the labeled enzyme . Studies are in progress to define the location of this nucleotide, which will be aided greatly by our recent clarification of the complete amino acid sequence of T2-deoxycytidylate deaminase.

Nucleic Acids Res, 1982 Oct 25, 10(20), 6475 - 85
Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments; Kramer W et al.; Gapped duplex DNA molecules of recombinant genomes of filamentous phage are constructed in vitro . Denatured restriction fragments covering (part of) the precisely constructed gap are hybridized to the gapped duplex DNA molecules to form ternary duplices . The two strands of the ternary duplex molecules carry different genetic markers within the region spanned by the restriction fragment leading to a one base pair mismatch or to an insertion loop of 93 nucleotides, respectively . The two strands also vary with respect to A-methylation in GATC sites . In cases of asymmetrical methylation, transfection of E . coli with these heteroduplex molecules leads to marker recoveries with a pronounced bias in favour of the marker encoded by the methylated strand . This effect at least partly explains the comparably low marker yields achieved in previous directed mutagenesis experiments using filamentous phage as the vector . The results suggest how these procedures can be optimized . Precise construction of a 93 bp insertion of 9.5% marker yield is described.

Science, 1982 Oct 22, 218(4570), 381 - 4
Herpes simplex virus type-1 glycoprotein D gene: nucleotide sequence and expression in Escherichia coli; Watson RJ et al.; The protein coding region of the herpes simplex virus type-1 glycoprotein D (gD) gene was mapped, and the nucleotide sequence was determined . The predicted amino acid sequence of the gD polypeptide was found to contain a number of features in common with other virus glycoproteins . Insertion of this protein coding region into a bacterial expressor plasmid enabled synthesis in Escherichia coli of an immunoreactive gD-related polypeptide . The potential of this system for preparation of a type-common herpes simplex virus vaccine is discussed.

J Am Vet Med Assoc, 1982 Oct 15, 181(8), 808 - 10
Pleuritis secondary to pneumonia or lung abscessation in 90 horses; Raphel CF et al.; Of 122 horses with pleural effusion, 90 (73.8%) had pleuritis secondary to pneumonia or lung abscessation . Fifty-one horses died or were euthanatized . The highest prevalence was in Thoroughbred and Standardbred racehorses . Eleven (12.2%) horses were postsurgical patients and 22 (24.4%) horses had been transported over 500 miles . There was no relationship between final outcome and the age, sex, breed, hematologic values, or laboratory findings pertaining to pleural fluid except for the bacterial isolation of Escherichia coli from the pleural fluid, as this was more frequently associated with death . Follow-up on 38 of the 39 horses that survived showed that 18 (46.2%) recovered and were able to return to performance equal to that prior to their illness . Ten (25.6%) were returned for breeding or pleasure use, with no attempt made to return them to racing . Follow-up was not available for 5 horses, 4 horses had just recently been discharged from the hospital, and 2 horses are racing poorer than prior to their illness.

Biochemistry, 1982 Oct 12, 21(21), 5280 - 8
Binding of spermidine to transfer ribonucleic acid; McMahon ME et al.; The binding of spermidine to yeast tRNAPhe and Escherichia coli tRNAGlu2 at low and high ionic strength was studied by equilibrium dialysis . Once corrected for the expected Donnan effect, the binding at low ionic strength obeys the simple relationship of equivalent binding sites, and cooperative binding of spermidine to tRNA could not be detected . At low ionic strength (0.013 M Na+ ion), tRNAPhe (yeast) has 13.9 +/- 2.3 strong spermidine binding sites per molecule with Kd = 1.39 X 10(-6) M and a few weak spermidine binding sites which were inaccessible to experimentation; tRNAGlu2 (E . coli) has 14.8 +/- 1.6 strong spermidine binding sites and 4.0 +/- 0.1 weak spermidine binding sites with Kd = 1.4 X 10(-6) M and Kd = 1.23 X 10(-4) M, respectively . At high ionic strength (0.12 M monovalent cation) and 0.01 M Mg2+, tRNAPhe (yeast) has approximately 13 strong spermidine binding sites with an apparent Kd = 3.4 X 10(-3) M while the dimeric complex tRNAPhe X tRNAGlu2 has 10.4 +/- 1.2 strong spermidine binding sites per monomer with an apparent Kd = 2.0 X 10(-3) M . In the presence of increasing Na+ ion or K+ ion concentration, spermidine binding data do not fit a model for competitive binding to tRNA by monovalent cations . Rather, analysis of binding data by the Debye-Huckel approximation results in a good fit of experimental data, indicating that monovalent cations form a counterion atmosphere about tRNA, thus decreasing electrostatic interactions . On the basis of equilibrium binding analyses, it is proposed that the binding of spermidine to tRNA occurs predominantly by electrostatic forces.

Biochemistry, 1982 Oct 12, 21(21), 5224 - 30
Structure-function relationships in Escherichia coli translational elongation factor G: modification of lysine residues by the site-specific reagent pyridoxal phosphate; Giovane A et al.; Translational elongation factor G (EF-G) of Escherichia coli was modified with the selective, site-specific lysine reagent pyridoxal phosphate (PLP) . The reaction results in the modification of a maximum of 12 lysine residues, one of which is essential for guanosine 5'-triphosphate (GTP) binding and whose modification is inhibited by the presence of GTP . Formation of a reversible adduct between 2,3-butanedione and an essential arginine similarly located in the GTP binding site {Rohrbach, M.S., & Bodley, J . W . (1977) Biochemistry 16, 1360-1363} also protects EF-G from PLP inactivation, suggesting that these two residues are spatially close to each other in the native factor . The essential lysine residue was found in the trypsin-resistant fragment T4 (Mr 41 000) . In addition to the lysine essential for GTP binding, at least one further lysine was found to be important for EF-G function, since GTP-protected, PLP-modified EF-G molecules fully competent in binding to 50S ribosomal subunits showed decreased activity in 50S- and 70S-dependent GTP hydrolysis . It is likely that a PLP-modified lysine impairs the interaction of the factor with 30S ribosomal subunits and/or a conformational change of the factor required for the hydrolysis of GTP.

Biochemistry, 1982 Oct 12, 21(21), 5129 - 35
Nuclear Overhauser assignment of the imino protons of the acceptor helix and the ribothymidine helix in the nuclear magnetic resonance spectrum of Escherichia coli isoleucine transfer ribonucleic acid: evidence for costacked helices in solution; Hare DR et al.; In a previous study we showed that, in the low-field nuclear magnetic resonance (NMR) spectrum of Escherichia coli tRNAVal1, the hydrogen-bonded imino protons of the four Watson-Crick base pairs in the dihydrouridine helix could be assigned on the basis of their proximity to the imino proton of s4U8 by means of sequential Nuclear Overhauser (NOE) connectivity (Hare & Reid, 1982) . In the present paper we have used the nearest-neighbor NOE technique to assign all the imino proton resonances of the acceptor helix and the ribothymidine helix of E . coli tRNAIle1 . As reference points we used the GU-type base pairs located at positions 5 and 49 in this molecule which are readily identifiable in the NMR spectrum by virtue of containing two imino protons in the same base pair . From UG5 the imino protons of base pairs 4,3,2,1 and 6,7 could be assigned by through-space NOE connectivity . Similarly the imino protons of 50,51,52,53 were assigned by their spatial relationship to G psi 49 . NOE connectivity also revealed a base pair stacked on the external side of GC53 which, by analogy with the crystal structure of yeast phenylalanine tRNA, is presumed to be the tertiary pair T54-A58 . This was confirmed by NOE connectivity from the thymine methyl resonance . In addition to assigning 17 of the imino resonances in the low-field NMR spectrum of isoleucine tRNA, these NOE studies show that the acceptor helix and the ribothymidine helix are stacked on each other in solution in that base pairs 7 and 49 are directly connected in space.

Nucleic Acids Res, 1982 Oct 11, 10(19), 6051 - 66
Modified polynucleotides . VI . Properties of a synthetic DNA containing the anti-herpes agent (E)-5-(2-bromovinyl)-2'-deoxyuridine; Sagi J et al.; A new modified polydeoxynucleotide, a copolymer of nucleotides of 2'-deoxyadenosine and the very efficacious anti-herpesvirus agent (E)-5-(2-bromovinyl)-2'-deoxyuridine was synthesized with E . coli DNA polymerase I enzyme . It is characterized by its physical (absorption and circular dichroism spectra, thermal transition, sedimentation analysis) and bioorganic (template activity, stability) properties . Compared to poly {d(A-T)}, the modified polydeoxynucleotide had a lower thermal stability but exhibited higher stability against DNases and higher template activity for DNA synthesis . Template activity for RNA synthesis of this template was, however, poor and extent of AMP and UMP incorporation was limited as well.

Nucleic Acids Res, 1982 Oct 11, 10(19), 6119 - 30
Nucleotide sequence of the fnr gene and primary structure of the Enr protein of Escherichia coli; Shaw DJ et al.; The nucleotide sequence of a 1.64 kb fragment of E . coli DNA containing the fnr gene (regulatory gene for fumarate and nitrate reduction) was determined using the dideoxy chain termination method . The fnr coding region (750 bp) was identified, and the initiation and termination points of fnr transcription were located by RNA:DNA hybridisation with single-stranded M13 probes . The DNA fragment also contained the 5' end of a separately transcribed gene of unknown function . The deduced molecular weight (27947) of the Fnr protein was in agreement with that of the protein identified by the maxicell procedure, and the primary structure contained regions of homology with several transcriptional regulator proteins.

Nucleic Acids Res, 1982 Oct 11, 10(19), 5949 - 65
Purification of ribonuclease H as a factor required for initiation of in vitro Co1E1 DNA replication; Itoh T et al.; Escherichia coli ribonuclease H was purified to near-homogeneity and identified as the only additional factor required for initiation of in vitro Co1E1 DNA replication from the unique origin by RNA polymerase and DNA polymerase I . Both ribonuclease H activity and stimulating activity for Co1E1 DNA synthesis comigrate with the single protein band in gel electrophoresis . These two activities coincide throughout the process of purification . Some DNA synthesis takes place on covalently closed-circular DNA molecules other than Co1E1 DNA with the three purified enzymes . This DNA synthesis is suppressed by an Escherichia coli single-strand DNA binding protein and/or a high concentration of ribonuclease H . Negative superhelicity of template DNA is required for efficient primer formation . No evidence that supports involvement of ribonuclease III in initiation of Co1E1 DNA replication or its regulation was found.

Nucleic Acids Res, 1982 Oct 11, 10(19), 5905 - 23
An analysis of cosmid clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glycoproteins are clustered in the genome; Van der Ploeg LH et al.; Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs) . Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC) . We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli . Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used . We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes . In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome . Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses . We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment.

J Biol Chem, 1982 Oct 10, 257(19), 11644 - 50
Escherichia coli glutaminyl-tRNA synthetase . II . Characterization of the glnS gene product; Hoben P et al.; Glutaminyl-tRNA synthetase has been purified by a simple, two-column procedure from an Escherichia coli K12 strain carrying the glnS structural gene on plasmid pBR322 . The primary sequence of this enzyme as derived from the DNA sequence (see accompanying paper) has been confirmed . Manual Edman degradation was used to identify the NH2-terminal sequence of the protein . Oligopeptides scattered throughout the primary sequence of glutaminyl-tRNA synthetase were sequenced by the gas chromatographic-mass spectrometric method and matched to the theoretical peptides derived from the translated DNA sequence . The expected carboxyl terminus at position 550 was verified by carboxypeptidase B digestion . The primary sequence of glutaminyl-tRNA synthetase contains no extensive sequence repeats . A search was made for sequence homologies between this enzyme and the few other aminoacyl-tRNA synthetases for which primary sequences are available . A single homologous region is shared by at least three of the synthetases examined here.

J Biol Chem, 1982 Oct 10, 257(19), 11474 - 8
ATP activation of DNA polymerase III holoenzyme from Escherichia coli . II . Initiation complex: stoichiometry and reactivity; Burgers PM et al.; DNA polymerase III holoenzyme (holenzyme) has an ATPase activity elicited only by a primed DNA template . Reaction of preformed ATP.holoenzyme complex with a primed template results in hydrolysis of the ATP bound to the holoenzyme, release of ADP and Pi, and formation of an initiation complex between holoenzyme and the primed template . Approximately two ATP molecules are hydrolyzed for each initiation complex formed, a value in keeping with the number bound in the ATP.holoenzyme complex . The possibility that the latter and the initiation complex contain two holoenzyme molecules is supported by the presence of two beta monomers in the initiation complex . Holoenzyme action in the absence of ATP resembles that of pol III (the holoenzyme core) or DNA polymerase III (holoenzyme lacking the beta subunit), with or without ATP, in sensitivity to salt and in processivity of elongation . The initiation complex formed by ATP-activated holoenzyme resists a level of KCl (150 mM) that completely inhibits nonactivated holoenzyme and the incomplete forms of the holoenzyme, and displays a processivity at least 20 times greater . Upon completing replication of available template, holoenzyme can dissociate and form an initiation complex with another primed template, provided ATP is available to reactivate the holoenzyme . By inference, no essential subunits are lost in the cycle of initiation, elongation and dissociation.

J Biol Chem, 1982 Oct 10, 257(19), 11468 - 73
ATP activation of DNA polymerase III holoenzyme of Escherichia coli . I . ATP-dependent formation of an initiation complex with a primed template; Burgers PM et al.; ATP (or dATP) stimulates DNA synthesis by DNA polymerase III holoenzyme (holoenzyme) on the synthetic template-primer poly(dA).oligo(dT)12 . Nonhydrolyzable ATP analogs and other natural (deoxy)ribonucleoside triphosphates are inactive . Because the nonhydrolyzable analog 5'-deoxyadenylylimidodiphosphate is efficiently used by holoenzyme for incorporation, the ATP (or dATP) requirement for activation of replication of natural DNA could be determined . Analysis of lag times in DNA synthesis and isolation of intermediates showed that ATP (or dATP) is required in the formation of an initiation complex between holoenzyme and primed DNA template, but not for subsequent DNA synthesis . ATP is bound to holoenzyme in the absence of DNA with a KD value of 0.8 microM; 2 to 3 molecules of ATP per molecule of holoenzyme are bound without apparent cooperativity . Binding of ATP to DNA polymerase III (holoenzyme minus beta subunit) is weak (KD greater than 5 microM) and binding to the beta subunit alone is not observed . However, holoenzyme reconstituted by mixing DNA polymerase III with beta subunit binds ATP as tightly (KD = 0.6 microM) as the original holoenzyme.

J Biol Chem, 1982 Oct 10, 257(19), 11261 - 7
The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP; Pingoud A et al.; Escherichia coli elongation factor (EF-Tu) binds aminoacyl-tRNAs (aa-tRNA) not only in the presence of GTP but also in the presence of GDP . Complex formation leads to a protection of the aa-tRNA against nonenzymatic deacylation and digestion by pancreatic ribonuclease, as well as to a protection of EF-Tu against proteolysis by trypsin . The equilibrium constant for the binding of Phe-tRNAPheyeast for example to EF-Tu.GDP has been determined to be 0.7 X 10(5) M-1 which is 2 orders of magnitude lower than the equilibrium constant for Phe-tRNAPheyeast binding to EF-Tu.GTP . In the presence of kirromycin, aminoacyl-tRNA binding to EF-Tu.GDP is not affected as much: Phe-tRNAPheyeast is bound with an equilibrium constant of 3 X 10(5) M-1 . While there is also a measurable interaction between EF-Tu.GTP and tRNA, such an interaction cannot be detected with EF-Tu.GDP and tRNA, not even at millimolar concentrations . A so far undetected complex formation between aminoacyl-tRNA and EF-Tu.GTP in the presence of pulvomycin, however, could be detected . The results are discussed in terms of the structural requirements of ternary complex formation and in the light of proofreading schemes involving A-site binding on the E . coli ribosome.

J Biol Chem, 1982 Oct 10, 257(19), 11639 - 43
Escherichia coli glutaminyl-tRNA synthetase . I . Isolation and DNA sequence of the glnS gene; Yamao F et al.; We have isolated a lambda-transducing phage carrying the gene (glnS) for Escherichia coli glutaminyl-tRNA synthetase . The location of the glnS gene within the 13.5-kilobase E . coli DNA transducing fragment was determined by genetic means . The glnS gene was recloned into plasmid pBR322 and its nucleotide sequence was established . The DNA sequence translates to a protein of 550 amino acids.

J Biol Chem, 1982 Oct 10, 257(19), 11497 - 502
Specific molecular activities of recombinant and hybrid leukocyte interferons; Rehberg E et al.; Hybrid interferon DNA recombinants were constructed from the IFLrA and IFLrD leukocyte interferon-coding sequences . Each of the hybrid interferons was purified with the use of a monoclonal antibody to human leukocyte interferon . Three amino acid residues were identified, one or all of which function to potentiate antiviral activity on feline cells and reduce activity on human cells . Because at sufficiently high concentrations human interferons can interact with mouse and rat receptors, it is apparent that the species barrier is only relative and that interferons can be forced into heterologous receptors by mass action . In addition, the specific molecular antiviral and antiproliferative activities (molecules of interferon/cell required for a specific effect) for each of these interferons were determined . The specific molecular activities permit an accurate comparison of the efficacy of different interferons for a specific effect . Because the ratios of antiproliferative/antiviral activity of these interferons vary over a 12-fold range, it appears that the antiviral and antiproliferative activities are promulgated through different mechanisms . To account for these results, it is proposed that there are at least two distinct interferon receptors on cells.

J Biol Chem, 1982 Oct 10, 257(19), 11340 - 5
Independent locations of kinase and 3'-phosphatase activities on T4 polynucleotide kinase; Soltis DA et al.; We have used two chemical modification reagents and three proteases to study the relationship between the two activities of T4 polynucleotide kinase . In each case, conditions were found where one of the two activities of the enzyme could be eliminated without greatly reducing the other . Taken together, these data indicate that the two activities are catalyzed by amino acid residues located in separate active sites on the polypeptide chain . Specific exopeptidase digestion indicates that the kinase activity lies in the NH2-terminal and the phosphatase in the COOH-terminal portion of the polypeptide chain . Partial trypsin digestion produces a 29,000-dalton fragment with no kinase activity and nearly normal 3'-phosphatase activity.

J Biol Chem, 1982 Oct 10, 257(19), 11332 - 9
Isolation and characterization of two mutant forms of T4 polynucleotide kinase; Soltis DA et al.; The purification of polynucleotide kinase from Escherichia coli infected by two different mutants in the T4 polynucleotide kinase (pseT) gene is described . The pseT 1 enzyme has virtually no 3' specific phosphatase activity and normal polynucleotide kinase activity . The pseT 47 enzyme has very little phosphatase activity and no kinase activity . However, enzyme isolated from a pseT 1, pseT 47 mixed infection appears to contain heterodimers with considerably more phosphatase activity . Thus, the pseT 47 mutation partially inactivates the phosphatase and totally inactivates the kinase . A study of the action of polynucleotide kinase on plasmid DNAs nicked to give a 3'-phosphate and a 5'-hydroxyl indicates that although the enzyme can catalyze both the removal of the 3'-phosphate and the insertion of a 5'-phosphate, there is no evidence for a concerted reaction involving both activities on the same polypeptide chain.

J Biol Chem, 1982 Oct 10, 257(19), 11796 - 801
Mechanism of interferon action . Kinetics of decay of the antiviral state and protein phosphorylation in mouse fibroblasts treated with natural and cloned interferons; Samuel CE et al.; The kinetics of decay of the antiviral state and protein phosphorylation induced with natural mouse interferon (IFN) and with cloned human IFN were examined in monolayer cultures of mouse Ll929 fibroblast cells . The antiviral state measured by single cycle virus yield reduction with either vesicular stomatitis virus or reovirus decayed significantly within 2 to 3 days following removal of IFN and by 5 to 8 days virus yields had returned to the level of untreated control cells . Trypsinization of IFN-treated cells did not detectably alter the rate of decay of the antiviral state; however, the decay occurred slightly more rapidly in actively growing as compared to stationary cell cultures . The decay of the IFN-induced protein kinase which catalyzes the phosphorylation of endogenous protein P1 and purified initiation factor eIF-2 alpha correlated with the decay of the antiviral state . The decay rates of the antiviral state and protein kinase observed in mouse L929 cells that had been treated with natural mouse IFN synthesized in Newcastle disease virus-induced L929 cells were comparable to the decay rates observed in L929 cells that had been treated with recombinant human IFN-alpha A/D synthesized in Escherichia coli . The induction and decay of the antiviral state and protein kinase following treatment with a single dose of IFN did not significantly affect the sensitivity of the cell population to a subsequent treatment with a single dose of IFN . However, continuous treatment of L929 cells with natural mouse IFN or recombinant human IFN prevented the decay of both the antiviral state and protein kinase but also ultimately lead to cell death . The results suggest that protein phosphorylation may play an important role in the mechanism of IFN action in mouse L929 fibroblasts.

J Biol Chem, 1982 Oct 10, 257(19), 11791 - 5
Mechanism of interferon action . Kinetics of induction of the antiviral state and protein phosphorylation in mouse fibroblasts treated with natural and cloned interferons; Samuel CE et al.; The induction of phosphorylation of both protein P1 and protein synthesis initiation factor eIF-2 alpha and the inhibition of virus replication were examined in mouse L929 fibroblasts treated with either natural mouse or individual cloned human interferons (IFN) . Natural mouse IFN synthesized in Newcastle disease virus-induced L929 cells and two cloned human leukocyte IFN subspecies synthesized in Escherichia coli, IFN-alpha D and IFN-alpha A/D, possessed antiviral activity in L929 cells as measured by single cycle virus yield reduction with both vesicular stomatitis virus and reovirus . Natural L929 IFN and cloned IFNs, alpha D and alpha A/D, also induced the protein kinase that catalyzed the phosphorylation of endogenous ribosome-associated protein P1 and the alpha subunit of purified initiation factor eIF-2 . Two other cloned human IFNs, alpha A and alpha D/A, were poor inducers of both the antiviral state and the phosphorylation of P1 and eIF-2 alpha in mouse L929 cells . The ability of individual human IFN-alpha subspecies to induce P1 and eIF-2 alpha phosphorylation in mouse L929 cells correlated with their ability to induce an antiviral state . Furthermore, the detailed kinetics of induction, in mouse L929 cells, of P1 and eIF-2 alpha phosphorylation and of the antiviral state by the heterologous cloned human IFN-alpha A/D were equivalent to the kinetics of induction by the homologous natural mouse L929 IFN . These results suggest that different subspecies of biologically active IFN induce equivalent antiviral activities and biochemical changes in mouse L929 cells, and that protein phosphorylation may play a major role in the antiviral mechanism of IFN action in mouse L929 fibroblasts.

Biosci Rep, 1982 Oct, 2(10), 751 - 60
Evidence for the presence of a cell-surface ribonuclease in mechanically prepared rat-liver cells in suspension; Sirdeshmukh R et al.; Rat-liver parenchymal cells obtained in suspension by a mechanical method are shown to contain a cell-surface nuclease(s) that rapidly degrades exogenously added total Escherichia coli RNA . However, no acid-soluble products are formed; all the degradation products in the incubation medium sediment in the 4-5S RNA region on a sucrose density gradient . A part of the degraded RNA seems to be taken up by the cells; the uptake of the degradation products, presumably derived from rRNAs, is more than that of purified 4-5S RNA . Most of the RNA taken up by the cell sediments in the 4-5S region; only a small proportion is degraded to acid-soluble material within the cell.

Ann Endocrinol (Paris), 1982 Oct-Nov, 43(5), 404 - 14
{Structure and expression of thyroglobulin gene}; Vassart G et al.; Thyroglobulin is composed of two 300000 dalton polypeptide chains, translated from an 8000 base mRNA . Preparation of a full length cDNA and its cloning in E . coli have lead to the demonstration that the polypeptides of thyroglobulin protomers were identical . Used as molecular probes, the cloned cDNA allowed the isolation of a fragment of thyroglobulin gene . Electron microscopic studies have demonstrated that this gene contains more than 90% intronic material separating small size exons (less than 200 bp) . Sequencing of bovine thyroglobulin structural gene is in progress . Preliminary results show evidence for the existence of repetitive segments . Availability of cloned DNA complementary to bovine and human thyroglobulin mRNA allows the study of genetic defects of thyroglobulin gene expression in the human and in various animal models.

Avian Dis, 1982 Oct-Dec, 26(4), 732 - 40
Cecal and hepatic granulomas of unknown etiology in chickens; Mutalib AA et al.; One hundred and seventy-nine outbreaks of cecal and hepatic granulomas were diagnosed in small flocks of 3-to-7-month-old chickens in Saskatchewan between 1968 and 1981 . The lesions were generally found at slaughter in the autumn . Outbreaks were widely distributed in farming areas in the province . The granulomas were either rough or smooth and were commonly confined to the liver and ceca but were also found in some outbreaks in the spleen, lung, mesentery, heart, kidney, and pancreas . The cause of the granulomas is unknown . Most granulomas were sterile on the basis of routine bacterial culture, but either Escherichia coli or a mixed flora of bacteria was isolated from some others . Acid-fast organisms and fungal elements have never been demonstrated using special histological stains.

Chem Biol Interact, 1982 Oct, 42(1), 85 - 95
Termination of DNA synthesis in vitro at apurinic sites but not at ethyl adducts on the template; Lockhart ML et al.; The effects of DNA lesions produced by the carcinogenic alkylating agents ethylnitrosourea and diethylsulfate on the extent of DNA synthesis have been studied in a system utilizing circular single-stranded phiX174 DNA as template and a 392-base restriction fragment as primer with E . coli polymerase I (Klenow fragment) . Apurinic sites produced by loss of unstable ethylated bases from the template terminate DNA synthesis at the first such site encountered, but ethyl adducts at most, if not all, locations permit readthrough.

Eur J Biochem, 1982 Oct, 127(2), 375 - 80
Chemical characterization of a rabbit leukocytic pyrogen; Pacak F et al.; Crude pyrogen was obtained by stimulating 1.4 x 10(12) peritoneal-exudate leukocytes from rabbits in vitro with Escherichia coli endotoxin . A pyrogen molecule was isolated by ammonium-sulfate precipitation, polyacrylamide gel electrophoresis at pH 5.5 and 8.9, and gel filtration on Bio-Gel P-100 . These procedures yielded a pyrogenic fraction which was electrophoretically and immunologically homogeneous . The pyrogenicity was 50 ng/kg of rabbit . This leukocyte pyrogen consisted of a protein with a molecular weight of approximately 14 000 . Amino-acid analysis showed high amounts of glutamic acid and glycine; methionine and tryptophan were present in small quantities . Arginine was found to be the N-terminal amino acid . The molecule contained about 0.8% neutral sugar and 0.7% lipids . Its approximate isoelectric point in citrate/phosphate buffer was 4.2 . The activity was lost by lyophilization and storage at - 18 degrees C for six months . The data presented here are the first detailed characterization of a rabbit leukocyte pyrogen . The properties in common to leukocytic pyrogen and interleukin 1 are discussed.

Int J Radiat Biol Relat Stud Phys Chem Med, 1982 Oct, 42(4), 435 - 48
Unscheduled DNA synthesis and elimination of DNA damage in liver cells of gamma-irradiated senescent mice; Gaziev AI et al.; The level of 'spontaneous' and gamma-radiation-induced DNA synthesis which is not inhibited with hydroxyurea (unscheduled synthesis) is considerably lower in hepatocytes of 18-22-month-old mice than that of 1.5-2-month-old mice . The dose-dependent increase (10-300 Gy) of unscheduled DNA synthesis (UDS) in hepatocytes of senescent mice is higher than in young animals . The elimination of damage in DNA of gamma-irradiated hepatocytes (100 Gy) was examined by using an enzyme system (M . luteus extract and DNA-polymerase I of E . coli) . It was found that the rate of elimination of the DNA damage in hepatocytes of 20-month-old mice is lower than that of 2-month-old mice although the activities of DNA-polymerase beta and apurinic endonuclease remain equal in the liver of both senescent and young mice . However, the nucleoids from gamma-irradiated liver nuclei of 2-month-old mice are relaxed to a greater extent (as judged by the criterion of ethidium-binding capacity) than those of 20-month-old mice . The results suggest that there are limitations in the functioning of repair enzymes and in their access to damaged DNA sites in the chromatin of senescent mouse liver cells.

J Gen Microbiol, 1982 Oct, 128 (Pt 10), 2243 - 9
Effect of glucose and amino acids on expression of K99 antigen in Escherichia coli; Girardeau JP et al.; K99 antigen production by enterotoxigenic Escherichia coli strains of bovine origin was investigated by slide agglutination and in vitro attachment to intestinal villi . Work with two strains (B41 and B44) showed that on minimal medium M2, K99 antigen was not repressed by a high concentration of glucose (2%, w/v) . Growth on synthetic or complex medium did not affect K99 antigen detection, which was independent of capsular antigens, and its synthesis was not repressed by Casamino acids or glucose . A survey of 12 strains revealed two groups: in one group K99 antigen production was constitutive on basal medium without glucose, and in the second group K99 antigen was produced only in the presence of glucose . Immunoelectrophoresis patterns, and the results of slide agglutination and attachment tests, were dependent upon K99 type, whereas haemagglutination patterns were not.

Antiviral Res, 1982 Oct, 2(5), 313 - 8
Antiviral and side effects of interferons produced by recombinant DNA techniques as tested in rhesus monkeys; Schellekens H et al.; Human interferon type alpha 2 (HuIFN-alpha 2) produced by Escherichia coli was found to be as active as natural leukocyte interferon in protecting rhesus monkeys against intradermal vaccinia virus infection . HuIFN-beta 1 produced in E . coli had similar but less pronounced activity . HuIFN-alpha 2 induced fever but not leukopenia, while HuIFN-beta 1 had opposite effects . Concurrent treatment with acetosalicylic acid and prednisolone/azothioprine combinations did not interfere with the efficacy of the human interferons.

Int J Pept Protein Res, 1982 Oct, 20(4), 331 - 6
Calorimetric study of tryptophan synthase alpha-subunit and two mutant proteins; Yutani K et al.; Heat-denaturation of tryptophan synthase alpha-subunit from E . coli and two mutant proteins (Glu 49 leads to Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein . The partial specific heat capacity in the native state at 20 degrees, Cp20, has been found to be (0.43 +/- 0.02) cal . k-1 . g-1, the unfolding heat capacity change, delta dCp, (0.10 +/- 0.01) cal . K-1 . g-1, and the unfolding enthalpy value extrapolated to 110 degrees, delta dh110, (9.3 +/- 0.5) cal . g-1 for the three proteins . The value of Cp20 was larger than those found for "fully compact protein" and that of delta dh110 was smaller . Unfolding Gibbs energy, delta dG at 25 degrees for Wild-type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal . mol-1 at pH 9.3, respectively . Unfolding enthalpy, delta dH, of the three proteins seemed to be the same and equal to (23.2 +/- 1.2) kcal . mol-1 at 25 degrees . As a consequence of the same value of delta dH and the different value in delta dG, substantial differences in unfolding entropy, delta dS, were found for the three proteins . The values of delta dG for the three proteins at 25 degrees coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.

Sci Sin {B}, 1982 Oct, 25(10), 1061 - 70
Effect of nifA gene product on expression of lacZ under nifH promoter in Escherichia coli; Kong QT et al.; Gene expression of the nitrogen fixation system from Klebsiello pneumonice was studied in Escherichia coli by using compatible plasmids as vectors . One constructed plasmid carried the nifH promoter fused to the structural gene for beta-galactosidase, lac Z . Another plasmid carried the promoter of a tetracycline-resistance gene fused to nifA . We found that anaerobic synthesis of beta-galactosidase was greatly enhanced by the presence of an active nifA gene, indicating that its product is a positive control factor for transcription of nifH . In addition, anaerobic expression of lacZ was repressed by ammonium or serine in the presence of nifA . Thus the regulatory mechanism under study is of physiological relevance.

Scand J Immunol, 1982 Oct, 16(4), 303 - 8
Isolation and identification of a cDNA clone coding for an HLA-DR transplantation antigen alpha-chain; Gustafsson K et al.; Membrane-bound mRNA was isolated from Raji cells and enriched for message coding for the HLA-DR transplantation antigen alpha-chain by sucrose gradient centrifugation . Double-stranded cDNA was constructed from this mRNA fraction, ligated to plasmid pBR322, and cloned into Escherichia coli . By hybrid selection, a plasmid, pDR-alpha-1, able to hybridize with mRNA coding for the HLA-DR alpha-chain was identified . From the nucleotide sequence of one end of the insert an amino acid sequence was predicted which is identical to part of the amino-terminal sequence of an HLA-DR alpha-chain preparation isolated from Raji cells . This clearly shows that pDR-alpha-1 carries almost the complete message for an HLD-DR alpha-chain . From the nucleotide sequence of this plasmid it will be possible to predict the primary structure of an HLA-DR alpha-chain.

J Bacteriol, 1982 Oct, 152(1), 363 - 71
Attenuation regulation in the thr operon of Escherichia coli K-12: molecular cloning and transcription of the controlling region; Lynn SP et al.; Recombinant plasmids were constructed which carry defined regions of the threonine (thr) operon regulatory region of Escherichia coli . In vitro transcription experiments utilizing plasmid or restriction fragment templates showed that two major RNA transcripts, which differ in length by one to a few bases, are transcribed from this region . The approximate length of the transcripts is 150 to 170 bases, and the site(s) of termination is near or within the thr attenuator . The efficiency of termination at the thr operon attenuator in vitro is approximately 90% . A regulatory mutation, thr79-20, which is a G-C insertion in the attenuator, reduces the frequency of transcription termination to 75% . In addition, in vivo RNA transcripts were identified which hybridize to the thr operon regulatory region . These transcripts appeared to be identical to the two major in vitro transcripts as judged by their mobilities on 8% polyacrylamide-8 M urea gels . This result indicates that the thr operon regulatory region is transcribed in vivo and that termination occurs near or within the thr attenuator.

J Bacteriol, 1982 Oct, 152(1), 345 - 50
Recombination properties of P1 dlac; Porter RD; The P1 dlac prophage plasmid of Escherichia coli K-12 has been utilized as the recipient DNA substrate in experiments with lambda plac5 transduction and with Hfr and F' conjugation . The P1 dlac plasmid does not recombine with lambda plac5 at the elevated levels seen for the F42lac plasmid . Recombination between lambda plac5 and P1 dlac is essentially indistinguishable from recombination between lambda plac5 and a chromosomal lac gene in tems of both level of recombination and recombination pathway (RecBC, RecE, and RecF) dependence . The initiation of recombination between P1 dlac and lac genes from an Hfr or F' donor is severalfold more efficient than it is for a recipient chromosomal lac gene.

Proc Natl Acad Sci U S A, 1982 Oct, 79(19), 5976 - 80
Clustering of spore-specific genes in Aspergillus nidulans; Orr WC et al.; We have investigated the chromosomal organization of genes that are expressed specifically in the asexual spores (conidia) of the Ascomycete fungus Aspergillus nidulans, using two experimental approaches . In the first, 30 different recombinant clones, containing long nuclear DNA inserts and at least one spore-specific gene, were selected randomly . The total number of spore-specific genes present in each clone was then determined by RNA blot analysis . In the second approach, several chromosomal recombinant DNA libraries, having average insert lengths ranging from 1 to 15 kilobases, were constructed . The fraction of clones in each library having one or more spore-specific poly(A)+RNA-coding regions was then determined by colony or plaque filter hybridization with radiolabeled, spore-specific, complementary DNA . The results from these experiments were compared to statistical predictions based on the assumption that the spore-specific genes are randomly distributed in the Aspergillus genome . In both cases, the experimental values deviated significantly from the predicted values, demonstrating that the spore-specific genes are nonrandomly arranged in the genome . Rather, they appear frequently to occur in tightly linked clusters.

Proc Natl Acad Sci U S A, 1982 Oct, 79(19), 5891 - 5
Structural variability of tRNA: small-angle x-ray scattering of the yeast tRNAphe-Escherichia coli tRNAGlu2 complex; Nilsson L et al.; The structure of the complex formed in solution between yeast tRNAPhe and Escherichia coli tRNAGlu2 has been studied by small-angle x-ray scattering . The complex has a radius of gyration of 4.0 nm and an electron-pair distance distribution that is incompatible with a model composed to two tRNAs joined at their complementary anticodons and exhibiting the L shape seen in the crystal . Instead a model in which the two tRNAs, still bound via the anticodons, assume a conformation with the acceptor arms folded toward the anticodon arms agrees with the observed scattering curves.

Proc Natl Acad Sci U S A, 1982 Oct, 79(19), 5798 - 802
Use of recombinant DNA technology to program eukaryotic cells to synthesize rat proinsulin: a rapid expression assay for cloned genes; Lomedico PT; To use recombinant DNA technology to functionally analyze mutations introduced into cloned eukaryotic genes, a rapid procedure is necessary to assay the steps along the gene expression pathway . Since cloned rat insulin genes are not transcribed efficiently after transfection into various cell lines, I have asked whether one could drive expression by placing the insulin gene inside a transcriptional unit that functions in all mammalian cells . By using a small simian virus 40 (SV40) fragment that contains initiation signals for replication and transcription, I connected the 5'-noncoding region of the SV40 tumor antigen gene to the 5'-noncoding region of the rat insulin II gene to create a pBR322-based recombinant . If one assays shortly after its introduction into mammalian cells, it can be shown that this recombinant plasmid programs the synthesis of correctly spliced and polyadenylylated insulin mRNA that functions in the synthesis and secretion of rat proinsulin . This system permits rapid analysis of cloned in vitro-engineered mutations and the programming of eukaryotic cells to manufacture proteins that they normally do not synthesize.

Avian Dis, 1982 Oct-Dec, 26(4), 787 - 97
Escherichia coli colonization of the trachea in poultry: comparison of virulent and avirulent strains in gnotoxenic chickens; Dho M et al.; In chickens, virulent Escherichia coli strains express their pathogenicity in the respiratory tract . A quantitative comparison of tracheal colonization by virulent and avirulent E . coli was carried out in gnotoxenic chickens after intestinal implantation . Two-week-old axenic chicks reared in isolators were inoculated per os with various associations of identified E . coli strains . No clinical sign of disease was observed in any of the chicks, despite the presence of virulent strains in all the intestines and most of the tracheas . The virulent organism reached greater population sizes in the trachea and feces of monocontaminated chicks and of chicks contaminated simultaneously with a virulent and an avirulent strain . In holoxenic chicks, identified virulent and avirulent strains were outnumbered by the E . coli population of the intestinal flora previously established and could not be recovered from the tracheas of most chicks.

Thorax, 1982 Oct, 37(10), 727 - 31
Technique, complications, and clinical value of endomyocardial biopsy in patients with heterotopic heart transplants; Cooper DK et al.; A review of 157 consecutive biopsies of donor endomyocardium in patients with heterotopic heart transplants is reported . The technique of percutaneous transvenous endomyocardial biopsy after this operation is described; manipulation of the catheter and bioptome into the junction of the donor superior vena cava and right atrium can be difficult when this anastomotic junction is small, as a result either of operative surgical technique or of subsequent contraction . The complication rate was 4%, but one patient may have died from infection resulting from biopsy when the bioptome had to be introduced at the groin . The histopathological changes seen in the biopsy specimens have been graded according to a scoring system to give the clinician a guide to the severity of rejection . Histopathological assessment was of clinical value in 96% of cases, but was inaccurate on two occasions, once because an opinion was given on what was in retrospect an inadequate sample . In patients undergoing persistent low-grade acute or chronic rejection there was difficulty in detecting or appreciating the true extent of myocardial fibrosis; this led to inadequate immunosuppressive treatment in two patients . Attention is drawn to the fact that ischaemic fibrosis resulting from the vascular changes of chronic rejection may spare the endomyocardium, which is kept viable by intracavitary blood, and that this may lead to a misleading histopathological report.

J Gen Microbiol, 1982 Oct, 128 (Pt 10), 2221 - 8
Amplification and product identification of the fnr gene of Escherichia coli; Shaw DJ et al.; The position of a gene (fnr) that is essential for growth of Escherichia coli with fumarate or nitrate as electron acceptor was located within an 11.5 kb HindIII fragment of bacterial DNA by deletion analysis with fnr transducing phages (lambda fnr) and by sub-cloning restriction fragments into multicopy plasmids . The functional gene was isolated in a 1.65 kb BamHI-HindIII fragment of a hybrid plasmid pGS24 . The fnr gene product was identified as a protein of Mr 31 000, by post-infection labelling and by the 'maxicell' method . Organisms containing the multicopy plasmid (pGS24) overproduced fumarate reductase to the same extent as cultures containing a comparable fumarate reductase plasmid (pNU31) during anaerobic growth; the fnr plasmid also overcame the repression of fumarate reductase synthesis that is normally observed during aerobic growth . Similar effects on nitrate reductase synthesis were also observed . The results support the view that the fnr gene product functions as a positive regulator or a specific sigma factor for expression of anaerobic energy-generating systems.

Arch Microbiol, 1982 Oct, 132(4), 365 - 71
Expression of pyruvate formate-lyase of Escherichia coli from the cloned structural gene; Pecher A et al.; It is shown here that a plasmid (p29) derived from the transducing phage lambda aspC2 (Christiansen and Pedersen 1981) codes for pyruvate formate-lyase . The identity of the 80 kilodaltons (kd) gene product of plasmid p29 with the pyruvate formate-lyase polypeptide was proven (i) by co-migration of the gene product expressed in the maxicell system with purified enzyme on O'Farrell gels, and (ii) by comparison of the peptide maps obtained from limited proteolysis . In vivo the 80 kd form of the enzyme was proteolytically converted to a 78 kd polypeptide . The two polypeptides (80 kd and 78 kd) and their charge isomers present in purified enzyme preparations are therefore products of a single gene . Aerobically grown cells of Escherichia coli contained a basal level of pyruvate formate-lyase which was derepressed 5- to 10-fold under anaerobiosis . Derepression also occurred during anaerobic growth on glycerol plus fumarate . Presence of plasmid p29 caused overproduction of pyruvate formatelyase, 11-fold upon anaerobic growth on glucose, 14-fold upon aerobic growth on glucose and 33-fold upon aerobic growth at the expense of D-lactate.

J Antibiot (Tokyo), 1982 Oct, 35(10), 1367 - 73
Function of plasmids in the production of aureothricin . I . Elimination of plasmids and alteration of phenotypes caused by protoplast regeneration in Streptomyces kasugaensis; Furumai T et al.; The spontaneous mutant 18a derived from Streptomyces kasugaensis MB273 exhibited pleiotropic effect such as loss of aerial mycelium formation, aureothricin (AT) production, and of citrullin biosynthesis, as well as changes in plasmid; the mutant required cystine for production of aureothricin . An improved method of protoplast regeneration was applied to S . kasugaensis MB 273-18a and a regeneration efficiency of 90% or more was obtained . Sixty to ninety percent of the colonies regenerated from the 18a protoplasts exhibited reversion of the pleiotropic mutation in 18a . Moreover, of 13 regenerated strains which showed these drastic phenotypic variations, it was found that their plasmid types varied . These types could be divided into two groups; the RI type (5 strains) which contained a large amount of pSK2, a small amount of pSK3 and no pSK1, and the RII type (8 strains) in which no closed-circular DNA was detected . From these results, the following conclusions were obtained . First, plasmid curing in RII type strains and also the variation of plasmid copy in the RI type strains occurred as the result of protoplast regeneration . Second, the structural genes for biosynthesis of AT probably exist on chromosome . Third, regeneration of 18a protoplasts causes the reversion of pleiotropic mutation with high frequency . A working hypothesis was proposed to explain these complex phenomena.

Eur J Biochem, 1982 Oct, 127(3), 587 - 95
Structure of ribosomal protein L6 from Escherichia coli . A fluorescence study; Steinhauser KG et al.; Protein L6 from the 50-S ribosomal subunit has been investigated using fluorimetric techniques . The intrinsic fluorophore Trp-61 and fluorescent labels (acetylaminoethyl-dansyl and acetylaminofluorescein) attached to the residue Cys-124 were used . It proved possible to incorporate fluorescence-labelled L6 into the 50-S ribosome . Trp-61 is exposed to solvent, as shown by its emission wavelength and by quenching experiments; the latter also show that it lies in a pocket with a high positive charge due to the basic residues in the N-terminal fragment . Cys-124 lies in a less strongly positive region . Upon incorporation into the 50-S subunit, the label on Cys-124 becomes less accessible for quenching but its positive potential rises, showing the absence of direct contact with 23-S RNA . Analysis of anisotropy data indicates a considerable degree of asphericity of free L6 . Energy transfer between Trp-61 and the dansyl label on Cys-124, measured by donor quenching and acceptor enhancement, reveals a separation of 3.5 +/- 0.4 nm (35 +/- 4 A) between fluorophores.

Eur J Biochem, 1982 Oct, 127(3), 449 - 57
Methionyl-tRNA synthetase from Escherichia coli . Primary structure of the active crystallised tryptic fragment; Barker DG et al.; A 3300-base segment of Escherichia coli chromosomal DNA, cloned into pBR322, will complement a methionine auxotroph in which the lesion is a defective methionyl-tRNA synthetase with a much reduced affinity for methionine . Crude extracts of these transformants contain elevated levels of a protein which has a subunit molecular weight of 66 000, methionyl-tRNA synthetase aminoacylation activity in vitro and which cross-reacts with anti-(methionyl-tRNA synthetase) antibodies . This polypeptide is very slightly larger than the well-characterised and crystallised tryptic fragment of methionyl-tRNA synthetase . A DNA sequence of 1750 residues at one end of the cloned insert codes for a non-terminated open reading frame in which we can locate a large number of methionyl-tRNA synthetase tryptic and chymotryptic peptides . We have also sequenced 300 nucleotides upstream of this coding segment where we find a large invert repeat in the putative methionyl-tRNA synthetase promoter region.

Can J Physiol Pharmacol, 1982 Oct, 60(10), 1281 - 6
Effects of indomethacin, acetazolamide, ethacrynate sodium, and atropine on intestinal secretion mediated by Escherichia coli heat-stable enterotoxin in pig jejunum; Ahrens FA et al.; Intraluminal perfusion of pig jejunum with Escherichia coli heat-stable enterotoxin reversed net absorption of water and electrolytes to net secretion . Addition of atropine (2 x 10(-5)M) to the perfusate reduced the secretory response to enterotoxin and enhanced sodium and chloride absorption in control segments . Indomethacin (1.4 x 10(-3)M), acetazolamide (2.2 x 10(-3)M), or ethacrynate sodium (3.1 x 10(-4)M) had no effect . Mucosal disaccharidase activity and Na-K-ATPase activity were not altered by enterotoxin . The results suggest that blockade of cholinergically mediated secretion in the small intestine attenuates the enterosorptive effects of heat-stable enterotoxin and may be useful therapeutically in the management of secretory diarrhea.

Can J Comp Med, 1982 Oct, 46(4), 426 - 9
The immunogenicity of K99 antigen in whole cell bacterins of Escherichia coli; Forman AJ et al.; K99 antigen in Escherichia coli whole cell bacterins was immunogenic in rabbits and mice . Mice vaccinated subcutaneously and bled three weeks later had a serum antibody response which was dose dependent . The dose response on dilution of bacterins was shown to be mainly due to dilution of K99 antigen, rather than the reduction in adjuvant or bacterial cell concentration . The procedure appears to be a satisfactory one for immunogenicity testing of bacterins containing K99 antigen.

Proc Natl Acad Sci U S A, 1982 Oct, 79(20), 6119 - 22
Energetics of plasmid-mediated arsenate resistance in Escherichia coli; Mobley HL et al.; Plasmid R773, which codes for resistances to arsenate, arsenite, and antimony, was introduced into Escherichia coli strain AN120, a mutant deficient in the H+-translocating ATPase of oxidative phosphorylation . Cultures depleted of endogenous energy reserves were loaded with 74AsO3-4, and arsenate efflux was measured after dilution into medium containing various energy sources and inhibitors . Rapid extrusion of arsenate occurred when glucose was added . Arsenate was extruded both against and down a concentration gradient . In this strain glucose allows formation of both ATP via substrate-level phosphorylation and an electrochemical proton gradient (or protonmotive force) via oxidation of the products of glycolysis . When oxidation was inhibited by cyanide, glucose metabolism still produced arsenate efflux . Energy sources such as succinate, which supplies a protonmotive force but not ATP, did not result in efflux . Measurement of intracellular ATP concentration under each set of conditions demonstrated a direct correlation between the rate of efflux and ATP levels . Osmotically shocked cells lost the ability to extrude arsenate; however, no arsenate-binding activity was detected in osmotic shock fluid from induced cells . These results suggest that the arsenate efflux system is coupled to cellular ATP rather than an electrochemical proton gradient, possibly by an arsenate-translocating ATPase.

J Neurol Neurosurg Psychiatry, 1982 Oct, 45(10), 898 - 904
Leukoencephalopathy associated with extensive burns; Gregorios JB; Unusual neuropathological changes were observed in two cases following extensive burns . These consisted of perivascular areas of demyelination distributed symmetrically in the brain and affecting the white matter predominantly . One case in addition had widespread petecchial and ring haemorrhages characteristic of brain purpura . Both patients sustained second and third degree burns in greater than 50% of the body surface area, developed metabolic acidosis, sepsis, disturbance in consciousness and multiple episodes of cardiorespiratory arrest prior to death . A toxic metabolic state related to a burn toxin released from the damaged tissue or from bacterial action to the tissue in addition to low platelet level is proposed as the major pathogenetic factor in the development of the neurological symptoms and the patients' demise.

Infect Immun, 1982 Oct, 38(1), 74 - 9
Synergistic protective effect of antibodies against Escherichia coli enterotoxin and colonization factor antigens; Ahren CM et al.; We studied the ability of antisera against different Escherichia coli surface antigens, both alone and in combination with anti-enterotoxin, to decrease fluid secretion induced by intestinal challenge with enterotoxigenic E . coli in rabbits . Antiserum against lipopolysaccharide protected significantly against O group homologous bacteria . Monospecific antisera against pilus-associated colonization factor antigens CFA/I and CFA/II were also effective, giving highly significant protection against enterotoxigenic E . coli strains bearing the corresponding colonization factor antigens . Protection was also observed with Fab fragments of the CFA/I antibodies . Addition of the anti-lipopolysaccharide serum to a protective antiserum against purified heat-labile enterotoxin resulted in an antisecretory effect which slightly exceeded the sum of the effects obtained with each preparation alone . The combination of antiserum against CFA/I or CFA/II with anti-enterotoxin gave protection that equaled the product of the effects obtained with each antiserum alone; i.e . the antisera cooperated synergistically.

Infect Immun, 1982 Oct, 38(1), 31 - 4
Polyclonal B-cell response to stimulation with Escherichia coli lipopolysaccharide in dietary protein restriction; Malave I et al.; The polyclonal B-cell response to Escherichia coli lipopolysaccharide was studied in C57BL/6 mice maintained after weaning on either a moderate protein-restricted diet with 8% casein or a normal diet . After in vitro or in vivo stimulation with the endotoxin, autoreactive and anti-hapten antibody-producing cells were quantitated by direct plaque assay, using bromelain-treated mouse erythrocytes and trinitrophenylated sheep erythrocytes as targets . Larger numbers of plaque-forming cells were generated in cultures of spleen cells from dietary-restricted than from normal mice stimulated with various doses of lipopolysaccharide . The number of background plaque-forming cells was also higher in nonstimulated spleen cell cultures from restricted animals . After injection of lipopolysaccharide in vivo, the number of cells producing antibodies to bromelain-treated mouse erythrocytes per 10(7) spleen cells was significantly increased in dietary-deficient mice . The results are discussed in relation to the different sensitivities of lymphocyte populations to protein deficiency and to the possible presence of high levels of endogenous polyclonal B-cell activators in the restricted mice.

Hoppe Seylers Z Physiol Chem, 1982 Oct, 363(10), 1241 - 6
Phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria, and from Escherichia coli: substrate specificity relationship with regard to ATP analogs; Gabius HJ et al.; Twelve structural analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria and E . coli . Three compounds are substrates for all five phenylalanyl-tRNA synthetase, three are completely inactive, while the other ATP analogs show differing properties with the different enzymes . Their Km, Ki and V values have been determined . The importance of the amino group in Position 6, the nitrogen in Position 7 and an unsubstituted Position 8 of the purine moiety as well as the supposed anti-conformation of the glycosidic bond and coordination of the magnesium cation to N-7 appear to be conserved through evolution . Bulky substituents on the 2' and 3' of the ribose moiety are generally not tolerated . Graduation of substrate properties of some analogs are similar for the intracellular heterotopic isoenzymes from yeast and hen liver.

Eur J Biochem, 1982 Oct, 127(2), 225 - 9
Chemical cross-linking of elongation factor G to both subunits of the 70-S ribosomes from Escherichia coli; Skold SE; Ribosomal proteins situated at or near the binding site of elongation factor G (EF-G) on the Escherichia coli ribosome have been identified by use of the bifunctional, cleavable cross-linker, dimethyl-4,9-diaza-5,8-dioxo-6,7-dihydroxy-dodecanebisimidate . Five different bimolecular EF-G x ribosomal-protein complexes were isolated electrophoretically . The ribosomal proteins found in each of these complexes were identified as the 50-S-subunit proteins L6, L7/L12 and L14 and the 30-S-subunit proteins S12 and S19 . In the presence of thiostrepton, which prevents binding of EF-G to the ribosome, there was a considerable decrease in the yield of each of these cross-linked complexes . The data suggest that EF-G is bound close to the ribosomal subunit interface.

Cell, 1982 Oct, 30(3), 915 - 23
Binding of the origin of replication of Escherichia coli to the outer membrane; Hendrickson WG et al.; The replication origin of the Escherichia coli chromosome binds with high affinity to outer membrane preparations . This binding requires a 460 bp stretch of origin DNA between positions -40 and 420 of the oriC map . Specific binding can be detected by the use of a membrane filter retention assay in the presence of excess calf thymus DNA . This binding is enhanced by divalent cations and takes place specifically at a few (0.7-3.0) membrane sites per cell . The apparent affinity of origin DNA for membranes is enhanced by two peptides, (55 kilodaltons (kd) and 75 kd), which remain attached to the DNA through treatment with 5.5 M cesium chloride.

Cell, 1982 Oct, 30(3), 865 - 71
Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon; Schumperli D et al.; Precise frameshift and nonsense mutations were introduced into the region preceding the galactokinase gene (galK) of Escherichia coli . These mutations after the position at which upstream translation terminates relative to the galK translation initiation signal . Constructions were characterized that allow ribosomes to stop selectively before, within or downstream from the galK initiation signal . The effects of these mutations on galK expression were monitored . Galactokinase levels are highest when upstream translation terminates within the galK initiation region . In contrast, when translation stops either upstream or down stream from the galK start site, galK expression is drastically reduced . These results demonstrate that the galK gene is translationally coupled to the gene immediately preceding galK in the gal operon (that is, galT), and that the coupling effect depends primarily on the position at which upstream translation terminates relative to the galK start site . Possible mechanisms and implications of this translational coupling phenomenon are discussed.

Cell, 1982 Oct, 30(3), 855 - 64
Fusion of the Escherichia coli tRNALeu1 promoter to the galK gene: analysis of sequences necessary for growth-rate-dependent regulation; Duester G et al.; We have fused DNA fragments derived from an Escherichia coli tRNALeu1 operon to the galK gene of E . coli to identify sequences necessary for the in vivo initiation of transcription and growth-rate-dependent regulation . Promoter sequences consisting of residues from -50 to +56 or -50 to +5 with respect to the in vivo site for initiation of transcription were introduced into chimeric plasmids upstream from the galK gene . Cells bearing these chimeric plasmids exhibited much higher levels of galactokinase than did cells bearing plasmids wherein the galactose promoter was fused to galK . This indicates that the tRNALeu1 promoter is substantially more efficient than the gal promoter . The tRNALeu1 promoter-galK chimeras exhibited marked growth-dependent regulation in a manner consistent with that reported for tRNA regulation . Since tRNALeu1 DNA spanning residues -50 to +5 was sufficient to provide growth rate regulation of galK, an inverted repeat centered at position +17 is not, under the conditions we used, required for this type of regulation.

Am Surg, 1982 Oct, 48(10), 552 - 4
Pulmonary extraction of biogenic amines during septic shock; Kerstein MD et al.; The effect of live Escherichia coli on the pulmonary extraction of the biogenic amines 14C 5-hydroxytryptamine, (5-HT) and 3H-epinephrine was investigated . The labeled isotopes were injected into a central venous catheter and collected from an aortic catheter . One hundred per cent of the labeled epinephrine was recovered in the control and septic state . Only 32.8 +/- 3.6% SEM of the 5-hydroxytryptamine was recovered before sepsis and 42.5 +/- 4.9% SEM after sepsis . During sepsis, mean arterial pressure fell to 58 mm Hg from 121 mm Hg . Pulmonary shunt increased from .7 +/- .05 SEM to .33 +/- .09 SEM.

Br J Surg, 1982 Oct, 69(10), 625 - 9
Endotoxin, prostaglandins and renal fibrin deposition in obstructive jaundice; Fletcher MS et al.; The delayed clearance of endotoxins in obstructive jaundice may cause renal impairment by inducing renal vasoconstriction and glomerular fibrin deposition as a consequence of intravascular coagulation . As endotoxins activate arachidonic acid metabolism we have examined the effects of selective inhibitors on mortality, plasma TXB2 and 6-oxo-PGF1 alpha production and renal fibrin deposition in rats with obstructive jaundice following endotoxin administration . Jaundiced rats had a high mortality following endotoxin--58 per cent at 4 h and 83 per cent at 24 h . Pretreatment with indomethacin 3 mg/kg i.p., dazoxiben 3 mg i.p . or prostacyclin 300 ng/kg i.v . produced significant improvements in survival . Endotoxaemia was associated with significant elevations of plasma TXB2 and early inhibition of plasma 6-oxo-PGF1 alpha generation . Renal fibrin deposition, assessed using indirect immunofluorescence and a 125I-labelled fibrinogen uptake ratio, occurred in jaundiced kidneys following endotoxin and could be prevented using indomethacin, dazoxiben and prostacylin . These results suggest that endotoxin-induced TXA2 production can cause renal fibrin deposition in obstructive jaundice, thus contributing in the pathogenesis of the renal impairment.

J Pediatr, 1982 Oct, 101(4), 519 - 23
Effect of iron saturation on the bacteriostasis of human serum: in vivo does not correlate with in vitro saturation; Baltimore RS et al.; Human serum inhibits the growth of a variety of human pathogens . One of the serum bacteriostatic components is transferrin, the major iron-binding protein . In the presence of transferrin, free iron, which is required for bacterial nucleoprotein synthesis, is unavailable and bacterial growth is inhibited . In an in vitro system, we tested the hypothesis that serum with highly saturated transferrin allows free iron to be available for rapid bacterial growth . We first confirmed the finding that addition of ionic iron sufficient to saturate transferrin in normal sera inhibits the bacteriostatic activity for Escherichia coli . In contrast, no differences were found in the growth rate of E . coli in sera from individuals representing the entire range of transferrin saturation found in humans (iron-deficient, normal, and thalassemic) . This finding supports the thesis that iron added in vitro is more easily extracted than in vivo, where it is tightly bound to transferrin, and does not support the contention that ordinary iron treatment predisposes infants to infection.

J Bacteriol, 1982 Oct, 152(1), 7 - 13
Effect of silver ions on transport and retention of phosphate by Escherichia coli; Schreurs WJ et al.; Silver ions inhibited phosphate uptake and exchange in Escherichia coli and caused efflux of accumulated phosphate as well as of mannitol, succinate, glutamine, and proline . The effects of Ag+ were reversed by thiols and, to a lesser extent, by bromide . In the presence of N-ethylmaleimide and several uncouplers, Ag+ failed to cause phosphate efflux, but still inhibited exchange of intracellular and extracellular phosphate, indicating an interaction at more than one site . It is unlikely that Ag+ caused metabolite efflux by acting solely as an uncoupler, as an inhibitor of the respiratory chain, or as a thiol reagent.

J Bacteriol, 1982 Oct, 152(1), 534 - 7
O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli; Mitra S et al.; O(6)-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine . We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3H-labeled O(6)-methylguanine as the only modified base . Although both wild-type and adaptation-deficient (ada) mutants of E . coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase.

J Bacteriol, 1982 Oct, 152(1), 506 - 9
Sequence analysis of the heat-labile enterotoxin subunit B gene originating in human enterotoxigenic Escherichia coli; Yamamoto T et al.; In this study, we determined the amino-terminal coding sequence, covering the signal peptide and the amino-terminus of the mature peptide, of the heat-labile enterotoxin subunit B (LT-B) gene originating in human enterotoxigenic Escherichia coli . Neither the signal sequence nor the amino-terminal sequence of the mature LT-B was identical to those sequences from porcine enterotoxigenic E . coli, but there was an extensive homology.

J Bacteriol, 1982 Oct, 152(1), 479 - 84
Plasmolysis during the division cycle of Escherichia coli; Olijhoek AJ et al.; Cells of Escherichia coli were plasmolyzed with sucrose . They were classified according to length by way of electron micrographs taken from samples prepared by agar filtration . The percentage of plasmolyzed cells increased about two- and threefold between mean cell sizes of newborn and separating cells . However, dividing cells were less frequently plasmolyzed than nondividing cells of the same length class . Analysis of cell halves (prospective daughters) in dividing cells showed that they behaved as independent cellular units with respect to plasmolysis . The results indicate that compressibility of the protoplast (given a certain plasmolysis space) is inversely related to cell size . That a dividing cell does not react as one osmotic compartment to osmotic stress may suggest that cell size-dependent strength of the cell membrane-cell wall association, rather than variation in turgor, plays a role during the cell division cycle.

J Bacteriol, 1982 Oct, 152(1), 372 - 83
Cloning of trg, a gene for a sensory transducer in Escherichia coli; Harayama S et al.; Clones of trg, a gene which codes for a chemotactic transducer, were isolated linked to ColE1 and pBR322 vectors . Studies with the hybrid plasmids demonstrated unequivocally that trg is the structural gene for methyl-accepting chemotaxis protein III . The Trg protein was found to be structurally complex, electrophoresing as a series of seven bands on high-resolution sodium dodecyl sulfate-polyacrylamide gels . The multiplicity of bands is a function of the activity of cheR, which codes for a methyltransferase, and of cheB, which codes for a demethylase . It appears that Trg, a quantitatively minor transducer, resembles the two major transducer proteins, Tsr and Tar, in that all three are multiply methylated and also multiply modified in a second way which requires an active cheB gene . However, preliminary analysis of the Trg protein indicated that it is significantly less related structurally to the Tsr or Tar protein than those two transducers are to each other . This implies that the features of multiple methylation and cheB-dependent modification are likely to be critical for the common physiological functions in chemotactic excitation and adaptation performed by all three transducers.

J Bacteriol, 1982 Oct, 152(1), 357 - 62
Molecular cloning and regulation of expression of the genes for initiation factor 3 and two aminoacyl-tRNA synthetases; Elseviers D et al.; A 22-kilobase fragment of the Escherichia coli chromosome which contains the genes for translation initiation factor 3, phenylalanyl-tRNA synthetase, and threonyl-tRNA synthetase was cloned into plasmid pACYC184 . The hybrid plasmid (designated pID1) complements a temperature-sensitive pheS lesion in E . coli NP37 . pID1-transformed NP37 overproduce initiation factor 3 and phenylalanyl-tRNA synthetase . Gene expression from pID1 was studied in vitro in a coupled transcription-translation system and in minicells . The results suggest that the genes for initiation factor 3 and phenylalanyl- and threonyl-tRNA synthetase are regulated by different mechanisms.

J Bacteriol, 1982 Oct, 152(1), 35 - 41
Changes in cell dimensions during amino acid starvation of Escherichia coli; Grossman N et al.; Electron microscopic analysis was used to study cells of Escherichia coli B and K-12 during and after amino acid starvation . The results confirmed our previous conclusion that cell division and initiation of DNA replication occur at a smaller cell volume after amino acid starvation . Although during short starvation periods, the number of constricting cells decreased due to residual division, it appears that during prolonged starvation, cells of E . coli B and K-12 were capable of initiating new constrictions . During amino acid starvation, cell diameter decreased significantly . The decrease was reversed only after two generation times after the resumption of protein synthesis and was larger in magnitude than that previously observed before division (F . J . Trueba and C . L . Woldringh, J . Bacteriol . 142:869-878, 1980) . This decrease in cell diameter correlates with synchronization of cell division which has been shown to occur after amino acid starvation.

J Bacteriol, 1982 Oct, 152(1), 223 - 31
Regulation of the ColV plasmid-determined iron (III)-aerobactin transport system in Escherichia coli; Braun V et al.; Regulation by iron was studied in Escherichia coli strains whose iron supply was entirely dependent on the iron(III)-aerobactin system determined by the ColV plasmid . By the insertion of phage Mu (Ap lac) into the ColV plasmid, mutants were selected that could no longer grow in iron-limited media . The inserted Mu (Ap lac) strongly reduced the amount of aerobactin and he cloacin receptor protein formed by the cells . Their production was no longer subject to regulation by iron . The Mu (Ap lac) insertion apparently led to a polar effect on the expression of the presumably closely linked genes that control the synthesis of aerobactin and the cloacin receptor protein . The expression of the beta-galactosidase gene on the inserted phage genome came under the control of the iron state of the cells . Under iron-limited growth conditions, the amount of beta-galactosidase synthesized was, depending on the strain studied, 6 to 30 times higher than under iron-sufficient growth conditions . In fur mutants with an impaired iron regulation of ll iron supply systems studied so far, high amounts of beta-galactosidase were synthesized independent of the cells' iron supply . The results demonstrate an iron-controlled promoter on the ColV plasmid which is subject to regulation by the chromosomal fur gene.

J Bacteriol, 1982 Oct, 152(1), 1 - 6
Molecular cloning of pheR in Escherichia coli K-12; Gowrishankar J et al.; The regulator gene pheR, which in Escherichia coli controls the expression of pheA, the structural gene for chorismate mutase P-prephenate dehydratase, was cloned on to multicopy plasmids directly from the E . coli chromosome; this was achieved with the aid of the tetracycline resistance transposon, Tn10, that had been inserted very close to the pheR gene . Subsequently, pheR was subcloned on a 1.1-kilobase-pair fragment on the plasmid vector pBR322; its position on the plasmid was localized by the method of gamma delta-mediated transpositional inactivation . The pheR gene product was identified in maxicells and found to be a protein of subunit molecular weight 19,000, suggesting that the coding segment of the gene is about 500 nucleotide pairs long.

Proc Natl Acad Sci U S A, 1982 Oct, 79(19), 5981 - 5
Orientation of cohesive end site cos determines the active orientation of chi sequence in stimulating recA . recBC-mediated recombination in phage lambda lytic infections; Kobayashi I et al.; Sequence chi, 5'G-C-T-G-G-T-G-G, locally enhances homologous recombination by recA and recBC proteins of Escherichia coli . Previous work showed that, in phage lambda, chi is more active in one orientation (leftward) than in the other (rightward) . Inverting cos, the sequence for the mature DNA ends of lambda, reverses this orientation dependence: the rightward chi becomes fully active, and the leftward chi becomes relatively inactive . We surmise that chi action in phage lambda is coupled with DNA packaging or injection.

Proc Natl Acad Sci U S A, 1982 Oct, 79(19), 5813 - 7
An amber suppressor tRNA gene derived by site-specific mutagenesis: cloning and function in mammalian cells; Laski FA et al.; We describe the synthesis, cloning, expression, and in vivo function of a suppressor tRNA gene in mammalian cells . By using "primer-directed mutagenesis" on a Xenopus laevis tyrosine tRNA gene cloned into the recombinant single-strand phage M13mp5, we have generated an amber suppressor tRNA gene that has a nucleotide change--GTA leads to CTA--in the anticodon sequence . The suppressor (Su) tRNA gene was introduced into monkey kidney cells (CV-1) by using simian virus 40 (SV40) DNA as vector (SV40-tRNATyrSu+) . CV-1 cells infected with virus containing the mutant, but not the wild-type, tRNA gene produce a functional amber suppressor tRNA as indicated by suppression of amber mutations in co-infecting adenovirus serotype 2-SV40 hybrids . Further evidence that suppression of these amber mutations is tRNA mediated was derived by isolation of total tRNA from CV-1 cells infected with the SV40-tRNATyr (Su+) recombinant and its use in demonstration of read through of an amber codon during in vitro translation of tobacco mosaic virus RNA in reticulocyte extracts . Interestingly, the amplification of an amber suppressor gene in CV-1 cells does not interfere with SV40 production, suggesting that suppression of amber codons may not be very deleterious to mammalian cell metabolism.

Gene, 1982 Oct, 19(3), 337 - 44
Expression of cloned calf prochymosin gene sequence in Escherichia coli; Nishimori K et al.; An expression plasmid for calf prochymosin (prorennin) cDNA was constructed . The plasmid (pCR301) contains the lacUV5 promoter in front of the fused gene in which the codons for the N-terminal four amino acids of prochymosin cDNA were replaced with those for the N-terminal ten amino acids of beta-galactosidase . Synthesis of the fused protein with the expected Mr was detected immunologically in Escherichia coli harboring pCR301 . The product seemed to be localized in the cell membrane of the bacterial host.

Gene, 1982 Oct, 19(3), 327 - 36
Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5; Beck E et al.; The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp . The derived gene product, an aminoglycoside 3'-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated Mr of 29053 . Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).

Gene, 1982 Oct, 19(3), 313 - 9
Cloning of alkaline phosphatase isozyme gene (iap) of Escherichia coli; Nakata A et al.; In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions . A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits . A chromosomal fragment which complemented the Iap- phenotype was cloned into pBR322 by a shotgun method . Transducing phage lambda iap was constructed in vitro from the chromosomal fragment containing the iap gene and lambda tna DNA . The integration site of the phage on chromosome was identified as the iap locus by P1 transduction, which meant that the cloned chromosomal DNA contained authentic iap gene . The restriction map of the hybrid plasmid was constructed . Based upon this information, several iap deletion plasmids as well as smaller iap+ plasmids were constructed . Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA . The cells carrying the iap+ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion . This indicates overproduction of the iap gene product.

Gene, 1982 Oct, 19(3), 259 - 68
The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers; Vieira J et al.; A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed . In one of these vectors a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM) . The drug-resistance marker can be cleaved out of this vector with any of the restriction enzymes that recognize a site of the flanking sequences of the RSM to generate an RSM with either various sticky ends or blunt ends . These fragments can be used for insertion mutagenesis of any target molecule with compatible restriction sites . Insertion mutants are selected by their resistance to kanamycin . When the drug-resistance marker is removed with PstI, a small in-frame insertion can be generated . In addition, two new MCSs having single restriction sites have been formed by altering the symmetrical structure of MCS7 . The resulting plasmids pUC8 and pUC9 allow one to clone doubly digested restriction fragments separately with both orientations in respect to the lac promoter . The terminal sequences of any DNA cloned in these plasmids can be characterized using the universal M13 primers.

Gene, 1982 Oct, 19(3), 251 - 7
Transposon Tn3-mediated replicon fusion; Miyoshi J et al.; To investigate inter-replicon transposition of Tn3, we used the cosmid-phage lambda packaging system coupled with density gradient fractionation and isolated recombinant molecules of different sizes . Cosmids derived from ampicillin-resistance-transducing phage were classified into four groups: (1) cosmid-Tn3 donor cointegrates considered as Tn3 transposition intermediates, (2) similar cointegrates carrying deletions of one copy of Tn3 and of adjacent cosmid DNA sequences, (3) cosmids carrying a single Tn3 insertion, and (4) cosmids carrying two independent Tn3 insertions . Genetic and biochemical studies indicated that cosmids isolated from ampicillin-resistance transductants were derived from the authentic cosmid-Tn3 donor cointegrate intermediates.

Acta Pharmacol Toxicol (Copenh), 1982 Oct, 51(4), 330 - 5
Reversal of enterotoxic diarrhoea by anaesthetic and membrane-stabilizing agents; Lonnroth I et al.; The effect of membrane-stabilizing and sedative drugs on enterotoxic diarrhoea was studied in mice . Fluid secretion was induced in ligated loops of the small intestine by challenge with cholera toxin (CT), heat-labile enterotoxin (LT) from Escherichia coli or dibuturyl-cyclic AMP (dB-cAMP) . Chlorpromazine, melperone, diazepam, mebumal, ketamine and ethanol all inhibited CT-induced hypersecretion . ED50 being 1.5, 4, 4, 35, 70 and 1500 mg/kg, respectively . The drugs also blocked CT-stimulation of adenylate cyclase, which mediates the action of CT . Concomitant with the antisecretory effect a sedative effect was induced, as judged by the motility and righting reflex of the animals . Hypersecretion by E . coli and LT was also totally blocked by chlorpromazine, diazepam, ketamine and ethanol . In contrast, the secretion by dB-cAMP which bypasses adenylate cyclase in its action, was not affected by diazepam and was only partly reversed by chlorpromazine, ketamine and ethanol . The reversal of dB-cAMP-secretion is probably due to enhanced absorption, rather than inhibition of adenylate-cyclase . The use of membrane-stabilizing drugs may represent a new principle for pharmacological treatment of diarrhoea.

Mol Cell Biol, 1982 Oct, 2(10), 1238 - 46
Deletions of N-terminal sequences of polyoma virus T-antigens reduce but do not abolish transformation of rat fibroblasts; Katinka M et al.; Polyoma virus transforms, upon infection or DNA transfection, nonpermissive Fisher rat fibroblasts . Cloned viral DNA was deleted of sequences around the Bg/I site at nucleotide 86 by Bal31 nuclease treatment and then recloned in Escherichia coli . The extent of deletion for each mutant was then determined by DNA sequencing . Deletions included the early transcription control signals; others stretched into the N-terminal coding sequences of the viral tumor antigens . The transformation efficiency of 16 mutants was tested by transfecting rat fibroblasts . Expression of the T antigens was analyzed by immunofluorescence detection after transfection of rat fibroblasts, mouse secondary embryo cells, and HeLa cells . We found that the absence of the early transcription control sequences (TATA and CAAT boxes) did not significantly alter the transformation capacity of the virus . On the other hand, deletion of the initiator methionine ATG codon or further into the coding sequences did abolish the transformation capacity in some mutants, whereas others maintained a reduced transforming activity, possibly by initiation of translation in a penultimate methionine.

Genetika, 1982 Oct, 18(10), 1636 - 44
{Comparative analysis of the P-1-group plasmids of incompatibility}; Azarian NG et al.; Wide host range plasmids (IncP-1) R906, R751 and R702 have several cleavage sites for BamHI, HindIII and EcoRI enzymes, in contrast to RP4 plasmid . Using these enzymes, deletion mutants of R906 plasmid have been obtained in vitro which only lost short DNA fragments (1 to 14 kb) . A narrow host range pAV1 plasmid of the same incompatibility group has been transformed into the cells of Escherichia coli . pAV1 is stably maintained in the new host and retains its narrow host range in the course of conjugation . Different restriction fragments of R702, R751, R906 and R906-derived deletion mutants hybridize with the nick-translated probe of RP4 DNA . It is suggested that the wide host range plasmids have a similarity in structural and functional organization.

Genetika, 1982 Oct, 18(10), 1631 - 5
{Cloning of the adenylate cyclase gene of Escherichia coli K-12}; Smirnov IuV et al.; The adenylate cyclase gene of Escherichia coli has been cloned on the plasmid vector pBR325 . The hybrid plasmid pTH4 obtained has a molecular weight of 6,4 megadalton and represents pBR325 plasmid with the insertion of 2,8 megadalton in the Pst1 site . The cya mutant bacteria carrying pTH4 recover their ability to utilize mannitol, lactose and other carbohydrates as carbon sources, and lose this ability again in the case of rare spontaneous excision of the DNA insert from the Pst1 site . The phenotypical effect of pTH4 in cya mutants can be only seen in the crp+ genome . The strains carrying pTH4 are also characterized by the ability of beta-galactosidase induction under conditions of catabolite repression . Besides, the bacteria containing cya+ allele on the plasmid do not grow on glycerol, which seems to be caused by toxic concentrations of methylglyoxal formed as a result of the increased intracellular level of cyclic adenosine monophosphate.

Genetika, 1982 Oct, 18(10), 1620 - 30
{Isolation and study of hybrid plasmids carrying Escherichia coli threonine operon genes}; Khurges EM et al.; A fragment of Escherichia coli chromosome containing the intact threonine operon or its distinct genes has been cloned on the pBR322 plasmid . This fragment has been mapped using some restriction endonucleases . Cloning results in an increased level of appropriate enzyme activity in cells containing hybrid plasmids . Those carrying the complete threonine operon are capable of accumulating threonine up to 5 g/l in culture medium during 48 h . When multi-copy plasmids are used for gene cloning, interpretation of experiments aimed at transformation of auxotrophic bacterial strains, might be complicated . For example, transformation of appropriate threonine auxotrophs by a hybrid plasmid carrying mutation in the threonine gene, might result in prototrophic phenotype . It is possible that the great amount of mutant enzyme molecules compensated their low activity . On the contrary, the presence of a gene within the plasmid, as shown by restriction and biochemical analysis, did not always ensure the growth on a minimal medium of auxotrophs transformed by this plasmid.

Genetika, 1982 Oct, 18(10), 1613 - 9
{Instability study of Escherichia coli strains containing hybrid plasmids with threonine operon fragments}; Kozlov IuI et al.; The stability of Escherichia coli strains carrying hybrid plasmids which contain ColE1-like replicon and threonine operon genes was studied . It was shown that the main reason for instability is the loss of a plasmid . The second reason for instability is the rec-dependent recombination that leads to formation of new plasmids . All experiments where instability of strains was observed, can be quantitatively described by the model that presumes a random loss of plasmids in cell population with a frequency of about 7.10(-4), which results in diappearance of plasmid-borne cells due to their low growth rate . Instability increases during the stationary phase but it is not easy to quantitatively estimate this process.

Genetika, 1982 Oct, 18(10), 1581 - 9
{Characteristics of hybrid plasmids carrying the genes of colicinogenic plasmid Collb-P9 responsible for colicin Ib synthesis and inhibition of phage T5 development}; Vorob'eva IP et al.; The EcoRI and HindII restriction endonucleases and pBR325 vector plasmid were used to obtain a set of hybrid plasmids containing ColIb-P9 fragments carrying the characters for colicin Ib synthesis and immunity and the ability to inhibit T5 phage growth . The genes responsible for colicin synthesis and immunity are closely linked and localized in the EcoRI fragment with a molecular weight of 1.85 MD (pIV41) or in the HindII fragment of 2.4 MD (pIV1) . The clones containing these plasmids show an increased level of both spontaneous and mitomycin C-induced colicin synthesis and an increased level of immunity due to a larger dosage of the genes . The genes controlling T5 growth inhibition are localized in other restriction fragments of ColIb DNA: the EcoRI fragment of 1.45 MD (pIV7) and the HindII fragment of 4.3 MD (pIV5) . We have demonstrated by means of hybrid plasmids that T5 growth inhibition is not connected with the colicin Ib synthesized in infected cells and is controlled by other specific product(s) of the ColIb plasmid genes . T5 phage growth was as efficient in clones containing plasmids with cloned colicin Ib genes as in a strain without plasmids . An investigation of the expression of the genes inhibiting T5 phage growth in an in vitro protein synthesis system has revealed a protein with a molecular weight of 36 000 which seems to take part in the process.

Proc Natl Acad Sci U S A, 1982 Oct, 79(20), 6156 - 60
Nucleotide sequence of the ilvB promoter-regulatory region: a biosynthetic operon controlled by attenuation and cyclic AMP; Friden P et al.; The DNA sequence of the promoter-regulatory region of the ilvB operon of Escherichia coli was determined . This region encodes a potential leader polypeptide containing 32 amino acids, 12 of which are the regulatory amino acids valine and leucine . Approximately 50 residues downstream from the coding region for the potential leader peptide is a site for terminating transcription . In vitro transcription experiments show that transcription terminates at this site and produces a leader mRNA of approximately equal to 188 nucleotides . A model for the multivalent regulation of this operon by valyl- and leucyl-tRNA is proposed on the basis of the mutually exclusive formation of five strong stem-and-loop structures in the leader mRNA . In addition, the -35 and -70 regions of this sequence show close structural homologies to areas in cyclic AMP receptor protein (CRP)-dependent promoters reported to be important for CRP function . In vitro transcription from the ilvB promoter was greatly increased by cyclic AMP and CRP . Taken together, these data strongly suggest that the ilvB biosynthetic operon is negatively controlled by multivalent transcription termination and is positively regulated by cyclic AMP and CRP.

J Virol, 1982 Oct, 44(1), 374 - 83
Virion DNA of ground squirrel hepatitis virus: structural analysis and molecular cloning; Ganem D et al.; The structure of the encapsidated DNA genome of ground squirrel hepatitis virus (GSHV) has been examined by restriction endonuclease cleavage, nucleic acid hybridization, and molecular cloning . GSHV virion DNA is a relaxed circular molecule of approximately 3,200 bases in length; most molecules harbor an extensive single-stranded region which is largely confined to one-half of the genome . The full-length viral DNA strand is covalently bound to protein . The single-stranded region can be repaired in vitro by the action of the endogenous virion polymerase, exogenously added DNA polymerase from avian myeloblastosis virus, or both . Restriction enzyme cleavage of viral DNA from different isolates demonstrated that multiple variants of GSHV exist in nature . The genomes of two such strains have been cloned in Escherichia coli, and their physical maps have been determined . Nucleic acid hybridization studies revealed that the strains share sequence homology with the DNA of human hepatitis B virus . Regions homologous to the coding regions for the surface and core antigens of human hepatitis B virus have been localized on the GSHV chromosome . Molecular cloning experiments have also led to the identification of a region of the viral genome which is altered in a procaryotic host.

Eur J Biochem, 1982 Oct, 127(2), 347 - 9
The intracellular concentration of pyrophosphate in the batch culture of Escherichia coli; Kukko E et al.; We applied our colorimetric PPi determination method {Heinonen, J., Honkasalo, S . and Kukko, E . (1981) Anal . Biochem . 117, 293-300} to bacterial cultures and measured the intracellular concentration of PPi in the batch culture of Escherichia coli . The cells growing in minimal medium contained about 2.5 nmol PPi/mg protein (0.5 mM) . There was no extracellular PPi . A similar concentration of PPi was found also in cells growing in minimal medium enriched by amino acid mixture.

Cell, 1982 Oct, 30(3), 883 - 92
Regulation of Tn5 by the right-repeat proteins: control at the level of the transposition reaction?
Isberg RR, Lazaar AL, Syvanen M.
The transposon Tn5 consists of inverted repeats, called IS50R and IS50L, each of which encode two proteins . We show here that the larger protein encoded on IS50R, protein 1, is absolutely required for transposition . Deletion or insertion mutants that fail to make this protein fail to promote gene movement . In addition, his protein acts in cis preferentially . We also show that the smaller protein encoded on IS50R, protein 2, is competent to inhibit transposition of a Tn5 freshly introduced into the cell on a lambda phage . In contrast, the proteins from IS50L possess neither of these two activities . By assaying expression of proteins that are hybrids between beta-galactosidase and IS50R proteins, we find that the regulation of transposition cannot be due to the inhibitor repressing synthesis of Tn5 proteins . Control experiments, in which we assay synthesis of IS50 proteins synthesized from a lambda::IS50R that has been infected into cells carrying the transposition inhibitor, confirm this conclusion.

Cell, 1982 Oct, 30(3), 873 - 82
Control of Tn5 transposition in Escherichia coli is mediated by protein from the right repeat; Johnson RC et al.; The right repeat in Tn5, which encodes protein absolutely required for transposition, is also capable of inhibiting Tn5 transposition . Analysis of Tn5 mutants indicates that the left repeat is defective in supplying the transposition-inhibition function because of the sequence difference between the repeats located at nucleotide 1443; that the transposition-inhibition activity is a function of the quantity of right-repeat protein synthesis; that the smaller of the right-repeat proteins, protein 2, is sufficient for supplying the transposition-inhibition function (but not for the transposase activity); and that the transposition-inhibition function can act in trans, as opposed to the transposase activity, which functions efficiently only in cis . Gene fusion experiments indicate that the transposition-inhibition activity cannot be explained by autogenous regulation of right-repeat protein synthesis . Finally, immunoprecipitation assays of right-repeat protein-lacZ fusion proteins indicate that protein 2 is synthesized in significantly greater amounts than protein 1 in whole cells . This synthetic ratio may ber important with respect to the control of Tn5 transposition.

J Bacteriol, 1982 Oct, 152(1), 549 - 52
Plasmid transfer to Bordetella pertussis: conjugation and transformation; Weiss AA et al.; Plasmids of the P and W incompatibility groups were introduced into Bordetella pertussis by conjugation . Plasmid DNA isolated from B . pertussis could be reintroduced by transformation . DNA isolated from Escherichia coli could not be introduced into B . pertussis by transformation if this DNA contained HindIII restriction sites . We have demonstrated that HindIII sites are modified by B . pertussis . Plasmids of the FI and FII incompatibility groups could not be introduced into B . pertussis by conjugation, and nonconjugative plasmids of the ColE1 and Q incompatibility groups could not be introduced by transformation . Our ability to introduce plasmids in the laboratory suggests that the apparent lack of plasmids in natural isolates of B . pertussis is not due to an inability to act as a plasmid recipient.

J Bacteriol, 1982 Oct, 152(1), 351 - 6
Cloning and identification of the product of the dnaE gene of Escherichia coli; Welch MM et al.; We successively subcloned the dnaE gene of Escherichia coli into pBR322, resulting in a plasmid that contains 4.6 kilobases of E . coli DNA . This plasmid can complement a dnaE temperature-sensitive mutation . A restriction map of the dnaE gene and the surrounding 10.7-kilobase region of the E . coli chromosome was determined . A unique HindIII restriction endonuclease site within the cloned segment of DNA was identified as a site required for expression of the dnaE gene . By using the maxicell plasmid-directed protein synthesizing system, we demonstrated that dnaE codes for the alpha subunit of DNA polymerase III.

J Bacteriol, 1982 Oct, 152(1), 208 - 14
Anaerobic incubation enhances the colony formation of a polA recB strain of Escherichia coli K-12; Morimyo M; Escherichia coli strain E247 (polA1 recB21) has reduced colony formation (even at the permissive temperature of 30 degrees C) because of a poor suppressor mutation (sup-126) . The colony formation was enhanced in the absence of oxygen about 3-fold at 30 degrees C and 10(6)-fold at 43 degrees C, suggesting that a polA recB strain was inviable due to oxygen toxicity . Colony formation was also increased by incubation in an agar medium containing the reducing agent thioglycolate and incubation in the presence of chloroform-killed Saccharomyces cerevisiae pet+ cells, but not pet cells . Since the E247 strain viability was inversely dependent on the oxygen pressure and since the strain was more sensitive to superoxide radical than either the polA or the recB mutant, it seems likely that the polA and recB genes play a role in repairing DNA damage during respiration.

Jpn J Antibiot, 1982 Oct, 35(10), 2354 - 63
{Clinical studies of piperacillin in the field of obstetrics and gynecology}; Seiga K et al.; We studied piperacillin (PIPC) clinically to evaluate its utility and safety, and obtained the following results . PIPC was given at a daily dose of 1 approximately 4 g for 3 approximately 10 days by bolus injection or dripping infusion to 18 patients with various infections in the field of obstetrics and gynecology . Four of 5 cases with intrauterine infection, 2 of 3 cases with focus infection of uterine cancer, 2 cases with perinatal intrauterine infection, 2 of 6 cases with adnexal infection and 2 cases with vulvar infection proved to respond effectively . The overall efficacy rate was 66.7% . No side effect or abnormal laboratory findings were observed except for 1 case with rash.

J Bacteriol, 1982 Oct, 152(1), 98 - 103
Application of hybrid plasmids carrying glycolysis genes to ATP production by Escherichia coli; Shimosaka M et al.; The closely linked structural genes of phosphofructokinase (pfkA) and triosephosphate isomerase (tpi) of Escherichia coli were separately cloned onto plasmid pBR322 . By gene dosage effects, transformed cells of E . coli C600 with these pBR322 hybrid plasmids showed 7- and 16-fold increases in the specific activities of phosphofructokinase and triosephosphate isomerase, respectively, over the specific activities in C600 . Dried preparations of E . coli cells dosed with these genes showed appreciably high ATP-regenerating activity.

J Bacteriol, 1982 Oct, 152(1), 521 - 3
Regulation of D-arabinose utilization in Escherichia coli K-12; Skjold AC et al.; Studies involving lambda phage transduction of the D-arabinose utilization gene (dar+) in Escherichia coli K-12 indicated the product of this gene to be a transdominant activator . An apparent anomaly regarding this hypothesis exists in that a diploid recessive lysogen (lambda dar-/dar-) can spontaneously become capable of growth on D-arabinose.

Arch Microbiol, 1982 Oct, 132(4), 304 - 7
A reappraisal of antigenic determinants for nitrogenase from Azotobacter and nitrate reductase from Escherichia coli; Byrne MD et al.; Previous work based on double immunodiffusion assays had shown that there are common antigenic determinants for nitrate reductase from Escherichia coli and component I of nitrogenase from Azotobacter vinelandii . Further work reported herein using a variety of immunoelectrophoretic techniques indicates that the cross-reaction between nitrate reductase and antiserum to component I of nitrogenase results from a contaminant antigen co-purified with nitrate reductase.

J Biochem (Tokyo), 1982 Oct, 92(4), 1173 - 7
Computer analysis of the sequence relationships among 4.5S RNA molecular species from various sources; Takeishi K et al.; Nucleotide sequence homology among 4.5S RNAs from various organisms was examined by computer analysis to evaluate their sequence relationships . Chloroplast 4.5S rRNAs of wheat and tobacco were not significantly related to Escherichia coli 4.5S RNA, but were closely related to the 3'-terminus of bacterial 23S rRNA . Significant sequence homology was found between rat Novikoff hepatoma 4.5S RNAI and mouse and hamster 4.5S RNAs, suggesting that these RNAs are products of a family of genes with diverged sequences . E . coli 4.5S RNA had no significant sequence homology with any rodent 4.5S RNAs as a whole sequence . The E . coli, mouse and hamster 4.5S RNAs, however, were found to share a homologous 14-nucleotide sequence at the center of the molecules, which is known to exist as a conserved sequence in both Alu and Alu-equivalent sequences of mammalian DNAs.

Eur J Biochem, 1982 Oct, 127(3), 531 - 7
The effect of a cytidine-to-uridine transition on the stability of Escherichia coli A19 5-S RNA; Digweed M et al.; We have been able to isolate several species of 5-S ribosomal RNA from Escherichia coli A19 . These molecules were separated on the basis of their differing stabilities during electrophoresis on 12% polyacrylamide gels in 7 M urea . This differing stability is shown, in one case, to be due to a different primary sequence . We have determined the sequence of the least stable of these molecules and have found only one difference to the published sequence of E . coli A19 5-S RNA, namely a uridine in place of a cytidine at position 92 . The consequent G x U base pair, formed in a normally highly stable G x C-rich region, is responsible for a drastic reduction in the stability of the molecule . This instability leads to a less constrained, more compact molecule which thus migrates faster in electrophoresis under denaturing conditions . This species of 5-S RNA is shown to make up 30% of the total 5-S RNA in the 50-S ribosomal subunits in this organism . Further structural studies were carried out using S1 nuclease digestion, sodium bisulphite modification and thermal melting analysis . All these methods indicate a 5-S RNA drastically destabilized in parts of its secondary and tertiary structure . Finally, the ability of the variant 5-S RNA to recognize and form a complex with its 50-S subunit binding proteins was examined and found to be impaired.

Eur J Biochem, 1982 Oct, 127(3), 525 - 9
Binding of tRNA in different functional states to Escherichia coli ribosomes as measured by velocity sedimentation; Schmitt M et al.; The binding of initiator and elongator tRNAs to 70-S ribosomes and the 30-S subunits was followed by velocity sedimentation in the analytical ultracentrifuge . fMet-tRNAfMet binds to A-U-G-programmed 30-S subunits, but not to free or misprogrammed particles . Both the formylmethione residue and the initiation factors increase the stability of the 30-S x A-U-G x fMet-tRNAfMet complex . fMet-tRNAfMet is bound only to the P site of the 70-S ribosome even in the absence of A-U-G . Two copies of tRNAPhe or Phe-tRNAPhe are bound to the ribosome with similar affinity . In contrast to a recent report {Rheinberger et al . (1981) Proc . Natl Acad . Sci . USA, 78, 5310-5314}, it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 x 10(4) M-1 . Phe-tRNAPhe when present as the ternary complex Phe-tRNAPhe . EF-Tu x guanosine 5'-{beta,gamma-methylene}triphosphate binds exclusively to the A site . The peptidyl-tRNA analogue, acetylphenylalanine-tRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site . The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome . The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis.

Eur J Biochem, 1982 Oct, 127(3), 459 - 71
Identification of nucleosides in hydrolysates of transfer RNA by high-resolution mass spectrometry; Pang H et al.; High-resolution mass spectrometry has been investigated as a technique for identification of modified nucleosides in unfractionated hydrolysates of transfer RNA . The method is based on recognition of predetermined sets of exact mass values which are characteristic of individual nucleoside components . Mass spectra are photographically recorded in a non-time-resolved fashion, so that differing rates of nucleoside vaporization are of no consequence . Experimental parameters of sensitivity, spectrometer resolution and rates of vaporization were studied . Under typical conditions, 1-4 micrograms of tRNA are hydrolyzed, converted to volatile trimethylsilyl derivatives, and mass spectra of the resulting mixture recorded during 3 min at resolution 20 000; 75-100% of the minor nucleosides are usually identified in a single recording . The method is complementary to conventional methods of identification which rely on chromatographic mobilities, and can in principle be generally applied to recognition of biologically or chemically modified bases in nucleic acid.

Infect Immun, 1982 Oct, 38(1), 41 - 5
Prediction of antigenic determinants and secondary structures of the K88 and CFA1 fimbrial proteins from enteropathogenic Escherichia coli; Klemm P et al.; Recently the amino acid sequences of the K88ab and the CFA1 fimbrial proteins from enteropathogenic Escherichia coli have become available . To elucidate the immunological interrelationships among the K88 family of antigenic variants, and to estimate the nature and position of the antigenic determinants in the K88 and CFA1 proteins, the positions of these features in the amino acid sequences have been predicted . This has been done by computerized algorithms, incorporating such factors as hydrophilicity and secondary structural potential . Some of the predicted determinants of K88 show good correlation with the known sequence differences between the antigenic variants of this protein . The presented results point to a possible use of synthetic vaccines against fimbriated pathogenic E . coli strains.

J Bacteriol, 1982 Oct, 152(1), 26 - 34
Induction and control of the autolytic system of Escherichia coli; Leduc M et al.; Various methods of inducing autolysis of Escherichia coli cells were investigated, some being described here for the first time . For the autolysis of growing cells only induction methods interfering with the biosynthesis of peptidoglycan were taken into consideration, whereas with harvested cells autolysis was induced by rapid osmotic or EDTA shock treatments . The highest rates of autolysis were observed after induction by moenomycin, EDTA, or cephaloridine . The different autolyses examined shared certain common properties . In particular, regardless of the induction method used, more or less extensive peptidoglycan degradation was observed, and 10(-2) M Mg2+ efficiently inhibited the autolytic process . However, for other properties a distinction was made between methods used for growing cells and those used for harvested cells . Autolysis of growing cells required RNA, protein, and fatty acid synthesis . No such requirements were observed with shock-induced autolysis performed with harvested cells . Thus, the effects of Mg2+, rifampicin, chloramphenicol, and cerulenin clearly suggest that distinct factors are involved in the control of the autolytic system of E . Coli . Uncoupling agents such as sodium azide, 2,4-dinitrophenol, and carbonyl-cyanide-m-chlorophenyl hydrazone used at their usual inhibiting concentration had no effect on the cephaloridine or shock-induced autolysis.

Proc Natl Acad Sci U S A, 1982 Oct, 79(19), 5828 - 32
Biosynthesis of aromatic compounds: 13C NMR spectroscopy of whole Escherichia coli cells; Ogino T et al.; 13C and 31P NMR spectra of wild-type Escherichia coli showed resonances from metabolic intermediates of glycolysis and ATP formation but no detectable signals from aromatic amino acids . However, tyrosine biosynthesis from D-{l-13C}glucose was observed in cells harboring a feedback-resistant allele of aroF, the gene encoding tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase {7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC4.1.2.15}, one of the isoenzymes that control carbon flow through the common aromatic biosynthetic pathway . A similar accumulation of tyrosine and phenylalanine is seen in cells carrying a multiple-copy plasmid that carries a wild-type aroF allele in addition to pheA and tyrA, the structural genes for controlling enzymes of the terminal pathways to phenylalanine and tyrosine biosynthesis . These in vivo measurements by a noninvasive probe suggest feedback inhibition as the quantitatively major mechanism controlling carbon flow in the common aromatic compound biosynthetic pathway . In strains accumulating aromatic amino acids, a transient accumulation of trehalose was detected, indicating that previously unknown changes in Escherichia coli metabolism accompany overproduction of aromatic compounds.

Biokhimiia, 1982 Oct, 47(10), 1747 - 51
{Stoichiometry of GTP hydrolysis during peptide synthesis on the ribosome . GTP hydrolysis uncoupled with ribosomal peptide synthesis and dependent on preparation of elongation factor T}; Smailov SK et al.; It is shown that preparations of EF-T are practically free from GTPase activity, if they were formed from EF-Tu and EF-Ts carefully purified from GTPase beforehand . Some Ef-T-dependent GTPase can arise after addition of tRNA or aminoacyl-tRNA preparations to a EF-T+ribosomes mixture . Poly(U) stimulates, as a rule, EF-T-dependent GTPase in the EF-T/ribosomes+tRNA mixture . Uncoupled EF-T-dependent GTPase of the EF-T+ribosomes+tRNA+poly(U) mixture is by 1--2 orders of magnitude less than uncoupled EF-G-dependent GTPase under the same conditions . A conclusion is made that uncoupled EF-T-dependent GTPase is an inevitable obstacle when studying the GTP expenditure in the process of ribosomal protein synthesis, if a traditional cell-free protein-synthesizing system is used for this purpose.

Biochemistry, 1982 Sep 28, 21(20), 5052 - 5
Inactivation of beta-Galactosidase by iodination of tyrosine-253; Huber RE et al.; Beta-Galactosidase is rapidly inactivated by iodination catalyzed by lactoperoxidase but is not inactivated in the presence of the substrate analogue, isopropyl beta-D-thiogalactoside (IPTG) . Enzyme activity is lost upon the incorporation of 1 mol of iodine per mol of monomer, without dissociation of the tetrameric structure . Tryptic digests of beta-galactosidase iodinated with 125I in the presence and absence of IPTG were separated by high-performance liquid chromatography and were compared . One fraction was found to be more highly labeled in the digest from the inactivated protein . After isolation of the peptide, amino acid analysis indicated it to be Asp-Tyr-Leu-Arg, residues 252-255 . Thus, Tyr-253 is the most reactive tyrosine in beta-galactosidase . This suggests that the conformation of this region of the protein may be altered by binding of IPTG to make Tyr-253 less accessible to iodination . Alternatively, Tyr-253 could be an active-site residue.

Biochim Biophys Acta, 1982 Sep 27, 698(3), 230 - 6
Codon:anticodon and anticodon:anticodon interaction: evaluation of equilibrium and kinetic parameters of complexes involving a g:u wobble; Labuda D et al.; In order to learn about the effect of the G:U wobble interaction we characterized to codon:anticodon binding between triplets: UUC, UUU and yeast tRNAPhe (anticodon GmAA) as well as the anticodon:anticodon binding between Escherichia coli tRNAGlu2, E . coli tRNALys (anticodons: mam5s2UUC, and mam5S2UUU, respectively) and tRNAPhe from yeast and E . coli (anticodon GAA) using equilibrium fluorescence titrations and temperature jump measurements with fluorescence and absorption detection . The difference in stability constants between complexes involving a G:U pair rather than a usual G:C basepair is in the range of one order magnitude and is mainly due to the shorter lifetime of the complex involving G:U in the wobble position . This difference is more pronounced when the codon triplet is structured, i.e., is built in the anticodon loop of a tRNA . The reaction enthalpies of the anticodon:anticodon complexes involving G:U mismatching were found to be about 4 kcal/mol smaller, and the melting temperatures more than 20 degrees C lower, than those of the corresponding complexes with the G:C basepair . The results are discussed in terms of different strategies that might be used in the cell in order to minimize the effect of different lifetimes of codon-tRNA complexes . Differences in these lifetimes may be used for the modulation of the translation efficiency.

Biochim Biophys Acta, 1982 Sep 27, 698(3), 252 - 9
Primary and secondary structure in a precursor of 5 S rRNA; Singh B et al.; In order to shed some light on the mechanism of action of RNAase E, we studied primary and secondary structure in 9 S (a precursor of 5 S rRNA), p5 and 5 S rRNAs . These molecules were digested with RNAases A and T1 in the presence of a high salt concentration; conditions under which single-stranded RNA is differentially digested . The double-stranded RNA fragments were isolated and analyzed . The analyses suggested that p5 and 5 S rRNA contain a stem of ten basepairs which consists of sequences from the 5' and 3' regions of the molecule, while 9 S contains a similar stem which is three basepairs longer and includes also a bulge of three unpaired bases . The stem in the 9 S RNA contains four sites where RNA processing cleavages occur . One of the cleavage sites which leads to the formation of p5 is in a junction between single-stranded and double-stranded RNA . The 9 S RNA molecule contains another large stem of at least 15 basepairs which is derived from the 3' end of the 9 S precursor molecular; it is apparently the transcription termination stem.

Nucleic Acids Res, 1982 Sep 25, 10(18), 5637 - 47
RNA polymerase: linear competitive inhibition by bis-(3' to 5')-cyclic dinucleotides,NpNp; Hsu CY et al.; We have investigated the possible role of the bis-(3' to 5')-cyclic dinucleotides UpUp and ApUp as kinetic inhibitors of the DNA dependent RNA polymerase enzyme of E . coli, using T7 delta D111 deletion mutant DNA and several synthetic DNA polymers as templates . We have established that UpUp is a linear competitive inhibitor of the initiation phase of the polymerization (Ki = 28 microM using T7 delta D111 DNA as a template), but that it has no effect when added during the elongation phase . The compound ApUp is an inhibitor of the reaction only when poly(dA-T).poly(dA-T) is used as a template, and UpUp is an inhibitor of the reaction when poly(dA).poly(dT) was employed as the DNA template.

J Biol Chem, 1982 Sep 25, 257(18), 10562 - 5
Isopentenylation of both cytoplasmic and mitochondrial tRNA is affected by a single nuclear mutation; Martin NC et al.; Cytoplasmic rRNA from the yeast mutant, mod5-1 is deficient in the modified base isopentenyladenosine and consequently migrates differently from isopentenylated wild type tRNAs on certain chromatographic systems . To determine if the mod5-1 mutation affects mitochondrial tRNA structure, the migration of mitochondrial tRNA from mod5-1 and wild type cells has been compared by reverse-phase chromatography . We conclude from this analysis that the single nuclear mutation that affects the isopentenylation of cytoplasmic tRNA also affects the isopentenylation of mitochondrial tRNA.

J Biol Chem, 1982 Sep 25, 257(18), 10759 - 65
Regulation of phospholipid synthesis in Escherichia coli . Composition of the acyl-acyl carrier protein pool in vivo; Rock CO et al.; The regulation of membrane lipid biogenesis was investigated by measuring the levels of the acyl-acyl carrier protein (acyl-ACP) intermediates in the biosynthetic pathway . In particular, the role of the sn-glycerol-3-phosphate acyltransferase was assessed by focusing on the size and composition of the long chain acyl-ACP pool . The ACP( pool was specifically labeled in vivo with beta-{3-3H}alanine and the ACP subspecies analyzed by reversed phase liquid chromatography and conformationally sensitive gel electrophoresis . The acyl-ACP pool was found to be a small fraction of the total ACP in normally growing cells and was particularly devoid of chain lengths that could serve as acyltransferase substrates . Inhibition of phospholipid synthesis at the acyltransferase step resulted in a rapid increase in the content of acyl-ACP, and analysis showed the presence of chain lengths that are acyltransferase substrates . Acyl-CoAs were not detected during interruption of acyl transfer activity . These results show that 1) acyl-ACPs are the acyl donors for phospholipid synthesis in vivo, 2) the acyltransferase does not play a role in the regulation of the lipid biosynthetic rate or the composition of phospholipid acyl moieties, 3) the primary regulatory site in phospholipid biosynthesis is at an early step in fatty acid biosynthesis, 4) feedback regulation by long chain acyl-ACP's is not a controlling mechanism for fatty acid synthesis under normal physiological circumstances, and 5) enzymes that utilize acyl-ACPs are involved in kinetic competition for the scarce acyl-ACP substrates.

Nucleic Acids Res, 1982 Sep 25, 10(18), 5447 - 65
Regulation of transcription from tandem and convergent promoters; Horowitz H et al.; We have examined transcription on templates containing the trp and lac UV5 promoters arranged in tandem or opposing orientations . These studies have revealed that the strengths of the two promoters are comparable, though the lac UV5 promoter is much more sensitive to the level of initiating purine present . Kinetic experiments have shown that a polymerase molecule poised at the lac promoter, or a lac repressor molecule bound to the lac operator, can temporarily block a polymerase molecule initiated from the trp promoter, though transcription eventually continues through . In the convergent construct, transcription from the lac promoter is hindered only when initiation is suboptimal due to low purine concentrations.

Nucleic Acids Res, 1982 Sep 25, 10(18), 5407 - 19
Cloning and sequence analysis of a cDNA plasmid for one of the rat liver glutathione S-transferase subunits; Tu CP et al.; We describe the construction and characterization of a cDNA plasmid for one of the rat liver glutathione S-transferase subunits . Poly(A)-RNA isolated from rat livers was enriched for glutathione S-transferase mRNA activity and used as templates to synthesize double stranded cDNA . The double stranded cDNAs were annealed to pBR322 through terminal deoxynucleotidyl transferase generated GC-tails followed by transformation into E . coli . Several candidate clones were selected by colony hybridization using polynucleotide kinase labeled liver and testis poly(A)-RNA probes . These candidate clones were further characterized by hybrid-selected translation of mRNA followed by immunoprecipitation and SDS gel electrophoresis . The positive clone, pGTR112 was mapped with restriction endonuclease analysis and sequenced by the chemical method of Maxam and Gilbert . The largest upen reading frame contains 142 amino acids very rich in Arg and Lys residues . The C-terminal residue phenylalanine of this open reading frame is consistent with what was reported for one of the ligandin subunits by Bhargava et al., (J . Biol . Chem . 253, 4116-4119, 1978) . Among the 352 nucleotides covered by both pGTR112 and pGST94 described by Kalinyak and Taylor (J . Biol . Chem . 257, 523-530, 1982), there are only 9 nucleotide differences resulting in four changes of amino acid sequences.

J Biol Chem, 1982 Sep 25, 257(18), 11113 - 20
In vitro transcription of the supB-E tRNA operon of Escherichia coli . Characterization of transcription products; Nakajima N et al.; The seven tRNA genes clustered in the supB-E region of the Escherichia coli chromosome were transcribed in vitro with purified RNA polymerase, using a restriction fragment from lambda psu degrees 2, a transducing phage carrying the chromosome region, as template . A single major transcript was synthesized, which was about 770 nucleotides long and contained all seven tRNA sequences . The terminal sequences of the transcript were determined and mapped on the DNA sequence of the supB-E region previously determined . The transcription start site is seven base pairs downstream from the Pribnow box sequence, as expected from the DNA sequence analysis and consistent with the findings on the trimeric tRNA precursor (pppG--tRNAMETM-tRNALeu-tRNAGln1) which was detected in an RNase P mutant and shown to be coded for by the supB-E region . Cleavage of the restriction fragment at the -35 region with another restriction endonuclease abolished the template activity of the fragment . Transcription of the supB-E tRNA operon was relatively unaffected by the presence of rho factor . Transcription termination occurs within a region of three bases between positions 770 and 772 from the transcription start site . Immediately upstream from the termination sites, there is a region of 26 nucleotides that could form a stem structure, thereby consistent with the general feature of rho-independent termination sites.

J Biol Chem, 1982 Sep 25, 257(18), 11078 - 86
Cloning and characterization of cDNA sequences corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin; Garfinkel LI et al.; A library of cDNA clones was constructed from adult rat skeletal muscle mRNA, from which a set of contractile protein clones was selected . These clones were identified by sequencing the cDNA inserts and comparing the derived amino acid sequences with published sequences of rabbit contractile proteins . In this manner, clones corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin were identified . A high degree of amino acid sequence conservation was found upon comparison of the rat and rabbit proteins . Using the cDNA clone panel, we analyzed the expression of abundant rat muscle mRNAs . We show that abundant rat muscle mRNAs can be classified into four developmentally regulated groups, based upon their expression at different stages of myogenesis . One class of mRNAs is expressed during all stages of muscle development . Since these mRNAs are also present in nonmuscle tissues, we conclude that they code for housekeeping proteins . The second class of mRNAs is present in both embryonic and adult muscle, while a third class of mRNAs is expressed only in adult muscle . A small number of mRNAs, which are present at greater levels in undifferentiated myoblasts than in adult muscle, comprise a fourth class . These results suggest the existence of at least four modes of gene control during myogenesis.

Biochim Biophys Acta, 1982 Sep 24, 691(1), 161 - 70
Escherichia coli B membrane stability related to cell growth phase . Measurement of temperature dependent physical state change of the membrane over a wide range; Souzu H; Escherichia coli B cytoplasmic and outer membrane from cells in different growth phases showed different chemical compositions . In freezing, logarithmic phase cells showed a marked permeability increase in the outer as well as the cytoplasmic membrane . Whereas, in the stationary phase cells no such change in membrane permeability was observed . Cooling of phospholipids and lipopolysaccharides with trans-parinaric acid showed a distinct fluorescence increase from room temperature to far below 0 degrees C . In the outer membrane the fluorescence similar to that of lipopolysaccharides was shown . The outer membranes of cells in different growth phases showed similar temperature-dependent fluorescence changes . The cytoplasmic membrane inhibited a temperature-dependent fluorescence similar to that of the phospholipids . The onset temperature of the increase in fluorescence differed with the cells at different growth phases . The presence of EDTA and MgCl2 modified the fluorescence changes in the membranes from cells in logarithmic phase . Whereas, in the membranes from cells in stationary phase no such effect was observed . These results suggest that the organizational stability of the membranes from cells in stationary phase is a fundamental basis of the membrane's resistance to the freezing damage.

Eur J Pharmacol, 1982 Sep 24, 83(3-4), 191 - 8
Pyrimido-pyrimidine derivative, RA642, a central pressor agent in cat endotoxin shock; Koyama S et al.; The effects of a pyrimido-pyrimidine derivative, RA642, in endotoxin shock were studied . Blood pressure and preganglionic splanchnic nerve (PSN) activity were measured in alpha-chloralosed cats . Within 30 min of intravenous administration of E . coli endotoxin (1 mg/kg), blood pressure and PSN activity were 68 +/- 8% and 47 +/- 10% of control and by 60 min were depressed by 54 +/- 8% and 42 +/- 8% . RA642 (0.25 mg/kg) injected i.v . 30 min after endotoxin, caused blood pressure to recover significantly to 83 +/- 6% of control within 5 min and be maintained at that level . PSN activity was similarly increased to and maintained at 62 +/- 9% of control . The efficacy of RA642 in reversing the lethal consequences of endotoxin shock were dramatic; all treated animals survived whereas the mortality rate of non-treated animals was 83% . This strongly suggests that the central pressor agent, RA642, may have important therapeutic applications in the management of endotoxin shock.

Nature, 1982 Sep 23, 299(5881), 312 - 6
Left-handed Z-DNA is induced by supercoiling in physiological ionic conditions; Singleton CK et al.; In physiological ionic conditions (200 mM NaCl), the (dC-dG)16 and (dC-dG)13 blocks in plasmid pRW751 are in a left-handed state when the negative superhelical density of the plasmid is greater than 0.972 . As the salt concentration decreases or when (dmC-dG) sequences are present, less negative supercoiling is required to induce the right- to left-handed DNA transition . Furthermore, the single strand-specific nuclease, S1, recognizes and cleaves aberrant structural features at the junction between neighbouring right- and left-handed DNA regions.

Nature, 1982 Sep 23, 299(5881), 365 - 7
Random components in mutagenesis; Foster PL et al.; The mutability of DNA varies enormously from one base pair to another . Part of this variation is due to the specificity of the reaction between mutagens and base, but much of the variation is due to unknown causes . A genetic system developed by Miller and colleagues allows the mutation frequencies of a large number of different base pairs in the lacI gene of Escherichia coli to be compared . For example, Coulondre and Miller found that the sites most readily mutated by UV light are almost 100 times more often mutated than the least susceptible sites . A recently completed study of mutagenesis with neocarzinostatin (NCS) in the lacI gene has prompted us to re-examine some previous studies, of mutagenesis in this gene . Our analysis, reported here, suggests that the mutations induced by certain mutagens fall into two classes: mutations in one class are clearly distributed non-randomly, that is, they are very common at some sites and significantly less common at others; mutations in the second class, however, occur at low frequency and appear to be randomly distributed . Both classes of mutations seem to occur only at damaged bases.

Biochim Biophys Acta, 1982 Sep 15, 681(3), 474 - 83
Membrane potential in a potassium transport-negative mutant of Escherichia coli K-12 . The distribution of rubidium in the presence of valinomycin indicates a higher potential than that of the tetraphenylphosphonium cation; Bakker EP; The membrane potential across the cytoplasmic membrane of EDTA-treated cells of a K+ transport-negative mutant of Escherichia coli K-12 was estimated from the equilibrium distribution of different lipid-soluble cations . With glucose as a substrate and at low K+ out, the membrane potential calculated from the distribution ratio of 86Rb+ in the presence of valinomycin (delta psi rb+) was considerably higher than that indicated by the {3H}tetraphenylphosphonium cation (delta psi TPP+) . The lipid-soluble anion phenyldicarbaundecaborane (PCB-) increased delta psi TPP+ close to delta psi Rb+ . To investigate whether these results were due to different binding of the cations to cellular components, residual Rb+ and TPP+ uptake was measured in cells permeabilized with 5% n-butanol (by volume) . In those cells the distribution ratios or Rb+, K+ and Na+ approached a value of 4, indicating that the uptake of all three ions was driven by a residual negative surface potential or transmembrane Donnan potential (internally negative) . The distribution ratio of TPP+ was 3--4-times higher than that of other cations and up to 10 mM TPP+ out was almost independent of the added TPP+ concentration . This extra uptake presumably represents binding of TPP+ to the cellular membranes . Thus, at pH 7.5, delta psi Rb+ was about 180--200 mV, whereas after correction for binding delta psi TPP+ was 110--150 and 150--170 mV in the absence and presence of PCB-, respectively . It is proposed that TPP+ indicates too low a potential, because by its strong binding it decreases the negative surface potential of the cytoplasmic membrane, and thereby inhibits its own further uptake . This is taken to mean that TPP+ distribution can be used as a qualitative probe only for the bacterial membrane potential.

Biochemistry, 1982 Sep 14, 21(19), 4657 - 64
Direct coordination of nucleotide with the intrinsic metal in Escherichia coli RNA polymerase . A nuclear magnetic resonance study with cobalt-substituted enzyme; Chatterji D et al.; Nuclear magnetic resonance studies were performed with Escherichia coli RNA polymerase (RPase) in which one of the two intrinsic Zn ions was substituted with Co(II) ion (Co-Zn RPase) . The Co ion was located in the beta subunit which contains the initiation site of the enzyme . The paramagnetic effect of Co-Zn RPase on the relaxation rates of rapidly exchanging water protons indicated that the Co ion was accessible to solvent . There were approximately two water molecules in the inner coordination sphere of the Co ion, one of which could be replaced by the substrate adenosine 5'-triphosphate (ATP) or the initiator adenylyl-(3' leads to 5')-adenine (ApA) but to a much less extent by uridine 5'-triphosphate . The effects of ATP and ApA did not require the presence of DNA or Mg (II) ions, and their Kd values were estimated to be 0.15 and 0.075 mM, respectively . These results showed that the Co ion was at the initiation site . From the measurements of the paramagnetic effects of Co-Zn RPase on the relaxation rates of 1H and 31P nuclei of ATP, the distances from the intrinsic Co ion to H2, H8, and H1' were determined to be 4.1 +/- 0.6, 3.6 +/- 0.5, and 6.8 +/- 0.8 A, respectively, and those to the alpha-, beta-, and gamma-phosphorus atoms were 10.5 +/- 0.7, 15.1 +/- 1.1, and 14.1 +/- 0.8 A, respectively . These spatial relationships clearly indicate that the Co ion is directly coordinated to the base moiety of ATP bound at the initiation site . Thus, the intrinsic metal in the beta subunit of RNA polymerase may play a regulatory role in the recognition of the initiating nucleotide and may orient the nucleotide in a stereospecific position for the initiation.

Biochemistry, 1982 Sep 14, 21(19), 4651 - 6
Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals; Chatterji D et al.; A simple in vitro substitution method involving a sequential denaturation--reconstitution process was developed to substitute selectively one of the two intrinsic Zn ions in Escherichia coli RNA polymerase with Co, Mn, Ni, or Cu ion . The resultant metal hybrid Co-Zn, Mn-Zn, Ni-Zn, and Cu-Zn RNA polymerases possess 100, 100, 60, and 17% of the enzymatic activity of the reconstituted Zn-Zn enzyme, respectively . The substituted metal was found to be located in the beta subunit of the polymerase which contains the substrate binding site . The biochemical and physical properties of these metal-substituted polymerases were compared with those of the native Zn enzyme . Co-Zn and Ni-Zn core polymerases exhibit characteristic absorption spectra in the near-UV and visible region, while Mn-Zn and Cu-Zn enzymes do not . The Co-Zn enzyme shows two major peaks at 400 nm (epsilon = 3000) and 475 nm (epsilon = 2700), while the Ni-Zn enzyme exhibits a major peak at 462 nm (epsilon = 8000) . The difference absorption spectrum of Ni-Zn core polymerase could be perturbed by the addition of substrate ATP but not by UTP in the absence of template and Mg(II) ion . These observations suggest that the substituted metal was located at the initiation site of the enzyme . The various metal hybrid enzymes do not differ appreciably in their abilities to incorporate noncomplementary nucleotide or deoxyribonucleotide into RNA product . It was found, however, that the difference in enzymatic activities of these metal hybrid enzymes resides at least partly in the initiation step of RNA synthesis.

Biochemistry, 1982 Sep 14, 21(19), 4700 - 6
Comparison of transfer ribonucleic acid structures using cobra venom and S1 endonucleases; Auron PE et al.; Cobra venom nuclease V1, which cleaves double-stranded RNA, has been used to study the structure of four Escherichia coli tRNAs: Phe, Glu2, Leu2, and Ile1 . The cleavage patterns are compared to those found for yeast tRNAPhe, the three-dimensional structure of which is known . The cleavage patterns of all the tRNA molecules are similar, and the most sensitive cleavage is found at the central base pair of the anticodon stem . Studies of E . coli tRNALeu2, which has a large variable loop, are consistent with the formation of a base-paired stem and loop structure that is not closely bound to the remainder of the molecule . A survey of the results suggests that the V1 molecule may interact with the minor groove of the double helix with an affinity for stacked bases and that it may require two or three stacked bases for optimal binding and cleavage.

Biochemistry, 1982 Sep 14, 21(19), 4772 - 6
Chemical modification of F1-ATPase by dicyclohexylcarbodiimide: application to analysis of the stoichiometry of subunits in Escherichia coli F1; Satre M et al.; N,N'-Dicyclohexylcarbodiimide (DCCD) covalently binds to the beta subunit of Escherichia coli F1-ATPase (BF1) . The ATPase activity is fully inhibited when 1 mol of DCCD is bound/mol of BF1, in spite of the fact that BF1 contains several beta subunits {Satre, M., Lunardi, J., Pougeois, R., & Vignais, P.V . (1979) Biochemistry 18, 3134-3140} . Advantage was taken of the reactivity of DCCD with respect to BF1 to determine the exact stoichiometry of the beta subunits in BF1 . Two methods were used . The first one was based on the fact that modification of the beta subunit by DCCD results in the disappearance of one negative charge, due to the binding of DCCD to a carboxyl group of the beta subunit . The nonmodified and the modified beta subunits were separated by electrofocusing, and the percentage of modified beta subunits was assessed as a function of the percentage of ATPase inactivation . The second method relied on direct comparison, after inactivation of BF1 by {14C}DCCD, of the specific radioactivities of the whole BF1 and the isolated beta subunits . Both methods indicate that each molecule of BF1 contains three beta subunits.

Biochemistry, 1982 Sep 14, 21(19), 4590 - 6
Simultaneous reconstitution of Escherichia coli membrane vesicles with D-lactate and D-amino acid dehydrogenases; Haldar K et al.; Purified preparations of D-amino acid dehydrogenase {Olsiewski, P.J., Kaczorowski, G . J., & Walsh, C . T . (1980) J . Biol . Chem . 225, 4487} and D-lactate dehydrogenase {Kohn, L.D., & Kaback, H.R . (1973) J . Biol . Chem . 248, 7012} bind independently to right-side-out and inverted Escherichia coli vesicles and to phosphatidylcholine liposomes without detectable competition . The reconstituted vesicles catalyze D-lactate- and D-alanine-dependent respiration (O2 uptake), proton translocation, and proton/lactose symport . The enzymes do not share common sites of association on either face of the E . coli membrane, and binding of both enzymes to the bilayer appears to be due to nonspecific affinity for the surface rather than specific binding to proteinaceous receptors . Each enzyme, however, appears to reduce a common proton translocating step in the membrane-bound respiratory chain, and substrate-derived electrons are transferred through a common rate-determining redox component that precedes the site of proton translocation . The results suggest that although binding is nonspecific, there is a common site for proton translocation in the membrane between the flavin-linked dehydrogenases and the cytochromes and that this site is accessible by distinct routes of electron transfer from primary dehydrogenases on either surface of the membrane.

Nucleic Acids Res, 1982 Sep 11, 10(17), 5209 - 21
Molecular structure of uvrC gene of Escherichia coli: identification of DNA sequences required for transcription of the uvrC gene; Sharma S et al.; We have carried out experiments to identify the regulatory regions of the uvrC gene of Escherichia coli . A uvrC+ plasmid, pUV7, containing the intact transcriptional unit for the uvrC gene, was used to subclone either the structural gene or combinations of the structural gene and 5'-flanking sequences . The plasmids so constructed were tested for ability to restore UV-resistant phenotype to uvrC- cells as an indication of expression of the uvrC gene . The chromosomal DNA in plasmid pUV7 was probed for strong binding with E . coli RNA polymerase in an attempt to identify a restriction fragment which bears the regulatory sequences for the uvrC transcriptional unit . The results indicate that DNA sequences at least 0.9 Kb upstream from the structural gene, but not the 5'-proximal sequences, regulate expression of the uvrC gene . Analysis of protein synthesis encoded by plasmid pUV7 and its derivatives suggest that there may be another gene that lies between the promoter and the uvrC gene and codes for a 27,000-Mr protein . The relation of this gene to uvrC function is not clear.

Nucleic Acids Res, 1982 Sep 11, 10(17), 5161 - 71
Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants; Kalderon D et al.; Deletion loop mutagenesis is a new, general method for site-directed mutagenesis that allows point mutations to the introduced within a sequence of DNA defined by a previously isolated deletion mutant . Wild type and deletion mutant DNA are cloned into a bacterial plasmid and each is cleaved with a different single cut restriction enzyme . Heteroduplexes are formed between the two DNAs to produce circular molecules containing a nick in each strand and a single-stranded deletion loop . The deletion loops are mutagenised using sodium bisulphite and the DNA transfected directly into a uracil repair deficient strain of Escherichia coli . Up to half of the resultant clones contain DNA produced by replication of the wild-type length strand and bear mutations exclusively within the target area . An example is given in which a deletion mutant lacking 21 nucleotides from the region coding for SV40 large-T was used . Eight of the possible nine target cytosine residues were mutagenised . The method described is specific, efficient and simple.

J Biol Chem, 1982 Sep 10, 257(17), 10222 - 7
Fatty acyl amidases from Dictyostelium discoideum that act on lipopolysaccharide and derivatives . I . Partial purification and properties; Verret CR et al.; Two amidases have been partially purified from the slime mold Dictyostelium discoideum; these act sequentially on the beta-hydroxymyristyl-amide groups present in the lipopolysaccharide derivative (4'-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta-(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine-1-phosphate (III) . Amidase-I, which specifically removes the myristyl chain near the 1-phosphate of compound III (apparent Km, 3.7 microM), has been purified 110-fold from a lysate of D . discoideum NC4 cultivated on Escherichia coli . The partially purified enzyme contains no other amidase or phosphatase activities; however, an esterase activity can be detected . The second amidase has been purified about 12-fold from the extracellular fluid of D . discoideum AX3 cultured axenically . This amidase hydrolyzes the distal amide linkage in III (apparent Km, approximately 20 microM) only after prior deacylation of the first site by amidase-I . The preparation is free from phosphatases and glycosidases that can act on lipopolysaccharide . The differential expression of the amidases in D . discoideum and some of their kinetic properties have been described . The amidases should prove useful in structure-function studies of lipopolysaccharide.

J Biol Chem, 1982 Sep 10, 257(17), 9922 - 5
Prolipoprotein signal peptidase in Escherichia coli is distinct from the M13 procoat protein signal peptidase; Tokunaga M et al.; We have previously reported a signal peptidase activity in Escherichia coli cell envelope which processes prolipoprotein modified with glyceride (Tokunaga, M., Tokunaga, H., and Wu, H . C . (1982) Proc . Natl . Acad . Sci . U . S . A . 79, 2255-2259) . To ascertain whether the processing enzyme for prolipoprotein is distinct from the signal peptidase for M13 procoat protein purified by Zwizinski and Wickner (Zwizinski, C., and Wickner, W . (1980) J . Biol . Chem . 255, 7973-7977), we have used antibody against purified procoat protein signal peptidase to study the processings of prolipoprotein and M13 procoat protein in vitro . the signal peptidase for modified prolipoprotein remained fully active in solubilized membrane preparations which had been treated with antibody against purified procoat protein signal peptidase whereas the activity towards procoat protein was completely abolished by immunoadsorption . Furthermore, both unmodified and glyceride-modified prolipoprotein were not cleaved by the highly purified signal peptidase preparation provided by Wickner . These data clearly indicate that prolipoprotein signal peptidase is distinct from the M13 procoat protein signal peptidase.

J Biol Chem, 1982 Sep 10, 257(17), 9895 - 7
Purification of the precursor form of maltose-binding protein, a periplasmic protein of Escherichia coli; Ito K; Cells of Escherichia coli K12 strain MM18 accumulate precursors of various exported proteins when the synthesis of the malE-lacZ hybrid protein is induced by maltose . Starting from a soluble cell extract of this strain, the precursor form of the maltose-binding protein, a periplasmic protein, was purified to homogeneity . The procedure includes affinity chromatography on cross-linked amylose and chromatofocusing . The purified precursor has an intact signal sequence at the NH2 terminus . It is soluble in aqueous buffer probably in monomeric state and is proteolytically processed by the leader peptidase, which was previously purified by Wickner and co-workers as an enzyme that processes M13 phage procoat protein.

J Biol Chem, 1982 Sep 10, 257(17), 10184 - 90
Energy-dependent inactivation and modification of a tryptophan biosynthetic enzyme in Escherichia coli; Mosteller RD et al.; The bifunctional tryptophan biosynthetic enzyme N-(5'-phosphoribosyl)anthranilate (PRA) isomerase/indole-3-glycerol phosphate (InGP) synthetase was purified from Escherichia coli cultures incubated under several conditions, some of which result in inactivation of the enzyme (starvation for ammonium or sulfate, chloramphenicol inhibition) . Recovery of enzyme activity during purification from cultures incubated under inactivating conditions suggested that activity was restored or that an inhibitor was removed . In these preparations, the enzymatic properties were altered slightly but not in a manner that could account for inactivation . Each preparation exhibited one major protein species (Mr approximately 50,000) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis but revealed several species during isoelectric focusing in the range of pH 5.5 to 6.0 . Comparison of the distribution of species indicated that modification to the more acidic forms had occurred in growing and nongrowing cells but not during stationary phase, glucose starvation, or energy depletion . The results suggested that inactivation and modification had occurred in vivo by an energy-dependent process . Several metabolites tested did not inhibit the purified enzyme . Attempts to detect an inhibitor in crude cell extracts were also not successful.

J Biol Chem, 1982 Sep 10, 257(17), 10119 - 27
Kinetic studies on the interaction of chain initiation factor 3 with 70 S Escherichia coli ribosomes and subunits; Goss DJ et al.; The magnesium ion dependencies of several of the reaction rates for assembly of the Escherichia coli protein synthesis initiation complex have been determined as well as the effects of IF-3 (initiation factor 3) on the overall reaction of 30 S and 50 S ribosomal subunits . The reaction kinetics were studied by light-scattering changes as well as by changes in the fluorescence anisotropy of dansylated-IF-3 . The full model for treating these processes consists of four reactions, three of which are thermodynamically independent . Conditions were chosen and techniques were employed that allowed three of the reactions to be studied individually . This allowed us to fit, with a single adjustable rate constant, all of the light-scattering changes that occurred upon flowing 30 S and 50 S ribosomal subunits against varying concentrations of both Mg2+ and IF-3 . Preparations of IF-3 were found to react toward 30 S subunits with either of two markedly different binding rates . We find that the simplest model that explains both the light-scattering and anisotropy data for all IF-3 experiments is one that includes as a necessary step the association of 30 S-IF-3 with 50 S subunits to form a 70 S-IF-3 complex.

J Biol Chem, 1982 Sep 10, 257(17), 10159 - 65
Energetic and structural inter-relationship between DNA supercoiling and the right- to left-handed Z helix transitions in recombinant plasmids; Stirdivant SM et al.; We have evaluated the B to Z conformational transitions in supercoiled recombinant plasmids containing different lengths of (dC-dG) described in the preceding paper . The sodium chloride-induced right- to left-handed transition in a small segment of the plasmids caused a relaxation of (-) supercoils which was monitored by electrophoretic mobility changes of individual topoisomers on agarose gels containing NaCl at concentrations up to 5.0 M . The number of supercoils relaxed was proportional to the length of the (dC-dG) segment in the plasmid in good agreement with theoretical values . A short B/Z junction region (less than 5 base pairs) was inferred . The stability of the Z conformation in (dC-dG) segments of the plasmids had a strong length dependency; shorter lengths were less stable . Ten base pairs of (dC-dG) was insufficient to allow a Z conformation under the conditions studied . Supercoiling imparts a substantial favorable free energy to the Z conformation, reducing the NaCl concentration necessary to cause the transition . The relationship of supercoiling with the NaCl concentration necessary to cause a B leads to Z transition suggests that supercoiling alone is sufficient to stabilize the Z conformation at physiological salt concentrations . These results support the notion that left-handed DNA has an important biological role.

J Biol Chem, 1982 Sep 10, 257(17), 10152 - 8
Left-handed DNA . Cloning, characterization, and instability of inserts containing different lengths of (dC-dG) in Escherichia coli; Klysik J et al.; Recombinant pBR322 derivatives were constructed containing tracts of (dC-dG) sequences which are 58, 32, 26, and 10 base pairs (bp) in length in conjunction with a 95-bp fragment containing the Escherichia coli lac operator-promoter . The biological properties of these plasmids were unusual since deletions in the (dC-dG) regions, but not in the pBR322 nor the lac segments, were frequently observed; segments of (dC-dG) longer than approximately 50 bp were not stable but suffered deletions . Segments of approximately 30 bp or shorter were stable in most cases . The (dC-dG) tracts seemed to enhance recA-mediated recombination when they were of suitable length and were cloned into certain sites in the recombinants . Also the (dC-dG)-containing plasmids were less supercoiled (by 6-12 turns) than expected, relative to control plasmids, after isolation from E . coli hosts . These recombinant plasmids were used in the following paper to evaluate the properties of segments containing the unorthodox left-handed conformations.

J Biol Chem, 1982 Sep 10, 257(17), 10378 - 86
The methyl-accepting chemotaxis proteins of Escherichia coli . Identification of the multiple methylation sites on methyl-accepting chemotaxis protein I; Kehry MR et al.; The methyl-accepting chemotaxis proteins (MCPs) are integral membrane proteins that undergo reversible methylation during adaptation of bacterial cells to environmental attractants and repellents . The numerous methylated forms of each MCP are seen as a pattern of multiple bands on polyacrylamide gels . We have characterized the methylation sites in MCPI by analyzing methyl-accepting tryptic peptides . At least two different tryptic peptides accept methyl esters; one methyl-accepting peptide contains methionine and lysine and may be methylated a maximum of four times . The second methyl-accepting tryptic peptide contains arginine and may be methylated twice . Base-catalyzed demethylations of tryptic peptides and analysis of the charge differences between the different methylated forms of MCPI show that MCPI molecules may be methylated a total of six times . The two methyl esters on the methyl-accepting arginine peptide appear to be preferentially methylated in most of the forms of MCPI in attractant-stimulated cells . The ability to acquire six methylations on MCPI allows the bacterial cells to adapt to a broad range of attractant and repellent concentrations.

J Biol Chem, 1982 Sep 10, 257(17), 10372 - 7
The structure of cloned DNA complementary to catfish pancreatic somatostatin-14 messenger RNA; Minth CD et al.; Pancreatic poly(A) RNA isolated from the channel catfish (Ictalurus punctatus) was enriched for sequences corresponding to somatostatin mRNA on isokinetic sucrose gradients . Double-stranded cDNA was synthesized and inserted into the Pst I site pBR322 via the poly(dG) . poly(dC) tailing method . Escherichia coli was transformed with this DNA, and colonies containing somatostatin cDNA sequences were identified by hybridization using a primer-extended somatostatin cDNA . The somatostatin cDNA was obtained by extending a 5'-labeled undecanucleotide primer complementary to somatostatin mRNA with reverse transcriptase using catfish poly(A) RNA as a template . The synthetic primer d(T-T-C-C-A-G-A-A-G-A-A) was deduced from the amino acid sequence Phe-Phe-Trp-Lys present in somatostatin-14 . Twenty positive colonies were obtained upon screening 2000 transformants . The restriction maps of the plasmid DNA obtained from the positive colonies were examined . Nineteen of these plasmids contained sequences corresponding to somatostatin-14, while one contained a sequence corresponding to somatostatin-22 . The nucleotide sequence of pancreatic somatostatin-14 is reported here . The cDNA contains 350 nucleotides in the 3' noncoding region, 345 nucleotides in the coding region, and 104 nucleotides in the 5'-untranslated region . The mRNA codes for a precursor to somatostatin which is 114 amino acids in length . The preprosomatostatin has a sequence of hydrophobic amino acids at the NH2 terminus, followed by a connecting peptide of approximately 75 amino acids . The sequence Arg-Lys precedes somatostatin-14 . Analysis of genomic DNA from the channel catfish reveals that somatostatin-14 and somatostatin-22 are present on different restriction fragments.

Nature, 1982 Sep 9, 299(5879), 185 - 6
The helicity of DNA in complexes with recA protein; Stasiak A et al.; The RecA protein of Escherichia coli is involved in recombination (for review see ref . 1) . The protein binds transiently to double-stranded DNA in the presence of ATP . In the presence of ATP gamma S, a non-hydrolysable analogue of ATP, recA-DNA complexes are stable . Duplex DNA in these complexes is stretched by a factor 1.5 (ref . 4), and the complexes appear in the electron microscope as helical filaments with a pitch of approximately 100 A and 6.2 recA units per turn covering 18.6 base pairs (bp) . RecA crystals have a space group of similar helical parameters . In order to understand the function of recA, it is necessary to describe the conformation of the DNA in the recA complex . Using a topological method, the present work determines the helicity of DNA in the complex . We find that the DNA helix follows the protein helix visible in the electron microscope and has 18.6 bp per turn, which corresponds to an unwinding of the DNA double helix by 15 degrees per bp.

Biochim Biophys Acta, 1982 Sep 9, 690(2), 282 - 9
PhoE protein pore of the outer membrane of Escherichia coli K12 is a particularly efficient channel for organic and inorganic phosphate; Korteland J et al.; This study was undertaken to investigate the proposed in vivo pore function of PhoE protein, an Escherichia coli K12 outer membrane protein induced by growth under phosphate limitation and to compare it with those of the constitutive pore proteins OmpF and OmpC . Appropriate mutant strains were constructed containing only one of the proteins PhoE, OmpF or OmpC, or none of these proteins at all . By measuring rates of nutrient uptake at low solute concentrations, the proposed pore function of PhoE protein was confirmed as the presence of the protein facilitates the diffusion of Pi through the outer membrane, such as a pore protein deficient strain behaves as a Km mutant . Comparison of the rates of permeation of Pi, glycerol 3-phosphate and glucose 6-phosphate through pores formed by PhoE, OmpF and OmpC proteins shows that PhoE protein is the most effective pore in facilitating the diffusion of Pi and phosphorus-containing compounds . The three types of pores were about equally effective in facilitating the permeation of glucose and arsenate . Possible reasons for the preference for Pi and Pi-containing solutes are discussed.

Biochem J, 1982 Sep 1, 205(3), 495 - 501
The size and conformation of Artemia (brine-shrimp) ribosomal RNA free in solution; Donceel K et al.; The RNA was isolated from the large ribosomal subunits of the brine shrimp Artemia, and its conformation free in solution was studied by determining its sedimentation and diffusion coefficients . A comparison was made of the hydrodynamic radius of the ribosomal subunit and its isolated RNA in various buffers . The conformation of the rRNA free in solution is more extended than when it is incorporated in the ribosome . This is not only the case when the rRNA solution lacks bivalent and polyvalent cations, but even in the presence of Mg2+ and spermidine, which cause a tightening of RNA . Thus the ribosomal proteins should induce a further tightening of the rRNA during the assembly of the ribosome . In the discussion, the reported data on Escherichia coli rRNA species are presented in such a way that large discrepancies between various studied are revealed, and that they can be compared with the data reported here on the larger rRNA of an eukaryote.

J Clin Microbiol, 1982 Sep, 16(3), 487 - 9
Rapid evaluation of female patients exposed to gonorrhea by use of the Limulus lysate test; Prior RB et al.; The Limulus amoebocyte lysate (LAL) assay was used to evaluate 115 females who were named as sexual contacts by men with culture-proven gonorrhea . These patients were treated for gonorrhea before laboratory confirmation, as recommended by the Centers for Disease Control, because of the lack of rapid screening tests and the serious consequences of undetected infection . For the LAL assay, endocervical samples were collected with depyrogenated cotton-tipped swabs, and the swabs were placed in 10 ml of diluent to assay for endotoxin; the negative predictive value of the LAL assay at this dilution was 100% . Incubation was carried out at 37 degrees C for 30 min; positive or negative results were indicated by gelation or lack of gelation, respectively . Lysate sensitivity was 0.3 ng/ml, with an Escherichia coli endotoxin standard . Single endocervical cultures and the LAL assay were both positive in 71 patients, but the Gram stain was positive in only 36 (50.7%) of these cases . For the 44 culture-negative cases, the LAL assay was negative in 21 (47.7%) . Thus, the LAL assay was able to selectively exclude approximately half of the culture-negative gonorrhea contacts and would have spared these patients inappropriate therapy and contact tracing, without excluding culture-positive gonorrhea cases.

Infect Immun, 1982 Sep, 37(3), 1086 - 92
Arousal of mucosal secretory immunoglobulin A antitoxin in rats immunized with Escherichia coli heat-labile enterotoxin; Klipstein FA et al.; Specific serum and mucosal antitoxin levels were determined by enzyme-linked immunosorbent assays in rats immunized with Escherichia coli heat-labile enterotoxin (LT) . Immunization by means of a parenteral prime followed by peroral boosts was the only approach that aroused titers of both serum immunoglobulin G (IgG) antitoxin and mucosal secretory IgA antitoxin that were increased fourfold or more over control values . Primary parenteral immunization was effective when given either intraperitoneally or subcutaneously with either Freund complete adjuvant or alum as the adjuvant . The magnitude of the nucosal secretory IgA antitoxin response and the degree of protection against challenge with either LT or viable LT-producing organisms were related to the number and dosage of peroral boosts . LT antigenicity, as determined by enzyme-linked immunosorbent assay, was progressively reduced by toxoiding it with increasing amounts of glutaraldehyde or a carbodiimide; when LT antigenicity was reduced by greater than 50%, the effectiveness of the toxoid in stimulating mucosal antitoxin and providing protection was compromised . Strong protection extended for more than 6 weeks only in rats immunized with a sufficient peroral dosage of LT to arouse mucosal secretory IgA antitoxin titers at least fourfold greater than those of controls . These observations indicate that the ability of LT to stimulate a mucosal secretory IgA antitoxin response is dependent on the antigenicity, route, and dosage of this immunogen; they suggest that the duration of protection in animals immunized by the peroral route is related to the extent of arousal of mucosal secretory IgA antitoxin.

J Nutr, 1982 Sep, 112(9), 1747 - 55
Influence of dietary proteins on the immune system of mice; Bounous G et al.; The effect of graded amounts of dietary lactalbumin (L) and casein (C) hydrolyzates on the immune responsiveness of C3H/HeN and DBA/2 strain mice has been investigated by measuring both the specific humoral immune response to sheep red blood cells (SRBC) and the nonspecific splenic cell responsiveness to phytohemagglutinin, concanavalin A and Escherichia coli lipopolysaccharide after stimulation with Mycobacterium bovis, strain BCG . The nutritional efficiency of these diets was similar at both 12 and 28% amino acid levels . The immune responses of mice fed the L diets were found to be significantly greater than those of mice fed the corresponding C diets, especially at the 28% level . Furthermore in the mice fed L diet, increasing the concentration of amino acid in the diet from 12 to 28% greatly enhanced immune responsiveness by both parameters measured . In the C-fed mice, a comparable enhancement of mitogen responsiveness with increasing amino acid level of diet was seen, but there was no change in the humoral immune response . The enhancement of immune responsiveness observed in mice fed the 28% L diet was moderately reduced by the addition of phenylalanine to the diet, indicating that the lower level of this amino acid in the L protein may be of some significance . These dietary effects on immune responsiveness were remarkably similar in both mouse strains tested.

J Infect Dis, 1982 Sep, 146(3), 436 - 9
Disturbed intrarenal distribution of gentamicin in experimental pyelonephritis due to Escherichia coli; Bergeron MG et al.; The intracortical, medullary, and papillary distribution of gentamicin was studied in normal and pyelonephritic rats . The animals were evaluated from 1 hr to 365 days after the end of therapy with either a single dose or two daily injections given every 12 hr for seven days . The serum levels of gentamicin at 1 hr were significantly (P less than 0.001) higher in the pyelonephritic rats than in the normal rats after one dose (26 vs . 12 microgram/ml) and 14 doses (25.7 vs . 8.8 microgram/ml) . Peak concentrations or gentamicin in all parts of infected kidneys were significantly (P less than 0.001) higher than in normal kidneys . Gentamicin was still detectable at levels of 1.2 microgram/g in the cortex of one pyelonephritic animal one year after the end of therapy, when the levels of both serum creatinine (1.1 mg/100 ml) and blood urea nitrogen (30 mg/100 ml) were much higher than at seven days after the end of therapy (0.5 and 19 mg/100 ml, respectively).

J Bacteriol, 1982 Sep, 151(3), 1591 - 4
Analysis of btuB receptor function by use of nonsense suppression; Hunter MG et al.; Informational suppression of btuB nonsense mutants allows the study of the effect of known, single amino acid substitutions on receptor function . We found that ligand uptake is largely unaffected by such amino acid changes . The few instances in which certain substitutions destroyed sensitivity to the two lethal agents (phage BF23, colicin E3) without affecting vitamin B12 uptake suggest a common region on the btuB receptor involved in the binding of these proteinaceous agents.

J Bacteriol, 1982 Sep, 151(3), 1553 - 9
Mutations in genes cpxA and cpxB alter the protein composition of Escherichia coli inner and outer membranes; McEwen J et al.; Mutations in chromosomal genes cpxA and cpxB altered the protein composition of the inner and outer bacterial membranes . Electrophoretic analyses of membrane proteins from isogenic strains differing only at their cpx loci and of spontaneous cpxA+ revertants of a cpxA cpxB double mutant showed that the alterations define a pattern that is uniquely attributable to the cpx mutations . Two major outer membrane proteins, the OmpF matrix porin and the murein lipoprotein, were deficient or absent from the outer membrane of mutant cells, whereas the quantities of two other major outer membrane proteins, the OmpC matrix porin and the OmpA protein, were not significantly altered . The cpx mutations did not generally alter the functional or chemical properties of the cell envelope . In the electron microscope, mutant cells appeared ovoid, but individual cells showed no surface irregularities to suggest gross defects in the cell envelope . These observations suggest that the primary effect of the mutations is to alter selectively the synthesis or translocation of certain envelope proteins.

J Bacteriol, 1982 Sep, 151(3), 1523 - 31
Heat-induced blebbing and vesiculation of the outer membrane of Escherichia coli; Katsui N et al.; Thermal damage to the outer membrane of Escherichia coli W3110 was studied . When E . coli cells were heated at 55 degrees C in 50 mM Tris-hydrochloride buffer at pH 8.0, surface blebs were formed on the cell envelope, mainly at the septa of dividing cells . Membrane lipids were released from the cells during the heating period, and part of the released lipids formed vesicle-like structures from the membrane . This vesicle fraction had a lipopolysaccharide to phospholipid ratio similar to that of the outer membrane of intact cells, whereas it had a lower content of protein than the isolated outer membrane . After heating bacterial cells at 55 degrees C for 30 min, the resulting leakage from the cells of a periplasmic enzyme, alkaline phosphatase, amounted to 52% of the total activity, whereas no release of a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase, was detected . The results obtained suggest that surface blebs formed by heat treatment almost completely consist of the outer membrane and that the blebs may be gradually released from the cell surface into the heating menstruum to partially form vesicles.

J Bacteriol, 1982 Sep, 151(3), 1391 - 6
A gene coding for a periplasmic protein is located near the locus for termination of chromosome replication in Escherichia coli; Harayama S et al.; Hybrid plasmids carrying trg, the genetic locus in closest proximity to terC, coded for several polypeptides in addition to the Trg protein . Polypeptides of 59,000 and 61,000 apparent molecular weight were the most prominent products synthesized in minicells containing the hybrid plasmids . Analysis of the effects of deletions generated by a restriction endonuclease identified a region of DNA immediately adjacent to trg as the putative gene coding for the two polypeptides . Studies with whole cells and minicells showed that the 59,000-dalton polypeptide is a periplasmic protein . Analysis by limited proteolysis indicated that the two polypeptides are related, and a number of observations support the notion that the 61,000-dalton protein is a precursor form of the 59,000-dalton mature exported protein . The identification and characterization of a protein, in addition to Trg, which is produced by a gene in close proximity to terC emphasizes the fact that the region does contain intact and active genes.

J Bacteriol, 1982 Sep, 151(3), 1320 - 5
Requirement of Fnr and NarL functions for nitrate reductase expression in Escherichia coli K-12; Stewart V; I used a chlC-lac operon fusion to study regulatory mutations which affect nitrate reductase expression in Escherichia coli . A NarL- mutant apparently lacks a nitrate-specific positive regulatory component . Furthermore, an fnr (nirR) mutation prevented enzyme induction under any conditions . These data are consistent with a two-step, positive control model for nitrate reductase regulation.

J Bacteriol, 1982 Sep, 151(3), 1314 - 9
Mapping of two loci affecting the synthesis and structure of a periplasmic protein involved in arginine and ornithine transport in Escherichia coli K-12; Celis RT; The map location of two genes, abpR and abpS, was established . The abpR locus is responsible for the synthesis and the abpS locus is responsible for the structure of the arginine-ornithine-binding protein, a required component of the arginine-ornithine transport system of Escherichia coli . Two loci that result in elevated synthesis of the arginine-ornithine-binding protein and in an altered protein were mapped by bacterial conjugation and transduction studies . The mapping showed that the two genes lie in close proximity near the argA genetic marker in the order, with respect to argA, of argA abpR abpS . The maximal influx of arginine into an abpR mutant, which produces the arginine-ornithine-binding protein in an elevated amount, was substantially higher than the value obtained with an isogenic wild-type strain (apbR+) . It also was observed that there was a close similarity between the affinity of the transport system for its substrate and the in vitro affinity of the binding protein for arginine both in the case of the isogenic wild type (abpS+) and a mutant (abpS6) carrying an altered protein . These results were consistent with the concept that the binding protein modulates the affinity of the transport system and suggest that it is the step of substrate recognition by the periplasmic protein which is rate-limiting in the entire process of transport at maximal influx.

J Bacteriol, 1982 Sep, 151(3), 1290 - 8
Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli; Goebel W et al.; We cloned the DNA containing the Escherichia coli hemolysin determinant on a small, high-copy plasmid . We generated plasmids containing fragments of this DNA and used them either alone or in two-plasmid complementation systems to define the limits of the structural genes . This system also allowed us to partially characterize the function of each of the gene products in the production and transport of hemolysin . Taken with previously published data, the present experiments indicate the following . (i) At least three cistrons, hlyC, hlyA, and hlyB (these were previously designated cisC, etc . {Noegel et al., Mol . Gen . Genet . 175:343-350, 1979}), contain the specific genetic information for the hemolytic phenotype, (ii) hlyA encodes a 107,000-kilodalton protein, which seems to be an inactive precursor of hemolysin . (iii) Normal amounts of hemolysin activity inactive precursor of hemolysin . (iii) Normal amounts of hemolysin activity require only the products of hlyA and hlyC . This activity was found in the periplasm; very little hemolysin activity was found in the cytoplasm, suggesting that the hlyC product is required for transport or activation of the hlyA product or both . (iv) Active hemolysin remains in the periplasm in the absence of hlyB function, hence the hlyB product seems to be necessary for the transport of hemolysin to the exterior of the cell . We further show that overproduction of the hlyA product is lethal, probably causing lysis of the cell.

J Bacteriol, 1982 Sep, 151(3), 1253 - 60
Tiamulin resistance mutations in Escherichia coli; Bock A et al.; Forty "two-step" and 13 "three-step" tiamulin-resistant mutants of Escherichia coli PR11 were isolated and tested for alteration of ribosomal proteins . Mutants with altered ribosomal proteins S10, S19, L3, and L4 were detected . The S19, L3, and L4 mutants were studied in detail . The L3 and L4 mutations did not segregate from the resistance character in transductional crosses and therefore seem to be responsible for the resistance . Extracts of these mutants also exhibited an increased in vitro resistance to tiamulin in the polyuridylic acid and phage R17 RNA-dependent polypeptide synthesis systems, and it was demonstrated that this was a property of the 50S subunit . In the case of the S19 mutant, genetic analysis showed segregation between resistance and the S19 alteration and therefore indicated that mutation of a protein other than S19 was responsible for the resistance phenotype . The isolated ribosomes of the S19, L3, and L4 mutants bound radioactive tiamulin with a considerably reduced strength when compared with those of wild-type cells . The association constants were lower by factors ranging from approximately 20 to 200 . When heated in the presence of ammonium chloride, these ribosomes partially regained their avidity for tiamulin.

J Bacteriol, 1982 Sep, 151(3), 1136 - 45
Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1; Riise E et al.; Deletions or insertions in the copB gene of plasmid R1 result in a copy mutant phenotype . The wild-type copB gene has been cloned on various plasmid vectors . The presence of such chimeric plasmids reduced the copy number of R1 copB mutant plasmids to normal or subnormal levels, indicating the expression of a trans-acting inhibitor activity from the copB chimeras . However, the cloned copB gene did not affect the copy number of wild-type R1, and no incompatibility was exerted by the cloned copB gene against wild-type R1 (or R100) . Although the copB gene is not normally required for the incompatibility exerted by copA, it is shown that the CopB function is required for expression of incompatibility by the copA gene from some types of chimeric plasmids . Mutant plasmids that have lost both Cop functions replicate in an uncontrolled fashion.

J Bacteriol, 1982 Sep, 151(3), 1129 - 35
Expression of a copy number control gene (copB) of plasmid R1 is constitutive and growth rate dependent; Light J et al.; The copy number control gene copB from plasmid R1 was fused to the lacZ gene in vitro, resulting in expression of a fused polypeptide consisting of the first 53 amino acids of the CopB polypeptide and the beta-galactosidase polypeptide minus its first 8 amino acids . Based on measurements of specific activities of this fused protein under various conditions, it was concluded that expression of copB is gene dosage dependent, unregulated by plasmid-coded functions, and proportional to growth rate between 0.4 and 2.0 doublings per h . The rate of expression of the copB gene is surprisingly high compared with other known cases of regulatory proteins.

Am J Vet Res, 1982 Sep, 43(9), 1661 - 4
Chemotactic factors for bovine leukocytes; Carroll EJ et al.; An agarose method was used to determine the nature of chemotaxins for bovine leukocytes as related to coliform mastitis . Freshly collected bovine serum or plasma was frequently chemotactic, probably due to spontaneous complement activation . Fresh serum was always rendered chemotactic when activated with zymosan or when heat-killed or viable coliform bacteria were incubated in the serum . The bacteria did not contribute to preexisting activity in the serum . Escherichia coli grown in synthetic media did not generate chemotaxins, and the synthetic peptide formyl-L-methionyl-L-leucyl-L-phenyl-alanine was not chemotactic in the agarose system for bovine leukocytes . Endotoxin-treated serum had variable activity.

J Biochem (Tokyo), 1982 Sep, 92(3), 835 - 8
Peripheral distribution of nervous system-specific S-100 protein in rat; Suzuki F et al.; S-100 protein, a nervous system-specific protein, was determined in a soluble extract of various rat tissues with a sensitive enzyme immunoassay method, which consisted of a solid-phase with immobilized anti-S-100 antibody and the antibody labeled with beta-D-galactosidase from Escherichia coli . The minimum detectable amount of S-100 protein was 3 pg/assay . Central nervous tissues (cerebrum, cerebellum, and brain stem) contained 1.4 to 2.8 micrograms S-100 protein/mg protein, whereas most of the peripheral tissues contained less than 0.05 microgram/ml of the specific protein . However, the level of S-100 protein was high in adipose tissue (0.5--1.1 micrograms/mg) and in trachea (about 0.5 microgram/mg), which involves cartilage . The S-100 protein levels in several tissues were significantly higher in female rats than in males at ages of 5 to 6 weeks.

Carbohydr Res, 1982 Sep 1, 107(1), 43 - 53
Attempted affinity-labelling of beta-D-galactosidase from Escherichia coli with 2,6:3,4-dianhydro-1-deoxy-D-talo-hept-1-enitol; Lehmann J et al.; The epoxides methyl 2,3-anhydro-beta-D-talopyranoside (1) and 2,6:3,4-dianhydro-1-deoxy-D-talo-hept-1-enitol (2), both prepared by improved methods, and 2,6:3,4-dianhydro-1-deoxy-D-gulo-hept-1-enitol (3) were applied as potential reagents for the affinity labelling of E . coli beta-D-galactosidase . Compounds 1 and 3 are ineffective as labelling reagents, whereas compound 2 irreversibly inhibits the enzyme activity . Deactivation is complete only when high concentrations (0.5M) of the inhibitor are applied over a relatively long period of time (24 h) . Saturation kinetics cannot be observed . Nevertheless, the competitive inhibitor isopropyl 1-thio-beta-D-galactopyranoside protects the enzyme from irreversible deactivation by 2, indicating that the latter also reacts with the active site . Treatment of beta-D-galactosidase with 2,6:3,4-dianhydro-1-deoxy-D-talo-{4-3H}hept-1-enitol under conditions that effect deactivation of the enzyme to only a minor extent causes labelling of the protein at the molar ratio of 48:1 . Specific, radioaffinity labelling of the active site of the enzyme cannot be thus achieved.

Proc Natl Acad Sci U S A, 1982 Sep, 79(18), 5450 - 4
Covalent crosslinking of tRNA1Val to 16S RNA at the ribosomal P site: identification of crosslinked residues; Prince JB et al.; N-Acetylvalyl-tRNA1Val (AcVal-tRNA1Val) was bound to the P site of uniformly 32P-labeled 70S ribosomes from Escherichia coli and crosslinked to 16S RNA in the 30S ribosomal subunit by irradiation with light of 300-400 nm . To identify the crosslinked nucleotide in 16S RNA . AcVal-tRNA1Val-16S {32P}RNA was digested completely with RNase T1 and the band containing the covalently attached oligonucleotides from tRNA and rRNA was isolated by polyacrylamide gel electrophoresis . The crosslinked oligonucleotide, and the 32P-labeled rRNA moiety released from it by photoreversal of the crosslink at 254 nm, were then analyzed by secondary hydrolysis with pancreatic RNase A and RNase U2.The oligonucleotide derived from 16S RNA was found to be the evolutionarily conserved sequence, U-A-C-A-C-A-C-C-G1401, and the nucleotide crosslinked to tRNA1Val, C1400 . The identity of the covalently attached residue in the tRNA was established by using AcVal-tRNA1Val-16S RNA prepared from unlabeled ribosomes . This complex was digested to completion with RNase T1 and the resulting RNA fragments were labeled at the 3' end with {5'-32P}pCp . The crosslinked T1 oligonucleotide isolated from the mixture yielded one major end-labeled component upon photoreversal . Chemical sequence analysis demonstrated that this product was derived from the anticodon-containing pentadecanucleotide of tRNA1Val, C-A-C-C-U-C-C-C-U-cmo5U-A-C-m6A-A-G39(cmo5U, 5-carboxymethoxyuridine) . A similar study of the crosslinked oligonucleotide revealed that the residue covalently bound to 16S was cmo5U34, the 5' or wobble base of the anticodon . The adduct is believed to result from formation of a cyclobutane dimer between cmo5U34 of tRNA1Val and C1400 of the 16S RNA.

Aust Vet J, 1982 Sep, 59(3), 93 - 5
Experimental colibacillosis in gnotobiotic piglets exposed to 3 enterotoxigenic serotypes; Tzipori S et al.; Three strains of enterotoxigenic Escherichia coli (ETEC) (064:KSNT, K88ac; 020:KSNT, K88ac and 08:K85ab, K99) originally cultured from outbreaks of diarrhoea in piglets a few hours old, were administered orally to gnotobiotic piglets . There was a marked age-related difference in the clinical response to infection between the 3 strains although they all produced heat-stable toxin . All 3 strains produced severe clinical signs of depression, anorexia, vomiting, diarrhoea, followed by dehydration and death in one-day-old piglets . In piglets infected at 3 days of age the two K88+ ETEC caused diarrhoea and death but the K99+ ETEC induced moderate diarrhoea only . In piglets infected at 7 days of age, the 064 strain produced severe diarrhoea and death, and 020 strain caused mild diarrhoea in 3 of 6 piglets with one death while the 08 strain caused no illness . Pathological changes in the intestinal tract associated with these infections were minimal, or absent . Immunofluorescent staining with homologous hyperimmune sera demonstrated adherence of the 3 ETEC strains to the brush border of small intestinal epithelial cells . Fluorescing organisms were observed in all infected piglets irrespective of the severity of clinical signs but the degree and extent of colonisation varied with the age of the piglets and the infecting strain . This may explain the difference in clinical response between the 3 strains.

J Gen Microbiol, 1982 Sep, 128 (Pt 9), 2097 - 103
Adhesive properties associated with the Vir plasmid: a transmissible pathogenic characteristic associated with strains of invasive Escherichia coli; Morris JA et al.; Escherichia coli strain S5 (O15:K+:H21) isolated from a septicaemic lamb and previously shown to possess a virulence plasmid, Vir, attached in vitro to calf epithelial tissue from the ileum, oesophagus and trachea in the presence of 0.5% (w/v) D-mannose . The Vir+ recombinant strains 711v and H209av, which had received the Vir plasmid(s) from strain S5, also attached to these epithelia but the parent strains 711 and H209a without the Vir plasmid were non-adhesive . The attachment of the Vir+ strain 711v to intestinal brush borders was inhibited by antiserum to live Vir+ strain H209av but not by antiserum to strain H209a lacking Vir . No adherence occurred with Vir+ organisms grown at 18 degrees C or after heating at 65 degrees C . Adhesion was unaffected by 0.5% (w/v) formaldehyde . Glucosamine, mannosamine, their N-acetyl derivatives and wheat germ lectin each inhibited attachment of Vir+ strain 711v to brush border epithelia.

J Gen Microbiol, 1982 Sep, 128 (Pt 9), 1969 - 80
Genes for l-sorbose utilization in Escherichia coli; Woodward MJ et al.; Amongst forty wild strains of Escherichia coli, nine used L-sorbose as a source of carbon and energy and two mutated to use it . Laboratory strains K12, B and C were L-sorbose-negative . Genes for L-sorbose utilization (sor+) were transferred to K12 from six wild strains; genes conferring the mutable phenotype were also transferred . All were cotransducible with metA at 90 min on the linkage map . The most probable gene order was met ace sor pgi mal . Complementation tests identified two genes for L-sorbose utilization . Genetical evidence showed that the catabolite repressor protein of K12 exerted positive control over sor+ genes introduced into K12 . The genes for phosphofructokinase (pfkA), the phosphocarrier protein (ptsH) and phosphotransferase enzyme I (ptsI) were required for utilization of L-sorbose . The frequency of transduction of sor+ was low when selection was made for sor+, because L-sorbose partially inhibited the growth of both L-sorbose-negative strains and K12 (sor+) strains . Uridine, thymidine and sorbitol each annulled the inhibition of growth and increased the frequency of transduction of sor+.

Arch Microbiol, 1982 Sep, 132(3), 270 - 5
An Escherichia coli mutant defective in the NAD-dependent succinate semialdehyde dehydrogenase; Skinner MA et al.; Escherichia coli mutants, unable to grow on 4-hydroxyphenylacetate, have been isolated and found to be defective in the NAD-dependent succinate semialdehyde dehydrogenase . When the mutants are grown with 4-aminobutyrate as sole nitrogen source an NAD-dependent succinate semialdehyde dehydrogenase seen in the parental strain is absent but, as in the parental strain, an NADP-dependent enzyme is induced . Growth of the mutants is inhibited by 4-hydroxyphenylacetate due to the accumulation of succinate semialdehyde . The mutants are more sensitive to inhibition by exogenous succinate semialdehyde than is the parental strain . Secondary mutants able to grow in the presence of 4-hydroxyphenylacetate but still unable to use it as sole carbon source were defective in early steps of 4-hydroxyphenylacetate catabolism and so did not form succinate semialdehyde from 4-hydroxyphenylacetate . The gene encoding the NAD-dependent succinate semialdehyde dehydrogenase of Escherichia coli K-12 was located at min 34.1 on the genetic map.

Am J Vet Res, 1982 Sep, 43(9), 1594 - 8
Effect of berberine on intestinal secretion mediated by Escherichia coli heat-stable enterotoxin in jejunum of pigs; Zhu B et al.; Intraluminal perfusion with Escherichia coli heat-stable enterotoxin (ST) reversed water and electrolyte movements from net absorption to net secretion in porcine jejunal segments . Addition of berberine hydrochloride (3.2 X 10(-5) M) to the perfusate reduced the jejunal secretory response of water, sodium, potassium, and chloride to ST and enhanced water and electrolyte absorption in control segments . At lower concentrations (1.1 X 10(-5) M), berberine reduced the secretory response in ST-exposed segments, but only the decrease of sodium flux was significant . In the presence of berberine, the mucosal enzyme activities of adenosine triphosphatase and disaccharidases were not significantly different between control and ST-exposed segments . Doses of 1, 2, 3, 4, 5, and 10 mg of berberine were injected into ligated loops of proximal part of the jejunum with 1 ml of ST filtrate . At doses of 2 or more mg/loop, berberine was effective in reducing water and electrolyte secretions induced by ST; the effect was dose-dependent . These findings indicate that berberine may be an effective antidiarrheal agent in E coli heat-stable enterotoxin mediated secretory diarrhea and provide a basis for the frequent empirical use of berberine alkaloid and berberine-containing plants in gastroenteritis and infectious diarrhea in Asian and other countries.

Zh Mikrobiol Epidemiol Immunobiol, 1982 Sep, (9), 64 - 8
{Plasmid preservation in Escherichia coli cells in batch and continuous cultivation}; Ermolenko ZM et al.; The growth kinetics of plasmid and nonplasmid strains of 2 isogenic E . coli K12 culture lines has been studied in experiments on batch and continuous cultivation . Under the optimum conditions the growth rate of R+- and R- cells is the same; under the conditions of limited glucose supply the plasmid strains grown slower, as their energy is partly spent on maintaining plasmids in the cell.

Res Vet Sci, 1982 Sep, 33(2), 248 - 55
Endotoxin-induced fever and associated haematological and blood biochemical changes in the goat: the effect of repeated administration and the influence of flurbiprofen; van Miert AS et al.; Flurbiprofen, a potent non-steroidal anti-inflammatory and antipyretic agent, was given as an intravenous infusion (2 mg/kg) followed by a bolus injection of 1 mg/kg six hours later . After drug administration body temperature and rumen contractions were slightly depressed, whereas urea values gradually increased; serum sorbitol dehydrogenase (SDH) activity, plasma iron concentration and the number of circulating lymphocytes were significantly lower . Intravenous injection of endotoxin from Escherichia coli O111B4 (0.1 microgram/kg) caused shivering, fever, inhibition of rumen contractions, changes in heart rate, lymphopenia, neutropenia followed by neutrophylic leucocytosis, changes in urea values, hypoferraemia, hypozincaemia and a decline in serum alkaline phosphatase (ALP) activity, whereas gamma-glutamyltranspeptidase, glutamic oxalacetic transaminase, lactic dehydrogenase and SDH values were not significantly altered . Pretreatment with flurbiprofen completely abolished the febrile reactions to endotoxin . The endotoxin-induced inhibition of rumen contractions was only delayed . The drug blocked the initial tachycardia to endotoxin but did not prevent the secondary biphasic increase in heart rate . Flurbiprofen failed to modify the endotoxin-induced decrease in both plasma zinc and serum ALP activity whereas the decline in plasma iron concentration was delayed . After drug pretreatment the changes in circulating white blood cells were more pronounced . These data demonstrate that most of the haematological, blood biochemical and clinical effects of endotoxin cannot be blocked by flurbiprofen, and that these effects are not due to the increase in body temperature alone . Tolerance induced by repetitive daily intravenous administration of endotoxin resulted in an almost complete abolition of all the effects . However, the plasma iron values from tolerant goats were significantly lower than those from non-tolerant animals, which demonstrates that the development of a refractory state can result in modification of this biochemical parameter.

Prikl Biokhim Mikrobiol, 1982 Sep-Oct, 18(5), 681 - 7
{Stability of the biocatalysts of L-aspartic acid synthesis based on immobilized Escherichia coli cells}; Zueva NN et al.; Experiments were carried out to investigate the process of a continuous enzymatic synthesis of L-aspartic acid from ammonium fumarate in uniform filling flow reactors . Escherichia coli (Soviet strain 85) cells immobilized in polyacrylamide gel granules reinforced by a solid carrier were used as biocatalysts . The conditions, under which a high aspartase activity of the biocatalyst and a stable hydrodynamic performance of the reactor were maintained, were determined . The main kinetic characteristics of a continuous performance of the reactor for 150 days were obtained.

Mol Biol (Mosk), 1982 Sep-Oct, 16(5), 984 - 90
{Interaction between tRNA-recognizing sites of phenylalanyl-tRNA synthetase from Escherichia coli MRE-600}; Gorshkova II et al.; Modification of phenylalanyl-tRNA synthetase by N-Br-Ac-{14C}Phe-tRNAPhe results in two products, one being stable in alkaline conditions and the other being instable . The instable product is formed in the presence of an equimolar excess of the reagent over the enzyme while the stable one--in the presence of a tenfold excess . The experimental results are described by a kinetic model which takes into account the existence of two routes of modification of product formation . The first route is from the complex containing one molecule of the reagent and the other from the complex containing two molecules of the reagent . It is assumed that the presence of the molecule of the reagent in one center of the dimeric enzyme induces a change in the acceptor point of the other center, which leads to local conformational changes of the enzyme molecule interacting with the substrate analogue.

Mol Biol (Mosk), 1982 Sep-Oct, 16(5), 1109 - 15
{Regulatory region of fr phage replicase cistron . II . Isolation and structure of specific fr RNA fragments}; Tsielens IE et al.; A new set of short RNA templates has been prepared for functional studies in initiation of translation in vitro . Number of individual RNA fragments which contain complete or part of the initiatory region of phage fr replicase cistron were isolated from complex fr RNA--fr coat protein . Their primary structure were determined by using standard fingerprint technique and rapid gel sequencing . Secondary structure of several RNA fragments and their binding activity with phage fr and MS2 coat proteins has been also studied.

J Virol, 1982 Sep, 43(3), 1132 - 7
Polar encapsidation of adenovirus DNA: cloning and DNA sequence of the left end of adenovirus type 3; Kosturko LD et al.; The left-end adenovirus type 3 DNA sequence is very similar to those of other subgroup B adenoviruses, especially in the area between the HinfI site (320 base pairs) and the early-region Ia gene . This segment of the genome has been implicated as necessary for the left-end polarity of adenovirus DNA encapsidation . This segment and the sequences flanking it are compared with the corresponding sequences of adenovirus type 5 and adenovirus type 12, and the extent and pattern of intersubgroup homologies are discussed.

J Biochem (Tokyo), 1982 Sep, 92(3), 971 - 3
Are purine bases enzymatically inserted into depurinated DNA in Escherichia coli?
Kataoka H, Sekiguchi M.
An enzyme activity to incorporate labeled dATP or dGTP preferentially into depurinated DNA, found in an extract of Escherichia coli, does not seem to represent "purine insertase," but may be ascribed to a combined action of DNA polymerase I and AP endonuclease(s) on the basis of the following findings . (1) The activity was found in polA+ but not in polA-cells . (2) The base moiety and the alpha-phosphate group of the nucleotide were equally incorporated into the DNA.

J Biochem (Tokyo), 1982 Sep, 92(3), 615 - 22
Isolation of DNA fragment containing phoS gene of Escherichia coli K-12; Iwakura M et al.; The DNA fragment containing the phoS gene, a regulatory gene for alkaline phosphatase, has been isolated from Escherichia coli K-12 chromosomal DNA by cutting off the DNA with Hind III restriction enzyme and by cloning the gene with plasmid vector pTP 4 which was constructed in this study . The isolated fragment was of about 12.3 kbp and seemed to contain the phoT, glmS, and bgl genes . The 12.3 kbp Hind III fragment was subjected to restriction enzymes EcoR I, BamH I, Sal I, and Pst I, and was found to possess two EcoR I, no BamH I, a Sal I, and four Pst I sites . Partial deletion using these restriction enzymes suggested that the about 6 kbp Hind III-Pst I fragment contained the phoS and phoT genes . Further analysis with other restriction enzymes revealed that the 6 kbp Hind III-Pst I fragment contained a BstE II, two Mlu I and four Hpa I sites . The deletion of these restriction sites using single-strand-specific nuclease S1 suggested that the BstE II and one of Mlu I sites were in the phoT gene, and the BstE II and two Mlu I sites were not in the phoS gene.

Genetika, 1982 Sep, 18(9), 1408 - 11
{Effect of polB mutation on the nature of the thymineless death of thy- cells of Escherichia coli K-12}; Naumova LA et al.; Cell survival, the rates of DNA and protein synthesis, stabilization and repair of single-stranded DNA breaks were measured for Escherichia coli K-12 pol+, polA1, polB1, polC(ts) cells during thymine deprivation . The results indicate that single-stranded breaks in DNA induced by thymine deprivation play no major role in thymineless death of thy- polB- cells, which is similar to thy recB mutant lacking exonuclease V . Probably, the dependence of exonuclease V activity on the presence of DNA polymerase II (the polB gene product) exists which determines molecular mechanisms of thymineless death of thy- polB- cells.

Eur J Biochem, 1982 Sep, 127(1), 165 - 8
Glucosyldiphosphoundecaprenol, the mannose acceptor in the synthesis of the O9 antigen of Escherichia coli . Biosynthesis and characterization; Weisgerber C et al.; We describe the biosynthesis in vitro of the mannose acceptor of the O9 mannan synthesis by Escherichia coli membranes and its analysis with chemical, enzymatic and physical means . Membranes from E . coli 1357 (O9:K29-:H-his,pmi,rfe) were incubated with 10 mM UDP-glucose and 20 mM magnesium chloride in large scale . The incubation mixtures were extracted with butan-1-ol and the extract was fractionated by ion-exchange chromatography on DEAE-cellulose . The presence of the mannose acceptor was detected in the column effluent by using aliquots of the fractions in membrane-reconstitution experiments . The purified mannose acceptor was hydrolyzed for 10 min in 0.1 M hydrochloric acid at 100 degrees C and the hydrolyzate was extracted with light petroleum . Mass spectrometric analysis of the material from the organic phase showed it to be undecaprenol . The aqueous phase contained phosphate and glucose (as determined with glucose oxidase peroxidase) in the ratio of 1.9, alpha-Galactosyldiphosphoundecaprenol and beta-glucosylphosphoundecaprenol were prepared for comparison in these experiments . The results obtained showed that the mannose acceptor in the synthesis of the O9 mannan of E . coli is alpha-glucosyldiphosphoundecaprenol.

Proc Natl Acad Sci U S A, 1982 Sep, 79(18), 5572 - 6
Dissection of a replication origin of Xenopus DNA; Chambers JC et al.; A previously cloned 503-base pair (bp) EcoRI segment of genomic DNA from Xenopus laevis selected for enhancement of replication of its vector plasmid was moved to the EcoRI site of pBR322 . This plasmid designated pJCC31 and five other clones, which were made by cleaving the 503-bp segment in relation to a dispersed repeated sequence and subcloning, were compared with pBR322 for replication by microinjection into Xenopus eggs . The replication measured by incorporation of a 32P-labeled nucleotide as well as semiconservative segregation and dilution of N6-methyladenine at the EcoRI sites showed pJCC31 to be about 15 times as efficient as pBR322 . The next most efficient subclone, pJCC31-2, contains an insert with a complete 320-bp dispersed repeated sequence bracketed by an 8-bp direct repeat . This observation, along with our previous report that repeated sequences of the Alu family in the human genome enhanced replication of the vector plasmid nearly as much as that of the presumptive Xenopus origin, leads to the hypothesis that members of a subset of the short dispersed repeated sequences in vertebrates function as origins for chromosomal replication . Preliminary studies also show that the presumptive Xenopus origin contains a RNA polymerase promoter that increases the transcription of the plasmid when it is microinjected into Xenopus oocytes.

Proc Natl Acad Sci U S A, 1982 Sep, 79(18), 5518 - 21
DNA methyltransferase-dependent transcription of the phage Mu mom gene; Hattman S; The phage Mu mom gene controls an unusual DNA modification . Expression of the mom function requires an active host (dam+) DNA adenine methylase {S-adenosyl-L-methionine:DNA (6-aminopurine)-methyltransferase}; in dam- hosts, Mu development is normal except that the viral DNA does not undergo the mom modification . The present communication compares transcription of the mom gene in dam+ versus dam- cells . 32P-labeled probes were prepared by nick-translation of a purified mom gene-containing restriction fragment and of virion DNA, respectively . These probes were hybridized with various RNAs blotted onto nitrocellulose filters (after fractionation by agarose gel electrophoresis) . The salient findings are: (i) mom-specific RNA was readily detected in dam+ lysogenic cells, but only after induction of the Mu prophage; (ii) the level of mom RNA was decreased at least to 1/20th in induced dam- Mu lysogens; and (iii) little difference, if any, was observed between dam+ and dam- cells with respect to total Mu transcripts produced after prophage induction . These results are in accord with the known pattern of mom gene expression and Mu development . They show that the host (dam+) DNA adenine methylase activity is required for transcription of the mom gene . This represents a unique example where a DNA methylase exerts a positive regulatory role in mRNA transcription; alternative mechanisms for this process will be discussed.

Proc Natl Acad Sci U S A, 1982 Sep, 79(18), 5480 - 4
Physical mechanism for regulation of proton solute symport in Escherichia coli; Konings WN et al.; The activity of the Escherichia coli transport proteins for lactose and proline can be altered by changing the redox state of the dithiols in these carriers . A series of lipophilic oxidizing agents has been shown to inhibit and subsequent addition of dithiothreitol to restore full activity . Both systems are irreversibly inhibited by N-ethylmaleimide, but prior addition of oxidizing agents protects against this inhibition . These data, as well as studies on the inhibitory effect of the dithiol-specific reagent phenylarsine oxide, show that the redox-sensitive step is the conversion of a dithiol to a disulfide . Measurement of the initial rate as a function of the lactose and L-proline concentrations shows that the oxidation of a dithiol to a disulfide increases the Km of the carriers for lactose and L-proline . The reduced (dithiol) form of the carrier has a low Km and the oxidized (disulfide) form has a high Km for its substrate . The changes in Km brought about by reduction and oxidation are the same as those that accompany the generation and dissipation, respectively, of an electrochemical proton gradient (delta mu H+) . These results support a mechanism in which an delta mu H+ or one of its components alters the ligand affinities of the carrier during a single transport cycle through conversion from the reduced to the oxidized form.

Proc Natl Acad Sci U S A, 1982 Sep, 79(17), 5184 - 8
Ribosomal protein S4 is an internal protein: Localization by immunoelectron microscopy on protein-deficient subribosomal particles; Winkelmann DA et al.; The location of protein S4 in the small ribosomal subunit has been identified by immunoelectron microscopy . Although intact small subunits are not reactive with antibodies directed against protein S4, subribosomal particles reconstituted without proteins S5 and S12 are reactive . By using these "incomplete" subparticles, we have mapped the position of S4 . It is located at a single site on the exterior (cytoplasmic) side of the subunit, at the partition that separates the one-third, or head, from two-thirds, or base, of the subunit . In this location, protein S4 is "beneath" proteins S5 and S12 . All three proteins are members of a complex on, or near, the external surface of the small ribosomal subunit that plays an important role in regulation of translational fidelity.

Infect Immun, 1982 Sep, 37(3), 966 - 74
Effect of Escherichia coli alpha-hemolysin on human peripheral leukocyte function in vitro; Cavalieri SJ et al.; To gain further evidence for the role of the Escherichia coli alpha-hemolysin in pathogenesis, its in vitro effects on human peripheral leukocyte function were studied . Leukocytes exposed to low doses of alpha-hemolysin responded with a marked chemiluminescence response, indicating activation of oxidative metabolism . This response was time and dose dependent . Pretreatment of leukocytes with doses of alpha-hemolysin at which nearly 80% of the cells survived decreased the ability of the cells to phagocytize bacteria and particles and to undergo chemotaxis . Premature activation of leukocytes and inhibition of phagocytosis and chemotaxis by alpha-hemolysin, if they occur in vivo, would greatly enhance the survival of an invading E . coli strain.

Infect Immun, 1982 Sep, 37(3), 891 - 4
Different pig phenotypes affect adherence of Escherichia coli to jejunal brush borders by K88ab, K88ac, or K88ad antigen; Bijlsma IG et al.; At least five different porcine phenotypes were distinguished with the three serological variants of the K88 antigen in the brush border adhesion test . Pigs of one phenotype (A) are susceptible to adherence of all three variants, pigs of three phenotypes are susceptible to only two (B and C) or one (D) of the K88 variants, and pigs of one phenotype (E) are entirely resistant to adhesion of K88 antigen did not interfere with the adhesion of K88ab- or K88ac-positive Escherichia coli, whereas in most cases K88ab and K88ac antigen completely blocked the adhesion of K88ad-positive E . coli . Likewise, K88ab antigen blocked the adhesion of K88ac-producing E . coli to both type A and type B brush borders, and vice versa.

Gene, 1982 Sep, 19(2), 221 - 4
Cloning and characterization of a plasmid DNA from anacystis nidulans 6301; Shinozaki K et al.; A plasmid DNA of Anacystis nidulans 6301 was isolated by CsCl-EtBr centrifugation . The Mr of the plasmid, named pBA1, was estimated to be 5.04 +/- 0.26 X 10(6) by electron microscopic analysis and 5.2 X 10(6) by agarose gel electrophoresis . The pBA1 DNA was opened at a unique site with BamHI and cloned in pBR322 vector propagated in Escherichia coli HB101 cells . The recombinant plasmid, named pBAS18, was digested with various restriction endonucleases and its cleavage map was constructed . Based on this result, the cleavage map of the pBA1 plasmid is presented.

Gene, 1982 Sep, 19(2), 211 - 9
Escherichia coli plasmid vectors containing synthetic translational initiation sequences and ribosome binding sites fused with the lacZ gene; Thomas DY et al.; The construction of a series of Escherichia coli plasmid vectors suitable for assaying the effects of gene control signals fused with the E . coli lacZ gene is reported . A synthetic deoxyoligonucleotide dodecamer 5'-CATGAATTCATG GTACTTAAGTAC-5' containing two translation initiation codons (ATG) separated by an EcoRI site was ligated with a lacZ gene derivative which lacks the codons for the first eight amino acids in plasmid pMC1403 (Casadaban et al., 1980) . Two ribosome-binding sequences were synthesised and inserted into the EcoRI site before an ATG, and the effects of these sequences on lacZ gene expression in vivo measured by assaying beta-galactosidase activity . The E . coli ribosomal RNA gene (rrnB) promoter, the tetracycline resistance gene promoter, and a lambda phage promoter were cloned using these plasmids . The plasmids are 9.9 kb in size, have ampicillin resistance as a selectable marker and are generally useful for the detection and in vivo assay of gene control regions.

Gene, 1982 Sep, 19(2), 191 - 200
Cloning of the ColE3-CA38 colicin and immunity genes and identification of a plasmid region which enhances colicin production; Watson RJ et al.; The colicin and immunity genes of plasmid ColE3-CA38 have been localized by characterization of bacteria carrying its cloned restriction fragments . They are within a 3.14-kb EcoRI segment, such that the immunity gene contains the KpnI site, and the colicin gene is adjacent to it within a 2.1-kb KpnI-HincII segment . The immunity gene and one end of the colicin gene are in the region of ColE3-CA38 which is not homologous to the closely related plasmid ColE2-P9 . A 0.64-kb PvuI-EcoRI segment of the plasmid adjacent to that containing the colicin and immunity genes was found to augment colicin production on solid media, and also affected the morphology of clearing zones produced by the cells when used as indicators in overlays of stabs of colicin E2 or E7 producers . The 0.64-kb segment was required in its native orientation relative to the 3.14-kb EcoRI segment to cause its effects.

Mol Biol (Mosk), 1982 Sep-Oct, 16(5), 1019 - 25
{Interactions of subunits of DNA gyrase}; Bogdanova ES et al.; Interaction of DNA gyrase A- and B-subunits during the process of DNA supercoiling was studied . For this purpose a E . coli Cour-1 mutant resistant to coumermycin and containing a mutation in the B-subunit of DNA gyrase was isolated and the influence of the DNA gyrase A-subunit specific inhibitor-nalidixic acid-on DNA supercoiling by wild-type and mutant enzymes was investigated . It turned out that the enzyme from the Cour-1 mutant strain was more sensitive to nalidixic acid than the DNA gyrase from the wild-type strain . Hence, the mutation affecting the B-subunit is capable to change A-subunit properties . That makes it possible to draw the conclusion about a close structural interaction of DNA gyrase subunits during DNA supercoiling.

J Virol, 1982 Sep, 43(3), 943 - 51
Molecular cloning of a highly leukemogenic, ecotropic retrovirus from an AKR mouse; Lenz J et al.; SL3-3 is a leukemogenic, ecotropic retrovirus produced by a T-cell line derived from a spontaneous lymphoma of an AKR mouse . We have isolated a molecular clone of its DNA provirus from infected NIH 3T3 fibroblasts . Cloned proviral DNA produced infectious virus upon transfection onto NIH 3T3 cells . Virus derived by transfection induced lymphomas at high frequency in AKR/J, C3H(f)/Bi, CBA/J, and NFS/N mice . Heteroduplex and RNase T1 fingerprinting analyses showed that the genomes of SL3-3 and the non-leukemogenic virus, Akv, contain no major substitutions relative to one another and differ by only a few base changes . These results unambiguously show that SL3-3 is a highly leukemogenic virus and that major rearrangements of the genome relative to Akv are not required for virulence.

J Virol, 1982 Sep, 43(3), 914 - 24
Molecular cloning of a family of retroviral sequences found in chimpanzee but not human DNA; Bonner TI et al.; A number of retrovirus-like sequences have been cloned from chimpanzee DNA which constitute the chimpanzee homologs of the endogenous colobus type C virus CPC-1 . One of the clones contains a nearly complete viral genome, but others have sustained deletions of 1 to 2 kilobases in the polymerase gene . The pattern of related sequences detected in other primate species is consistent with the genetic transmission of these sequences for millions of years . However, the appropriately related sequences have not been detected in human, gibbon, or orangutan DNAs . These results suggest either that this family of sequences has been deleted from humans, gibbons, and orangutans, or that the genes were recently acquired in the chimpanzee and gorilla lineages.

Cell, 1982 Sep, 30(2), 499 - 508
Persistence of freely replicating SV40 recombinant molecules carrying a selectable marker in permissive simian cells; Tsui LC et al.; We have demonstrated that a SV40-pBR322 recombinant vector (pSV2-gpt) carrying a bacterial gene of selectable phenotype (Eco-gpt) may persist extrachromosomally in COS1 cells, a simian cell line that endogenously produces SV40 large T antigen . The amount of circular (supercoiled) recombinant DNA was estimated to be between 5 and 2000 copies per cell among several pSV2-transformed COS1 clonal lines examined . Complete pSV2 molecules were found in the majority of the transformants, although some of the pSV2 DNAs recovered were shown to have deletions in the pBR322 region . Our results indicate that removal of the pBR322 "inhibitory sequence" in pSV2 is not necessary for stable maintenance of these recombinant molecules in COS1 cells . In addition, large amounts of pSV2-related high molecular weight DNAs, probably concatemers of pSV2, were detected in the transformed lines.

Proc Natl Acad Sci U S A, 1982 Sep, 79(18), 5616 - 20
DNA repair in Escherichia coli: identification of the uvrD gene product; Maples VF et al.; A 2.9-kilobase (kb) Pvu II DNA fragment that contains the uvrD gene of Escherichia coli K-12 has been cloned in both low-copy and multiple-copy plasmid vehicles . The low-copy uvrD plasmid (pVMK49) complements a variety of uvrD, uvrE, and recL mutations . In contrast, the same strains carrying the 2.9-kb fragment in a multiple-copy plasmid (pVMK45) remain sensitive to ultraviolet light (UV) . Additionally, pVMK45 transformants of wild-type E . coli are sensitive to UV and methyl methanesulfonate and appear to be recombination deficient . The cloned uvrD gene does not complement the dominant uvrD3 allele . The 2.9-kb Pvu II insert in these plasmids encodes a single 76,000-dalton protein, which, on the basis of insertional inactivation experiments with the Tn1000 transposon, must be the uvrD gene product . These data confirm earlier genetic analysis which suggested that recL, uvrE, and uvrD were all allelic . The direction of transcription of the uvrD gene has also been determined.

Proc Natl Acad Sci U S A, 1982 Sep, 79(18), 5475 - 9
Primary structure of the replication initiation protein of plasmid R6K; Germino J et al.; The cistron of the replication initiation protein of plasmid R6K has been cloned into the single-strand DNA vectors M13mp8 and M13mp9 and its complete nucleotide sequence has been determined . The amino acid sequence of the initiator protein as predicted from its nucleotide sequence shows that the protein is lysine rich and weakly basic and has a molecular weight of 35,000, which is in close agreement with that estimated from the mobility in NaDodSO4/acrylamide gels . The secondary structure of the protein, approximately by the probabilistic methods of Chou and Fasman {Chou, P . & Fasman, G . (1978) Adv . Enzymol . 47, 45-148}, suggests an NH2-terminal domain of primarily positively charged alpha-helical structure, a core region of interspersed short stretches of random coils and beta-sheets and -turns, and a COOH-terminal domain of alpha-helix.

Proc Natl Acad Sci U S A, 1982 Sep, 79(18), 5470 - 4
Map of distamycin, netropsin, and actinomycin binding sites on heterogeneous DNA: DNA cleavage-inhibition patterns with methidiumpropyl-EDTA.Fe(II); Van Dyke MW et al.; We report a direct technique for determining the binding sites of small molecules on naturally occurring heterogeneous DNA . Methidiumpropyl-EDTA.Fe(II) {MPE.Fe(II) cleaves double helical DNA with low sequence specificity . Using a combination of MPE.Fe(II) cleavage of drug-protected DNA fragments and Maxam-Gilbert gel methods of sequence analysis, we have determined the preferred binding sites on a Rsa I-EcoRI restriction fragment from pBR322 for the intercalator actinomycin D and the minor groove binders netropsin and distamycin A . Netropsin and distamycin A gave identical DNA cleavage-inhibition patterns and bound preferentially to A+T-rich regions with a minimal protected site of four base pairs . We were able to observe the effect of increasing concentration on site selection by netropsin and distamycin A . Actinomycin D afforded a completely different cleavage-inhibition pattern, with 4- to 16-base-pair-long protected regions centered around one or more G.C base pairs.

Proc Natl Acad Sci U S A, 1982 Sep, 79(17), 5347 - 51
Incompatibility of plasmids containing the replication origin of the Escherichia coli chromosome; Yamaguchi K et al.; Plasmids containing the replication origin of the Escherichia coli chromosome (oriC plasmids) are unstable in certain recA strains of E . coli . However, they can be maintained more stably in other recA strains . This stable maintenance has allowed us to study the incompatibility properties of oriC plasmids . We have found that two oriC plasmids are incompatible: they cannot be stably coinherited in individual dividing cells . An oriC plasmid is excluded from growing bacteria at a much faster rate in the presence of a hybrid plasmid made from an oriC plasmid and a high-copy-number vector plasmid than in the presence of another oriC plasmid . By inserting various segments around the oriC region into high-copy-number vectors, we have shown that two different regions in the vicinity of the oriC region determine incompatibility . One region, which we named incA, includes the region essential for autonomous replication of the oriC plasmid . The other, incB, is adjacent to incA but is not required for autonomous replication.

Proc Natl Acad Sci U S A, 1982 Sep, 79(17), 5215 - 9
In vitro transcription of the thymidine kinase gene of herpes simplex virus; Read GS et al.; We transcribed in vitro a cloned 3.5-kilobase fragment of herpes simplex virus type 1 DNA that contains the gene for the viral thymidine kinase . Extracts from uninfected HeLa cells produced five in vitro transcripts, one of which initiated at the in vivo start site for the thymidine kinase mRNA (an early viral message) . A second in vitro transcript initiated at or near the start site for a major late in vivo viral mRNA . The remaining three in vitro transcripts may correspond to minor in vivo mRNA species . Sequences similar to the "T-A-T-A" and "C-A-A-T" boxes, which may be involved in the control of transcription of a variety of viral and cellular genes, were found to precede the initiation site of each of the five in vitro transcripts . Considerable overlap of transcription units was observed.

Infect Immun, 1982 Sep, 37(3), 903 - 6
Seroepidemiology of heat-labile enterotoxigenic Escherichia coli and Norwalk virus infections in Panamanians, Canal Zone residents, Apache Indians, and United States Peace Corps volunteers; Ryder RW et al.; Serum antibody titrations against the heat-labile enterotoxin (LT) of Escherichia coli were carried out on Panamanians, U.S . citizens resident in the Panama Canal Zone, Apache Indians living on the reservation in Whiteriver, Arizona, and Peace Corps volunteers before they traveled overseas . Antibody titers to Norwalk virus were also carried out on serum from Panamanian and Canal Zone residents . A high prevalence of low-titer LT antibodies was found in infants and adults from Panama, the Canal Zone, and Whiteriver . Panamanian children aged 1 to 5 years had the highest LT antibody titers . Peace Corps volunteers had a low prevalence and titer of LT antibodies . Prevalence and titer of antibodies to Norwalk virus were generally higher in Panamanians compared with Canal Zone residents of the same age . In the populations we studied, various modes of transmission and mechanisms of immunity likely explain the differences which we observed in antibody prevalence and titer to these two enteric pathogens.

Infect Immun, 1982 Sep, 37(3), 858 - 68
Genetic and molecular studies of plasmids coding for colonization factor antigen I and heat-stable enterotoxin in several escherichia coli serotypes; Willshaw GA et al.; Plasmids coding for colonization factor antigen I (CFA/I) and heat-stable enterotoxin (ST) were identified in 10 strains of human enterotoxigenic Escherichia coli . The strains, which belonged to serogroups O63, O114, O128, and O153, were isolated in Bangladesh, Latin America, Spain, and South Africa . Two strains produced heat-labile enterotoxin in addition to ST . CFA/I-ST plasmids were mobilized from two O128 strains into E . coli K-12 with the R factor R1-19K- . Like the prototype CFA/I-ST plasmid NTP113, mobilized previously from an E . coli O78 strain into K-12, these two plasmids were non-autotransferring . All 10 CFA/I-ST plasmids were incompatible with NTP113 and had molecular weights ranging from 59 X 10(6) to 72 X 10(6) . The molecular properties of seven of these plasmids were compared with those of six CFA/I-ST plasmids previously mobilized from O78 strains from Ethiopia, South Africa, and Bangladesh and with those of one plasmid coding for CFA/I, ST and heat-labile enterotoxin from a South African strain of serogroup O63 . Digestion with the restriction endonuclease HindIII showed that several plasmids had very similar fragment patterns and two were identical . Generally, a larger proportion of HindIII fragments were of common size in digests of plasmids identified in strains from related geographical areas, regardless of serogroup . However, all except one plasmid shared five or six HindIII fragments of the same size, one of which had been shown previously to be involved in CFA/I production . There was at least 90% DNA homology between CFA/I-ST plasmids with a molecular weight of about 58 X 10(6) from O78 strains from different sources . Most of the DNA sequences of these plasmids were present in a larger CFA/I-ST plasmid (72 X 10(6) from an O128 strain . The results of genetic and molecular studies suggest that CFA/I and ST production is determined by very similar plasmids in different serogroups of human enterotoxigenic E . coli from several sources.

J Bacteriol, 1982 Sep, 151(3), 1637 - 40
Relative map location of the rep and rho genes of Escherichia coli; Tessman I et al.; The rep gene of Escherichia coli was mapped between ilvC and rho by three-factor P1 transductional crosses and also by complementation with a set of lambda transducing phages that contain known amounts of bacterial DNA linked to ilvC . The physical distance between ilvC and rep and between rep and rho were calculated with an accuracy of +/- 0.4 kilobase to be 0 less than or equal to ilvC-rep less than or equal to 3.4 kilobases and 2.0 less than or equal to rep-rho less than or equal to 6.0 kilobases . It was shown that rho-15 is Gro+ for phage ST-1 . An ilv::Tn10 mutation was located in ilvY.

J Bacteriol, 1982 Sep, 151(3), 1346 - 57
Regulation of adenylate cyclase synthesis in Escherichia coli: studies with cya-lac operon and protein fusion strains; Bankaitis VA et al.; We have isolated cya-lac operon and protein fusions in Escherichia coli K-12, and we used these to study the regulation of cya, the structural gene for adenylate cyclase . Data obtained from these fusion strains suggest that neither cyclic AMP (cAMP) nor the cAMP receptor protein plays a major role in transcriptional or translational regulation of cya expression . Modulation of intracellular cAMP concentrations elicited only weak repression of cya-lac fusion activity under conditions of high intracellular cAMP, relative to fusion activity under conditions of low intracellular cAMP . The functional cAMP receptor protein was required for this effect . Incorporation of delta crp into cya-lac fusion strains did not affect fusion expression in glucose-grown cells as compared with similarly cultured isogenic crp+ strains . Furthermore, 20 independently obtained mutants derived from a cya-lacZ protein fusion strain exhibiting a weak Lac+ phenotype were isolated, and it was determined that the mutants had beta-galactosidase activities ranging from 2- to 77-fold greater than those of the parental strain . None of the mutations responsible for this increase in fusion activity map in the crp locus . We used these mutants to aid in the identification of a 160,000-dalton cya-lacZ hybrid protein . Finally, chromosome mobilization experiments, using cya-lac fusion strains, allowed us to infer a clockwise direction of transcription for the cya gene relative to the standard E . coli genetic map.

J Bacteriol, 1982 Sep, 151(3), 1279 - 89
Mapping nonselectable genes of Escherichia coli by using transposon Tn10: location of a gene affecting pyruvate oxidase; Chang YY et al.; Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated by screening for the production of 14CO2 from {1-14C}pyruvate by the method of Tabor et al . (J . Bacteriol . 128:485-486, 1976) . One of these lesions (designated poxA) decreased the pyruvate oxidase activity to 10 to 15% of the normal level but grew well . To map this nonselectable mutation, we isolated strains having transposon Tn10 inserted into the chromosome close to the poxA locus and mapped the transposon . These insertions were isolated by the following procedure: (i) pools of Tn10 insertions into the chromosomes of two different Hfr strains were prepared by transposition from a lambda::Tn10 vector; (ii) these Tn10-carrying strains were then mated with a poxA recipient strain, and tetracycline-resistant (Tetr) recombinants were selected; (iii) the Tetr recombinants were then screened for 14CO2 production from {1-14C}pyruvate . This method was shown to give a greater than 40-fold enrichment of insertions of Tn10 near the poxA gene as compared with transduction . Calculations indicate that a similar enrichment should be expected for other genes . The enrichment is due to the much greater map interval over which strong linkage between selected and unselected markers is found in conjugational crosses as compared with transductional crosses . The use of Hfr conjugative transfer allows isolation of transposon insertions closely linked to a nonselectable gene by scoring hundreds rather than thousands of colonies . Using a Tn10 insertion greater than 98% cotransduced with the poxA locus, we mapped the poxA gene on the E . coli genetic map . The poxA locus is located at 94 min, close to the psd locus . The clockwise gene order is ampA, poxA, psd, purA . The poxA mutation is recessive and appears to be a regulatory gene.

J Bacteriol, 1982 Sep, 151(3), 1086 - 94
Cointegrate formation between homologous plasmids in Escherichia coli; Peterson BC et al.; Conjugation experiments were performed in which the donor was Escherichia coli K-12 strain KP245 containing either R plasmid NR1 plus an ampicillin-resistant derivative of ColE1 (*ColE1::Tn3, called RSF2124) or NR1 plus RSF2124 carrying a cloned EcoRI fragment of NR1 . The recipient was the polA amber mutant JG112, in which RSF2124 cannot replicate . Ampicillin-resistant transconjugants can arise only when the genes for ampicillin resistance are linked to NR1 or are transposed to the host chromosome . When EcoRI fragment A of NR1 (20.5 kilobases) was cloned to RSF2124, the frequency of cotransfer of ampicillin resistance with tetracycline resistance was 25 to 60% . Plasmid DNA from these ampicillin-resistant transconjugant cells was analyzed by gel electrophoresis and was shown to be a cointegrate of NR1 and the RSF2124 derivative . Analysis of plasmid DNA isolated from donor cultures showed that the cointegrates were present before conjugation, which indicates that the mating does not stimulate cointegrate formation . When the cloned fragment was EcoRI fragment H of NR1 (4.8 kilobases), the frequency of cotransfer of ampicillin resistance with tetracycline resistance was about 4%, and the majority of the ampicillin-resistant transconjugants were found to contain cointegrate plasmids . When the donor contained NR1 and RSF2124, the frequency of cotransfer of ampicillin resistance was less than 0.1%, and analysis of plasmid DNA from the ampicillin-resistant transconjugants showed that Tn3 had been transposed onto NR1 . These data suggest that plasmids which share homology may exist in cointegrate form to a high degree within a host cell.

J Gen Microbiol, 1982 Sep, 128 (Pt 9), 2199 - 202
Deletion analysis of essential genes of Escherichia coli: investigation of the btuB-rpoBC interval; McKee RA et al.; A method is described for deletion mapping of essential genes in Escherichia coli . It involves the isolation of secondary-site insertions of lambda cI857plac5 into an F' plasmid . The transposed lacZ gene is useful both for the ready screening of plasmid-phage cointegrates and for rapid analysis of deletions that extend from the site of phage integration into the bacterial genes carried on the substituted plasmid . Such deletions may be used to 'hook-up' bacterial cistrons to the powerful lac promoter . We report the application of this technique to the study of the btuB-rpoBC interval, a cluster of genes encoding components of the transcription-translation apparatus.

J Bacteriol, 1982 Sep, 151(3), 1363 - 71
Differential polypeptide synthesis of the proton-translocating ATPase of Escherichia coli; Brusilow WS et al.; We investigated the regulation of the synthesis of the eight polypeptides of the Escherichia coli proton-translocating ATPase . A plasmid carrying the eight genes of the unc operon was used to direct in vivo and in vitro protein synthesis of the eight polypeptides . Analysis of these data indicates that the ATPase polypeptides are synthesized in unequal amounts both in vitro and in vivo . We identified several regions within the unc operon at which expression of a gene is either increased or decreased from that of the preceding gene . Since genetic information indicates a single polycistronic mRNA for all eight genes of this operon, the observed differential synthesis of the polypeptides is most likely the result of translational regulation . The effect of varying the temperature suggests that the secondary structure in the mRNA may affect the rate of translation initiation in the region between uncE and uncF.

Cancer Res, 1982 Sep, 42(9), 3480 - 5
Role of depurination in mutagenesis by chemical carcinogens; Schaaper RM et al.; The effect of modifying phi chi 174 viral DNA by the chemical carcinogens beta-propiolactone, N-acetoxyacetylaminofluorene and anti-benzo{a}pyrene diol-epoxide was investigated by transfecting the modified DNA into Escherichia coli spheroplasts . Modification of the DNA in vitro by each of these agents was mutagenic for the phi chi 174 amber mutants am3 and am86 . Mutagenicity depended on the induction of the "SOS" response in the host spheroplasts . Heating beta-propiolactone-treated DNA at neutral pH caused strong inactivation such that the number of lethal hits was increased 4-fold . Sucrose gradient analysis showed the induction of alkali-labile sites in the heated DNA . The "nicked circle assay" with double-stranded phi chi 174 DNA showed greater than 70% of these sites to be apurinic sites . Concomitantly with the production of these new sites, a strong increase in the mutation frequency was observed . This mutagenesis also depended upon the induction of the error-prone SOS response in the spheroplasts, as was previously shown to be the case for mutagenesis at putative apurinic sites induced directly by acid-heat treatment . These results suggest that depurination may be of importance to the mechanism of mutagenesis by beta-propiolactone and other carcinogens.

Biken J, 1982 Sep, 25(3), 121 - 30
In vivo evidence for nusA and nusB gene function in general transcription of the Escherichia coli genome; Miyashita T et al.; The requirements for nusA and nusB gene products for the effective transcription of E . coli genes, including the trp operon, were demonstrated by analysis of RNA transcripts produced in nusA, nusB and nusAnusB mutants . In the nusA1 mutant, the levels of overall synthesis of both bulk RNA and trp mRNA were reduced more at 37 C than at 30 C, consistent with the idea of temperature-sensitive nus protein function (Friedman et al., 1973) . In the nusB27 and nusA1nusB27 mutants (Friedman et al., 1976), decrease in the overall rates of these RNA synthesis was notable even at 30 C . In nus mutants harboring trp delta LD1412, in which the trp operon attenuator site is deleted, the reduction in the trp mRNA level was less severe . Thus, the effect of nus mutations seems to result in part from more frequent transcription termination at the attenuator site . Also in nusA1nusB27 mutants, in which the distal portion of the trp operon is deleted, the trp mRNA level was not reduced much . Thus, transcription in the nus- mutants seems to be frequently and prematurely arrested at sites along the trp operon.

J Gen Microbiol, 1982 Sep, 128 (Pt 9), 2211 - 4
A dimeric complex of ruthenium: a new inhibitor of respiration-driven calcium transport in Escherichia coli K12; Gibson JF et al.; Succinate-driven calcium uptake by everted membrane vesicles from Escherichia coli was sensitive to 2 mM-KCN and a dimeric, mixed valence complex of ruthenium, Ru2 (NH3)6Br5(H2O) (100 micro M), but relatively insensitive to 100 micro M-Ruthenium Red . The sensitivity of uptake to KCN and the slight inhibition caused by Ruthenium Red could be attributed to the different potencies of these compounds as respiratory inhibitors . Respiration was completely insensitive to the dimeric ruthenium complex, however, suggesting that this compound is an inhibitor of respiration-driven calcium transport.

Gene, 1982 Sep, 19(2), 179 - 83
The nucleotide sequence of cDNA coding for preproinsulin from the primate Macaca fascicularis; Wetekam W et al.; DNA complementary to preproinsulin messenger RNA from the primate Macaca fascicularis has been cloned into the PstI endonuclease site of the plasmid pBR322 . One clone contains the entire preproinsulin coding region as well as 59 nucleotides of the 5'-untranslated region . The results predict an amino acid sequence for the Macaca fascicularis preproinsulin and establish for the first time that the primary structures of human and primate insulins are identical . The two amino acid exchanges between human and primate preproinsulins are restricted to the pre- and the C-peptide, respectively.

Biochem J, 1982 Sep 1, 205(3), 521 - 8
Construction and partial characterization of a recombinant DNA probe for locust vitellogenin messenger RNA; James TC et al.; Double-stranded DNA complementary to poly(A)-containing RNA from the fat body of adult female locusts, Locusta migratoria, was synthesized . Hybrid molecules containing this cDNA was constructed in the PstI site of the plasmid pAT 153 by the technique of dC . dG tailing and amplified in Escherichia coli K-12 strain HB 101 . Ten colonies of bacteria were identified as carrying recombinant plasmids containing DNA complementary to locust vitellogenin mRNA by (a) 'Northern' blot hybridization analysis and (b) hybrid selection of vitellogenin mRNA and immunological detection of the products of translation of the mRNA . Of the ten recombinant plasmids, one, termed plasmid 4E, containing a cDNA insert of about 650 nucleotides, was characterized in greater detail and a partial restriction map obtained . Using this hybrid plasmid it was possible to derive a value for the average content of vitellogenin mRNA in the adult female locust fat body as 1.5 x 10(5) molecules/cell, and to establish that the haploid genome of L . migratoria contains only one or two genes coding for vitellogenin.

Mol Biol (Mosk), 1982 Sep-Oct, 16(5), 977 - 83
{Reverse transcription of heterologous RNA by RNA-dependent DNA polymerase from Escherichia coli}; Vorob'eva NV et al.; The appropriate conditions for the reverse transcription of rabbit globin mRNA by E . coli RNA-dependent DNA polymerase has been studied . By reducing the ionic strength, increasing the incubation temperature from 37 to 43 degrees and predenaturing the template it was possible to increase the cDNA size . The molar ratio of Mn2+ and dNTP optimal for cDNA synthesis is approximately 1.5-1.7:1 . The increase of dNTP concentration from 0.05 to 0.4 mM each, under conditions of Mn2+ deficiency, results in the decrease of the cDNA size to 4S, obviously, the inhibitory effect of dNTP is not complexed with Mn2+ . The synthesis of cDNA is inhibited also by the excess of Mn2+ . Hybridization of cDNA with globin mRNA protects the former from S1-nuclease . Optimization of the conditions for reverse transcription of heterologous RNa by E . coli RNA-dependent DNA polymerase led to the increase of the cDNA length up to approximately 550 nucleotides which is about 70% of the RNA template length.

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