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