FEBS Lett, 1988 Aug 1, 235(1-2), 35 - 9 An improved purification of lactose permease; Dornmair K; The integral membrane protein lactose permease of Escherichia coli was purified to homogeneity in detergent micelles . No other proteins could be detected in the final product . The molar ratio of lactose permease to lipid was less than 0.2 in detergent solution, thus the preparation was essentially lipid-free . All molecules were functionally active as shown by substrate binding. FEBS Lett, 1988 Aug 1, 235(1-2), 267 - 70 The amino-terminal sequence of the Xenopus laevis mitochondrial SSB is homologous to that of the Escherichia coli protein; Mahoungou C et al.; Two closely related forms of the single-stranded DNA binding protein purified from Xenopus laevis oocytes mitochondria have been identified . Their amino terminal sequences exhibit homology with the Escherichia coli SSB protein. FEBS Lett, 1988 Aug 1, 235(1-2), 189 - 93 Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase; Jordan PM et al.; The dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was specifically labelled with 13C by growth of the bacteria in the presence of 5-amino{5-13C}levulinic acid . Using 13C-NMR spectroscopy, the structure of the cofactor was confirmed as a dipyrromethane made up of two linked pyrrole rings each derived from porphobilinogen . The chemical shift data indicate that one of the pyrrole rings of the cofactor is covalently linked to the deaminase enzyme through a cysteine residue . Evidence from protein chemistry studies suggest that cysteine-242 is the covalent binding site for the cofactor. Epidemiol Infect, 1988 Aug, 101(1), 83 - 91 First recognized community outbreak of haemorrhagic colitis due to verotoxin-producing Escherichia coli O 157.H7 in the UK; Morgan GM et al.; The first recognized outbreak of haemorrhagic colitis due to Escherichia coli O 157.H7 in the United Kingdom affected at least 24 persons living in East Anglia over a 2-week period . The illnesses were characterized by severe abdominal pain and bloody diarrhoea of short duration . Eleven patients were admitted to hospital and there was one death . Patients were mainly adult women who had not eaten out of the home in the 2 weeks before onset . Unlike previously reported outbreaks hamburgers were not the vehicle of infection, and a case-control study suggested that handling vegetables, and particularly potatoes, was the important risk factor. Surgery, 1988 Aug, 104(2), 343 - 9 Fatty acid intake and Kupffer cell function: fish oil alters eicosanoid and monokine production to endotoxin stimulation; Billiar TR et al.; Diets high in n-3 fatty acids appear to have an anti-inflammatory effect, which is thought to be due to decreased macrophage prostaglandin (PG) and thromboxane (Tx) production after incorporation of these fatty acids into cell membrane phospholipids . The effect of n-3 fatty acids incorporation on macrophage monokine release in response to septic stimuli is not well established . Kupffer cells, the fixed macrophages of the liver, were obtained from rats fed diets with fat sources derived from corn oil (CO, control), fish oil (FO, high in n-3 fatty acids), or safflower oil (SO, high in n-6 fatty acids) for 2 or 6 weeks . After exposure to bacterial lipopolysaccharide, Kupffer cells from rats fed FO for 2 or 6 weeks produced less PG and Tx than Kupffer cells from rats fed CO or SO . After 2 weeks of defined diets, interleukin-1 (IL-1) and tumor necrosis factor release were not affected by dietary fat source . In contrast, after 6 weeks of feeding, Kupffer cells from both the FO and the SO groups released less IL-1 and tumor necrosis factor when triggered by lipopolysaccharide than Kupffer's cells from animals fed the control diet that contained CO . These data suggest that altered monokine release from macrophages may contribute to the anti-inflammatory effect of diets high in n-3 fatty acids . Also shown in our results is that prolonged changes in membrane phospholipid content induced by dietary fat source can influence not only PG and Tx production but monokine release as well. Surg Gynecol Obstet, 1988 Aug, 167(2), 92 - 8 Endotoxin and pulmonary cell injury; Bloom RJ et al.; The physiopathologic similarity between adult respiratory distress syndrome (ARDS) secondary to sepsis and endotoxin-induced pulmonary abnormalities has provided extensive descriptive information confirming bacterial endotoxin as a factor initiating the heterogeneous pulmonary changes in ARDS . The present studies have used an established in vitro model for pulmonary cell injury to examine bacterial endotoxin 1, as a direct cytotoxic agent on the two major alveolar cell types, pulmonary endothelium and epithelium; 2, as a stimulant of neutrophil-mediated pulmonary cell injury, and 3, to examine effector mechanisms of cell-mediated damage by studying the potential effectiveness of antioxidants and antiproteolytic agents in the inhibition of this process . Endotoxin direct toxicity and stimulation of neutrophil-mediated pulmonary cell injury was observed in both pulmonary cell populations in systems free of activated serum complement . Endothelial cells were observed to be more susceptible to both the direct effect of endotoxin and to neutrophil-mediated injury when compared with epithelial cell derived monolayers . The addition of an antiprotease (soybean trypsin inhibitor {STI}) was superior to antioxidants (catalase, superoxide dismutase) in reducing the neutrophil-mediated endothelial toxicity (stimulated 51CR per cent release) observed . A 92 per cent degree of protection was observed with the highest dose of STI (5 milligrams per milliliter) used . Proteases released by activated neutrophils on endotoxin stimulation appear to be the predominant toxic species responsible for endothelial injury in this system. Proc Natl Acad Sci U S A, 1988 Aug, 85(15), 5497 - 501 Antibody to sigma 32 cross-reacts with DnaK: association of DnaK protein with Escherichia coli RNA polymerase; Skelly S et al.; A polyclonal antibody to sigma 32, the heat shock sigma factor, has been used to show the presence of low levels of sigma 32 in Escherichia coli RNA polymerase preparations (E sigma 70), which explains the observed in vitro activity of E sigma 70 towards heat shock genes . The sigma 32 antibody cross-reacts with DnaK, and DnaK has been found associated with purified preparations of both E sigma 70 and the heat shock RNA polymerase, E sigma 32. Proc Natl Acad Sci U S A, 1988 Aug, 85(15), 5444 - 8 Alternative DNA loops regulate the arabinose operon in Escherichia coli; Huo L et al.; The araCBAD regulatory region of Escherichia coli contains two divergently oriented promoters and three sites to which AraC, the regulatory protein of the operon, can bind . This paper presents the results of in vivo dimethyl sulfate "footprinting" experiments to monitor occupancy of the three AraC sites and measurements of activity of the two promoters . These measurements were made both in the absence of the inducer arabinose and at various times after arabinose addition to growing cells containing the wild-type ara regulatory region or the regulatory region containing various deletions and point mutations . The data lead to the conclusion that two different DNA loops can form in the ara regulatory region . These loops are generated by AraC protein molecules binding to two different DNA sites and binding to each other . One of these loops predominates in the absence of arabinose and plays a major role in repressing activity of one of the promoters . Upon the addition of arabinose the amount of the first loop type, the repression loop, decreases and the amount of a second loop increases . Formation of this second loop precludes the counterproductive formation of the repression loop. Mol Cell Biol, 1988 Aug, 8(8), 3303 - 10 Step-arrest mutants of FLP recombinase: implications for the catalytic mechanism of DNA recombination; Parsons RL et al.; The site-specific recombinase (FLP) encoded by the yeast plasmid 2 micron circle belongs to the integrase (of phage lambda) family of recombinases . The sparse homology within the members of this family contrasts with the invariance of three residues, His-396, Arg-399, and Tyr-433 (the numbers correspond to the family alignment positions), among them . We report here results on substrate recognition and catalysis by FLP proteins altered at these residues . Mutations of the conserved His and Tyr that aborted the reaction at specific steps of catalysis permitted genetic dissection of the possible biochemical steps of recombination . We provide indirect evidence that recombination by FLP proceeds through a Holliday junction intermediate. Mol Gen Genet, 1988 Aug, 213(2-3), 541 - 4 Increased expression of the Escherichia coli umuDC operon restores SOS mutagenesis in lexA41 cells; Ennis DG et al.; The lexA41 allele of Escherichia coli encodes a semidefective mutant repressor that is also resistant to RecA facilitated cleavage . Cells harboring the lexA41 allele were found previously to repress only a subset of operons in the SOS regulon . lexA41 cells cannot promote SOS mutagenesis, presumably because one or more operons required for mutagenesis are repressed by this mutant repressor . Using the lac regulatory system to increase the expression of the umuDC operon, we were able to restore mutagenesis in the lexA41 mutant . We conclude that the products of the umuDC operon appear to be uniquely limiting in this mutant. Mol Gen Genet, 1988 Aug, 213(2-3), 491 - 8 Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli; Caillet-Fauquet P et al.; In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA . Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA . The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected . This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e . non-targeted, replication errors . This recA730 mutator effect is suppressed by a mutation in the umuC gene . We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair . We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands . The recA730 mutation increases spontaneous mutagenesis of phage lambda poorly . UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA+ strains . This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation . Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations. FEBS Lett, 1988 Aug 1, 235(1-2), 252 - 6 Purification and characterization of phospholipase A2 inhibitory proteins from pig thyroid gland; Antonicelli F et al.; A 32 kDa phospholipase A2 inhibitory protein was isolated from pig thyroid gland after calcium precipitation and fast protein liquid anion-exchange chromatography . SDS-polyacrylamide gel electrophoresis revealed the purity of the protein . The protein activity was assessed by the inhibition of pancreatic phospholipase A2 on {3H}oleic acid-labelled Escherichia coli membranes as substrate and on the prostaglandin E2 production of cultured thyroid cells . The amino acid composition and the isoelectric point were quite similar to those of endonexin previously described in other tissues or cells . The cross-reactivity of a polyclonal antibody against a 32 kDa lipocortin from human peripheral blood mononuclear cells with our thyroidal 32 kDa protein confirmed its lipocortin nature . Before the purification by fast protein liquid chromatography, the Ca2+ pellet contained lipocortin I (35 kDa and its core protein 33 kDa) identified by its cross-reactivity with a polyclonal antibody. Arch Biochem Biophys, 1988 Aug 1, 264(2), 519 - 24 Influence of ligands on the aggregation of the normal and mutant forms of phosphofructokinase 2 of Escherichia coli; Guixe V et al.; The aggregation states of Escherichia coli phosphofructokinase 2 (Pfk-2) and of a mutant enzyme (Pfk-2*) altered in the inhibitory allosteric site for MgATP were measured in the presence and in the absence of substrates and products of the reaction . When sucrose gradient ultracentrifugation experiments were performed in the absence of added ligands, both enzymes sedimented as dimers . Likewise, at low concentrations of both substrates (0.1 mM) the aggregation state of Pfk-2 and Pfk-2* corresponded to a dimer . However, in the presence of 1 mM MgATP alone, Pfk-2 sedimented as a tetramer, whereas Pfk-2* sedimented as a dimer . At a low fructose 6-phosphate concentration (0.1 mM) and an inhibitory concentration of MgATP (4 mM), Pfk-2 sedimented as a tetramer . However, at the same MgATP concentration but at a higher fructose-6-P concentration (1 mM), a condition under which Pfk-2 is not inhibited by the Mg-nucleotide complex, the enzyme sedimented as a dimer . Pfk-2* is not inhibited under these conditions and sedimented as a dimer in each case . Thus, the effectiveness of MgATP in promoting the aggregation of Pfk-2 and Pfk-2* parallels the inhibitability of the enzymes by the nucleotide complex . However, ATP4-, a potent inhibitor of Pfk-2 and Pfk-2* that binds to the catalytic site of the enzymes, had no effect upon their aggregation states . Possibly Pfk-2* is not able to form a tetramer because of an alteration in the regulatory site for the Mg-nucleotide complex. J Virol, 1988 Aug, 62(8), 2951 - 9 Purification and functional properties of simian virus 40 large and small T antigens overproduced in insect cells; Murphy CI et al.; The insect baculovirus Autographa californica nuclear polyhedrosis virus was used as an expression vector for the simian virus 40 (SV40) small t (t) and large T (T) antigens . Spodoptera frugiperda (SF9) cells infected with recombinant viruses encoding these proteins produced approximately 1 to 2 micrograms of t and up to 30 micrograms of T per 3 X 10(6) cells . The former was highly soluble after Nonidet P-40 extraction of the infected cells, unlike its Escherichia coli-produced counterpart . Both SF9-produced proteins were of authentic size and could be readily immunoprecipitated by specific antibodies . Single-step immunoaffinity chromatography was used to purify the two proteins to near homogeneity, with yields averaging 70% in each case . Experiments to test the biological activity of the baculovirus SV40 proteins showed that SF9 t was capable of associating with two of the cellular proteins reported to bind to t in SV40-infected mammalian cells . Moreover, SF9 T had ATPase activity comparable to that of T produced in monkey cells, exhibited helicase activity and SV40 origin-specific DNA binding, and was active in the SV40 DNA replication assay in vitro . Thus, the SV40 T antigens produced in insect cells can be used in future studies of their biochemical roles in vitro and in vivo. Microcirc Endothelium Lymphatics, 1988 Aug, 4(4), 293 - 309 Hepatic microvascular regulatory mechanisms . X . Effects of alpha-one or -two adrenoceptor blockade on glucoregulation in normotensive endotoxic rats with optimal perfusion and flowrates; Reilly FD et al.; Circulating-blood glucose, hepatic glycogen distribution, and the glycogen contents of liver and skeletal muscle, were determined for 60 min in 31 fed and anesthetized Sprague-Dawley rats . These rats received an endoportal infusion of 15 mg per kg b.w . E . coli endotoxin (026:B6) or of sterile saline solution as a control . Either substance was given intravenously at 9:30 a.m . following an intraperitoneal injection at 9:00 a.m . of 0.1 mg per kg b.w . prazosin or 0.3 mg per kg b.w . yohimbine or of the carrier, distilled water . Infused endotoxin elevated blood glucose without affecting hepatic glycogen distribution and total glycogen contents of liver and skeletal muscle when compared to control . Prazosin inhibited endotoxin-induced hyperglycemia, and prazosin plus endotoxin provoked centrilobular glycogen depletion and decreased total hepatic glycogen content . However, no significant alteration in the glycogen content of skeletal muscle accompanied blockade of glucogenesis . Prazosin administered by itself produced no changes in hepatic and muscle glycogen . Although yohimbine blocked endotoxin-induced hyperglycemia, yohimbine, or yohimbine plus endotoxin, produced no significant change in the glycogen contents of liver and skeletal muscle . Blockade in the latter case was associated with some depletion of glycogen in hepatocytes dispersed randomly throughout the unit lobule and in cells located centrivenously . These results suggested that endotoxin-induced hyperglycemia is evoked by activation of alpha-1 and -2 adrenergic receptors . Since no detectible change in hepatic glycogen distribution and in the contents of liver and muscle glycogen accompanied glucogenesis, glycogen catabolism and deposition are postulated to proceed simultaneously and at equivalent rates by 60 min following the experimental induction of endotoxemia . Blockade of alpha (one or two) adrenoceptors is hypothesized to inhibit endotoxin-induced hyperglycemia by facilitating glucose utilization and not by stimulating glycogenesis or by antagonizing glycogenolysis in the liver or skeletal muscle. J Bioenerg Biomembr, 1988 Aug, 20(4), 469 - 80 Mechanism of F1-ATPase studied by the genetic approach; Futai M et al.; E . coli F1-ATPase has been studied mainly by the genetic approach . Mutations in either the alpha or beta subunit modified the kinetics of multisite and uni-site hydrolysis of ATP . The mechanism of F1-ATPase and the essential amino acid residues of beta subunits are discussed. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Aug, 269(2), 218 - 36 Relative cell surface hydrophobicity of Escherichia coli strains with various recognized fimbrial antigens and without recognized fimbriae; Gonzalez EA et al.; Bacterial surface hydrophobicity as well as mannose-sensitive haemagglutinating (MSHA) and mannose-resistant haemagglutinating (MRHA) activities were studied in Escherichia coli strains with and without recognized fimbrial antigens grown under different conditions . Relative bacterial surface hydrophobicity was measured by the salt aggregation test . Four kinds of bacterial aggregations depending on fimbrial antigens were observed: bacteria with CFA/I, CFA/II, CFA/III and K88 aggregated in a particulated form, bacteria with type 1 pilus in a granular form, and bacteria with K99 in a tiny granular form . Some strains, mainly when grown under non-optimal conditions at 18 degrees C or when heated at 80 degrees C, aggregated in a non-typical filamentous form . Among MRHA- MSHA+ (type 1 pilus positive), two classes of bacteria were detected: hydrophobic bacteria aggregating in 0.2-0.4 M ammonium sulphate, and non-hydrophobic bacteria aggregating in 2.0 to 1.6 M ammonium sulphate . The hydrophobicity levels in strains possessing different recognized fimbrial antigens, when grown under optimal conditions to express fimbriae and their typical haemagglutination pattern, were of a decreasing order, viz . CFA/III = CFA/I greater than CFA/II greater than MRHA-MSHA+ hydrophobic = MRHA+ greater than P987 greater than K99-F41 = K88 greater than MRHA- MSHA+ non-hydrophobic greater than MRHA- MSHA- without recognized fimbriae. Mol Gen Genet, 1988 Aug, 213(2-3), 278 - 84 The aspartate transcarbamylase domain of a mammalian multifunctional protein expressed as an independent enzyme in Escherichia coli; Maley JA et al.; Although aspartate transcarbamylase (ATCase) is an independent, monofunctional enzyme in Escherichia coli, mammalian ATCase is one of the globular enzymatic domains of the multifunctional CAD protein . We subcloned fragments of the hamster CAD cDNA and assayed polypeptide products expressed in E . coli for ATCase activity in order to isolate a stretch of cDNA which encodes only the ATCase domain . Three such expression constructs contain fragments of hamster CAD cDNA similar in length to the gene encoding the E . coli ATCase catalytic subunit (pyrB) . These constructs yield stable proteins with ATCase activity, ascertained by both in vivo and in vitro assays; the clones also possess sequence homology with the pyrB gene at both the 5' and 3' ends . The clone producing the most active ATCase contains cDNA which is analogous to the entire pyrB gene, plus a small amount of CAD sequence upstream of this region . Because these constructs produce independently folded, active ATCase from a piece of cDNA the size of the E . coli pyrB gene, they open the door for the in-depth investigation of the isolated mammalian enzyme domain utilizing recombinant DNA technology . This approach is potentially useful for the analysis of domains of other multifunctional proteins. Am J Vet Res, 1988 Aug, 49(8), 1325 - 8 Prevalence of fimbrial antigens and enterotoxins in nonclassical serogroups of Escherichia coli isolated from newborn pigs with diarrhea; Fairbrother JM et al.; Ninety-nine nonclassical serogroups isolated from newborn pigs with neonatal diarrhea were tested for fimbrial antigens F4(K88), F5(K99), F6(987P), F41, and F165, and for heat-labile enterotoxin, heat-stable enterotoxin a (STa), heat-stable enterotoxin b, verocytotoxin, and cytolethal-distending toxin . Thirty-two strains, belonging mostly to serogroups O64:K"V142,", O9:K103, and O20:K101, were F5-positive and usually produced STa, although 5 strains produced only heat-stable enterotoxin b . Fifteen strains, belonging mostly to serogroups O64:K"V142" and O20:K101, were F41 positive and usually produced STa . Twenty-three stains, belonging mostly to serogroups O64:K"V142," O9:K103, and O20:K101, were F6-positive and usually produced STa . Several strains produced more than one fimbrial antigen, either F5 and F41, F5 and F6, F6 and F41, F6 and F165, or F5, F6, F41, and F165 . None of the strains produced heat-labile enterotoxin, verocytotoxin, or cytolethal-distending toxin . The indirect immunofluorescence test was much more sensitive than was the slide agglutination test for the detection of each of the fimbrial antigens F5, F6, F41, and F165 on strains grown in vitro . The production of F6 by certain strains for which only a low proportion of cells were F6-positive in vitro, as demonstrated by immunofluorescence, was confirmed by experimental infection of newborn pigs. Genetics, 1988 Aug, 119(4), 771 - 8 Effects of chromosomal inversion on cell fitness in Escherichia coli K-12; Hill CW et al.; In an effort to learn what factors might mitigate the establishment of Escherichia coli variants bearing major chromosomal rearrangements, we have examined the effects on cell growth of two inversions between rRNA operons . One of these inversions, IN(rrnD-rrnE), had been propagated in a commonly used subline of E . coli K-12 for approximately 30 yr before its discovery, a fact that illustrates the absence of obvious detrimental effects associated with the inversion . We found that culturing under conditions requiring repeated transition from stationary phase to rapid growth led to the replacement of IN(rrnD-rrnE) cells by cells that had undergone either of two types of additional chromosomal inversion: one type fully restored the wild-type order, while the other partially restored it . The partial reinversion was also between rrn operons, but it left a small transposition . The tendency for overgrowth by these revertants persisted through several rounds of periodic selection . In contrast, the other inversion, IN(rrnG-rrnE), was associated with severe, detrimental effects . The effects of IN(rrnG-rrnE) were also alleviated by full or partial reinversion . The probable relationship between the severity of the effects caused by the inversions and the degree of displacement of the replication origin is discussed . Spontaneous inversion events between rrn operons separated by 18% of the chromosome were estimated to occur at a frequency of roughly 10(-5) . If extended to natural situations, the growth disadvantage together with the relatively high frequency of reinversion suggest that clones of cells with an inversion between these rrn operons would be readily overgrown by revertants. J Bacteriol, 1988 Aug, 170(8), 3640 - 9 Isolation and characterization of Escherichia coli mutants that lack the heat shock sigma factor sigma 32; Zhou YN et al.; The product of the Escherichia coli rpoH (htpR) gene, sigma 32, is required for heat-inducible transcription of the heat shock genes . Previous studies on the role of sigma 32 in growth at low temperature and in gene expression involved the use of nonsense and missense rpoH mutations and have led to ambiguous or conflicting results . To clarify the role of sigma 32 in cell physiology, we have constructed loss-of-function insertion and deletion mutations in rpoH . Strains lacking sigma 32 are extremely temperature sensitive and grow only at temperatures less than or equal to 20 degrees C . There is no transcription from the heat shock promoters preceding the htpG gene or the groESL and dnaKJ operons; however, several heat shock proteins are produced in the mutants . GroEL protein is present in the rpoH null mutants, but its synthesis is not inducible by a shift to high temperature . The low-level synthesis of GroEL results from transcription initiation at a minor sigma 70-controlled promoter for the groE operon . DnaK protein synthesis cannot be detected at low temperature, but can be detected after a shift to 42 degrees C . The mechanism of this heat-inducible synthesis is not known . We conclude that sigma 32 is required for cell growth at temperatures above 20 degrees C and is required for transcription from the heat shock promoters . Several heat shock proteins are synthesized in the absence of sigma 32, indicating that there are additional mechanisms controlling the synthesis of some heat shock proteins. J Bacteriol, 1988 Aug, 170(8), 3448 - 58 Genes encoding the alpha, gamma, delta, and four F0 subunits of ATP synthase constitute an operon in the cyanobacterium Anabaena sp . strain PCC 7120; McCarn DF et al.; A cluster of genes encoding subunits of ATP synthase of Anabaena sp . strain PCC 7120 was cloned, and the nucleotide sequences of the genes were determined . This cluster, denoted atp1, consists of four F0 genes and three F1 genes encoding the subunits a (atpI), c (atpH), b' (atpG), b (atpF), delta (atpD), alpha (aptA), and gamma (atpC) in that order . Closely linked upstream of the ATP synthase subunit genes is an open reading frame denoted gene 1, which is equivalent to the uncI gene of Escherichia coli . The atp1 gene cluster is at least 10 kilobase pairs distant in the genome from apt2, a cluster of genes encoding the beta (atpB) and epsilon (atpE) subunits of the ATP synthase . This two-clustered ATP synthase gene arrangement is intermediate between those found in chloroplasts and E . coli . A unique feature of the Anabaena atp1 cluster is overlap between the coding regions for atpF and atpD . The atp1 cluster is transcribed as a single 7-kilobase polycistronic mRNA that initiates 140 base pairs upstream of gene 1 . The deduced translation products for the Anabaena sp . strain PCC 7120 subunit genes are more similar to chloroplast ATP synthase subunits than to those of E . coli. J Bacteriol, 1988 Aug, 170(8), 3350 - 8 Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein; Hanson MS et al.; Type 1 pili of Escherichia coli contain three integral minor proteins with apparent molecular weights (Mr) of 28,000 (28K protein), 16,500, and 14,500 attached to rods composed of Mr-17,000 pilin subunits (Hanson and Brinton, Nature {London} 322:265-268) . We describe here an improvement on our earlier method of pilus purification, which gives higher yields and higher purity . Also reported are methods allowing fractionation of intact type 1 pili into rods of pure pilin and free minor proteins, as well as fractionation of the 28K tip adhesion protein from the 16.5K and 14.5K proteins . We have determined the amino acid composition and amino-terminal sequence of the adhesion protein . This sequence shows limited homology with the amino-terminal sequences of several E . coli pilins, including type 1. Infect Immun, 1988 Aug, 56(8), 2180 - 8 CS31A, a new K88-related fimbrial antigen on bovine enterotoxigenic and septicemic Escherichia coli strains; Girardeau JP et al.; The nature of the common surface antigen of six hemagglutinating and adhesive piliated Escherichia coli strains isolated from diarrheic or septicemic calves was studied . By electron microscopy studies, the E . coli surface antigen designated CS31A was found on bacterial cells and in purified form to consist of thin (2-nm) "fibrillar" fimbriae . E . coli 31A, which was cured of a 105-megadalton plasmid, failed to express CS31A fimbriae, but retained the ability to hemagglutinate and to adhere in vitro on intestinal cells . Conversely, E . coli K-12, harboring the 105-megadalton plasmid originating from strain 31A, produced CS31A fimbriae but was not able to hemagglutinate or adhere on intestinal cells . A single fimbrial subunit of 29 kilodaltons was observed when purified fimbriae from the 105-megadalton plasmid-containing E . coli K-12 strain was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis or eluted by gel filtration after dissociation by 8.5 M guanidium hydrochloride from an S300 Sephacryl column . Western immunoblot analysis and the N-terminal sequence and amino acid composition of CS31A indicate structural and immunological relatedness between CS31A and K88 protein subunits. Infect Immun, 1988 Aug, 56(8), 1846 - 57 Characterization of the plasmid from Escherichia coli RDEC-1 that mediates expression of adhesin AF/R1 and evidence that AF/R1 pili promote but are not essential for enteropathogenic disease; Wolf MK et al.; RDEC-1, an Escherichia coli strain that adheres to rabbit mucosa and causes an attaching, effacing lesion, expresses the pilus adhesin AF/R1 which determines in vitro attachment to rabbit intestinal brush borders . In order to determine the role of AF/R1 pili in the pathogenesis of enteropathogenic diarrhea in rabbits, we localized the genes for AF/R1 expression, constructed an AF/R1- strain, and compared the virulence of the AF/R1+ and AF/R1- strains with particular attention to the development of attaching, effacing lesions . We introduced Tn5 into the 86-megadalton (MDa) conjugative plasmid known to mediate expression of AF/R1 pili and transferred the derivative plasmids into laboratory strain HB101 . Transconjugant M5 was found to contain the 86-MDa plasmid from RDEC-1 and to express AF/R1 pili . Pilus expression on M5 was confirmed by reaction with antiserum raised against purified AF/R1 pili and allowed the bacteria to adhere to the rabbit ileum in an in vitro assay . Three Tn5 insertions in the 86-MDa plasmid were obtained which resulted in loss of AF/R1 expression . Part of the plasmid was mapped, including a region necessary for AF/R1 pilus expression . AF/R1- mutant strain M34 was constructed, and its pathogenesis was investigated . M34 produced disease in rabbits but was less virulent than the parent . The characteristic effacing lesions of RDEC-1 and enteropathogenic E . coli developed in the intestine of rabbits infected with either M34 or RDEC-1, although with M34 they were much less frequent and did not involve the small bowel . We conclude that AF/R1 pilus expression is not essential for the attaching, effacing lesion but serves as an accessory virulence factor which promotes an initial interaction of RDEC-1 with normal epithelial cells. Mol Cell Biol, 1988 Aug, 8(8), 3382 - 90 Requirement for the simian virus 40 small tumor antigen in tumorigenesis in transgenic mice; Choi YW et al.; To examine the role of simian virus 40 (SV40) large T and small t antigens in tumorigenesis in animals, we generated transgenic mice which expressed either both the SV40 large T and small t antigens or the SV40 large T antigen alone under the control of the mouse mammary tumor virus long terminal repeat . The mouse mammary tumor virus long terminal repeat directs the expression of transgenes in ductal epithelial cells of several organs, including the mammary gland, lung, and kidney, and in lymphoid cells . The mice which expressed both the T and t tumor antigens developed lung and kidney adenocarcinomas, while those which expressed large T alone did not . Both types of mice developed malignant lymphomas with similar frequencies and latency periods . Our results show that the SV40 small t antigen cooperates with the large T antigen in inducing tumors in slowly dividing epithelial cells in the lung and kidney. Mol Cell Biol, 1988 Aug, 8(8), 3267 - 71 Fidelity of DNA synthesis in a mammalian in vitro replication system; Hauser J et al.; We have used the simian virus 40 (SV40)-based shuttle vector pZ189 in a forward-mutation assay to determine the fidelity of DNA replication in the in vitro DNA replication system developed by J.J . Li and T.J . Kelly (Proc . Natl . Acad . Sci . USA 81:6973-6977, 1984) . We find that very few base substitution errors (approximately 1/180,000 bases incorporated) are made during in vitro replication of the pZ189 vector in a system derived from CV-1 monkey cells . This replication is completely dependent on added SV40 T antigen and presumably reflects synthesis that is initiated at the SV40 replication origin . The observed level of fidelity is far greater than that reported for in vitro replication of DNA by conventionally purified eucaryotic DNA polymerases alpha and beta . Thus, there must be additional cellular factors in the crude in vitro system that serve to enhance the fidelity of DNA replication. Mol Cell Biol, 1988 Aug, 8(8), 3160 - 7 A constitutive promoter directs expression of the nerve growth factor receptor gene; Sehgal A et al.; Expression of nerve growth factor receptor is normally restricted to cells derived from the neural crest in a developmentally regulated manner . We analyzed promoter sequences for the human nerve growth factor receptor gene and found that the receptor promoter resembles others which are associated with constitutively expressed genes that have housekeeping and growth-related functions . Unlike these other genes, the initiation of transcription occurred at one major site rather than at multiple sites . The constitutive nature of the nerve growth factor receptor promoter may account for the ability of this gene to be transcribed in a diverse number of heterologous cells after gene transfer . The intron-exon structure of the receptor gene indicated that structural features are precisely divided into discrete domains. Mol Cell Biol, 1988 Aug, 8(8), 2999 - 3007 Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha; Defeo-Jones D et al.; Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors . To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha . These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays . Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity . Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins . However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity . Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis . However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold) . These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity. Mol Cell Biol, 1988 Aug, 8(8), 2989 - 98 Complete nucleotide sequence of a mouse VL30 retro-element; Adams SE et al.; The complete nucleotide sequence of a mouse retro-element is presented . The cloned element is composed of 4,834 base pairs (bp) with long terminal repeats of 568 bp separated by an internal region of 3,698 bp . The element did not appear to have any open reading frames that would be capable of encoding the functional proteins that are normally produced by retro-elements . However, some regions of the genome showed some homology to retroviral gag and pol open reading frames . There was no region in VL30 corresponding to a retroviral env gene . This implies that VL30 is related to retrotransposons rather than to retroviruses . The sequence also contained regions that were homologous to known reverse transcriptase priming sites and viral packaging sites . These observations, combined with the known transcriptional capacity of the VL30 promoter, suggest that VL30 relies on protein functions of other retro-elements, such as murine leukemia virus, while maintaining highly conserved cis-active promoter, packaging, and priming sites necessary for its replication and cell-to-cell transmission. Genetika, 1988 Aug, 24(8), 1333 - 42 {Cloning of the gpp gene of Escherichia coli and the use of recBC, sbcB cells for inserting its mutant allele into the chromosomal structure}; Belitskii BR et al.; The gpp gene involved in the pppGpp conversion into ppGpp in Escherichia coli cells was cloned and localized within the multicopy pBR322 plasmid . Amplification of the gpp gene leads to the decline of the intracellular level of pppGpp, which implies enhanced activity of the corresponding enzyme, guanosine pentaphosphatase . To inactivate the cloned gene, a fragment of the pUC4K plasmid containing the kan gene was inserted within the gpp gene . The functional chromosomal allele of the gpp gene was replaced by its inactivated gpp::kan allele, taking advantage of homologous recombination during the transformation of recBC, sbcB cells with the intact hybrid plasmid . This procedure is accompanied by plasmid elimination and may be used for the replacement of other loci of bacterial chromosome with appropriate cloned alleles. Biochem J, 1988 Aug 1, 253(3), 809 - 18 Alterations in the binding site of the cyclic AMP receptor protein at the Escherichia coli galactose operon regulatory region; Gaston K et al.; Gene manipulation techniques have been used to alter the binding site for the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP) at the regulatory region of the Escherichia coli galactose (gal) operon . The effects of these changes on CRP-dependent stimulation of expression from the galP1 promoter in vivo have been measured, and gel binding assays have been used to measure the affinity of cAMP-CRP for the modified sites . Firstly we have deleted progressively longer sequences from upstream of the gal CRP site in order to locate the functional limit of the site . A deletion to -49, removing the first base that corresponds to the consensus sequence for a CRP binding site, is sufficient to reduce CRP binding and block CRP-dependent stimulation of P1 . Secondly, we used synthetic oligonucleotides to invert the asymmetric nucleotide sequence at the gal CRP binding site or to make the sequence symmetric . Inversion of the site has little effect on CRP binding, the architecture of open complexes at P1 revealed by DNAase I footprinting, or the stimulation of transcription from P1 . Making the site symmetric increases the affinity for CRP by over 50-fold and leads to increased transcription from P1, whilst hardly altering the DNAase I footprint of open complexes . Our results confirm that the strength of binding of CRP depends on the nature of the site and show that it is this that principally accounts for differences in CRP-dependent stimulation of transcription. Biochem J, 1988 Aug 1, 253(3), 801 - 7 Mutations in the cyclic AMP binding site of the cyclic AMP receptor protein of Escherichia coli; Gronenborn AM et al.; Mutants in the cyclic AMP binding site of the cyclic AMP receptor protein (CRP) of Escherichia coli have been constructed by oligonucleotide-directed mutagenesis . They have been phenotypically characterized and their ability to enhance the expression of catabolite-repressible operons has been tested . In addition, the binding of cyclic nucleotides to the mutants has been investigated . It is shown that the six mutants made fall into one of three classes: (i) those that bind cyclic AMP better than the wild type protein (Ser-62----Ala) and result in greater transcription enhancement; (ii) those that bind cyclic AMP similarly to wild type (Ser-83----Ala, Ser-83----Lys, Thr-127----Ala, Ser-129----Ala); and (iii) those that do not bind cyclic AMP at all (Arg-82----Leu) . Implications of these findings with respect to present models of the cyclic nucleotide binding pocket of CRP are discussed. Proc Natl Acad Sci U S A, 1988 Aug, 85(16), 6037 - 41 Tn10 transposition promotes RecA-dependent induction of a lambda prophage; Roberts D et al.; We present evidence that Tn10 transposition, or a closely correlated event, induces expression of bacterial SOS functions . We have found that lambda prophage induction is increased in Escherichia coli lambda lysogens containing increased Tn10 transposase function plus single or multiple copies of an appropriate pair of transposon ends . This increase occurs by the normal pathway for prophage induction, which involves RecA-mediated cleavage of the phage lambda repressor protein . We also present evidence that Tn10 promotes induction of expression of the E . coli sfiA gene . Tn10 transposes by a nonreplicative mechanism . We propose that the signal for RecA protease activation and SOS induction is generated by degradation of the transposon donor molecule and suggest that SOS induction is biologically important in helping a cell undergoing transposition to repair and/or recover from damage to the transposon donor chromosome. Genetics, 1988 Aug, 119(4), 759 - 69 Homologous recombination involving a large heterology in Escherichia coli; Yamamoto K et al.; Recombination between two different deletion alleles of a gene (neo) for neomycin and kanamycin resistance was studied in an Escherichia coli sbcA- recB-C- strain . The two homologous regions were in an inverted orientation on the same plasmid molecule . Kanamycin-resistant plasmids were selected and analyzed . The rate of recombination to form kanamycin-resistant plasmids was decreased by mutations in the recE, recF and recJ genes, but was not decreased by a mutation in the recA gene . It was found that these plasmids often possessed one wild-type kanamycin-resistant allele (neo+) while the other neo allele was still in its original (deletion) form . Among kanamycin-resistant plasmids with one wild-type and one parental allele it was often found that the region between the inverted repeats had been flipped (turned around) with respect to sites outside the inverted repeats . These results were interpreted as follows . Gene conversion, analogous to gene conversion in eukaryotic meiosis, is responsible for a unidirectional transfer of information from one neo deletion allele to the other . The flipping of the region between the inverted repeats is interpreted as analogous to the crossing over associated with gene conversion in eukaryotic meiosis . In contrast with a rec+ strain, these products cannot be explained by two rounds of reciprocal crossing over involving a dimeric form as an intermediate . In the accompanying paper we present evidence that gene conversion by double-strand gap repair takes place in the same E . coli strain. Virology, 1988 Aug, 165(2), 596 - 600 Expression of Theiler's murine encephalomyelitis virus protease 3C and polymerase 3D in Escherichia coli and characterization of monospecific sera; Ozden S et al.; Defined DNA fragments of cloned Theiler's murine encephalomyelitis virus genome were used to construct procaryotic recombinant plasmids expressing viral genes 3C and 3D . In these plasmids (pEX-EMBL vectors), viral sequences are fused in-phase behind the Escherichia coli lac Z' gene which is under the control of the inducible lambda Pr promoter . Partially purified fusion proteins were used to immunize Balb/c mice . Sera monospecific for the viral protease 3C and polymerase 3D were obtained . These sera detected their corresponding antigens in situ in infected BHK cells using immunocytochemical reactions. J Bacteriol, 1988 Aug, 170(8), 3786 - 8 Determination of gene products and coding regions from the murE-murF region of Escherichia coli; Maruyama IN et al.; We report the cloning of murE and murF genes and the identification of their gene products . The murE and murF genes encode diaminopimelic acid- and D-alanyl-D-alanine-adding enzymes, respectively, and both genes are involved in cell wall peptidoglycan biosynthesis in Escherichia coli. J Bacteriol, 1988 Aug, 170(8), 3747 - 9 Lipoprotein 28, an inner membrane protein of Escherichia coli encoded by nlpA, is not essential for growth; Yamaguchi K et al.; Lipoprotein 28, an inner membrane protein of Escherichia coli encoded by nlpA, was found to be nonessential for cell growth . A deletion strain in which most of the coding region of the nlpA gene was replaced by a kanamycin resistance gene was able to grow under various conditions . No discernible differences between the wild type and the deletion strain were observed in terms of cell morphology and sensitivity to various chemicals, except for kanamycin. J Bacteriol, 1988 Aug, 170(8), 3675 - 81 Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+; Thoms B et al.; Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations . We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain . We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants . (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background . (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional . The level of expression of recJ in a lexA(Ind) strain suffices for full suppression . Suppression by recA441 at 30 degrees C also depends on recJ+ . The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation . This improvement did not require recJ+ . We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function. J Bacteriol, 1988 Aug, 170(8), 3479 - 84 Deletion and insertion mutations in the rpoH gene of Escherichia coli that produce functional sigma 32; Calendar R et al.; Escherichia coli K-12 strain 285c contains a short deletion mutation in rpoD, the gene encoding the sigma 70 subunit of RNA polymerase . The sigma 70 protein encoded by this allele (rpoD285) unstable, and this instability leads to temperature-sensitive growth . Pseudorevertants of 285c that can grow at high temperature contain mutations in the rpoH gene (encoding the heat shock sigma factor sigma 32), and their mutant sigma 70 proteins have increased stability . We characterized the alterations in three of these rpoH alleles . rpoH111 was a point mutation resulting in a single amino acid substitution . rpoH107 and rpoH113, which are known to be incompatible with rpoD+, altered the restriction map of rpoH . rpoH113 was deleted for 72 base pairs of the rpoH gene yet retained some sigma 32 activity . rpoH107 had two IS1 elements that flanked an unknown DNA segment of more than 6.4 kilobases inserted in the rpoH promoter region . The insertion decreased the amount of rpoH mRNA to less than 0.5% of the wild-type level at 30 degrees C . However, the mRNA from several heat shock promoters was decreased only twofold, suggesting that the strain has a significant amount of sigma 32. J Bacteriol, 1988 Aug, 170(8), 3404 - 14 Nucleotide sequence of the secA gene and secA(Ts) mutations preventing protein export in Escherichia coli; Schmidt MG et al.; The DNA sequence of the secA gene, essential for protein export in Escherichia coli, was determined and found to encode a hydrophilic protein of 901 amino acid residues with a predicted molecular weight of 101,902, consistent with its previously determined size and subcellular location . Sequence analysis of 9 secA(Ts) mutations conferring general protein export and secA regulatory defects revealed that these mutations were clustered in three specific regions within the first 170 amino acid residues of the SecA protein and were the result of single amino acid changes predicted to be severely disruptive of protein structure and function . The DNA sequence immediately upstream of secA was shown to encode a previously inferred gene, gene X . Sequence analysis of a conditionally lethal amber mutation, am109, previously inferred to be located proximally in the secA gene, revealed that it was located distally in gene X and was conditionally lethal due to its polar effect on secA expression . This and additional evidence are presented indicating that gene X and secA are cotranscribed. FEBS Lett, 1988 Aug 1, 235(1-2), 16 - 24 A novel superfamily of nucleoside triphosphate-binding motif containing proteins which are probably involved in duplex unwinding in DNA and RNA replication and recombination; Gorbalenya AE et al.; A statistically significant similarity was demonstrated between the amino acid sequences of 4 Escherichia coli helicases and helicase subunits, a family of non-structural proteins of eukaryotic positive-strand RNA viruses and 2 herpesvirus proteins all of which contain an NTP-binding sequence motif . Based on sequence analysis and secondary structure predictions, a generalized structural model for the ATP-binding core is proposed . It is suggested that all these proteins constitute a superfamily of helicases (or helicase subunits) involved in NTP-dependent duplex unwinding during DNA and RNA replication and recombination. Eur J Biochem, 1988 Aug 1, 175(2), 379 - 85 Effect of histone H1, poly(ethyleneglycol) and DNA concentration on intermolecular and intramolecular ligation by T4 DNA ligase; Sobczak J et al.; The efficiency of ligation of linear DNA and the relative amounts of intramolecular versus intermolecular ligation may be triggered by a number of additive agents . The results show that it is possible to mimic the effect of poly(ethyleneglycol) 6000 by simply increasing DNA concentration about 15-fold: both the rate and the extent of the reaction are greatly enhanced, and intermolecular ligation is largely favored . However, in this case the stimulation by salts, which occurs in poly(ethyleneglycol) solutions, is not observed; we suggest that salts enhance the hydrophobic interactions between ligase and DNA that take place in the presence of poly(ethyleneglycol) . We also show that histone H1, which is involved in the formation of chromatin fibers, is able to stimulate intermolecular ligation by T4 ligase . This effect is more specific than a simple neutralisation of the phosphate groups of the DNA by positive charges of the histone; it still occurs at 125 mM NaCl and in the presence of the four core histones . The implications of the finding concerning the mode of action of histone H1 on DNA are discussed. Eur J Biochem, 1988 Aug 1, 175(2), 221 - 8 Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli . Cloning and characterization of quinolinate synthesis genes nadA and nadB; Flachmann R et al.; The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized . Quinolinate (pyridine-2,3-dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD . Gene nadA was identified by complementation in three different nadA mutant strains . Sequence analysis provided an 840-bp open reading frame coding for a 31,555-Da protein . Gene nadB was identified by complementation in a nadB mutant strain and by the L-aspartate oxidase activity of its gene product . Sequence analysis showed a 1620-bp open reading frame coding for a 60,306-Da protein . For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences . The nadB gene product, L-aspartate oxidase, was purified to homogeneity and the N-terminal sequence of 19 amino acids was determined . The enzyme was shown to be specific for L-aspartate . High-copy-number vectors, carrying either gene nadA, nadB or nadA + nadB, increased quinolinate production 1.5-fold, 2.0-fold and 15-fold respectively . Both gene products seem to be equally rate-limiting in quinolinate synthesis. Am J Med Sci, 1988 Aug, 296(2), 98 - 102 Corticosteroids inhibit endotoxin-induced lung lymph neutrophil stimulating activity in sheep; Lucht WD et al.; The Adult Respiratory Distress Syndrome (ARDS) most frequently is the result of sepsis . Accumulation of neutrophils in lung interstitium is a well-documented phenomenon, but the nature of their presence remains obscure . We hypothesized that endotoxin causes the release of substances into lung lymph that activate neutrophils and that methylprednisolone may prevent sequestration and activation of neutrophils . We used the sheep lung lymph fistula-endotoxin model of ARDS to test this hypothesis . Unanesthetized animals were given either 0.5 microgram/kg of E . coli endotoxin intravenously alone or, on a different experimental day, an identical dose of endotoxin preceded by a 1 gm bolus of methylprednisolone plus a 1 gm/hr continuous infusion . Endotoxin infusion caused the release of substances into lung lymph that were capable of stimulating normal sheep neutrophils to aggregate, migrate, and release superoxide . This activity appeared within 1 hour of endotoxin and persisted for at least 4 hours . Pretreatment by methylprednisolone did not prevent the early activity but did significantly reduce such activity 3-4 hours after endotoxin, when the permeability defects caused by endotoxin are most pronounced . We speculate that endotoxin-stimulated production of humoral neutrophil-activating substances in the lung may play a role in the pathogenesis of acute lung injury. J Virol, 1988 Aug, 62(8), 2994 - 3002 Regulation of human papillomavirus type 11 enhancer and E6 promoter by activating and repressing proteins from the E2 open reading frame: functional and biochemical studies; Chin MT et al.; E2-C, a protein consisting mainly of the carboxy-terminal 45% of the human papillomavirus type 11 (HPV-11) E2 protein, was expressed from the Rous sarcoma virus long terminal repeat in mammalian cells . It competitively repressed the stimulatory action of the full-length E2 protein on the HPV-11 enhancer located in the upstream regulatory region, as assayed by the expression of a reporter gene from the simian virus 40 (SV40) early promoter in transiently transfected monkey CV-1 cells . A mutation in the initiation codon for E2-C protein eliminated repression . In the human cervical carcinoma cell line C-33A, which apparently lacks endogenous HPV DNA, the HPV-11 enhancer-SV40 promoter and the HPV-11 enhancer in its normal association with the E6 promoter had high constitutive activity . In these cells, E2 proteins had little or no stimulatory effect on the transcriptional activity of the HPV-11 enhancer-SV40 promoter . In contrast, the HPV-11 enhancer-E6 promoter was stimulated by the HPV-11 E2 protein but repressed by the bovine papillomavirus type 1 E2 protein, an effect due either to a quantitative difference in E2 expression levels or to a qualitative difference in the trans-activating abilities of the two E2 proteins . In this cell line, the HPV-11 E2-C protein suppressed both the constitutive activity and the HPV-11 E2 trans activation . The E2-C protein was also produced from an expression vector in Escherichia coli . The E2-C protein present in crude E . coli lysates or purified by DNA affinity chromatography associated in vitro with a specific sequence, ACCN6GGT, in filter-binding assays . Moreover, the protein generated DNase I footprints spanning this motif identical to those of bacterially expressed full-length E2 proteins . This DNA sequence motif is necessary and sufficient for E2 binding in vitro and enhancer trans activation in vivo (H . Hirochika, R . Hirochika, T . R . Broker, and L . T . Chow, Genes Dev . 2:54-67, 1988) . Mutations in this sequence that abolished interactions with E2 also precluded binding to the E2-C protein . These data strongly suggest that the full-length E2 protein consists of two functional domains: the amino-terminal half for trans activation and the carboxy-terminal half for DNA binding . The mechanism by which E2-C represses E2-dependent enhancer activity most likely involves competition with E2 for binding to a common transcriptional regulatory site.(ABSTRACT TRUNCATED AT 400 WORDS) J Virol, 1988 Aug, 62(8), 2970 - 7 An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis; Goldstein DJ et al.; An insertional mutagen was developed which consists of the lacZ gene of Escherichia coli under the control of the regulatory elements of the large subunit of ribonucleotide reductase (ICP6) . This ICP6::lacZ cassette was used to create a mutation in a gene designated UL52 (D . J . McGeoch, M . A . Dalrymple, A . Dolan, D . McNab, L . J . Perry, P . Taylor, and M . D . Challberg, J . Virol . 62:444-453, 1988), which is predicted to encode a 114,000-molecular-weight protein . To isolate and propagate this mutant, we generated a cell line, BL-1, by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 KOS strain BamHI L fragment (coordinates 0.708 to 0.745) . An ICP6::lacZ insertion mutant, hr114, was capable of growing in BL-1 cells but not in normal Vero cells . In addition, hr114 was defective in the synthesis of viral DNA and late proteins; however, this mutant appeared to exhibit normal early gene expression . Thus, the results presented in this report show that the UL52 gene product is required for viral DNA synthesis . Furthermore, our studies indicate that the ICP6::lacZ cassette will provide a useful tool for obtaining mutants of other herpes simplex virus genes. J Virol, 1988 Aug, 62(8), 2696 - 700 Activity of avian retroviral protease expressed in Escherichia coli; Kotler M et al.; The 3' end of the avian sarcoma leukosis virus (ASLV) gag gene encodes a 124-amino-acid protease (PR) responsible for processing the gag and pol polyprotein precursors into the mature virion structural proteins and the reverse transcriptase . Here we report the synthesis of the mature ASLV PR and a nucleocapsid (NC)-PR gag precursor fragment in Escherichia coli . E . coli extracts containing mature PR correctly cleaved a synthetic decapeptide homologous to a known ASLV cleavage site . Also, the NC-PR precursor fragment appeared to be correctly processed to produce NC and PR in the bacterial cells . This cleavage was blocked by a mutation in the putative active site of PR . These results strongly support the hypothesis that PR is involved in cleaving itself from the gag precursor. J Gen Microbiol, 1988 Aug, 134 ( Pt 8), 2189 - 99 Molecular cloning of the plasmid-located determinants for CS1 and CS2 fimbriae of enterotoxigenic Escherichia coli of serotype O6:K15:H16 of human origin; Boylan M et al.; The plasmid pCS001, isolated from an enterotoxigenic strain of Escherichia coli, mediates expression of the CS1 or CS2 and CS3 fimbrial adhesins in appropriate E . coli hosts . To characterize this further, HindIII-generated DNA fragments of this plasmid were cloned into the vector plasmid pBR322 . A chimaera, called pCS200, which mediated expression of the CS1 or CS2 fimbrial antigen but not of CS3 fimbrial antigen in appropriate host strains, was obtained . The DNA inserted into the vector sequences of plasmid pCS200 comprised HindIII fragments of 4.7 kbp and 0.8 kbp . Plasmid pCS200-carrying wild-type E . coli hosts of serotype O6:K15:H16 that expressed the CS1 or CS2 antigen also caused mannose-resistant agglutination of bovine red blood cells, suggesting that functional fimbriae were present on the bacterial surface . As previously observed with strain K12 recipients of CS-fimbriae-associated plasmids mobilized from wild-type enterotoxigenic E . coli, K12 recipients of the chimaeric plasmid pCS200 did not express the CS1 or CS2 fimbrial antigen . An oligonucleotide probe, synthesized on the basis of the published N-terminal amino acid sequence of the CS2 fimbrial subunit, hybridized to plasmid pCS200, indicating that the gene for the structural subunit of this fimbria resided on the plasmid. Mol Gen Genet, 1988 Aug, 213(2-3), 379 - 87 Studies in vivo on Escherichia coli RNA polymerase mutants altered in the stringent response; Baracchini E et al.; Previous studies on two Escherichia coli rpoB mutants, carrying single amino acid substitutions at approximate amino acid positions 736 and 906 in the beta subunit, showed that these alterations in the RNA polymerase resulted in an apparent reduced response to valine-induced amino acid starvation in vivo and prevented ppGpp-mediated inhibition of transcriptional initiation at stable RNA promoters in vitro . These observations suggested that the mutations had altered either the ppGpp binding site or the promoter selectivity of the enzyme . The in vivo analysis presented here indicates that these mutants encode an RNA polymerase that responds normally to changes in the level of ppGpp; their apparent relaxedness is due to a reduced accumulation of ppGpp during isoleucine starvation . Thus, there is no indication that the mutations have altered ppGpp binding sites . These observations and the difference between in vitro and in vivo results can be explained by the assumption that the mutations produce an extended ppGpp-dependent pausing of RNA polymerase during the transcription of unstable RNA . Comparison of the vivo and in vitro effects of ppGpp on rrn transcription further suggests that these reflect different phenomena, although in both cases ppGpp inhibits rrn transcription. Mol Gen Genet, 1988 Aug, 213(2-3), 214 - 22 Basal ppGpp level adjustment shown by new spoT mutants affect steady state growth rates and rrnA ribosomal promoter regulation in Escherichia coli; Sarubbi E et al.; This work describes an approach towards analyzing the regulatory effects of variation of guanosine 3',5'-bispyrophosphate (ppGpp) basal levels in Escherichia coli during steady state growth . A series of strains was derived by mutating the spoT gene (which encodes the major cellular ppGppase) so as to obtain systematic increments in ppGpp basal levels . These strains differ genetically at the spoT locus and, in some cases, also at the relA locus because of the severity of spoT mutant alleles . Measurements of ppGpp revealed a ten-fold range of basal levels during growth on minimal medium . The empirical relationship between ppGpp concentration and growth rate is a simple linear inverse correlation . Tandem rrnA ribosomal RNA promoters, present on a multicopy plasmid, are shown to be differentially regulated over this range of basal levels . The upstream P1 promoter activity shows an inverse exponential relation to ppGpp concentration whereas the downstream P2 promoter is only weakly affected . We conclude that there are systematic regulatory consequences associated with small changes in ppGpp basal levels during steady state growth that probably are part of a continuum with more dramatic effects observed during the stringent response to amino acid deprivation. Proc Natl Acad Sci U S A, 1988 Aug, 85(16), 6152 - 6 Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of factor VIII fragments expressed in Escherichia coli; Scandella D et al.; Epitopes for antibodies that inhibit factor VIII procoagulant protein were analyzed by deletion mapping of factor VIII protein fragments expressed in Escherichia coli . A human factor VIII cDNA clone was used to generate E . coli expression vectors encoding fragments containing the 80-kDa factor VIII light chain (A3, C1, and C2 domains) and the 44-kDa carboxyl-terminal half of the factor VIII heavy chain (A2 domain) . A series of deletions of each fragment was constructed and tested by immunoblotting for the binding of alloantibody and autoantibody inhibitors . Analysis of derivatives of the 80-kDa fragment showed that six inhibitors recognized a major epitope(s) within the carboxyl-terminal 17.3 kDa of factor VIII . These inhibitors also recognized weaker epitopes nearby and one inhibitor recognized epitopes scattered throughout the 80-kDa fragment . Deletions within the heavy chain fragment revealed one epitope-containing region confined to the amino-terminal 18.3 kDa recognized by six inhibitors . Bacterially produced factor VIII fragments containing the major epitopes were capable of neutralizing inhibitors in vitro but fragments containing weaker or no epitopes did not . These data suggest a potential therapeutic use of factor VIII fragments for neutralization of inhibitor antibodies. J Gen Virol, 1988 Aug, 69 ( Pt 8), 2115 - 22 Location of a neutralizing epitope for the haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus; Chambers P et al.; The binding site of a monoclonal antibody to the haemagglutinin-neuraminidase (HN) polypeptide of Newcastle disease virus (NDV) has been located . Complementary DNA or synthetic oligonucleotides corresponding to portions of the HN gene were cloned into the Escherichia coli vector pUC19 and fragments of the HN protein were thereby fused to the alpha-peptide of beta-galactosidase . Western blot analysis of E . coli lysates containing expressed fragments of the HN cDNA or synthetic oligonucleotides identified an antibody-binding peptide (Asp-Glu-Gln-Asp-Tyr-Gln-Ile-Arg; amino acid residues 346 to 353) . Nucleotide sequence analysis of an antibody-resistant mutant of NDV revealed a Glu (wild-type) to Lys (mutant) substitution within the above sequence . The methods described could be useful for the location of continuous epitopes of other polypeptides. J Clin Invest, 1988 Aug, 82(2), 514 - 23 Mechanism of action of Escherichia coli heat stable enterotoxin in a human colonic cell line; Huott PA et al.; Escherichia coli heat stable enterotoxin (STa) caused Cl- secretion across T84 cell monolayers in a dose-dependent manner only when applied to the apical membrane surface and not when applied to the basolateral surface . Measurement of cAMP, cGMP, and free cytosolic Ca2+ in response to STa suggested that cGMP alone mediated the Cl- secretory response . Studies utilizing blockers of the Na+,K+-ATPase pump, a Na+,K+,Cl- cotransport system, a K+ channel, and a Cl- channel suggest that all of them participate in the Cl- secretory process induced by STa . The results suggest that the Cl- secretory response induced by STa is mediated by cGMP after the enterotoxin binds to its receptor on the apical membrane . The enterotoxin, by increasing cGMP, opens a K+ channel on the basolateral membrane as well as a Cl- channel on the apical membrane . The activation of these ion exit mechanisms, together with activations of the Na+,K+,Cl- cotransporter and the Na+,K+-ATPase pump drives Cl- exit through the Cl- channel on the apical membrane. J Bacteriol, 1988 Aug, 170(8), 3611 - 7 Genetic identification of the pore domain of the OmpC porin of Escherichia coli K-12; Misra R et al.; We have isolated and characterized 31 mutations in the ompC gene which allow Escherichia coli to grow on maltotriose (Dex+) in the absence of the LamB and OmpF porins . These ompC(Dex) mutations include single-base-pair substitutions, small deletions, and small insertions . DNA sequence analysis shows that all of the alterations occur within the coding region for the first 110 amino acids of mature OmpC . The 26 independent point mutations repeatedly and exclusively alter residues R37, R74, and D105 of mature OmpC . In each case, a charged amino acid is changed to an uncharged residue . Biochemical and physiological tests suggest that these alterations increase the size of the pore channel . Starting with three different ompC(Dex) strains with alterations affecting R74, we isolated mutants that could grow on maltohexose (Hex+) . These mutants each contained a second alteration in the ompC gene involving residues R37, D105, or R124 . The combined effects on pore function of the two mutations appear to be additive . These experiments suggest that we have identified the important residues of OmpC peptide involved in pore function . On the basis of these mutations and general rules for membrane protein folding, a model for the topology of the OmpC protein is proposed. J Bacteriol, 1988 Aug, 170(8), 3390 - 5 Characterization of the upstream region of the formate dehydrogenase operon of Methanobacterium formicicum; Patel PS et al.; The fdhA and fdhB genes of Methanobacterium formicicum, which code for the alpha and beta subunits of formate dehydrogenase, were cotranscribed as part of a large transcript . By using Northern (RNA) gel blot analysis, the transcription start site was located within a 1.6-kilobase BglII-NcoI fragment 4.3 kilobases upstream from the fdhA gene . The precise transcription start site within the fragment was determined with the aid of primer extension analysis and S1 nuclease protection studies . A putative promoter sequence for structural genes of methanogenic archaebacteria is proposed based on a comparison of DNA sequences of the upstream region of methanogen operons for which transcription initiation sites are known . Comparison of the DNA sequence of the upstream region of the fdh operon of M . formicicum with the sequence upstream of the fdhF gene of Escherichia coli revealed regions of considerable identity. J Bacteriol, 1988 Aug, 170(8), 3359 - 66 Excision repair of thymine glycols, urea residues, and apurinic sites in Escherichia coli; Laspia MF et al.; The genetic requirements for the excision repair of thymine glycols, urea residues, and apurinic (AP) sites were examined by measuring the survival in Escherichia coli mutants of phi X174 replicative form (RF) I transfecting DNA containing selectively introduced lesions . phi X RF I DNA containing thymine glycols was inactivated at a greater rate in mutants deficient in endonuclease III (nth) than in wild-type hosts, suggesting that endonuclease III is involved in the repair of thymine glycols in vivo . phi X RF I DNA containing thymine glycols was also inactivated at a greater rate in mutants that were deficient in both exonuclease III and endonuclease IV (xth nfo) than in wild-type hosts, suggesting that a class II AP endonuclease is required for the in vivo processing of thymine glycols . phi X duplex-transfecting DNA containing urea residues or AP sites was inactivated at a greater rate in xth nfo double mutants than in wild-type, but not single-mutant, hosts, suggesting that exonuclease III or endonuclease IV is required for the repair of these damages and that either activity can substitute for the other . These data are in agreement with the known in vitro substrate specificities of endonuclease III, exonuclease III, and endonuclease IV. Eur J Biochem, 1988 Aug 1, 175(2), 375 - 8 Control of the tRNA-tufB operon in Escherichia coli . 3 . Feedback inhibition of tufB expression by an EF-Tu with a deletion in the guanine-nucleotide-binding domain; Van Delft JH et al.; The expression of tufB, one of the two EF-Tu-encoding genes in Escherichia coli, is under autogenous control . Feedback inhibition of tufB expression by plasmid-borne EF-Tu has been used to answer the question of whether or not the integrity of the guanine-nucleotide-binding domain of EF-Tu is required for the autoregulatory role of the factor protein . We show that a large deletion of tufB, causing the elimination of an 81-amino-acid segment from the plasmid-borne EF-Tu, does not abolish tufB repression . We conclude that the autoregulation of the cellular EF-Tu level is not dependent on an intact guanine-nucleotide-binding domain and does not require binding of GTP to EF-Tu . The repressor activity of the deletion derivative of EF-Tu can be measured despite a rapid disappearance of the (altered) mutant protein from the soluble cytoplasmic fraction of the cell . Degradation and assembly in larger complexes are responsible for this disappearance. Eur J Biochem, 1988 Aug 1, 175(2), 363 - 74 Control of the tRNA-tufB operon in Escherichia coli . 2 . Mechanisms of the feedback inhibition of tufB expression studied in vivo and in vitro; Van Delft JH et al.; The mechanism underlying feedback inhibition of tufB expression has been studied in vivo by gene-dosage experiments and by gene and operon fusions involving lacZ . Raising the cellular EF-Tu content, by introducing a multicopy plasmid encoding EF-TuA into the cell, repressed the level of EF-TuB but left the content of tRNA(Thr)3, encoded by the tRNA-tufB operon, unaffected . This indicates that autoregulation of chromosomal tufB expression does not occur by modulating transcription initiation at the promoter of the tRNA-tufB operon . This conclusion is further substantiated by experiments with a tRNA':lacZ operon fusion . The molecular ratio of chromosome-borne tufA and tufB transcripts also remained unaltered under conditions of excess EF-Tu, though experiments with a tRNA-tufB':lacZ operon fusion showed a decrease of tufB transcripts . Our data further exclude drastic effects of the autogenous repressor on processing of the contranscript of the operon into monocistronic tufB RNA and on alteration of EF-TuB turnover . Two possible mechanisms remain, which cannot yet be decided between . One is modulation of EF-Tu by transcription termination either directly or indirectly by affecting antitermination . The second is translational repression . In vitro translation of transcripts derived from SP6 clones did not reveal any feedback inhibition of EF-TuB synthesis . Surprisingly, addition of EF-Tu to a coupled transcription/translation systems was found to block transcription initiation at the primary promoter of the tRNA-tufB operon by over 90% . Although this in vitro effect of EF-Tu could not be demonstrated in vivo, possibly because of a difference in higher-order structure between plasmid-borne and chromosome-borne DNA, it indicates that under certain conditions EF-Tu binds very specifically to the tRNA-tufB operon promoter or its upstream region. Eur J Biochem, 1988 Aug 1, 175(2), 355 - 62 Control of the tRNA-tufB operon in Escherichia coli . 1 . rRNA gene dosage effects and growth-rate-dependent regulation; Van Delft JH et al.; 'Ribosome feedback' effects on the expression of the genes specifying tRNA and EF-Tu in E . coli have been studied at increased rrnB doses (rRNA gene doses) . We confirm previous observations that the introduction into the cell of a multicopy plasmid carrying the rrnB operon reduces the cellular content of most tRNAs, including those encoded by the tRNA-tufB operon, but leaves the 5S rRNA content unaffected . Increase of the dosage of intact, but not of deleted rRNA genes, causes a slight drop in total EF-Tu that can be fully accounted for by a decrease in EF-TuB level . The drop in EF-TuB content (approx . 25%) is much smaller than that in tRNA content (approx . 80%) . The synthesis rate of total EF-Tu is hardly affected, indicating that the turnover of EF-Tu has not changed . The ratio of tRNA over tuf RNA synthesis rates remains the same after elevation of rrnB dosage . Considering the large decrease in tRNA content this means that both RNA synthesis rates decrease to approximately the same extent . The relatively small drop in EF-Tu synthesis must be due, therefore, to an enhancement of the number of EF-Tu molecules synthesized per mRNA molecule . Apparently a post-transcriptional mechanism, regulating EF-Tu synthesis, becomes operative under these conditions . Growth-rate-dependent regulation of the tRNA-tufB operon has been studied using lysogens carrying tRNA':lacZ and tRNA-tufB':lacZ operon fusions and a tufB':lacZ' gene fusion . These experiments show that the cellular contents of tRNA, tufB RNA and EF-TuB vary in direct proportion to the growth rate . This indicates that growth rate control of tRNA-tufB operon transcription resembles that of stable RNA operons and not of r-protein operons, and that the read-through of the terminator at the end of the tRNA gene cluster remains unaltered. Surgery, 1988 Aug, 104(2), 280 - 6 Tumor necrosis factor and endotoxin induce similar metabolic responses in human beings; Michie HR et al.; After injury, infection, or major operations a number of predictable metabolic responses occur . It has been proposed that the cytokine tumor necrosis factor (TNF)/cachectin is a primary mediator of these host responses . To test this hypothesis, we studied 16 tumor-bearing humans with normal renal and hepatic function, who received 24-hour continuous intravenous infusions of escalating doses of recombinant TNF (4 to 636/micrograms/m2/24 h) . Serial measurements were made of vital signs and plasma concentrations of TNF, interleukin-1, adrenocorticotropic hormone, cortisol, iron, glucose, and C-reactive protein . Low doses of TNF had minimal metabolic effects, but infusions of greater than or equal to 545 micrograms/m2/24 hr (n = 8) resulted in fever, pituitary, and stress hormone release and acute phase changes . These alterations were compared with the changes that occurred in healthy humans (n = 13) receiving intravenous bolus injections of Escherichia coli endotoxin (4 ng/kg) . TNF infusion in doses greater than or equal to 545 micrograms/m2/24 hr produced peak plasma TNF concentrations and metabolic responses that were similar to those after endotoxin injection . Interleukin-1 concentrations remained basal after TNF or endotoxin administration . TNF may represent the primary afferent signal that initiates many of the metabolic responses associated with sepsis and endotoxemia. Infect Immun, 1988 Aug, 56(8), 1974 - 80 Genetic control and properties of coli surface antigens of colonization factor antigen IV (PCF8775) of enterotoxigenic Escherichia coli; McConnell MM et al.; Enterotoxigenic Escherichia coli producing coli surface antigen 4 (CS4), CS5, and CS6 of colonization factor antigen IV were examined . This factor was originally called putative colonization factor 8775 (PCF8775) . All of the coli surface antigens were plasmid coded and were usually carried on the same plasmid as the genes coding for heat-stable toxin (ST) or heat-labile toxin (LT); thus, CS5-CS6-ST, CS6-ST, and CS6-LT plasmids were found . In strains of serotype O25:H42, the genes coding for CS4 and CS6 were on a plasmid separate from that containing the genes coding for ST and LT . CS4 and CS5 were fimbrial antigens with a subunit molecular mass of about 17.0 and 21.0 kilodaltons (kDa), respectively . CS6 was found as a single polypeptide with a molecular mass of about 14.5 kDa in strains of serotypes O25:H42, O27:H7, and O27:H20 when heated extracts were run on sodium dodecyl sulfate-polyacrylamide gels . CS6-positive extracts of strains of serogroups O148, O159, and O167 showed two bands with molecular masses between 14.5 and 16.0 kDa. J Virol, 1988 Aug, 62(8), 2867 - 73 An Okazaki piece of simian virus 40 may be synthesized by ligation of shorter precursor chains; Nethanel T et al.; It is generally accepted that an aphidicolin-sensitive DNA polymerase elongates the eucaryotic RNA primer (iRNA) into a mature Okazaki piece reaching ca . 200 nucleotides . Yet, as shown here, nascent DNA chains below 40 nucleotides accumulated in simian virus 40 (SV40) DNA replicating in isolated nuclei in the presence of aphidicolin . These products resembled precursors of longer Okazaki pieces synthesized in the absence of aphidicolin (termed here DNA primers) in size distribution, lagging-replication-fork polarity, and content of iRNA . Within the isolated SV40 replicative intermediate, DNA primers could be extended in a reaction catalyzed by the Escherichia coli DNA polymerase I large fragment . This increased their length by an average of 21 deoxyribonucleotide residues, indicating that single-stranded gaps of corresponding length existed 3' to the DNA primers . Incubation with T4 DNA ligase converted most of the extended DNA primers into products resembling long Okazaki pieces . These data led us to propose that the synthesis of an SV40 Okazaki piece could be itself discontinuous and could comprise the following steps: (i) iRNA synthesis by DNA primase, (ii) iRNA extension into a DNA primer by an aphidicolin-resistant activity associated with DNA primase-DNA polymerase alpha, (iii) removal of iRNA moieties between adjacent DNA primers, (iv) "gap filling" between DNA primers by the aphidicolin-sensitive DNA polymerase alpha, and (v) ligation of DNA primer units onto a growing Okazaki piece . Eventually, a mature Okazaki piece is ligated onto a longer nascent DNA chain. Gene, 1988 Jul 30, 67(2), 159 - 68 A directed nucleotide-sequencing approach for single-stranded vectors based on recloning intermediates of a progressive DNA synthesis reaction; Burton FH et al.; A simple method for site-directed nucleotide sequencing is presented that uses a novel procedure for generating nested 'deletions' within inserts of single-stranded clones . In this method, single-stranded template, sequencing primer, and the Klenow fragment of Escherichia coli DNA polymerase I are used to initiate progressive DNA synthesis of the entire insert of the clone . By time-dependent sampling and pooling of intermediates from the synthesis reaction a series of nested double-stranded DNA subfragments of the insert can be created . Nested subclones are then produced by S1-endonuclease treatment and oriented subcloning methods . First, smaller quantities of template DNA can be used, equivalent to a fraction of a small DNA sequencing prep . Second, it works with single-stranded M13 phage DNA rather than requiring the preparation of double-stranded replicative form DNA as in ExoIII-based methods . Third, the 'deletions' it generates can span areas of simple nucleotide sequence or secondary structure that often halt digestion in the single-stranded exonuclease-based method . Last, the method is adaptable to a larger variety of insert cloning sites than the ExoIII-based method . The main disadvantage of the method is that, due to the lower efficiency of subcloning larger DNA fragments, subclone inserts larger than 3 kb are generated only infrequently. Gene, 1988 Jul 30, 67(2), 287 - 94 A series of shuttle vectors using chloramphenicol acetyltransferase as a reporter enzyme in yeast; Mannhaupt G et al.; Reports from numerous laboratories have shown that the gene coding for the bacterial enzyme chloramphenicol-3-O-acetyltransferase can be used as a reporter gene (cat) in mammalian and plant systems to analyze gene activity at the transcriptional level when combined with endogenous regulatory signals; the enzyme activity can be quantified by a chromatographic or a photometric assay . To adapt this simple and highly sensitive test for the yeast system, we constructed a series of yeast vectors containing the cat gene together with selectable markers for Escherichia coli and yeast; integrating, autonomously replicating and centromere-carrying plasmids were used . We show that the cat gene lacking the endogenous promoter is expressed at low levels in yeast transformants . To demonstrate functional expression of the cat gene placed under the control of a yeast promoter, we chose the PHO5 regulatory region . We found that cat expression was induced via the PHO5 promoter in a manner as observed for the endogenous PHO5 gene, whereas in the repressed state cat expression remained low . Using these vectors, it should be feasible to analyze other sequences conferring promoter activity or other control functions in yeast. Gene, 1988 Jul 30, 67(2), 147 - 58 Effects of dominant-negative lac repressor mutations on operator specificity and protein stability; Betz JL et al.; The specific binding of dominant-negative (I-d) lactose (lac) repressors to wild-type (wt) as well as mutant (Oc) lac operators has been examined to explore the sequence-specific interaction of the lac repressor with its target . Mutant lacI genes encoding substitutions in the N-terminal 60 amino acids (aa) were cloned in a derivative of plasmid pBR322 . Twelve of these lacI-d missense mutations were transferred from F'lac episomes using general genetic recombination and molecular cloning, and nine lacI missense mutations were recloned from M13-lacI phages {Mott et al., Nucl . Acids Res . 12 (1984) 4139-4152} . The mutant repressors were examined for polypeptide size and stability, for binding the inducer isopropyl-beta-D-thiogalactoside (IPTG), as well as binding to wt operator . The mutant repressors' affinities for wt operator ranged from undetectable to about 1% that of wt repressor, and the mutant repressors varied in transdominance against repressor expressed from a chromosomal lacIq gene . Six of the I-d repressors were partially degraded in vivo . All repressors bound IPTG with approximately the affinity of wt repressor . Repressors having significant affinity for wt operator or with substitutions in the presumed operator recognition helix (aa 17-25) were examined in vivo for their affinities for a series of single site Oc operators . Whereas the Gly-18-, Ser-18- and Leu-18-substituted repressors showed altered specificity for position 7 of the operator {Ebright, Proc . Natl . Acad . Sci . USA 83 (1986) 303-307}, the His-18 repressor did not affect specificity . This result may be related to the greater side-chain length of histidine compared to the other amino acid substitutions. Gene, 1988 Jul 30, 67(2), 169 - 82 New cloning vectors and techniques for easy and rapid restriction mapping; Tartof KD et al.; We have modified plasmid, phage lambda and cosmid cloning vectors to be of general use for easily and unambiguously determining restriction maps of recombinant DNA molecules . Each vector is constructed so that it contains the rarely found NotI restriction site joined to a short synthetic linker sequence that is followed by a multiple cloning site . DNA cloned into these vectors may be restriction-mapped by either of two methods . In one technique, the cloned DNA is completely digested with NotI, followed by partial digestion with any other restriction enzyme . After electrophoresis and transfer to a nylon membrane, the fragments are hybridized to a labeled probe complementary to the NotI linker . In the second technique, referred to as recession hybridization detection, cloned DNA is digested with NotI and then briefly treated with exonuclease III to recess the 3' ends . After hybridizing a labeled complementary oligodeoxynucleotide to the single-stranded 5' end containing the linker sequence, the DNA is partially digested with another restriction enzyme, electrophoresed and the gel is exposed to x-ray film . With either method the size of each labeled fragment corresponds directly to the distance that a restriction site is located from the NotI linker terminus . Methods for obtaining partial restriction enzyme digests have been devised so that as many as 20 different enzymes may be conveniently mapped on a single gel in little more than a day . The vectors and techniques described may also be adapted to automated or semi-automated devices that read fragment lengths and calculate the resulting restriction map.(ABSTRACT TRUNCATED AT 250 WORDS) Science, 1988 Jul 29, 241(4865), 551 - 7 Parallel stranded DNA; van de Sande JH et al.; A series of four hairpin deoxyoligonucleotides was synthesized with a four-nucleotide central loop (either C or G) flanked by the complementary sequences d(T)10 and d(A)10 . Two of the molecules contain either a 3'-p-3' or 5'-p-5' linkage in the loop, so that the strands in the stem have the same, that is, parallel (ps) polarity . The pair of reference oligonucleotides have normal phosphodiester linkages throughout and antiparallel (aps) stem regions . All the molecules adopt a duplex helical structure in that (i) the electrophoretic mobilities in polyacrylamide gels of the ps and aps oligomers are similar . (ii) The ps hairpins are substrates for T4 polynucleotide kinase, T4 DNA ligase, and Escherichia coli exonuclease III . (iii) Salt-dependent thermal transitions are observed for all hairpins, but the ps molecules denature 10 degrees C lower than the corresponding aps oligomers . (iv) The ultraviolet absorption and circular dichroism spectra are indicative of a base-paired duplex in the stems of the ps hairpins but differ systematically from those of the aps counterparts . (v) The bis-benzimidazole drug Hoechst-33258, which binds in the minor groove of B-DNA, exhibits very little fluorescence in the presence of the ps hairpins but a normal, enhanced emission with the aps oligonucleotides . In contrast, the intercalator ethidium bromide forms a strongly fluorescent complex with all hairpins, the intensity of which is even higher for the ps species . (vi) The pattern of chemical methylation is the same for both the ps and aps hairpins . The combined results are consistent with the prediction from force field analysis of a parallel stranded right-handed helical form of d(A)n.d(T)n with a secondary structure involving reverse Watson-Crick base pairs and a stability not significantly different from that of the B-DNA double helix . Models of the various hairpins optimized with force field calculations are described. Biochemistry, 1988 Jul 26, 27(15), 5515 - 9 Enzyme IIMtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier; Pas HH et al.; The cysteines of the membrane-bound mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine . After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified . N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity {Roossien, F . F., & Robillard, G . T . (1984) Biochemistry 23, 211-215} . NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine . Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384 . Positive identification of the activity-linked cysteine was accomplished by inactivation with {14C}iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing. Biochemistry, 1988 Jul 26, 27(15), 5778 - 85 Effect of zinc ions on tRNA structure: imino proton NMR spectroscopy; Flanagan JM et al.; The structure of tRNA in solution was explored by NMR spectroscopy to evaluate the effect of divalent cations, especially zinc, which has a profound effect on the chromatographic behaviour of tRNAs in certain systems . The divalent ions Mg2+ and Zn2+ have specific effects on the imino proton region of the 1H NMR spectrum of valine transfer RNA (tRNA(Val} of Escherichia coli and of phenylalanine transfer RNA (tRNA(Phe} of yeast . The dependence of the imino proton spectra of the two tRNAs was examined as a function of Zn2+ concentration . In both tRNAs the tertiary base pair (G-15).(C-48) was markedly affected by Zn2+ (shifted downfield possibly by as much as 0.4 ppm); this is the terminal base pair in the augmented dihydrouridine helix (D-helix) . Base pair (U-8).(A-14) in yeast tRNA(Phe) or (s4U-8).(A-14) in tRNA1(Val), which are stacked on (G-15).(C-48), was not affected by Zn2+, except when 1-2 Mg2+ ions per tRNA were also present . Another imino proton that may be affected by Zn2+ in both tRNAs is that of the tertiary base pair (G-19).(C-46) . The assignment of this resonance in yeast tRNA(Phe) is tentative since it is located in the region of highly overlapping resonances between 12.6 and 12.3 ppm . This base pair helps to anchor the D-loop to the T psi C loop.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Jul 26, 27(15), 5678 - 85 Penetration of the signal sequence of Escherichia coli PhoE protein into phospholipid model membranes leads to lipid-specific changes in signal peptide structure and alterations of lipid organization; Batenburg AM et al.; In order to obtain more insight in the initial steps of the process of protein translocation across membranes, biophysical investigations were undertaken on the lipid specificity and structural consequences of penetration of the PhoE signal peptide into lipid model membranes and on the conformation of the signal peptide adopted upon interaction with the lipids . When the monolayer technique and differential scanning calorimetry are used, a stronger penetration is observed for negatively charged lipids, significantly influenced by the physical state of the lipid but not by temperature or acyl chain unsaturation as such . Although the interaction is principally electrostatic, as indicated also by the strong penetration of N-terminal fragments into negatively charged lipid monolayers, the effect of ionic strength suggests an additional hydrophobic component . Most interestingly with regard to the mechanism of protein translocation, the molecular area of the peptide in the monolayer also shows lipid specificity: the area in the presence of PC is consistent with a looped helical orientation, whereas in the presence of cardiolipin a time-dependent conformational change is observed, most likely leading from a looped to a stretched orientation with the N-terminus directed toward the water . This is in line also with the determined peptide-lipid stoichiometry . Preliminary 31P NMR and electron microscopy data on the interaction with lipid bilayer systems indicate loss of bilayer structure. Biochemistry, 1988 Jul 26, 27(15), 5507 - 15 Propagation of allosteric changes through the catalytic-regulatory interface of Escherichia coli aspartate transcarbamylase; Xu W et al.; Each of two previously isolated strains of Escherichia coli containing a single nonsense codon within the pyrB gene was suppressed with four different nonsense suppressors . The kinetic analysis using crude extracts of these nonsense-suppressed strains indicated that the mutant aspartate transcarbamylases had altered cooperativity and affinity for aspartate as judged by the substrate concentration at half of the maximal velocity . Both pyrB genes were cloned and then sequenced . In both cases, a single base change was identified which converted a glutamine GAC codon into a TAC nonsense codon . Both mutations occurred in the catalytic chain of aspartate transcarbamylase and were identified at positions 108 and 246 . The glutamine at position 108 in the wild-type structure is located at the interface between the catalytic and regulatory chains and is involved in a number of interactions with backbone and side chains of the regulatory chain . The glutamine at position 246 in the wild-type structure is located in the 240s loop of the enzyme . Two additional mutant versions of aspartate transcarbamylase were created by site-directed mutagenesis to further investigate the 108-position in the structure, a glutamine to tyrosine substitution at position 108 of the catalytic chain, and an asparagine to glycine change at position 113 of the regulatory chain, a residue which interacts directly with glutamine-108 in the wild-type structure . Both mutant enzymes have reduced affinity for aspartate . However, the Tyr-108 mutant enzyme exhibits a reduced Hill coefficient while the Gly-113 enzyme exhibits an increased Hill coefficient . The response to the allosteric effectors ATP and CTP is also changed for both the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Jul 26, 27(15), 5499 - 506 Mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli: pH and deuterium isotope effects with NADPH as the variable substrate; Morrison JF et al.; The variations with pH of the kinetic parameters and primary deuterium isotope effects for the reaction of NADPH with dihydrofolate reductase from Escherichia coli have been determined . The aims of the investigations were to elucidate the chemical mechanism of the reaction and to obtain information about the location of the rate-limiting steps . The V and V/KNADPH profiles indicate that a single ionizing group at the active center of the enzyme must be protonated for catalysis, whereas the Ki profiles show that the binding of NADPH to the free enzyme and of ATP-ribose to the enzyme-dihydrofolate complex is pH independent . From the results of deuterium isotope effects on V/KNADPH, it is concluded that NADPH behaves as a sticky substrate . It is this stickiness that raises artificially the intrinsic pK value of 6.4 for the Asp-27 residue of the enzyme-dihydrofolate complex {Howell, E . E., Villafranca, J . E., Warren, M . S., Oatley, S . J., & Kraut, J . (1986) Science (Washington, D.C.) 231, 1123} to an observed value of 8.9 . Thus, the binary enzyme complex is largely protonated at neutral pH . The elevation of the intrinsic pK value of 6.4 for the ternary enzyme-NADPH-dihydrofolate complex to 8.5 is not due to the kinetic effects of substrates . Rather, it is the consequence of the lower, pH-independent rate of product release and the faster pH-dependent catalytic step . At neutral pH, the proportion of enzyme present as a protonated ternary enzyme-substrate complex is sufficient to keep catalysis faster than product release.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Jul 26, 27(15), 5493 - 9 Dihydrofolate reductase from Escherichia coli: the kinetic mechanism with NADPH and reduced acetylpyridine adenine dinucleotide phosphate as substrates; Stone SR et al.; Kinetic studies on the reaction catalyzed by dihydrofolate reductase from Escherichia coli have been undertaken with the aim of characterizing further the kinetic mechanism of the reaction . For this purpose, the kinetic properties of substrates were determined by measurement of (a) initial velocities over a wide range of substrate concentrations and (b) the stickiness of substrates in ternary enzyme complexes . Stickiness is defined as the rate at which a substrate reacts to give products relative to the rate at which that substrate dissociates . Stickiness was determined by varying the viscosity of reaction mixtures and the concentration of one substrate in the presence of a saturating concentration of the other substrate . The results indicate that NADPH is sticky in the enzyme-NADPH-dihydrofolate complex, while dihydrofolate is much less sticky in this complex . At higher concentrations, NADPH functions as an activator through the formation of an enzyme-NADPH-tetrahydrofolate from which tetrahydrofolate is released more rapidly than from an enzyme-tetrahydrofolate complex . Higher concentrations of dihydrofolate also cause enzyme activation, and it appears that this effect is due to the ability of dihydrofolate to displace tetrahydrofolate from a binary enzyme complex through the formation of a transitory enzyme-tetrahydrofolate-dihydrofolate complex . As NADPH and dihydrofolate function as activators and as NADPH behaves as a sticky substrate, the kinetic mechanism of the dihydrofolate reductase reaction with the natural substrates is steady-state random . By contrast with NADPH, reduced 3-acetylpyridine adenine dinucleotide phosphate exhibits only slight stickiness and does not function as an activator.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Jul 26, 27(15), 5628 - 35 Effects of Escherichia coli Nus A protein on transcription termination in vitro are not increased or decreased by DNA sequences sufficient for antitermination in vivo; Sigmund CD et al.; The ability of Escherichia coli Nus A protein to recognize specific DNA or RNA sequences in vitro was tested by using transcription templates containing a variety of promoters, transcription terminators, and antitermination-conferring regions . We conclude that the effects of Nus A on termination are not qualitatively or quantitatively altered by sequences present in promoters, Rho-dependent terminators, or antitermination-conferring regions . Nus A was also shown to increase termination at the rrnC Rho-independent T1 terminator by a mechanism that is independent of the promoter or sequences involved in antitermination . Altogether, these observations argue against a direct Nus A-nucleic acid interaction affecting termination in vitro . Together with the results described in the accompanying paper {Sigmund, C . D., & Morgan, E . A . (1988) Biochemistry (preceding paper in this issue)}, these results suggest that the effects of Nus A on termination in vitro may not be related to the in vivo functions of Nus A. Biochemistry, 1988 Jul 26, 27(15), 5622 - 7 Nus A protein affects transcriptional pausing and termination in vitro by binding to different sites on the transcription complex; Sigmund CD et al.; We examined the in vitro concentration dependence of the effects of Nus A on transcription termination and pausing to determine if Nus A affects both pausing and termination in vitro by binding to a single site on the transcription complex . Nus A was shown to cause maximal increases of pausing at a concentration approximately equimolar to RNA polymerase . However, the effects of Nus A on termination require much higher Nus A concentrations than are required for pausing . It is therefore likely that the effects of Nus A on pausing and termination result from the binding of Nus A to different sites on the transcription complex . Since proteins that probably bind RNA nonspecifically were also shown to strongly reduce termination at a Rho-dependent terminator, Nus A may decrease Rho-dependent termination by binding nonspecifically to RNA . This proposal is consistent with most of the available data on the in vitro effects of Nus A and provides a mechanistic basis for previously unexplained details of Nus A caused decreases in Rho-dependent termination . We further speculate that most or all of the in vivo roles of Nus A may involve the enhancement of pausing. Biochemistry, 1988 Jul 26, 27(15), 5544 - 52 Mechanism of adenylate kinase . Histidine-36 is not directly involved in catalysis, but protects cysteine-25 and stabilizes the tertiary structure; Tian GC et al.; Several previous reports on muscle adenylate kinase (AK) have suggested that histidine-36 (His-36) is located in the binding site of adenosine 5'-triphosphate (ATP) and is involved in catalysis . We have tested the role of His-36 using site-specific mutagenesis on chicken muscle AK expressed in Escherichia coli . Three mutant proteins (H36Q, H36N, and H36G) were obtained by substituting His-36 with glutamine, asparagine, and glycine, respectively . Steady-state kinetic studies showed that the mutants have similar kinetic properties to those of the wild-type (WT) AK, which suggested that His-36 is not directly involved in catalysis . However, His-36 is likely to interact with or protect cysteine-25 (Cys-25) on the basis of the following evidence: The crystal structure of porcine muscle AK revealed a close proximity between His-36 and Cys-25; the mutants were unstable during purification (the order of stability was WT greater than H36Q greater than H36N greater than H36G); the H36G mutant readily dimerized; the sulfhydryl groups of mutants became more reactive (WT less than H36Q less than H36N) toward 5,5'-dithiobis(2-nitrobenzoic acid) . Furthermore, His-36 was found to stabilize the tertiary structure of AK on the basis of guanidine hydrochloride induced denaturation studies, which showed that the conformational stability decreases in the order WT greater than H36Q greater than H36N.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Jul 26, 27(15), 5477 - 85 Heme pocket interactions in cytochrome c peroxidase studied by site-directed mutagenesis and resonance Raman spectroscopy; Smulevich G et al.; Resonance Raman spectra are reported for FeII and FeIII forms of cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis and cloning in Escherichia coli . These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn, Trp-191----Phe) and distal (Trp-51----Phe, Arg-48----Leu and Lys) side of the heme . These spectra are used to assess the spin and ligation states of the heme, via the porphyrin marker band frequencies, especially v3, near 1500 cm-1, and, for the FeII forms, the status of the Fe-proximal histidine bond via its stretching frequency . The FeII-His frequency is elevated to approximately 240 cm-1 in CCP(MI) and in all of the distal mutants, due to hydrogen-bonding interactions between the proximal His-175 N delta and the carboxylate acceptor group on Asp-235 . The FeII-His RR band has two components, at 233 and 246 cm-1, which are suggested to arise from populations having H-bonded and deprotonated imidazole; these can be viewed in terms of a double-well potential involving proton transfer coupled to protein conformation . The populations shift with changing pH, possibly reflecting structure changes associated with protonation of key histidine residues, and are influenced by the Leu-48 and Phe-191 mutations . A low-spin FeII form is seen at high pH for the Lys-48, Leu-48, Phe-191, and Phe-51 mutants; for the last three species, coordination of the distal His-52 is suggested by a approximately 200-cm-1 RR band assignable to Fe(imidazole)2 stretching.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Jul 26, 27(15), 5755 - 62 Structure and function of the sigma-70 subunit of Escherichia coli RNA polymerase . Monoclonal antibodies: localization of epitopes by peptide mapping and effects on transcription; Strickland MS et al.; Murine monoclonal antibodies reactive with the major sigma subunit (sigma-70) of Escherichia coli RNA polymerase were obtained by standard hybridoma techniques . Western blot analyses established that seven antibodies had unique specificities after various chemical and enzymatic methods were used to fragment sigma . Peptides were purified by HPLC using size-exclusion, reverse-phase, or ion-exchange chromatography . The epitopes for six of these antibodies have been localized to specific peptides . These peptides were further characterized by amino acid composition and N-terminal sequencing . Sigma, which has a molecular weight of 70.2K, runs as 83K on SDS gels in this study . This anomalous behavior has been localized to the very acidic N-terminal half of the molecule . One antibody is unable to bind to native sigma . Two others do not bind well to sigma when it is contained in holoenzyme, indicating that their epitopes are in regions of sigma which are inaccessible in the holoenzyme complex . All three of these antibodies fail to inhibit in vitro transcription by holoenzyme . The other four antibodies all can inhibit in vitro transcription. Nucleic Acids Res, 1988 Jul 25, 16(14B), 6897 - 913 Ribosomal protein S14 genes in broad bean mitochondrial DNA; Wahleithner JA et al.; Broad bean (Vicia faba) mtDNA contains an open reading frame with a predicted amino acid sequence that is 41% homologous to the ribosomal protein S14 (RPS14) of Escherichia coli, and which is located 1232 ntp upstream from a gene for cytochrome b (cob) . A second putative rpS14 gene occurs in broad bean mtDNA, 344 ntp upstream from a gene for ATPase subunit 9 (atp9) . However, the atp9-linked rpS14 gene is 12 codons shorter than the cob-linked rpS14 gene . Sequence homology is found upstream (for 218 ntp) but not downstream from the two rpS14 genes . Transcripts were detected in broad bean mtRNA only for the cob-linked rpS14 gene . All RNA molecules that include a transcript of the rpS14 gene also include a transcript of the cob gene . Sequences homologous to the broad bean mitochondrial rpS14 gene were detected in soybean mtDNA, but not in corn mtDNA . Relationships between the amino acid sequences of RPS14s encoded in broad bean mtDNA, in chloroplast DNAs of various angiosperms, and in E . coli are consistent with the view that the ancestral lines of these three kinds of DNA diverged from each other within a relatively short time period. Nucleic Acids Res, 1988 Jul 25, 16(14B), 6825 - 37 Identification of regions essential for extrachromosomal replication and maintenance of an endogenous plasmid in Dictyostelium; Ahern KG et al.; Initial experiments with the endogenous 12.3 kb Dictyostelium discoideum plasmid Ddp1 led to the generation of a large shuttle vector, Ddp1-20 . In addition to Ddp1, this vector contains pBR322 and a gene fusion that confers G418 resistance in Dictyostelium cells . We have shown that Ddp1-20 replicates extrachromosomally in Dictyostelium cells and can be grown in Escherichia coli cells (1) . We have now examined deletions within this vector to identify the elements essential for extrachromosomal replication and stable maintenance of the plasmid . We find that a 2.2 kb fragment is sufficient to confer stable, extrachromosomal replication with a reduction in copy number from about 40 to approximately 10-15 copies per cell . Vectors containing additional Ddp1 sequences have a higher copy number . The 2.2 kb region contains none of the complete, previously identified transcription units on Ddp1 expressed during vegetative growth or development . These results suggest that gene products expressed by Ddp1 are not essential for replication, stability, or partitioning of the plasmid between daughter cells . Vectors carrying only the 2.2 kb fragment plus the gene fusion conferring G418 resistance transform Dictyostelium cells with high efficiency using either calcium phosphate mediated transformation or electroporation . Finally, we have examined the relative levels of expression of actin promoters driving neoR genes when in extrachromosomal or integrating vectors. J Biol Chem, 1988 Jul 25, 263(21), 10304 - 13 Kinetics of the formation and isomerization of methotrexate complexes of recombinant human dihydrofolate reductase; Appleman JR et al.; The kinetics of inhibitor binding to highly purified recombinant human dihydrofolate reductase (rHDHFR) have been examined . Methotrexate (MTX) binds rapidly (kon = 1.0 x 10(8) M-1 s-1) and tightly (koff/kon = 210 pM) to the preformed complex of rHDHFR with NADPH . The initial association reaction between rHDHFR.NADPH an