Mol Microbiol, 1992 Mar, 6(6), 735 - 42 The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein (PAL); Lazzaroni JC et al.; The excC mutants of Escherichia coli are hypersensitive to drugs such as cholic acid and release periplasmic proteins into the extracellular medium . A 1884 bp fragment carrying the excC gene was isolated and sequenced . It contains the 3' end of the tolB gene which maps at min 17 on the E . coli map and an open reading frame which encodes the 18,748 Da ExcC protein . The protein is composed of a hydrophobic region of 22 residues and displayed an overall hydrophilic configuration . It was shown that the ExcC protein is indeed the PAL (peptidoglycan-associated lipoprotein) described by Mizuno (1979) . The pal gene had not yet been characterized on the E . coli linkage map since no obvious phenotype could be identified for mutations in this gene . A topologic analysis of the PAL protein using PAL-PhoA translational fusions showed that PAL is associated with the outer membrane only by its N-terminal moiety . The carboxy-terminal part of the protein is necessary for correct interaction of PAL with the peptidoglycan layer. Mol Microbiol, 1992 Mar, 6(6), 715 - 22 An amino-proximal domain required for the localization of FtsQ in the cytoplasmic membrane, and for its biological function in Escherichia coli; Dopazo A et al.; The location of FtsQ, an Escherichia coli protein essential for cell division, is, under physiological conditions, in the cytoplasmic membrane facing towards the periplasmic space . An amino-proximal hydrophobic domain is required for FtsQ to reach its location and for its activity in the cell . Overexpression of modified forms of FtsQ is deleterious for the cell. Mol Microbiol, 1992 Mar, 6(6), 683 - 8 Elongation factor Tu: a molecular switch in protein biosynthesis; Weijland A et al.; Elongation factor Tu (EF-Tu), the most abundant protein in Escherichia coli, is a guanine nucleotide-binding protein that in the 'on' state acts as a carrier of amino acyl-tRNA to the ribosome . Our knowledge of this essential component of translation has brought substantial progress in the past decade thanks to the co-ordinated application of biochemical, physico-chemical and genetic methods . Crystallographic analysis at 2.6 A resolution and site-directed mutagenesis have revealed structural and functional similarities between the guanine nucleotide-binding domains of EF-Tu and human H-ras p21 protein . The regulation of the expression of the two EF-Tu-encoding genes in E . coli, particularly that of tufB, has been shown to involve diverse mechanisms . Several aspects of the functions of EF-Tu in the elongation cycle have been reinvestigated, leading to new insights . These studies have emphasized the manifold aspects of the mechanisms regulating the activity of EF-Tu in the bacterial cell. Biotechniques, 1992 Mar, 12(3), 430 - 4 Large-scale purification of plasmid DNA by anion-exchange high-performance liquid chromatography; Hines RN et al.; Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA . Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today . In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E . coli cell lysates . Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run . The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E . coli transformation and DNA-mediated gene transfection of eukaryotic cells. Biotechniques, 1992 Mar, 12(3), 320 - 3 A rapid and simple chemiluminescent assay for Escherichia coli beta-galactosidase; Beale EG et al.; We describe a chemiluminescent assay for E . coli beta-galactosidase using Lumi-Gal 530, a commercial formulation containing a stable phenylgalactose-substituted dioxetane as the substrate . Removal of the galactose moiety leads to the generation of an unstable dioxetane which decomposes to provide the observed chemiluminescence which is measured with a luminometer . Advantages of the assay are that it is simple, inexpensive and has 20-fold greater sensitivity than the standard spectrophotometric assay . Additional advantages are that the dioxetane is quite stable in the commercial formulation, and beta-galactosidase functions efficiently and is not degraded during the course of an assay . As luminometers are becoming commonplace in molecular biology laboratories, this assay provides a preferable alternative to the spectrophotometric assay. J Dairy Sci, 1992 Mar, 75(3), 739 - 46 High cortisol concentrations and mediation of the hypogalactia during endotoxin-induced mastitis; Shuster DE et al.; A series of experiments was conducted to define the role of cortisol in the hypogalactia during endotoxin-induced mastitis . In the first experiment, three of six nonmastitic cows were given a continuous infusion of trilostane, a 3 beta-hydroxysteroid dehydrogenase inhibitor that blocks enhanced cortisol synthesis . Trilostane had no effect in these cows . In the second experiment, six midlactation cows were given 10 micrograms of endotoxin in each of two homolateral quarters to induce mastitis . Three of these cows also received trilostane . Increased serum cortisol following endotoxin infusion was blocked by trilostane treatment, whereas serum glucose and rectal temperatures were unaffected . Preventing the cortisol increase failed to reduce hypogalactia in endotoxin-infused or uninfused quarters . Decreases in milk production and increases in measures of mammary inflammation were greater in trilostane-treated cows, indicating that endogenous cortisol may moderate the cow's inflammatory response . In the third experiment, three of six nonmastitic cows were injected intramuscularly with 150 IU of ACTH . Serum cortisol concentration exceeded 70 ng/ml for at least 3 h in cows receiving ACTH . This cortisol concentration, comparable with concentrations found during endotoxin mastitis, did not inhibit milk production . Together, these data demonstrate that the acute cortisol increase does not mediate the hypogalactia associated with endotoxin-induced mastitis. Ann R Coll Surg Engl, 1992 Mar, 74(2), 119 - 23; discussion 123-5 Chronic pancreatitis with biliary obstruction; Huizinga WK et al.; In a 4-year review of 509 patients with chronic pancreatitis, the incidence of clinically manifest fixed common bile duct (CBD) stenosis was 9% (45 patients) . In 76% this was alcohol related, and pancreatic calcification was present in 51% . All patients presented with unrelenting jaundice and five (11%) had cholangitis . The mean serum bilirubin (165 +/- 108, normal 0-17 mumol/l), alkaline phosphatase (1790 +/- 1143, normal 73-207 U/l) and gamma glutamyl transferase (798 +/- 660, normal 7-64 U/l) were markedly raised . Diabetes occurred in 8 (18%) . A biliary drainage operation was performed in 43 patients and 11 had concomitant pancreaticojejunostomy . Endoscopic retrograde cholangiopancreatography (ECRP) provided valuable information preoperatively in outlining both biliary and pancreatic disease in selecting patients for dual ductal drainage . Minor complications not related to biliary anastomosis occurred in 14% . Four patients died (9%), two from pseudocyst-related haemorrhage . Jaundice was successfully relieved in all and did not recur during follow-up . No secondary biliary cirrhosis was encountered, but varying degrees of portal fibrosis were present in 75% of liver biopsies . The commonest biliary pathogen was E . coli . It is recommended that a biliary bypass operation be performed when the diagnosis is radiologically confirmed and no improvement occurs within 1 month. Mol Biochem Parasitol, 1992 Mar, 51(1), 17 - 27 Characterisation of the gene encoding a candidate vaccine antigen of Theileria parva sporozoites; Nene V et al.; We have cloned and characterised the gene encoding the 67-kilodalton stage-specific surface antigen, p67, of Theileria parva (Muguga) sporozoites . The gene which is present in a single copy, is divided into 2 exons by an intron 29 bp long and is transcribed into mRNA of about 2500 nucleotides . The gene is present in all stocks of T . parva and there is a related gene in Theileria annulata . The deduced amino acid sequence of 709 residues predicts that p67 is a membrane protein and that it lacks tandemly repeated sequences . Recombinant p67 has been expressed in Escherichia coli as a fusion protein with Sj-26, a glutathione-S-transferase of Schistosoma japonicum . Antibodies to purified recombinant proteins containing residues 9-316 or 397-709 of p67 bind to p67 in immunoblots and neutralise sporozoite infectivity in vitro . Recombinant p67 is, therefore, a candidate antigen for development of an anti-sporozoite vaccine for East Coast fever in cattle. Curr Genet, 1992 Mar, 21(3), 249 - 54 Transcripts and translation products of a mitochondrial plasmid of Claviceps purpurea; Gessner-Ulrich K et al.; Expression of the linear mitochondrial (mt) plasmid pClK1 of strain K of the ascomycete Claviceps purpurea was studied at the RNA and protein level . Using strand-specific probes two major transcripts were detected, corresponding to ORF1 and ORF2 of the plasmid, which most likely code for a DNA-polymerase and an RNA-polymerase, respectively . Primer extension experiments showed that both transcripts start at identical positions from opposite sides within the terminal inverted repeat (TIR) . The initiation sequence corresponds to the proposed general mt initiation consensus-sequence . Conserved parts of the putative polymerase ORFs were expressed in an E . coli system and used to prepare antisera . In Western experiments the presence of corresponding proteins was demonstrated in a strain carrying plasmid pClK1, whereas a plasmid-free strain lacked these polypeptides . The sizes of these proteins are in good accordance with the sizes derived from the coding capacity of ORF1 and 2. J Steroid Biochem Mol Biol, 1992 Mar, 41(3-8), 249 - 72 DNA-binding by the glucocorticoid receptor: a structural and functional analysis; Dahlman-Wright K et al.; The glucocorticoid receptor belongs to a family of ligand activated nuclear receptors . This family includes, in addition to the receptors for steroid hormones, receptors for thyroid hormone, retinoic acid and 1,25-dihydroxy vitamin D3 as well as some receptors with as yet unknown ligands . The glucocorticoid receptor DNA-binding domain has been expressed in E . coli . The purified protein binds to the same DNA sequences as the native receptor and is therefore suitable for biochemical and structural studies of the DNA-binding function of the receptor protein . This protein has been shown to bind as a dimer to its DNA-binding site . Protein-protein interactions facilitate DNA-binding and a segment responsible for these interactions has been identified close to the C-terminal zinc-binding site . The family of nuclear receptors, with their related DNA-binding sites, provides an opportunity to study determinants for DNA sequence recognition . A segment close to the N-terminal zinc ion has been shown to be responsible for the target specificity of glucocorticoid and estrogen receptors . DNA-binding domains of nuclear receptors include nine conserved cysteine residues which have been shown to coordinate two zinc ions and zinc has been shown to be required for the structural integrity and DNA-binding ability of the glucocorticoid receptor DNA-binding domain . A motif for DNA recognition, based around zinc ions, was first described for transcription factor IIIA and nuclear receptors were believed to recognize DNA via a similar motif . However, the three-dimensional structure determination of the glucocorticoid receptor DNA-binding domain shows that its structure is clearly different from that of the TFIIIA type zinc-binding domains. Poult Sci, 1992 Mar, 71(3), 440 - 7 Restricted feeding and broiler performance: age at initiation and length of restriction; Ballay M et al.; Broiler cockerels were provided either ad libitum access or restricted to alternate-day access to feed initiated at different ages and lasting for different durations . Regimens consisted of ad libitum feeding; alternate-day feeding; alternate-day feeding from 0 to 6, 6 to 12, or 12 to 18 days of age (DOA); alternate-day feeding from 0 to 12, 0 to 6, and 12 to 18, or 6 to 18 DOA; and alternate-day feeding from 0 to 18 DOA . By 39 DOA, chicks restricted for only one 6-day period reached body weights equivalent to those of chicks eating ad libitum . Body weights of chicks restricted for more than 6 days during the first 18 days after hatch were lower than those of chicks eating ad libitum at 39 DOA . Restriction for more than 6 days improved feed efficiency . Chicks restricted for the whole experiment had superior feed efficiency but low body weights . Antibody responses to sheep erythrocyte antigen were similar among feeding regimens . Response to Escherichia coli inoculation was generally less severe in chicks provided restricted access to feed, and overall mortality was highest in chicks eating ad libitum . Feeding regimens had little effect on organ weights relative to body weight, amount of abdominal fat (either on an absolute basis or relative to body weight), or percentage lipid in the abdominal fat pad. Am J Trop Med Hyg, 1992 Mar, 46(3), 307 - 13 Antibodies to a recombinant glutamate-rich Plasmodium falciparum protein: evidence for protection of individuals living in a holoendemic area of Liberia; Hogh B et al.; A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli . The encoded 783 amino acid residues contain two areas of repeated amino acid sequences . Antibodies against recombinant GLURP489-1271, as well as against a synthetic peptide corresponding to GLURP899-916, and against a synthetic peptide representing the major glutamate rich repeat sequence from the P . falciparum ring erythrocyte surface antigen (Pf155/RESA) (EENV)6 were examined in 423 individuals (age range 30 days-78 years) living in a malaria holoendemic area of Liberia . In the 5-9-year-old age group, subjects with anti-GLURP489-1271 antibody concentrations greater than the mean value of the group had lower parasite densities than those with low antibody concentrations (P = 0.0151) . High levels of anti-GLURP899-916 antibodies did not correlate with low parasite densities . However, high levels of anti-(EENV)6 antibodies were associated with significantly lower parasite densities in the 2-4-year-old age group (P = 0.0189) . There was no correlation between the anti-GLURP489-1271 and anti-(EENV)6 antibody responses . The data provide indirect evidence for a protective role of antibodies reacting with recombinant GLURP489-1271 as well as with the synthetic peptide (EENV)6 from the Pf155/RESA. Am J Physiol, 1992 Mar, 262(3 Pt 2), F354 - 60 Anoxia induces phospholipase A2 activation in rabbit renal proximal tubules; Portilla D et al.; Phospholipase A2 (PLA2) activation during anoxic cell injury was determined by use of a variety of approaches in rabbit proximal renal tubules . Arachidonic acid (AA) mass release increased from 4 +/- 1 (normoxia control) to 40 +/- 6 ng/mg protein after 20 min and 106 +/- 16 ng/mg protein after 40 min of anoxia . PLA2 activity was measured by estimating the amount of sn-2 fatty acid released from either 14C-labeled Escherichia coli membranes or {14C}phosphatidylethanolamine (PE) micelles incubated with membrane and cytosolic fractions obtained from normoxic or anoxic tubules . At pH 7.4 and 1 mM Ca, PLA2 activity increased in the 20-min anoxic membrane fractions from 8.1 +/- 2.3 (normoxic) to 15.2 +/- 2.1 pmol.min-1.mg protein-1 (anoxic) . When the proximal tubules were homogenized in the absence of Ca, the anoxia-induced PLA2 activity was found to be soluble . Preincubation with pancreatic PLA2 antibody inhibited 50% of both basal and anoxia-stimulated PLA2 activity . Two protein bands (40- and 21-kDa species) immunoreactive to PLA2 antibody were detected in the membrane fraction . A sixfold increase in the immunoreactivity of the 40-kDa band was detected after 40 min of anoxia of proximal tubules . These results suggest that anoxia induces an intracellular PLA2 activity in kidney cells that could be immunologically related to pancreatic PLA2. Mol Immunol, 1992 Mar, 29(3), 371 - 8 An HLA-A2/beta 2-microglobulin/peptide complex assembled from subunits expressed separately in Escherichia coli; Parker KC et al.; The human class I histocompatibility antigen HLA-A2 has been assembled from subunits expressed separately in E . coli . A peptide that is known to be recognized by human cytotoxic T lymphocytes (CTLs) in association with HLA-A2 is a necessary component of the reconstitution mixture . The N-terminal extracellular fragment of the HLA-A2 heavy chain is initially synthesised as an insoluble aggregate . The aggregate is solubilized in denaturant, mixed with the influenza nucleoprotein 85-94 decapeptide (NP peptide), and diluted into a solution containing human beta 2-microglobulin (beta 2 m) isolated from the E . coli periplasm . The HLA-A2 heavy chain becomes soluble in physiological solutions if both beta 2m and the NP peptide are present . The reconstituted HLA-A2 complex is recognised by a monoclonal antibody that is specific for the native HLA-A2/beta 2m heterodimer, and is also recognised by a monoclonal antibody that recognises beta 2m . When other peptides known from CTL studies to associate with HLA-A2 are used, a significantly lower yield of reconstituted complex is obtained . The isoelectric point of the reconstituted complex depends on which peptide is used, confirming that the peptide is a component of the reconstituted complex. Mol Gen Genet, 1992 Mar, 232(2), 295 - 303 Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC); Coleman J; An Escherichia coli strain deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity has previously been isolated, and the gene (plsC) has been shown to map near min 65 on the chromosome . I precisely mapped the location of plsC on the chromosome, and determined its DNA sequence . plsC is located between parC and sufI, and is separated from sufI by 74 bp . Upstream of plsC is parC, separated by 233 bp, which includes an active promoter . parC, plsC, and sufI are all transcribed in the counterclockwise direction on the chromosome, possibly in an operon with multiple promoters . The amino-terminal sequence of the partially purified protein, combined with the DNA sequence, reveal 1-acyl-sn-glycerol-3-phosphate acyltransferase to be a 27.5 kDa highly basic protein . The plsC gene product, 1-acyl-sn-glycerol-3-phosphate acyltransferase, is localized to the cytoplasmic membrane of the cell . The amino-terminal sequence of the purified protein reveals the first amino acid to be a blocked methionine residue, most probably a formyl-methionine . The amino acid sequence of 1-acyl-sn-glycerol-3-phosphate acyltransferase has a short region of homology to two other E . coli acyltransferases that utilize acyl-acyl carrier protein as the acyl donor, sn-glycerol-3-phosphate acyltransferase and UDP-N-acetyl-glucosamine acyltransferase (involved in lipid A biosynthesis). Mol Gen Genet, 1992 Mar, 232(2), 199 - 205 Intramolecular homologous recombination of linearized plasmids in Escherichia coli K12; Chua KL et al.; The efficacy of linear DNA as a substrate for general homologous recombination was demonstrated using BamHI-linearized pKLC8.5, a plasmid that carries internal direct repeats flanking the unique BamHI site . An analogous plasmid, pKLC2.31, was used in a parallel and comparative study of intramolecular homologous recombination in circular DNA substrates . When the rec+ wild-type strain, AB1157, and its isogenic rec- derivatives were transformed with linear pKLC8.5 DNA, intramolecular homologous recombination was independent of recA, recB, recN, recO and exonuclease III (xth-1) functions . Although the recBCsbcA and recBCsbcBC cells were both very recombination proficient, only linear but not circular DNA was used as substrate for intramolecular homologous recombination in the recBCsbcA cells . In both the recBCsbcA and recBCsbcBC genetic backgrounds, the recombination frequencies for linearized pKLC8.5 DNA were 100% . A notable difference between the two strains was that none of the recBCsbcA transformants obtained with circular pKLC8.5 DNA were Tcs recombinants, whereas 11% of the corresponding recBCsbcBC transformants were Tcs recombinants . The sbcB mutation was responsible for the recombination proficiency of the recBCsbcBC cells . Unlike the case in recBCsbcA cells, intramolecular homologous recombination of linear DNA in the recBCsbcBC cells was dependent on recA and recF as well as recN and recO gene functions, but was independent of recJ and recL gene functions. J Ky Med Assoc, 1992 Mar, 90(3), 117 - 9 Respiratory complications of renal infection; Miller JD et al.; We present a case report of pleural effusion and hypoxia developing in a nonpregnant woman with a renal abscess . Our review of the literature reveals that the other reported cases of respiratory complications of renal infections have all occurred in pregnant women. Mol Gen Genet, 1992 Mar, 232(1), 40 - 8 Topological and functional studies on HlyB of Escherichia coli; Gentschev I et al.; The topology of HlyB, a protein located in the inner membrane of Escherichia coli and involved in the secretion of alpha-haemolysin (HlyA), was determined by the generation of HlyB-PhoA and HlyB-LacZ fusion proteins . The data obtained by this biochemical method together with computer predictions suggest that HlyB is inserted in the cytoplasmic membrane by six stable hydrophobic, alpha-helical transmembrane segments . These segments extend from amino acid positions 158 to 432 of HlyB . The cytoplasmic loops between these transmembrane segments are relatively large and carry an excess of positively charged amino acids, while the periplasmic loops are rather small . In addition to these six transmembrane segments, two additional regions in the 78 N-terminal amino acids of HlyB appear to be also inserted in the cytoplasmic membrane . However, the association of these two segments with the cytoplasmic membrane seems to be less tight, since active PhoA and LacZ fusions were obtained by insertion into the same positions of these segments . A LacZ-HlyAs, fusion protein carrying, at the C-terminus of LacZ, the 60-amino acid signal sequence of HlyA was not secreted in the presence of HlyB/HlyD . However, transport of this fusion protein into the cytoplasmic membrane appeared to be initiated, as suggested by the tight association of this protein with the inner membrane . A similar close association of LacZ-HlyAs with the inner membrane was also observed in the presence of HlyB alone but not in its absence . These data suggest that HlyB recognizes the HlyA signal sequence and initiates the transport of HlyA into the membrane. Mol Gen Genet, 1992 Mar, 232(1), 1 - 6 Dissection of functional domains in Escherichia coli DNA photolyase by linker-insertion mutagenesis; Yamamoto K; The phr gene, which encodes protein of 472 amino acid residues, is required for light-dependent photoreactivation and enhances light-independent excision repair of ultraviolet light (UV)-induced DNA damage . In this study, dodecamer HindIII linker insertions were introduced into the cloned phr gene and the functional effects of the resulting mutations on photoreactivation and light-independent dark repair in vivo were studied . Among 22 mutants obtained, 7 showed no photoreactivation as well as no enhancement of light-independent repair . Four of these were located in amino acid residues between Gln333 and Leu371 near the 3' end of the gene, two were located in a small region at Glu275 to Glu280 near the middle of the gene and the remaining one was between Pro49 and Arg50 . Three mutants that had insertions located in the 42 bp segment from 399 to 441 bp of the phr coding sequence (corresponding to amino acid residues Ile134 to Lys149) lost the light-independent repair effect but retained photoreactivation . These results suggest that (i) Escherichia coli DNA photolyase contains several critical sites that are distributed over much of the enzyme molecule, and (ii) a functional domain required for the effect on light-independent repair is at least in part distinct from that necessary for light-dependent photoreactivation. Mol Microbiol, 1992 Mar, 6(5), 621 - 7 Involvement of FtsZ in coupling of nucleoid separation with septation; Tetart F et al.; The cell-cycle parameters of an Escherichia coli strain expressing essential division gene ftsZ at one-fifth of its normal level, because of antisense regulation by DicF RNA, have been analysed . Inhibition of FtsZ expression affects neither the generation time nor the replication initiation mass, the C period, or the constriction period, but it does dramatically retard the initiation of constriction relative to replication termination . Separation of the nucleoids is equally postponed, indicating that division is not coupled to termination of replication, but to partitioning . The severe inhibition of nucleoid separation by DicF RNA, and its suppression by overproduction of FtsZ, suggest a role for FtsZ in the control of separation, and consequently in the coupling of separation and division . We suggest that the normal pattern of nucleoid separation previously found in cells deficient in ftsZ function was a consequence of the loss of a negative effect exerted by FtsZ on separation . In agreement with this view, we find that nucleoid separation is temporarily inhibited after arrest of FtsZ synthesis, but is later resumed as FtsZ is further diluted into the elongating filaments. Mol Microbiol, 1992 Mar, 6(5), 605 - 13 Zygotic induction of plasmid ssb and psiB genes following conjugative transfer of Incl1 plasmid Collb-P9; Jones AL et al.; The Incl1 conjugative plasmid Collb-P9 carries a psiB gene that prevents induction of the SOS response in host bacteria . This locus is located 2.5 kb downstream of the ssb (single-stranded DNA-binding protein) gene in the leading region . This portion of Collb is strikingly similar to part of the leading region of the otherwise distinct F plasmid . Expression of psiB and ssb is increased when the host cell is exposed to an SOS-inducing treatment or the Collb transfer system is derepressed . Moreover, expression of both genes on a derepressed plasmid is strongly enhanced in conjugatively infected recipient cells . Carriage of the psiB gene by Collb is shown to prevent a low level of SOS induction following conjugation . Plasmid ssb and psiB genes may function to promote installation of the replicon in the new cell. Mol Microbiol, 1992 Mar, 6(5), 581 - 9 Effects of template topology on RNA polymerase pausing during in vitro transcription of the Escherichia coli rrnB leader region; Krohn M et al.; Transcription elongation catalysed by DNA-dependent RNA polymerase does not occur at a constant rate . Instead, during the transcription of many genes pausing occurs at defined template positions . Pausing is known to be influenced by the intracellular NTP concentration, the secondary structure of the growing transcript or by transcription factors like NusA . We have investigated the effects of the template topology of transcriptional pauses in the presence and absence on purified NusA protein . Taking advantage of a method for quantifying transcriptional pauses we have studied pausing behaviour during in vitro transcription of the early region of a plasmid-encoded ribosomal RNA operon . Plasmid templates with different superhelical densities (sigma between +0.0017 and -0.055) were employed in transcription elongation assays . If linearized or relaxed templates are used, some of the characteristic pauses can no longer be detected . For the stronger pauses we could demonstrate a direct correlation between pause strength and the negative superhelical densities of the templates used . This correlation is observed regardless of whether or not pauses are dependent upon NusA . Changes in the average transcription elongation rate, caused by variations in the NTP concentration or the temperature, do not appear to have a comparable effect on transcription pausing . The results are consistent with the assumption that the template topology has a regulatory function in transcription elongation of rRNA genes in Escherichia coli. Mol Microbiol, 1992 Mar, 6(5), 569 - 72 Z-DNA as a probe for localized supercoiling in vivo; Rahmouni AR; Biological processes such as transcription are expected to generate local variations in DNA supercoiling . The existence of localized supercoiling was recently demonstrated in Escherichia coli by using the supercoil-driven B-to-Z transition as a superhelicity probe . This new methodology is described and its extension to other biological systems discussed. J Clin Microbiol, 1992 Mar, 30(3), 732 - 4 Development of an immunoassay using recombinant maltose-binding protein-STa fusions for quantitating antibody responses against STa, the heat-stable enterotoxin of Escherichia coli; Aitken R et al.; A set of fusion proteins containing heat-stable enterotoxin (STa) and maltose-binding protein were engineered . These molecules were readily purified and used as solid-phase antigens in an enzyme-linked immunosorbent assay to monitor anti-STa responses in mice immunized with a recombinant vaccine composed of STa and the B subunit of heat-labile enterotoxin. Gene, 1992 Mar 1, 112(1), 67 - 76 Phenobarbital and sulfonylurea-inducible operons encoding herbicide metabolizing cytochromes P-450 in Streptomyces griseolus; Patel NV et al.; We have identified the promoters for two inducible genes, in Streptomyces griseolus, that encode herbicide-metabolizing cytochromes P-450 . They are in the class of promoters that have -35 and -10 sequences similar to those used in Escherichia coli by RNA polymerase E sigma 70 . Transcription from either promoter was shown to be induced by sulfonylurea (chlorimuron ethyl) or phenobarbital . Mapping of mRNA showed that each cytochrome P450-encoding gene was transcribed on a separate multicistronic mRNA that encodes cytochrome P-450 (suaC or subC), ferredoxin (suaB or subB) and at least one other open reading frame . An inducible, site-specific DNA-binding activity was identified that bound to two similar 8-bp inverted repeat sequences within or near the sua promoter (suaP) . A noninducible DNA-binding activity, distinct from that which bound to suaP, was found that bound to an 11-bp inverted repeat at the sub transcription start point. Gene, 1992 Mar 1, 112(1), 29 - 35 The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion; Barnes WM; KlenTaq DNA polymerase is an N-terminally truncated Thermus aquaticus (Taq) DNA polymerase I . As expressed from a gene construct in Escherichia coli, translation initiates at Met236, bypassing the 5'----3' exonuclease domain of the DNA polymerase-encoding gene . A sensitive forward mutation assay was used to measure the relative number of mutations introduced into the entire lacZ gene by the polymerase chain reaction (PCR) under various conditions which allow the amplification of such a large DNA span . Two selectable markers, one at each end of the test lacZ fragment, were employed to avoid the plating and scoring of PCR artefacts such as primer initiation in the midst of the lacZ gene, and cloning artefacts such as empty vector plasmid . The measured relative mutation rate was twofold lower for KlenTaq as compared to the full-length Taq DNA polymerase. Genetics, 1992 Mar, 130(3), 451 - 60 Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus; Plessis A et al.; The Saccharomyces cerevisiae mitochondrial endonuclease I-SceI creates a double-strand break as the initiating step in the gene conversional transfer of the omega+ intron to omega- DNA . We have expressed a galactose-inducible synthetic I-SceI gene in the nucleus of yeast that also carries the I-SceI recognition site on a plasmid substrate . We find that the galactose-induced I-SceI protein can be active in the nucleus and efficiently catalyze recombination . With a target plasmid containing direct repeats of the Escherichia coli lacZ gene, one copy of which is interrupted by a 24-bp cutting site, galactose induction produces both deletions and gene conversions . Both the kinetics and the proportion of deletions and gene conversions are very similar to analogous events initiated by a galactose-inducible HO endonuclease gene . We also find that, in a rad52 mutant strain, the repair of double-strand breaks initiated by I-SceI and by HO are similarly affected: the formation of deletions is reduced, but not eliminated . Altogether, these results suggest either that the two endonucleases act in the same way after double-strand break formation or that the two endonucleases are not involved in subsequent steps. Genetics, 1992 Mar, 130(3), 411 - 28 Simultaneous gain and loss of functions caused by a single amino acid substitution in the beta subunit of Escherichia coli RNA polymerase: suppression of nusA and rho mutations and conditional lethality; Sparkowski J et al.; Transcript elongation and termination in Escherichia coli is modulated, in part, by the nusA gene product, an acidic protein that interacts not only with RNA polymerase itself but also with ancillary factors, namely the host termination protein Rho and phage lambda antitermination protein, N . The E . coli nusA1 mutant fails to support lambda development due to a specific defect in N-mediated antitermination . Certain rifampicin-resistant (rifR) variants of the nusA1 host support lambda growth . We report here the isolation and pleiotropic properties of one such rifR mutant, ts8, resulting from a single amino acid substitution mutation in rpoB, the structural gene for polymerase beta subunit . ts8 is a recessive lethal mutation that blocks cell growth at 42 degrees . Pulse-labeling and analysis of newly synthesized proteins indicate that the mutant cell is proficient in RNA synthesis at high temperature . Apparently, ts8 causes a loss of some specialized function of RNA polymerase without a gross defect in general transcription activities . ts8 is an allele-specific suppressor of nusA1 . It does not suppress nusAsal, nusB5 and nusE71 mutations nor does it bypass the requirement for a functional N gene and the nut site for antitermination and lambda growth . A mutation in the N gene, punA1, that restores lambda growth in the nusA1 mutant host but not in the nusAsal host, compensates for the nusAsal allele in the ts8 mutant . This combined effect of two allele-specific suppressors suggests that they enhance some aspect of polymerase-NusA-N interaction and function . ts8 suppresses the rho15 mutation, but not the rho112 mutation, indicating that it might render RNA polymerase susceptible to the action of a defective Rho protein . Marker rescue analysis has localized ts8 to a 910-bp internal segment of rpoB that encodes the Rif domain . By amplification, cloning and sequencing of this segment of the mutant chromosome we have determined that ts8 contains Phe in place of Ser522, caused by a C to T transition . By gene conversion, we have established that the simultaneous gain and loss of three functions of polymerase is caused by this single amino acid substitution . Clearly, a site in the beta subunit critical for the functioning of both termination and antitermination factors is altered by ts8 . The alteration, we imagine, might make this site on polymerase receptive to some factors but repulsive to others. J Bacteriol, 1992 Mar, 174(6), 2032 - 8 Localization of diphtheria toxin nuclease activity to fragment A; Lessnick SL et al.; We describe a series of experiments that aimed to establish whether nuclease activity is actually associated with diphtheria toxin (DTx) and its A subunit (DTA), as we originally reported (M . P . Chang, R . L . Baldwin, C . Bruce, and B . J . Wisnieski, Science 246:1165-1168, 1989) . Here we show that (i) trypsinization of DTx does indeed produce nucleolytically active DTA, (ii) reduction of electroeluted, unreduced, cleaved DTx (58 kDa) yields nuclease-active DTA (24 kDa), and (iii) fractionation of DTx and DTA by anion-exchange chromatography leads to coelution of nuclease activity with both forms of the toxin, even though each form elutes at a distinct salt concentration . In addition, we show that Escherichia coli-derived DTA also expresses nuclease activity . These studies confirm our initial assertion that the nuclease activity observed in DTx preparations is intrinsic to the DTA portion of DTx. J Bacteriol, 1992 Mar, 174(6), 2028 - 31 Cross-linkage and cross-linking of peptidoglycan in Escherichia coli: definition, determination, and implications; Driehuis F et al.; The glycan chains in peptidoglycan or murein are cross-linked by transpeptidation of the peptide side chains . To assess the fraction of side chains involved in cross-bridges, distinction has been made between cross-linkage and cross-linking . The first expression refers to the situation in unlabeled (or fully labeled) peptidoglycan, and the second refers to pulse-labeled peptidoglycan . It is argued that for the determination of the cross-linking value, the mode of insertion as denoted by the so-called acceptor/donor radioactivity ratio should be taken into account. J Bacteriol, 1992 Mar, 174(6), 1974 - 82 An Escherichia coli dnaE mutation with suppressor activity toward mutator mutD5; Schaaper RM et al.; The Escherichia coli mutator mutD5 is a conditional mutator whose strength is moderate when the strain is growing in minimal medium but very strong when it is growing in rich medium . The primary defect of this strain resides in the dnaQ gene, which encodes the epsilon (exonucleolytic proofreading) subunit of the DNA polymerase III holoenzyme . In one of our mutD5 strains we discovered a mutation that suppressed the mutability of mutD5 . Interestingly, the level of suppression was strong in minimal medium but weak in rich medium . The mutation was localized to the dnaE gene, which encodes the alpha (polymerase) subunit of the DNA polymerase III holoenzyme . This mutation, termed dnaE910, also conferred improved growth of the mutD5 strain and caused increased temperature sensitivity in both wild-type and dnaQ49 backgrounds . The reduction in mutator strength by dnaE910 was also observed when this allele was placed in a mutL, a mutT, or a dnaQ49 background . The results suggest that dnaE910 encodes an antimutator DNA polymerase whose effect might be mediated by improved insertion fidelity or by increased proofreading via its effect on the exonuclease activity. J Bacteriol, 1992 Mar, 174(6), 1956 - 64 Expression of argU, the Escherichia coli gene coding for a rare arginine tRNA; Saxena P et al.; The Escherichia coli argU gene encodes the rare arginine tRNA, tRNA(UCUArg), which decodes the similarly rare AGA codons . The argU promoter is, with two exceptions, a typical, strongly expressed stable RNA gene promoter which is stimulated by an upstream activator sequence . Unlike other tRNA operons, however, argU expression is severely inhibited by sequences downstream of the transcription start point . In vivo, nucleotides +2 to +45 inhibited expression by 25- to 100-fold when measured by fusion of argU promoter regions to the chloramphenicol acetyltransferase reporter gene or by quantitative primer extension analysis . In vitro, linearized argU promoter fragments on which the argU region ended at +1 supported 5- to 10-fold-more transcription than when the argU region ended at +45 . This difference in degree of inhibition between in vivo and in vitro conditions suggests that several factors, some of which could be absent in vitro, might limit expression in vivo . Alternatively, one mechanism might limit expression both in vivo and in vitro but function more efficiently in vivo . A second difference from strongly expressed stable RNA promoters is the fact the argU gene is relatively insensitive to growth rate regulation, at least when assayed on a multicopy plasmid. Genes Dev, 1992 Mar, 6(3), 511 - 9 Sequence requirements for efficient translational frameshifting in the Escherichia coli dnaX gene and the role of an unstable interaction between tRNA(Lys) and an AAG lysine codon; Tsuchihashi Z et al.; Synthesis of the gamma-subunit of DNA polymerase III holoenzyme depends on precise and efficient translational frameshifting to the -1 frame at a specific site in the dnaX gene of Escherichia coli . In vitro mutagenesis of this frameshift site demonstrated the importance of an A AAA AAG heptanucleotide sequence, which allows two adjacent tRNAs to retain a stable interaction with mRNA after they slip to the -1 position . The AAG lysine codon present in the 3' half of this heptanucleotide was a key element for highly efficient frameshifting . A tRNA(Lys) with a CUU anticodon, which has a strong affinity for AAG lysine codons, is present in eukaryotic cells but absent in E . coli . Expression in E . coli of a mutant tRNA(Lys) with a CUU anticodon specifically inhibited the frameshifting at the AAG codon, suggesting that the absence of this tRNA in E . coli contributes to the efficiency of the dnaX frameshift. Eur J Immunol, 1992 Mar, 22(3), 867 - 70 Cell selection strategies for making antibodies from variable gene libraries: trapping the memory pool; Hawkins RE et al.; The B cells of immunized animals can be used as a source of variable region (V) gene libraries . Such libraries offer a way of making antibodies directly in bacteria: rearranged V genes are amplified using the polymerase chain reaction, cloned and expressed as soluble fragments in bacteria, and then screened for antigen binding . Here we have used a model system to investigate antigen-selected B cells as a source of V gene libraries . Mice were immunized with (4-hydroxyl-3-nitrophenyl)acetyl (NP)-chicken gammaglobulin, and the splenocytes harvested seven days after primary immunization . We prepared a heavy chain variable (VH) gene library from the DNA of cells selected on antigen-coated magnetic beads, and two other libraries from the DNA or mRNA of unselected cells . The VH gene libraries were combined with the V lambda 1 gene (as this light chain dominates the primary response to NP), expressed as Fv fragments in Escherichia coli and screened for binding to (4-hydroxy-3-iodo-5-nitrophenyl)acetyl-bovine serum albumin . The frequency of antigen-binding clones was much greater (greater than 50 fold) in the library from the DNA of antigen-selected cells (17/282) or from the mRNA of unselected cells (29/282) compared to the DNA from unselected cells (0/940) . Sequencing of the antigen-binding clones revealed that they almost invariably used the V-186.2 heavy chain, as expected from analysis of primary response hybridomas . The D segments from the mRNA library were entirely DFL16.1 (29/29), as in primary response hybridomas, whereas those from the DNA of selected cells were more diverse, using in addition to DFL16.1, other D segments (5/17) as in later response hybridomas . This suggests that the DNA library from selected cells is derived at least in part from cells destined for the memory compartment . Given the long life of memory cells, they may prove a useful source of antibody libraries in the absence of recent immunization. EMBO J, 1992 Mar, 11(3), 1205 - 16 Activation of distant replication origins in vivo by DNA looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance; Miron A et al.; We have isolated mutants of the pi initiator protein of the plasmid R6K that are defective in DNA looping in vitro but retain their normal DNA binding affinity for the primary binding sites (iterons) at the gamma origin/enhancer . One such looping defective mutant called R6 was determined to be a proline to leucine change at position 46 near the N terminus of the pi protein . Using a set of genetic assays that discriminate between the activation of the gamma origin/enhancer from those of the distantly located alpha and beta origins, we show that the looping defective initiator protein fails to activate the alpha and beta origins but derepresses initiation from the normally silent gamma origin in vivo . The results conclusively prove that DNA looping is required to activate distant replication origins located at distances of up to 3 kb from the replication enhancer. EMBO J, 1992 Mar, 11(3), 1055 - 63 The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins; Bianchi ME et al.; The mammalian nuclear protein HMG1 contains two segments that show a high sequence similarity to each other . Each of the segments, produced separately from the rest of the protein in Escherichia coli, binds to DNA with high specificity: four-way junction DNA of various sequences is bound efficiently, but linear duplex DNA is not . Both isolated segments exists as dimers in solution, as shown by gel filtration and chemical crosslinking experiments . HMG1-like proteins are present in yeast and in protozoa: they consist of a single repetition of a motif extremely similar to the DNA binding segments of HMG1, suggesting that they too might form dimers with structural specificity in DNA binding . Sequences with recognizable similarity to either of the two DNA binding segments of HMG1, called HMG boxes, also occur in a few eukaryotic regulatory proteins . However, these proteins are reported to bind to specific sequences, suggesting that the HMG box of proteins distantly related to HMG1 might differ significantly from the HMG box of HMG1-like proteins. Clin Pediatr (Phila), 1992 Mar, 31(3), 130 - 6 The changing spectrum of neonatal meningitis over a fifteen-year period; Shattuck KE et al.; One hundred seventy-seven cases of neonatal meningitis treated at the University of Texas Medical Branch at Galveston over a 15-year period (1974-1988) were reviewed . Over this period, the incidence of bacterial meningitis decreased, the incidence of aseptic meningitis remained stable, and the diagnosis of enteroviral meningitis increased in frequency . During 1984-1988, enterovirus was the most common cause of meningitis in neonates older than seven days and accounted for one third of all cases of neonatal meningitis . Half of all neonates with bacterial meningitis had negative blood cultures . We recommend that 1) diagnostic lumbar puncture remain part of the routine assessment of the neonate with suspected sepsis, and 2) CSF be cultured for enterovirus as well as for bacteria when a neonate older than seven days presents with suspected sepsis. Carcinogenesis, 1992 Mar, 13(3), 349 - 54 Mutation spectra of the two guanine adducts of the carcinogen 4-nitroquinoline 1-oxide in Escherichia coli . Influence of neighbouring base sequence on mutagenesis; Daubersies P et al.; When the chemical carcinogen 4-nitroquinoline 1-oxide binds to DNA in vitro, two major adducts are formed, both at guanine residues, but at different positions, i.e . the C8 or the N2 position . Well-defined adducts (either C8 or N2 guanine adducts) can be formed in vitro by reacting DNA with 4-actoxyaminoquinoline 1-oxide (Ac-4HAQO) under different reaction conditions . Forward mutations induced by each of both main 4NQO adducts in the tetracycline resistance gene of pBR322 were determined . In total, 30 independent 4NQO-induced mutations were characterized, showing mainly base-pair substitution mutations and some frameshift mutations . We have observed that the 5' neighbouring base influences the specificity of dGuo-N2-AQO induced base-pair substitutions mutagenesis; a similar effect does not occur with dGuo-C8-AQO . This study reveals the importance of the N2 guanine adduct in the mutagenesis induced by 4NQO in vivo. Biochem J, 1992 Mar 1, 282 ( Pt 2), 505 - 10 Design of two chimaeric human-rat class alpha glutathione transferases for probing the contribution of C-terminal segments of protein structure to the catalytic properties; Bjornestedt R et al.; Two chimaeric human-rat class Alpha glutathione transferases were constructed by fusion of DNA segments derived from the plasmids pTGT2-AT and pGTB38 and expression of the corresponding proteins in Escherichia coli . The recombinant proteins H1R1/1 and H1R1/2 encoded by plasmids pH1R1/1 and pH1R1/2 are composed of a segment of the human class Alpha subunit 1 from the N-terminus to His-143 and Pro-207 respectively, followed by the complementary C-terminal portion of the rat class Alpha subunit 1 sequence . Compared with the parental human enzyme, H1R1/1 is altered in 20 positions due to the introduction of 79 residues from the rat enzyme, while H1R1/2 is altered in five positions out of 15 in the C-terminal region . The design of mutant H1R1/1 is equivalent to introduction of exons 6 and 7 of the rat subunit 1 gene in place of the homologous human nucleotide sequence . The two chimaeric proteins are enzymatically active with several substrates, even though the activity in most cases is somewhat decreased in comparison with the wild-type human enzyme . Inhibition studies show that the kinetic properties mimic those of the human enzyme, indicating that the N-terminal two-thirds of the primary structure plays the major role in governing the catalytic properties . The results of this study demonstrate that recombination of segments of primary structure between homologous enzymes may serve as a useful cassette technique for design of novel catalytically active proteins. Mol Cell Biol, 1992 Mar, 12(3), 1366 - 74 Multiple SH2-mediated interactions in v-src-transformed cells; Koch CA et al.; The Src homology 2 (SH2) domain is a noncatalytic region which is conserved among a number of signaling and transforming proteins, including cytoplasmic protein-tyrosine kinases and Ras GTPase-activating protein (GAP) . Genetic and biochemical data indicate that the SH2 domain of the p60v-src (v-Src) protein-tyrosine kinase is required for full v-src transforming activity and may direct the association of v-Src with specific tyrosine-phosphorylated proteins . To test the ability of the v-Src SH2 domain to mediate protein-protein interactions, v-Src polypeptides were expressed as fusion proteins in Escherichia coli . The bacterial v-Src SH2 domain bound a series of tyrosine-phosphorylated proteins in a lysate of v-src-transformed Rat-2 cells, including prominent species of 130 and 62 kDa (p130 and p62) . The p130 and p62 tyrosine-phosphorylated proteins that complexed v-Src SH2 in vitro also associated with v-Src in v-src-transformed Rat-2 cells; this in vivo binding was dependent on the v-Src SH2 domain . In addition to binding soluble p62 and p130, the SH2 domains of v-Src, GAP, and v-Crk directly recognized these phosphotyrosine-containing proteins which had been previously denatured and immobilized on a filter . In addition, the SH2 domains of GAP and v-Crk bound to the GAP-associated protein p190 immobilized on a nitrocellulose membrane . These results show that SH2 domains bind directly to tyrosine-phosphorylated proteins and that the Src SH2 domain can bind phosphorylated targets of the v-Src kinase domain.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Cell Biol, 1992 Mar, 12(3), 1087 - 95 Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association; Werner M et al.; The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro . By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C . All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype . We also obtained two conditional thermosensitive mutants affecting this region . One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro . This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31. J Gen Virol, 1992 Mar, 73 ( Pt 3), 639 - 51 Autoprocessing of the human immunodeficiency virus type 1 protease precursor expressed in Escherichia coli from a synthetic gene; Valverde V et al.; A gene encoding an N-terminally extended precursor of 107 residues of the human immunodeficiency virus type 1 protease (PR107) was chemically synthesized and cloned into a bacterial expression vector, under the control of the araB promoter . PR107 was expressed alone or fused in phase to the amino or carboxy terminus of the bacterial beta-galactosidase (beta-gal) . The yield of protease and beta-gal was found to be significantly higher when the gene for PR107 was cloned upstream of the Escherichia coli lacZ gene (PR107-beta-gal) . Comparisons of the level of cloned protein expression between protease precursor and mature form suggested that this enhanced expression was due to the additional 5' sequence of the PR107 gene, and occurred at the post-transcriptional level . Autoprocessing of protease precursor and its release from the beta-gal fusion protein were analysed using wild-type and mutated cleavage sites . Mutations were introduced at amino acids downstream of the F-P scissile bond, at positions P4' and P5' in the C-terminal site (TLNF*PISP), and at position P3' in a consensus N-terminal site (TLNF*PQITL) placed at the protease-beta-gal junction . The data obtained suggested that (i) autoprocessing at the carboxy-terminal F-P bond was not significantly influenced by the presence of the N-terminal precursor sequence, (ii) P4' and P5' substitutions in the C-terminal site had no effect on cleavage, and (iii) P3' in the N-terminal site tolerated a wide variety of substitutions. Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1963 - 7 Cloning and nucleotide sequence of the cDNA encoding human 2-oxoglutarate dehydrogenase (lipoamide); Koike K et al.; 2-Oxoglutarate dehydrogenase (lipoamide) (( OGDH: 2-oxoglutarate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2 )) is a component enzyme of the 2-oxoglutarate dehydrogenase complex . We have cloned a human cDNA encoding OGDH from a fetal liver cDNA library by plaque hybridization with a mixture of oligonucleotide probes designed from the amino acid sequences of porcine OGDH . This cDNA spans 4156 bases and contains an open reading frame of 3009 nucleotides encoding a presequence of 40 amino acid residues and a mature protein of 963 amino acid residues (Mr = 108,642) . The size of the mRNA is approximately 4.2 kilobases . Comparison of the deduced amino acid sequence of the human OGDH with experimentally determined segments of porcine OGDH comprising 308 amino acid residues shows 93% sequence identity . The human OGDH has 37% sequence identity with 933 amino acid residues of the Escherichia coli OGDH and 40% sequence identity with 1014 residues of the yeast OGDH. Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1842 - 6 cDNA cloning and functional expression of the Schistosoma mansoni protective antigen triose-phosphate isomerase; Shoemaker C et al.; M.1 monoclonal antibody has previously been shown to passively transfer partial resistance to schistosome infection within mice and to recognize a 28-kDa antigen that has peptide sequence homology with triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) . We have now isolated the complete coding DNA for Schistosoma mansoni TPI and confirmed that this cDNA encodes the 28-kDa antigen recognized by M.1 . The predicted translation product has strong homology with other TPIs, particularly from higher eukaryotes, and the sequence homology is greatest in regions known to form the active site . The complete coding DNA has been expressed within an Escherichia coli host to produce high levels of soluble, recombinant S . mansoni TPI protein . The product is recognized and purified by the M.1 antibody and is a functional TPI with an intrinsic specific activity comparable to that of rabbit and yeast TPI. Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1740 - 4 The c-rel protooncogene product c-Rel but not NF-kappa B binds to the intronic region of the human interferon-gamma gene at a site related to an interferon-stimulable response element; Sica A et al.; Interferon-gamma (IFN-gamma) is an important immunoregulatory protein that is expressed usually only in large granular lymphocytes and T cells . The gene encoding IFN-gamma was previously found to contain an intronic enhancer element that was not tissue-specific in its activity, despite the restricted expression of the intact IFN-gamma-encoding gene . Using nuclear extracts from the human T-cell line Jurkat, we have now identified two protein-binding regions in this intronic enhancer element . One of the protected regions has strong partial identify to the NF-kappa B site present in the promoter region of the human interleukin 2-encoding gene . Based on this observation and recent reports of the interaction of the c-rel protooncogene product (c-Rel) with NF-kappa B sites, we determined whether c-Rel could interact with the intronic enhancer element in the human IFN-gamma genomic DNA . Most surprisingly, gel-shift analysis, using c-Rel expressed in Escherichia coli established that c-Rel binds specifically to the IFN-gamma intronic DNA but not to the interleukin 2-like NF-kappa B site . Additional studies with antibodies prepared against c-Rel peptides verified specificity of the interaction of c-Rel with this binding site . In addition, using an affinity-purified p50 subunit of the NF-kappa B complex, we observed that the p50 protein did not bind to this additional c-Rel-binding site . Furthermore, nucleotide sequence analysis of this DNA region revealed a strong similarity of the additional c-Rel-binding site to a previously identified IFN-stimulable response element . These data show that c-Rel can interact with DNA regions distinct from that recognized by NF-kappa B and may, in fact, be involved in transcriptional regulation of the IFN-stimulable genes via the IFN-stimulable response element. Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1534 - 8 A streptavidin-metallothionein chimera that allows specific labeling of biological materials with many different heavy metal ions; Sano T et al.; We have designed a streptavidin-metallothionein chimeric protein in which the streptavidin moiety provides a means of binding the metallothionein moiety tightly to specific biological targets . A gene fusion of streptavidin with mouse metallothionein I was efficiently expressed in Escherichia coli, and the expressed chimeric protein was purified to homogeneity by a simple procedure . The purified chimera, consisting of four identical subunits, bound one biotin and approximately seven Cd2+ ions per subunit (19.5 kDa) . This indicates that both the streptavidin and the metallothionein moieties are fully functional . The high binding affinity of the chimera both for biotin and for heavy metal ions allows the specific labeling or conjugation of any biological material containing unhindered biotin with a variety of different heavy metal ions and their isotopes, thereby opening the way for simultaneous assay systems for a large number of biological targets. Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1524 - 8 Functional complementation of internal deletion mutants in the lactose permease of Escherichia coli; Bibi E et al.; Using the lactose permease of Escherichia coli, a well-characterized membrane protein with 12 transmembrane domains, we demonstrated that certain paired in-frame deletion constructs complement each other functionally . Although cells expressing the deletion mutants individually are unable to catalyze active lactose accumulation, cells simultaneously expressing specific pairs of deletions catalyze transport up to 60% as do cells expressing wild-type permease . Moreover, complementation clearly does not occur at the level of DNA but probably occurs at the protein level . Remarkably, functional complementation is observed only with pairs of permease molecules containing large deletions and is not observed with missense mutations or point deletions . Although the mechanism of functional complementation is obscure, the findings indicate that certain pairs of permease molecules containing specific internal deletions can interact to form a functional complex in the same way phenomenologically as do independently expressed polypeptides corresponding to different N- and C-terminal portions of the permease. J Cell Biol, 1992 Mar, 116(6), 1369 - 80 Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain; Hemmings L et al.; To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells . Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195 . When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends . This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding . The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E . coli . Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189 . Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding . Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin . Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells . Furthermore, an E . coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro. Infect Immun, 1992 Mar, 60(3), 892 - 8 Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae; Gerlach GF et al.; An expression library was constructed from Actinobacillus pleuropneumoniae serotype 7 . Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum . One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified . Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in the A . pleuropneumoniae wild type after iron-restricted growth only . The recombinant protein bound transferrin after blotting onto nitrocellulose . Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established . Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography . Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so . Southern blot analysis of several other A . pleuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4. Eur J Biochem, 1992 Mar 1, 204(2), 875 - 83 Human-immunodeficiency-virus-type-1-encoded Vpu protein is phosphorylated by casein kinase II; Schubert U et al.; Vpu as a human-immunodeficiency-virus-type-1-encoded 81-amino-acid integral-membrane protein was expressed in Escherichia coli using the inducible ptrc promoter of an ATG fusion vector . Recombinant Vpu is associated with membranes of E . coli and could be partially solubilized by detergents . Recombinant Vpu was phosphorylated in vitro with purified porcine casein kinase II (CKII) as well as with a CKII-related protein kinase found in cytoplasmic extracts of human and hamster cells . Recombinant Vpu associated with E . coli membranes has turned out to be the best substrate for in vitro phosphorylation with CKII . This reaction can be inhibited by heparin and the ATP analogue 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB), both known to be potent inhibitors of CKII . Radiolabelled gamma ATP and gamma GTP were used as phosphate donors in vitro phosphorylation of recombinant Vpu . In vivo phosphorylation of Vpu in HIV-1-infected H9 cells was also inhibited by DRB . We concluded therefrom that the Vpu protein is phosphorylated by the ubiquitous CKII in HIV-1-infected human host cells . Two seryl residues in the sequence of Vpu (position 52 and 56) correspond to the consensus S/TXXD/E for CKII . These potential phosphorylation sites are located within a well-conserved dodecapeptide of Vpu (residues 47-58), which is found in different HIV-1 strains as well as in a Vpu-like protein of SIVCPZ . Monoclonal and polyclonal antibodies directed against two different epitopes of Vpu were used for immunoprecipitation of Vpu from HIV-1-infected cells and for detection of Vpu in Western blot analyses . Vpu from HIV-1-infected cells as well as recombinant Vpu expressed in E . coli were determined by SDS/PAGE using 6 M urea to be 9 kDa, which corresponds to the calculated molecular mass of Vpu. Eur J Biochem, 1992 Mar 1, 204(2), 649 - 55 Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues; Caccia P et al.; The production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes . Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability . Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups . Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF) . Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group . The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form . Cm-FGF and unmodified rhbFGF showed similar affinity both for heparin and for high-affinity receptors . Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDS/PAGE analysis . Interestingly, Cm-FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts . This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications. Eur J Biochem, 1992 Mar 1, 204(2), 583 - 9 Different conformational forms of Escherichia coli and rat liver 5S rRNA revealed by Pb(II)-induced hydrolysis; Ciesiollka J et al.; Different stable forms of Escherichia coli and rat liver 5S rRNA have been probed by Pb(II)-induced hydrolysis . In the native A forms of 5S rRNA, Pb2+ reveal single-stranded RNA stretches and regions of increased conformational flexibility or distorted by the presence of bulged nucleotides . Hydrolysis of urea/EDTA-treated E . coli 5S rRNA (B form) shows the presence of two strong helical domains; helix A retained from the A form and a helix composed of RNA regions G33-C42 and G79-C88 . Other RNA regions resistant to hydrolysis may be involved in alternative base pairing, causing conformational heterogeneity of that form . Pb(II)-induced hydrolysis distinguishes two different forms of rat liver 5S rRNA; the native A form and the form obtained by renaturation of 5S rRNA in the presence of EDTA . Pb(II)-hydrolysis data suggest that both forms are highly structured . In the latter form, the orientation of the bulged C66 is changed with respect to helix B . At the same time, a new helical segment is possibly formed, composed of nucleotides from helix C and loop c on one side and from helix E and loop d' on the other. Crit Care Med, 1992 Mar, 20(3), 402 - 8 Endorphin mediation of mesenteric blood flow after endotoxemia in sheep; Navaratnam N et al.; BACKGROUND AND METHODS: The administration of endotoxin in small dosages to sheep results in an acute decrease followed by an increase in cardiac output . It has previously been determined that the initial decrease in output was the result of a reduction in blood flow to the mesenteric areas . These changes were associated with increased blood concentrations of beta endorphin . The present study was accomplished to determine if the systemic cardiovascular response to endotoxin could be affected by the administration of an opiate receptor-blocking agent . Female range sheep (n = 12) were prepared for chronic study by implantation of cardiopulmonary catheters and a flow probe on the cephalic mesenteric artery . Endotoxin (Escherichia coli, 1 microgram/kg) was administered to these animals . Half of the sheep were treated with naloxone (2 mg/kg + 2 mg/kg.hr), and the remainder with an equivalent volume of sodium chloride (0.9%) . RESULTS: In untreated sheep, cardiac indices decreased by 15% to 20% (5.1 +/- 0.1 to 4.2 +/- 0.4 L/min.m2) between 0 and 1 hr and 2 and 5 hrs after endotoxin (4.5 +/- 0.2 L/min.m2), and then increased to a value 40% (7.2 +/- 0.6 L/min.m2) above baseline by 12 hrs . Flow in the cephalic mesenteric artery decreased in a pattern similar to the reduction in cardiac index (962 +/- 152 {time, T = 0} to 379 +/- 111 {T = 0.8} and 384 +/- 88 mL/min {T = 4.0}, p less than .05) but did not increase to the same extent (1008 +/- 153 mL/min {T = 4.0}, p greater than .05) . There was a concomitant increase in the plasma beta-endorphin concentrations as the blood flow decreased (5 +/- 4 {T = 0} to 40 +/- 15 pg/mL {T = 0.8; untreated group}, p less than .05; and 10 +/- 4 to 50 +/- 7 pg/mL {T = 0.8; naloxone-treated group}, p less than .05) . In the naloxone-treated group, the same pattern of cardiac output change was noted with endotoxin; however, reduction of mesenteric artery flow was only 30% (1118 +/- 129 to 908 +/- 122 mL/min, p less than .05) of the value seen in the untreated animals (962 +/- 152 to 379 +/- 111 mL/min, p less than .05) . These changes in mesenteric blood flow were statistically significant from one another . As the cardiac output increased in the sheep treated with the opiate antagonist, mesenteric blood flows increased 20% above the baseline value (1391 +/- 199 mL/min, p less than .05) . CONCLUSIONS: The decrease in cardiac output noted with endotoxin can be accounted for by the decrease in the blood flow in the cephalic mesenteric artery . This phenomenon can be attributed, at least in part, to the release of endorphins . There is both a vasodilation and constriction during endotoxemia in the ovine model. Lab Invest, 1992 Mar, 66(3), 362 - 9 Induction of severe vascular leakage by low doses of Escherichia coli hemolysin in perfused rabbit lungs; Ermert L et al.; S . aureus alpha-toxin and E . coli hemolysin (Hly) represent two prototypes of pore-forming cytolysins . Both are established virulence factors and have been implicated in the development of septic lung failure . Low doses of these agents cause thromboxane-mediated vasoconstriction and edema formation in isolated perfused rabbit lungs . In a preceding investigation, we observed that alpha-toxin causes overt endothelial cell damage in these lungs, as demonstrable by electron microscopy (Seeger W, Birkemeyer RG, Ermert L, Suttorp N, Bhakdi S, Duncker HR: Lab Invest 63:341, 1990) . Here, we present results of a parallel study conducted with E . coli hemolysin . Thromboxane-dependent pulmonary hypertension was suppressed by the addition of acetylsalicylic acid to the perfusion fluid in all cases . Administration of 0.2 hemolytic units (HU; i.e., 20 ng/ml protein) resulted in progressive weight gain after a lag period of 10 to 15 minutes, and 30 minutes after toxin application the gravimetrically determined capillary filtration coefficients (Kfc) were increased greater than 10-fold . Perfusion was terminated when the total lung weight gain surpassed 20 gm . 0.12 HU/ml E . coli hemolysin caused 2- to 3-fold increased capillary filtration coefficients values within 110 minutes, concomitant with intermediate quantities of edema formation (9.7 +/- 2.7 gm) . Potassium liberation in the absence of lactate dehydrogenase release occurred in all toxin treated lungs . Electron microscopic examination after perfusion fixation revealed interstitial edema formation in areas remote from the blood-gas exchange barrier . Increased numbers of endothelial plasmalemmal vesicles were visualized at the very onset of edema formation in lungs exposed to 0.2 HU/ml, and after a 110-minute exposure to 0.12 HU/ml of the toxin, but not in lungs exhibiting severe edema (greater than 20 gm weight gain) . In contrast to our previous results with alpha-toxin, endothelial cells displayed normal electron density here and were not detached from the fused basal lamina . Hence, although both pore formers provoke severe vascular leakage in our experimental model, the underlying pathways probably divert fundamentally from each other. J Cell Physiol, 1992 Mar, 150(3), 632 - 9 Susceptibility to verotoxin as a function of the cell cycle; Pudymaitis A et al.; Infection with Verotoxin producing Escherichia coli (VTEC) has been implicated in hemolytic uremic syndrome, the leading cause of pediatric renal failure . Verotoxin (VT) binds to globotriaosylceramide (Gal alpha 1-4Gal beta 1-4GlcCer Gb3) in susceptible cells . Gb3 is required for cytotoxicity and toxin-resistant cells deficient in Gb3 can be sensitized to VT cytotoxicity by incorporation of exogenous Gb3 into the cells . However, the absolute Gb3 content of cell lines does not necessarily correspond directly with the degree of sensitivity to VT . The present study demonstrates that susceptibility to VT is a function of cell growth and that stationary phase cells are resistant to VT . Using chemically synchronized Vero cells, we have also found a tenfold difference in susceptibility to VT during the cell cycle . Our experiments define a maximal sensitivity "window" of 1-2 hours from the G1/S boundary . This corresponds to increased VT binding without change in overall Gb3 content . Cell surface labelling indicated that cyclic turnover and exposure of Gb3 may be the critical parameter in determining VT sensitivity . Such changes during the cell cycle may also be of relevance in vivo in determining toxin pathology during VTEC infections and the physiology of plasma membrane Gb3. J Bacteriol, 1992 Mar, 174(5), 1686 - 9 Identification of the pheS5 mutation, which causes thermosensitivity of Escherichia coli mutant NP37; Kast P et al.; The pheS5 mutation responsible for the thermosensitive phenylalanyl-tRNA synthetase of the classical Escherichia coli NP37 was cloned by a recombination event and identified by DNA sequence analysis . The mutation was subsequently verified by direct sequencing of amplified NP37 DNA generated by an asymmetric polymerase chain reaction . The resulting amino acid exchange, Gly-98 to Asp-98 in the phenylalanyl-tRNA synthetase alpha subunit, might cause subunit disaggregation due to electrostatic repulsion. J Bacteriol, 1992 Mar, 174(5), 1682 - 5 Quantitation of Dam methyltransferase in Escherichia coli; Boye E et al.; An antiserum against Escherichia coli Dam methyltransferase has been developed in rabbits and employed to detect and quantitate the enzyme in immunoblots . A wild-type, rapidly growing E . coli cell (doubling time = 30 min) was found to contain about 130 molecules of Dam methyltransferase. J Bacteriol, 1992 Mar, 174(5), 1612 - 8 SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of complementary-strand DNA synthesis; Higashitani N et al.; We report that the SOS response is induced in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of the complementary (minus)-strand synthesis . One such mutant, R377, which lacks the entire region of the minus-strand origin, failed to synthesize any detectable amount of primer RNA for minus-strand synthesis . In addition, the rate of conversion of parental single-stranded DNA of the mutant to the double-stranded replicative form in infected cells was extremely slow . Upon infection, R377 induced the SOS response in the cell, whereas the wild-type phage did not . The SOS induction was monitored by (i) induction of beta-galactosidase in a strain carrying a dinD::lacZ fusion and (ii) increased levels of RecA protein . In addition, cells infected with R377 formed filaments . Another deletion mutant of the minus-strand origin, M13 delta E101 (M . H . Kim, J . C . Hines, and D . S . Ray, Proc . Natl . Acad . Sci . USA 78:6784-6788, 1981), also induced the SOS response in E . coli . M13Gori101 (D . S . Ray, J . C . Hines, M . H . Kim, R . Imber, and N . Nomura, Gene 18:231-238, 1982), which is a derivative of M13 delta E101 carrying the primase-dependent minus-strand origin of phage G4, did not induce the SOS response . These observations indicate that single-stranded DNA by itself induces the SOS response in vivo. J Bacteriol, 1992 Mar, 174(5), 1544 - 53 Locating essential Escherichia coli genes by using mini-Tn10 transposons: the pdxJ operon; Takiff HE et al.; The mini-Tn10 transposon (delta 16 delta 17Tn10) confers tetracycline resistance . When inserted between a gene and its promoter, it blocks transcription and prevents expression of that gene . Tetracycline in the medium induces divergent transcription of the tetA and tetR genes within the transposon, and this transcription extends beyond the transposon in both directions into the bacterial genes . If the mini-Tn10 inserts between an essential bacterial gene and its promoter, the insertion mutation can cause conditional growth which is dependent on the presence of tetracycline . Two essential genes in adjacent operons of Escherichia coli have been detected by screening for tetracycline dependence among tetracycline-resistant insertion mutants . These essential genes are the era gene in the rnc operon and the dpj gene in the adjacent pdxJ operon . The pdxJ operon has not been described previously . It consists of two genes, pdxJ and dpj . Whereas the dpj gene is essential for E . coli growth in all media tested, pdxJ is not essential . The pdxJ gene encodes a protein required in the biosynthesis of pyridoxine (vitamin B6). J Bacteriol, 1992 Mar, 174(5), 1537 - 43 pH dependence and gene structure of inaA in Escherichia coli; White S et al.; The weak-acid-inducible locus inaA in Escherichia coli was mapped to 48.6 min by P1 cotransduction of inaA Mud lac fusions and linked Tn10 insertions . The inaA1::lac fusion tested negative for phenotypes characteristic of mutations in the nearby locus ubiG . Sequence analysis of a fragment amplified by polymerase chain reaction located the inaA1::lac fusion joint within an open reading frame 311 nucleotides downstream of nrdB, transcribed in the opposite direction, encoding a 168-amino-acid polypeptide . Constitutive mutant strains identified on lactose MacConkey revealed a novel regulatory locus unlinked to inaA, which mapped at 34 min (designated inaR) . Expression of inaA1::lac increased slightly with external acidification; the presence of benzoate, a membrane-permeant weak acid, greatly increased the acid effect . The expression at various combinations of benzoate and external pH correlated with the decrease in intracellular pH . The uncouplers salicylate and dinitrophenol also caused acid-dependent induction of inaA, but substantial induction was seen at external pH values higher than the internal pH; this effect cannot be caused by internal acidification . Nondissociating analogs of benzoate and salicylate, benzyl alcohol and salicyl alcohol, did not induce inaA . Expression of inaA was inversely related to growth temperature over the range of 30 to 45 degrees C . The inaA1::lac fusion was transferred to a strain defective for K+ uptake (kdpABC trkA trkD) in which pH homeostasis was shown to depend on the external K+ concentration . In this construct, inaA1::lac retained pH-dependent induction by benzoate but was not induced at low K+ concentrations . Induction of inaA appears to involve several factors in addition to internal pH . inaR may be related to the nearby locus marA/soxQ, which is inducible by acidic benzyl derivatives. J Bacteriol, 1992 Mar, 174(5), 1528 - 36 Aspartate and maltose-binding protein interact with adjacent sites in the Tar chemotactic signal transducer of Escherichia coli; Gardina P et al.; The Tar protein of Escherichia coli is a chemotactic signal transducer that spans the cytoplasmic membrane and mediates responses to the attractants aspartate and maltose . Aspartate binds directly to Tar, whereas maltose binds to the periplasmic maltose-binding protein, which then interacts with Tar . The Arg-64, Arg-69, and Arg-73 residues of Tar have previously been shown to be involved in aspartate sensing . When lysine residues are introduced at these positions by site-directed mutagenesis, aspartate taxis is disrupted most by substitution at position 64, and maltose taxis is disrupted most by substitution at position 73 . To explore the spatial distribution of ligand recognition sites on Tar further, we performed doped-primer mutagenesis in selected regions of the tar gene . A number of mutations that interfere specifically with aspartate taxis (Asp-), maltose taxis (Mal-), or both were identified . Mutations affecting residues 64 to 73 or 149 to 154 in the periplasmic domain of Tar are associated with an Asp- phenotype, whereas mutations affecting residues 73 to 83 or 141 to 150 are associated with a Mal- phenotype . We conclude that aspartate and maltose-binding protein interact with adjacent and partially overlapping regions in the periplasmic domain of Tar to initiate attractant signalling. J Bacteriol, 1992 Mar, 174(5), 1454 - 61 Multicopy suppression: an approach to understanding intracellular functioning of the protein export system; Ueguchi C et al.; Escherichia coli genes were cloned onto a multicopy plasmid and selected by the ability to restore growth and protein export defects caused by a temperature-sensitive secY or secA mutation . When secA51 was used as the primary mutation, only clones carrying groE, which specifies the chaperonin class of heat shock protein, were obtained . Selection using secY24 yielded three major classes of genes . The first class encodes another heat shock protein, HtpG; the most frequently obtained second class encodes a neutral histonelike protein, H-NS; and the third class, msyB, encodes a 124-residue protein of which 38 residues are acidic amino acids . Possible mechanisms of suppression as well as the significance and limitations of the multicopy suppression approach are discussed. Arch Biochem Biophys, 1992 Mar, 293(2), 377 - 81 Expression cloning in Escherichia coli and preparative isolation of the reductase coacting with chalcone synthase during the key step in the biosynthesis of soybean phytoalexins; Welle R et al.; The cDNA for the reductase involved in the biosynthesis of 6'-deoxychalcone (4,2',4'-trihydroxychalcone), the first specific intermediate in the pathway to soybean phytoalexins, was cloned into the expression vector pKK233-2 and transformed into Escherichia coli . Using this source, about 5 mg of homogeneous reductase was isolated from 45 g of cells . The protein purification protocol differs completely from the scheme applied to soybean cell cultures . Size, N-terminal and specific enzyme activities were identical for the plant and E . coli protein . The pure protein is fairly stable, retaining 70% of initial activity after storage at 5 degrees C during 4 weeks . This protein is used for crystallization and in the study of its protein-protein interaction with chalcone synthase. Tsitol Genet, 1992 Mar-Apr, 26(2), 17 - 20 {Ultrastructural features of changes in capillaries and atrial cardiomyocytes induced by endotoxin injection}; Kharlanova NG et al.; The experiments on the rabbits and dogs have revealed that in the initial period of endotoxin shock the number of secretory granules in the atrial cardiomyocytes decreases and vascular permeability increases due to transendothelial micropinocytosis . In the intermediate period of endotoxin shock the ultrastructural alterations progress . The development of ultrastructural manifestations of endotoxemia greatly depends on the condition of endocrine heart function. Radiobiologiia, 1992 Mar-Apr, 32(2), 194 - 7 {The lethal and mutagenic action of 3H incorporated into the 2' position of the deoxyribose in the DNA of the extracellular phage lambda}; Konevega LV et al.; The lethal and mutagenic effects of 3H decay in 2' position of deoxyribose residues in DNA of extracellular lambda phage were studied, {2'-3H}-deoxyadenosine (3H-dA) or {2'-3H}-thymidine (3H-dT) being used as labelled DNA precursors . As estimated by the efficiency of the lethal and mutagenic actions of 3H decay in position 2' was significantly lower than that of the decay in the incorporated 3H-pyrimidines . The genetic effects of 3H decay in 2' position may be attributed to the radiation effect of beta-particles on DNA . In UV-irradiated E . coli cells, with the induced SOS repair, the mutagenic effect of 3H-dA in phage lambda is significantly higher than that of 3H-dT . This is perhaps related to the formation in DNA of AP-sites, resulting from 3H-decay in 2' position, and to the predominant incorporation of adenosine residues opposite to AP-sites during SOS repair. J Gen Microbiol, 1992 Mar, 138 ( Pt 3), 597 - 603 Transport of ferric-aerobactin into the periplasm and cytoplasm of Escherichia coli K12: role of envelope-associated proteins and effect of endogenous siderophores; Wooldridge KG et al.; Purified {14C}aerobactin, supplied exogenously to non-growing bacteria, was translocated via the periplasm into the cytoplasm of Escherichia coli K12 strains expressing the aerobactin receptor protein IutA . No significant uptake was observed into either compartment of strains lacking the iutA gene or specifically defective in tonB . Uptake into both compartments was markedly reduced, but not abolished, in an exb mutant . Accumulation of {14C}aerobactin in the periplasm of fhuD, fhuB or fhuC mutant strains was not significantly lower than in the wild-type strain, but entry into the cytoplasm was greatly reduced in all cases . Uptake of aerobactin by strains wild-type for all transport functions occurred most efficiently in strains either lacking or specifically defective in the genetic determinants for aerobactin biosynthesis; significantly lower levels of exogenous 14C-labelled siderophore were observed in both compartments of strains producing aerobactin . Aerobactin-mediated 59Fe uptake, however, was not inhibited by the presence of endogenous aerobactin . Endogenous enterochelin did not affect aerobactin uptake. Mol Microbiol, 1992 Mar, 6(6), 781 - 5 Overproduction of a selenocysteine-containing polypeptide in Escherichia coli: the fdhF gene product; Chen GT et al.; The fdhF gene of Escherichia coli codes for the selenocysteine-including protein subunit of formate dehydrogenase H . The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon . The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e . the selC gene product . The ability of E . coli cells to overproduce a selenopolypeptide was examined using the fdhF gene as a model system . Surprisingly, E . coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein . This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter . FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen . The product was specifically labelled with 75Se, proving that it consisted of a selenoprotein . The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H. J Bacteriol, 1992 Mar, 174(6), 1948 - 55 Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine; Lin R et al.; The leucine regulon coordinates the expression of several Escherichia coli genes according to the presence of exogenous leucine, which interacts with the lrp gene product, Lrp . We isolated and characterized 22 strains with lambda placMu insertions in Lrp-regulated genes . Lrp and leucine influenced gene expression in a surprising variety of ways . We identified two genes that are regulated by Lrp and not affected by L-leucine . We therefore rename this the leucine-lrp regulon . Genes coding for glycine cleavage and leucine biosynthesis enzymes have been identified as members of the leucine-lrp regulon . We suggest that the lrp gene product activates genes needed for growth in minimal medium, and we show that the gene is repressed by its own product and is highly repressed during growth in rich medium. EMBO J, 1992 Mar, 11(3), 909 - 16 Residue 21 of human granulocyte-macrophage colony-stimulating factor is critical for biological activity and for high but not low affinity binding; Lopez AF et al.; The functional role of the predicted first alpha-helix of human granulocyte-macrophage colony-stimulating factor (GM-CSF) was analysed by site-directed mutagenesis and multiple biological and receptor binding assays . Initial deletion mutagenesis pointed to residues 20 and 21 being critical . Substitution mutagenesis showed that by altering Gln20 to Ala full GM-CSF activity was retained but that by altering Glu21 for Ala GM-CSF activity and high affinity receptor binding were decreased . Substitution of different amino acids for Glu21 showed that there was a hierarchy in the ability to stimulate the various biological activities of GM-CSF with the order of potency being Asp21 greater than Ser21 greater than Ala21 greater than Gln21 greater than Lys21 = Arg21 . To distinguish whether position 21 was important for GM-CSF binding to high or low affinity receptors, GM-CSF (Arg21) was used as a competitor for {125I}GM-CSF binding to monocytes that express both types of receptor . GM-CSF (Arg21) exhibited a greatly reduced capacity to compete for binding to high affinity receptors, however, it competed fully for {125I}GM-CSF binding to low affinity receptors . Furthermore, GM-CSF (Arg21) was equipotent with wild-type GM-CSF in binding to the cloned low affinity alpha-chain of the GM-CSF receptor . These results show that (i) this position is critical for high affinity but not for low affinity GM-CSF receptor binding thus defining two functional parts of the GM-CSF molecule; (ii) position 21 of GM-CSF is critical for multiple functions of GM-CSF; and (iii) stimulation of proliferation and mature cell function by GM-CSF are mediated through high affinity receptors. J Radiat Res (Tokyo), 1992 Mar, 33 Suppl, 95 - 108 Resolution and characterization of polymorphic DNA by SSCP and chemical cleavage methodologies; Yatagai F et al.; A variety of techniques have been developed to detect single-base changes for the two different purposes . One is the detection of mutational events without phenotypic selection, and another is the rapid and conventional identification of mutations such as the specific base changes related to activation of oncogene, genetic diseases, etc . In this study, the utility of the two methods, single strand conformation polymorphism (SSCP) and chemical cleavage, was explored using 13 E . coli lacI- mutations cloned onto M13 phase . The 167 base region encompassing mutations was amplified by PCR as dsDNA . Following denaturation, these PCR products were analyzed by non-denaturing polyacrylamide gel electrophoresis (SSCP) and the separation of the ssDNA fragment carrying the altered sequence from the original sequence was found to be dependent on the location and type of the change . Hetero duplexes of changed/original sequences were also prepared by hybridization of the above PCR products . Mismatched C and T bases were modified by hydroxylamine and osmium tetroxide, respectively, and subsequently treated with piperidine to analyze the cleaved DNA fragments on a polyacrylamide gel (Chemical Cleavage) . The cleavage efficiency was also found to be influenced by the type of mismatch and its surrounding sequence . Such observed characteristics should contribute to a better appreciation for these types of mutational systems, which in turn should lead to insight into the mechanisms of mutagenesis. East Afr Med J, 1992 Mar, 69(3), 135 - 9 Escherichia coli associated with acute measles and diarrhoea at Kenyatta National Hospital, Kenya; Sang FC et al.; Three hundred and three children under 5 years old with acute measles and diarrhoea (cases) and 300 other age-matched children with diarrhoea (controls) were examined for enteroadherent E . coli (EAEC) and other agents including rotavirus and Cryptosporidium . EAEC was determined by tissue culture of HEP-2 cells . Other agents were determined by conventional methods . EAEC was identified from both cases and control accounting for 10.3% (31/303) and 15.2% (46/300) respectively . Other bacterial agents were: 10.3% (31/303) from cases and 12.8% (39/300) from controls . A higher detection rate of enteroparasites was found among cases 15% (45/300) than controls 8.9% (27/300) whereas rotavirus was the reverse, 3% (9/303) in cases and 30.3% (92/300) in controls . To our knowledge characterization of EAEC has not been done before and therefore might be attributing factor to some of our unexplained diarrhoeal cases. J Pediatr Surg, 1992 Mar, 27(3), 333 - 6; discussion 336-8 Unique characteristics of the neonatal intestinal mucosal barrier; Smith SD et al.; The purpose of this study was to compare the newborn and weanling intestinal mucosa to determine differences in: (1) the electrophysiologic characteristics of the mucosal barrier; (2) the effects of glutamine supplementation on these physiological characteristics; and (3) transmucosal bacterial passage . The Ussing chamber was used to study ileal mucosa from newborn (1 to 4 days old) and weanling (21 days old) piglets . After the seromuscularis was stripped off the bowel wall, the mucosa was mounted in the chamber and perfused with Hanks Balanced Salt Solution (HBSS) or HBSS + 20 mmol/L of glutamine . Following initial stabilization, potential difference (PD) and resistance (R) were measured at 30-minute intervals for 2 hours . Transmucosal bacterial passage was measured by quantitative cultures of the mucosal and serosal reservoirs obtained 2 hours after adding 10(8) E coli C-25 to the mucosal reservoir . Six groups of membranes were studied: (1) newborn and HBSS; (2) weanling and HBSS; (3) newborn and HBSS + glutamine; (4) weanling and HBSS + glutamine; (5) newborn - HBSS + glutamine + E coli; and (6) weanling - HBSS + glutamine + E coli . Newborn ileal mucosa had significantly lower PD and R compared with weanling at all time points . Glutamine led to a significant increase in PD in both newborn and weanling . Newborn mucosa had a significantly increased incidence of transmucosal bacterial passage (4/7) compared with weanling (0/10) . These findings suggest that: (1) newborn mucosal barrier has uniquely different electrophysiologic characteristics; (2) glutamine improves the metabolic activity as measured by PD in both newborn and weanling; and (3) the newborn mucosal barrier allows increased transmucosal passage of bacteria. Plant Cell, 1992 Mar, 4(3), 241 - 51 The cucumber long hypocotyl mutant lacks a light-stable PHYB-like phytochrome; Lopez-Juez E et al.; A novel cDNA sequence homologous to a phytochrome B (phyB) gene that was isolated in a library from tobacco tissue has been used in an Escherichia coli expression system to raise anti-phytochrome B (anti-PHYB) polypeptide-specific monoclonal antibodies . The specificity of these antibodies has been tested by cross-reactivity against purified pea light-labile type 1 and light-stable type 2 phytochromes, with some antibodies reacting with the type 2 and none with the type 1 phytochromes . One such antibody, monoclonal mAT1, has been employed to analyze the phytochrome molecular species present in a photomorphogenic long hypocotyl (lh) mutant of cucumber . The results indicated that the mutant contains wild-type levels of the light-labile type 1 phytochrome polypeptide (PHYA), which has an apparent molecular mass of approximately 120 kD, but shows less than 1% (detection limit) of a light-stable polypeptide recognized by mAT1 in wild-type seedlings . This protein, not detectable in the lh mutant, has the properties of light-stable type 2 phytochrome, has an apparent molecular mass of 116 to 117 kD, and remains at constant levels under continuous low-fluence-rate red light . Therefore, we conclude that the lh mutant lacks at least one type 2 phytochrome-like polypeptide, most probably a phyB gene product . The correlation between the lack of this protein and the deficiency or absence of physiological responses to a light-stable phytochrome species in this mutant helps to identify the physiological roles played by the products of different subfamilies within the phytochrome gene family. Med Lab Sci, 1992 Mar, 49(1), 12 - 5 Escherichia coli: rapid identification by chromogenic tests; Dealler SF et al.; A system has been assessed for the identification of Esch . coli using a rapid triple chromogenic test which relies on the ability of the organism to produce a beta-galactosidase, a beta-glucuronidase, and indole . Coliforms which had been fully identified were tested by this system . Of 512 non-Esch . coli strains there were no false positives, whereas of 514 Esch . coli strains 486 (94.5%) were found to give positive results . Two hundred and twenty-one coliforms that had been isolated from blood cultures were also tested using the colistrip in advance of, or without knowledge of the API 20E result . The test was found to be 100% specific and 94% sensitive for the 105 Esch . coli strains . The test was rapid, simple to perform and economical. Zhonghua Yu Fang Yi Xue Za Zhi, 1992 Mar, 26(2), 92 - 3 {A SOS induction test screening study for vegetables inhibiting mutagenicity caused by antineoplastic drugs}; Zhao ZZ et al.; Using mutational and anti-mutational synchronous in SOS inductest (+/- S9), We found that 7 out of 11 kinds of commonly eaten vegetables had the ability to inhibit mutagenicity caused by chemical drugs such as Mitomycin C, Bleomycinia, Fluorouracil, Cis-Diaminodichloroplatinum, Arabinosylcytosin and mustargen, They were garlic, green Chinese onion, onion, garlic bulb, tomato, cucumber and water radish . The other 4 lacking this ability were rape, chinese toon, ginger and asparagus lettuce stalk . We believe that our results can be helpful in the preparation . of cancer patients' diet, who are receiving chemotherapy and in the prevention of cancer. J Biomol NMR, 1992 Mar, 2(2), 183 - 94 The effect of selective deuteration on magnetization transfer in larger proteins; Pachter R et al.; The effects of selective deuteration on calculated NOESY intensities have been analyzed for the structure of the E . coli trp aporepressor, a 25 kDa protein . It is shown that selectively deuterated trp aporepressor proteins display larger calculated NOESY intensities than those for the same interproton distances in the natural abundance protein . The relatively larger magnetization transfer is demonstrated by a comparison of the NOE build-up curves for specific proton pairs, and for the calculated NOE intensities of short-range NOEs to backbone amide protons . This increase in intensity is especially pronounced for the NHi-NHi+1 cross peaks in the alpha-helical regions, and particularly for amide protons of two sequential deuterated residues . The effect is shown to be further intensified for longer mixing times . It is also shown that in all cases, each amide proton exhibits stronger NOEs to its own side chain, with an enhanced effect for deuterated derivatives . This theoretical analysis demonstrates that an evaluation of the relative NOE intensities for different selectively deuterated analogs may be an important tool in assigning NMR spectra of large proteins . These results also serve as a guide for the interpretation of NOEs in terms of distances for structure calculations based on data using selectively deuterated proteins. J Biomol NMR, 1992 Mar, 2(2), 149 - 60 Multidimensional 1H and 15N NMR investigation of glutamine-binding protein of Escherichia coli; Tjandra N et al.; Specific and uniform 15N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His156, Trp32 and Trp220 residues . These assignments have been made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associated with the active transport of L-glutamine across the cytoplasmic membrane of Escherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment . The assignment of H epsilon 2 of His156 refines the earlier model where this particular proton forms an intermolecular hydrogen bond to the delta-carbonyl of L-glutamine, while assignments of both Trp32 and Trp220 show the variation in local structures which ensure the specificity in ligand binding and protein-protein interaction . Using 3D NOESY-HMQC NMR, amide connectivities can be traced along 8-9 amino acid residues at a time . This paper illustrates the usefulness of combining 15N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25,000. Mol Gen Mikrobiol Virusol, 1992 Mar-Apr, (3-4), 12 - 7 {Isolation and purification of Brucella antigens synthesized by Escherichia coli K12 cells}; Chibisova VA et al.; The homogeneous preparations of the brucella protein antigens were isolated from the hybrid producer strains Escherichia coli 6SE579 and 6SE800 by the cold osmotic shock technique and further purification on immunosorbents . The 18 + 38 and 38 kDa antigens were obtained . The antiserum specific to brucella 38 kDa antigen was obtained and used for isolation of the 18 kDa antigen from the producer strain 6SE579 synthesizing two brucella antigens . The immunosorbent developed on the basis of BrCn-agarose conjugated with antibodies from the serum has permitted isolation of 18 kDa protein antigen preparation . Thus, the combined technique of cold osmotic shock and affinity chromatography on immunosorbents permits one to isolate highly purified individual antigens of brucella from Escherichia coli K12 producer cells. J Pharmacobiodyn, 1992 Mar, 15(3), 121 - 9 Highly sensitive enzyme-linked immunosorbent assay for marograstim (KW-2228), a mutant of human granulocyte colony-stimulating factor; Kuwabara T et al.; An enzyme-linked immunosorbent assay (ELISA) for marograstim (KW-2228) was established . This ELISA proved to be highly sensitive with the detection limit of 0.01 ng/ml (about 0.5 fmol/ml) of KW-2228 and the assay range between 0.01 and 2 ng/ml . When 0.02 to 2 ng/ml of KW-2228 added to human plasma was determined, the variation coefficiencies of intra-day and inter-day assays were 0.4 to 7.6% and 5.2 to 15.8%, respectively, with good recoveries . These results indicate that this ELISA will be applicable to pharmacokinetic studies on KW-2228 . With respect to the specificity, recombinant human granulocyte colony-stimulating factor (rhG-CSF) produced in Escherichia coli as well as KW-2228 which does not have sugar chains in its structure showed slightly less immunoreactivity toward the antibody raised against KW-2228 . The rhG-CSF produced in Chinese hamster ovary cells (CHO) having sugar chains showed the lower immunoreactivity . The antigenic domains of KW-2228 were evaluated using a number of variants of hG-CSF . The variants having different amino acids from KW-2228 in the 1st to 5th residue of the N-terminus showed almost equal immunoreactivities to KW-2228 . The immunoreactivities of the variants lacking 7 to 18 amino acids of N-terminus were less than 50% of that of KW-2228 . No immunoreactivity was observed for the variants deleted in the area of 70th to 130th amino acids from the N-terminus . In addition, the immunoreactivity of a variant lacking the 10 amino acids from the C-terminus was 20% of that of KW-2228.(ABSTRACT TRUNCATED AT 250 WORDS) Protein Eng, 1992 Mar, 5(2), 139 - 46 A preliminary 3-D model of the tertiary fold of the polymerase domain of HIV-1 reverse transcriptase; Narasimhan LS et al.; Development of a 3-D model of the reverse transcriptase from type 1 human immunodeficiency virus (HIV-1 RT), a key enzyme in the pathogenesis of the virus, presents a significant challenge . Three-dimensional structural information is not available for any close homolog, the only 3-D structural data being that of the Klenow fragment (KF) of Escherichia coli DNA polymerase I, for which coordinates of only the alpha-carbons are available . A recently published study of the sequences of a large number of polymerases led to the identification of three common sequence patterns, nominally motif A, motif B and motif C, and to the hypothesis that the various DNA and RNA polymerases including E . coli DNA polymerase I and HIV-1 RT share a common structural motif around their respective polymerase active sites . The preliminary results of recent structural studies on two other polymerases also support this hypothesis . Based on the assumption of structural homology in the active site regions of their polymerase domains, the HIV-1 RT and KF sequences were aligned using pattern-based secondary structure predictions as a guide and motifs A, B and C as 'anchor points' . However, as suggested by the results of chemical modification experiments, it was assumed that the order of the motifs in KF, viz . A, B and C, differed from that of the related motifs A, C and B' in HIV-1 RT, a rearrangement that could have been brought about by an exon shuffling type of mechanism.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Microbiol, 1992 Mar, 6(6), 771 - 80 Differential levels of fertility inhibition among F-like plasmids are related to the cellular concentration of finO mRNA; van Biesen T et al.; The FinOP system of F-like plasmids consists of an antisense RNA (FinP) and a 22 kDa protein (FinO) which act in concert to prevent the translation of TraJ, the positive regulator of the transfer operon . Earlier studies suggested that two different variants of finO were responsible for differential levels of fertility inhibition among F-like plasmids . We have shown that these variations are due to the presence of an additional open reading frame (orf286) upstream of the finO gene of conjugative plasmids that are highly repressed for transfer . When orf286 and finO are linked in cis, the level of FinO expression is increased because of a rise in the cellular concentration of finO mRNA . orf286 frameshift and deletion mutants also gave the same concentration of finO transcript, suggesting that the effect is due to mRNA stabilization . We suggest that the levels of fertility inhibition exhibited by F-like plasmids are a function of their cellular FinO concentration. Arch Emerg Med, 1992 Mar, 9(1), 1 - 8 The effect of low molecular weight dextran on haemodynamics and respiratory function during endotoxin-induced shock; Christenson JT et al.; The effects of low molecular weight dextran (LMWD) infusion, on gas exchange and haemodynamics were evaluated in sheep during endotoxin shock . The infusion of LMWD was started after signs of shock and lung injury were evident . After a stabilization period 10 micrograms kg-1 E . Coli endotoxin was infused i.v. . Endotoxin infusion resulted in an marked increase in pulmonary artery pressure (PAP) and decrease in mean arterial pressure (MAP), respiratory compliance, arterial oxygen tension (PaO2) and oxygen delivery index (DO2l) . After 3 h MAP, PaO2, DO2l and compliance improved significantly in LMWD treated animals . The PAP had also decreased significantly in the LMWD-treated animals, but remained high in the controls (P less than 0.01) . It was concluded that LMWD infusion improves haemodynamics and gas-exchange in sheep during endotoxin shock. Mol Gen Genet, 1992 Mar, 232(2), 221 - 30 Light-regulated expression of the psbD gene family in Synechococcus sp . strain PCC 7942: evidence for the role of duplicated psbD genes in cyanobacteria; Bustos SA et al.; The genome of the cyanobacterium Synechococcus sp . strain PCC 7942 contains two psbD genes encoding the D2 protein of the photosystem II reaction center: psbDI, which is cotranscribed as a discistronic message with psbC (the gene encoding CP43, a chlorophyll-a binding protein), and psbDII, which is monocistronic . Northern blot analysis of psbD transcripts showed that the two genes responded differently when wild-type cells were shifted from moderate to high light intensity . Whereas psbDII transcripts increased 500% relative to unshifted control cells, psbDI-psbC transcripts remained unchanged . The beta-galactosidase activities expressed from translational fusions between the psbD genes and the Escherichia coli lacZ reporter gene displayed responses similar to those seen in the RNA . D2 protein levels in thylakoid membranes from wild-type cells increased to 250% of those of the unshifted control cells 12 h after a shift to high light intensities . In contrast, in a mutant strain (AMC016) that carries an inactive psbDII gene, D2 levels decreased by 50% under identical conditions . These results suggested that induction of psbDII gene expression by light can serve as a supplementary system for maintaining a functional photosystem II reaction center at high light intensity . This hypothesis was corroborated by mixed-culture experiments, in which AMC016 cells competed poorly with wild-type cells at high light intensity . These data suggest for the first time that differential expression of members of a cyanobacterial gene family serves to maintain a functional PSII reaction center under diverse environmental conditions. Mol Microbiol, 1992 Mar, 6(5), 615 - 20 Regulation of the expression of the cell-cycle gene ftsZ by DicF antisense RNA . Division does not require a fixed number of FtsZ molecules; Tetart F et al.; We show that the 53-nucleotide RNA molecule encoded by gene dicF blocks cell division in Escherichia coli by inhibiting the translation of ftsZ mRNA . Such a role for dicF had been predicted on the basis of the complementarity of DicF RNA with the ribosome-binding region of the ftsZ mRNA . An analysis of ftsZ expression at its chromosomal locus, and of an ftsZ-lacZ translational fusion controlled by promoters ftsZ1p and ftsZ2p only, indicates that ftsZ is not autoregulated . Partial inhibition of FtsZ synthesis leads to