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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 increased cell size . However, the number of FtsZ molecules per cell can be reduced threefold without affecting the division rate significantly . Our results suggest that septation is not triggered by a fixed number of newly synthesized FtsZ molecules per cell.

J Bacteriol, 1992 Mar, 174(6), 1777 - 82
Premature termination of in vivo transcription of a gene encoding a branched-chain amino acid transport protein in Escherichia coli; Williamson RM et al.; Previous studies have suggested that control of expression of genes of the LIV-I permease system for the high-affinity transport of branched-chain amino acids in Escherichia coli involves modulation in the frequency of mRNA elongation . Mutation of the Rho transcription termination factor and shortages of charged leucyl-tRNA have been shown to alter LIV-I transport activity . Rho-dependent transcription termination regulated by shortages of charged leucyl-tRNA at sites preceding structural genes has been proposed to account for their role in regulation of LIV-I transport . Transcription of the livJ-binding protein gene, encoding one of the periplasmic components of the LIV-I system, was analyzed in vivo with strains which lack repression of the LIV-I genes and harbor a temperature-sensitive allele for either leucyl-tRNA synthetase or Rho factor . Analysis of mRNA synthesis by DNA-RNA hybridization in the various mutant strains indicated that both shortages of leucyl-tRNA caused by inactivation of the temperature-sensitive leucyl-tRNA synthetase and inactivation of the Rho factor were associated with increased synthesis of livJ mRNA . Nuclease protection and gel electrophoresis studies detected prematurely terminated transcripts corresponding in size to the leader region of livJ mRNA . Accumulations of these short transcripts were suppressed in strains harboring temperature-sensitive alleles for either leucyl-tRNA synthetase or Rho factor . These results provide support for the hypothesis that expression of livJ involves Rho-dependent transcription termination in which antitermination is associated with the intracellular availability of aminoacyl leucyl-tRNA.

EMBO J, 1992 Mar, 11(3), 1195 - 203
Replication control in plasmid R1: duplex formation between the antisense RNA, CopA, and its target, CopT, is not required for inhibition of RepA synthesis; Wagner EG et al.; The replication frequency of plasmid R1 is regulated by an antisense RNA, CopA, which inhibits the synthesis of the rate-limiting initiator protein RepA . The inhibition requires an interaction between the antisense RNA and its target, CopT, in the leader of the RepA mRNA . This binding reaction has previously been studied in vitro, and the formation of a complete RNA duplex between the two RNAs has been demonstrated in vitro and in vivo . Here we investigate whether complete duplex formation is required for CopA-mediated inhibition in vivo . A mutated copA gene was constructed, encoding a truncated CopA which is impaired in its ability to form a complete CopA/CopT duplex, but which forms a primary binding intermediate (the 'kissing complex') . The mutated CopA species (S-CopA) mediated incompatibility against wild-type R1 plasmids and inhibited RepA-LacZ fusion protein synthesis . Northern blot, primer extension and S1 analyses indicated that S-CopA did not form a complete duplex with CopT in vivo since bands corresponding to RNase III cleavage products were missing . An in vitro analysis supported the same conclusion . These data suggest that formation of the 'kissing complex' suffices to inhibit RepA synthesis, and that complete CopA/CopT duplex formation is not required . The implications of these findings are discussed.

Am J Pathol, 1992 Mar, 140(3), 649 - 57
Biologic and immunohistochemical analysis of interleukin-6 expression in vivo . Constitutive and induced expression in murine polymorphonuclear and mononuclear phagocytes; Terebuh PD et al.; Interleukin-6 (IL-6) is considered an important multifunctional cytokine involved in the regulation of a variety of cellular responses, including the induction of acute-phase protein synthesis, lymphocyte activation, and hematopoiesis . In vitro studies have identified many cells that can produce IL-6, but the cellular sources under physiologic conditions have yet to be identified . Using immunoaffinity purified goat anti-murine IL-6, the authors performed immunohistochemical studies to localize cells expressing IL-6 in selected organs of normal and endotoxin challenged NIH-Swiss outbred mice . In the blood, findings were correlated with cell-associated bioactivity using the standard B9 cell proliferation assay . In normal mice, constitutive expression was seen in granulocytes, monocytes and their precursors as well as in bone marrow and splenic stromal macrophages . Hepatic macrophages were negative, as were lymphocytes, megakaryocytes, erythroid precursors, and endothelial cells . In the absence of significant serum levels of IL-6, cell-associated IL-6 bioactivity was detected in circulating polymorphonuclear leukocytes (PMNs), but not lymphocytes . After endotoxin challenge, there was a threefold increase in PMN IL-6 content from 1 to 3 hours followed by almost complete depletion at 6 hours . This correlated well with a threefold increase of IL-6 mRNA in the bone marrow followed by a decrease at 6 hours . This pattern also correlated with serum levels of IL-6, which peaked at 3 hours and dropped significantly by 6 hours . By 24 hours, cell-associated IL-6 showed recovery with no increase in serum levels . In vivo findings showing IL-6 expression in bone marrow macrophages support in vitro studies suggesting a role for IL-6 in hematopoiesis . Furthermore, PMNs as well as macrophages are likely important sources of IL-6 during inflammatory and septic states.

J Gen Virol, 1992 Mar, 73 ( Pt 3), 695 - 700
Epitope mapping on fragments of beet necrotic yellow vein virus coat protein; Commandeur U et al.; The location of five SDS-stable epitopes on the coat protein (CP) of beet necrotic yellow vein virus was determined by reacting Escherichia coli-expressed free CP, as well as fusion proteins (FP) containing fragments of the CP, with polyclonal and monoclonal antibodies on Western blots . Epitope 1, which has previously been found to be exposed on only one extremity of the virus particle, was located in the region between amino acids (aa) 1 and 7, i.e . on the N terminus of the CP . It was blocked when the N terminus of the CP was linked to a portion of the beta-galactosidase sequence in an FP . Epitope 3, which has previously been found to be exposed on the opposite extremity of the particle, was located in the region between aa 37 and 59 . Epitope 4, which is exposed along the entire length of the particle, occurs on the C terminus of CP (aa 183 to 188) . Two previously unknown epitopes were identified in the regions between aa 115 and 125 and 125 and 140, respectively . The former was located on the same extremity of the particle as epitope 3, the latter became accessible only after denaturation of the particle . Nothing is known about the probably non-adjacent aa sequences that participate in the formation of the two SDS-labile epitopes (epitopes 2 and 5) which are found on one extremity and along the entire length of the particle, respectively.

Mutat Res, 1992 Mar, 281(3), 221 - 5
Construction of a umuDC operon substitution mutation in Escherichia coli; Woodgate R; Using a specialized transducing lambda phage, the umuDC operon of Escherichia coli was deleted and replaced with the chloramphenicol acetyltransferase gene . The delta (umuDC)595::cat mutation was subsequently transferred by generalized P1 transduction into a variety of genetic backgrounds . It is concluded that the UmuDC proteins, which are normally required for inducible mutagenesis, are not essential for cell survival.

Infect Immun, 1992 Mar, 60(3), 1254 - 7
Colonization factor antigen CFA/IV (PCF8775) of human enterotoxigenic Escherichia coli: nucleotide sequence of the CS5 determinant; Clark CA et al.; Human enterotoxigenic Escherichia coli isolates expressing the colonization factor antigen CFA/IV (previously designated PCF8775) produce plasmid-encoded CS5 fimbriae . The nucleotide sequence of the region encoding the major CS5 fimbrial subunit was determined . The subunit is synthesized as a precursor of 203 amino acids (20.85 kDa) with a mature protein of 181 amino acids corresponding to a size of 18.6 kDa . The CS5 subunit shows homology to the corresponding component of porcine enterotoxigenic E . coli F41, particularly within the signal sequence and at the carboxy terminus.

Infect Immun, 1992 Mar, 60(3), 1210 - 6
Cloning and expression of the gene for the Avi-3 antigen of Mycobacterium avium and mapping of its epitopes; Yamaguchi R et al.; The Avi-3 antigen, which is found only in Mycobacterium avium culture sonic extracts, is species specific and results in strong skin test activity in guinea pigs sensitized with heat-killed M . avium . Its gene was cloned by using a previously developed single-probe method and was sequenced . The gene encoded a 194-amino-acid polypeptide with a molecular weight of 21,500 . A recombinant Avi-3 antigen expressed in Escherichia coli reacted with monoclonal and polyclonal antibodies raised against the native Avi-3 antigen . To identify epitopes on this protein for immunodiagnostic purposes, various parts of the Avi-3 antigen were expressed as beta-galactosidase fusion proteins, using pUR and pURS expression vectors . The clones screened by both antibody reactivity and T-cell proliferative activity defined fragments with coexisting B- and T-cell epitopes . A B-cell epitope (Asn-176 to Ala-186) and two T-cell epitopes (Glu-75 to Ile-86 and Arg-155 to Leu-164) were thus defined . The synthetic polymerized peptides of the T-cell epitopes were proven to elicit a delayed cutaneous hypersensitivity reaction in guinea pigs . This mapping method would be useful in the development of a subunit vaccine consisting of an immunodominant B-cell epitope linked to a T-cell epitope in the vicinity.

J Virol, 1992 Mar, 66(3), 1571 - 8
High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay; Tchenio T et al.; We devised an indicator gene for retrotransposition, nlsLacZRT, which contains the Escherichia coli lacZ gene fused to a nuclear location signal (nlsLacZ), engineered in such a way that the gene is expressed only if the structure in which it has been inserted transposes itself through an RNA intermediate . A cloned murine leukemia retrovirus with an ecotropic host range (Moloney murine leukemia virus), rendered defective by a large deletion encompassing the three viral gag, pol, and env open reading frames, was marked with this indicator gene and introduced by transfection into heterologous feline cells . No beta-galactosidase activity could be detected among the clonal cell population, unless the defective provirus was complemented in trans by the gag-pol gene products . Under these conditions, cell variants which disclosed an easily detectable nuclear blue coloration upon in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining were observed . Fluorescence-activated cell sorting of the beta-galactosidase-positive cells, followed by Southern blot analysis, demonstrated an unambiguous correlation between nlsLacZRT activation and retrotransposition of the marked provirus . Transposition occurs at a high frequency (up to 10(-4) events per cell per generation), which is dependent on the level of expression of the gag-pol gene and is concomitant with the release of noninfectious retroviruslike particles which are the hallmarks, but not the intermediates, of the intracellular transposition process.

Biosci Biotechnol Biochem, 1992 Mar, 56(3), 376 - 9
Overexpression and purification of asparagine synthetase from Escherichia coli; Sugiyama A et al.; Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUC18, in E . coli . The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region . The enzyme, comprising ca . 15% of the total soluble protein in the E . coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies . The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene . Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.

Biotechnology (N Y), 1992 Mar, 10(3), 315 - 9
Production and biological activity of hybrid growth hormone-releasing hormone propeptides; Smith DP et al.; Growth hormone-releasing hormone (GHRH), a hypothalamic hormone that stimulates the synthesis and release of growth hormone (GH) from anterior pituitary cells, has been previously produced by synthetic peptide chemistry and recombinant DNA procedures . GHRH is capable of stimulating growth as well as eliciting other anabolic effects on animals and thus may have potential applications in agriculture and human medicine . However, economical production of GHRH by recombinant DNA process has been difficult since GHRH is degraded rapidly by endogenous E . coli proteases . We report here an efficient process to produce hybrid GHRH analogs of higher molecular weight . These hybrid GHRH propeptides (proGHRH) are comprised of an analog of GHRH (44 aa) and the human GHRH carboxy-terminal peptide (33 aa) . In E . coli K-12 RV308, the expression levels of the proGHRH analogs were estimated to be 10% of the total cellular protein . An in vitro assay to measure the release of rat growth hormone by GHRH analogs using crude E . coli lysates was also developed . This assay showed that the proGHRH analogs produced in E . coli efficiently stimulated GH release from rat anterior pituitary cells . One proGHRH analog, {alao}-proGHRH, was purified ans shown to efficiently elevate plasma GH levels in wether lambs . Our data indicate that the hybrid proGHRH peptides, unlike other hormone propeptides such as proinsulin, are remarkably bioactive.

Protein Sci, 1992 Mar, 1(3), 363 - 9
Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins; Viitanen PV et al.; In vitro experiments employing the soluble proteins from Escherichia coli reveal that about half of them, in their unfolded or partially folded states, but not in their native states, can form stable binary complexes with chaperonin 60 (groEL) . These complexes can be isolated by gel filtration chromatography and are efficiently discharged upon the addition of Mg.ATP . Binary complex formation is substantially reduced if chaperonin 60 is presaturated with Rubisco-I, the folding intermediate of Rubisco, but not with native Rubisco . Binary complex formation is also reduced if the transient species that interact with chaperonin 60 are permitted to progress to more stable states . This implies that the structural elements or motifs that are recognized by chaperonin 60 and that are responsible for binary complex formation are only present or accessible in the unfolded states of proteins or in certain intermediates along their respective folding pathways . Given the high-affinity binding that we have observed in the present study and the normal cellular abundance of chaperonin 60, we suspect that the folding of most proteins in E . coli does not occur in free solution spontaneously, but instead takes place while they are associated with molecular chaperones.

Protein Sci, 1992 Mar, 1(3), 342 - 55
Time-resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: conformational analysis of intermediates and transition-state complexes; Atkins WM et al.; Single tryptophan-containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency-domain fluorescence spectroscopy in the presence of various substrates and inhibitors . At pH 6.5, the Mn-bound wild-type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme . The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp-57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp-158 . These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry 30, 3406-3416) . In addition, Trp-57 and Trp-158 both exhibit an ATP-induced increase in the relative fraction of the long lifetime component, whereas only Trp-57 is affected by this ligand at pH 7.5 . The transition-state analogue L-methionine-(R,S)-sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp-158 . This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein . Addition of glutamate to the ATP complex, which affords the gamma-glutamylphosphate-ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp-158, but has no effect on Trp-57 . Similar results were obtained when ATP was added to the MSOX complex; Trp-57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns . Decay kinetics of Trp-158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX-ATP inactivated complex . These results provide a model for the sequence of structural and dynamic changes that take place at the Trp-57 loop and the central loop (Trp-158) during several intermediate stages of catalysis.

Mol Gen Genet, 1992 Mar, 232(1), 145 - 53
Characterization of the F plasmid bifunctional conjugation gene, traG; Firth N et al.; The Escherichia coli F plasmid gene, traG, is required for two stages of the conjugation process: pilus biosynthesis and mating aggregate stabilization . The nucleotide sequence of traG has been determined and the topology of its product in the cytoplasmic membrane analysed using protease accessibility experiments . Complementation analysis employing plasmid deletions revealed a correlation between an N-terminal periplasmic segment of the protein product (TraGp) and its pilus assembly activity . Production of an anti-TraGp antiserum has facilitated the detection of TraGp*, a possible internal cleavage product of TraGp . Although its function is unknown . TraGp* is located in the periplasm and has been shown to possess sequences required for aggregate stabilization . The detection of TraGp* raises the possibility that the two functions of traG are carried out by separate products.

J Bacteriol, 1992 Mar, 174(6), 1869 - 74
Structures of chaperonins from an intracellular symbiont and their functional expression in Escherichia coli groE mutants; Ohtaka C et al.; An intracellular symbiont harbored by the aphid bacteriocyte, a specialized fat body cell, synthesizes in vivo substantially only one protein, symbionin, which is a member of the chaperonin-60 family of molecular chaperones . Nucleotide sequence determination of the symbionin region of the endosymbiont genome revealed that it contains the two-cistron operon sym . Just like the Escherichia coli groE operon, the sym operon was dually led by a heat shock and an ordinary promoter sequence . According to the nucleotide sequence, symbionin was 85.5% identical to GroEL of E . coli at the amino acid sequence level . SymS, another protein encoded in the sym operon, which is a member of chaperonin-10, was 79.6% identical to GroES . Complementation experiments with E . coli groE mutants showed that the chaperonin-10 and chaperonin-60 genes from the endosymbiont are expressed in E . coli and that they can function as molecular chaperones together with endogenous GroEL and GroES, respectively.

Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1865 - 9
Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor; Cull MG et al.; We have constructed a large library of random peptides fused to the C terminus of the lac repressor . The DNA binding activity of the repressor protein physically links the peptides to the plasmid encoding them by binding to lac operator sequences on the plasmid . This linkage allows efficient enrichment for specific peptide ligands in the random population of peptides by affinity purification of the peptide-repressor-plasmid complexes with an immobilized receptor . After transformation of Escherichia coli with recovered plasmids, the library can be amplified for additional rounds of affinity enrichment or specific plasmids can be sequenced to determine the primary structure of the peptides . We used a monoclonal antibody specific for the peptide dynorphin B as a model receptor to screen a random dodecamer library . After only two rounds of enrichment, the majority of the plasmids in the selected population encoded fusion peptides that bound specifically to the antibody . These peptides contain a consensus sequence similar to a segment of dynorphin B (RQFKVV) . This technique should be useful to find peptide ligands for a variety of biological receptors.

Infect Immun, 1992 Mar, 60(3), 998 - 1007
Passive protective effect of chicken egg yolk immunoglobulins against experimental enterotoxigenic Escherichia coli infection in neonatal piglets; Yokoyama H et al.; Passive protection of neonatal piglets against fatal enteric colibacillosis was achieved with powder preparations of specific antibodies against K88, K99, and 987P fimbrial adhesins of enterotoxigenic Escherichia coli . The antibody powders were obtained by spray drying the water-soluble protein fraction of egg yolks from immunized hens after the lipid components were precipitated with an aqueous dispersion of acrylic resins (Eudragit L30D-55; Rohm pharma) . The anti-K88, -K99, and -987P antibody preparations reacted specifically against the corresponding fimbrial antigens in an enzyme-linked immunosorbent assay . The orally administered antibodies protected in a dose-dependent fashion against infection with each of the three homologous strains of E . coli in passive immunization trials with a colostrum-deprived piglet model of enterotoxigenic E . coli diarrhea . Scanning electron microscopy revealed adherence of enterotoxigenic E . coli in intestinal epithelial surfaces of control piglets, whereas in treated piglets treated with high-titer antibodies, a resistance to bacterial adhesion was observed . An enzyme immunoassay with avidin-biotin complex demonstrated specific local antibody activity in target areas of the small intestines . In vitro, E . coli K88+, K99+, and 987P+ strains adhered equally to porcine duodenal and ileal epithelial cells but failed to do so in the presence of homologous anti-fimbrial antibodies . Absorption of egg yolk antibodies with fimbrial immunosorbent removed the anti-fimbrial antibody fraction and reduced significantly the protective nature of the antibody preparation in a passive immunization experiment, suggesting that anti-fimbrial antibodies were the active components.

Gastroenterology, 1992 Mar, 102(3), 816 - 22
Escherichia coli enterotoxin (STa) binds to receptors, stimulates guanyl cyclase, and impairs absorption in rat colon; Mezoff AG et al.; To determine the contribution of the colon in Escherichia coli heat-stable enterotoxin-mediated diarrheal disease, toxin binding, guanyl cyclase activation, and toxin-induced water flux in the rat colon and ileum were compared . Scatchard analysis suggested a single class of heat-stable enterotoxin receptors with an affinity constant of binding of 10(9) L/mol in both colonocytes and ileocytes; however, the number of toxin receptors per cell was 3.5-fold greater in coloncytes than ileocytes (8.32 +/- 1.33 x 10(5) vs . 2.33 +/- 0.28 x 10(5) receptors per cell; P = 0.02) . Heat-stable enterotoxin stimulated guanyl cyclase activation in an identical dose-dependent manner in proximal colonic and ileal membranes, with similar sensitivity and maximum response . Heat-stable enterotoxin also inhibited net water flux to a similar degree in both colon and ileum (-47.8 vs . -48.4 microL.cm-1.h-1, respectively) at a dose of 8 nmol/L . At this dose in the colon, because of a higher baseline of absorption, absorption continued, but at a diminished level . At this dose in the ileum, heat-stable enterotoxin induced net secretion . These data are consistent with the concept that heat-stable enterotoxin-induced diarrheal disease results from a decreased absorptive capacity in the colon in the face of increased small intestinal fluid secretion.

Sarcoidosis, 1992 Mar, 9(1), 29 - 34
Tumour necrosis factor production by alveolar macrophages in pulmonary sarcoidosis and tuberculosis; Foley NM et al.; Tumour Necrosis Factor alpha (TNF/Cachectin) is a cytokine produced mainly by macrophages, which has been shown to cause endothelial cell damage, pyrexia and weight loss, clinical features of tuberculosis, but not of sarcoidosis which is in many other respects a similar disease . 1,25 di-hydroxy Vitamin D and gamma interferon, factors which are present in vivo in both tuberculosis and sarcoidosis, enhance the ability of macrophages to release TNF in vitro . We have studied the ability of pulmonary alveolar macrophages (PAM) harvested by broncho-alveolar lavage (BAL) to produce TNF in response to stimulation with E . coli endotoxin lipopolysaccharide (LPS) . 25 patients undergoing bronchoscopy and BAL were studied: 9 with sarcoidosis, 7 with tuberculosis (TB) and 9 (non-neoplastic) disease controls . TNF was assayed by Enzyme Linked Immunosorbent Assay (ELISA) in lavage fluid and cell culture supernatants . No TNF was detected in lavage fluid from any of the groups . PAMs from control patients released no detectable TNF spontaneously, but released 59 +/- 31 units after LPS stimulation . Cells from patients with sarcoidosis and tuberculosis released TNF spontaneously in vitro (TB 226 +/- 106 units; Sarcoidosis 293 +/- 176) . TNF release by these cells was not increased further by addition of an optimal concentration of LPS . Thus, the pulmonary macrophages of patients with sarcoidosis and tuberculosis released significantly more TNF than those of controls.

Gene, 1992 Mar 1, 112(1), 37 - 43
Ligation-independent cloning of glutathione S-transferase fusion genes for expression in Escherichia coli; Haun RS et al.; A plasmid vector has been constructed that allows the ligation-independent cloning of cDNAs in any reading frame and directs their synthesis in Escherichia coli as glutathione S-transferase-linked fusion proteins . The cloning procedure does not require restriction enzyme digestion of the target sequence and does not introduce any additional sequences between the thrombin cleavage site and the foreign protein . Extended single-stranded tails complementary between the vector and insert, generated by the (3'----5') exonuclease activity of T4 DNA polymerase, obviate the need for in vitro ligation prior to bacterial transformation . This cloning procedure is rapid and highly efficient, and has been used successfully to construct a series of fusion proteins to investigate the sequence requirements for efficient thrombin cleavage.

Res Microbiol, 1992 Mar-Apr, 143(3), 251 - 61
Mutational analysis of the enzyme IIIGlc of the phosphoenolpyruvate phosphotransferase system in Escherichia coli; Zeng GQ et al.; The phosphoenolpyruvate phosphotransferase system (PTS) component EIIIGlc is responsible for transport and phosphorylation of glucose via EIIGlc . It also regulates the catabolism of other carbon sources, such as lactose and maltose, by modulating both the intracellular concentrations of the corresponding inducers and of cAMP . Mutational analysis of EIIIGlc was performed in order to identify crucial residues mediating the interactions between EIIIGlc and its target proteins . Such mutations were isolated by in vitro hydroxylamine mutagenesis of the cloned EIIIGlc gene, crr . Five mutated EIIIGlc impaired in the function of inducer exclusion were obtained . However, these mutations did not abolish the function of EIIIGlc in the transport and phosphorylation of glucose, nor in activation of adenylate cyclase . A single amino acid change was found for each mutation, which is located in a restricted area of the polypeptide chain: Gly47-->Ser47 for the HA2 and HA5 mutations, Ala76-->Thr76 for HA4 mutation and Ser78-->Phe78 for HA3 mutation, indicative of quaternary interactions between the corresponding region of EIIIGlc and its target protein(s).

Vopr Virusol, 1992 Mar-Apr, 37(2), 113 - 6
{The identification of alphaviruses by using cDNA probes complementary to the 3'-terminal of the viral genome}; Vinskaia AI et al.; Venezuelan equine encephalomyelitis, Eastern equine encephalomyelitis and Sindbis viruses, as well as members of various serological groups of alphaviruses were identified using synthesized molecular probes complementary to 3'-terminus of the molecule of the appropriate genomic RNA: 32P-single-stranded kDNA and E . coli-cloned 32P-double-stranded kDNA . A high species-specificity of kDNA-probes was demonstrated . The sensitivity of the method was 1 ng of viral RNA overlayed on the nitrocellulose filter.

J Biomol NMR, 1992 Mar, 2(2), 137 - 47
1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids; Oda Y et al.; Two-dimensional (2D) 1H NMR experiments using deuterium labeling have been carried out to investigate the solution of ribonuclease HI (RNase HI) from Escherichia coli (E . coli), which consists of 155 amino acids . To simplify the 1H NMR spectra, two fully deuterated enzymes bearing several protonated amino acids were prepared from an RNase HI overproducing strain of E . coli grown in an almost fully deuterated medium . One enzyme was selectively labeled by protonated His, Ile, Val, and Leu . The other was labeled by only protonated His and Ile . The 2D 1H NMR spectra of these deuterated RNase HI proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme . The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons . The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme . In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.

Hua Xi Yi Ke Da Xue Xue Bao, 1992 Mar, 23(1), 21 - 3
{Experimental study of use of shuttle vector to develop a system of detecting mutagenesis in mammalian cells}; Lin F et al.; The paper presents a series of experimental studies of the use of shuttle vector to develop a short-time mutagenesis system of detecting gene mutation in the mammalian cell at DNA level . The system is based on the finding that in the EB virus the shuttle vector pMCi5 carries LacI gene . The LacI is used as a target of induced mutagenesis in mouse cells . Use pMCi5 plasmid to transform the 3T3 mouse cell, expose it to a chemical mutagen, ethyl methanesulfonate (EMS); transfer the treated plasmid DNA to E . coli strain MC1061F' 150Kan; and then plate the transformation on a special culture medium containing X-gal for rapid detection and analysis of LacI mutation . Results: The spontaneous mutant frequency of the system is less than 1.21 x 10(-4) . After the treatment with 300 micrograms/ml EMS, the induced mutant frequency increases to 3.8 x 10(-3) . No gross alteration has been discovered in seven LacI mutants at DNA level . No point mutation has occurred in the EcoRI site by analysis with restriction enzyme EcoRI.

Plasmid, 1992 Mar, 27(2), 93 - 104
RecP, a new minor pathway of general recombination in Escherichia coli encoded by plasmid R1drd-19; Chernin LS et al.; Plasmid R1drd-19 markedly improves the recombination deficiency of recB and recBrecC mutants of Escherichia coli K12 as measured by Hfr crosses and increases their resistance to uv inactivation . The effect correlates with the production of an ATP-dependent ds DNA exonuclease in recB/R1drd-19 cells . This paper further investigates the suppressive effect of plasmid R1drd-19 on the recB mutation of E . coli . The gene(s) responsible for the effect was localized to the 13.1-kb EcoRI-C fragment of the resistance transfer factor (RTF) portion of R1drd-19 . The plasmid-encoded activity does not merely replace the RecBCD enzyme failure but differs in several significant ways . It promotes a hyper-recombinogenic phenotype, as judged by the phenomenon of super oligomerization of the tester pACYC184 plasmid in recB/R1drd-19 cells and two inter- and intramolecular plasmid recombination test systems . It is probably not inhibited by lambda Gam protein and does not restrict plating of T4gp2 mutant . No significant homology between the E . coli chromosomal fragment carrying recBrecCrecD genes and the EcoRI-C fragment of R1drd-19 was observed . It is suggested that the plasmid-encoded recombination activity is involved in a new minor recombination pathway (designated RecP, for Plasmid) . RecP resembles in some traits the RecBCD-independent pathways RecE and RecF but differs in activity and perhaps substrate specificity from the main RecBCD pathway.

Mol Endocrinol, 1992 Mar, 6(3), 409 - 15
Amino acid substitutions in the DNA-binding domain of the human androgen receptor are a frequent cause of receptor-binding positive androgen resistance; Zoppi S et al.; In some subjects with genetic and endocrine evidence of androgen resistance, no defect is demonstrable in the binding of androgen to its receptor in cultured genital skin fibroblasts . We have defined the molecular defect in the androgen receptor in four unrelated subjects in this category (termed receptor positive) with the phenotype of compete or incomplete testicular feminization . In these patients we detected amino acid substitutions in either exon 2 or exon 3, which encodes the DNA-binding domain of the androgen receptor . In one patient with incomplete testicular feminization, two separate mutations were present in exon 3 . Introduction of these amino acid substitutions into the androgen receptor-coding segment leads to the expression of receptor proteins that bind ligand in a normal fashion but do not activate the transcription of the androgen-responsive mouse mammary tumor virus promoter . Mobility shift assays using androgen receptor fusion proteins produced in E . coli indicate that these mutations impair binding of the receptor to specific DNA sequences . In the subject with incomplete testicular feminization, a Ser-Gly substitution at amino acid residue 595 is able to partially restore DNA-binding activity to a mutant receptor protein that carries an Arg-Pro substitution at position 615 . These findings indicate that mutations in amino acid residues crucial to the binding of the androgen receptor to target DNA sequences are a common cause of receptor-binding positive androgen resistance and that variable impairment of DNA binding can lead to distinctive phenotypes.

Jpn J Cancer Res, 1992 Mar, 83(3), 264 - 8
Serodiagnostic assay of hepatitis C virus infection using viral proteins expressed in Escherichia coli; Mori S et al.; Infection with hepatitis C virus (HCV) was analyzed by an enzyme-linked immunosorbent assay based on recombinant viral proteins encoded by regions of the putative viral core, NS3, NS4 and NS5, which were expressed in E . coli . Results showed that 106 of 124 cases (85.5%) of non-A, non-B chronic hepatitis and 43 of 45 cases (95.5%) of hepatocellular carcinoma, negative for HBV marker, were positive for antibodies against at least one of these viral proteins . One of 87 healthy individuals with normal alanine aminotransferase activity was positive for antibody against only the viral core, but was negative for HCV RNA . The serum of one patient with chronic hepatitis was positive for one of these proteins, but negative for HCV RNA . These findings in combination with results on detection of HCV RNA in the sera of patients with non-A, non-B chronic hepatitis indicated that 105 of 124 cases (84.6%) were positive for HCV infection . Sera that were negative for HCV antibodies against all these proteins were also negative for HCV RNA assayed by reverse transcription followed by the polymerase chain reaction . Screening of HCV infection by detecting viral antibodies in circulating blood using all these viral proteins is useful for reducing the number of ambiguous results in screening for viral infection . Thus, this assay system may be useful diagnostic purposes.

New Biol, 1992 Mar, 4(3), 238 - 46
Direct gene transfer to the liver with herpes simplex virus type 1 vectors: transient production of physiologically relevant levels of circulating factor IX; Miyanohara A et al.; We have used gene transfer vectors derived from a replication-defective mutant of herpes simplex virus type 1 (HSV-1) expressing the hepatitis B virus surface antigen (HBsAg), Escherichia coli beta-galactosidase (beta-gal), or canine factor IX (cFIX) from the immediate early promoter of human cytomegalovirus (hCMV) to infect mouse liver by direct injection or through the portal vein . By either route, high levels of transgene expression were demonstrated by the detection of immunoreactive HBsAg or cFIX in the circulation and by histochemical detection of beta-gal activity in situ . The results were striking in that the serum level of cFIX reached 10% of the normal murine levels . Although the level of transgene expression from the hCMV promoter was transient, a significant number of persistent vectors could be rescued from the livers of recipient mice up to 2 months after inoculation . Replacement of the hCMV promoter with the HSV-1 latency-associated transcript (LAT) promoter resulted in reduced but prolonged expression of both HBsAg and cFIX . The very high level of factor IX expression suggests that clinically useful gene transfer may eventually be feasible through direct vector delivery to the liver.

Brain Res Mol Brain Res, 1992 Mar, 13(1-2), 7 - 17
Molecular cloning of calmodulin mRNA species which are preferentially expressed in neurons in the rat brain; Ni B et al.; A cDNA clone designated NGB, which was isolated from a rat brain expression library, detected two mRNA species of 1.8 and 4.0 kb which are highly enriched in brain tissue . cDNAs NGB1 and NGB2 corresponding to these two mRNAs have been isolated and characterized . Sequence data showed that both mRNA species contain the same open reading frames but differ in their 3' untranslated regions . The open reading frame encodes a calmodulin protein of 148 amino acids . Both mRNA species are derived from the rat CaMI gene by utilization of different polyadenylation addition sites . Analysis of the 3' untranslated sequence which is unique to the larger mRNA species revealed a putative AU-rich 'destabilizer' sequence which is thought to be involved in mechanisms of selective mRNA breakdown . In situ hybridization studies revealed that the two calmodulin mRNAs are expressed strongly in neuronal cells in the adult rat brain . Levels of the two mRNA species increased during early postnatal development.

J Appl Bacteriol, 1992 Mar, 72(3), 233 - 43
The PhoE porin and transmission of the chemical stimulus for induction of acid resistance (acid habituation) in Escherichia coli; Rowbury RJ et al.; Escherichia coli K12 becomes resistant to killing by acid (habituates to acid) in a few minutes at pH 5.0 . Habituation involves protein synthesis-dependent and -independent stages; both must occur at an habituating pH . The habituation sensor does not detect increased delta pH (or decreased delta psi) nor an increased difference between pHo and periplasmic pH but probably detects a fall in either external or periplasmic pH . Phosphate ions inhibit habituation, at any stage, probably by interfering with outer membrane passage of hydrogen ions . Most outer membrane components tested are not required for habituation but phoE deletion mutants habituated poorly and are acid-resistant . Strains derepressed for phoE, in contrast, showed increased acid sensitivity . These and other results suggest that habituation involves hydrogen ions or protonated carriers crossing the outer membrane preferentially via the PhoE pore, a process inhibited by phosphate and other anions . Stimulation by phosphate of the poor growth of E . coli at pH 5.0 is in accord with the above . Acetate did not enhance acid killing of pH 5.0 cells, suggesting that their resistance does not depend on maintaining pHi near to neutrality at an acidic pHo level.

J Virol Methods, 1992 Mar, 36(3), 277 - 82
Establishment of a genomic bank of bovine herpesvirus 1 using a novel positive selection plasmid vector; Christensen LS et al.; A small positive selection cloning vector, designated pSiig1, suitable for the construction of genomic banks in E . coli is described and used for the establishment of a bank of the bovine herpesvirus 1 genome . Hybrid transformants are directly selected on agar plates containing ampicillin . The vector is based on the replicon of R1 and has a lambda PR promotor inserted upstream of the replication control genes . The vector has an uncontrolled (runaway) replication and is lethal to the host cell unless the PR promotor is brought under trans-acting control of the lambda cI repressor or runaway replication is blocked by an insertion between the PR promotor and the replicon . The vector contains a unique Bg/II site between PR and the replicon which is suitable for insertion of genomic DNA.

Mol Gen Genet, 1992 Mar, 232(1), 117 - 25
The promoter region of the Escherichia coli pepD gene: deletion analysis and control by phosphate concentration; Henrich B et al.; A series of deletions removing progressively larger parts of the 5' flanking region of the Escherichia coli pepD gene was constructed . After fusing the resulting promoter fragments to the chromosomal malPQ operon, their activities were determined by assaying for amylomaltase, the product of the malQ gene . Transcription from the pepD promoter region in exponentially growing cells was estimated to be about 5 times less efficient than transcription from the induced lac promoter . Approximately 115 bp preceding the translation start site of the pepD gene are important for regular promoter functioning, whereas the more distal sequences could be deleted without any significant effects . In bacterial cultures containing limiting amounts of inorganic phosphate, the rate of de novo synthesis of peptidase D, simultaneously with the derepression of alkaline phosphatase, increased about fivefold as a consequence of phosphate starvation . This regulation was shown to occur at the transcriptional level by the use of chromosomal pepD promoter-malPQ fusions . The inducibility by phosphate limitation was conserved in all of the deletion clones in which the pepD promoter region was still functional . As demonstrated by the use of phoB, R, and M mutants, the modulation of pepD expression is independent of the genetic system controlling the pho regulon.

Mol Microbiol, 1992 Mar, 6(5), 599 - 604
SecB-binding does not maintain the translocation-competent state of prePhoE; de Cock H et al.; The role of SecB protein in the export of the precursor of outer membrane protein PhoE and mutant forms of this precursor was studied in vitro . When synthesized in the absence of SecB, translocation-competent prePhoE was observed post-translationally, but addition of SecB was required for efficient translocation into inner membrane vesicles . The translocation competency of in vitro synthesized prePhoE diminished with a similar half-life during incubations in the presence or absence of SecB . The loss of translocation competency of prePhoE, synthesized in the presence of SecB, was not due to dissociation of prePhoE-SecB complexes as could be demonstrated in co-immunoprecipitation experiments with anti-SecB serum . Apparently, SecB does not maintain the translocation-competent conformation of prePhoE, but is mainly required for efficient targeting of this precursor to the export apparatus.

Gene, 1992 Mar 1, 112(1), 97 - 100
Purification and N-terminal amino acid sequences of two polypeptides encoded by the mcrB gene from Escherichia coli K-12; Zheng L et al.; This report provides a purification method for the two proteins, 51 kDa and 33 kDa, both encoded by the same mcrB gene of the McrBC restriction system in Escherichia coli K-12 . The two proteins were produced in large quantity using a T7 expression system and copurified to near homogeneity by DEAE-Sepharose and Affi-Gel blue column chromatography . The N-terminal amino acid sequences of these purified McrB proteins were the same as those predicted from the mcrB DNA sequence by Ross et al . {J . Bacteriol . 171 (1989b) 1974-1981} . The 33-kDa protein totally overlaps the C-terminal part of the 51-kDa protein.

J Bacteriol, 1992 Mar, 174(6), 2043 - 6
Anaerobic induction of the alkylation-inducible Escherichia coli aidB gene involves genes of the cysteine biosynthetic pathway; Matijasevic Z et al.; The Escherichia coli aidB gene is a component of the adaptive response to alkylation damage . This gene is subject to two different forms of induction: an ada-dependent alkylation induction and an ada-independent induction that occurs when cells are grown anaerobically (M . R . Volkert, L . I . Hajec, and D . C . Nguyen, J . Bacteriol . 171:1196-1198, 1989; M . R . Volkert, and D . C . Nguyen, Proc . Natl . Acad . Sci . USA 81:4110-4114, 1984) . In this study, we isolated and characterized strains bearing mutations that specifically affect the anaerobic induction pathway . This pathway requires a functional cysA operon, which encodes sulfate permease . Mutations in cysA block this pathway of aidB induction . In contrast, mutations in either cysH, cysD, cysN, or cysC result in elevated levels of aidB expression during aerobic growth . These results indicate that the sulfate transport genes perform a role in anaerobic induction of the aidB gene and suggest that growth under anaerobic conditions may modify either the function or the expression of gene products encoded by the cysA operon.

EMBO J, 1992 Mar, 11(3), 847 - 53
Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force; Driessen AJ; The SecY/E protein of Escherichia coli was coreconstituted with the proton pump bacteriorhodopsin and cytochrome c oxidase yielding proteoliposomes capable of sustaining a protonmotive force (delta p) of defined polarity and composition . Proteoliposomes support the ATP- and SecA-dependent translocation of proOmpA which is stimulated by a delta p, inside acid and positive . delta p of opposite polarity, inside alkaline and negative, suppresses translocation while SecA-mediated ATP hydrolysis continues unabated . delta psi and delta pH are equally effective in promoting or inhibiting translocation . Membrane-spanning translocation intermediates move backwards in the presence of a reversed delta p . These results support a model {Schiebel, E., Driessen, A.J.M., Hartl, F.-U . and Wickner, W . (1991) Cell, 64, 927-939} in which the delta p defines the direction of translocation after ATP hydrolysis has released proOmpA from its association with SecA . The polarity effect of the delta p challenges models involving delta p-dependent membrane destabilization and provides further evidence for a role of the delta p as driving force in precursor protein translocation.

J Gen Virol, 1992 Mar, 73 ( Pt 3), 667 - 72
Demonstration of a hepatitis C virus-specific antigen predicted from the putative core gene in the circulation of infected hosts; Takahashi K et al.; An ELISA was used to detect a protein derived from the core gene of the hepatitis C virus (HCV) in human plasma . The solid phase antibody in the assay was a murine monoclonal antibody against a synthetic peptide deduced from the putative core gene of HCV (residues 39 to 74) . An enzyme-labelled affinity-purified human antibody directed at another region within the HCV core (residues 5 to 23) was the second antibody tracer . The ELISA had a sensitivity capable of detecting a few ng/ml of the HCV core polypeptide expressed in Escherichia coli . Core antigen activity in plasma of infected hosts was detected after treatment of HCV RNA-rich fractions from buoyant density centrifugation with the detergent Tween 80 . There was a direct correlation between core antigen ELISA values of a plasma fraction and intensities of polymerase chain reaction signals for HCV RNA . These observations are consistent with the proposal that the N-terminal sequence of the predicted polyprotein of HCV is a nucleocapsid protein, and that improved core antigen assays may correlate with viraemia.

J Gen Virol, 1992 Mar, 73 ( Pt 3), 539 - 47
The myristylated virion proteins of herpes simplex virus type 1: investigation of their role in the virus life cycle; MacLean CA et al.; Herpes simplex virus type 1 (HSV-1) gene UL11 encodes a myristylated virion protein . In this paper we have characterized the UL11 product further and investigated its role in the virus life cycle . Wild-type HSV-1 strain 17syn+ expresses three electrophoretically distinguishable UL11 polypeptide species . Analysis of single plaque isolates demonstrated that two virus populations exist within the 17syn+ stock: a major population encoding only the two higher Mr species, and a minor population encoding the lowest Mr species alone . DNA sequence analysis suggests that the latter polypeptide differs from the former ones at a single amino acid residue only . The UL11 polypeptides are synthesized as delayed early gene products and are phosphorylated in vitro . Following subcellular fractionation of infected cells, they are found predominantly associated with membranes . Within the virus particle, they appear to reside within the tegument . An insertion mutant containing the lacZ gene from Escherichia coli within the UL11 open reading frame is viable in tissue culture, although it gives smaller plaques and is impaired for growth compared to the wild-type parent or revertant viruses; it does not have a temperature-sensitive or host-range phenotype . Thus, although required for efficient replication, the myristylated HSV-1 virion protein, in contrast to those of many other viruses, is not essential for virus growth in tissue culture.

J Gen Virol, 1992 Mar, 73 ( Pt 3), 521 - 30
Characterization of the varicella-zoster virus gene 61 protein; Stevenson D et al.; The protein predicted to be encoded by varicella-zoster virus (VZV) gene 61 exhibits limited amino acid sequence similarity to the herpes simplex virus type 1 nuclear phosphoprotein Vmw110, which functions as a transcriptional activator . The gene 61 protein was expressed in its entirety, or as an amino- or carboxy-terminal fragment in Escherichia coli and vaccinia virus recombinants, and monospecific rabbit antisera were raised against an E . coli fusion between beta-galactosidase and the majority of the gene 61 protein . Use of the antisera showed that the gene 61 protein is present in VZV-infected cell nuclei as a heterogeneous phospho-protein of Mr62K to 65K . Phosphorylation occurs in the amino- and, to a lesser extent, carboxy-terminal portions of the protein . The carboxy-terminal region directs transport of the protein to the nucleus, whereas the amino-terminal region, which contains a potential zinc-binding domain, is responsible for a punctate distribution . Preliminary mapping data indicated that gene 61 is transcribed as a 1.8 kb mRNA which initiates about 65 bp upstream from the translation initiation codon, at a position located appropriately with respect to potential regulatory elements.

Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1636 - 40
Expression of nerve growth factor in vivo from a defective herpes simplex virus 1 vector prevents effects of axotomy on sympathetic ganglia; Federoff HJ et al.; Sympathetic neurons in the superior cervical ganglion (SCG) of adult rats depend on target-derived nerve growth factor (NGF) for maintenance of tyrosine hydroxylase (TH) levels and the noradrenergic neurotransmitter system . Axotomy of a SCG results in NGF deprivation, causing a decline in TH activity; continuous local application of NGF can prevent this decline in TH activity . We now report that injection of a defective herpes simplex virus 1 vector that expresses NGF (pHSVngf) into a SCG can prevent the decline in TH activity that follows axotomy . SCG of adult rats were injected with either pHSVngf virus or pNFlac virus, which expresses Escherichia coli beta-galactosidase . Analysis of RNA from pHSVngf-infected SCG indicated that the NGF gene was efficiently transcribed and processed . Furthermore, 4 days after pHSVngf injection animals underwent axotomy of the virus-injected SCG . After another 10 days, animals were sacrificed and both the injected-axotomized and contralateral control ganglia were assayed for TH activity . Axotomy of SCG injected with pNFlac virus produced a 50% decline in TH activity relative to control ganglia (P = 0.02) . In contrast, SCG injected with pHSVngf virus did not show a decline in TH activity following axotomy; instead, these ganglia manifested an 18% increase in TH levels relative to control ganglia . These data demonstrate that herpes simplex virus 1 vectors can be used to modify neuronal physiology in vivo; specifically, expression of a critical gene product by neural cells that do not normally produce it has potential applications for gene therapy.

Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1544 - 7
Artificial mobile DNA element constructed from the EcoRI endonuclease gene; Eddy SR et al.; There exist several examples of mobile group I introns . These introns appear to use a straightforward mechanism to achieve highly site-specific and efficient insertion into homologous intronless genes . Because the only intron-specific function required by the prevailing model for the mechanism of intron mobility is the introduction of a site-specific double-stranded break in the intronless recipient DNA molecule, we reasoned that it should in principle be possible to construct artificially mobile DNA sequences . We have constructed an artificial mobile element from the gene for the restriction enzyme EcoRI that is capable of site-specific insertion at rates near those of authentic mobile introns . The generality of the mobility mechanism may enable high-efficiency targeted gene replacements or disruptions in a variety of organisms.

J Bacteriol, 1992 Mar, 174(5), 1690 - 3
Cloning and identification of the Escherichia coli murB DNA sequence, which encodes UDP-N-acetylenolpyruvoylglucosamine reductase; Pucci MJ et al.; The murB gene, which complemented the UDP-N-acetylenolpyruvoylglucosamine reductase (EC 1.1.1.158) mutation in Escherichia coli ST5, was cloned from an E . coli chromosomal library . murB was subcloned on a 2.8-kb PvuII fragment into pUC19 and sequenced . A 1,029-bp open reading frame encoded a 342-amino-acid polypeptide of 37,859 Da . A DNA sequence homology search revealed that murB had almost 100% homology with a previously reported unidentified open reading frame, ORFII, at 89.9 min . Physical and genetic mapping results were consistent with this map position, and minicell analyses of murB subclones showed a plasmid-encoded protein of approximately 37,000 Da, which closely matched the calculated size of the murB protein.

J Bacteriol, 1992 Mar, 174(5), 1522 - 7
Molecular analysis of the signaling pathway between EnvZ and OmpR in Escherichia coli; Waukau J et al.; OmpR is a DNA-binding protein that regulates transcription of ompF and ompC . The activity of OmpR is controlled by the inner membrane osmosensor, EnvZ . In order to study the signaling process between EnvZ and OmpR, we analyzed two different envZ strains: the envZ473 strain, in which OmpC is constitutively produced and OmpF is fully repressed, and the envZ3 strain, in which the production of OmpC is greatly reduced and OmpF is not fully repressed by high-osmolarity growth conditions . Using direct sequencing of DNA derived from the polymerase chain reaction amplification method, we identified the mutation in the envZ473 strain as a Val-241-to-Gly substitution and the mutation in the envZ3 as an Ala-219-to-Val substitution . The relative DNA-binding affinity of OmpR derived from the envZ473 strain was dramatically increased for the upstream sequence of both ompF and ompC . In contrast, OmpR derived from the envZ3 strain was not converted to the high-affinity form . The intracellular levels of OmpR-phosphate, as analyzed by the in vivo phosphorylation approach, significantly increased in the envZ473 strain, while in the envZ3 strain the levels were considerably reduced, relative to those found in the parent strain . The intracellular level of OmpR protein in the envZ473 strain was also found to be markedly elevated relative to that of the parent strain . These results are discussed in relation to the role of phosphorylation and relative DNA-binding affinity of OmpR in the expression of ompF and ompC.

Exp Neurol, 1992 Mar, 115(3), 400 - 13
Persistence of plasmid DNA and expression in rat brain cells in vivo; Jiao S et al.; The purpose of this study was to determine whether plasmid DNA is able to persist in nondividing or slowly dividing brain cells in vivo . A new cationic lipid formulation which contains 70 mol% of DOTMA (N{1-(2,3-dioleyloxy)propyl}-N, N, N-trimethylammonium) and 30 mol% of cholesterol was used to transfect reporter genes into fetal brain cells in culture that were then transplanted into adult host brains . Gene expression was localized both to glial and neuronal cells after transfection of fetal brain cells with pRSVLac-Z, the gene coding for Escherichia coli beta-galactosidase protein . After the transfection of pRSVL plasmid which contains the firefly luciferase gene into fetal brain cells that were transplanted, substantial amounts of luciferase and pRSVL DNA were present in the host brains for 1 to 2 months . These results have implications for intracerebral viral infections and gene therapy of brain disorders.

Exp Neurol, 1992 Mar, 115(3), 303 - 16
Introduction of a foreign gene (Escherichia coli lacZ) into rat neostriatal neurons using herpes simplex virus mutants: a light and electron microscopic study; Huang Q et al.; Introducing genes into adult neurons in vivo may be a useful experimental tool for studying and modifying neuronal function . In this study two herpes simplex virus type 1 (HSV-1) mutants were used to examine the capability of different types of neostriatal neurons to express a foreign gene introduced through viral infection . In these HSV-1 mutants (7134 and RH105) the Escherichia coli gene, lacZ, under the control of viral promoters active during the early phase of infection, was substituted for viral genes (ICPO and TK, respectively) needed for efficient replication in the nervous system . Adult male rats received unilateral injections of HSV-1 mutant 7134 or RH105 into the neostriatum . Animals survived for 1 to 70 days with no apparent adverse physiological or behavioral effects . At the injection site, both mutant viruses produced focal tissue necrosis and reactive gliosis . Histochemical detection of the lacZ gene product, beta-galactosidase (beta Gal), revealed extensive labeling of neurons with mutant 7134 and relatively limited neuronal labeling with the mutant RH105 . Mutant 7134, which is capable of some replication in cells, conferred beta Gal expression in cells over an area that was twofold greater than the necrotic area . In contrast, mutant RH105, which cannot replicate in cells, produced a zone of beta Gal-labeled cells only two-thirds the area of the necrotic core . Both medium- and large-sized neostriatal neurons were positive for beta Gal, and a higher proportion of large cells were labeled as compared to other neuronal populations in the normal striatum . A few glial cells were also beta Gal-positive . Retrograde transport of virus to the substantia nigra pars compacta and to the cortex was minimal and occurred only with mutant 7134 . No evidence was seen for anterograde transport . Immunohistochemical localization of beta Gal at the ultrastructural level after inoculation with mutant 7134 revealed that both types of medium-sized neurons (spiny and aspiny types), as well as large neurons, were infected 3 days following inoculation . Immunoreactive neurons ranged from severely pathologic to remarkably healthy . Some of the axon terminals that contacted beta Gal-immunoreactive dendrites and spines were degenerated . These results demonstrate that in the adult rat replication-deficient HSV-1 vectors injected intrastriatally can be used to express a foreign gene in at least three types of neostriatal neurons, while maintaining the long-term survival and general health of the injected animals . The neurotoxicity induced by HSV-1 mutants may still be considerable, however, and ways of minimizing neuropathological effects need to be addressed.

Arch Biochem Biophys, 1992 Mar, 293(2), 246 - 53
Anomalous effect of uncouplers on respiratory chain-linked transhydrogenation in Escherichia coli membranes: evidence for a localized proton pathway?
Chang DY, Hou C, Bragg PD.
Energization of the pyridine nucleotide transhydrogenase in everted membrane vesicles from Escherichia coli JM83 was compared with the process in vesicles of the same strain transformed with the plasmid pDC21 overexpressing this enzyme . Proton translocation was assayed by the quenching of the fluorescence of the probe quinacrine . Agents able to discharge transmembrane proton gradients such as nigericin and the uncouplers 3,3',4',5-tetrachlorosalicylanilide and carbonyl cyanide m-chlorophenylhydrazone inhibited ATP-dependent transhydrogenation of NADP by NADH and discharged transmembrane proton gradients generated by transhydrogenation of AcNAD by NADPH, by oxidation of NADH, and by hydrolysis of ATP . This was observed in everted membrane vesicles of both strains JM83 and JM83pDC21 . These strains differed significantly in the response of the NADH oxidation-dependent transhydrogenase . This reaction was inhibited by nigericin and uncouplers in membrane vesicles of JM83 but there was little inhibition or the reaction was stimulated in JM83pDC21, in spite of the discharge of the NADH oxidation-generated proton gradient measured by quinacrine fluorescence in the latter strain . It is proposed that the transhydrogenase is energized by direct or local (nonbulk phase) proton translocation in membranes of this strain . Uncouplers might facilitate these routes but would not discharge them . The generality of these observations was shown using other strains . NADH oxidase activity was severalfold lower in membrane vesicles of JM83pDC21 compared with JM83 . The levels of ubiquinone and cytochromes, and the activities of NADH dehydrogenases I and II, and of cytochrome oxidase, were similar in the two strains . It is concluded that the NADH oxidase activity of JM83pDC21 is low because of the reduced rate of collision between electron-transferring complexes of the respiratory chain due to the large amount of transhydrogenase protein in the membranes of this strain . The large amount of transhydrogenase favors direct, nonbulk phase proton transfer . Transhydrogenase activity was stimulated by Ca2+, Mg2+, or Mn2+.

J Virol, 1992 Mar, 66(3), 1468 - 75
Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses; Battini JL et al.; The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor . In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB . We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted . These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay . Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged . The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function . Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins . Downstream sequences contribute to the stabilization of the receptor-specific structure.

Cancer Res, 1992 Mar 1, 52(5), 1082 - 6
Mutagenic specificity of oxygen radicals produced by human leukemia cells; Reid TM et al.; An important source of endogenous oxygen radicals are phagocytic cells such as neutrophils and macrophages . The human leukemia cell line HL-60 can be induced to differentiate into a neutrophil-like cell population . Among the properties of these differentiated cells is the ability to produce reactive oxygen species when stimulated by tumor promoters . Mutagenesis induced by HL-60-generated free radicals was assessed using the M13mp2 forward mutation assay . Single-stranded M13mp2 DNA was coincubated with phorbol ester-stimulated HL-60 cells, after which mutations were scored by transfecting the DNA into SOS-induced Escherichia coli . The mutation frequency was increased 6-fold above background in DNA incubated with HL-60 cells . The majority of the mutations were single-base substitutions . However, approximately 6% of the mutations were tandem double substitutions that occurred in runs of adjacent cytidines . Overall, the mutations were clustered at apparent "hot spots," many of which were similar to sites seen using iron to generate oxygen radicals . These results suggest that human cells able to produce oxygen radicals in response to tumor promoters might play a significant role in the generation of tumors.

Protein Sci, 1992 Mar, 1(3), 356 - 62
A functional protein hybrid between the glucose transporter and the N-acetylglucosamine transporter of Escherichia coli; Hummel U et al.; The glucose and N-acetylglucosamine-specific transporters (IIGlc/IIIGlc and IIGlcNAc) of the bacterial phosphotransferase system mediate carbohydrate uptake across the cytoplasmic membrane concomitant with substrate phosphorylation . The two transporters have 40% amino acid sequence identity . Eight chimeric proteins between the two transporters were made by gene reconstruction . All hybrid proteins could be expressed, some inhibited cell growth, and one was active . The active hybrid transporter consists of the transmembrane domain (residues 1-386) of the IIGlc subunit and the two hydrophilic domains (residues 370-648) of IIGlcNAc . The N-terminal hydrophilic domain of IIGlcNAc contains the transiently phosphorylated cysteine-412 . The hybrid protein is specific for glucose, which indicates that the sugar specificity determinant is in the transmembrane domain and that the cysteine from which the phosphoryl group is transferred to the substrate is not part of the binding site . The protein sequence (LKTPGRED) at which the successful fusion occurred has the characteristic properties of an interdomain oligopeptide linker (Argos, P., 1990, J . Mol . Biol . 211, 943-958).

Plant J, 1992 Mar, 2(2), 193 - 202
Characterization of cDNAs encoding two isoforms of granule-bound starch synthase which show differential expression in developing storage organs of pea and potato; Dry I et al.; We have isolated cDNA clones to two isoforms of granule-bound starch synthase (GBSS) from pea embryos and potato tubers . The sequences of both isoforms are related to that of glycogen synthase from E . coli and one, GBSSI, is very similar to the waxy protein of maize and other species . In pea, GBSSII carries a novel 203-amino-acid domain at its N-terminus . Genes encoding both proteins are expressed during pea embryo development, but GBSSII is most highly expressed earlier in development than GBSSI . Similarly, GBSSI and GBSSII are differentially expressed in developing potato tubers . Expression of both isoforms is much lower in other organs of pea than in embryos . GBSSII is expressed in every organ tested while GBSSI is not expressed in roots, stipules or flowers . The possible consequences of this differential use of GBSS isoforms are discussed.

J Helminthol, 1992 Mar, 66(1), 25 - 32
Molecular cloning of the cDNA encoding a 42 kDa antigenic polypeptide of Anisakis simplex larvae; Sugane K et al.; The gene encoding an antigenic polypeptide of Anisakis simplex larvae was studied using recombinant DNA techniques . cDNA synthesized from poly(A)-rich mRNA from A . simplex larvae was ligated into phage vector lambda gtll DNA and packaged in vitro . The phages were propagated on Escherichia coli and a lambda gtll expression library was constructed . A cDNA clone encoding a 42 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method . A clone containing cDNA for a 42 kDa protein was isolated . The gene encoding this 42 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe . The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 42 kDa polypeptide . The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.

Lancet, 1992 Feb 29, 339(8792), 521 - 3
Intestinal epithelial cell protein phosphorylation in enteropathogenic Escherichia coli diarrhoea; Manjarrez-Hernandez HA et al.; The ability of enteropathogenic Escherichia coli (EPEC) to cause diarrhoea in man is associated with the formation of characteristic histopathological lesions in small-intestine enterocytes, with gross cytoskeletal damage and loss of brush-border microvilli . Investigation of enterocyte protein phosphorylation in response to EPEC infection showed that the major phosphorylated protein, identified by immunoprecipitation, is myosin light-chain--an important cytoskeletal protein known to affect actin organisation in non-muscle cells . High enterocyte concentrations of actin and myosin were observed at sites of bacterial infection . Our findings indicate that enterocyte cytoskeletal changes in response to EPEC may be directly triggered by bacterial adherence through signal transduction pathways that stimulate protein kinase activity.

Biochem Biophys Res Commun, 1992 Feb 28, 183(1), 343 - 9
Effects of the flrA regulatory locus on biosynthesis and excretion of amino acids in Escherichia coli B/r; Nummer BA et al.; We have partially characterized phenotypic effects of an unusual amino acid regulatory locus, flrA, in E . coli B/r that alters the expression of the ilv and leu operons {Kline, E.L (1972) J . Bacteriol . 110:1127-1134} . This study demonstrated that a primary effect of the flrA7 mutation in haploid strains was overproduction of valine . In diploid strains (FflrA+/flrA7) this mutation resulted in excretion of valine, isoleucine, leucine, aspartate, threonine, glutamate, histidine and lysine . Increased excretion of amino acids by mutant strains might be explained by a membrane alteration or by flrA encoding a positive regulatory factor that affects the ilv operon and has pleiotropic effects on other amino acid operons.

Biochim Biophys Acta, 1992 Feb 28, 1130(1), 41 - 6
The use of oligonucleotide probes containing 2'-deoxy-2'-fluoronucleosides for regiospecific cleavage of RNA by RNase H from Escherichia coli; Schmidt S et al.; Protected 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-fluorocytidine suitable for incorporation into oligonucleotides via the phosphoramidite approach have been prepared . Five modified and two unmodified oligonucleotides have been synthesized to investigate the regiospecific cleavage of a 5S RNA from Escherichia coli by RNase H . In order to show whether the modified oligonucleotides are able to hybridize with the RNA the physico-chemical properties (melting curves, CD spectra) of analogous DNA/oligodeoxyribonucleotide duplexes have been examined . The modified oligonucleotides are shown to form stable duplexes with a DNA-matrix which exist in an A-like form . Two of the modified probes containing four 2'-deoxy-2'-fluorocytidines or two 2'-deoxy-2'-fluorouridines direct the splitting by RNase H of only one phosphodiester bond of the RNA.

Biochim Biophys Acta, 1992 Feb 28, 1130(1), 120 - 2
Cloning and nucleotide sequence of the Brucella abortus groE operon; Gor D et al.; The cloning and sequencing of the Brucella abortus groES and groEL genes are reported . The genes are adjacent on the Brucella chromosome, and presumably comprise a functional operon . Putative promoter and terminator sequences are also identified . The groES gene exhibits 60%, and the groEl gene 69%, sequence identity with the corresponding Escherichia coli genes.

Biochim Biophys Acta, 1992 Feb 28, 1130(1), 68 - 74
Regulation of the Escherichia coli lac operon expressed in human cells; Biard DS et al.; We have investigated the use of various Epstein-Barr virus (EBV)-based vectors bearing the two components of the Escherichia coli lac operator-repressor (lacO, lacI) complex . Our aim was to develop a model system of gene expression by looking at the transcription of the bacterial beta-galactosidase coding gene (lacZ) in 293 human embryonic kidney cells . Several vectors have been built carrying different promoters upstream of the lacI and lacZ genes and in which natural or synthetic operator sequences were inserted in the 5' part of the lacZ gene . In transient expression assays we achieved efficient lacZ gene repression which could be released by the specific inducer isopropyl beta-D-thiogalactoside (IPTG) . A stable transformed cell line carrying two EBV-derived plasmids with the building blocks of the lac operator/repressor system was established . This cell line allowed us to achieve a wide range of lacZ gene regulation . In this cell line IPTG alone could remove the repression to trigger a 5-fold increase of lacZ expression . Heavy metal ions, which induced the mouse metallothionein I promoter located upstream of the lacZ gene, added together with IPTG gave rise to a 40-fold induction of lacZ expression.

J Biol Chem, 1992 Feb 25, 267(6), 4223 - 35
The intracellular targeting and membrane topology of 3-hydroxy-3-methylglutaryl-CoA reductase; Olender EH et al.; We present evidence that the amino-terminal 39 residue region of 3-hydroxy-3-methylglutaryl- (HMG) CoA reductase, which includes the putative first transmembrane span, is a signal sequence for targeting HMG-CoA reductase to the endoplasmic reticulum . This evidence is based upon fractionation, endoglycosidase-H sensitivity and protease protection assays on an in vitro transcription/translocation system programmed with a mutant cDNA of HMG-CoA reductase that is deleted for sequences coding for all of the putative transmembrane spans except the first . We show that the protein product of this mutant cDNA is associated with microsomes, glycosylated, or protected from proteolysis only in the presence of Signal Recognition Particle . Also, we present evidence for a topological model of HMG-CoA reductase that consists of eight transmembrane spans . This evidence is based upon a concanavalin A binding assay for in vivo glycosylation of an engineered glycosylation site in each of a series of mutants of the fusion protein, HMGal (Skalnik, D . G., Narita, H., Kent, C., and Simoni, R . D . (1988) J . Biol . Chem . 263, 6836-6841) . This series of mutants was designed such that for each linker segment between transmembrane spans, a mutant was constructed with an engineered glycosylation site introduced into that linker segment . We show that only the mutants with glycosylation sites in the linker segments between transmembrane spans 1 and 2, 3 and 4, and 5 and 6 are glycosylated . These results support an eight transmembrane span model for the topology of HMG-CoA reductase and are inconsistent with a seven-transmembrane span model.

J Biol Chem, 1992 Feb 25, 267(6), 4074 - 83
Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork . V . Primase action regulates the cycle of Okazaki fragment synthesis; Wu CA et al.; Replication forks formed during rolling-circle DNA synthesis supported by a tailed form II DNA substrate in the presence of the primosome, the single-stranded DNA binding protein, and the DNA polymerase III holoenzyme (Pol III HE) that had been reconstituted from the purified subunits, beta, tau, and the gamma.delta complex, at limiting (with respect to nucleotide incorporation) concentrations of the Pol III core (alpha, epsilon, and theta) produced aberrantly small Okazaki fragments, while the synthesis of the leading strand was unperturbed . These small Okazaki fragments were not arrayed in tandem along the lagging-strand DNA template, but were separated by large gaps . Similarly structured synthetic products were not manufactured by replication forks reconstituted with higher, saturating concentrations of the Pol III core . Replication forks producing these small fragments could respond, by modulating the size of the Okazaki fragments produced, to variations in the concentration of NTPs or the primase, conditions that affect the frequency of priming on the lagging strand, but not to variation in the concentration of dNTPs, conditions that affect the frequency of utilization of the primers . Significantly longer Okazaki fragments (greater than 7 kilobases) could be produced in the presence of a limiting amount of Pol III core at low concentrations of the primase . These observations indicated that the production of small Okazaki fragments was not a result of a debilitated lagging-strand Pol III core, but rather a function of the time available for nascent strand synthesis during the cycle of events that are required for the manufacture of an Okazaki fragment and that it was the association of primase with the replication fork that keyed this cycle.

J Biol Chem, 1992 Feb 25, 267(6), 4045 - 53
Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork . II . Frequency of primer synthesis and efficiency of primer utilization control Okazaki fragment size; Zechner EL et al.; To investigate the role of the priming apparatus at the replication fork in determining Okazaki fragment size, the products of primer synthesis generated in vitro during rolling-circle DNA replication catalyzed by the DNA polymerase III holoenzyme, the single-stranded DNA binding protein, and the primosome on a tailed form II DNA template were isolated and characterized . The abundance of oligoribonucleotide primers and the incidence of covalent DNA chain extension of the primer population was measured under different reaction conditions known to affect the size of the products of lagging-strand DNA synthesis . These analyses demonstrated that the factors affecting Okazaki fragment length could be distinguished by either their effect on the frequency of primer synthesis or by their influence on the efficiency of initiation of DNA synthesis from primer termini . Primase and the ribonucleoside triphosphates were found to stimulate primer synthesis . The observed trend toward smaller fragment size as the concentration of these effectors was raised was apparently a direct consequence of the increased frequency of primer synthesis . The beta subunit of the DNA polymerase III holoenzyme and the deoxyribonucleoside triphosphates did not alter the priming frequency; instead, the concentration of these factors influenced the ability of the lagging-strand DNA polymerase to efficiently utilize primers to initiate DNA synthesis . Maximum utilization of the available primers correlated with the lowest mean value of Okazaki fragment length . These data were used to draw general conclusions concerning the temporal order of enzymatic steps that operate during a cycle of Okazaki fragment synthesis on the lagging-strand DNA template.

J Biol Chem, 1992 Feb 25, 267(6), 4030 - 44
Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork . I . Multiple effectors act to modulate Okazaki fragment size; Wu CA et al.; The coordinated action of many enzymatic activities is required at the DNA replication fork to ensure the error-free, efficient, and simultaneous synthesis of the leading and lagging strands of DNA . In order to define the essential protein-protein interactions and model the regulatory pathways that control Okazaki fragment synthesis, we have reconstituted the replication fork of Escherichia coli in vitro in a rolling circle-type DNA replication system . In this system, in the presence of the single-stranded DNA binding protein, the helicase/primase function on the lagging-strand template is provided by the primosome, and the synthesis of DNA strands is catalyzed by the DNA polymerase III holoenzyme . These reconstituted replication forks synthesize equivalent amounts of leading- and lagging-strand DNA, move at rates comparable to those measured in vivo (600-800 nucleotides/s at 30 degrees C), and can synthesize leading strands in the range of 150-500 kilobases in length . Using this system, we have studied the cycle of Okazaki fragment synthesis at the replication fork . This cycle is likely to have several well defined decision points, steps in the cycle where incorrect execution by the enzymatic machinery will result in an alteration in the product of the reaction, i.e . in the size of the Okazaki fragments . Since identification of these decision points should aid in the determination of which of the enzymes acting at the replication fork control the cycle, we have endeavored to identify those reaction parameters that, when varied, alter the size of the Okazaki fragments synthesized . Here we demonstrate that some enzymes, such as the DnaB helicase, remain associated continuously with the fork while others, such as the primase, must be recruited from solution each time synthesis of an Okazaki fragment is initiated . We also show that variation of the concentration of the ribonucleoside triphosphates and the deoxyribonucleoside triphosphates affects Okazaki fragment size, that the control mechanisms acting at the fork to control Okazaki fragment size are not fixed at the time the fork is assembled but can be varied during the lifetime of the fork, and that alteration in the rate of the leading-strand DNA polymerase cannot account for the effect of the deoxyribonucleoside triphosphates.

J Biol Chem, 1992 Feb 25, 267(6), 3841 - 6
An analysis of the side chain requirement at position 177 within the lactose permease which confers the ability to recognize maltose; Gram CD et al.; In previous work (Brooker, R . J., and Wilson, T . H . (1985) Proc . Natl . Acad . Sci . U . S . A . 82, 3959-3963), lactose permease mutants were isolated which possessed an enhanced recognition for maltose . In some of these mutants, the wild-type alanine residue at position 177 was changed to valine or threonine . To gain further insight into the side chain requirement at position 177 that confers maltose recognition, further substitutions of isoleucine, leucine, phenylalanine, proline, and serine have been made via site-directed mutagenesis . Permeases containing alanine or serine exhibited poor maltose recognition whereas those containing isoleucine, leucine, phenylalanine, proline, or valine showed moderate or good recognition . As far as galactosides are concerned, the Val-177, Pro-177, and Ser-177 mutants were able to transport lactose as well as, or slightly better than, the wild-type strain . The other mutants displayed moderately reduced levels of lactose transport . For example, the Phe-177 mutant, which was the most defective, showed a level of downhill transport which was approximately 20% that of the wild-type strain . In uphill transport assays, all of the position 177 mutants were markedly defective in their ability to accumulate beta-D-thiomethylgalactopyranoside against a concentration gradient . Finally, the position 177 mutants were analyzed for their ability to catalyze an H+ leak . Interestingly, even though the wild-type permease does not leak H+ across the bacterial membrane, all of the position 177 mutants were shown to transport H+ in the absence of sugars . For most of the mutants, this H+ leak was blocked by the addition of beta-D-thiodigalactoside . Overall, these results are discussed with regard to the effects of position 177 substitutions on the sugar recognition site and H+ transport.

J Biol Chem, 1992 Feb 25, 267(6), 3731 - 4
Carbonic anhydrase in Escherichia coli . A product of the cyn operon; Guilloton MB et al.; The product of the cynT gene of the cyn operon in Escherichia coli has been identified as a carbonic anhydrase . The cyn operon also includes the gene cynS, encoding the enzyme cyanase . Cyanase catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide . The carbonic anhydrase was isolated from an Escherichia coli strain overexpressing the cynT gene and characterized . The purified enzyme was shown to contain 1 Zn2+/subunit (24 kDa) and was found to behave as an oligomer in solution; the presence of bicarbonate resulted in partial dissociation of the oligomeric enzyme . The kinetic properties of the enzyme are similar to those of carbonic anhydrases from other species, including inhibition by sulfonamides and cyanate . The amino acid sequence shows a high degree of identity with the sequences of two plant carbonic anhydrases . but not with animal and algal carbonic anhydrases . Since carbon dioxide formed in the bicarbonate-dependent decomposition of cyanate diffuses out of the cell faster than it would be hydrated to bicarbonate, the apparent function of the induced carbonic anhydrase is to catalyze hydration of carbon dioxide and thus prevent depletion of cellular bicarbonate.

J Biol Chem, 1992 Feb 25, 267(6), 4084 - 96
The signal for Golgi retention of bovine beta 1,4-galactosyltransferase is in the transmembrane domain; Teasdale RD et al.; The expression and localization of bovine beta 1,4-galactosyltransferase (Gal T) has been studied in mammalian cells transfected with Gal T cDNA constructs, and the role of the amino-terminal domains of Gal T in Golgi localization examined . Here we demonstrate that the transmembrane (signal/anchor) domain of bovine Gal T contains a positive Golgi retention signal . Bovine Gal T was characterized in transfected cells with anti-bovine Gal T antibodies, affinity-purified from a rabbit antiserum using a bacterial recombinant fusion protein . These affinity-purified antibodies recognized native bovine Gal T and showed minimum cross-reactivity with Gal T from non-bovine sources . Bovine Gal T cDNA was expressed, as active enzyme, transiently in COS-1 cells and stably in murine L cells, and the product was shown to be localized to the Golgi complex by immunofluorescence using the polypeptide-specific antibodies . A low level of surface bovine Gal T was also detected in the transfected L cells by flow cytometry . The removal of 18 of the 24 amino acids from the cytoplasmic domain of bovine Gal T did not alter the Golgi localization of the product transiently expressed in COS-1 cells or stably expressed in L cells . Both the full-length bovine Gal T and the cytoplasmic domain deletion mutant were N-glycosylated in the transfected L cells, indicating both proteins have the correct N(in)/C(out) membrane orientation . Deletion of both the cytoplasmic and signal/anchor domains of bovine Gal T and incorporation of a cleavable signal sequence resulted in a truncated soluble bovine Gal T that was rapidly secreted (within 1 h) from transfected COS-1 cells . Replacement of the signal/anchor domain of bovine Gal T with the signal/anchor domain of the human transferrin receptor resulted in the transport of the hybrid molecule to the cell surface of transfected COS-1 cells . Furthermore, a hybrid construct containing the signal/anchor domain of Gal T with ovalbumin was efficiently retained in the Golgi complex, whereas ovalbumin anchored to the membrane by the transferrin receptor signal/anchor was expressed at the cell surface of transfected COS-1 cells . Overall, these studies show that the hydrophobic, signal/anchor domain of Gal T is both necessary and sufficient for Golgi localization.

Nucleic Acids Res, 1992 Feb 25, 20(4), 811 - 7
Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K; Wu F et al.; A dnaA 'null' strain could not support replication of intact plasmid R6K or derivatives containing combinations of its three replication origins (alpha, gamma, beta) . DnaA binds in vitro to sites in two functionally distinct segments of the central gamma origin . The 277-bp core segment is common to all three origins and contains DnaA box 2, which cannot be deleted without preventing replication . Immediately to the left of the core lies the 106-bp origin enhancer, which contains DnaA box 1 . When the origin enhancer is deleted, the core alone can still initiate replication if levels of wt pi protein are decreased or if copy-up pi mutant proteins are provided in trans . DnaA does not effect expression of R6K replication initiator protein pi, although several DnaA boxes were identified in the coding segment of the pir gene, which encodes pi . Together these data suggest that a single DnaA box, 2, is sufficient for initiation from the gamma origin and might be sufficient for initiation from the gamma origin and might be sufficient and required for the activity of the alpha and beta origins as well . Implications of the DnaA protein binding to two domains of the gamma origin and the role of the 106-bp origin enhancer in replication are discussed.

Nucleic Acids Res, 1992 Feb 25, 20(4), 879 - 87
Homologies between the contiguous and fragmented rRNAs of the two Plasmodium falciparum extrachromosomal DNAs are limited to core sequences; Feagin JE et al.; Plasmodium falciparum contains two extrachromosomal DNAs, a 6 kb linear element and a 35 kb circular DNA; both encode rDNA sequences . The 6 kb element rDNAs comprise fragments of both large and small subunit rRNAs . Comparison of these with corresponding rDNA sequences from the 35 kb DNA and E . coli show that sequences conserved between the three are largely confined to highly conserved core regions; in fact, most of the 6 kb rDNA sequences correspond to core regions . Both the 6 kb element and 35 kb rDNAs show less conservation to each other than to E . coli sequences, suggesting that the two extrachromosomal DNAs of P . falciparum are not closely related . The characteristics of the fragmented rRNAs from the 6 kb element suggest they are functional, possibly in mitochondrial ribosomes.

Nucleic Acids Res, 1992 Feb 25, 20(4), 719 - 26
Both fis-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent; Newlands JT et al.; Transcription from the Escherichia coli rrnB P1 promoter is increased by a cis-acting sequence which extends upstream of the -35 hexamer to about -150 with respect to the transcription initiation site, the Upstream Activation Region (UAR) . Activation by the UAR involves two components: (1) a trans-acting protein, Fis, which binds to three sites in the UAR between -60 and -150, and (2) the UAR sequences themselves which affect RNA polymerase (RNAP) activity independent of other proteins . We refer to the latter as Factor-Independent Activation (FIA) . In addition to its interactions with the -10 and -35 hexamers typical of E . coli promoters, RNAP makes contacts to the -53 region of rrnB P1, which may be related to the FIA effect . We constructed a series of insertion mutants containing integral and non-integral numbers of helical turns at position -46, between the Fis binding sites and the -35 region, and the resulting promoter activities were measured in vitro and in vivo . The data suggest that both Fis-dependent and factor-independent activation are face of the helix dependent: the Fis binding site and the sequences responsible for factor-independent activation must be correctly oriented relative to RNA polymerase in order to activate transcription . These results, in conjunction with other evidence, support a model for the involvement of direct Fis-RNAP interactions in upstream activation . We also demonstrate that RNAP interacts with the -53 region of the rrnB P1 UAR even when these sequences are displaced upstream of the RNAP binding site, and that these interactions correlate with factor-independent activation.

Nucleic Acids Res, 1992 Feb 25, 20(4), 689 - 97
Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor; Elhag GA et al.; Poly(A)+ mRNA isolated from Nicotiana tabacum (cv . Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11 . Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced . Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli . The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids . The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12 . The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12 . A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA . Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12 . Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco.

Nucleic Acids Res, 1992 Feb 25, 20(4), 665 - 9
Differential expression of genes encoding the hypusine-containing translation initiation factor, eIF-5A, in tobacco; Chamot D et al.; Two Nicotiana plumbaginifolia cDNA clones, NeIF-5A1 and NeIF-5A2, encoding eukaryotic translation initiation factor eIF-5A (formerly called eIF-4D) were cloned by heterologous screening with Dictyostelium and human eIF-5A probes . eIF-5A is the only protein known to contain a unique amino acid modification, hypusine . Comparison of the Nicotiana deduced amino acid sequences with those of other eIF-5A polypeptides reveals conservation throughout the coding sequence, especially in the region of the hypusine residue . Transcript analysis reveals that NeIF-5A1 is preferentially expressed in photosynthetic tissues, while NeIF-5A2 is constitutively expressed in all plant tissues examined . A polyclonal antibody was raised against NeIF-5A1 overexpressed in E . coli . NeIF-5A1 antiserum crossreacts with an 18 kDa polypeptide doublet in all tobacco tissues examined . At least one polypeptide of ca . 18 kDa from a diversity of higher and lower plants crossreacts with NeIF-5A1 antiserum.

J Biol Chem, 1992 Feb 25, 267(6), 4166 - 70
SecY, SecE, and band 1 form the membrane-embedded domain of Escherichia coli preprotein translocase; Brundage L et al.; The preprotein translocase of Escherichia coli is a multisubunit enzyme with two domains, the peripheral membrane protein SecA and the membrane-embedded SecY/E protein . SecY/E has been isolated as a complex of three polypeptides, SecY, SecE, and band 1 . We now present four lines of evidence that the active species of SecY/E is composed of a tightly associated complex of these three subunits: 1) antibodies to SecY efficiently precipitate SecY/E activity as well as all three polypeptides; 2) the proportions of SecY, SecE, and band 1 in the immunoprecipitates are the same as in the starting fraction; 3) the immunoprecipitable complex is not disrupted by treatment with either high salt or urea but is disrupted by brief incubation at 20 degrees C, and the kinetics of dissociation of both band 1 and SecE from SecY at 20 degrees C parallel the loss of translocation ATPase activity; 4) upon immunoprecipitation of similar units of activity of translocase from detergent solutions from either wild-type membranes or a SecY and SecE overproducer strain, the SecE and band 1 subunits are recovered in the same proportions . These data establish that the subunits of SecY/E are firmly associated and that it is the associated complex which is active for translocation.

Nucleic Acids Res, 1992 Feb 25, 20(4), 847 - 50
Processing in the 5' region of the pnp transcript facilitates the site-specific endonucleolytic cleavages of mRNA; Takata R et al.; The primary transcript of pnp, the gene encoding polynucleotide phosphorylase in Escherichia coli, is processed in the 5' end region by ribonuclease III (RNase III) . The unprocessed transcript shows enhanced stability compared with the processed transcript . We report here that, unlike the processed transcript, the unprocessed pnp transcript did not accept endonucleolytic attack at, at least, five cleavage sites . Sequencing analysis of the four cleavage products shows no sequence specific to all these sites, but AU rich stretches were observed at three sites.

Nucleic Acids Res, 1992 Feb 25, 20(4), 783 - 9
Formation of phosphonester bonds catalyzed by DNA polymerase; Victorova LS et al.; 3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma- diphosphate and 2'-deoxythymidine-5'-(alpha-methylphosphonyl)-beta, gamma- diphosphate have been synthesized . Both compounds are incorporated into DNA chains during catalysis by reverse transcriptases of human immunodeficiency (HIV) and avian myeloblastosis (AMV) viruses, DNA polymerase beta from rat liver, terminal deoxynucleotidyl transferase from calf thymus and (at a very low rate) is by E . coli DNA polymerase I, Klenow fragment . The first compound is a termination substrate while the second is capable of multiple incorporation into the DNA chains . For instance, reverse transcriptase catalysis resulted in the appearance of 8 residues of second compound . DNA polymerases alpha and epsilon from human placenta incorporated none of the above compounds into DNA chains, although an inhibition of DNA synthesis by both compounds was observed with all enzymes mentioned . The 3'----5'-exonuclease activity of DNA polymerase I, Klenow fragment, hydrolyzed DNA fragments containing phosphonomethyl internucleoside groups, while such DNA fragments were resistant to the E . coli exonuclease III.

Biochemistry, 1992 Feb 25, 31(7), 1897 - 903
Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli; Shiota S et al.; A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library . On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa . The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally . The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT . Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs . Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT . Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli . The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein . Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.

J Biol Chem, 1992 Feb 25, 267(6), 4199 - 206
A single locus encodes both phenylalanine hydroxylase and tryptophan hydroxylase activities in Drosophila; Neckameyer WS et al.; We have used a full-length clone encoding rabbit tryptophan hydroxylase (TRH) to isolate the Drosophila homologue (DTPH) . Southern analysis of Drosophila genomic DNA reveals a pattern indicative of a single gene . The single transcript is expressed in adult head and body mRNA but is also detected in mRNA from early embryos . The embryonic transcript is ubiquitously expressed and appears to concentrate in yolk granules . In situ hybridization of TRH-homologous antisense RNA probe to sectioned tissue from third instar larvae demonstrated the presence of this transcript in fat body and cuticular tissue . Developmental immunoblot analysis using antibodies raised against a beta-galactosidase-Drosophila fusion protein revealed a 45-kDa embryonic protein also detected in female abdomens and a 50-kDa protein found in larval and adult stages . Immunocytochemical analysis of the Drosophila protein in the larval central nervous system showed that it appeared to be present in both serotonin- and catecholamine-containing neurons . A nonfusion protein generated in Escherichia coli hydroxylates both tryptophan and phenylalanine . We propose that there are only two aromatic amino acid hydroxylase genes in Drosophila: one encoding tyrosine hydroxylase, DTH, and DTPH, a gene encoding both tryptophan and phenylalanine hydroxylase activities.

J Biol Chem, 1992 Feb 25, 267(6), 4177 - 82
DNA chain termination activity and inhibition of human immunodeficiency virus reverse transcriptase by carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine triphosphate; Orr DC et al.; Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir, NSC 614846) is an anti-retroviral agent that may be useful in the treatment of AIDS . We have examined the ability of (-)-enantiomeric carbovir triphosphate to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (EC 2.7.7.49) . A comparison of inhibition kinetics was made with 3'-azido-2',3'-dideoxythymidine triphosphate and phosphonoformate . Inhibition of the reverse transcriptase was evaluated using poly(rA).oligo(dT)12-18, poly(rC).oligo(dG)12-18, or influenza virion RNA template with a specific oligodeoxynucleotide as primer . (-)-Carbovir 5'-triphosphate was shown to be a potent inhibitor of HIV-1 reverse transcriptase with an apparent Ki similar to that of 3'-azido-2',3'-dideoxythymidine triphosphate . Chain elongation studies utilizing an MS2 RNA template showed that (-)-carbovir 5'-triphosphate terminated transcription at positions identical to those where dideoxy-GTP terminated . This indicates that (-)-carbovir 5'-monophosphate is incorporated into the newly synthesized DNA and terminates transcription at that point . We conclude that (-)-carbovir 5'-triphosphate is a potent inhibitor of the HIV-1 reverse transcriptase enzyme and that (-)-carbovir most likely inhibits HIV by activity at the triphosphate level by a combination of direct competition for binding of the natural deoxynucleoside triphosphates to the reverse transcriptase and chain termination.

Eur J Pharmacol, 1992 Feb 25, 212(1), 93 - 6
Effect of endotoxin on circulating cyclic GMP in the rat; Schuller F et al.; The aim of this study was to investigate the possible use of plasmatic cyclic GMP as an index of L-arginine/nitric oxide (L-Arg/NO) pathway activation by E . coli endotoxin in vivo . Endotoxin (20 mg kg-1 i.p.) caused a time-dependent increase in plasmatic cyclic GMP in anaesthetised rats which corresponded with the time course of L-Arg/NO pathway activation in aortas from the same rats, but was not prevented by a specific inhibitor of this pathway, NG-nitro-L-arginine methyl ester (1 mg kg-1 or 20 mg kg-1 h-1 i.v.) . Elevated plasmatic cyclic GMP was however also associated with an increased plasma concentration of atrial natriuretic peptide (ANP) in endotoxin-treated rats . We conclude that plasma cyclic GMP cannot be used as a direct marker of L-Arg/NO pathway activation by endotoxin but may instead be a reflection of an endotoxin-induced increase in plasma ANP activity.

J Biol Chem, 1992 Feb 25, 267(6), 4207 - 14
Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme; Roman LJ et al.; A fluorescence assay was used to measure the processivity of Escherichia coli recBCD enzyme helicase activity . Under standard conditions, recBCD enzyme unwinds an average of 30 +/- 3.2 kilobase pairs (kb)/DNA end before dissociating . The average processivity (P obs) of DNA unwinding under these conditions is 0.99997, indicating that the probability of unwinding another base pair is 30,000-fold greater than the probability of dissociating from the double-stranded DNA . The average number of base pairs unwound per binding event (N) is sensitive to both mono- and divalent salt concentration and ranges from 36 kb at 80 mM NaCl to 15 kb at 280 mM NaCl . The processivity of unwinding increases in a hyperbolic manner with increasing ATP concentration, yielding a KN value for ATP of 41 +/- 9 microM and a limiting value of 32 +/- 1.8 kb/end for the number of base pairs unwound . The importance of the processivity of recBCD enzyme helicase activity to the recBCD enzyme-dependent stimulation of recombination at Chi sites observed in vivo is discussed.

J Biol Chem, 1992 Feb 25, 267(6), 3847 - 51
The glucose transporter of Escherichia coli . Mutants with impaired translocation activity that retain phosphorylation activity; Buhr A et al.; The glucose transporter of the bacterial phosphotransferase system couples translocation with phosphorylation of the substrate in a 1:1 stoichiometry . It is a complex consisting of a transmembrane subunit (IIGlc) and a hydrophilic subunit (IIIGlc) . Both subunits are transiently phosphorylated . IIIGlc is phosphorylated at a histidyl residue by the cytoplasmic phosphoryl carrier protein phospho-heat-stable phosphoryl carrier protein; IIGlc is phosphorylated at a cysteinyl residue by phospho-IIIGlc . The IIGlc subunit consists of two domains . The N-terminal hydrophobic domain is presumed to span the membrane several times; the C-terminal cytoplasmic domain includes the phosphorylation site . IIGlc phosphorylates glucose and methyl-alpha-D-glucopyranoside in transit across the inner membrane but can also phosphorylate intracellular glucose . Ten mutants resistant against extracellular toxic methyl-alpha-D-glucopyranoside yet capable of phosphorylating intracellular glucose were isolated . Strong impairment of transport activity in these mutants was accompanied by only a slight decrease of phosphorylation activity . Amino acid substitutions occurred at six sites that are clustered in three presumably hydrophilic loops in the transmembrane domain of IIGlc: M17T, M17I, G149S, K150E, S157F, H339Y, and D343G . We presume that the three polypeptide segments are directly involved in sugar translocation and/or binding but are of little importance for phosphorylation activity, folding, and membrane localization of IIGlc.

FEBS Lett, 1992 Feb 24, 298(2-3), 145 - 8
A ribosomal protein is specifically recognized by saporin, a plant toxin which inhibits protein synthesis; Ippoliti R et al.; Many plants express enzymes which specifically remove an adenine residue from the skeleton of the 28 S RNA in the major subunit of the eukaryotic ribosome (ribosome inactivating proteins, RIPs) . The site of action of RIPs (A4324 in the rRNA from rat liver) is in a loop structure whose nucleotide sequence all around the target adenine is also conserved in those species which are completely or partially insensitive to RIPs . In this paper we identify a covalent complex between saporin (the RIP extracted from Saponaria officinalis) and ribosomal proteins from yeast (Saccharomyces cerevisiae), by means of chemical crosslinking and immunological or avidin-biotin detection . The main complex (mol . wt . congruent to 60 kDa) is formed only with a protein from the 60 S subunit of yeast ribosomes, and is not detected with ribosomes from E . coli, a resistant species . This observation supports the hypothesis for a molecular recognition mechanism involving one or more ribosomal proteins, which could provide a 'receptor' site for the toxin and favour optimal binding of the target adenine A4324 to the active site of the RIP.

FEBS Lett, 1992 Feb 24, 298(2-3), 126 - 8
Construction and characterization of a fusion protein with epidermal growth factor and the cell-binding domain of fibronectin; Kawase Y et al.; An efficient expression system was constructed for C-EGF, a fusion protein made of a fragment of the cell-binding domain of human fibronectin (FN) bound with epidermal growth factor (EGF) . C-EGF was produced in Escherichia coli HB101 cells carrying the recombinant plasmid pCE102 as inclusion bodies, which were solubilized and refolded after purification . C-EGF had both cell-adhesive and EGF activities, so it might be more effective than EGF in therapeutic applications . This fusion system would be useful for the construction of a recombinant drug delivery system for cells that have fibronectin receptors (integrins).

Biochim Biophys Acta, 1992 Feb 21, 1099(2), 170 - 4
Chemical structures critical for the induction of FMN-dependent NADH-quinone reductase in Escherichia coli; Unemoto T et al.; An FMN-dependent NADH-quinone reductase is induced in Escherichia coli by growing the cells in the presence of menadione (2-methyl-1,4-naphthoquinone) . Since the properties of induced enzyme are very similar to those of NAD(P)H: (quinone-acceptor) oxidoreductase (EC 1.6.99.2), known as DT-diaphorase, from animal cells, structural requirements of quinone derivatives as an inducer of NADH-quinone reductase in E . coli were examined . Among quinone derivatives examined, it was found that 2-alkyl-1,4-quinone structure with C-3 unsubstituted or substituted with Br is critical as a common inductive signal . Michael reaction acceptors which have been reported to be strong inducers of DT-diaphorase in mouse hepatoma cells were not always effective inducers in E . coli . However, several compounds, such as 2-methylene-4-butyrolactone, methylacrylate and methyl vinyl ketone, showed a slight inductive activity . The efficient inducers of NADH-quinone reductase in E . coli contain 1,4-quinone structure as a part of the inductive signal . These compounds belong to Michael acceptors and are likely to conjugate with thiol compounds such as glutathione.

J Med Chem, 1992 Feb 21, 35(4), 663 - 76
Crystal-structure-based design and synthesis of benz{cd}indole-containing inhibitors of thymidylate synthase; Varney MD et al.; The X-ray crystal-structure-based design, synthesis, and biological activity of a novel family of benz{cd}indole-containing inhibitors of thymidylate synthase (TS) are described . The structure-activity of the lead compound was studied by conceptually dividing the molecule into four regions and independently optimizing the substituents for each region . Combination of favored substituents for each region led to inhibitors with Ki's against the human enzyme in the range of 10-20 nM . Thymidine shift experiments suggested that the cytotoxic properties of the best enzyme inhibitors were due to TS targeting in cells . The inhibitors were synthesized from substituted 6-aminobenz{cd}indol-2(1H)-ones by alkylation with both a simple alkyl group and a substituted benzylic portion . The 2,6-diaminobenz{cd}indoles were prepared from the corresponding lactams by conversion to the thiolactam, alkylation to the methylated thiolactam, and then displacement with a substituted or unsubstituted amine.

Science, 1992 Feb 21, 255(5047), 994 - 6
The specificity of translational control switched with transfer RNA identity rules; Graffe M et al.; The interaction of Escherichia coli threonyl-transfer RNA (tRNA) synthetase with the leader sequence of its own messenger RNA inhibits ribosome binding, resulting in negative translational feedback regulation . The leader sequence resembles the substrate (tRNA(Thr)) of the enzyme, and the nucleotides that mediate the correct recognition of the leader and the tRNA may be the same . A mutation suggested by tRNA identity rules that switches the resemblance of the leader sequence from tRNA(Thr) to tRNA(Met) causes the translation of the threonyl-tRNA synthetase messenger RNA to become regulated by methionyl-tRNA synthetase . This identity swap in the leader messenger RNA indicates that tRNA identity rules may be extended to interactions of synthetases with other RNAs.

Cell, 1992 Feb 21, 68(4), 775 - 85
Expression cloning of the TGF-beta type II receptor, a functional transmembrane serine/threonine kinase; Lin HY et al.; A cDNA encoding the TGF-beta type II receptor protein has been isolated by an expression cloning strategy . The cloned cDNA, when transfected into COS cells, leads to overexpression of an approximately 80 kd protein that specifically binds radioiodinated TGF-beta 1 . Excess TGF-beta 1 competes for binding of radioiodinated TGF-beta 1 in a dose-dependent manner and is more effective than TGF-beta 2 . The predicted receptor structure includes a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a predicted cytoplasmic serine/threonine kinase domain . A chimeric protein containing the intracellular domain of the type II receptor and expressed in E . coli can phosphorylate itself on serine and threonine residues in vitro, indicating that the cytoplasmic domain of the type II receptor is a functional kinase . This result implicates serine/threonine phosphorylation as an important mechanism of TGF-beta receptor-mediated signaling.

J Mol Biol, 1992 Feb 20, 223(4), 857 - 71
Specific interaction of the first three zinc fingers of TFIIIA with the internal control region of the Xenopus 5 S RNA gene; Liao XB et al.; A DNA plasmid encoding the first 101 amino acid residues of the Xenopus 5 S RNA gene-specific transcription factor IIIA (TFIIIA) was derived by polymerase chain reaction amplification of this region from the cDNA for TFIIIA . This polypeptide includes the first three zinc fingers of the TFIIIA DNA binding domain . The polypeptide was expressed in Escherichia coli and purified to greater than 95% homogeneity . The three finger polypeptide binds the internal control region of the 5 S RNA gene with sequence specificity and high affinity . Binding is metal-dependent and treatment of the polypeptide with EDTA abolishes binding . Polypeptide-DNA complexes exhibit a dissociation constant of 5.6(+/- 0.9) nM, while that for full-length Xenopus TFIIIA is 2.2(+/- 0.4) nM, measured under the same conditions . This suggests that the majority of the free energy of TFIIIA binding resides in these amino-terminal zinc fingers . The polypeptide protects 21 base-pairs of the internal control region from attack by DNase I, with protection from nucleotides +75 to +95 of the 120 base-pair gene . This region includes the C-block promoter element and several guanine residues that are essential for TFIIIA binding . Methylation interference experiments suggest that the mode of binding of the polypeptide and TFIIIA are similar . The minimal DNA sequences required for polypeptide binding were determined using a series of synthetic double-stranded deoxyribo-oligonucleotides . A 13 base-pair oligonucleotide spanning nucleotides +80 to +92 of the 5 S RNA gene retained specific and high-affinity binding, although the latter was reduced sixfold relative to longer DNA fragments . Polypeptides containing fingers 1 and 2, or fingers 2, 3 and 4 of TFIIIA do not exhibit sequence-specific DNA binding . Overall, these studies provide strong support for a model in which the first three zinc fingers of TFIIIA bind with high affinity between nucleotides +80 and +92 of the internal control region of the 5 S RNA gene.

J Mol Biol, 1992 Feb 20, 223(4), 823 - 9
Genetical and biochemical evidence for the involvement of the coprotease domain of Escherichia coli RecA protein in recombination; Cazaux C et al.; RecA amino acid residue 204 is involved in the coprotease domain of the protein responsible for the induction of mutagenic repair . Two mutations were created at this site leading to the addition of either a methyl or an isopropyl group on the original glycine . Analyses of both the in vivo and the in vitro properties of these mutated proteins demonstrated that this residue 204 is involved in many RecA activities, suggesting that this site could allosterically direct conformational changes in the protein or could be situated in a region interacting with many RecA cofactors.

J Mol Biol, 1992 Feb 20, 223(4), 817 - 22
Intramolecular dG.dG.dC triplex detected in Escherichia coli cells; Kohwi Y et al.; The formation of an intramolecular dG.dG.dC triplex in Escherichia coli cells is demonstrated at single-base resolution . The intramolecular dG.dG.dC triplex structure was probed in situ for E . coli cells containing plasmid DNAs with varying lengths of poly(dG).poly(dC) tracts employing chloroacetaldehyde . This chemical probe reacts specifically with unpaired DNA bases . The triplex structure formed with the poly(dG).poly(dC) tracts of 35 and 44 base-pairs, but not with 25 base-pairs . The triplex was detected only one to two hours after the chloramphenicol treatment: the period at which the extracted plasmid DNA revealed the maximal superhelical density.

J Mol Biol, 1992 Feb 20, 223(4), 1177 - 82
Crystallization of beta-galactosidase from Escherichia coli; Jacobson RH et al.; Two crystal forms of beta-galactosidase have been obtained from Escherichia coli . One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A . The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees . The monoclinic form seems better suited to detailed structural analysis . The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections . On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit . The Patterson function strongly suggests that two of the tetramers are related to the other two by translation . The data are consistent with the tetramers having 222 point symmetry, but this is not proven.

J Mol Biol, 1992 Feb 20, 223(4), 1155 - 65
Two-dimensional crystals of Escherichia coli maltoporin and their interaction with the maltose-binding protein; Stauffer KA et al.; We have reconstituted Escherichia coli maltoporin into phospholipid membranes at low lipid-to-protein ratios to produce two-dimensional crystals of this membrane protein . Electron microscopy of negatively stained membranes showed three different types of arrays, two of them hexagonal and the third rectangular, all diffracting to approximately (2 nm)-1 . Furthermore, we have core-constituted maltoporin with the maltose-binding protein from E . coli, a soluble periplasmic protein that has been proposed to interact with maltoporin . One of the hexagonal arrays was found to bind maltose-binding protein molecules in a regular way, while the maltose-binding protein binding sites were not accessible in the other crystal forms . Difference maps from averaged decorated arrays and undecorated controls showed three symmetry-related maltose-binding protein binding sites per maltoporin trimer, of which not more than one is likely to be occupied at a given time . Using multivariate statistical analysis to select similar unit cells of the decorated maltoporin array, we have obtained a map showing the rough outline of a maltose-binding protein molecule interacting with the pore formed by a maltoporin trimer.

J Mol Biol, 1992 Feb 20, 223(4), 837 - 43
Two independent amsacrine-resistant human myeloid leukemia cell lines share an identical point mutation in the 170 kDa form of human topoisomerase II; Lee MS et al.; Cloning and sequencing of cDNA segments of human TOP2 gene encoding the 170 kDa form of human DNA topoisomerase II show that Arg486 of the enzyme has been mutated to a lysine in the enzyme from two human leukemia cell lines HL-60/AMSA and KBM-3/AMSA, which were independently selected for resistance to the antitumor drug amsacrine (4'-{9-acridinylamino}-methanesulfon-m-anisidide, mAMSA) . Sequence identity comparisons between eukaryotic DNA topoisomerase II and bacterial gyrase (bacterial DNA topoisomerase II) indicate that the position of the common mutation observed in mAMSA-resistant human TOP2 corresponds to that of the point mutation nal-31 in the Escherichia coli gyrase B gene, which confers resistance to nalidixic acid . Because mAMSA and nalidixic acid are known to act on their respective targets by a common mechanism of trapping the covalent enzyme-DNA intermediates, these results provide strong evidence that the 170 kDa form of human DNA topoisomerase II is a major cellular target of mAMSA, and that Arg486 of this enzyme is involved in mAMSA-mediated trapping of the covalent enzyme-DNA complex.

J Mol Biol, 1992 Feb 20, 223(4), 1029 - 52
Structural details of ribonuclease H from Escherichia coli as refined to an atomic resolution; Katayanagi K et al.; The crystal structure of RNase H from Escherichia coli has been determined by the multiple isomorphous replacement method, and refined by the stereochemically restrained least-squares procedure to a crystallographic R-factor of 0.196 at 1.48 A resolution . In the final structure, the root-mean-square (r.m.s.) deviation for bond lengths is 0.017 A, and for angle distances 0.036 A . The structure is composed of a five-stranded beta-sheet and five alpha-helices, and reveals the details of hydrogen bonding, electrostatic and hydrophobic interactions between intra- and intermolecular residues . The refined structure allows an explanation of the particular interactions between the basic protrusion, consisting of helix alpha III and the following loop, and the remaining major domain . The beta-sheet, alpha II, alpha III and alpha IV form a central hydrophobic cleft that contains all six tryptophan residues, and presumably serves to fix the orientation of the basic protrusion . Two parallel adjacent helices, alpha I and alpha IV, are associated with a few triads of hydrophobic interactions, including many leucine residues, that are similar to the repeated leucine motif . The well-defined electron density map allows detailed discussion of amino acid residues likely to be involved in binding a DNA/RNA hybrid, and construction of a putative model of the enzyme complexed with a DNA/RNA hybrid oligomer . In this model, a protein region, from the Mg(2+)-binding site to the basic protrusion, covers roughly two turns of a DNA/RNA hybrid double helix . A segment (11-23) containing six glycine residues forms a long loop between the beta A and beta B strands . This loop, which protrudes into the solvent region, lies on the interface between the enzyme and a DNA/RNA hybrid in the model of the complex . The mean temperature factors of main-chain atoms show remarkably high values in helix alpha III that constitutes the basic protrusion, suggesting some correlation between its flexibility and the nucleic acid binding function . The Mg(2+)-binding site, surrounded by four invariant acidic residues, can now be described more precisely in conjunction with the catalytic activity . The arrangement of molecules within the crystal appears to be dominated by the cancelling out of a remarkably biased charge distribution on the molecular surface, which is derived in particular from the separation between the acidic Mg(2+)-binding site and the basic protrusion.

Nature, 1992 Feb 20, 355(6362), 740 - 3
Crystal structure of a dUTPase; Cedergren-Zeppezauer ES et al.; The enzyme dUTPase catalyses the hydrolysis of dUTP and maintains a low intracellular concentration of dUTP so that uracil cannot be incorporated into DNA . dUTPase from Escherichia coli is strictly specific for its dUTP substrate, the active site discriminating between nucleotides with respect to the sugar moiety as well as the pyrimidine base . Here we report the three-dimensional structure of E . coli dUTPase determined by X-ray crystallography at a resolution of 1.9 A . The enzyme is a symmetrical trimer, and of the 152 amino acid residues in the subunit, the first 136 are visible in the crystal structure . The tertiary structure resembles a jelly-roll fold and does not show the 'classical' nucleotide-binding domain . In the quaternary structure there is a complex interaction between the subunits that may be important in catalysis . This possibility is supported by the location of conserved elements in the sequence.

Biochemistry, 1992 Feb 18, 31(6), 1849 - 58
31P NMR spectra of oligodeoxyribonucleotide duplex lac operator-repressor headpiece complexes: importance of phosphate ester backbone flexibility in protein-DNA recognition; Karslake C et al.; The 31P NMR spectra of various 14-base-pair lac operators bound to both wild-type and mutant lac repressor headpiece proteins were analyzed to provide information on the backbone conformation in the complexes . The 31P NMR spectrum of a wild-type symmetrical operator, d(TGTGAGCGCTCACA)2, bound to the N-terminal 56-residue headpiece fragment of a Y7I mutant repressor was nearly identical to the spectrum of the same operator bound to the wild-type repressor headpiece . In contrast, the 31P NMR spectrum of the mutant operator, d(TATAGAGCGCTCATA)2, wild-type headpiece complex was significantly perturbed relative to the wild-type repressor-operator complex . The 31P chemical shifts of the phosphates of a second mutant operator, d(TGTGTGCGCACACA)2, showed small but specific changes upon complexation with either the wild-type or mutant headpiece . The 31P chemical shifts of the phosphates of a third mutant operator, d(TCTGAGCGCTCAGA)2, showed no perturbations upon addition of the wild-type headpiece . The 31P NMR results provide further evidence for predominant recognition of the 5'-strand of the 5'-TGTGA/3'-ACACT binding site in a 2:1 protein to headpiece complex . It is proposed that specific, strong-binding operator-protein complexes retain the inherent phosphate ester conformational flexibility of the operator itself, whereas the phosphate esters are conformationally restricted in the weak-binding operator-protein complexes . This retention of backbone torsional freedom in strong complexes is entropically favorable and provides a new (and speculative) mechanism for protein discrimination of different operator binding sites . It demonstrates the potential importance of phosphate geometry and flexibility on protein recognition and binding.

Biochemistry, 1992 Feb 18, 31(6), 1656 - 64
Mutational analysis of carbamyl phosphate synthetase . Substitution of Glu841 leads to loss of functional coupling between the two catalytic domains of the synthetase subunit; Guillou F et al.; The synthetase subunit of Escherichia coli carbamyl phosphate synthetase has two catalytic nucleotide-binding domains, one involved in the activation of HCO3- and the second in phosphorylation of carbamate . Here we show that a Glu841----Lys841 substitution in a putative ATP-binding domain located in the carboxyl half of the synthetase abolishes overall synthesis of carbamyl phosphate with either glutamine or NH3 as the nitrogen source . Measurements of partial activities indicate that while HCO3(-)-dependent ATP hydrolysis at saturating concentrations of substrate proceeds at higher than normal rates, ATP synthesis from ADP and carbamyl phosphate is nearly completely suppressed by the mutation . These results indicate Glu841 to be an essential residue for the phosphorylation of carbamate in the terminal step of the catalytic mechanism . The Lys841 substitution also affects the kinetic properties of the HCO3- activation site . Both kcat and Km for ATP increase 10-fold, while Km for HCO3- is increased 100-fold . Significantly, NH3 decreases rather than stimulates Pi release from ATP in the HCO3(-)-dependent ATPase reaction . The increase in kcat of the HCO3(-)-dependent ATPase reaction, and an impaired ability of the Lys841 enzyme to catalyze the reaction of NH3 with carboxy phosphate, strongly argues for interactions between the two catalytic ATP sites that couple the formation of enzyme-bound carbamate with its phosphorylation.

Biochemistry, 1992 Feb 18, 31(6), 1621 - 30
A fully active variant of dihydrofolate reductase with a circularly permuted sequence; Buchwalder A et al.; The amino acid sequence of mouse dihydrofolate reductase was permuted circularly at the level of the gene . By transposing the 3'-terminal half of the coding sequence to its 5' terminus, the naturally adjacent amino and carboxyl termini of the native protein were fused, and one of the flexible peptide loops at the protein surface was cleaved . The steady-state kinetic constants, the dissociation constants of folate analogues, and the degree of activation by both mercurials and salt as well as the resistance toward digestion by trypsin were almost indistinguishable from those of a recombinant wild-type protein . Judged by these criteria, the circularly permuted variant has the same active site and overall structure as the wild-type enzyme . The only significant difference was the lower stability toward guanidinium chloride and the lower solubility of the circularly permuted variant . This behavior may be due to moving a mononucleotide binding fold from the interior of the sequence to the carboxyl terminus . Thus, dihydrofolate reductase requires neither the natural termini nor the cleaved loop for stability, for the conformational changes that accompany catalysis as well as the binding of inhibitors, and for the folding process.

Biochemistry, 1992 Feb 18, 31(6), 1595 - 602
Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding; Krudy GA et al.; Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops . The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3 . No downfield-shifted Gly resonance was observed from the naturally inactive site I . Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65 . Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites . In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances . Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65 . The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Feb 18, 31(6), 1579 - 84
Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli; Pack P et al.; We have designed dimeric antibody fragments that assemble in Escherichia coli . They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment . The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide . The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers . All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind . The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments . This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E . coli.

Biochemistry, 1992 Feb 18, 31(6), 1695 - 700
A conformational transition involved in antagonistic substrate binding to the allosteric phosphofructokinase from Escherichia coli; Deville-Bonne D et al.; The binding of fructose 6-phosphate, ATP or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate), ADP, and phosphoenolpyruvate to Escherichia coli phosphofructokinase has been studied by changes in the protein fluorescence and/or equilibrium dialysis . The results lead to the following conclusions: (1) tetrameric phosphofructokinase can bind four ATP but only two fructose-6-phosphate, and this binding occurs without cooperativity; (2) only two conformational states, T and R, with respectively a high and a low fluorescence, seem accessible to phosphofructokinase, which exists as a mixture of one-third R and two-third T states in the absence of ligand; (3) the substrate fructose 6-phosphate and the allosteric activator ADP bind preferentially to the low-fluorescence R state, while the other substrate, ATP {or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate)}, and the allosteric inhibitor phosphoenolpyruvate bind to the high-fluorescence T state; (4) the binding of a given ligand is cooperative, with a Hill coefficient of 2, only when this binding is accompanied by a complete shift from one state to the other; for instance, the binding of the ATP analogue adenylyl 5'-(beta,gamma-methylenediphosphonate) to the T state is cooperative only in the presence of fructose 6-phosphate which favors the R state . This behavior is qualitatively consistent with a concerted transition, but quite different from that described earlier for phosphofructokinase from steady-state activity measurements (Blangy et al., 1968) . This discrepancy suggests that the allosteric properties of phosphofructokinase are due in part to ligand binding and in part to the kinetics of the enzymatic reaction.

Biochemistry, 1992 Feb 18, 31(6), 1677 - 85
C-terminal cysteines of Tn501 mercuric ion reductase; Moore MJ et al.; Mercuric ion reductase (MerA) catalyzes the reduction of Hg(II) to Hg(0) as the last step in the bacterial mercury detoxification pathway . A member of the flavin disulfide oxidoreductase family, MerA contains an FAD prosthetic group and redox-active disulfide in its active site . However, the presence of these two moieties is not sufficient for catalytic Hg(II) reduction, as other enzyme family members are potently inhibited by mercurials . We have previously identified a second pair of active site cysteines (Cys558 Cys559 in the Tn501 enzyme) unique to MerA, that are essential for high levels of mercuric ion reductase activity {Moore, M . J., & Walsh, C . T . (1989) Biochemistry 28, 1183; Miller, S . M., et al . (1989) Biochemistry 28, 1194} . In this paper, we have examined the individual roles of Cys558 and Cys559 by site-directed mutagenesis of each to alanine . Phenotypic analysis indicates that both merA mutations result in a total disruption of the Hg(II) detoxification pathway in vivo, while characterization of the purified mutant enzymes in vitro shows each to have differential effects on catalytic function . Compared to wild-type enzyme, the C558A mutant shows a 20-fold reduction in kcat and a 10-fold increase in Km, for an overall decrease in catalytic efficiency of 200-fold in kcat/Km . In contrast, mutation of Cys559 to alanine results in less than a 2-fold reduction in kcat and an increase in Km of only 4-5 fold for an overall decrease in catalytic efficiency of only ca . 10-fold in vitro . From these results, it appears that Cys558 plays a more important role in forming the reducible complex with Hg(II), while both Cys558 and Cys559 seem to be involved in efficient scavenging (i.e., tight binding) of Hg(II).

Biochemistry, 1992 Feb 18, 31(6), 1610 - 21
NMR analysis of helix I from the 5S RNA of Escherichia coli; White SA et al.; The structure of helix I of the 5S rRNA from Escherichia coli has been determined using a nucleolytic digest fragment of the intact molecule . The fragment analyzed, which corresponds to bases (-1)-11 and 108-120 of intact 5S rRNA, contains a G-U pair and has unpaired bases at its termini . Its proton resonances were assigned by two-dimensional NMR methods, and both NOE distance and coupling constant information have been used to calculate structural models for it using the full relaxation matrix algorithm of the molecular dynamics program XPLOR . Helix I has A-type helical geometry, as expected . Its most striking departure from regular helical geometry occurs at its G-U, which stacks on the base pair to the 5' side of its G but not on the base pair to its 3' side . This stacking pattern maximizes interstrand guanine-guanine interactions and explains why the G-U in question fails to give imino proton NOE's to the base pair to 5' side of its G . These results are consistent with the crystal structures that have been obtained for wobble base pairs in tRNAPhe {Mizuno, H., & Sundaralingam, M . (1978) Nucleic Acids Res . 5, 4451-4461} and A-form DNA {Rabbinovich, D., Haran, T., Eisenstein, M., & Shakked, Z . (1988) J . Mol . Biol . 200, 151-161} . The conformations of the terminal residues of helix I, which corresponds to bases (-1)-11 and 108-120 of native 5S RNA, are less well-determined, and their sugar puckers are intermediate between C2' and C3'-endo, on average.

FEBS Lett, 1992 Feb 17, 298(1), 1 - 5
Ordered binding model as a general mechanistic mechanism for secondary active transport systems; Yamato I; The mechanistic mechanism of secondary active transport processes has not been fully elucidated . Based on substrate binding studies dependent on coupling cation concentrations of the glutamate, melibiose, lactose and proline transport carriers in Escherichia coli, the ordered binding mechanism was proposed as the energy coupling mechanism of the transport systems . This ordered binding mechanism satisfactorily explained the properties of the secondary active transport systems . Thus, this mechanism as the general energy coupling mechanism for the transport systems is discussed.

FEBS Lett, 1992 Feb 17, 298(1), 84 - 8
Colicin A and colicin E1 lysis proteins differ in their dependence on secA and secY gene products; Cavard D; The export of colicin A and of colicin E1 is not equally affected in both secA and secY mutants of Escherichia coli: release of colicin A occurs slowly while that of colicin E1 is blocked . Processing and functioning of Cal, the colicin A lysis protein, seem to be slightly or not at all modified in these mutants, whereas synthesis and assembly of CelA, the colicin E1 lysis protein, are highly inhibited . These variations observed in the dependence of the two lysis proteins on secA and secY gene products are interpreted as being either the cause or the consequence of the differences observed in their rate of biogenesis.

FEBS Lett, 1992 Feb 17, 298(1), 69 - 73
Over-expression and refolding of beta-subunit from the chloroplast ATP synthase; Chen Z et al.; We established a bacterial system for high-level over-expression of the spinach chloroplast atpB gene which encodes the ATP synthase beta subunit . Upon induction, atpB was expressed as at least 50% to 70% of total cell protein . Although the over-expressed beta polypeptide formed insoluble inclusion bodies, more than fifty percent of it was restored to a functional form by solubilizing the inclusion bodies with 4 M urea and slowly removing the urea by stepwise dialysis . The resulting beta subunit exhibited specific and selective nucleotide binding properties identical to those of the native beta subunit.

Proc Natl Acad Sci U S A, 1992 Feb 15, 89(4), 1453 - 7
Termination efficiency at rho-dependent terminators depends on kinetic coupling between RNA polymerase and rho; Jin DJ et al.; Rho-dependent terminators constitute one of two major classes of terminators in Escherichia coli . Termination at these sites requires the concerted action of RNA polymerase and rho protein . We present evidence that the efficiency of termination at these sites is governed by kinetic coupling of the rate of transcription of RNA polymerase and the rate of action of rho protein . Termination experiments in vitro indicate that termination efficiency at a rho-dependent terminator is an inverse function of the rate of elongation of RNA polymerase, and each of the mutant phenotypes can be accounted for by the altered rate of elongation of the mutant RNA polymerase . Experiments in vivo show that fast-moving mutant RNA polymerases are termination deficient, while slow-moving mutant RNA polymerases are termination proficient and can suppress the termination deficiency of a slow-acting mutant rho protein . Because of the close coupling of rho action with RNA polymerase, small changes in the elongation rate of RNA polymerase can have very large effects on termination efficiency, providing the cell with a powerful way to modulate termination at rho-dependent terminators.

Proc Natl Acad Sci U S A, 1992 Feb 15, 89(4), 1403 - 7
The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs; Takagaki Y et al.; Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs . We have shown previously that this factor is composed of three subunits with estimated molecular masses of 77, 64, and 50 kDa and that the 64-kDa subunit can be UV-crosslinked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner . We have now isolated cDNAs encoding the 64-kDa subunit of human cleavage stimulation factor . The 64-kDa subunit contains a ribonucleoprotein-type RNA binding domain in the N-terminal region and a repeat structure in the C-terminal region in which a pentapeptide sequence (consensus MEARA/G) is repeated 12 times and the formation of a long alpha-helix stabilized by salt bridges is predicted . An approximately 270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (approximately 20% for each) . When cloned 64-kDa subunit was expressed in Escherichia coli, an N-terminal fragment containing the RNA binding domain bound to RNAs in a polyadenylylation-signal-independent manner, suggesting that the RNA binding domain is directly involved in the binding of the 64-kDa subunit to pre-mRNAs.

Proc Natl Acad Sci U S A, 1992 Feb 15, 89(4), 1310 - 4
Carcinogen-induced frameshift mutagenesis in repetitive sequences; Lambert IB et al.; We have constructed plasmids pS3G-1 and pSG4 that contain single acetylaminofluorene adducts within contiguous runs of three (5'-CCCG1G2G3-3') and four (5'-CG1GGG4T-3') guanine residues, respectively . In Escherichia coli, the frequency of induced -1 frameshift mutations was strongly dependent on the position of modification: pS3G-G3 was approximately 100-fold and 10-fold more mutagenic than pS3G-G1 and pS3G-G2, respectively; pSG4-G4 was approximately 600-fold more mutagenic than pSG4-G1 . Mutagenesis was SOS-dependent and was markedly reduced in bacteria that were proficient in nucleotide excision repair as compared to a repair-deficient uvrA6 mutant . DNA sequencing showed that -1 frameshift events in pS3G-1 consisted of either targeted mutations (greater than 90% of induced mutations) within the guanine sequence or semitargeted mutations (greater than 10%) in the 5' flanking repetitive cytosine sequence . Semitargeted events, which were observed when acetylaminofluorene modification was at G1 and G2, show that a lesion can reduce the fidelity of replication at positions 5' to its location on the template strand . No semitargeted frameshifts were observed in plasmid pSG4, which lacks a repetitive sequence 5' to the adduct . Our results are consistent with a model for frameshift mutagenesis in which the acetylaminofluorene adduct (i) allows accurate incorporation of cytosine opposite the bulky lesion during DNA synthesis and (ii) impedes elongation of primer/template termini formed opposite the adduct or 5' to the adduct on the template strand, providing increased opportunity for the formation of slipped frameshift intermediates.

Proc Natl Acad Sci U S A, 1992 Feb 15, 89(4), 1286 - 90
Molecular characterization of two plant flavonol sulfotransferases; Varin L et al.; cDNA clones coding for flavonol 3- and 4'-sulfotransferases (STs) were isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA extracted from terminal buds of Flaveria chloraefolia . Sequence analysis revealed full-length cDNA clones with open reading frames of 933 and 960 base pairs, which encode polypeptides containing 311 and 320 amino acids, respectively . This corresponds to a molecular mass of 36,442 Da for the 3-ST and 37,212 Da for the 4'-ST . Expression of these clones in Escherichia coli led to the synthesis of beta-galactosidase-ST fusion proteins having the same substrate and position specificities as those for the 3- and 4'-flavonol ST enzymes isolated from the plant . Comparison of the deduced amino acid sequence of the two clones revealed an overall identity of 69% in 311 amino acid residues . The two flavonol STs of F . chloraefolia also shared significant sequence similarities with steroid and aryl STs found in animal tissues and with the senescence marker protein 2 isolated from rat liver, suggesting an evolutionary link between plant and animal STs.

Proc Natl Acad Sci U S A, 1992 Feb 15, 89(4), 1266 - 70
Reconstitution of enzymatic activity from fragments of M1 RNA; Guerrier-Takada C et al.; Certain fragments of M1 RNA, the catalytic subunit of RNase P from Escherichia coli, either have no enzymatic activity at all or have altered substrate specificity compared with that of the intact catalytic RNA . After simple mixing in vitro, many of these fragments of M1 RNA can reassociate with other fragments to form complexes that have enzymatic activity typical of wild-type M1 RNA . Furthermore, inactive M1 RNA molecules with internal deletions can be complemented in vitro by other inactive derivatives of M1 RNA that have nonoverlapping deletions . Thus, two inactive molecules of M1 RNA can interact to form an active RNA enzyme . Functional attributes can be assigned to various regions of M1 RNA when the reconstitution process is combined with assays for activity with different substrates.

Proc Natl Acad Sci U S A, 1992 Feb 15, 89(4), 1159 - 63
Mechanism of SOS mutagenesis of UV-irradiated DNA: mostly error-free processing of deaminated cytosine; Tessman I et al.; We measured the kinetics of growth and mutagenesis of UV-irradiated DNA of phages S13 and lambda that were undergoing SOS repair; the kinetics strongly suggest that most of SOS mutagenesis arises from the deamination of cytosine in cyclobutane pyrimidine dimers, producing C----T transitions . This occurs because the SOS mechanism bypasses T--T dimers promptly, while bypass of cytosine-containing dimers is delayed long enough for deamination to occur . The mutations are thus primarily the product of a faithful mechanism of lesion bypass by a DNA polymerase and are not, as had been generally thought, the product of an error-prone mechanism . All of these observations are explained by the A-rule, which is that adenine nucleotides are inserted noninstructionally opposite DNA lesions.

Experientia, 1992 Feb 15, 48(2), 178 - 201
Proteases and protein degradation in Escherichia coli; Maurizi MR; In E . coli, protein degradation plays important roles in regulating the levels of specific proteins and in eliminating damaged or abnormal proteins . E . coli possess a very large number of proteolytic enzymes distributed in the cytoplasm, the inner membrane, and the periplasm, but, with few exceptions, the physiological functions of these proteases are not known . More than 90% of the protein degradation occurring in the cytoplasm is energy-dependent, but the activities of most E . coli proteases in vitro are not energy-dependent . Two ATP-dependent proteases, Lon and Clp, are responsible for 70-80% of the energy-dependent degradation of proteins in vivo . In vitro studies with Lon and Clp indicate that both proteases directly interact with substrates for degradation . ATP functions as an allosteric effector promoting an active conformation of the proteases, and ATP hydrolysis is required for rapid catalytic turnover of peptide bond cleavage in proteins . Lon and Clp show virtually no homology at the amino acid level, and thus it appears that at least two families of ATP-dependent proteases have evolved independently.

Eur J Biochem, 1992 Feb 15, 204(1), 137 - 46
Sequence-specific NMR assignments of the trp repressor from Escherichia coli using three-dimensional 15N/1H heteronuclear techniques; Borden KL et al.; Sequence-specific 15N and 1H assignments for the trp holorepressor from Escherichia coli are reported . The trp repressor consists of two identical 107-residue subunits which are highly helical in the crystal state {Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C . L . & Sigler, P . B . (1985) Nature 317, 782-786} . The high helical content and the relatively large size of the protein (Mr = 25,000) make it difficult to assign even the main-chain resonances by conventional homonuclear two-dimensional NMR methods . However, we have now assigned the main-chain resonances of 94% of the residues by using three-dimensional 15N/1H heteronuclear experiments on a sample of protein uniformly labelled with 15N . The additional resolution obtained by spreading out the signals into three dimensions proved indispensable in making these assignments . In particular, we have been able to resolve signals from residues in the N-terminal region of the A helix for the first time in solution . The observed NOE results confirm that the repressor is highly helical in solution, and contains no extended chain conformations.

J Biol Chem, 1992 Feb 15, 267(5), 3466 - 72
Regulated expression of alpha 2,6-sialyltransferase by the liver-enriched transcription factors HNF-1, DBP, and LAP; Svensson EC et al.; Tissue-specific carbohydrate structures are thought to result from the selective expression of specific terminal glycosyltransferases responsible for their synthesis . However, little is known about the regulation of the expression of these enzymes . Previous analysis of the distribution of one such enzyme, beta-galactoside alpha 2,6-sialyltransferase, revealed that its expression is tissue restricted, with highest levels being found in the liver . Examination of the gene suggested that its expression is regulated at the level of transcription by multiple promoters, one of which is strongly active in the liver . In this present work, an analysis of the liver-restricted promoter was undertaken to identify the promoter elements necessary for liver-restricted expression . Footprinting studies, 5' deletion analysis, and site-directed mutagenesis identified two cis-elements which were potentially important in the tissue-specific expression of this promoter . One of these elements contains a consensus binding site for the liver-enriched transcription factor hepatocyte nuclear factor-1 alpha, while the other is a consensus binding site for the liver-specific factors D-binding protein and liver-enriched transcriptional activator protein . Expression vectors containing cDNAs of these factors are capable of trans-activating transcription of the alpha 2,6ST promoter, demonstrating their ability to regulate transcription of this promoter . Together, these results suggest that tissue-specific glycosylation can be regulated at the level of transcription by the same factors involved in the expression of a number of other tissue-specific genes.

J Biol Chem, 1992 Feb 15, 267(5), 3455 - 9
Conversion of monocyte chemoattractant protein-1 into a neutrophil attractant by substitution of two amino acids; Beall CJ et al.; The small cytokine monocyte chemoattractant protein-1 has structural similarity to the neutrophil chemoattractant interleukin-8, but each protein is specific in attracting its own target cell . To investigate the structural basis of this cell type specificity, we have developed an Escherichia coli expression system for the monocyte chemoattractant and mutagenized selected amino acid residues to ones found at the corresponding positions of interleukin-8 . We find that a double mutation of tyrosine 28 and arginine 30 to leucine and valine, respectively, causes a drastic decrease in chemotactic activity toward monocytes with the appearance of a novel (interleukin-8-like) neutrophil chemotactic activity . Computer graphic analysis predicts that, with the double substitution, a putative receptor binding groove of the monocyte chemoattractant protein would become topographically similar to that of interleukin-8 . We therefore postulate that one or both of these amino acid residues are part of the binding contact of these small cytokines and their receptors.

J Biol Chem, 1992 Feb 15, 267(5), 3429 - 37
Human estrogen receptor mutants with altered estrogen and antiestrogen ligand discrimination; Pakdel F et al.; A structural characteristic of many antiestrogens (AEs) is a bulky side chain with basic or polar functional groups that may interact with charged and polar amino acids near the hormone-binding site in the estrogen receptor . Recently, we have identified Cys530 as the residue of the human estrogen receptor that is the site of covalent labeling by aziridine analogs of estrogens (Es) and AEs (Harlow, K . W., Smith, D . N., Katzenellenbogen, J . A., Greene, G . L., and Katzenellenbogen, B . S . (1989) J . Biol . Chem . 264, 17476-17485) . Since the aziridine function is on the bulky side chain of these ligands, Cys530 must be at or near the site of those side chain interactions . To probe these interactions, we have, by site-directed mutagenesis, made mutant human estrogen receptors in which charged and polar amino acids near Cys530 are changed (Glu523 to Gln, Lys529 and Lys531 to Gln, Asn532 to Asp, and Asp538 to Asn) so as to alter charge with minimal steric alteration . These receptors were expressed in mammalian (Chinese hamster ovary or COS-1) cells and assayed for their binding affinity for Es and AEs, their interaction with estrogen-responsive element DNA, and their ability to activate or suppress transcription of estrogen-responsive reporter genes . Two of the estrogen receptor mutants, KKN-QQD (mutation of Lys529, Lys531, and Asn532 to Gln529, Gln531, and Asp532, respectively) and KK-QQ (mutation of Lys529 and Lys531 to Gln529 and Gln531, respectively), in which the local charge is changed from +2 to -1 or 0, respectively, display an affinity for estradiol (E2) 5-10 times lower than that of the wild-type receptor, which is attributable to an enhanced rate of E2 dissociation . Although these mutant receptors have reduced affinity for a variety of Es, they retain unaltered affinity for AE . The profiles of transcriptional activation of reporter genes by various concentrations of E2 show that these two mutants (KKN-QQD and KK-QQ) require 40- and 15-fold higher E2 concentrations, respectively, to achieve half-maximal activity . In contrast, mutants E523Q and D538N, with changes at amino acids further from Cys530, were unaltered in their hormone binding and transactivation activity by E or AE . Interestingly, the AEs 4-hydroxytamoxifen, LY 117018, U 23469M, and ICI 164384 were 15-30-fold more effective in inhibiting E2-stimulated transcription by mutants KKN-QQD and KK-QQ compared to the wild-type receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1992 Feb 15, 267(5), 3409 - 15
Selective inactivation of the Arg-Gly-Asp-Ser (RGDS) binding site in von Willebrand factor by site-directed mutagenesis; Beacham DA et al.; In order to assess the requirement for the Arg-Gly-Asp-Ser (RGDS) consensus adhesion sequence in von Willebrand factor (vWF) for vWF binding to platelets and endothelial cells, point mutations were introduced into this sequence by site-directed mutagenesis . A glycine to alanine substitution yielded RADS-vWF, while an aspartate to glutamate substitution resulted in RGES-vWF . Recombinant RADS-vWF and RGES-vWF, purified from transformed Chinese hamster ovary cells, were compared with recombinant wild type vWF (WT-vWF) in functional assays with platelets and human umbilical vein endothelial cells (HU-VECs) . High molecular weight RADS-vWF and RGES-vWF multimers inhibited binding of 125I-vWF to a mixture of insolubilized native type I and III collagen and competed effectively with 125I-vWF for binding to formalin-fixed platelets in the presence of ristocetin, indicating functional collagen and platelet glycoprotein Ib binding . However, RADS-vWF and RGES-vWF were unable to displace the binding of 125I-vWF to thrombin or ADP-activated platelets . The attachment of HUVECs to either RADS-vWF or RGES-vWF coated surfaces was reduced and spreading was almost completely inhibited, compared with WT-vWF . We conclude that point mutations of the RGDS sequence in vWF selectively impair binding to platelet glycoprotein IIb/IIIa and the HUVEC vitronectin receptor.

J Biol Chem, 1992 Feb 15, 267(5), 3200 - 4
Global conformational changes in allosteric proteins . A study of Escherichia coli cAMP receptor protein and muscle pyruvate kinase; Heyduk E et al.; One of the basic features in allosteric regulation involves long range transduction of information . Based on crystallographic data on protein systems that are regulated by allosteric mechanisms, a global conformational change has always been observed . It is, therefore, important and useful to correlate the cooperativity of global structural change with the mode of binding of the regulatory ligand . Two systems were chosen for study, namely Escherichia coli cAMP receptor protein and muscle pyruvate kinase, which show negative and positive cooperativity in the binding of allosteric ligands, respectively . Quantitative titration of the global structural change, monitored by a high precision analytical gel chromatography technique, was conducted as a function of allosteric effector concentration . The results obtained for cAMP receptor protein show that the protein undergoes contraction upon binding of cAMP . The decreases in Stokes radius associated with complex formation are 0.1 +/- 0.1 and 0.7 +/- 0.1 A when one and two cAMP-binding sites are filled, respectively . The results for the pyruvate kinase system show a concerted structural change that quantitatively match the predicted behavior based on equilibrium constants derived from the analysis of steady state kinetic data by a two-state model . Hence, for these two systems, these results show that negative and positive cooperativity are correlated with sequential and concerted modes of structural change, respectively.

J Biol Chem, 1992 Feb 15, 267(5), 3173 - 8
Involvement of phage T5 tail proteins and contact sites between the outer and inner membrane of Escherichia coli in phage T5 DNA injection; Guihard G et al.; The penetration of phage T5 DNA into the Escherichia coli envelope takes place through ion channels (Boulanger, P., and Letellier, L . (1992) J . Biol . Chem . 267, 3168-3172) . To identify putative phage protein(s) involved in the formation of these channels, E . coli cells were infected at 37 degrees C with radioactively labeled phage and their envelopes were fractionated . After a flotation gradient, proteins belonging to the phage tail were recovered both in fractions containing the contact sites between the inner and outer membranes and in the outer membrane . The electrophoretic banding pattern of phage proteins indicates that the contact sites were enriched in the protein pb2 . Moreover, infected cells were significantly enriched in contact sites as compared to intact cells . There was no enrichment of contact sites and very little radioactivity was found in this fraction and in the outer membrane when the cells were infected at 4 degrees C (i.e . under conditions where the phage does not inject its DNA) . These results suggest that both contact sites and pb2 may play a central role in the translocation of phage T5 DNA.

J Biol Chem, 1992 Feb 15, 267(5), 3088 - 95
A mutation in the consensus ATP-binding sequence of the RecD subunit reduces the processivity of the RecBCD enzyme from Escherichia coli; Korangy F et al.; We have constructed a mutant form of the RecBCD enzyme from Escherichia coli with a lysine to glutamine change in the consensus ATP-binding sequence in the RecD subunit (Korangy, F., and Julin, D.A . (1992a, 1992b) J . Biol . Chem., 1727-1732; 1733-1740) . We compare here the kinetics of double-stranded DNA-dependent ATP hydrolysis by the mutant (RecBCD-K177Q) and wild-type enzymes . We included heparin to trap enzyme not bound to DNA, or the single-stranded DNA-binding (SSB) protein from Escherichia coli to prevent the enzyme from binding to single-stranded DNA products and partially single-stranded reaction intermediates . The ATP hydrolysis kinetics in either case show a rapid burst phase followed by a slower second phase . The wild-type enzyme hydrolyzes an amount of ATP about equal to the DNA nucleotide concentration in the rapid phase . The amount of ATP hydrolyzed by the RecBCD-K177Q enzyme in the burst is about 8-10-fold lower than the wild-type, in the presence of either heparin or SSB . The burst magnitude of the wild-type enzyme with heparin is proportional to the size of the DNA from about 1,420 to 22,400 base pairs whereas that of the mutant is independent of the DNA size . The wild-type enzyme completely degrades a 6,250-base pair DNA substrate with no partially degraded molecules visible on agarose gels . RecBCD-K177Q enzyme reaction mixtures in the presence of SSB protein contain a heterogeneous mixture of partially degraded molecules of 2,000-5,000 base pairs . These results indicate that the RecBCD-K177Q enzyme is less processive than the wild-type enzyme.

J Biol Chem, 1992 Feb 15, 267(5), 3052 - 9
Fluorescence energy transfer between the primer and the beta subunit of the DNA polymerase III holoenzyme; Griep MA et al.; We report here our initial success in using fluorescence energy transfer to map the position of the subunits of the DNA polymerase III holoenzyme within initiation complexes formed on primed DNA . Using primers containing a fluorescent derivative 3 nucleotides from the 3'-terminus and acceptors of fluorescence energy transfer located on Cys333 of the beta subunit, a donor-acceptor distance of 65 A was measured . Coupling this distance with other information enabled us to propose a model for the positioning of beta within initiation complexes . Examination of the fluorescence properties of a labeled primer with the unlabeled beta subunit and other assemblies of DNA polymerase III holoenzyme subunits allowed us to distinguish all of the known intermediates of the holoenzyme-catalyzed reaction . Specific fluorescence changes could be assigned for primer annealing, Escherichia coli single-stranded DNA-binding protein binding, 3'----5' exonucleolytic hydrolysis of the primer, DNA polymerase III* binding, initiation complex formation upon the addition of beta in the presence of ATP, and DNA elongation . These fluorescence changes are sufficiently large to support future detailed kinetic studies . Particularly interesting was the difference in fluorescence changes accompanying initiation complex formation as compared to binding of DNA polymerase III holoenzyme subunit assemblies . Initiation complex formation resulted in a strong fluorescence enhancement . Binding of DNA polymerase III* led to a fluorescence quenching, and transfer of beta to primed DNA by the gamma delta complex did not change the fluorescence . This demonstrates a rearrangement of subunits accompanying initiation complex formation . Monitoring fluorescence changes with labeled beta, we have determined that beta binds with a stoichiometry of one monomer/primer terminus.

J Biol Chem, 1992 Feb 15, 267(5), 3030 - 7
Human stomach aldehyde dehydrogenase cDNA and genomic cloning, primary structure, and expression in Escherichia coli; Hsu LC et al.; An aldehyde dehydrogenase isozyme, ALDH3, which is strongly expressed in the stomach, may play a role in the oxidation of toxic aldehydes . Using reverse genetic approach, we cloned and characterized the cDNA and the gene for the ALDH3 . The full length cDNA is 1624 base pairs (bp) in length and contains an open reading frame encoding 453 amino acid residues . The deduced amino acid sequence shows a high degree of resemblance to that of rat hepatocarcinoma ALDH . The human ALDH3 gene spans about 8 kb in length and consists of 10 exons . The putative TATA and CCAAT boxes are located in the consensus upstream distance from the transcription initiation site . Southern blot analysis of total genomic DNA argues against the proposed two-gene model for the ALDH3 isozymes (Yin, S.-J., Cheng, T.-C., Chang, C.-P., Chen, Y.-J., Chao, Y.-C., Tang, H.-S., Chang, T.-M., and Wu, C.-W . (1988) Biochem . Genet . 26, 343-360) . Northern blot hybridization and analysis of PCR amplification products of cellular RNA demonstrated the existence of a high level of ALDH3 mRNA in human stomach and hepatoma cells, but a very low level in the normal liver . Expression of ALDH3 cDNA in Escherichia coli yielded a protein of 55 kDa, which exhibited kinetic properties similar to that found in ALDH3 isozyme purified from human stomach and liver, and was hybridizable with rabbit anti-human-hepatoma ALDH serum.

J Biol Chem, 1992 Feb 15, 267(5), 3024 - 9
Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase; Bagchi IC et al.; Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa light chains of myosin . In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R . (1989) J . Biol . Chem . 264, 15843-15849), we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity . Initial mutagenesis experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme . We have now carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids that are critical for CaM binding and activation . Our studies reveal that the individual mutation of several hydrophobic amino acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete loss of CaM binding and activation of smMLCK . The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated enzymes . We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and electrostatic interactions which appear to be conserved among various target enzymes of CaM.

J Biol Chem, 1992 Feb 15, 267(5), 2955 - 9
The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA-helix-destabilizing properties; Ghisolfi L et al.; Nucleolin, a major nucleolar protein implicated in preribosome assembly and transcriptional regulation, possesses a C-terminal domain unusually rich in glycine, arginine, and phenylalanine residues . A polypeptide (p10), corresponding to this domain, has been synthesized by means of an Escherichia coli expression system and purified to homogeneity . Nitrocellulose binding assays have clearly shown that this domain of nucleolin is capable of interacting with RNA, and indeed all nucleic acids tested, in an efficient but nonspecific manner . A combination of circular dichroism and infrared spectroscopy provide strong evidence that repeated beta-turns are a major structural component of this polypeptide, which is entirely consistent with its amino acid composition and above all the presence of repeat motifs such as RGGF . Circular dichroism technique also shows that the interaction of p10 with RNA involves an unstacking of the nucleotide bases and an unfolding of the RNA secondary structure . While the role of the C-terminal domain of nucleolin in vivo has yet to be established, our findings suggest that it may act to unfold regions of ribosomal RNA so that a second domain of nucleolin has access to its specific binding site.

J Biol Chem, 1992 Feb 15, 267(5), 2909 - 14
Identification of chromophore binding domains of yeast DNA photolyase; Malhotra K et al.; Photolyases contain two chromophores, flavin plus either methenyltetrahydrofolate (MTHF) or 8-OH-5-deazaflavin (HDF) . Amino acid sequence comparison reveals that all photolyases sequenced to date have extensive sequence homology in the carboxyl-terminal half; in the amino-terminal region the folate and deazaflavin class enzymes are more homologous to other members of the same class . This modular arrangement of sequence homologies suggests that the amino-terminal half of photolyase is involved in MTHF or HDF binding whereas the carboxyl-terminal half carries the flavin binding site . In this study we attempted to identify such structural domains of yeast photolyase by partial proteolysis and gene fusion techniques . Partial digestion with chymotrypsin yielded an amino-terminal 34-kDa fragment containing tightly bound MTHF and a carboxyl-terminal 20-kDa polypeptide which lacked chromophore or DNA binding activity . However, a fusion protein carrying the carboxyl-terminal 275 amino acids of yeast photolyase bound specifically to FAD but not to MTHF or DNA . We conclude that the amino-terminal half of yeast photolyase constitutes the folate binding domain and that the carboxyl-terminal half carries the flavin binding site.

J Biol Chem, 1992 Feb 15, 267(5), 2845 - 8
The P2 element of the td intron is dispensable despite its normal role in splicing; Salvo JL et al.; The P2 region of group I introns has been proposed to be involved in the correct positioning of the P1 5'-splice site duplex in the catalytic core (Michel, F., and Westhof, E . (1990) J . Mol . Biol . 216, 585-610) . The behavior of delta P2 deletion mutants of the td intron is consistent with this hypothesis . The delta P2 mutants are capable of site-specific hydrolysis, indicating that the conformation of the ribozyme is not grossly altered, but they are incapable of transesterification reactions at the splice sites, as would be predicted if P1 is not appropriately aligned within the catalytic core . Nevertheless, the function of the P2 element can be bypassed in specific pseudorevertants isolated by genetic selection from the delta P2 mutants . These results, together with phylogenetic data, support the existence of alternate strategies to create a functional P1-core interaction.

Cancer Res, 1992 Feb 15, 52(4), 1018 - 25
Disseminating tumor cells and their interactions with leukocytes visualized in the brain; Lampson LA et al.; Brain tumors are increasingly prevalent . Recent advances focus attention on individual, disseminated tumor cells that cannot be imaged or eliminated . Cells of the immune system may be ideally suited to attack individual tumor cells, but more basic understanding is needed . We describe a rat model, using the lacZ reporter gene, that allows identification of individual tumor cells, and tumor-leukocyte interactions in vivo . The model demonstrates how widely tumor can disseminate, without secondary tumorigenesis or recruitment of nonneoplastic cells . It demonstrates that leukocytes have access to disseminating tumor . Among its many applications, this work lays a foundation for developing cell-mediated immunotherapy against individual brain tumor cells.

Biochem J, 1992 Feb 15, 282 ( Pt 1), 155 - 64
The catalytic consequences of experimental evolution . Studies on the subunit structure of the second (ebg) beta-galactosidase of Escherichia coli, and on catalysis by ebgab, an experimental evolvant containing two amino acid substitutions; Elliott AC et al.; 1 . The ratio of ebgA-gene product of ebgC-gene product in the functional aggregate of ebg beta-galactosidases was determined to be 1:1 by isolation of the enzyme from bacteria grown on uniformly radiolabelled amino acids and separation of the subunits by gel-permeation chromatography under denaturing conditions . 2 . This datum, taken together with a recalculation of the previous ultracentrifuge data {Hall (1976) J . Mol . Biol . 107, 71-84}, analytical gel-permeation chromatography and electron microscopy, strongly suggests an alpha 4 beta 4 quaternary structure for the enzyme . 3 . The second chemical step in the enzyme turnover sequence, hydrolysis of the galactosyl-enzyme intermediate, is markedly slower for ebgab, having both Asp-97----Asn and Trp-977----Cys changes in the large subunit, than for ebga (having only the first change) and ebgb (having only the second), and is so slow as to be rate-determining even for an S-glycoside, beta-D-galactopyranosyl thiopicrate, as is shown by nucleophilic competition with methanol . 4 . The selectivity of galactosyl-ebgab between water and methanol on a molar basis is 57, similar to the value for galactosyl-ebgb . 5 . The equilibrium constant for the hydrolysis of lactose at 37 degrees C is 152 +/- 19 M, that for hydrolysis of allolactose is approx . 44 M and that for hydrolysis of lactulose is approx . 40 M . 6 . A comparison of the free-energy profiles for the hydrolyses of lactose catalysed by the double mutant with those for the wild-type and the single mutants reveals that free-energy changes from the two mutations are not in general independently additive, but that the changes generally are in the direction predicted by the theory of Burbaum, Raines, Albery & Knowles {(1989) Biochemistry 28, 9283-9305} for an enzyme catalysing a thermodynamically irreversible reaction . 7 . Michaelis-Menten parameters for the hydrolysis of six beta-D-galactopyranosylpyridinium ions and ten aryl beta-galactosides by ebgab were measured . 8 . The derived beta 1g values are the same as those for ebgb (which has only the Trp-977----Cys change) and significantly different from those for ebgo (the wild-type enzyme) and ebga . 9 . The alpha- and beta-deuterium secondary isotope effects on the hydrolysis of the galactosyl-enzyme of 1.08 and 1.00 are difficult to reconcile with the pyranose ring in this intermediate being in the 4C1 conformation.

Eur J Biochem, 1992 Feb 15, 204(1), 85 - 91
Hybrid myosin light chains containing a calcium-specific site from troponin C; da Silva AC et al.; Recombinant DNA approaches have allowed us to probe the mechanisms by which the regulatory light chains (RLCs) regulate myosin function by identifying the functional importance of specific regions of the RLC molecule . For example, we have demonstrated that the presence of high-affinity Ca2+/Mg(2+)-binding site in the N-terminal domain of the RLC is essential for the regulation of myosin-actin interaction {Reinach, F . C., Nagai, K . & Kendrick-Jones, J . (1986) Nature 322, 80-83} . To explore further the role of this metal-binding site in the RLC and generate an RLC with a Ca(2+)-specific site, we constructed four chicken skeletal muscle myosin regulatory light chain hybrid 'genes' . In these, the first domain containing the high-affinity Ca2+/Mg(2+)-binding site in the RLC was replaced with that containing the lower-affinity, Ca(2+)-specific, regulatory site from troponin C (TnC) . In two of these hybrids, we replaced only the Ca(2+)-binding EF hand, while in the other two the EF hand and the N-terminal helix of TnC were transplanted . These hybrids were expressed in Escherichia coli in high yields and the purified proteins were used in calcium-binding experiments to assay the affinity and specificity of the sites and incorporated into scallop myosin to assay their regulatory behaviour . The results obtained show that the calcium-binding site from TnC, when transplanted into the RLC backbone, had a low affinity although most of its specificity appeared to be retained . As a result, although the TnC/RLC hybrids bound to scallop myosin and were able to activate the MgATPase activity of scallop acto-myosin, they were unable to regulate it . These results are in agreement with our previous findings that occupancy of the Ca2+/Mg2+ site in the RLC is essential for regulation . Our results suggest that the specificity and affinity of the calcium-binding site in troponin C is dependent on both intra- and inter-domain interactions within troponin C and that these latter interactions appear to be missing when this binding site is transplanted into the light chain backbone.

J Biol Chem, 1992 Feb 15, 267(5), 3482 - 9
Deletions in hydrophilic domains of subunit a from the Escherichia coli F1F0-ATP synthase interfere with membrane insertion or F0 assembly; Lewis MJ et al.; The a subunit is a membrane component of the F1F0-ATP synthase from Escherichia coli . Regions of a which appear important for membrane insertion or F0 assembly have been identified by analysis of both deletion mutants and fusion proteins which link the mutant a subunits to alkaline phosphatase . This analysis suggests the hydrophilic, amino-terminal domain of a is required for proper membrane targeting and/or insertion of the nascent polypeptide . In addition, the subcellular fractionation of four different a subunit-beta-galactosidase fusion proteins suggests this domain is localized to the periplasm, in agreement with a proposed topological model of the protein (Lewis, M.J., Chang, J.A., and Simoni, R.D . (1990) J . Biol . Chem . 265, 10541-10550) . Deletions within the next three putative loops of a appear to have no significant effect on membrane targeting or insertion . Rather, they seem to interfere with the subsequent assembly of a functional enzyme.

Gene, 1992 Feb 15, 111(2), 183 - 97
Synthetic human tRNA(UUULys3) and natural bovine tRNA(UUULys3) interact with HIV-1 reverse transcriptase and serve as specific primers for retroviral cDNA synthesis; Weiss S et al.; Full-length and 5'-truncated variants of human (h) tRNA(UUULys3) were synthesized by in vitro transcription using SP6 RNA polymerase . Bovine(b) tRNA(SUULys3) was purified from calf liver . Both full-length tRNA species were shown to be biologically active in an aminoacylation assay . Gel retardation assays revealed that both full-length tRNA species, as well as a 5'-truncated h-tRNA(UUULys3) molecule containing 24 nucleotides (nt) at the 3' end (Lys24), interact with human immunodeficiency virus (HIV)-1 reverse transcriptase (RT) . Competition studies with these three tRNA species demonstrate that the 3' end of h-tRNA(UUULys3) contributes to the interaction with HIV-1 RT . Escherichia coli tRNA(UUULys) and tRNA(UUCGlu2) were also able to interact with the enzyme, whereas unrelated RNA molecules such as E . coli 5S rRNA did not bind to RT . Both b-tRNA(SUULys3) and h-tRNA(UUULys3) molecules, as well as the 5'-truncated variants, could be demonstrated to prime cDNA synthesis specifically using a HIV-1 RNA template, prepared by in vitro transcription, indicating that other viral or cellular proteins are not essential for this process . E . coli tRNA(UUULys) and tRNA(UUCGlu2), although able to interact with HIV-1 RT, failed to prime retroviral transcription . Products of cDNA synthesis were characterized by polymerase chain reaction, demonstrating that at least 18 nt at the 3' ends of h-tRNA(UUULys3) and b-tRNA(SUULys3) are still present in the cDNA product, whereas the 5' ends of both primer molecules were removed by the RNase H activity of HIV-1 RT.

Eur J Biochem, 1992 Feb 15, 204(1), 51 - 6
Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12; Andersen PS et al.; The upp gene coding for uracil phosphoribosyltransferase was subcloned on a 5-kb EcoRI restriction fragment along with the purMN operon . By a combination of complementation, deletion and minicell analyses, the upp gene was located adjacent to and divergently transcribed from the purMN operon . All three gene products could be identified in minicell extracts . The cloned upp gene shows an elevated expression upon uracil starvation . The nucleotide sequence and transcription start of the gene were determined . The sequence yields an open reading frame of 624 nucleotides encoding a protein of 22.5 kDa which is in agreement with the previously determined subunit Mr of the purified enzyme . A putative 5-phosphoribosyl-alpha-1-diphosphate (PRPP) binding site has been identified which is similar to the PRPP binding site of the yeast uracil phosphoribosyltransferase.

J Biol Chem, 1992 Feb 15, 267(5), 3168 - 72
Ion channels are likely to be involved in the two steps of phage T5 DNA penetration into Escherichia coli cells; Boulanger P et al.; Phage T5 injects its DNA into Escherichia coli cells in two steps; 8% of the chromosome is first injected, and then there is a pause during which proteins encoded by this DNA fragment are synthesized allowing the remaining DNA to be injected . Using a potassium-selective electrode, we show that the injection of the two DNA fragments is associated with an efflux in two steps, of cytoplasmic potassium . The rate of efflux is linearly related to the number of added phages suggesting that each phage induces the formation of at least one channel in the inner membrane . The first efflux occurs even in depolarized cells suggesting that the insertion and the opening of the channel can take place in the absence of the electrochemical gradient of protons (delta mu H+) . The channel is in a closed configuration during the time required for the synthesis of the phage-encoded proteins; this closing and the second efflux are prevented by the depolarization of the cell . The insertion of the channel in the inner membrane requires a fluid membrane . The results obtained suggest that the function of this channel is to translocate phage T5 DNA.

J Biol Chem, 1992 Feb 15, 267(5), 2849 - 52
Effect of overproduction of heat shock chaperones GroESL and DnaK on human procollagenase production in Escherichia coli; Lee SC et al.; The effect of overexpression of the heat shock chaperone genes dnaK and groESL on heterologous protein production in Escherichia coli was examined, using a set of related human procollagenase proteins . A diverse range of effects on protein solubility, secretion, and accumulation was observed, and these effects were highly dependent on the particular chaperone/procollagenase pairing involved . Both chaperones caused a large increase in the apparent solubility of a fusion of the LamB signal peptide to procollagenase . GroESL had no effect on the accumulation of mature (secreted) procollagenase, while DnaK suppressed secretion considerably . In the absence of a signal peptide, overexpression of either chaperone resulted in a dramatic increase in both solubility and accumulation of procollagenase . The 10-fold increase in accumulation was associated with an increase in in vivo protein half-life.

Gene, 1992 Feb 15, 111(2), 245 - 8
Production in Escherichia coli of porcine type-I collagenase as a fusion protein with beta-galactosidase; O'Hare MC et al.; Porcine type-I collagenase (Colg-1) was produced as a fusion protein in Escherichia coli using the pAX5 expression vector . The fusion protein consists of beta-galactosidase at the N terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to Colg-1 . Recombinant collagenase (reColg-1) was biologically active in the form of a fusion protein and could be released by treatment with factor Xa to yield Colg-1 with the authentic N terminus (phenylalanine) found in vivo . The results show that reColg-1 produced in E . coli is folded correctly, cleaves type-I collagen into 1/4 and 3/4 fragments at the characteristic Colg-sensitive site, and is produced at high enough levels to generate a source of recombinant enzyme for x-ray crystallography studies.

Gene, 1992 Feb 15, 111(2), 215 - 22
Isolation of a clone which induces expression of the gene encoding the human tumor necrosis factor receptor; Garret M et al.; Tumor necrosis factor (TNF) is a cytokine with pleiotropic effects upon cell growth, inflammation and immunologic responsiveness . High-affinity TNF receptors (TNFRs) of 55 and 75 kDa are found in many cell types . Using an Epstein-Barr virus (EBV)-based mammalian expression library, we have isolated a clone from human lymphoblastoid transfectants that induces overexpression of the TNFR-encoding gene (TNFR) . Transfectants overproducing the TNFR were isolated by multiple rounds of sorting on a fluorescence-activated cell sorter using fluorescent TNF ligand binding as the selection procedure . Among the sorted transfectants were cells producing approx . 150,000 receptors per cell (Kd of approx . 1 nM) . These cells have multiple copies of the TNFR gene present as extrachromosomal plasmids . These cells also overproduced the mRNA for TNFR . Low-Mr EBV episomes were isolated from these overproducing cells and used to transform Escherichia coli . One of the colonies isolated contained a plasmid encoding a portion of the noncoding region of the TNFR gene . Transfection of human lymphoblastoid cells with this DNA gave rise to high-level production of TNFR . Fluorescent TNF bound to these transfectants is fully and specifically displaced by an excess of TNF . The rescued clone contains approx . 10 kb of human genomic DNA including the 3'-untranslated region of TNFR and several Alu sequences; apparently during the selection procedure in human cells, recombination occurred to rescue a portion of the TNFR gene . Transient transfection was used to narrow down the region responsible for TNFR induction to 5.2 kb . The mechanism by which this clone induces TNFR expression has not been determined.

Biochem J, 1992 Feb 15, 282 ( Pt 1), 299 - 303
Stimulation of glycolysis as an activation signal in rat peritoneal macrophages . Effect of glucocorticoids on this process; Bustos R et al.; 1 . Peritoneal macrophages were prepared from control, Escherichia coli-treated and triamcinolone acetonide-treated rats . Control and E . coli-treated rats produced resident and activated macrophages respectively . Glycolysis in these cells was studied by the fructose 2,6-bisphosphate (Fru-2,6-P2) content, lactate release and 6-phosphofructo-1-kinase (PFK-1) and 6-phosphofructo-2-kinase (PFK-2) activities . 2 . In activated macrophages, lactate release and Fru-2,6-P2 content were increased several-fold compared with those in resident cells . Moreover, the response of these parameters to phorbol 12-myristate 13-acetate in activated macrophages was greater than for resident cells . 3 . PFK-2 activity was moderately increased (about 3-fold), but PFK-1 activity was increased 5-fold in activated macrophages compared with resident cells . Partially purified preparations of PFK-1 were sensitive to Fru-2,6-P2, with K0.5 about 0.25 microM in both control and activated cells . However, the Vmax . of PFK-1 from activated cells was increased . In addition, AMP stimulated PFK-1, but the kinetic pattern was different from that described for Fru-2,6-P2 . Moreover there was no difference in the stimulation by AMP of PFK-1 from resident and activated cells . 4 . Fru-2,6-P2 content and lactate release in macrophages from triamcinolone acetonide-treated rats were decreased in both resident and activated cells . Also, the glucocorticoid inhibited PFK-1 and PFK-2 activities in both resident and activated macrophages . PFK-1 from triamcinolone acetonide-treated rats was not stimulated by Fru-2,6-P2, whereas the effect of AMP was unchanged . The effects of glucocorticoid seem to be specific for phagocytic cells, since the glucocorticoid treatment increased PFK-1 and PFK-2 activities in liver.

Biochem J, 1992 Feb 15, 282 ( Pt 1), 255 - 9
The orientation of the three haems of the 'in situ' ubiquinol oxidase, cytochrome bd, of Escherichia coli; Ingledew WJ et al.; The Escherichia coli cytochrome bd complex incorporates three haems as prosthetic groups . In the ferric form these are a predominantly high-spin chlorin (haem d), a high-spin haem b (b595) and a low-spin haem b (b558) . The orientations of these three haems have been determined by e.p.r . studies on oriented multilayer preparations of cytoplasmic membrane fragments . The low-spin haem b (b558) and the high-spin haem d are oriented with their haem planes perpendicular to the membrane plane . The high-spin haem b595 is oriented with its haem plane at approx . 55 degrees to the membrane plane . A minor low-spin component, attributable to a low-spin subpopulation of the haem d, is also oriented with its haem plane perpendicular to the membrane plane.

Biochem J, 1992 Feb 15, 282 ( Pt 1), 139 - 45
Phosphorylation of the C-terminal domain of the Na+/H+ exchanger by Ca2+/calmodulin-dependent protein kinase II; Fliegel L et al.; The Na+/H+ exchanger is a pH-regulatory protein that extrudes one H+ ion in exchange for one Na+ ion when intracellular pH declines . A number of studies have shown phorbol ester stimulation of activity in intact cells, leading to the idea that the exchanger is regulated by protein kinase C-mediated phosphorylation in vivo . cDNA encoding the protein has been cloned, and a recent model suggests a large internal cytoplasmic C-terminal domain that may be a site of regulation of the exchanger {Sardet, Franchi & Pouyssegur (1989) Cell 56, 271-280} . We examined this region of the protein using a rabbit cardiac Na+/H+ exchanger cDNA clone . cDNA of the Na+/H+ exchanger, coding for the C-terminal 178 amino acid residues, was cloned into the expression vector pEX-1 and expressed as a fusion protein with beta-galactosidase . The fusion protein reacted with an antibody produced against a synthetic peptide of the C-terminal 13 amino acid residues of the Na+/H+ exchanger, confirming the identity of the expressed protein . Control and experimental pEX-1-Na+/H+ exchanger protein was purified on a p-aminophenyl beta-D-thiogalactopyranoside-agarose column . Purified Ca2+/calmodulin-dependent protein kinase II readily phosphorylated the Na+/H+ exchanger protein in a Ca(2+)- and calmodulin-dependent manner in vitro, but this region of the protein was not a substrate for purified protein kinase C or for the catalytic subunit of cyclic AMP-dependent protein kinase . Control-expressed beta-galactosidase was phosphorylated to a maximal level of 0.77 +/- 0.17 mol of Pi/mol (mean +/- S.E.M., n = 6) whereas the fusion protein was phosphorylated to a maximal level of 4.09 +/- 0.39 mol of Pi/mol (n = 6), suggesting one site of phosphorylation in beta-galactosidase and three in the C-terminal domain of the Na+/H+ exchanger . Examination of the deduced amino acid sequence of this part of the exchanger reveals three consensus sequences for Ca2+/calmodulin-dependent protein kinase II . These results suggest that the exchanger may be directly regulated in vivo by calmodulin-dependent protein kinase II but not by protein kinase C or cyclic AMP-dependent protein kinase.

Proc Natl Acad Sci U S A, 1992 Feb 15, 89(4), 1244 - 8
IS5: a mobile enhancer of transcription in Escherichia coli; Schnetz K et al.; The cryptic bgl operon of Escherichia coli is activated by the spontaneous insertion of mobile DNA elements . Screening of a collection of such mutations revealed insertion of the 1195-base-pair element IS5 into various positions both upstream and downstream of the bgl promoter P0 . Activation of the operon was in all cases attributable to enhancement of P0 activity . Introduction of internal deletions into IS5 almost completely abolished P0 enhancement, demonstrating that enhancement is not simply the result of mutational inactivation of some inhibitory sequences . Intact copies of IS5 in trans restored the enhancing activity of the deletion derivatives . The trans-activator is encoded by IS5 gene ins5A, an essential transposition function . Activation of gene expression by means of interaction of a defective mobile element in cis with functions encoded by a nondefective element in trans has so far been described only for a maize controlling element.

Arch Biochem Biophys, 1992 Feb 14, 293(1), 93 - 102
Expression and function of heterologous forms of malate dehydrogenase in yeast; Steffan JS et al.; The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms . To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase . The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo . Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells . Efficient expression of the E . coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1 . Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein . Results of in vitro import experiments suggest that the percursor form of the E . coli protein associates with yeast mitochondria but is not efficiently internalized . Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain . However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.

Arch Biochem Biophys, 1992 Feb 14, 293(1), 140 - 6
Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin; Furutani M et al.; The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared . Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only . EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not . The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins . The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin . The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them . EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin . In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin . The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation . In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.

Anal Biochem, 1992 Feb 14, 201(1), 152 - 7
Detection and possible origins of aminomalonic acid in protein hydrolysates; Copley SD et al.; Aminomalonic acid (Ama) was first detected in alkaline hydrolysates of proteins in 1984 . In this work we describe our search for the origin of aminomalonic acid in alkaline hydrolysates of proteins . We have developed a technique for quantitation of aminomalonic acid based upon gas chromatography/mass spectrometry . Using this technique, we find approximately 0.3 Ama/1000 amino acids in hydrolysates of Escherichia coli protein . We have demonstrated that Ama is not formed from any of the 20 major amino acids during the hydrolysis procedure . Furthermore, the amount of Ama found does not depend on the presence of small amounts of O2 during the hydrolysis . Thus far, we have not been able to demonstrate an artifactual origin for Ama . The results described above suggest that Ama may indeed be a constituent of proteins before the hydrolysis procedure . Possible origins of Ama include errors in protein synthesis and oxidative damage to amino acid residues in proteins.

J Immunol Methods, 1992 Feb 14, 147(1), 119 - 24
Establishment of an enzyme release assay for cytotoxic T lymphocyte activity; Ohmori H et al.; In the present report, we describe the establishment of a cell line that can be used as the target for measuring the activity of cytotoxic T lymphocytes (CTL) by an enzyme release assay . We transfected P3/NS1-Ag4-1 (NS-1), a myeloma cell line derived from BALB/c mice with Escherichia coli beta-galactosidase (beta-Gal) gene, and isolated a stable transformant designated as NS-1/Z that expressed a high level of the enzyme activity intracellularly . The effector cells showing cytotoxicity against NS-1/Z were induced when the spleen cells of AKR or C3H mice were cultured with mitomycin C-treated BALB/c spleen cells for 4 days . When 2 x 10(4) NS-1/Z cells were incubated with varying numbers of effector cells, beta-Gal activity was released from the target cells depending on the number of effector cells and the time of incubation for up to 8 h . A highly sensitive enzyme assay was performed by using a fluorescent substrate, 4-methylumbelliferyl-beta-D-galactoside . The cytotoxicity was specific for H-2 haplotype of the stimulator cells, and was abolished by treating the effector cells with anti-Lyt 2 plus complement . The sensitivity of the enzyme release assay was comparable to that of 51Cr release assay . These results indicate that NS-1/Z can be used as a target cell line for the non-radioactive measurement of CTL activity.

Biochem Biophys Res Commun, 1992 Feb 14, 182(3), 981 - 6
Importance of histidine residue 25 of rat heme oxygenase for its catalytic activity; Ishikawa K et al.; A truncated, soluble, and enzymatically active rat heme oxygenase lacking its membrane-associative, C-terminal segment was expressed in E . coli strain JM109 . The roles of its four histidine residues were examined by determining the enzymatic activities of mutant enzymes in which each of these residues in turn was replaced by alanine . Mutation of histidine residue 25 to alanine resulted in marked decrease in activity for heme breakdown, indicating that this histidine residue has an important role in the heme oxygenase reaction.

Biochem Biophys Res Commun, 1992 Feb 14, 182(3), 1499 - 505
Characterization of the unprocessed and processed forms of rab6 expressed in baculovirus/insect cell systems; Yang C et al.; Rab6 protein (rab6p) belongs to a family of ras-like GTP-binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells . We have constructed a recombinant baculovirus in order to express rab6p in insect cells . We report here the characterization of four forms of this protein which are found in cytosolic and membrane fractions of infected Sf9 cells . The two major forms are a cytosolic 24 kD protein which represents the unprocessed precursor form of rab6p and a membrane-bound isoprenylated 23 kD protein which represents the processed form . Two other minor forms were also detected: a cytosolic isoprenylated 23 kD protein which may represent a pool in equilibrium with the 23 kD membrane-bound form and a 24 kD non-isoprenylated membrane-bound form which may represent an intermediate in the processing of rab6p.

Biochem Biophys Res Commun, 1992 Feb 14, 182(3), 1482 - 90
Mutagenesis studies on the amino acid residues involved in the iron-binding and the activity of human 5-lipoxygenase; Ishii S et al.; Human 5-lipoxygenase contains a non-heme iron essential for its activity . In order to determine which amino acid residues are involved in the iron-binding and the lipoxygenase activity, nine amino acid residues in highly homologous regions among the lipoxygenases were individually replaced by means of site-directed mutagenesis . Mutant 5-lipoxygenases in which His-367 or His-550 was replaced by either Asn or Ala, His-372 by either Asn or Ser, or Glu-376 by Gln were completely devoid of the activity . Though mutants containing an alanine residue instead of His-390 or His-399 lacked the activity, the corresponding asparagine substituted mutants exhibited . The other mutants retained the enzyme activity . These results strongly suggest that His-367, His-372, His-550 and Glu-376 are crucial for 5-lipoxygenase activity and coordinate to the essential iron.

Biochem Biophys Res Commun, 1992 Feb 14, 182(3), 1218 - 25
Requirement for GLY-60 of Escherichia coli adenylyl cyclase for ATP binding and catalytic activity; Amin N et al.; The region of Escherichia coli adenylyl cyclase spanned by glycine-55 to threonine-65 was tested for its importance for enzyme activity . Site-directed mutagenesis was used to replace glycine-55 and glycine-60 as well as lysine-59, leucine-63 and threonine-65 with other amino acids . While substitution of glycine-55 with aspartic acid produced no significant change in kinetic parameters, the change of glycine-60 to aspartic acid or asparagine eliminated binding to 8-azido-ATP and decreased the Vmax (two orders of magnitude) and Km (factor of four-five) . Smaller effects on kinetic parameters were observed with substitutions of lysine-59, leucine-63 or threonine-65.

Biochem Biophys Res Commun, 1992 Feb 14, 182(3), 1040 - 6
Sequence of a putative glutathione synthetase II gene and flanking regions from Anaplasma centrale; Peters JM et al.; The complete nucleotide sequence of a putative glutathione synthetase gene (gsh II) has been determined from Anaplasma centrale . The predicted 308 amino acid protein has a molecular weight of 34,222 and is 32% identical to the enzyme, glutathione synthetase (EC 6.3.2.3), encoded by Escherichia coli gsh II . The previously proposed ATP-binding site is not highly conserved . The putative glutathione synthetase gene (gsh II) is preceded by an unassigned open reading frame . Downstream of gsh II is the 5' region of an open reading frame encoding a protein with significant similarity to bacterial D-alanine:D-alanine ligases (ADP forming) (EC 6.3.2.4) . The predicted partial amino acid sequence is 33% identical to the amino acid sequence of the protein encoded by the E . coli ddl gene.

Science, 1992 Feb 14, 255(5046), 838 - 41
Structure of transcription elongation complexes in vivo; Kainz M et al.; The opening of duplex DNA in the elongation phase of transcription by Escherichia coli RNA polymerase in vivo was detected at a regulatory site where a prolonged pause in transcription occurs . Single-stranded DNA in the transcription bubble was identified by its reactivity with potassium permanganate (KMnO4) . The elongation structure in vivo was similar to that of transcription complexes made in vitro with some differences . The observed reactivity to KMnO4 of the DNA template strand was consistent with the existence of an RNA-DNA hybrid of about 12 nucleotides.

Science, 1992 Feb 14, 255(5046), 809 - 12
The elongation-termination decision in transcription; von Hippel PH et al.; At any template position, the decision to extend the transcript by one residue or to release the nascent RNA represents a kinetic competition between elongation and termination pathways . This competition is discussed in terms of alternative Eyring transition state barriers; changes in termination efficiency correspond to small changes in the relative heights of these barriers . Elongation complexes are stable at nonterminator positions; a model is presented to explain the destabilization of these complexes at intrinsic termination sites . Functionally analogous effects can operate at rho-dependent terminators . Mechanisms for modulation of termination efficiency by regulatory proteins are described.

Biochem Biophys Res Commun, 1992 Feb 14, 182(3), 1130 - 8
cDNA cloning for and preparation of antibodies against subunit d of H(+)-ATP synthase in rat mitochondria; Motojima K et al.; The cDNA for subunit d of rat mitochondrial H(+)-ATP synthase was cloned from a brain cDNA library . The protein contains internally repeat structures that have sequences similar to those of other ATP-related proteins . The antibodies raised against the protein A-subunit d fusion protein expressed in E . coli specifically recognized only the protein in the mitochondria . The Na2 CO3 fractionation followed by immunoblotting analyses suggest that at least a part of the protein is inserted into the membrane.

Anal Biochem, 1992 Feb 14, 201(1), 178 - 84
A novel serum assay for breast epithelial antigen using a fusion protein; Ceriani RL et al.; Serum levels of breast epithelial antigens (BrE-Ags) are presently used in the follow-up of breast cancer patients . Available assays do not have optimal sensitivity and rely on reagents that could vary in their source and purity . A novel competitive solid-phase radioimmunoassay was developed for BrE-Ags that consists of the NP5 fusion protein, produced in Escherichia coli, that is composed of beta-galactosidase and polypeptide sequence obtained from a breast carcinoma cell line cDNA library, and anti-human milk fat globule monoclonal antibody Mc5 . The fusion protein carries an altered epitope sequence (mimotope) that is similar, but not identical, to that found in the native antigen . This new competitive assay configuration has two essential features, a solid-phase affinity step that purifies the fusion protein carrying the mimotope for Mc5 and a competitive step that provides quantitation . Serum values for this assay show high specificity and sensitivity for breast cancer patients when compared to normal subjects and post-surgical breast cancer patients during their disease-free period.

J Immunol Methods, 1992 Feb 14, 147(1), 1 - 11
Use of recombinant fusion proteins for generation and rapid characterization of monoclonal antibodies . Application to the Kunitz domain of human beta amyloid precursor protein; Wunderlich D et al.; Production of peptides by recombinant DNA techniques is an efficient alternative to chemical synthesis of peptides . Proteins and peptides produced by recombinant DNA methods in E . coli are routinely used as antigens for the production of antibodies . However, most small peptides are rapidly degraded within the E . coli cell, and therefore, must initially be expressed as components of larger, more stable fusion proteins . The peptide of interest must be cleaved from the fusion protein, and purified prior to immunization to eliminate epitopes contributed by the fusion partner . We have now established methods for the production and characterization of monoclonal antibodies using partially purified, uncleaved fusion proteins . We have also described a method for efficient production and detection of the fusion protein, an EIA for rapid differential screening of hybridoma supernatants, and a strategy for epitope mapping of the antibodies . These methods have been applied to the production and characterization of monoclonal antibodies specific for a 75-amino-acid internal segment of the Alzheimer amyloid precursor protein, and should be applicable to a wide variety of other peptides and proteins.

Biochem Biophys Res Commun, 1992 Feb 14, 182(3), 1402 - 7
Purification of recombinant SH2/SH3 proteins of phospholipase C-gamma 1 and -gamma 2 and their inhibitory effect on PIP2-hydrolysis induced by both types of phospholipase C-gamma; Homma Y et al.; In order to examine physiological function of the SH2/SH3 region of phospholipase C-gamma (Z region), we independently expressed cDNA fragments corresponding to the SH2/SH3 region of PLC-gamma 1 and PLC-gamma 2 in Escherichia coli . Although these recombinant proteins were recovered in particulate fractions by centrifugation of cell extracts, they were successfully solubilized by guanidium hydrochloride and then purified to homogeneity by heparin column chromatography . The molecular mass of the proteins was 45 kDa (derived from PLC-gamma 1 and designated as rP45Z) and 38 kDa (derived from PLC-gamma 2 and designated as rP38Z), which was consistent with that as expected from inserted cDNA . We determined the effect of purified rP45Z or rP38Z on PIP2-hydrolyzing activity of either PLC-gamma 1 or PLC-gamma 2 and found that these proteins strongly suppressed the rate of PLC-dependent PIP2-hydrolysis . Furthermore, both rP45Z and rP38Z were phosphorylated at tyrosine residue by epidermal growth factor receptors and their inhibitory effect on PIP2-hydrolysis was significantly decreased by this phosphorylation . These results indicate that the Z region might be involved in autoregulation of PLC-gamma as intrinsic negative regulator.

FEMS Microbiol Rev, 1992 Feb, 8(2), 109 - 35
Structure-function relationships among the nickel-containing hydrogenases; Przybyla AE et al.; The enzymology of the heterodimeric (NiFe) and (NiFeSe) hydrogenases, the monomeric nickel-containing hydrogenases plus the multimeric F420-(NiFe) and NAD(+)-(NiFe) hydrogenases are summarized and discussed in terms of subunit localization of the redox-active nickel and non-heme iron clusters . It is proposed that nickel is ligated solely by amino acid residues of the large subunit and that the non-heme iron clusters are ligated by other cysteine-rich polypeptides encoded in the hydrogenase operons which are not necessarily homologous in either structure or function . Comparison of the hydrogenase operons or putative operons and their hydrogenase genes indicate that the arrangement, number and types of genes in these operons are not conserved among the various types of hydrogenases except for the gene encoding the large subunit . Thus, the presence of the gene for the large subunit is the sole feature common to all known nickel-containing hydrogenases and unites these hydrogenases into a large but diverse gene family . Although the different genes for the large subunits may possess only nominal general derived amino acid homology, all large subunit genes sequenced to date have the sequence R-X-C-X-X-C fully conserved in the amino terminal region of the polypeptide chain and the sequence of D-P-C-X-X-C fully conserved in the carboxyl terminal region . It is proposed that these conserved motifs of amino acids provide the ligands required for the binding of the redox-active nickel . The existing EXAFS (Extended X-ray Absorption Fine Structure) information is summarized and discussed in terms of the numbers and types of ligands to the nickel and the various redox species of nickel defined by EPR spectroscopy . New information concerning the ligands to nickel is presented based on site-directed mutagenesis of the gene encoding the large subunit of the (NiFe) hydrogenase-1 of Escherichia coli . Based on considerations of the biochemical, molecular and biophysical information, ligand environments of the nickel in different redox states of the (NiFe) hydrogenase are proposed.

Biochemistry, 1992 Feb 11, 31(5), 1498 - 504
Engineering surface charge . 2 . A method for purifying heterodimers of Escherichia coli glutathione reductase; Deonarain MP et al.; Two gor genes encoding different mutants of Escherichia coli glutathione reductase have been expressed in the same E . coli cell, leading to the creation of a hybrid form of the enzyme dimer . One of the gor genes carried, in addition to various directed mutations, a 5' extension that encodes a benign penta-arginine "arm" added to the N-terminus of the glutathione reductase polypeptide chain {Deonarain, M.P., Scrutton, N.S., & Perham, R.N . (1992) Biochemistry (preceding paper in this issue)} . This made possible, by means of ion-exchange chromatography or nondenaturing polyacrylamide gel electrophoresis, the facile separation of the hybrid enzyme from the two parental forms . Moreover, the two subunits in the hybrid enzyme could be made to carry different mutations . In this way, glutathione reductases with only one active site per dimer were generated: the effects of replacing tyrosine-177 with glycine in the NADPH-binding site, which greatly diminishes the Km for glutathione and switches the kinetic mechanism from ping-pong to ordered sequential, and of replacing His-439 with glutamine in the glutathione-binding site, which greatly diminishes the Km for NADPH, were both found to be restricted to the one active site carrying the mutations . This system of generating separable enzyme hybrids is generally applicable and should make it possible now to undertake a more systematic study of catalytic mechanism and assembly for the many enzymes with quaternary structure.

Biochemistry, 1992 Feb 11, 31(5), 1491 - 7
Engineering surface charge . 1 . A method for detecting subunit exchange in Escherichia coli glutathione reductase; Deonarain MP et al.; The gene gor encoding Escherichia coli glutathione reductase was mutated to create a positively charged N-terminal extension consisting of five arginine residues followed by a factor Xa cleavage site to the enzyme polypeptide chain . The modified protein assembled in vivo to yield a dimeric enzyme with kinetic parameters indistinguishable from those of wild-type glutathione reductase . The N-terminal extension could not be released by treatment with factor Xa but could be removed by exposure to trypsin, again without effect on the enzyme activity . The modified enzyme was readily separated from the wild-type enzyme by means of ion-exchange chromatography or nondenaturing polyacrylamide gel electrophoresis . Incubation of the modified and wild-type enzymes, separately or as a mixture, with NADH led to their partial inactivation, and activity was restored by exposure to 1 mM reduced glutathione . No hybrid dimer was formed in the mixture of modified and wild-type enzymes, as judged by polyacrylamide gel electrophoresis, strongly suggesting that the inactivation induced by NADH was not due to dissociation of the parental dimers . The addition of otherwise benign positively or negatively charged extensions to the N- or C-terminal regions of the constituent polypeptide chains of oligomeric enzymes offers a simple route to detecting hybrid formation and the causative subunit dissociation and exchange.

Biochemistry, 1992 Feb 11, 31(5), 1370 - 5
Substitution of Torpedo acetylcholine receptor alpha 1-subunit residues with snake alpha 1- and rat nerve alpha 3-subunit residues in recombinant fusion proteins: effect on alpha-bungarotoxin binding; Chaturvedi V et al.; A fusion protein consisting of the TrpE protein and residues 166-211 of the Torpedo acetylcholine receptor alpha 1 subunit was produced in Escherichia coli using a pATH10 expression vector . Residues in the Torpedo sequence were changed by means of oligonucleotide-directed mutagenesis to residues present in snake alpha 1 subunit and rat nerve alpha 3 subunit which do not bind alpha-bungarotoxin . The fusion protein of the Torpedo sequence bound 125I-alpha-bungarotoxin with high affinity (IC50 = 2.5 x 10(-8) M from competition with unlabeled toxin, KD = 2.3 x 10(-8) M from equilibrium saturation binding data) . Mutation of three Torpedo residues to snake residues, W184F, K185W, and W187S, had no effect on binding . Conversion of two additional Torpedo residues to snake, T191S and P194L, reduced alpha-bungarotoxin binding to undetectable levels . The P194L mutation alone abolished toxin binding . Mutation of three Torpedo alpha 1 residues to neuronal alpha 3-subunit residues, W187E, Y189K, and T191N, also abolished detectable alpha-bungarotoxin binding . Conversion of Try-189 to Asn which is present in the snake sequence (Y189N) abolished toxin binding . It is concluded that in the sequence of the alpha subunit of Torpedo encompassing Cys-192 and Cys-193, Try-189 and Pro-194 are important determinants of alpha-bungarotoxin binding . Tyr-189 may interact directly with cationic groups or participate in aromatic-aromatic interactions while Pro-194 may be necessary to maintain a conformation conductive to neurotoxin binding.

Biochemistry, 1992 Feb 11, 31(5), 1270 - 9
The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli; Glockshuber R et al.; The formation of the disulfide bonds in the variable domains VH and VL of the antibody McPC603 was found to be essential for the stability of all antigen binding fragments investigated . Exposure of the Fv fragment to reducing conditions in vitro resulted in irreversible denaturation of both VH and VL . In vitro refolding of the reduced Fv fragment was only possible when the disulfide bonds were allowed to form under oxidizing conditions . The analysis of a series of mutants of the Fv fragment, the Fab fragment and the single-chain Fv fragment, all secreted into the periplasm of Escherichia coli, in which each of the cysteine residues of the variable domains was replaced by a series of other amino acids, showed that functional antigen binding fragments required the presence of both the disulfide bond in VH and the one in VL . These results were also used to devise an alternative expression system based on the production of insoluble fusion proteins consisting of truncated beta-galactosidase and antibody domains, enzymatic cleavage, and refolding and assembly in vitro . This strategy should be useful for providing access to unstable antibody domains and fragments.

Eur J Pharmacol, 1992 Feb 11, 211(2), 269 - 72
L-arginine induces relaxation of small mesenteric arteries from endotoxin-treated rats; Schneider F et al.; The effect of arginine (Arg) was studied on norepinephrine- (1 microM) precontracted small mesenteric arteries removed from rats treated with E . coli lipopolysaccharide (LPS) . The addition of L- (but not D-) Arg (1 mM) relaxed, within 3 min, the small mesenteric arteries from LPS-treated but not from control rats, the maximal relaxation (65.3 +/- 11%) being reached with less than 100 microM L-Arg . NG-Nitro-L-arginine methyl ester (1 mM) and methylene blue (10 microM) restored contractions to the level reached before addition of L-Arg . These results show that LPS induces the production of an L-Arg-derived, nitric oxide-like, relaxing factor in small mesenteric arteries.

Biochim Biophys Acta, 1992 Feb 11, 1129(3), 278 - 86
Biochemical and biological activity of phosphorylated and non-phosphorylated ras p21 mutants; Chung HH et al.; In contrast to all cellular ras oncogenes which carry a single activating mutation at codon 12, 13 or 61, all known retroviral ras oncogenes have two mutations at codons 12 and 59 . To understand the role of the mutation at codon 59, we have constructed plasmids containing genes for Harvey ras: p21(Gly-12,Thr-59) and p21(Val-12,Thr-59) . Escherichia coli expressed proteins and their respective phosphorylated (Pi) and non-phosphorylated (non-Pi) proteins were purified to 95% homogeneity by ion-exchange chromatography and gel filtration . GTPase, autophosphorylation and nucleotide exchange activities of the mutants were studied . When the mutants were microinjected into Xenopus oocytes, the non-phosphorylated forms of p21(Gly-12,Thr-59) and p21(Val-12,Thr-59) showed high activity . Surprisingly, their phosphorylated forms were inactive . These results suggest that threonine at position 59 endows the protein with transforming activity but that phosphorylation of the residue inhibits biological activity . A structural interpretation of the observation is presented.

Nucleic Acids Res, 1992 Feb 11, 20(3), 425 - 32
The kinetics and specificity of cleavage by RNase P is mainly dependent on the structure of the amino acid acceptor stem; Kirsebom LA et al.; Cleavage by RNase P of the tRNA(His precursor yields a mature tRNA with an 8 base pair amino acid acceptor stem instead of the usual 7 base pair stem . Here we show, both in vivo and in vitro, that this is mainly dependent on the primary structure and length of the acceptor stem in the precursor . Furthermore, the tRNA(His) precursor used in this study was processed with a change in both kinetic constants, Km and kcat, in comparison to the kinetics of cleavage of the precursor to tRNA(Tyr)Su3 . Cleavage of a chimeric tRNA precursor showed that these altered kinetics were due to a difference in the primary structure and in the length of the acceptor stems of these two tRNA precursors . We also studied the cleavage reaction as a function of base substitutions at positions -1 and/or +73 in the precursor to tRNA(His) . Our results suggest that the nucleotide at position +73 in tRNA(His) plays a significant role in the kinetics of cleavage of its precursor, possibly in product release . In addition, it appears that the C5 protein of RNase P is involved in the interaction between the enzyme and its substrate in a substrate-dependent manner, as previously suggested.

Nucleic Acids Res, 1992 Feb 11, 20(3), 421 - 3
The acceptor stem in pre-tRNAs determines the cleavage specificity of RNase P; Holm PS et al.; As the result of an unusual RNase P specificity, some special, mature tRNAs have acceptor stems with eight instead of the common seven base pairs . The data from numerous studies suggest that some features in the tRNA domain of pre-tRNAs are important for this behaviour . Here, we show that only five base pairs in the acceptor stem of bacterial histidine tRNAs are required to obtain the changed cleavage site in an unrelated eukaryotic serine tRNA.

Nucleic Acids Res, 1992 Feb 11, 20(3), 525 - 32
Transcriptional enhancer related DNA sequences: anomalous 1H NMR NOE crosspeaks; Donlan ME et al.; A dynamic heterogeneity which correlates with the function of the operator DNA in the lactose operon of E . coli . was previously observed (1) as a local minimum in the thymine imino proton T1 centered at a GTG/C-CAC sequence . Since this triplet occurs frequently in DNA regulatory regions, it was proposed that these sequences may be part of a structural element for specific protein interaction . We examine here three additional biologically significant 17 base pair duplexes containing GTG/CAC triplets: (1) a sequence from the mouse heavy chain immunoglobulin enhancer, (2) a sequence from the critical core of the Simian Virus 40 (SV40) enhancer, and (3) a sequence from pBR322 plasmid used as control for experiments with the SV40 DNA sequences . The 1H NMR resonance assignment for nearly all the nonexchangeable protons for both eukaryotic enhancer duplexes with the exception of the H5'/H5" protons was accomplished to use for structural analysis of these duplexes . The data presented show several NOE's associated with the GTG/CAC triplets which suggest structural variation from uniform B-DNA . In addition, anomalous broad crosspeaks for the fixed thymine methyl to its own H6 proton in combination with the imino proton kinetics associated with these triplets reinforces the original observation of a sequence dependent dynamic variation.

Biochemistry, 1992 Feb 11, 31(5), 1363 - 70
Binding of verocytotoxin 1 to its receptor is influenced by differences in receptor fatty acid content; Pellizzari A et al.; Globotriaosylceramide {(Gal alpha 1-4Gal beta 1-4Glc-ceramide (Gb3)} was separated from human kidney, and the fatty acid composition was determined . Semisynthetic Gb3 molecular species of corresponding fatty acid chain length were prepared and compared for verotoxin (VT) binding affinity by TLC overlay, and a quantitative binding assay was performed in the presence of auxiliary lipids . Our results indicate that, within the natural range, fatty acid chain length has little effect on verotoxin binding but that Gb3 molecular species containing different fatty acids can interact to provide a higher affinity toxin receptor than any of the individual component receptor species . Receptor function as assayed by TLC overlay was not always found to correlate with binding in a lipid environment . Short-chain fatty acid Gb3 molecular species could not function as VT receptors under these conditions . Evidence is presented to suggest that fatty acid chain length can have a stereoselective effect on carbohydrate conformation.

FEBS Lett, 1992 Feb 10, 297(3), 247 - 9
Interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase and its functional consequences; Khoroshilova NA et al.; E . coli D-glyceraldehyde-3-phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble E . coli 3-phosphoglycerate kinase with a stoichiometry of 1.77 +/- 0.61 kinase molecules per tetramer of the dehydrogenase and an apparent Kd of 1.03 +/- 0.68 microM (10 mM sodium phosphate, 0.15 M NaCl) . No interaction was detected between E . coli D-glyceraldehyde-3-phosphate dehydrogenase and rabbit muscle 3-phosphoglycerate kinase . The species-specificity of the bienzyme association made it possible to develop a kinetic approach to demonstrate the functionally significant interaction between E . coli D-glyceraldehyde-3-phosphate dehydrogenase and E . coli 3-phosphoglycerate kinase, which consists of an increase in steady-state rate of the coupled reaction.

Nature, 1992 Feb 6, 355(6360), 561 - 4
Lactose binding to heat-labile enterotoxin revealed by X-ray crystallography; Sixma TK et al.; Recognition of the oligosaccharide portion of ganglioside GM1 in membranes of target cells by the heat-labile enterotoxin from Escherichia coli is the crucial first step in its pathogenesis, as it is for the closely related cholera toxin . These toxins have five B subunits, which are essential for GM1 binding, and a single A subunit, which needs to be nicked by proteolysis and reduced, yielding an A1-'enzyme' and an A2-'linker' peptide . A1 is translocated across the membrane of intestinal epithelial cells, possibly after endocytosis, upon which it ADP-ribosylates the G protein Gs alpha . The mechanism of binding and translocation of these toxins has been extensively investigated, but how the protein is orientated on binding is still not clear . Knowing the precise arrangement of the ganglioside binding sites of the toxins will be useful for designing drugs against the diarrhoeal diseases caused by organisms secreting these toxins and in the development of oral vaccines against them . We present here the three-dimensional structure of the E . coli heat-labile enterotoxin complexed with lactose . This reveals the location of the binding site of the terminal galactose of GM1, which is consistent with toxin binding to the target cell with its A1 fragment pointing away from the membrane . A small helix is identified at the carboxy terminus of A2 which emerges through the central pore of the B subunits and probably comes into contact with the membrane upon binding, whereas the A1 subunit is flexible with respect to the B pentamer.

J Biol Chem, 1992 Feb 5, 267(4), 2443 - 50
Long range effects of amino acid substitutions in the catalytic chain of aspartate transcarbamoylase . Localized replacements in the carboxyl-terminal alpha-helix cause marked alterations in allosteric properties and intersubunit interactions; Peterson CB et al.; A single alpha-helical polypeptide segment of 21 amino acids near the carboxyl terminus of the catalytic chain of aspartate transcarbamoylase from Escherichia coli has been shown recently to be important for the in vivo folding of the chains and assembly of the enzyme (Peterson, C . B., and Schachman, H . K . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 458-462) . Calorimetric measurements on purified mutant enzymes showed that single amino acid replacements within this secondary structural element affect the overall thermal stability of the oligomeric enzyme and the energetics of the interactions between polypeptide chains within the holoenzyme . Studies presented here demonstrate that marked changes in cooperativity occur due to single amino acid substitutions . Replacement of Gln288 by either Ala or Glu leads to a striking increase in the Hill coefficient of the holoenzymes and a substantial increase in the aspartate concentration corresponding to one-half Vmax . In contrast, the isolated catalytic trimers harboring these same substitutions were similar in activity to the wild-type subunit, with the same affinity for aspartate as indicated by the values of Km . Substituting Ala for the only charged residue in the helix, Arg296, caused a marked reduction in enzyme activity, as well as a greatly reduced stability of the holoenzyme due to a substantial weakening of the interactions between the catalytic and regulatory subunits . A subunit exchange method was used to demonstrate the changes in interchain interactions resulting from the amino acid substitutions and to show the additional weakening upon the binding of the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, at the active sites . Taken together, the results on this series of mutant enzymes illustrate how the effects of single amino acid replacements in one element of secondary structure are propagated throughout the molecule to positions remote from the site of the substitution.

J Biol Chem, 1992 Feb 5, 267(4), 2414 - 20
Comparison of the enzymatic and biochemical properties of human insulin-degrading enzyme and Escherichia coli protease III; Ding L et al.; The enzymatic and biochemical properties of human insulin-degrading enzyme and Escherichia coli protease III have been compared . Both enzymes were found to degrade insulin in such a way that its receptor binding activity was rapidly lost but its precipitability in trichloracetic acid was only slightly decreased . Both enzymes were also found to be inhibited by chelating agents . The bacterial enzyme, which could be purified in large amounts, was found to contain 0.6 mol of zinc per mol of enzyme but no detectable manganese . The mammalian enzyme but not the bacterial one was inhibited by a sulfhydryl alkylating agent . The two enzymes also differed in substrate specificity . The mammalian enzyme degraded insulin much better than insulin-like growth factor II, whereas the bacterial enzyme degraded them equally . The mammalian enzyme could be labeled by cross-linking to insulin = bombyxin II much greater than insulin-like growth factor I and II much greater than relaxin, while the bacterial enzyme was labeled by insulin-like growth factor II greater than insulin = insulin-like growth factor I much greater than relaxin much greater than bombyxin . Finally, sucrose gradient centrifugation and cross-linking studies both in vitro and in vivo indicated that active human enzyme partially existed as a homo- or heterodimer, whereas the bacterial enzyme was active as a monomer.






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