Microbiology, 1996 Sep, 142 ( Pt 9), 2595 - 602 Defect in export and synthesis of the periplasmic galactose receptor MglB in dnaK mutants of Escherichia coli, and decreased stability of the mglB mRNA; el Yaagoubi A et al.; The high-affinity galactose permease, which comprises the periplasmic galactose receptor MglB, the membrane translocator MglC and the membrane-associated ATPase MglA, displayed a reduced activity in a dnaK temperature-sensitive mutant of Escherichia coli . This reduced transport activity correlated with a reduction in the quantity of MglB . At 42 degrees C, an accumulation of pre-MglB in the dnaK temperature-sensitive mutant reflected a defect in MglB export . In addition, an accumulation of pre-MglB in secB, secA and secY mutants suggested that SecB and the Sec translocase are also involved in export of the periplasmic galactose receptor . At 30 degrees C, there was no accumulation of pre-MglB in the dnaK mutant, but there was still a decreased amount of MglB in the periplasm . The reduction in MglB expression was not the result of a decrease in its stability, nor was it the result of a general defect in translation or transcription, since the MglA protein (which is expressed from the same operon as MglB) was synthesized in normal amounts . Two mRNAs are implicated in the expression of the mgl genes, a polycistronic mglBAC mRNA, and a more stable and more abundant mglB mRNA, produced by 3'-5' degradation of the mglBAC mRNA (R . W . Hogg, C . Voelker & I . von Carlowitz, 1991, Mol Gen Genet 229, 453-459) . The mglB mRNA is protected against exonucleases by a REP (Repetitive Extragenic Palindrome) sequence located at its 3' extremity, which is responsible for the higher expression of MglB compared to MglA and MglC . The decreased MglB expression in the dnaK mutant at 30 degrees C in the present work correlated with a reduced stability of the mglB mRNA, which may have resulted from a defective stabilization by the REP sequence, or from a defect in translation of the mglB gene. Microbiology, 1996 Sep, 142 ( Pt 9), 2543 - 8 The gene for gamma-glutamylcysteine synthetase from Thiobacillus ferrooxidans has low homology to its Escherichia coli equivalent and is linked to the gene for citrate synthase; Powles R et al.; The gene for gamma-glutamylcysteine synthetase (gshA) from Thiobacillus ferrooxidans was isolated from a family of cosmids by its ability to complement an Escherichia coli gshA trxA double mutant which was unable to grow on minimal medium lacking glutathione . The predicted sequence of the gamma-glutamylcysteine synthetase was found to have only 18% amino acid sequence identity to the equivalent enzyme from E . coli . In spite of this low sequence homology, concentrations of GSH in a cell extract prepared from the E . coli gshA trxA mutant containing the cloned gene were almost as high as in a cell extract prepared from a wild-type E . coli strain . The gshA gene was found to be physically and transcriptionally linked to the T . ferrooxidans gene for citrate synthase (gltA) . The T . ferrooxidans and E . coli citrate synthases shared 37% amino acid sequence identity and the cloned T . ferrooxidans citrate synthase gene was able to complement an E . coli gltA mutant. Microbiology, 1996 Sep, 142 ( Pt 9), 2471 - 9 Isolation of Borrelia burgdorferi genes encoding homologues of DNA-binding protein HU and ribosomal protein S20; Tilly K et al.; Linear DNA with covalently closed ends is the predominant form of DNA in the spirochaete Borrelia burgdorferi . All bacteria examined to date have small DNA-binding proteins related to the Escherichia coli IHF and HU proteins that appear to play roles in DNA compaction and replication, but such proteins had not been isolated from bacteria with linear genomes . We found a single gene in B . burgdorferi (named hbb) whose product (named Hbb) complements the defects for gamma DNA packaging found in E . coli strains mutant in the genes for IHF and HU . The sequence of the predicted B . burgdorferi protein is similar to those of HU and IHF-like proteins in other bacteria . The gene appears to be in an operon with the order rpsT-hbb-orfH, where the rpsT gene is a homologue of the E . coli gene encoding ribosomal protein S20 and the orfH gene encodes a protein of unknown function . This operon is located upstream of the previously identified B . burgdorferi rho homologue. Microbiology, 1996 Sep, 142 ( Pt 9), 2439 - 47 The Mycobacterium tuberculosis purine biosynthetic pathway: isolation and characterization of the purC and purL genes; Jackson M et al.; Genes from the Mycobacterium tuberculosis purine biosynthetic pathway were identified using purine auxotrophic mutants of Mycobacterium smegmatis obtained by Tn611 transposon mutagenesis . Two approaches were followed in parallel . The first consisted of the complementation of the M . smegmatis purine auxotrophs using a M . tuberculosis H37Rv shuttle cosmid library . In the second approach, specific probes corresponding to the regions adjacent to the insertion sites of Tn611 in the M . smegmatis genome were used to screen a M . tuberculosis plasmid library by colony hybridization for inserts carrying homologous DNA fragments . Nucleotide sequence analysis of two M . tuberculosis genes isolated by these methods revealed high similarities with purC and purL genes from other bacterial and fungal sources . Transcriptional start sites were mapped for both genes, which revealed similar-10 boxes but with a higher GC content than the Escherichia coli sigma 70 consensus. Microbiology, 1996 Sep, 142 ( Pt 9), 2429 - 37 Stringent and growth-rate-dependent control of the gua operon of Escherichia coli K-12; Davies IJ et al.; The promoter of the gua operon has been located by transcript mapping using primer extension with reverse transcriptase . The surrounding nucleotide sequence has features characteristic of promoters under stringent and growth-rate-dependent regulation, namely a GC-rich discriminator next to the -10 hexamer, an upstream AT-rich sequence (the UP element) and potential FIS-binding sites . Transcriptional activity of the gua promoter was examined using transcriptional fusions to lacZ placed at a single chromosomal location . Expression from gua was reduced under stringent conditions in vivo, and varied with growth rate . Growth-rate control was independent of guanine-mediated repression . A fusion in which the GC-rich discriminator was mutated by insertion of an AT-rich oligonucleotide was used to demonstrate the importance of this region in control . Both stringent and growth-rate-dependent controls were abolished by the mutation . Other potential regulatory signals in the vicinity of the gua promoter are a pur operator (binding site for the PurR repressor), a gua operator, a DnaA-binding site and a CRP/FNR-binding sequence . The gua promoter lies back-to-back with the promoter for xseA (exonuclease VII), the two promoters being separated by only 20 bp. Carcinogenesis, 1996 Sep, 17(9), 2085 - 8 The Escherichia coli MutS DNA mismatch binding protein specifically binds O(6)-methylguanine DNA lesions; Rasmussen LJ et al.; DNA mismatch repair defects in certain cell types confer resistance to the cytotoxic effects of alkylating agents, suggesting that a normally functioning DNA mismatch repair pathway can actually mediate alkylation-induced cell death . In eukaryotic cells this phenomenon is only observed in cells lacking adequate DNA methyltransferase for the repair of O6-methylguanine (O6MeG) DNA lesions . It has been proposed that O6MeG may act as a substrate for DNA mismatch repair when paired with cytosine and when mispaired with thymine and that repeated futile DNA mismatch repair at O6MeG DNA lesions is cytotoxic . Here we show that the Escherichia coli MutS DNA mismatch repair binding protein does indeed bind specifically to O6MeG DNA lesions . In contrast, MutS does not bind DNA containing another O-alkylated base, namely O4-methylthymine, or another kind of modified guanine, namely 8-oxoguanine . These results provide direct biochemical evidence for the involvement of DNA mismatch repair in specifically processing O6MeG DNA lesions. Carcinogenesis, 1996 Sep, 17(9), 1997 - 2002 Mutational specificity of aflatoxin B1 . Comparison of in vivo host-mediated assay with in vitro S9 metabolic activation; Prieto-Alamo MJ et al.; An intrasanguineous host-mediated assay was used to determine the pattern of mutagenesis induced by the carcinogen aflatoxin B1 in the lacI gene of Escherichia coli recovered from rat liver . To investigate the influence of different types of metabolic activation, the mutation spectrum induced by AFB1 activated in vitro by a commercially prepared S9 microsomal fraction from Aroclor 1254-treated rats was also obtained . A total of 281 forward mutations affecting the N-terminal region of the lacI gene were characterized by DNA sequencing analysis . AFB1 induced similar type of mutations with similar site specificity when activated by the standard S9 fraction or by employing a rat host-mediated assay . These results indicate the ability of the in vitro S9 fraction to mimic the in vivo metabolism, suggesting that the same active metabolite, presumably AFB1 8,9-epoxide, is responsible for generating a similar pattern of DNA damage, as reflected in the similarity of mutational spectra . For both activation systems, most mutations (>90%) were base substitutions that occurred primarily at G:C pairs . Somewhat over one-half of G:C targeted substitutions were GC>TA transversions, other mutations being evenly divided between G:C>AT transitions and GC>CG transversions . The mutational specificity exhibited by activated AFB1 can be explained by incorporation of different bases opposite a single type of non-instructive lesion during error-prone DNA synthesis . To what extent the mutations are due to the main adduct (AFB1-N7-Gua), its imidazole-ring-opened derivative or an apurinic site remains unknown. Plant Physiol, 1996 Sep, 112(1), 371 - 8 Biotin synthase from Arabidopsis thaliana . cDNA isolation and characterization of gene expression; Patton DA et al.; The full-length BIO2 cDNA from Arabidopsis thaliana was isolated using an expressed sequence tag that was homologous to the Escherichia coli biotin synthase gene (BioB) . Comparisons of the deduced amino acid sequence from BIO2 with bacterial and yeast biotin synthase homologs revealed a high degree of sequence similarity . The amino terminus of the predicted BIO2 protein contains a stretch of hydrophobic residues similar in composition to transit peptide sequences . BIO2 is a single-copy nuclear gene in Arabidopsis that is expressed at high levels in the tissues of immature plants . Expression of BIO2 was higher in the light relative to dark and was induced 5-fold during biotin-limited conditions . These results demonstrate that expression of at least one gene in this pathway is regulated in response to developmental, environmental, and bio-chemical stimuli. Plant Physiol, 1996 Sep, 112(1), 89 - 97 Evidence that a 77-kilodalton protein from the starch of pea embryos is an isoform of starch synthase that is both soluble and granule bound; Edwards A et al.; In this paper we provide further evidence about the nature of a 77-kD starch synthase (SSII) that is both soluble and bound to the starch granules in developing pea (Pisum sativum L.) embryos . Mature SSII gives rise to starch synthase activity when expressed in a strain of Escherichia coli lacking glycogen synthase . In transgenic potatoes (Solanum tuberosum L.) expressing SSII, the protein is both soluble and bound to the starch granules . These results confirm that SSII is a starch synthase and indicate that partitioning between the soluble and granule-bound fraction of storage organs is an intrinsic property of the protein . A 60-kD isoform of starch synthase found both in the soluble and granule-bound fraction of the pea embryos is probably derived by the processing of SSII and is a different gene product from GBSSI, the exclusively granule-bound 59-kD isoform of starch synthase that is similar to starch synthases encoded by the waxy genes of cereals and the amf gene of potatoes . Consistent with this, expression in E . coli of an N-terminally truncated version of SSII gives rise to starch synthase activity. Invest Ophthalmol Vis Sci, 1996 Sep, 37(10), 2022 - 8 Adenovirus-mediated gene transfer to retinal ganglion cells; Cayouette M et al.; PURPOSE: To evaluate the capacity of a replication-defective adenoviral vector to transport retrogradely from the superior colliculus to the nuclei of retinal ganglion cells and to express in these cells vector-encoded transgene . METHODS: A replication-deficient adenovirus encoding an expression cassette for the Escherichia coli gene lacZ was injected into the right superior colliculus of mice . Brain sections and both eyes were tested histochemically and/or immunohistochemically for E . coli beta-galactosidase (LacZ) activity at 3, 7, 14, and 30 days after injection . RESULTS: lacZ expression was detected in the retinal ganglion cells of the contralateral eye at 7, 14, and 30 days after injection, but no expression was observed in the retina at 3 days after injection . No signs of retinal pathology was observed histologically . LacZ-positive cells also were found in other afferent systems to the superior colliculus, such as the reticular formation, layer V of the ipsilateral visual cortex, and pars reticulata of the ipsilateral substantia nigra . CONCLUSIONS: Injection of an adenovirus vector into the superior colliculus is an effective means to transfer and express a gene in retinal ganglion cells while avoiding damage to the eye tissues; thus, it represents a potentially useful tool to manipulate gene expression selectively in the retina. Arthritis Rheum, 1996 Sep, 39(9), 1588 - 95 Epitope analysis of the major reactive region of the 100-kd protein of PM-Scl autoantigen; Ge Q et al.; OBJECTIVE: To localize the epitope(s) bound by anti-PM-Scl antibodies in the N-terminal half of the 100-kd protein, the major antigen of the PM-Scl complex . METHODS: Investigations were performed by immunoblotting 20 anti-PM-Scl positive sera against bacterially expressed, polymerase chain reaction-derived deletion mutants of the S1 fragment (amino acids 11-437), enzyme-linked immunosorbent assay (ELISA) screening against synthesized serial octapeptides, and ELISA screening, with anti-PM-Scl positive sera, against a synthesized 21-amino acid peptide covering the active region . RESULTS: Anti-PM-Scl positive sera retained full immunoblot activity with fragment 207-436 and most activity with fragment 11-241, but had markedly decreased activity against fragments 236-436 and 11-212, indicating a major epitope in the aa 207-241 region . Fusion proteins with smaller fragments localized this activity between aa 226 and aa 246 . Of 42 anti-S1-positive, anti-PM-Scl positive sera tested by ELISA against a synthetic peptide of this region, 36 were definitely positive, 4 borderline, and 2 negative . Similar activity was seen with a peptide from which proline 228 was deleted . Three additional epitope areas were found in S1, but each reacted with only a few sera . Anti-PMScl positive sera did not react with any octapeptide spanning the major epitope area (aa 207-246) . CONCLUSION: The main immunoblot epitope of the PM-Scl 100-kd protein is within a central area of 21 aa (aa 226-246), but is longer than the usual linear epitope . This peptide may be useful in patient testing . Three minor epitopes in S1 may also be recognized by some sera. Protein Expr Purif, 1996 Sep, 8(2), 247 - 53 Recombinant human cytidine deaminase: expression, purification, and characterization; Vincenzetti S et al.; The complementary DNA (cDNA) coding for human cytidine deaminase (CDA) was obtained using two specific primers to screen RNA from peripheral blood polymorphonuclear leukocytes by reverse transcriptase PCR . The cDNA fragment was ligated into the expression vector pTrc99-A and expressed in Escherichia coli following induction with isopropyl-1-thio-beta-D-galactopyranoside (IPTG) . The nucleotide sequence of the cDNA corresponded to that published by Laliberte and Momparler (Cancer Res . 54, 5401-5407, 1994) . It contained a 438-bp open reading frame encoding a polypeptide of 146 amino acids with a predicted molecular mass of 16.2 kDa . The protein expressed in E . coli showed high cytidine deaminase activity and its molecular mass was estimated to be 57 kDa by gel filtration and 16 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) . Both values are in agreement with those already published and suggest that human CDA contains three or four identical subunits . Cross-linking experiment indicated that the enzyme is a tetramer . The recombinant CDA has been purified to homogeneity by a rapid procedure consisting of heat inactivation followed by affinity chromatography . The final enzyme preparation showed a specific activity of 105 U/mg, corresponding to about 88-fold purification with respect to the crude extract and was judged to be >98% pure by SDS-PAGE . Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis revealed the presence of 1 atom of Zn per subunit . Since CDA causes the deamination of several antitumoral cytidine-analog drugs, the recombinant enzyme was characterized kinetically and several pyrimidine nucleoside analogs were tested as potential substrates and inhibitors . The results obtained agreed closely with those previously reported for the purified human placenta CDA. Protein Expr Purif, 1996 Sep, 8(2), 238 - 46 Purification and functional characterization of wild-type and mutant HIV-1 and HIV-2 Tat proteins expressed in Escherichia coli; Orsini MJ et al.; We describe a single method to purify milligram amounts of authentic wild-type and mutant HIV-1 and HIV-2 Tat proteins overexpressed in Escherichia coli . This method takes advantage of the highly basic, positively charged RNA binding domain present in both HIV-1 and HIV-2 Tat, which also facilitated purification of HIV-1 and HIV-2 mutant Tat proteins . In contrast to previously described methods, our method does not require use of denaturing or reducing agents, since Tat is present in the soluble fraction after bacterial lysis . The activity of purified wild-type and mutant HIV-1 and HIV-2 Tat proteins was determined using cell-based uptake, in vitro transcription, and TAR binding assays . As expected, both HIV-1 and HIV-2 Tat efficiently transactivated the HIV-2 LTR, whereas HIV-2 Tat transactivated the HIV-1 LTR less efficiently than HIV-1 Tat . Purified HIV-2 Tat proteins in which the glutamic acid residue at position 77 was changed to either glycine or glutamine transactivated the HIV-1 LTR more efficiently than wild-type Tat-2, providing additional evidence that the net charge of this region may be responsible for nonreciprocal transactivation between Tat-1 and Tat-2 . Our results demonstrate that this method can be used to rapidly purify authentic wild-type and mutant Tat proteins which are suitable for cell-based and in vitro functional studies. Protein Expr Purif, 1996 Sep, 8(2), 215 - 26 Isolation and characterization of human tissue kallikrein produced in Escherichia coli: biochemical comparison to the enzymatically inactive prokallikrein and methionyl kallikrein; Lu HS et al.; This report describes bacterial expression, isolation, and characterization of human tissue kallikrein recombinantly produced in Escherichia coli . Successful production of enzymatically active recombinant human kallikrein requires the following processes: expression, solubilization and refolding of prokallikrein, thermolysin activation, and chromatographic separation . All experimental data confirmed that bacterially derived human kallikrein is properly folded and exhibits expected biochemical functions . As confirmed by SDS-PAGE and reverse-phase HPLC, recombinant kallikrein is apparently pure and is devoid of reduced or other partially folded kallikrein forms . Recombinant kallikrein behaves as a monomeric molecule in solution and exhibits full enzymatic activity in hydrolyzing peptide substrates . The molecule can bind to aprotinin to form kallikrein-inhibitor complex at a 1:1 molar ratio . Peptide mapping analysis derived from pepsin digestion of recombinant kallikrein assigned five disulfide bonds which match those of porcine kallikrein predicted from X-ray structure . Peptides containing unpaired cysteines or mispaired disulfide bonds were not detected . Both properly folded prokallikrein and methionyl kallikrein, containing a propeptide and an initiator methionine at their N-termini, respectively, were also produced and isolated . These two molecules are structurally similar to recombinant kallikrein, but are not enzymatically active. Protein Expr Purif, 1996 Sep, 8(2), 183 - 90 Cloning of chalcone-flavanone isomerase cDNA from Pueraria lobata and its overexpression in Escherichia coli; Terai Y et al.; Chalcone-flavanone isomerase (CHI) cDNA was isolated from Pueraria lobata by combination of cDNA library screening using Phaseolus vulgaris CHI cDNA as a probe and polymerase chain reaction techniques . Analysis of nucleotide sequence of the cloned cDNA revealed a 675-bp open reading frame encoding a 225-amino-acid polypeptide with a molecular weight of 23,803 Da . The CHI cDNA coding region was cloned into pET-3d expression vector and successfully overexpressed in Escherichia coli cells as active CHI enzyme . The recombinant CHI was then purified to apparent homogeneity by DEAE-cellulose column chromatography . Replacement of Cys-119 with Ala was carried out by site-directed mutagenesis and the result that the mutant CHI showed CHI enzyme activity confirmed that Cys-119 is not involved in the CHI catalytic active site. Protein Expr Purif, 1996 Sep, 8(2), 160 - 6 Recombinant human phenylethanolamine N-methyltransferase: overproduction in Escherichia coli, purification, and characterization; Caine JM et al.; The gene encoding phenylethanolamine N-methyltransferase (PNMT) has been amplified from a human adrenal medulla cDNA library . Following ligation of the gene into a pET3a-derived expression vector and transformation into Escherichia coli BL21(DE3)pLysS, PNMT was expressed, yielding about 10% of the soluble protein . The enzyme was purified to homogeneity by ammonium sulfate fractionation followed by ion-exchange chromatography and gel filtration . The Km for phenylethanolamine and S-adenosyl-L-methionine were determined to be 130 and 16 microM, respectively . The enzyme could be inhibited by reagents expected to modify cysteine, arginine, tyrosine, and histidine residues, but not by methyl acetimidate, a reagent expected to modify lysine residues. Protein Expr Purif, 1996 Sep, 8(2), 151 - 9 Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies; Nandi A et al.; The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli . The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography . The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line . No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions . Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography . Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor . Purified antibodies recognized a single protein of Mr 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells . These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E . coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation . The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction. Protein Expr Purif, 1996 Sep, 8(2), 145 - 50 Expression of a recombinant kringle V of human apolipoprotein(a): antibody characterization and species specificity; Chenivesse X et al.; Lipoprotein(a) is a macromolecular complex consisting of a low-density lipoprotein-like particle with an additional glycoprotein, apolipoprotein(a) {apo(a)}, linked to apolipoprotein B-100 via a disulfide bond . Apo(a) is highly homologous to plasminogen . We have cloned the sequence corresponding to the kringle V domain of apo(a) from human liver cDNA using an experimental approach involving use of the polymerase chain reaction . The protein product of this clone was expressed in the cytoplasmic compartment of Escherichia coli as a MalE fusion protein . Fusion apo(a) Kr V was isolated from cytoplasmic extracts and purified by amylose-agarose affinity chromatography by eluting with 10 mM maltose . The fusion protein was injected into sheep in order to generate a polyclonal anti-apo(a) Kr V antibody . The antibody raised reacted against both reduced Lp(a) and the C-terminal domain of apo(a), corresponding to a sequence extending from Kr 33 to the C-terminal residue, but did not react with the N-terminal domain containing the repeated Kr IV sequences . The presence of the Kr V sequence was detected in every human apo(a) size isoform tested but only in apo(a) from human and chimpanzee among a panel of apo(a) proteins derived from different animal species. Protein Expr Purif, 1996 Sep, 8(2), 137 - 44 Purification and characterization of recombinant osteocalcin fusion protein expressed in Escherichia coli; Kakonen SM et al.; Human osteocalcin (hOC) is a 49-amino-acid peptide produced mainly by bone osteoblasts . The amount of hOC in the circulation reflects the status of bone metabolism and it is used to monitor various bone-related diseases . The aim of this study was to produce recombinant human osteocalcin (rhOC) in Escherichia coli and use it for designing new osteocalcin fluorescence immunoassays . Recombinant DNA technology was used to fuse synthetic hOC coding sequences to an affinity handle system based on glutathione S-transferase (GST) gene . GST-rhOC fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form . The affinity-purified fusion protein was cleaved with activated protease factor X releasing the rhOC portion . The structure of rhOC was confirmed by mass spectrometry and amino acid sequencing . The fusion protein and its proteolytic cleavage product proved to be immunoreactive as shown by Western blotting analysis and by a new osteocalcin immunoassay based on time-resolved fluorescence . When osteocalcin was tested for its ability to bind to hydroxyapatite, there were no differences between the recombinant forms and native human osteocalcin purified from bone, suggesting that the Gla residues might be important only in oriented high-affinity binding. Plant J, 1996 Sep, 10(3), 505 - 13 A self-fertile mutant of Phalaris produces an S protein with reduced thioredoxin activity; Li X et al.; Gametophytic self-incompatibility in the Phalaris coerulescens is controlled by two unlinked genes, S and Z . Isolation of the S gene from the pollen of this grass species indicated that the C terminus has significant homology with thioredoxin H proteins . The protein from the C terminus, expressed in Escherichia coli, exhibits thioredoxin-life activity . This paper demonstrates that the C terminus of the S protein from an S complete mutant shows significant reduction in thioredoxin activity when compared with the wild-type form . Both pollen and stigma have lost self-compatibility in this mutant . Close examination of the lesions, which were found only in the C terminus of the mutant gene suggests that the substitution of a serine by an arginine is responsible for the reduced enzymatic activity . The association between reduced activity and the loss of the self-incompatibility provides evidence for a role of thioredoxin activity in the self-incompatibility reaction of this species. J Gen Virol, 1996 Sep, 77 ( Pt 9), 2077 - 84 Characterization of the NTPase activity of Japanese encephalitis virus NS3 protein; Kuo MD et al.; Japanese encephalitis (JE) virus NS3 protein and two N-terminally truncated (delta 1-148 and delta 1-323) forms of NS3 were engineered and expressed in E . coli as fusion proteins with a histidine tag at the N terminus . The purified recombinant proteins his-NS3 and his-NS3(delta 1-148) were found to possess NTPase activity which was stimulated by single-stranded RNA, whereas NS3(delta 1-323) did not . The requirements for MgCl2 and MnCl2 and the salt and pH ranges necessary for optimal activity of the enzyme were determined and shown to be slightly different from those of the NTPases of other flaviviruses . Poly(U) and poly(C) were better than poly(A) at stimulating the NTPase activities, in contrast to other flaviviral NTPases . The substrate preference was in the order GTP > ATP >> UTP > CTP . Interestingly, we found that Ca2+ could not substitute for Mg2+; on the contrary, it inhibited NTPase activity . The removal of the N-terminal 148 amino acids enhanced NTPase activity, but further deletion of the region (amino acids 148-323) completely abolished the activity . Therefore, amino acids 148-323 contain a critical region required for NTPase activity. J Trauma, 1996 Sep, 41(3), 430 - 7; discussion 437-8 Endotoxin tolerance after severe injury and its regulatory mechanisms; Keel M et al.; OBJECTIVE: To study the responsiveness of peripheral blood mononuclear cells to lipopolysaccharide (LPS) after severe trauma and its regulatory mechanisms . MATERIALS AND METHODS: The release of proinflammatory reacting cytokines (tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-6, IL-8, interferon (IFN)-gamma) into whole blood from 12 patients on day 1, 5, 10, and 14 after severe trauma (Injury Severity Score, 39.3 +/- 2.8 points) and 10 healthy volunteers was studied after stimulation with LPS, concanavalin A, phorbol myristate acetate (PMA), and the addition of recombinant IFN-gamma . MAIN RESULTS: Trauma caused a significant reduction of LPS and concanavalin A induced release of inflammation activating cytokines into whole blood, including IFN-gamma . However, the diminished release of proinflammatory cytokines could be increased with recombinant IFN-gamma or even attenuated after stimulation of peripheral blood mononuclear cells with the protein kinase C activator PMA . CONCLUSIONS: Trauma leads to reduced responsiveness of blood monocytes to LPS and a decreased secretion of proinflammatory reacting lymphokines . Because activation of the protein kinase C pathway with PMA or the addition of IFN-gamma significantly increased cytokine response, endotoxin tolerance is not caused by inhibition of protein synthesis, but to disturbances in the signal transduction pathway and its regulating mediators. J Med Microbiol, 1996 Sep, 45(3), 200 - 5 Polymorphism at the dnaK locus of Brucella species and identification of a Brucella melitensis species-specific marker; Cloeckaert A et al.; The dnaK gene and surrounding sequences from reference strains of the six Brucella species were amplified by the polymerase chain reaction (PCR) with primers chosen according to the published sequence of the B . ovis dnaK gene and studied for polymorphism with nine restriction endonucleases . The restriction patterns were identical for all species with all restriction endonucleases tested except for B . melitensis strain 16M that showed a different pattern with EcoRV, consistent with the presence of a single site instead of two for the other Brucella species . The absence of the second EcoRV site for B . melitensis 16M was confirmed by DNA sequence analysis . The second EcoRV site in other Brucella species was located in a 12-bp segment, which was missing from the published dnaK sequence of B . ovis, between the stop codon of the dnaK gene and its putative transcription terminator sequence . The difference between B . ovis strain 63/290 and B . melitensis 16M was due to an additional base-pair in B . melitensis 16M . Subsequently, 71 other field, vaccinal and reference strains of the six Brucella species and their different biovars were studied for restriction fragment length polymorphism (RFLP) of the dnaK locus with EcoRV . The presence of a unique EcoRV site was specific to B . melitensis strains . Southern blot analysis of whole genomic DNA digested with EcoRV and with the dnaK gene used as probe also detected a distinct pattern for B . melitensis . These results indicate that both PCR-RFLP and Southern blot analysis of the dnaK locus can be used to distinguish B . melitensis strains from the other Brucella species and may be useful for typing and diagnostic purposes as well. Br J Rheumatol, 1996 Sep, 35(9), 828 - 38 Induction by chronic autoimmune arthritis in DBA/1 mice by oral administration of type II collagen and Escherichia coli lipopolysaccharide; Terato K et al.; We have developed a new model of autoimmune arthritis in DBA/1 mice by feeding chick type II collagen (CII) for 2-3 week intervals over a 15 week period . Clinically evident arthritis occurred in 8/10 mice receiving native CII (nCII; 100 micrograms/mouse) alone at 9-13 weeks . Arthritis was aggravated by the further ingestion of CII, while remission occurred after withdrawal of the CII . Heat-denatured CII (dCII; 200 micrograms/mouse) was also arthritogenic if co-administered with ovoinhibitor (OVI; 2 mg/mouse), a proteinase inhibitor . Co-oral administration of lipopolysaccharide (LPS; 10 micrograms/mouse) with CII enhanced the antibody production and T-cell responses to CII, and induced a more chronic arthritis that progressed spontaneously without further administration of CII or LPS . Long-term oral administration of LPS alone also induced a mild arthritis characterized by destruction of bone rather than cartilage . These observations suggest that abnormal gastrointestinal absorption of dietary mimic antigens and intestinal bacterial toxins can potentially disrupt self-tolerance mechanisms, thereby precipitating or exacerbating autoimmune disease in genetically susceptible individuals. Biochem J, 1996 Sep 1, 318 ( Pt 2), 665 - 71 Interleukin 1 beta and cAMP trigger the expression of GTP cyclohydrolase I in rat renal mesangial cells; Pluss C et al.; Endogenous synthesis of tetrahydrobiopterin (BH4) is an important requirement for cytokine-stimulated nitric oxide (NO) production in mesangial cells . We have shown that inducible NO synthase is expressed in mesangial cells in response to two principal classes of activating signals, inflammatory cytokines such as interleukin 1 beta (IL-1 beta) and agents that elevate cellular levels of cAMP {Kunz, Muhl, Walker and Pfeilschifter (1994) Proc . Natl . Acad . Sci . U.S.A . 91, 5387-5391} . In the present paper we demonstrate that IL-1 beta and cAMP similarly increase the steady-state mRNA levels of GTP cyclohydrolase I (EC 3,5,4,16), the rate-limiting enzyme in BH4 biosynthesis, as measured by a sensitive and quantitative nuclease protection assay . Stimulation of cells with a combination of IL-1 beta plus cAMP revealed an additive induction profile of GTP cyclohydrolase I mRNA . Message stability studies established that GTP cyclohydrolase I mRNA induced by cAMP has a longer half-life than the IL-1 beta-induced message . Moreover, cAMP exposure markedly prolonged the half-life of GTP cyclohydrolase I mRNA, from 1.5 to 3.4 h . In a next step we generated a rabbit polyclonal antibody against rat GTP cyclohydrolase I expressed in Escherichia coli and demonstrated that IL-1 beta and cAMP elevated GTP cyclohydrolase I protein levels in mesangial cells . Furthermore, IL-1 beta and cAMP led to a marked increase in GTP cyclohydrolase I activity and to increased accumulation of biopterin in mesangial cells . Combinations of IL-1 beta and cAMP resulted in a synergistic stimulation of GTP cyclohydrolase I activity . This may suggest that, in addition to transcriptional and post-transcriptional regulation, there is a prominent post-translational modulation of enzyme activity. Biochem J, 1996 Sep 1, 318 ( Pt 2), 443 - 9 Location of essential sequence elements at the Escherichia coli melAB promoter; Keen J et al.; The Escherichia coli melAB promoter has been cloned on a short DNA fragment and subjected to deletion mutagenesis, random mutagenesis and site-directed mutagenesis . In previous work we had shown that expression from the melAB promoter is triggered by melibiose and that this requires the MelR transcription activator . Melibiose-dependent expression is suppressed by deletions that remove both DNA-binding sites for MelR and by point mutations in the -10 hexamer, the -35 hexamer and the region just upstream of the -35 hexamer . The point mutations identify promoter elements that are essential for triggering the melAB promoter . The importance of these elements was confirmed by site-directed mutagenesis . The results show that the organization of the melAB promoter is fundamentally different from the organization of other bacterial promoters controlled by homologues of MelR. RNA, 1996 Sep, 2(9), 919 - 27 Selection of tRNA(Asp) amber suppressor mutants having alanine, arginine, glutamine, and lysine identity; Martin F et al.; Elements that confer identity to a tRNA in the cellular environment, where all aminoacyl-tRNA synthetases are competing for substrates, may be delineated by in vivo experiments using suppressor tRNAs . Here we describe the selection of active Escherichia coli tRNAAsp amber mutants and analyze their identity . Starting from a library containing randomly mutated tRNA(CUA)Asp genes, we isolated four amber suppressors presenting either lysine, alanine, or glutamine activity . Two of them, presenting mainly alanine or lysine activity, were further submitted to a second round of mutagenesis selection in order to improve their efficiency of suppression . Eleven suppressors were isolated, each containing two or three mutations . Ten presented identities of the two parental mutants, whereas one had switched from lysine to arginine identity . Analysis of the different mutants revealed (or confirmed for some nucleotides) their role as positive and/or negative determinants in AlaRS, LysRS, and ArgRS recognition . More generally, it appears that tRNAAsp presents identity characteristics closely related to those of tRNALys, as well as a structural basis for acquiring alanine or arginine identity upon moderate mutational changes; these consist of addition or suppression of the corresponding positive or negative determinants, as well as tertiary interactions . Failure to isolate aspartic acid-inserting suppressors is probably due to elimination of the important G34 identity element and its replacement by an antideterminant when changing the anticodon of the tRNAAsp to the CUA triplet. J Bacteriol, 1996 Sep, 178(18), 5522 - 8 Kinetics of expression of the Escherichia coli cad operon as a function of pH and lysine; Neely MN et al.; The Escherichia coli cadBA genes are regulated at the transcriptional level by external pH and lysine . The membrane-localized CadC protein is required for activation of this operon under inducing conditions, which include acidic external pH, lysine, and oxygen limitation . To better understand the mechanism by which CadC functions, the kinetics of cadBA expression as a function of pH and lysine were examined . By primer extension assays, cadBA expression was detected within 4 min following exposure of cells to one of the inducing stimuli (low pH or lysine), provided that the cells had first been grown to steady state in the presence of the other inducing stimulus . The induction time was three to four times longer when both inducing stimuli were added simultaneously . cadBA expression was shut off within 4 min following a shift from acidic to neutral pH . Treatment of cells with chloramphenicol prevented induction by acidic pH and lysine . Transcription of lysP (encodes a lysine transporter) was also examined, since it is a negative regulator of cadBA expression in the absence of lysine . lysP expression was repressed by lysine but not influenced by pH . Putative transcription start sites for lysP and cadC were determined . Together, these data suggest that CadC senses the lysine- and pH-induced signals separately and that one of the roles of lysine in inducing cadBA may be to repress expression of lysP, thus eliminating the repressing effects of LysP. J Bacteriol, 1996 Sep, 178(18), 5447 - 51 Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of four species of sigma subunit under various growth conditions; Jishage M et al.; By a quantitative Western immunoblot analysis, the intracellular levels of two principal sigma subunits, sigma 70 (sigma D, the rpoD gene product) and sigma 38 (sigma S, the rpoS gene product), and of two minor sigma subunits, sigma 54 (sigma N, the rpoN gene product) and sigma 28 (sigma F, the rpoF gene product), were determined in two Escherichia coli strains, W3110 and MC4100 . The results indicated that the levels of sigma 54 and sigma 28 are maintained at 10 and 50%, respectively, of the level of sigma 70 in both strains growing at both exponential and stationary phases, but in agreement with the previous measurement for strain MC4100 (M . Jishage and A . Ishihama, J . Bacteriol . 177:6832-6835, 1995), the level of sigma 38 was undetectable at the exponential growth phase but increased at 30% of the level of sigma 70 at the stationary phase . Stress-coupled change in the intracellular level was observed for two sigma subunits: (i) the increase in sigma 38 level and the decrease in sigma 28 level upon exposure to heat shock at the exponential phase and (ii) the increase in sigma 38 level under high-osmolality conditions at both the exponential and stationary phases. J Bacteriol, 1996 Sep, 178(18), 5438 - 46 Identification of sulfate starvation-regulated genes in Escherichia coli: a gene cluster involved in the utilization of taurine as a sulfur source; van der Ploeg JR et al.; Genes whose expression is regulated by sulfate starvation in Escherichia coli were identified by generating random translational lacZ fusions in the chromosome with the lambda placMu9 system . Nine lacZ fusion strains which expressed beta-galactosidase after growth under sulfate starvation conditions but not after growth in the presence of sulfate were found . These included two strains with insertions in the dmsA and rhsD genes, respectively, and seven strains in which the insertions were located within a 1.8-kb region downstream of hemB at 8.5 minutes on the E . coli chromosome . Analysis of the nucleotide sequence of this region indicated the presence of four open reading frames designated tauABCD . Disruption of these genes resulted in the loss of the ability to utilize taurine (2-aminoethanesulfonate) as a source of sulfur but did not affect the utilization of a range of other aliphatic sulfonates as sulfur sources . The TauA protein contained a putative signal peptide for transport into the periplasm; the TauB and TauC proteins showed sequence similarity to ATP-binding proteins and membrane proteins, respectively, of ABC-type transport systems; and the TauD protein was related in sequence to a dichlorophenoxyacetic acid dioxygenase . We therefore suggest that the proteins encoded by tauABC constitute an uptake system for taurine and that the product of tauD is involved in the oxygenolytic release of sulfite from taurine . The transcription initiation site was detected 26 to 27 bp upstream of the translational start site of tauA . Expression of the tauD gene was dependent on CysB, the transcriptional activator of the cysteine regulon. J Bacteriol, 1996 Sep, 178(18), 5422 - 30 Analysis of the in vivo activation of hemolysin (HlyA) from Escherichia coli; Ludwig A et al.; Hemolysin (HlyA) from Escherichia coli containing the hlyCABD operon separated from the nonhemolytic pro-HlyA upon two-dimensional (2-D) polyacrylamide gel electrophoresis . The migration distance indicated a net loss of two positive charges in HlyA as a result of the HlyC-mediated activation (modification) . HlyA activated in vitro in the presence of {U-14C}palmitoyl-acyl carrier protein comigrated with in vivo-activated hemolysin on 2-D gels and was specifically labelled, in agreement with the assumption that the activation is accomplished in vitro and in vivo by covalent fatty acid acylation . The in vivo-modified amino acid residues were identified by peptide mapping and 2-D polyacrylamide gel electrophoresis of mutant and truncated HlyA derivatives, synthesized in E . coli in the presence and absence of HlyC . These analyses indicated that the internal residues Lys-564 and Lys-690 of HlyA, which have recently been shown by others to be fatty acid acylated by HlyC in vitro, are also the only modification sites in vivo . HlyA activated in E . coli was quantitatively fatty acid acylated at both sites, and the double modification was required for wild-type hemolytic activity . Single modifications in mutant and truncated HlyA derivatives suggested that both lysine residues are independently fatty acid acylated by a mechanism requiring additional sequences or structures flanking the corresponding acylation site . The intact repeat domain of HlyA was not required for the activation . The pore-forming activities of pro-HlyA and singly modified HlyA mutants in planar lipid bilayer membranes suggested that the activation is not essential for transmembrane pore formation but rather required for efficient binding of the toxin to target membranes. J Bacteriol, 1996 Sep, 178(18), 5382 - 7 Increased unsaturated fatty acid production associated with a suppressor of the fabA6(Ts) mutation in Escherichia coli; Rock CO et al.; Plasmids that corrected the temperature-sensitive unsaturated fatty acid auxotrophy of strain M6 {fabA6 (Ts)} were isolated from an Escherichia coli genomic library . Subcloning and physical mapping localized the new gene (called sfa for suppressor of fabA) at 1,070 kb on the E . coli chromosome . DNA sequencing revealed the presence of a 227-bp open reading frame which directed the synthesis of a peptide of approximately 8 kDa, which correlated with the correction of the fabA6(Ts) phenotype . However, the sfa gene was an allele-specific suppressor since plasmids harboring the sfa gene corrected the growth phenotype of fabA6(Ts) mutants but did not correct the growth of fabA2(Ts) or fabB15(Ts) unsaturated fatty acid auxotrophs . Overexpression of the sfa gene in fabA6(Ts) mutants restored unsaturated fatty acid content at 42 degrees C, and overexpression in wild-type cells resulted in a substantial increase in the unsaturated fatty acid content of the membrane . Thus, the suppression of the fabA6(Ts) mutation by sfa was attributed to its ability to increase the biosynthesis of unsaturated fatty acids. J Bacteriol, 1996 Sep, 178(18), 5370 - 81 Use of phoA and lacZ fusions to study the membrane topology of ProW, a component of the osmoregulated ProU transport system of Escherichia coli; Haardt M et al.; The Escherichia coli ProU system is a member of the ATP-binding cassette (ABC) superfamily of transporters . ProU consists of three components (ProV, ProW, and ProX) and functions as a high-affinity, binding protein-dependent transport system for the osmoprotectants glycine betaine and proline betaine . The ProW protein is the integral inner membrane component of the ProU system . Its hydropathy profile predicts seven transmembrane spans and a hydrophilic amino terminus of approximately 100 residues, and it suggests the presence of an amphiphilic alpha-helix (L-61 to F-97) in close proximity to the first strongly hydrophobic segment of ProW . We have studied the membrane topology of the ProW protein by the phoA and lacZ gene fusion approach . A collection of 10 different proW-phoA fusions with alkaline phosphatase activity and 8 different proW-lacZ fusions with beta-galactosidase activity were isolated in vivo after TnphoAB and TnlacZ mutagenesis of a plasmid-encoded proW gene . The recovery of both enzymatically active ProW-PhoA and ProW-LacZ hybrid proteins indicates that segments of ProW are exposed on both sides of the cytoplasmic membrane . To compare the enzymatic activities of each of the indicator proteins joined at a particular site in ProW, we switched the phoA and lacZ reporter genes in vitro in each of the originally in vivo-isolated gene fusions . A mirror-like pattern in the enzyme activity of the resulting new ProW-PhoA and ProW-LacZ hybrid proteins emerged, thus providing positive signals for the location of both periplasmic and cytoplasmic domains in ProW . The protease kallikrein digests the amino-terminal tail of a ProW-LacZ hybrid protein in spheroplasts, suggesting that the amino terminus of ProW is located on the periplasmic side of the cytoplasmic membrane . From these data, a two-dimensional model for ProW was constructed; this model consists of seven transmembrane alpha-helices and an unusual amino-terminal tail of approximately 100 amino acid residues that protrudes into the periplasmic space. J Bacteriol, 1996 Sep, 178(18), 5347 - 52 Identification of the mpl gene encoding UDP-N-acetylmuramate: L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase in Escherichia coli and its role in recycling of cell wall peptidoglycan; Mengin-Lecreulx D et al.; A gene, mpl, encoding UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelat e ligase was recognized by its amino acid sequence homology with murC as the open reading frame yjfG present at 96 min on the Escherichia coli map . The existence of such an enzymatic activity was predicted from studies indicating that reutilization of the intact tripeptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate occurred and accounted for well over 30% of new cell wall synthesis . Murein tripeptide ligase activity could be demonstrated in crude extracts, and greatly increased activity was produced when the gene was cloned and expressed under control of the trc promoter . A null mutant totally lacked activity but was viable, showing that the enzyme is not essential for growth. Arch Biochem Biophys, 1996 Sep 1, 333(1), 308 - 15 The interaction of NADPH-P450 reductase with P450: an electrochemical study of the role of the flavin mononucleotide-binding domain; Estabrook RW et al.; The electrochemically reduced mediator cobalt sepulchrate requires the presence of a flavoprotein for the rapid transfer of electrons to cytochrome P450 . This electrochemical method has been used here to show the interaction of NADPH-P450 reductase (either the detergent-solubilized form, d-OR, or the proteolytic-cleaved truncated form, t-OR), as well as Escherichia coli flavodoxin (FLD), with P450c17 by measuring the rate of 17 alpha-hydroxylation of progesterone . When NADPH is used as electron donor with a reconstituted system composed of d-OR and P450c17, the addition of t-OR, flavodoxin, or cytochrome c inhibited the rate of formation of 17 alpha-hydroxyprogesterone . These results suggest the presence of a common protein binding site on the surface of d-OR, t-OR, and flavodoxin which plays a role in the interaction of the flavoproteins with the P450 . It is speculated that a domain composed of acidic amino acids, located near the flavin mononucleotide-binding region of the flavoproteins, may serve as this site . No inhibition by t-OR, flavodoxin, or cytochrome c is observed when comparable experiments are carried out using the artificial recombinant fusion protein rF450{mBov17A/mRatOR}L1 containing the heme-domain of P450c17 linked to the flavin-domains of NADPH-P450 reductase. Arch Biochem Biophys, 1996 Sep 1, 333(1), 251 - 9 Site-directed mutagenesis of a highly conserved aspartate in the putative 10-formyl-tetrahydrofolate binding site of yeast C1-tetrahydrofolate synthase; Kirksey TJ et al.; C1-tetrahydrofolate (THF) synthase is a eukaryotic trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase . Although the 10-formyl-THF synthetase reaction (a reversible ATP-dependent formylation of THF) has been studied extensively, little is known about specific residues involved in the catalytic mechanism . In this study, we have examined the role of a highly conserved aspartate residue, Asp449 of yeast cytoplasmic C1-THF synthase . Asp449 is part of a putative folate binding site found in many proteins that bind 10-formyl-THF . The corresponding aspartate has been identified as a critical catalytic residue in Escherichia coli and human GAR transformylase, which catalyzes a 10-formyl-THF-dependent formyl transfer . In order to determine if Asp449 has a similar catalytic role in the 10-formyl-THF synthetase reaction, three mutant proteins were produced by site-directed mutagenesis in which Asp449 of yeast cytoplasmic C1-THF synthase was changed to Asn, Glu, or Ala . The mutant proteins were expressed in yeast, purified, and characterized with respect to kinetic properties and enzyme stability . All three of the mutant enzymes retained substantial 10-formyl-THF synthetase activity, indicating that Asp449 is not a critical catalytic residue . However, our data suggest that it does play a role in folate binding, probably by contributing to the proper conformation of the active site . Thus, these results suggest that the 10-formyl-THF binding site differs significantly between the GAR transformylase and 10-formyl-THF synthetase families, and that the conserved aspartate plays different roles in the two enzymes. Arch Biochem Biophys, 1996 Sep 1, 333(1), 96 - 102 Expression of rat liver tryptophan 2,3-dioxygenase in Escherichia coli: structural and functional characterization of the purified enzyme; Ren S et al.; The hepatic hemoprotein tryptophan 2,3-dioxygenase (TDO) is the key regulatory enzyme that, through irreversible degradation, controls the flux of tryptophan through physiologically relevant pathways . This enzyme is composed of four identical subunits and in its fully assembled tetrameric form requires 2 mol of heme (Fe(+2)-protoporphyrin IX)/mol of protein for functional competence . Using a full-length cDNA for the rat liver TDO subunit (pUC119/TDO) as the template, TDO cDNA was amplified by polymerase chain reaction (PCR) and incorporated into the expression vector pTrc99A after introduction of convenient restriction sites as well as modification of the second codon AGT to GCT to optimize its bacterial expression . DH5 alpha F' strain Escherichia coli cells transfected with this pTrc99A/TDO construct expressed soluble, functionally active, tetrameric TDO protein in high yields . The enzyme was isolated from 30,000g supernatant of cell lysates, purified by ion-exchange chromatography, and its spectral and catalytic properties were assessed in terms of its substrate and prosthetic moiety specificities . In almost all aspects, the bacterially expressed enzyme was found to be identical to that of the rat liver . Heterologous expression of the fully functional enzyme, we trust, will enable future elucidation of its structure-function relationships. Arch Biochem Biophys, 1996 Sep 1, 333(1), 85 - 95 Recombinant human phenylalanine hydroxylase: novel regulatory and structural properties; Kowlessur D et al.; Recombinant human liver phenylalanine hydroxylase (PAH) expressed in Escherichia coli has been purified to homogeneity . The recombinant enzyme exists in solution as a mixture of 80% tetramers and 20% dimers . A study of the kinetic properties of the enzyme indicates that compared to the recombinant and the native rat liver enzymes, the recombinant human enzyme is in an activated state . This conclusion is supported by the finding that its catalytic activity is only marginally stimulated by incubation with either phenylalanine or lysolecithin . In contrast, the native and the recombinant rat liver enzymes are activated 8- to 25-fold, respectively, when preincubated with phenylalanine or lysolecithin . In the absence of activators, the ratio of the hydroxylase activity in the presence of 6-methyl-5,6,7,8-tetrahydropterin compared to the activity in the presence of (6R)-5,6,7,8-tetrahydrobiopterin (BH4), which is an index of the state of activation of the enzyme, is 4 for the human recombinant PAH compared to a value of 12 for the recombinant rat liver enzyme . Furthermore, the Km for phenylalanine in the presence of BH4 is 0.050 mM, a value that is one-fifth that of the recombinant rat liver enzyme . Covalent modification of the human enzyme by phosphorylation with protein kinase A provides further evidence that the human enzyme is in a substantially activated state . Phosphorylation, which results in the incorporation of 0.6 mol of phosphate/mol of subunit, leads to only a modest activation of 1.5-fold compared to about a 3-fold activation seen after phosphorylation of the native and the recombinant rat liver enzymes . Moreover, the recombinant human liver enzyme is less sensitive than the rat liver enzyme to stimulation by lysolecithin when tryptophan is the substrate . Just as is true for the rat liver enzyme, the apparent Km values for tryptophan and pheylalanine vary with the pterin cofactor employed . The ability of 7-tetrahydrobiopterin (7-BH4) to substitute for the natural cofactor tetrahydrobiopterin has been studied in vitro . The apparent Km for 7-BH4 for the recombinant human enzyme is 0.2 mM and the Km for phenylalanine is 0.05 mM . The hydroxylase reaction is severely inhibited by 7-BH4 in the presence of physiological concentrations of BH4 . This inhibition can be overcome by a decrease in the concentration of phenylalanine . The implications of these novel properties of human PAH for phenylalanine homoestasis in man are discussed. Arch Biochem Biophys, 1996 Sep 1, 333(1), 75 - 84 Adenylate kinase from Sulfolobus acidocaldarius: expression in Escherichia coli and characterization by Fourier transform infrared spectroscopy; Bonisch H et al.; Adenylate kinase from the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius has been overexpressed in Escherichia coli . The highly purified enzyme was characterized by Fourier transform infrared spectroscopy (FTIR) . Analysis of FTIR spectra and estimation of secondary structure revealed a global protein structure similar to that of other adenylate kinases . Thermal unfolding of the protein with an estimated Tm value near 90 degrees C is irreversible due to protein aggregation . The enzyme exhibits long-term stability up to 80 degrees C, which is an excellent adaptation to the physiological growth temperature of 75-80 degrees C . Half-widths of secondary-structure-sensitive bands and hydrogen-deuterium exchange experiments revealed that in comparison to adenylate kinase from porcine muscle cytosol the Sulfolobus enzyme is characterized by a significantly more compact and rigid protein core structure, which is likely to contribute specifically to the extreme thermostability of the protein. Genes Dev, 1996 Sep 1, 10(17), 2222 - 33 A hierarchy of SSB protomers in replication protein A; Philipova D et al.; Replication Protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (SSB) found in all eukaryotic cells . RPA is known to be required for many of the same reactions catalyzed by the homotetrameric SSB of bacteria, but its origin, subunit functions, and mechanism of binding remain a mystery . Here we show that the three subunits of yeast RPA contain a total of four domains with weak sequence similarity to the Escherichia coli SSB protomer . We refer to these four regions as potential ssDNA-binding domains (SBDs) . The p69 subunit, which is known to bind ssDNA on its own, contains two SBDs that together confer stable binding to ssDNA . The p36 and p13 subunits each contain a single SBD that does not bind stably, but corresponds to the minimal region required for viability in yeast . Photocross-linking of recombinant protein to ssDNA indicates that an SBD consists of approximately 120 amino acids with two centrally located aromatic residues . Mutation of these aromatic residues inactivates ssDNA binding and is a lethal event in three of the four domains . Finally, we present evidence that the p36 subunit binds ssDNA, as part of the RPA complex, in a salt-dependent reaction similar to the wrapping of ssDNA about E . coli SSB . The results are consistent with the notion that RPA arose by duplication of an ancestral SSB gene and that tetrameric ssDNA-binding domains and higher order binding are essential features of cellular SSBs. Genes Dev, 1996 Sep 1, 10(17), 2145 - 57 Conjugating plasmids are preferred targets for Tn7; Wolkow CA et al.; Most transposons display target site selectivity, inserting preferentially into sites that contain particular features . The bacterial transposon Tn7 possesses the unusual ability to recognize two different classes of target sites . Tn7 inserts into these classes of target sites through two transposition pathways mediated by different combinations of the five Tn7-encoded transposition proteins . In one transposition pathway, Tn7 inserts into a unique site in the bacterial chromosome, attTn7, through specific recognition of sequences in attTn7; the other transposition pathway ignores the attTn7 target . Here we examine targets of the non-attTn7 pathway and find that Tn7 preferentially inserts into bacterial plasmids that can conjugate between cells . Furthermore, Tn7 appears to recognize preferred targets through the conjugation process, as we show that Tn7 inserts poorly into plasmids containing mutations that block plasmid transfer . We propose that Tn7 recognizes preferred targets through features of the conjugation process, a distinctive target specificity that offers Tn7 the ability to spread efficiently through bacterial populations. Appl Environ Microbiol, 1996 Sep, 62(9), 3466 - 9 Prevalence of enterohemorrhagic Escherichia coli in raw and treated municipal sewage; Grant SB et al.; Municipal sewage was screened for DNA encoding Shiga-like Toxin (SLT) II, a key protein involved in the virulence of enterohemorrhagic Escherichia coli . PCR analysis of sewage concentrates showed that DNA encoding SLT II was present in a single sample of untreated sewage and absent in all other samples tested (n = 6) . Thermotolerant E . coli cultured from the sewage (n = 1,520) also tested negative for SLT II by colony hybridization. Appl Environ Microbiol, 1996 Sep, 62(9), 3462 - 5 Typing of bovine attaching and effacing Escherichia coli by multiplex in vitro amplification of virulence-associated genes; China B et al.; Attaching and effacing Escherichia coli is a new causal agent of diarrhea in calves . Its major virulence factors are the intimin protein, encoded by the eaeA gene, and the Shiga-like toxins, encoded by slt genes . Because the sequences of these genes are available, we selected specific primers to amplify each virulence gene so as to develop a new identification test based on multiplex amplification of virulence-associated genes . Of 30 tested strains, 14 were eaeA+, 15 were eaeA+ slt-I+, 1 was eaeA+ slt-I+ slt-II+, and 1 was eaeA+ slt-II+ . The method proved in our hands to be fast and specific and in perfect correlation with the hybridization method. J Cell Biol, 1996 Sep, 134(5), 1333 - 44 A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice; Bou-Gharios G et al.; We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse pro alpha 2 (I) collagen gene . These sites were detected in cells that produce type I collagen but not in cells that do not express these genes . A construction containing the sequences from -17 kb to +54 bp of the mouse pro alpha 2 (I) collagen gene, cloned upstream of either the Escherichia coli beta-galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments . Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs . High levels of expression could also be detected in some osteoblastic cells . When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal pro alpha 2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites . Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse pro alpha 1(I) collagen promoter, which is silent in transgenic mice . Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro alpha 2(I) collagen gene. Dig Dis Sci, 1996 Sep, 41(9), 1853 - 62 Immunogenicity and safety of recombinant Helicobacter pylori urease in a nonhuman primate; Stadtlander CT et al.; Groups of squirrel monkeys (Saimiri spp.), predetermined to be free of Helicobacter infections in the gastric mucosa, were immunized orally with 0.5-4.5 mg of Helicobacter pylori recombinant urease (rUrease) and 25-500 micrograms of Escherichia coli heat-labile enterotoxin (LT) adjuvant . Oral immunization with rUrease resulted in a markedly elevated serum immunoglobulin G (IgG) antibody response with peak levels at 45 days after immunization . No significant gastric inflammation or cytotoxicity was evident in rUrease immunized monkeys as determined by light and electron microscopy . Twenty-five micrograms of LT was a sufficient and safe adjuvant dosage, whereas higher dosages resulted in diarrhea and lethargy . Animals developed a serum IgG antibody response to LT that did not impede the production of anti-rUrease antibody levels . The results of this investigation indicate that rUrease is immunogenic in a nonhuman primate. Development, 1996 Sep, 122(9), 2697 - 707 Drosophila Paired regulates late even-skipped expression through a composite binding site for the paired domain and the homeodomain; Fujioka M et al.; The even-skipped (eve) pair-rule gene plays a key role in the establishment of the anterior-posterior segmental pattern of the Drosophila embryo . The continuously changing pattern of eve expression can be resolved into two phases . Early expression consists of seven broad stripes in the blastoderm embryo, while late expression, which occurs after cellularization, consists of narrow stripes with sharp anterior borders that coincide with the odd-numbered parasegment boundaries . Previous studies have shown that these two phases are controlled by separate classes of cis elements in the eve promoter . Early stripes are expressed by multiple stripe-specific elements under the control of maternal-effect genes and gap genes, while late stripes are expressed by a single regulatory element, the 'late element', under the control of pair-rule genes including eve itself . We report here that paired (prd), a pair-rule gene which had been considered to be below eve in the regulatory hierarchy of pair-rule genes, in fact plays a critical role in the regulation of late eve expression . Transgenic analysis shows that this regulation is largely mediated by an evolutionarily conserved sequence within the late element termed PTE (Paired Target Element) . In vitro analysis shows that the Prd protein binds strongly to this sequence . Interestingly, PTE contains juxtaposed binding sites for the two DNA-binding domains of the Prd protein, the paired domain and the homeodomain . Mutagenesis of either binding site leads to significant reduction in the activity of the late element, indicating that both DNA-binding domains in the Paired protein are required for regulation. Hepatology, 1996 Sep, 24(3), 691 - 6 Endotoxin stimulates hydrogen peroxide detoxifying activity in rat hepatic endothelial cells; Spolarics Z et al.; The study aimed to assess the effect of lipopolysaccharide (LPS) in vivo (from Escherichia coli, 2 mg/kg body weight intraperitoneally) on the production and elimination of hydrogen peroxide (H2O2) in rat hepatic endothelial and Kupffer cells . Twenty-two hours after the injection of LPS, hepatic cells were isolated by collagenase and pronase digestion followed by centrifugal elutriation, and cell-associated H2O2 was determined by flow cytometry analysis using 2',7'-dichloroflorescin diacetate (DCF-diacetate) . LPS treatment did not alter the basal or phorbol myristate acetate-stimulated levels of H2O2-related fluorescence in endothelial cells; however, it doubled phorbol myristate acetate-stimulated fluorescence in Kupffer cells . Administration of varying concentrations of H202 (range, 10(-7) - 10(-4) mol/L) in vitro caused a significantly delayed increase in fluorescence in endothelial cells from endotoxemic rats as compared with cells from saline-injected animals . The 50% effective concentration of H202 was found at 1.1 x 10(-6) and 8.1 x 10(-6) mol/L on endothelial cells after saline and LPS treatment, respectively . No differences were detected in H2O2-stimulated fluorescence between resting and LPS-stimulated Kupffer cells . Administration of varying glucose concentrations in vitro significantly decreased the H2O2-stimulated fluorescence in endothelial and Kupffer cells from LPS-injected animals . Inhibition of nitric oxide synthase by in vitro administration of NG-monomethyl-L-arginine (L-NNMMA) did not alter the H2O2- or phorbol myristate acetate-stimulated responses in endothelial and Kupffer cells . As shown earlier, LPS stimulates the gene expression of GLUT1 glucose transporter, glucose-6-phosphate dehydrogenase (G6PD), superoxide dismutases, and glutathione peroxidase in hepatic endothelial cells . The present data indicate that the LPS-induced metabolic alterations are accompanied by an increased H2O2-detoxifying capacity in hepatic endothelial cells . This may represent a protective mechanism against exogenous oxidative stress caused by activated hepatic phagocytes during inflammation . Our observations are consistent with primed production of reactive oxygen species (ROS) in LPS-activated Kupffer cells. Hepatology, 1996 Sep, 24(3), 592 - 5 Influence of dietary fat on the effect of endotoxin on murine hepatic peroxisomes; DeLamatre JG et al.; This study investigates the effects of either a high-fat diet or endotoxin on peroxisome metabolism as assessed by measuring catalase activity, catalase mass, and peroxisomal beta-oxidation . Three mouse strains C3H/HeJ, BALB/c, and C57BL/6J were fed either a low-fat or a high-fat diet and injected intraperitoneally with 1 microg Escherichia coli lipopolysaccharide . These parameters were not different in C3H/HeJ mice fed a high-fat diet compared with controls fed a low-fat diet . Total liver catalase activity and peroxisomal beta-oxidation were higher in BALB/c mice fed high-fat diet compared with low-fat controls . Total liver catalase activity, catalase mass and peroxisomal beta-oxidation increased to the greatest extent in C57BL/6J mice fed a high-fat diet . Endotoxin treatment did not alter any of the parameters in mice fed a low-fat diet . Among mice fed a high-fat diet, endotoxin did not affect hepatic catalase or peroxisomal beta-oxidation in the C3H/HeJ mice, decreased catalase activity in BALB/c mice (28%), and greatly decreased both catalase (66%) and peroxisomal beta-oxidation (69%) in C57BL/6J mice . The decrease in catalase activity in C57BL/6J mice was apparently because of an inactivation of the enzyme as determined by the activity/mass ratio . Thus, endotoxin is showed to inhibit both catalase and peroxisomal beta-oxidation in mice and the sensitivity to endotoxin is greatest in C57BL/6J mice fed a high-fat diet. Curr Genet, 1996 Sep, 30(4), 305 - 11 Isolation by genetic complementation of two differentially expressed genes for beta-isopropylmalate dehydrogenase from Aspergillus niger; Williams BA et al.; We have constructed an Aspergillus niger cDNA library with a yeast expression vector . The library DNA complemented a leucine auxotroph of Saccharomyces cerevisiae (strain BWG1-7a) at a frequency of 4x10(-4) . Plasmids rescued from the yeast prototrophs also complemented Escherichia coli (strain MC1066) deficient in leucine biosynthesis . Sequence determination of the rescued plasmids revealed two genes for beta-isopropylmalate dehydrogenase, which we called leu2A and leu2B . Genomic-blot analysis suggested that both leu2A and leu2B were derived from single-copy genes . Northern-blot hybridization showed that in nutrient-rich medium a leu2A transcript accumulated during germination and log-phase growth while the leu2B transcript appeared late in the growth phase . In minimal medium, only leu2A expression was greatly stimulated . We examined the codon preference of these two genes . Whereas leu2A shows a bias in codon usage typical of A . niger genes, leu2B does not . These results indicate the presence in A . niger of two highly divergent, differentially regulated, isozymes for beta-isopropylmalate dehydrogenase. Diabetes, 1996 Sep, 45(9), 1238 - 44 Effect of mitochondrial and/or cytosolic glycerol 3-phosphate dehydrogenase overexpression on glucose-stimulated insulin secretion from MIN6 and HIT cells; Ishihara H et al.; The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH) . Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in beta-cells . We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively . Therefore, we selected the MIN6 cell line as a beta-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a beta-cell model with impaired insulin secretion and lower activities of these enzymes . The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells . Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli beta-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells . Infection with a recombinant adenovirus with mGPDH cDNA (Adex1CAmGPDH) caused 2.1-fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively . Infection with a recombinant adenovirus with cGPDH cDNA (Adex1CAcGPDH) caused a more than 50-fold increase in activity in both cell lines . Glycerol phosphate shuttle flux, as estimated by {2-3H}glycerol conversion to {3H}H2O, was increased to 120-130% by infection with Adex1CAmGPDH, but not with Adex1CAcGPDH infection, in both MIN6 and HIT cells . No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with Adex1CAmGPDH together with Adex1CAcGPDH . Furthermore, neither {U-14C}glucose oxidation nor the insulin secretory response to glucose was affected in either cell line . Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells . The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated. Mol Cell Biol, 1996 Sep, 16(9), 4691 - 9 A new member of the hsp90 family of molecular chaperones interacts with the retinoblastoma protein during mitosis and after heat shock; Chen CF et al.; A gene encoding a new heat shock protein that may function as a molecular chaperone for the retinoblastoma protein (Rb) was characterized . The cDNA fragment was isolated by using the yeast two-hybrid system and Rb as bait . The open reading frame of the longest cDNA codes for a protein with substantial sequence homology to members of the hsp90 family . Antibodies prepared against fusions between glutathione S-transferase and portions of this new heat shock protein specifically recognized a 75-kDa cellular protein, hereafter designated hsp75, which is expressed ubiquitously and located in the cytoplasm . A unique LxCxE motif in hsp75, but not in other hsp90 family members, appears to be important for binding to the simian virus 40 T-antigen-binding domain of hypophosphorylated Rb, since a single mutation changing the cysteine to methionine abolishes the binding . In mammalian cells, Rb formed complexes with hsp75 under two special physiological conditions: (i) during M phase, when the envelope that separates the nuclear and cytoplasmic compartments broke down, and (ii) after heat shock, when hsp75 moved from its normal cytoplasmic location into the nucleus . In vitro, hsp75 had a biochemical activity to refold denatured Rb into its native conformation . Taken together, these results suggest that Rb may be a physiological substrate for the hsp75 chaperone molecule . The discovery of a heat shock protein that chaperones Rb identifies a mechanism, in addition to phosphorylation, by which Rb is regulated in response to progression of the cell cycle and to external stimuli. Mol Cell Biol, 1996 Sep, 16(9), 4673 - 82 Cyclin-binding motifs are essential for the function of p21CIP1; Chen J et al.; The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage . We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2 . The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo . Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts . A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts . Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell . Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo . The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex. J Bacteriol, 1996 Sep, 178(17), 5333 - 4 Experimental rejection of a nonadaptive explanation for increased cell size in Escherichia coli; Mongold JA et al.; Populations of Escherichia coli that have been serially propagated for thousands of generations in glucose minimal medium show heritable increases in both cell size and growth rate . We sought to test the hypothesis that the increased cell size of the derived genotypes could be explained solely by their faster growth . The regression of cell size on growth rate differed significantly between populations having ancestral and derived genotypes, with the latter producing larger cells over almost the entire range of growth rates . Thus, the physiological coupling between cell size and growth rate has been evolutionarily altered. J Bacteriol, 1996 Sep, 178(17), 5316 - 9 Dynamic aspects of colicin N translocation through the Escherichia coli outer membrane; El Kouhen R et al.; Colicin N is a bacteriocin that kills sensitive Escherichia coli cells . After binding to the cell surface-exposed receptor, a short period exists when a significant number of the cell-associated colicin N molecules are sensitive to external enzymes . Two colicin N populations are discriminated by proteases: the susceptible pool bound to OmpF porin on the cell surface and another population corresponding to protease-inaccessible colicin N . During translocation, colicin N reaches the periplasmic space and proteolytic cleavage of the colicin occurs only when the outer membrane barrier is permeabilized. J Bacteriol, 1996 Sep, 178(17), 5309 - 15 Genetic control of the resistance to phage C1 of Escherichia coli K-12; Likhacheva NA et al.; Escherichia coli K-12 lytic phage C1 was earlier isolated in our laboratory . Its adsorption is controlled by at least three bacterial genes: dcrA, dcrB, and btuB . Our results provide evidence that the dcrA gene located at 60 min on the E . coli genetic map is identical to the sdaC gene . This gene product is an inner membrane protein recently identified as a putative specific serine transporter . The dcrB gene, located at 76.5 min, encodes a 20-kDa processed periplasmic protein, as determined by maxicell analysis, and corresponds to a recently determined open reading frame with a previously unknown function . The btuB gene product is known to be an outer membrane receptor protein responsible for adsorption of BF23 phage and vitamin B12 uptake . According to our data the DcrA and DcrB proteins are not involved in these processes . However, the DcrA protein probably participates in some cell division steps. J Bacteriol, 1996 Sep, 178(17), 5182 - 7 A positive control mutant of the transcription activator protein FIS; Gosink KK et al.; The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli . We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo . The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop . In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS . These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending. J Bacteriol, 1996 Sep, 178(17), 5138 - 43 asmB, a suppressor locus for assembly-defective OmpF mutants of Escherichia coli, is allelic to envA (lpxC); Kloser AW et al.; A novel genetic scheme allowed us to isolate extragenic suppressor mutations that restored mutant OmpF assembly . One group of these mutations, termed asmB for assembly suppressor mutation B, permitted mutant OmpF assembly in a non-allele-specific manner . Genetic mapping analyses placed the asmB mutations at the 2-min region of the Escherichia coli K-12 chromosome . Further analyses revealed that the asmB mutations map within the envA (lpxC) gene, which encodes an enzyme needed for the synthesis of the lipid A moiety of lipopolysaccharide (LPS) . Nucleotide sequence analysis showed that the asmB mutations caused a change from F-50 to S (F50S substitution) (asmB2 and asmB3) or a G210S substitution (asmB1) in EnvA . Cells bearing the asmB alleles displayed increased sensitivity to various hydrophobic compounds and detergents, suggesting an alteration within the outer membrane . Direct examination (of the LPS showed that its amounts were reduced by the asmB mutations, with asmB1 exerting a greater effect than asmB2 or asmB3 . Thus, it appears that the asmB mutations achieve mutant OmpF assembly suppression by reducing LPS levels, which in turn may alter membrane fluidity. J Bacteriol, 1996 Sep, 178(17), 5105 - 11 Generation of an endogenous DNA-methylating agent by nitrosation in Escherichia coli; Taverna P et al.; Escherichia coli ada ogt mutants, which are totally deficient in O6-methylguanine-DNA methyltransferases, have an increased spontaneous mutation rate . This phenotype is particularly evident in starving cells and suggests the generation of an endogenous DNA alkylating agent under this growth condition . We have found that in wild-type cells, the level of the inducible Ada protein is 20-fold higher in stationary-phase and starving cells than in rapidly growing cells, thus enhancing the defense of these cells against DNA damage . The increased level of Ada in stationary cells is dependent on RpoS, a stationary-phase-specific sigma subunit of RNA polymerase . We have also identified a potential source of the mutagenic agent . Nitrosation of amides and related compounds can generate directly acting methylating agents and can be catalyzed by bacteria} enzymes . E . coli moa mutants, which are defective in the synthesis of a molybdopterin cofactor required by several reductases, are deficient in nitrosation activity . It is reported here that a moa mutant shows reduced generation of a mutagenic methylating agent from methylamine (or methylurea) and nitrite added to agar plates . Moreover, a moa mutation eliminates much of the spontaneous mutagenesis in ada ogt mutants . These observations indicate that the major endogenous mutagen is not S-adenosylmethionine but arises by bacterially catalyzed nitrosation. J Bacteriol, 1996 Sep, 178(17), 5100 - 4 Identification of a new inhibitor of essential division gene ftsZ as the kil gene of defective prophage Rac; Conter A et al.; A gene function carried by a plasmid, causing arrest of cell division in Escherichia coli, has been identified as the product of a short open reading frame of the prophage Rac, previously designated orfE, expressed only under conditions of prophage induction . Because Rac carries a killing function expressed under conditions of zygotic induction, an orfE-defective Rac+ strain was constructed . This strain had lost the killing function, indicating that orfE is kil . Division inhibition by kil was specifically relieved by overexpression of essential division gene ftsZ . The kil gene product acts independently of the min operon, and its effects are increased in conditions of high cyclic AMP (cAMP) receptor protein-cAMP complex levels in the cell . Furthermore, at high levels of expression, kil product distorts the rod shape of the cells . These features distinguish kil-encoded protein from the inhibitory product of gene dicB, which occupies a similar genetic location in Kim (Qin), another defective prophage of Escherichia coli. J Bacteriol, 1996 Sep, 178(17), 5080 - 5 Interaction between FtsZ and inhibitors of cell division; Huang J et al.; The interaction between inhibitors of cell division and FtsZ were assessed by using the yeast two-hybrid system . An interaction was observed between FtsZ and SulA, a component of the SOS response, and the interacting regions were mapped to their conserved domains . This interaction was reduced by mutations in sulA and by most mutations in ftsZ that make cell refractory to sulA . No interaction was detected between FtsZ and MinCD, an inhibitory component of the site selection system . However, interactions were observed among various members of the Min system, and MinE was found to reduce the interaction between MinC and MinD . The implications of these findings for cell division are discussed. Cancer Res, 1996 Sep 1, 56(17), 3926 - 33 Characterization of purified human recombinant cytochrome P4501A1-Ile462 and -Val462: assessment of a role for the rare allele in carcinogenesis; Zhang ZY et al.; Human cytochrome P4501A1 (CYP1A1) occurs extrahepatically and is polymorphic, the common form having Ile at position 462 and the rare form having Val . The rare allele has been associated with enhanced susceptibility to lung cancer . To resolve its role in cancer we have constructed CYP1A1-Val462 cDNA by site-directed mutagenesis from CYP1A1-Ile462, as confirmed by sequencing and allele-specific PCR . Both alleles were expressed in Escherichia coli, and CYP1A1-Ile462 and -Val462 were purified to electrophoretic homogeneity . The secondary structures of both forms were virtually identical, with high alpha helix content, as assessed by circular dichroism . The P450s stereoselectively and regioselectively catalyzed the metabolism of (R)- and (S)- warfarin, in reconstituted systems, with very similar profiles . Both P450s produced (R)-6- and 8-hydroxy-warfarin with Km values of 0.40 +/- 0.06 and 0.43 +/- 0.05 mM, respectively, and Vmax values of 84.0 +/- 6.8 and 137.7 +/- 8.9 pmol/min/nmol CYP1A1-Val462, respectively, 1.0 +/- 0.1 and 1.0 +/- 0.1 mM, respectively, and 46.7 +/- 2.5 and 80.0 +/- 4.4 pmol/min/nmol CYP1A1-Ile462, respectively . Reconstituted CYP1A1-Val462 catalyzed ethoxyresorufin metabolism at a slightly but significantly higher rate than did CYP1A1-Ile462; Vmax values were 4.4 +/- 0.6 and 3.1 +/- 0.3 nmol/min/nmol CYP1A1, respectively . However, with the carcinogen benzo(a) pyrene as substrate, reconstituted CYP1A1-Ile462 together with epoxide hydrolase produced 7,8- and 9,10-dihydrodiols at comparable rates than did CYP1A1-Val462 . Thus, the apparently greater susceptibility of the CYP1A1-Val462 genotype to lung cancer is probably not related to greater extents of carcinogen bioactivation. J Neurochem, 1996 Sep, 67(3), 917 - 26 Expression and deletion mutagenesis of tryptophan hydroxylase fusion proteins: delineation of the enzyme catalytic core; D'Sa CM et al.; cDNAs encoding the full-length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1-98) or catalytic (amino acids 99-444) domains of the enzyme, were cloned and expressed as glutathione S-transferase fusion proteins in E . coli . The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione-agarose . The full-length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity . The regulatory domain was devoid of activity . The full-length enzyme and the catalytic core, while adsorbed to glutathione-agarose beads, obeyed Michaelis-Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase . Both active forms of the glutathione S-transferase-tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity (Vmax) than the brain enzyme . Analysis of full-length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species . These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99-444 of the full-length enzyme, contains the sequence motifs needed for subunit assembly . Both wild-type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron . The tryptophan hydroxylase catalytic core is also as active as the full-length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic core of tryptophan hydroxylase. Mol Biol Evol, 1996 Sep, 13(7), 978 - 89 Nuclear-encoded rDNA group I introns: origin and phylogenetic relationships of insertion site lineages in the green algae; Bhattacharya D et al.; Group I introns are widespread in eukaryotic organelles and nuclear-encoded ribosomal DNAs (rDNAs) . The green algae are particularly rich in rDNA group I introns . To better understand the origins and phylogenetic relationships of green algal nuclear-encoded small subunit rDNA group I introns, a secondary structure-based alignment was constructed with available intron sequences and 11 new subgroup ICI and three new subgroup IB3 intron sequences determined from members of the Trebouxiophyceae (common phycobiont components of lichen) and the Ulvophyceae . Phylogenetic analyses using a weighted maximum-parsimony method showed that most group I introns form distinct lineages defined by insertion sites within the SSU rDNA . The comparison of topologies defining the phylogenetic relationships of 12 members of the 1512 group I intron insertion site lineage (position relative to the E . coli SSU rDNA coding region) with that of the host cells (i.e., SSU rDNAs) that contain these introns provided insights into the possible origin, stability, loss, and lateral transfer of ICI group I introns . The phylogenetic data were consistent with a viral origin of the 1512 group I intron in the green algae . This intron appears to have originated, minimally, within the SSU rDNA of the common ancestor of the trebouxiophytes and has subsequently been vertically inherited within this algal lineage with loss of the intron in some taxa . The phylogenetic analyses also suggested that the 1512 intron was laterally transferred among later-diverging trebouxiophytes; these algal taxa may have coexisted in a developing lichen thallus, thus facilitating cell-to-cell contact and the lateral transfer . Comparison of available group I intron sequences from the nuclear-encoded SSU rDNA of phycobiont and mycobiont components of lichens demonstrated that these sequences have independent origins and are not the result of lateral transfer from one component to the other. Infect Immun, 1996 Sep, 64(9), 3905 - 7 Influence of Actinobacillus pleuropneumoniae and its metabolites on porcine alveolar epithelial cells; van de Kerkhof A et al.; The effect of Actinobacillus pleuropneumoniae and its metabolites on the viability of porcine alveolar epithelial cells was studied by using a neutral-red uptake test . Alveolar epithelial cells were obtained from 5-week-old colostrum-deprived pigs . The purity of these cells as assessed by the modified Papanicolaou stain was 90 to 95% . Incubation of these cells with 10(6) CFU of a biotype 1 serotype 1 strain resulted in death of the alveolar epithelial cells within 1.5 h . A cytotoxic effect was also seen when alveolar epithelial cells were incubated with sterile culture supernatants of biotype 1 serotype 1, biotype 1 serotype 10, and biotype 2 serotype 2 strains or with ApxI, ApxII, or ApxIII produced by recombinant Escherichia coli . Incubation of alveolar epithelial cells with a knockout mutant of the biotype 1 serotype 1 parent strain which is unable to secrete Apx toxins or with its supernatant did not result in death of these cells . These results indicate that cytotoxicity is at least in part due to production of Apx toxins. Infect Immun, 1996 Sep, 64(9), 3614 - 9 Immunization of Aotus nancymai with recombinant C terminus of Plasmodium falciparum merozoite surface protein 1 in liposomes and alum adjuvant does not induce protection against a challenge infection; Burghaus PA et al.; Merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is an antimalarial vaccine candidate . The highly conserved 19-kDa C-terminal processing fragment of MSP-1 (MSP-1(19)) is of particular interest since it contains epitopes recognized by monoclonal antibodies which inhibit the invasion of erythrocytes in vitro . The presence of naturally acquired anti-MSP-1(19) antibodies in individuals exposed to malaria has been correlated with reduced morbidity, and immunization with an equivalent recombinant P . yoelii antigen induces substantial protection against this parasite in mice . We have expressed P . falciparum MSP-1(19) in Escherichia coli as a correctly folded protein and immunized Aotus nancymai monkeys by using the protein incorporated into liposomes and adsorbed to alum . After vaccination, the sera from these animals contained anti-MSP-1(19) antibodies, some of which competed for binding to MSP-1(19) with monoclonal antibodies that inhibit parasite invasion of erythrocytes in vitro . However, after challenge with either a homologous or a heterologous strain of parasite, all animals became parasitemic and required treatment . The immunization did not induce protection in this animal model. Infect Immun, 1996 Sep, 64(9), 3555 - 64 Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies; Mechin MC et al.; As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins . Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG . With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located . Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG . In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein . The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A . To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies . Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit . Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A . These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides. J Virol, 1996 Sep, 70(9), 6468 - 73 Proteoglycans secreted by packaging cell lines inhibit retrovirus infection; Le Doux JM et al.; Using a model recombinant retrovirus encoding the Escherichia coli lacZ gene, we have found that medium conditioned with NIH 3T3 cells and packaging cell lines derived from NIH 3T3 cells inhibits infection . Most of the inhibitory activity was greater than 100 kDa and was sensitive to chondroitinase ABC digestion, which is consistent with the inhibitor being a chondroitin sulfate proteoglycan . Proteoglycans secreted by NIH 3T3 cells and purified by anion-exchange chromatography inhibited amphotropic retrovirus infection . Pretreatment of amphotropic retrovirus stocks with chondroitinase ABC boosted the level of transduction efficiency by more than twofold . The implications of these findings with respect to retrovirus-cell interactions and the production of high-titer retroviral stocks are discussed. J Virol, 1996 Sep, 70(9), 6459 - 62 Sequence analysis of the human DNA flanking sites of human immunodeficiency virus type 1 integration; Stevens SW et al.; Human immunodeficiency virus type 1 (HIV-1) tagged with the Escherichia coli supF gene has been used to clone integrated HIV-1 proviruses . Sequence analysis of the 600 to 800 bp of human DNA adjacent to 29 clones revealed a propensity for HIV-1 to integrate near the Alu class of human repetitive elements. J Virol, 1996 Sep, 70(9), 6378 - 83 The 126- and 183-kilodalton proteins of tobacco mosaic virus, and not their common nucleotide sequence, control mosaic symptom formation in tobacco; Bao Y et al.; Nucleotide substitutions at two positions within the open reading frame encoding the 126-kDa protein in the attenuated masked (M) strain of tobacco mosaic tobamovirus (TMV) to those found in the virulent U1-TMV genome led to the induction of near U1-TMV-like symptoms on leaves of Nicotiana tabacum L . cv . Xanthi nn by progeny virus (M . H . Shintaku, S . A . Carter, Y . Bao, and R . S . Nelson, Virology 221:218-225, 1996) . In this study, further site-directed mutations were made at these positions within the M strain cDNA to determine whether the protein or nucleotide sequence directly controlled the symptom phenotype . The protein and not the nucleotide sequence directly controlled the symptom phenotype when amino acid 360 within the 126-kDa protein sequence was altered and likely controlled the symptom phenotype when amino acid 601 was altered . The effects of the substitutions at amino acid position 360 on viral protein accumulation were studied by pulse-labeling proteins in infected protoplasts . Accumulation of the 126- and 183-kDa proteins was less for an attenuated mutant than for two virulent mutants, but the viral movement protein and coat protein accumulated to levels reported to be sufficient for normal systemic symptom development . The size of necrotic local lesions on N . tabacum L . cv . Xanthi NN was negatively correlated with symptom development and accumulation of the 126-kDa protein for these mutants . With reference to this last finding, an explanation of the cause of the differing symptoms induced by these viruses is presented. J Virol, 1996 Sep, 70(9), 6269 - 77 Autoantigens interact with cis-acting elements of rubella virus RNA; Pogue GP et al.; Rubella virus (RV) infections in adult women can be associated with acute and chronic arthritic symptoms . In many autoimmune individuals, antibodies are found targeting endogenous proteins, called autoantigens, contained in ribonucleoprotein complexes (RNPs) . In order to understand the molecular mechanisms involved in the RV-associated pathology, we investigated the nature of cellular factors binding RV RNA and whether such RNPs were recognized by antibodies in infected individuals . Previously, we noted that cellular proteins associated with the RV 5'(+) stem-loop (SL) RNA are recognized by serum with Ro reactivity . To better understand the nature of the autoantigens binding RV cis-acting elements, serum samples from individuals with various autoimmune diseases were tested for their ability to immunoprecipitate RNPs containing labeled RV RNAs . A subset of serum samples recognizing autoantigen La, or Ro and La, immunoprecipitated both the RV 5'(+)SL and 3'(+)SL RNA-protein complexes . Autoantigens binding the RV 5'(+)SL and 3'(+)SL RNAs differed in molecular mass, specificities for respective RNA binding substrates, and sensitivity to alkaline phosphatase treatment . The La autoantigen was found to interact with the RV 5'(+)SL RNA as determined by immunological techniques and binding reactions with mixtures containing recombinant La protein . To test whether there is a correlation between La binding to an RV RNA element and the appearance of an anti-La response, we measured anti-La titers in RV-infected individuals . Significant anti-La activity was detected in approximately one-third of RV-infected individuals 2 years postinfection. J Virol, 1996 Sep, 70(9), 6162 - 8 Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme; Yu L et al.; Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase activities . The enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively . The N-terminal 60-kDa of the D1 subunit (from residues 1 to 545) is an autonomous domain which catalyzes the triphosphatase and guanylyltransferase reactions . Mutations in the D1 subunit that specifically inactivate the guanylyltransferase without affecting the triphosphatase component have been described (P . Cong and S . Shuman, Mol . Cell . Biol . 15:6222-6231, 1995) . In the present study, we identified two alanine-cluster mutations of D1(1-545), R77A-K79A and E192A-E194A, that selectively inactivated the triphosphatase, but not the guanylyltransferase . Concordant mutational inactivation of RNA triphosphatase and nucleoside triphosphatase functions (to approximately 1% of wild-type specific activity) suggests that both gamma-phosphate cleavage reactions occur at a single active site . The R77A-K79A and E192A-E194A mutant enzymes were less active than wild-type D1(1-545) in the capping of triphosphate-terminated poly(A) but could be complemented in vitro by D1(1-545)-K260A, which is inert in nucleotidyl transfer but active in gamma-phosphate cleavage . Whereas wild-type D1(1-545) formed only the standard GpppA cap, the R77A-K79A and E192A-E194A enzymes synthesized an additional dinucleotide, GppppA . This finding illuminates a novel property of the vaccinia virus capping enzyme, the use of triphosphate RNA ends as an acceptor for nucleotidyl transfer when gamma-phosphate cleavage is rate limiting. J Virol, 1996 Sep, 70(9), 6151 - 6 Amino acids essential for RNase H activity of hepadnaviruses are also required for efficient elongation of minus-strand viral DNA; Chen Y et al.; The hepadnavirus P gene contains amino acid sequences which share homology with all known RNases H . In this study, we made four mutants in which single amino acids of the duck hepatitis B virus (DHBV) RNase H region were altered . In two of them, amino acids at locations comprising the putative catalytic site were changed, while the remaining mutants had alterations at amino acids conserved among hepadnaviruses . Transfection of these mutant genomes into permissive cells resulted in synthesis of several discrete viral nucleic acid species, ranging in apparent sizes from approximately 500 to 3,000 bp, numbered I, II, III, IV, and V . While the locations of the species were similar in all mutants, the proportions of the species varied among the mutants . Analysis of the nucleic acid species revealed that they were hybrid molecules of RNA and minus-strand DNA, indicating that the RNase H activity was missing or greatly reduced in these mutants . Primer extension experiments showed that the mutant viruses initiated minus-strand viral DNA synthesis normally . The 3' termini of minus-strand DNA in species II, III, and IV were mapped just downstream of nucleotides 1659, 1220, and 721, respectively . Species V contained essentially full-length minus-strand viral DNA . A parallel amino acid change in the putative catalytic site of the HBV RNase H domain resulted in accumulation of low-molecular-weight hybrid molecules consisting of RNA and minus-strand DNA and similar in size and pattern to those seen with DHBV . These studies demonstrate experimentally the involvement of the C-terminal portion of the P gene in RNase H activity in both DHBV and human hepatitis B virus and indicate that the amino acids essential for RNase H activity of hepadnavirus P protein are also important for the efficient elongation of minus-strand viral DNA. Mamm Genome, 1996 Sep, 7(9), 654 - 6 Construction and analysis of an hn-cDNA library derived from the p-arm of pig chromosome 12; Anderson Dear DV et al.; Our aim is to find unidentified genes on specific pig chromosomes or chromosome fragments . Our approach has involved the construction of a heterogeneous nuclear complementary (hn-c) DNA library of the p-arm of pig Chromosome (Chr) 12, the only pig chromosome present in the pig x hamster hybrid cell line 8990 . Total RNA was extracted from the cells and first-strand synthesis of hn-cDNA carried out with random and oligo dT primers . Pig hn-cDNA was isolated by amplification of first-strand synthesized hn-cDNA with primers specific for Short Interspersed Repeat Elements (SINEs) via the polymerase chain reaction (PCR) . Hn-cDNAs were size selected and cloned in E . coli XL-1 blue cells with PCR-Script as the vector . The library consisted of 6000 clones . Clone inserts were amplified by PCR with vector-specific primers, and randomly picked inserts greater than 600 bp were sequenced . Homology searches were carried out with the FASTA search program on the GenEmbl database . Thirty clones were sequenced, and of these three showed strong homologies to GenEmbl sequences: (1) to sheep, mouse, human, and rat mammary gland factor (MGF); (2) to MLN-50, a gene that is amplified in human familial breast cancer and is present on human Chr 17; the latter is homologous to pig chromosome 12; (3) to a family of unassigned overlapping human ESTs . Of the other sequenced clones, seven were over 80% homologous with pig SINE sequences; three were over 75% homologous to human LINE sequences; six displayed open reading frames over a mean distance equivalent to 50 amino acids, although these showed no significant similarities with sequences in the databases.Using this approach, we have been able to identify several new genes on the p-arm of pig Chr 12 . This is the first report of gene isolation from a library derived from a pig chromosome fragment. J Mol Evol, 1996 Sep, 43(3), 236 - 40 Heterogeneity of the glutathione transferase genes encoding enzymes responsible for insecticide degradation in the housefly; Syvanen M et al.; One of the four glutathione-S-transferases (GST) that is overproduced in the insecticide-resistant Cornell-R strain of the housefly (Musca domestica) produces an activity that degrades the insecticide dimethyl parathion and conjugates glutathione to lindane . In earlier work, it was shown that the resistant Cornell-R carries an amplification, probably a duplication, of one or more of its GST loci and that this amplification is directly related to resistance . Using polymerase chain reaction (PCR) amplification with genomic DNA, multiple copies of the gene encoding the parathion-degrading activity (called MdGst-3) were subcloned from both the ancestral, insecticide-susceptible strain BPM and from the insecticide-resistant Cornell-R . In BPM, three different MdGst-3 genes were identified while in Cornell-R, 12 different MdGst-3 sequences were found that, though closely related to ancestral genes, had diverged by a few nucleotides . This diversity in MdGst-3 genomic sequences in Cornell-R is reflected in the expressed sequences, as sampled through a cDNA bank . Population heterozygosity cannot account for these multiple GST genes . We suggest that selection for resistance to insecticides has resulted in not only amplification of the MdGst-3 genes but also in the divergence of sequence between the amplified copies. Curr Microbiol, 1996 Sep, 33(3), 200 - 8 Role of type 1 fimbriae in the adhesion of Escherichia coli to salivary mucin and secretory immunoglobulin A; Moshier A et al.; Saliva is known to modulate the adhesion of bacteria in the oral cavity . The present work was performed to assess the effect of salivary components on the adhesion of Escherichia coli to a model oral surface . Several genetically engineered E . coli strains were used to examine the role of type 1 fimbriation in the interaction of these strains with salivary components in solution or adsorbed to hydroxyapatite . High (MG1) and low (MG2) molecular weight salivary mucins, and secretory immunoglobulin A (sIgA), were found to interact with the surface of E . coli, and these interactions were independent of the expression of fimbriae or capsule . In contrast, fimbriated strains of E . coli adhered to a greater extent to saliva-coated synthetic hydroxyapatite (HAP) than did nonfimbriated strains . Testing of salivary components separated by gel filtration chromatography revealed that only high-molecular-weight components promoted adhesion of E . coli to HAP . Additional studies found that purified MG2 and sIgA promoted the adhesion of E . coli to HAP . Expression of type 1 fimbriae enhanced ad