J Biol Chem, 1998 Aug 21, 273(34), 21594 - 602 Molecular cloning and characterization of a transcription regulator with homology to GC-binding factor; Reed AL et al.; GC-binding factor (GCF) represses transcription of certain genes and is encoded by a 3.0-kilobase mRNA (Kageyama, R., and Pastan, I . (1989) Cell 59, 815-825) . The GCF cDNA hybridizes to two additional mRNA species, 4.2 and 1.2 kilobases . We have used differential hybridization to identify a cDNA clone (termed GCF2) for the 4 . 2-kilobase mRNA and find that it is highly expressed in HUT-102 cells . The open reading frame consists of 2256 nucleotides and encodes a protein of 752 amino acids with a calculated molecular mass of 83 kilodaltons . GCF2 expressed in vitro using reticulocyte lysates and Escherichia coli migrates as a 160-kilodalton protein in SDS-polyacrylamide gel electrophoresis but has a molecular mass of 83 kilodaltons as determined by mass spectrum analysis . GCF2 binds to epidermal growth factor receptor promoter fragments, and the major binding site is located between nucleotides -249 and -233 . Cotransfection assays show that GCF2 acts to repress transcription from the epidermal growth factor receptor promoter in constructs containing the major GCF2 binding site and not when the site had been mutated . Thus, GCF2 is a newly identified transcriptional repressor with aberrant electrophoretic mobility. J Biol Chem, 1998 Aug 21, 273(34), 21585 - 93 Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III . Direct identification of Lys-212 as the active nucleophilic residue; Ikeda S et al.; The human endonuclease III (hNTH1), a homolog of the Escherichia coli enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA . Its cDNA was cloned, and the full-length enzyme (304 amino acid residues) was expressed as a glutathione S-transferase fusion polypeptide in E . coli . Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH2 terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a {4Fe-4S}cluster, as in E . coli Nth . The enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex . The protein had a molar extinction coefficient of 5.0 x 10(4) and a pI of 10 . With the DHU-containing oligonucleotide duplex as substrate, the Km was 47 nM, and kcat was approximately 0.6/min, independent of whether DHU paired with G or A . The enzyme carries out beta-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal . The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptide-oligonucleotide adduct . Furthermore, replacing Lys-212 with Gln inactivated the enzyme . However, replacement with Arg-212 yielded an active enzyme with about 85-fold lower catalytic specificity than the wild-type protein . DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5'-boundary) in the complementary strand . Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm. J Biol Chem, 1998 Aug 21, 273(34), 21463 - 72 Kinetic evidence that a radical transfer pathway in protein R2 of mouse ribonucleotide reductase is involved in generation of the tyrosyl free radical; Schmidt PP et al.; Class I ribonucleotide reductases consist of two subunits, R1 and R2 . The active site is located in R1; active R2 contains a diferric center and a tyrosyl free radical (Tyr.), both essential for enzymatic activity . The proposed mechanism for the enzymatic reaction includes the transport of a reducing equivalent, i.e . electron or hydrogen radical, across a 35-A distance between Tyr . in R2 and the active site in R1, which are connected by a hydrogen-bonded chain of conserved, catalytically essential amino acid residues . Asp266 and Trp103 in mouse R2 are part of this radical transfer pathway . The diferric/Tyr . site in R2 is reconstituted spontaneously by mixing iron-free apoR2 with Fe(II) and O2 . The reconstitution reaction requires the delivery of an external reducing equivalent to form the diferric/Tyr . site . Reconstitution kinetics were investigated in mouse apo-wild type R2 and the three mutants D266A, W103Y, and W103F by rapid freeze-quench electron paramagnetic resonance with >/=4 Fe(II)/R2 at various reaction temperatures . The kinetics of Tyr . formation in D266A and W103Y is on average 20 times slower than in wild type R2 . More strikingly, Tyr . formation is completely suppressed in W103F . No change in the reconstitution kinetics was found starting from Fe(II)-preloaded proteins, which shows that the mutations do not affect the rate of iron binding . Our results are consistent with a reaction mechanism using Asp266 and Trp103 for delivery of the external reducing equivalent . Further, the results with W103F suggest that an intact hydrogen-bonded chain is crucial for the reaction, indicating that the external reducing equivalent is a H . Finally, the formation of Tyr . is not the slowest step of the reaction as it is in Escherichia coli R2, consistent with a stronger interaction between Tyr . and the iron center in mouse R2 . A new electron paramagnetic resonance visible intermediate named mouse X, strikingly similar to species X found in E . coli R2, was detected only in small amounts under certain conditions . We propose that it may be an intermediate in a side reaction leading to a diferric center without forming the neighboring Tyr. Virology, 1998 Aug 15, 248(1), 83 - 94 Complementation of a replication-defective mutant of equine herpesvirus type 1 by a cell line expressing the immediate-early protein; Garko-Buczynski KA et al.; Equine herpesvirus type 1 (EHV-1) possesses a sole, diploid immediate-early (IE) gene that encodes a major regulatory protein of 1487 amino acids capable of modulating expression of both early and late EHV-1 promoters and capable of trans-repressing its own promoter . In this study, a rabbit kidney cell line (IE13.1) that constitutively expresses the EHV-1 IE protein was generated by cotransfection of rabbit kidney (RK-13) cells with the viral IE gene and a neomycin resistance marker . The IE protein expressed by this cell line was shown (1) to be expressed by and to localize to the nucleus of virtually all cells as demonstrated by indirect immunofluorescence, (2) to be the full-size IE polypeptide as judged by Western immunoblot analyses with an anti-IE protein-specific antibody, and (3) to be functional as shown by the transactivation of two representative EHV-1 early promoters linked to the chloramphenicol acetyltransferase reporter gene in transient transfection assays . The IE13.1 cell line was able to complement a recombinant virus in which both copies of the IE gene were replaced by insertion of the Escherichia coli lacZ gene . This IE deletion mutant, designated KyADeltaIE, was not able to replicate in equine, rabbit, or mouse cells but was capable of replication in the IE13.1 cells that provided the IE protein in trans . Rescue of the KyADeltaIE virus was achieved by recombination with a marker plasmid that harbors the wild-type IE gene, and the rescued virus (KyADeltaIER) was able to grow on noncomplementary cells . Overall, these results offer direct evidence that the IE gene is essential for EHV-1 replication and provide reagents useful for the analysis of IE protein function . Virology, 1998 Aug 15, 248(1), 6 - 11 Characterization of the nucleic acid binding properties of tomato spotted wilt virus nucleocapsid protein; Richmond KE et al.; Tomato spotted wilt tospovirus (TSWV) is the type member of the plant-infecting viruses of the genus Tospovirus in the family Bunyaviridae . The three TSWV RNAs are encapsidated with nucleocapsid (N) protein to form ribonucleoprotein (RNP) which serves as the template for viral transcription and replication . Regions of the open reading frame coding for the N protein on the small (S) RNA were subcloned into pET protein expression vectors and expressed in Escherichia coli BL21 (DE3) cells . Full-length N, N amino and carboxy halves, and two N carboxy-terminal regions were expressed and isolated by metal chelate affinity chromatography . The N protein, both of its halves and the extreme carboxy-terminal region, bound cooperatively and irrespective of sequence to radiolabeled single-stranded RNA produced by runoff transcription of clones of either TSWV S RNA or cowpea chlorotic mottle virus RNA3 . N protein did not bind to radiolabeled double-stranded TSWV RNA . The density of the synthetic RNase-sensitive N protein-RNA complexes was 1.32 g/ml, similar to the density of authentic Bunyaviridae RNPs . These studies are the first to indicate differences in the nucleic acid binding abilities of Tospovirus and Hantavirus nucleocapsid proteins, the only characterized nucleocapsid proteins of the family Bunyaviridae . Biol Chem, 1998 Jul, 379(7), 857 - 66 Efficient in vitro translational termination in Escherichia coli is constrained by the orientations of the release factor, stop signal and peptidyl-tRNA within the termination complex; McCaughan KK et al.; There have been contrasting reports of whether the positioning of a translational stop signal immediately after a start codon in a single oligonucleotide can act as a model template to support efficient in vitro termination . This paradox stimulated this study of what determines the constraints on the positioning of the components in the termination complex . The mini mRNA, AUGUGAA, was unable to support efficient in vitro termination in contrast to separate AUG/UGA(A) codons, unless the ribosomal interaction of the stop signal with the decoding factor, release factor 2, was stimulated with ethanol or with nucleotide-free release factor 3, or by using (L11-)-ribosomes which have a higher affinity for release factor 2, or unless the fMet-tRNA was first bound to 30S subunits independently of the mini mRNA . An additional triplet stop codon could restore activity of the mini mRNA, indicating that its recognition was not sterically restrained by the stop signal already within it . This suggests that in an initiation complex an adjoining start/stop signal is not positioned to support efficient decoding by release factor unless it is separated from the start codon . Site-directed crosslinking from mRNAs to components of the termination complex has shown that mRNA elements like the Shine-Dalgarno sequence and the codon preceding the stop signal can affect the crosslinking to release factor, and presumably the orientation of the signal to the factor. Biol Chem, 1998 Jul, 379(7), 807 - 18 Small angle scattering in ribosomal structure research: localization of the messenger RNA within ribosomal elongation states; Junemann R et al.; Besides EM and biochemical studies small angle scattering (SAS) examinations have contributed significantly to our current knowledge about the ribosomal structure . SAS does not only allow the validation of competing models but permits independent model building . However, the major contribution of SAS to ribosomal structure research derived from its ability to reveal the spatial distribution of the individual ribosomal components (57 in the E . coli ribosome) within the ribosomal structure . More recently, an improved scattering method (proton-spin contrast variation) made it possible also to address the question of mapping functional ligands in defined ribosomal elongation states . Here, we review the contributions of SAS to the current understanding of the ribosome . Furthermore we present the direct localization of a small mRNA fragment within 70S elongation complexes and describe its movement upon the translocation reaction . The successful mapping of this fragment comprising only about 0.6% of the total mass of the complex proves that proton-spin contrast-variation is a powerful tool in modern ribosome research. Mol Cell Endocrinol, 1998 Apr 30, 139(1-2), 199 - 207 Use of recombinant herpes simplex virus type 1 vectors for gene transfer into tumour and normal anterior pituitary cells; Goya RG et al.; In this paper we demonstrate the use of recombinant viral vectors derived from herpes simplex virus type 1 (HSV1) to transfer reporter genes in vitro into rat anterior pituitary cells grown in primary cultures and the anterior pituitary tumour cell lines GH3 and AtT20 . The three vectors used were, tsK/beta-galactosidase (beta-gal), tsK/CRH and tsK/TIMP, the corresponding transgene products respectively being E . coli beta-gal, pre-procorticotropin releasing hormone (ppCRH), and the chimeric protein TIMP/Thy1 (tissue inhibitor of metalloproteinases (TIMP)/linked to the carboxy terminus of Thy1 which confers the addition of a glycolipid glycosyl-phosphatidylinositol anchor in the ER) . Double labelling immunofluorescence experiments to detect reporter proteins and transduced cell types indicated that the three vectors could transfer and express the reporter genes in normal and tumour anterior pituitary cells . Virus infection of pituitary cells was characterised, and it was shown that infection with tsK/beta-gal at multiplicities of infection (MOI)=10, 100% of tumour and non-endocrine anterior pituitary cells expressed beta-gal, whereas 75% endocrine anterior pituitary cells expressed the transgene . Long-term expression studies after infection with tsK/beta-gal indicated that anterior pituitary cells in primary cultures expressed the transgene for significant longer periods than tumour anterior pituitary cells . Growth arrest by serum starvation markedly decreased the frequency of transgene expression in anterior pituitary cells following infection with tsK/beta-gal . Transgenic products expressed from tsK were targeted to their correct intracellular domain in both anterior pituitary cells in primary cultures and in pituitary tumour cell lines . We conclude that transgenes can be delivered into anterior pituitary cells in primary culture and pituitary tumour cell lines using tsK derived HSV1 vectors . The prospect of employing viral vectors to transfer genes into endocrine cells opens up the potential exploration of various molecular aspects of pituitary cell function both in vitro and in vivo, as well as the use of gene transfer into the pituitary for potentially therapeutic applications, such as the treatment of pituitary tumours. Arthritis Rheum, 1998 Aug, 41(8), 1505 - 10 Autoantibodies to DEK oncoprotein in a patient with systemic lupus erythematosus and sarcoidosis; Dong X et al.; A patient was identified with an unusual autoimmune syndrome consisting of systemic lupus erythematosus and sarcoidosis . Her serum contained extremely high levels of autoantibodies to the DEK protooncogene product . The patient's serum was used to clone a dek complementary DNA, which was expressed as a histidine-tagged fusion protein in Escherichia coli . Using affinity-purified recombinant DEK protein, anti-DEK autoantibodies were found in the patient's serum at a titer of 1:10(6) by enzyme-linked immunosorbent assay (ELISA) . Longitudinal studies revealed marked variations in anti-DEK autoantibody levels over time . Although it has been suggested that anti-DEK autoantibodies are a marker for pauciarticular juvenile rheumatoid arthritis with iridocyclitis, the present data suggest that they may be associated with other disease subsets as well . The quantitative ELISA technique will be useful for defining these subsets further and for examining the relationship between anti-DEK titers and disease activity. Acta Vet Hung, 1998, 46(1), 95 - 100 Influence of Escherichia coli endotoxin induced fever on the pharmacokinetics and dosage regimen of oxytetracycline in cross-bred calves; Singh RP et al.; The pharmacokinetics and dosage regimen of oxytetracycline were determined in healthy and febrile cross-bred calves following its single intravenous administration (10 mg kg-1) . Fever was induced by a single intravenous injection of E . coli endotoxin (1 microgram kg-1 i.v.) . The elimination half-life (t1/2 beta) and the apparent volume of distribution {vd(area)} were slightly increased in febrile calves, as compared to healthy animals . The values of t1/2 beta and Vd(area) were 3.22 +/- 0.20 h and 0.49 +/- 0.02 L . kg-1 in healthy calves and 4.06 +/- 0.32 h and 0.70 +/- 0.09 L kg-1, respectively, in febrile calves . An intravenous dosage regimen suitable for maintaining the minimum therapeutic plasma concentration of > or = 2 micrograms/ml in febrile animals would be 10 mg kg-1 repeated at 12-h intervals. Vox Sang, 1998, 74 Suppl 2, 233 - 41 Modified hemoglobin-based blood substitutes: crosslinked, recombinant and encapsulated hemoglobin; Chang TM; Native hemoglobin in the form of stroma-free hemoglobin cannot be used as blood substitute . Hemoglobin has to be modified either molecularly or encapsulated . First generation molecularly modified ultrapure hemoglobins are now in clinical trial--some in Phase III . There are a number of these . Polyhemoglobin is formed by crosslinking hemoglobin molecules intermolecularly and intramolecularly . A crosslinked single hemoglobin molecule is formed by crosslinking hemoglobin intramolecularly . Recombinant hemoglobin from E.coli is formed by fusion of the subunits of each hemoglobin molecule . Conjugated hemoglobin is formed by crosslinking each hemoglobin molecule to soluble polymers . A second generation system formed by crosslinking hemoglobin-superoxide dismutase-catalase is being developed . A third generation hemoglobin-based blood substitute is based on microencapsulated hemoglobin, artificial red blood cells, that more closely resemble a complete red blood cell. Biotechnol Annu Rev, 1996, 2, 391 - 408 Novel molecular biology approaches to acellular vaccines; Rappuoli R et al.; Bacterial toxins are commonly detoxified by chemical treatment in order to use them in human vaccines . We have used site-directed mutagenesis of toxin genes to obtain bacteria that produce naturally nontoxic mutants of bacterial toxins, such as pertussis toxin (PT), cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) . Genetically detoxified PT showed a superior safety and immunogenicity in animal models, phase I and phase II clinical trials, and a superior protective efficacy in the early and late stage of a phase III efficacy trial, proving in a definitive and extensive way that genetic detoxification of bacterial toxins can, and should, replace chemical treatment . The results obtained with genetically inactivated LT and CT indicate that genetic detoxification of bacterial toxins can be used not only to produce vaccines for systemic immunization that are superior to the ones produced by conventional technologies, but suggest that these type of molecules may be the prototype molecules for the design and construction of innovative vaccines with a totally new design, such as mucosally delivered preventive and therapeutic vaccines. Biotechnol Annu Rev, 1996, 2, 259 - 79 ADPglucose pyrophosphorylase: basic science and applications in biotechnology; Preiss J; The enzymatic reactions of bacterial glycogen and plant starch synthesis are similar and some of the properties of the biosynthetic enzymes are compared . Regulation occurs at the synthesis of ADPglucose and in almost all cases, ADPglucose pyrophosphorylase, is allosterically activated about 10- to over 40-fold by glycolytic intermediates and inhibited by AMP, ADP or Pi . The activator specificity of the ADPglucose pyrophosphorylase varies with respect to the source of enzyme and can be correlated to the major assimilation pathway occurring in the organism . For example, ADPglucose pyrophosphorylases from plants and other oxygenic photosynthetic organisms are activated by 3-phosphoglycerate . Organisms using glycolysis for carbon assimilation have ADPglucose pyrophosphorylases with fructose-1,6-bis-phosphate as the major activator . Chemical modification and site-directed mutagenesis studies that have determined the activator binding sites for some enzymes are described . The structural genes of Escherichia coli ADPglucose pyrophosphorylase allosteric mutants which no longer require activator for activity have been isolated . Transformation of plant systems with an allosteric bacterial mutant gene (but not with the wild-type gene) increases their starch content . Transformed potato tubers can have 25-60% more starch than the normal tuber indicating the importance of allosteric regulation of ADPglucose synthesis . The increase of a normal plant product by transformation of the plant with a gene encoding the rate-limiting enzyme in starch synthesis is an important biotechnological advance and suggests the possibilities of changing starch composition (extent of branching and chain sizes) via transformation with the starch synthase and branching enzyme genes. Biochem Biophys Res Commun, 1998 Jul 30, 248(3), 920 - 6 Ion pairs involved in maintaining a thermostable structure of glutamate dehydrogenase from a hyperthermophilic archaeon; Rahman RN et al.; Intersubunit ion pairs are considered to be involved for maintaining a stable structure of the glutamate dehydrogenase (GDH) from hyperthermophiles . In order to demonstrate an effect of intersubunit ion pairs on the structural stability, two kinds of mutation (T138E, Thr at position 138 was replaced by Glu; E158Q, Glu at position 158 was replaced by Gln) which add and remove ion pairs, respectively, were introduced into Pk-gdhA gene encoding GDH from Pyrococcus kodakaraensis KOD1 . Addition of one ion pair (Pk-GDHA-T138E) increased the optimum temperature and thermostability . In contrast, Pk-GDH-E158Q showed lower optimum temperature and less thermostability than wild type GDH . Structure analysis of GDHs was performed by circular dichroism (CD) and indicated that all recombinant enzymes (Pk-GDH, Pk-GDH-T138E, Pk-GDH-E158Q) possess different structures from that of natural GDH . Upon heat treatment (60 degrees C, 2 h), the structures of Pk-GDH and Pk-GDH-T138E were converted to another form close to the natural structure . However, no structural conversion by heat treatment was observed in Pk-GDH-E158Q . These results indicate that intersubunit ion pairs play an important role in forming thermostable structure of Pk-GDH. Biochem Biophys Res Commun, 1998 Jul 30, 248(3), 608 - 12 cDNA cloning and genomic organization of peroxisome proliferator-inducible long-chain acyl-CoA hydrolase from rat liver cytosol; Yamada J et al.; The cDNA for a peroxisome proliferator-inducible long-chain acyl-CoA hydrolase from rat liver cytosol, referred to as rLACH2, was isolated and its genomic structure was determined . The cDNA encoded a 419-amino-acid polypeptide with a calculated molecular weight of 46,011 . Sequence analysis identified an active-site serine motif (Gly-x-Ser-x-Gly) common to carboxylesterases and lipases . When expressed in Escherichia coli, the cDNA directed expression of a protein immunoreactive to an anti-rLACH2 antibody with a molecular mass of 47 kDa, identical to that of purified rLACH2 . Northern blot analysis showed marked induction of rLACH2 mRNA in the liver after feeding rats with di(2-ethylhexyl)phthalate, a peroxisome proliferator . The rLACH2 gene spanned about 19 kb and comprised 3 exons, the intron/exon boundaries of which were consistent with the donor/acceptor splice rule . A putative peroxisome proliferator response element (AGGTCATGGTTCA) was identified in the 5'-flanking region, suggesting the involvement of peroxisome proliferator-activated receptors in the regulation of rLACH2 gene expression. Biochem Biophys Res Commun, 1998 Jul 30, 248(3), 591 - 6 The NADP-reducing hydrogenase of Desulfovibrio fructosovorans: evidence for a native complex with hydrogen-dependent methyl-viologen-reducing activity; de Luca G et al.; The NADP-reducing hydrogenase of Desulfovibrio fructosovorans represents a novel class of {Fe} hydrogenases which is encoded by the well-characterized hndABCD operon containing the genes hndA, hndB, hndC, and hndD . Expression of this operon, monitored by measuring the NADP-reducing activity, was found to be maximum during the exponential phase of growth on fructose and then decreased when the concentration of the carbon and energy source became limiting . The optimum pH for the H2-driven NADP reduction was 8, and the apparent K(m) and Vmax were determined to be 0.09 mM and 13 x 10(-3) u/mg, respectively . Heterologous expression of the hnd genes in Escherichia coli was carried out to raise antisera against the different subunits of the NADP-reducing hydrogenase . The antisera were used to detect the four subunits in cell extract of D . fructosovorans after separation by SDS- and native PAGE . The four subunits of the NADP-reducing hydrogenase were demonstrated to be associated in a complex which exhibited H2-driven methyl viologen reduction . Furthermore, on native gel, a form lacking HndD, with no hydrogen-dependent methyl viologen reductase activity was also shown to be present in D . fructosovorans. Biochem Biophys Res Commun, 1998 Jul 30, 248(3), 548 - 53 Merlin differs from moesin in binding to F-actin and in its intra- and intermolecular interactions; Huang L et al.; The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a protein with homology to the cell membrane/F-actin linking proteins, moesin, ezrin and radixin . Unlike these closely related proteins, merlin lacks a C-terminal F-actin binding site detectable by actin blot overlays, and the GFP-tagged merlin C-terminal domain co-distributes with neither stress fibers nor cortical actin in NIH3T3 cells . Merlin also differs from the other three proteins in its inter- and intramolecular domain interactions, as shown by in vitro binding and yeast two-hybrid assays . As is true for ezrin, moesin and radixin, the N- and C-terminal domains of merlin type 1 bind to each other . However, full-length merlin and its N- and C-terminal domains, as well as the C-terminal domain of ezrin, interact with other full-length merlin type 1 molecules, and its C-terminal domain interacts with itself . Merlin 1 function in cells may thus depend on intra- and intermolecular interactions and their modulation, which include interactions with other members of this protein family. Jpn J Cancer Res, 1998 Jun, 89(6), 641 - 8 Identification of glutathione S-transferase p-1 as the class pi form dominantly expressed in mouse hepatic adenomas; Ookawa K et al.; To clarify which of the two genes for pi class glutathione S-transferases (GSTs) (p-1 and p-2) is dominantly expressed in mouse hepatic adenomas, the relative mRNA levels were examined by means of the reverse transcription-polymerase chain reaction (RT-PCR) . Hepatic adenomas were induced in male and female B6C3F1 mice by diethylnitrosamine treatment . Northern blot analysis revealed that pi class mRNA levels were decreased in adenomas of male mice, but increased in those of females, with reference to the respective surrounding non-adenoma tissues . In contrast to the marked sex difference in surrounding tissues, pi class GST mRNA levels in adenomas were almost the same in both males and females . To evaluate p-1 and p-2 mRNA levels separately, the products of RT-PCR employing primers common for both cDNAs were digested with the endonuclease BanI (specific for p-2) and then resolved by electrophoresis . The p-1 mRNA was thus found to be dominant in adenomas of both female and male mice . The p-2 mRNA levels were increased in the lesions as compared with those in the surrounding non-adenoma tissues . Recombinant p-1 and p-2 proteins were expressed in Escherichia coli . Unlike p-1, the p-2 protein did not show any significant activity towards 1-chloro-2,4-dinitrobenzene and did not bind to S-hexylglutathione-Sepharose despite immunological cross-reactivity . The dominant pi class form in adenomas could also be identified as p-1 by its binding to S-hexylglutathione-Sepharose . Single radial immunodiffusion analyses confirmed that the p-1 protein levels were in line with the mRNA findings, i.e., 1.9+/-0.3 mg/g adenoma as compared to 6.5+/-1.2 mg/g non-adenoma tissue for males and 2.2+/-0.6 mg/g as compared to 0.7+/-0.2 mg/g for females . The results thus indicated that the change of pi class forms in adenomas is caused mainly by alteration in the p-1 level and the contribution of p-2 is minimal. Br J Cancer, 1998 Aug, 78(3), 312 - 20 Hydroquinones cause specific mutations and lead to cellular transformation and in vivo tumorigenesis; Joseph P et al.; Benzo(a)pyrene and benzene are human carcinogens . The metabolic activation of these compounds into ultimate mutagenic and carcinogenic metabolites is prerequisite for their carcinogenic effects . In this report, the mutagenicity and carcinogenicity of hydroquinones of benzo(a)pyrene and benzene was investigated to address two important questions: (1) do hydroquinones contribute to benzo(a)pyrene and benzene carcinogenicity; and (2) how safe is it to increase the levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), a key enzyme in the generation of hydroquinone . The supF tRNA of the plasmid pSP189 was used as the mutational target in a cell-free and Chinese hamster ovary (CHO) cell system to study hydroquinone mutagenicity . RNA and protein-free pSP189 DNA was incubated in a cell-free system with benzo(a)pyrene-3,6-quinone and purified NQO1 or with benzoquinone hydroquinone to generate adducted pSP189 DNA . The adducted pSP189 DNA was transfected in human embryonic kidney cells Ad293 . In the CHO cell system, monolayer cultures of CHO cells and CHO cells overexpressing NQO1 or P450 reductase were transfected with pSP189 vector DNA, treated with benzo(a)pyrene-3,6-quinone . The adducted and replicated pSP189 DNA was rescued from transfected Ad293 (cell-free system) and CHO cells (CHO cell system), digested with the restriction enzyme Dpn1 to remove unreplicated DNA followed by transformation in Escherichia coli MBM7070 . The mutant colonies {white/pale blue on 5-bromo-4-chloro-3-indolyl beta-D-galactoside/isopropyl beta-D-thiogalactoside (X-gal/IPTG) plates} were selected, regrown and analysed by DNA sequencing . Mutagenesis experiments demonstrated that hydroquinones cause sequence-specific frameshift mutations involving deletion of a single cytosine from the DNA sequence 5'-172-CCCCC176-3' or a single guanosine from the complementary strand sequence 5'-GGGGG-3' in the supF tRNA gene . This mutation was specific to the hydroquinones, as it was not observed with quinones and other components of the redox cycling (semiquinones and reactive oxygen species) . Exposure of BALBc/3T3 cells to hydroquinones resulted in cellular transformation leading to the loss of contact inhibition and regulation of cell growth . The transformation efficiency of BALBc/3T3 cells exposed to hydroquinones was significantly increased by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), indicating that hydroquinones are excellent initiators that require additional co-carcinogens or promoters to exert an effect . The hydroquinone + TPA as well as hydroquinone-transformed BALBc/3T3 cells, when injected s.c . in severe combined immunodeficient (SCID) mice, produced tumours at 100% frequency . These results establish that hydroquinones lead to mutagenicity and carcinogenicity. Biol Pharm Bull, 1998 Jul, 21(7), 667 - 72 Quinolone-resistant mutations of DNA gyrase increase sensitivity to acriflavine; Funatsuki K et al.; DNA gyrases were constructed to possess the quinolone-resistant (D87N in GyrA or K447E in GyrB) and acrB (S759R-R760C in GyrB) mutations and their sensitivities to acriflavine and oxolinic acid were examined . Both quinolone-resistant mutations in GyrA and GyrB increased acriflavine sensitivities in the supercoiling assay irrespective of the co-presence of the acrB mutation . In the DNA binding assay, however, the hypersensitvity caused by the GyrB (K447E) mutation was observed only in the co-presence of the acrB mutation; the presence of the acrB mutation, which not affecting acriflavine sensitivity, reduces the extent of DNA binding, as reported previously . Thus, the quinolone-resistant mutation site in GyrB is likely to be involved in DNA binding which is not detectable in acrB+ gyrase . Furthermore, oxolinic acid was found to enhance DNA binding of the gyrase having GyrB (acrB-K447E), supporting a recent proposal that quinolone binding to the DNA-gyrase complex does not require DNA breakage. Biol Pharm Bull, 1998 Jul, 21(7), 657 - 61 Alteration in levels of unsaturated fatty acids in mutants of Escherichia coli defective in DNA replication; Suzuki E et al.; We previously reported that mutations in the dnaA gene which encodes the initiator of chromosomal DNA replication in Escherichia coli caused an alteration in the levels of unsaturated fatty acids of phospholipids in membranes . In this study, we examined fatty acid compositions in other mutants which are defective in DNA replication . As in the case of temperature-sensitive dnaA mutants, temperature-sensitive dnaC and dnaE mutants, which have defects in initiation and elongation, respectively, of DNA replication showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) compared with the wild-type strain, especially at high temperatures . On the other hand, temperature-sensitive mutants defective in cellular processes other than DNA replication, such as RNA synthesis and cell division, did not show a lower level of unsaturation of fatty acids compared with the wild-type strain . These results suggest that the inhibition of DNA replication causes a lower level of unsaturation of fatty acids in Escherichia coli cells. Vopr Virusol, 1998 May-Jun, 43(3), 124 - 6 {Presence of HTLV-I antibodies in patients with sexually transmitted diseases}; Sankov MN et al.; The prevalence of human T-lymphotropic type I virus (HTLV-I) among patients with sexually-transmitted diseases is studied in Russia . Primary screening of antibodies to HTLV-I in the sera was carried out by enzyme immunoassay with recombinant gag and env HTLV-I-specific antigens synthesized in Escherichia coli . For secondary screening, Serodia HTLV-I and Vironostika Microelisa System kits were used . None of the 1271 serum samples collected in the towns of Perm and Ekaterinburg was HTLV-I-positive . This indicates a lower, in comparison with other European countries, level of infection with this virus, at least in this region of Russia . Testing of sera from 79 HIV-infected patients revealed none infected with HTLV-I. J Pediatr Gastroenterol Nutr, 1998 Aug, 27(2), 166 - 71 Human colostrum contains IgA antibodies reactive to enteropathogenic Escherichia coli virulence-associated proteins: intimin, BfpA, EspA, and EspB; Loureiro I et al.; BACKGROUND: In Brazil, enteropathogenic Escherichia coli diarrhoea is endemic among infants born into low economic levels, and it is one of the main causes of morbidity and mortality in this group . Binding of enteropathogenic E . coli to the brush border mucosa triggers a cascade of transmembrane and intracellular signals, causing cytoskeletal reorganization and formation of a specific lesion, termed the attaching and effacing lesion . Several enteropathogenic E . coli gene products have been implicated in formation of attaching and effacing lesions . Evaluation of pathogen-specific protective factors shows that breast feeding is effective against enteropathogenic E . coli infection . To investigate the nature of the protection, defatted colostrum and secretory immunoglobulin A obtained from mothers living in Sao Paulo were investigated for the ability to recognise selected enteropathogenic E . coli-associated virulence factors . METHODS: Western blot analysis was used to investigate the IgA repertoire in pooled colostrum that is reactive with specific enteropathogenic E . coli proteins . Whole enteropathogenic E . coli bacterial cell extracts, nonpathogenic E . coli strains overexpressing specific virulence factors, and purified polypeptides were used as antigen sources in this study . RESULTS: Reaction of the colostrum samples in Western blots of whole bacterial cell extracts and selected purified enteropathogenic E . coli proteins showed that they contained a secretory immunoglobulin A reactive with all the virulence-associated proteins studied . CONCLUSION: These results suggest that maternal antibodies may protect infants from enteropathogenic E . coli infection by interfering with adherence processes (anti-intimin and anti-bundle-forming pili antibodies) and cell signaling (anti-enteropathogenic Escherichia coli-secreted protein A and B antibodies. Biofizika, 1998 May-Jun, 43(3), 470 - 4 {Role of Arc-system for the control of synthesis of respiratory enzymes in regulation of K+-transporting system in glycolysing Escherichia coli}; Trchunian AA et al.; It is shown that the uptake of K+ ions by anaerobically grown E . coli bacteria, which perform glycolysis with the production of H2 in exchange for H+ ions, which are extruded from the cells, occurs with a fixed stoichiometry of N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes (2H+/K+) . This process is observed in the precursor strain and arcB mutant, and is destroyed in arcA mutant . The K(+)-uptake by the latter mutant, which proceeds with a moderate affinity (KM 2.0 mM) and is triggered by a positive shock, is sensitive to external osmotonicity . The K(+)-uptake by the arcA mutant is also inhibited by N,N'-dicyclohexylcarbodiimide and has a variable stoichiometry of N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes and does not significantly change in the presence of valinomycin and with varying temperature, whereas the intracellular activity of K+ ions is lower . The Arc-system for the control of synthesis of respiratory enzymes in E . coli participates in the regulation of the K(+)-transporting TrkA system, which directly interacts with F0F1 H-ATPasa; this system in the arcA mutant operates independent of F0F1 and interacts with the latter by mediation of the proton gradient. Biofizika, 1998 May-Jun, 43(3), 433 - 7 {Conformational analysis of complexes of RNA polymerase from Escherichia coli with DNA}; Kamzolova SG et al.; Conformational behaviour of T2 DNA during its complex formation with E.coli RNA polymerase was studied by spin label technique . T2 DNA was selectively modified at its readily melting AT-rich sites by bromoacetooxypiperidine-1-oxyl-radical . Specific conformational changes are induced in DNA structure by RNA polymerase attachment . The changes are observed under the conditions when open transcriptionally competent complexes are formed . The readily melting sites of T2 DNA were shown to be involved in the formation of functionally active promoters. Mol Cell, 1998 Jul, 2(1), 55 - 64 Crucial role of the RNA:DNA hybrid in the processivity of transcription; Sidorenkov I et al.; We present an approach for studying the role of complementary nucleic acid interactions in transcription elongation by E . coli RNA polymerase (RNAP) . The method involves in vitro reconstitution of a catalytically active elongation complex (EC) by the addition of RNAP to a single-strand DNA oligonucleotide containing the preannealed RNA primer, followed by incorporation of the complementary nontemplate DNA oligonucleotide . In all parameters tested, the reconstituted complex is indistinguishable from normal EC obtained by promoter-specific initiation . Using RNA primers of different lengths, which were fully or partially complementary to the DNA, we determined the minimal transcript length and the degree of its template pairing that is required to stabilize protein/ nucleic acid interactions in EC to the high level characteristic of normal transcription . Our data show that a hybrid at least 9 nt long, formed between the template DNA and 3'-proximal RNA transcript, is necessary for the high processivity of EC during RNA chain elongation. Mol Microbiol, 1998 Jul, 29(1), 359 - 67 TolB protein of Escherichia coli K-12 interacts with the outer membrane peptidoglycan-associated proteins Pal, Lpp and OmpA; Clavel T et al.; The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity . Transmembrane domains of TolQ, TolR and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane . TolB and the central domain of TolA interact in vitro with the outer membrane porins . In this study, both genetic and biochemical analyses were carried out to analyse the links between TolB, Pal and other components of the cell envelope . It was shown that TolB could be cross-linked in vivo with Pal, OmpA and Lpp, while Pal was associated with TolB and OmpA . The isolation of pal and tolB mutants disrupting some interactions between these proteins represents at first approach to characterizing the residues contributing to the interactions . We propose that TolB and Pal are part of a multiprotein complex that links the peptidoglycan to the outer membrane . The Tol-Pal proteins might form transenvelope complexes that bring the two membranes into close proximity and help some outer membrane components to reach their final destination. Mol Microbiol, 1998 Jul, 29(1), 331 - 41 Coupled structure changes of SecA and SecG revealed by the synthetic lethality of the secAcsR11 and delta secG::kan double mutant; Suzuki H et al.; An Escherichia coli strain carrying either the secAcsR11 or delta secG::kan mutation is unable to grow at low temperature owing to cold-sensitive protein translocation but grows normally at 37 degree C . However, introduction of the two mutations into the same cells caused a severe defect in protein translocation and the cells were unable to grow at any temperature examined, indicating that secG is essential for the secAcsR11 mutant . The mutant SecA (csSecA) was found to possess a single amino acid substitution in the precursor-binding region and was defective in the interaction with the precursor protein . Furthermore, the membrane insertion of SecA and the membrane topology inversion of SecG, both of which took place upon the initiation of protein translocation, were significantly retarded even at 37 degree C, when csSecA was used instead of the wild-type SecA . The insertion of the wild-type SecA was also significantly defective when SecG-depleted membrane vesicles were used in place of SecG-containing ones . No insertion of csSecA occurred into SecG-depleted membrane vesicles . Examination of in vitro protein translocation at 37 degree C revealed that SecG is essential for csSecA-dependent protein translocation . We conclude that SecG and SecA undergo a coupled structure change, that is critical for efficient protein translocation. Mol Mar Biol Biotechnol, 1998 Sep, 7(3), 173 - 80 Usefulness of the medaka beta-actin promoter investigated using a mutant GFP reporter gene in transgenic medaka (Oryzias latipes); Hamada K et al.; The activity of the medaka beta-actin promoter as a ubiquitous expression vector in transgenic medaka was examined using complementary DNA of the green fluorescent protein (GFP) . Plasmid pOBA-GFP contained both the medaka beta-actin promoter and cDNA of the wild-type GFP, while pOBA-hGFP contained the medaka beta-actin promoter and cDNA of the mutant GFP in which serine was substituted for threonine at position 65 and codon usage was humanized to promote translation in vertebrate cells . The ApaI-SmaI fragment of both plasmids was microinjected into the nuclei of oocytes or the cytoplasm of embryos at the one-cell stage . The gene expression was detected, using a fluorescent stereomicroscope, from early stages of development to 1 week after hatching . The expression of the wild-type GFP was detected in early embryos, in the yolk sac and in small portions of the muscle and epidermis . This expression pattern was similar to that of the Escherichia coli beta-galactosidase reporter gene (lacZ), driven by the medaka beta-actin promoter, which was examined in our previous studies . The mutant GFP was expressed in early embryos and in many tissues such as the epidermis, blood vessels, muscle, notochord, fin ray, gut, eyes, and yolk sac, and the fluorescence was much stronger than that of the wild-type GFP . Thus, the usefulness of the medaka beta-actin promoter as a ubiquitous expression vector was confirmed using the mutant GFP as a reporter gene. Plant Physiol, 1998 Aug, 117(4), 1411 - 21 Characterization of two glutamate decarboxylase cDNA clones from Arabidopsis; Turano FJ et al.; Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh . were characterized . The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography . Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58- and 56-kD peptides, respectively . The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35- and 13-fold, respectively, by Ca2+/calmodulin but not by Ca2+ or calmodulin alone . Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis . The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only . Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested . The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mM NH4Cl, 5 mM NH4NO3, 5 mM glutamic acid, or 5 mM glutamine as the sole nitrogen source compared with samples from plants treated with 10 mM KNO3 . The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability . Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs . The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism. Plant Physiol, 1998 Aug, 117(4), 1393 - 400 Molecular characterization of an Arabidopsis gene encoding hydroperoxide lyase, a cytochrome P-450 that is wound inducible; Bate NJ et al.; Hydroperoxide lyase (HPL) cleaves lipid hydroperoxides to produce volatile flavor molecules and also potential signal molecules . We have characterized a gene from Arabidopsis that is homologous to a recently cloned HPL from green pepper (Capsicum annuum) . The deduced protein sequence indicates that this gene encodes a cytochrome P-450 with a structure similar to that of allene oxide synthase . The gene was cloned into an expression vector and expressed in Escherichia coli to demonstrate HPL activity . Significant HPL activity was evident when 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid was used as the substrate, whereas activity with 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid was approximately 10-fold lower . Analysis of headspace volatiles by gas chromatography-mass spectrometry, after addition of the substrate to E . coli extracts expressing the protein, confirmed enzyme-activity data, since cis-3-hexenal was produced by the enzymatic activity of the encoded protein, whereas hexanal production was limited . Molecular characterization of this gene indicates that it is expressed at high levels in floral tissue and is wound inducible but, unlike allene oxide synthase, it is not induced by treatment with methyl jasmonate. Plant Physiol, 1998 Aug, 117(4), 1341 - 9 Intracellular beta-carbonic anhydrase of the unicellular green alga Coccomyxa . Cloning of the cdna and characterization of the functional enzyme overexpressed in Escherichia coli; Hiltonen T et al.; Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes . We have cloned and sequenced a full-length cDNA encoding an intracellular beta-CA from the unicellular green alga Coccomyxa . Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids . The predicted polypeptide is similar to beta-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30% . The Coccomyxa cDNA was overexpressed in E . coli, and the enzyme was purified and biochemically characterized . The mature protein is a homotetramer with an estimated molecular mass of 100 kD . The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog . However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants . Fractionation studies further showed that Coccomyxa CA is extrachloroplastic. Plant Physiol, 1998 Aug, 117(4), 1317 - 23 Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase; Norris SR et al.; Plastoquinone and tocopherols are the two major quinone compounds in higher plant chloroplasts and are synthesized by a common pathway . In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway . Mutation of the PDS1 locus disrupts the activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the first committed step in the synthesis of both plastoquinone and tocopherols in plants . Although plants homozygous for the pds1 mutation could be rescued by growth in the presence of homogentisic acid, the product of HPPDase, we were unable to determine if the mutation directly or indirectly disrupted HPPDase activity . This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli . pHPPD encodes a 50-kD polypeptide with homology to previously identified HPPDases, including 37 highly conserved amino acid residues clustered in the carboxyl region of the protein . Expression of pHPPD in E . coli catalyzes the accumulation of homogentisic acid, indicating that it encodes a functional HPPDase enzyme . Mapping of pHPPD and co-segregation analysis of the pds1 mutation and the HPPD gene indicate tight linkage . Constitutive expression of pHPPD in a pds1 mutant background complements this mutation . Finally, comparison of the HPPD genomic sequences from wild type and pds1 identified a 17-bp deletion in the pds1 allele that results in deletion of the carboxyterminal 26 amino acids of the HPPDase protein . Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene. Plant Physiol, 1998 Aug, 117(4), 1165 - 70 RNA polymerase subunits encoded by the plastid rpo genes are not shared with the nucleus-encoded plastid enzyme; Serino G et al.; Plastid genes in photosynthetic higher plants are transcribed by at least two RNA polymerases . The plastid rpoA, rpoB, rpoC1, and rpoC2 genes encode subunits of the plastid-encoded plastid RNA polymerase (PEP), an Escherichia coli-like core enzyme . The second enzyme is referred to as the nucleus-encoded plastid RNA polymerase (NEP), since its subunits are assumed to be encoded in the nucleus . Promoters for NEP have been previously characterized in tobacco plants lacking PEP due to targeted deletion of rpoB (encoding the beta-subunit) from the plastid genome . To determine if NEP and PEP share any essential subunits, the rpoA, rpoC1, and rpoC2 genes encoding the PEP alpha-, beta'-, and beta"-subunits were removed by targeted gene deletion from the plastid genome . We report here that deletion of each of these genes yielded photosynthetically defective plants that lack PEP activity while maintaining transcription specificity from NEP promoters . Therefore, rpoA, rpoB, rpoC1, and rpoC2 encode PEP subunits that are not essential components of the NEP transcription machinery . Furthermore, our data indicate that no functional copy of rpoA, rpoB, rpoC1, or rpoC2 that could complement the deleted plastid rpo genes exists outside the plastids. J Cell Sci, 1998 Sep, 111 ( Pt 17), 2635 - 44 The putative sponge aggregation receptor . Isolation and characterization of a molecule composed of scavenger receptor cysteine-rich domains and short consensus repeats; Blumbach B et al.; Porifera (sponges) are the oldest extant metazoan phylum . Dissociated sponge cells serve as a classic system to study processes of cell reaggregation . The reaggregation of dissociated cells is mediated by an extracellularly localized aggregation factor (AF), based on heterophilic interactions of the third order; the AF bridges two cells by ligating a cell-surface-bound aggregation receptor (AR) . In the present study we report cloning, expression and immunohistochemical localization of a polypeptide from the marine sponge Geodia cydonium, which very likely represents the AR . The presumed AR gene gives rise to at least three forms of alternatively spliced transcripts of 6.5, 4.9 and 3.9 kb, as detected by northern blotting . Two cDNA clones corresponding to the shorter forms were already reported earlier; here we present an analysis of the largest . All three putative polypeptides feature scavenger receptor cysteine-rich (SRCR) domains . The largest form, SRCR-SCR-Car, is a cell-surface receptor of molecular mass 220 kDa, which is assumed to be the cell-adhesion receptor AR; the second form, SRCR-Re, is also a putative receptor of 166 kDa, while the third form, SRCR-Mo, is a soluble molecule of 129 kDa . The SRCR-SCR-Car molecule consists of fourteen SRCR domains, six short consensus repeats (SCRs), a C-terminal transmembrane domain and a cytoplasmic tail; its fourteenth SRCR domain features an Arg-Gly-Asp tripeptide . To obtain monoclonal antibodies, a 170-amino-acid-long polypeptide that is found in all three forms of the SRCR-containing proteins was expressed in E . coli . In a western blot of sponge cells lysate the monoclonal antibody raised against the recombinant polypeptide recognized two major immuno-reacting polypeptides (220 and 117 kDa) and two minor bands (36 and 32 kDa) . The antibody was found to react with antigen(s) predominantly localized on the plasma membranes of cells, especially those of spherulous cells . In a functional assay Fab' fragments of the antibodies suppressed AF-mediated cell-cell reaggregation . Additionally, a recombinant SRCR-soluble fragment effectively inhibited AF-mediated cell-cell reaggregation . We conclude that the 220 kDa SRCR-containing protein of the sponge G . cydonium is very likely the AR. Acta Biochim Pol, 1998, 45(1), 191 - 202 Repair of DNA alkylation damage; Bouziane M et al.; Alkylation damage of DNA is one of the major types of insults which cells must repair to remain viable . One way alkylation damaged ring nitrogens are repaired is via the Base Excision Repair (BER) pathway . Examination of mutants in both BER and Nucleotide Excision Repair show that there is actually an overlap of repair by these two pathways for the removal of cytotoxic lesions in Escherichia coli . The enzymes removing damaged bases in the first step in the BER pathway are DNA glycosylases . The coding sequences for a number of methylpurine-DNA glycosylases (MPG proteins) were cloned, and a comparison of the amino-acid sequences shows that there are some similarities between these proteins, but nonetheless, compared to other DNA glycosylases, MPG proteins are more divergent . MPG proteins have been purified to homogeneity and used to identify their substrates ranging from methylating agents to deamination products to oxidatively damaged bases . The ligation-mediated polymerase chain reaction has been used to study the formation of alkylation damage, and its repair in mammalian cells . We have studied DNA damage in the PGK1 gene for a series of DNA alkylating agents including N-methyl-N'-nitro-N-nitrosoguanidine, Mechlorethamine, and Chlorambucil and shown that the damage observed in the PGK1 (phosphoglycerate kinase 1) gene depends on the alkylating agent used . This report reviews the literature on the MPG proteins, DNA glycosylases removing 3-methyladenine, and the use of these enzymes to detect DNA damage at the nucleotide level. Acta Biochim Pol, 1998, 45(1), 127 - 32 Effect of DNA-interacting drugs on phage T7 RNA polymerase; Piestrzeniewicz M et al.; 9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation . Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s . Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties . T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E . coli enzyme while less sensitive to actinomycin D . Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase . Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands. RNA, 1998 Aug, 4(8), 1019 - 23 A new assay for tRNA aminoacylation kinetics; Wolfson AD et al.; An improved quantitative assay for tRNA aminoacylation is presented based on charging of a nicked tRNA followed by separation of an aminoacylated 3'-fragment on an acidic denaturing polyacrylamide gel . Kinetic parameters of tRNA aminoacylation by Escherichia coli AlaRS obtained by the new method are in excellent agreement with those measured by the conventional method . This assay provides several advantages over the traditional methods of measuring tRNA aminoacylation: (1) the fraction of aminoacyl-tRNA is measured directly; (2) data can be obtained at saturating amino acid concentrations; and (3) the assay is significantly more sensitive. RNA, 1998 Aug, 4(8), 928 - 36 tRNA nucleotide 47: an evolutionary enigma; Cermakian N et al.; A previous analysis of tRNA sequences suggested a correlation between the absence of a nucleotide at position 47 (nt 47) in the extra loop and the presence of a U13:G22 base pair in the D-stem . We have evaluated the significance of this correlation by determining the in vivo activity of tRNAs containing either a C13:G22 or a U13:G22 pair in tRNA molecules with or without nt 47 . Although this correlation might reflect some malfunction of tRNAs lacking nt 47, but containing the C13:G22, assays of the in vivo suppressor activity showed that this tRNA is actually more active than the tRNA with the features found in the database, i.e., a U13:G22 base pair and no nt 47 . Moreover, analogous constructs with a GGC anticodon permitted the growth of an Escherichia coli strain deleted for tRNA(Ala)GGC genes equally well . On the other hand, long-term growth experiments with competing E . coli strains harboring the tRNA lacking nt 47, either with the C13:G22 or the U13:G22 base pair demonstrated that the U13:G22 tRNA overtook the C13:G22 strain even when the starting proportion of strains favored the C13:G22 strain . Thus, the preference for the U13:G22 tRNA lacking nt 47 in the sequence database is most likely due to factors that come into play during extended growth or latency rather than to the ability of the tRNA to engage in protein synthesis. Nitric Oxide, 1997 Feb, 1(1), 31 - 8 Gender differences in nitric oxide production by alveolar macrophages in ethanol plus lipopolysaccharide-treated rats; Spitzer JA; Alveolar macrophages (AMs) constitute an important first line of host defense against infection in the lung, and NO is an essential component of the microbicidal activity of cytokine-activated macrophages . Previously we studied the respiratory burst, protein kinase C activity, and NO generation in tumor necrosis factor (TNF) and lipopolysaccharide (LPS)-treated AMs and gender differences in phagocytosis in ethanol (EtOH)-intoxicated rats . Now we have investigated NO production by AMs in EtOH plus LPS-treated male and female rats . Rats were infused iv with EtOH for 3 h to a blood level of approximately 180 mg/dl . At 90 min of infusion, Escherichia coli LPS (750 microg/kg) was injected i.v . Controls received saline (SAL) + LPS . AMs were isolated by bronchoalveolar lavage and cultured for 20 h in the absence and presence of LPS, interferon-y (IFN), and LPS + IFN . Nitrite was determined in the medium and was taken as an index of NO production . EtOH alone resulted in no significant differences compared with SAL infusion . LPS treatment caused a decrease in basal and an increase in LPS and IFN-stimulated generation of NO in males and females . EtOH + LPS treatment vs EtOH showed no significant differences . There are gender differences in both spontaneous and in vitro stimulated NO production by AMs . AMs of female rats treated with SAL + LPS released significantly more NO spontaneously than AMs of equally treated male rats . Cells of SAL + LPS-treated male rats activated in vitro by LPS and IFN-gamma produced significantly greater amounts of NO than AMs of female rats . These differences in activated induction of NO production were abrogated by ethanol treatment. Plant Mol Biol, 1998 Aug, 37(6), 1013 - 22 Molecular cloning and expression of a cDNA sequence encoding histidinol phosphate aminotransferase from Nicotiana tabacum; El Malki F et al.; A Nicotiana tabacum cDNA sequence encoding histidinol phosphate aminotransferase (HPA) was isolated by functional complementation of an Escherichia coli histidine auxotroph (UTH780) . The enzymatic assay has confirmed that the isolated cDNA encodes a functional HPA protein . Amino acid sequence alignment of the HPA protein from N . tabacum, Saccharomyces cerevisiae and E . coli revealed that, despite the low degree of identity, some residues were found to be highly conserved . The predicted protein contains a transit peptide sequence at the amino-terminal end, suggesting a chloroplastic localization of the HPA enzyme . Western blot analysis demonstrated that the deduced HPA protein and the mature HPA protein have an apparent molecular mass of about 45 kDa and 40 kDa respectively . Gene copy number estimation by Southern analysis indicates the presence of at least two genes per haploid genome coding for this protein in Nicotiana sp . From northern analysis results, the gene seems to be highly expressed in green tissues and the detected transcript showed a single band of expected molecular size. J Chromatogr B Biomed Sci Appl, 1998 Jun 26, 711(1-2), 217 - 22 Enzyme release from heat-stressed cell membranes as a function of hydrophobicity evaluated by using aqueous two-phase systems; Umakoshi H et al.; The release behavior of a periplasmic enzyme, acid phosphatase, from heat-stressed Escherichia coli cells was characterized by using kinetic analyses when the cells were treated by Triton X-100-EDTA . The hydrophobicity of the cell surface and the release-rate of the enzyme were not influenced by heat treatment at temperatures between 30 and 50 degrees C . However, these values varied above 55 degrees C . The release-rate constants were found to correspond to the net and local hydrophobicity of the outer membrane surface, evaluated by aqueous two-phase partitioning. J Chromatogr B Biomed Sci Appl, 1998 Jun 26, 711(1-2), 185 - 94 Aqueous two-phase systems as an alternative process route for the fractionation of small inclusion bodies; Walker SG et al.; Aqueous two-phase protocols have been established which successfully generate highly purified preparations of small inclusion bodies (IBs) from whole cell homogenates . Particle size analysis of disruptates confirmed that intense disruption (concomitant with maximal product release) was compromised by the corelease of contaminating solutes and the micronisation of cell debris yielding a similar particle size range to the IBs (100-200 nm) . PEG 300-phosphate systems enabled partial recovery of IBs in the top phase of ATPS . In contrast, PEG 8000-phosphate systems partitioned IBs more efficiently as a discrete sediment within the lower phase, whilst the majority of micronised debris remained in the interphase . The alpha-glucosidase IB yield and purity in ATPS was bettered only by analytical sucrose density gradient centrifugation, which is not readily scaleable for application in process operations . The successful recovery of such small IBs from complex homogenates highlights a generic role that ATPS techniques might play in the recovery and purification of new bioparticulate products (viral and plasmid gene therapy vectors, particulate protein vaccines etc.). Cancer Res, 1998 Aug 1, 58(15), 3209 - 14 Inhibition of vascular endothelial growth factor-induced receptor activation with anti-kinase insert domain-containing receptor single-chain antibodies from a phage display library; Zhu Z et al.; A single-chain antibody phage display library was constructed from spleen cells of mice immunized with a soluble form of a human vascular endothelial growth factor (VEGF) receptor, kinase insert domain-containing receptor (KDR) . After two rounds of biopanning, >90% of the clones recovered were specifically reactive to KDR . Subsequent selection identified two clones that blocked VEGF binding to KDR . The clones were expressed in Escherichia coli and purified as soluble single-chain Fv (scFv) antibodies . The affinities of the scFv for binding to KDR were determined by BIAcore analysis (2.1 x 10(-9)-5.9 x 10(-9) M) . One scFv, p1C11, was shown to inhibit VEGF-induced KDR phosphorylation and VEGF-stimulated DNA synthesis in human umbilical vein endothelial cells . There is much experimental evidence to suggest that the VEGF/KDR/Flk-1 pathway plays an important role in tumor angiogenesis, a process that is essential for tumor growth and metastasis . The antibodies discussed here, which block VEGF binding to KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved. Nat Struct Biol, 1998 Aug, 5(8), 682 - 6 Structure of interleukin 16 resembles a PDZ domain with an occluded peptide binding site; Muhlhahn P et al.; The structure of a folded core of IL-16 is similar to that of intracellular protein modules called PDZ domains . IL-16 is thus the first extracellular protein found to have a PDZ-like fold . However, it does not exhibit normal peptide binding properties of PDZ domains . This is due to alterations of the structure at the 'PDZ-like binding site' of IL-16 (the GLGF cleft): the GLGF cleft of IL-16 is much smaller than those of PDZ-domains and is additionally blocked with a tryptophan side chain at its center . Our experiments indicate also that IL-16 nonspecifically aggregates in solution; but formation of a homo-tetrameric protein is not required, in contrast to previous suggestions, for its chemo-attractant activity. J Mol Neurosci, 1998 Apr, 10(2), 129 - 41 Increased activity of matrix metalloproteinase-2 in human glial and neuronal cell lines treated with HIV-1 gp41 peptides; Chong YH et al.; Part of the neurodegenerative cascade in AIDS dementia may involve overexpression of matrix metalloproteinases (MMPs) . Here, we examined the possible effect of HIV-1 gp41, which has been shown as a key determinant associated with pathogenesis of AIDS dementia, on the activity of MMPs using human neuronal and glial cell lines . Zymographic analysis revealed that treatment with the gp41 peptide (aa 583-599) for 24 h markedly elevated the activity of MMP with Mr 66 kDa in the cultured media of glioblastoma cell line T98G in a concentration-dependent manner as well as of neuroblastoma cell line SK-N-SH despite of lower magnitude of the activity . In contrast, the immediately adjacent gp41 peptide (aa 598-613) as well as the reverse peptide (aa 598-583) had a little effect . Recombinant gp41 protein containing extracellular domain also elicited a similar effect, although with a lesser extent . This 66 kDa MMP was confirmed as gelatinase A (MMP-2) based on the results of its activity dependent on Ca2+ and inhibited in the presence of 1,10-phenanthroline or EDTA, as well as its specific immunoreactivity on the Western blot . N-acetyl cysteine (NAC) downregulated this gp41 peptide-induced MMP-2 activity in T98G . The soluble form of amyloid precursor protein (sAPP), which is synthesized in the Escherichia coli system, also inhibited the MMP-2 activity in vitro . Taken together, these results implicate that high production of HIV-1 gp41 or its metabolites containing aa 583-599 within central nervous system (CNS) could result in the increased activity of MMP-2 and that the extracellular deficiency of reducing agent or decreased level of sAPP within CNS could exacerbate this gp41-induced MMP-2 activity. Mem Inst Oswaldo Cruz, 1998 Mar-Apr, 93(2), 247 - 54 Molecular and antigenic characterization of the Leishmania (Viannia) panamensis kinetoplastid membrane protein-11; Ramirez JR et al.; The kinetoplastid membrane protein 11 (KMP-11) has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane . Two oligonucleotide primers were synthesized to PCR-amplify the entire encoding region of New World Leishmania species . The Leishmania (Viannia) panamensis amplification product was clone, sequenced and the putative amino acid sequence determined . A remarkably high degree of sequence homology was observed with the corresponding molecule of L . (L) donovani and L . (L) infantum (97% and 96%, respectively) . Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene . the L . (V) panamensis ORF was subsequently clone in a high expression vector and the recombinant protein was induced and purified from Escherichia coli cultures . Immunoblot analysis showed that 80%, 77% and 100% sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein . In a similar assay, 86% of asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11 . We proposed that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America. J Mol Biol, 1998 Aug 21, 281(3), 485 - 99 A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A; Su Y et al.; A mutation in the dimer interface of Escherichia coli glycinamide ribonucleotide transformylase (GarTfase) disrupts the observed pH-dependent association of the wild-type enzyme, but has no observable effect on the enzyme activity . Here, we assess whether a pH effect on the enzyme's conformation is sufficient by itself to explain the pH-dependence of the GarTfase reaction . A pH-dependent conformational change is observed between two high-resolution crystal structures of the Glu70Ala mutant GarTfase at pH 3.5 (1.8 A) and 7.5 (1.9 A) . Residues 110 to 131 in GarTfase undergo a transformation from a disordered loop at pH 3.5, where the enzyme is inactive, to an ordered loop-helix structure at pH 7.5, where the enzyme is active . The ordering of this flexible loop-helix has a direct effect on catalytic residues in the active site, binding of the folate cofactor and shielding of the active site from solvent . A main-chain carbonyl oxygen atom from Tyr115 in the ordered loop forms a hydrogen bond with His108, and thereby provides electronic and structural stabilization of this key active site residue . Kinetic data indicate that the pKa of His108 is in fact raised to 9 . 2 . The loop movement can be correlated with elevation of the His pKa, but with further stabilization, probably from Asp144, after the binding of folate cofactor . Leu118, also in the loop, becomes positioned near the p-amino benzoic acid binding site, providing additional hydrophobic interactions with the cofactor 10-formyl tetrahydrofolate . Thus, the pH-dependence of the enzyme activity appears to arise from local active site rearrangements and not from differences due to monomer-dimer association . J Mol Biol, 1998 Aug 21, 281(3), 465 - 73 Visualization of the binding site for the transcript cleavage factor GreB on Escherichia coli RNA polymerase; Polyakov A et al.; The structure of Escherichia coli core RNA polymerase (RNAP) complexed with the transcript cleavage factor GreB was determined from electron micrographs of negatively stained, flattened helical crystals . A binding assay was developed to establish that GreB was incorporated into the RNA polymerase crystals with high occupancy through interactions between the globular C-terminal domain and the RNA polymerase . Comparison of the core RNAP:GreB structure with the previously determined structure of core RNAP located the GreB binding site on one face of the RNA polymerase, next to but not in the 25 A-diameter channel of RNA polymerase . J Mol Biol, 1998 Aug 21, 281(3), 453 - 63 Mutational analysis of the global regulator KorA of broad-host-range plasmid RK2; Kostelidou K et al.; KorA protein encoded in the central control region of IncP plasmid RK2 binds to seven operators on the plasmid genome and acts as a global repressor of genes for replication and stable inheritance functions . At trfAp, the promoter for plasmid replication genes, KorA also causes derepression of trbAp, the promoter for trbA, encoding another global regulator (TrbA), which controls genes required for conjugative transfer . Both KorB, a second global repressor encoded in the central control region, and TrbA also act in the trfAp-trbAp region to down-regulate trfAp, but neither of these extra repressors allows derepression of trbAp . To initiate a functional dissection of KorA, we used random mutagenesis and a positive selection system to identify korA mutants which no longer repressed trfAp . Nine single amino acid changes were obtained, which did not affect polypeptide length or apparent stability . These clustered either in the N-terminal region of the protein (region I) or in the putative HTH motif (region II) . No changes were obtained in the C-terminal region (region III) . Four truncated KorA proteins, with deletions either from the N-terminal or the C-terminal end, were also screened together with the single mutants . Both the band-shift assay with trfAp DNA and the in vivo promoter-probe assays with either trfAp or trbAp showed that none of the region II mutants could bind to DNA and repress the promoter . The region I mutants with a conservative amino acid substitution retained some DNA binding and repressor activity, as well as the ability to dimerise . However, an in vivo system to detect trans-dominance of the mutants indicated that one region I point mutant together with the two N-terminally truncated mutants had lost their dimerisation ability . Deletions into the basic C terminus of KorA did not abolish dimerisation . The results implicate region I in dimerisation, region II in DNA binding and region III in a yet unspecified role, possibly interaction with other proteins such as KorB . J Mol Biol, 1998 Aug 14, 281(2), 363 - 77 Allosteric regulation and substrate channeling in multifunctional pyrimidine biosynthetic complexes: analysis of isolated domains and yeast-mammalian chimeric proteins; Serre V et al.; The initial steps of pyrimidine biosynthesis in yeast and mammals are catalyzed by large multifunctional proteins of similar size, sequence and domain structure, but appreciable functional differences . The mammalian protein, CAD, has carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities . The yeast protein, ura2, catalyzes the first two reactions and has a domain, called pDHO, which is homologous to mammalian DHOase, but is inactive . In CAD, only CPSase is regulated, whereas both CPSase and ATCase in the yeast protein are inhibited by UTP . These functional differences were explored by constructing a series of mammalian yeast chimeras . The isolated ATCase domain is catalytically active, but is not regulated . The inclusion of the yeast sequences homologous to the mammalian regulatory domain (B3) and the intervening pDHO domain did not confer regulation . Chimeric proteins in which the homologous regions of the mammalian protein were replaced by the corresponding domains of ura2 exhibited full catalytic activity, as well regulation of the CPSase, but not the ATCase, activities . The yeast B3 subdomain confers UTP sensitivity on the mammalian CPSase, suggesting that it is the locus of CPSase regulation in ura2 . Taken together, these results indicate that there are regulatory site(s) in ura2 . Channeling is impaired in all the chimeric complexes and completely abolished in the chimera in which the pDHO domain of yeast is replaced by the mammalian DHO domain. J Mol Biol, 1998 Aug 14, 281(2), 285 - 99 Structural and theoretical studies suggest domain movement produces an active conformation of thymidine phosphorylase; Pugmire MJ et al.; Two new crystal forms of Escherichia coli thymidine phosphorylase (EC 2.4.2.4) have been found; a monoclinic form (space group P21) and an orthorhombic form (space group I222) . These structures have been solved and compared to the previously determined tetragonal form (space group P43212) . This comparison provides evidence of domain movement of the alpha (residues 1 to 65, 163 to 193) and alpha/beta (residues 80 to 154, 197 to 440) domains, which is thought to be critical for enzymatic activity by closing the active site cleft . Three hinge regions apparently allow the alpha and alpha/beta-domains to move relative to each other . The monoclinic model is the most open of the three models while the tetragonal model is the most closed . Phosphate binding induces formation of a hydrogen bond between His119 and Gly208, which helps to order the 115 to 120 loop that is disordered prior to phosphate binding . The formation of this hydrogen bond also appears to play a key role in the domain movement . The alpha-domain moves as a rigid body, while the alpha/beta-domain has some non-rigid body movement that is associated with the formation of the His119-Gly208 hydrogen bond . The 8 A distance between the two substrates reported for the tetragonal form indicates that it is probably not in an active conformation . However, the structural data for these two new crystal forms suggest that closing the interdomain cleft around the substrates may generate a functional active site . Molecular modeling and dynamics simulation techniques have been used to generate a hypothetical closed conformation of the enzyme . Analysis of this model suggests several residues of possible catalytic importance . The model explains observed kinetic results and satisfies requirements for efficient enzyme catalysis, most notably through the exclusion of water from the enzyme's active site . J Mol Biol, 1998 Aug 14, 281(2), 241 - 52 Initiation factors IF1 and IF2 synergistically remove peptidyl-tRNAs with short polypeptides from the P-site of translating Escherichia coli ribosomes; Karimi R et al.; A novel function of initiation factors IF1 and IF2 in Escherichia coli translation has been identified . It is shown that these factors efficiently catalyse dissociation of peptidyl-tRNAs with polypeptides of different length from the P-site of E . coli ribosomes, and that the simultaneous presence of both factors is required for induction of drop-off . The factor-induced drop-off occurs with both sense and stop codons in the A-site and competes with peptide elongation or termination . The efficiency with which IF1 and IF2 catalyse drop-off decreases with increasing length of the nascent polypeptide, but is quite significant for hepta-peptidyl-tRNAs, the longest polypeptide chains studied . In the absence of IF1 and IF2 the rate of drop-off varies considerably for different peptidyl-tRNAs, and depends both on the length and sequence of the nascent peptide . Efficient factor-catalysed drop-off requires GTP but not GTP hydrolysis, as shown in experiments without guanine nucleotides, with GDP or with the non-cleavable analogue GMP-PNP.Simultaneous overexpression of IF1 and IF2 in vivo inhibits cell growth specifically in some peptidyl-tRNA hydrolase deficient mutants, suggesting that initiation factor-catalysed drop-off of peptidyl-tRNA can occur on a significant scale in the bacterial cell . Consequences for the bacterial physiology of this previously unknown function of IF1 and IF2 are discussed . J Surg Res, 1998 May, 76(2), 159 - 64 Prostaglandin I2 analogue, iloprost, down regulates mitogen-activated protein kinases of macrophages; Lo CJ et al.; OBJECTIVE: Vascular endothelial cells (EC) play a pivotal role in diffuse organ injury seen in ARDS and MOFS . On exposure to cytokines or endotoxin (LPS) EC are stimulated to express adhesion molecules as well as proinflammatory and procoagulant activity . However, the potential feedback control of EC on macrophages (M-theta) is not clear . We studied the cellular mechanism of iloprost, a PGI2 analogue, in regulation of TNF production by LPS-stimulated M-theta . METHODS: Rabbit alveolar M-theta and mouse M-theta RAW 264.7 cells were exposed to Escherichia coli LPS in the presence of various concentrations of iloprost . TNF production was measured by L929 bioassays . To further study the cellular mechanism of iloprost on M-theta activation, RAW 264.7 cells were stimulated by LPS (10 micrograms/ml) in the presence of either iloprost or specific mitogen-activated protein kinase (MAPK) inhibitors, either PD98059 or SB202190 . P44/P42 and P38 MAPK activation were evaluated by Western blot assays with anti-phospho MAPK antibodies . RESULTS: LPS induced M-theta TNF production, which was inhibited by iloprost . Iloprost also attenuated the activation of P44/P42 and P38 induced LPS . Inhibition of P44/P42 with PD98059 or P38 with SB202190 significantly reduced TNF production by LPS-stimulated RAW cells . CONCLUSIONS: The regulatory mechanism of EC on M-theta activation is dependent on PGI2 . The effect of PGI2 on M-theta is, at least in part, mediated through inhibiting MAPKs. Biochemistry, 1998 Aug 11, 37(32), 11385 - 92 Kinetic mechanism of UDP-hexose synthase, a point variant of hexose-1-phosphate uridylyltransferase from Escherichia coli; Ruzicka FJ et al.; Galactose-1-phosphate (galactose-1-P) uridylyltransferase from Escherichia coli catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double-displacement mechanism through a uridylyl-enzyme intermediate, in which the uridine-5'-phosphoryl group is covalently bonded to Nepsilon of His 166 . The point variant H166G displays a UDP-hexose synthase activity, in that it catalyzes the reaction of uridine 5'-phosphoimidazolide (UMPIm) with glucose-1-P to form UDP-glucose and imidazole . Inasmuch as the wild-type uridylyltransferase catalyzes its cognate reaction with ping-pong kinetics, an intrinsically ordered substrate binding mechanism, the kinetic mechanism of the UDP-hexose synthase activity of H166G became of interest . The synthase activity follows sequential kinetics {Kim, J., Ruzicka, F., and Frey, P . A . (1990) Biochemistry 29, 10590-10593} . In this work, product inhibition patterns for the synthase activity of H166G indicate random equilibrium binding of substrates . Comparison of the synthase activities of the variants H166G and H166A showed that the glycine variant is about 340- and 600-fold more active than the alanine variant in the forward and reverse directions, respectively . The kinetic consequences of varying the amino acid at position 166 were largely kcat effects, with more modest Km effects . Comparison of the synthase activities of these variants with that of the wild-type enzyme in the production of glucose-1-P showed that the loss of the beta-carbon of His 166 in the complex H166G-UMPIm increases the activation energy for uridylyl group transfer by 2.4 kcal mol-1, and the presence of two additional hydrogen atoms in the complex H166A-UMPIm increases the activation energy by 6.2 kcal mol-1 . It is concluded that the active site is much less tolerant of additional steric bulk in the locus of the beta-carbon of His 166 than it is of the loss of the beta-carbon . The sensitivities to additional steric bulk around other positions of the His 166-imidazole ring are much less severe, as indicated by the reactivities of methylated analogues of UMPIm in the synthase reaction of H166G . Uridine 5'-phospho-N-methylimidazolide is more reactive as a synthase substrate than UMPIm, and this is attributed to the positive charge of the imidazole ring . The fact that the imidazole ring of the wild-type covalent uridylyl-enzyme retains its proton and is positively charged is supported by the pH-rate profile for hydrolysis of the intermediate. Biochemistry, 1998 Aug 11, 37(32), 11255 - 63 Functional role of the N-terminal region of the Lon protease from Mycobacterium smegmatis; Roudiak SG et al.; Lon protease homologues contain a poorly conserved N-terminal region of variable length . To better understand the role of the N-terminal region of Lon in the complicated reaction cycle of ATP-dependent protein degradation, we expressed and characterized mutants of the Lon protease from Mycobacterium smegmatis (Ms-Lon) lacking 90, 225, and 277 N-terminal residues (N-G91, N-E226, and N-I278, respectively) . N-I278 displayed neither peptidase nor ATPase activity despite the fact that it was stable and soluble in vivo, had a near-wild-type CD spectrum, and the deleted residues included neither the catalytic nucleophile for peptide bond hydrolysis (S675) nor the ATP binding regions . N-G91 and N-E226 retained peptidase activities against small unstructured peptides that were stimulated, to near-wild-type levels, by the Ms-Lon substrate protein alpha-casein . By contrast, N-G91 and N-E226 retained basal ATPase activities, but these activities were only stimulated weakly by alpha-casein . Ms-Lon, N-E226, and N-G91 all exhibited low-level peptidase activity in assays containing nonhydrolyzed nucleotide analogues . However, these peptidase activities were stimulated strongly by alpha-casein in the case of Ms-Lon but weakly by alpha-casein in the cases of N-G91 and N-E226 . Strikingly, despite the near-wild-type peptidase activities of N-G91 and N-E226, both were severely impaired in their degradation of the Ms-Lon protein substrates alpha-casein in vitro and RcsA in vivo . Overall, N-G91 and N-E226 displayed catalytic properties similar to Escherichia coli Lon (Ec-Lon) in the presence of the PinA inhibitor, suggesting that PinA inhibits Ec-Lon protease by inhibiting the function of Ec-Lon's N-terminal region . In vivo protease assays further revealed that, in contrast to the inactive Ms-Lon point mutant S675A, N-G91 and N-E226 did not reduce the cellular activity of RcsA . This same defect was observed previously for Ms-Lons with multiple mutations in their peptidase active sites . We conclude that proteolytically inactive mutants of Ms-Lon retain the ability to reduce the cellular activity of RcsA but that both the N-terminal region and the peptidase active site region of Ms-Lon are required for this activity of wild-type Ms-Lon . The inabilities of N-G91 and N-E226 to degrade larger protein substrates and to reduce the cellular activity of RcsA were not the result of drastic alterations in their quaternary structures . Gel filtration profiles of N-G91 and N-E226 revealed that each was primarily tetrameric, with an increased percentage of dimeric species and a decreased percentage of trimeric species relative to Ms-Lon . The observed shifts in the dimer/trimer ratios of the N-terminal truncation mutants suggest that the Ms-Lon tetramer contains two types of subunit-subunit interactions. Biochemistry, 1998 Aug 11, 37(32), 11202 - 14 Rho-dependent termination within the trp t' terminator . I . Effects of rho loading and template sequence; Zhu AQ et al.; About one-half of the terminators of the Escherichia coli genome require transcription termination factor rho to function . Here we use the very "diffuse" trp t' terminator of E . coli to show that both template sequence and transcript secondary structure are involved in controlling the template positions and efficiencies of rho-dependent termination . Termination begins in the wild-type trp t' terminator sequence approximately 97 bps downstream of the promoter under our standard reaction conditions, and termination efficiencies for individual positions on three related templates have been determined in the form of quantitative patterns of rho-dependent RNA release . Comparison of these patterns shows that the rho-dependent termination efficiency at individual template positions depends primarily on the nucleotide sequence at and near the putative 3' end of the transcript, although these efficiencies can also be influenced by RNA sequence elements located further upstream . The amplitudes of the peaks of the RNA release patterns at specific template positions are controlled primarily by the effectiveness of the binding of the rho hexamer to the "rho loading site" of the transcript . Introduction of a stable element of secondary structure into the nascent RNA within the loading site both shifts the position of initial rho-dependent termination downstream and decreases the amplitudes of the peaks of the RNA release pattern at the corresponding sequences . These results and others are consistent with the view that rho-dependent terminators contain two essential components: (i) an upstream rho loading site on the RNA that is 70-80 nucleotide residues in length, essentially devoid of secondary structure, and which contains sufficient numbers of rC residues to activate the RNA-dependent ATPase of rho; and (ii) a downstream sequence within which termination actually occurs . In this study we use the trp t' terminator to characterize the involvement of each of these sequence components in detail in order to provide the parameters required to define a quantitative mechanistic model for the function of rho in transcript termination. Immunol Lett, 1998 Jul, 62(3), 145 - 9 Immunoglobulin G subclass responses in mice immunized with plasmid DNA encoding the CFA/I fimbria of enterotoxigenic Escherichia coli; Alves AM et al.; Balb/c mice immunized either with a plasmid vector (pRECFA), encoding the CFA/I fimbrial adhesin of enterotoxigenic Escherichia coli (ETEC), or purified CFA/I fimbriae induced similar antibody responses in regard to the kinetics of serum total immunoglobulins and IgG . Nonetheless, the IgG subclass responses in mice vaccinated with DNA were composed predominantly by IgG2a (ranging from 39 to 74% of the total anti-CFA/I IgG) and, to a lesser extent, by IgG (ranging from 15 to 24% of the total anti-CFA/I IgG) during a 12 week observation period . On the other hand, mice immunized with purified CFA/I presented mainly an IgG1 antibody response (ranging from 39 to 67% of the total anti-CFA/I IgG) . These results indicated that, irrespective of the immunogenic properties and-or origin of the encoded antigen, immunization with pRECFA elicited an specific IgG subclass response which may affect the ability of DNA vaccines to induce a protective immune response against CFA/I mediated colonization of ETEC bacterial cells. Trends Biochem Sci, 1998 Jul, 23(7), 247 - 51 Role of the human RAD51 protein in homologous recombination and double-stranded-break repair; Baumann P et al.; Eukaryotic cells possess several mechanisms for repairing double-stranded breaks in DNA . One mechanism involves genetic recombination with an intact sister duplex . The recent identification of the RAD51 protein, a eukaryotic homologue of Escherichia coli RecA, represents a landmark discovery in our understanding of the key reactions in this repair pathway . RAD51 is similar to RecA, both biochemically and structurally: it promotes homologous pairing and strand exchange within a regular nucleoprotein filament . The isolation of yeast and human RecA homologues shows that homologous recombination and recombinational repair have been conserved throughout evolution . The goal is now to identify other factors involved in recombinational repair and to define their roles in this essential process. Pac Symp Biocomput . 1998;:279-90. Deriving ribosomal binding site (RBS) statistical models from unannotated DNA sequences and the use of the RBS model for N-terminal prediction; Hayes WS et al.; Accurate prediction of the position of translation initiation (N-terminal prediction) is a difficult problem . N-terminal prediction from DNA sequence alone is ambiguous is several candidate start sites are close to each other . Protein similarity search is usually unable to indicate the true start of a gene as it would require a strong protein sequence similarity at the N-terminal portion of a protein where conservative regions are rarely situated . With the aid of the GeneMark program for gene identification, we extract DNA sequence fragments presumably containing ribosome binding sites (RBS) from unannotated complete genomic sequences . These DNA segments are aligned to generate the RBS model using the Gibbs' sampling method . N-terminal prediction is then performed by using the RBS model in conjunction with the GeneMark start codon prediction to aid in determining the true N-terminal site. Pac Symp Biocomput . 1998;:54-65. Rules for the evolution of gene circuitry; Savageau MA; Cells possess the genes required for growth and function in a variety of contexts . In any given context there is a corresponding pattern of gene expression in which some genes are OFF and others ON . The ability of cells to switch genes ON and OFF in a coordinate fashion to produce the required patterns of expression is the fundamental basis for complex processes like normal development and pathogenesis . The molecular study of gene regulation has revealed a plethora of mechanisms and circuitry that have evolved to perform what appears to be the same switching function . To some this implies the absence of rules . However, simple rules capable of relating molecular design to the natural environment have begun to emerge through the analysis of elementary gene circuits . Two of these rules are reviewed in this paper . These simple rules have the ability to unify understanding across several different levels of biological organization--molecular, physiological, developmental, ecological. Microb Comp Genomics, 1998, 3(2), 133 - 40 Yeast "operons"; Zhang X et al.; To achieve coordinate gene regulation, yeast (Saccharomyces cerevisiae) appears to have exploited two distinct multifunction "operon" schemas: one, by concatenating originally separate functional domains into single polypeptides, and two, by linking opposite strand genes through common promoter elements . For example, distinct functions found in bacterial operons are often concatenated in yeast . A selective advantage, similar to that for bringing multiple related functions into a single peptide, may also explain the large numbers of yeast opposite-strand, ORF pairs sharing a common regulatory region. J Biol Chem, 1998 Aug 14, 273(33), 21282 - 90 The biosynthesis of mycolic acids in Mycobacterium tuberculosis . Enzymatic methyl(ene) transfer to acyl carrier protein bound meromycolic acid in vitro; Yuan Y et al.; A closely related family of enzymes from Mycobacterium tuberculosis has been shown by heterologous expression to catalyze the modification of mycolic acids through the addition of a methyl (or methylene) group derived from S-adenosyl-L-methionine (SAM) . Overproduction of all six of these enzymes in Escherichia coli and subsequent in vitro reactions with heat-inactivated acceptor fractions derived from Mycobacterium smegmatis in the presence of {methyl-3H}SAM demonstrated that the immediate substrate to which methyl group addition occurs was a family of very long-chain fatty acids . Inhibitors of methyl transfer, such as S-adenosyl-L-homocysteine and sinefungin, were shown to inhibit this reaction but had no effect on whole cells of either M . smegmatis or M . tuberculosis . Purified mycolic acids from M . tuberculosis were pyrolyzed, and the resulting meroaldehyde was oxidized and methylated to produce full-length methyl meromycolates . These esters were shown to comigrate with a fraction of the acceptor from the in vitro reactions, suggesting that methyl group addition occurs up to the level of the meromycolate . Protease and other treatments destroyed the activity of the acceptor fraction, which was also found to be extremely sensitive to basic pH . Antibody to the acyl carrier protein AcpM, which has recently been shown to be the carrier of full-length meromycolate produced by a unique type II fatty acid synthase system, inhibited the cell-free methyl(en)ation of these acids . These results suggest that mycolate modification reactions occur parallel with the synthesis of the AcpM-bound meromycolate chain. J Virol, 1998 Sep, 72(9), 7137 - 43 An enhanced packaging system for helper-dependent herpes simplex virus vectors; Stavropoulos TA et al.; Helper-dependent herpes simplex virus (HSV) vectors (amplicons) show considerable promise to provide for long-term transduced-gene expression in most cell types . The current packaging system of choice for these vectors involves cotransfection with a set of five overlapping cosmids that encode the full HSV type 1 (HSV-1) helper virus genome from which the packaging (pac) elements have been deleted . Although both the helper virus and the HSV amplicon can replicate, only the latter is packaged into infectious viral particles . Since the titers obtained are too low for practical application, an enhanced second-generation packaging system was developed by modifying both the helper virus and the HSV amplicon vector . The helper virus was reverse engineered by using the original five cosmids to generate a single HSV-bacterial artificial chromosome (BAC) clone in Escherichia coli from which the pac elements were deleted to generate a replication-proficient but packaging-defective HSV-1 genome . The HSV amplicon was modified to contain the simian virus 40 origin of replication, which acts as an HSV-independent replicon to provide for the replicative expansion of the vector . The HSV amplicon is packaged into infectious particles by cotransfection with the HSV-BAC helper virus into the 293T cell line, and the resulting cell lysate is free of detectable helper virus contamination . The combination of both modifications to the original packaging system affords an eightfold increase in the packaged-vector yield. J Bacteriol, 1998 Aug, 180(16), 4314 - 8 Importance of glutathione for growth and survival of Escherichia coli cells: detoxification of methylglyoxal and maintenance of intracellular K+; Ferguson GP et al.; The role of the tripeptide glutathione in the growth and survival of Escherichia coli cells has been investigated . Glutathione-deficient mutants leak potassium and have a reduced cytoplasmic pH . These mutants are more sensitive to methylglyoxal than the parent strain, indicating that in the absence of glutathione-dependent detoxification, acidification of the cytoplasm cannot fully protect cells . However, increasing the intracellular pH of the glutathione-deficient strain resulted in enhanced sensitivity to methylglyoxal . This suggests that acidification of the cytoplasm can provide some protection to E . coli cells in the absence of glutathione . In the presence of the Kdp system, glutathione-deficient mutants are highly sensitive to methylglyoxal . This is due to the higher intracellular pH in these cells . In the absence of methylglyoxal, the presence of the Kdp system in a glutathione-deficient strain also leads to an extended lag upon dilution into fresh medium . These data highlight the importance of glutathione for the regulation of the K+ pool and survival of exposure to methylglyoxal. J Bacteriol, 1998 Aug, 180(16), 4294 - 9 Involvement of the gapA- and epd (gapB)-encoded dehydrogenases in pyridoxal 5'-phosphate coenzyme biosynthesis in Escherichia coli K-12; Yang Y et al.; We show that epd (gapB) mutants lacking an erythrose 4-phosphate (E4P) dehydrogenase are impaired for growth on some media and contain less pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) than their epd+ parent . In contrast to a previous report, we found that gapA epd double mutants lacking the glyceraldehyde 3-phosphate and E4P dehydrogenases are auxotrophic for pyridoxine . These results implicate the GapA and Epd dehydrogenases in de novo PLP and PMP coenzyme biosynthesis. J Bacteriol, 1998 Aug, 180(16), 4287 - 90 The Escherichia coli starvation gene cstC is involved in amino acid catabolism; Fraley CD et al.; Escherichia coli strains mutant in the starvation gene cstC grow normally in a mineral salts medium but are impaired in utilizing amino acids as nitrogen sources . They are also compromised in starvation survival, where amino acid catabolism is important . The cstC gene encodes a 406-amino-acid protein that closely resembles the E . coli ArgD protein, which is involved in arginine biosynthesis . We postulate that CstC is a counterpart of ArgD in an amino acid catabolic pathway . The cstC upstream region contains several regulatory consensus sequences . Both sigmaS and sigma54 promoters are probably involved in cstC transcription and appear to compete with each other, presumably to match cstC expression to the cellular amino acid catabolic needs. J Bacteriol, 1998 Aug, 180(16), 4252 - 7 Localization and function of early cell division proteins in filamentous Escherichia coli cells lacking phosphatidylethanolamine; Mileykovskaya E et al.; Escherichia coli cells that contain the pss-93 null mutation are completely deficient in the major membrane phospholipid phosphatidylethanolamine (PE) . Such cells are defective in cell division . To gain insight into how a phospholipid defect could block cytokinesis, we used fluorescence techniques on whole cells to investigate which step of the cell division cycle was affected . Several proteins essential for early steps in cytokinesis, such as FtsZ, ZipA, and FtsA, were able to localize as bands to potential division sites in pss-93 filaments, indicating that the generation and localization of potential division sites was not grossly affected by the absence of PE . However, there was no evidence of constriction at most of these potential division sites . FtsZ and green fluorescent protein (GFP) fusions to FtsZ and ZipA often formed spiral structures in these mutant filaments . This is the first report of spirals formed by wild-type FtsZ expressed at normal levels and by ZipA-GFP . The results suggest that the lack of PE may affect the correct interaction of FtsZ with membrane nucleation sites and alter FtsZ ring structure so as to prevent or delay its constriction. J Bacteriol, 1998 Aug, 180(16), 4227 - 32 DNA bending by AraC: a negative mutant; Saviola B et al.; We sought a mutation in the DNA binding domain of the arabinose operon regulatory protein, AraC, of Escherichia coli that allows the protein to bind DNA normally but not activate transcription . The mutation was isolated by mutagenizing a plasmid overproducing a chimeric leucine zipper-AraC DNA binding domain and screening for proteins that were trans dominant negative with regard to wild-type AraC protein . The mutant with the lowest transcription activation of the araBAD promoter was studied further . It proved to alter a residue that had previously been demonstrated to contact DNA . Because the overproduced mutant protein still bound DNA in vivo, it is deficient in transcription activation for some reason other than absence of DNA binding . Using the phase-sensitive DNA bending assay, we found that wild-type AraC bends DNA about 90 degrees whereas the mutant bends DNA by a smaller amount. J Bacteriol, 1998 Aug, 180(16), 4192 - 8 Fnr, NarP, and NarL regulation of Escherichia coli K-12 napF (periplasmic nitrate reductase) operon transcription in vitro; Darwin AJ et al.; The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP . Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation . First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation . Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein . In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region . In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation . The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins . These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not . This suggests that Fnr and NarP may act synergistically to activate napF operon expression . As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro. J Bacteriol, 1998 Aug, 180(16), 4171 - 6 Biochemical and genetic characterization of a gentisate 1, 2-dioxygenase from Sphingomonas sp . strain RW5; Werwath J et al.; A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp . strain RW5 was cloned and sequenced . The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa . Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp . strain KP7 (T . Iwabuchi and S . Harayama, J . Bacteriol . 179:6488-6494, 1997) . This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases . The gene was subcloned and hyperexpressed in E . coli . The resulting product was purified to homogeneity and partially characterized . Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa . The enzyme thus appears to be a homotetrameric protein . The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester . Values of affinity constants (Km) and specificity constants (Kcat/Km) of the enzyme were determined to be 15 microM and 511 s-1 M-1 x 10(4) for gentisate and 754 microM and 20 s-1 M-1 x 10(4) for 3, 6-dichlorogentisate . Three further open reading frames (ORFs) were found downstream of gtdA . The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively. J Bacteriol, 1998 Aug, 180(16), 4140 - 5 Tetrachloroethene dehalogenase from Dehalospirillum multivorans: cloning, sequencing of the encoding genes, and expression of the pceA gene in Escherichia coli; Neumann A et al.; The genes encoding tetrachloroethene reductive dehalogenase, a corrinoid-Fe/S protein, of Dehalospirillum multivorans were cloned and sequenced . The pceA gene is upstream of pceB and overlaps it by 4 bp . The presence of a sigma70-like promoter sequence upstream of pceA and of a rho-independent terminator downstream of pceB indicated that both genes are cotranscribed . This assumption is supported by reverse transcriptase PCR data . The pceA and pceB genes encode putative 501- and 74-amino-acid proteins, respectively, with calculated molecular masses of 55,887 and 8,354 Da, respectively . Four peptides obtained after trypsin treatment of tetrachloroethene (PCE) dehalogenase were found in the deduced amino acid sequence of pceA . The N-terminal amino acid sequence of the PCE dehalogenase isolated from D . multivorans was found 30 amino acids downstream of the N terminus of the deduced pceA product . The pceA gene contained a nucleotide stretch highly similar to binding motifs for two Fe4S4 clusters or for one Fe4S4 cluster and one Fe3S4 cluster . A consensus sequence for the binding of a corrinoid was not found in pceA . No significant similarities to genes in the databases were detected in sequence comparisons . The pceB gene contained two membrane-spanning helices as indicated by two hydrophobic stretches in the hydropathic plot . Sequence comparisons of pceB revealed no sequence similarities to genes present in the databases . Only in the presence of pUBS 520 supplying the recombinant bacteria with high levels of the rare Escherichia coli tRNA4Arg was pceA expressed, albeit nonfunctionally, in recombinant E . coli BL21 (DE3). J Bacteriol, 1998 Aug, 180(16), 4116 - 22 The cyanobacterium Synechocystis sp . strain PCC 6803 expresses a DNA methyltransferase specific for the recognition sequence of the restriction endonuclease PvuI; Scharnagl M et al.; By use of restriction endonucleases, the DNA of the cyanobacterium Synechocystis sp . strain PCC 6803 was analyzed for DNA-specific methylation . Three different recognition sites of methyltransferases, a dam-like site including N6-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain . slr0214, a Synechocystis gene encoding a putative methyltransferase that shows significant similarities to C5-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization . Mutations in slr0214 were generated by the insertion of an aphII gene cassette . Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed . The recognition sequence of the methyltransferase was identified as 5'-CGATCG-3', corresponding to the recognition sequence of PvuI . The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells . Mutation of slr0214 also led to changed growth characteristics of the cells compared to wild-type cells . These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in Synechocystis . The Slr0214 protein was also overexpressed in Escherichia coli, and the purified protein demonstrated methyltransferase activity and specificity for PvuI recognition sequences in vitro . We propose the designation M.Ssp6803I {corrected} (Synechocystis methyltransferase I) for the slr0214-encoded enzyme. J Bacteriol, 1998 Aug, 180(16), 4102 - 10 Rhs elements comprise three subfamilies which diverged prior to acquisition by Escherichia coli; Wang YD et al.; The Rhs elements are complex genetic composites widely spread among Escherichia coli isolates . One of their components, a 3.7-kb, GC-rich core, maintains a single open reading frame that extends the full length of the core and then 400 to 600 bp beyond into an AT-rich region . Whereas Rhs cores are homologous, core extensions from different elements are dissimilar . Two new Rhs elements from strains of the ECOR reference collection have been characterized . RhsG (from strain ECOR-11) maps to min 5.3, and RhsH (from strain ECOR-45) maps to min 32.8, where it lies in tandem with RhsE . Comparison of strain K-12 to ECOR-11 indicates that RhsG was once present in but has been largely deleted from an ancestor of K-12 . Phylogenetic analysis shows that the cores from eight known elements fall into three subfamilies, RhsA-B-C-F, RhsD-E, and RhsG-H . Cores from different subfamilies diverge 22 to 29% . Analysis of substitutions that distinguish between subfamilies shows that the origin of the ancestral core as well as the process of subfamily separation occurred in a GC-rich background . Furthermore, each subfamily independently passed from the GC-rich background to a less GC-rich background such as E . coli . A new e