J Biol Chem, 2000 Feb 11, 275(6), 4192 - 8 Inhibition of protein phosphatase-1 by clavosines A and B . Novel members of the calyculin family of toxins; McCready TL et al.; Site-directed mutagenesis was used to investigate the mechanism of interaction between the catalytic subunit of human protein phosphatase-1 (PP-1cgamma) and members of the calyculin family of toxins . Clavosines A and B are related to calyculins but are glycosylated with a trimethoxy rhamnose group . We provide experimental evidence implicating Tyr-134 as an important residue in PP-1cgamma that mediates interactions with the calyculins . Mutation of Tyr-134 to Phe, to prevent hydrogen bond formation, resulted in a slight increase in sensitivity of PP-1cgamma to clavosines A and B and calyculin A . In contrast, a Y134A mutant was 10-fold less sensitive to inhibition by all three inhibitors . The greatest effect on inhibition was found by substituting an Asp for Tyr-134 in the phosphatase . Clavosine B inhibited PP-1cgamma Y134D with a 310-fold decrease in potency . Clavosine A and calyculin A were also markedly poorer inhibitors of this mutant . These results suggest that a hydrogen bond between Tyr-134 and the calyculins is unlikely to be essential for inhibitor binding to the phosphatase . The clavosines and calyculin A were tested for their ability to inhibit other mutants of PP-1cgamma (including Ile-133, Val-223, and Cys-291) . Our mutagenesis studies provide an experimental basis for assessing models of calyculin binding found in the literature (Lindvall, M . K., Pihko, P . M., and Koskinen, A . M . (1997) J . Biol . Chem . 272, 23312-23316; Gupta, V., Ogawa, A . K., Du, X., Houk, K . N., and Armstrong, R . W . (1997) J . Med . Chem . 40, 3199-3206; Gauss, C . M., Sheppeck, I . J., Nairn, A . C., and Chamberlain, R . (1997) Bioorg . Med . Chem . 5, 1751-1773) . A new model for clavosine and calyculin A binding to PP-1c is presented that is consistent with previous structure-function experiments and which accommodates key structural features of the clavosines, including the novel rhamnose moiety. J Biol Chem, 2000 Feb 11, 275(6), 4183 - 91 Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7); Yu WH et al.; Many matrix metalloproteinases (MMPs) are tightly bound to tissues; matrilysin (MMP-7), although the smallest of the MMPs, is one of the most tightly bound . The most likely docking molecules for MMP-7 are heparan sulfate proteoglycans on or around epithelial cells and in the underlying basement membrane . This is established by extraction experiments and confocal microscopy . The enzyme is extracted from homogenates of postpartum rat uterus by heparin/heparan sulfate and by heparinase III treatment . The enzyme is colocalized with heparan sulfate in the apical region of uterine glandular epithelial cells and can be released by heparinase digestion . Heparan sulfate and MMP-7 are expressed at similar stages of the rat estrous cycle . The strength of heparin binding by recombinant rat proMMP-7 was examined by affinity chromatography, affinity coelectrophoresis, and homogeneous enzyme-based binding assay; the K(D) is 5-10 nM . Zymographic measurement of MMP-7 activity is greatly enhanced by heparin . Two putative heparin-binding peptides have been identified near the C- and N-terminal regions of proMMP-7; however, molecular modeling suggests a more extensive binding track or cradle crossing multiple peptide strands . Evidence is also found for the binding of MMP-2, -9, and -13 . Binding of MMP-7 and other MMPs to heparan sulfate in the extracellular space could prevent loss of secreted enzyme, provide a reservoir of latent enzyme, and facilitate cellular sensing and regulation of enzyme levels . Binding to the cell surface could position the enzyme for directed proteolytic attack, for activation of or by other MMPs and for regulation of other cell surface proteins . Dislodging MMPs by treatment with compounds such as heparin might be beneficial in attenuating excessive tissue breakdown such as occurs in cancer metastasis, arthritis, and angiogenesis. J Biol Chem, 2000 Feb 11, 275(6), 4112 - 7 Mutational analysis of Escherichia coli topoisomerase IV . III . Identification of a region of parE involved in covalent catalysis; Bahng S et al.; The products of three dominant-negative alleles of parE, encoding the ATP-binding subunit of topoisomerase IV (Topo IV), were purified and their activities characterized when reconstituted with ParC to form Topo IV . The ability of the ParE E418K, ParE G419D, and ParE G442D mutant Topo IVs to bind DNA, hydrolyze ATP, and close their ATP-dependent clamp was relatively unaffected . However, their ability to relax negatively supercoiled DNA was compromised significantly . This could be attributed to severe defects in covalent complex formation between ParC and DNA . Thus, these residues, which are far from the active site Tyr of ParC, contribute to covalent catalysis . This indicates that a dramatic conformational rearrangement of the protein likely occurs subsequent to the binding of the G segment at the DNA gate and prior to its opening. J Biol Chem, 2000 Feb 11, 275(6), 4104 - 11 Mutational analysis of Escherichia coli topoisomerase IV . II . ATPase negative mutants of parE induce hyper-DNA cleavage; Nurse P et al.; ParE is the ATP-binding subunit of topoisomerase IV (Topo IV) . During topoisomerization, the ATP-binding and hydrolysis cycle must be coordinated with the cycle of DNA cleavage and religation . We have isolated three dominant-negative mutant alleles of parE that encode ParE proteins that fail to hydrolyze ATP when reconstituted with ParC to form Topo IV . ParE G110S Topo IV and ParE S123L Topo IV failed to bind ATP at all, whereas ParE T201A could bind ATP . All three mutant Topo IV proteins exhibited an elevated level of spontaneous DNA cleavage that could be associated with a decreased rate of DNA resealing . In ParE T201A Topo IV, this defect appeared to result from an increased likelihood that the tetrameric enzyme would fall apart after DNA cleavage . Thus, while ATP is not required for DNA cleavage, the properties of these mutant enzymes suggests that ATP-hydrolysis informs DNA religation. J Biol Chem, 2000 Feb 11, 275(6), 4099 - 103 Mutational analysis of Escherichia coli topoisomerase IV . I . Selection of dominant-negative parE alleles; Mossessova E et al.; In order to define regions of ParE, one of the two subunits of topoisomerase IV, that are involved in catalysis during topoisomerization, we developed a selection procedure to isolate dominant-negative parE alleles . Both wild-type parC and mutagenized parE were expressed from a tightly-regulated lac promoter on a moderate-copy plasmid . Mutated parE alleles were rescued from those plasmids that caused IPTG-dependent cell death . The mutant ParE proteins could be divided into two groups when reconstituted with ParC to form topoisomerase IV, those that elicited hyper-DNA cleavage and those that affected covalent complex formation. J Biol Chem, 2000 Feb 11, 275(6), 4072 - 80 Characterization of a novel alanine-rich protein located in surface microdomains in Trypanosoma brucei; Nolan DP et al.; Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA ( approximately 1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP) . Analysis of the amino acid sequence predicted the presence of a typical NH(2)-terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor . A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense . BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass ( approximately 50-70 kDa) significantly higher than predicted from the amino acid sequence ( approximately 26 kDa) . Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface . Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction . These data suggested that BARP may be sequestered in lipid rafts. J Biol Chem, 2000 Feb 11, 275(6), 4060 - 5 The bifunctional active site of S-adenosylmethionine synthetase . Roles of the basic residues; Taylor JC et al.; S-adenosylmethionine (AdoMet) synthetase catalyzes a unique two-step enzymatic reaction leading to formation of the primary biological alkylating agent . The crystal structure of Escherichia coli AdoMet synthetase shows that the active site, which lies between two subunits, contains four lysines and one histidine as basic residues . In order to test the proposed charge and hydrogen bonding roles in catalytic function, each lysine has been changed to an uncharged methionine or alanine, and the histidine has been altered to asparagine . The resultant enzyme variants are all tetramers like the wild type enzyme; however, circular dichroism spectra show reductions in helix content for the K245*M and K269M mutants . (The asterisk denotes that the residue is in the second subunit.) Four mutants have k(cat) reductions of approximately 10(3)-10(4)-fold in AdoMet synthesis; however, the k(cat) of K165*M variant is only reduced 2-fold . In each mutant, there is a smaller catalytic impairment in the partial reaction of tripolyphosphate hydrolysis . The K165*A enzyme has a 100-fold greater k(cat) for tripolyphosphate hydrolysis than the wild type enzyme, but this mutant is not activated by AdoMet in contrast to the wild type enzyme . The properties of these mutants require reassessment of the catalytic roles of these residues. J Biol Chem, 2000 Feb 11, 275(6), 4055 - 9 Identification of a highly diverged class of S-adenosylmethionine synthetases in the archaea; Graham DE et al.; S-adenosylmethionine is the primary alkylating agent in all known organisms . ATP:L-methionine S-adenosyltransferase (MAT) catalyzes the only known biosynthetic route to this central metabolite . Although the amino acid sequence of MAT is strongly conserved among bacteria and eukarya, no homologs have been recognized in the completed genome sequences of any archaea . In this study, MAT has been purified to homogeneity from the archaeon Methanococcus jannaschii, and the gene encoding it has been identified by mass spectrometry . The peptide mass map identifies the gene encoding MAT as MJ1208, a hypothetical open reading frame . The gene was cloned in Escherichia coli, and expressed enzyme has been purified and characterized . This protein has only 22 and 23% sequence identity to the E . coli and human enzymes, respectively, whereas those are 59% identical to each other . The few identical residues include the majority of those constituting the polar active site residues . Each complete archaeal genome sequence contains a homolog of this archaeal-type MAT . Surprisingly, three bacterial genomes encode both the archaeal and eukaryal/bacterial types of MAT . This identification of a second major class of MAT emphasizes the long evolutionary history of the archaeal lineage and the structural diversity found even in crucial metabolic enzymes. J Biol Chem, 2000 Feb 11, 275(6), 3970 - 6 Rad51 uses one mechanism to drive DNA strand exchange in both directions; Namsaraev EA et al.; The Rad51 protein of Saccharomyces cerevisiae, like its bacterial counterpart RecA, promotes strand exchange between circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in vitro . However, the two proteins differ in the requirement for initiating joint molecules and in the polarity of branch migration . Whereas RecA initiates joint molecules from any type of ends on the dsDNA and branch migration proceeds exclusively in the 5'- to 3'-direction with respect to the single strand DNA substrate, initiation mediated by Rad51 requires a complementary 3' or 5' overhanging end of the linear dsDNA and branch migration proceeds in either direction . Here we report that the rates of Rad51-mediated branch migration in either the 5'- to 3'- or 3'- to 5'-directions are affected to the same extent by temperature and MgCl(2) . Furthermore, branch migration in both directions is equally impeded by insertions of non-homologous sequences in the dsDNA, inserts of 6 base pairs or more being completely inhibitory . We have also found that the preference of strand exchange in the 5'- to 3'-direction does not change if RPA is replaced by Escherichia coli SSB or T4 gene 32 proteins, suggesting that the preference for the direction of strand exchange is intrinsic to Rad51 . Based on these results, we conclude that Rad51-promoted branch migration in either direction occurs fundamentally by the same mechanism, quite probably by stabilizing successively formed heteroduplex base pair. J Biol Chem, 2000 Feb 11, 275(6), 3931 - 5 Invariance of the nucleoside triphosphate pools of Escherichia coli with growth rate; Petersen C et al.; The ATP and GTP pools of Escherichia coli have recently been reported to increase approximately 10-fold with increasing growth rates in the range from 0.4 to 1.4 generations/hour (Gaal, T., Bartlett, M . S., Ross, W., Turnbough, C . L., and Gourse, R . L . (1997) Science 278, 2092-2097) . Moreover, it was proposed that this variation of the nucleotide pools, particularly the ATP pool, might be responsible for the well known growth rate-dependent regulation of rRNA synthesis in E . coli . To test this hypothesis we have measured the nucleoside triphosphate pools as a function of growth rate for several E . coli strains . We found that the size of all four RNA precursor pools are essentially invariant with growth rate, in the range from 0.5 to 2.3 generations/hour . Nevertheless we observed the expected growth rate-dependent increase of RNA accumulation in these strains . In light of these results, it seems unlikely that nucleotide pool variations should be responsible for the growth rate-dependent regulation of rRNA synthesis. J Biol Chem, 2000 Feb 11, 275(6), 3873 - 8 The ATP hydrolytic activity of purified ZntA, a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli; Sharma R et al.; ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II) . ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity . The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized . The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione . The maximal ATPase activity of ZntA was approximately 3 micromol/(mg x min) obtained with the Pb(II)-thiolate complex . In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo . These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux . Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date. J Biol Chem, 2000 Feb 11, 275(6), 3737 - 40 Formation of the Ras dimer is essential for Raf-1 activation; Inouye K et al.; Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood . In this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cell-free system, when incorporated into liposome . Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively . These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1 . To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome . A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y . (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras . Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1 . From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation. Comp Immunol Microbiol Infect Dis, 2000 Jan, 23(1), 1 - 7 Serotypes of verotoxin-producing (Shiga toxin-producing) Escherichia coli isolated from healthy sheep; Bettelheim KA et al.; Using PCR techniques Shiga toxin-producing strains of Escherichia coli were isolated from the faeces of 45 out of 101 healthy sheep . These strains were serotyped and found to include O5:H-, O91:H- and O163:H19, which had previously been reported as being associated with human disease including haemolytic uraemic syndrome. APMIS, 1999 Dec, 107(12), 1069 - 78 Serological response to Helicobacter pylori recombinant antigens in Chilean infected patients with duodenal ulcer, non-ulcer dyspepsia and gastric cancer; Opazo P et al.; We have previously cloned 10 Helicobacter pylori antigen genes from a Chilean strain including: cytotoxin VacA, a truncated region of CagA (called A17), a species-specific protein (Ag26), urease subunits (UreA, UreB), a flagellin, (FlaB), heat shock proteins (HspA and HspB), an adhesin (HpaA) and a lipoprotein (Lpp20) . Immunogenicity of these antigens was tested by immunoblot with sera of Chilean infected patients, revealing that HpaA, A17, HspB and VacA were more frequently recognized (86%, 82%, 68% and 68%, respectively) . According to the clinical condition, it was determined that Lpp20 was preferentially recognized by sera from non-ulcer dyspepsia patients (80%), A17 and VacA by patients with duodenal ulcer (92% and 83% respectively), and HspB by patients with duodenal ulcer (83%) and gastric cancer (90%) . An ELISA was developed with a purified mixture of A17 and VacA antigens to test the different groups of patients . It was found that sera from duodenal ulcer patients showed higher values than those from non-ulcer dyspepsia patients, but this difference was not significant (p<0.2) . Moreover, sera from gastric cancer patients showed values lower than those from non-ulcer dyspepsia patients (p<0.019) . These results indicate that, in the Chilean population, antibodies raised against VacA and A 7 are not markers either for duodenal ulcer or for gastric cancer. Mol Gen Genet, 2000 Jan, 262(6), 1052 - 9 In vitro recruitment of the RfaH regulatory protein into a specialised transcription complex, directed by the nucleic acid ops element; Bailey MJ et al.; An unusual regulatory mechanism that controls transcription elongation in long fertility and virulence operons in bacteria is effected by two specialised components, the RfaH protein and the nucleic acid ops element . Without direct interaction, ops acts to reduce the concentration of RfaH required to stimulate distal gene transcription, and we have proposed that ops recruits RfaH to the transcription machinery . To provide direct experimental evidence for this view, we used gel fitration to identify potential RfaH complexes assembled in Escherichia coli cell extracts that carry out RfaH-dependent transcription . This novel molecular weight shift assay revealed that RfaH-dependent transcription elongation occurs concomitantly with recruitment of RfaH into a high molecular weight transcription complex, and that this recruitment is specifically directed by the ops element . Assembly of this complex required RNA polymerase and nucleotide hydrolysis, but not processive transcription . Neither assembly of the complex nor RfaH-dependent transcription was observed in in vitro reactions containing only ops, RfaH and purified core (alphabetabeta') RNA polymerase; both processes required the combination of subcellular fractions containing the RNA polymerase complex, the cytoplasmic membrane and ribosomes . The data confirm that the ops element directs recruitment of RfaH into a multi-component RNA polymerase complex that resists transcription termination. J Dairy Sci, 2000 Jan, 83(1), 48 - 51 Short communication: effects of isolation stress on mammary tight junctions in lactating dairy cows; Stelwagen K et al.; Eighteen cows had been selected for their responsiveness to psychological stress during the first lactation and were classified as having low (n = 10) or high (n = 8) cortisol concentrations in response to isolation-induced stress . In the present study these cows, now in their second lactation, were used to determine the effect of social isolation stress on the permeability of mammary tight junctions . During the experiment, each cow was isolated from the rest of the herd for 55 h . After the 1st h of isolation, each cow received a bolus infusion of endotoxin in one hind quarter in order to challenge tight junctions . Blood samples were taken throughout to measure lactose, which was used as an indicator of tight-junction leakiness . After 1 h of isolation, stress caused an increase in tight junction permeability in both groups, which was further enhanced by the endotoxin treatment . Although the permeability did not differ significantly between the two groups, it was consistently higher in the high-cortisol group, which was also the most stress-responsive group . Thus, psychological stress may adversely affect milk quality by allowing serum components to leak into milk. Nature, 2000 Jan 20, 403(6767), 335 - 8 A synthetic oscillatory network of transcriptional regulators; Elowitz MB et al.; Networks of interacting biomolecules carry out many essential functions in living cells, but the 'design principles' underlying the functioning of such intracellular networks remain poorly understood, despite intensive efforts including quantitative analysis of relatively simple systems . Here we present a complementary approach to this problem: the design and construction of a synthetic network to implement a particular function . We used three transcriptional repressor systems that are not part of any natural biological clock to build an oscillating network, termed the repressilator, in Escherichia coli . The network periodically induces the synthesis of green fluorescent protein as a readout of its state in individual cells . The resulting oscillations, with typical periods of hours, are slower than the cell-division cycle, so the state of the oscillator has to be transmitted from generation to generation . This artificial clock displays noisy behaviour, possibly because of stochastic fluctuations of its components . Such 'rational network design may lead both to the engineering of new cellular behaviours and to an improved understanding of naturally occurring networks. J Steroid Biochem Mol Biol, 1999 Dec 15, 71(3-4), 123 - 31 Correlation between PAP-dependent steroid binding activity and substrate specificity of mouse and human estrogen sulfotransferases; Qian Y et al.; Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as an activated sulfate donor . A finding of undetermined significance in the study of EST has been that the guinea pig EST is able to bind pregnenolone and estradiol with high affinity in the presence of PAP, the reaction by-product of the sulfate donor PAPS . This finding has raised the possibility that EST may have other physiological functions independent of its enzymatic activity as a sulfotransferase . To determine if the PAP-dependent steroid binding activity is a common property shared by other estrogen sulfotransferases, we have expressed the mouse and human EST in bacteria and used the purified protein to address this question . We found that, in the presence of PAP, both recombinant mouse and human EST were able to bind estradiol with high affinity but only the human EST was able to bind pregnenolone . In addition, we show that human but not the mouse EST was also able to bind dehydroepiandrosterone, a property that was not described for the guinea pig EST . Furthermore, we demonstrate that the promiscuity of human EST in steroid binding is mirrored by a correspondingly low substrate specificity in its enzymatic activity as a sulfotransferase . Reversely, the lack of stable binding of pregnenolone and dehydroepiandrosterone by the mouse EST is paralleled by a lack of sulfotransferase activity of this enzyme toward these two steroids . Mutagenesis of mouse EST within a domain critical for PAPS binding abolished both its sulfotransferase and PAP-dependent estrogen binding activity . These data suggest that stable binding of steroids such as pregnenolone or estrogen is not an independent property of estrogen sulfotransferases but rather is related to their catalytic activity. Adv Exp Med Biol, 1999, 473, 163 - 71 Ultrastructure and DNA fragmentation analysis of arterioles in swine infected with Shiga toxin-producing Escherichia coli; Matise I et al.; Shiga toxins (Stx) produced by E . coli are potent cytotoxins that affect the vascular system . In humans, systemic toxemia causes renal glomerular damage manifested as hemolytic uremic syndrome . In swine, Stx-producing E . coli (STEC) cause edema disease that is characterized microscopically by segmental arteriolar smooth muscle cell (SMC) lesions . Our objectives were to characterize ultrastructurally and by TUNEL the type of death (apoptosis or necrosis) that occurs in SMCs during edema disease . Increased DNA fragmentation consistent with apoptosis was detected by TUNEL in arterioles of challenged pigs 14-15 days post inoculation . Ultrastructurally 3 grades of SMC lesions were distinguished: 1) Partial loss of SMCs, intercellular space filled with granular cellular debris admixed with membrane bound vacuoles; 2) Complete loss of SMCs; only granular cellular debris and clear vacuoles remained within basement membrane; 3) Inflammation of media; SMCs replaced by a rim of cellular debris located in the periphery of vessel wall . The most common lesion detected was grade 1 (9 ilea and 4 brains) . We did not find apoptotic nuclear changes in SMCs or apoptotic inclusion bodies within resident cells . Our study indicates, that (1) Stx produced during edema disease does not cause SMC apoptosis 14-15 dpi; (2) SMCs undergo an array of changes from degeneration to necrosis. Adv Exp Med Biol, 1999, 473, 147 - 54 K88 adhesins of enterotoxigenic Escherichia coli and their porcine enterocyte receptors; Francis DH et al.; The three antigenic variants of the K88 fimbrial adhesin (K88ab, K88ac, and K88ad) of enterotoxigenic Escherichia coli (ETEC) each exhibit unique specificity with regard to their hemagglutination characteristics . The variants are also unique in the specificity of their binding to the brush borders of enterocytes isolated from pigs with different genetic backgrounds . Diversity in enterocyte binding specificity suggests the existence of several K88 receptors, expressed individually or in various combinations on porcine enterocytes . Three candidate receptors have been identified that may explain the adhesion of K88 fimbrial variants to various porcine enterocytes . These receptors are an intestinal mucin-type sialoglycoprotein (IMTGP), an intestinal transferrin (GP74), and an intestinal neutral glycosphingolipid (IGLad) . The IMTGP binds K88ab and K88ac, but not K88ad . The GP74 binds K88ab, but not K88ac or K88ad, and the IGLad binds K88ad, but not K88ab or K88ac . Each of the candidate receptors has been found in brush borders that are adhesive for the fimbriae that bind the respective receptor . They have not been found in brush borders that are not adhesive for those same fimbriae . The presence of IMTGP was highly correlated with susceptibility of neonatal gnotobiotic pigs to ETEC expressing K88ab or K88ac. Adv Exp Med Biol, 1999, 473, 137 - 45 Age-dependent variation in the density and affinity of Escherichia coli heat-stable enterotoxin receptors in mice; al-Majali AM et al.; Enterotoxigenic strains of Escherichia coli that produce heat-stable enterotoxin (STa), are a major cause of diarrheal disease worldwide . Resistance to diarrheal disease in human infants and newborn animals has been attributed to a gradual turnover in the intestinal brush border membrane receptors to bacterial pili . In this study, we demonstrated age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa . Flow cytometry and radiolabeled-STa (125I-STa) assays were used as more reliable quantitative measures for the characterization of STa-enterocyte receptor interaction . These assays indicated a stronger interaction of STa with its putative receptor on the enterocytes of the 2-day-old suckling mice than with enterocytes from 1-week, 2-week and 2-month-old mice . Scatchard plot analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at a higher number on enterocytes from the 2-day-old mice than enterocytes of the older mice . Additionally, receptors from the 2-day-old mice had a greater affinity for STa ligand than receptors from the older mice . Density of STa receptors on enterocytes and their affinity to STa may determine the extent of binding and severity of secretory response . This may further explain the increased susceptibility of newborn animals and human infants to STa-mediated diarrheal disease. Adv Exp Med Biol, 1999, 473, 129 - 36 The locus for enterocyte effacement (LEE) of enteropathogenic Escherichia coli (EPEC) from dogs and cats; Goffaux F et al.; Enteropathogenic Escherichia coli (EPEC) produce attaching and effacing lesions . The genes responsible for this lesion are clustered on the chromosome forming a 35.5 kilobase pathogenesis island called LEE . The LEE was identified, characterized and completely sequenced from the human EPEC strain E2348/69 . The LEE carries genes coding for: a type III secretion system (genes esc and sep), the translocated intimin receptor (gene tir), the outer membrane protein intimin (gene eae) and the E . coli secreted proteins EspA, EspB, and EspD (genes esp) . In addition to man and farm animals, EPEC are also isolated from dogs and cats . We studied structurally and functionally the LEE of dog and cat EPEC . First, we used four probes scattered along the LEE to identify the presence of a LEE in canine and feline EPEC isolates . Second, by PCR, we checked the presence of genes homologous to eae, sep, esp, and tir genes in these strains . Third, since the four types of eae and tir genes were described, we developed a multiplex PCR in order to determine the type of eae and tir genes present in each strain . Fourth, we determined by PCR the site of the LEE insertion on the chromosome . Fifth, we tested several of the canine EPEC in their capacity to induce attaching and effacing lesions in the rabbit intestinal loop assay . We can conclude from this study: first, that the a LEE-like structure is present in all tested strains and that it contains genes homologous to esp, sep, tir, and eae genes; second, that there is some preferential associations between the type of eae gene and the type of tir gene present in a strain; third, that the majority of the tested strains contained a LEE located elsewhere on the chromosome in comparison to the human EPEC strain E2348/69; and fourth that dog EPEC were able to induce attaching and effacing lesions in rabbit ileal loop assay. Adv Exp Med Biol, 1999, 473, 125 - 8 Reproduction of lesions and clinical signs with a CNF2-producing Escherichia coli in neonatal calves; Van Bost S et al.; CNF2-producing necrotoxigenic E . coli (NTEC2) are associated with diarrhoea and septicaemia in calves . We orally inoculated neonatal calves with a NTEC2 strain in order to reproduce clinical signs and lesions . We observed diarrhoea in each inoculated calf, bacteraemia (80%), the presence of CNF2+ bacteria in the lungs (80%) and in the liver (20%) . The observed lesions were inflammation of the entire gut, hypertrophy of the mesenteric lymph nodes and hepatisation of the lungs . We were unable to detect characteristic lesions that are classical signs of septicaemia. Adv Exp Med Biol, 1999, 473, 113 - 23 Insulin modulates intestinal response of suckling mice to the Escherichia coli heat-stable enterotoxin; al-Majali AM et al.; Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied . Four groups (8-10 in each group) of two day old Swiss Webster suckling mice were used . Five, 10, 25, and 50 micrograms of insulin were given orally to half the mice in each group respectively . The rest of the mice in each group were given normal saline as intra-litter controls . After 7 days, the suckling mouse assay for STa was performed on three mice from each insulin-treated and control groups . Enterocyte suspensions were prepared from mice in all groups . Intestinal tissue samples were taken for electron microscopy . Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry . The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin treated groups compared to control mice (p < 0.05) . Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an upregulatory effect on the STa receptor level . Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa . Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa . This study may provide further insights into the mechanism of STa/receptor interaction in diarrhea in newborn animals and human infants. Microbiology, 2000 Jan, 146 ( Pt 1), 155 - 63 Elucidation of anthracyclinone biosynthesis by stepwise cloning of genes for anthracyclines from three different Streptomyces spp; Kantola J et al.; The anthracycline skeleton is biosynthesized by aromatic (type II) polyketide synthases . Furthermore, three post-polyketide steps are needed to form the basic aglycone of anthracyclines . Auramycinone was produced in Streptomyces lividans by introducing nine structural genes from three different anthracycline-producing Streptomyces species . The genes used to construct the auramycinone biosynthesis cluster were derived from nogalamycin-, daunomycin- and aclacinomycin-producing Streptomyces strains . The biosynthetic stages were divided into polyketide and post-polyketide steps on the assumption that the first stable intermediate would be nogalonic acid, named analogously to aklanonic acid, the precursor of several anthracyclines . Single genes were cloned in the expression construct in the order determined by the proposed biosynthetic pathway . This facilitated investigation of the products formed in the heterologous host after addition of each separate gene to the construct . The results thus elucidate the biosynthesis steps, products and the genes responsible for the reactions needed to build up an anthracyclinone. FASEB J, 2000 Feb, 14(2), 407 - 17 In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain; Huang D et al.; Our aims were to examine whether oxidative DNA damage was elevated in brain cells of male C57BL/6 mice after oxidative stress, and to determine whether neuronal nitric oxide synthase (nNOS) was involved in such damage . Oxidative stress was induced by occluding both common carotid arteries for 90 min, followed by reperfusion . Escherichia coli exonuclease III (Exo III) removes apyrimidinic or apurinic (AP) sites and 3'-phosphate termini in single-strand breaks, and converts these lesions to 3'OH termini . These ExoIII-sensitive sites (EXOSS) can then be postlabeled using digoxigenin-11-dUTP and Klenow DNA polymerase-I, and detected using fluorescein isothiocyanate-IgG against digoxigenin . Compared with the non-ischemia controls, the density of EXOSS-positive cells was elevated at least 20-fold (P < 0.01) at 15 min of reperfusion, and remained elevated for another 30 min . EXOSS mainly occurred in the cell nuclei of the astrocytes and neurons . Signs of cell death were detected at 24 h of reperfusion and occurred mostly in the neurons . Both DNA damage and cell death in the cerebral cortical neurons were abolished by treatment with 3-bromo-7-nitroindazole (30 mg/kg, intraperitoneal), which specifically inhibited nNOS . Our results suggest that nNOS, its activator (calcium), and peroxynitrite exacerbate oxidative DNA damage after brain ischemia.-Huang, D., Shenoy, A., Cui, J., Huang, W., Liu, P . In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain. Biochem J, 2000 Feb 15, 346 Pt 1, 223 - 32 Structure-function studies of tryptophan mutants of equinatoxin II, a sea anemone pore-forming protein; Malovrh P et al.; Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes . It contains five tryptophan residues in three different regions of the molecule . In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp(45), EqtII Trp(116/117) and EqtII Trp(149), were prepared in an Escherichia coli expression system {here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein} . They all possess a single intrinsic fluorescent centre . All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same . EqtII Trp(116/117) resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties . Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane . The secondary structure of the other two mutants, EqtII Trp(45) and EqtII Trp(149), was altered to a certain extent . EqtII Trp(149) was the most dissimilar from the wild-type, displaying a higher content of random-coil structure . It also retained the lowest number of nitrogen-bound protons after exchange with (2)H(2)O, which might indicate a reduced compactness of the molecule . Tryptophans in EqtII Trp(45) and EqtII Trp(149) were more exposed to water, and also remained as such in the membrane-bound form. Biochem J, 2000 Feb 15, 346 Pt 1, 163 - 8 Properties of Leishmania major dUTP nucleotidohydrolase, a distinct nucleotide-hydrolysing enzyme in kinetoplastids; Camacho A et al.; We have previously reported the presence, in the parasitic protozoan Leishmania major, of an enzyme involved in controlling intracellular dUTP levels . The gene encoding this enzyme has now been overexpressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity . Biochemical and enzymic analyses of the Leishmania enzyme show that it is a novel nucleotidohydrolase highly specific for deoxyuridine 5'-triphosphate . The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates . The reaction products are dUMP and PP(i) when dUTP is the substrate and dUMP and P(i) in the case of dUDP . The enzyme is sensitive to inhibition by the reaction product dUMP but not by PP(i) . dUTPase activity is highly dependent on Mg(2+) concentrations and markedly sensitive to the phosphatase inhibitor, NaF . In summary, Leishmania dUTPase appears to be markedly different to other proteins characterized previously that accomplish the same function. Biochem J, 2000 Feb 15, 346 Pt 1, 1 - 8 Thioredoxin reductase; Mustacich D et al.; The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases . TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds . The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR . There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified . The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated . The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form . Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR . Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases. Nat Biotechnol, 2000 Feb, 18(2), 168 - 71 Trehalose expression confers desiccation tolerance on human cells; Guo N et al.; Many organisms that withstand desiccation express the disaccharide trehalose . We have now expressed the otsA and otsB genes of Escherichia coli, which encode trehalose biosynthetic enzymes, in human primary fibroblasts using a recombinant adenovirus vector . Infected cells produced increased amounts of trehalose with increasing multiplicity of infection (MOI) . Human primary fibroblasts expressing trehalose could be maintained in the dry state for up to five days . Fourier transform infrared spectroscopy indicated that dry, but viable, human cells contained no detectable water . This study shows that mammalian cells can be engineered to retain viability in the absence of water. J Mol Biol, 2000 Feb 11, 296(1), 57 - 86 Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides; Knappik A et al.; By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used . A consensus sequence was derived for each family and optimized for expression in Escherichia coli . In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs . Molecular modeling verified that all canonical classes were present . We could show that all master genes are expressed as soluble proteins in the periplasm of E . coli . A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question . Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences . The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 % . Selection experiments against many antigens yielded a diverse set of binders with high affinities . Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries . The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes . J Mol Biol, 2000 Feb 11, 296(1), 19 - 31 Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe; Biswas R et al.; The protein subunit of Escherichia coli ribonuclease P (which has a cysteine residue at position 113) and its single cysteine-substituted mutant derivatives (S16C/C113S, K54C/C113S and K66C/C113S) have been modified using a sulfhydryl-specific iron complex of EDTA-2- aminoethyl 2-pyridyl disulfide (EPD-Fe) . This reaction converts C5 protein, or its single cysteine-substituted mutant derivatives, into chemical nucleases which are capable of cleaving the cognate RNA ligand, M1 RNA, the catalytic RNA subunit of E . coli RNase P, in the presence of ascorbate and hydrogen peroxide . Cleavages in M1 RNA are expected to occur at positions proximal to the site of contact between the modified residue (in C5 protein) and the ribose units in M1 RNA . When EPD-Fe was used to modify residue Cys16 in C5 protein, hydroxyl radical-mediated cleavages occurred predominantly in the P3 helix of M1 RNA present in the reconstituted holoenzyme . C5 Cys54-EDTA-Fe produced cleavages on the 5' strand of the P4 pseudoknot of M1 RNA, while the cleavages promoted by C5 Cys66-EDTA-Fe were in the loop connecting helices P18 and P2 (J18/2) and the loop (J2/4) preceding the 3' strand of the P4 pseudoknot . However, hydroxyl radical-mediated cleavages in M1 RNA were not evident with Cys113-EDTA-Fe, perhaps indicative of Cys113 being distal from the RNA-protein interface in the RNase P holoenzyme . Our directed hydroxyl radical-mediated footprinting experiments indicate that conserved residues in the RNA and protein subunit of the RNase-P holoenzyme are adjacent to each other and provide structural information essential for understanding the assembly of RNase P . J Mol Biol, 2000 Jan 28, 295(4), 927 - 38 X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity; Albert A et al.; The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and lithium toxicity and its overexpression improves salt tolerance in yeast and plants . Hal2p is a metabolic phosphatase which catalyses the hydrolysis of 3'-phosphoadenosine-5'-phosphate (PAP) to AMP . It is, the prototype of an evolutionarily conserved family of PAP phosphatases and the engineering of sodium insensitive enzymes of this group may contribute to the generation of salt-tolerant crops . We have solved the crystal structure of Hal2p in complex with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at 1.6 A resolution . A functional screening of random mutations of the HAL2 gene in growing yeast generated forms of the enzyme with reduced cation sensitivity . Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions determining cation sensitivity . Hal2p belongs to a larger superfamily of lithium-sensitive phosphatases which includes inositol monophosphatase . The hydrophobic interaction mutated in Hal2p is conserved in this superfamily and its disruption in human inositol monophosphatase also resulted in reduced cation sensitivity . J Mol Biol, 2000 Jan 28, 295(4), 831 - 52 Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein . Application of the matrix projection operator technique to analyze stopped-flow kinetics; Bujalowski W et al.; Kinetics of the Escherichia coli primary replicative helicase DnaB protein binding to a single-stranded DNA, in the presence of the ATP non-hydrolyzable analog AMP-PNP, have been performed, using the fluorescence stopped-flow technique . This is the first direct determination of the mechanism of the ssDNA recognition by a hexameric helicase . Binding of the fluorescent etheno-derivative of a ssDNA to the enzyme is characterized by a strong increase of the nucleic acid fluorescence, which provides an excellent signal to quantitatively study the mechanism of ssDNA recognition by the helicase . The kinetic experiments have been performed with a ssDNA 20-mer, depsilonA(pepsilonA)(19), that encompasses the entire, total ssDNA-binding site of the helicase and with the 10-mer depsilonA(pepsilonA)(9), which binds exclusively to the ssDNA strong subsite within the total ssDNA-binding site . Association of the DnaB helicase with the 20-mer is characterized by three relaxation times, which indicates that the binding occurs by the minimum three-step mechanism where the bimolecular binding step is followed by two isomerization steps . This mechanism is described by the equation: Helicase+ssDNAk1/(k1)<-->(k-1)(H-ssDNA)1(k2)<-->(k-2)(H-ssDNA)2 (k3)<-->(k-3)(H-ssDNA)3 . The value of the bimolecular rate constant, k(1), is four to six orders of magnitude lower than the value expected for the diffusion-controlled reaction . Moreover, quantitative amplitude analysis suggests that the major conformational change of the ssDNA takes place in the formation of the (H-ssDNA)(1) . These results indicate that the determined first step includes formation of the collision and an additional transition of the protein-ssDNA complex, most probably the local opening of the protein hexamer . The data indicate that the binding mechanism reflects the interactions of the ssDNA predominantly through the strong ssDNA-binding subsite . The analysis of the stopped-flow kinetics has been performed using the matrix-projection operator technique, which provides a powerful method to address stopped-flow kinetics, particularly, the amplitudes . The method allowed us to determine the specific fluorescence changes accompanying the formation of all the intermediates . The sequential nature of the determined mechanism indicates the lack of the kinetically significant conformational equilibrium of the DnaB hexamer as well as a transient dissociation of the hexamer prior to the ssDNA binding . The significance of these results for the functioning of the DnaB helicase is discussed . J Mol Biol, 2000 Jan 28, 295(4), 815 - 29 Mutations influencing the frr gene coding for ribosome recycling factor (RRF); Janosi L et al.; A total of 52 null, six reversion, and five silent mutations of frr (the gene encoding for ribosome recycling factor (RRF)) of Escherichia coli are discussed along with 12 temperature-sensitive (ts) mutations and 14 intergenic suppressor strains of ts RRF . The null mutations were classified into six different categories . A computer-based secondary structure analysis showed three domains; domain A which has the N-terminal helix, domain B which contains coil, alpha-helix and beta-strand structure, and domain C which is a C-terminal helix . The ts mutations fell into domains A and C but not in domain B . More than a half of the null mutations fell into domain B while the silent mutations fell outside domain B . Substitution of Arg132 in domain C by other amino acids was observed among five independently isolated null mutants . It is suggested that domain B is important for maintaining the RRF structure, while the region including Arg132 is one of the active sites . A total of 14 intergenic suppressor strains of ts RRF were grouped into four categories, depending on which temperature-sensitive alleles were suppressed . J Mol Biol, 2000 Jan 28, 295(4), 803 - 14 DNA polymerase switching: II . Replication factor C abrogates primer synthesis by DNA polymerase alpha at a critical length; Mossi R et al.; A crucial event in DNA replication is the polymerase switch from the synthesis of a short RNA/DNA primer by DNA polymerase alpha/primase to the pro?cessive elongation by DNA polymerase delta . In order to shed light on the role of replication factor C (RF-C) in this process, the effects of RF-C on DNA polymerase alpha were investigated . We show that RF-C stalls DNA polymerase alpha after synthesis of approximately 30 nucleotides, while not inhibiting the polymerase activity per se . This suggested that RF-C and the length of the primer may be two important factors contributing to the polymerase switch . Furthermore the DNA binding properties of RF-C were tested . Band shift experiments indicated that RF-C has a preference for 5' recessed ends and double-stranded DNA over 3' ends . Finally PCNA can be loaded onto a DNA template carrying a RNA primer, suggesting that a DNA moiety is not necessarily required for the loading of the clamp . Thus we propose a model where RF-C, upon binding to the RNA/DNA primer, influences primer synthesis and sets the conditions for a polymerase switch after recruiting PCNA to DNA . J Mol Biol, 2000 Jan 28, 295(4), 777 - 89 Probing a tRNA core that contributes to aminoacylation; Hamann CS et al.; The contribution of the tRNA "core" to aminoacylation is beginning to be recognized . One example is the core region of Escherichia coli tRNA(Cys), which has been shown by biochemical studies to be important for aminoacylation . This core has several layers of unusual base-pairs, which are revealed by the recent crystal structure of the tRNA complexed with the elongation factor EF-Tu and an analog of GTP . One of these layers consists of a 9:{13:22} base-triple, rather than the 46:{13:22} or 45:{13:22} base-triple that is commonly observed in tRNA structure . Because 13:22 is an important element in aminoacylation of E . coli tRNA(Cys), a better understanding of its structure in the tRNA core will shed light on its role in aminoacylation . In this study, we used the phage T7 transcript of the tRNA as a substrate . We probed the structure of 13:22 by dimethyl sulfate and tested its partner in a base-triple by generating mutations that could be assayed for aminoacylation . The results of this study in general are in a better agreement with a 46:{13:22} base-triple that we previously proposed . Although these results are not interpreted as direct proof for the 46:{13:22} base-triple, they shed new light on features of the tRNA core that are important for aminoacylation . J Mol Biol, 2000 Jan 28, 295(4), 767 - 75 A DNA-binding domain swap converts the invertase gin into a resolvase; Schneider F et al.; DNA resolvases and invertases are closely related, yet catalyze recombination within two distinct nucleoprotein structures termed synaptosomes and invertasomes, respectively . Different protein-protein and protein-DNA interactions guide the assembly of each type of recombinogenic complex, as well as the subsequent activation of DNA strand exchange . Here we show that invertase Gin catalyzes factor for inversion stimulation dependent inversion on isolated copies of sites I from ISXc5 res, which is typically utilized by the corresponding resolvase . The concomitant binding of Gin to sites I and III in res, however, inhibits recombination . A chimeric recombinase, composed of the catalytic domain of Gin and the DNA-binding domain of ISXc5 resolvase, recombines two res with high efficiency . Gin must therefore contain residues proficient for both synaptosome formation and activation of strand exchange . Surprisingly, this chimera is unable to assemble a productive invertasome; a result which implies a role for the C-terminal domain in invertasome formation that goes beyond DNA binding . J Mol Biol, 2000 Jan 28, 295(4), 737 - 44 Localized, stereochemically sensitive hydrophobic packing in an early folding intermediate of dihydrofolate reductase from Escherichia coli; O'Neill JC Jr et al.; Mutational analysis was performed to probe the development of hydrophobic clusters during the early events in the folding of dihydrofolate reductase . Replacements were made in several hydrophobic subdomains to examine the roles of hydrophobicity and stereochemistry in the formation of kinetic intermediates . Amide protons in two of these clusters, including residues I91, I94, and I155, have been shown to be protected against solvent exchange within 13 ms of folding . Additional hydrophobic clusters were probed by substitutions at residues I2, I61, and L112; these residues are not protected from exchange until later in the folding reaction . Valine and leucine replacements at positions I91, I94, and I155 significantly diminish the formation of the burst phase kinetic intermediate, relative to the wild-type protein . In contrast, I2 and I61 are insensitive to these substitutions in the first 5 ms of the folding reaction, as is the replacement of L112 with either isoleucine or valine . These results demonstrate that the tightly packed core of dihydrofolate reductase is acquired in a non-uniform fashion, beginning in the submillisecond time frame . The progressive development of specific side-chain packing in localized hydrophobic clusters may be a common theme for complex protein folding reactions . Am Nat, 2000 Jan, 155(1), 24 - 35 Long-Term Experimental Evolution in Escherichia coli . VIII . Dynamics of a Balanced Polymorphism; Rozen DE et al.; We describe the short- and long-term dynamics of a phenotypic polymorphism that arose in a population of Escherichia coli while it was serially propagated for almost 20,000 generations in a glucose-limited minimal medium . The two types, designated L and S, differ conspicuously in the size of the colonies they form on agar plates as well as the size of their individual cells, and these differences are heritable . The S type reached a detectable frequency (>1%) at generation 6,000, and it remained above that frequency throughout the subsequent generations . In addition to morphological differences, L and S diverged in important ecological properties . With clones isolated at 18,000 generations, L has a maximal growth rate in fresh medium that is approximately 20% higher than that of S . However, experiments with conditioned media demonstrate that L and S secrete one or more metabolites that promote the growth of S but not of L . The death rate of L during stationary phase also increases when S is abundant, which suggests that S may either secrete a metabolite that is toxic to L or remove some factor that enables the survival of L . One-day competition experiments with the clones isolated at generation 18,000 show that their relative fitness is frequency dependent, with each type having an advantage when rare . When these two types are grown together for a period of several weeks, they converge on an equilibrium frequency that is consistent with the 1-d competition experiments . Over the entire 14,000-generation period of coexistence, however, the frequency of the S type fluctuated between approximately 10% and 85% . We offer several hypotheses that may explain the fluctuations in this balanced polymorphism, including the possibility of coevolution between the two types. Mech Ageing Dev, 1999 Nov, 111(2-3), 89 - 95 Role of superoxide, NO and oxygen in the regulation of energy metabolism and suppression of senile diseases; Inoue M et al.; Although nitric oxide (NO) rapidly reacts with molecular oxygen under air atmospheric conditions, thereby losing its biological functions, the lifetime of this gaseous radical increases under physiologically low intracellular oxygen tensions . To understand the pathophysiological roles of NO and related molecules in aerobic life, we analyzed the effect of oxygen tensions on the NO-dependent processes in resistance arteries, isolated mitochondria, intact cells and enteric bacteria . Kinetic analysis revealed that NO enhanced the generation of cGMP and induced vasorelaxation of resistance arteries more potently under physiologically low oxygen tensions than under hyperbaric conditions . NO reversibly inhibited the respiration of isolated mitochondria, intact cells and Escherichia coli; the inhibitory effect was more marked under hypoxic conditions than under hyperbaric conditions . Kinetic analysis revealed that NO has pivotal action to increase arterial supply of molecular oxygen for the generation of ATP in peripheral tissues and to suppress energy production in mitochondria and cells in an oxygen-dependent manner . These functions of NO are enhanced by decreasing oxygen tension in situ and suppressed by locally generated superoxide radicals . Thus, cross-talk of NO, superoxide and molecular oxygen constitutes a supersystem by which the energy metabolism in cells and tissues is beautifully regulated in a site-specific manner depending on the relative concentrations of these three radical species. Phytochemistry, 2000 Jan, 53(1), 55 - 8 Antifungal nitro compounds from skunk cabbage (Lysichitum americanum) leaves treated with cupric chloride; Hanawa F et al.; Two nitro compounds, 2-(4-methoxyphenyl)-1-nitroethane named as lysichitalexin and 2-(4-hydroxyphenyl)-1-nitroethane were isolated as stress metabolites from the leaves of Lysichitum americanum Hulten and St . John treated with cupric chloride . Their structures were determined by spectroscopic methods and chemical reactions . The former compound showed antifungal activities against Fusarium oxysporum and Cladosporium herbarum . Both compounds were isolated for the first time from this species and the former was isolated from natural sources for the first time . This is the first report on stress metabolites from a member of the Araceae. J Biotechnol, 2000 Jan 21, 76(2-3), 215 - 26 Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein; Jonasson P et al.; An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product . The process has been used for production in E . coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer . The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm . A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium . This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein . Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues . The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography . The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56% . The suitability of this process also for the production of other recombinant proteins is discussed. Free Radic Biol Med, 2000 Jan 1, 28(1), 55 - 63 DNA damage in arsenite- and cadmium-treated bovine aortic endothelial cells; Liu F et al.; Reactive oxygen species have been shown to be involved in the mutagenicity, clastogenicity, and apoptosis of mammalian cells treated with arsenic or cadmium . As these endpoints require several hours of cellular processing, it is not clear that reactive oxygen species damage DNA directly or interfere with DNA replication and repair . Using single-cell alkaline electrophoresis, we have detected DNA strand breaks (DSBs) in bovine aortic endothelial cells by a 4-h treatment with sodium arsenite (As) and cadmium chloride (Cd) in sublethal concentrations . As-induced DSBs could be decreased by nitric oxide (NO) synthase inhibitors, superoxide scavengers, and peroxynitrite scavengers and could be increased by superoxide generators and NO generators . Treatment with As also increased nitrite production . These results suggest that As-increased NO may react with O2*- to produce peroxynitrite and cause DNA damage . The results showing that Cd increased cellular H2O2 levels and that Cd-induced DSBs could be modulated by various oxidant modulators suggest that Cd may induce DSBs via O2*-, H2O2, and *OH . Nevertheless, the DSBs in both As- and Cd-treated cells seem to come from the excision of oxidized bases such as formamidopyrimidine and 8-oxoguanine, as the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg) increased DSBs in cells treated with As, 3-morpholinosydnonimine (a peroxynitrite-generating agent), Cd, or H2O2. Nat Struct Biol, 2000 Feb, 7(2), 108 - 13 Crystal structures of Escherichia coli phytase and its complex with phytate; Lim D et al.; Phytases catalyze the hydrolysis of phytate and are able to improve the nutritional quality of phytate-rich diets . Escherichia coli phytase, a member of the histidine acid phosphatase family has the highest specific activity of all phytases characterized . The crystal structure of E . coli phytase has been determined by a two-wavelength anomalous diffraction method using the exceptionally strong anomalous scattering of tungsten . Despite a lack of sequence similarity, the structure closely resembles the overall fold of other histidine acid phosphatases . The structure of E . coli phytase in complex with phytate, the preferred substrate, reveals the binding mode and substrate recognition . The binding is also accompanied by conformational changes which suggest that substrate binding enhances catalysis by increasing the acidity of the general acid. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1062 - 7 Biosynthesis of terpenoids: YchB protein of Escherichia coli phosphorylates the 2-hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol; Luttgen H et al.; A comparative analysis of all published complete genomes indicated that the putative orthologs of the unannotated ychB gene of Escherichia coli follow the distribution of the dxs, dxr, and ygbP genes, which have been shown to specify enzymes of the deoxyxylulose phosphate pathway of terpenoid biosynthesis, thus suggesting that the hypothetical YchB protein also is involved in that pathway . To test this hypothesis, the E . coli ychB gene was expressed in a homologous host . The recombinant protein was purified to homogeneity and was shown to phosphorylate 4-diphosphocytidyl-2C-methyl-D-erythritol in an ATP-dependent reaction . The reaction product was identified as 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate by NMR experiments with various (13)C-labeled substrate samples . A (14)C-labeled specimen of this compound was converted efficiently into carotenoids by isolated chromoplasts of Capsicum annuum . The sequence of E . coli YchB protein is similar to that of the protein predicted by the tomato cDNA pTOM41 (30% identity), which had been implicated in the conversion of chloroplasts to chromoplasts. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1002 - 7 Optimization of the helper-dependent adenovirus system for production and potency in vivo; Sandig V et al.; Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences provide for safe and highly efficient gene transfer with long-lasting transgene expression . High titer stocks of HD vectors can be generated by using the cre-recombinase system . However, we have encountered difficulties with this system, including rearranged HD vectors and variable efficiency of HD vector rescue . These problems represent a major hindrance, particularly with regard to large-scale production . To overcome these limitations, we have modified the system in two ways: We constructed a new helper virus with a modified packaging signal and enhanced growth characteristics . We also redesigned the vector backbones by including noncoding adenovirus sequences adjacent to the right inverted terminal repeat and by incorporated a number of different segments of noncoding DNA of human origin as "stuffer." Comparison of these vectors showed that the nature of the stuffer sequence affects replication of the HD vector . Optimization of the system resulted in a more robust and consistent production of HD vectors with low helper contamination and high in vivo potency. Genetics, 2000 Feb, 154(2), 533 - 42 Mechanisms of dinucleotide repeat instability in Escherichia coli; Bichara M et al.; The high level of polymorphism of microsatellites has been used for a variety of purposes such as positional cloning of genes associated with diseases, forensic medicine, and phylogenetic studies . The discovery that microsatellites are associated with human diseases, not only as markers of risk but also directly in disease pathogenesis, has triggered a renewed interest in understanding the mechanism of their instability . In this work we have investigated the role of DNA replication, long patch mismatch repair, and transcription on the genetic instability of all possible combinations of dinucleotide repeats in Escherichia coli . We show that the (GpC) and (ApT) self-complementary sequence repeats are the most unstable and that the mode of replication plays an important role in their instability . We also found that long patch mismatch repair is involved in avoiding both short deletion and expansion events and also in instabilities resulting from the processing of bulges of 6 to 8 bp for the (GpT/ApC)- and (ApG/CpT)- containing repeats . For each dinucleotide sequence repeat, we propose models for instability that involve the possible participation of unusual secondary structures. Genetics, 2000 Feb, 154(2), 513 - 22 Palindromes as substrates for multiple pathways of recombination in Escherichia coli; Cromie GA et al.; A 246-bp imperfect palindrome has the potential to form hairpin structures in single-stranded DNA during replication . Genetic evidence suggests that these structures are converted to double-strand breaks by the SbcCD nuclease and that the double-strand breaks are repaired by recombination . We investigated the role of a range of recombination mutations on the viability of cells containing this palindrome . The palindrome was introduced into the Escherichia coli chromosome by phage lambda lysogenization . This was done in both wt and sbcC backgrounds . Repair of the SbcCD-induced double-strand breaks requires a large number of proteins, including the components of both the RecB and RecF pathways . Repair does not involve PriA-dependent replication fork restart, which suggests that the double-strand break occurs after the replication fork has passed the palindrome . In the absence of SbcCD, recombination still occurs, probably using a gap substrate . This process is also PriA independent, suggesting that there is no collapse of the replication fork . In the absence of RecA, the RecQ helicase is required for palindrome viability in a sbcC mutant, suggesting that a helicase-dependent pathway exists to allow replicative bypass of secondary structures. Genetics, 2000 Feb, 154(2), 503 - 12 Antagonism of ultraviolet-light mutagenesis by the methyl-directed mismatch-repair system of Escherichia coli; Liu H et al.; Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2.MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to "matched" photoproducts in DNA . We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E . coli lacZ codon 461, which revert, respectively, via CCC --> CTC and CTT --> CTC transitions . F' lacZ targets were mated from mut(+) donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation . In umu(+) mut(+) recipients, a range of UV fluences induced lac(+) revertant frequencies of 4-25 x 10(-8); these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients . Since this effect on mutation frequency was unaltered by an Mfd(-) defect, it appears not to involve transcription-coupled excision repair . In mut(+) umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m(2)) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5-10 x 10(-8) . Thus, at UV doses too low to induce SOS functions, such as Umu(2)'D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial. Br J Ophthalmol, 2000 Feb, 84(2), 205 - 11 Apoptosis is a prominent feature of acute anterior uveitis in the Fischer 344 rat; Smith JR et al.; AIMS: To examine the hypothesis that apoptosis of infiltrating cells contributes to spontaneous resolution of uveitis in clinically relevant rodent models . METHODS: Experimental melanin induced uveitis (EMIU) was induced in Fischer 344 rats by immunisation with 250 microg bovine ocular melanin . Endotoxin induced uveitis (EIU) was induced by injection of 200 microg Escherichia coli lipopolysaccharide . Formalin fixed, paraffin embedded ocular cross sections were stained by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL) to identify apoptotic cells . Indirect immunoperoxidase staining of paraformaldehyde lysine periodate fixed tissue cross sections was used to demonstrate expression of inducible nitric oxide synthase (iNOS) . RESULTS: TUNEL positive mononuclear cells were observed in the anterior uvea during both EMIU and EIU at all selected time points . However, whereas the majority of mononuclear cells appeared apoptotic from the outset of disease, neutrophils were notably TUNEL negative at all time points examined . Many infiltrating neutrophils expressed iNOS . CONCLUSION: Apoptosis occurs early in the course of rat EMIU and EIU, and may contribute to resolution of these diseases . In general, infiltrating mononuclear cells die rapidly, while neutrophils survive, producing inducible nitric oxide synthase which may contribute to disease pathogenesis. Immunol Lett, 1999 Dec 1, 70(3), 151 - 5 Expression of an epitopic region of AspfI, an allergen/antigen/cytotoxin of Aspergillus fumigatus; Sarma PV et al.; The gene for an 18 kD allergen/cytotoxin of Aspergillus fumigatus was cloned in pUC-19 vector and expressed in Escherichia coli JM109 . Digestion of this gene with AluI resulted in four fragments of 216bp, 120bp, 39bp and 21bp . These fragments were cloned in the Sma-I site of pUC-19 . The recombinants thus, generated after transformation in E . coli JM109, were screened using monoclonal antibodies raised against the AspfI . The fusion protein containing 120 bp AluI fragment was recognised by the MoAb indicating presence of epitope(s) in the 120 bp region . The study indicates a viable strategy for identification and expression of an immunologically active domain of a major allergen/antigen of A . fumigatus for the first time. Nat Med, 2000 Feb, 6(2), 164 - 70 Protection from septic shock by neutralization of macrophage migration inhibitory factor; Calandra T et al.; Identification of new therapeutic targets for the management of septic shock remains imperative as all investigational therapies, including anti-tumor necrosis factor (TNF) and anti-interleukin (IL)-1 agents, have uniformly failed to lower the mortality of critically ill patients with severe sepsis . We report here that macrophage migration inhibitory factor (MIF) is a critical mediator of septic shock . High concentrations of MIF were detected in the peritoneal exudate fluid and in the systemic circulation of mice with bacterial peritonitis . Experiments performed in TNFalpha knockout mice allowed a direct evaluation of the part played by MIF in sepsis in the absence of this pivotal cytokine of inflammation . Anti-MIF antibody protected TNFalpha knockout from lethal peritonitis induced by cecal ligation and puncture (CLP), providing evidence of an intrinsic contribution of MIF to the pathogenesis of sepsis . Anti-MIF antibody also protected normal mice from lethal peritonitis induced by both CLP and Escherichia coli, even when treatment was started up to 8 hours after CLP . Conversely, co-injection of recombinant MIF and E . coli markedly increased the lethality of peritonitis . Finally, high concentrations of MIF were detected in the plasma of patients with severe sepsis or septic shock . These studies define a critical part for MIF in the pathogenesis of septic shock and identify a new target for therapeutic intervention. Nat Genet, 2000 Feb, 24(2), 188 - 91 Mutations in the gene encoding peroxisomal alpha-methylacyl-CoA racemase cause adult-onset sensory motor neuropathy; Ferdinandusse S et al.; Sensory motor neuropathy is associated with various inherited disorders including Charcot-Marie-Tooth disease, X-linked adrenoleukodystrophy/adrenomyeloneuropathy and Refsum disease . In the latter two, the neuropathy is thought to result from the accumulation of specific fatty acids . We describe here three patients with elevated plasma concentrations of pristanic acid (a branched-chain fatty acid) and C27-bile-acid intermediates . Two of the patients suffered from adult-onset sensory motor neuropathy . One patient also had pigmentary retinopathy, suggesting Refsum disease, whereas the other patient had upper motor neuron signs in the legs, suggesting adrenomyeloneuropathy . The third patient was a child without neuropathy . In all three patients we discovered a deficiency of alpha-methylacyl-CoA racemase (AMACR) . This enzyme is responsible for the conversion of pristanoyl-CoA and C27-bile acyl-CoAs to their (S)-stereoisomers, which are the only stereoisomers that can be degraded via peroxisomal beta-oxidation . Sequence analysis of AMACR cDNA from the patients identified two different mutations that are likely to cause disease, based on analysis in Escherichia coli . Our findings have implications for the diagnosis of adult-onset neuropathies of unknown aetiology. EMBO J, 2000 Feb 1, 19(3), 400 - 9 The organized chromatin domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome; Ducker CE et al.; In yeast alpha cells the a cell-specific genes STE6 and BAR1 are packaged as gene-sized chromatin domains of positioned nucleosomes . Organized chromatin depends on Tup1p, a corepressor that interacts with the N-terminal regions of H3 and H4 . If Tup1p functions to organize or stabilize a chromatin domain, the protein might be expected to be present at a level stoichiometric with nucleosomes . Chromatin immunoprecipitation assays using Tup1p antibodies showed Tup1p to be associated with the entire genomic STE6 coding region . To determine stoichiometry of Tup1p associated with the gene, a yeast plasmid containing varying lengths of the STE6 gene including flanking control regions and an Escherichia coli lac operator sequence was constructed . After assembly into chromatin in vivo in Saccharomyces cerevisiae, minichromosomes were isolated using an immobilized lac repressor . In these experiments, Tup1p was found to be specifically associated with repressed STE6 chromatin in vivo at a ratio of about two molecules of the corepressor per nucleosome . These observations strongly suggest a structural role for Tup1p in repression and constrain models for organized chromatin in repressive domains. Pediatr Nephrol, 2000 Jan, 14(1), 73 - 83 Enterohemorrhagic Escherichia coli infections: following transmission routes; Verweyen HM et al.; Infections with enterohemorrhagic Escherichia coli (EHEC) are the major cause of hemolytic-uremic syndrome (HUS), the most-common cause of acute renal failure in childhood . The mortality rate of HUS (0%-5% in most recent series and 10%-30% in individual reports) and residual chronic renal sequelae (in up to 50% of patients in long-term follow-up studies) emphasize the seriousness of HUS for public health . Several studies have described possible sources of EHEC infection . However, in the majority of cases the pathogen cannot be identified in food or animals and the routes of transmission remain unclear . In this review article the hypothesized routes of transmission are summarized . The medical data bases "Medline" and "Current contents" were screened for the years January 1966 through November 1998 . The difficulties in following the chain of EHEC infection are discussed . A precise evaluation of the environmental aspects of the patient is a precondition for further analysis. Biophys J, 2000 Feb, 78(2), 1036 - 41 Torque-speed relationship of the flagellar rotary motor of Escherichia coli; Chen X et al.; The output of a rotary motor is characterized by its torque and speed . We measured the torque-speed relationship of the flagellar rotary motor of Escherichia coli by a new method . Small latex spheres were attached to flagellar stubs on cells fixed to the surface of a glass slide . The angular speeds of the spheres were monitored in a weak optical trap by back-focal-plane interferometry in solutions containing different concentrations of the viscous agent Ficoll . Plots of relative torque (viscosity x speed) versus speed were obtained over a wide dynamic range (up to speeds of approximately 300 Hz) at three different temperatures, 22.7, 17.7, and 15.8 degrees C . Results obtained earlier by electrorotation (, Biophys . J . 65:2201-2216) were confirmed . The motor operates in two dynamic regimes . At 23 degrees C, the torque is approximately constant up to a knee speed of nearly 200 Hz, and then it falls rapidly with speed to a zero-torque speed of approximately 350 Hz . In the low-speed regime, torque is insensitive to changes in temperature . In the high-speed regime, it decreases markedly at lower temperature . These results are consistent with models in which torque is generated by a powerstroke mechanism (, Biophys . J . 76:580-587). Biochemistry, 2000 Feb 8, 39(5), 1169 - 79 Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity . Allosteric activation of ATP:citrate lyase by phosphorylated sugars; Potapova IA et al.; Recombinantly expressed human ATP:citrate lyase was purified from E . coli, and its kinetic behavior was characterized before and after phosphorylation . Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein . Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits . Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic . The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity . Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction . Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation. J Mol Biol, 2000 Feb 4, 295(5), 1265 - 73 Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin; Skrynnikov NR et al.; Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation . In many multidomain proteins these conformational changes involve reorientation of domains within the protein . Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution . Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent . The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with beta-cyclodextrin has been determined on the basis of one-bond (15)N-H(N), (15)N-(13)C', (13)C(alpha)-(13)C', two-bond (13)C'-H(N), and three-bond (13)C(alpha)-H(N) dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively . This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings . Consistent structures have been derived in this manner starting from four different crystal forms of MBP . The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(+/-1) degrees domain closure . We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5 degrees uncertainty in orientation of internuclear vectors within domains) . In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein . J Mol Biol, 2000 Feb 4, 295(5), 1237 - 49 High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence; Czapinska H et al.; A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and structural consequences of truncating the binding loop residues to alanine . In addition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitution at position P(1) and an unrelated Met52Leu change . In spite of the binding loop modification, the affinity for trypsin is only 30 times lower than that of the wild-type protein . At pH 7.5 the protein can be crystallized on the time-scale of hours, yielding very stable crystals of a new (tetragonal) form of BPTI . Conventional source X-ray data collected to 1.4 A at room temperature allowed anisotropic structure refinement characterized by R=0.1048 . The structure reveals all 58 residues, including the complete C terminus, which is in a salt-bridge contact with the N terminus . The Cys14-Cys38 disulfide bridge is observed in two distinct chiralities . This bridge, together with an internal water molecule, contributes to the stabilization of the binding loop . The Ala mutations have only an insignificant and localized effect on the binding loop, which retains its wild-type conformation (maximum deviation of loop C(alpha) atoms of 0.7 A at Ala13) . Four (instead of the typical three) additional water molecules are buried in an internal cleft and connected to the surface via a sulfate anion . Three more SO(4)(2-) anions are seen in the electron density, one of them located on a 2-fold axis . It participates in the formation of a dimeric structure between symmetry-related BPTI molecules, in which electrostatic and hydrogen bonding interactions resulting from the mutated Lys15Arg substitution are of central importance . This dimeric interaction involves direct recognition loop-recognition loop contacts, part of which are hydrophobic interactions of the patches created by the alanine mutations . Another 2-fold symmetric interaction between the BPTI molecules involves the formation of an antiparallel intermolecular beta-sheet that, together with the adjacent intramolecular beta-hairpin loops, creates a four-stranded structure . J Mol Biol, 2000 Feb 4, 295(5), 1225 - 36 Structural comparison of the PhoB and OmpR DNA-binding/transactivation domains and the arrangement of PhoB molecules on the phosphate box; Okamura H et al.; PhoB is a transcriptional activator that binds to the phosphate box in the promoters of the phosphate genes of Escherichia coli . PhoB contains two functional domains, an N-terminal phosphorylation domain and a C-terminal DNA-binding/transactivation domain . Here, the three-dimensional structure of the DNA-binding/transactivation domain has been determined by NMR . It consists of an N-terminal four-stranded beta-sheet, a central three helical bundle and a C-terminal beta-hairpin . The second and third helices form a helix-turn-helix (HTH) variant containing a longer turn than the corresponding turn of the classical HTH motif . The overall architecture is very close to that of the OmpR DNA-binding/transactivation domain, however, the conformation of the long turn region of PhoB, a putative interaction site for the RNA polymerase sigma subunit, is entirely different from that of the corresponding turn of OmpR, which interacts with the alpha subunit . In addition, the third helix of PhoB is three amino acid residues longer than the corresponding helix of OmpR . The binding site of PhoB is a TGTCA sequence and the phospahte box contains the two binding sites . NMR studies of the complexes of the PhoB DNA-binding/transactivation domain bound to several different DNA molecules have revealed that two PhoB molecules bind in a tandem array on the phosphate box . In each complex of PhoB the third helix of the DNA-binding/transactivation domain is likely to recognize the TGTCA sequence from the major groove of DNA and the C-terminal beta-hairpin contacts on the minor groove of the 3' site out of the TGTCA sequence in a non-specific manner . The long turn region facing outward is likely to interact with the sigma subunit . J Mol Biol, 2000 Feb 4, 295(5), 1163 - 72 Two active-site tyrosyl residues of protein TrwC act sequentially at the origin of transfer during plasmid R388 conjugation; Grandoso G et al.; Protein TrwC is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation . Site-directed mutagenesis was used to change to phenylalanine each of a set of four conserved tyrosyl residues in the sequence of the N-terminal relaxation domain of the protein . Simultaneous mutation of both Y18 and Y26 was required to abolish in vitro cleavage and strand-transfer reactions catalyzed by protein TrwC on oligonucleotides containing the nic site . Thus, both Y18 and Y26 could be involved independently in the formation of oligonucleotide-protein covalent complexes that constitute presumed intermediates of these reactions . This hypothesis was confirmed by the observation of Y18 and Y26-specific peptide-oligonucleotide adducts after protease digestion of TrwC and mutant derivatives . Finally mutation Y18F, but not mutation Y26F, abolished nic-cleavage of a supercoiled DNA containing the R388 origin of transfer (oriT) . These data allowed the construction of a model for conjugative DNA processing in which Y18 specifically catalyzes the initial cleavage reaction, while Y26 is used for the second strand-transfer reaction, which terminates conjugation . The model suggests a control mechanism that can be effective at each conjugative replication cycle . J Mol Biol, 2000 Feb 4, 295(5), 1113 - 8 Evidence for helical unwinding of an RNA substrate by the RNA enzyme RNase P: use of an interstrand disulfide crosslink in substrate; Pomeranz Krummel DA et al.; To gain an understanding of structural changes induced in substrates by Escherichia coli ribonuclease P (RNase P), we have incorporated an interstrand disulfide crosslink proximal to the cleavage site in a model substrate . RNase P is able to process the reduced, non-crosslinked form of this substrate as well as a substrate in which the free thiol molecules have been alkylated with iodoacetamide . However, the oxidized, crosslinked form is cleaved at a significantly lower rate . Therefore, helical unwinding of the analog of the aminoacyl stem of the substrate near its site of cleavage may be necessary for efficient processing by E . coli RNase P . Pflugers Arch, 2000, 439(3 Suppl), R119 - 21 Tissue expression and immunolocalization of a novel human cathepsin P; Pungercar J et al.; The mRNA of a novel human cathepsin P is expressed at high levels in lung, liver and heart . Using antibodies raised against recombinant cathepsin P produced in Escherichia coli, a single protein band of 33 kDa was detected by immunoblotting an extract of human liver . By immunofluorescence, positive signals were observed in hepatocytes and Kupffer cells of liver, and the distal tubule cells of kidney showing mainly perimembranous distribution, indicating a role, as yet unknown, for this novel putative protease that is distinct from other cathepsins of the papain family. Pflugers Arch, 2000, 439(3 Suppl), R113 - 5 Early events in TNFa-p55 receptor interations--experiments with TNF dimers; Menart V et al.; The first essential step in TNF signal transduction is believed to be clustering of the membrane bound receptors around the trimeric TNF molecule . To check if one receptor binding site would be enough to trigger the signal, we tried to prepare several types of TNF dimer . For this purpose, two TNF analogs bearing different cysteine mutations at the inner subunit binding surfaces were designed, expressed in E . coli and prepared in pure form . By mixing equimolar quantities of these analogs under appropriate conditions, two different types of dimer were prepared . The first, Dim/S2, proved to be composed mainly of a disulfide-linked dimer, which was still capable of trapping the third subunit of either of the precursor analogs, thus showing relatively high residual cytotoxicity . To avoid trimeric structures, Dim/S2 was further transformed into Dim/Iaa2 by alkylation of -SH groups of the newly introduced cysteines, allowing binding of only two TNF subunits through native contact surfaces . These dimers showed substantially reduced cytotoxicity on the L929 cell line . In addition, it appears that Dim/Iaa2 is able to competitively inhibit cytotoxicity of native TNF, as assessed on the L-M cell line. Anticancer Res, 1999 Jul-Aug, 19(4B), 2925 - 8 Viscotoxin-free aqueous extracts from European mistletoe (Viscum album L.) stimulate activity of human granulocytes; Stein GM et al.; BACKGROUND: Extracts from European mistletoe (Viscum album L., VAL) are used for complementary cancer treatment . Viscotoxins (VT) and whole plant extracts with high amounts of the VT have been shown to stimulate functional activity of granulocytes . MATERIALS AND METHODS: We stimulated neutrophils from healthy donors in vitro with aqueous VT-free VAL extracts and mistletoe lectins (ML) in the presence of E.coli and studied phagocytosis (via incorporation of FITC-labelled E.coli) and respiratory burst (via oxidation of dihydrorhodamine 123 to rhodamine 123) by flow cytometry . RESULTS: The VT-free VAL extract significantly stimulated granulocyte activity, and this effect correlated with the content of the ML, although the ML exerted no influence at relevant concentrations . Co-incubation of the cells with VAL in the presence of VT further increased granulocyte response . CONCLUSIONS: From these data it is suggested that (1) a non-VT non-ML component of the VAL extracts activated granulocytes and (2) different activation pathways may be involved in the stimulation by the whole plant extract and the VT. Anticancer Res, 1999 Jul-Aug, 19(4B), 2869 - 73 Recombinant vaccinia virus expressing cytokine GM-CSF as tumor vaccine; Chatterjee SK et al.; The efficacy of a recombinant vaccinia virus (rvv-GM-CSF) expressing the granulocyte macrophage colony stimulating factor (GM-CSF) as tumor vaccine was evaluated in the murine B16-F10 melanoma model . The vaccine was prepared by infection of irradiated tumor cells with rvv-GM-CSF . Control vaccine was B-16 cells infected with a recombinant vaccinia virus expressing Escherichia coli beta-galactosidase (rvv-lacZ) . Pre-vaccination of naive C57BL/6 mice later inoculated with tumor cells and treatment of mice bearing tumors with GM-CSF vaccine inhibited tumor development and prolonged survival . Lung metastasis of B-16 was also inhibited by treatment with GM-CSF vaccine . The vaccine effects appeared to be tumor cell specific . The efficacy of the vaccine was comparable to a retroviral vaccine (MFG-muGM-CSF) in this system . The vaccine was also effective when rvv-GM-CSF was directly injected into the tumor . These data suggest that this vaccine approach has potential for use in cancer treatment, especially for patients with easily accessible tumors. Trends Genet, 2000 Feb, 16(2), 88 - 92 Eukaryotic DNA replication: a model for a fixed double replisome; Falaschi A; To duplicate their genomes, eukaryotic cells have to overcome some formidable chemical and topological hurdles, considering the number of nucleotides that have to be polymerized faithfully and the sheer physical size of the DNA molecules that have to be disentangled and partitioned in an orderly way . This article tackles one particular aspect of the process: the organization of the apparatus that advances the replicative growing forks along the DNA molecule . Here, I suggest a solution to the difficulty of separating the daughter molecules in an orderly way and propose an alternative to the current models, which reconciles the use of a single polarity of synthesis by the DNA polymerases with the need for parallel polymerization of two strands of opposite polarity. J Biol Chem, 2000 Feb 4, 275(5), 3713 - 21 Elastase in intestinal mucus enhances the cytotoxicity of Shiga toxin type 2d; Kokai-Kun JF et al.; Shiga toxin variant type 2d (Stx2d) produced by some strains of Shiga toxin-producing Escherichia coli is composed of an enzymatically active A subunit and a B (binding) pentamer . The cytotoxicity of Stx2d is increased (activated) 10-1000-fold for Vero cells when the toxin is incubated with mucus obtained from the small intestine of mice . In this study we isolated an Stx2d activator and identified it as a mouse elastase with strong homology to human elastase IIIB . Moreover, commercially available porcine pancreatic elastase preparations also activated Stx2d cytotoxicity although with a lower specific activity than isolated mouse elastase . Elastase directly nicked the Stx2d A subunit to A(1) and A(2), an event that did not correlate with activation . However, elastase also reduced the size and changed the isoelectric point of the A(2) peptide, as determined by SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis followed by Western immunoblot analysis . This elastase-mediated size and charge shift in the A(2) peptide of Stx2d occurred concurrently with activation of the toxin . Both the reduction in size of the Stx2d A(2) peptide by incubation with elastase as well as the associated activation of Stx2d cytotoxicity were fully inhibited by elastatinal, an elastase-specific inhibitor. J Biol Chem, 2000 Feb 4, 275(5), 3661 - 6 Identification of RNA polymerase beta' subunit segment contacting the melted region of the lacUV5 promoter; Brodolin K et al.; Identification of the RNA polymerase functional regions involved in interactions with promoter is a basis for understanding the mechanism of transcription initiation . We have used formaldehyde cross-linking to identify a region of Escherichia coli RNA polymerase beta' subunit contacting lacUV5 promoter in open complex . Treatment of open complex with formaldehyde results in cross-linking of beta' and sigma(70) subunits at positions -5 and -3 on the nontemplate strand of the promoter DNA . These cross-links reflect specific interactions between RNA polymerase and promoter established in open complex . The positions of formaldehyde cross-links in the beta' subunit were mapped to the N-terminal segment (Cys(198)-Met(237)), which is contiguous to the evolutionary conserved region B . The proximity of the beta' and sigma cross-links suggest that the N-terminal region of the beta' subunit, interacting with single-stranded promoter DNA, can cooperate with the sigma subunit in the process of open complex formation. J Biol Chem, 2000 Feb 4, 275(5), 3583 - 92 Mapping of subunit-subunit contact surfaces on the beta' subunit of Escherichia coli RNA polymerase; Katayama A et al.; The RNA polymerase core enzyme of Escherichia coli with the catalytic activity of RNA polymerization is assembled sequentially under the order: 2alpha --> alpha(2) --> alpha(2)beta --> alpha(2)betabeta' . The core enzyme gains the activities of promoter recognition and transcription initiation after binding the sigma subunit . The subunit-subunit contact surfaces of beta' subunit (1407 residues) were analyzed by testing complex formation between various beta' fragments and either the alpha(2)beta complex or the sigma(70) subunit . Results indicate that two regions, one central region between residues 515 and 842 and the other COOH-terminal proximal region downstream from residue 1141, are involved in binding the alpha(2)beta complex; and the NH(2)-terminal proximal region between residues 201 and 345 plays a major role in binding the sigma(70) subunit . However, both alpha(2)beta binding sites have weak activity of the sigma(70) subunit; likewise, the sigma(70) subunit-contact surface has weak binding activity of the alpha(2)beta complex . The sites involved in the catalytic function of RNA polymerization are all located within two spacer regions sandwiched between these three subunit-subunit contact surfaces. J Biol Chem, 2000 Feb 4, 275(5), 3360 - 4 Interactions of the sulfonylurea receptor 1 subunit in the molecular assembly of beta-cell K(ATP) channels; Mikhailov MV et al.; We have investigated protein interactions involved in pancreatic beta-cell ATP-sensitive potassium channel assembly . These channels, which are of key importance for control of insulin release, are a hetero-oligomeric complex of pore-forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits with two nucleotide-binding domains (NBD1 and NBD2) . We divided SUR1 into two halves at Pro-1042 . Expression of either the individual N- or C-terminal domain in a baculovirus expression system did not lead to glibenclamide binding activity, although studies with green fluorescent protein fusion proteins showed that both half-molecules were inserted into the plasma membrane . However, significant glibenclamide binding activity was observed when the half-molecules were co-expressed (even when NBD2 was deleted from the C-terminal half-molecule) . Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity . We conclude that the glibenclamide-binding site includes amino acid residues from both halves of the molecule, that there is strong interaction between different regions of SUR1, that NBD2 is not essential for glibenclamide binding, and that interactions between Kir6.2 and SUR1 participate in ATP-sensitive potassium channel assembly . Investigation of NBD1-green fluorescent protein fusion protein distribution inside insect cells expressing C-terminal halves of SUR1 demonstrated strong interaction between NBD1 and NBD2 . We also expressed and purified NBD1 from Escherichia coli . Purified NBD1 was found to exist as a tetramer indicating strong homomeric attractions and a possible role for NBD1 in SUR1 assembly. J Biol Chem, 2000 Feb 4, 275(5), 3201 - 5 The ferrous dioxygen complex of the oxygenase domain of neuronal nitric-oxide synthase; Couture M et al.; The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown . To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy to generate and identify the ferrous dioxygen complex of the oxygenase domain of nNOS (Fe(2+)O(2) nNOSoxy) . We detect a line at 1135 cm(-1) in the resonance Raman spectrum of the intermediate formed from 0.6 to 3.0 ms after the rapid mixing of the ferrous enzyme with oxygen that is shifted to 1068 cm(-1) with (18)O(2) . This line is assigned as the O-O stretching mode (nu(O-O)) of the oxygenated complex of nNOSoxy . Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or N(omega)-hydroxy-L-arginine, in the presence or absence of (6R)-5,6, 7,8-tetrahydro-L-biopterin, reveal that the nu(O-O) line is insensitive to the presence of the substrate and the pterin . The optical spectrum of this ferrous dioxygen species, with a Soret band wavelength maximum at 430 nm, confirms the identification of the previously reported oxygenated complexes generated by stopped flow techniques. J Biol Chem, 2000 Feb 4, 275(5), 3192 - 200 Regulation of Pap phase variation . Lrp is sufficient for the establishment of the phase off pap DNA methylation pattern and repression of pap transcription in vitro; Weyand NJ et al.; The pyelonephritis-associated pili (pap) operon in Escherichia coli is regulated by an epigenetic mechanism involving the formation of specific DNA methylation patterns characteristic of transcriptionally active (phase ON) and inactive (phase OFF) cells . The formation of pap DNA methylation patterns in vivo was previously shown to require the leucine-responsive regulatory protein (Lrp) and DNA adenine methylase (Dam) . To monitor the binding of Lrp to pap DNA, an in vitro methylation protection assay was developed . Binding of Lrp to a Dam target site proximal to the papBA promoter (designated GATC(prox)) blocked methylation of this site and specifically repressed transcription . The DNA methylation pattern and transcription state are identical to those observed in vivo in phase OFF cells . To determine if binding of Lrp at GATC(prox) was necessary for repression of papBA transcription, we analyzed a pap mutation (pap-13) that reduced the affinity of Lrp for the GATC(prox) region . Binding of Lrp to pap-13 DNA was shifted to a promoter distal Dam target site (designated GATC(dist)) . Lrp blocked methylation of GATC(dist) in the pap-13 mutant, but did not repress papBA transcription . Together, these results show that binding of Lrp to the GATC(prox) region is sufficient for the establishment of the phase OFF DNA methylation pattern and repression of papBA transcription. J Biol Chem, 2000 Feb 4, 275(5), 3137 - 43 Molecular and biochemical evidence for the involvement of calcium/calmodulin in auxin action; Yang T et al.; The use of (35)S-labeled calmodulin (CaM) to screen a corn root cDNA expression library has led to the isolation of a CaM-binding protein, encoded by a cDNA with sequence similarity to small auxin up RNAs (SAURs), a class of early auxin-responsive genes . The cDNA designated as ZmSAUR1 (Zea mays SAURs) was expressed in Escherichia coli, and the recombinant protein was purified by CaM affinity chromatography . The CaM binding assay revealed that the recombinant protein binds to CaM in a calcium-dependent manner . Deletion analysis revealed that the CaM binding site was located at the NH(2)-terminal domain . A synthetic peptide of amino acids 20-45, corresponding to the potential CaM binding region, was used for calcium-dependent mobility shift assays . The synthetic peptide formed a stable complex with CaM only in the presence of calcium . The CaM affinity assay indicated that ZmSAUR1 binds to CaM with high affinity (K(d) approximately 15 nM) in a calcium-dependent manner . Comparison of the NH(2)-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic alpha-amphiphilic helix similar to the CaM binding region of ZmSAUR1 . CaM binds to the two synthetic peptides from the NH(2)-terminal regions of Arabidopsis SAUR-AC1 and soybean 10A5, suggesting that this is a general phenomenon for all SAURs . Northern analysis was carried out using the total RNA isolated from auxin-treated corn coleoptile segments . ZmSAUR1 gene expression began within 10 min, increased rapidly between 10 and 60 min, and peaked around 60 min after 10 microM alpha-naphthaleneacetic acid treatment . These results indicate that ZmSAUR1 is an early auxin-responsive gene . The CaM antagonist N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride inhibited the auxin-induced cell elongation but not the auxin-induced expression of ZmSAUR1 . This suggests that calcium/CaM do not regulate ZmSAUR1 at the transcriptional level . CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the post-translational level . Our data provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction. J Biol Chem, 2000 Feb 4, 275(5), 3017 - 20 The DnaX-binding subunits delta' and psi are bound to gamma and not tau in the DNA polymerase III holoenzyme; Glover BP et al.; The DnaX complex subassembly of the DNA polymerase III holoenzyme is comprised of the DnaX proteins tau and gamma and the auxiliary subunits delta, delta', chi, and psi, which together load the beta processivity factor onto primed DNA in an ATP-dependent reaction . delta' and psi bind directly to DnaX whereas delta and chi bind to delta' and psi, respectively (Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O'Donnell, M . (1995) J . Biol . Chem . 270, 13348-13357) . Until now, it has been unclear which DnaX protein, tau or gamma, in holoenzyme binds the auxiliary subunits delta, delta', chi,and psi . Treatment of purified holoenzyme with the homobifunctional cross-linker bis(sulfosuccinimidyl)suberate produces covalently cross-linked gamma-delta' and gamma-psi complexes identified by Western blot analysis . Immunodetection of cross-linked species with anti-delta' and anti-psi antibodies revealed that no tau-delta' or tau-psi cross-links had formed, suggesting that the delta' and psi subunits reside only on gamma within holoenzyme. Genes Dev, 2000 Jan 15, 14(2), 212 - 23 Dynamic organization of chromosomal DNA in Escherichia coli; Niki H et al.; We have revealed the subcellular localization of different DNA segments that are located at approximately 230-kb intervals on the Escherichia coli chromosome using fluorescence in situ hybridization (FISH) . The series of chromosome segments is localized within the cell in the same order as the chromosome map . The large chromosome region including oriC shows similar localization patterns, which we call the Ori domain . In addition, the localization pattern of the large segment including dif is characteristic of the replication terminus region . The segment also shows similar localization patterns, which we call the Ter domain . In newborn cells, Ori and Ter domains of the chromosome are differentially localized near opposite cell poles . Subsequently, in the B period, the Ori domain moves toward mid-cell before the initiation of replication, and the Ter domain tends to relocate at mid-cell . An inversion mutant, in which the Ter domain is located close to oriC, shows abnormal subcellular localization of ori and dif segments, resulting in frequent production of anucleate cells . These studies thus suggest that the E . coli chromosome is organized to form a compacted ring structure with the Ori and Ter domains; these domains participate in the cell cycle-dependent localization of the chromosome. Biochem Biophys Res Commun, 2000 Feb 5, 268(1), 136 - 40 Effects of C5 protein on Escherichia coli RNase P catalysis with a precursor tRNA(Phe) bearing a single mismatch in the acceptor stem; Park BH et al.