Yeast, 1994 Sep, 10(9), 1141 - 55 Identification of a set of yeast genes coding for a novel family of putative ATPases with high similarity to constituents of the 26S protease complex; Schnall R et al.; There is accumulating evidence for a large, highly conserved gene family of putative ATPases . We have identified 12 different members of this novel gene family (the YTA family) in yeast and determined the nucleotide sequences of nine of these genes . All of the putative gene products are characterized by the presence of a highly conserved domain of 300 amino acids containing specialized forms of the A and B boxes of ATPases . YTA1, YTA2, YTA3 and YTA5 exhibit significant similarity to proteins involved in human immunodeficiency virus Tat-mediated gene expression but more significantly to subunits of the human 26S proteasome . YTA1 and YTA2 are essential genes in yeast . Remarkably, the cDNA of human TBP-1 can compensate for the loss of YTA1 . Preliminary experiments indicate that YTA1 is a component of the 26S protease complex from yeast . Our findings lead us to propose that YTA1, YTA2, YTA3 and YTA5 function as regulatory subunits of the yeast 26S proteasome . YTA10, YTA11 and YTA12 share significant homology with the Escherichia coli FtsH protein, and together with YTA4 and YTA6 may constitute a separate subclass within this family of putative ATPases. Shock, 1994 Sep, 2(3), 185 - 91 The roles of nitric oxide and hydrogen peroxide production in lipopolysaccharide-induced intestinal damage; Wang JF et al.; The importance of gastrointestinal injury in endotoxin-induced shock and multiple organ failure is of great interest . In this paper we describe a method to assess the degree of intravascular congestion and bleeding into the wall of the intestine by determining the hemoglobin content of the tissue . After validating this method, we used it to study the mechanism of jejunal injury induced by intravenous injection of Escherichia coli lipopolysaccharide (LPS, 50 mg/kg bw), the role of nitric oxide release in maintaining the integrity of endothelial cells, and the participation of H2O2 production in the LPS-induced intestinal damage in rats . Our results show that after the administration of LPS at the dose of 50 mg/kg intravenously, the hemoglobin content of the jejunum (17.8 mg/100 mg tissue) increased 7.7-fold over that of control animals (2.3 mg/100 mg), reflecting a serious degree of congestion, bleeding, and damage in the gastrointestinal tract . Administration of nitro-L-arginine methyl ester (L-NAME) not only enhanced this injury, but also markedly decreased the dose of LPS necessary to induce intestinal damage . Infusion of L-arginine (300 mg/kg bolus plus infusion 600 mg/kg.h intravenously) protected the intestine against LPS or LPS plus L-NAME . Inhibition of basal nitric oxide release by L-NAME produced significant changes in cardiovascular variables, but failed to induce a significant bleeding damage . However, when inhibition of NO release was combined with enhanced H2O2 production by a small dose of LPS, a serious bleeding damage was observed . This was accompanied by a marked decrease in mesenteric blood flow and cardiac output . High dose of LPS induced the above effects, and thus could be responsible for the bleeding damage, while low dose of LPS that fails to inhibit nitric oxide, did not induce any intestinal bleeding . It seems that inhibition of NO release and stimulation of H2O2 production are both involved in the LPS-induced bleeding damage. Shock, 1994 Sep, 2(3), 179 - 84 Heat shock attenuates endothelium-dependent vasodilation in skeletal muscle microcirculation; Lubbe AS; Endothelium-derived relaxing factors (EDRFs) mediate vasodilation of small arterioles in skeletal muscle under various (patho)physiological conditions: Escherichia coli sepsis, systemic hypoxia, and topical acetylcholine (ACH) application . To test if heat shock changes EDRF-dependent reactivity of arterioles to ACH, we used closed-circuit videomicroscopy in the in vivo cremaster muscle of rats whose systemic temperatures had been slowly raised to and maintained at 41 degrees C . We also tested for ACH responses after increasing cremaster muscle temperatures and maintaining those at 40 degrees C . The experiments showed that EDRF-dependent vasodilation of small arterioles to acetylcholine was substantially attenuated in response to systemic and local heat treatment . In two other animal groups, concentration-dependent vasodilation of small arterioles to sodium-nitroprusside was not as much attenuated in the response to local tissue temperature elevation . This suggests that locally elevated tissue or systemically elevated body temperatures can change generation or efficacy of EDRFs in the post-hyperthermia phase in the skeletal muscle microcirculation. Shock, 1994 Sep, 2(3), 164 - 72 Hepatic Na(+)-independent amino acid transport in endotoxemic rats: evidence for selective stimulation of arginine transport; Inoue Y et al.; The effects of endotoxin on the activities of the major Na(+)-independent amino acid transporters in rat liver (Systems n, asc, L, bo,+, and y+) were studied using using hepatic plasma membrane vesicles (HPMVs) . Rats were treated with a single dose of Escherichia coli endotoxin (E . coli lipopolysaccharide 0127:B8 (LPS), 7.5, 15, or 30 mg/kg BW) and HPMVs were prepared by Percoll density gradient centrifugation at various timepoints after LPS administration . Vesicle purity and integrity was established by assay of enzyme markers and identical equilibrium uptakes . The activities of the Na(+)-independent amino acid transport systems y+ and bo,+ (arginine), asc (alanine and cysteine), L (leucine), and n (glutamine) were evaluated by measuring the uptake of radiolabeled amino acids using a rapid mixing/filtration technique . Amino acid uptake by HPMVs consisted of saturable and nonsaturable components . Prior treatment with endotoxin did not alter the activities of Systems n, asc, or L but resulted in a time- and dose-dependent stimulation of saturable arginine transport . Arginine transport increased within 2 h of LPS administration and exhibited a return towards basal levels by 24 h . Nonsaturable uptake (diffusion) in HPMVs was unaltered by LPS treatment . Kinetic analysis of arginine transport demonstrated the presence of both a high affinity and a low affinity carrier . Treatment with LPS resulted in a 73% increase in the Vmax of the high affinity carrier (System y+) and a 25% increase in the Vmax of the low affinity transporter (System bo,+) . The data indicate selective stimulation of Na(+)-independent arginine transport in the liver during endotoxemia which may serve to support important arginine-dependent pathways during sepsis. Afr J Med Med Sci, 1994 Sep, 23(3), 291 - 9 The purification and characterization of intracellular invertase obtained from pathogenic Escherichia coli; Olusanya O et al.; All the non-pathogenic strains of Escherichia coli tested failed to synthesize invertase . However, among the pathogenic E . coli, only 11% of them synthesized the enzyme . Invertase synthesis was best at pH 8.0, when the sole nitrogen source was peptone . The enzyme was induced by sucrose but repressed by glucose and fructose . The enzyme was partially purified by ammonium sulphate precipitation, followed by dialysis and gel permeation chromatography . The partially purified invertase possessed a molecular weight of 125,000 KD and an apparent km of approximately 2.94mM for sucrose . The enzyme was stimulated by Ca++ and Mg++, inhibited by Cu++, U++, IAA and exhibited optimum activity at pH 6.5 at 40 degrees C. Biochem J, 1994 Sep 1, 302 ( Pt 2), 347 - 53 Arylamine N-acetyltransferase in Balb/c mice: identification of a novel mouse isoenzyme by cloning and expression in vitro; Kelly SL et al.; Three genes encoding arylamine N-acetyltransferase were identified in Balb/c mice . All three genes were cloned from genomic DNA, sequenced and expressed in a bacterial expression system . Two of the genes corresponded to Nat-1 and Nat-2 which have been previously identified in A/J and C57B1/6 strains of mice (Martell et al., 1991) . The new gene, designated Nat-3, can be distinguished from the other mouse Nat genes both by specific amplification using PCR and by restriction-endonuclease digestion . The products of all three genes are demonstrated to catalyse acetylation of aminofluorene and anisidine following expression in Escherichia coli. Mol Microbiol, 1994 Sep, 13(6), 1133 - 42 Decay of the IS10 antisense RNA by 3' exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack; Pepe CM et al.; RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn10/IS10 transposition folds into a simple stem-loop structure . The unusually high metabolic stability of RNA-OUT is dependent, in part, on the integrity of its stem-domain: mutations that disrupt stem-domain structure (Class II mutations) render RNA-OUT unstable, and restoration of structure restores stability . Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA-OUT . We show here that stem-domain integrity determines RNA-OUT's resistance to 3' exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3' exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) . RNase II and PNPase are individually able to degrade various RNA-OUT species, albeit with different efficiencies: RNA-OUT secondary structure provides greater resistance to RNase II than to PNPase . Surprisingly, RNA-OUT is threefold more stable in wild-type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens PNPase attack on RNA-OUT . We discuss how this might occur . We also show that wild-type RNA-OUT stability changes only two-fold across the normal range of physiological growth temperatures (30-44 degrees C) in wild-type cells, which has important implications for IS10 biology. J Biochem (Tokyo), 1994 Sep, 116(3), 560 - 74 Characterization of gangliosides of epithelial cells of calf small intestine, with special reference to receptor-active sequences for enteropathogenic Escherichia coli K99; Teneberg S et al.; Glycolipids were prepared from epithelial cells of the small intestine of a newborn calf and assayed for Escherichia coli K99 binding activity on thin-layer chromatograms and in microtiter wells . The bacteria did not bind to any of the non-acid glycolipids, while in the acid fraction several binding-positive glycolipids were detected . The acid glycolipids were isolated and characterized by mass spectrometry, proton NMR spectroscopy and other methods . The following gangliosides were identified, mainly from the epithelial cells from the upper part of the small intestine: NeuAc alpha 2-3Gal beta 1-4Glc beta 1-Cer (NeuAc-GM3), NeuGc alpha 2-3Gal beta 1-4Glc beta 1-Cer (NeuGc-GM3), GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GM2), Gal beta 1-3GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GM1), and NeuGc alpha 2-3Gal beta 1-3GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GD1a) . A positive binding was demonstrated to NeuGc-GM3, NeuGc-GM2, and NeuGc-GD1a, while NeuAc-GM3 and NeuGc-GM1 were negative . The binding pattern differed somewhat for total acid glycolipids of epithelial cells from three different parts of the small intestine . Based on binding preferences of E . coli K99 to a number of glycolipids of various origins, in comparison with calculated minimum energy conformations, a binding epitope was delineated. Plasmid, 1994 Sep, 32(2), 195 - 207 In vitro replication of cyanobacterial plasmids from Synechocystis PCC 6803; Yang X et al.; Little knowledge of DNA replication in cyanobacteria is available . In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803 . This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column . DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation . DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain . The in vitro replication products consist mostly of the supercoiled form of the plasmids . Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide . These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803 . In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis . Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication. Plasmid, 1994 Sep, 32(2), 131 - 67 Mathematical model of temperature-sensitive plasmid replication; Leipold RJ et al.; The copy number of a series of plasmids constructed at Odense University is regulated by the lambda PR/PRM promoters and the temperature-sensitive cI857 repressor . At low temperatures, these plasmids exhibit the low copy number of the parent plasmid R1 (5-6 per cell) . At high temperatures, the plasmids exhibit runaway replication, reaching copy numbers of greater than 1,000 per cell . A detailed mathematical model of the temperature-sensitive replication of these plasmids has been developed incorporating three features: replication of the parent plasmid, regulation of the lambda PR/PRM promoters by the cI repressor, and thermal denaturation of the cI857 repressor . Models of the first two of these features have been described by others . We revised and extended those models, described the thermal denaturation of the cI857 repressor, and integrated these features to give a comprehensive model of temperature-sensitive plasmid replication . Model predictions were compared to experimental measurements of both steady-state copy numbers as a function of temperature and the change in copy number following temperature shifts up and down . The model accurately describes the qualitative behavior of the system and gives reasonable quantitative results . This is particularly significant since all the parameter values used in this model were determined independently: that is, there was no adjustment of parameter values to match our experimental data . The regulatory system that gives rise to the temperature-sensitive replication of these plasmids is widely used in biotechnology applications, so the elements of the model related to this regulation should be applicable to a wide variety of systems. Protein Eng, 1994 Sep, 7(9), 1103 - 8 Construction of an enzymatically active ribonuclease H domain of human immunodeficiency virus type 1 reverse transcriptase; Stahl SJ et al.; The isolated ribonuclease (RNase) H domain of human immunodeficiency virus type 1 (HIV-1) is enzymatically inactive . The incorporation of the putative substrate binding site of Escherichia coli RNase HI (amino acid residues 76-102, the alpha c-helix and adjacent loop region) into the equivalent position of the RNase H domain of HIV-1 resulted in a highly active hybrid protein dependent on Mn2+ . Similar restoration of RNase H activity has been observed when histidine residues are added to either the N- or C-terminus of the HIV-1 RNase H domain . The hybrid HIV-1/E . coli RNase H protein is approximately 10-fold more active than HIV-1 reverse transcriptase and 30-fold more active than the histidine-tagged proteins, indicating that the alpha c-helix and adjacent loop region of E . coli RNase HI is an excellent substrate binding region because of its sequence and/or location . The RNase H hybrid produced the same specific cleavage in the model tRNA(Lys3) primer removal assay as HIV-1 reverse transcriptase, showing that substrate binding and specificity are separable and that the specificity determinants are at least partially, if not totally, contained in the amino acid sequence of the hybrid protein derived from HIV-1 reverse transcriptase. Biofizika, 1994 Sep-Oct, 39(5), 915 - 8 {Detection of the generation of nitric oxide from L-arginine in the murine brain in vivo using EPR}; Mikoian VD et al.; Nitric oxide (NO) was shown by EPR method to be generated via L-arginine-dependent way in brain of mice in vivo . The complexes of diethyldithiocarbamate (DETC) or pirrolidinedithiocarbamate (PDTC) with endogenous or exogenous Fe2+ were used as a traps of NO, which are capable to bind NO resulted in the formation of mononitrosyl iron complexes with DETC or PDTC (MNIC-DETC or PDTC) recovered by EPR method . These complexes were detected in mouse brain in concentration of 2.5 nmole/g of wet tissue for 30 min only when exogenous Fe2+ was injected in to mice . The level of MNIC-DETC (PDTC) was 5 fold increased in brain of mice pretreated for 4 hrs with lipopolysaccharide from Escherichia coli, which induced the inflammation processes . The inhibitor of NO-synthase, NG-nitro-L-arginine attenuated the formation of MNIC-DETC (PDTC) in mouse brain in vivo . Exogenous Fe2+ is suggested to induced the synthesis of NO-synthase in mouse brain. J Clin Microbiol, 1994 Sep, 32(9), 2235 - 41 Early detection of anti-HCc antibody in acute hepatitis C virus (HCV) by western blot (immunoblot) using a recombinant HCV core protein fragment; Yeh CT et al.; Crude extract from Escherichia coli which expressed a recombinant protein containing amino acids 2 to 127 of the hepatitis C virus (HCV) core protein was used to detect the antibody against HCV core protein (anti-HCc) . After electrophoretic separation of proteins from the extract, Western blot (immunoblot) analysis was performed with the serum samples . This method was compared with a commercially available second-generation enzyme immunoassay (EIA) which employed synthetic peptides corresponding to highly antigenic segments of both structural and nonstructural portions of HCV . Also, reverse transcription PCR for HCV RNA was used for comparison . Seventy-two serum samples from three groups of patients were tested . Groups I and II represented healthy subjects and subjects with acute hepatitis A or B, respectively . Group III included patients with newly acquired acute hepatitis C . By Western blot analysis, 31 of 31 (100%) samples from group I were negative for anti-HCc antibody, whereas 4 of 22 (18%) samples from group II were positive for anti-HCc . One of these four samples was also positive for anti-HCV antibody by the second-generation EIA (1 of 22 {4.5%}) . Among 19 patients diagnosed with newly acquired acute hepatitis C, 4 (21%) were positive for anti-HCV by the second-generation EIA, whereas 12 of 19 (63%) were positive for anti-HCc by Western blot analysis . Of EIA-positive subjects, 4 of 4 (100%) were also positive for anti-HCc by Western blot analysis, whereas among EIA-negative subjects, 8 of 15 (53%) were positive . For HCV RNA detected by reverse transcription PCR, 15 of 19 (80%) of this group of samples were positive . Strikingly, the peak bilirubin level for patients with EIA-negative and Western blot-positive results is significantly higher than that for patients with consistent EIA and Western blot results (22.7 versus 7.2 mg/dl) . A series of serum samples from a patient with concurrent hepatitis B and C viral infection was also studied by both tests . Although anti-HCc persisted throughout the course of infection, anti-HCV by EIA converted from negative to positive 20 days after admission and then converted back to negative 30 days later. Antimicrob Agents Chemother, 1994 Sep, 38(9), 1909 - 14 Subunit specificity of mutations that confer resistance to nonnucleoside inhibitors in human immunodeficiency virus type 1 reverse transcriptase; Boyer PL et al.; We constructed plasmid vectors that simultaneously express both the p66 and p51 subunits of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in Escherichia coli . These vectors allow us to generate HIV-1 RT heterodimers in which either the p66 or the p51 subunit has the wild-type sequence and the other subunit has a specific amino acid substitution . We used these vectors to express HIV-1 RT heterodimers containing several different amino acid substitutions reported to confer resistance to nonnucleoside inhibitors . Most of the amino acid substitutions conferred resistance to nonnucleoside inhibitors R86183 (TIBO) and TSAO-m3T only when present in the p66 subunit of the p66-p51 heterodimer; heterodimers that contained a wild-type p66 subunit and a mutant p51 subunit remained sensitive to the inhibitors . However, there was one mutation, E138K, that conferred drug resistance when the mutation was present in the p51 subunit . The corresponding heterodimer with the E138K mutation in the p66 subunit and a wild-type p51 subunit remained sensitive to the inhibitors . Analysis of the three-dimensional structure of HIV-1 RT indicated that residue 138 of the p51 subunit is in the nonnucleoside inhibitor-binding pocket while residue 138 of the p66 subunit is not . The mutagenesis results, combined with structural data, support the idea that the nonnucleoside inhibitors exert their effects by binding to a hydrophobic pocket in the RT heterodimer and that mutations which give rise to drug resistance directly interfere with the interactions between the nonnucleoside inhibitors and HIV-1 RT. Genetics, 1994 Sep, 138(1), 6 - 10 Presence of different O antigen forms in three isolates of one clone of Escherichia coli; Liu D et al.; Escherichia coli strains ECOR2, ECOR3 and K-12 are very closely related in genotype as indicated by multilocus enzyme electrophoresis . We show that they have very different rfb regions indicating that recombination has occurred in this region, and we suggest that it may be associated with niche adaptation. Kidney Int, 1994 Sep, 46(3), 683 - 9 Inducible nitric oxide synthase mRNA and activity in glomerular mesangial cells; Shultz PJ et al.; Previous studies have suggested that glomerular mesangial cells produce nitric oxide (NO), using measurements of the NO decomposition products, NO2- and NO3- . We have now directly measured NO in the headspace above rat mesangial cell cultures, using a chemiluminescence analyzer . In addition, we examined mesangial cell RNA for inducible NO synthase (iNOS) . We found no detectable NO in the headspace or iNOS mRNA in unstimulated mesangial cells . However, after four hours of incubation with LPS (10 micrograms/ml), iNOS mRNA was apparent and after six hours, significant increases in NO were detected . Both of these parameters continued to increase for at least 24 hours . Significant increases in NO2-/NO3- in the media and cGMP in the mesangial cells were also detected after 24 hours of incubation with LPS . The induction of iNOS mRNA by LPS was markedly inhibited by actinomycin D and dexamethasone, as was the accumulation of NO2-/NO3- in the media . Cycloheximide significantly inhibited NO2-/NO3- in the media of LPS-treated cells, but had little effect on induction of iNOS mRNA by LPS . We conclude that rat mesangial cells possess an iNOS, with activity and regulation similar to that described in macrophages . Furthermore, we demonstrate the activity of this enzyme by direct measurement of NO and its decomposition products, NO2- and NO3- . We suggest that production of NO by glomerular mesangial cells could occur, even when macrophage infiltration is not present, and could, thereby, modulate glomerular and tubular functions within the kidney. Int Arch Allergy Immunol, 1994 Sep, 105(1), 70 - 4 In vivo biological activity of recombinant Der p II allergen of house-dust mite; Lynch NR et al.; The cDNA coding for one of the major allergens of Dermatophagoides pteronyssinus, Der p II, has been cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase . The recombinant material (r Der p II) was tested in the skin of house-dust allergic patients, and was found to be almost as active as the purified native allergen (n Der p II) in stimulating immediate hypersensitivity reactions . The positivity to r Der p II in these patients, who were selected for their skin-test reactivity to house-dust extract (HDE) and whole mite extract (WME), was over 60%, and 80% of their sera contained detectable IgE antibody against the recombinant allergen . The level of specific IgE antibody against r Der p II was a mean of 40% of that against WME in the sera of patients who were skin test positive to both allergen preparations . In contrast to the house-dust allergic patients, none of a group of allergic patients whose clinical history did not implicate house dust, and who were skin test negative to HDE or WME, were positive to r Der p II . Of interest was the observation that only 37% of a group of subjects who were skin test positive to HDE and WME, but who reported no clinical history of allergic disease upon natural exposure to house dust, reacted to r Der p II . These results demonstrate the biological activity of r Der p II, and indicate, in persons skin test positive to house-dust mite extract, an association between reactivity to Der p II and the manifestation of clinical symptoms. Mol Immunol, 1994 Sep, 31(13), 955 - 66 Potential therapeutic recombinant proteins comprised of peptides containing recombined T cell epitopes; Rogers BL et al.; The complete primary structure of Fel d I2 has been determined and shown to be comprised of two separate polypeptide chains (designated chain 1 and 2) . Overlapping peptides covering the entire sequence of both chains of Fel d I have been used to map the major areas of human T cell reactivity . The present study describes three non-contiguous T cell reactive regions of < 30 aa in length that were assembled in all six possible configurations using PCR and recombinant DNA methods . These six recombinant proteins comprised of defined non-contiguous T cell epitope regions artificially combined into single polypeptide chains have been expressed in E . coli, highly purified, and examined for their ability to bind to human cat-allergic IgE and for human T cell reactivity . Several of these recombined T cell epitope-containing polypeptides exhibit markedly reduced IgE binding as compared to the native Fel d I . Importantly, the human T cell reactivity to individual T cell epitope-containing regions is maintained even though each was placed in an unnatural position as compared to the native molecule . In addition, T cell responses to potential junctional epitopes were not detected . It was also demonstrated in mice that s.c . injection of T cell epitope-containing polypeptides inhibits the T cell response to the individual peptides upon subsequent challenge in vitro . Thus, these recombined T cell epitope-containing polypeptides, which harbor multiple T cell reactive regions but have significantly reduced reactivity with allergic human IgE, constitute a novel potential approach for desensitization to important allergens. EMBO J, 1994 Sep 1, 13(17), 3953 - 63 Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA; Harris ME et al.; Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA . The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3-D structure . In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments . A molecular mechanics-based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models . Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations . Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre-tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons. Mutat Res, 1994 Sep, 315(2), 85 - 94 Excision of ultraviolet-induced photoproducts of 5-methylcytosine from DNA; Vairapandi M et al.; Transition mutations at DNA 5-methylcytosines, congregated at CpG islands, are implicated in the etiogenesis of human diseases . Formation of 5-methylcytosine hydrate (5-methyl-6-hydroxy-5,6-dihydrocytosine) by hydration of the 5,6 double bond of 5-methylcytosine has been suggested as an intermediate in a possible mechanism of deamination to thymine . Ultraviolet irradiation of DNA yields pyrimidine hydrates, which are removed by repair glycosylases . We have identified 5-methylcytosine photoproducts following their excision from DNA by E . coli endonuclease III . Poly(dG-{3H}5-medC):poly(dG-{3H}5-medC) was irradiated and reacted with the enzyme . Radiolabeled photoproduct releases were directly proportional to irradiation doses and enzyme concentrations . These were identified as cis-thymine hydrate (6-hydroxy-5,6-dihydrothymine) and trans-thymine hydrate . Recovery of thymine hydrates is consistent with hydration of pyrimidines . Subsequent heating (which converts thymine hydrates to thymines) and chemical sequencing of an irradiated, 3' end-labeled, synthetic DNA strand demonstrated the appearance of thymine at the 5-methylcytosine site . These results demonstrate a mechanism for deamination of DNA 5-methylcytosine via hydration of the 5,6 double bond, putatively yielding 5-methylcytosine hydrate; this deaminates to thymine hydrate, and loss of water yields thymine formation at the 5-methylcytosine site . Identification of these DNA 5-methylcytosine modified moieties indicates a possible molecular mechanism for the frequent transition mutations found at CpG loci. Mutat Res, 1994 Sep, 315(2), 105 - 12 Mutagenesis resulting from DNA damage by lipid peroxidation in the supF gene of Escherichia coli; Akasaka S et al.; In vitro incubation of rat microsomal lipids with NADPH and Fe3+ in the presence of cytochrome P450 reductase produces lesions in double-stranded pZ189 plasmid DNA, the mutagenic potential of which was analyzed after transfection into Escherichia coli host cells that had been induced for SOS functions by ultraviolet irradiation . The extent of lipid peroxidation, when monitored by the formation of thiobarbituric acid reaction substances, was increased with increased addition of lipids in the reaction mixture . Mutagenesis was determined with the forward mutation assay using the supF gene of pZ189 as a target . When treated pZ189 DNA was used to transfect host cells, a seven-fold increase in mutation frequency for SOS uninduced hosts and a 12-fold increase in mutation frequency for SOS induced hosts was observed at 50% survival compared to that observed with untreated DNA . Sequence analysis shows that incubation of pZ189 DNA in the lipid peroxidation reaction mixture results mostly in single base substitutions, the most frequent base change being G:C-->C:G transversion, followed by G:C-->T:A transversion . The fact that, in the SOS induced hosts, the spectrum obtained by lipid peroxidation is similar to that of hydrogen peroxide suggests the possible involvement for mutagenesis of superoxide produced during lipid peroxidation, but not lipid peroxidation end products such as aldehyde or alkane . Treatment of pZ189 DNA with increasing extents of lipid peroxidation did not yield increasing formation of 8-hydroxyguanine . The results suggest that the origins of G:C-->C:G and G:C-->T:A transversions may be (an) as yet unidentified lesion(s) generated by lipid peroxidation. Mutat Res, 1994 Sep 1, 309(2), 225 - 33 The groE gene products of Escherichia coli are dispensable for mucA+B(+)-dependent UV mutagenesis; Donnelly CE et al.; UV mutagenesis in Escherichia coli requires the groES+EL+ chaperonins as well as the umuD+C+ SOS-regulated genes . GroES and GroEL appear to be required to stabilize UmuC . The mucA+B+ genes, which are encoded on a broad host range plasmid, are functionally analogous and structurally similar to the umuD+C+ genes of E . coli . While these gene pairs are quite similar, differences have been reported in the functioning of these gene products . We tested whether mucA+B+ function requires the groE+ gene products as well . We show that mucA+B(+)-induced UV mutagenesis, UV resistance, phage reactivation and cold sensitivity do not require the groE+ heat shock genes . These findings suggest that the requirement of UmuC for groES+EL+ function is not shared by its analog, MucB. Mutat Res, 1994 Sep 1, 309(2), 147 - 63 DNA sequence analysis of gamma-radiation (anoxic)-induced and spontaneous lacId mutations in Escherichia coli K-12; Sargentini NJ et al.; An extensive spectrum of ionizing radiation mutagenesis was determined by sequencing 318 137Cs gamma-radiation (anoxic)-induced episomal lacId mutations in Escherichia coli strain NR9102 . The most commonly found radiation-induced mutations were base substitutions (44% transversions and 41% transitions) . The radiation-induced spectrum consisted of: 23% G.C-->A.T, 18% A.T-->G.C, 17% G.C-->T.A, 14% G.C-->C.G, 8% A.T-->T.A, 6% A.T-->C.G, 8% single-base deletions, 5% multiple mutations, 3% multi-base deletions, and essentially no single- or multi-base additions . This spectrum compared better with spectra for other systems obtained by in vivo irradiation than with one obtained by in vitro irradiation . Multiple mutations, which were unique to the radiation-induced spectrum, generally consisted of one active and one closely linked silent mutation, and are suggested to result from an altered replication complex of reduced fidelity . Mutation rates were 4.1 x 10(-8) lac-constitutive mutations/gene/Gy and 1.2 x 10(-10) base substitutions/base pair/Gy . Thirty-two percent more radiation-induced mutations occurred at G.C vs . A.T base pairs . A strand asymmetry was noted for G.C-->C.G and A.T-->T.A transversions . A nearest-neighbor analysis showed that C (vs . A, G, or T), on either side of the mutation site, substantially enhanced most types of base substitutions . Similarly, G and C flanked both sides of single-base deletion sites twice as frequently as would be expected from the base composition of the mutation target . For comparative purposes, we sequenced 411 spontaneous lac-constitutive mutants of which 269 were lacId mutants, and there was good agreement between these and previously published mutational spectra . The spontaneous and radiation-induced mutational spectra differed substantially for virtually every class of mutation . For example, the set of spontaneous dominant lac-constitutive mutations contained many more mutations that did not map in the normal region for lacId mutations (i.e., 35% vs . 3%) and were presumed to be lacO-constitutive mutations . A sampling of these presumptive lacOc mutations was also sequenced: 17/22 (spontaneous) and 1/9 (radiation) were found to be lacOc long deletions, one from each set were base substitutions, and the remaining mutations showed the wild-type lacO sequence . Like the radiation-induced spectrum, the spontaneous spectrum showed enhanced mutagenesis at G.C sites, strand asymmetry, and enhanced mutagenesis when G or C were the nearest neighbors. Clin Immunol Immunopathol, 1994 Sep, 72(3), 350 - 6 The influence of DNA size on the binding of anti-DNA antibodies in the solid and fluid phase; Pisetsky DS et al.; To elucidate the interaction of anti-DNA antibodies with DNA, the reactivity of lupus sera with single-stranded fragments from calf thymus, Escherichia coli, and salmon testes DNA was investigated . These fragments were generated by digestion with the restriction enzyme HinfI and ranged in size from approximately 100-4000 bases . By ELISA using polystyrene microtiter plates, fragments from all three species were weakly antigenic compared to intact DNA . These fragments, however, were all antigenic when tested as inhibitors in competition-binding assays . The weak antigenicity of fragments could not be explained by poor adherence to the plates since fragments and intact DNA showed similar levels of binding as assessed using biotinylated preparations . Together, these results demonstrate that the antigenicity of DNA fragments is dramatically altered by solid-phase binding and suggest that constraints on topological or conformational rearrangements of DNA in the solid phase limit antibody interaction. Mol Cell Biochem, 1994 Aug 31, 137(2), 109 - 16 Purification and characterization of a deoxy-ribonuclease acting on native and UV irradiated DNA from young and aging rat brain; Suvarchala E et al.; A deoxyribonuclease has been purified to electrophoretic homogeneity from young and old rat brain . The enzyme is an endonuclease, with an optimum pH 5.0 . Divalent cations are not needed for the activity . The DNase showed highest activity towards Native DNA either as such or UV irradiated with little activity on denatured DNA, apurinic DNA or DNA pretreated with mitomycin C or actinomycin D . The enzyme hydrolyzes double stranded poly (dA-dT).(dA-dT) but not other homologous or heterologous synthetic polynucleotides . The enzyme does not excise pyrimidine dimers preferentially but acts at a site away from the dimer . The DNase was partially purified from nuclei also and both the nuclear and extra nuclear enzymes showed similar properties . The specific activity of brain DNase decreases markedly with age . DNase preparations from both young and old rats showed similar apparent molecular weight (62KD) and many other properties like elution profiles and the N-terminal amino acid . However the old enzyme was more susceptible to temperature and proteolytic digestion . These results are taken to indicate a possible role for this enzyme in recognizing conformational distortions in DNA and that altered molecules of this enzyme accumulate in aging brain. J Biotechnol, 1994 Aug 31, 36(3), 221 - 30 High level expression of hepatitis B virus preS1 peptide in Escherichia coli; Rhyum SB et al.; PreS1 region gene fragment encoding the N-terminal 56 amino acid (aa) of hepatitis B virus (HBV, adr subtype), which encodes B- and T-cell epitopes and an hepatocyte receptor binding site, was synthesized by PCR and fused to the 3'-end of MalE gene encoding maltose-binding protein (MBP) to yield expression plasmid pMalpreS1-56 . The plasmid was introduced into Escherichia coli DH5 alpha and expressed at 37 degrees C under the control of inducible tac promoter . The resulting fusion protein was highly expressed in a soluble form, about 40% of total cellular proteins, but it bound only partially to an amylose column . Therefore, the soluble preS1 fusion protein was purified to near homogeneity by two passages of anion-exchange chromatography followed by gel filtration . The yield of the fusion protein was 70 mg per 1 culture that had been induced by IPTG for 6 h . The purified fusion protein was specifically cleaved by a Factor Xa digestion to release the preS1 peptide, which was then purified by gel filtration to homogeneity . The purity, integrity, antigenicity and immunogenicity of the purified preS1 peptide was confirmed by glycerol-SDS-PAGE, Western analysis, N-terminal amino acid sequencing and an indirect ELISA. Biochem Pharmacol, 1994 Aug 30, 48(5), 937 - 47 Purine nucleoside phosphorylase: inhibition by purine N(7)- and N(9)-acyclonucleosides; and substrate properties of 7-beta-D-ribofuranosylguanine and 7-beta-D-ribofuranosylhypoxanthine; Bzowska A et al.; A series of 10 N(7)- and N(9)-acyclonucleosides of guanine and 8-substituted guanines (8-Br, 8-SH and 8-NH2), and two N(7)-acyclonucleosides of hypoxanthine, were tested for their ability to inhibit purine nucleoside phosphorylase (PNP) (E.C . 2.4.2.1) from human erythrocytes and rabbit kidney . The acyclic chains contained a nitrogen in place of a carbon at the 3', 4' or 5' position and, in one case, an ether oxygen at the 2' position . Most striking was the finding that one of the N(7)-acyclonucleoside analogues, 7-{(1,3-dihydroxypropyl-2)amino}ethylguanine, proved to be a 3-fold more effective inhibitor than its corresponding N(9) counterpart, with Ki = 5 vs 14 microM for the human enzyme and 0.7 vs 2.3 microM for the rabbit enzyme . Both analogues, as well as the others examined, inhibited phosphorolysis competitively with respect to nucleoside substrates (inosine with the human enzyme and guanosine with the rabbit enzyme) . The foregoing logically led to the finding that the 7-beta-D-ribosides of guanine (N7Guo) and hypoxanthine (N7Ino) were weak substrates of PNP from human erythrocytes, calf spleen and E . coli . With the human enzyme the pseudo-first-order rate constants (Vmax/Km) for phosphorolysis of N7Guo and N7Ino were 0.08 and 0.02% that for Ino . The Michaelis constants (Km) for N7Guo were 27 (calf PNP), 108 (human PNP) and 450 microM (E . coli PNP) . For N7Ino the corresponding Km values were 1.52, 1.26 and 0.64 mM . Four previously well-characterized N(9)-acyclonucleoside inhibitors of calf spleen PNP were found to inhibit phosphorolysis of N7Ino by the same enzyme 2-10-fold more effectively than the parent Ino . The overall results, along with the known excellent substrate properties of N(7)-alkyl- Guo and Ino (Bzowska et al . J Biol Chem 263, 9212-9217, 1988), were examined in relation to present concepts regarding binding of substrates and inhibitors at the active site(s) of these enzymes. Proc Natl Acad Sci U S A, 1994 Aug 30, 91(18), 8670 - 4 Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer; Lamour V et al.; An important step ensuring the fidelity in protein biosynthesis is the aminoacylation of tRNAs by aminoacyl-tRNA synthetases . The accuracy of this process rests on a family of 20 enzymes, one for each amino acid . One exception is the formation of Gln-tRNA(Gln) that can be accomplished by two different pathways: aminoacylation of tRNA(Gln) with Gln by glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) or transamidation of Glu from Glu-tRNA(Gln) mischarged by glutamyl-tRNA synthetase (GluRS; EC 6.1.1.17) . The latter pathway is widespread among bacteria and organelles that, accordingly, lack GlnRS . However, some bacterial species, such as Escherichia coli, do possess a GlnRS activity, which is responsible for Gln-tRNA(Gln) formation . In the cytoplasm of eukaryotic cells, both GluRS and GlnRS activities can be detected . To gain more insight into the evolutionary relationship between GluRS and GlnRS enzyme species, we have now isolated and characterized a human cDNA encoding GlnRS . The deduced amino acid sequence shows a strong similarity with other known GlnRSs and with eukaryotic GluRSs . A molecular phylogenetic analysis was conducted on the 14 GlxRS (GluRS or GlnRS) sequences available to date . Our data suggest that bacterial GlnRS has a eukaryotic origin and was acquired by a mechanism of horizontal gene transfer. Proc Natl Acad Sci U S A, 1994 Aug 30, 91(18), 8527 - 31 Homology-associated nonhomologous recombination in mammalian gene targeting; Sakagami K et al.; Nonhomologous (illegitimate) recombination of DNA underlies many changes in the genome . It involves no or little homology between recombining DNAs and has been considered unrelated with homologous recombination, which requires long homology . In mouse cells, however, we found recombination products whose sequences suggest that homologous interaction between DNAs caused nonhomologous recombination with another DNA . The intermediates of homologous recombination were apparently trapped at various stages and shunted to nonhomologous recombination . In one product, the nonhomologous recombination disrupted gene conversion . In another, it took place exactly at the end of long homology shared between two DNAs . This finding explains why gene targeting needs long uninterrupted homology and why mammalian homologous recombination is often nonconservative . We discuss possible consequences and roles of this type of homology-driven gene destruction mechanism. Proc Natl Acad Sci U S A, 1994 Aug 30, 91(18), 8329 - 33 Mos oncogene product associates with kinetochores in mammalian somatic cells and disrupts mitotic progression; Wang XM et al.; The mos protooncogene has opposing effects on cell cycle progression . It is required for reinitiation of meiotic maturation and for meiotic progression through metaphase II, yet it is an active component of cytostatic factor . mos is a potent oncogene in fibroblasts, but high levels of expression are lethal . The lethality of mos gene expression in mammalian cells could be a consequence of a blockage induced by its cytostatic factor-related activity, which may appear at high dosage in mitotic cells . We have directly tested whether expression of the Mos protein can block mitosis in mammalian cells by microinjecting a fusion protein between Escherichia coli maltose-binding protein and Xenopus c-Mos into PtK1 epithelial cells and analyzing the cells by video time-lapse and immunofluorescence microscopy . Time-course analyses showed that Mos blocked mitosis by preventing progression to a normal metaphase . Chromosomes frequently failed to attain a bipolar orientation and were found near one pole . Injection of a kinase-deficient mutant Mos had no effect on mitosis, indicating that the blockage of mitotic progression required Mos kinase activity . Antitubulin immunostaining of cells blocked by Mos showed that microtubules were present but that spindle morphology was abnormal . Immunostaining for the Mos fusion protein showed that both wild-type and kinase mutant proteins localized at the kinetochores . Our results suggest that mitotic blockage by Mos may result from an action of the Mos kinase on the kinetochores, thus increasing chromosome instability and preventing normal congression. Proc Natl Acad Sci U S A, 1994 Aug 30, 91(18), 8319 - 23 Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli: antibodies for orotate decarboxylation; Smiley JA et al.; Antibodies capable of decarboxylating orotate were sought by immunization with a hapten designed to elicit antibodies with combining sites that resemble the orotate-binding and catalytic portion of the active site of the enzyme orotidine 5'-monophosphate (OMP) decarboxylase (orotidine-5'-monophosphate carboxy-lyase, EC 4.1.1.23) . Active recombinant antibody fragments (Fabs) were selected from a combinatorial cDNA library by complementation of a pyrF strain of Escherichia coli and growth of the library-expressing cells on pyrimidine-free medium . In this biological screen, a sufficiently active antibody from the library would decarboxylate orotate to produce uracil, a pyrimidine source for the auxotroph, and would provide the cells with a growth advantage compared to cells without an active antibody . Six recombinant Fabs yielded identifiable colonies in a screen of 16,000 transformants . To enhance its stability and expression level, one of the six positive fragments was converted into single-chain form . In this form, the antibody fragment conferred a definite growth advantage to the auxotroph that was eliminated when the hapten was included in the medium . The purified single-chain antibody displayed orotate decarboxylase activity in vitro, as determined by a 14CO2 displacement assay . The specific activity of the antibody is approximately 10(-7) times that of naturally occurring OMP decarboxylase, but this antibody-catalyzed rate is estimated to be 10(8) times the background rate . The results offer the potential to use these methods to obtain catalytic antibodies for other biosynthetic reactions as well as to assess the effectiveness of the hapten transition state or active site analog in eliciting antibody catalysts. Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 326 - 30 SecA of Escherichia coli traverses lipid bilayer of phospholipid vesicles; Ahn T et al.; SecA protein of Escherichia coli, when added externally to the vesicles composed of phosphatidylethanolamine, dioleoylphosphatidylglycerol and cardiolipin, was found to be fragmented by trypsin encapsulated within the vesicles . In the presence of ATP or its non-hydrolyzing analogue, ATP-gamma S, the number of fragments and extent of hydrolysis occurred much less than in the absence of these compounds . When ADP was added, however, the hydrolysis products were similar to those when no nucleotide was present . Quenching of SecA fluorescence by vesicle-entrapped iodide corroborated the digestion results . These experiments demonstrated that the SecA protein traverses the lipid bilayer and its membrane topology depends on the kind of nucleotide present. Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 225 - 30 Production of an enzymatically active E1 component of human pyruvate dehydrogenase complex in Escherichia coli: supporting role of E1 beta subunit in E1 activity; Jeng J et al.; A co-expression plasmid containing the coding sequence of both the human liver pyruvate dehydrogenase (PDH) E1 alpha and E1 beta subunits was constructed . Functionally active PDH E1 protein was produced when this co-expression plasmid was introduced into the host Escherichia coli cell, BL21 (DE3)/plysS . In contrast, the production of E1 alpha alone resulted in a catalytically inactive protein, suggesting an important role of the E1 beta subunit in constituting enzyme activity . The PDH E1 protein produced in E . coli was capable of being phosphorylated by PDH-specific kinase . This co-expression system will provide a useful tool for studying the biochemical properties of human PDH E1. Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 121 - 7 Inhibition of rhodopsin phosphorylation by non-myristoylated recombinant recoverin; Kawamura S et al.; Bovine recoverin regulates rhodopsin phosphorylation and controls photoreceptor light sensitivity in a Ca(2+)-dependent manner . Recoverin is post-translationally modified with lipids (myristic acid or related lipids) at its N-terminus . Since with this lipid modification (N-myristoylation), recoverin associates with rod outer segment membranes in a Ca(2+)-dependent manner, N-myristoylation has been suggested to be important for the function of this protein . To study the role of this modification, we obtained recombinant non-myristoylated recoverin in E . coli and studied its functional properties . Here, we report that recombinant non-myristoylated recoverin inhibits rhodopsin phosphorylation at Ca2+ concentrations of 30 nM-10 microM in a similar way as native N-myristoylated recoverin does . Thus, our result showed that N-myristoylation is not essential for the Ca(2+)-dependent inhibition of rhodopsin phosphorylation by recoverin. Biochemistry, 1994 Aug 30, 33(34), 10470 - 6 Effects of phosphorylation, Mg2+, and conformation of the chemotaxis protein CheY on its binding to the flagellar switch protein FliM; Welch M et al.; CheY is the response regulator of bacterial chemotaxis . Previously, we showed that CheY binds to the flagellar switch protein FliM and that this binding is increased upon phosphorylation of CheY {Welch, M., Oosawa, K., Aizawa, S.-I., & Eisenbach, M . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 8787-8791} . Here, we demonstrate that it is the phosphorylated conformation of CheY, rather than the phosphate group itself, that is recognized and bound by FliM . We found that subsequent to the phosphorylation of CheY, Mg2+ was not required for the binding of CheY to FliM . However, phosphorylation of CheY did cause a change in the coordination properties of Mg2+ in the acid pocket of the protein . This change in the coordination of Mg2+ required the presence of the absolutely conserved residue Lys109 . When Lys109 was substituted by arginine, the resulting CheY protein was unable to adopt an active conformation upon phosphorylation, and the protein was not bound by FliM . Surprisingly, the CheY13DK mutant protein, which is active in vivo but cannot be phosphorylated in vitro, exhibited only a low level of FliM binding activity, suggesting that its ability to cause clockwise rotation in the cell is not due to a constitutively high level of FliM binding . On the basis of these findings, we propose a mechanism for CheY activation by phosphorylation. Biochemistry, 1994 Aug 30, 33(34), 10220 - 8 The unfolding of trp aporepressor as a function of pH: evidence for an unfolding intermediate; Eftink MR et al.; The urea-induced unfolding of trp aporepressor from Escherichia coli has been studied as a function of pH from 2.5 to 12.0 at 25 degrees C . At pH 7 and above, the unfolding transition, as monitored by changes in the fluorescence intensity at 360 nm, shows a single transition . At low pH, the transition again appears to be a single transition . In the range of 3.5-6.0, the transition is biphasic, indicating the existence of a folding intermediate . The transitions have also been studied using circular dichroism and size exclusion chromatography . The data were fitted by a model in which the dimeric protein first unfolds to form structured monomers, followed by the unfolding of the monomers . From fits with this "folded monomers" model, the free energy change for the dimer<-->monomer dissociation becomes less positive as pH is decreased; the free energy change for the unfolding of the monomers is essentially independent of pH . An alternate model is one in which the dimer first undergoes a transition to a partially unfolded dimeric state, with this intermediate then denaturing to unfolded monomers . Both models give adequate fits to the data obtained at a single protein concentration . From a study of the concentration dependence of the urea-induced unfolding at pH 5, the "folded monomers" model is found to be more consistent with the data . Size exclusion chromatography data support the description of the intermediate state, which is the most populated state at low pH in the absence of urea, as being a relatively compact monomer.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Aug 30, 33(34), 10215 - 9 Pausing of the restriction endonuclease EcoRI during linear diffusion on DNA; Jeltsch A et al.; Linear diffusion is a mechanism to accelerate association rates beyond their three-dimensional diffusional limit . It is employed by the restriction endonuclease EcoRI as well as many other proteins interacting with specific DNA sequences to locate their target sites on the macromolecular substrate . In order to investigate biochemical and biophysical details of the linear diffusion process, we have developed a competitive cleavage assay which allows us to assess with great accuracy the influence of sequence, sequence context, and other structural features on the linear diffusion of EcoRI on DNA . We show here that linear diffusion is not a hopping but a sliding movement in which EcoRI follows the helical pitch of the DNA, because it does not "overlook" any cleavage site . Linear diffusion is slowed when EcoRI encounters sites on the DNA which resemble its recognition site ("star" sites) . Pauses of up to 20 s are induced, depending on sequence and orientation of the star site . These data suggest that EcoRI can bind to DNA in two binding modes: one tight, specific, and immobile, leading to DNA cleavage, and another one loose and nonspecific, allowing for linear diffusion . Depending on the similarity between the recognition sequence and the DNA sequence being encountered by EcoRI, there will be a continuous transition between these binding modes . Other proteins bound to the DNA and irregular DNA structures such as bent DNA or a triple helix constitute a barrier that cannot easily be passed by EcoRI. Biochemistry, 1994 Aug 30, 33(34), 10249 - 56 Three-dimensional structure of the biotin carboxylase subunit of acetyl-CoA carboxylase; Waldrop GL et al.; Acetyl-CoA carboxylase is found in all animals, plants, and bacteria and catalyzes the first committed step in fatty acid synthesis . It is a multicomponent enzyme containing a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase functionality . Here we report the X-ray structure of the biotin carboxylase component from Escherichia coli determined to 2.4-A resolution . The structure was solved by a combination of multiple isomorphous replacement and electron density modification procedures . The overall fold of the molecule may be described in terms of three structural domains . The N-terminal region, formed by Met 1-Ile 103, adopts a dinucleotide binding motif with five strands of parallel beta-sheet flanked on either side by alpha-helices . The "B-domain" extends from the main body of the subunit where it folds into two alpha-helical regions and three strands of beta-sheet . Following the excursion into the B-domain, the polypeptide chain folds back into the body of the protein where it forms an eight-stranded antiparallel beta-sheet . In addition to this major secondary structural element, the C-terminal domain also contains a smaller three-stranded antiparallel beta-sheet and seven alpha-helices . The active site of the enzyme has been identified tentatively by a difference Fourier map calculated between X-ray data from the native crystals and from crystals soaked in a Ag+/biotin complex . Those amino acid residues believed to form part of the active site pocket include His 209-Glu 211, His 236-Glu 241, Glu 276, Ile 287-Glu 296, and Arg 338.2+ represents the first X-ray model of a biotin-dependent carboxylase. Proc Natl Acad Sci U S A, 1994 Aug 30, 91(18), 8582 - 6 An arcane role of DNA in transcription activation; Ryu S et al.; The mechanism by which the cAMP receptor protein (CRP) activates transcription has been investigated using the lac promoter of Escherichia coli . For transcription activation, an interaction between DNA-bound CRP and RNA polymerase is not sufficient . CRP must bind to a site in the same DNA and close to the promoter . CRP action requires an intact spacer DNA to provide a rigid support in building a CRP-RNA polymerase protein bridge or to allow a conformational change in the DNA to be transmitted to the lac promoter using the protein bridge as a structural support. Proc Natl Acad Sci U S A, 1994 Aug 30, 91(18), 8660 - 4 Control of transcription processivity in phage lambda: Nus factors strengthen the termination-resistant state of RNA polymerase induced by N antiterminator; DeVito J et al.; During transcription of phage lambda early operons, the N gene product alters host RNA polymerase (RNAP) so that transcription proceeds through multiple stop signals . Here, we reproduce the essence of N activity with purified components in synthetic transcription units that contain lambda pL promoter and the N-recognition site, nutL, followed by a variety of intrinsic terminators . We show that three host factors (NusA, NusE, and NusG) are essential for N to allow appreciable transcription through multiple terminators and that this persistent antitermination is stimulated by a fourth factor, NusB . Remarkably, in the absence of all four factors, N suppresses various terminators placed near the nut site . This basal antitermination activity of N is enhanced by NusA and is diminished by high salt and temperature . We postulate that N interacts with RNAP directly, inducing the termination-resistant state . While NusA facilitates this interaction, the other factors strengthen it sufficiently over time and distance so that RNAP bypasses multiple terminators . The dispensability of NusB for persistent antitermination in vitro, but not in vivo, raises the possibility that NusB performs two functions: it increases the stability of N antitermination complex and also counteracts an inhibitory factor in the cell. Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 430 - 5 Extracellular ATP potentiates nitric oxide synthase expression induced by lipopolysaccharide in RAW 264.7 murine macrophages; Tonetti M et al.; Inducible nitric oxide synthase (iNOS) activity in the murine macrophage cell line RAW 264.7 was increased from two- to four-fold after co-exposure of the cells to low doses of bacterial lipopolysaccharide (LPS) and micromolar ATP, compared to LPS alone . Extracellular ATP and its analogs "per se", i.e . without LPS, were not able to induce iNOS activity . The stimulating effect of UTP too, the concentration range of activity (1-100 mM nucleotides) and the rank of potency (ATP-gamma-S = AMP-PNP > ATP = ADP >> AMP-CPP = UTP) seem to indicate an involvement of P2y-type purinergic receptors . GTP, CTP and adenosine were virtually ineffective . These data suggest that binding of extracellular nucleotides to purinergic receptors may increase nitric oxide production by macrophages . This effect might occur in pathological conditions (i.e . inflammation/infection or trauma) where significant amounts of intracellular ATP can be released due to cellular damage. Biochemistry, 1994 Aug 30, 33(34), 10286 - 93 Iron(II) bleomycin-mediated degradation of a DNA-RNA heteroduplex; Morgan MA et al.; The effect of iron(II) bleomycin on a DNA-RNA heteroduplex was investigated using a substrate formed by reverse transcription of Escherichia coli 5S ribosomal RNA . Both strands of the heteroduplex were cleaved by FeII.BLM A2 at comparable concentrations; complete digestion of both strands was observed using 5 microM FeII.BLM A2 . The DNA strand of the heteroduplex was cleaved predominantly at 5'-G-pyr-3' sites; the sites of cleavage of the DNA strand were a subset of those observed for the corresponding DNA strand of a DNA duplex of identical sequence . The sites of cleavage of the RNA strand of the heteroduplex involved both purines and pyrimidines and were found to be different than the sites of cleavage of the 5S rRNA alone, demonstrating that cleavage of the former must actually have involved heteroduplex recognition by FeII.BLM A2 . Both the DNA and RNA strands of the heteroduplex were cleaved by FeII.BLM A2 in the presence of physiological concentrations of Mg2+, consistent with the possibility that DNA-RNA heteroduplexes may be therapeutically relevant targets for bleomycin. FEBS Lett, 1994 Aug 29, 351(1), 49 - 52 The precursor of the Streptomyces R61 DD-peptidase containing a C-terminal extension is inactive; Fanuel L et al.; The Streptomyces R61 DD-peptidase gene encodes a 26-residue C-terminal extension which is not found in the mature protein . When the gene was expressed in Escherichia coli, the extension was not cleaved and the precursor protein was not enzymatically active . It also reacted with penicillins significantly more slowly than the mature protein . The introduction of a 'stop' codon after that corresponding to the C-terminal residue of the mature protein resulted in the production of an active protein in the periplasm of E . coli. J Biol Chem, 1994 Aug 26, 269(34), 21915 - 8 The P1 reactive site methionine residue of ecotin is not crucial for its specificity on target proteases . A potent inhibitor of pancreatic serine proteases from Escherichia coli; Seong IS et al.; The importance of the P1 reactive site for the specificity of ecotin on target proteases was examined by site-directed mutagenesis . The replacement of Met at the P1 site with Ile, Arg, Glu, or Tyr showed little or no effect on the ability of ecotin to inhibit trypsin . Similar results were obtained for chymotrypsin, except that its replacement with Glu caused about 40% reduction of the inhibitory activity of ecotin . On the other hand, the replacement of the Met residue with Arg, Tyr, or Glu dramatically reduced its ability to inhibit elastase, while that with Ile showed little or no effect . Nevertheless, elastase could be completely inhibited upon incubation with excess amounts of the mutant ecotin containing Arg, Glu, or Tyr . Moreover, all the mutant forms of ecotin could be cleaved at the mutated P1 site upon incubation with trypsin at pH 3.75 . In addition, the replacement of a Cys residue in the disulfide bridge with Ser showed little or no effect on the ability of ecotin to inhibit trypsin, chymotrypsin, or elastase . However, the mutant ecotin containing Ser was more sensitive to inactivation by heating at 100 degrees C than the wild-type inhibitor . Furthermore, the wild-type ecotin whose disulfide bond had been reduced and alkylated was also more easily inactivated by heat treatment than the untreated control . These results strongly suggest that the P1 site of ecotin is not crucial for its specificity on target proteases and that the disulfide bridge in ecotin appears to play an important role in maintenance of its structural stability. J Biol Chem, 1994 Aug 26, 269(34), 21828 - 34 Photoaffinity labeling of DNA template-primer binding site in Escherichia coli DNA polymerase I . Identification of involved amino acids; Pandey VN et al.; We have used two self-annealing template-primers (TPs) to covalently cross-link the Klenow fragment of Escherichia coli DNA polymerase I in its polymerase mode . The specificity of cross-linking is demonstrated by the observation that other template-primers, but not the template or primer alone, readily compete with self-annealing TPs . The enzyme-TP covalent complex is catalytically active and can incorporate one nucleotide on the primer terminus of the immobilized template-primer . Using a peptide mapping approach, we have identified a 17-amino acid tryptic peptide spanning residues 759-775 as a major constituent of the TP binding domain . Amino acid sequence analysis further revealed that Ile-765, Tyr-766 in the O-helix and Ser-769, Phe-771 in the O1-helix of the three-dimensional crystal structure of the Klenow fragment constitute the attachment site for TP. J Biol Chem, 1994 Aug 26, 269(34), 21820 - 7 Cloning, expression, and characterization of human apolipoprotein(a) kringle IV37; LoGrasso PV et al.; A portion of kringle IV37 (KIV37) of apolipoprotein (a), (apo(a)), was polymerase chain reaction-cloned from human liver cDNA . The protein product of this clone was expressed in Escherichia coli as a poly histidine fusion protein . Based on recovery of purified fusion apo(a) KIV37 protein expression levels were estimated to be 10 mg/g of E . coli cell paste . Mass spectral analysis showed the molecular mass of fusion apo(a) KIV37 to be 12,260 +/- 1 daltons . Almost all fusion apo(a) KIV37 was expressed as inclusion bodies and had to be refolded . Fusion apo(a) KIV37 was isolated from the inclusion bodies and purified by lysine-Sepharose affinity chromatography by eluting with 0.2 M epsilon-aminocaproic acid . The fusion protein was treated with thrombin to yield a homogeneous, functional apo(a) KIV37 domain composed of 92 amino acids having a molecular mass of 10,510 +/- 1 daltons . N-terminal protein sequencing and amino acid analysis have confirmed the sequence and composition of apo(a) KIV37 . The molar extinction coefficient, epsilon, for apo(a) KIV37 was determined to be 3.1 x 10(4) M-1 cm-1, and the pI was measured to be 6.7 +/- 0.1 . In addition, the dissociation constants, Kd, for a series of 11 lysine analogs have been determined by measuring the change in intrinsic fluorescence of apo(a) KIV37 upon saturable binding with these compounds . Kd values ranged from 4.2 +/- 0.9 microM for trans-4-(aminomethyl)cyclohexanecarboxylic acid to 4.6 +/- 0.4 mM for L-arginine . Apo(a) KIV37 binds to plasmin-treated fibrinogen with an EC50 value of 14 +/- 1.2 microM and prevents the binding of Lp(a) to plasmin-treated fibrinogen with an IC50 value of 16 +/- 6 microM . Lp(a) binds to the plasmin-treated fibrinogen surface with an EC50 value of approximately 1.0 +/- 0.3 nM . These studies demonstrate that apo(a) KIV37 can be expressed at high levels, refolded properly, and used as a fully functional lysine-binding domain . In addition, these results also demonstrate that apo(a) KIV37 provides the major interaction of Lp(a) with fibrinogen . One additional weak binding site in Lp(a) is adequate to describe overall Lp(a) binding to fibrinogen. J Biol Chem, 1994 Aug 26, 269(34), 21812 - 9 Upstream interactions of functional mammalian tRNA gene transcription complexes probed using a heterologous DNA-binding protein; Tapping RI et al.; An oligonucleotide containing the recognition site for the Escherichia coli lac repressor was inserted at various positions in the 5' flanking region of a human serine tRNA gene, and the consequences of binding lac repressor on in vitro transcription by RNA polymerase III were investigated . lac repressor prebound to operator sites centered at positions -9, -15, -35, and -37 upstream of the mature tRNA coding region completely inhibited transcription by interfering with the formation or stability of transcription complexes . lac repressor also inhibited transcription of tDNA derivatives containing operator sites at -9 and -15 when added following assembly of transcription complexes or during ongoing synthesis, but had no effects on the other tDNA derivatives if added subsequent to complex assembly . lac repressor prebound at position -43 and -46 partially inhibited transcription and redirected initiation to sites farther downstream . These effects required the continued presence of bound repressor protein . Our findings demonstrate that the human RNA polymerase III transcription complex extends at least 35 nucleotides upstream of the coding region and suggest that the spatial constraints imposed by a protein bound this far upstream can alter start site selection . Moreover, the flanking region encompassing the transcription start site remains accessible to DNA-binding proteins following assembly of the initiation complex and throughout multiple rounds of transcription. J Mol Biol, 1994 Aug 26, 241(4), 524 - 33 Determinants of receptor specificity of coliphages of the T4 family . A chaperone alters the host range; Hashemolhosseini S et al.; E . coli phages of the T4 family (T4, TuIa, TuIb) recognize their cellular receptors with a C-terminal region of protein 37 . This protein, common to all three phages, is present as a dimer located at the distal part of the long tail fibers and possesses a C-terminal domain consisting of 40 to 70 highly conserved C-terminal residues, followed by a variable region of 50 to 80 residues which is again followed by a highly conserved area . Protein 38, not being a component of the mature virion, is required for dimerization of protein 37; this represents a non-covalent association of a structural protein . Seven host range mutants of TuIa or TuIb were analyzed which were able to use proteinaceous receptors other than those recognized by their parents . All had suffered amino acid substitutions within the variable region . It is concluded that in all probability it is this region which interacts directly with the cellular receptors . Conditional mutants of T4 are known which, when propagated at the non-permissive temperature (42 degrees C), yield phage of normal morphology but these are more or less unable to adsorb to cells . The causative amino acid substitutions were found both downstream and upstream from the variable area . Distortion of it in the mutants could suggest a "snap-back" conformation of the tail fiber; the conserved C-terminal region may fold back and expose the variable region as a loop at the tip of the fiber . One of the phage mutants (L93), when grown at the permissive temperature, had lost the ability to use the OmpC porin (a receptor for T4) as a receptor . A secondary mutant, able to do so, was isolated . An additional mutation, leading to one amino acid substitution, had occurred in gene 38 . This mutant gene acted in trans and caused a much enhanced temperature-sensitivity of infectivity without conferring temperature-sensitivity per se, i.e . the mutant protein 38 apparently altered the conformation of the receptor-recognizing area of the dimer of protein 37 . A gene from phage lambda, about 40% identical to gene 38 of T4, complements gene 38 amber mutants . The corresponding protein also restored the ability of L93 to recognize OmpC but did not cause any such temperature-sensitivity . Hence, protein 38, classifying as a chaperone, appears to act instructively in conveying steric information to the target polypeptide. J Biol Chem, 1994 Aug 26, 269(34), 21907 - 14 Interactions of Escherichia coli endonuclease IV and exonuclease III with abasic sites in DNA; Takeuchi M et al.; Duplex oligodeoxynucleotides with synthetic analogs of abasic sites were used to study the specificity of the abasic endonucleases of Escherichia coli . The apparent Km values of exonuclease III for the tetrahydrofuranyl, propanyl, and deoxyribosyl substrates varied only somewhat (20-140 nM) in either Mg2+ or Ca2+ and were similar to those for endonuclease IV . In Mg2+, exonuclease III had a turnover number 4-13-fold higher than measured for endonuclease IV (ranging 5.6-18 min-1), but was lowered in Ca2+ to values similar to those for endonuclease IV . The rate of cleavage of tetrahydrofuranyl (F) substrate by both enzymes was unaffected by the base in the opposite strand or its replacement by a tetrahydrofuranyl moiety . A C:C mismatch on the 5' but not the 3' side of F strongly inhibited cleavage by exonuclease III in Ca2+, while mismatches on both sides were required to diminish endonuclease IV cleavage significantly . A phosphorothioate ester linked 5' to the tetrahydrofuranyl moiety inhibited both enzymes, with the Rp stereoisomer most effective . Endonuclease IV bound stably to duplex substrates containing the Rp phosphorothioate in the presence of poly(dI-dC) . Although the apurinic/apyrimidinic-cleaving activities of endonuclease IV and exonuclease III have some common features they also differ in their specific interactions with DNA containing abasic sites. J Biol Chem, 1994 Aug 26, 269(34), 21741 - 7 An active recombinant p15 RNase H domain is functionally distinct from the RNase H domain associated with human immunodeficiency virus type 1 reverse transcriptase; Evans DB et al.; An active p15 RNase H domain, consisting of amino acids 427-560 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and a genetically engineered penta-histidine N-terminal affinity tag, was expressed in Escherichia coli and purified to apparent homogeneity by immobilized metal affinity chromatography . The purified p15 RNase H domain exhibited no substrate preference for {3H}poly(rG).poly(dC) compared to {3H}poly(rA).poly(dT), in contrast with the HIV-1 RT-associated RNase H, which showed a 30-fold preference for the former substrate . Unlike the HIV-1 RT-associated RNase H, when challenged with unlabeled substrate, the recombinant p15 RNase H domain was relatively nonprocessive in RNA degradative activity of the {3H}poly(rA).poly(dT) duplex . Kinetic studies using p15 RNase H showed substrate inhibition with an apparent K(i) value of 0.12 micron for the {3H}poly(rA).poly(dT) hybrid . Substrate inhibition was not observed for the HIV-1 RT-associated RNase H . The results show that the isolated p15 HIV-1 RNase H domain is functionally distinct from the recombinant HIV-1 RT-associated RNase H. Nucleic Acids Res, 1994 Aug 25, 22(16), 3433 - 9 Interactions between yeast TFIIIB components; Huet J et al.; Yeast transcription factor TFIIIB is a multicomponent factor comprised of the TATA-binding protein TBP and of associated factors TFIIIB70 and B" . Epitope-tagged or histidine-tagged TFIIIB70 could be quantitatively removed from TFIIIB by affinity chromatography . TBP and B" (apparent mass 160-200 kDa) could be easily separated by gel filtration or ion-exchange chromatography . While only weak interactions were detected between TBP and B", direct binding of {35S}-labeled TBP to membrane-bound TFIIIB70 could be demonstrated in absence of DNA . On tRNA genes, there was no basal level of transcription in the complete absence of TBP . The two characterized TFIIIB components (recombinant rTFIIIB70 and rTBP) and a fraction cochromatographing with B" activity were found to be required for TFIIIC-independent transcription of the TATA-containing U6 RNA gene in vitro . Therefore, beside the TFIIIC-dependent assembly process, each TFIIIB component must have an essential role in DNA binding or RNA polymerase recruitment. Nucleic Acids Res, 1994 Aug 25, 22(16), 3317 - 21 Characterization of minisatellites in Arabidopsis thaliana with sequence similarity to the human minisatellite core sequence; Tourmente S et al.; A strategy based on random PCR amplification was used to isolate new repetitive elements of Arabidopsis thaliana . One of the random PCR product analyzed by this approach contained a tandem repetitive minisatellite sequence composed of 33 bp repeated units . The genomic locus corresponding to this PCR product was isolated by screening a lambda genomic library . New related loci were also isolated from the genomic library by screening with a 14 mer oligonucleotide representing a region conserved among the different repeated units . Alignment of the consensus sequence for each minisatellite locus allowed the definition of an Arabidopsis thaliana core sequence that shows strong sequence similarities with the human core sequence and with the generalized recombination signal Chi of Escherichia coli . The minisatellites were tested for their ability to detect polymorphism, and their chromosomal position was established. Nucleic Acids Res, 1994 Aug 25, 22(16), 3312 - 6 Human xeroderma pigmentosum group G gene encodes a DNA endonuclease; Habraken Y et al.; Because of defective nucleotide excision repair of ultraviolet damaged DNA, xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers . Cell fusion studies have identified seven XP complementation groups, A to G . Previous studies have implicated the products of these seven XP genes in the recognition of ultraviolet-induced DNA damage and in incision of the damage-containing DNA strand . Here, we express the XPG-encoded protein in Sf9 insect cells and purify it to homogeneity . We demonstrate that XPG is a single-strand specific DNA endonuclease, thus identifying the catalytic role of the protein in nucleotide excision repair . We suggest that XPG nuclease acts on the single-stranded region created as a result of the combined action of the XPB helicase and XPD helicase at the DNA damage site. Biochim Biophys Acta, 1994 Aug 25, 1214(1), 1 - 10 Expression of human liver fatty acid-binding protein in Escherichia coli and comparative analysis of its binding characteristics with muscle fatty acid-binding protein; Maatman RG et al.; Human liver fatty acid-binding protein (L-FABP) has been efficiently expressed in Escherichia coli . The cDNA encoding human liver FABP was under the control of T7 RNA polymerase promoter in the expression vector pET-3b . Expression required overnight induction with isopropyl beta-D-thiogalactopyranoside in the presence of the bacterial RNA polymerase inhibitor, rifampicin . The protein could be purified by (NH4)2SO4 fractionation, anion-exchange and gel filtration chromatography, and was recognized by anti-(human L-FABP) antiserum . The binding characteristics of delipidated recombinant human L-FABP and muscle FABP (M-FABP) for fatty acids of different chain length and saturation grade, and for various hydrophobic ligands, were determined by radiochemical analysis and also by fluorescence for L-FABP . The apparent binding affinity of the ligands was calculated by using displacement curves of oleic acid and dansylamino-undecanoic acid (DAUDA) . L-FABP showed a preference for the binding of long-chain saturated and unsaturated fatty acids up to C24:1, whereas the M-FABP has a preference for unsaturated fatty acids, especially those with 18 C atoms . L-FABP also binds palmitoyl derivatives and many other hydrophobic ligands--however, generally with a lower affinity than fatty acids . M-FABP binds--besides with fatty acids--only with oestradiol and testosterone with high affinity . Fatty acids with fluorescent reporter groups are also more tightly bound by L-FABP . A direct assay and displacement study of oleic acid gave the same Kd value of DAUDA for L-FABP . Fluorescence enhancement and displacement studies indicate that the binding of fluorescent fatty acids is determined by both the fluorescent reporter group and the acyl carbon chain. Nature, 1994 Aug 25, 370(6491), 666 - 8 Structural similarity between the p17 matrix protein of HIV-1 and interferon-gamma; Matthews S et al.; The human immunodeficiency virus (HIV) matrix protein, p17, forms the outer shell of the core of the virus, lining the inner surface of the viral membrane . The protein has several key functions . It orchestrates viral assembly via targeting signals that direct the gag precursor polyprotein, p55, to the host cell membrane and it interacts with the transmembrane protein, gp41, to retain the env-encoded proteins in the virus . In addition, p17 contains a nuclear localization signal that directs the preintegration complex to the nucleus of infected cells . This permits the virus to infect productively non-dividing cells, a distinguishing feature of HIV and other lentiviruses . We have determined the solution structure of p17 by nuclear magnetic resonance (NMR) with a root-mean square deviation for the backbone of the well-defined regions of 0.9 A . It consists of four helices connected by short loops and an irregular, mixed beta-sheet which provides a positively charged surface for interaction with the inner layer of the membrane . The helical topology is unusual; the Brookhaven protein database contains only one similar structure, that of the immune modulator interferon-gamma. Nature, 1994 Aug 25, 370(6491), 662 - 6 Crystal structure of the extracellular region of human tissue factor; Harlos K et al.; Tissue factor is a cell-surface glycoprotein receptor which initiates the blood coagulation cascade after vessel injury by interacting with blood clotting factor VII/VIIa and which is implicated in various pathological processes . When bound to tissue factor, factor VII is readily converted to the active protease factor VIIa by trace amounts of factors Xa, IXa or VIIa . Human tissue factor consists of 263 residues, the first 219 of which comprise the extracellular region . We have determined the crystal structure of the extracellular region at a resolution of 2.2 A . Tissue factor consists of two immunoglobulin-like domains associated through an extensive, novel, interdomain interface region . The binding site for factor VII lies at the interface region and involves residues from domain 1 and an extended loop (binding 'finger') of domain 2 . This is the first reported structure of a representative of the class 2 cytokine receptor family, which also includes interferon-alpha, interferon-gamma (refs 2, 3) and interleukin-10 (ref . 4) receptors. Biochemistry, 1994 Aug 23, 33(33), 9912 - 21 Identity switches between tRNAs aminoacylated by class I glutaminyl- and class II aspartyl-tRNA synthetases; Frugier M et al.; High-resolution X-ray structures for the tRNA/aminoacyl-tRNA synthetase complexes between Escherichia coli tRNAGln/GlnRS and yeast tRNAAsp/AspRS have been determined . Positive identity nucleotides that direct aminoacylation specificity have been defined in both cases; E . coli tRNAGln identity is governed by 10 elements scattered in the tRNA structure, while specific aminoacylation of yeast tRNAAsp is dependent on 5 positions . Both identity sets are partially overlapping and share 3 nucleotides . Interestingly, the two enzymes belong to two different classes described for aminoacyl-tRNA synthetases . The class I glutaminyl-tRNA synthetase and the class II aspartyl-tRNA synthetase recognize their cognate tRNA from opposite sides . Mutants derived from glutamine and aspartate tRNAs have been created by progressively introducing identity elements from one tRNA into the other one . Glutaminylation and aspartylation assays of the transplanted tRNAs show that identity nucleotides from a tRNA originally aminoacylated by a synthetase from one class are still recognized if they are presented to the enzyme in a structural framework corresponding to a tRNA aminoacylated by a synthetase belonging to the other class . The simple transplantation of the glutamine identity set into tRNAAsp is sufficient to obtain glutaminylatable tRNA, but additional subtle features seem to be important for the complete conversion of tRNAGln in an aspartylatable substrate . This study defines C38 in yeast tRNAAsp as a new identity nucleotide for aspartylation . We show also in this paper that, during the complex formation, aminoacyl-tRNA synthetases are at least partially responsible for conformational changes which involve structural constraints in tRNA molecules. Biochemistry, 1994 Aug 23, 33(33), 9898 - 903 Direct photolinkage of GTP to the vaccinia virus mRNA (guanine-7-) methyltransferase GTP methyl acceptor site; Niles EG et al.; Direct UV photolinkage of {alpha-32P}GTP to the methyl acceptor site of the vaccinia virus (guanine-7-) methyltransferase was attempted in order to identify the GTP binding region of this enzyme . Low-efficiency photolinkage of GTP to the carboxyl terminal domain of the large subunit, D1R498-844, was achieved and shown to be specific by several criteria . The half-saturation value for GTP was determined to be 35 microM which is equivalent to the catalytic Km for the substrate . GTP photolinkage was shown to be inhibited by GpppA, a substrate for the methyltransferase reaction, better than GMepppA, the reaction product . The addition of MgCl2, known to prevent GTP from serving as a methyl group acceptor in this reaction, was found to eliminate GTP photolinkage . Finally, AdoHcy, a potent product inhibitor of AdoMet binding, failed to inhibit GTP photolinkage, demonstrating that GTP was not linked to the AdoMet binding site . Chemical cleavage of the GTP-labeled enzyme permitted the identification of multiple radioactive peptides, demonstrating the existence of multiple interaction sites in the carboxyl terminal domain of the D1R subunit . The addition of the small D12L subunit has been shown to activate the (guanine-7-) methyltransferase activity in D1R498-844 30-50-fold . The efficiency of GTP photolinkage to the isolated D1R498-844 domain, however, was found to be only marginally effected by the addition of the D12L subunit, demonstrating that this enhancement of mRNA (guanine-7-) methyltransferase activity mediated by D12L was not achieved by altering the strength of GTP binding. Biochemistry, 1994 Aug 23, 33(33), 9881 - 8 Protein-DNA interactions and alterations in the DNA structure upon UvrB-DNA preincision complex formation during nucleotide excision repair in Escherichia coli; Visse R et al.; The UvrB-DNA preincision complex is a key intermediate in the repair of damaged DNA by the UvrABC endonuclease from Escherichia coli . DNaseI footprinting of this complex on DNA with a cis-{Pt(NH3)2{d(GpG)-N7(1),N7(2)}} adduct provided global information on the protein binding site on this substrate {Visse, R., et al . (1991) J . Biol . Chem . 266, 7609-7617} . By applying a method developed by Fairall and Rhodes {Fairall, L., & Rhodes, D . (1992) Nucleic Acids Res . 20, 4727-4731}, who have used the size and shape of DNasI for the interpretation of a footprint, we were able to define in more detail the region where UvrB-DNA interactions in the preincision complex occur . The potential interactions with phosphate groups could be reduced to less then 14 in the damaged and to 12 in the nondamaged strand . The main UvrB-DNA interactions seem restricted to the major groove on both sides of the lesion . As a consequence UvrB crosses the minor groove just downstream of the damage . Such a binding of UvrB orients the protein away from the damage . The more detailed interpretation of UvrB-DNA interactions was supported by methylation protection experiments . The structure of the DNA in the preincision complex formed on cis-{Pt(NH3)2{GpG-N7(1),N7(2)}} is altered as could be shown diethylpyrocarbonate sensitivity of adenines just downstream of the lesion . However the adenines just downstream of another cisplatin adduct, cis-{Pt(NH3)2{d(GpCpG)-N7(1),N7(3)}}, did not become diethylpyrocarbonate sensitive in the preincision complex although this complex is incision proficient.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Aug 23, 33(33), 9875 - 80 Effect of pH and temperature on the stability of UV-induced repairable pyrimidine hydrates in DNA; O'Donnell RE et al.; UV irradiation of cytosine yields 6-hydroxy-5,6-dihydrocytosine (cytosine hydrate) whether the cytosine is in solution as base, nucleoside, or nucleotide or on the DNA backbone . Cytosine hydrate decomposes by elimination of water, yielding cytosine, or by irreversible deamination, yielding uracil hydrate, which, in turn, decomposes by dehydration yielding uracil . To determine how pH and temperature affect these decomposition reactions, alternating poly(dG-{3H}dC) copolymer was irradiated at 254 nm and incubated under different conditions of pH and temperature . The cytosine hydrate and uracil hydrate content of the DNA was determined by the use of Escherichia coli endonuclease III, which releases pyrimidine hydrates from DNA by virtue of its DNA glycosylase activity . Uracil content was determined by using uracil-DNA glycosylase . The rate of decomposition of cytosine hydrate to cytosine was determined at 4 temperatures at pH 3.1, 5.4, and 7.4 . The Ea was determined from the rates by using the Arrhenius equation and proved to be the same at pH 5.4 and 7.4, although the decomposition rate at pH 5.4 was faster at all temperatures . At pH 3.1, the Ea was reduced . These results suggest that the dehydration reaction is affected by two discrete protonations, most probably of the N-3 and the OH group of C-6 of cytosine hydrate . The deamination of cytosine hydrate to uracil hydrate was maximal at pH 3.1 at all temperatures . The doubly protonated cytosine hydrate probably is the common intermediate for both competing decomposition reactions, explaining why cytosine hydrate is prone to deamination at acid pH.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Aug 23, 33(33), 9826 - 30 Analysis of hydrogen bonding in enzyme-substrate complexes of chloramphenicol acetyltransferase by infrared spectroscopy and site-directed mutagenesis; Murray IA et al.; Chloramphenicol acetyltransferase (CAT) reversibly transfers an acetyl group between CoA and the 3-hydroxyl of either chloramphenicol (Cm) or 1-acetylchloramphenicol (1AcCm) . The products of the forward reactions, 3-acetylchloramphenicol (3-AcCm) and 1,3-diacetylchloramphenicol (1,3Ac2-Cm), are the substrates for the reverse reaction . The role of the 3-acetyl carbonyl group in the binding of the substrates 3AcCm and 1,3Ac2Cm to CAT has been investigated using infrared spectroscopy . Comparison of difference spectra (3-{12C = O}acetyl- minus 3-{13C = O}acetyl-) obtained for the binary complexes of 3AcCm with wild-type CAT, and with a variant wherein serine-148 is replaced by alanine (S148A), reveals a large (9 cm-1) down frequency shift for the 3-acetyl carbonyl stretch in the wild-type complex, indicative of a hydrogen bond between this carbonyl and the hydroxyl group of Ser-148 . The carbonyl bandwidth in the wild-type complex is reduced by 33% compared to that for the complex with S148A, indicating restriction of carbonyl mobility and dispersion in the former, an observation consistent with the proposed hydrogen bond between the ester carbonyl and the hydroxyl of Ser-148 . Repetition of the experiment using 1,3Ac2Cm as the ligand reveals a frequency shift of only 3 cm-1 between wild-type and S148A complexes, indicating only a small change in the strength of carbonyl interaction.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Aug 23, 33(33), 10207 - 14 Role of subunit IV in the cytochrome b-c1 complex from Rhodobacter sphaeroides; Chen YR et al.; Rhodobacter sphaeroides mutants lacking subunit IV (M(r) = 14,384) of the cytochrome b-c1 complex (representative mutant strain, RS delta IV-2) have been constructed by site-specific recombination between the wild-type genomic subunit IV structural gene (fbcQ) and a suicide plasmid containing a defective fbcQ sequence . RS delta IV-2 gives rise to a photosynthetically competent phenotype after a period of adaptation . The chemical compositions, spectral properties, and cytochrome b-c1 complex activities in subunit IV-deficient chromatophores from adapted RS delta IV-2 are similar to those in wild-type chromatophores . However, the apparent Km for Q2H2 for the b-c1 complex in subunit IV-deficient chromatophores from adapted RS delta IV-2 cells is about four times higher than that in chromatophores from wild-type cells . The cytochrome b-c1 complex activity in subunit IV-deficient chromatophores of adapted RS delta IV-2 cells is more labile to detergent treatment than that from wild-type cells . The specific activities of dodecylmaltoside-solubilized fractions of RS delta IV-2, based on cytochrome b, are only one-fourth that of the untreated chromatophores . Introducing a wild-type fbcQ operon on a stable low copy number plasmid, pRK415, into RS delta IV-2 restores photosynthetic growth behavior, the apparent Km value for Q2H2, and tolerance to detergent treatment to that of wild-type cells . Cytochrome b-c1 complex purified from adapted RS delta IV-2 contains only three subunits . It has only 25% of the activity of the four-subunit enzyme . This low activity is accompanied by an increase of the apparent Km for Q2H2 from 3 to 13 microM, suggesting that subunit IV may be involved in quinone binding in addition to its structural role. Biochemistry, 1994 Aug 23, 33(33), 10120 - 6 Escherichia coli glycerol kinase: role of a tetramer interface in regulation by fructose 1,6-bisphosphate and phosphotransferase system regulatory protein IIIglc; Liu WZ et al.; Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) is a key element in a signal transduction pathway that couples expression of genes required for glycerol metabolism to the relative availability of glycerol and glucose . Its catalytic activity is inhibited by protein-protein interactions with IIIglc, a phosphotransferase system protein, and by fructose 1,6-bisphosphate (FBP); each of these allosteric effectors constitutes a positive signal that glucose is available . Loss of glucose inhibition of glycerol metabolism was used to screen for regulatory mutants of glycerol kinase after hydroxylamine mutagenesis of the cloned glpK gene . Two mutant enzymes were identified and shown by DNA sequencing to contain the mutations alanine 65 to threonine (A65T) and aspartate 72 to asparagine (D72N) . Initial velocity studies show the mutations do not significantly affect the catalytic properties, hence active-site structures, of the enzymes . Both mutations decrease inhibition by FBP; A65T eliminates the inhibition while D72N appears to decrease the affinity for FBP and the extent of the inhibition . However, neither mutation significantly affects inhibition by IIIglc . Gel-permeation chromatography studies show that both of the mutations alter the dimer-tetramer assembly reaction of the enzyme and the effect of FBP in increasing the molecular weight . The effects of the mutations on the assembly reaction are consistent with the locations of these two amino acid residues in the X-ray structure, which shows them to be associated with an alpha-helix that constitutes one of the two subunit-subunit interfaces within the tetramer.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Aug 23, 33(33), 10007 - 12 Kinetics of the quaternary structure change of aspartate transcarbamylase triggered by succinate, a competitive inhibitor; Tsuruta H et al.; The quaternary structural change of Escherichia coli aspartate transcarbamylase (ATCase) was studied by time-resolved X-ray solution scattering following the binding of carbamoyl phosphate and of succinate, a competitive inhibitor of the natural substrate L-aspartate . Stopped-flow experiments at sub-zero temperatures in the presence of 30% ethylene glycol allowed us to monitor the evolution of the scattering pattern, including the characteristic scattering peak in an s (=2 sin theta/lambda) range of 0.01-0.06 A-1 . The inhibitor binding promotes a quaternary structure change from the T state toward the R state, and as expected for a simple ligand binding process, ATCase remains in the R state, unlike the physiological enzyme reaction {Tsuruta, H., et al . (1990) FEBS Lett . 263, 66-68} . After equilibrium had been established, the final scattering pattern was recorded . When the succinate concentration was sufficiently high, this pattern was the same as that given by ATCase saturated with the bisubstrate analogue N-phosphonoacetyl-L-aspartate (PALA) . This implies that, under cryogenic conditions, succinate and carbamoyl phosphate promote the same quaternary structure change as PALA, which is in good agreement with the crystallographic studies of Gouaux and Lipscomb {Gouaux, J.E., & Lipscomb, W.N . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 4205-4208} . Scattering patterns recorded during the course of the structural transition were satisfactorily reproduced by a linear combination of the initial and final patterns, suggesting that there is no significant concentration of quaternary structure intermediates between the T and R states . This is consistent with a concerted structural transition of ATCase.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Aug 23, 33(33), 9845 - 55 Solution structure of a POU-specific homeodomain: 3D-NMR studies of human B-cell transcription factor Oct-2; Sivaraja M et al.; The POU DNA-binding motif defines a conserved family of eukaryotic transcription factors involved in regulation of gene expression . This bipartite motif consists of an N-terminal POU-specific domain (POUs), a flexible linker, and a C-terminal POU-specific homeodomain (POUHD) . Here we describe the solution structure of a POU-specific homeodomain . An NMR model is obtained from Oct-2, a human B-cell specific transcription factor which participates in the regulation of immunoglobulin genes . A fragment of Oct-2 containing POUHD and an adjoining linker was expressed in Escherichia coli and characterized by three-dimensional nuclear magnetic resonance (3D-NMR) spectroscopy . Complete 1H and 15N resonance assignment of the POUHD moiety is presented . The POUHD solution structure, as calculated by distance geometry and simulated annealing (DG/SA), is similar to that of canonical homeodomains . A salient difference between solution and crystal structures is observed in the C-terminal segment of alpha-helix 3 (the HTH recognition helix), which is not well ordered in solution . Because this segment presumably folds upon specific DNA binding, its flexibility in solution may reduce the intrinsic DNA affinity of POUHD in the absence of POUs. FEBS Lett, 1994 Aug 22, 350(2-3), 258 - 62 Direct observation of the iron binding sites in a ferritin; Hempstead PD et al.; X-Ray analysis of the ferritin of Escherichia coli (Ec-FTN) and of Ec-FTN crystals soaked in (NH4)2Fe(SO4)2 has revealed the presence of three iron-binding sites per subunit . Two of these form a di-iron site in the centre of the subunit as has been proposed for the 'ferroxidase centres' of human ferritin H chains . This di-iron site, lying within the 4-alpha-helix bundle, resemble those of ribonucleotide reductase, methane monoxygenase and haemerythrin . The third iron is bound by ligands unique to Ec-FTN on the inner surface of the protein shell . It is speculated that this state may represent the nucleation centre of a novel type of Fe(III) cluster, recently observed in Ec-FTN. FEBS Lett, 1994 Aug 22, 350(2-3), 249 - 52 Differential inhibition of eukaryotic DNA polymerases by halenaquinol sulfate, a p-hydroquinone sulfate obtained from a marine sponge; Shioda M et al.; Halenaquinol sulfate, a p-hydroquinone sulfate obtained from a marine sponge, inhibited the activity of eukaryotic DNA polymerases in varying degrees; the Ki values for DNA polymerases, alpha, beta, delta and epsilon were 1.3, 80, 17.5 and 2.0 microM, respectively, whereas it was less effective against E . coli DNA polymerase I . The inhibition occurred competitively with each of dATP and dTTP, but non-competitively with dCTP, dGTP and the template DNA . Thus, halenaquinol sulfate is demonstrated to be a potential inhibitor of DNA polymerases alpha and epsilon, and be a useful tool for analyzing the dNTP binding sites of DNA polymerases. FEBS Lett, 1994 Aug 22, 350(2-3), 245 - 8 Inclusion body formation by interleukin-1 beta depends on the thermal sensitivity of a folding intermediate; Wetzel R et al.; We show that sequence and growth temperature effects on IB formation in the small, monomeric beta-barrel protein interleukin-1 beta (IL-1 beta) can be quantitatively reproduced in an in vitro system in which IL-1 beta is refolded from denaturant at different temperatures . The results suggest that temperature and mutational effects on IB formation may be based on intrinsic properties of the protein sequence rather than interactions with chaperones or other cellular factors . We also report striking correlations of IB formation with mutation-dependent changes in residue hydrophobicity . The nature of these trends differs considerably with residue position, however, suggesting that they are mediated by particular local environments created by an ordered structure. Eur J Pharmacol, 1994 Aug 22, 261(3), 265 - 72 Role of platelet activating factor in acute pancreatitis induced by lipopolysaccharides in rabbits; Emanuelli G et al.; In the present study we demonstrated that a single injection of endotoxin (lipopolysaccharides, E . Coli 0111-B4) into the superior pancreaticoduodenal artery of rabbits induced a dose-dependent acute necrotizing pancreatitis . The lesions observed by light microscopy were significant for 10 micrograms lipopolysaccharides and were maximal for 20 micrograms . After 24 h the main findings were edema, acinar cell vacuolisation, polymorphonuclear neutrophil infiltration and tissue necrosis . The pancreatic lesions developed strictly in the area supplied by the artery injected with lipopolysaccharides, without significant intestinal involvement . Since platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine, PAF; 50-500 ng), a phospholipid mediator of endotoxin-induced inflammation and shock, was previously shown to cause an acute necrotizing pancreatitis in rabbits, the role of PAF in the development of acute pancreatitis induced by lipopolysaccharides was studied by evaluating: (1) the synergism between doses of lipopolysaccharides (5-10 micrograms), which produced a mild tissue injury, and doses of PAF (10 ng) not producing, per se, any significant injury, and (2) the effect of three structurally unrelated PAF receptor antagonists . The results obtained demonstrated that 10 ng of PAF significantly potentiated pancreatic tissue damage induced by 10 micrograms of lipopolysaccharides.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1994 Aug 19, 241(3), 480 - 2 Purification, characterization and crystallization of human platelet profilin expressed in Escherichia coli; Fedorov AA et al.; Human platelet profilin was expressed in Escherichia coli using a T7 based expression vector . The recombinant material is similar to authentic human platelet profilin based on the measured Kd for rabbit skeletal muscle actin . Crystals of the recombinant material were obtained from both PEG 8000 and (NH4)2SO4 . These crystals are isomorphous and belong to the monoclinic space group C2, a = 75.0, b = 32.0, c = 62.5, beta = 123 degrees . These crystals contain one molecule in the asymmetric unit and diffract to at least 2.0 A. J Mol Biol, 1994 Aug 19, 241(3), 378 - 89 Structure-function relationship of the Lrp-binding region upstream of lysU in Escherichia coli; Gazeau M et al.; In Escherichia coli, one of the two genes encoding lysyl-tRNA synthetase, lysU, belongs to the regulon controlled by the leucine-responsive regulatory protein (Lrp) . To map the site of Lrp action, mutants escaping regulation in rich medium were generated through random mutagenesis of the lysU promoter region . The mutations showed parallel effects on the strength of Lrp-DNA association, as measured in vitro by gel retardation experiments, and on the degree of repression of lysU expression by Lrp in vivo . In addition, DNase I and hydroxyl radical footprinting experiments indicated that several Lrp molecules bind to a DNA region of over 110 bp in a highly cooperative manner . This region, which encompasses the -35 box of the lysU promoter, was the target of all the mutations affecting the strength of the Lrp-DNA association . These mutations are frequently located in short A + T-rich runs distributed along the Lrp binding region with a periodicity of one helix turn . Because we could find such a regular alternance of A + T runs upstream of several other Lrp-regulated genes, we suggest that this pattern is one feature indicative of the binding of Lrp. J Biol Chem, 1994 Aug 19, 269(33), 21234 - 8 Overexpression of hexokinase I in isolated islets of Langerhans via recombinant adenovirus . Enhancement of glucose metabolism and insulin secretion at basal but not stimulatory glucose levels; Becker TC et al.; Glucose metabolism and glucose-stimulated insulin secretion are thought to be controlled at the level of glucose phosphorylation in pancreatic islet beta-cells . In the current study we have investigated the importance of glucose phosphorylation by using recombinant adenovirus as a gene delivery system for isolated rat islets . Treatment of islets with a virus containing the cDNA encoding the Escherichia coli beta-galactosidase gene (AdCMV-beta GAL) resulted in efficiencies of gene transfer of 70.3 +/- 2.5 and 61.2 +/- 2.2% in two independent experiments . Treatment of islets with a virus containing the cDNA encoding rat hexokinase I (AdCMV-HKI) resulted in a 10.7-fold increase in immunodetectable hexokinase protein and a similar increase in enzyme activity . A large percentage of the overexpressed hexokinase activity was associated with a cell fraction enriched in mitochondria . These changes in enzyme level were accompanied by a 2-fold increase in insulin release and {5-3H}glucose usage at basal glucose concentrations (3 mM) . The rate of glucose usage at 20 mM glucose and the magnitude of the insulin secretory response to this stimulatory level of the sugar were unchanged relative to control islets . Overexpression of hexokinase I in isolated islets therefore creates a phenotype of elevated basal insulin release similar to that seen in islets from obese and insulin-resistant mammals . The discrepancy between the large increase in hexokinase activity and the small increase in glucose usage and insulin release may indicate, however, that other steps in glucose metabolism become rate-limiting after only modest increases in glucose-phosphorylating activity. J Biol Chem, 1994 Aug 19, 269(33), 21078 - 85 Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region; Lei J et al.; Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans . It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases . We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains . The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation . The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism . Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913 . This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase . By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core . Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated. J Biol Chem, 1994 Aug 19, 269(33), 20826 - 8 The sigma subunit conserved region 3 is part of "5'-face" of active center of Escherichia coli RNA polymerase; Severinov K et al.; Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase holoenzyme in open promoter complexes were cross-linked to the beta and sigma 70 subunits . Using limited proteolysis and chemical degradation, the cross-link site in sigma 70 was mapped to a segment between amino acids Glu508 and Met561 containing the C-terminal part of conserved region 3 . This result, when reconciled with genetic data on the interaction of sigma 70 conserved regions 2 and 4 with the -10 and -35 promoter regions, respectively, allows us to model the orientation of the sigma 70 subunit domains within the open promoter complex. Gene, 1994 Aug 19, 146(1), 47 - 56 Phosphorylation of the AfsR protein involved in secondary metabolism in Streptomyces species by a eukaryotic-type protein kinase; Matsumoto A et al.; A global regulatory protein, AfsR, involved in secondary metabolism, was found to be phosphorylated by a membrane-associated phosphokinase, named AfsK, of Streptomyces coelicolor A3(2) and S . lividans . The N-terminal portion of AfsK, deduced from the nucleotide (nt) sequence of the afsK gene, which was located downstream from the afsR gene, showed significant sequence similarity to the catalytic domain of eukaryotic Ser/Thr protein kinases (PKs) . Consistent with this, experiments with AfsK produced by use of an Escherichia coli host-vector system revealed a self-catalyzed phosphate incorporation into both Ser and Tyr residues of AfsK . The recombinant AfsK phosphorylated the purified AfsR at both Ser and Thr residues . Disruption of the chromosomal afsK gene with the phage vector KC515 resulted in significant, but not complete, loss of actinorhodin production . This result implies the involvement of afsK in the regulation of secondary metabolism . The presence of an additional PK able to phosphorylate AfsR is predicted, because the afsK-disrupted strain still contained an activity able to phosphorylate Ser and Thr residues of AfsR . Southern hybridization experiments showed that nt sequences homologous to afsK, as well as afsR, were distributed among many Streptomyces spp . It is thus concluded that a signal transduction system similar to that found in higher organisms is involved in the regulation of secondary metabolism in the bacterial genus Streptomyces. Gene, 1994 Aug 19, 146(1), 131 - 2 A merR homologue at 74 minutes on the Escherichia coli genome; Christie GE et al.; The nucleotide sequence between the trkA gene and the alpha ribosomal protein operon of Escherichia coli was determined . This 1592-bp region contains four open reading frames, one of which shows striking similarity to the MerR family of transcriptional regulatory proteins. Gene, 1994 Aug 19, 146(1), 129 - 30 Nucleotide sequence of the Escherichia coli groE promoter; Lindler LE; The promoter region of the Escherichia coli groE operon was cloned using the polymerase chain reaction (PCR) . The 118-bp nucleotide sequence of the cloned groE promoter was determined on both strands of DNA . Two bp were found between the -10 and -35 regions of this promoter that have not been reported in previous publications of this sequence. Gene, 1994 Aug 19, 146(1), 117 - 21 Improved shuttle vectors for Haloferax volcanii including a dual-resistance plasmid; Holmes M et al.; Two new Haloferax-Escherichia shuttle vectors are described, pMDS20 and pMLH3 . These vectors contain the E . coli ColE1 plasmid ori region and ampicillin-resistance(ApR)-conferring bla gene, and the Haloferax pHK2 replicon region and novobiocin-resistance(NbR)-encoding gyrB gene, enabling maintenance and selection in both hosts . Plasmid pMLH3 has, in addition, a H . volcanii mevinolin-resistance (MvR) determinant and restriction sites allowing insertional inactivation of either marker, to facilitate the identification of Haloferax transformants harbo