Escherichia coli

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 harbouring cloned sequences . Sequencing of gyrA, within the NbR determinant, and the pHK2 ori region has been completed so the complete sequence of both pMDS20 and pMLH3 is now known.

J Mol Biol, 1994 Aug 19, 241(3), 341 - 52
Transcription activation by the Escherichia coli cyclic AMP receptor protein . Receptors bound in tandem at promoters can interact synergistically; Busby S et al.; Starting with a semi-synthetic Escherichia coli promoter with a binding site for the cyclic AMP receptor protein (CRP) centred between base-pairs 41 and 42 upstream from the transcription start site, a second upstream CRP-binding site, centred between base-pairs 90 and 91, was introduced . CRP binding to this second upstream site results in a several-fold greater stimulation of CRP-dependent transcription initiation, compared to activation at the starting promoter with just one CRP-binding site . Activation of transcription by the upstream CRP molecule is blocked by the HL159 substitution, suggesting that the upstream-bound CRP makes a direct contact with RNA polymerase . Footprinting experiments suggest that RNA polymerase contacts the promoter DNA between the two CRP-binding sites, most likely due to interactions involving the C-terminal part of the alpha subunit . Synergy between tandem bound CRP molecules in transcription activation requires that the two CRP-binding sites be separated by around 40 or 50 base-pairs, but is not found at intermediate spacings . An experiment in which the upstream CRP-binding site is replaced by a site for the related transcription factor, FNR, shows that heterologous synergistic interactions between FNR and CRP are possible.

Nature, 1994 Aug 18, 370(6490), 533 - 9
Structure of ribonucleotide reductase protein R1; Uhlin U et al.; Ribonucleotide reductase is the only enzyme that catalyses de novo formation of deoxyribonucleotides and is thus a key enzyme in DNA synthesis . The radical-based reaction involves five cysteins . Two redox-active cysteines are located at adjacent antiparallel strands in a new type of ten-stranded alpha/beta-barrel, and two others at the carboxyl end in a flexible arm . The fifth cysteine, in a loop in the centre of the barrel, is positioned to initiate the radical reaction.

Biochim Biophys Acta, 1994 Aug 17, 1207(2), 187 - 93
Endogenous polypeptide-chain length and partial sequence of aspartate transcarbamoylase from wheat, characterised by immunochemical and cDNA methods; Bartlett TJ et al.; Aspartate transcarbamoylase (ATCase) is purified from wheat germ as a monofunctional trimer of 36 kDa chains . The possibility that this may be a proteolytic fragment of a large endogenous complex in which ATCase is covalently fused to other pyrimidine-pathway enzymes (such as exists in animals or fungi) was tested . Examination of a rabbit antiserum raised against the purified enzyme confirmed the presence of anti-(wheat ATCase) antibodies by several independent methods . Extracts of wheat seedlings prepared under non-proteolysing conditions were challenged by the antiserum, and in some cases by purified anti-(36 kDa ATCase) antibodies, using immunoblotting techniques . The 36 kDa species was the dominant immunopositive polypeptide . However, the extract also contained small amounts of two larger (45 and 55 kDa) immunopositive polypeptides, as well as traces of polypeptides smaller than 36 kDa, which were assumed to be minor proteolytic products . Neither of the 45 or 55 kDa polypeptides is large enough to also incorporate a carbamoyl phosphate synthetase or dihydroorotase of the sizes found in other organisms . They may be targeted precursors of ATCase with intact leader sequences . A screen of a wheat cDNA expression library by the anti-(ATCase) serum yielded a single positive clone which was shown, by DNA sequencing, to be a concatemer of two cDNAs, one of which encoded a partial ATCase . Northern analysis using this clone as probe identified two transcripts of about 1.3 and 1.0 kbp, but showed no evidence of a transcript of 2 kbp or greater which would be required to encode a bifunctional polypeptide . These results confirm that wheat ATCase is not translationally fused to dihydroorotase or carbamoylphosphate synthetase, as it is in animals and fungi . The deduced amino-acid sequence of the partial wheat ATCase is compared with the catalytic chain of the ATCase from Escherichia coli and with other ATCases.

Biochim Biophys Acta, 1994 Aug 17, 1207(2), 165 - 72
Identification of a conditionally essential heat shock protein in Escherichia coli; Peruski LF Jr et al.; Protein D48.5 was recognized as a heat-inducible protein of Escherichia coli during the screening of a group of random, temperature-inducible Mud-Lac fusion mutants . Physiological and genetic analysis demonstrated that (i) the structural gene for this protein, designated htpI, is a member of the sigma 32-dependent heat shock regulon, (ii) at 37 degrees C the synthesis of protein D48.5 is nearly constitutive, increasing slightly with growth rate in media of different composition, and (iii) this protein is essential for growth at high temperature.

Carbohydr Res, 1994 Aug 17, 261(2), 215 - 22
Structure of the O83-specific polysaccharide of Escherichia coli O83:K24:H31; Jann B et al.; The polysaccharide moiety of the O83 antigen (lipopolysaccharide, LPS) consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and D-glucuronic acid in the molar ratios 1:2:1:1 . Methylation analysis of the polysaccharide and derived oligosaccharides as well as one- and two-dimensional 1H and 13C NMR spectroscopy of the polysaccharide at pD 1 and 6 showed that the O83 polysaccharide has the primary structure-->6)-alpha-D-Glc p-(1-->4)-beta-D-Glc pA-(1-->6)-beta-D-Gal p-(1-->4)-beta-D- Gal p-(1-->4)-beta-D-Glc pNAc-(1-->.

Biochemistry, 1994 Aug 16, 33(32), 9643 - 50
Mapping protein domains involved in macromolecular interactions: a novel protein footprinting approach; Heyduk E et al.; A novel direct approach, analogous to DNA footprinting, for mapping protein domains involved in macromolecular interactions is presented in this paper and applied to cAMP receptor protein (CRP) interactions with the allosteric ligand (cAMP) and DNA . In this approach, a protein-macromolecule complex is subjected to a nonspecific cleavage by Fe-EDTA . The cleavage products are resolved by SDS-PAGE and transferred to a PVDF membrane . Transferred polypeptides are visualized by immunostaining with antibodies specific to the N-terminal peptide of the protein . The mobility of the bands visualized in such a way is directly proportional to the distance of the cleavage sites from the N-terminus, and thus the positions of the sites protected from cleavage by a bound macromolecule can be determined . Thus, protein domains involved in macromolecular interactions can be mapped . In the case of CRP, the cleavage conditions were established which resulted in, on the average, less than one cleavage event/protein molecule and which preserved satisfactory levels of protein and DNA activity . When applied to CRP-DNA interactions, the protein footprinting approach correctly identified domains of CRP that were known to be involved in the recognition of DNA . The obtained results showed also that the binding of CRP to the DNA binding site perturbed the region of CRP involved in intersubunit interactions . An allosteric ligand (cAMP) appeared to perturb the same region of CRP . This stresses out the importance of intersubunit interactions in cAMP modulation of protein DNA binding affinity . The protein footprinting methodology presented in this paper should be broadly generalizable to any protein-macromolecule system.

Biochemistry, 1994 Aug 16, 33(32), 9566 - 77
Mixed Cu+ and Zn2+ coordination in the DNA-binding domain of the AMT1 transcription factor from Candida glabrata; Thorvaldsen JL et al.; AMT1 is the transcription factor required for Cu-induced expression of metallothionein genes in the yeast Candida glabrata . The copper-binding, DNA-binding domain of AMT1 has been purified after expression of an AMT1 synthetic gene in bacteria and was confirmed as active in a gel shift assay . The Cu-activated AMT1 was shown to contain a Cu(+)-thiolate tetracopper center and a single Zn2+ site . AMT1 is purified as a Cu-Zn protein from bacterial cultures grown in the presence of CuSO4 . Chemical analysis suggested that 4.2 +/- 0.2 and 1.2 +/- 0.2 molar equiv copper and zinc ions bound, respectively . Electrospray mass spectrometry was used to verify that a uniform species was present with 4 Cu+ ions and 1 Zn2+ ion bound per AMT1 molecule . Cu+ binding to form a tetracopper center occurs cooperatively as shown by electrospray MS of apoAMT1 samples reconstituted with increasing equivalency of Cu+ . Copper-thiolate coordination was indicated by Cu-S charge-transfer transitions in the ultraviolet, luminescence typical of Cu-thiolate clusters and EXAFS . Analysis of the EXAFS of CuZnAMT1 revealed predominantly trigonal Cu+ coordination and the presence of a polycopper cluster by virtue of a short Cu-Cu distance of 2.7 A . Zn K-edge EXAFS of Cu4Zn1AMT1 and electronic spectroscopy of AMT1 with Co2+ substituted for the single Zn2+ ion are consistent with tetrahedral Zn2+ coordination with thiolate ligands . The Cu-activated AMT1 exhibited a conformation distinct from that of metal-free AMT1 as shown by circular dichroism . DNA binding by AMT1 was dependent on the tetracopper center but was independent of occupancy of the Zn2+ site . This is the first report of a single, uniform tetracopper center in a metal-activated transcription factor.

Biochemistry, 1994 Aug 16, 33(32), 9552 - 60
Efficiency of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli; Korangy F et al.; We have measured the rates and efficiencies of DNA unwinding (the number of ATP molecules hydrolyzed per DNA base pair unwound) catalyzed by the RecBC,RecBCD-K177Q (a site-directed mutant in the putative ATP-binding site in the RecD subunit), and RecBCD enzymes from Escherichia coli . The DNA unwinding rate was measured with a coupled assay in which unwound DNA is degraded by the combined action of the RecJ enzyme and exonuclease I . The rates of DNA unwinding by the RecBC and RecBCD-K177Q enzymes are reduced by about 4-fold compared to the case of the RecBCD enzyme . The efficiency of ATP hydrolysis was determined in two ways . First, it was calculated from the ratio of the ATP hydrolysis rate to the rate of DNA unwinding . In the second method, ATP hydrolysis was measured under conditions where all of the DNA substrate becomes completely unwound . The efficiency is the ratio of the total amount of ATP hydrolyzed to the amount of DNA substrate present in the reaction . The average efficiencies measured kinetically and by the complete unwinding experiment are as follows: 2.30 and 1.74 ATP/base pair (RecBCD enzyme); 1.44 and 1.28 (RecBC); and 1.20 and 1.07 (RecBCD-K177Q) . The RecBC and RecBCD-K177Q enzymes are therefore able to couple ATP hydrolysis to DNA unwinding at least as efficiently as the RecBCD holoenzyme . The lower ATP per base pair ratios found for RecBC and RecBCD-K177Q indicate that the RecD subunit hydrolyzes ATP during DNA unwinding by the RecBCD enzyme.

Biochemistry, 1994 Aug 16, 33(32), 9405 - 13
The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties; Mildner AM et al.; Site-directed mutagenesis of autolysis sites in the human immunodeficiency virus type 1 (HIV-1) protease was applied in an analysis of enzyme specificity; the protease served, therefore, as both enzyme and substrate in this study . Inspection of natural substrates of all retroviral proteases revealed the absence of beta-branched amino acids at the P1 site and of Lys anywhere from P2 through P2' . Accordingly, several mutants of the HIV-1 protease were engineered in which these excluded amino acids were substituted at their respective P positions at the three major sites of autolysis in the wild-type protease (Leu5-Trp6, Leu33-Glu34, and Leu63-Ile64), and the mutant enzymes were evaluated in terms of their resistance to autodegradation . All of the mutant HIV-1 proteases, expressed as inclusion bodies in Escherichia coli, were enzymatically active after refolding, and all showed greatly diminished rates of cleavage at the altered autolysis sites . Some, however, were not viable enzymatically because of poor physical characteristics . This was the case for mutants having Lys replacements of Glu residues at P2' and for another in which all three P1 leucines were replaced by Ile . However, one of the mutant proteases, Q7K/L33I/L63I, was highly resistant to autolysis, while retaining the physical properties, specificity, and susceptibility to inhibition of the wild-type enzyme . Q7K/L33I/L63I should find useful application as a stable surrogate of the HIV-1 protease . Overall, our results can be interpreted relative to a model in which the active HIV-1 protease dimer is in equilibrium with monomeric, disordered species which serve as the substrates for autolysis.

Biochemistry, 1994 Aug 16, 33(32), 9371 - 5
Yeast mitochondrial phosphate transport protein expressed in Escherichia coli . Site-directed mutations at threonine-43 and at a similar location in the second tandem repeat (isoleucine-141); Wohlrab H et al.; Yeast mitochondrial phosphate transport activity has been reconstituted from the import receptor (MIR) expressed as inclusion bodies in Escherichia coli . This result undermines the suggestion {Murakami, H., et al . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 3358-3362} that the MIR has been misidentified as the phosphate transport protein (PTP) . PTP was solubilized with N-lauroylsarcosinate and Triton X-100 and purified with a yield of about 2 mg/L of induced bacterial culture . This PTP, reconstituted in liposomes, catalyzes phosphate uptake with a Vmax {24.5 degrees C, net (zero trans), pHi = 8.0, pHe = 6.8} of 0.61 mmol of phosphate min-1 (mg of PTP)-1 and a Km of 1.30 mM . This Vmax is higher and the Km about the same as that obtained with PTP purified from mitochondria . Replacement of Thr43 and Ile141 by other amino acids results in three types of PTP: (a) 2.5-5.0% Vmax of wild-type PTP (PTPwt) (Thr43Cys; Thr43Ser; Ile141Cys), (b) < 0.1% Vmax (detection limit of assay) of PTPwt (Thr43Ala; Thr43Asp), and (c) proton transport uncoupled from phosphate transport (Ile141Cys) . Km changes are not significant . Activity of Thr43Cys confirms results obtained with mitochondrially expressed protein . Thus, yeast PTP requires Thr43 and mammalian PTP the similarly located Cys42 for high transport activity . Thr43 and Ile141 are each situated between two basic residues (LysThrArg vs ArgIleArg) . Cys substitutions in either of these positions confer the same high N-ethylmaleimide sensitivity to the yeast PTPwt as displayed by the mammalian PTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1994 Aug 16, 1187(1), 73 - 9
Expression of the 23 kDa protein from the oxygen-evolving complex of higher plants in Escherichia coli; Seidler A; The 23 kDa protein from the oxygen-evolving complex of higher plants regulates the binding of one Ca2+ ion, which is an essential cofactor of water splitting . In this report its expression in Escherichia coli is described . The 23 kDa protein was expressed and secreted to the periplasm where it accumulated in a soluble form . After purification by cation exchange chromatography the recombinant protein was found to NaCl-washed Photosystem II . Detailed analysis of oxygen-evolving activity demonstrates its function in Ca2+ binding identical to the spinach 23 kDa protein . This expression system opens the way for mutational analysis and isotopic labelling in order to study its function in water splitting.

Biochim Biophys Acta, 1994 Aug 16, 1187(1), 67 - 72
Residues interacting with serine-174 and alanine-295 in the beta-subunit of Escherichia coli H(+)-ATP synthase: possible ternary structure of the center region of the subunit; Miki J et al.; The mutation of serine-174 to phenylalanine that causes a defect in the Escherichia coli F1-ATPase beta-subunit is suppressed by further mutations; Gly-149 to Ser, Ala-295 to Thr, Ala-295 to Pro, or Leu-400 to Gln (Miki, J., Fujiwara, K., Tsuda, M., Tsuchiya, T . and Kanazawa, H . (1990) J . Biol . Chem . 265, 21567-21572) . We analyzed the effects of these second site mutations and of a newly identified Asn-158 to Tyr mutation on the activities of the ATPase without the original Ser-174 to Phe mutation . The beta-subunit with each amino acid replacement was expressed in the mutant strain JP17, which does not have a beta-subunit . Cells transformed with the plasmid carrying Ala-295 to Pro mutation alone did not grow on minimal medium agar supplemented with succinate as the sole carbon source, and showed 3% of the wild-type ATPase activity, suggesting that this mutation caused structural alterations affecting the catalytic function of the enzyme . Conversely transformants with other mutations grew well and had higher ATPase activities, suggesting that these mutations did not cause extensive structural alterations . From the transformants with the plasmid carrying the Ala-295 to Pro mutation, seven revertants capable of cell growth on succinate plates were isolated and reversion mutations were identified at residues 140, 159, 166, 171, 172 and 184 of the beta-subunits . The results suggested that Ser-174 and Ala-295 do not necessarily interact directly, but that the regions including these suppression mutation sites close to Ser-174, and Ala-295 interact with each other for the proper functioning of the ATPase . The ternary structure of the region surrounded by the residues which were identified as the reversion mutation sites for Ser-174 to Phe and Ala-295 to Pro mutations is important for the catalytic function of this enzyme.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8273 - 7
Lambda foo: a lambda phage vector for the expression of foreign proteins; Maruyama IN et al.; This work describes a lambda phage expression system, lambda foo, that produces foreign proteins fused to the surface of the virus particle . The lambda foo vector has multiple cloning sites for the insertion of a foreign DNA fragment and color selection for recombinants . Foreign proteins are fused to the C terminus of a truncated phage tail protein, pV, by a peptide linker . Conditional chain termination allows the assembly and fusion of multisubunit proteins . We have attached the complete Escherichia coli beta-galactosidase and the plant Bauhinia purpurea agglutinin by cloning their genes into the vector . The constructs express functionally active proteins on the phage particle surface and have been purified by affinity chromatography with an antibody for beta-galactosidase and a mucin as a ligand for Bauhinia purpurea agglutinin.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8253 - 7
N,N'-dicyclohexylcarbodiimide cross-linking suggests a central core of helices II in oligomers of URF13, the pore-forming T-toxin receptor of cms-T maize mitochondria; Rhoads DM et al.; URF13 is a mitochondrially encoded, integral membrane protein found only in maize carrying the cms-T cytoplasm . URF13 is associated with cytoplasmic male sterility, Texas type, and causes susceptibility to the fungal pathogens Bipolaris maydis race T and Phyllosticta maydis . URF13 is predicted to contain three transmembrane alpha-helices and is a receptor for the pathotoxins (T-toxins) produced by B . maydis race T and P . maydis . Binding of T-toxin to URF13 leads to membrane permeability . Cross-linking of URF13 oligomers with N,N'-dicyclohexylcarbodiimide (DCCD) protects Escherichia coli cells expressing URF13 and cms-T mitochondria from the permeability caused by T-toxin or methomyl . Using mutated forms of URF13 expressed in E . coli cells, we determined the molecular mechanism of DCCD protection . We separately changed Lys-37 in helix II to isoleucine (K37I-URF13) and Lys-32 in the helix I/helix II loop region to alanine (K32A-URF13) . DCCD treatment of K37I-URF13-expressing cells did not protect the cells from permeability caused by T-toxin or methomyl . DCCD cross-linking was greatly reduced in K37I-URF13 and in D39V-URF13-expressing cells, but it was unaffected in K32A-URF13-expressing cells . Binding of methomyl or T-toxin decreases DCCD cross-linking of URF13 oligomers expressed in either E . coli or cms-T mitochondria . We conclude that Asp-39 in helix II is cross-linked by DCCD to Lys-37 in helix II of an adjacent URF13 molecule and that this cross-linking protects against toxin-mediated permeabilization . Our results also indicate that helices II form a central core in URF13 oligomers.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8244 - 7
Cold denaturation of a repressor-operator complex: the role of entropy in protein-DNA recognition; Foguel D et al.; The mechanisms by which regulatory proteins recognize specific DNA sequences are not fully understood . Here we examine the basis for the stability of a protein-DNA complex, using hydrostatic pressure and low temperature . Pressure converts the DNA-binding Arc repressor protein from a native state to a denatured, molten-globule state . Our data show that the folding and dimerization of Arc repressor in the temperature range 0-20 degrees C are favored by a large positive entropy value, so that the reaction proceeds in spite of an unfavorable positive enthalpy . On binding operator DNA, Arc repressor becomes extremely stable against denaturation . However, the Arc repressor-operator DNA complex is cold-denatured at subzero temperatures under pressure, demonstrating that the favorable entropy increases greatly when Arc repressor binds tightly to its operator sequence but not a nonspecific sequence . We show how an increase in entropy may operate to provide the protein with a mechanism to distinguish between a specific and a nonspecific DNA sequence . It is postulated that the formation of the Arc-operator DNA complex is followed by an increase in apolar interactions and release of solvent which would explain its entropy-driven character, whereas this solvent would not be displaced in nonspecific complexes.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8087 - 91
Cleavage of the nascent transcript induced by TFIIS is insufficient to promote read-through of intrinsic blocks to elongation by RNA polymerase II; Cipres-Palacin G et al.; RNA polymerases encounter a variety of types of blocks to elongation during transcription in eukaryotic cells . At least one protein, TFIIS, can promote read-through of many types of blocks to elongation by RNA polymerase II, and this protein stimulates cleavage of the nascent transcript in stalled elongation complexes as a prelude to read-through . The C-terminal half of the TFIIS protein is sufficient for stimulating the cleavage and read-through reactions in vitro . To study how TFIIS changes the response of RNA polymerase II elongation complexes to such blocks, targeted amino acids in the C terminus of HeLa TFIIS were mutated to alanines . Two mutant TFIIS proteins as well as the unmutated C-terminal half of the TFIIS protein were purified following overexpression in Escherichia coli . Each protein was examined for read-through activity and ability to stimulate transcript cleavage in ternary elongation complexes . Mutant TFIIS5 (E174A, E175A) was reduced in read-through and cleavage activities relative to the unmutated, truncated TFIIS (delta TFIIS) . Mutant TFIIS7 (K187A, K189A) was able to stimulate cleavage nearly at the rate and to the extent of the TFIIS5 mutant . In contrast to what was observed with TFIIS5, no detectable read-through was observed in the presence of the TFIIS7 mutant during the course of the reaction . Thus, there is no simple, direct correlation between the ability of TFIIS to promote cleavage and its ability to promote read-through by RNA polymerase II . These results suggest that although TFIIS is necessary to mediate the cleavage reaction that precedes the read-through event, the cleavage event itself is not sufficient to allow read-through by RNA polymerase II.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8027 - 31
A single 43-bp sopC repeat of plasmid mini-F is sufficient to allow assembly of a functional nucleoprotein partition complex; Biek DP et al.; Stable maintenance of the low-copy-number mini-F plasmid in Escherichia coli is dependent on a functional partition system . The sop partition region encodes proteins SopA and SopB and a cis-acting element sopC, which contains multiple sites to which SopB binds . We have found that SopB protein acting at sopC in vivo is associated with a marked effect on plasmid DNA supercoiling, which may reflect the formation of a wrapped nucleoprotein complex . In this study, we demonstrate that a functional partition complex can form with a single 43-bp SopB binding site . Our experiments suggest that SopB bound at a single site nucleates the binding of additional SopB protein and wrapping of adjacent DNA sequences, such that approximately equal numbers of supercoils are restrained regardless of the number of tandem sopC repeats present . It is likely that this unusual nucleoprotein complex allows interaction of the plasmid with the partition apparatus.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 7970 - 4
Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag-Pol polyprotein; Louis JM et al.; Upon renaturation, the polyprotein MBP-delta TF-Protease-delta Pol, consisting of HIV-1 protease and short native sequences from the trans-frame protein (delta TF) and the polymerase (delta Pol) fused to the maltose-binding protein (MBP) of Escherichia coli, undergoes autoprocessing to produce the mature protease in two steps . The initial step corresponds to cleavage of the N-terminal sequence to release the protein intermediate Protease-delta Pol, which has enzymatic activity comparable to that of the mature enzyme . Subsequently, the mature enzyme is formed by a slower cleavage at the C terminus . The rate of increase in enzymatic activity is identical to that of the appearance of MBP-delta TF and the disappearance of the MBP-delta TF-Protease-delta Pol . Initial rates are linearly dependent on the protein concentration, indicating that the N-terminal cleavage is first-order in protein concentration . The reaction is competitively inhibited by pepstatin A and has a pH rate profile similar to that of the mature enzyme . These results and molecular modeling studies are discussed in terms of a mechanism in which a dimeric full-length fusion protein must form prior to rate-limiting intramolecular cleavage of the N-terminal sequence that leads to an increase in enzymatic activity.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 7884 - 8
Independent in vitro assembly of a ribonucleoprotein particle containing the 3' domain of 16S rRNA; Samaha RR et al.; Small (30S) subunits of Escherichia coli ribosomes are composed of 21 proteins and a 1542-nucleotide 16S rRNA, whose secondary structure is divided into three domains . An in vitro transcript of the 3' domain of 16S rRNA (residues 923-1542), assembles efficiently with 30S ribosomal proteins to form a compact ribonucleoprotein (RNP) particle . Isolated particles examined under the electron microscope have a globular appearance, similar in size and shape to the head of the 30S ribosomal subunit . Two-dimensional gel analysis of the particles indicates the presence of proteins S3, S7, S9, S10, S13, S14, and S19 and smaller amounts of S2, all of which have been localized to the head of the 30S subunit by immunoelectron microscopy and neutron diffraction and belong to the S7 assembly family . Interestingly, protein S4, which is believed to interact exclusively with the 5' domain, is also reproducibly found associated with the particles in significant amounts . Chemical probing of the RNA in the assembled particle reveals characteristic cleavage protection patterns, showing that the proteins assemble with the 3'-domain RNA similarly to the way in which they assemble with 16S rRNA, although some of the later steps of assembly appear to be incomplete . These results show that the 3' domain of 16S rRNA can indeed assemble independently of the rest of the 30S subunit into a particle that resembles its structure in the ribosome . In addition, the assembled particles are able to bind spectinomycin with an affinity comparable to that of 30S subunits.

Biochemistry, 1994 Aug 16, 33(32), 9428 - 37
Mapping the lipoyl groups of the pyruvate dehydrogenase complex by use of gold cluster labels and scanning transmission electron microscopy; Yang YS et al.; This paper describes the organization of lipoyl moieties within the pyruvate dehydrogenase (PDH) complex from Escherichia coli as studied in the scanning transmission electron microscope (STEM) . The PDH complex is a multienzyme complex consisting of E1, pyruvate dehydrogenase, E2, dihydrolipoyl transacetylase, and E3, dihydrolipoyl dehydrogenase . The core of the complex is the cubic 24-subunit E2 component, which contains the lipoyl moieties bonded to lipoyl-bearing domains . E1 and E3 are associated along the edges (E1) and on the faces (E3) of the core . The lipoyl moieties were reduced with NADH and alkylated with a p-maleimidobenzoyl undecagold cluster complex . The gold labels were found to be bound very nearly specifically by dihydrolipoyl transacetylase (E2) . Undecagold clusters were imaged directly by the STEM and also digitally mapped by radial mass analysis . The mass of the E2E3 subcomplex is about half that of the PDH complex . The PDH complex and GC-PDH are both about 420 A in diameter, as determined by radial mass analysis, and the E2E3 subcomplex and GC-E2E3 are 320 and 350 A, respectively . The outer boundary of the E2E3 subcomplex was clearly shown in STEM micrographs by the undecagold labels in GC-E2E3 . Data obtained from radial mass analysis of GC-E2E3 and the unlabeled E2E3 subcomplex also showed that the size of the subcomplex is extended by the lipoyl-bearing domains surrounding the central E2 core . The capabilities of lipoyl moieties to undergo translocation over long distances through structural mobility in the lipoyl-bearing domains was confirmed by the observation that many of the lipoyl groups in E2E3 subcomplexes relax outward into space vacated by the removal of E1 during the preparation of the subcomplex from PDH complex . Radial mass analysis of the PDH complex and GC-PDH indicates that lipoyl groups are distributed over a large region of the PDH complex, extending from the central core to 170-180 A from the center of the complex, with the highest density at about 75 A from the particle centers, near the interface between E2 and the associated components E1 and E3.

Ann N Y Acad Sci, 1994 Aug 15, 730, 329 - 31
Role of nitric oxide in hepatic injury following acute endotoxemia; Rodriguez del Valle M et al.; Hepatocytes from control and endotoxemic rats were cultured for 40 hours in 96-well dishes in medium containing 0-5 micrograms/ml of lipopolysaccharide in the presence or absence of 50 U/ml IFN gamma . Nitric oxide production was quantified by the accumulation of nitrite in the culture medium by the Greiss reaction . Hepatocyte proliferation and protein synthesis were measured by (3H)thymidine (TdR) and (3H)leucine (Leu) incorporation, respectively . Results are the mean +/- standard error of triplicate wells from four experiments.

Mol Gen Genet, 1994 Aug 15, 244(4), 444 - 50
Escherichia coli mutY-dependent mismatch repair involves DNA polymerase I and a short repair tract; Tsai-Wu JJ et al.; Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function . The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines . The size of the mutY-dependent repair tract was measured by the specific incorporation of alpha-{32P}dCTP into different restriction fragments of the repaired DNA . The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3' side of the mismatched adenine . This repair synthesis is carried out by DNA polymerase I.

Mol Gen Genet, 1994 Aug 15, 244(4), 439 - 43
Analysis of putative DNA amplification genes in the element AUD1 of Streptomyces lividans 66; Piendl W et al.; The amplifiable AUD1 element of Streptomyces lividans 66 consists of two copies of a 4.7 kb sequence flanked by three copies of a 1 kb sequence . The DNA sequences of the three 1 kb repeats were determined . Two copies (left and middle repeats) were identical: (1009 bp in length) and the right repeat was 1012 bp long and differed at 63 positions . The repeats code for open reading frames (ORFs) with typical Streptomyces codon usage, which would encode proteins of about 36 kD molecular weight . The sequences of these ORFs suggest that they specify DNA-binding proteins and potential palindromic binding sites are found adjacent to the genes . The putative amplification protein encoded by the right repeat was expressed in Escherichia coli.

Mol Gen Genet, 1994 Aug 15, 244(4), 331 - 40
Two genes that encode Ca(2+)-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana; Urao T et al.; Two cDNA clones, cATCDPK1 and cATCDPK2, encoding Ca(2+)-dependent, calmodulin-independent protein kinases (CDPK) were cloned from Arabidopsis thaliana and their nucleotide sequences were determined . Northern blot analysis indicated that the mRNAs corresponding to the ATCDPK1 and ATCDPK2 genes are rapidly induced by drought and high-salt stress but not by low-temperature stress or heat stress . Treatment of Arabidopsis plants with exogenous abscisic acid (ABA) had no effect on the induction of ATCDPK1 or ATCDPK2 . These findings suggest that a change in the osmotic potential of the environment can serve as a trigger for the induction of ATCDPK1 and ATCDPK2 . Putative proteins encoded by ATCDPK1 and ATCDPK2 which contain open reading frames of 1479 and 1488 bp, respectively, are designated ATCDPK1 and ATCDPK2 and show 52% identity at the amino acid sequence level . ATCDPK1 and ATCDPK2 exhibit significant similarity to a soybean CDPK (51% and 73%, respectively) . Both proteins contain a catalytic domain that is typical of serine/threonine protein kinases and a regulatory domain that is homologous to the Ca(2+)-binding sites of calmodulin . Genomic Southern blot analysis suggests the existence of a few additional genes that are related to ATCDPK1 and ATCDPK2 in the Arabidopsis genome . The ATCDPK2 protein expressed in Escherichia coli was found to phosphorylate casein and myelin basic protein preferentially, relative to a histone substrate, and required Ca2+ for activation.

Eur J Biochem, 1994 Aug 15, 224(1), 249 - 55
A single amino acid mutation enhances the thermal stability of Escherichia coli malate dehydrogenase; Goward CR et al.; The stability of wild-type Escherichia coli malate dehydrogenase was compared with a mutant form of the enzyme with the amino acid residue at position 102 changed from arginine to glutamine . The mutation occurs on the underside of a mobile loop which closes over the active-site cleft on formation of the enzyme/cofactor/substrate ternary complex . The mutant enzyme is kinetically compromised while the wild-type enzyme is highly specific for oxaloacetate . The mutant enzyme was shown to be more resistant to irreversible thermal denaturation by thermal inactivation experiments and high-sensitivity differential scanning calorimetry than the wild-type enzyme . In contrast, resistance of both enzymes to reversible unfolding in guanidinium chloride was similar . Circular dichroic spectropolarimetry shows the secondary structures of the enzymes are similar but there is a demonstrable difference in tertiary structure . From the position of the mutation, it is conjectured that the substitution on a mobile surface loop results in partial closure of the loop and greater resistance to thermal inactivation of the mutant enzyme . However, molecular modelling combined with circular dichroic spectropolarimetry indicate that the mutation may have a more widespread effect on the structure than simply partial closure of the mobile surface loop as the environment of distant tyrosine residues is altered . Resistance of the wild-type enzyme to thermal inactivation can be increased by cofactor addition, which may have the effect of partial closure of the mobile surface loop, but has little effect on the mutant enzyme.

Eur J Biochem, 1994 Aug 15, 224(1), 173 - 8
Flavodoxin is required for conversion of dethiobiotin to biotin in Escherichia coli; Ifuku O et al.; We have reported {Ifuku, O., Kishimoto, J., Haze, S., Yanagi, M . & Fukushima, S . (1992) Biosci . Biotechnol . Biochem . 56, 1780-1785} the enzymic conversion of dethiobiotin to biotin (catalyzed by the enzyme encoded by bioB) in cell-free extract of Escherichia coli which had been genetically engineered for high bioB expression . An unidentified protein(s) in addition to the bioB gene product is obligatory for this reaction . We have found that this protein was precipitated from the cell-free extract with poly(ethyleneimine), and we have purified it to homogeneity by a procedure which includes ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration, and Mono Q chromatography . The apparent molecular mass of the purified protein was estimated to be about 21 kDa by SDS/PAGE . The N-terminal amino acid sequence of the purified protein was identical with that of E . coli flavodoxin . We conclude that flavodoxin is required for conversion of dethiobiotin to biotin in E . coli . Studies with purified flavodoxin and the fraction containing the bioB gene product suggested that protein(s) in addition to the bioB gene product and flavodoxin is also obligatory for the reaction.

Eur J Biochem, 1994 Aug 15, 224(1), 109 - 14
A misfolded but active dimer of bovine seminal ribonuclease; Kim JS et al.; Bovine seminal ribonuclease (BS-RNase) is an unusual homolog of RNase A . Isolated from bulls as a dimer, BS-RNase has special biological properties including antispermatogenic, antitumor and immunosuppressive activities . The structural bases for these properties are unknown . Four forms of BS-RNase were isolated after folding and air oxidation of the denatured and reduced protein produced in Escherichia coli: two dimers (M = M and M x I, where x signifies an active site composed of residues from both subunits) and two monomers (M and I) . Considerable ribonuclease activity was generated by air oxidation of an equimolar mixture of two inactive mutant proteins ({H12D}BS-RNase and {H119D}BS-RNase) prepared by site-directed mutagenesis . This activity came from a dimer (M x I) with a composite active site . 1H-NMR spectroscopy revealed that this dimer contained one correctly folded subunit (M), and one incorrectly folded subunit (I) . Form I, which is a poor catalyst, was activated by ribonuclease S-protein, suggesting that the C-terminal portion of I was not folded properly . Electrospray-ionization mass spectrometry and sulfhydryl group titration indicated that I contains a single oxidized sulfhydryl group, which cannot participate in a disulfide bond . These results show that quaternary structure in BS-RNase is attained by the initial formation of two monomers, M and I, which then combine with another M to form M = M and M x I, respectively . Adventitious oxidation can thus lead to the formation of a misfolded but active enzyme (M x I).

EMBO J, 1994 Aug 15, 13(16), 3902 - 8
A mutational analysis of the two motifs common to adenine methyltransferases; Willcock DF et al.; All methyltransferases that use S-adenosyl methionine as the methyl group donor contain a sequence similar to (D/E/S)XFXGXG which has been postulated to form part of the cofactor binding site . In N6-adenine DNA methyltransferases there is a second motif, (D/N)PP(Y/F), which has been proposed to play a role similar to the catalytically essential PC motif conserved in all C5-cytosine DNA methyltransferases . We have made a series of amino acid changes in these two motifs in the EcoKI N6-adenine DNA methyltransferase . The mutant enzymes have been purified to homogeneity and characterized by physical biochemical methods . The first G is the most conserved residue in motif I . Changing this G to D completely abolished S-adenosyl methionine binding, but left enzyme structure and DNA target recognition unaltered, thus documenting the S-adenosyl methionine binding function of motif I in N6-adenine methyltransferases . Substitution of the N with D, or F with either G or C, in motif II abolished enzyme activity, but left S-adenosyl methionine and DNA binding unaltered . Changes of F to Y or W resulted in partial enzyme activity, implying that an aromatic residue is important for methylation . The substitution of W for F greatly enhanced UV-induced cross-linking between the enzyme and S-adenosyl methionine, suggesting that the aromatic residue is close in space to the methyl-group donor.

Biochem J, 1994 Aug 15, 302 ( Pt 1), 215 - 21
Human GTP cyclohydrolase I: only one out of three cDNA isoforms gives rise to the active enzyme; Gutlich M et al.; GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis . Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner . Three different cDNA forms have been reported for human GTP cyclohydrolase I {Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem . Biophys . Res . Commun . 187, 359-365} . We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al . (1992) . The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli . Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate . The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction . Recombinant human GTP cyclohydrolase I showed sigmoidal substrate kinetics and maximum activity at 60 degrees C . These findings are well in line with the published properties of the enzyme isolated from rat liver . The data indicate that cytokine-mediated induction of GTP cyclohydrolase I is not due to the expression of enzyme isoforms.

FEBS Lett, 1994 Aug 15, 350(1), 87 - 90
A pulsed field gradient isotope-filtered 3D 13C HMQC-NOESY experiment for extracting intermolecular NOE contacts in molecular complexes; Lee W et al.; A pulsed field gradient three-dimensional isotope-filtered 13C HMQC-NOESY experiment has been developed to characterize intermolecular contacts in a 37 kDa macromolecular ternary complex consisting of uniformly 13C labeled trp-repressor, its natural abundance co-repressor, L-tryptophan, and natural abundance operator DNA . The pulse scheme makes use of pulsed field gradients for the removal of artifacts and dephasing of unwanted magnetization during isotope filtering, and employs a strategy to minimize the time that magnetization resides in the transverse plane . The experiment provides solely intermolecular NOE contacts between protons of the labeled protein and protons of the unlabeled species, and has proven to be especially useful in eliminating ambiguities between intra- and intermolecular NOEs in the isotope-edited 3D 13C HMQC-NOESY spectrum of the complex.

Biochem Biophys Res Commun, 1994 Aug 15, 202(3), 1468 - 75
Molecular cloning and expression of a cDNA encoding NGAL: a lipocalin expressed in human neutrophils; Bundgaard JR et al.; NGAL, a protein recently isolated from human neutrophils, is a novel member of the lipocalins . NGAL binds a derivative of the bacterial chemotactic peptide formylmethionyl-leucyl-phenylalanine and may have important immunomodulatory functions . We here report the cloning of a cDNA for NGAL covering a 63 bp 5' untranslated region and the coding region of 591 bp . The cDNA encodes a protein of 197 amino acids, with a 19 amino acid leader sequence and a mature protein of 178 amino acids . Alignment of the cDNA sequence of NGAL to the rat analogue, alpha 2-microglobulin related protein, demonstrates a very high degree of conservation of this lipocalin . Northern blotting of a variety of tissues revealed that NGAL is mainly expressed in myeloid cells, where a signal of approximately 850 bp is observed . A faint signal was observed in fetal and adult human lung tissue . The molecular cloning of the NGAL cDNA allowed the recombinant production of NGAL in E . coli.

Biochem Biophys Res Commun, 1994 Aug 15, 202(3), 1445 - 51
Mutational analysis of the structure-function relationship in interferon-alpha; Di Marco S et al.; A number of mutants of the recombinant human hybrid interferon-alpha 8{60}alpha 1{92}alpha 8 have been expressed in Escherichia coli, purified to homogeneity and characterized . The introduction of one or two negative charges results in an anomalously lower electrophoretic mobility than that expected from the molecular mass . The mutations Lys84-->Glu, Cys86-->Tyr, Cys86-->Ser, Thr87-->Ile, Tyr90-->Asp and Lys84-->Glu/Tyr90-->Asp (double mutant) result in a marked decrease of the biological activity in murine cells compared to the unmutated protein . Comparisons with published structural and homology models of interferon-beta and -alpha, imply that these residues are located primarily on the external surface of the carboxylterminus of helix C . We propose a model of interferon-receptor interaction in which these residues define a potential binding site.

Biochem Biophys Res Commun, 1994 Aug 15, 202(3), 1329 - 36
Mutants of smooth muscle myosin light chain kinase at tryptophan 800; Matsushima S et al.; The importance of Trp 800 in the calmodulin-binding site of myosin light chain kinase was investigated . Truncation mutants from Leu 447 to the C-terminus were expressed in E . coli and these were modified by point mutations of Trp 800 to Gly, Cys, Leu and Tyr . Trp at this position was more effective than any of the other residues . The Leu mutant was partially active and its Km for calmodulin decreased from about 10 nM to 175 nM . The Tyr mutant had detectable activity but the other two mutants were inactive and did not bind calmodulin . Thus Trp at position 800 is critical . The activity of the Leu mutant at high calmodulin concentrations was less than the wild-type mutant, about 20% . This suggests that the binding of calmodulin does not release inhibition in an all-or-none mechanism and that other intramolecular interactions are important.

Cancer Res, 1994 Aug 15, 54(16), 4468 - 71
Deficiency of queuine, a highly modified purine base, in transfer RNAs from primary and metastatic ovarian malignant tumors in women; Baranowski W et al.; The tRNAs from rapidly growing tissues, particularly from neoplasia, often exhibit queuine deficiency . In order to check whether different kinds of ovarian tumors display queuine deficiencies we have analyzed tRNA samples from 16 ovarian malignancies . The tRNAs from histologically normal myometrium (4 samples) and myoma (6 samples) were taken as healthy tissue and benign tumor references . Queuine deficiency was determined by an exchange assay using {8-3H}guanine and tRNA:guanine transglycosylase from Escherichia coli . The mean values of queuine deficiencies in tRNAs were: 10.95 +/- 2.21 (SD) pmol/A260 in gonadal and germ cell tumors (5 cases); 23.75 +/- 7.89 pmol/A260 in primary epithelial tumors (9 cases); and 34.58 +/- 7.18 pmol/A260 in metastatic tumors (2 cases) . These values displayed statistically significant differences (P = 0.0003, Kruskal-Wallis test) . The queuine deficiencies in tRNAs significantly increased when moving from well-differentiated through moderately differentiated to poorly differentiated tumors, with the highest values found in poorly differentiated metastatic tumors (P = 0.0002, Kruskal-Wallis test) . Queuine deficiency determination in tRNAs is proposed as a factor for clinical outcome prognosis of ovarian malignancies.

Anal Biochem, 1994 Aug 15, 221(1), 57 - 60
Colorimetric detection of DNA polymerase activity after sodium dodecyl sulfate polyacrylamide gel electrophoresis; Venegas J et al.; A nonradioactive method is developed to detect DNA polymerase activity after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis containing gapped DNA as template . The technique is based on the use of digoxigenin- or biotin-labeled deoxynucleotides during DNA synthesis, and their detection by means of an anti-digoxigenin antibody-alkaline phosphatase conjugate or by a streptavidin-alkaline phosphatase conjugate . The detection of the DNA polymerase catalytic subunit is achieved after incubation of the gels with colorimetric alkaline phosphatase substrates . The technique is able to detect nanogram amounts of Escherichia coli DNA polymerase I and picogram amounts of its Klenow fragment . The results with other DNA polymerases and E . coli extracts suggest that this colorimetric detection system could be used for the analysis of an extended range of DNA polymerase enzymes . The method presented in this report offers an alternative to the already described radioactive techniques for detection of DNA polymerase activity after SDS-polyacrylamide gel electrophoresis.

Anal Biochem, 1994 Aug 15, 221(1), 118 - 26
A protein/peptide assay using peptide-resin adduct: application to the calmodulin/RS20 complex; Guimard L et al.; To obtain equilibrium and kinetic constants of a protein/peptide complex, we have developed a rapid procedure which uses peptides specifically linked to a resin . With this peptide-resin adduct, bound and free 125I-labeled protein could be easily separated by simple centrifugation . The feasibility of the method was demonstrated with the calmodulin/RS20 complex, where RS20 is the putative calmodulin binding peptide of the smooth muscle myosin light chain kinase (smMLCK) . In addition to the wild-type calmodulin (SYNCAM) expressed in Escherichia coli, we also examined calmodulin mutants with charge reversals called SYNCAM12A (DEE 118-120-->KKK) and SYNCAM18A (EEE 82-84-->KKK and DEE 118-120-->KKK) . The kinetic analysis of the interaction between SYNCAM and RS20 associated with titration experiments allowed us to measure dissociation constants (KD) in the range of 10(-9) M, in good agreement with previously published data . Moreover, the binding assays showed that SYN-CAM18A did not interact with RS20, whereas SYN-CAM12A did with a KD around 10(-8) M . The lack of binding of SYNCAM18A to RS20 provides an explanation for the lack of smMLCK activation by SYNCAM18A . Altogether, these results demonstrate that peptide-resin can be used as a tool for separating bound from free protein, thus enabling a rapid and reliable quantification of the protein/peptide interaction.

Biochem J, 1994 Aug 15, 302 ( Pt 1), 291 - 5
Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans; Moreau A et al.; On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated . Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression . Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site . These two amino acids probably represent the catalytic residues . Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein . Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax . 14-fold when compared with the wild-type enzyme . The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate . Mutation D124E increased the Km and decreased the Vmax . 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax . 75-fold . The mutations had no effect on the cleavage mode of xylopentaose.

J Biotechnol, 1994 Aug 15, 36(2), 145 - 55
High-level expression of Mycoplasma arginine deiminase in Escherichia coli and its efficient renaturation as an anti-tumor enzyme; Misawa S et al.; The arginine deiminase (AD) gene was cloned from Mycoplasma arginini and expressed in the cytosol of Escherichia coli as inclusion bodies with an expression level of at least 20% of the total bacterial proteins . The inclusion bodies were solubilized with 6 M guanidine hydrochloride (Gdn-HCl) under reducing conditions, in order to avoid incorrect disulfide-bond formation of the recombinant (r-) AD molecules, and renaturation was performed under various refolding conditions . The optimum renaturation conditions were found to be incubation for 90 h at pH 7.5 and 15 degrees C . The resulting completely refolded r-AD was purified to homogeneity by anion-exchange and arginine-affinity chromatography and its activity yield was 72.5% . The specific activity of the purified r-AD was comparable to and its amino acid composition was identical to those of Mycoplasma AD, and NH2-terminal sequence analysis revealed that its methionine residue corresponding to the translation initiation codon had been removed completely . Anti-tumor activity analyses showed that r-AD inhibited the growth of two mouse cell lines, hepatoma MH134 and fibrosarcoma Meth A, strongly in vitro at concentrations in excess of 10 ng ml-1 . Moreover, when MH134-implanted mice were given single intravenous injections of r-AD at doses of 50 mg kg-1 and higher, their survival times were prolonged significantly . These results, taken together, indicate that the enzymatic properties and biological actions of r-AD were highly consistent with those of Mycoplasma AD.

Anal Biochem, 1994 Aug 15, 221(1), 94 - 102
A cloning and epsilon-epitope-tagging insert for the expression of polymerase chain reaction-generated cDNA fragments in Escherichia coli and mammalian cells; Olah Z et al.; An intercompatible gene-tagging insert sequence was designed to conveniently introduce epitope-tagged polypeptides into bacteria and mammalian cells . The presence of rare restriction enzyme sites located between the ATG codon and the sequence encoding the introduced epsilon-tag creates a general cloning site which allows efficient cloning of virtually any desired cDNA fragment produced by the polymerase chain reaction (PCR) . The tagging insert sequence encodes a KGF-SYFGEDLMP peptide, derived from the last 12 amino acids of the protein kinase C epsilon gene, to serve as a C-terminal epitope tag of the expressed protein . While the insert can be readily adapted for insertion into any expression vector, this paper details the introduction and characterization of the epsilon-epitope-tagging insert into the bacterial pTrcHis A (epsilon TrcHis A) vector and into the metallothionein promoter-driven eukaryotic (epsilon MTH) expression vector . The expressed epsilon-tagged proteins can be readily detected with a commercially available antibody specific for the epsilon-peptide . Immunoscreening of Escherichia coli colonies transformed with the PCR-generated cDNA inserted into the epsilon TrcHis A vector enables rapid, direct biochemical characterization of the PCR product . The biochemically characterized gene constructs from the epsilon TrcHis A plasmid can be inserted into the epsilon MTH vector by a single subcloning step using the introduced compatible cohesive ends . This epsilon-epitope-tagging insert provides investigators with a versatile, uncomplicated, and reliable method of expressing an epitope-tagged PCR product in the cell type of interest.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1994 Aug 15, 224(1), 21 - 8
Accumulation of reactive oxygen species and oxidation of cytokinin in germinating soybean seeds; Gidrol X et al.; Seed germination is an important developmental switch when quiescent seed cells initiate oxidative phosphorylation for further development and differentiation . During early imbibition of soybean seeds (Glycine max L . cv . Weber), a superoxide dismutase (SOD) activity peak was observed, in embryonic axes, after 6 h imbibition . Peroxidase activities, including catalase, were significantly increased after 12 h inhibition and during germination phase III . Catalase was the most efficient enzyme in catabolizing H2O2 in embryonic axes . When stored at 42 degrees C and 100% relative humidity, seeds were stressed and lost their viability in a time-dependent manner . A significant increase in the Cu, Zn-superoxide-dismutase activity, and to a lesser extent, Mn superoxide dismutase activity was observed during germination in low-viability (stressed) seeds as compared to high-viability (unstressed) seeds . Northern blot analysis confirmed that superoxide dismutase induction resulted from an accumulation of its transcripts in response to the production of O2- . The induction of catalase did not occur in low-viability seeds, resulting in dramatic accumulation of H2O2 . Using capillary electrophoresis, HPLC and NMR we found that the endogenous cytokinin, zeatin riboside, was present in large quantities in the high-viability seeds, but it was oxidized into adenine in the low-viability seeds . In vitro superoxide anion could also oxidize the cytokinin . Zeatin riboside, but not adenine, was found to act as a scavenger of superoxide anions and may help to maintain seed viability by detoxifying reactive oxygen species . Germination of stressed seeds was partially restored by the addition of exogenous cytokinin (zeatin riboside) . Protection against oxidative stress by cytokinin seemed to be a general phenomenon, as Escherichia coli cells were also protected against superoxide stress in the presence of cytokinin.

Eur J Biochem, 1994 Aug 15, 224(1), 191 - 6
Structural studies of the O-antigenic polysaccharides of Escherichia coli O3 and the enteroaggregative Escherichia coli strain 17-2; Medina EC et al.; The polysaccharide part of the lipopolysaccharide isolated from an enteroaggregative Escherichia coli isolated from a young child with diarrhoea in Santiago, Chile (strain 17-2), has been investigated . Sugar and methylation analyses of native and partially degraded polysaccharide together with 1H-NMR and 13C-NMR spectroscopies revealed that the polysaccharide is composed of pentasaccharide repeating units . The structure of the repeating unit of E . coli strain 17-2 O-polysaccharide is: {formula: see text} The structure of the O-polysaccharide from E . coli O3 was shown to be identical to that of E . coli strain 17-2 by sugar and methylation analyses and by 1H-NMR and 13C-NMR spectroscopies.

Cancer Lett, 1994 Aug 15, 83(1-2), 261 - 8
Angiogenic activity of the recombinant hst-1 protein; Yoshida T et al.; The hst-1 transforming gene encodes a protein which belongs to the FGF family of growth factors . We showed previously that a human hst-1 protein produced in silkworm cells has in vitro mitogenic activity to vascular endothelial cells . Here we report effective synthesis of an unfused human hst-1 protein in E . coli and a potent in vivo angiogenic activity of this hst-1 protein by two in vivo assays for angiogenesis, chick chorioallantoic membrane assay and rat cornea assay . The NIH3T3 transformant transfected with the hst-1 gene appeared to develop a highly-vascularized tumor on nude mice . These data showed that the hst-1 protein has an angiogenic activity in vivo as well as in vitro.

Blood, 1994 Aug 15, 84(4), 1157 - 63
Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2; Barron-Casella EA et al.; The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene . To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N-terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1 . To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form . When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2) . These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay . Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds . Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets . This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes . Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

J Mol Biol, 1994 Aug 12, 241(2), 292 - 4
Crystallization and preliminary X-ray diffraction studies on the Apo form of orotate phosphoribosyltransferase from Escherichia coli; Aghajari N et al.; Three different crystal forms of the apoenzyme orotate phosphoribosyltransferase, with M(r) = 23,552 from Escherichia coli have been grown . The crystals, which are all suitable for X-ray diffraction analysis, have been grown by the hanging drop vapour diffusion method . The first form crystallizes in the orthorhombic space group P2(1)2(1)2, with cell dimensions: a = 136.34 A, b = 75.98 A and c = 40.32 A; the second form in the monoclinic space group P2, with unit cell dimensions: a = 101.61 A, b = 40.49 A, c = 79.05 A and beta = 87.33 degrees; and the third form in P2(1)2(1)2(1), the cell dimensions being a = 70.27 A, b = 103.53 A, c = 53.83 A.

J Mol Biol, 1994 Aug 12, 241(2), 281 - 2
Preliminary crystallographic study of Escherichia coli RuvC protein . An endonuclease specific for Holliday junctions; Ariyoshi M et al.; Single crystals of the RuvC protein, an Escherichia coli endonuclease specific for Holliday junctions, were grown by the microdialysis method . The crystals belong to the space group P2(1), with unit cell dimensions a = 72.8 A, b = 139.6 A, c = 32.4 A and beta = 93.0 degrees, and contain four molecules in an asymmetric unit . Diffraction data to a Bragg spacing of 2.5 A resolution has been obtained using a synchrotron X-ray source.

J Mol Biol, 1994 Aug 12, 241(2), 275 - 7
Crystallization of histidyl-tRNA synthetase from Escherichia coli; Francklyn C et al.; Histidyl-tRNA synthetase from Escherichia coli was over-expressed and purified by Q Sepharose and hydroxyapatite chromatography . Crystals of the complex containing histidyl-tRNA synthetase, ATP and histidine have been grown by vapor diffusion against reservoirs containing 0.1 M Tris (pH 7.4), 0.5 M NaCl and 10% polyethylene glycol 6000 . Under these conditions, two crystal forms are obtained . The triclinic form has unit cell dimensions a = 61.3 A, b = 108.5 A, c = 110.2 A, alpha = 115.1 degrees, beta = 90.2 degrees and gamma = 97.2 degrees . The monoclinic form, space group P2(1), has cell dimensions a = 61.2 A, b = 109.7 A, c = 196.7 A and beta = 98.1 degrees . Both crystal forms diffract up to 2.7 A and are stable in the synchrotron beam . Assuming a dimeric mass of 96,000 daltons and Vm value of 3.4 A3/dalton, the asymmetric unit in both forms contains two dimers with a solvent content of approximately 60% . A 3.7 A resolution native dataset with an Rmerge on intensities of 7.9% has been collected from the monoclinic crystal form.

J Mol Biol, 1994 Aug 12, 241(2), 150 - 65
In vitro interaction of nitrate-responsive regulatory protein NarL with DNA target sequences in the fdnG, narG, narK and frdA operon control regions of Escherichia coli K-12; Li J et al.; The narL gene product is a nitrate-responsive activator and repressor of anaerobic respiratory gene expression . Mutational studies and sequence comparisons have suggested that NarL protein binding sites contain heptameric sequences related to the consensus, TACYNMT (where Y = C or T, M = A or C, and N = any nucleotide) . There are four NarL heptamers in the -105 region of the fdnGHI (formate dehydrogenase-N) operon, and mutational analysis supports the role of these heptamers in nitrate induction . To examine NarL-DNA interactions, we purified the NarL protein as a maltose binding protein (MBP) fusion protein (MBP-NarL) . A constitutive mutant form with a single substitution (V88A) in the amino-terminal (response regulator) region was used . The MBP-NarL (V88A) protein protected all four heptamers in the fdnG operon control region from DNase I cleavage . Identical footprints were observed with NarL (V88A) protein that had been proteolytically cleaved free from the MBP domain . Binding of MBP-NarL (V88A) protein to the four heptamers in the -105 region of the fdnG operon appeared to be cooperative, and occupancy of the central heptamers was necessary for occupancy of the flanking heptamers . In addition to the V88A substitution, a low molecular weight phosphodonor, such as acetyl phosphate, was required for observable footprints . This indicates that phosphorylation of the NarL protein enhances its affinity for its multiple DNA targets in the fdnG operon, perhaps by increasing protein-protein interactions rather than protein-DNA interactions . We also performed footprinting studies at the narGHJI (nitrate reductase), narK (nitrite efflux), and frdABCD (fumarate reductase) operon control regions . Extensive areas of each control region were protected from DNase I attack by phosphorylated MBP-NarL (V88A) protein . The narG operon control region was protected from positions -50 to -110, and, at higher protein concentrations, also around position -200 . Mutational analysis indicates that the NarL heptamer centered at position -89, in addition to the previously-identified -200 region, is involved in nitrate induction . Comparisons of the four operon control regions studied indicate that the NarL heptamers are arranged with diverse orientations and spacing.

J Mol Biol, 1994 Aug 12, 241(2), 143 - 9
Homology modeling of divergent proteins; Sudarsanam S et al.; A method is presented for homology modeling of proteins bearing weak sequence identity to proteins of known tertiary structure . To accommodate non-identical amino acids in the core region, the backbone of the structurally conserved core of the model protein is allowed to deviate from that of the template protein . We have expanded FOLDER, a distance geometry-based homology modeling method, to allow for such displacements in the structurally conserved core . Models are built by rigidly constraining the interatomic distances within a structurally conserved segment and by allowing the interatomic distances between these segments to vary by a "divergence factor" . We test this method by simulating models of the beta-barrel domain D1 of CD4 and a four-helix bundle protein cytochrome b562 using the crystal structures of Bence-Jones protein and cytochrome c' as templates, respectively . In both cases, previously published structure-based sequence alignments were used for simulating models . The root-mean-square (r.m.s.) deviation of the backbone atoms in the common core between the templates and models was found to be a function of the imposed divergence factor . Our results demonstrate that this r.m.s . deviation results from the relative displacements of structurally conserved segments to accommodate the amino acid replacements in the core of the model protein . To test the integrity of the simulated structures we compared them with their respective crystal structures . The r.m.s . deviation of the backbone atoms in the core regions of the simulated models and their respective crystal structures is approximately 1.4 A . The r.m.s . deviation for all the backbone atoms in the models, including those in the structurally variable regions, which are modeled de novo, is 2.4 A for CD4 and 3.2 A for cytochrome b562 when compared with their respective X-ray structures.

J Mol Biol, 1994 Aug 12, 241(2), 133 - 5
Different peptide binding specificities of hsp70 family members; Gragerov A et al.; A set of heptapeptides was used to compare the relative peptide affinities of three proteins of the hsp70 family: bacterial DnaK, mammalian cytosolic hsc70, and BiP from mammalian ER . Each hsp displays a characteristic pattern of relative affinities . DnaK and hsc70 are more similar to each other than to BiP . A difference in peptide binding specificity may be an important determinant in adjusting an hsp70 family member to its particular cellular function.

J Biol Chem, 1994 Aug 12, 269(32), 20727 - 32
Molecular cloning and expression of a gamma-interferon-inducible activator of the multicatalytic protease; Realini C et al.; The multicatalytic protease (MCP) can be activated by two distinct multisubunit complexes . One is the regulatory component of the 26 S protease, which contains at least 15 distinct subunits . The other is a hexameric activator composed of 31- and 29-kDa subunits . A cDNA for the smaller subunit has been cloned and sequenced . The cDNA encodes a protein of 249 amino acids . Embedded between sequences typical of globular protein domains is a stretch of 28 "alternating" lysine and glutamic acid residues . Similar regions, which we call KEKE motifs, are also found in two MCP subunits, in subunit 12 of the 26 S protease and in a variety of chaperonins including hsp90, hsp70, and calnexin . Expression of the activator cDNA in Escherichia coli produced a functional protein virtually indistinguishable from MCP activator purified directly from red blood cells . The recombinant protein formed three isoelectric species on two-dimensional polyacrylamide gel electrophoresis, and it reacted with antibodies to red blood cell activator . Recombinant activator also bound the multicatalytic protease and stimulated cleavage at the carboxyl terminus of hydrophobic or charged residues . Synthesis of the activator subunit was induced by gamma interferon treatment of HeLa cells . These last two findings have implications for antigen presentation by class I major histocompatibility receptors.

J Biol Chem, 1994 Aug 12, 269(32), 20707 - 17
Cloning of cDNAs from fetal rat liver encoding glutathione S-transferase Yc polypeptides . The Yc2 subunit is expressed in adult rat liver resistant to the hepatocarcinogen aflatoxin B1; Hayes JD et al.; Fetal rat liver possesses substantial levels of glutathione S-transferase (GST) activity toward aflatoxin B1-8,9-epoxide . The enzyme responsible for this activity is an Alpha-class GST heterodimer comprising Yc1 and Yc2 subunits . The cDNAs encoding these polypeptides have been cloned and shown to share approximately 91% identity over 920 base pairs, extending from nucleotide -23 to the AATAAA polyadenylation signal . GST Yc2Yc2 expressed in Escherichia coli was found to exhibit 150-fold greater activity toward aflatoxin B1-8,9-epoxide than GST Yc1Yc1 . Comparison between the structures of Alpha-class GST suggests that tyrosine at residue 108 and/or aspartate at residue 208 is responsible for the high aflatoxin B1 detoxication capacity of Yc2 . Immunoblotting and enzyme assays have shown that liver from adult female rats contains about 10-fold greater levels of Yc2 than is found in liver from adult male rats . This sex-specific expression of Yc2 in adult rat liver may contribute to the relative insensitivity of female rats to aflatoxin B1 . Dietary administration of oltipraz, a synthetic antioxidant which protects against aflatoxin-hepatocarcinogenesis, serves as an inducer of GST Yc2.

J Biol Chem, 1994 Aug 12, 269(32), 20700 - 6
Mutational analysis of a multifunctional protein, with mRNA 5' cap-specific (nucleoside-2'-O-)-methyltransferase and 3'-adenylyltransferase stimulatory activities, encoded by vaccinia virus; Schnierle BS et al.; The vaccinia virus-encoded protein VP39 is a poly(A) polymerase subunit that stimulates the formation of long poly(A) tails as well as a cap-specific mRNA (nucleoside-2'-O-)-methyltransferase . We have carried out mutagenesis studies aimed at locating regions of VP39 which are important for these activities . The open reading frame encoding VP39 was expressed in Escherichia coli as a glutathione S-transferase fusion protein . The affinity-purified protein had both mRNA modification activities, before and after removal of the glutathione S-transferase domain . Truncation, charge cluster-->Ala scanning, and Cys-->Ser substitution mutations of VP39 were made, and the proteins were synthesized, purified, and analyzed . Deletion of the RNA binding domain, experimentally localized within the carboxyl-terminal 112 amino acids, resulted in the loss of both mRNA modification activities . Eleven of the 21 charge cluster-->Ala mutated proteins had low to nondetectable methyltransferase activity . Four of those 11 also lacked adenylyl-transferase stimulatory function, whereas the remainder had amino acid substitutions that selectively affected methyltransferase activity . However, no mutated proteins lacking adenylyltransferase stimulatory function but possessing methyltransferase activity were isolated by the procedures used . Neither of the 2 cysteine residues in VP39 was necessary for either mRNA modification activity.

J Biol Chem, 1994 Aug 12, 269(32), 20475 - 81
Domains of the tetrafunctional protein acting in glyoxysomal fatty acid beta-oxidation . Demonstration of epimerase and isomerase activities on a peptide lacking hydratase activity; Preisig-Muller R et al.; Peroxisomes from different eukaryotic organisms house a multifunctional protein acting in fatty acid beta-oxidation . In plant glyoxysomes, one of the isoforms of this protein contains the activities of L-3-hydroxyacyl-CoA hydrolyase (EC 4.2.1.17), L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.211), D-3-hydroxyacyl-CoA epimerase, and delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) . This was demonstrated after molecular cloning of a cDNA coding for a protein of 79047 Da and its bacterial expression . Chromatographic purification yielded a monomeric protein exhibiting all four activities . In addition, mutant forms were prepared, and peptides representing single domains were purified . Peptides containing the N-terminal region showed D-3-hydroxyacyl-CoA epimerase and delta 3,delta 2-enoyl-CoA isomerase activities but lacked 2-trans-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activities . Using the N-terminal fragment, we demonstrated that the D-3-hydroxyacyl-CoA converting activity is actually an epimerase rather than part of a combined water eliminating and water attaching system . The C-terminal half of the multifunctional protein represents the dehydrogenase domain.

J Biol Chem, 1994 Aug 12, 269(32), 20462 - 7
Cooperativity and stoichiometry of substrate binding to the catalytic sites of Escherichia coli F1-ATPase . Effects of magnesium, inhibitors, and mutation; Weber J et al.; The fluorescence of residue Trp beta 331 in beta Y331W mutant Escherichia coli F1-ATPase was used as reporter probe to investigate the effects of magnesium ions, inhibitors, and mutation on substrate (ATP) binding stoichiometry and cooperativity . It was found that Mg2+ is required for catalytic site binding cooperativity . In the absence of magnesium, ATP bound to three independent catalytic sites, each with Kd = 76 microM . In contrast, MgATP bound to three catalytic sites with Kd1 < 50 nM, Kd2 = 0.5 microM, and Kd3 = 25 microM . There was no significant ATPase activity in the absence of Mg2+ . Catalysis is therefore correlated with substrate binding cooperativity and the formation of the high-affinity catalytic site 1 . Catalytic site 3 had properties similar to those of the isolated beta-subunit nucleotide-binding site . The inhibitors dicyclohexylcarbodiimide and N-ethylmaleimide (in alpha S373C/beta Y331W mutant F1) gave potent inhibition of multisite ATPase activity without significantly affecting MgATP binding stoichiometry or cooperativity . Therefore each seems to selectively attenuate positive catalytic cooperativity . The same conclusions held for the alpha S373F mutation (in alpha S373F/beta Y331W mutant F1) . 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole, however, reduced the catalytic site MgATP binding stoichiometry from three to two, and appears to inhibit catalysis by sterically blocking catalytic site 3.

J Biol Chem, 1994 Aug 12, 269(32), 20446 - 55
Isolation and characterization of mutants of Tus, the replication arrest protein of Escherichia coli; Skokotas A et al.; Mutations in the tus gene of Escherichia coli, which encodes the replication arrest protein Tus, were isolated using a selection scheme based on the plasmid pHV750T2+, which transforms tus mutants at a much higher frequency than wild type strains . Seven mutants containing single nucleotide substitutions were isolated, and all of these mutants showed reduced or complete loss of DNA binding and replication arrest activity . Two of the mutant proteins, containing a valine (A173V) or threonine (A173T) in place of the alanine normally found at amino acid 173, were purified and characterized further . A173T had a 4100-fold lower affinity for Ter sites than wild type Tus and was unable to halt DNA replication in vivo or inhibit DnaB-catalyzed strand displacement in an in vitro helicase assay . A173V showed a 130-fold lower affinity for Ter sites than wild type Tus but was still able to arrest DNA replication in vivo, suggesting that protein-protein interactions were responsible for Tus-mediated arrest of DNA replication . In addition, we found that A173V was a weak inhibitor of DnaB-catalyzed strand displacement in vitro, yet halted DNA replication in vivo at 75% of the efficiency of wild type Tus . We concluded from these observations that the standard in vitro helicase assay was inadequate for measuring Tus activity.

J Biol Chem, 1994 Aug 12, 269(32), 20376 - 9
Archaebacterial elongation factor 1 alpha carries the catalytic site for GTP hydrolysis; Masullo M et al.; The elongation factor 1 alpha from the archaebacterium Sulfolobus solfataricus (aEF-1 alpha) possesses an intrinsic GTPase activity that is triggered by NaCl up to 5.2 M and requires the presence of at least 1 mM MgCl2 or MnCl2 . Chloride salts of other monovalent cations are inefficient, whereas other sodium salts are much less efficient or not efficient at all as compared with NaCl . This aEF-1 alpha GTPase (GTPaseNa) reaches a maximum in a broad pH range and is not affected by other nucleoside triphosphates but is competitively inhibited by GDP . The turnover of GTPaseNa is rate limited by the breakdown of GTP . The Km for GTP is in the range of 2.2-9.3 microM, depending on the NaCl concentration and temperature . The highest catalytic efficiency is reached at 87 degrees C, which is the optimum temperature for growth of S . solfataricus . The energetic parameters of GTPaseNa are similar to those reported in the literature for the GTPase of Escherichia coli elongation factor Tu (EF-Tu) triggered by 2 M KCl, thus suggesting that the GTPase activity supported by either EF-Tu or aEF-1 alpha undergoes a similar mechanism of activation by salt at high concentration . A molecular mechanism for this activation is proposed.

J Biol Chem, 1994 Aug 12, 269(32), 20312 - 7
Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L; Wang X et al.; Phosphatidylcholine-hydrolyzing phospholipase D (PLD; EC 3.1.4.4) has been proposed to play an important role in the signal transduction pathways in animals and in various cellular processes in plants . To unravel the structure of PLD and further the investigation of its modes of regulation and cellular function, we have isolated a PLD cDNA from castor bean (Ricinus communis L . var . Hale) by using oligonucleotide probes based on the N-terminal amino acid sequence of purified PLD protein . The nucleotide sequence of the castor bean PLD cDNA clone contains an open reading frame of 2424 bases encoding an 808-amino acid protein of 92,400 daltons . Expression of this PLD cDNA clone in Escherichia coli resulted in the accumulation of a functional PLD able to catalyze hydrolysis and transphosphatidylation reactions, demonstrating that the introduction of this single gene product was sufficient to confer PLD activity and that both the hydrolysis and transphosphatidylation reactions are catalyzed the single PLD protein . Comparison of the deduced protein sequence of the clone to the N-terminal amino acid sequence of purified PLD revealed that the mature PLD protein is preceded by a 30-amino acid leader peptide . Removal of this leader peptide resulted in accumulation of non-functional PLD and also increased PLD degradation in E . coli, suggesting that this leader peptide may be involved in proper folding of PLD . The primary structure of the castor bean PLD exhibits no significant similarities with sequences of other cloned lipolytic enzymes.

J Biol Chem, 1994 Aug 12, 269(32), 20299 - 304
Identification of a protein kinase associated with the cytoplasmic domain of the p60 tumor necrosis factor receptor; Darnay BG et al.; Tumor necrosis factor (TNF) has been shown to bind two distinct receptors, designated p60 and p80, with high affinity, resulting, within minutes, in phosphorylation of several proteins . The receptors themselves do not exhibit protein kinase activity nor have any associated proteins been identified . We employed the glutathione-S-transferase (GST) fusion protein system consisting of the cytoplasmic domain of p60 (GST-p60CD delta 1) as a probe to help us identify receptor-associated proteins from human histiocytic lymphoma U-937 cells . We found that a protein of approximately 52 kDa (pp52) bound to GST-p60CD delta 1 from {35S}methionine- and 32P-labeled cells . The associated protein was phosphorylated on serine and threonine residues . Furthermore, we identified serine/threonine kinase activity associated with p60CD delta 1 that required either Mn2+ or Mg2+ for optimal activity . The preferred substrates for this kinase, in addition to p60CD delta 1, included casein and histone H1, but not histone H2B, myelin basic protein, enolase, or the cytoplasmic domain of p80 . As was the case in U-937 cells, p60CD delta 1-associated kinase activity was also identified in human breast adenocarcinoma MCF-7 cells and human foreskin fibroblasts . TNF stimulation of MCF-7 and foreskin fibroblasts for 5-15 min induced approximately 50 and 240% increases in phosphorylation of p60CD delta 1, respectively . Thus, our results provide the first evidence for protein kinase activity that is specifically associated with the cytoplasmic domain of the p60 form of the TNF receptor and causes its phosphorylation . This p60 TNF receptor-associated protein and the associated kinase described here are referred to as p60-TRAP and p60-TRAK, respectively.

J Biol Chem, 1994 Aug 12, 269(32), 20217 - 20
Zinc-dependent cell growth conferred by mutant tRNA synthetase; Landro JA et al.; We present evidence that zinc bound near the C terminus of a long tRNA synthetase polypeptide, and at a location far in the sequence from the catalytic domain, is needed to sustain cell growth and is, therefore, essential for enzyme function . Several class I and class II tRNA synthetases contain bound zinc, including the 939-amino acid class I Escherichia coli isoleucyl-tRNA synthetase, which has two zinc atoms coordinated to cysteine sulfhydryls . The functional significance of these bound zinc atoms has been unclear . Like other class I tRNA synthetases, the isoleucine enzyme has a class-defining conserved N-terminal domain that contains the catalytic site . The C-terminal domain is variable in sequence and structure and not conserved among all of the class I enzymes . Using split proteins, we localized a zinc binding site to the C-terminal end of isoleucyl-tRNA synthetase . Serine substitutions of single cysteines at a thiol-containing putative zinc binding site that is less than 40 amino acids from the C terminus confer a zinc-dependent growth phenotype on cells harboring the mutant enzymes . We propose that zinc bound near the C terminus is part of a structure that interacts directly or indirectly with the active site . A structure at the C terminus that provides a functional link between the conserved N-terminal catalytic and non-conserved C-terminal domain may be common to several class I enzymes.

J Chromatogr A, 1994 Aug 12, 677(1), 45 - 52
Novel DNA-Sepharose purification of the FadR transcription factor; DiRusso C et al.; A DNA sequence bound by the FadR transcription factor of Escherichia coli was covalently attached to Sepharose by two different approaches: by chemical coupling or by template-directed enzymatic synthesis using a DNA polymerase . The two kinds of DNA-Sepharose were packed into small columns and used for the purification of the FadR protein; chromatography was without using competitor DNA and the supports contained single-copy, non-repetitive DNA sequences . Comparison showed that the enzymatically prepared support, while having less bound DNA, bound more FadR protein than did the chemically prepared support . This probably results from the lack of detrimental DNA modification by the gentle enzymatic procedure . The chemically prepared support was of lower capacity but yielded purer FadR protein when compared under the same elution conditions . This may be explained by the simpler DNA sequence which could be coupled chemically; less contaminating proteins were bound by the simpler DNA sequence . However, the enzymatically prepared support could also yield comparable purity if the protocol was modified to include additional washes with salt containing buffers . In all cases, FadR was eluted from the DNA using high-salt (0.8 M) mobile phase; ligand-specific elution of FadR using a fatty acyl-coenzyme A thiol ester was ineffective . Affinity chromatography on DNA-Sepharose provided a more rapid, simple purification of FadR than conventional purification techniques and yielded biologically active protein.

J Biol Chem, 1994 Aug 12, 269(32), 20369 - 75
Delineation of the catalytic core of phenylalanine hydroxylase and identification of glutamate 286 as a critical residue for pterin function; Dickson PW et al.; Rat phenylalanine hydroxylase was expressed in Escherichia coli . High level expression was achieved when the transformed E . coli were incubated at 27 degrees C for 24 h . A series of truncated fragments were expressed . The smallest fragment that gave an active soluble protein was from Leu142 to Phe410 . This fragment corresponds closely to the region where there is highest homology between the three aromatic amino acid hydroxylases . The circular dichroism spectra of the phenylalanine hydroxylase catalytic core suggested that it contains around 50% alpha-helix . The core fragment is monomeric in dilute solutions but self-associates at higher concentrations . The E . coli expression system was used to generate a number of mutations in phenylalanine hydroxylase from position 264 to 290 . This region had been previously shown to be important for pterin binding . Characterization of the mutant phenylalanine hydroxylase molecules identified Glu286 as an amino acid critical for pterin function in phenylalanine hydroxylase.

J Mol Biol, 1994 Aug 12, 241(2), 246 - 62
Thermodynamics of folding a pseudoknotted mRNA fragment; Gluick TC et al.; A sequence in the leader and first gene of the Escherichia coli alpha mRNA folds into a complex pseudoknot structure that is required for binding of a translational repressor . The thermal denaturation of a 112 nt RNA containing this structure has been followed by calorimetry and UV hyperchromicity . To determine the partially folded intermediates in unfolding, the denaturation of 13 mutants and of several fragments with successive deletions of helices were investigated as well . An unfolding pathway with seven states is proposed as the simplest mechanism that accounts for the data, and has several implications . (1) The lowest temperature transition appears only in the presence of moderate concentrations of Mg2+ or high concentrations of K+ (delta H approximately 45 kcal/mol), and is the unfolding of tertiary structures, rather than secondary structure . Under some conditions it is destabilized by increasing salt concentration . (2) Two of the intermediates unfolding at higher temperature must have non-canonical or tertiary interactions in addition to the known secondary structure . (3) Two alternative structures compete for formation of the complete pseudoknot, and form as the pseudoknot unfolds . Thus structures not present in the completely folded pseudoknot affect the overall thermodynamics, and probably the kinetics, of unfolding . (4) Approximately 16 kcal/mol of free energy is required to completely expose the coding region to ribosomes at 37 degrees C, though approximately 6.5 kcal/mol is regained by refolding of upstream regions after the pseudoknot is unfolded . The substantial energy needed to unfold the pseudoknot may affect the rate of translation from this ribosome binding site . A simple model of RNA folding in which an optimum secondary structure forms first, followed by tertiary interactions that further stabilize the secondary structure, does not hold in this RNA.

Nucleic Acids Res, 1994 Aug 11, 22(15), 3194 - 201
Quantitative studies of the effect of HTLV-I Tax protein on CREB protein--DNA binding; Anderson MG et al.; The human T-cell leukemia virus type I (HTLV-I) Tax protein increases the DNA binding activity of a number of different host cell transcription factors, including the cyclic AMP response element binding protein (CREB) . We have performed quantitative studies of CREB binding in the presence and absence of Tax in an attempt to gain insight into the mechanism of the Tax effect . Enhancement of binding occurred over a wide range of CREB concentrations, but sharply increased at the lowest concentrations tested . The data are best explained by a two-step binding model where Tax changes the apparent equilibrium constants for both a CREB-CREB dimerization step and a (CREB)2-DNA binding step . We used the model to perform a quantitative analysis of the binding of CREB to DNA that had been mutated at positions flanking the core CREB recognition site . Results suggest that there are altered or more extensive DNA-protein contacts at these positions in the presence of Tax . We also used the model to analyze differences in the interaction of Tax with nonphosphorylated and protein kinase A-phosphorylated CREB protein . There was no significant change in the behavior of CREB upon phosphorylation.

Nucleic Acids Res, 1994 Aug 11, 22(15), 3174 - 80
Hydrophobicity, expressivity and aromaticity are the major trends of amino-acid usage in 999 Escherichia coli chromosome-encoded genes; Lobry JR et al.; Multivariate analysis of the amino-acid compositions of 999 chromosome-encoded proteins from Escherichia coli showed that three main factors influence the variability of amino-acid composition . The first factor was correlated with the global hydrophobicity of proteins, and it discriminated integral membrane proteins from the others . The second factor was correlated with gene expressivity, showing a bias in highly expressed genes towards amino-acids having abundant major tRNAs . Just as highly expressed genes have reduced codon diversity in protein coding sequences, so do they have a reduced diversity of amino-acid choice . This showed that translational constraints are important enough to affect the global amino-acid composition of proteins . The third factor was correlated with the aromaticity of proteins, showing that aromatic amino-acid content is highly variable.

Nucleic Acids Res, 1994 Aug 11, 22(15), 3124 - 30
Suppression of the serT42 mutation with modified tRNA(1Ser) and tRNA(5Ser) genes; Yamada Y et al.; Serine tRNA gene derivatives with altered anticodons were introduced to the temperature-sensitive serT42 mutant, whose tRNA(1Ser) shows a base substitution of A10 for wild type G10 . When a low copy number vector-system was used, the growth and beta-galactosidase synthetic activity of the serT42 mutant were restored by complementation with the tRNA(5Ser) (T34) gene or the tRNA(1Ser) (G34) gene as well as the tRNA(1Ser) (wt) gene, but not with tRNA(5Ser) (wt), tRNA(1Ser) (A34) or tRNA(1Ser) (C34) genes at 42 degrees C . When multicopy vectors were used, the transformation even with tRNA(1Ser) (A10) gene restored the growth and beta-galactosidase synthetic activity at 42 degrees C . The tRNA(1Ser) (A10) showed no thermosensitivity in serine acceptor activity by in vitro assay . At 42 degrees C, the amount of tRNA(1Ser) (A10) in the serT42 mutant was almost the same as those in the wild type . The nucleotides in the tRNA(1Ser) (A10) were found to be fully modified like those in the wild type tRNA(1Ser) . Both of the tRNAs transcribed from tRNA(5Ser) (T34) and tRNA(1Ser) (G34) genes showed serine acceptor activity . Modified nucleosides of these tRNAs were also analyzed.

Nucleic Acids Res, 1994 Aug 11, 22(15), 3018 - 25
Contacts between 16S ribosomal RNA and mRNA, within the spacer region separating the AUG initiator codon and the Shine-Dalgarno sequence; a site-directed cross-linking study; Rinke-Appel J et al.; mRNA analogues containing several 4-thiouridine (thio-U) residues at selected positions were prepared by T7-transcription . The spacer region between the Shine-Dalgarno sequence and the AUG codon consisted of four or eight bases with a single thio-U at a variable position; alternatively, cro-mRNA analogues were used carrying the thio-U substituted spacer sequence UUGU . The mRNAs were bound to E . coli ribosomes, and--after irradiation--the sites of cross-linking to 16S RNA were analysed . Three cross-links to the 16S RNA from the spacer region were observed, namely to positions 665, 1360, and a site close to nucleotide 1530 . The cross-links were formed in different amounts in the presence or absence of tRNA(fMet), and were observed from thio-U residues located at various positions within the spacer sequence, although in the presence of tRNA they were in general stronger from positions close to the Shine-Dalgarno end of the spacer . The cross-linking behaviour in this upstream area of the mRNA is thus rather different in character from the previously published pattern in the downstream area . From considerations of structural conservation in small subunit RNA, we propose that both the upstream and downstream cross-links to 16S RNA reflect a universal mRNA path through the ribosome.

Nucleic Acids Res, 1994 Aug 11, 22(15), 2963 - 9
Seryl-tRNA synthetase from Escherichia coli: implication of its N-terminal domain in aminoacylation activity and specificity; Borel F et al.; Escherichia coli seryl-tRNA synthetase (SerRS) a dimeric class II aminoacyl-tRNA synthetase with two structural domains charges specifically the five iso-acceptor tRNA(ser) as well as the tRNA(sec) (selC product) of E . coli . The N-terminal domain is a 60 A long arm-like coiled coil structure built of 2 long antiparallel a-h helices, whereas the C-terminal domain is a alpha-beta structure . A deletion of the N-terminal arm of the enzyme does not affect the amino acid activation step of the reaction, but reduces dramatically amino-acylation activity . The Kcat/Km value for the mutant enzyme is reduced by more than 4 orders of magnitude, with a nearly 30 fold increased Km value for tRNA(ser) . An only slightly truncated mutant form (16 amino acids of the tip of the arm replaced by a glycine) has an intermediate aminoacylation activity . Both mutant synthetases have lost their specificity for tRNA(ser) and charge also non-cognate type 1 tRNA(s) . Our results support the hypothesis that class II synthetases have evolved from an ancestral catalytic core enzyme by adding non-catalytic N-terminal or C-terminal tRNA binding (specificity) domains which act as determinants for cognate and anti-determinants for non-cognate tRNAs.

Nucleic Acids Res, 1994 Aug 11, 22(15), 2958 - 62
A cluster of constitutive mutations affecting the C-terminus of the redox-sensitive SoxR transcriptional activator; Nunoshiba T et al.; Activation of Escherichia coli oxidative stress regulon genes (sodA, zwf, fumC, nfo, etc.) is mediated by a two-stage regulatory system: the redox-sensitive SoxR protein transcriptionally activates the soxS gene, whose product then stimulates transcription of the regulon genes . Previous experiments showed that limited 3' truncation of soxR gene causes constitutive soxRS expression . DNA sequence analysis of the soxR genes from the soxRS-constitutive strains isolated originally (Greenberg et al . (1990) Proc . Natl . Acad . Sci . USA 87, 6181-6185) revealed that three alleles encode amino acid substitutions or a chain termination clustered near the C-terminus of SoxR . Two other single-amino-acid substitutions in constitutive alleles mapped to the helix-turn-helix motif and to a region of unknown function in the center of the polypeptide, respectively . No constitutive mutation was found within the region encoding the cysteines of the SoxR FeS center, in the soxR or soxS promoters, or in the soxS structural gene . Since an in-frame deletion of just nine SoxR residues (136-144; full-length SoxR = 154 residues) gave rise to a powerful constitutive allele, it appears that a small segment of the SoxR C-terminus maintains the protein in the inactive state . Conservely, an intact C-terminus is evidently not required for gene activation by SoxR.

Nucleic Acids Res, 1994 Aug 11, 22(15), 2902 - 7
Difference and similarity of DNA sequence recognized by VDR homodimer and VDR/RXR heterodimer; Nishikawa J et al.; Nuclear receptors for the thyroid hormone and vitamin A and D cooperate with the retinoid X receptor (RXR) in activating the transcription . Although the hormone response elements for these receptors have been proposed in which spacing of the direct repeated motifs determine the specificity (so called 3-4-5 rule), vitamin D response elements (VDREs) in the natural context consist of often imperfect direct repeats . Vitamin D receptor (VDR) alone can bind to the mouse osteopontin (mSPP-1) VDRE, which contains a direct repeat separated by 3 nucleotides, but not to the rat osteocalcin (rOST) VDRE having inexact direct repeat . The presence of RXR not only allows the VDR to bind to the rOST VDRE, but also increases the binding affinity for the mSPP-1 VDRE . The RXR/VDR heterodimer exhibits the similar affinity constants for the mSPP-1 VDRE and the rOST VDRE, in spite of the apparently different affinities for two VDREs of the VDR homodimer . A random oligonucleotide selection procedure revealed that the consensus sequence selected by the RXR homodimer is the direct repeat spaced by one A residue . In contrast, the sequences preferentially selected by the VDR homodimer and the VDR/RXR heterodimer are similar, which are the direct repeats spaced by 3 nucleotides . The difference and similarity of DNA sequence recognition are discussed.

Nucleic Acids Res, 1994 Aug 11, 22(15), 2894 - 901
Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 of the cyclic nucleotide binding pocket; Lee EJ et al.; The cyclic 3', 5' adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute cysteine or glycine for serine 83; cysteine, glycine, isoleucine, or serine for threonine 127; and threonine or alanine for serine 128 . Cells that expressed the binding pocket residue-substituted forms of CRP were characterized by measurements of beta-galactosidase activity . Purified wild-type and mutant CRP preparations were characterized by measurement of cAMP binding activity and by their capacity to support lacP activation in vitro . CRP structure was assessed by measurement of sensitivity to protease and DTNB-mediated subunit crosslinking . The results of this study show that cAMP interactions with serine 83, threonine 127 and serine 128 contribute to CRP activation and have little effect on cAMP binding . Amino acid substitutions that introduce hydrophobic amino acid side chain constituents at either position 127 or 128 decrease CRP discrimination of cAMP and cGMP . Finally, cAMP-induced CRP structural change(s) that occur in or near the CRP hinge region result from cAMP interaction with threonine 127; substitution of threonine 127 by cysteine, glycine, isoleucine, or serine produced forms of CRP that contained, independently of cAMP binding, structural changes similar to those of the wild-type CRP:cAMP complex.

Nucleic Acids Res, 1994 Aug 11, 22(15), 2876 - 81
Self-methylation of BspRI DNA-methyltransferase; Szilak L et al.; The DNA (cytosine-5)-methyltransferase (m5C-MTase) M.BspRI is able to accept the methyl group from the methyl donor S-adenosyl-L-methionine (AdoMet) in the absence of DNA . Transfer of the methyl group to the enzyme is a slow reaction relative to DNA methylation . Self-methylation is dependent on the native conformation of the enzyme and is inhibited by S-adenosyl-L-homocysteine, DNA and sulfhydryl reagents . Amino acid sequencing of proteolytic peptides obtained from M.BspRI, which had been methylated with {methyl-3H}AdoMet, and thin layer chromatography of the modified amino acid identified two cysteines, Cys156 and Cys181 that bind the methyl group in form of S-methylcysteine . One of the acceptor residues, Cys156 is the highly conserved cysteine which plays the role of the catalytic nucleophile of m5C-MTases.

Nucleic Acids Res, 1994 Aug 11, 22(15), 2970 - 5
Assembly of DNA polymerase delta and epsilon holoenzymes depends on the geometry of the DNA template; Podust LM et al.; To study in details the assembly of DNA polymerases delta and epsilon holoenzymes a circular double-stranded DNA template containing a gap of 45 nucleotides was constructed . Both replication factor C and proliferating cell nuclear antigen were absolutely required and sufficient for assembly of DNA polymerase delta holoenzyme complex on DNA . On such a circular DNA substrate replication protein A (or E . coli single-strand DNA binding protein) was neither required for assembly of DNA polymerase delta holoenzyme complex nor for the gap-filling reaction . A circular structure of the DNA substrate was found to be absolutely critical for the ability of auxiliary proteins to interact with DNA polymerases . The linearization of the circular DNA template resulted in three dramatic effects: (i) DNA synthesis by DNA polymerase delta holoenzyme was abolished, (ii) the inhibition effect of replication factor C and proliferating cell nuclear antigen on DNA polymerase alpha was relieved and (iii) DNA polymerase epsilon could not form any longer a holoenzyme with replication factor C and proliferating cell nuclear antigen . The comparison of the effect of replication factor C and proliferating cell nuclear antigen on DNA polymerases alpha, delta and epsilon indicated that the auxiliary proteins appear to form a mobile clamp, which can easily slide along double-stranded DNA.

Biochemistry, 1994 Aug 9, 33(31), 9365 - 70
Mechanism-based inactivation of L-aspartase from Escherichia coli; Schindler JF et al.; The substrate analogue L-aspartate beta-semialdehyde (L-ASA) has been identified as a mechanism-based inactivator of L-aspartase from Escherichia coli . The enzyme catalyzes the deamination of L-ASA to yield fumaric acid semialdehyde (FAA) and NH4+, with the product FAA partitioning between subsequent release or irreversible enzyme inactivation . Complete protection against L-ASA inactivation is observed in the presence of the product fumarate and a divalent metal ion . However, protection against inactivation by the product FAA also requires the presence of an enzyme activator . In addition to functioning as a mechanism-based inactivator, L-ASA has also been shown to serve as an activator of L-aspartase . The mechanism of inactivation by FAA involves the attack of an active site nucleophilic at the alpha-carbon of FAA to yield a stable Michael type enzyme adduct . Subsequent formation of a hydrazone upon treatment of the enzyme adduct with 2,4-dinitrophenylhydrazine confirms the presence of the unreacted aldehydic group of FAA . Examination of a group of product analogues with different substituents has demonstrated a correlation between the electron-withdrawing ability of these functional groups and the rate of inactivation of L-aspartase.

Biochemistry, 1994 Aug 9, 33(31), 9351 - 7
Substrate-dependent mechanisms in the catalysis of human immunodeficiency virus protease; Polgar L et al.; The most preferred residue in the substrates of human immunodeficiency virus (HIV-1) protease is glutamic acid in the P2' position . The catalytic importance of this charged residue has been studied to obtain a detailed insight into the mechanism of action, which will promote drug design to combat the virus . To this end, we have synthesized Lys-Ala-Arg-Val-Leu*Phe(NO2)-Glu-Ala-Nle (substrate E) and its counterpart containing the neutral Gln (substrate Q) in place of Glu . Kinetic analyses have shown that the specificity rate constants (kcat/Km) display bell-shaped pH dependencies for both substrates, but the pH-independent limiting value is 35-40-fold higher with substrate E than with substrate Q . In contrast to the pH-rate profiles of kcat/Km, there is a striking difference between the pH dependencies of Km and kcat for the two substrates . This indicates different ground state and transition state stabilizations in the two reactions . Solvent kinetic deuterium isotope effects show that the rate-limiting step for the hydrolysis of substrate E is a chemical step coupled with proton transfer whereas with substrate Q it is a physical step, presumably a conformational change . Accordingly, the charged residue in P2' alters the rate-limiting step and the nature of the enzyme-substrate complex, resulting in different mechanisms for the two substrates.

Biochemistry, 1994 Aug 9, 33(31), 9327 - 32
Thermodynamics of unfolding of the all beta-sheet protein interleukin-1 beta; Makhatadze GI et al.; The thermal denaturation of interleukin-1 beta in solution has been studied by differential scanning calorimetry at various pH values . It is shown that the thermal transition of interleukin-1 beta is completely reversible below pH 2.5, only partly reversible in the pH range 2.5-3.5, and irreversible above pH 3.5 . Analysis of the reversible unfolding of interleukin-1 beta shows that the heat denaturation is well approximated by a two-state transition and is accompanied by a significant increase of heat capacity . The partial heat capacity of denatured interleukin-1 beta is very close to that expected for the completely unfolded protein . This permitted us to assign the thermodynamic characteristics of interleukin-1 beta denaturation to its complete unfolding and to correlate them with structural features of the protein . The contributions of hydrogen bonding and hydrophobic interactions to the stability of interleukin-1 beta are analyzed and compared to those for other globular proteins . It is shown that the Gibbs energy of a hydrogen bond in a beta-sheet structure is greater than in alpha-helices.

Biochemistry, 1994 Aug 9, 33(31), 9169 - 77
Response of repair-competent and repair-deficient Escherichia coli to three O6-substituted guanines and involvement of methyl-directed mismatch repair in the processing of O6-methylguanine residues; Pauly GT et al.; Plasmids containing a site-specifically incorporated O6-methyl- (m6G), O6-ethyl- (e6G), or O6-benzylguanine (b6G) within the ATG initiation codon of the lacZ' gene were used to transform Escherichia coli that were repair proficient or deficient in one or both of the E . coli O6-alkylguanine-DNA alkyltransferases, the uvr(ABC) excision repair system, the recA-mediated recombination system, or the methylation-directed mismatch repair system . Colonies were scored phenotypically for adduct-induced mutations . With plasmids containing either e6G or b6G, the frequency of adduct-induced mutation was low and independent of the repair proficiency of the strain transformed . Plasmids containing an m6G residue elicited similar responses in all but the mismatch repair-deficient strain . The generally low mutagenicity of all the O6-substituted guanines was interpreted as reflecting an adduct-induced arrest of replication of the modified strand while the unmodified complementary strand was replicated normally . Studies of the involvement of mismatch repair in m6G mutagenesis showed that m6G:T base pairs were more readily processed than m6G:C base pairs, indicating that mismatch repair involving m6G residues occurs after replication . These data support a model in which the E . coli methylation-directed mismatch repair system diverts plasmids containing promutagenic m6G:T base pairs into replication-arrested complexes providing another line of defense against O6-methylguanine mutagenicity in addition to O6-alkylguanine-DNA alkyltransferase repair and excision repair mechanisms.

Biochemistry, 1994 Aug 9, 33(31), 9062 - 9
X-ray structure of nucleoside diphosphate kinase complexed with thymidine diphosphate and Mg2+ at 2-A resolution; Cherfils J et al.; We report the crystal structure of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum with thymidine diphosphate (dTDP) and Mg2+ bound at the active site . The structure has been refined to an R-factor of 18.3% at 2-A resolution . The base stacks on the aromatic ring of Phe 64 near the protein surface and is wedged between the side chains of Phe 64 and Val 116 . The sugar and the pyrophosphate are deeper inside the protein and make numerous H-bonds with protein side chains . There is no backbone interaction with the nucleotide . A Mg2+ ion bridges the alpha- and beta-phosphates and interacts with the protein via water molecules . NDP kinase shows little specificity toward ribonucleotides and deoxyribonucleotides . This property, required by the enzyme biological function, can now be analyzed by comparing the crystal structures of free, ADP-ligated, and dTDP-ligated enzymes . The most significant differences are located in residues 60-64, which adapt their conformation to allow Phe 64 to stack on both types of bases . Nonspecific binding is achieved by the absence of polar interaction between the base and protein atoms . The ribose of ADP and the deoxyribose of dTDP occupy similar positions, their hydroxyl groups interacting with Lys 16 and Asn 119 . The H-bond between Lys 16 and the O2' hydroxyl of ADP is replaced by a similar interaction with a water molecule in the dTDP complex . The beta-phosphate position is the same for ADP and dTDP, suggesting that the mechanism of phosphate transfer is the same for all substrates ofNDP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Aug 9, 33(31), 9343 - 50
Evidence supporting catalytic roles for aspartate residues in phosphoribulokinase; Charlier HA Jr et al.; The DNA encoding Rhodobacter sphaeroides phosphoribulokinase (PRK) has been modified to allow ligation into pET-3d . Using the resulting expression plasmid, PRK was overexpressed in Escherichia coli and isolated in milligram quantities . Homogeneous preparations of the enzyme exhibit properties comparable to those of PRK expressed using a previously described pUC19-derived construct {Sandbaken et al., Biochemistry 31, 3715-3719} . Mutagenesis experiments have been designed to produce conservative substitutions that eliminate the carboxyl groups of each of four conserved acidic residues (D42, E131, D169, and E178) . Using the newly developed expression system, the resulting PRK variants have been expressed, isolated, and characterized . Expression levels and recoveries upon affinity chromatography purification are similar to the results obtained with wild-type PRK . Apparent substrate affinities of these mutant proteins do not differ greatly from values observed for wild-type PRK . In contrast, these PRK variants display a wide range of Vmax values, ranging from wild-type activity (approximately 200 units/mg; E178A) to levels that are diminished by 4 (D169A) to 5 (D42A, D42N) orders of magnitude . That the large diminutions in catalytic activity are significant and do not merely reflect gross perturbations in protein structure is suggested not only by the modest effects on substrate affinity but also by the allosteric properties of D169A, D42A, and D42N . The activities of these proteins, like that of wild-type PRK, are markedly stimulated by the positive effector NADH . The magnitude of the Vmax perturbations suggests that D42 and D169 are candidates for the role of active site base or activator cation ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1994 Aug 8, 349(3), 433 - 8
Construction and in vivo analysis of new split lactose permeases; Wrubel W et al.; The capacity of incomplete segments of Escherichia coli lactose permease to form transport-competent complexes in vivo was further tested . Two series of mutant lacY genes were constructed . One encoded N-terminal lactose permease segments of different length . The proteins specified by the other group contained deletions of different length and location within the N-terminal region . Several pairs of such mutant proteins reconstituted active lactose transport . For certain combinations duplications of protein segments were compatible with the formation of an active carrier . Duplication of helices could also be tolerated, when part of a single polypeptide chain.

FEBS Lett, 1994 Aug 8, 349(3), 397 - 402
The characterisation of the shikimate pathway enzyme dehydroquinase from Pisum sativum; Deka RK et al.; Peptides accounting for 157 residues of the bifunctional shikimate pathway enzyme, dehydroquinase/shikimate dehydrogenase, of Pisum sativum were sequenced . Three of the peptides were homologous to regions in Escherichia coli dehydroquinase and two to E . coli shikimate dehydrogenase . The pea dehydroquinase activity was inhibited by treatment with dehydroquinate plus sodium borohydride, establishing it as a type I dehydroquinase . Synthetic oligonucleotides designed from the amino acid sequence were used as PCR primers to amplify fragments of P . sativum cDNA . DNA sequence analysis showed that these amplified products were derived from dehydroquinase/shikimate dehydrogenase cDNA . The complete amino acid sequence of the dehydroquinase domain has been defined; it is homologous to all other type I dehydroquinases and is N-terminal.

Gene, 1994 Aug 5, 145(2), 267 - 72
Cloning of a cDNA encoding a human protein which binds a sequence in the c-myc gene similar to the interferon-stimulated response element; Stasiv YZ et al.; A human cDNA clone encoding a c-myc promoter-binding protein (IRLB) was selected by screening a human fibroblast lambda gt11 phage library with the hexamer oligodeoxyribonucleotide (oligo) 5'-GGCGGGAAAAAGAACGGA, corresponding to the protein-binding element of human c-myc similar to the interferon-stimulated response element (ISRE) . The lambda gt11 phage clone, encoding a fusion protein which bound the probe oligo, was used to create an strain of Escherichia coli . The deduced amino-acid sequence of the cloned protein contains a putative alpha-helix which is expected to act as the DNA-binding domain . DNase footprinting analysis and oligo-binding specificity assays showed that the cloned factor recognizes the ISRE-like element of the P2 promoter region of human c-myc.

Gene, 1994 Aug 5, 145(2), 221 - 6
Processing of hepatitis C viral polyprotein in Escherichia coli; Komoda Y et al.; Two proteinase activities, encoded by hepatitis C virus (HCV), Cpro-1 and Cpro-2 . Cpro-1 and Cpro-2 appear to process the precursor polyprotein from which they originate . Mutant HCV polypeptides containing the region for these proteinases were produced in Escherichia coli as fusion proteins . The N- and C-terminal ends of the HCV polypeptides were fused with the E . coli maltose-binding protein (MBP) and E . coli dihydrofolate reductase (DHFR), respectively . The proteinase activities cleaved the fusion polypeptides by the same processing pathway used in eukaryotic protein production systems . The N-terminal amino acid (aa) sequences of the processed fusion proteins were determined . A comparison of those N-terminal sequences with the aa sequence of the HCV precursor polyprotein showed that the N-terminal and C-terminal cleavage sites of p70(NS3), one of the HCV nonstructural (NS) proteins, were the same as those identified in other processing studies: cleavages were estimated to be between aa 1026 and 1027 and between aa 1657 and 1658 of the HCV precursor protein, which are known to be cleaved by Cpro-1 and Cpro-2, respectively . Cpro-1 and Cpro-2 both functioned in E . coli and possessed authentic characteristic features.

Gene, 1994 Aug 5, 145(2), 179 - 87
The effect of multiple copies of the upstream region on expression of the Aspergillus niger glucoamylase-encoding gene; Verdoes JC et al.; The regulation of transcription of the glucoamylase-encoding gene (glaA) of Aspergillus niger was studied . To facilitate this study a reporter strain containing a fusion of the glaA promoter (PglaA) of A . niger to the beta-glucuronidase-encoding gene (uidA) of Escherichia coli was constructed . To analyze whether regulatory proteins are involved in the regulation of glaA, multiple copies of PglaA were introduced into this reporter strain . Analysis of the resulting strains revealed that introduction of an increasing number of PglaA copies resulted in lower expression of the uidA reporter gene and the endogenous glaA gene in cultures cultivated on different inducing carbon sources . However, repression by xylose was not influenced by the copy number of PglaA . These results indicate that the expression of genes under control of PglaA are regulated by specific trans-acting regulatory protein(s) . Deletion analysis of PglaA indicated that regulatory proteins interact with DNA sequences within 0.5-kb upstream from the ATG, whereas sequences between about 0.8- and 0.5-kb upstream from the ATG are required for high-level expression of glaA.

Gene, 1994 Aug 5, 145(2), 163 - 9
Non-cloning amplification of specific DNA fragments from whole genomic DNA digests using DNA 'indexers'; Unrau P et al.; A highly systematic, non-cloning method of distinguishing and isolating every fragment in a class-IIS or interrupted palindrome restriction digest has been developed in our laboratory . These enzymes produce informative, non-identical cohesive ends which can be selectively modified by ligation to individual synthetic oligodeoxyribonucleotides with the corresponding complementary ends . In this way, polymerase chain reaction and sequencing primer sites and labels can be introduced specifically into a single fragment in a total genomic digest . Known and unknown fragments from genomes of the complexity of Escherichia coli can be isolated directly in sequencable form without the necessity of synthesizing unique primers . Human DNA has also been assessed in this way . Problems intrinsic to cloning (selective fragment loss, mutation and sequence rearrangement) are avoided . Systematic characterization of DNA fragments by their cohesive ends and length provides tremendous power and flexibility for analysis of any DNA molecule without specific clones, probes or libraries . We report proof of principle of this remarkable system and indicate potential applications in DNA sequence tagged site and restriction mapping, sequencing, restriction-fragment-length polymorphism analysis and DNA diagnostics.

J Mol Biol, 1994 Aug 5, 241(1), 128 - 30
Crystallization of inhibitor complexes of an N-terminal 24 kDa fragment of the DNA gyrase B protein; Lewis RJ et al.; A 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein has been crystallized in the presence of novobiocin . One crystal form has been obtained that is orthorhombic, P2(1)2(1)2(1), with unit cell dimensions a = 40.3 A, b = 47.7 A, c = 111.9 A . The asymmetric unit of this crystal form contains one molecule (Vm = 2.24 A3/Da) . Complete native data have been collected to 2.5 A resolution . This same protein fragment has also been crystallized in the presence of GR122222X, an inhibitor that is structurally related to cyclothialidine . These crystals also exhibit P2(1)2(1)2(1) symmetry but have unit cell dimensions of a = 68.8 A, b = 68.6 A, c = 48.6 A . The Vm value of this crystal form is 2.39 A3/Da, assuming one molecule in the asymmetric unit, and native data have been collected to 2.0 A resolution . Molecular replacement studies of both complexes are underway.

J Biol Chem, 1994 Aug 5, 269(31), 20179 - 88
Expression and characterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-specific phospholipase C-delta 1; Yagisawa H et al.; It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-immobilized matrix was the delta 1 isoform of phosphatidylinositol-specific phospholipase C (PLC) . We expressed rat PLC-delta 1 in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P3 binding . The lysate was applied to Ins(1,4,5)P3-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100-kDa protein showed high Ins(1,4,5)P3 binding . The lysate was also purified to near homogeneity using a glutathione-Sepharose 4B affinity system . Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme . PLC-delta 1 consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme . Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P3 binding domain in the molecule . Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding . A 76-kDa proteolytic product of the expressed PLC-delta 1 which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P3 binding activity . A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-delta 1, which contains 6 basic amino acids, binds to an Ins(1,4,5)P3-immobilized matrix . Moreover, Ins(1,4,5)P3 binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate . These results, taken together, indicate that the amino-terminal domain of PLC-delta 1 is important for the binding of both Ins(1,4,5)P3 and phosphatidylinositol 4,5-bisphosphate.

J Biol Chem, 1994 Aug 5, 269(31), 20149 - 58
Characterization of KpsT, the ATP-binding component of the ABC-transporter involved with the export of capsular polysialic acid in Escherichia coli K1; Pavelka MS Jr et al.; The 17-kilobase kps gene cluster of Escherichia coli K1 contains all the information necessary for the expression of capsular polysaccharide . Region 3 of the cluster encodes two genes, kpsM and kpsT, whose products belong to the ATP-Binding Cassette (ABC)-transporter protein family . The KpsMT system is involved with the export of capsular polysaccharide in E . coli . Earlier work indicated that interaction between KpsT and ATP is important for transport . In this study, we report that KpsT, a peripheral inner membrane protein, can be photolabeled by the ATP analog, 8-N3{gamma-32P}ATP . The derivatization of KpsT by this reagent is inhibited by cold ATP or ATP gamma S . Furthermore, the protein seems to require a membrane environment for efficient photolabeling, but does not require any other kps gene products . Results obtained from saturation mutagenesis of the ATP-binding consensus sequence of KpsT and the phenotypes of strains with defined mutations in the chromosomal gene, are consistent with the view that ATP-binding by KpsT is required for transport of polymer across the inner membrane . The structure of KpsT was compared to a model developed for other ABC-transport proteins, and important functional regions were determined . The results obtained from chemical mutagenesis of kpsT are consistent with the model and revealed characteristics particular to capsule transporters.

J Biol Chem, 1994 Aug 5, 269(31), 20090 - 4
Sequence-specific cleavage of oligoribonucleotide capable of forming a stem and loop structure; Hosaka H et al.; The precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Sp1 RNA) has the capacity to cleave itself at specific positions ((UpA (137-138) and CpA (170-171)) within a possible loop and stem structure . This specific cleavage requires at least a monovalent cation and nonionic detergents . To confirm that the sequence-specific cleavage occurs autolytically, we studied the selective hydrolysis of RNA using a chemically synthesized 13-mer (GUUUCGUACAAAC) having sequences corresponding to G131-C143 of p2Sp1 RNA . The cleavage occurred at two identical sites (UpA and CpA) of a hairpin loop under physiological conditions and was affected by monovalent or divalent metals, nonionic detergent, salt, pH, and temperature . The hairpin loop domain and specific sequences are necessary for the cleavage of RNA 13-mer (GUUUCGUACAAAC).

J Biol Chem, 1994 Aug 5, 269(31), 19888 - 96
An amphipathic sequence determinant of membrane protein topology; Seligman L et al.; We developed a screen involving alkaline phosphatase gene fusions to identify mutations altering the membrane topology of a bacterial chemoreceptor (Escherichia coli Tsr) . We identified three informative classes of mutations causing increased export of the protein's normally cytoplasmic carboxyl-terminal domain . The first class consisted of deletions eliminating all or most of the membrane-spanning sequence (TM2) immediately amino-terminal to the cytoplasmic domain . The second class consisted of mutations altering a highly amphipathic sequence at the beginning of the domain . The third class of mutation was a deletion of an upstream spanning sequence (TM1) . The amphipathic sequence appears to be a novel determinant of membrane topology whose function is not due to its positive residue density . The amphipathic character of the sequence is relatively well-conserved in chemoreceptors and their relatives . Although deletions removing the amphipathic sequence or TM1 alone caused only partial carboxyl-terminal domain export, a double mutation removing both caused efficient export . This result suggests that the two sequences function independently to promote normal membrane insertion . The independent functioning of the two sequences may help ensure that Tsr insertion is normally a high fidelity process.

J Biol Chem, 1994 Aug 5, 269(31), 19803 - 9
The low molecular weight protein which co-purifies with alpha-latrotoxin is structurally related to crustacean hyperglycemic hormones; Gasparini S et al.; LMWP is the low molecular weight protein which copurifies with alpha-latrotoxin, the main neurotoxin from the black widow venom . It contains 70 residues and three disulfides . We found that its primary structure, including its 6 half-cystines, can be aligned with the amino acid sequences of crustacean hyperglycemic hormones (CHHs) which contain 72-73 residues and three disulfides . To further investigate this structural relationship, we produced a recombinant analog of LMWP in which the unique Met was changed in Leu (LMWPM35L) . LMWPM35L was produced as a folded fusion protein in the periplasm of Escherichia coli and was generated in vitro by treating the fusion protein with cyanogen bromide . We showed that LMWPM35L and CHHs have an identical disulfide pairing pattern and possess some alpha-helical structure, as deduced from a comparison of their circular dichroism spectra . In addition, LMWPM35L and CHHs are consensually predicted to possess a helical structure within the region 13-17 . Together, the data indicate that CHHs are structurally related to LMWPM35L and presumably also to LMWP . Finally, preliminary studies showed that LMWPM35L is not toxic to mice and does not form channels in lipid bilayers, two well-known properties of alpha-latrotoxin preparations.

J Biol Chem, 1994 Aug 5, 269(31), 19713 - 8
Molecular cloning and characterization of yeast rho GDP dissociation inhibitor; Masuda T et al.; We have previously isolated rho GDP dissociation inhibitor (rho DGI) from bovine brain and characterized it . Bovine rho GDI is a protein of a M(r) of 23,421 with 204 amino acids . rho GDI inhibits the GDP/GTP exchange reaction of post-translationally lipid-modified small GTP-binding proteins (G proteins) of the rho family, including the rho, rac, and cdc42 subfamilies, and keeps them in the GDP-bound inactive form . In the present study, we first purified rho GDI from the cytosol fraction of the yeast Saccharomyces cerevisiae and isolated its gene . Yeast rho GDI gene had an open reading frame without introns encoding a protein of a M(r) of 23,138 with 202 amino acids . Yeast rho GDI protein was 36% identical with bovine rho GDI . Yeast rho GDI expressed in Escherichia coli was active not only on yeast rho1 but also on mammalian rho family members which were post-translationally modified . Disruption of rho GDI did not induce apparent phenotypes, whereas overexpression of yeast or bovine rho GDI resulted in the inhibition of cell growth . These results indicate that rho GDI exists and regulates the function of the rho family members in yeast.

Science, 1994 Aug 5, 265(5173), 793 - 6
Discontinuous mechanism of transcription elongation; Nudler E et al.; During transcription elongation, three flexibly connected parts of RNA polymerase of Escherichia coli advance along the template so that the front-end domain is followed by the catalytic site which in turn is followed by the RNA product binding site . The advancing enzyme was found to maintain the same conformation throughout extended segments of the transcribed region . However, when the polymerase traveled across certain DNA sites that seemed to briefly anchor the front-end domain, cyclic shifting of the three parts, accompanied by buildup and relief of internal strain, was observed . Thus, elongation proceeded in alternating laps of monotonous and inchworm-like movement with the flexible RNA polymerase configuration being subject to direct sequence control.

J Chromatogr A, 1994 Aug 5, 676(2), 337 - 45
One-step affinity purification of bacterially produced proteins by means of the "Strep tag" and immobilized recombinant core streptavidin; Schmidt TG et al.; The "Strep tag" is a nine amino acid peptide with intrinsic streptavidin-binding activity . If this sequence is genetically fused to the C-terminus of a polypeptide the recombinant protein can be directly purified by affinity chromatography from the host cell extract on immobilized streptavidin . However, variations were observed in the suitability of different commercial streptavidin-agarose preparations for this purpose . Therefore, the influence of the source of streptavidin, the coupling chemistry, and the nature of the affinity chromatography resin was investigated . A procedure was developed for the production of recombinant core streptavidin in Escherichia coli, followed by its efficient refolding and purification with an overall yield of up to 140 mg functional protein per 11 bacterial culture . When coupled to activated CH-Sepharose 4B this truncated form of streptavidin showed a performance in the affinity chromatography of Strep tag fusion proteins that was superior to all other combinations tested . In contrast to its conventional preparation from Streptomyces strains the recombinant core streptavidin was produced without a proteolytic processing step . Thus, deleterious effects during the streptavidin affinity purification of proteins due to residual proteolytic activity in the immobilized streptavidin were avoided . The versatility of the optimized purification system was demonstrated with five different Strep tag fusion proteins.

Gene, 1994 Aug 5, 145(2), 261 - 5
The human DNA-binding protein, PO-GA, is homologous to the large subunit of mouse replication factor C: regulation by alternate 3' processing of mRNA; Lu Y et al.; We have previously cloned a human gene encoding a 128-kDa protein which we termed PO-GA {Lu et al., Biochem . Biophys . Res . Commun . 193 (1993) 779-786} . In the present report, we compared PO-GA to recent DNA database entries and determined that PO-GA was 80% identical, at the amino-acid level, to the large subunit of replication factor C (activator 1) cloned from mouse {Burbelo et al., Proc . Natl . Acad . Sci . USA 91 (1994) in press} . This indicates that PO-GA probably represents the corresponding subunit of human replication factor C . In addition, PO-GA has high homology to a putative Drosophila transcription factor . All three proteins contain a nuclear translocation signal and an ATP/ADP-binding motif, and are highly conserved in regions with homology to Escherichia coli and yeast DNA ligases . We determined that PO-GA mRNA species of 5.3 and 4.5 kb can be detected in most human tissues, but levels are especially high in ovary . Analysis of the sequence of a new PO-GA cDNA clone that we obtained reveals a previously undetected 650-bp 3'-UTR extension . This region contains several A+U-rich regions potentially involved in regulation of mRNA stability . This fragment only hybridizes to the larger 5.3-kb mRNA . Comparison of cDNA sequences revealed that the two mRNA species arise as a result of alternate use of poly(A)-addition sites . Since the ratio of the two mRNA species is variable in different tissues, we speculate that alternative 3' processing of the PO-GA mRNA is utilized as a mechanism of regulating cellular levels of mRNA.

J Biol Chem, 1994 Aug 5, 269(31), 19766 - 76
Identification of a chaperonin binding site in a chloroplast precursor protein; Dessauer CW et al.; Although chaperonin-assisted protein folding has been studied in vitro by a number of investigators, the feature(s) of the unfolded polypeptide that are recognized and bound by chaperonins is not known . We have addressed this question using the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pS) as a substrate for GroEL . The protein was expressed in Escherichia coli as a C-terminal fusion to protein A . Protein A-pS and any associated cellular proteins were then purified by affinity chromatography . GroEL could be eluted from the fusion protein by incubation with ATP and either GroES or casein, consistent with results of in vitro folding assays . At least half of the transit sequence of pS is required to maintain this high stoichiometry of chaperonin binding . Using deletion mutagenesis from the C terminus of pS, we defined the smallest truncation of pS, PAxpS90T, that binds GroEL with high avidity (dissociation constant = 53 nM) . A series of site-specific mutations targeting the C-terminal 15-20 amino acids of PAxpS90T was constructed and analyzed for the ability to bind GroEL . Our results show that complex formation between GroEL and pS is not dependent upon any single feature such as overall hydrophobicity, a net positive charge, or secondary structure but may be dependent upon a combination of these features.

Gene, 1994 Aug 5, 145(2), 231 - 5
Characterization of a Y-Box factor from Aplysia californica; Skehel PA et al.; A cDNA isolated from the marine invertebrate Aplysia californica encodes a protein containing a domain with a high degree of homology to the Y-Box-binding factors . The expression of this gene is unaffected by the facilitatory neurotransmitter, 5-hydroxytryptamine . When expressed in Escherichia coli, the encoded protein is shown to bind RNA in vitro.

J Mol Biol, 1994 Aug 5, 241(1), 1 - 6
Characterization of the Borrelia burgdorferi RNase P RNA gene reveals a novel tertiary interaction; Mattsson JG et al.; Characterization of the RNase P RNA gene derived from Borrelia burgdorferi reveals covariation of the conserved nucleotides at positions corresponding to nucleotides 128 and 230 in Escherichia coli RNase P RNA (M1 RNA) . Single base substitutions at either of these positions in M1 RNA resulted in a lack of complementation of the temperature-sensitive phenotype associated with rnpA49 in vivo whereas complementation was observed for the double mutant M1 RNA or wild-type M1 RNA . Our in vitro data showed that M1 RNA harbouring a substitution at 128 or 230 cleaved a tRNA precursor both in the absence and presence of C5 with reduced efficiency compared to the wild-type and the double mutant M1 RNA . We conclude that the nucleotides at positions 128 and 230 establish a long-range tertiary interaction in RNase P RNA . Our data also suggest that this interaction together with the identity of the nucleotide at position 230 is important for Pb2+ induced cleavage at specific positions in M1 RNA.

Biochim Biophys Acta, 1994 Aug 4, 1213(3), 309 - 18
Characterization of mouse phosphatidylinositol transfer protein expressed in Escherichia coli; Geijtenbeek TB et al.; The cDNA encoding mouse phosphatidylinositol transfer protein (PI-TP) was isolated by means of reverse transcriptase polymerase chain reaction . The nucleotide sequence of this cDNA has a high similarity (98%) with that of rat PI-TP; the predicted amino acid sequence is 99.6% identical to that of rat PI-TP . The cDNA encoding mouse PI-TP was cloned into the expression vector pET3d and the Escherichia coli strain BL21(DE3) was transformed with the resulting plasmid . After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside, PI-TP was efficiently expressed in the E . coli strain . It was estimated that 5% of the total soluble cell protein consisted of PI-TP . The recombinant mouse PI-TP was purified from the bacterial lysate in four steps: ammonium sulphate precipitation, anion-exchange chromatography, heparin-Sepharose affinity and gel filtration chromatography . Fractionation on the heparin-Sepharose affinity column yielded two forms: PI-TP Hepa1 and Hepa2 . These two proteins have the same molecular mass of 35 kDa, both contain a phosphatidylglycerol molecule and both are recognized by anti-PI-TP antibody . Both recombinant proteins have an isoelectric point of 5.4 as compared to 5.5 for bovine brain PI-TP . Sequence analysis of the first 25 N-terminal amino acid residues showed that both forms are identical, except that PI-TP Hepa1 contains the initiator methionine which is lacking from PI-TP Hepa2 . The two PI-TP forms have similar phospholipid-binding and transfer activity, comparable to that of bovine brain PI-TP . Both forms and bovine brain PI-TP are phosphorylated equally well in a Ca2+/phospholipid-dependent way by protein kinase C.

Eur J Pharmacol, 1994 Aug 3, 270(4), 291 - 300
Parainfluenza virus type 3 induced alterations in tachykinin NK1 receptors, substance P levels and respiratory functions in guinea pig airways; Kudlacz EM et al.; We have investigated the effects of parainfluenza virus type 3 (PI-3) on sensory neuropeptide levels, tachykinin receptors and their functions in guinea pig airways during the course of respiratory viral infection . PI-3 infected guinea pigs were hyperresponsive to methacholine and substance P aerosols as determined by earlier onset of dyspnea in these animals as compared with control on post-inoculation day (PID) 7 but not 19 . In addition, plasma protein extravasation produced in response to the tachykinin was increased in infected airways during the first week post inoculation . Infected guinea pig trachea did not respond any differently to methacholine when smooth muscle contraction and {3H}inositol phosphate accumulation were measured although the magnitude of substance P effects using in vitro tests was significantly greater than control on post-inoculation day 7 but not 19 . Trachea from PI-3 infected animals were characterized by reductions in substance P-like immunoreactivity, tachykinin NK1 receptor number and agonist affinity during the first post-inoculation week . Substance P levels or tachykinin NK1 receptor numbers or affinity were not altered in trachea of guinea pigs 4 days after treatment with lipopolysaccharide . These data suggest substance P release occurs during critical periods of respiratory viral infection which are temporally correlated with airway hyperresponsiveness . Despite apparent down-regulation of tachykinin NK1 receptors, substance P-mediated functions remained enhanced suggesting some alterations in post-receptor mechanisms.

Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7752 - 6
Mutagenic replication in human cell extracts of DNA containing site-specific N-2-acetylaminofluorene adducts; Thomas DC et al.; We have analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of bidirectional replication of double-stranded DNA in a human cell extract . Plasmid vectors were constructed containing the simian virus 40 origin of replication and single AAF adducts at one of three guanines in the Nar I sequence GGCGCC in a lacZ reporter gene . The presence of an AAF adduct diminishes replication efficiency in HeLa cell extracts by 70-80% . Replication product analyses reveal unique termination sites with each damaged vector, suggesting that when the replication fork encounters an AAF adduct, it often stops before incorporation opposite the adduct . We also observed a higher proportion of products representing replication of the undamaged strand compared to the damaged strand . This suggests that the undamaged strand is replicated more readily, either by uncoupling the first fork to encounter the lesion or by replication using the fork arriving from the other direction . Also included among replication products are covalently closed monomer-length molecules resistant to cleavage at the AAF-modified Nar I site . This resistance is characteristic of substrates containing the AAF adduct, suggesting that translesion bypass had occurred . Transformation of Escherichia coli cells with the replicated damaged DNA yielded lacZ alpha revertant frequencies significantly above values obtained with undamaged DNA or with damaged DNA not replicated in vitro . This increase was only seen with the substrate modified at the third guanine position . Analysis of mutant DNA demonstrated the loss of a GC dinucleotide at the Nar I sequence . Generation of this position-dependent AAF-induced frameshift error in a human replication system is consistent with previous observations in E . coli suggesting that, after incorporation of dCMP opposite modified guanine in the third position, realignment of the template-primer occurs to form an intermediate with two unpaired nucleotides in the template strand.

Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7618 - 22
The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA; Stasiak A et al.; The RuvB protein is induced in Escherichia coli as part of the SOS response to DNA damage . It is required for genetic recombination and the postreplication repair of DNA . In vitro, the RuvB protein promotes the branch migration of Holliday junctions and has a DNA helicase activity in reactions that require ATP hydrolysis . We have used electron microscopy, image analysis, and three-dimensional reconstruction to show that the RuvB protein, in the presence of ATP, forms a dodecamer on double-stranded DNA in which two stacked hexameric rings encircle the DNA and are oriented in opposite directions with D6 symmetry . Although helicases are ubiquitous and essential for many aspects of DNA repair, replication, and transcription, three-dimensional reconstruction of a helicase has not yet been reported, to our knowledge . The structural arrangement that is seen may be common to other helicases, such as the simian virus 40 large tumor antigen.

Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7613 - 7
A structural model for fidelity in transcription; Eichhorn GL et al.; Distances between the metal ions bound to the product terminus i site and the substrate i + 1 site of Escherichia coli RNA polymerase range from 5.0 to 5.6 A when the substrate is complementary to a template base and from 6.5 to 7.0 A for a noncomplementary relationship . The metal bound to the substrate at the i + 1 site exhibits a constant distance to the three phosphates on the substrate regardless of complementarity, but the distance to base and ribose protons changes . The differences in these geometric parameters are explained by the ability of the enzyme to assume two conformations, one to place correct nucleotide substrates in optimal position for bond formation and the other to prevent incorrect nucleotides from assuming such a position . In this scheme a metal-triphosphate complex can move toward or away from the terminal 3' OH group of the growing RNA chain, to assure fidelity of transcription.

Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7468 - 72
Extrachromosomal elements in tobacco plastids; Staub JM et al.; The plastid genome of higher plants is a circular double-stranded DNA molecule which is present in multiple identical copies . We report here an 868-bp plastid DNA minicircle, NICE1, that formed in tobacco (Nicotiana tabacum) plastids during transformation, as an unexpected product of homologous recombination . Such extrachromosomal elements are normally absent in plastids of higher plants . We have constructed shuttle plasmids containing NICE1 sequences which are maintained extrachromosomally when reintroduced into plastids by particle bombardment . Furthermore, recombination between homologous sequences in the shuttle plasmids and the main plastid genome occurs . Recombination products were characterized after recovery of the shuttle plasmids in Escherichia coli and of recombinant plastid genomes in the progeny of transformed plants . Our findings indicate that shuttle plasmids can be used to engineer plastid genes without concomitant integration of foreign DNA and to recover plastid mutations in E . coli.

Biochim Biophys Acta, 1994 Aug 2, 1218(3), 481 - 4
tRNA regions which contact with the ribosomal poly(U)-programmed P-site; Nekhai SA et al.; Equilibrium binding affinity of yeast tRNA(Phe) for Escherichia coli poly(U)-programmed 70S ribosomal P-site was compared with corresponding affinities of several tRNA(Phe) 3'- and 5'-end-truncated derivatives, all containing the anticodon arm . Our findings strongly suggest that besides three 3'-terminal-CCA nucleotides (C74, C75 and A76), only the tRNA(Phe) anticodon arm (N28-N42) contains ribosomal P-site contact centers and that there are no such centers in the intermediate regions N1-N27 and N43-N73.

Biochemistry, 1994 Aug 2, 33(30), 9024 - 31
Mutation of tryptophan 128 in T4 endonuclease V does not affect glycosylase or abasic site lyase activity; Latham KA et al.; Mutation of various residues within the carboxy-terminal 11 amino acids of endonuclease V, an enzyme made up of 138 amino acids that initiates the repair of cyclobutane pyrimidine dimers in DNA, has demonstrated the importance of this region in dimer-specific binding . In a previous study, substitution of a serine residue for tryptophan 128 resulted in a protein with decreased abasic site lyase activity without a concomitant decrease in DNA glycosylase activity {Nakabeppu, Y., et al . (1982) J . Biol . Chem . 257, 2556-2562} . To assess the importance of the tryptophan at position 128, six mutants were constructed by site-directed mutagenesis, including W128Y, W128V, W128I, W128G, W128S, and W128T . Upon characterization, these six mutants were found qualitatively to complement the repair deficiency of ultraviolet (UV) light irradiated Escherichia coli cells (recA-, uvrA-) to levels comparable to that of wild-type endonuclease V . The activities of the mutant proteins were characterized using UV-irradiated plasmid DNA and oligonucleotides containing either a site-specific cyclobutane pyrimidine dimer or an abasic site . In all cases, the six mutants displayed glycosylase and abasic site lyase activities comparable to those of wild-type endonuclease V, indicating that Trp-128 is not crucial for dimer-specific binding or catalysis.

Biochemistry, 1994 Aug 2, 33(30), 8905 - 11
Nucleoside modifications stabilize Mg2+ binding in Escherichia coli tRNA(Val): an imino proton NMR investigation; Yue D et al.; The structures of in vitro transcribed Escherichia coli tRNA(Val), which lacks base modifications, and the native tRNA, which contains them, are very similar in the presence of excess Mg2+ (Kintanar, Yue, and Horowitz, unpublished results) . To further probe the effects of base modifications on the structure of tRNA, the Mg2+ ion dependence of the downfield region of the 1H NMR spectrum of in vitro transcribed E . coli tRNA(Val) in aqueous phosphate buffer was investigated . The spectra indicate a remarkable conformational change in unmodified E . coli tRNA(Val) coincident with binding or release of Mg2+ . Assignment of the imino proton resonances in the low Mg2+ form of the tRNA transcript allows a detailed description of the conformational change . There is near total disruption of the D stem and tertiary interactions in the absence of bound Mg2+ . A new strong interaction between the U67-A6 base pair and the G50-U64 wobble pair is observed, indicating a substantial structural rearrangement at the junction of the acceptor and T stems . The binding constants of the strong Mg2+ binding sites in the D loop and near the D stem in unmodified tRNA(Val) are at least 2 orders of magnitude less than in tRNAVal containing base modifications . The metal ion binding site in the anticodon loop is somewhat stronger than metal ion binding sites in the D loop and stem in unmodified tRNA(Val), but it is still weaker than all strong Mg2+ binding sites in native tRNA(Val) . Thus, one role of the base modifications found in tRNA is to stabilize or strengthen the Mg2+ binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Aug 2, 33(30), 8962 - 8
In vitro reconstitution of the 24-meric E2 inner core of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex: requirement for chaperonins GroEL and GroES; Wynn RM et al.; We have investigated the in vitro reconstitution of the 24-meric inner core domain (E2c) of the transacylase (E2) component of bovine branched-chain alpha-keto acid dehydrogenase complex . The yield of recombinant E2c (amino acid residues 161-421 of bovine E2) expressed in Escherichia coli was markedly increased by fusing the bacterial maltose-binding protein (MBP) to the amino terminus of bovine E2c . Following factor Xa digestion to remove the MBP moiety, E2c was completely unfolded in 4.5 M guanidine HCl (Gdn.HCl) . The denatured E2c monomers (apparent M(r) = 27,000) were diluted 100-fold at 25 degrees C into a refolding buffer containing 5 mM Mg-ATP and a 4-fold molar excess of chaperonins GroEL and GroES . Full E2 activity was recovered in 45 min . Omission of the chaperonins in the refolding buffer failed to recover any E2 activity . Recovery of E2 activity obeyed hyperbolic kinetics as a function of the chaperonin-to-E2c molar ratio and showed a requirement for hydrolysis of Mg-ATP . A stable GroEL-E2c complex was isolated which, in the presence of GroES and Mg-ATP, generated active E2c 24-mers . Dissociation of recombinant E2c 24-mers into active trimers was achieved by incubation in 1.5 M Gdn.HCl at 25 degrees C . The E2c trimers with an apparent M(r) of 84,000 were isolated by sucrose density gradient centrifugation in the presence of the chaotropic reagent . Removal of 1.5 M Gdn.HCl resulted in the spontaneous reassembly of trimers into the native 24-mer structure independent of chaperonins.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Biochem, 1994 Aug, 26(8), 991 - 1001
Post-translational and transcriptional regulation of polyamine biosynthesis in Escherichia coli; Panagiotidis CA et al.; Ornithine and arginine decarboxylases (ODC and ADC) of Escherichia coli are inhibited post-translationally by antizyme and ribosomal proteins S20 and L34 . The inhibition of either enzyme is relieved when excess of the other decarboxylase is added . Using this approach, in vitro as well as in vivo, we demonstrate that the extent of the post-translational inhibition of ODC and ADC in E . coli is at least 65 and 50%, respectively . The inhibited enzyme levels increase even further upon exposure of cells to polyamines . The post-translational mode of regulation can counteract a 4-fold increase of ODC protein in the cell . The negative transcriptional regulation of ODC and ADC expression by polyamines is mediated by transcription factors and not by direct polyamine effects on the promoters of their genes . Three proteins interacting with the ODC promoter region were found by southwestern blot analysis.

FEMS Microbiol Lett, 1994 Aug 1, 121(1), 71 - 6
Variability of peptidoglycan surface density in Escherichia coli; Caparros M et al.; Murein synthesis in Escherichia coli can be partially inhibited by D-methionine without concomitant alterations in growth and morphology . D-Methionine-treated cultures grow steadily for an indefinite time, therefore murein surface density should be reduced . Determination of this parameter in control and D-methionine-treated cells showed a severe reduction in the latter . Murein surface density increases drastically in resting cells, irrespective of the presence of D-methionine . Mutants in ponB are hypersensitive to D-methionine . Analysis of ponB strains revealed an important reduction in murein surface density . An approximately two-fold reduction in average surface density is apparently compatible with normal growth and division.

FEMS Microbiol Lett, 1994 Aug 1, 121(1), 39 - 46
Acyl-coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum: effect of amino acid substitutions at Ser227, Ser230 and Ser309 on proenzyme cleavage and activity; Tobin MB et al.; Using a high level Escherichia coli expression system for the Penicillium chrysogenum penDE gene, we have produced acyl-coenzyme A: isopenicillin N acyltransferase (AT) enzymes containing amino acid substitutions at three conserved Ser residues . Chosen for study based on amino acid sequence homologies to other proteins, Ser227, Ser230 and Ser309 were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism . Substitutions at Ser230 had no effect on proenzyme cleavage, acyl-coenzyme A: IPN acyltransferase (IAT) or acyl-coenzyme A:6-aminopenicillanic acid acyltransferase (AAT) activities . While Ser227-->Cys had no effect, Ser227-->Ala produced uncleaved proenzyme lacking both AAT and IAT activities, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity . Substitution of Ser309-->Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity . Recombinant AT (recAT) proenzyme containing Ser309-->Ala was cleaved; however, IAT and AAT activities were not observed . This separation of proenzyme cleavage from IAT and AAT activities has not been previously observed, and suggests that Ser309 is involved in substrate acylation.

Mol Pharmacol, 1994 Aug, 46(2), 338 - 45
Site-directed mutagenesis of putative substrate recognition sites in cytochrome P450 2B11: importance of amino acid residues 114, 290, and 363 for substrate specificity; Hasler JA et al.; Eleven amino acid residues unique to dog cytochrome P450 (P450) 2B11, compared with rat 2B1 and 2B2, rabbit 2B4 and 2B5, and mouse 2B10, in the putative substrate recognition sites {J . Biol . Chem . 267:83-90 (1992)} were mutated to the residues found in 2B1 or 2B5 . The mutants were expressed initially in COS cells and screened for activity toward androstenedione and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) . P450 2B11 mutants V107I, M199L-N200E-V204R, V234I, A292L, Q473R, and I475S showed no differences from wild-type P450 2B11 in metabolite profiles with either substrate . Mutants V114I, D290I, and L363V exhibited altered androstenedione metabolite profiles and were expressed in Escherichia coli for further study with androstenedione, testosterone, 7-ethoxycoumarin, (R)- and (S)-warfarin, and 245-HCB . With V114I, hydroxylation of steroids and warfarin and 2-hydroxylation of 245-HCB were decreased, whereas 7-ethoxycoumarin O-dealkylation and 3-hydroxylation of 245-HCB were unaltered . For D290I, activities toward all substrates were decreased, except for 16 beta-hydroxylation of testosterone . The activity of L363V was increased 5-6-fold for 16 alpha-hydroxylation of androstenedione and testosterone but was decreased to 40-50% of wild-type activity with 7-ethoxycoumarin and warfarin and to 6-8% of control for 2-hydroxylation of 245-HCB . Alignment of P450 2B11 with P450 101 and super-imposition of the 11 mutated 2B11 residues on a P450 101 three-dimensional model suggest that only residues 114, 290, and 363 represent substrate contact residues, in excellent agreement with the experimental results . The data indicate the importance of the three residues 114, 290, and 363 in substrate specificity and regio- and stereoselectivity of P450 2B11 and also demonstrate that the effects of the mutations vary considerably with different substrates.

Plant Mol Biol, 1994 Aug, 25(5), 865 - 76
Sequencing and characterization of the kinesin-related genes katB and katC of Arabidopsis thaliana; Mitsui H et al.; Complementary DNAs of two kinesin-related genes, katB and katC, were isolated from Arabidopsis thaliana and sequenced . The carboxyl-terminal regions of the polypeptides encoded by these genes, especially the presumptive ATP-binding and microtubule-binding domains, share significant sequence homology with the mechanochemical motor domain of the kinesin heavy chain . The predicted secondary structures of KatB and KatC proteins include a large globular domain in the carboxyl-terminal region and a small globular domain in the amino-terminal region that are separated by a long alpha-helical coiled-coil with heptad repeats . A truncated KatC polypeptide (KatC(207-754)), which includes the carboxyl-terminal region of KatC, was expressed in Escherichia coli and was shown to possess microtubule-stimulated ATPase activity and to bind to microtubules in an ATP-sensitive manner, both of which are characteristics of kinesin and kinesin-like proteins.

J Trauma, 1994 Aug, 37(2), 249 - 54
Pulmonary reaction during intramedullary fracture management in traumatic shock: an experimental study; Wozasek GE et al.; Immediate nailing of shaft fractures in severely injured patients causes fat embolization . This method therefore is considered potentially dangerous, since fat intravasation in association with multiple trauma and subsequent endotoxemia might lead to pulmonary dysfunction . We therefore studied the pathophysiologic events of intramedullary nailing in the lungs of sheep with chronic instrumentation including lung lymph fistula . In the 7 animals in group I closed nailing of the intact tibia and femur was performed . Group II (n = 7) animals sustained hypovolemic shock and retransfusion prior to nailing, while group III (n = 11) animals were treated like those in group II and further challenged on the following two days with endotoxin . Group III was compared with group IV (n = 6), in which endotoxin was given only once without additional trauma . Nailing in group I led to a significant increase of the MPAP from 10.8 to 13.8 mm Hg postoperatively (p < 0.05), but no increase in lung permeability . Only additional hypovolemia, retransfusion and nailing as performed in groups II and III showed significant increase of the lymph flow (QI) from 4.4 mL/h to 12.4 mL/h and the protein clearance (Pclear) from 3 to 6.3 . A significant difference of the pulmonary permeability between group I and II was only observed postoperatively . There was no difference in the lung response between group III and IV . This ovine study corroborates that although nailing causes a moderate increase in pulmonary pressure, it does not lead to increased lung permeability . Only additional hemorrhagic shock, even when adequately resuscitated, leads to lung disturbance postoperatively . The subsequent endotoxin challenge does not aggravate lung injury.

EMBO J, 1994 Aug 1, 13(15), 3423 - 9
Trimerization and crystallization of reconstituted light-harvesting chlorophyll a/b complex; Hobe S et al.; The major light-harvesting complex (LHCII) of photosystem II, the most abundant chlorophyll-containing complex in higher plants, is organized in trimers . In this paper we show that the trimerization of LHCII occurs spontaneously and is dependent on the presence of lipids . LHCII monomers were reconstituted from the purified apoprotein (LHCP), overexpressed in Escherichia coli, and pigments, purified from chloroplast membranes . These synthetic LHCII monomers trimerize in vitro in the presence of a lipid fraction isolated from pea thylakoids . The reconstituted LHCII trimers are very similar to native LHCII trimers in that they are stable in the presence of mild detergents and can be isolated by partially denaturing gel electrophoresis or by centrifugation in sucrose density gradients . Moreover, both native and reconstituted LHCII trimers exhibit signals in circular dichroism in the visible range that are not seen in native or reconstituted LHCII monomers, indicating that trimer formation either establishes additional pigment-pigment interactions or alters pre-existing interactions . Reconstituted LHCII trimers readily form two-dimensional crystals that appear to be identical to crystals of the native complex.

Eur J Biochem, 1994 Aug 1, 223(3), 823 - 30
Determination of common structural features in Escherichia coli promoters by computer analysis; Lisser S et al.; Escherichia coli promoters show a large degree of sequence variation . However, they are all recognized specifically by RNA polymerase as the sites for transcription initiation, suggesting that they share common basic structural features distinguishing them from the rest of the sequence . Our hypothesis is that the promoter is determined not only by the two consensus sequences at -10 and -35, but also by the surrounding nucleotides, and that it is not only the identity of the nucleotides that is important for promoter function but the presence of specific physical-chemical and structural characteristics that are sequence dependent . This approach is supported by accumulating evidence indicating the role that the DNA conformation may play in modulating protein-DNA interaction . In this study, four intrinsic sequence-dependent characteristics are examined in E . coli promoter regions: helix stability, helix flexibility, and two conformational parameters represented by the DNA tendencies for B-->Z and B-->A transition . The promoter is defined by the consensus sequences and their vicinity and the examined properties are compared between promoter and random sequences . It is demonstrated that both the consensus and flanking regions are less stable, more flexible and show a higher tendency for the B conformation in comparison to random sequences . Discriminant analysis is used to evaluate the relative contributions of the various characteristics.

Eur J Biochem, 1994 Aug 1, 223(3), 799 - 803
Cross-linking of nucleic acids to proteins . Modified poly(A) as mRNA for Escherichia coli ribosomes; Wojtech E et al.; Poly(adenylic acid) was modified by methylchlorotetrolic ester in a reproducible and defined content of the derivatized bases . The nucleic acid derivative is protein reactive and was coupled to 70S ribosomes from Escherichia coli, in order to identify proteins along the mRNA pathway . The binding of the label becomes specific under the direction of tRNA(Lys) and is then almost exclusively located on the small subunit . The proteins S1, S12, S18 and S21 were labeled, as shown by an antibody assay . The yield of the affinity label was 5.4%, as calculated from the labeled nucleic acid . This compares favourably with the yields from photolabile compounds.

Eur J Biochem, 1994 Aug 1, 223(3), 1069 - 77
Expression, purification and crystallization of human phosphotyrosine phosphatase 1B; Hoppe E et al.; Protein phosphotyrosine phosphatases are believed to be involved in the regulation of the activity of cellular proteins, such as receptor tyrosine kinases, by controlling their phosphorylation status . One of the best described and characterized protein of this class of enzymes is the phosphotyrosine phosphatase 1B . To obtain sufficient quantities for structural investigations, truncated forms of PTP1B encompassing the catalytic domain were over-expressed in Escherichia coli and purified to apparent homogeneity by conventional chromatography . The activity of these purified enzymes has been compared with the wild-type enzyme expressed in mammalian cells . By measuring the activities against p-nitrophenyl phosphate, the pH dependence of this activity, and responses to different modulators, it could be demonstrated that the truncated forms of PTP1B retained the same characteristics as the full-length mammalian enzyme, but are not subject to inhibition of enzymic activity mediated by the C-terminus . Due to their improved solubility, it can be assumed that the catalytic domains are advantageous for crystallization studies in comparison to the natural enzyme . In a screening for crystallization conditions, we obtained protein crystals indicating that the quality of the purified protein is sufficient for crystallographic studies.

Carcinogenesis, 1994 Aug, 15(8), 1657 - 62
Detection of DNA point mutations with DNA mismatch repair enzymes; Hsu IC et al.; We have developed a simple and reliable procedure to screen gene mutations using DNA mismatch repair (MR) specific mut Y enzyme of Escherichia coli and thymidine DNA glycosylase from HeLa cells . The mut Y enzyme cleaves A of G/A mismatches in DNA duplex and thymidine glycosylase cleaves T at G/T mismatches . Previously, we showed the determination of G:C-->T:A mutations in the N-ras gene in two human tumor samples with mut Y G/A MR enzyme . As low as 1-2% mutant DNAs in a sample of mutant and wild-type DNA can be detected with a synthetic DNA to create G/A mispairing for the assay . In this paper, we simplify the assay, include G/T MR thymidine glycosylase from HeLa cells and evaluate the application for screening DNA point mutations of p53 and ras genes . In this method, DNA fragments amplified from normal and mutated genes by polymerase chain reaction (PCR) were mixed and annealed to create DNA mismatches for cleavage by mismatch repair enzymes . The cleaved products and the substrates were separated by gel electrophoresis and detected by autoradiography . In theory, the enzymes that cut G/A or G/T mispairs will detect the mutations of G:C-->A:T, A:T-->G:C, G:C-->T:A and T:A-->G:C . Several human tumor samples were examined for p53 or K-ras mutations with G/A and G/T mismatch repair enzymes . The reliability of mutation detection was evaluated by comparing the results with reported mutations or confirmed by DNA sequencing of the same PCR-amplified DNA fragments . Our data showed that, following mismatch repair enzyme cleavage, all mutated DNA samples yielded cleaved products with sizes as expected . In addition, our assay is able to characterize the nature of mutation by 5' end-labeling of 32P on mutant or wild-type DNA fragments . The low background, reliability and the determination of the sites of mutations as well as the types of DNA base changes indicate the advantages of the method over other techniques in testing DNA mutants.

Ann Surg, 1994 Aug, 220(2), 155 - 63
Does the route of feeding modify the inflammatory response?
Santos AA, Rodrick ML, Jacobs DO, Dinarello CA, Wolff SM, Mannick JA, Wilmore DW.
OBJECTIVE: The authors compared the responses to endotoxin in enterally and parenterally fed human volunteers . BACKGROUND: Recent investigations have reported that the response to endotoxin in humans is greater in individuals who receive parenteral nutrition rather than enteral feeding . It was proposed that this difference was related to gut barrier dysfunction during intravenous nutrition . To evaluate this hypothesis, the authors analyzed the responses of human subjects to an intravenously administered bolus of endotoxin after enteral or parenteral nutrition . METHODS: Fifteen randomly selected healthy volunteers were studied during two separate investigations; ten studies were performed in ten subjects who received enteral nutrition, and nine studies were carried out in five additional subjects who received parenteral nutrition . After 2 days of enteral feedings or 7 days of parenteral feedings, endotoxin was administered by intravenous injection; temperature, symptom score, and duration then were measured serially . Blood samples were obtained for leukocyte and platelet count, and plasma concentrations of corticotrophin, cortisol, epinephrine, norepinephrine, tumor necrosis factor, and interleukin-6 . Mononuclear cell response to phytohemagglutinin was determined at 0, 4, and 24 hours . RESULTS: In the parenteral group, a diminished response was observed in platelet count and plasma interleukin-6 levels compared with volunteers who received enteral nutrition . The duration of symptoms tended to be reduced in the parenterally fed group, although this did not achieve significance . Other responses were not significantly different between the two groups . CONCLUSION: The responses to endotoxin in human subjects who received parenteral nutrition were similar compared with subjects who received enteral nutrition, although platelet count and plasma interleukin-6 concentration were diminished.

Am J Gastroenterol, 1994 Aug, 89(8), 1142 - 6
Endoscopic management of cholangitis: critical review of an alternative technique and report of a large series; Siegel JH et al.; OBJECTIVE: To assess the outcome of endoscopic techniques as the solitary treatment modality for the complete management of ascending, bacterial cholangitis, compared with results of radiological and surgical methods as historical controls . METHODS: Endoscopic techniques were used to decompress bile ducts obstructed by stones (898 patients) or stenosis (49 patients) . Endoscopic sphincterotomy (ES) was performed in 839 patients, and either 7-Fr straight stents (79), or nasobiliary tubes (29), were utilized as initial therapy in 108 patients . Of these latter patients, 68 subsequently underwent ES and stone removal, 17 had ES, lithotripsy, and stone removal, 18 were left with stents in place, and 5 were lost to follow-up . Follow-up was conducted by direct patient contact, by telephone, or through the referring physicians . RESULTS: All patients were managed by endoscopic techniques . There were four deaths (0.42%) in the first 30 days (none before 2 wk); no deaths were related to the procedures but were attributed to intercurrent medical problems . Two patients underwent surgery: one pancreatitis, one perforation . Complications were infrequent, occurring in 6% of patients . Bleeding occurred in 3%, pancreatitis in 2.8%, and perforation 0.2% . CONCLUSIONS: Endoscopic management of cholangitis is as effective as surgical or radiological methods for managing bacterial cholangitis, a potentially fatal syndrome, but ERCP and ES have been shown to be safer . Endoscopy is the preferred index technique both for establishing a definitive diagnosis and providing therapy.

J Cell Biol, 1994 Aug, 126(4), 945 - 54
N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion; Whiteheart SW et al.; The NEM-sensitive fusion protein, NSF, together with SNAPs (soluble NSF attachment proteins) and the SNAREs (SNAP receptors), is thought to be generally used for the fusion of transport vesicles to their target membranes . NSF is a homotrimer whose polypeptide subunits are made up of three distinct domains: an amino-terminal domain (N) and two homologous ATP-binding domains (D1 and D2) . Mutants of NSF were produced in which either the order or composition of the three domains were altered . These mutants could not support intra-Golgi transport, but they indicated that the D2 domain was required for trimerization of the NSF subunits . Mutations of the first ATP-binding site that affected either the binding (K266A) or hydrolysis (E329Q) of ATP completely eliminated NSF activity . The hydrolysis mutant was an effective, reversible inhibitor of Golgi transport with an IC50 of 125 ng/50 microliters assay . Mutants in the second ATP-binding site (binding, K549A; hydrolysis, D604Q) had either 14 or 42% the specific activity of the wild-type protein, respectively . Using coexpression of an inactive mutant with wild-type subunits, it was possible to produce a recombinant form of trimeric NSF that contained a mixture of subunits . The mixed NSF trimers were inactive, even when only one mutant subunit was present, suggesting that NSF action requires each of the three subunits in a concerted mechanism . These studies demonstrate that the ability of the D1 domain to hydrolyze ATP is required for NSF activity and, therefore is required for membrane fusion . The D2 domain is required for trimerization, but its ability to hydrolyze ATP is not absolutely required for NSF function.

J Cell Biol, 1994 Aug, 126(4), 853 - 62
Yeast NPI46 encodes a novel prolyl cis-trans isomerase that is located in the nucleolus; Shan X et al.; We have identified a gene (NPI46) encoding a new prolyl cis-trans isomerase within the nucleolus of the yeast Saccharomyces cerevisiae . The protein encoded by NPI46 was originally found by us in a search for proteins that recognize nuclear localization sequences (NLSs) in vitro . Thus, NPI46 binds to affinity columns that contain a wild-type histone H2B NLS but not a mutant H2B NLS that is incompetent for nuclear localization in vivo . NPI46 has two domains, a highly charged NH2 terminus similar to two other mammalian nucleolar proteins, nucleolin and Nopp140, and a COOH terminus with 45% homology to a family of mammalian and yeast proline isomerases . NPI46 is capable of catalyzing the prolyl cis-trans isomerization of two small synthetic peptides, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, as measured by a chymotrypsin-coupled spectrophotometric assay . By indirect immunofluorescence we have shown that NPI46 is a nucleolar protein . NPI46 is not essential for cell viability.

J Bacteriol, 1994 Aug, 176(16), 5163 - 6
Mutations in the Bordetella pertussis bvgS gene that confer altered expression of the fhaB gene in Escherichia coli; Goyard S et al.; The bvg locus of Bordetella pertussis, required for coordinate regulation of virulence genes in response to environmental signals, encodes two proteins, BvgS and BvgA, that belong to the bacterial two-component signal transduction systems . We have isolated spontaneous mutations of the bvg locus in Escherichia coli and analyzed their effects on the expression of fhaB::lacZY transcriptional fusions . The mutations, localized in the linker and transmitter domain of BvgS, result in increased activation of fhaB and/or in insensitivity to a modulating agent, nicotinic acid.

J Bacteriol, 1994 Aug, 176(16), 5101 - 7
Between feast and famine: endogenous inducer synthesis in the adaptation of Escherichia coli to growth with limiting carbohydrates; Death A et al.; Escherichia coli adapted to growth with low carbohydrate concentrations bypassed the requirement for exogenous inducer with at least three well-studied sugar regulons . Induction of mgl and gal genes became independent of added galactose in bacteria approaching stationary phase or during continuous culture with micromolar glucose in the medium . Bacteria became independent of exogenous induction because endogenous galactose and cyclic AMP (cAMP) pools were sufficient for high expression of mgl and gal genes under glucose limitation . Limitation-stimulated induction of mgl was dependent on a functional galETK operon for synthesis of the inducer galactose . Intracellular galactose levels were maximal not during starvation (or slow steady-state growth rates approaching starvation) but at fast growth rates with micromolar glucose . The extent of mgl/gal induction correlated better with inducer availability than with cAMP concentrations under all conditions tested . Endogenous inducer accumulation represents an adaptation to low-nutrient environments, leading to derepression of high-affinity transport systems like Mgl essential for bacterial competitiveness at low nutrient concentrations.

J Bacteriol, 1994 Aug, 176(16), 5093 - 100
Chi-dependent formation of linear plasmid DNA in exonuclease-deficient recBCD+ strains of Escherichia coli; Zaman MM et al.; Escherichia coli strains carrying mutations in sbcB (exonuclease I) or xthA (exonuclease III) accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids containing the chi sequence, 5'-GCTGGTGG-3' . Chi-dependent formation of high-molecular-weight plasmid DNA is dependent on recA and recF functions . In addition, chi stimulation occurs only in cis . Our data are consistent with models in which RecA and RecF proteins bind to and protect the DNA ends produced by RecBCD-chi interaction.

J Bacteriol, 1994 Aug, 176(16), 5059 - 67
Conduction of pEC22, a plasmid coding for MR.EcoT22I, mediated by a resident Tn3-like transposon, Tn5396; Elhai J et al.; pEC22 is a small plasmid that encodes the restriction-modification system MR.EcoT22I . Restriction and functional analysis of the plasmid identified the positions of genes encoding that system . The plasmid is able to be conducted by conjugal plasmids, a process mediated by a transposon contained within pEC22 . This cryptic transposon, called Tn5396, was isolated from pEC22 and partially sequenced . The sequence of Tn5396 is for the most part typical of transposons of the Tn3 family and is most similar to that of Tn1000 . The transposon differs from closely related transposons in that it lacks well-conserved sequences in the inverted-repeat region and has an unusually long terminal inverted repeat . Consideration of regions of internal sequence similarity in this and other transposons in the Tn3 family supports a theory of the mechanism by which the ends of Tn3-like transposons may maintain substantial identity between their inverted repeats over the course of evolutionary time.

J Bacteriol, 1994 Aug, 176(16), 5044 - 51
Regulation of plasmid pE194 replication: control of cop-repF operon transcription by Cop and of repF translation by countertranscript RNA; Kwak JH et al.; The cop-rep region of plasmid pE194 contains two tandem structural genes, cop and repF, as well as the plus and minus origins of replication . The two structural genes comprise an operon whose expression is repressed by the binding of Cop protein to a 28-bp inverted complementary repeat sequence that overlaps the cop-repF promoter . From its position relative to the promoter and the experimentally determined footprint made by the Cop protein, the 28-bp inverted complementary repeat sequence is presumed to function as the cop operator . The intercistronic region between cop and repF is 80 nucleotides (nt) long and is transcribed bidirectionally: in the forward direction as part of the synthesis of the cop-repF message (ca . 900 nt), and in the reverse direction to yield a countertranscript ca . 65 nt long . The proposed countertranscript RNA (ctRNA) can form a single stem-and-loop structure that includes the single SphI sequence of plasmid pE194 as part of the loop-forming segment . Enlargement of the proposed loop from 6 to 14 nt by insertion of a SphI-BamHI adapter at the SphI site or contraction of the proposed loop down to 4 nt, by cutting with SphI followed by blunting with S1 nuclease, yields mutants with an increased copy number . By gel retardation and DNaseI footprinting analysis, Cop protein was shown to bind to the promoter region of cop; no binding by Cop protein at the 5' end of repF was detected . Two major transcripts were synthesized in vitro by using cop-repF region DNA as a template, the tandem cop-repF transcript, and the ctRNA . Addition of purified Cop protein to an vitro transcription reaction mixture reduced only the rate of cop-repF transcription but not that of ctRNA . These observations suggest that regulations of repF occurs at two levels: (i) with Cop protein acting as a repressor of cop-repF mRNA transcription and (ii) with ctRNA acting as a repressor of RepF translation.

J Bacteriol, 1994 Aug, 176(16), 5011 - 21
Isolation and characterization of novel plasmid-encoded umuC mutants; Woodgate R et al.; Most inducible mutagenesis in Escherichia coli is dependent upon the activity of the UmuDC proteins . The role of UmuC in this process is poorly understood, possibly because of the limited number of genetically characterized umuC mutants . To better understand the function of the UmuC protein in mutagenic DNA repair, we have isolated several novel plasmid-encoded umuC mutants . A multicopy plasmid that expressed UmuC at physiological levels was constructed and randomly mutagenized in vitro by exposure to hydroxylamine . Mutated plasmids were introduced into the umu tester strain RW126, and 16 plasmids that were unable to promote umuC-dependent spontaneous mutator activity were identified by a colorimetric papillation assay . Interestingly, these plasmid mutants fell into two classes: (i) 5 were expression mutants that produced either too little or too much wild-type UmuC protein, and (ii) 11 were plasmids with structural changes in the UmuC protein . Although hydroxylamine mutagenesis was random, most of the structural mutants identified in the screen were localized to two regions of the UmuC protein; four mutations were found in a stretch of 30 amino acids (residues 133 to 162) in the middle of the protein, while four other mutations (three of which resulted in a truncated UmuC protein) were localized in the last 50 carboxyl-terminal amino acid residues . These new plasmid umuC mutants, together with the previously identified chromosomal umuC25, umuC36, and umuC104 mutations that we have also cloned, should prove extremely useful in dissecting the genetic and biochemical activities of UmuC in mutagenic DNA repair.

J Bacteriol, 1994 Aug, 176(16), 4985 - 92
Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli; Schroder I et al.; The NarX, NarQ, and NarL proteins make up a nitrate-responsive regulatory system responsible for control of the anaerobic respiratory pathway genes in Escherichia coli, including nitrate reductase (narGHJI), dimethyl sulfoxide/trimethylamine-N-oxide reductase (dmsABC), and fumarate reductase (frdABCD) operons among others . The two membrane-bound proteins NarX and NarQ can independently sense the presence of nitrate and transfer this signal to the DNA-binding regulatory protein NarL, which controls gene expression by transcriptional activation or repression . To establish the role of protein phosphorylation in this process and to determine whether the NarX and NarQ proteins differ in their interaction with NarL, the cytoplasmic domains of NarX and NarQ were overproduced and purified . Both proteins were autophosphorylated in the presence of {gamma-32P}ATP and MgCl2 but not with {alpha-32P}ATP . Whereas these autophosphorylation reactions were unaffected by the presence of nitrate, molybdate, GTP, or AMP, ADP was an inhibitor . The phosphorylated forms of 'NarX and 'NarQ were stable for hours at room temperature . Each protein transferred its phosphoryl group to purified NarL protein, although 'NarQ-phosphate catalyzed the transfer reaction at an apparently much faster rate than did 'NarX-phosphate . In addition, NarL was autophosphorylated with acetyl phosphate but not with ATP as a substrate . NarL-phosphate remained phosphorylated for at least 3 h . However, addition of 'NarX resulted in rapid dephosphorylation of NarL-phosphate . In contrast, 'NarQ exhibited a much slower phosphatase activity with NarL-phosphate . These studies establish that the cytoplasmic domains of the two nitrate sensors 'NarX and 'NarQ differ in their ability to interact with NarL.

J Bacteriol, 1994 Aug, 176(16), 4958 - 65
Cell cycle arrest of a Caulobacter crescentus secA mutant; Kang PJ et al.; Cell differentiation is an inherent component of the Caulobacter crescentus cell cycle . The transition of a swarmer cell, with a single polar flagellum, into a sessile stalked cell includes several morphogenetic events . These include the release of the flagellum and pili, the proteolysis of chemotaxis proteins, the biogenesis of the polar stalk, and the initiation of DNA replication . We have isolated a group of temperature-sensitive mutants that are unable to complete this process at the restrictive temperature . We show here that one of these strains has a mutation in a homolog of the Escherichia coli secA gene, whose product is involved in protein translocation at the cell membrane . This C . crescentus secA mutant has allowed the identification of morphogenetic events in the swarmer-to-stalked cell transition that require SecA-dependent protein translocation . Upon shift to the nonpermissive temperature, the mutant secA swarmer cell is able to release the polar flagellum, degrade chemoreceptors, and initiate DNA replication, but it is unable to form a stalk, complete DNA replication, or carry out cell division . At the nonpermissive temperature, the cell cycle blocks prior to the de novo synthesis of flagella and chemotaxis proteins that normally occurs in the predivisional cell . Although interactions between the chromosome and the cytoplasmic membrane are believed to be a functional component of the temporal regulation of DNA replication, the ability of this secA mutant to initiate replication at the nonpermissive temperature suggests that SecA-dependent events are not involved in this process . However, both cell division and stalk formation, which is analogous to a polar division event, require SecA function.

J Bacteriol, 1994 Aug, 176(16), 4825 - 37
A monocysteine approach for probing the structure and interactions of the UmuD protein; Lee MH et al.; UmuD participates in a variety of protein-protein interactions that appear to be essential for its role in UV mutagenesis . To learn about these interactions, we have initiated an approach based on the construction of a series of monocysteine derivatives of UmuD and have carried out experiments exploring the chemistry of the unique thiol group in each derivative . In vivo and in vitro characterizations indicate that these proteins have an essentially native structure . In proposing a model for the interactions of UmuD in the homodimer, we have made the following assumptions: (i) the conformations of the mutant proteins are similar to that of the wild type, and (ii) the differences in reactivity of the mutant proteins are predominantly due to the positional effects of the single cysteine substitutions . The model proposes the following . The region including the Cys-24-Gly-25 cleavage site, Val-34, and Leu-44 are closer to the interface than the other positions tested as suggested by the relative ease of dimer cross-linking of the monocysteine derivatives at these positions by oxidation with iodine (I2) and by reaction with bis-maleimidohexane . The mutant with a Ser-to-Cys change at position 60 (SC60) is similar in iodoacetate reactivity to the preceding derivatives but cross-links less efficiently by I2 oxidation . This suggests that Ser-60, the site of the putative nucleophile in the cleavage reaction, is located further from the dimer interface or in a cleft region . Both Ser-19, located in the N-terminal fragment of UmuD that is removed by RecA-mediated cleavage, and Ser-67 are probably not as close to the dimer interface, since they are cross-linked more easily with bis-maleimidohexane than with I2 . The SC67 mutant phenotype also suggests that this position is less important in RecA-mediated cleavage but more important in a subsequent role for UmuD in mutagenesis . Ala-89, Gln-100, and Asp-126 are probably not particularly solvent accessible and may play important roles in protein architecture.

FEBS Lett, 1994 Aug 1, 349(2), 281 - 5
Ability of MBP or RBP signal peptides to influence folding and in vitro translocation of wild-type and hybrid precursors; Rosemond MJ et al.; Maltose-binding protein (MBP), whose export in E . coli is dependent upon the chaperone SecB, and ribose-binding protein (RBP), whose export is SecB-independent, have been used to generate hybrid secretory proteins . Here, in vitro techniques were used to analyze MBP, RBP, RBP-MBP (RBP signal and MBP mature), and MBP-RBP (MBP signal and RBP mature) . In protease-protection experiments, RBP folded considerably faster than MBP, RBP-MBP, or MBP-RBP . Only the folding properties of proteins containing the MBP mature moiety were influenced by SecB . In post-translational translocation assays, MBP exhibited the highest translocation efficiency . The hybrids RBP-MBP and MBP-RBP showed intermediate levels, and RBP translocation was not detected in these assays . These experiments demonstrate the influence of the signal peptide in determining folding properties and translocation efficiency of precursor secretory proteins.

Surgery, 1994 Aug, 116(2), 367 - 76; discussion 376-7
Activation of hepatic proliferation-associated transcription factors by lipopolysaccharide; Beauchamp RD et al.; BACKGROUND . The hepatic acute-phase response is the result of reprogramming of gene expression in the liver . Similar acute-phase responses occur in regenerating liver after partial hepatectomy and are preceded by increases in the expression of a set of transcriptional regulatory proteins that are encoded by "immediate-early" genes . The purpose of this study was to determine whether acute systemic inflammation after lipopolysaccharide injection induces hepatic immediate-early genes that are induced by partial hepatectomy . METHODS . Two- to 4-month-old Balb/c mice received intraperitoneal Escherichia coli lipopolysaccharide (0111:B4; 100 micrograms), and total liver RNA, nuclear protein extracts, or total liver protein lysates were obtained at 0, 1, 3, 12, and 24 hours . RNA blot hybridization analysis was used to determine steady-state messenger RNA levels for c-jun, jun-B, jun-D, c-fos, fos-B, fra-1, nup475, and zif268 . Specific nuclear protein-binding activity was determined by gel mobility shift assay . The protein c-Jun was detected by antibody-blocking experiments, and Jun-B was detected by gel supershift assay of the activating protein (AP-1) complex . Steady-state Jun-B levels were determined by immunoblot analysis . RESULTS . Intraperitoneal injection of lipopolysaccharide is followed by induction (from fivefold to 13-fold) of c-jun, jun-B, c-fos, zif268, and nup475 messenger RNAs in the liver . Lipopolysaccharide induced increases in AP-1 and Zif268 consensus DNA-binding activity in mouse liver . The proteins c-Jun and Jun-B are detected in the AP-1 complex after administration of lipopolysaccharide . CONCLUSIONS . The induction of hepatic immediate-early genes after lipopolysaccharide is similar to that that follows partial hepatectomy . These transcription factors likely have important roles in the reprogramming of gene expression that leads to the acute-phase response.

Surgery, 1994 Aug, 116(2), 356 - 65; discussion 365-6
Antibody to tumor necrosis factor attenuates endotoxin-stimulated amino acid transport in rat liver; Inoue Y et al.; BACKGROUND . Endotoxemia stimulates amino acid consumption by the liver, but the regulation of this response is poorly understood . We studied the effect of Escherichia coli endotoxin (lipopolysaccharide) on hepatic carrier-mediated plasma membrane amino acid transport and the role of the cytokine tumor necrosis factor-alpha (TNF) in regulating this transport activity . METHODS . We investigated the activities of the Na(+)-dependent amino acid transport systems A, ASC, and N in hepatic plasma membrane vesicles prepared from rats treated with endotoxin in vivo . Vesicle purity and functionality were evaluated by assaying marker enzymes and by the presence of classic overshoots . RESULTS . Endotoxin treatment did not alter sodium transport but resulted in time- and dose-dependent 6-fold (system A), 3.5-fold (system N), and 3-fold (system ASC) increases in transport activity secondary to an increase in carrier maximum velocity . Lipopolysaccharide treatment did not alter transporter affinity or plasma membrane sodium transport . Transport activity increased within 2 hours of endotoxin administration, peaked at 4 hours after exposure to lipopolysaccharide, and returned to basal levels within 24 hours . Pretreatment of animals with an anti-TNF monoclonal antibody diminished the endotoxin-induced enhancement in transport activity by 50% to 75% by decreasing carrier maximum velocity . In contrast, when the antibody was given after endotoxin challenge, transport activity was not attenuated . CONCLUSIONS . The marked acceleration in hepatic amino acid uptake that occurs during endotoxemia is secondary to an increased Na(+)-dependent hepatocyte plasma membrane transport activity and is mediated, in large part, by the cytokine TNF.

Surgery, 1994 Aug, 116(2), 307 - 12
Uncoupling of coronary microvascular beta 2-adrenoceptors by Escherichia coli endotoxemia; Wang SY et al.; BACKGROUND . Cardiovascular responses to the adrenergic stimulation are depressed in clinical and experimental endotoxemia . However, the effect of Escherichia coli endotoxemia on coronary microvascular beta-adrenergic function remains to be determined . The purpose of the present study was to test the hypothesis that endotoxemia impairs the beta-adrenoceptor- and adenosine 3'5'-cyclic monophosphate-mediated relaxation in the porcine coronary microcirculation . METHODS . Coronary arterioles (80 to 170 microns internal diameter) were isolated from pigs 3 hours after intravenous administration of E . coli endotoxin (150 micrograms/kg, over 1 hour, n = 8) or Ringer's lactate (control, n = 8) . Arterioles were studied in vitro in a pressurized, partially contracted, no-flow state by videomicroscopy . RESULTS . Precontracted (30% to 50% of baseline diameter with acetylcholine) control coronary arterioles dilated in response to either the nonselective beta-adrenoceptor agonist, isoproterenol, the Gs-protein activator, sodium fluoride, or the adenylate cyclase activator, forskolin . After 3 hours of endotoxemia, the relaxation responses to isoproterenol and sodium fluoride were significantly reduced, but the relaxation response to forskolin was preserved . The beta 2-adrenoceptor blocker, ICI-118, 551, markedly reduced the relaxation of control microvessels induced by isoproterenol, whereas the beta 1-adrenoceptor blocker, atenolol, caused only a slight reduction in isoproterenol-induced relaxation . CONCLUSIONS . beta 2-Adrenoceptors appear to predominate over beta 1-adrenoceptors in the coronary microcirculation . E . coli endotoxemia impairs beta 2-adrenoceptor-mediated relaxation in the porcine coronary microcirculation, apparently because of changes proximal to adenylate cyclase in the signal transduction pathway.

Surgery, 1994 Aug, 116(2), 197 - 203; discussion 203-4
In vivo adenoviral-mediated human p53 tumor suppressor gene transfer and expression in rat liver after resection; Drazan KE et al.; BACKGROUND . The aim of this study was to establish a clinically relevant model for gene transfer to liver with an adenoviral vector encoding wild-type p53 as a first step toward use of this class of gene products in the treatment of primary and metastatic liver tumors . METHODS . Full-size or 50% hepatectomized rat livers were subjected to asanguineous portal perfusion with a replication-defective adenoviral vector encoding wild-type p53 (Ad5p53), whereas control animals received adenoviral vector encoding Escherichia coli beta-galactosidase (beta-gal) (Ad5LacZ) or Ringer's lactate only . Liver biopsy specimens, blood samples, and liver weight were serially obtained . Gene transfer and expression were confirmed by X-Gal staining for gamma-gal, DNA/RNA polymerase chain reaction, (PCR) and Western blots for p53 and beta-gal . Liver integrity was assessed by histologic findings, serum transaminase levels, and synthetic function . RESULTS . The gene transfer rate in whole liver and after hepatectomy ranged from 20% to 40% . DNA PCR showed Ad sequences in livers transduced with Ad5p53 and Ad5LacZ . RNA PCR and Western blot confirmed expression and production of recombinant wild-type p53 . Liver regeneration was not affected by p53 gene transduction . Liver histologic findings and synthetic function were not different between transduced and control groups . CONCLUSIONS . Ad5p53 gene transfer to full-size or hepatectomized livers is efficient . Liver regeneration and hepatocyte function are unaffected by overexpression of p53 . Adenovirus-mediated tumor-suppressor transduction of the liver is a safe and promising adjuvant in cancer gene therapy.

Scand J Immunol, 1994 Aug, 40(2), 165 - 70
Cloning, sequencing and expression in Escherichia coli of the gene for alpha antigen from Mycobacterium scrofulaceum; Takano M et al.; The gene for the extracellular alpha antigen of Mycobacterium scrofulaceum (S-alpha) was cloned by using the alpha antigen gene fragments of Mycobacterium bovis BCG as probes . The complete nucleotide sequence was determined . The gene was expressed in Escherichia coli . The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein . The deduced amino acid sequences were highly homologous to the alpha antigen of the species of other mycobacteria . Interestingly, the alpha antigens of MAIS complex (Mycobacterium avium-Mycobacterium intracellulare-M . scrofulaceum) were closely homologous even at the C-terminal regions which were variable among those of M . bovis BCG, Mycobacterium leprae and Mycobacterium kansasii.

J Med Microbiol, 1994 Aug, 41(2), 133 - 8
Promotion of Escherichia coli adherence to rubber slices by adsorbed fibronectin; Yu JL et al.; Biomaterial-associated infections are a problem in the use of endoprosthetic materials in the palliative treatment of malignant obstructive jaundice . Fibronectin has been reported to mediate adherence of bacteria to host tissue and biomaterials . Adsorption of fibronectin to rubber--representing material used for biliary drainage--and subsequent adherence of Escherichia coli strain PSS1 and E . coli strain NG7C (which binds to immobilised fibronectin) were investigated . Quantitative adsorption of fibronectin to rubber slices was studied with 125I-labelled, purified human plasma fibronectin . In buffer solutions, fibronectin showed a high affinity for rubber slices . Adherence of the E . coli strains to uncoated rubber slices was similar and was significantly inhibited by the presence of plasma components and bile . Adherence of E . coli PSS1 to fibronectin-coated slices was poor . In contrast, E . coli NG7C adhered efficiently to coated slices in proportion to the amount of adsorbed fibronectin; adherence was not reduced by the presence of albumin or bile, or the fibronectin-binding ligands gelatin, heparin and fibrinogen . However, pre-digestion of coated slices with trypsin significantly reduced adherence.

J Gen Virol, 1994 Aug, 75 ( Pt 8), 2041 - 6
Expression of equine herpesvirus type 1 glycoprotein gp14 in Escherichia coli and in insect cells: a comparative study on protein processing and humoral immune responses; Osterrieder N et al.; The extracellular portion (amino acids 1 to 844) of the equine herpesvirus type 1 (EHV-1) glycoprotein gp14, the homologue of gB of herpes simplex virus, was expressed in Escherichia coli and in insect cells using a recombinant baculovirus . Immunoblot analysis revealed that the recombinant E . coli expressed a fusion protein of M(r) 135K which was composed of the truncated gp14 and the maltose-binding protein (MBP) provided by the vector and a 90K protein lacking the MBP moiety . Both proteins were sequestered within the cells in form of inclusion bodies . Infection of insect cells with the recombinant baculovirus resulted in the production of a 115K to 118K glycoprotein which was cleaved intracellularly into two subunits of M(r) 55K and 63K to 65K . The cleaved subunits were secreted into the cell culture supernatant and formed disulphide-linked dimers of M(r) 120K to 122K . The recombinant proteins produced in E . coli and in insect cells elicited EHV-1-specific antibodies in goats as demonstrated by Western blot analysis . The gp14 expressed in insect cells induced antibodies with virus-neutralizing activity . In contrast, the truncated gp14 expressed by E . coli failed to elicit neutralizing antibodies . The results suggest that post-translational modification of the EHV-1 gp14 may be important for the expression of epitopes necessary for the induction of neutralizing antibodies.

J Gen Virol, 1994 Aug, 75 ( Pt 8), 1953 - 60
E5a gene of human papillomavirus type 11 is required for initiation but not for maintenance of transformation in NIH 3T3 cells; Chen SL et al.; We have previously shown that the E5a gene of human papilloma virus type 11 (HPV-11/HPV-6c) is a transforming oncogene . In order to dissect the biological consequences of E5a gene expression we utilized the lac operator/repressor system to manipulate E5a gene expression . Cells were cotransfected with the lac repressor gene and the E5a gene that had been inserted downstream of a simian virus 40 (SV40) promoter containing the lac operator sequence . The expression of E5a gene could therefore be repressed by binding of lac repressor to the lac operator sequence in proximity to this SV40 regulatory region . The transfected cells were cultured in the presence of the inducer IPTG and under G418 selection . IPTG derepressed E5a gene expression by binding to the repressor and reducing its affinity for the lac operator sequence . In these studies, we found that E5a-transformed cells still maintained the transformed phenotype as judged by growth density, cell morphology and anchorage-independent growth when E5a gene expression was repressed . We also found that c-jun expression was induced 3 h after E5a expression was induced by IPTG and c-jun expression was not shut down after repression of E5a expression . This is the first demonstration that the E5a gene of HPV-11 initiates transformation of NIH 3T3 cells but is dispensable for maintenance of the transformed phenotype.

J Gen Virol, 1994 Aug, 75 ( Pt 8), 1889 - 99
Structural and antigenic analysis of the nucleoprotein of bovine ephemeral fever rhabdovirus; Walker PJ et al.; The nucleotide sequence of the bovine ephemeral fever virus (BEFV) genome has been determined from the 3' terminus to the end of the nucleoprotein (N) gene . The 3' leader sequence comprises 50 nucleotides and shares a common terminal three nucleotides (3'-UGC-) and a downstream U-rich domain with vesicular stomatitis virus (VSV) and rabies virus . The N gene comprises 1328 nucleotides from the transcription initiation consensus sequence (AACAGG) to the conserved transcription termination-poly(A) sequence {CATG(A)7} and encodes a polypeptide of 431 amino acids with an estimated M(r) of 49,159 and a pI of 5.4 . The deduced amino acid sequence of the BEFV N protein is similar to those of other mammalian rhabdoviruses and is more closely related in sequence to vesiculoviruses (VSV Indiana and New Jersey, Piry, Chandipura) than to lyssaviruses (rabies and Mokola) . An almost full-length clone, 1301 bp in length, of the BEFV N gene and clones derived from 5'-terminal (559 bp) and 3'-terminal (742 bp) fragments were expressed in Escherichia coli as glutathione-S-transferase fusion proteins . A panel of 12 BEFV N protein-specific monoclonal antibodies was shown to react in immunoblots with fusion proteins containing the almost full-length N protein and the C-terminal fragment, but not the N-terminal fragment . Two of these antibodies also reacted with baculovirus-expressed rabies virus N protein . Polyclonal mouse ascitic fluids derived from BEFV, rabies virus and several other related viruses were also shown to cross-react in immunoblots with purified preparations of rabies virus and BEFV N proteins.

J Bacteriol, 1994 Aug, 176(15), 4790 - 3
Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable {4Fe-4S} ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus; Heltzel A et al.; The gene for ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, sequenced, and expressed in Escherichia coli . The coding region confirmed the determined amino acid sequence . Putative archaeon-type transcriptional regulatory elements were identified . The fdxA gene appears to be an independent transcriptional unit . Recombinant ferredoxin was indistinguishable from the protein purified from P . furiosus in its thermal stability and in the potentiometric and spectroscopic properties of its {4Fe-4S} cluster.

J Bacteriol, 1994 Aug, 176(15), 4656 - 63
The DNA replication fork blocked at the Ter site may be an entrance for the RecBCD enzyme into duplex DNA; Horiuchi T et al.; In Escherichia coli, eight kinds of chromosome-derived DNA fragments (named Hot DNA) were found to exhibit homologous recombinational hotspot activity, with the following properties . (i) The Hot activities of all Hot DNAs were enhanced extensively under RNase H-defective (rnh) conditions . (ii) Seven Hot DNAs were clustered at the DNA replication terminus region on the E . coli chromosome and had Chi activities (H . Nishitani, M . Hidaka, and T . Horiuchi, Mol . Gen . Genet . 240:307-314, 1993) . Hot activities of HotA, -B, and -C, the locations of which were close to three DNA replication terminus sites, the TerB, -A, and -C sites, respectively, disappeared when terminus-binding (Tau or Tus) protein was defective, thereby suggesting that their Hot activities are termination event dependent . Other Hot groups showed termination-independent Hot activities . In addition, at least HotA activity proved to be dependent on a Chi sequence, because mutational destruction of the Chi sequence on the HotA DNA fragment resulted in disappearance of the HotA activity . The HotA activity which had disappeared was reactivated by insertion of a new, properly oriented Chi sequence at the position between the HotA DNA and the TerB site . On the basis of these observations and positional and orientational relationships between the Chi and the Ter sequences, we propose a model in which the DNA replication fork blocked at the Ter site provides an entrance for the RecBCD enzyme into duplex DNA.

J Bacteriol, 1994 Aug, 176(15), 4617 - 26
Regulation of the Escherichia coli nrd operon: role of DNA supercoiling; Sun L et al.; An in vitro RNA transcription assay was used to investigate the regulation of the expression of the nrd promoter . Using a linear DNA template, we found that Fis protein, which has a positive effect on expression of the nrd promoter in an nrd-lacZ fusion in vivo, had a moderate negative effect in vitro . However, with a supercoiled DNA template as substrate, we found that Fis had a concentration-dependent positive effect on nrd transcription in vitro . This positive effect was not present on two templates that had 35- or 37-bp insertions between the Fis binding site and the nrd promoter . In the absence of Fis protein, a dramatic decrease in transcription was observed in templates with reduced supercoiling generated by the treatment with wheat germ topoisomerase I . Templates with insertions of 35 bp into an HpaII site at -102 or 37 bp into the MnlI site at -33 bp from the start of transcription failed to exhibit the DNA supercoiling sensitivity of the nrd promoter . Analysis of cells containing either of these two nrd-lacZ fusion constructs that has an insertion at the regulatory region by flow cytometry indicated that these two constructs, unlike the parental construct, were not cell cycle regulated.

J Bacteriol, 1994 Aug, 176(15), 4565 - 71
Requirements for translocation of periplasmic domains in polytopic membrane proteins; Uhland K et al.; Periplasmic domains of cytoplasmic membrane proteins require export signals for proper translocation . These signals were studied by using a MalF-alkaline phosphatase fusion in a genetic selection that allowed the isolation of mislocalization mutants . In the original construct, alkaline phosphatase is fused to the second periplasmic domain of the membrane protein, and its activity is thus confined exclusively to the periplasm . Mutants that no longer translocated alkaline phosphatase were selected by complementation of a serB mutation . A total of 11 deletions in the amino terminus were isolated, all of which spanned at least the third transmembrane segment . This domain immediately precedes the periplasmic domain to which alkaline phosphatase was fused . Our results obtained in vivo support the model that amino-terminal membrane-spanning segments are required for translocation of large periplasmic domains . In addition, we found that the inability to export the alkaline phosphatase domain could be suppressed by a mutation, prlA4, in the secretion apparatus.

J Bacteriol, 1994 Aug, 176(15), 4483 - 91
The short form of CheA couples chemoreception to CheA phosphorylation; Wolfe AJ et al.; Escherichia coli cells express two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct in-frame initiation sites in the gene cheA . The long form, CheAL, plays a crucial role in chemotactic signal transduction . As a histidine protein kinase, it first autophosphorylates at amino acid His-48; then, it phosphorylates two other chemotaxis proteins, CheY and CheB . The short form, CheAS, lacks the amino-terminal 97 amino acids of CheAL and, therefore, does not contain the site of autophosphorylation . However, it does retain a functional kinase domain . As a consequence, CheAS can mediate transphosphorylation of kinase-deficient CheAL variants . Here we demonstrate in vitro that CheAS also can mediate transphosphorylation of a CheAL variant that lacks the C-terminal segment, a portion of the protein which is thought to interact with CheW and the chemoreceptors . The presence of CheW and the chemoreceptor Tsr enhances this activity and results in modulation of the transphosphorylation rate in response to the Tsr ligand, L-serine . Because CheAS can mediate this activity, it can restore chemotactic ability to Escherichia coli cells that express this truncated CheAL variant.

Crit Care Med, 1994 Aug, 22(8), 1211 - 8
Effect of a recombinant endotoxin-neutralizing protein on endotoxin shock in rabbits; Garcia C et al.; OBJECTIVES: Limulus anti-lipopolysaccharide factor, an 11.8-kilodalton peptide isolated from amebocytes of Limulus polyphemus inhibits the biologic activities of endotoxin in vitro, including gelation of Limulus amebocyte lysate . A recombinant version of Limulus anti-lipopolysaccharide factor, termed endotoxin neutralizing protein, has now been expressed in yeast . Endotoxin-neutralizing protein was evaluated for its potential prophylactic and therapeutic effects in rabbits challenged with Escherichia coli endotoxin . DESIGN: Controlled animal trial . SETTING: Animal research laboratory . SUBJECTS: A total of 112 New Zealand white rabbits were studied . INTERVENTIONS: Rabbits were challenged with an LD80 dose of E . coli endotoxin (100 micrograms/kg); control animals (n = 52) were treated with saline solution at the time of endotoxin challenge; experimental animals received endotoxin-neutralizing protein 2.5 mg/kg prechallenge (n = 20), 5.0 mg/kg prechallenge (n = 20), or 5.0 mg/kg 30 mins postchallenge (n = 20).

J Clin Invest, 1994 Aug, 94(2), 703 - 8
Messenger RNA levels of XPAC and ERCC1 in ovarian cancer tissue correlate with response to platinum-based chemotherapy; Dabholkar M et al.; Nucleotide excision repair is a DNA repair pathway that is highly conserved in nature, with analogous repair systems described in Escherichia coli, yeast, and mammalian cells . The rate-limiting step, DNA damage recognition and excision, is effected by the protein products of the genes ERCC1 and XPAC . We therefore assessed mRNA levels of ERCC1 and XPAC in malignant ovarian cancer tissues from 28 patients that were harvested before the administration of platinum-based chemotherapy . Cancer tissues from patients whose tumors were clinically resistant to therapy (n = 13) showed greater levels of total ERCC1 mRNA (P = 0.059), full length transcript of ERCC1 mRNA (P = 0.026), and XPAC mRNA (P = 0.011), as compared with tumor tissues from those individuals clinically sensitive to therapy (n = 15) . In 19 of these tissues, the percentage of alternative splicing of ERCC1 mRNA was assessed . ERCC1 splicing was highly variable, with no difference observed between responders and nonresponders . The alternatively spliced species constituted 2-58% of the total ERCC1 mRNA in responders (median = 18%) and 4-71% in nonresponders (median = 13%) . These data suggest greater activity of the DNA excision repair genes ERCC1 and XPAC in ovarian cancer tissues of patients clinically resistant to platinum compounds . These data also indicate highly variable splicing of ERCC1 mRNA in ovarian cancer tissues in vivo, whether or not such tissues are sensitive to platinum-based therapy.

Infect Immun, 1994 Aug, 62(8), 3424 - 33
Cloning and sequence analysis of a chymotrypsinlike protease from Treponema denticola; Arakawa S et al.; A clone expressing a Treponema denticola chymotrypsinlike protease from recombinant plasmid pSA2 was identified in a genomic library of T . denticola ATCC 35405 . Nucleotide sequencing of the insert identified an open reading frame, designated the prtB gene, which codes for the protease . Two potential inverted repeat sequences are present both upstream and downstream from the prtB gene . The prtB gene would code for a putative protein of 273 amino acids with a calculated molecular mass of 30.4 kDa and an estimated pI of 7.0 . The G+C content of the gene is 40.3% . The results of maxicell analysis are consistent with the expression of a 30-kDa protease from the prtB gene . Preliminary characterization of the protease indicated that it was inhibited by the protease inhibitors phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and N-tosyl-L-phenylalanine chloromethyl ketone but not by N alpha-p-tosyl-L-lysine chloromethyl ketone . Purification of the protease was accomplished with the PinPoint protein purification system following construction of site-directed mutagenized plasmid pXa-3:2 . The purified protease degraded human and bovine serum albumins as well as casein . Furthermore, hemolysis of sheep erythrocytes by the protease was observed . Northern (RNA) blot analysis of mRNA extracted from strain 35405 indicated a single 1.9-kb mRNA species containing the prtB transcript . In addition, the results of primer extension analysis indicated that transcription was initiated primarily at a T residue . However, no corresponding -10 and -35 sequences related to Escherichia coli promoter sequences were identified . The availability of the purified protein and its gene will aid in evaluating the potential role of the protease in the physiology and virulence of T . denticola since proteases may play a key role in oral treponemal pathogenicity.

Infect Immun, 1994 Aug, 62(8), 3337 - 47
Evidence that verotoxins (Shiga-like toxins) from Escherichia coli bind to P blood group antigens of human erythrocytes in vitro; Bitzan M et al.; The interaction of verotoxins (VTs) with human erythrocytes (RBCs) in vitro was investigated, with particular reference to the role of P blood group glycolipids that are structurally related to the known VT receptors . RBC binding of purified VT1, VT2, VT2c, and VT2e was detected by direct and indirect immunofluorescence . Glycolipids were extracted from defined RBCs, separated by thin-layer chromatography, and assessed for VT binding in an overlay assay by adding toxin and specific antibodies . All VTs bound to P1 phenotype (Pk, P, and P1 antigens) and P2 phenotype (Pk and P antigens) RBCs but not to p phenotype (lacking the Pk, P, and P1 antigens) RBCs . Binding of VT1 and VT2 was approximately 10-fold greater to P1 and the rare Pk2 (Pk antigen but no P1 or P antigen) phenotype cells than to P2 phenotype RBCs, whereas VT2e bound equally well to P1 and P2 phenotype cells . The VT1 and VT2 immunofluorescence results correlated with the detection of P1 and/or increased amounts of Pk (globotriaosylceramide) antigen; VT2e immunofluorescence correlated with the detection of P (globotetraosylceramide) antigen . The Pk band pattern and VT binding observed in the thin-layer chromatogram of human P1 and P phenotype RBC extracts varied from that of human kidney and Pk1 phenotype (Pk and P1 antigens) RBCs . We conclude that each VT binds to human RBCs in vitro by utilizing specific P blood group glycolipids as receptors . On P1 and P phenotype RBCs, the accessibility of the Pk antigen for VTs appeared to be restricted . The occurrence of VT-RBC binding in natural VT-producing Escherichia coli disease and its relevance for the pathophysiology of hemolytic uremic syndrome remain to be established.

Infect Immun, 1994 Aug, 62(8), 3329 - 36
Host specificity of enteropathogenic Escherichia coli from rabbits: lack of correlation between adherence in vitro and pathogenicity for laboratory animals; Robins-Browne RM et al.; The pathogenicity of four attaching and effacing strains of enteropathogenic Escherichia coli originally isolated from diarrheic rabbits was investigated by inoculating them perorally into rabbits, guinea pigs, and mice . The ability of the four strains to adhere to cultured epithelial cells, erythrocytes, and intestinal brush borders from various animal species, including rabbits, guinea pigs, and mice, varied considerably . Only one strain carried AF/R1 fimbriae, which are believed to determine the host specificity of these bacteria . Despite these differences, the pattern of behavior of the four strains in experimentally infected animals was similar . Each strain caused fatal diarrhea in rabbits (although the virulence of individual strains for rabbits differed significantly), and none was virulent for guinea pigs or mice . None of the strains colonized the intestinal tract of guinea pigs, but all were able to cause attaching-effacing lesions in ligated loops of guinea pig small intestine . By contrast, all four strains colonized mice, in particular the distal intestine, but none induced attaching-effacing lesions in mouse intestinal loops . These findings suggest that there may be previously unrecognized host-restricted adhesins in enteropathogenic E . coli and indicate that adherence to erythrocytes or intestinal brush borders in vitro does not necessarily reflect colonizing ability or pathogenicity in vivo.

Infect Immun, 1994 Aug, 62(8), 3282 - 8
Escherichia coli serogroup O111 includes several clones of diarrheagenic strains with different virulence properties; Campos LC et al.; Genetic variation among isolates of Escherichia coli O111 obtained mostly from patients with diarrhea in Brazil was assessed by multilocus enzyme electrophoresis to characterize chromosomal genotypes and by gene probes and adherence assays to characterize virulence properties . Among the 152 isolates, we resolved 16 distinct electrophoretic types (ETs), which differed on average at 40% of the enzyme loci . We identified four major bacterial O111 clones of different disease classes: ET 12, which includes the bulk of the enteropathogenic E . coli strains, typically showing localized adherence and intimate attachment in tissue culture assays; ET 1, which includes strains with a different set of virulence markers; ET 9, which includes strains that show intimate attachment but lack localized adherence and Shiga-like toxin genes; and ET 8, which includes strains that are Shiga-like toxin producers and have the corresponding traits of enterohemorrhagic E . coli . Enteroaggregative strains constituted ET 10 and also occurred in ET 1 . Isolates of the major clones were found in South and North America and matched in ET and virulence factors to previously described diarrheagenic clones that are widely disseminated in the human population . Because the major clones are genetically distantly related and exhibit different combinations of virulence factors, we hypothesize that they have distinct mechanisms of pathogenesis . The results indicate that genetic divergence of bacteria with the O111 antigen, as measured by allelic variation in enzyme loci, is accompanied by divergence in virulence properties of clones so that identification and classification of pathogenic E . coli strains cannot be based solely on serotyping or a single virulence factor.

Infect Immun, 1994 Aug, 62(8), 3270 - 5
Immunogenicity of the Plasmodium falciparum glutamate-rich protein expressed by vaccinia virus; Theisen M et al.; The glurp gene of Plasmodium falciparum F32 has been inserted into a vaccinia virus, and the recombinant virus was designated VVG4 . Expression of glurp in VVG4-infected Vero cells was analyzed by immunoprecipitation and revealed a primary GLURP product of approximately 220,000 Da; GLURP was detected both intracellularly and in culture supernatants . To study the immunogenicity of vaccinia virus-expressed GLURP, mice were immunized with VVG4 and serum samples were analyzed for antibody reactivity with three polypeptides, covering almost the entire GLURP molecule; these three polypeptides were produced in recombinant form in Escherichia coli . The immune response was primarily directed against a carboxy-terminal repeat region . The mouse anti-GLURP serum recognized authentic GLURP by immunoprecipitation analysis from P . falciparum grown in vitro . These results demonstrate that vaccinia virus-expressed glurp product can induce a humoral immune response against GLURP derived from blood-stage parasites.

Arch Biochem Biophys, 1994 Aug 1, 312(2), 554 - 65
The high-level expression in Escherichia coli of the membrane-bound form of human and rat cytochrome b5 and studies on their mechanism of function; Holmans PL et al.; A T7 expression system is described for the high-level production in Escherichia coli of the membrane-bound form of human and rat cytochrome b5 . The cDNAs of b5 have been engineered to contain a coding sequence for a four-member histidine domain at the amino-terminus of the recombinant protein permitting the use of a nickel-chelate affinity column for rapid purification of the detergent-solubilized hemoprotein . Results are presented demonstrating the ability of the purified recombinant b5 proteins to stimulate the rate of oxidation of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17A, and to stimulate the 6 beta-hydroxylation of testosterone, catalyzed by human P450 3A4 . These P450-catalyzed reactions have been used to compare the properties of different forms of b5 . Purified b5 can serve as a "coupling protein" as illustrated by its inhibition of NADPH oxidation, catalyzed by a fusion protein containing the heme domain of P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhibition of hydrogen peroxide formation . Kinetic studies show the formation of a complex of the flavoprotein, NADPH-P450 reductase, with b5 for the rapid transfer of electrons from NADPH.

Arch Biochem Biophys, 1994 Aug 1, 312(2), 516 - 23
Functional expression and characterization of the assimilatory-type sulfite reductase from Desulfovibrio vulgaris (Hildenborough); Tan J et al.; By use of a conjugational transfer method, the gene encoding the assimilatory-type sulfite reductase (ASiR) from Desulfovibrio vulgaris (Hildenborough) has been functionally expressed in Desulfovibrio hosts using the broad-host-range plasmid pDSK519 . Production is increased greater than 50-fold relative to natural expression levels . Recombinant enzyme has been characterized by standard biochemical methods, nuclear magnetic resonance, electron paramagnetic resonance, electronic absorption, and activity measurements . It cannot be distinguished from the native enzyme on the basis of spectroscopic characteristics or enzyme activity.

Arch Biochem Biophys, 1994 Aug 1, 312(2), 501 - 10
Molecular cloning of isoflavone reductase from pea (Pisum sativum L.): evidence for a 3R-isoflavanone intermediate in (+)-pisatin biosynthesis; Paiva NL et al.; Isoflavone reductase (IFR) reduces achiral isoflavones to chiral isoflavanones during the biosynthesis of chiral pterocarpan phytoalexins . A cDNA clone for IFR from pea (Pisum sativum) was isolated using the polymerase chain reaction and expressed in Escherichia coli . Analysis of circular dichroism (CD) spectra of the reduction product sophorol obtained using the recombinant enzyme indicated that the isoflavanone possessed the 3R stereochemistry, in contrast to previous reports indicating a 3S-isoflavanone as the product of the pea IFR . Analysis of CD spectra of sophorol produced using enzyme extracts of CuCl2-treated pea seedlings confirmed the 3R stereochemistry . Thus, the stereochemistry of the isoflavanone intermediate in (+)-pisatin biosynthesis in pea is the same as that in (-)-medicarpin biosynthesis in alfalfa, although the final pterocarpans have the opposite stereochemistry . At the amino acid level the pea IFR cDNA was 91.8 and 85.2% identical to the IFRs from alfalfa and chickpea, respectively . IFR appears to be encoded by a single gene in pea . Its transcripts are highly induced in CuCl2-treated seedlings, consistent with the appearance of IFR enzyme activity and pisatin accumulation.

Arch Biochem Biophys, 1994 Aug 1, 312(2), 436 - 46
Expression of modified human cytochrome P450 1A1 in Escherichia coli: effects of 5' substitution, stabilization, purification, spectral characterization, and catalytic properties; Guo Z et al.; Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals . Purification of active enzyme from human tissues has not been successful . We report the expression and purification of the recombinant enzyme from Escherichia coli . A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E . coli cells . Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5'-terminal codons were maximized for AT content and minimized for the potential of secondary structure formation of the mRNA transcript . alpha-Naphthoflavone was found to protect against denaturation by detergents during solubilization and was added to buffers used for purification . The recombinant P450 1A1 was purified to electrophoretic homogeneity after two ion-exchange chromatography steps in approximately 50% yield . N-Terminal amino acid sequence analysis verified the expected first 21 residues, with the exception of the terminal Met . The isolated human ferric P450 1A1 was predominantly in the high spin state, in contrast to the orthologous rat and rabbit enzymes . Recombinant P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation and benzo{a}pyrene 3-hydroxylation with Km values of 0.58 and 15 microM and Vmax values of 8.3 and 2.5 nmol min-1 (nmol P450 1A1)-1, respectively . The successful expression and purification of human P450 1A1 should increase the availability of this enzyme and the generation of antibodies for further biochemical and other biological studies.

Arch Biochem Biophys, 1994 Aug 1, 312(2), 385 - 91
Effects of nitroxide stable radicals on juglone cytotoxicity; Zhang R et al.; Nitroxides stable radicals are unreactive toward most diamagnetic molecules, but readily undergo one-electron redox reactions with paramagnetic species such as free radicals and transition metals, thus serving as cell-permeable antioxidants . The cytotoxicity of juglone (5-hydroxy-1,4-naphthoquinone), like that of other naphthoquinones, requires bioreduction to yield the semiquinone which in turn reduces oxygen to O2.- . Therefore, nitroxides are expected to mitigate cytotoxicity of quinone-based xenobiotics, such as naphthoquinones . In the present study, in vitro scission of isolated DNA was induced upon juglone reduction by glutathione and Fe(II) ions, however, not by xanthine oxidase or cytochrome c reductase . The DNA scission was inhibited by nitroxides, catalase and chelating agents, though not by superoxide dismutase . Juglone was more toxic toward bacterial cells under hypoxia than under air . Nitroxides < or = 2 mM protected bacterial cells from juglone-induced toxicity under both aerobic and hypoxic conditions . The cytoprotective effect of lipophilic nitroxide was greater than that of hydrophilic ones . Catalase and metal chelating agents decreased juglone-induced cell killing, whereas H2O2 increased it . The mechanisms underlying the nitroxides protective effect involve (a) the reoxidation of reduced transition metal ions, (b) the selective radical-radical reaction with juglone semiquinone, and possibly (c) under aerobic condition catalytic removal of extra- and intracellular O2.- . The present results suggest also that the cell membrane rather than DNA is the main target of juglone toxicity.

Mol Cell Biol, 1994 Aug, 14(8), 5114 - 22
Simian virus 40 origin- and T-antigen-dependent DNA replication with Drosophila factors in vitro; Kamakaka RT et al.; DNA replication of double-stranded simian virus 40 (SV40) origin-containing plasmids, which has been previously thought to be a species-specific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Drosophila melanogaster . This reaction is dependent upon both large T antigen, the SV40-encoded replication initiator protein and DNA helicase, and a functional T-antigen binding site at the origin of DNA replication . The efficiency of replication with extracts derived from Drosophila embryos is approximately 10% of that observed with extracts prepared from human 293 cells . This activity is not a unique property of embryonic extracts, as cytoplasmic extracts from Drosophila tissue culture cells also support T-antigen-mediated replication of SV40 DNA . By using highly purified proteins, DNA synthesis is initiated by Drosophila polymerase alpha-primase in a T-antigen-dependent manner in the presence of Drosophila replication protein A (RP-A; also known as single-stranded DNA-binding protein), but neither human RP-A nor Escherichia coli single-stranded DNA-binding protein could substitute for Drosophila RP-A . In reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymerase alpha-primase . These results collectively indicate that many of the specific functional interactions among T antigen, polymerase alpha-primase, and RP-A are conserved from primates to Drosophila species . Moreover, the observation that SV40 DNA replication can be performed with Drosophila factors provides a useful assay for the study of bidirectional DNA replication in Drosophila species in the context of a complete replication reaction.

J Virol, 1994 Aug, 68(8), 5239 - 46
Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli; Henry LJ et al.; The adenovirus fiber protein is used for attachment of the virus to a specific receptor on the cell surface . Structurally, the protein consists of a long, thin shaft that protrudes from the vertex of the virus capsid and terminates in a globular domain termed the knob . To verify that the knob is the domain which interacts with the cellular receptor, we have cloned and expressed the knob from adenovirus type 5 together with a single repeat of the shaft in Escherichia coli . The protein was purified by conventional chromatography and functionally characterized for its interaction with the adenovirus receptor . The recombinant knob domain bound about 4,700 sites per HeLa cell with an affinity of 3 x 10(9) M-1 and blocked adenovirus infection of human cells . Antibodies raised against the knob also blocked virus infection . By gel filtration and X-ray diffraction analysis of protein crystals, the knob was shown to consist of a homotrimer of 21-kDa subunits . The results confirm that the trimeric knob is the ligand for attachment to the adenovirus receptor.

J Virol, 1994 Aug, 68(8), 5232 - 8
Selected mutations of the duck hepatitis B virus P gene RNase H domain affect both RNA packaging and priming of minus-strand DNA synthesis; Chen Y et al.; The genome of all hepadnaviruses has an open reading frame called the P gene, which encodes a polypeptide of 90 to 97 kDa . The product or products of this P gene are involved in multiple functions of the viral life cycle . These functions include a priming activity which initiates minus-strand DNA synthesis, a polymerase activity which synthesizes DNA by using either RNA or DNA templates (reverse transcriptase), a nuclease activity which degrades the RNA strand of RNA-DNA hybrids (RNase H), and involvement in packaging the RNA pregenome into nucleocapsids . In a previous study, we found that a single point mutation at position 711 in the duck hepatitis B virus (DHBV) P gene product RNase H domain prevented viral RNA packaging . In the present experiments, we have mutated additional conserved amino acids in the DHBV RNase H domain and examined the ability of viral genomes containing these mutations to package RNA and replicate viral DNA . Charged and sulfur group amino acids adjacent to Cys-711 were mutated . None of these mutants was defective in either RNA packaging or viral replication . We also tested a number of mutations on the basis of common elements in the crystal structures of Escherichia coli and human immunodeficiency virus reverse transcriptase RNase H enzymes and on the basis of the similarities of their amino acid sequences to those of the RNase H domains of DHBV and HBV . Our results revealed that the entire beta 4 strand and amino acids Leu-712, Leu-697, and Val-719 in the putative hydrophobic cores of the beta 4, alpha A, and alpha B regions, respectively, are involved in pregenomic RNA encapsidation . This suggests that the basic structure of the RNase H domain in the DHBV P gene product is required for viral RNA packaging . We used the in vitro DHBV minus-strand DNA priming system developed by Wang and Seeger (G.-H . Wang and C . Seeger, Cell 71:663-670, 1992) to test the effect of RNase H packaging mutations on P gene product enzymatic activity . While all packaging-defective mutants tested maintained DNA priming activity, levels were decreased 5- to 20-fold compared with that of the wild-type genome . This observation suggests that the hepadnavirus RNase H domain plays a role in optimizing priming of minus-strand DNA synthesis.

J Neurochem, 1994 Aug, 63(2), 612 - 6
Induction of manganese superoxide dismutase by cytokines and lipopolysaccharide in cultured mouse astrocytes; Mokuno K et al.; To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes . Treatment of astrocytes with 10(2) to 10(4) U/ml tumor necrosis factor-alpha for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions . Treatment with 1.0 microgram/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent . Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1 alpha at concentrations of 10(2) or 10(3) U/ml . However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes . By contrast, interferon-gamma did not change the levels of either Mn or Cu/Zn SOD in the cells . The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues . The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.

Cancer Res, 1994 Aug 1, 54(15), 3963 - 6
Treatment of malignant gliomas using ganciclovir-hypersensitive, ribonucleotide reductase-deficient herpes simplex viral mutant; Mineta T et al.; We have demonstrated that attenuated mutants of herpes simplex virus (HSV) have therapeutic potential for malignant brain tumors . In this report, we tested a ribonucleotide reductase-deficient (RR-) HSV mutant as an experimental treatment for malignant brain tumors . The HSV-RR- mutant hrR3, containing an Escherichia coli lacZ gene insertion in the ICP6 gene that encodes the large subunit of RR, was used in this study . We examined the cytopathic effect of hrR3 (0.1 plaque-forming unit/cell) on the U-87MG human glioblastoma cell line in vitro . Only 0.2% of U-87 cells were alive 67 h postinfection . Drug sensitivity assays demonstrated that hrR3 is hypersensitive to the antiherpetic agent ganciclovir . For in vivo studies, 10 animals harboring U-87MG tumors were randomly divided and treated intraneoplastically with either 5 x 10(6) plaque-forming units of hrR3 or medium alone . The viral treatment group showed significant inhibition of tumor growth (P < 0.01; one-sided Wilcoxon rank test) . Expression of the lacZ gene in hrR3, visualized by 5-bromo-4-chrolo-3-indolyl-beta-D-galactopyranoside histochemistry, could be detected in treated tumors . The therapeutic potential of this HSV-RR- mutant for malignant gliomas is discussed.

Virology, 1994 Aug 1, 202(2), 912 - 20
Identification of hepatitis B virus core protein regions exposed or internalized at the surface of HBcAg particles by scanning with monoclonal antibodies; Pushko P et al.; Hepatitis B virus (HBV) core antigen (HBcAg) particles purified from Escherichia coli were probed in a competition enzyme immunoassay (EIA) with a panel of 16 murine monoclonal antibodies (MAbs) directed to different forms of core protein . The linear binding sites of the MAbs were mapped by combination of solid-phase and competition EIA using synthetic peptides covering the complete sequence of HBV core protein . Relative accessibilities of the linear binding sites at the HBcAg surface were investigated by comparing reactivities in solution of the MAbs to (i) two genetic variants of particulate HBcAg, (ii) denatured core protein, and (iii) synthetic peptides mimicking the appropriate linear binding sites . Further, accessibilities of HBV preS1 and preS2 epitopes (introduced into core protein at positions 77 or 144) at the surface of chimeric HBcAg particles were investigated . The previously described surface localization of core protein region 78-83 at the core particle surface was confirmed . In addition, another region, encompassing residues 127-133, was found to occupy a surface position at particulate HBcAg, whereas regions 9-20 and 133-145 were exposed after denaturation of the core protein and at synthetic peptides but not at particulate HBcAg.

Virology, 1994 Aug 1, 202(2), 771 - 81
Construction of recombinant avian infectious laryngotracheitis virus expressing the beta-galactosidase gene and DNA sequencing of the insertion region; Guo P et al.; Avian infectious laryngotracheitis virus (ILTV), a herpesvirus, is a highly contagious pathogen that causes an upper respiratory tract infection in chickens . It is one of the major problems in the poultry industry worldwide . Current vaccines are not satisfactory due to the induction of latent infection . Here we describe a system for the construction of recombinant ILTV . A 4-kbp ILTV EcoRI DNA fragment was cloned into plasmid pUC13 and sequenced . Computer prediction revealed two potential open reading frames with 216 and 259 amino acid residues, respectively . The 259-residue polypeptide was serine-rich . The beta-galactosidase (beta-gal) gene of E . coli was cloned into the XhoI/Bg/II site of this DNA fragment, integrated into the ILTV genome via homologous recombination, expressed under the control of the immediate-early cytomegalovirus promoter, and caused the formation of blue plaques in the presence of X-gal . The insertion of a foreign gene into the ILTV genome and the successful expression of the incorporated gene demonstrated the potential for the construction of attenuated recombinant ILTV vaccines and the development of ILTV as vectors for polyvalent vaccines against avian upper respiratory tract infections.

Virology, 1994 Aug 1, 202(2), 642 - 50
Characterization of the Shope fibroma virus DNA ligase gene; Parks RJ et al.; The Shope fibroma virus (SFV) DNA ligase gene has been cloned and sequenced, and the biochemical requirements of the gene product have been determined in vitro . The SFV ligase gene maps to the BamHI L1/L2 boundary and spans 1.7 kb . The gene is predicted to encode a 559-amino-acid protein of M(r) = 63,139 which shares 45% amino acid identity with Orthopoxvirus ligases . The C-terminal two-thirds of the protein appears to encode the catalytic domain and shares distant homology with many ligases . The N-terminal homology is shared between only Orthopoxviruses and Leporipoxviruses and suggests that DNA ligases may be composite structures consisting of two independently evolved protein domains . Although the the gene encodes features characteristic of both early and late poxviral genes, Northern analysis showed that SFV ligase is expressed as a late gene product . In order to prove the identity of the protein it was expressed as a glutathione S-transferase fusion in Escherichia coli, affinity purified, and shown to be a Mg2+.ATP-dependent ligase in vitro . The recombinant protein can also form a covalent ligase.AMP complex characteristic of ATP-dependent DNA ligases . The SFV ligase gene can be disrupted and is thus not essential for viral growth in culture . This was shown by recombining a PCR product, encoding a P7.5 promoter and E . coli guanine phosphoribosyltransferase gene (gpt) into the open reading frame, and selecting for gpt+ viruses . This work provides insights into the evolution of Orthopoxviruses and Leporipoxviruses and strains suitable for a detailed analysis of the role DNA ligases play in poxviral recombination.

Virology, 1994 Aug 1, 202(2), 1070 - 5
Molecular characterization of the AL3 protein encoded by a bipartite geminivirus; Pedersen TJ et al.; The genome of tomato golden mosaic virus (TGMV) is composed of two circular, single-stranded DNA molecules that together contain 6 open reading frames (ORFs) . Three of these ORFs (designated AL1, AL2, and AL3) overlap and are specified by multiple polycistronic mRNAs . No RNA specifying the AL3 ORF alone has been detected, suggesting that the AL3 gene product is translated from an internal ORF . A recombinant histidine-tagged-AL3 fusion protein was purified from Escherichia coli and used to raise a polyclonal antiserum . Analysis of protein extracts from healthy plants and plants infected with TGMV by SDS-PAGE and immunoblotting showed that a protein corresponding to the predicted AL3 gene product is produced only in infected plants . This protein comprises approximately 0.05% of the cellular proteins and is present in the soluble and organelle fractions . These results are discussed with respect to the expression and role of the AL3 protein in the viral life cycle.

Virology, 1994 Aug 1, 202(2), 1018 - 23
Mouse hepatitis virus gene 5b protein is a new virion envelope protein; Yu X et al.; Highly purified radiolabeled mouse hepatitis virus (MHV) A59 contained a previously overlooked protein which coelectrophoreses with the gene 5b product immunoprecipitated from infected cells . The gene 5b protein is post-translationally acylated . Rabbit antibody raised against a recombinant gene 5b protein expressed in Escherichia coli neutralized viral infectivity in the presence of complement, although not in the absence of complement . Immunofluorescent staining of MHV-infected cells with two anti-peptide antibodies revealed that the gene 5b product is membrane-associated and is transported to the cell surface, findings consistent with the prediction of a membrane-spanning segment in the gene 5b polypeptide . These results suggest strongly that the gene 5b polypeptide represents a new MHV virion envelope protein which is homologous to the TGEV ORF 4 and IBV 3c proteins.

J Urol, 1994 Aug, 152(2 Pt 1), 506 - 9
Adenoviral-mediated gene transfer to bladder in vivo; Morris BD Jr et al.; This study was designed to examine the potential for gene therapy in bladder in vivo using adenoviral vectors . Gene transfer to rat bladders was accomplished via direct intravesical instillation using a replication-defective adenoviral vector containing a marker gene encoding for Escherichia coli beta-galactosidase (beta-gal) . Successful gene transfer was confirmed by analyzing bladder samples for DNA and RNA using polymerase chain reaction (PCR) with primers specific for beta-gal and adeno sequences, detecting beta-gal in full-thickness bladder wall using specific histochemical staining (X-gal) and documenting recombinant protein production . Bladder architecture was preserved, without evidence of distant spread of virus as assessed by PCR . Gene expression was evident for at least 7 days . In summary, bladder cells can be genetically altered using replication-deficient adenoviral vectors via simple intravesical instillation of vector . Introduction of exogenous genetic material is a potentially powerful therapeutic modality for immunomodulation of bladder neoplasms.

J Appl Physiol, 1994 Aug, 77(2), 567 - 73
Regional lung hematocrit variation and assessment of acute lung injury; Kanazawa M et al.; Estimating blood content in the lung remains a key step in calculating lung water volume and microvascular permeability . We studied the effect of regional lung hematocrit (Hct) variation on assessment of acute lung injury . Escherichia coli endotoxin was administered in guinea pigs intravenously . Lung injury was evaluated by measuring the wet-to-dry weight ratio (W/D) and transvascular 125I-labeled albumin leakage for 3 h {tissue-to-plasma 125I-albumin ratio (T/P)} in five tissue samples from each animal . Residual blood content was corrected using either 51Cr-red blood cells as a blood cell marker, 99mTc-albumin as a plasma marker, or both, injected 10 min before the guinea pigs were killed . Lung Hct, estimated from the marker counts of lung and peripheral blood samples, was lower than peripheral blood Hct; intraindividual variation, represented by the standard deviation in each subject, was 0.024 +/- 0.015 for the control group (coefficient of variation 8.0 +/- 5.1%) and 0.026 +/- 0.013 for the endotoxin group (coefficient of variation 8.5 +/- 4.1%) . Uncorrected W/D for residual blood content was greater than the corrected W/D . 99mTc-albumin correction gave values closer to the W/D corrected by both markers . T/P corrected by 99mTc-albumin showed smaller data variations than the values obtained with 51Cr-red blood cell correction, which was affected by variations in lung Hct . We recommend using a plasma marker to correct for blood content in assessing acute lung injury by W/D and T/P.

Indian J Biochem Biophys, 1994 Aug, 31(4), 339 - 43
DNA topoisomerase I from Mycobacterium smegmatis; Bhaduri T et al.; DNA topoisomerase I has been purified from Mycobacterium smegmatis to near homogeneity using different column chromatographic techniques . The enzyme activity relaxes form I DNA into form IV DNA, requiring Mg2+, but not ATP or any other cofactors for its activity . Several properties of the enzyme were found to be similar to that of the prototype enzyme, Escherichia coli topoisomerase I.

Mol Mar Biol Biotechnol, 1994 Aug, 3(4), 192 - 9
An efficient expression vector for transgenic medaka construction; Takagi S et al.; The transparency and external fertilization of the eggs of medaka (Oryzias latipes) make them ideally suitable for investigating molecular interactions that occur during vertebrate development . Genetically engineered medaka is a potential tool for such studies . It requires several types of suitable expression vectors . To obtain abundant and ubiquitous expression of foreign genes in medaka embryos, we have designed an expression vector that contains the proximal promoter and enhancer elements and polyadenylation signal of the medaka beta-actin gene . The utility of this "all-medaka" expression vector was examined using the Escherichia coli lacZ gene as a reporter gene . Most of the injected embryo showed high gene expression, and several embryos showed ubiquitous expression even at six days after injection . Of nine individuals derived from the injected embryos and grown until adult stage, one produced expression-positive F1 fish . The transgene was identified in these F1 using polymerase chain reaction (PCR) . These data revealed that the expression vector based on the expression cassette from the medaka beta-actin gene should be useful for making transgenic medaka . The cloned gene in this cassette vector is stably transmittable and efficiently expressible.

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