Curr Opin Biotechnol, 1995 Oct, 6(5), 507 - 16 Effects of overexpressing folding modulators on the in vivo folding of heterologous proteins in Escherichia coli; Wall JG et al.; Interest continues to increase in the use of folding modulators to overcome problems with heterologous protein folding in Escherichia coli . Currently, this approach, though highly successful with a number of individual proteins, remains a somewhat hit-and-miss affair . Ongoing research directed at unraveling the precise role and specificity of these folding modulators should generate a clearer understanding of the potential and limitations of overexpressing folding catalysts in vivo . This will facilitate the development, in the not too distant future, of a more structured and rational approach to improving the folding of heterologous gene products in E . coli. Curr Opin Biotechnol, 1995 Oct, 6(5), 494 - 500 Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli; Kane JF; Within Escherichia coli and other species, a clear codon bias exists among the 61 amino acid codons found within the population of mRNA molecules, and the level of cognate tRNA appears directly proportional to the frequency of codon usage . Given this situation, one would predict translational problems with an abundant mRNA species containing an excess of rare low tRNA codons . Such a situation might arise after the initiation of transcription of a cloned heterologous gene in the E . coli host . Recent studies suggest clusters of AGG/AGA, CUA, AUA, CGA or CCC codons can reduce both the quantity and quality of the synthesized protein . In addition, it is likely that an excess of any of these codons, even without clusters, could create translational problems. Plant Mol Biol, 1995 Oct, 29(2), 303 - 15 Post-translational processing of the highly processed, secreted periplasmic carbonic anhydrase of Chlamydomonas is largely conserved in transgenic tobacco; Roberts CS et al.; The periplasmic carbonic anhydrase (CA) gene CAH1 of Chlamydomonas reinhardtii codes for a highly processed secreted glycoprotein . The primary translation product of the CAH1 gene is targeted to the ER, where it is proteolytically processed to yield two different subunits, glycosylated, assembled into an active heterotetramer, and secreted . After replacing the target leader sequence with that from tobacco anionic peroxidase, expression of this gene in transgenic tobacco plants was investigated . SDS-PAGE gels of the purified protein from tobacco, showed that it migrated as a series of discrete bands (two large and one small) with slightly faster mobility than the comparable bands in the purified algal protein . The expressed protein in the plant was active, and staining with thymol and sulfuric acid confirmed that it was also glycosylated . The periplasmic CA1 (peri-CA1) also was found to be enriched in the intercellular fluid of transgenic tobacco, indicating it was secreted . The specific activity of the enzyme and its sensitivity to sulfonamide inhibitors were similar to that of the native algal enzyme . These results suggest that the post translational processing of Chlamydomonas peri-CA1 is largely conserved in a higher plant. Plant Mol Biol, 1995 Oct, 29(2), 267 - 78 Identification of a cDNA that encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii; Brown AP et al.; Two different techniques were used to isolate potential cDNAs for acyl-CoA: 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPA-AT) enzymes from Limnanthes douglasii . Both heterologous screening with the maize pMAT1 clone and in vivo complementation of the Escherichia coli mutant JC201 which is deficient in LPA-AT activity, were carried out . Clones identified by these procedures were different . Homology searches demonstrated that the clone isolated by heterologous probing, pLAT1, encodes a protein which is most similar to the maize (open reading frame in pMAT1) and yeast SLC1 proteins, which are putative LPA-AT sequences . This L . douglasii sequence shows much lower homology to the E . coli LPA-AT protein PlsC, which is the only LPA-AT sequence confirmed by over-expression studies . The clone isolated by complementation, pLAT2, encodes a protein with homology to both SLC1 and PlsC . It was not possible to over-express the complementing protein encoded by pLAT2 but further experimentation on membranes from complemented JC201 demonstrated that they possess a substrate specificity distinctly different from PlsC and similar to Limnanthes sp . microsome specificity . This data strongly supports the contention that pLAT2 is an LPA-AT clone . Northern blot analysis revealed different expression patterns for the two genes in pLAT1 and pLAT2 . Transcription of the gene encoding the insert of pLAT2 occurred almost exclusively in developing seed tissue, whilst the cDNA of pLAT1 hybridised to poly(A)+ mRNA from seed, stem and leaf, demonstrating more widespread expression throughout the plant . Southern blot analysis indicated that the cDNA of pLAT2 was transcribed from a single-copy gene while that for pLAT1 was a member of a small gene family. Neuron, 1995 Oct, 15(4), 941 - 9 An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula; Swartz KJ et al.; The Kv2.1 voltage-activated K+ channel, a Shab-related K+ channel isolated from rat brain, is insensitive to previously identified peptide inhibitors . We have isolated two peptides from the venom of a Chilean tarantula, G . spatulata, that inhibit the Kv2.1 K+ channel . The two peptides, hanatoxin1 (HaTx1) and hanatoxin2 (HaTx2) are unrelated in primary sequence to other K+ channel inhibitors . The activity of HaTx was verified by synthesizing it in a bacterial expression system . The concentration dependence for both the degree of inhibition at equilibrium (Kd = 42 nM) and the kinetics of inhibition (kon = 3.7 x 10(4) M-1s-1; koff = 1.3 x 10(-3) s-1), are consistent with a bimolecular reaction between HaTx and the Kv2.1 K+ channel . Shaker-related, Shaw-related, and eag K+ channels were relatively insensitive to HaTx, whereas a Shal-related K+ channel was sensitive . Regions outside the scorpion toxin binding site (S5-S6 linker) determine sensitivity to HaTx . HaTx introduces a new class of K+ channel inhibitors that will be useful probes for studying K+ channel structure and function. Biochem J, 1995 Oct 1, 311 ( Pt 1), 209 - 17 Characterization of bovine tracheobronchial phenol sulphotransferase cDNA and detection of mRNA regulation by cortisol; Schauss SJ et al.; Phenol sulphotransferases esterify both endogenous and foreign hydroxylated aromatic compounds with sulphate . Since these enzymes participate in both hormone and drug metabolism, elucidating their regulation at both the enzymic and molecular levels may provide new understanding in several metabolic pathways . The primary structure of a bovine phenol sulphotransferase has been determined by isolation of the corresponding cDNA . Two partial bovine cDNAs were first isolated by probing a tracheal epithelial cell lambda gt11 cDNA library with a rat phenol sulphotransferase cDNA . These clones provided the sequences of the 5' and 3' ends of the predicted coding region . A contiguous cDNA was subsequently isolated by PCR using 5' and 3' oligonucleotide primers and the cDNA library as the template . The sequence of the resulting approx . 1 kbp cDNA predicted an amino acid sequence that included sequences determined for several tryptic peptides of the purified protein . Antiserum directed to a synthetic N-terminal peptide predicted by the cDNA sequence showed reactivity with the purified enzyme . High-level Trc-promoter-driven expression of the recombinant bovine enzyme was achieved in Escherichia coli . The bovine cDNA was used to determine relative steady-state levels of phenol sulphotransferase transcripts in bovine lung tissues; distal lung parenchymal RNA levels were 6-10-fold greater than those in tracheobronchial epithelium . Using a bronchial epithelial cell culture model, however, cortisol was observed to increase mRNA levels by 5-fold in both a dose- and time-dependent manner; this corresponds to previously reported glucocorticoid stimulation of phenol sulphotransferase activity in this system {Beckmann, Illig and Bartzatt (1994) J . Cell Physiol . 160, 603-610}. Arch Biochem Biophys, 1995 Oct 1, 322(2), 445 - 52 Identification of a topology control domain in the tetracycline resistance protein; Miller KW et al.; Two N-terminal fusion proteins combining Escherichia coli maltose-binding protein (MBP) and the 12-transmembrane-segment pBR322 tetracycline resistance protein (Tet) have been constructed to determine the strength and location of topology control signals within the N-terminal portion of the Tet protein . The fusions contain either a secretable (wild-type) or a nonsecretable (MBP delta 2-26) MBP domain joined to the normally cytoplasmic N-terminus of the Tet protein . The effects of MBP targeting on Tet topology were investigated by analyzing the susceptibility of fusion strains to tetracycline and by proteolysis of the fusion proteins in inverted membrane vesicles and spheroplasts . The fusion protein containing MBP delta 2-26 conferred tetracycline resistance to the host strain and gave a normal pattern of Tet digestion fragments, indicating that its Tet domain is oriented and folded properly in the membrane . In contrast, the fusion containing secretable MBP was catalytically inactive apparently due to transfer of the Tet N-terminus to the periplasm with MBP . However, protease treatment of this fusion revealed that MBP secretion seems to affect only the topology of segments 1 and/or 2 of the Tet domain . Therefore, a strong topology control sequence appears to be located in the first cytoplasmic loop of the protein. Arch Biochem Biophys, 1995 Oct 1, 322(2), 369 - 77 Purification and characterization of recombinant cytochrome P450TYR expressed at high levels in Escherichia coli; Halkier BA et al.; The multifunctional tyrosine N-hydroxylase, cytochrome P450TYR (CYP79), from Sorghum bicolor catalyzing the conversion of tyrosine to p-hydroxyphenyl-acetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin, has been expressed in Escherichia coli using the isopropyl-beta-D-thiogalactopyranoside-inducible vector pSP19g10L, containing the cDNA encoding CYP79 . The expression construct was optimized by reducing the length of the N-terminal hydrophobic core of the signal sequence of cytochrome P450TYR and by exchanging the first eight codons with the first eight codons of bovine P45017 alpha . The highest yielding construct provided 200-500 nmol P450TYR/liter cell culture . The recombinant P450TYR was gently and efficiently extracted from E . coli spheroblasts by temperature-induced phase partitioning of Triton X-114 in the presence of 30% glycerol and isolated by DEAE and reactive red chromatography . In reconstitution experiments using saturating amounts of sorghum NADPH-cytochrome P450 reductase, the Km and turnover rate for isolated recombinant P450TYR was 0.22 +/- 0.06 mM and 49.2 +/- 3.8 min-1, respectively, whereas a turnover rate as high as 350 min-1, was obtained using E . coli membranes . Addition of 3 mM glutathione stimulated the activity of reconstituted P450TYR and of sorghum microsomes although the effect was highly variable . Phenylalanine, the precursor of several cyanogenic glucosides, gave a type I binding spectrum, but was not metabolized by P450TYR, demonstrating the high substrate specificity of this P450 . Administration of radioactively labeled p-hydroxyphenylacetaldoxime to E . coli cells, showed E . coli metabolized p-hydroxyphenylacetaldoxime independent of the expression of P450TYR. Virology, 1995 Oct 1, 212(2), 641 - 9 Recombinant duck interferon: a new reagent for studying the mode of interferon action against hepatitis B virus; Schultz U et al.; Although interferon is widely used to treat chronic hepatitis B virus infections, its mode of action against hepadnaviruses is largely unknown . This deficit is due mainly to the lack of suitable model systems . The duck system could not be used because purified duck interferon was not available in sufficient quantities . We have now cloned a DNA fragment that contains an intronless gene for duck interferon . The primary translation product consists of 191 amino acids, the N-terminal 30 residues of which constitute a signal peptide . Mature duck interferon is 50% identical to the recently cloned chicken interferon . Sequence homology to mammalian interferons is marginal, but conservation of four cysteine residues and inducibility by virus indicate a distant relationship between duck interferon and mammalian type I interferons . Purified recombinant duck interferon from Escherichia coli is biologically active: it activates the interferon-inducible Mx gene, prevents cell destruction by cytolytic RNA viruses, and has a strong inhibitory effect on duck hepatitis B virus in cultured primary duck hepatocytes . This new reagent should help to define the interferon-sensitive step of the hepadnavirus life cycle . Furthermore, the duck system can now be used for systematic studies of the in vivo effectiveness of interferon in chronic hepatitis B virus infections. Scand J Immunol, 1995 Oct, 42(4), 487 - 92 Secretion of MPB64 antigen by a recombinant clone of Mycobacterium smegmatis: characterization and application for the diagnosis of tuberculosis; Tasaka H et al.; MPB64, a specific antigen to Mycobacterium tuberculosis complex (TB complex), was produced and secreted by a clone of M . smegmatis-MPB64 where the structural gene of MPB64 was inserted using a new mycobacteria, E . coli shuttle plasmid pNIS vector . Antibodies against the recombinant MPB64 (rMPB64) were used for the reverse particle latex agglutination (RPLA) test to detect the MPB64 antigen rapidly . RPLA tests were applied to the shock cultures and the clinical isolates of mycobacteria to identify TB complex . RPLA with anti-MPB64 antibody-coated latex beads completely distinguished TB complex from other mycobacteria . Thus, it is suggested that RPLA with anti-MPB64 antibody would be a new, easy and inexpensive method for the rapid diagnosis of tuberculosis. Mutat Res, 1995 Oct, 338(1-6), 189 - 201 Factors affecting somatic mutation frequencies in vivo; Zhang XB et al.; The factors that influence the spontaneous mutant frequencies in mammalian tissues have been ranked on the basis of data from our laboratory together with published data . Some of the data come from the endogenous hprt and Dlb-1 loci, but most come from transgenic mice carrying the bacterial lacI and lacZ genes in recoverable lambda phage vectors . Since there is evidence that these bacterial loci are selectively neutral, the mutant frequency observed is the integral of the mutation rates from the formation of the zygote . The factors that affect the inferred mutation rate, in decreasing order of importance are: site of integration of the transgene, age, tissue, and strain . Insufficient data exist to determine the influence of gender (probably small) and inter-laboratory variables (probably at least as important as age) . The two most surprising results are (1) that about half of all mutations arise during development (and half of these in utero) and (2) that most somatic tissues, whether queiscent or actively proliferating, have similar mutant frequencies and similar increases during adult life. Mol Cell Biol, 1995 Oct, 15(10), 5376 - 88 Expression and regulation by interferon of a double-stranded-RNA-specific adenosine deaminase from human cells: evidence for two forms of the deaminase; Patterson JB et al.; A 6,474-nucleotide human cDNA clone designated K88, which encodes double-stranded RNA (dsRNA)-specific adenosine deaminase, was isolated in a screen for interferon (IFN)-regulated cDNAs . Northern (RNA) blot analysis revealed that the K88 cDNA hybridized to a single major transcript of approximately 6.7 kb in human cells which was increased about fivefold by IFN treatment . Polyclonal antisera prepared against K88 cDNA products expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins recognized two proteins by Western (immunoblot) analysis . An IFN-induced 150-kDa protein and a constitutively expressed 110-kDa protein whose level was not altered by IFN treatment were detected in human amnion U and neuroblastoma SH-SY5Y cell lines . Only the 150-kDa protein was detected in mouse fibroblasts with antiserum raised against the recombinant human protein; the mouse 150-kDa protein was IFN inducible . Immunofluorescence microscopy and cell fractionation analyses showed that the 110-kDa protein was exclusively nuclear, whereas the 150-kDa protein was present in both the cytoplasm and nucleus of human cells . The amino acid sequence deduced from the K88 cDNA includes three copies of the highly conserved R motif commonly found in dsRNA-binding proteins . Both the 150-kDa and the 110-kDa proteins prepared from human nuclear extracts bound to double-stranded but not to single-stranded RNA affinity columns . Furthermore, E . coli-expressed GST-K88 fusion proteins that included the R motif possessed dsRNA-binding activity . Extracts prepared either from K88 cDNA-transfected cells or from IFN-treated cells contained increased dsRNA-specific adenosine deaminase enzyme activity . These results establish that K88 encodes an IFN-inducible dsRNA-specific adenosine deaminase and suggest that at least two forms of dsRNA-specific adenosine deaminase occur in human cells. Mol Cell Biol, 1995 Oct, 15(10), 5329 - 38 Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product; Onel K et al.; The REC1 gene of Ustilago maydis has an uninterrupted open reading frame, predicted from the genomic sequence to encode a protein of 522 amino acid residues . Nevertheless, an intron is present, and functional activity of the gene in mitotic cells requires an RNA processing event to remove the intron . This results in a change in reading frame and production of a protein of 463 amino acid residues . The 3'-->5' exonuclease activity of proteins derived from the REC1 genomic open reading frame, the intronless open reading frame, and several mutants was investigated . The mutants included a series of deletions constructed by removing restriction fragments at the 3' end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation sensitivity . All of these proteins were overproduced in Escherichia coli as N-terminal polyhistidine-tagged fusions that were subsequently purified by immobilized metal affinity chromatography and assayed for 3'-->5' exonuclease activity . The results indicated that elimination of the C-terminal third of the protein did not result in a serious reduction in 3'-->5' exonuclease activity, but deletion into the midsection caused a severe loss of activity . The biological activity of the rec1-1 allele, which encodes a truncated polypeptide with full 3'-->5' exonuclease activity, and the rec1-5 allele, which encodes a more severely truncated polypeptide with no exonuclease activity, was investigated . The two mutants were equally sensitive to the lethal effect of UV light, but the spontaneous mutation rate was elevated 10-fold over the wild-type rate in the rec1-1 mutant and 100-fold in the rec1-5 mutant . The elevated spontaneous mutation rate correlated with the ablation of exonuclease activity, but the radiation sensitivity did not . These results indicate that the C-terminal portion of the Rec1 protein is not essential for exonuclease activity but is crucial in the role of REC1 in DNA damage repair. J Mol Evol, 1995 Oct, 41(4), 440 - 8 Sex in Escherichia coli does not disrupt the clonal structure of the population: evidence from random amplified polymorphic DNA and restriction-fragment-length polymorphism; Desjardins P et al.; Analysis of the Escherichia coli population by multilocus enzyme electrophoresis (MLEE) has established its clonal organization, but there is increasing evidence that horizontal DNA transfer occurs in E . coli . We have assessed the genetic structure of the species E . coli and determined the extent to which recombination can affect the clonal structure of bacteria . A panel of 72 E . coli strains from the ECOR collection was characterized by random amplified polymorphic DNA (RAPD) and restriction-fragment-length polymorphism (RFLP) of the ribosomal RNA gene (rrn) regions . These strains have been characterized by MLEE and are assumed to reflect the range of genotypic variation in the species as a whole . Statistical analysis, including factorial analysis of correspondence (FAC) and hierarchical classifications, established that the data obtained with the three genetic markers are mutually corroborative, thus providing compelling evidence that horizontal transfer does not disrupt the clonal organization of the population . However, there is a gradient of correlation between the different classifications which ranges from the highly clonal structure of B2 group strains causing extraintestinal infections in humans to the less-stringent structure of B1 group strains that came mainly from nonprimate mammals . This group (B1) appears to be the framework from which the remaining non-A group strains have emerged . These results indicate that RAPD analysis is well suited to intraspecies characterization of E . coli . Lastly, treating the RAPD data by FAC allowed description of subgroup-specific DNA fragments which can be used, in a strategy comparable to positional cloning, to isolate virulence genes. J Neurochem, 1995 Oct, 65(4), 1771 - 9 Characteristics of the chromaffin granule aspartic proteinase involved in proenkephalin processing; Azaryan AV et al.; Proteolytic processing of neuropeptide precursors is required for production of active neurotransmitters and hormones . In this study, a chromaffin granule (CG) aspartic proteinase of 70 kDa was found to contribute to enkephalin precursor cleaving activity, as assayed with recombinant ({35S}Met) preproenkephalin . The 70-kDa CG aspartic proteinase was purified by concanavalin A-Sepharose, Sephacryl S-200, and pepstatin A agarose affinity chromatography . The proteinase showed optimal activity at pH 5.5 . It was potently inhibited by pepstatin A, a selective aspartic proteinase inhibitor, but not by inhibitors of serine, cysteine, or metalloproteinases . Lack of inhibition by Val-D-Leu-Pro-Phe-Val-D-Leu--an inhibitor of pepsin, cathepsin D, and cathepsin E--distinguishes the CG aspartic proteinases from classical members of the aspartic proteinase family . The CG aspartic proteinase cleaved recombinant proenkephalin between the Lys172-Arg173 pair located at the COOH-terminus of (Met)enkephalin-Arg6-Gly7-Leu8, as assessed by peptide microsequencing . The importance of full-length prohormone as substrate was demonstrated by the enzyme's ability to hydrolyze 35S-labeled proenkephalin and proopiomelanocortin and its inability to cleave tri- and tetrapeptide substrates containing dibasic or monobasic cleavage sites . In this study, results provide evidence for the role of an aspartic proteinase in proenkephalin and prohormone processing. J Infect Dis, 1995 Oct, 172(4), 1119 - 22 Escherichia coli endotoxin-mediated endothelial injury is modulated by glutathione ethyl ester; Morris PE et al.; The mechanisms involved in endotoxin-induced endothelial injury are not fully understood . Oxidant stress is thought to play a role in cell damage after endotoxin exposure . Glutathione may ameliorate these affects . Glutathione ethyl ester (GSE) was used in bovine pulmonary artery endothelial cell (BPAEC) cultures to determine the potential for attenuation of endotoxin-induced injury . GSE (0.05-25 mM) was preincubated with BPAEC for 4 h before endotoxin exposure . Fresh media containing GSE and Escherichia coli endotoxin (0.05 microgram/mL) were then placed on the BPAEC and incubated for 18 h . GSE, at doses of 5 and 25 mM, attenuated endotoxin-induced injury, as reflected by a significant reduction in lactate dehydrogenase release . This was paralleled by a significant increase in endotoxin-stimulated prostaglandin E2 and prostacyclin release . Thus, GSE attenuates endotoxin-induced injury of BPAEC in culture and alters BPAEC prostaglandin metabolism. J Clin Invest, 1995 Oct, 96(4), 1715 - 21 Recombinant peanut allergen Ara h I expression and IgE binding in patients with peanut hypersensitivity; Burks AW et al.; Peanut allergy is a significant health problem because of the frequency, the potential severity, and the chronicity of the allergic sensitivity . Serum IgE from patients with documented peanut hypersensitivity reactions and a peanut cDNA expression library were used to identify clones that encode peanut allergens . One of the major peanut allergens, Ara h I, was selected from these clones using Ara h I specific oligonucleotides and polymerase chain reaction technology . The Ara h I clone identified a 2.3-kb mRNA species on a Northern blot containing peanut poly (A)+ RNA . DNA sequence analysis of the cloned inserts revealed that the Ara h I allergen has significant homology with the vicilin seed storage protein family found in most higher plants . The isolation of the Ara h I clones allowed the synthesis of this protein in E . coli cells and subsequent recognition of this recombinant protein in immunoblot analysis using serum IgE from patients with peanut hypersensitivity . With the production of the recombinant peanut protein it will now be possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general J Bacteriol, 1995 Oct, 177(19), 5707 - 10 Glucose starvation stimulon of Escherichia coli: role of integration host factor in starvation survival and growth phase-dependent protein synthesis; Nystrom T; The DNA-binding protein IHF was found to be required for starvation survival and for the induction of 14 proteins of the glucose starvation stimulon . Many of these proteins have been shown previously to be general responders to diverse stress conditions . Overexpression of IHF during balanced growth was not sufficient to induce these proteins, but it resulted in an increased synthesis of rpoH-dependent heat shock proteins. J Bacteriol, 1995 Oct, 177(19), 5612 - 21 Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction; Brosh RM Jr et al.; Site-directed mutagenesis has been employed to address the functional significance of the highly conserved aspartic and glutamic acid residues present in the Walker B (also called motif II) sequence in Escherichia coli DNA helicase II . Two mutant proteins, UvrDE221Q and UvrDD220NE221Q, were expressed and purified to apparent homogeneity . Biochemical characterization of the DNA-dependent ATPase activity of each mutant protein demonstrated a kcat that was < 0.5% of that of the wild-type protein, with no significant change in the apparent Km for ATP . The E221Q mutant protein exhibited no detectable unwinding of either partial duplex or blunt duplex DNA substrates . The D220NE221Q mutant, however, catalyzed unwinding of both partial duplex and blunt duplex substrates, but at a greatly reduced rate compared with that of the wild-type enzyme . Both mutants were able to bind DNA . Thus, the motif II mutants E221Q and D220NE221Q were able to bind ATP and DNA to the same extent as wild-type helicase II but demonstrate a significant reduction in ATP hydrolysis and helicase functions . The mutant uvrD alleles were also characterized by examining their abilities to complement the mutator and UV light-sensitive phenotypes of a uvrD deletion mutant . Neither the uvrDE221Q nor the uvrDD220NE221Q allele, supplied on a plasmid, was able to complement either phenotype . Further genetic characterization of the mutant uvrD alleles demonstrated that uvrDE221Q confers a dominant negative growth phenotype; the uvrDD220NE221Q allele does not exhibit this effect . The observed difference in effect on viability may reflect the gene products' dissimilar kinetics for unwinding duplex DNA substrates in vitro. J Bacteriol, 1995 Oct, 177(19), 5561 - 6 Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus; Colombo S et al.; Mammalian metallocarboxypeptidases play key roles in major biological processes, such as digestive-protein degradation and specific proteolytic processing . A Sulfolobus solfataricus gene (cpsA) encoding a recently described zinc carboxypeptidase with an unusually broad substrate specificity was cloned, sequenced, and expressed in Escherichia coli . Despite the lack of overall sequence homology with known carboxypeptidases, seven homology blocks, including the Zn-coordinating and catalytic residues, were identified by multiple alignment with carboxypeptidases A, B, and T . S . solfataricus carboxypeptidase expressed in E . coli was found to be enzymatically active, and both its substrate specificity and thermostability were comparable to those of the purified S . solfataricus enzyme. J Bacteriol, 1995 Oct, 177(19), 5554 - 60 Functional analysis of the ffh-trmD region of the Escherichia coli chromosome by using reverse genetics; Persson BC et al.; We have analyzed the essentiality or contribution to growth of each of four genes in the Escherichia coli trmD operon (rpsP, 21K, trmD, and rplS) and of the flanking genes ffh and 16K by a reverse genetic method . Mutant alleles were constructed in vitro on plasmids and transferred by recombination to the corresponding lambda phage clone (lambda 439) and from the phage clone to the E . coli chromosome . An ability to obtain recombinants only in cells carrying a complementing plasmid indicated that the mutated gene was essential, while an ability to obtain recombinants in plasmid-free cells indicated nonessentiality . In this way, Ffh, the E . coli homolog to the 54-kDa protein of the signal recognition particle of mammalian cells, and ribosomal proteins S16 and L19 were shown to be essential for viability . A deletion of the second gene, 21K, of the trmD operon reduced the growth rate of the cells fivefold, indicating that the wild-type 21-kDa protein is important for viability . A deletion-insertion in the same gene resulted in the accumulation of an assembly intermediate of the 50S ribosomal subunit, as a result of polar effects on the expression of a downstream gene, rplS, which encodes ribosomal protein L19 . This finding suggests that L19, previously not considered to be an assembly protein, contributes to the assembly of the 50S ribosomal subunits . Strains deleted for the trmD gene, the third gene of the operon, encoding the tRNA (m1G37)methyltransferase (or TrmD) showed a severalfold reduced growth rate . Since such a strain grew much slower than a strain lacking the tRNA(m(1)G37) methyltransferase activity because of a point mutation, the TrmD protein might have a second function in the cell . Finally, a 16-kDa protein encoded by the gene located downstream of, and convergently transcribed to, the trmD operon was found to be nonessential and not to contribute to growth. J Bacteriol, 1995 Oct, 177(19), 5517 - 22 Inhibition of the release factor-dependent termination reaction on ribosomes by DnaJ and the N-terminal peptide of rhodanese; Kudlicki W et al.; A peptide consisting of the 17 N-terminal amino acids of native bovine rhodanese in combination with the chaperone DnaJ specifically inhibits release factor- and stop codon-dependent hydrolysis of N-formylmethionine from N(formyl)-methionyl-tRNA bound with AUG to salt-washed ribosomes . Neither the peptide nor DnaJ by itself causes this inhibition . The N-terminal peptide and DnaJ both singularly and combined do not affect the peptidyltransferase reaction per se . The total amount of rhodanese synthesized in the cell-free coupled transcription-translation system is reduced by the peptide, with concomitant accumulation of full-length enzymatically inactive rhodanese polypeptides on ribosomes . In combination with DnaJ, the N-terminal polypeptide inhibits the termination and release of full-length rhodanese peptides that have accumulated on Escherichia coli ribosomes during the course of uninhibited coupled transcription-translation in the cell-free system . This inhibition appears to involve release factor 2-mediated termination at the UGA termination codon in the coding sequence for rhodanese . It is suggested that the N-terminal peptide inhibits the binding of the release factor to ribosomes . These data appear to provide the first report of differential inhibition of the termination reaction on ribosomes without inhibition of the peptidyltransferase reaction and peptide elongation. J Bacteriol, 1995 Oct, 177(19), 5506 - 16 Identification and characterization of genes (xapA, xapB, and xapR) involved in xanthosine catabolism in Escherichia coli; Seeger C et al.; We have characterized four genes from the 52-min region on the Escherichia coli linkage map . Three of these genes are directly involved in the metabolism of xanthosine, whereas the function of the fourth gene is unknown . One of the genes (xapA) encodes xanthosine phosphorylase . The second gene, named xapB, encodes a polypeptide that shows strong similarity to the nucleoside transport protein NupG . The genes xapA and xapB are located clockwise of a gene identified as xapR, which encodes a positive regulator belonging to the LysR family and is required for the expression of xapA and xapB . The genes xapA and xapB form an operon, and their expression was strictly dependent on the presence of both the XapR protein and the inducer xanthosine . Expression of the xapR gene is constitutive and not autoregulated, unlike the case for many other LysR family proteins . In minicells, the XapB polypeptide was found primarily in the membrane fraction, indicating that XapB is a transport protein like NupG and is involved in the transport of xanthosine. J Bacteriol, 1995 Oct, 177(19), 5393 - 400 The gene for the longest known Escherichia coli protein is a member of helicase superfamily II; Reuven NB et al.; The Escherichia coli rnt gene, which encodes the RNA-processing enzyme RNase T, is cotranscribed with a downstream gene . Complete sequencing of this gene indicates that its coding region encompasses 1,538 amino acids, making it the longest known protein in E . coli . The gene (tentatively termed lhr for long helicase related) contains the seven conserved motifs of the DNA and RNA helicase superfamily II . An approximately 170-kDa protein is observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-labeled extracts prepared from cells in which lhr is under the control of an induced T7 promoter . This protein is absent when lhr is interrupted or when no plasmid is present . Downstream of lhr is the C-terminal region of a convergent gene with homology to glutaredoxin . Interruptions of chromosomal lhr at two different positions within the gene do not affect the growth of E . coli at various temperatures in rich or minimal medium, indicating that lhr is not essential for usual laboratory growth . lhr interruption also has no effect on anaerobic growth . In addition, cells lacking Lhr recover normally from starvation, plate phage normally, and display normal sensitivities to UV irradiation and H2O2 . Southern analysis showed that no other gene closely related to lhr is present on the E . coli chromosome . These data expand the known size range of E . coli proteins and suggest that very large helicases are present in this organism. Invest Ophthalmol Vis Sci, 1995 Oct, 36(11), 2296 - 303 Sequence, molecular properties, and chromosomal mapping of mouse lumican; Funderburgh JL et al.; PURPOSE . Lumican is a major proteoglycan of vertebrate cornea . This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization . METHODS . Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe . Tissue expression and size of lumican mRNA were determined using Northern hybridization . Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican . Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross . RESULTS . Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins . The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation . A 1.9-kb lumican mRNA is present in cornea and several other tissues . Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea . Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo . The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10 . The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology . CONCLUSIONS . This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene. Invest Ophthalmol Vis Sci, 1995 Oct, 36(11), 2211 - 5 In vivo gene transfer into murine corneal endothelial and trabecular meshwork cells; Budenz DL et al.; PURPOSE . To determine whether a reporter gene can be introduced into adult mammalian corneal endothelial and trabecular meshwork cells in vivo using a recombinant replication-deficient adenovirus . METHODS . Purified replication-deficient adenovirus containing the cytomegalovirus-promoted Escherichia coli reporter gene, lacZ, was injected into the vitreous cavities or anterior chambers of 30 adult CD-1 mice using the contralateral eyes as controls . LacZ expression was assessed histochemically in enucleated eyes from 2 to 21 days after injection using the beta-Galactosidase (beta-Gal) assay . RESULTS . LacZ expression was demonstrated in corneal endothelial and trabecular meshwork cells for as long as 14 days after injection . beta-Gal activity was also observed in lens and iris epithelial cells . There was no toxicity of the adenoviral vector demonstrated histologically, and no nonocular tissues expressed lacZ as measured by beta-Gal assay . CONCLUSIONS . A functional gene can be transferred in vivo into adult mammalian corneal endothelial and trabecular meshwork cells using a replication-defective adenoviral vector . Gene expression is relatively short-lived compared to that demonstrated previously in other ocular tissues (photoreceptors and retinal pigment epithelium) . Adenoviral vectors may be a viable means for short-term delivery of therapeutic genes in vivo to cells in the anterior segment of the eye. Infect Immun, 1995 Oct, 63(10), 4195 - 8 Prevalence and association of the longus pilus structural gene (lngA) with colonization factor antigens, enterotoxin types, and serotypes of enterotoxigenic Escherichia coli; Giron JA et al.; Human enterotoxigenic Escherichia coli (ETEC) produces a plasmid-encoded type IV pilus termed longus (for long pilus) . Regardless of the geographic origins of ETEC strains, the longus structural gene lngA was found to have the highest level of association with ETEC producing colonization factor antigen (CFA) CFA/II, followed by ETEC producing CFA/I and CFA/IV . ETEC bearing the less prevalent CFA/III and putative colonization factors and ETEC negative for CFA and putative colonization factor also contained lngA-related sequences . lngA was found in a considerable number of ETEC serotypes and was more often associated with ETEC producing heat-stable enterotoxins than with ETEC producing both heat-labile and heat-stable enterotoxins or heat-labile enterotoxin alone . lngA was found more often in strains isolated from children with diarrhea than in strains from healthy children, suggesting an association with intestinal disease . We conclude that longus is a widely distributed antigenic determinant in ETEC that is highly associated with known plasmid-encoded virulence factors, namely, CFAs and enterotoxins . A longus-specific probe may be a helpful epidemiological tool to assist in the identification of ETEC. Infect Immun, 1995 Oct, 63(10), 4143 - 9 Expression of the gene cluster associated with the Escherichia coli pilus adhesin K99; Lee JH et al.; The biogenesis of the pilus adhesin K99 is dependent on the expression of eight contiguous genes, fanA to fanH . Transposon mutants were prepared by using TnlacZ and TnphoA, and selected transposon mutants were used to measure expression of each K99 gene . Expression of the K99 genes is likely controlled at the transcription level, since in general, there were no differences between the results obtained with the two transposons . fanC was the most highly expressed, and fanD was expressed at very low levels . The expression of TnlacZ fusions in fanA and fanB fusions was high . Deletion of fanA, fanB, and part of fanC abolished the expression of fanD but had no effect on the distal genes fanE to fanH . To locate the DNA regions required for expression of fanE to fanH, deletion mutations were prepared and the effects on expression of fanE to fanH were determined . The deletion of a segment between fanD and fanE abolished fanE and fanF expression but did not affect fanG and fanH . The deletion of a portion of fanF (approximately 1 kb proximal to fanG) abolished the expression of fanG and fanH . These results indicate the presence of regulatory elements proximal to fanE and to fanG . Putative promoters were identified in these regions by DNA homology and by primer extension . A stem-loop structure that may act as a transcriptional attenuator of fanF was also found at the beginning of fanF . These data confirm our previous model of K99 transcriptional organization. Infect Immun, 1995 Oct, 63(10), 4003 - 10 Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex; Laqueyrerie A et al.; Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response . To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria . A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F . Romain, A . Laqueyrerie, P . Militzer, P . Pescher, P . Chavarot, M . Lagranderie, G . Auregan, M . Gheorghiu, and G . Marchal, Infect . Immun . 61:742-750, 1993) . Since the cognate antibodies recognize the very same antigens present in M . tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M . tuberculosis . An M . tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins . A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified . An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da . The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein . Southern hybridization analysis of digested total genomic DNA from M . tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a single copy on the genome . The N-terminal identity or homology of the M . tuberculosis and M . bovis BCG purified molecules and their similar global and deduced amino acid compositions demonstrated the perfect correspondence between the molecular and chemical analyses . The presence of a high percentage of proline (21.7%) was confirmed and explained the apparent higher molecular mass (45/47 kDa) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulting from the increased rigidity of molecules due to proline residues.(ABSTRACT TRUNCATED AT 400 WORDS) Infect Immun, 1995 Oct, 63(10), 3994 - 4002 Molecular and biochemical characterization of a Coccidioides immitis-specific antigen; Pan S et al.; Results of earlier investigations have indicated that the saprobic phase of Coccidioides immitis produces a heat-stable, 19-kDa antigen with serine proteinase activity which has been suggested to be specific for this pathogenic fungus . In the present study we have determined the N-terminal and partial internal amino acid sequences of the purified, 19-kDa antigen, cloned the gene which encodes this polypeptide, and confirmed that the secreted proteinase is a Coccidioides-specific antigen (CS-Ag) . Both the genomic and cDNA sequences are reported and reveal that the csa gene which encodes this antigen has no introns . A 543-bp open reading frame encodes a 181-amino-acid-containing protein with a predicted molecular mass of 19.8 kDa and an isoelectric point of 8.3 . The csa gene was localized on chromosome I of three representative C . immitis clinical isolates on the basis of Southern hybridizations . Expression of the csa gene in Escherichia coli using the pET21a plasmid vector yielded a recombinant protein that was recognized in immunoblot assays by antibody raised to the purified 19-kDa CS-Ag . Secretion of the native antigen is suggested to occur by cleavage of a putative 23-residue signal peptide . The native CS-Ag showed a low degree of glycosylation . Analysis of the carbohydrate composition of the CS-Ag revealed xylose, mannose, galactose, and glucose . However, the purified antigen showed no affinity for concanavalin A . A PCR method with specificity and high sensitivity for detection of C . immitis genomic DNA, using a pair of synthetic oligonucleotide primers whose sequences were based on that of the csa gene, was developed . A 520-bp product was amplified only when C . immitis genomic DNA was used as the template . The lower limits of DNA detection using this PCR method were 1 pg of C . immitis genomic DNA by ethidium bromide staining and 100 fg after Southern hybridization . The csa gene-based PCR method for detection of C . immitis DNA is useful for culture identification and may have clinical applications for the diagnosis of coccidioidal infections. Infect Immun, 1995 Oct, 63(10), 3920 - 6 Pertussis toxin export genes are regulated by the ptx promoter and may be required for efficient translation of ptx mRNA in Bordetella pertussis; Baker SM et al.; The gene products from an 8-kb region adjacent to the 3' end of the ptx operon are required by Bordetella pertussis for the export of pertussis holotoxin . At least one of these gene products (PtlC) is specifically required for the export of assembled holotoxin from the periplasmic space . ptlC mutants exhibit a 20-fold reduction in the amount of holotoxin present in the culture supernatant but have no effect upon the assembly or steady-state level of holotoxin present in the periplasmic space . Impaired export of holotoxin from the ptlC strain blocks expression of toxin at a posttranscriptional level, and wild-type levels of ptx mRNA are detected in the mutant strain . The transcription of ptl is subject to modulation by MgSO4 in the same manner as ptx is; however, in B . pertussis strains containing an E . coli tac promoter in place of the native ptx promoter, wild-type levels of ptx mRNA are present and holotoxin is synthesized and exported even in the presence of MgSO4 . Promoter mapping of the region extending from the ptxS3 coding region to the ptlC coding region failed to detect the ptl transcription initiation site . Additional RNase protection experiments with ptx promoter deletion and substitution strains indicate that the ptl operon is transcribed from the ptx promoter as part of a > 11-kb mRNA. Infect Immun, 1995 Oct, 63(10), 3827 - 34 Adoptive transfer of T lymphocytes to T-cell-depleted mice inhibits Escherichia coli translocation from the gastrointestinal tract; Gautreaux MD et al.; Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal (GI) tract to extraintestinal sites, such as the mesenteric lymph node (MLN), spleen, liver, kidneys, and blood . Previously, we reported that depletion of CD4+ and/or CD8+ T cells promotes bacterial translocation from the GI tract to the MLN . In the present study, CD4+ and/or CD8+ T cells, harvested from donor mice, were adoptively transferred to mice previously depleted of T cells by thymectomy plus intraperitoneal injection of rat anti-mouse T-cell monoclonal antibodies . The adoptively transferred CD4+ and/or CD8+ T cells inhibited the translocation of Escherichia coli from the GI tract . Migration of the adoptively transferred T cells to the spleens and MLNs of the recipient mice was determined by utilizing Thy 1.1+ donor cells adoptively transferred into mice whose cells express the Thy 1.2 marker . These results provide further evidence of the importance of T cells in the host immune defense against bacterial translocation from the GI tract. Infect Immun, 1995 Oct, 63(10), 3796 - 803 Molecular cloning of the cDNA and gene for an elastinolytic aspartic proteinase from Aspergillus fumigatus and evidence of its secretion by the fungus during invasion of the host lung; Lee JD et al.; Hydrolysis of structural proteins in the lung by extracellular proteinases secreted by Aspergillus fumigatus is thought to play a significant role in invasive aspergillosis . This fungus was found previously to secrete an elastinolytic serine proteinase and a metalloproteinase . We report that A . fumigatus also secretes an aspartic proteinase (aspergillopepsin F) that can catalyze hydrolysis of the major structural proteins of basement membrane, elastin, collagen, and laminin . The pH optimum for the enzymatic activity was 5.0 with elastin-Congo red as the substrate, and the activity was not significantly inhibited by pepstatin A, diazoacetyl norleucine methylester, and 1,2-epoxy-3-(p-nitrophenoxy) propane . The cDNA and gene encoding this aspartic proteinase were cloned and sequenced . The open reading frame, interrupted by three introns, would encode a protein of 393 amino acids composed of a putative 21-amino-acid signal peptide and a 49-amino-acid propeptide preceding the 323-amino-acid mature protein . The amino acid sequence of A . fumigatus aspartic proteinase has 70, 66, and 67% homology to the sequences of those from Aspergillus oryzae, Aspergillus awamori, and Aspergillus saitoi, respectively . The active-site motif (DTG) and the catalytic aspartic residues characteristic of aspartic proteinases are found in the presently described enzyme, indicating that it belongs to a family of aspartic proteinases . Polyclonal antibodies were produced in rabbits against both the mature and precursor forms of the aspartic proteinase expressed in Escherichia coli . Immunoblotting with both antibodies detected a 39-kDa mature enzyme in the culture supernatant of A . fumigatus . The aspartic proteinase activity was inhibited by the antibodies, suggesting that the aspartic proteinase in the culture supernatant corresponds to the product of the cloned gene . Immunogold electron microscopy showed that the aspartic proteinase was secreted by A . fumigatus invading neutropenic mouse lung and its secretion was directed toward the germ tubes of penetrating hyphae. Genes Dev, 1995 Oct 1, 9(19), 2350 - 63 Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis; Wu Z et al.; Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program . In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts . Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner . Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes . Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program . Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD) . The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed . C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator . Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes. FASEB J, 1995 Oct, 9(13), 1267 - 76 Mechanism and structure of thioredoxin reductase from Escherichia coli; Williams CH Jr; The flavoprotein thioredoxin reductase catalyzes the reduction of the small redox protein thioredoxin by NADPH . Thioredoxin reductase contains a redox active disulfide and is a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes that includes lipoamide dehydrogenase, glutathione reductase, trypanothione reductase, mercuric reductase, and NADH peroxidase . The structure of thioredoxin reductase has recently been determined from X-ray crystallographic data . In this paper, we attempt to correlate the structure with a considerable body of mechanistic data and to arrive at a mechanism consistent with both . The path of reducing equivalents in catalysis by glutathione reductase and lipoamide dehydrogenase is clear . To envisage the path of reducing equivalents in catalysis by thioredoxin reductase, a conformational change is required in which the NADPH domain rotates relative to the FAD domain . The rotation moves the nascent dithiol from its observed position adjacent to the re surface of the flavin ring system toward the protein surface for dithiol-disulfide interchange with the protein substrate thioredoxin and moves the nicotinamide ring of NADPH adjacent to the flavin ring for efficient hydride transfer . Reverse rotation allows reduction of the redox active disulfide by the reduced flavin . This requires that the enzyme pass through a ternary complex; the kinetic evidence for such a complex is discussed. Nat Struct Biol, 1995 Oct, 2(10), 906 - 10 Localized perturbations in CheY structure monitored by NMR identify a CheA binding interface; Swanson RV et al.; Phosphotransfer between the autophosphorylating histidine kinase CheA and the response regulator CheY represents a crucial step in the bacterial chemotaxis signal transduction pathway . The 15N-1H correlation spectrum of CheY complexed with an amino-terminal fragment of CheA exhibits specific localized differences in chemical shifts when compared to the spectrum of uncomplexed CheY . When mapped onto the three-dimensional structure of CheY, these changes define a region distinct from the active site . A single amino-acid substitution within this binding region on CheY, alanine to valine at position 103, significantly decreases the affinity of CheY for CheA . The binding face described by these changes partially overlaps a flagellar switch binding surface previously defined by mutagenesis. Int Arch Allergy Immunol, 1995 Oct, 108(2), 113 - 8 Inhibition of HEp-2 cell invasion by enteroinvasive Escherichia coli by human colostrum IgA; Carbonare SB et al.; This paper demonstrates that human colostrum can inhibit the invasion of HEp-2 cells by enteroinvasive Escherichia coli (EIEC) of serotypes O28:H- and O29:H-, and that IgA antibodies mediate the inhibitory process . Seventy three of 77 (95.9%) colostrum samples prevented invasion of HEp-2 cells by E . coli O28:H- . Most of these samples contained high levels of IgA reactive to EIEC in immunoenzymatic assays . IgA eluted from an affinity chromatography column strongly inhibited HEp-2 invasion by EIEC, whereas IgA-depleted colostrum had no inhibitory effect on bacterial invasion . Immunoblots of colostrum samples with high (> 60%) invasion-inhibiting levels were performed with water extracts of invasive and noninvasive strains . Bacterial antigens from the invasive strain were recognized and the size of some was consistent with the invasion plasmid antigens (Ipas) A, B, C, and D, with stronger reactions with Ipas A and C . Colostrum samples with high inhibitory levels showed a strong reaction in Western blot assays, in contrast to the faint bands observed with poor-inhibitory samples . The results obtained in the present study suggest that colostrum IgA may protect infants against invasive E . coli infections. J Virol, 1995 Oct, 69(10), 6563 - 6 A screen in Escherichia coli for nucleoside analogs that target human immunodeficiency virus (HIV) reverse transcriptase: coexpression of HIV reverse transcriptase and herpes simplex virus thymidine kinase; Kim B et al.; Human immunodeficiency virus (HIV) reverse transcriptase substitutes for temperature-sensitive DNA polymerase I (Pol Its) in Escherichia coli, providing a screen for anti-HIV reverse transcriptase nucleoside analogs in bacteria . Since phosphorylation of nucleosides in E . coli is limited to thymidine and its derivatives, we coexpressed herpes simplex virus thymidine kinase, an enzyme that phosphorylates a wide variety of nucleoside analogs, together with HIV reverse transcriptase . Coexpression of herpes simplex virus thymidine kinase and HIV reverse transcriptase rendered Pol Its cells sensitive to dideoxycytidine . Studies with different nucleoside analogs indicate that this bacterial screening system is able to select and identify nucleoside analogs that specifically target HIV reverse transcriptase. J Virol, 1995 Oct, 69(10), 6273 - 9 Feline immunodeficiency virus reverse transcriptase: expression, functional characterization, and reconstitution of the 66- and 51-kilodalton subunits; Amacker M et al.; The two subunits of the feline immunodeficiency virus (FIV) reverse transcriptase (RT) were cloned and functionally expressed in Escherichia coli . The recombinant proteins are enzymatically active as homodimers (p66 and p51) as well as a heterodimer p66/p51 . The biochemical properties of the FIV RT are very similar to those of the counterpart of the human immunodeficiency virus type 1 in being an RNA-dependent and DNA-dependent DNA polymerase . When a double-stranded DNA containing a small gap of 26 nucleotides was tested, we found a new activity of the FIV RT p66/p51 heterodimer--the cat viral enzyme could perform strand displacement DNA synthesis of approximately 300 bases . The FIV RT homodimer p66 alone could carry out limited strand displacement DNA synthesis, but this activity was stimulated by the p51 subunit at a molar ratio of one molecule of p66 to five molecules of p51 . On the other hand, the homodimeric p51 itself was unable to fill a small gap of 26 nucleotides in a double-stranded DNA substrate and was not active by itself in strand displacement DNA synthesis . These data are in agreement with an earlier finding of strand displacement DNA synthesis by human immunodeficiency virus type 1 RT (M . Hottiger, V.N . Podust, R.L . Thimmig, C.S . McHenry, and U . Hubscher . J . Biol . Chem . 269:986-991, 1994) . Our data therefore suggest a general and important function of lentiviral p51 subunits in strand displacement DNA synthesis which appears to be required in later stages of the lentiviral replication cycle, when DNA-dependent DNA synthesis occurs on double-stranded DNA. Kansenshogaku Zasshi, 1995 Oct, 69(10), 1159 - 61 {A case of Acanthamoeba keratitis after operation for cataract}; Takatsu M et al.; Acanthamoeba keratitis occurs mainly in contact lens users . We experienced a patient with Acanthamoeba keratitis after operation for cataract . A 70-year-old male, who suffered from suppurative keratitis with impairment of visual acuity and eye pain in the left eye after the operation, was admitted to our hospital . After admission he received treatment with oral and topical antibiotics without any improvement . Neither bacterial or fungal pathogens was detected from corenal skrappings . Blue stained Acanthamoeba cysts were detected with the Parker ink KOH preparation from punctured fluid of the anterior chamber of the eye . Acanthamoeba cysts were also cultured on a nonnurient agar plate with Escherichia coli . Then he was treated with oral and topical miconazole and topical fluconazole . His visual acuity did not improve because of the lag of appropriate treatment . Therefore, attention must be paid for the existence of Acanthamoeba keratitis after ophthalmologic operations. Kansenshogaku Zasshi, 1995 Oct, 69(10), 1135 - 40 {Serological diagnosis for human parvovirus B19 infection by an enzyme immunoassay kit with recombinant antigens synthesized in a baculovirus expression system}; Yoto Y et al.; Propagation of human parvovirus B19 (B19) in cell cultures are not applicable to the source of viral antigens for serological assays at present . Enzyme immunoassay (EIA) kits with recombinant B19 capsids by E . coli or baculovirus expression system have been developed . We tested serum samples from the patients with erythema infectiosum and aplastic crisis by EIA kit with recombinant antigens synthesized in a baculovirus expression system (Denka Seiken Co., Tokyo, Japan) . The antigens used in the kit are self-assembled recombinants containing both VP-1 and VP-2 with the same proportion as found in native B19 capsids . B19 IgM is detected by antibody capture methods and IgG by indirect methods . All of the samples were positive for B19 DNA by nested PCR . Thirty-six (97%) of the 37 patients with erythema infectiosum and all (100%) of the 4 patients with aplastic crisis were positive for B19 IgM . The EIA kit with recombinant antigens synthesized in a baculovirus expression system has proved to be reliable and useful for the diagnosis of B19 infection. Nippon Ika Daigaku Zasshi, 1995 Oct, 62(5), 469 - 81 Cell damage and liberation of nitric oxide synthase in rat heart induced by endotoxin administration; Fukui M et al.; To evaluate the relationship between cardiovascular injury and the pathological aspect of the liberation of the nitric oxide synthase in endotoxin shock, Wistar rats were injected intraperitoneally with 10 mg/kg E . coli endotoxin and the cardiovascular changes were observed chronologically by immunohistochemical and electron microscopic techniques at 2, 4, 8 and 12 h respectively after endotoxin administration . Light microscopically, irregular contraction of cardiomyocytes was observed in the early stage . After 8 h of endotoxin administration, occasional disintegration of the myocytes as well as lysis of myofibrils became prominent, and diminished stainings of myoglobin and actin were seen in these myocytes . Ultrastructurally, increased pinocytotic vesicles were observed in endothelial cells associated with widened intercellular spaces, and vacuolization of endothelial and medial cells of the vasculature . In cardiomyocytes, swelling of mitochondria and interstitial edema were noted at 12 h after endotoxin administration . Immunohistochemically, an increased permeability of albumin was noted in the myocytes and vasculature and nitric oxide synthase was localized on the cytomembrane of the endothelium of the coronary arteries in control rats . After endotoxin administration, the reaction products of nitric oxide synthase increased in the endothelial and medial cells, and cardiomyocytes . These results suggest that the increased activity of nitric oxide synthase is an important factor in the excessive production of nitric oxide and in promoting the pathologic changes in the cardiovascular system after endotoxin administration. Plant J, 1995 Oct, 8(4), 603 - 12 Derepression of the activity of genetically engineered heat shock factor causes constitutive synthesis of heat shock proteins and increased thermotolerance in transgenic Arabidopsis; Lee JH et al.; ATHSF1 is a heat shock transcription factor (HSF) of Arabidopsis that is constitutively expressed but its activity for DNA binding, trimer formation and transcriptional activation of heat shock (hs) genes is repressed at normal temperatures . In this study the functional properties of chimeric HSF-glucuronidase (GUS) fusion proteins were tested . Ectopic expression of HSF-GUS or GUS-HSF in transgenic Arabidopsis plants resulted in a derepression of HSF functions as shown by trimer formation, specific DNA binding, and the constitutive expression of heat shock proteins (HSPs) at normal temperature . A novel GUS activity-staining protocol was used to show the specific binding of trimeric HSF fusion proteins to DNA and following hs, an interaction between chimeric HSF-GUS and authentic HSF proteins . The chimeric HSFs were insensitive to the negative regulation that counteracts activation of the authentic HSF at normal temperature . Heterotrimer complexes were reconstituted in vitro from recombinant ATHSF1 and HSF-GUS proteins expressed in Escherichia coli and using this protocol, the temperature-dependent activation of wt HSF was monitored in vivo and in vitro . Transgenic plants expressing constitutively active HSF-GUS fusion proteins are also constitutive for HSPs . Approximately 20% of the maximum heat-inducible levels of HSP18 were already present at normal temperature . The level of basic thermotolerance was significantly enhanced in these plants . The results indicate that genetic engineering using protein fusion is a very effective means to derepress the activity of an important regulatory protein in plants, that consequently activates a constitutive hs response in the absence of heat stress and eventually alters the thermotolerance phenotype. RNA, 1995 Oct, 1(8), 841 - 51 Structural and functional accommodation of nucleotide variations at a conserved tRNA tertiary base pair; Sterner T et al.; The U8:A14 tertiary base pair of transfer RNAs (tRNAs) stabilizes the sharp turn from the acceptor stem to the dihydrouridine stem . This tertiary base pair is important for the overall L-shaped tRNA structure . Inspection of tRNA sequences shows that U8:A14 is highly conserved . However, variations of U8:A14 are found in natural sequences . This raises the question of whether all 16 permutations of U8:A14 can be accommodated by a single tRNA sequence framework and by the bacterial translational apparatus . Here we expressed the wild type and 15 variants of U8:A14 of an alanine tRNA amber suppressor in Escherichia coli and tested the ability of each to suppress an amber mutation . We showed that 12 of the 15 variants are functional suppressors (sup+) and 3 are nonfunctional (sup-) . Of the 12 functional suppressors, the G8:G14 variant is the most efficient suppressor, whose suppression efficiency is indistinguishable from that of the wild type . Analysis of tRNA structure with chemical probes and the lead-cleavage reaction, however, showed a distinct difference between the G8:G14 variant and the wild type . Thus, two different structures of E . coli tRNAAla/CUA share an identical functional phenotype in protein synthesis . The remaining 11 sup+ variants with reduced suppression efficiencies are likely to have other structural variations . We suggest that the variations of these sup+ mutants are structurally and functionally accommodated by the bacterial translational apparatus . In contrast, the three sup- mutants harbor variations that alter the backbone structure in the corner of the L . These variations are likely to reduce the stability of the tRNA inside the cell or, among others, to interfere with the ability of the tRNA to functionally interact with elongation factor Tu and with the ribosome. RNA, 1995 Oct, 1(8), 783 - 93 Escherichia coli protein StpA stimulates self-splicing by promoting RNA assembly in vitro; Zhang A et al.; An Escherichia coli gene, stpA, has been identified and cloned based on its ability to suppress the Td- phenotype of a resident, splicing-defective phage T4 td (thymidylate synthase) gene . The stpA gene, which was localized to 60.24 min on the E . coli chromosome, encodes a 15.3-kDa protein . Overproduction of StpA in vivo led to an increase in td pre-mRNA levels and modest enhancement of td mRNA:pre-mRNA ratios . Consistent with its in vivo effect, purified StpA promoted RNA splicing in vitro, and facilitated RNA annealing and strand exchange with model substrates . These results suggest that StpA promotes splicing of the intron by binding RNA nonspecifically, resolving misfolded precursor molecules and facilitating association of critical base pair elements . Furthermore, proteinase K treatment of StpA-assembled precursors prior to the initiation of the splicing reaction still resulted in splicing enhancement, indicating that StpA is not required for the catalytic step, unlike the Neurospora splicing effector CYT-18, whose presence was necessary for catalysis to proceed . Together these results suggest that StpA has chaperone activity in vitro, with the property of promoting assembly of the precursors into an active conformation, in contrast to splicing effectors that stabilize the catalytically active intron structure. J Toxicol Sci, 1995 Oct, 20 Suppl 1, 125 - 31 {Mutagenicity studies of iodixanol, a new non-ionic isotonic contrast medium}; Igarashi M et al.; A new non-ionic isotonic contrast medium, iodixanol (a diastereomeric mixture of 5,5'-{(2-hydroxytrimethylene) bis (acetylimino)} bis {N, N'-bis (2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzene-dicarboxamide}) was studied for mutagenicity by using the Ames method, in vitro cytogenetics and micronucleus test . Iodixanol had no mutagenic effect on S . typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) or E . coli (WP2uvrA) in the reverse mutation assay with or without metabolic activations . In the cytogenetic study, iodixanol had no effect on the Chinese hamster cells with or without metabolic activations . Intravenous injections of iodixanol for two times at a dose of 800, 1,600 or 3,200 mgI/kg in the mouse micronucleus test did not increase the incidence of micronucleated polychromatic erythrocytes . These results show that iodixanol has no mutagenic potential. Int J Exp Pathol, 1995 Oct, 76(5), 369 - 79 Suppression of pannus-like extension of synovial cells by lipid-derivatized chondroitin sulphate: in vitro and in vivo studies using Escherichia coli-induced arthritic rabbits; Sugiura N et al.; In rheumatoid arthritis, pannus formation resulting from synovial inflammation is a major factor in cartilage destruction . The ability of arthritic synovial cells to undergo pannus formation depends upon their initial adhesion to the partially deformed cartilage surfaces . Our recent studies using various lipid-derivatized glycosaminoglycans have revealed a preeminent inhibitory activity of phosphatidyl ethanol amine-derivatized chondroitin sulphate (CS-PE) toward cell-matrix adhesion . Here we evaluate whether CS-PE may protect articular cartilage from pannus extension in different in vitro and in vivo model systems using Escherichia coli 0:14-induced arthritis in rabbits and the articular cartilage explants, synovial tissues, and synovial cells obtained from them . These studies showed that CS-PE suppressed the in vivo pannus-like extension on cartilage surfaces, as well as the in vitro extension of the synovial cell layer on both CS-PE treated culture plates and cartilage explants . The results suggest that native chondroitin sulphate proteoglycans in the surface of normal articular cartilage play an important role in protecting the tissues from pannus extension and that the CS-PE immobilized onto partially eroded cartilage can mimic the inhibitory action of native chondroitin sulphate proteoglycans. Arch Microbiol, 1995 Oct, 164(4), 301 - 7 An essential role for DsbA in cytochrome c synthesis and formate-dependent nitrite reduction by Escherichia coli K-12; Metheringham R et al.; An Escherichia coli K-12 mutant, isolated on the basis of its inability to catalyze formate-dependent nitrite reduction, was characterized . The mutant was defective in the synthesis of all known c-type cytochromes during anaerobic growth . The mutation was localized by conjugation, transduction, and Southern blotting experiments to the dsbA gene at minute 87 on the E . coli chromosome and was complemented by the wild-type allele . Both DsbA and the recently described DipZ protein were shown to be essential for cytochrome c synthesis, suggesting that they act sequentially in a pathway for cytochrome c assembly in the E . coli periplasm. Arch Microbiol, 1995 Oct, 164(4), 243 - 54 From growth to autolysis: the murein hydrolases in Escherichia coli; Holtje JV; Murein hydrolases cleave bonds in the bacterial exoskeleton, the murein (peptidoglycan) sacculus, a covalently closed bag-shaped polymer made of glycan strands that are crosslinked by peptides . During growth and division of a bacterial cell, these enzymes are involved in the controlled metabolism of the murein sacculus . Murein hydrolases are believed to function as pacemaker enzymes for the enlargement of the murein sacculus since opening of bonds in the murein net is needed to allow the insertion of new subunits into the sacculus . Furthermore, they are responsible for splitting the septum during cell division . The murein turnover products that are released during growth are further degraded by these (1 --> 6)-anhydromuramic acid derivatives by an intramolecular transglycosylation reaction. Appl Environ Microbiol, 1995 Oct, 61(10), 3567 - 72 Unidirectional motility of Escherichia coli in restrictive capillaries; Liu Z et al.; In a 6-microns capillary filled with buffer and in the absence of any chemotactic stimuli, Escherichia coli K-12 cells swim persistently in only one direction . This behavior of E . coli can be simply explained by means of the length and relative rigidity of their flagella . Single-cell motility parameters--swimming speed, turn angle, and run length time--were measured . Compared with the motility parameters measured in bulk phase, turn angle was influenced because of the effect of the geometrical restriction. Plant Physiol, 1995 Oct, 109(2), 687 - 96 Multiple isoforms of Arabidopsis casein kinase I combine conserved catalytic domains with variable carboxyl-terminal extensions; Klimczak LJ et al.; Three cDNA clones encoding isoforms of casein kinase I (CKI) were isolated from Arabidopsis thaliana . One full-length clone, designated CKI1, contained an open reading frame of 1371 bp encoding a protein of 51,949 D with an isoelectric point of 9.7 . In addition to the highly conserved catalytic domain (of about 300 amino acids), the Arabidopsis CKI isoforms contain 150 to 180 amino acid carboxyl-terminal extensions, which show among themselves a lower level of sequence conservation . These extensions do not show any sequence similarity to nonplant CKI isoforms, such as rat testis CKI delta, which is their closest isolated homolog, or to yeast CKI isoforms . Three additional isoforms of Arabidopsis CKI were found in the data bases of expressed sequence tags and/or were isolated serendipitously in nonspecific screening procedures by others . One of them also shows a carboxyl-terminal extension, but of only 80 amino acids . Casein kinase activity was detected in the soluble fraction of Escherichia coli strains expressing the CKI1 protein . This activity showed the crucial properties of CKI, including the ability to phosphorylate the D4 peptide, a specific substrate of CKI, and inhibition by N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, a specific CKI inhibitor . Like several recombinant CKI isoforms from yeast, CKI1 was able to phosphorylate tyrosine-containing acidic polymers. Photochem Photobiol, 1995 Oct, 62(4), 651 - 6 Green fluorescent protein; Chalfie M; Several bioluminescent coelenterates use a secondary fluorescent protein, the green fluorescent protein (GFP), in an energy transfer reaction to produce green light . The most studied of these proteins have been the GFPs from the jellyfish Aequorea victoria and the sea pansy Renilla reniformis . Although the proteins from these organisms are not identical, they are thought to have the same chromophore, which is derived from the primary amino acid sequence of GFP . The differences are thought to be due to changes in the protein environment of the chromophore . Recent interest in these molecules has arisen from the cloning of the Aequorea gfp cDNA and the demonstration that its expression in the absence of other Aequorea proteins results in a fluorescent product . This demonstration indicated that GFP could be used as a marker of gene expression and protein localization in living and fixed tissues . Bacterial, plant and animal (including mammalian) cells all express GFP . The heterologous expression of the gfp cDNA has also meant that it could be mutated to produce proteins with different fluorescent properties . Variants with more intense fluorescence or alterations in the excitation and emission spectra have been produced. J Biotechnol, 1995 Sep 29, 42(2), 177 - 84 Optimising the recovery of recombinant thermostable proteins expressed in mesophilic hosts; Kirk N et al.; The purification of a thermostable Caldocellum saccharolyticum beta-glucosidase expressed in Escherichia coli was investigated using heat precipitation of unclarified cell homogenates . Heat treatment at 70 degrees C was capable of purification with respect to cell debris, small particulates and the majority of cell protein, although E . coli proteins were even more efficiently removed at 80 degrees C and above . For thermostable proteins expressed in E . coli, a precipitation temperature of 80 degrees C or greater is recommended for optimal removal of contaminant proteins . In small-scale heating trials, heating rate was found to influence enzyme yield significantly . Losses were minimised when 'flash-heating' was employed . The successful single-step removal of particulates, labile protein and nucleic acids was achieved by simultaneous heat-treatment and polyethyleneimine addition, although the purification achieved was additive rather than synergistic. J Biotechnol, 1995 Sep 29, 42(2), 133 - 43 Production in Escherichia coli of a functional murine and murine::human chimeric F(ab')2 fragment and mature antibody directed against human placental alkaline phosphatase; Flamez D et al.; We report the production in Escherichia coli of a murine antibody IgG2b, a murine::human chimeric antibody IgG3 and the corresponding F(ab')2 fragments, all directed against human placental alkaline phosphatase, a tumor marker . The cDNA of the heavy chain of the mature antibodies and their fragments were linked up to the bacterial alkaline phosphatase signal sequence and were placed under control of the inducible tac promoter . Coexpression with the murine kappa light chain resulted in production of functional dimeric, trimeric and tetrameric, mature antibodies and F(ab')2 fragments in the periplasm of E . coli in a yield of 200-300 micrograms l-1 . High amounts of light and heavy chains were present also in the insoluble fraction. J Biol Chem, 1995 Sep 29, 270(39), 23060 - 4 Peptide environment of the peptidyl transferase center from Escherichia coli 70 S ribosomes as determined by thermoaffinity labeling with dihydrospiramycin; Bischof O et al.; In an attempt to gain information about the peptidyl transferase center at the peptide level we cross-linked the spiramycin derivative dihydrospiramycin to its functional binding site in the 70 S ribosome of Escherichia coli . In this manner ribosomal proteins S12, S14, L17, L18, L27 and L35 were found specifically affinity-labeled . Proteolytic fragmentation of these proteins, separation by C18 reversed-phase high performance liquid chromatography of the peptide mixtures, and subsequent sequence analysis of labeled peptides revealed peptide regions at positions Ala1-Lys9 and Tyr116-Lys119 of S12, Leu47-Asp53 of protein S14, Ser6-Lys35 of protein L17, Ala57-Lys63 of protein L18, Ala5-Lys18 and Val66-Lys71 of protein L27, and Thr5-Lys11 of protein L35 . This approach is a valuable tool to characterize the binding site of spiramycin as well as the peptidyl transferase center at the molecular level. J Biol Chem, 1995 Sep 29, 270(39), 22962 - 7 Inactive GroEL monomers can be isolated and reassembled to functional tetradecamers that contain few bound peptides; Ybarra J et al.; For the first time, it has been shown that GroEL can be converted from tetradecamers (14-mers) to monomers under conditions commonly used for the preparation of this chaperonin . The essential requirements are the simultaneous presence of nucleotides such as MgATP or MgADP and a solid-phase anion-exchange medium . The monomers that are formed are metastable in that they only reassemble to a small degree in the absence of additives . These results are in keeping with previous studies on high pressure dissociation that showed the separated monomers display conformational plasticity and can undergo conformational relaxation when relieved of the constraints of the quaternary structure in the oligomer (Gorovits, B., Raman, C . S., and Horowitz, P . M . (1995) J . Biol . Chem . 270, 2061-2066) . The monomers display greatly enhanced hydrophobic exposure to the probe 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, although they are not active in folding functions, and they are unable to form complexes with partially folded rhodanese . The monomers can be completely reassembled to 14-mers by incubation in 1 M ammonium sulfate . There is no evidence of intermediates in the reassembly process . Compared with the original oligomers, the reassembled 14-mers have (a) very low levels of polypeptide contaminants and tryptophan-like fluorescence, two problems that previously hampered spectroscopic studies of GroEL structure and function; (b) functional properties that are very similar to the original material; (c) considerably decreased hydrophobic exposure in the native state; and (d) a similar triggered exposure of hydrophobic surfaces after treatment with urea or spermidine . This study demonstrates that the quaternary structure of GroEL is more labile than previously thought . These results are consistent with suggestions that nucleotides can loosen subunit interactions and show that changes in quaternary structure can operate under conditions where GroEL function has been demonstrated. J Biol Chem, 1995 Sep 29, 270(39), 22939 - 45 Messenger RNA recognition by fragments of ribosomal protein S4; Baker AM et al.; Ribosomal protein S4 from Escherichia coli binds a large domain of 16 S ribosomal RNA and also a pseudoknot structure in the alpha operon mRNA, where it represses its own synthesis . No similarity between the two RNA binding sites has been detected . To find out whether separate protein regions are responsible for rRNA and mRNA recognition, proteins with N-terminal or C-terminal deletions have been overexpressed and purified . Protein-mRNA interactions were detected by (i) a nitrocellulose filter binding assay, (ii) inhibition of primer extension by reverse transcriptase, and (iii) a gel shift assay . Circular dichroism spectra were taken to determine whether the proteins adopted stable secondary structures . From these studies it is concluded that amino acids 48-104 make specific contacts with the mRNA, although residues 105-177 (out of 205) are required to observe the same toeprint pattern as full-length protein and may stabilize a specific portion of the mRNA structure . These results parallel ribosomal RNA binding properties of similar fragments (Conrad, R . C., and Craven, G . R . (1987) Nucleic Acids Res . 15, 10331-10343, and references therein) . It appears that the same protein domain is responsible for both mRNA and rRNA binding activities. J Biol Chem, 1995 Sep 29, 270(39), 22895 - 906 Probing the roles of active site residues in D-xylose isomerase; Whitaker RD et al.; The roles of active site residues His54, Phe94, Lys183, and His220 in the Streptomyces rubiginosus D-xylose isomerase were probed by site-directed mutagenesis . The kinetic properties and crystal structures of the mutant enzymes were characterized . The pH dependence of diethylpyrocarbonate modification of His54 suggests that His54 does not catalyze ring-opening as a general acid . His54 appears to be involved in anomeric selection and stabilization of the acyclic transition state by hydrogen bonding . Phe94 stabilizes the acyclic-extended transition state directly by hydrophobic interactions and/or indirectly by interactions with Trp137 and Phe26 . Lys183 and His220 mutants have little or no activity and the structures of these mutants with D-xylose reveal cyclic alpha-D-xylopyranose . Lys183 functions structurally by maintaining the position of Pro187 and Glu186 and catalytically by interacting with acyclic-extended sugars . His220 provides structure for the M2-metal binding site with properties which are necessary for extension and isomerization of the substrate . A second M2 metal binding site (M2') is observed at a relatively lower occupancy when substrate is added consistent with the hypothesis that the metal moves as the hydride is shifted on the extended substrate. J Biol Chem, 1995 Sep 29, 270(39), 22890 - 4 Direct kinetic evidence for triplet state energy transfer from Escherichia coli alkaline phosphatase tryptophan 109 to bound terbium; Schlyer BD et al.; The addition of excess Tb3+ to metal-depleted Escherichia coli alkaline phosphatase results in enhanced luminescence from enzyme-bound terbium, which increases with sample deoxygenation and exhibits a tryptophan-like excitation spectrum . Following pulsed excitation at 280 nm, the time-resolved terbium emission shows a negative prefactor associated with a submillisecond rise time, which is independent of the concentration of dissolved oxygen . The absence of a build-up phase and similarity in lifetime in the decay kinetics of directly excited (488 nm) terbium allows for the assignment of the submillisecond component in the 280 nm excited sample to bound terbium . The results of the steady state and time-resolved experiments suggest that the time evolution of alkaline phosphatase-bound terbium emission is determined by energy transfer (kET approximately 360 and 120 s-1) from the triplet state of tryptophan to terbium followed by terbium decay . This model is based on the observations that 1) the tryptophan phosphorescence lifetime (previously assigned to Trp109) corresponds to the longer component of the terbium emission and 2) the long-lived emission is enhanced, as is the Trp109 phosphorescence, by deoxygenation . An energy transfer mechanism involving the Trp109 triplet state is shown to be inconsistent with a dipole-dipole process and is best understood as a through-space electron exchange over a donor-acceptor distance of 9-10 A. J Biol Chem, 1995 Sep 29, 270(39), 22855 - 8 Substrate requirements for transglutaminases . Influence of the amino acid residue preceding the amine donor lysine in a native protein; Grootjans JJ et al.; Thirteen recombinant alpha A-crystallin mutants were constructed that differed in the type of amino acid residue directly preceding the sole amine donor lysine for transglutaminases in this protein . The capacity of these mutants to be cross-linked to amine acceptor substrates by tissue transglutaminase and factor XIII was assessed . Two different biotinylated glutamine-containing oligopeptides were used as amine acceptor probes . It appears that the type of residue preceding the amine donor lysine has a considerable influence on the substrate potential of alpha A-crystallin for transglutaminases . This influence shows qualitatively similar trends for tissue transglutaminase and factor XIII and is irrespective of the amine acceptor probe . In general, glycine or aspartic acid before the amine donor lysine has the strongest adverse effects on substrate reactivity, and proline, histidine, and tryptophan are less favorable . Valine, arginine, and phenylalanine, and to a more variable or somewhat lesser extent also serine, alanine, leucine, tyrosine, and asparagine, have an enhancing effect . This pattern of preference is largely in agreement with that observed for the limited number of characterized amine donor lysines in protein substrates for transglutaminases . It can be concluded that tissue transglutaminase and factor XIII have a rather broad yet clearly differentiated tolerance with respect to the residue preceding the amine donor lysine substrate in native proteins. J Biol Chem, 1995 Sep 29, 270(39), 22850 - 4 Beta-gamma subunit interaction is required for catalysis by H(+)-ATPase (ATP synthase) . Beta subunit amino acid replacements suppress a gamma subunit mutation having a long unrelated carboxyl terminus; Jeanteur-De Beukelaer C et al.; The mechanisms of energy coupling and catalytic co-operativity are not yet understood for H(+)-ATPase (ATP synthase) . An Escherichia coli gamma subunit frameshift mutant (downstream of Thr-gamma 277) could not grow by oxidative phosphorylation because both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and Futai, M . (1990) J . Biol . Chem . 265, 5043-5048) . The defect(s) of the gamma frameshift was obvious, because the mutant subunit had a carboxyl terminus comprising 16 residues different from those in the wild type . However, in this study, we surprisingly found that an Arg-beta 52-->Cys or Gly-beta 150-->Asp replacement could suppress the deleterious effects of the gamma frameshift . The membranes of the two mutants (gamma frameshift/Cys-beta 52 with or without a third mutation, Val-beta 77-->Ala) exhibited increased oxidative phosphorylation, together with 70-100% of the wild type ATPase activity . Similarly, the gamma frameshift/Asp-beta 150 mutant could grow by oxidative phosphorylation, although this mutant had low membrane ATPase activity . These results suggest that the beta subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the gamma mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme. J Biol Chem, 1995 Sep 29, 270(39), 22831 - 5 Diverse effects of mutation on the activity of the Escherichia coli export chaperone SecB; Kimsey HH et al.; The Escherichia coli SecB protein binds newly synthesized precursor maltose-binding protein (preMBP) and promotes its rapid export from the cytoplasm . Site-directed mutagenesis of two regions of SecB was carried out to better understand factors governing the SecB.preMBP interaction . 30 aminoacyl substitution mutants were analyzed, revealing two distinct classes of secB mutants . Substitutions at the alternating positions Phe-74, Cys-76, Val-78, or Gln-80 reduced the ability of SecB to form stable complexes with preMBP, but caused only mild defects in the rate of MBP export from living cells . The pattern revealed by this class of mutants suggests that a primary binding site for preMBP is hydrophobic and contains beta-sheet secondary structure . In contrast, substitutions at Asp-20, Glu-24, Leu-75, or Glu-77 caused a severe slowing in the rate of MBP export but did not disrupt SecB.preMBP complex formation . These largely acidic residues may function to regulate the opening of a preprotein binding site, allowing both high affinity preprotein binding and rapid dissociation of SecB.preprotein complexes at the membrane translocation site. J Biol Chem, 1995 Sep 29, 270(39), 22752 - 7 The NH2-terminal half of the Tn10-specified tetracycline efflux protein TetA contains a dimerization domain; McMurry LM et al.; The 43.1-kDa tetracycline-cation/proton antiporter TetA from Tn10 comprises two equal-sized domains, alpha and beta (amino-terminal and carboxyl-terminal halves, respectively) . An inactivating mutation in the alpha domain can complement a mutation on a second polypeptide in the beta domain to restore partial tetracycline resistance in bacterial cells, suggesting that intermolecular interactions permit this transport protein to act as a multimer . In the present studies, multimer formation was examined in mixtures of dodecylmaltoside extracts of membranes from Escherichia coli cells containing different TetA derivatives . TetA, TetA alpha, and TetA beta were each fused genetically to a six-histidine carboxyl-terminal tail . The ability of these fusions, immobilized on a nickel affinity column, to bind wild type TetA or other Tet fusions was determined . An interaction between alpha domains on different polypeptides which resulted in multimerization was seen . The binding was specific for Tet protein and did not occur with other membrane proteins or another polyhistidine fusion protein . No alpha-beta interactions were detected by this method, although they are postulated to occur in the intact cell based on the alpha-beta genetic complementations . A dimeric model for TetA having intermolecular alpha-alpha and alpha-beta interactions is presented. J Biol Chem, 1995 Sep 29, 270(39), 22747 - 51 NAD(+)-dependent ADP-ribosylation of T lymphocyte alloantigen RT6.1 reversibly proceeding in intact rat lymphocytes; Maehama T et al.; Rat T lymphocyte alloantigen 6.1 (RT6.1), which was synthesized as the fusion protein with a maltose-binding protein in Escherichia coli, displayed NAD(+)-dependent auto-ADP-ribosylation in addition to an enzyme activity of NAD+ glycohydrolase . Such ADP-ribosylation of RT6.1 was also observed in lymphocytes isolated from rat tissues as follows . When intact rat lymphocytes expressing RT6.1 mRNA were incubated with {alpha-32P}NAD+, its radioactivity was incorporated into a cell surface protein with the M(r) of 31,000 . The radiolabeled 31-kDa protein was released from the cell surface by treatment of the cells with phosphatidylinositol-specific phospholipase C and immunoprecipitated with anti-RT6.1 antiserum . The radioactivity incorporated into the 31-kDa protein was recovered as 5'-{32P}AMP upon incubation with snake venom phosphodiesterase and also removed by NH2OH treatment . These results suggested that the NAD(+)-dependent modification of the 31-kDa protein was due to ADP-ribosylation of glycosylphosphatidylinositol-anchored RT6.1 at an arginine residue . When intact lymphocytes, in which RT6.1 had been once modified by {32P}ADP-ribosylation, were further incubated in the absence of NAD+, there was reduction of the radioactivity in the {32P}ADP-ribosylated RT6.1 . The reduced radioactivity was recovered from the incubation medium as {32P}ADP-ribose . This reduction was effectively inhibited by the addition of ADP-ribose to the reaction mixture . Moreover, readdition of NAD+ caused the ADP-ribosylation of RT6.1 again . Thus, the ADP-ribosylation of RT6.1 appeared to proceed reversibly in intact rat lymphocytes. J Biol Chem, 1995 Sep 29, 270(39), 22721 - 30 Fos leucine zipper variants with increased association capacity; Porte D et al.; The Fos wild-type leucine zipper is unable to support homodimerization . This finding is generally explained by the negative net charge of the Fos zipper leading to the electrostatic repulsion of two monomers . Using a LexA-dependent in vivo assay in Escherichia coli, we show here that additional antideterminants for Fos zipper association are the residues in position a within the Fos zipper interface . If the wild-type Fos zipper is fused to the DNA binding domain of the LexA repressor (LexA-DBD), no excess repression is observed as compared with the LexA-DBD alone, in agreement with the incapacity of the wild-type Fos zipper to promote homodimerization . If hydrophobic amino acids (Ile, Leu, Val, Phe, Met) are inserted into the five a positions of a LexA-Fos zipper fusion protein, substantial transcriptional repression is recovered showing that Fos zipper homodimerization is not only limited by the repulsion of negatively charged residues but also by the nonhydrophobic nature of the a positions . The most efficient variants (harboring Ile or Leu in the five a positions) show an about 80-fold increase in transcriptional repression as compared with the wild-type Fos zipper fusion protein . In the case of multiple identical substitutions, the overall improvement is correlated with the hydrophobicity of the inserted side chains, i.e . Ile Leu > Val > Phe > Met . However at least for Val, Phe, and Met the impact of a given residue type on the association efficiency depends strongly on the heptad, i.e . on the local environment of the a residue . This is particularly striking for the second heptad of the Fos zipper, where Val is less well tolerated than Phe and Met . Most likely the a1 residue modulates the interhelical repulsion between two glutamic acid side chains in positions g1 and e2 . Most of the hydrophobic Fos zipper variants are also improved in heteroassociation with a Jun leucine zipper, such that roughly half of the additional free energy of homodimerization is imported into the heterodimer . A few candidates (including the Fos wild-type zipper) deviate from this correlation, showing considerable excess heteroassociation. Biochem Pharmacol, 1995 Sep 28, 50(7), 1015 - 20 Correlation between catalytic activity and protein content for the polymorphically expressed dihydropyrimidine dehydrogenase in human lymphocytes; Fernandez-Salguero P et al.; A TLC procedure was developed to determine dihydropyrimidine dehydrogenase (DPD) activity in human peripheral lymphocytes . The assay, which used radiolabeled uracil as a substrate, was validated using recombinant pig DPD in which it was demonstrated to yield kinetic constants similar to those found by methods that rely on either spectroscopic determination of NADPH oxidation or HPLC . DPD activity was measured in a group of human lymphocyte extracts, including an extract from a subject that actually presented toxicity to 5-fluorouracil treatment . Measurements of DPD protein content using western immunoblots revealed a significant correlation with activity levels in human lymphocytes . Thus, this correlation could be used to determine not only the levels of expression of this enzyme, which is the cause of an inherited genetic deficiency in pyrimidine catabolism, but also to estimate the degree of sensitivity to pyrimidine-based cancer drugs such as 5-fluorouracil. Nature, 1995 Sep 28, 377(6547), 309 - 14 A base pair between tRNA and 23S rRNA in the peptidyl transferase centre of the ribosome; Samaha RR et al.; Interaction of the conserved CCA terminus of tRNA with rRNA in the peptidyl transferase P site has been studied by in vitro genetics . A watson-Crick G-C pair between G2252 in a conserved hairpin loop of 23S rRNA and C74 at the acceptor end of tRNA is required for proper functional interaction of the CCA end of tRNA with the ribosomal P site . These findings establish a direct role for 23S rRNA in protein synthesis. Biochim Biophys Acta, 1995 Sep 27, 1252(1), 69 - 78 Isolation and biochemical characterization of highly purified Escherichia coli molecular chaperone Cpn60 (GroEL) by affinity chromatography and urea-induced monomerization; Blennow A et al.; Isolated Escherichia coli molecular chaperone Cpn60 (GroEL) has been further purified from tightly bound substrate polypeptides by two different procedures: (i) group-specific affinity chromatography by using the triazine dye Procion yellow HE-3G as affinity ligand, and (ii) urea-induced monomerization and subsequent chromatography . Procion yellow binds specifically to aromatic amino-acid side chains present in the majority of proteins, but has no affinity to GroEL because of its low content of aromatic residues . Some GroEL-bound polypeptides are buried within the aqueous cavity of the GroEL oligomer, whereas others are exposed on its surface and available for affinity-ligand interactions and the complex is thereby retarded on Procion yellow columns . Pure substrate-free GroEL was obtained after ion-exchange chromatography of GroEL monomers followed by reassembly of the purified monomers into functional GroEL oligomers . The final preparation contained no substrate polypeptides bound to GroEL as judged by electrophoretic analysis and lack of tryptophan fluorescence . GroEL preparations also displayed two equally strong bands on native electrophoresis suggesting the presence of two conformers . Monomers of GroEL showed heterogeneity with respect to isoelectric point and molecular mass when analysed by MALDI-MS and electrophoresis under native and denaturing conditions respectively . By use of MALDI-MS, highly accurate molecular masses of wild-type and a truncated form of GroEL were determined and verified, by comparison with their respective gene sequences. Proc Natl Acad Sci U S A, 1995 Sep 26, 92(20), 9402 - 6 Identification of the overtone of the Fe-CO stretching mode in heme proteins: a probe of the heme active site; Wang J et al.; Two CO-isotope sensitive lines have been detected in the overtone region of the resonance Raman spectra of CO-bound hemeproteins . One line is assigned as the overtone of the Fe-CO stretching mode and is located in the 1000- to 1070-cm-1 region . The other line is found in the 1180- to 1210-cm-1 region and is assigned as a combination between a porphyrin mode, nu 7, and the Fe-CO stretching mode . The high intensities of these lines, which in the terminal oxidase class of proteins are of the same order as those of the fundamental stretching mode, indicate that the mechanism of enhancement for modes involving the Fe-CO moiety is different from that for the modes of the porphyrin macrocycle and call for reexamination of Raman theory of porphyrins as applied to axial ligands . The anharmonicity of the electronic potential function was evaluated, revealing that in the terminal oxidases the anharmonicity is greater than in the other heme proteins that were examined, suggesting a distinctive interaction of the bound CO with its distal environment in this family . Furthermore, the anharmonicity correlates with the frequency of the C-O stretching mode, demonstrating that both of these parameters are sensitive to the Fe-CO bond energy . The overtone and combination lines involving the bound CO promise to be additional probes of heme protein structural properties. Proc Natl Acad Sci U S A, 1995 Sep 26, 92(20), 9318 - 22 Stopped-flow NMR spectroscopy: real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase; Hoeltzli SD et al.; Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan . The 19F NMR assignments are known in the native, unliganded form and the unfolded form . We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding . The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (approximately 20%) of the intensities of the unfolded resonances appears . We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility . Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure . Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k approximately 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-flow circular dichroism (representing 80% of total amplitude) . These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate . Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above . The NMR data show that approximately 20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein. Proc Natl Acad Sci U S A, 1995 Sep 26, 92(20), 9279 - 83 Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution; Tolman JR et al.; The measurement of dipolar contributions to the splitting of 15N resonances of 1H-15N amide pairs in multidimensional high-field NMR spectra of field-oriented cyanometmyoglobin is reported . The splittings appear as small field-dependent perturbations of normal scalar couplings . Assignment of more than 90 resonances to specific sequential sites in the protein allows correlation of the dipolar contributions with predictions based on the known susceptibility and known structure of the protein . Implications as an additional source of information for protein structure determination in solution are discussed. Proc Natl Acad Sci U S A, 1995 Sep 26, 92(20), 9274 - 8 Molecular cloning, characterization, and functional expression of rat oxidosqualene cyclase cDNA; Abe I et al.; A cDNA encoding rat oxidosqualene lanosterol-cyclase {lanosterol synthase; (S)-2,3-epoxysqualene mutase (cyclizing, lanosterol-forming), EC 5.4.99.7} was cloned and sequenced by a combination of PCR amplification, using primers based on internal amino acid sequence of the purified enzyme, and cDNA library screening by oligonucleotide hybridization . An open reading frame of 2199 bp encodes a M(r) 83,321 protein with 733 amino acids . The deduced amino acid sequence of the rat enzyme showed significant homology to the known oxidosqualene cyclases (OSCs) from yeast and plant (39-44% identity) and still retained 17-26% identity to two bacterial squalene cyclases (EC 5.4.99.-) . Like other cyclases, the rat enzyme is rich in aromatic amino acids and contains five so-called QW motifs, highly conserved regions with a repetitive beta-strand turn motif . The binding site sequence for the 29-methylidene-2,3-oxidosqualene (29-MOS), a mechanism-based irreversible inhibitor specific for the vertebrate cyclase, is well-conserved in all known OSCs . The hydropathy plot revealed a rather hydrophilic N-terminal region and the absence of a hydrophobic signal peptide . Unexpectedly, this microsomal membrane-associated enzyme showed no clearly delineated transmembrane domain . A full-length cDNA was constructed and subcloned into a pYEUra3 plasmid, selected in Escherichia coli cells, and used to transform the OSC-deficient uracil-auxotrophic SGL9 strain of Saccharomyces cerevisiae . The recombinant rat OSC expressed was efficiently labeled by the mechanism-based inhibitor {3H}29-MOS. Proc Natl Acad Sci U S A, 1995 Sep 26, 92(20), 9264 - 8 Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability; Korolev S et al.; The crystal structure of the large fragment of the Thermus aquaticus DNA polymerase (Klentaq1), determined at 2.5-A resolution, demonstrates a compact two-domain architecture . The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I) . Although the N-terminal domain of Klentaq1 differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor . The structure of Klentaq1 reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme. Proc Natl Acad Sci U S A, 1995 Sep 26, 92(20), 9196 - 200 Association of the "major histocompatibility complex subregion" I-J determinant with bioactive glycosylation-inhibiting factor; Nakano T et al.; Murine suppressor T-cell hybridoma cells (231F1) secrete not only bioactive glycosylation-inhibiting factor (GIF) but also an inactive peptide comparable to bioactive GIF peptide in its molecular size and reactivity with anti-GIF; the amino acid sequence of the inactive peptide is identical to that of the bioactive homologue . The inactive GIF peptide in culture supernatant of both the 231F1 cells and a stable transfectant of human GIF cDNA in the murine suppressor T hybridoma selectively bound to Affi-Gel 10, whereas bioactive GIF peptides from the same sources failed to bind to the gel . The inactive cytosolic human GIF from the stable transfectant and Escherichia coli-derived recombinant human GIF also had affinity for Affi-Gel 10 . Both the bioactive murine GIF peptide from the suppressor T hybridoma and bioactive recombinant human GIF from the stable transfectant bound to the anti-I-J monoclonal antibody H6 coupled to Affi-Gel . However, bioactive hGIF produced by a stable transfectant of human GIF cDNA in BMT10 cells failed to be retained in H6-coupled Affi-Gel . These results indicate that the I-J specificity is determined by the cell source of the GIF peptide and that the I-J determinant recognized by monoclonal antibody H6 does not represent a part of the primary amino acid sequence of GIF . It appears that the epitope is generated by a posttranslational modification of the peptide. Proc Natl Acad Sci U S A, 1995 Sep 26, 92(20), 9186 - 90 Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage; Wu J et al.; Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity chromatography and derivatized with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper {OP(Cu)} . Studies with purified, OP(Cu)-labeled Leu-330 --> Cys permease in dodecyl-beta-D-maltopyranoside demonstrate that after incubation in the presence of ascorbate, cleavage products of approximately 19 and 6-8 kDa are observed on immunoblots with anti-C-terminal antibody . Remarkably, the same cleavage products are observed with permease embedded in the native membrane . Comparison with the C-terminal half of the permease expressed independently as a standard indicates that the 19-kDa product results from cleavage near the cytoplasmic end of helix VII, whereas the 6- to 8-kDa fragment probably results from fragmentation near the cytoplasmic end of helix XI . Results are entirely consistent with a tertiary-structure model of the C-terminal half of the permease derived from earlier site-directed fluorescence and site-directed mutagenesis studies . Similar studies with OP(Cu)-labeled Cys-148 permease exhibit cleavage products at approximately 19 kDa and at 15-16 kDa . The larger fragment probably reflects cleavage at a site near the cytoplasmic end of helix VII, whereas the 15- to 16-kDa fragment is consistent with cleavage near the cytoplasmic end of helix VIII . When OP(Cu) is moved 100 degrees to position 149 (Val-149 --> Cys p