Scand J Immunol, 1990 Aug, 32(2), 163 - 76 Major autoantigenic sites of the (U1) small nuclear ribonucleoprotein-specific 68-kDa protein; Netter HJ et al.; A 68-kDa protein associated with (U1)snRNP is a major target for human autoantibodies to small ribonucleoprotein particles (snRNP) prevalent in a variety of inflammatory rheumatic diseases . The epitopes recognized by these antibodies were mapped by expression of subfragments of p68 cDNA in Escherichia coli and testing of the corresponding recombinant proteins for immunoreactivity with sera of patients with autoimmune diseases . Three of four antigenic regions were analysed in detail . The immunodominant autoantigenic region was found to coincide with the RNA-binding domain of the p68 protein and was shown to contain a nested set of overlapping discontinuous epitopes . Two additional non-overlapping major antigenic domains were localized in the carboxy-terminal half of the p68 protein . Each of these two carboxy-terminal domains was shown to contain more than one conformation-dependent epitope . Taking into account previous mapping studies, the data demonstrate that p68 contains at least four antigenic regions, each of which harbours multiple epitopes which are recognized in a patient-specific manner. Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6331 - 5 Patterns of expression of position-dependent integrated transgenes in mouse embryo; Bonnerot C et al.; The abilities to introduce foreign DNA into the genome of mice and to visualize gene expression at the single-cell level underlie a method for defining individual elements of a genetic program . We describe the use of an Escherichia coli lacZ reporter gene fused to the promoter of the gene for hypoxanthine phosphoribosyl transferase that is expressed in all tissues . Most transgenic mice (six of seven) obtained with this construct express the lacZ gene from the hypoxanthine phosphoribosyltransferase promoter . Unexpectedly, however, the expression is temporally and spatially regulated . Each transgenic line is characterized by a specific, highly reproducible pattern of lacZ expression . These results show that, for expression, the integrated construct must be complemented by elements of the genome . These elements exert dominant developmental control on the hypoxanthine phosphoribosyltransferase promoter . The expression patterns in some transgenic mice conform to a typological marker and in others to a subtle combination of typology and topography . These observations define discrete heterogeneities of cell types and of certain structures, particularly in the nervous system and in the mesoderm . This system opens opportunities for developmental studies by providing cellular, molecular, and genetic markers of cell types, cell states, and cells from developmental compartments . Finally this method illustrates that genes transduced or transposed to a different position in the genome acquire different spatiotemporal specificities, a result that has implications for evolution. Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6181 - 5 Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli; Greenberg JT et al.; Escherichia coli responds to superoxide-generating agents by inducing approximately 40 proteins . We have identified a genetic locus, soxR (superoxide response), that positively regulates 9 of these proteins during superoxide stress . Induction under soxR control is at the transcriptional level, as shown with lac fusions to five paraquat-inducible promoters . Members of the soxR regulon include at least three proteins with demonstrable antioxidant roles: Mn-containing superoxide dismutase (which destroys superoxide radicals), endonuclease IV (which repairs radical-induced damages in DNA), and glucose-6-phosphate dehydrogenase (which produces NADPH) . Induction of the soxR regulon also leads to diminished levels of the major outer membrane protein OmpF and alteration of the small-subunit ribosomal protein S6 . These latter changes confer resistance to a variety of antibiotics . The soxR regulon may thus operate as an inducible defense against xenobiotics in general. Mutat Res, 1990 Aug, 244(4), 287 - 93 Influence of RNA synthesis on DNA-repair replication in human cell extracts; Frosina G et al.; An in vitro system allowing human cell extracts to carry out excision repair in ultraviolet light-irradiated, closed circular DNA was used to study the influence of transcription on repair synthesis . RNA synthesis from plasmid DNA containing bacterial transcription units was obtained by addition of Escherichia coli RNA polymerase and ribonucleoside triphosphates to the repair-incubation mixture . No increase in UV-stimulated repair replication was observed under transcriptional conditions; in fact, an inhibition of repair replication occurred, possibly due to impairment of DNA polymerization . This is unlike the in vivo situation where preferential DNA repair of transcribed genes has been found after UV-irradiation . Possible factors important for preferential repair of expressed genes in mammalian cells are discussed. J Bacteriol, 1990 Aug, 172(8), 4708 - 9 Location of a gene (ssrA) for a small, stable RNA (10Sa RNA) in the Escherichia coli chromosome; Oh BK et al.; The gene for 10Sa RNA, which is a major small, stable RNA in Escherichia coli, is a unique gene in the E . coli chromosome . The 10Sa RNA gene (ssrA) has been located between 2,760 and 2,761 kilobases on the E . coli genome. J Bacteriol, 1990 Aug, 172(8), 4641 - 51 Peptidase D gene (pepD) of Escherichia coli K-12: nucleotide sequence, transcript mapping, and comparison with other peptidase genes; Henrich B et al.; The nucleotide sequence of a 2.3-kilobase-pair DNA fragment of Escherichia coli that contains the transcription signals and the coding region of the pepD gene specifying aminopeptidase D was determined . The location and extent of the open reading frame were verified by partial amino acid sequencing of the purified pepD product . By use of a promoter-screening vector, initiation signals for pepD transcription were located in the 5'-flanking region of the open reading frame . Analysis of pepD transcripts by S1 mapping, primer extension, and Northern (RNA) hybridization revealed two species of monocistronic mRNA with different 5' ends and a common 3' end . Calculation of the degree of codon usage bias in the coding region suggested that the efficiency of pepD translation is relatively low . As deduced from the predicted amino acid sequence, peptidase D is a slightly hydrophilic protein of 485 amino acid residues that contains no extended domains of marked hydrophobicity . Structural and functional features of the pepD gene are discussed and compared with other already sequenced peptidase genes of E . coli. J Bacteriol, 1990 Aug, 172(8), 4472 - 81 The ompA 5' untranslated RNA segment functions in Escherichia coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency; Emory SA et al.; The 5' untranslated region (UTR) of the long-lived Escherichia coli ompA message can function in vivo as an mRNA stabilizer . Substitution of this ompA mRNA segment for the corresponding segment of the labile bla gene transcripts prolongs their lifetime by a factor of 6 . We show here that the function of this ompA mRNA stabilizer requires the presence of a 115-nucleotide ompA RNA segment that lies upstream of the ribosome-binding site . Although deletion of this segment reduced the half-life of the ompA transcript by a factor of 5, its absence had almost no effect on the translational efficiency of ompA mRNA . Like the ompA transcript, but unlike bla mRNA, hybrid ompA-bla messages containing the complete ompA 5' UTR were significantly less stable under conditions of slow bacterial growth . We conclude that the stabilizing activity of the ompA 5' UTR is growth rate regulated and that the mechanism of mRNA stabilization by this RNA segment is not related to the spacing between translating ribosomes. J Bacteriol, 1990 Aug, 172(8), 4197 - 205 soxR, a locus governing a superoxide response regulon in Escherichia coli K-12; Tsaneva IR et al.; The nfo (endonuclease IV) gene of Escherichia coli is induced by superoxide generators such as paraquat (methyl viologen) . An nfo'-lacZ operon fusion was used to isolate extragenic mutations affecting its expression . The mutations also affected the expression of glucose 6-phosphate dehydrogenase, Mn2(+)-superoxide dismutase (sodA), and three lacZ fusions to soi (superoxide-inducible) genes of unknown function . The mutations were located 2 kilobases clockwise of ssb at 92 min on the current linkage map . One set of mutations, in a new gene designated soxR, caused constitutive overexpression of nfo and the other genes . It included insertions or deletions affecting the carboxyl end of a 17-kilodalton polypeptide . In a soxR mutant, the expression of sodA, unlike that of nfo, was also regulated independently by oxygen tension . Two other mutants were isolated in which the target genes were noninducible; they had an increased sensitivity to killing by superoxide-generating compounds . One had a Tn10 insertion in or near soxR; the other had a multigene deletion encompassing soxR . Therefore, the region functions as a positive regulator because it encodes one or more products needed for the induction of nfo . Regulation is likely to be at the level of transcription because the mutations were able to affect the expression of an nfo'-lac operon fusion that contained the ribosome-binding site for lacZ . Some mutant plasmids that failed to suppress (or complement) constitutivity in trans had insertion mutations several hundred nucleotides upstream of soxR in the general region of a gene for a 13-kilodalton protein encoded by the opposite strand, raising the possibility of a second regulatory gene in this region . The result define a new regulon, controlled by soxR, mediating at least part of the global response to superoxide in E . coli. Infect Immun, 1990 Aug, 58(8), 2719 - 24 Synthetic peptide segments from the Escherichia coli porin OmpF constitute leukocyte activators; Vordermeier HM et al.; After characterization of the porin OmpF and selection of molecular structures responsible for leukocyte activation by using computer-assisted epitope analysis, the analogs OmpF (153-174) (containing amino acids 153 to 174), OmpF (157-174), and OmpF (275-285) were synthesized and tested . Like the native protein, the segments were mitogenic for BALB/c splenocytes and induced B lymphocyte differentiation into antibody-producing plasma cells and tumor cytotoxicity of macrophages against the fibroblast cell line L929 . We thus demonstrated that defined peptide segments are responsible for the leukocyte-activating properties of a major bacterial surface protein. Infect Immun, 1990 Aug, 58(8), 2637 - 43 Identification and mapping of an immunogenic region of Mycoplasma hyopneumoniae p65 surface lipoprotein expressed in Escherichia coli from a cloned genomic fragment; Kim MF et al.; A previously characterized lipid-modified amphiphilic surface protein of Mycoplasma hyopneumoniae, p65, has been defined by its reaction with a surface-binding monoclonal antibody (MAb) and by its exclusive partitioning into the detergent phase during Triton X-114 phase fractionation (K . S . Wise and M . F . Kim, J . Bacteriol . 169:5546-5555, 1987) . In the current study, polyclonal mouse antibody (PAb) to gel-purified p65 was used to identify recombinant phage plaques expressing p65-related epitopes . Several characteristic partial tryptic fragments of p65 were recognized by both PAb and p65 and MAb to p65, but the PAb population specifically eluted from recombinant phage plaques bound only epitopes restricted to the largest of these fragments . Graded carboxypeptidase-Y digestion of intact M . hyopneumoniae generated C terminally truncated peptides that were recognized by PAb to p65 and MAb to p65, indicating that the C terminus and much of the adjoining region of p65 were present and accessible on the external face of the membrane . However, antibody eluted from recombinant phage plaques bound only to the largest truncated polypeptide, suggesting that a recombinant product corresponding to the C-terminal region of p65 was expressed in Escherichia coli . A 19-kilodalton recombinant protein (p19), which was recognized by PAb to p65 but not by MAb to p65, was detected in recombinant phage lysates . Serum antibodies from swine taken after, but not before, experimentally induced M . hyopneumoniae pneumonia preferentially recognized the native, amphiphilic p65 lipoprotein and also bound specifically to the p19 recombinant product . This confirmed that the p65 lipoprotein is a major immunogen of M . hyopneumoniae recognized during disease and identified its C-terminal region as an immunogenic domain. Enzyme Microb Technol, 1990 Aug, 12(8), 612 - 5 Endonuclease-free, protoplast-forming enzyme preparation and its application in fungal transformation; Mink M et al.; An endonuclease-free, protoplast-forming enzyme complex was prepared from the "snail enzyme." The purified preparation has high protoplast-forming activity comparable to the crude enzyme complex without destroying circular plasmid DNA . Furthermore, a higher transformation rate was achieved by the application of the endonuclease-free enzyme complex in both yeast and filamentous fungal vector-host systems. Enzyme Microb Technol, 1990 Aug, 12(8), 584 - 90 Selective flocculation with chitosan in Escherichia coli disintegrates: effects of pH and nuclease treatment; Agerkvist I et al.; The flocculation of cell debris from a beta-galactosidase constitutive E . coli with chitosan as a flocculant was studied to investigate the possibility of obtaining a selective flocculation in cell disintegrates with high product recoveries . The flocculation removed 98% of the cell debris by 30 min sedimentation under gravity, which should be compared to a separation of the cell debris without flocculation of only 70% by centrifugation at 15,000 g . Optimal flocculation dosages varied between 12 and 43 mg chitosan g-1 dry weight of cells, depending on pH . The yield of the product beta-galactosidase reached 60% at optimal pH . Hydrolysis of the nucleic acids by DNAase and RNAase decreased the optimal flocculation dosages considerably . The study showed that the flocculation is somewhat selective, since chitosan also removed 85% of the nucleic acids and 50% of the proteins, which contributed to the purification of the protein solution. Biochemistry, 1990 Jul 31, 29(30), 7024 - 32 Mechanistic studies of ionizing radiation and oxidative mutagenesis: genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome; Wood ML et al.; T4 RNA ligase was used to construct a deoxypentanucleotide containing a single 8-hydroxyguanine (7-hydro-8-oxoguanine; G8-OH) residue, which is one of the putatively mutagenic DNA adducts produced by oxidants and ionizing radiation . The pentamer d(GCTAG8-OH)p was prepared by the ligation of a chemically synthesized acceptor molecule, d(GCTA), to an adducted donor, 8-hydroxy-2'-deoxyguanosine 5',3'-bisphosphate . The acceptor was efficiently converted to the reaction product (greater than 95%), and the final product yield was 50% . Following 3'-dephosphorylation, the pentamer was characterized by UV spectroscopy, by high-pressure liquid chromatography, and by gas chromatography-mass spectrometry of the nucleosides released by enzymatic hydrolysis . Both d(GCTAG8-OH) and an unmodified control were 5'-phosphorylated by using {gamma -32P}ATP and incorporated covalently by DNA ligase into a five-base gap at a unique NheI restriction site in the otherwise duplex genome of an M13mp19 derivative . The ligation product contained G8-OH at the 3' residue of an in-frame amber codon (5'-TAG-3') (genome position 6276) of the phage lacZ alpha gene . The adduct was part of a nonsense codon in a unique restriction site in order to facilitate the identification and selection of mutants generated by the replication of the modified genome in Escherichia coli . Both control and adducted pentamers ligated into the genome at 50% of the maximum theoretical efficiency, and nearly all (approximately 90%) of the site-specifically adducted products possessed pentanucleotides that were covalently linked at both 5' and 3' termini . The G8-OH lesion in the NheI site inhibited the cleavage of the site by a 200-fold excess of NheI.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Jul 31, 29(30), 7011 - 20 Folding and stability of trp aporepressor from Escherichia coli; Gittelman MS et al.; Equilibrium and kinetic studies of the urea-induced unfolding of trp aporepressor from Escherichia coli were performed to probe the folding mechanism of this intertwined, dimeric protein . The equilibrium unfolding transitions at pH 7.6 and 25 degrees C monitored by difference absorbance, fluorescence, and circular dichroism spectroscopy are coincident within experimental error . All three transitions are well described by a two-state model involving the native dimer and the unfolded monomer; the free energy of folding in the absence of denaturant and under standard-state conditions is estimated to be 23.3 +/- 0.9 kcal/mol of dimer . The midpoint of the equilibrium unfolding transition increases with increasing protein concentration in the manner expected from the law of mass action for the two-state model . We find no evidence for stable folding intermediates . Kinetic studies reveal that unfolding is governed by a single first-order reaction whose relaxation time decreases exponentially with increasing urea concentration and also decreases with increasing protein concentration in the transition zone . Refolding involves at least three phases that depend on both the protein concentration and the final urea concentration in a complex manner . The relaxation time of the slowest of these refolding phases is identical with that for the single phase in unfolding in the transition zone, consistent with the results expected for a reaction that is kinetically reversible . The two faster refolding phases are presumed to arise from slow isomerization reactions in the unfolded form and reflect parallel folding channels. Biochemistry, 1990 Jul 31, 29(30), 6977 - 86 Pairwise specificity and sequential binding in enzyme catalysis: thymidylate synthase; Finer-Moore JS et al.; The structures of thymidylate synthase (TS) from Escherichia coli, in ternary complexes with substrate and an analogue of the cofactor, are the basis of a stereochemical model for a key reaction intermediate in the catalyzed reaction . This model is used to compare the reaction chemistry and chirality of the transferred methyl group with structures of the components, to identify those residues that participate, and to propose a stereochemical mechanism for catalysis by TS . Effects of chemical modification of specific amino acid residues and site-directed mutations of residues are correlated with structure and effects on enzyme mechanism . The ordered binding sequence of substrate deoxyuridine monophosphate and methylenetetrahydrofolate can be understood from the structure, where each forms a large part of the binding site for the other . The catalytic site serves to orient the reactants, which are sequestered along with many water molecules within a cavernous active center . Conformational changes during the reaction could involve nearby residues in ways that are not obvious in this complex. Biochemistry, 1990 Jul 31, 29(30), 6964 - 77 Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate; Montfort WR et al.; The structure of Escherichia coli thymidylate synthase (TS) complexed with the substrate dUMP and an analogue of the cofactor methylenetetrahydrofolate was solved by multiple isomorphous replacement and refined at 1.97-A resolution to a residual of 18% for all data (16% for data greater than 2 sigma) for a highly constrained structure . All residues in the structure are clearly resolved and give a very high confidence in total correctness of the structure . The ternary complex directly suggests how methylation of dUMP takes place . C-6 of dUMP is covalently bound to gamma S of Cys-198(146) during catalysis, and the reactants are surrounded by specific hydrogen bonds and hydrophobic interactions from conserved residues . Comparison with the independently solved structure of unliganded TS reveals a large conformation change in the enzyme, which closes down to sequester the reactants and several highly ordered water molecules within a cavernous active center, away from bulk solvent . A second binding site for the quinazoline ring of the cofactor analogue was discovered by withholding addition of reducing agent during crystal storage . The chemical change in the protein is slight, and from difference density maps modification of sulfhydryls is not directly responsible for blockade of the primary site . The site, only partially overlapping with the primary site, is also surrounded by conserved residues and thus may play a functional role . The ligand-induced conformational change is not a domain shift but involves the segmental accommodation of several helices, beta-strands, and loops that move as units against the beta-sheet interface between monomers. Eur J Biochem, 1990 Jul 31, 191(2), 325 - 31 Transcriptional control of the cysG gene of Escherichia coli K-12 during aerobic and anaerobic growth; Peakman T et al.; The 74-min region of the Escherichia coli chromosome includes five open reading frames of known sequence . The first and last of these genes, nirB and cysG, are transcribed in the same direction and both are essential for NADH-dependent nitrite reductase activity . The functions of the other genes, nirD, nirE and nirC, which are located between nirB and cysG, are unknown . The nirB gene is transcribed from a promoter which is anaerobically induced, expression being dependent on the transcription activator protein, Fnr . Here we show that the nirD, nirE, nirC and cysG genes are also expressed from the nirB promoter . After subcloning cysG, a second promoter was located less than 100 bases upstream of cysG . Two groups of transcription start points separated by 40 bases were detected in this region by S1 mapping . Rates of transcription from the isolated cysG promoter were the same during aerobic growth and anaerobic growth in the presence or absence of nitrite . However, when the nirB gene and its promoter were cloned back upstream from the cysG promoter, the rate of transcription was higher during anaerobic growth than during aerobic growth and was further induced by nitrite . These increases were totally dependent on a functional fnr gene and were shown by S1 mapping experiments to be due to transcriptional read-through from the Fnr-dependent nirB promoter . No promoter activity was associated with DNA fragments between the BamHI site located within the N-terminal coding region of the nirB gene and the cysG promoter located at the C-terminus of nirC. Eur J Biochem, 1990 Jul 31, 191(2), 315 - 23 Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coli K-12 chromosome; Peakman T et al.; The DNA sequence and derived amino-acid sequence of a 5618-base region in the 74-min area of the Escherichia coli chromosome has been determined in order to locate the structural gene, nirB, for the NADH-dependent nitrite reductase and a gene, cysG, required for the synthesis of the sirohaem prosthetic group . Three additional open reading frames, nirD, nirE and nirC, were found between nirB and cysG . Potential binding sites on the NirB protein for NADH and FAD, as well as conserved central core and interface domains, were deduced by comparing the derived amino-acid sequence with those of database proteins . A directly repeated sequence, which includes the motif -Cys-Xaa-Xaa-Cys-, is suggested as the binding site for either one {4Fe-4S} or two {2Fe-2S} clusters . The nirD gene potentially encodes a soluble, cytoplasmic protein of unknown function . No significant similarities were found between the derived amino-acid sequence of NirD and either NirB or any other protein in the database . If the nirE open reading frame is translated, it would encode a 33-amino-acid peptide of unknown function which includes 8 phenylalanyl residues . The product of the nirC gene is a highly hydrophobic protein with regions of amino-acid sequence similar to cytochrome oxidase polypeptide 1. Biochem Biophys Res Commun, 1990 Jul 31, 170(2), 705 - 12 Expression of a catalytically active polypeptide of calmodulin-dependent protein kinase II alpha subunit in Escherichia coli; Ohsako S et al.; Two cDNAs, one containing the entire coding region of alpha subunit of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the other containing only its protein kinase domain, were separately ligated into the bacterial expression vector pET3a and expressed in Escherichia coli . The activity of the recombinant alpha subunit protein was dependent on Ca2+/calmodulin, whereas the activity of the recombinant protein containing only the protein kinase domain (recombinant alpha-I protein) was absolutely independent of Ca2+/calmodulin . These proteins showed similar enzymatic properties to brain CaM kinase II with some minor differences . These results directly demonstrated that the protein kinase domain alone without the rest of the subunit was sufficient to exhibit its activity. FEBS Lett, 1990 Jul 30, 268(1), 301 - 5 Expression and characterization of human lamin C; Moir RD et al.; We have expressed human lamin C cDNA in E . coli using a modification of the pLcII vector system . Protein produced in this way had seven additional amino acids at its N-terminus, but retained key lamin structural and assembly properties . The modified vector we produced may prove useful when difficulties are encountered in removal of the cII fusion peptide by factor X cleavage in the pLcII system . Shadowed preparations of expressed lamin C showed the presence of 50-nm rod-like particles that closely resembled those observed for native material . Isolated molecules had two globular domains at one end, indicating that they were constructed from two parallel polypeptide chains . The expressed material also formed paracrystals with a characteristic 22.5 nm axial repeat, indicating that its assembly properties had also been retained . We also used site-specific mutagenesis to engineer a lamin fragment that lacked the C-terminal non-helical domain of the molecule . This material formed paracrystals similar to those obtained with the intact molecule, indicating that the large C-terminal non-helical domain did not contain information vital for lamin assembly. FEBS Lett, 1990 Jul 30, 268(1), 213 - 6 The environments of Trp-248 and Trp-330 in tryptophan indole-lyase from Escherichia coli; Phillips RS et al.; The two tryptophan residues, Trp-248 and Trp-330, in tryptophan indole-lyase (tryptophanase) from E . coli have been separately mutated to phenylalanine using site-directed mutagenesis . Both single tryptophan mutant enzymes have full catalytic activity, but exhibit different fluorescence and near-UV circular dichroism spectra . These results indicate that Trp-330 is more deeply buried than is Trp-248, and is in a more asymmetric environment . Neither residue reacts with N-bromosuccinimide (NBS), although tryptophan indole-lyase is inactivated by NBS . These results demonstrate that the tryptophan residues in tryptophan indole-lyase are not catalytically essential. FEBS Lett, 1990 Jul 30, 268(1), 17 - 20 Grapevine stilbene synthase cDNA only slightly differing from chalcone synthase cDNA is expressed in Escherichia coli into a catalytically active enzyme; Melchior F et al.; Stilbene synthase is responsible for the formation of resveratrol and other stilbenes which function in grapevine as phytoalexins . A full-length stilbene synthase cDNA was prepared from grapevine mRNA and sequenced . The insert in pSV25 coding for a polypeptide with 392 amino acids was inserted into the vectors pKK233-2 and pDS12/RBSII-2, respectively . Expression of the cDNA in Escherichia coli yielded an enzymatically active dimer exhibiting solely stilbene synthase activity . The protein was characterized by enzyme activity and Western blot analysis. FEBS Lett, 1990 Jul 30, 268(1), 146 - 8 Inactivation of thioredoxin by sulfite ions; Wurfel M et al.; Oxidized thioredoxin undergoes sulfitolysis of its single disulfide bond at low concentrations of sulfite ions and protein and in the absence of denaturing agents . The reaction, which has an optimum at pH 8, was studied using {35S}sulfite and E . coli thioredoxin as model . The product, thioredoxin-S-sulfonate, has a half-life of several hours in solution . It is unable to activate chloroplast NADP malate dehydrogenase . Thioredoxin sulfitolysis may therefore be a physiologically important factor in mediating the phytotoxic effects of sulfur dioxide in plants. Biochim Biophys Acta, 1990 Jul 30, 1049(3), 278 - 85 Comparison of secretory expression in Escherichia coli and Streptomyces of Streptomyces subtilisin inhibitor (SSI) gene; Taguchi S et al.; To elucidate differences in the mechanism of gene expression between Streptomyces and Escherichia coli, the regulatory region for expression of the gene for a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), was altered to express efficiently in E . coli . This was carried out by inserting a pre-SSI-encoding region downstream of the tac promoter and ribosome-binding site in a multi-copy plasmid . When the resultant plasmid pMKSI161-9 was introduced into E . coli JM105, SSI protein was found to be expressed and secreted into the periplasmic space by Western blot analysis . When introduced into 'leaky' E . coli strains, this protein was detected in the medium as well as in the periplasmic space in bacteria . NH2-terminal sequencing analysis of the SSI purified from E . coli JM105 indicated two processing sites, Ala(-4)/Ala(-3)-Pro(-2)-Gly and Ala(-4)-Ala-3/Pro(-2)-Gly-1, of pre-SII . These sites were different from those in Streptomyces albogriseolus S-3253 and Streptomyces lividans 66 . The inhibitor activity of the processed protein toward subtilisin BPN' was almost the same as that of authentic SSI. Biochim Biophys Acta, 1990 Jul 30, 1049(3), 339 - 42 Interactions of berberine with poly(A) and tRNA; Nandi R et al.; The interaction of berberine chloride with poly(A) and tRNA has been studied by various spectroscopic techniques . Binding parameters determined from spectrophotometric and spectrofluorimetric measurements by Scatchard analysis indicate a very high effective binding capacity of berberine to poly(A) as compared to DNA or tRNA . The circular dichroism studies show that binding of berberine to poly(A) causes a significant change in the circular dichroic spectrum of poly(A) itself, as manifested by (i) a decrease of both positive and negative bands and (ii) appearance of a conservative type of extrinsic circular dichroic spectrum in the wavelength range of 300-400 nm, while it does not cause any significant alteration to the A form structure of tRNA . It is concluded that berberine interacts stronger with poly(A) than DNA or tRNA . The results are interpreted in terms of its reported biological activities. Science, 1990 Jul 27, 249(4967), 404 - 6 Random peptide libraries: a source of specific protein binding molecules; Devlin JJ et al.; Libraries of random peptide sequences were constructed and screened to identify peptides that specifically bind to proteins . In one of these about 2 X 10(7) different 15-residue peptide sequences were expressed on the surface of the coliphage M13 . Each phage encoded a single random sequence and expressed it as a fusion complex with pIII, a minor coat protein present at five molecules per phage . Phage encoding nine different streptavidin-binding peptide sequences were isolated from this library . The core consensus sequence was His-Pro-Gln and binding of these phage to streptavidin was inhibited by biotin . This type of library makes it possible to identify peptides that bind to proteins (or other macromolecules) that have no previously known affinity for peptides. Science, 1990 Jul 27, 249(4967), 386 - 90 Searching for peptide ligands with an epitope library; Scott JK et al.; Tens of millions of short peptides can be easily surveyed for tight binding to an antibody, receptor or other binding protein using an "epitope library." The library is a vast mixture of filamentous phage clones, each displaying one peptide sequence on the virion surface . The survey is accomplished by using the binding protein to affinity-purify phage that display tight-binding peptides and propagating the purified phage in Escherichia coli . The amino acid sequences of the peptides displayed on the phage are then determined by sequencing the corresponding coding region in the viral DNA's . Potential applications of the epitope library include investigation of the specificity of antibodies and discovery of mimetic drug candidates. Nature, 1990 Jul 26, 346(6282), 394 - 6 Spontaneous shuffling of domains between introns of phage T4; Bryk M et al.; The three self-splicing introns in phage T4 (in the td, sunY and nrdB genes) (Fig . 1a) each have the conserved group I catalytic RNA core structure (Fig . 1b), out of which is looped an open reading frame . Although the core sequences are very similar (approximately 60% identity), the open reading frames seem to be unrelated . Single crossover recombination events between homologous core sequences in the closely linked td and nrdB introns have led to 'exon shuffling . Here we describe spontaneous double crossovers between the unlinked td and sun Y introns that result in shuffling of an intron structure element, P7.1 (refs 3 and 4) . The intron domain-switch variants were isolated as genetic suppressors of a splicing-defective P7.1 deletion in the td intron . This unprecedented example of suppression through inter-intron sequence substitution indicates that the introns are in a state of genetic flux and implies the functional interchangeability of the two analogous but nonidentical P7.1 elements . The implications of such recombination events are discussed in the light of the evolution of the introns themselves as well as that of their host genomes. Nucleic Acids Res, 1990 Jul 25, 18(14), 4083 - 8 Alternative base pairing between 5'- and 3'-terminal sequences of small subunit RNA may provide the basis of a conformational switch of the small ribosomal subunit; Kossel H et al.; The compiled sequences of small subunit ribosomal RNAs have been screened for base complementary between 5'- and 3'-terminal regions . Highly conserved complementary sequences are found which allow formation of a helix between the two ends of 5 or 6 base pairs . This helix is composed of sequences from the loop region of the first 5'-terminal stem and from sequences immediately distal to the last stem (the Me2A-stem) of the 3' terminus and therefore allows a coaxial stacking with either of these two flanking stems . Formation of the 5'/3'-helical arrangement is, however, only possible at the cost of dissolving the 'pseudo-knot' helix between the 5'-terminal region and the internal region of small subunit RNA . It is postulated that the mutually exclusive conformational states are in dynamic equilibrium and that they correlate with distinct functional states of the small ribosomal subunit . The 'pseudo-knot' containing conformation with the 3'-terminal sequences more exposed is likely to represent the initiating state, whereas the 5'/3' terminal paired 'closed' conformation may represent the elongating state in which interaction with fortuitous ribosomal binding sequences of mRNAs is avoided. J Biol Chem, 1990 Jul 25, 265(21), 12728 - 33 Protein composition of Vitreoscilla hemoglobin inclusion bodies produced in Escherichia coli; Hart RA et al.; The protein composition of inclusion bodies produced in recombinant Escherichia coli overproducing Vitreoscilla hemoglobin (VHb) was analyzed by one-dimensional and two-dimensional electrophoresis techniques . Results indicate the presence of two types of cytoplasmic aggregates of differing morphology in single bacterial cells . These aggregates also differ in their relative content of VHb and pre-beta-lactamase and are separable by differential centrifugation . Results further suggest that the cytoplasmic protein elongation factor Tu is integrated into VHb inclusion bodies . The presence of the outer membrane proteins OmpA and OmpF in inclusion body preparations is attributed to cell envelope contamination rather than specific involvement in inclusion bodies . The specificity of in vivo protein aggregation is discussed. J Biol Chem, 1990 Jul 25, 265(21), 12546 - 52 Clp P represents a unique family of serine proteases; Maurizi MR et al.; The amino acid sequence of Clp P, the proteolytic subunit of the ATP-dependent Clp protease of Escherichia coli, closely resembles a protein encoded by chloroplast DNA, which is well conserved between chloroplasts of different plant species . The homology extends over almost the full length of the sequences of both proteins and consists of approximately 46% identical and approximately 70% similar amino acids . Antibodies against E . coli Clp P cross-reacted with proteins with Mr of 20,000-30,000 in bacteria, lower eukaryotes, plants, and animal cells . Since the regulatory subunit of Clp protease, Clp A, also has a homolog in plants, as well as in other bacteria and in lower eukaryotes, it is likely that ATP-dependent proteolysis in chloroplasts is catalyzed in part by a Clp-like protease and that both components of Clp-like proteases are widespread in living cells . We have identified Ser-111 as the active site serine in E . coli Clp P modified by diisopropyl fluorophosphate . Mutational alteration of Ser-111 or His-136 eliminates proteolytic activity of Clp P . Both residues are found in highly conserved regions of the protein . The sequences around the active site residues suggest that Clp P represents a unique class of serine protease . Amino-terminal processing of cloned Clp P mutated at either Ser-111 or His-136 occurs efficiently when wild-type clpP is present in the chromosome but is blocked in clpP- hosts . Processing of Clp P appears, therefore, to involve an intermolecular autocatalytic cleavage reaction . Since processing of Clp P occurs in clpA- cells, the autoprocessing activity of Clp P is independent of Clp A. J Biol Chem, 1990 Jul 25, 265(21), 12536 - 45 Sequence and structure of Clp P, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli; Maurizi MR et al.; The ATP-dependent Clp protease of Escherichia coli contains two dissimilar components: the Clp A regulatory polypeptide, with two ATP binding sites and intrinsic ATPase activity, and the Clp P subunit, which contains the proteolytic active site . The DNA sequence of the clpP gene predicts a protein of 207 amino acids (Mr 21,679), which is in close agreement with the size determined by sodium dodecyl sulfate-gel electrophoresis of purified Clp P . Clp P has a native Mr of approximately 240,000, and electron micrographs of the protein show superimposed disk-like structures with a central cavity, similar in appearance to purified proteasomes from eukaryotic cells . Clp P is synthesized with a 14-amino acid leader which is rapidly cleaved in vivo to yield the 193-amino acid protein which has activity in vitro . The clpP gene is at 10 min on the E . coli map, close to that for the ATP-dependent Lon protease of E . coli and far from the gene for clpA . Primer extension experiments indicate that transcription initiates immediately upstream of the coding region for Clp P, with a major transcription start at 120 bases in front of the start of translation . Insertion mutations in clpP have been isolated and transferred to the chromosome; strains devoid of Clp P are viable in the presence or absence of Lon protease . Mutations in clpP stabilize the same Clp A-beta-galactosidase fusion protein specifically stabilized by clpA mutations, providing the first genetic evidence that Clp A and Clp P act together in vivo. J Biol Chem, 1990 Jul 25, 265(21), 12337 - 41 Cloning metabolic pathway genes by complementation in Escherichia coli . Isolation and expression of Plasmodium falciparum glucose phosphate isomerase; Kaslow DC et al.; Genetic complementation of an Escherichia coli double mutant was used to isolate and express the gene coding for Plasmodium falciparum glucose phosphate isomerase . The gene contains a 1773-base pair open reading frame, has no introns, and maps to P . falciparum chromosome 14 . 34% of the deduced amino acid sequence is identical to human glucose phosphate isomerase, with highest similarity in regions of the proposed active sites . The putative initiation site of translation was determined by deletional and oligonucleotide mediated, site-specific mutageneses . Our data suggest that key metabolic enzymes of Plasmodia can be cloned and expressed in E . coli without prior knowledge of the primary amino acid or nucleic acid structure. J Biol Chem, 1990 Jul 25, 265(21), 12287 - 92 UhpT, the sugar phosphate antiporter of Escherichia coli, functions as a monomer; Ambudkar SV et al.; We have characterized the minimal functioning unit of UhpT, the secondary carrier that mediates exchange of phosphate and glucose 6-phosphate in Escherichia coli . Membranes of a UhpT overproducing strain were solubilized with 1.25% octyl beta-D-glucopyranoside, in the presence of 0.1% E . coli phospholipid and with 20% glycerol as the osmolyte stabilant . That soluble UhpT could bind its natural substrates was indicated by the protections afforded by sugar phosphates against thermal inactivation or chemical modification with pyridoxal 5'-phosphate . Moreover, the degree of protection correlated with the strength of interaction between UhpT and the test substrate (2-deoxyglucose 6-phosphate = glucose 6-phosphate greater than galactose 6-phosphate = glucose 1-phosphate much greater than glucose 6-sulfate) . Other experiments demonstrated that soluble UhpT existed as a monomer . For example, during both high performance liquid chromatography and conventional gel permeation chromatography, the elution pattern of UhpT activity was measured directly by a rapid reconstitution technique . In both cases, and in the presence and absence of substrate, UhpT activity traveled as a single component of Mr 53,000, corresponding closely to the sequence prediction of 50,600 . Finally, reconstitution was studied at protein to lipid ratios low enough to achieve between 0.075 and 1.5 UhpT monomers/proteoliposome . Specific activity was constant throughout this range, a finding consistent with the idea of a functional monomer . Mitochondria and chloroplasts provide the only other anion exchange carriers described at this level of biochemical resolution, and these organelle antiporters function as dimers . By contrast, work summarized here places their bacterial counterpart, UhpT, in the same class as the lactose carrier of E . coli and the glucose carrier of the human erythrocyte, both of which function as monomers . Consideration of this pattern in conjunction with the known hydropathy profiles of these proteins suggests a novel scheme for the classification of all secondary carriers, with implications for both the structure and origin of these transport proteins. Biochim Biophys Acta, 1990 Jul 25, 1018(2-3), 124 - 7 Recent studies of the cytochrome o terminal oxidase complex of Escherichia coli; Chepuri V et al.; The cytochrome o complex is the predominant terminal oxidase in the aerobic respiratory chain of Escherichia coli when the bacteria are grown under conditions of high aeration . The oxidase is a ubiquinol oxidase and reduces molecular oxygen to water . Electron transport through the enzyme is coupled to the generation of a protonmotive force . The purified cytochrome o complex contains four or five subunits, two protoheme IX (heme b) prosthetic groups, plus at least one Cu . The subunits are all encoded by the cyo operon . Sequence comparisons show that the cytochrome o complex is closely related to the aa3-type cytochrome c oxidase family . Gene fusions have been used to define the topology of each of the gene products . Subunits I, II, III and IV are proposed to have 15, 2, 5 and 3 transmembrane spans, respectively . The fifth gene product (cyoE) encodes a protein with 7 membrane spanning segments, and this may also be a subunit of this enzyme . Fourier transform infrared spectroscopy has been used to monitor CO bound in the active site where oxygen is reduced . These data provide definitive proof that the cytochrome o complex has a heme-copper binuclear center, similar to that present in the aa3-type cytochrome c oxidases . Site-directed mutagenesis is being utilized to define which amino acids are ligands to the heme iron and copper prosthetic groups. Nucleic Acids Res, 1990 Jul 25, 18(14), 4105 - 10 Protein sequence comparisons show that the 'pseudoproteases' encoded by poxviruses and certain retroviruses belong to the deoxyuridine triphosphatase family; McGeoch DJ; Amino acid sequence comparisons show extensive similarities among the deoxyuridine triphosphatases (dUTPases) of Escherichia coli and of herpesviruses, and the 'protease-like' or 'pseudoprotease' sequences encoded by certain retroviruses in the oncovirus and lentivirus families and by poxviruses . These relationships suggest strongly that the 'pseudoproteases' actually are dUTPases, and have not arisen by duplication of an oncovirus protease gene as had been suggested . The herpesvirus dUTPase sequences differ from the others in that they are longer (about 370 residues, against around 140) and one conserved element ('Motif 3') is displaced relative to its position in the other sequences; a model involving internal duplication of the herpesvirus gene can account effectively for these observations . Sequences closely similar to Motif 3 are also found in phosphofructokinases, where they form part of the active site and fructose phosphate binding structure; thus these sequences may represent a class of structural element generally involved in phosphate transfer to and from glycosides. J Biol Chem, 1990 Jul 25, 265(21), 12400 - 3 Substitution of 2 base pairs (1 base pair per DNA half-site) within the Escherichia coli lac promoter DNA site for catabolite gene activator protein places the lac promoter in the FNR regulon; Zhang XP et al.; The consensus DNA site for Escherichia coli catabolite gene activator protein (CAP) is 5'-AAATGTGATCTAGATCACATTT-3' . The proposed consensus DNA site for E . coli FNR is 5'-AAATTTGATATATATCAAATTT-3' . In this report, we show that substitution of 2 base pairs (1 base pair per DNA half-site) within the E . coli lac DNA site for CAP suffices to remove the lac promoter from the CAP regulon and to place the lac promoter in the FNR regulon . FNR stimulates transcription of the derivative of the lac promoter having G:C----T:A substitutions at base pair 5 each DNA half-site (13-fold stimulation) . FNR does not stimulate transcription of the wild-type lac promoter, or of derivatives of the lac promoter having G:C----A:T or G:C----C:G substitutions at base pair 5 of each DNA half-site . Stimulation of transcription is strictly dependent on anaerobiosis . FNR-stimulated transcription initiates at the same base pair as does CAP-dependent transcription of the wild-type lac promoter. J Biol Chem, 1990 Jul 25, 265(21), 12300 - 5 Effects of transcription and translation on gyrase-mediated DNA cleavage in Escherichia coli; Koo HS et al.; Gyrase-mediated DNA cleavage on plasmid DNAs was measured in Escherichia coli treated with oxolinic acid . On pBR322 DNA, gyrase cleavage sites were concentrated in the region between the 3'-ends of the tetA and bla genes . The preferential cleavage in this region was dependent on RNA transcription and the divergent orientation of these two transcription units . The enhanced gyrase cleavage also required translation; chloramphenicol treatment or the insertion of a translation terminator within the 5'-proximal region of the tetA gene abolished the enhanced cleavage . We suggest that the enhanced gyrase cleavage may reflect the changes in local DNA supercoiling during RNA transcription as gyrase cleavage in vitro was shown to be sensitive to the supercoiling state of DNA . The effects of transcription and translation on gyrase cleavage can best be explained by the twin-supercoiled-domain model of transcription (Liu, L . F., and Wang, J . C . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 7024-7027). J Biol Chem, 1990 Jul 25, 265(21), 12280 - 6 Differential metabolism of diradyl glycerol molecular subclasses and molecular species by rabbit brain diglyceride kinase; Ford DA et al.; Elevations in the mass of ether-linked diglycerides (i.e . 1-O-alk-1'-enyl-2-acyl-sn-glycerol (AAG) and 1-O-alkyl-2-acyl-sn-glycerol (Alkyl AG)) during cellular activation are prolonged in comparison to their 1,2-diacyl-sn-glycerol (DAG) counterparts . Since the metabolic removal of DAG is determined, in large part, by the rate of its phosphorylation by diglyceride kinase, we quantified differences in the activity of diglyceride kinase utilizing individual subclasses of diradyl glycerols as substrate . Rabbit brain microsomal diglyceride kinase activity was over 30-fold greater utilizing DAG as substrate (25.8 nmol.mg-1.min-1) in comparison to AAG (0.8 nmol.mg-1.min-1) . No alterations in the affinity of microsomal diglyceride kinase for ATP were present (Km approximately 0.5 mM) utilizing each diradyl glycerol subclass . Similar subclass specificities for diglyceride kinase (i.e . DAG greater than Alkyl AG much greater than AAG) were present in brain and liver cytosol as well as in liver microsomes utilizing multiple assay conditions . In sharp contrast, Escherichia coli diglyceride kinase phosphorylated DAG, Alkyl AG, or AAG diradyl glycerol molecular subclasses at identical rates . Furthermore, although DAG was rapidly hydrolyzed by diglyceride lipase, catabolism of AAG or Alkyl AG by plasmalogenase, alkyl ether hydrolase, or diglyceride/monoglyceride lipase was undetectable . Collectively, these results demonstrate the importance of the differential catabolism of each diradyl glycerol molecular subclass as a primary determinant of their biologic half-lives . Since individual subclasses of diglycerides have distinct physical properties and physiologic functions, these results underscore the importance of lipid subclass specific metabolism in tailoring individual cellular responses during activation. Biochim Biophys Acta, 1990 Jul 25, 1018(2-3), 203 - 5 A plasmid-encoded anion-translocating ATPase; Rosen BP et al.; An anion-translocating ATPase has been identified as the product of the arsenical resistance operon of resistance plasmid R773 . When expressed in Escherichia coli this ATP-driven oxyanion pump catalyzes extrusion of the oxyanions arsenite, antimonite and arsenate . Maintenance of a low intracellular concentration of oxyanion produces resistance to the toxic agents . The pump is composed of two polypeptides, the products of the arsA and arsB genes . This two-subunit enzyme produces resistance to arsenite and antimonite . A third gene, arsC, expands the substrate specificity to allow for arsenate pumping and resistance. J Biol Chem, 1990 Jul 25, 265(21), 12146 - 8 Specific suppression of heterotropic interactions in phosphofructokinase by the mutation of leucine 178 into tryptophan; Serre MC et al.; The leucine residue at position 178 in the allosteric phosphofructokinase from Escherichia coli has been changed into a tryptophan residue by oligonucleotide-directed mutagenesis . The modified enzyme has been purified to homogeneity, and its enzymatic properties show that this single mutation suppresses the heterotropic interactions without affecting the homotropic ones . The mutant has the same saturation curve by fructose 6-phosphate as the wild type, showing that its active site binds this substrate with the same affinity and cooperativity . The regulatory site of the mutant enzyme can bind the effectors, the activator GDP, or the inhibitor phosphoenolpyruvate, as measured by protection against irreversible thermal denaturation . However, the binding of either effector does no longer influence the activity . This specific suppression of the coupling between the regulatory and active sites is not predicted by the concerted model which postulates that the same structural transition between two states R and T is responsible for both homotropic and heterotropic interactions . Leu-178 belongs to neither the active nor the regulatory site but appears as an important residue in the conformational change(s) involved in the regulation by allosteric effectors. Nucleic Acids Res, 1990 Jul 25, 18(14), 4175 - 8 A simple vector modification to facilitate oligonucleotide-directed mutagenesis; Setzer DR et al.; We describe a simple modification of commonly used single-stranded cloning vectors that permits the efficient recovery of mutant DNA molecules in oligonucleotide-directed mutagenesis experiments, even when the absolute efficiency of mutagenesis is very low . The modification consists of the insertion of a short synthetic DNA fragment into the vector's polylinker and permits the identification of mutant clones based on a standard chromogenic plate assay for bacterial colonies or phage plaques producing functional beta-galactosidase . Other useful properties of the original vector are retained in the modified version . In vitro mutagenesis reactions are carried out with two oligonucleotides, one to introduce the mutation of interest, and the second to correct a frameshift mutation introduced into the beta-galactosidase gene of the modified vector . We have found that these two sequence changes are closely linked following transformation of an appropriate E . coli strain with the products of the in vitro mutagenesis reaction, and have thereby recovered desired mutations at a frequency of about 50% even when the overall mutagenesis efficiency is less than 1% . By alternately correcting and re-introducing the beta-galactosidase frameshift mutation, we have shown that multiple rounds of mutagenesis can be carried out on the same template with a high efficiency of mutant recovery in each step . Modifications similar or identical to those we describe here should be feasible for most commonly used single-stranded cloning vectors and should increase the usefulness of these vectors by providing an additional option for oligonucleotide-directed mutagenesis to be used in conjunction with or in lieu of other commonly used approaches. Nucleic Acids Res, 1990 Jul 25, 18(14), 4139 - 42 On the use of T7 RNA polymerase transcripts for physical investigation; Szewczak AA et al.; A few years ago we made some observations which raised questions about the accuracy with which T7 RNA polymerase transcribes templates in vitro, and the suitability of its in vitro products for biophysical study (1) . The experiments described below demonstrate that there is no reason for concern; the products of T7 RNA polymerase transcription in vitro are as suitable for biophysical characterization as RNAs synthesized in vivo . It is likely that aggregation involving the transcribed portions of the T7 RNA polymerase promoter caused our initial observations. Biochemistry, 1990 Jul 24, 29(29), 6789 - 98 Mechanistic studies on trans-2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (Ent A) in the biosynthesis of the iron chelator enterobactin; Sakaitani M et al.; The enzyme 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (2,3-diDHB dehydrogenase, hereafter Ent A), the product of the enterobactin biosynthetic gene entA, catalyzes the NAD(+)-dependent oxidation of the dihydroaromatic substrate 2,3-dihydro-2,3-dihydroxybenzoate (2,3-diDHB) to the aromatic catecholic product 2,3-dihydroxybenzoate (2,3-DHB) . The catechol 2,3-DHB is one of the key siderophore units of enterobactin, a potent iron chelator secreted by Escherichia coli . To probe the reaction mechanism of this oxidation, a variety of 2,3-diDHB analogues were synthesized and tested as substrates . Specifically, we set out to elucidate both the regio- and stereospecificity of alcohol oxidation as well as the stereochemistry of NAD+ reduction . Of those analogues tested, only those with a C3-hydroxyl group (but not a C2-hydroxyl group) were oxidized to the corresponding ketone products . Reversibility of the Ent A catalyzed reaction was demonstrated with the corresponding NADH-dependent reduction of 3-ketocyclohexane- and cyclohexene-1-carboxylates but not the 2-keto compounds . These results establish that Ent A functions as an alcohol dehydrogenase to specifically oxidize the C3-hydroxyl group of 2,3-diDHB to produce the corresponding 2-hydroxy-3-oxo-4,6-cyclohexadiene-1-carboxylate (Scheme II) as a transient species that undergoes rapid aromatization to give 2,3-DHB . Stereospecificity of the C3 allylic alcohol group oxidation was confirmed to be 3R in a 1R,3R dihydro substrate, 3, and hydride transfer occurs to the si face of enzyme-bound NAD+. J Chromatogr, 1990 Jul 20, 512, 3 - 12 Binding of sodium dodecyl sulphate to an integral membrane protein and to a water-soluble enzyme . Determination by molecular-sieve chromatography with flow scintillation detection; Wallsten M et al.; We have determined the binding of sodium dodecyl sulphate (SDS) to the human red cell glucose transporter (polypeptide, Mr 54,117) and to a water-soluble enzyme, N-5'-phosphoribosylanthranilate isomerase-indole-3-glycerol-phosphate synthase (PRAI-IGPS) from Escherichia coli (Mr 49,484) . {35S}SDS was equilibrated with each protein on molecular-sieve chromatography at a series of SDS concentrations . The binding ratios of SDS to protein were determined by flow scintillation detection and automated amino acid analyses . Unexpectedly the glucose transporter, which is a transmembrane protein, bound about the same amount of SDS per gram of protein as did the enzyme . At 1.6 mM SDS, slightly below the critical micelle concentration (CMC) (1.8 mM) in the eluent, the binding ratio was 1.6 g SDS/g protein for both the glucose transporter and PRAI-IGPS . At 2.0 mM SDS (above the CMC) the glucose transporter showed a binding ratio of 1.7 g SDS/g protein . The corresponding value for the enzyme was about 1.5 g/g . The SDS-glucose transporter complex seems to be more compact than the SDS-enzyme complex as judged by molecular-sieve chromatography and by SDS-polyacrylamide gel electrophoresis . Recent neutron scattering results have shown a protein-decorated triple-micelle structure for the SDS-PRAI-IGPS complex . Hypothetically, the more compact SDS-glucose transporter complex may therefore consist of a dual-micelle structure . The molecular-sieve gel beads bound considerable amounts of SDS . The SDS binding to the gel matrix and to the proteins increased with increasing SDS concentration up to at least 1.6-2.0 mM SDS . In the case of the water-soluble enzyme a shoulder was observed in the binding curve at 1 mM SDS, probably reflecting a change in the conformation of the complex. Eur J Biochem, 1990 Jul 20, 191(1), 155 - 61 Solution NMR studies of colicin E1 C-terminal thermolytic peptide . Structural comparison with colicin A and the effects of pH changes; Wormald MR et al.; The aqueous solution structure of the C-terminal thermolytic peptide of colicin E1 has been investigated using both one- and two-dimensional NMR techniques . The NMR data are consistent with a fold for the peptide very similar to that reported for the colicin A C-terminal peptide in the crystalline state, although some differences have been noted . The one-dimensional NMR spectrum of the peptide has been used to follow changes in both the structure and dynamics of the peptide on changing pH . The in vitro functionally competent form of the peptide (present in solution only below pH 6) does not differ in structure significantly from the higher pH form . However, small local conformational changes are observed together with an increase in mobility in some of the more hydrophilic regions . This suggests that the effect of lower pH is to change the ease with which the major conformational changes during insertion into a membrane can occur. J Mol Biol, 1990 Jul 20, 214(2), 397 - 406 Inhibition of DNA gyrase activity in an in vitro transcription-translation system stimulates gyrA expression in a DNA concentration dependent manner . Evidence for the involvement of factors which may be titrated; Carty M et al.; Stimulation of gyrA expression in an in vitro transcription-translation system by novobiocin, a DNA gyrase inhibitor, depends on the DNA concentration in the extract . At low DNA concentrations (less than 20 micrograms/ml) we note significant stimulation (2 to 25 x) upon the addition of novobiocin; at high DNA concentrations, stimulation is minimal . This observation is not due to the limited capacity of the system to transcribe or relax DNA . Using an extract prepared from a novobiocin-resistant strain of Escherichia coli, we were able to show that DNA gyrase mediates the novobiocin-enhanced expression . In an experiment with a fixed level of a gyrA-lac template, we found that the addition of a second non-Lac template increased expression from the gyrA-lac template, while concomitantly decreasing the extent of novobiocin stimulation . These observations are consistent with an inhibitory factor that can be titrated by increasing the DNA concentration and whose effects are minimized when the DNA template is in a relaxed conformation . The results of a mixed-extract experiment using two extracts that differ in their activities and degrees of novobiocin stimulation are also consistent with an inhibitory factor that mediates the relaxation-induced stimulation of transcription. Eur J Biochem, 1990 Jul 20, 191(1), 195 - 201 The quaternary structure of Escherichia coli inorganic pyrophosphatase is essential for phosphorylation; Sklyankina VA et al.; The hexameric inorganic pyrophosphatase (PPase) is irreversibly inactivated by phosphoric acid monoesters . The inactivation kinetics are consistent with the formation of a dissociable complex of the phosphoric acid monoester with the enzyme, followed by phosphorylation of the dicarboxylic amino acid of its active site . PPi and its analogues, binding at the regulatory site, release the inhibitor from the active site and thus restore PPase activity . Chemically identical subunits in the hexameric PPase interact, promoting their cooperativity in a reaction with phosphoric acid monoesters . The trimeric and monomeric PPase, exhibiting full catalytic activity, form a dissociable complex with the phosphoric acid monoesters but, in contrast to the hexameric PPase, do not form a covalent bond with them . This indicates that the native hexameric structure is essential for the irreversible inactivation of Escherichia coli PPase by phosphoric acid monoesters . Possible nontraditional pathways for activity regulation of PPase are discussed. Eur J Biochem, 1990 Jul 20, 191(1), 105 - 13 DNA linking potential generated by gyrase; Kozyavkin SA et al.; Whether or not DNA gyrase can supercoil DNA so that alternative structures will arise in it is the major question of this work . We have shown gyrase to produce in pAO3 DNA a superhelix density sufficient for cruciform formation . However, the transition does not take place because of too slow kinetics . A change of ionic conditions in favour of more intense DNA supercoiling by gyrase shifts the midpoint of the equilibrium transition to the cruciform structure toward more supercoiled topoisomers . The width of the equilibrium transition to the cruciform as a function of linking number has been revealed to be an order of magnitude larger in buffers containing magnesium and spermidine than in buffers with monovalent cations only . We ascribe this effect to the influence that the counter ions surrounding the DNA molecule have on its elasticity, the coefficient of elasticity being dependent on superhelix density sigma . Thus, the free energy of supercoiling (a) depends on the ionic conditions and (b) is not a quadratic function of sigma in the physiological range of parameters . We propose a description of DNA as a system of links that can be either closed or open; we also introduce a new concept of the DNA linking potential akin to the chemical and electric potentials . The linking potential is a suitable parameter for describing the equilibrium distribution of links in heterogeneous DNA, the coexistence of various DNA structures, the equilibrium input and output of DNA links by enzymes, and the nonequilibrium movement of links along DNA chains . Within the framework of this approach DNA gyrase is considered as the source of the DNA linking potential. J Chromatogr, 1990 Jul 20, 512, 325 - 35 Peptide mapping on HIV polypeptides expressed in Escherichia coli . Quality control of different batches and identification of tryptic fragments containing residues of aromatic amino acids or cysteine; Renlund S et al.; Peptide mapping was used for the quality control of different batches of the recombinant HIV proteins p24 core and p24-gp41, expressed in Escherichia coli . These proteins comprise gag and env region polypeptides of the virus and may serve as suitable components in the diagnosis of HIV infections . The proteins were digested with trypsin and the mixtures were subjected to peptide mapping to prove batch equivalence of p24-gp41 and to isolate fragments of the p24-gp41 digest that differ from those of the p24 core digest . The proteins were reduced with dithiothreitol and the cysteine residues were derivatized by addition of 4-vinylpyridine . Peptide mapping was performed by means of reversed-phase high-performance liquid chromatography . Batch equivalence was proved by comparison of the maps . Peaks present in one map but not in the other were considered to be due to sequence differences or variability in digestion. J Mol Biol, 1990 Jul 20, 214(2), 407 - 22 Identification and characterization of a testis-specific isoform of a chaperonin in a moth, Heliothis virescens; Miller SG et al.; Two relatively abundant proteins having subunit molecular weights of 60,000 and 63,000 (p60 and p63, respectively) have been purified as a 16 to 18S complex from sperm mitochondria of a moth . Heliothis virescens . Although the function of these proteins had heretofore not been established, interest in the p63 polypeptide stemmed from its sperm-specific expression and its striking occurrence as a net charge variant among several insect species surveyed, using two-dimensional gel electrophoresis . Genomic and cDNA clones corresponding to the p63 protein have now been isolated and their sequencing has revealed extensive amino acid sequence identity with both the Escherichia coli GroEL protein and its eukaryotic homologues, the chaperonins . Immunoblot studies with a Tetrahymena chaperonin antiserum demonstrated that the p60 protein, which is expressed in all cell types, is structurally related to p63 and is itself a chaperonin subunit . While the chaperonin complex from Heliothis sperm shares certain properties with GroEL, including the ability to hydrolyze ATP and organization of its subunits into a seven-member ring, electron microscopic analysis revealed that its higher-order structure differed from GroEL (and other lower eukaryotic chaperonins) in that the native particle comprises one such ring rather than a doublet . It is not yet known whether the two chaperonin isoforms coexpressed in moth sperm assemble separately or give rise to hybrid particles . In either case, the existence of multiple chaperonin subunits in sperm leaves open the possibility that some aspect of mitochondrial biogenesis that is dependent upon the activity of these proteins is qualitatively or quantitatively different in this cell type. J Natl Cancer Inst, 1990 Jul 18, 82(14), 1191 - 7 In vivo tumor targeting of a recombinant single-chain antigen-binding protein; Colcher D et al.; We describe here the first in vivo targeting of tumors with a single-chain antigen-binding protein . The molecule, which was constructed and expressed in Escherichia coli, is a novel recombinant protein composed of a variable light-chain (VL), amino acid sequence of an immunoglobulin tethered to a variable heavy-chain (VH) sequence by a designed peptide . We show that this protein, derived from the DNA sequence of the variable regions of the antitumor monoclonal antibody B6.2, has the same in vitro antigen-binding properties as the B6.2 Fab' fragment . Comparative pharmacokinetic studies in athymic mice demonstrate much more rapid alpha and beta phases of plasma clearance for the single-chain antigen-binding protein than for the Fab' fragment, as well as an extremely rapid whole-body clearance . Half-life values for alpha and beta phases of single-chain antigen-binding protein clearance were 2.4 minutes and 2.8 hours, respectively, versus 14.8 minutes and 7.5 hours for Fab' . Furthermore, the single-chain antigen-binding protein molecule did not show accumulation in the kidney as did the Fab' molecule or, as previously shown, the F(ab')2 molecule . Despite its rapid clearance, the single-chain antigen-binding protein showed uptake in a human tumor xenograft comparable to that of the Fab' fragment, resulting in tumor to normal tissue ratios comparable to or greater than those obtained with the Fab' fragment . These studies thus demonstrate the in vivo stability of recombinant single-chain antigen-binding proteins and their potential in some diagnostic and therapeutic clinical applications in cancer and other diseases. J Natl Cancer Inst, 1990 Jul 18, 82(14), 1199 - 202 Dictyostelium nucleoside diphosphate kinase highly homologous to Nm23 and Awd proteins involved in mammalian tumor metastasis and Drosophila development; Wallet V et al.; Two complementary DNAs (cDNAs) previously isolated, one by functional screening and the other by immunological screening of a Dictyostelium discoideum expression library, encode two proteins, Gip17 and Guk7.2, sharing 71% homology . In the present study, we found that the expression of their messenger RNAs (mRNAs) is developmentally regulated, with a sharp decrease during the first hours of differentiation . The Gip17 protein was purified to homogeneity from D . discoideum amoebas and from recombinant Escherichia coli and was conclusively identified as a nucleoside diphosphate (NDP) kinase . NDP kinases play a major role in synthesis of nucleoside triphosphates and, in many systems, are found associated with guanosine triphosphate (GTP)-binding proteins . We found the Gip17 protein to be 77% homologous to the human Nm23 protein and 75% homologous to the Drosophila melanogaster Awd protein . The levels of murine and human nm23 mRNA and Nm23 protein are significantly reduced in tumor cells of high metastatic potential, suggesting that Nm23 is involved in suppression of mammalian tumor metastasis, and mutants of the awd gene exhibit widespread development abnormalities, suggesting that Awd is involved in D . melanogaster development . The high percentage of homology of the Gip17 and Guk7.2 proteins with the Nm23 and Awd proteins indicates that Nm23 and Awd also have nucleoside diphosphate kinase activity . Possible modulations in the activity of this metabolic enzyme could be related to the altered metabolism of tumor cells and the control of metastatic potential . Our results point to an unexpected role of NDP kinase in development, growth control, and oncogenic transformation. Biochemistry, 1990 Jul 17, 29(28), 6678 - 87 Active-site mapping and site-specific mutagenesis of glycinamide ribonucleotide transformylase from Escherichia coli; Inglese J et al.; The affinity reagent N10-(bromoacetyl)-5,8-dideazafolate has previously been shown to inactivate glycinamide ribonucleotide transformylase (EC 2.1.2.2) from Escherichia coli in an active-site-directed manner with a 1:1 stoichiometry {Inglese et al . (1990) Biochemistry 29, 1436-1443} . After a series of mild proteolytic digestions, the dideazafolate label was localized to an active-site peptide attached by an ester linkage to the highly conserved residue Asp 144 . Subsequent site-specific mutagenesis of Asp 144 to Asn 144 resulted in a catalytically inactive enzyme that retained the ability to bind substrates and inhibitors . The Asn 144 mutant could be further labeled with the affinity reagent in an active-site-directed stoichiometric fashion; however, the site of modification in this case was His 119 . These results imply that Asp 144 may function as a general base within the catalytic center of the transformylase and is in close proximity to His 119 in the folded protein. Biochemistry, 1990 Jul 17, 29(28), 6609 - 18 Structure of yeast triosephosphate isomerase at 1.9-A resolution; Lolis E et al.; The structure of yeast triosephosphate isomerase (TIM) has been solved at 3.0-A resolution and refined at 1.9-A resolution to an R factor of 21.0% . The final model consists of all non-hydrogen atoms in the polypeptide chain and 119 water molecules, a number of which are found in the interior of the protein . The structure of the active site clearly indicates that the carboxylate of the catalytic base, Glu 165, is involved in a hydrogen-bonding interaction with the hydroxyl of Ser 96 . In addition, the interactions of the other active site residues, Lys 12 and His 95, are also discussed . For the first time in any TIM structure, the "flexible loop" has well-defined density; the conformation of the loop in this structure is stabilized by a crystal contact . Analysis of the subunit interface of this dimeric enzyme hints at the source of the specificity of one subunit for another and allows us to estimate an association constant of 10(14)-10(16) M-1 for the two monomers . The analysis also suggests that the interface may be a particularly good target for drug design . The conserved positions (20%) among sequences from 13 sources ranging on the evolutionary scale from Escherichia coli to humans reveal the intense pressure to maintain the active site structure. Biochim Biophys Acta, 1990 Jul 17, 1018(1), 61 - 6 Crosslinking and radiation inactivation analysis of the subunit structure of the pyridine nucleotide transhydrogenase of Escherichia coli; Hou C et al.; The pyridine nucleotide transhydrogenase of Escherichia coli consists of two types of subunit (alpha: Mr 53,906; beta: Mr 48,667) . The purified and membrane-bound enzymes were crosslinked with a series of bifunctional crosslinking agents and by catalyzing the formation of inter-chain disulfides in the presence of cupric 1,10-phenanthrolinate . Crosslinked dimers alpha 2, alpha beta and beta 2, and the trimer alpha 2 beta were obtained . A small amount of tetramer, probably alpha 2 beta 2, was also formed . Radiation inactivation was used to determine the molecular size of the transhydrogenase . The radiation inactivation size (217,000) and chemical crosslinking are consistent with the structure (Mr 205,146) being the oligomer that is responsible for biological activity. Biochemistry, 1990 Jul 17, 29(28), 6670 - 7 Proteolytic activities of human fibroblast collagenase: hydrolysis of a broad range of substrates at a single active site; Fields GB et al.; The action of human fibroblast collagenase (HFC) on six substrates of markedly different size, sequence, and conformation, including rat type I collagen, rat alpha 1(I) gelatin, beta-casein, and the three synthetic oligopeptides Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, Asp-Val-Ala-Gln-Phe-Val-Leu-Thr-Pro-Gly, and Pro-Val-Gln-Pro-Ile-Gly-Pro-Gln, has been examined . The first peptide is a model for the collagenase cleavage site in the alpha 1(I) chain of type I collagen, while the latter two peptides are models for the autolytic activation and degradation sites in pro-HFC, respectively . The goal of these studies was to assess whether HFC hydrolyzes all of these disparate substrates at the same active site . Individual kinetic parameters for the hydrolysis of all six substrates have been determined . Gel zymography experiments using collagen, gelatin, and casein as substrates show that all three activities are associated solely with HFC rather than impurities . Recombinant HFC expressed in Escherichia coli also exhibits caseinase activity, reinforcing the view that this activity is not due to a contaminating protease from fibroblasts . The ratios of these activities agree within experimental error for several independent HFC preparations and do not change when two additional affinity purification steps are employed . The inhibition of the hydrolysis of these substrates by both 1,10-phenanthroline and Boc-Pro-Leu-Gly-NHOH is identical within experimental error . A series of assays carried out in the presence of pairs of these substrates clearly shows that they compete for the same active site.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Jul 17, 29(28), 6687 - 98 Zinc interactions with regulatory dimers from Escherichia coli aspartate transcarbamoylase; Jefferson JR et al.; Zn2+ is tetrahedrally bonded to the 4 nonadjacent thiols of each regulatory chain (Mr 17,000) near r-c contacts between catalytic (c) and regulatory chains (r) in aspartate transcarbamoylase (ATCase; c6r6) . This paper reports on Zn2+ interactions with r dimer in the absence of stabilizing r-c contacts . After r2 and c3 subunits were separated, -SH groups of r2 were titrated with p-(hydroxymercuri)benzenesulfonate (PMPS) at pH 7.0 . The concomitant release of Zn2+ (2 equiv/r dimer) was quantitated with 4-(2-pyridylazo)resorcinol (PAR) and was a linear function of PMPS added until 8 mercaptide bonds per r2 were formed . Breakage of 1 of 4 Zn2(+)-sulfur bonds in a Zn2+ binding cluster therefore makes the other three bonds more labile . From stopped-flow measurements, the PMPS-promoted Zn2+ release from r2 or mercaptide bond formation with 10- to 20-fold excess PMPS/r2-SH at pH 7.0 was first order with an Arrhenius activation energy Ea = 10 kcal/mol and a half-time t 1/2 = 9 +/- 2 ms at 20 degrees C without inhibitory anions present . The rate of mercurial-promoted Zn2+ release from r2 is at least 77 times faster than that from intact c6r6 {Hunt, J.B., Neece, S.H., Schachman, H.K., and Ginsburg, A . (1984) J . Biol . Chem . 259, 14793}; this indicates that Zn2+ binding clusters are more accessible to attack by PMPS than are those in ATCase . The addition of a 25-fold excess of the multidentate fluorescent chelator quin-2 to r2 gave a rate of Zn2+ dissociation that was 1/210th of that observed with excess mercurial . Furthermore, the Zn(PAR)1 complex was identified as the active species in the transfer of Zn2+ from Zn(PAR)2 to aporegulatory subunits, with kappa = (8 +/- 3) x 10(5) M-1 s-1 at pH 7.0 and 15 degrees C for this second-order association reaction . Although kinetic results are dependent on the mechanisms involved, an affinity constant K'A = (1.3 +/- 0.6) x 10(12) M-1 for Zn2+ binding to r dimer at pH 7.0 and 20 degrees C in the absence and presence of 100 mM KCl could be determined spectrally by rapid equilibration with the high-affinity, sensitive metalloindicators indo-1 and quin-2 . This K'A value is based on the assumptions that Zn2+ binding sites in r2 are equivalent (noninteracting) and that apo-r2 does not dissociate; if apo-r2 dissociates, K'A approximately 10(14) M-1 . Within experimental error, the K'A value was independent of {indo-1}/{r2} ratios from 36 to 3 with 0.3-8 microM r2.(ABSTRACT TRUNCATED AT 400 WORDS) Gene, 1990 Jul 16, 91(2), 281 - 5 Primary structure of bovine lactate dehydrogenase-A isozyme and its synthesis in Escherichia coli; Ishiguro N et al.; Eleven cDNA clones encoding lactate dehydrogenase(LDH)-A isozyme were isolated from a bovine lymphocyte cDNA library, and the nucleotide sequences of three of the clones (pLDH5, pLDH9 and pLDH12) were determined . With the exception of variation in the 5' portion, two cDNA clones (pLDH9 and pLDH12) appeared to contain the full-length cDNA of 1786 bp, consisting of the protein-coding sequence (996 bp), the 5'- and the 3'-untranslated regions and the poly(dA) tail . The predicted amino acid (aa) sequence of bovine LDH-A (332 aa) showed 96.7% homology with that of pig LDH-A . The protein-coding cDNA region (1650 bp) was inserted into an Escherichia coli expression vector ptac11 and expressed . The protein synthesized in E . coli showed enzyme activity of LDH and was identified by cellogel electrophoresis as LDH-5 isozyme whose subunit M chain is the product of the LDH-A gene. Gene, 1990 Jul 16, 91(2), 275 - 9 The effect of phage T7 lysozyme on the production of biologically active porcine somatotropin in Escherichia coli from a gene transcribed by T7 RNA polymerase; O'Mahony DJ et al.; We have analyzed the inducible synthesis of recombinant porcine somatotropin (rPST) from the phage T7 gene 10 promotor on the vector pET3a {Rosenberg et al., Gene 56 (1987) 125-135} . Low-level synthesis of phage T7 lysozyme is crucial for high-level synthesis (40%) of rPST, which is greatly reduced if T7 lysozyme synthesis is absent or too high . The synthesis of rPST mRNA is optimized in those constructs coding for low levels of T7 lysozyme, with a reduction in mRNA levels in constructs coding for higher levels of T7 lysozyme or no lysozyme . The rPST can be readily purified following a single chromatographic step and is biologically active as determined by the tibia test following administration to hypophysectomized rats. FEBS Lett, 1990 Jul 16, 267(2), 239 - 41 Pressure-induced dissociation of tight couple ribosomes; Gross M et al.; Ribosomes from Escherichia coli have been shown to undergo subunit dissociation at elevated hydrostatic pressure . This holds for both crude and highly purified ribosomes . No inhibitory effect could be detected by addition of either the S100 supernatant, or tRNA, polyuridylic acid, and spermine . Light scattering experiments at pressures up to 1000 bar reveal different susceptibility of tight couple and loose couple ribosomes toward pressure dissociation . Tight couples are subjected to EF-Tu-catalyzed binding of aminoacyl-tRNA, thus yielding a model system of the elongating ribosome before the peptidyl transfer step . High pressure dissociation of this compound suggests that enzymatic binding converts tight couples into loose couples . A hypothesis referring to conformational changes during the elongation cycle is presented. Biochem Biophys Res Commun, 1990 Jul 16, 170(1), 104 - 10 Synthesis of a new helical protein: the effect of secondary structure rearrangement on structure formation; Tanaka T et al.; A new helical protein was designed and synthesized to alter the sequential connectivity of the 4 helices in human growth hormone and to delete the long surface loop structures . The protein accumulated as an insoluble form in E . coli was solubilized and purified to apparent homogeneity in the presence of 7M urea, and refolded by the aid of 1% n-octyl-beta-D-glucopyranoside . The circular dichroism spectrum was typical of a highly helical protein . The molecular weight estimated by gel permeation chromatography and the red-shift of the fluorescence maximum by urea-induced denaturation suggest that the protein folds into a compact globular form . The new protein obtained, however, was destabilized relative to the original human growth hormone. Biochem Biophys Res Commun, 1990 Jul 16, 170(1), 1 - 9 Temperature dependence of transcriptional activity of yeast 3-phosphoglycerokinase promoter in Escherichia coli; Mondal AK et al.; We have studied the activity of yeast PGK promoter in E . coli at different temperatures using lacZ as the reporter gene . The B-galactosidase activity was found to be less at 42 degrees C than at 30 degrees C . Northern blot analysis showed that the level of lacZ transcript was less in recombinant cells grown at 42 degrees C, whereas plasmid copy number per cell was more as compared to recombinant cells grown at 30 degrees C . Data suggest that the yeast PGK promoter is less active at 42 degrees C and that this activity is regulated at the level of transcription. FEBS Lett, 1990 Jul 16, 267(2), 193 - 8 Nucleotide sequence of four genes encoding ribosomal proteins from the 'S10 and spectinomycin' operon equivalent region in the archaebacterium Halobacterium marismortui; Arndt E; Four genes encoding ribosomal proteins HmaS17, HmaL14, HmaL24 and HS3, have been identified in the lambda EMBL3 clone PP*7 from a genomic library of the archaebacterium Halobacterium marismortui . The clone contains genes from the 'S10 and spectinomycin' operon equivalent region . Three of the deduced proteins are homologous to the corresponding Escherichia coli and Methancoccus vannielii S17, L14 and L24 proteins, as well as to eukaryotic proteins from rat or yeast . HS3 was identified as an extra protein corresponding to the gene product for orfc in M . vannielii and the eukaryotic ribosomal protein RS4 from rat . The equivalence of HmaL24 (HL16) and E . coli L24, which share only 28% identical amino acid residues, could now be shown by localizing the HmaL24 gene at the same position in the cluster. FEBS Lett, 1990 Jul 16, 267(2), 207 - 12 Does the Kunitz domain from the Alzheimer's amyloid beta protein precursor inhibit a kallikrein responsible for post-translational processing of nerve growth factor precursor? Castro M, Marks CB, Nilsson B, Anderson S. Alternative splicing of the Alzheimer's amyloid beta protein precursor (ABPP) message leads to the production of several variants of this precursor polypeptide . Two of these variants contain a domain that is highly homologous to members of the Kunitz class of protease inhibitors . In order to initiate a study of the physiological role of this domain, we have produced active ABPP Kunitz inhibitor by constructing and expressing a synthetic gene in E . coli . Nerve growth factor (NGF) deficiency has been suggested as a possible cause of the neural degeneration characteristic of Alzheimer's disease, and trypsin and gamma-NGF are the two enzymes that have been shown to be capable of processing beta-NGF precursor to active, mature beta-NGF in vitro, therefore, the specificity of purified recombinant ABPP Kunitz inhibitor was analyzed with respect to these two proteases . Binding of isolated ABPP Kunitz domain both to trypsin (Ki,app less than 10 nM and to gamma-NGF (Ki,app = 300 nM) was observed . This difference in binding to the two proteases correlates with the approximately 20-fold higher rate observed for in vitro processing of the beta-NGF precursor by trypsin compared to processing by gamma-NGF, indicating that perhaps the inhibitor mimics the interaction of the beta-NGF precursor with proteases . The kallikrein actually responsible for beta-NGF precursor processing in vivo is unknown, but these results suggest that it is capable of being significantly inhibited by exposure to the ABPP Kunitz domain. J Biol Chem, 1990 Jul 15, 265(20), 11417 - 20 In situ labeling of the adipocyte lipid binding protein with 3-{125I}iodo-4-azido-N-hexadecylsalicylamide . Evidence for a role of fatty acid binding proteins in lipid uptake; Waggoner DW et al.; Differentiating 3T3-L1 cells have been used to investigate the process of fatty acid uptake, its cellular specificity, and the involvement of cytoplasmic carrier proteins . The profile of fatty acid uptake in both differentiated and undifferentiated cells was biphasic, consisting of an initial rapid phase (0-20 s) followed by a second slower phase (60-480 s) . In both cell types the initial phase of fatty acid (FA) uptake was temperature-insensitive whereas the rate of uptake during the second phase decreased 4-fold when measurements were made at 4 degrees C . The rate of {9,10-3H}oleate uptake in 3T3-L1 adipocytes was 10-fold greater than in the fibroblastic precursor cells . The acquisition of a differentially expressed cytoplasmic fatty acid binding protein (adipocyte lipid binding protein (ALBP} occurs coincident with the increased ability of these cells to take up FAs . Uptake experiments with 3-{125I}iodo-4-azido-N-hexadecylsalicylamide demonstrated that this photoactivatable FA analogue accumulated intracellularly in a time-, temperature-, and cell-specific fashion . Moreover, when 3T3-L1 adipocytes were presented with 3-{125I}iodo-4-azido-N-hexadecylsalicylamide and then irradiated, a single cytoplasmic 15-kDa protein was labeled . The in situ-labeled 15-kDa protein was identified as ALBP by its ability to be immunoprecipitated with anti-ALBP antisera . Taken together these results indicate that fatty acids traverse the plasma membrane and are bound by ALBP in the cytoplasmic compartment . It is likely that lipid uptake in other cell systems, such as liver, heart, intestine, and nerve tissue, proceeds by a similar process and that this represents a general mechanism for cell-specific FA uptake and utilization. Biochem J, 1990 Jul 15, 269(2), 443 - 50 Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue; Russell GC et al.; The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase multienzyme complex (PDH complex) . A thermoinducible expression system was developed to amplify a variety of genetically restructured PDH complexes, including those containing three, two, one and no lipoyl domains per E2p chain . Although large quantities of the corresponding complexes were produced, they had only 20-50% of the predicted specific activities . The activities of the E1p components were diminished to the same extent, and this could account for the shortfall in overall complex activity . Thermoinduction was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E2p component . This substitution abolished dihydrolipoamide acetyltransferase activity of the complex without affecting other E2p functions . The results support the view that His-602 is an active-site residue . The inactivation could mean that the histidine residue performs an essential role in the acetyltransferase reaction mechanism, or that the reaction is blocked by an irreversible modification of the cysteine substituent . Complementation was observed between the H602C PDH complex and a complex that is totally deficient in lipoyl domains, both in vitro, by the restoration of overall complex activity in mixed extracts, and in vivo, from the nutritional independence of strains that co-express the two complexes from different plasmids. Biochem J, 1990 Jul 15, 269(2), 335 - 40 Sequence of cDNA for rat cystathionine gamma-lyase and comparison of deduced amino acid sequence with related Escherichia coli enzymes; Erickson PF et al.; A cDNA clone for cystathionine gamma-lyase was isolated from a rat cDNA library in lambda gt11 by screening with a monospecific antiserum . The identity of this clone, containing 600 bp proximal to the 3'-end of the gene, was confirmed by positive hybridization selection . Northern-blot hybridization showed the expected higher abundance of the corresponding mRNA in liver than in brain . Two further cDNA clones from a plasmid pcD library were isolated by colony hybridization with the first clone and were found to contain inserts of 1600 and 1850 bp . One of these was confirmed as encoding cystathionine gamma-lyase by hybridization with two independent pools of oligodeoxynucleotides corresponding to partial amino acid sequence information for cystathionine gamma-lyase . The other clone (estimated to represent all but 8% of the 5'-end of the mRNA) was sequenced and its deduced amino acid sequence showed similarity to those of the Escherichia coli enzymes cystathionine beta-lyase and cystathionine gamma-synthase throughout its length, especially to that of the latter. J Biol Chem, 1990 Jul 15, 265(20), 11885 - 9 Structure/function analysis of interleukin-2-toxin (DAB486-IL-2) . Fragment B sequences required for the delivery of fragment A to the cytosol of target cells; Williams DP et al.; We have used cassette and deletion mutagenesis to analyze the structural features of fragment B-related sequences in the fusion toxin DAB486-IL-2 (where IL-2 represents interleukin-2) that are necessary for the efficient delivery of fragment A to the cytosol of target cells . We demonstrate that whereas an intact disulfide bond between Cys461 and Cys471 may be required for the cytotoxic action of native diphtheria toxin, this bond is not required for the cytotoxic action of DAB486-IL-2 . The in-frame deletion of the 97 amino acids from Thr387 to His485 of DAB486-IL-2 increases both the potency and the apparent dissociation constant (Kd) of the resulting fusion toxin for high affinity interleukin-2 receptor-bearing target cells . In contrast, the inframe deletion of either the 191 amino acids between Asp291 and Gly483 or the 85 amino acids between Asn204 and Ile290 results in a 1000-fold loss in potency . These regions contain the putative membrane-spanning regions and the amphipathic membrane surface-associating regions of fragment B, respectively . These results indicate that the efficient delivery of the ADP-ribosyltransferase from DAB486-IL-2 to the cytosol requires the membrane-associating domains of fragment B . This function has been postulated to play a role in the diphtherial intoxication of eukaryotic cells . However, unlike native diphtheria toxin, fragment B sequences distal to Thr387 do not enhance the potency of DAB486-IL-2. J Biol Chem, 1990 Jul 15, 265(20), 11667 - 75 Regulation of yeast LEU2 . Total deletion of regulatory gene LEU3 unmasks GCN4-dependent basal level expression of LEU2; Brisco PR et al.; We have constructed a total deletion of the regulatory gene LEU3 . Comparing the deletion mutant with a leu3 spontaneous mutant, we find that both types of mutants have lost the ability to regulate a LEU2'-lacZ translational fusion by the LEU3-alpha-isopropylmalate-dependent mechanism, which we confirm to be the major regulatory mechanism for LEU2 . Surprisingly, cells containing the total leu3 deletion are more leaky (i.e . grow better in the absence of extraneous leucine) than cells containing a spontaneous leu3 mutation . Accompanying the growth rate difference is a difference in the expression of the LEU2-lacZ fusion: the specific activity of beta-galactosidase amounts to about 8% of a wild type control in a leu3 total deletion mutant, but drops to about 2% in a leu3 spontaneous mutant . The spontaneous mutant differs from the total deletion mutant in that it produces an inactive protein which is still able to bind to the LEU2 upstream activating sequence . We conclude that a basal level control of LEU2 becomes manifest in the absence of LEU3 and is interfered with when LEU3 protein binds to the LEU2 promoter . This conclusion is supported by the finding that a mutant which contains an intact LEU3 gene but is unable to generate alpha-isopropylmalate also interferes with basal level expression of LEU2 . Basal level expression depends upon the GCN4 protein, even though LEU2 is not subject to derepression by the general amino acid control system . Changes in the steady-state concentration of LEU2 mRNA show the same trend as changes in the specific activity of the LEU2-lacZ fusion protein, suggesting that regulation of LEU2 expression at both the basal and nonbasal levels is largely transcriptional . The role of alpha-isopropylmalate in the regulation of LEU2 expression appears to be that of a co-activator . Employing mobility shift assays, we show that specific interaction between the LEU3 protein and a 30-base pair DNA fragment carrying the upstream activating sequence of LEU2 takes place irrespective of the presence or absence of alpha-isopropylmalate. J Biol Chem, 1990 Jul 15, 265(20), 11549 - 54 19F nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol binding protein II produced in Escherichia coli . An analysis of four tryptophan substitution mutants and their interactions with all-trans-retinol; Li E et al.; Rat cellular retinol binding protein (CRBP II) is a 134-amino acid intracellular protein synthesized in the polarized absorptive cells of the intestine . We have previously used 19F nuclear magnetic resonance (NMR) spectroscopy to survey the structural effects of ligand binding on the apoprotein . For these studies, all 4 Trp residues of rat CRBP II were efficiently labeled with 6-fluorotryptophan (6-F-Trp) by inducing its expression in a tryptophan auxotroph of Escherichia coli . Resonances corresponding to 2 of its Trp residues underwent large downfield shifts upon binding of all-trans-retinol and retinal, while resonances corresponding to the other 2 Trp residues underwent only minor perturbations in chemical shifts . To identify which Trp residues undergo changes in their environment upon ligand binding, we have constructed four CRBP II mutants where Trp9, Trp89, Trp107, or Trp110 have been replaced by another hydrophobic amino acid . By comparing the 19F NMR spectrum of each 6-F-Trp-labeled mutant with that of wild type 6-F-Trp CRBP II, we demonstrate that the 19F resonance corresponding to Trp107 undergoes the largest change in chemical shift upon ligand binding (2.0 ppm downfield) . This is consistent with the position of this residue predicted from molecular modeling studies . The 19F resonance corresponding to Trp9 also undergoes a downfield change in chemical shift of 0.5 ppm associated with retinol binding even though it is predicted to be removed from the ligand binding site . By contrast, the resonances assigned to Trp89 and Trp110 undergo only minor perturbations in chemical shifts . These results have allowed us to identify residue-specific probes for evaluating the interactions of all-trans-retinol (and other retinoids) with this intracellular binding protein. J Biol Chem, 1990 Jul 15, 265(20), 11436 - 43 Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli; Adams MD et al.; The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined . Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport . Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes . These two proteins confer specificity on the LIV-I and LS transport systems . livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF . The protein products of the livHMGF genes are shared by the two systems . An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times . The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane . Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins . A deletion strain that does not express any of the liv genes was constructed . This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained. Biochem J, 1990 Jul 15, 269(2), 303 - 8 Mutations within the uncE gene affecting assembly of the F1F0-ATPase of Escherichia coli; Fimmel AL et al.; Site-directed mutagenesis has been used to construct two mutations within the uncE gene, which codes for the c-subunit of the F1F0-ATPase, resulting in the substitution of glycine-27 by leucine and of glycine-32 by leucine . Strains carrying each mutation are unable to grow on minimal medium containing succinate as the sole carbon source and possess an uncoupled growth yield . Membranes prepared from strains carrying each mutation possess low levels of ATPase activity and are proton-impermeable . The c-subunit in each mutant strain appears to assemble into the F0-ATPase and disrupt the normal assembly of the F1-ATPase . The results are discussed in relation to a previously proposed model for the F0 sector {Cox, Fimmel, Gibson & Hatch (1986) Biochim . Biophys . Acta 849, 62-69}. J Biol Chem, 1990 Jul 15, 265(20), 11734 - 9 Polyphosphate kinase from Escherichia coli . Purification and demonstration of a phosphoenzyme intermediate; Ahn K et al.; Polyphosphate kinase (PPK) polymerizes the terminal phosphate of ATP to a long chain polyphosphate (poly(P) or (Pi)n) in a freely reversible reaction (Kornberg, S . R . (1957) Biochim . Biophys . Acta 26, 294-300), nATP in equilibrium nADP + (Pi)n, PPK, now purified to homogeneity, is a tetramer of 69-kDa subunits . Addition of a primer in the synthetic reaction is not required, nor does ATP or inorganic orthophosphate (Pi) serve in this role . PPK is autophosphorylated under the conditions of poly(P) synthesis; Pi is linked by a nitrogen-phosphate bond as judged by its acid lability and alkali stability . Incorporation of phosphate from the isolated phosphoenzyme into poly(P) upon the addition of ATP in the synthetic reaction and its incorporation into ATP upon the addition of ADP indicate phosphoenzyme to be an intermediate in the reaction . At an ATP level of 5 microM, well below its Km of 2 mM, a pronounced lag in poly(P) synthesis can be removed by tetrapolyphosphate but not by Pi, PPi, or tripolyphosphate . The basis for this stimulatory effect is not clear inasmuch as tetrapolyphosphate does not promote the dephosphorylation of the presumed phosphoenzyme intermediate. J Biol Chem, 1990 Jul 15, 265(20), 11622 - 7 Deletion analysis of the mini-P1 plasmid origin of replication and the role of Escherichia coli DnaA protein; Wickner S et al.; The mini-P1 plasmid origin of replication is contained on a 246 base pair (bp) piece of DNA . At one end there are five 19-bp binding sites for the P1 initiator protein, RepA, and near the other end there are two 9-bp DnaA protein-binding sites . To further define the limits of the origin, we cloned the origin region in M13 and constructed deletions of either end . We sequenced the DNA and tested the replicative form I DNA of the deletion phages for their ability to support RepA-dependent DNA replication in an in vitro system . The origin that is functional in vitro could be reduced to 202 bp . It includes three intact and one incomplete RepA-binding sites at one end and the two DnaA-binding sites at the other end . When the two naturally occurring DnaA-binding sites were replaced with one or two synthetic sites, only the construction containing two sites was active in vitro . We found that the minimal origin that is functional in vivo contains all of the five RepA and the two DnaA-binding sites . Mini-P1 plasmid replication both in vivo and in vitro requires two initiator proteins, the Escherichia coli DnaA protein and the P1 RepA protein . We have found that the ADP form of DnaA is as active as the ATP form of the protein in the in vitro replication of mini-P1 . In contrast, only the ATP form is active for in vitro replication of plasmids carrying the E . coli origin (Bramhill, D., and Kornberg, A . (1988) Cell 52, 743-755). J Biol Chem, 1990 Jul 15, 265(20), 11942 - 7 cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein; Fu YH et al.; cys-3, the positive-acting master sulfur regulatory gene of Neurospora crassa, turns on the expression of an entire set of unlinked structural genes which encode sulfur-catabolic enzymes . cys-3 encodes a protein of 236 amino acid residues and contains a potential bipartite DNA-binding domain which consists of a leucine zipper and an adjacent highly basic region . Gel band mobility shift and DNA footprint experiments were used to demonstrate that the CYS3 protein, expressed in Escherichia coli, binds to three distinct sites in the 5' upstream DNA of cys-14, the structural gene for sulfate permease II . The CYS3 protein also binds to one distinct sequence element upstream of the cys-3 gene itself, which suggests an autoregulatory role for this protein . Two mutant CYS3 proteins, altered in the basic region of the DNA-binding domain, failed to bind to either the cys-14 or the cys-3 upstream recognition elements. J Biol Chem, 1990 Jul 15, 265(20), 11560 - 6 An obligatory pH-mediated isomerization on the {Asn-160}recA protein-promoted DNA strand exchange reaction pathway; Muench KA et al.; We recently described a mutant recA protein in which glycine 160 of the recA polypeptide was replaced by an asparagine residue (Bryant, F . R . (1988) J . Biol . Chem . 263, 8716-8723) . Although the {Asn-160}recA protein has a ssDNA-dependent ATPase activity that is similar to that of the wild-type recA protein, the mutant protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions (pH 7.5, 1 mM ATP) . We have found that the {Asn-160}recA protein is able to carry out the three-strand exchange reaction at pH 6.0 to 6.7, but that the strand exchange activity is abolished at higher pH . The induction of strand exchange activity at low pH correlates directly with a pH-mediated activation of an ATP-dependent isomerization of the {Asn-160}recA protein . This ATP-dependent isomerization is characterized by the conversion of the {Asn-160}recA protein to a form that is not displaced from ssDNA by the Escherichia coli SSB protein . In contrast to the pronounced pH sensitivity of the {Asn-160}recA protein, the wild-type recA protein undergoes ATP-dependent isomerization, and is able to carry out the three-strand exchange reaction, over the range of pH 6.0 to 8.4 . These results show that the {Asn-160} mutation disrupts the ATP-dependent isomerization of the recA protein and suggest that protonation of the {Asn-160}recA protein (or the {Asn-160}recA-ssDNA complex) relieves this mechanistic defect . Furthermore, the direct correlation between ATP-dependent isomerization and the strand exchange activity of the {Asn-160}recA protein strongly suggests that the ATP-dependent isomerization is an obligatory step in the recA protein-promoted strand exchange mechanism. Int J Cancer, 1990 Jul 15, 46(1), 35 - 8 Circulating antibodies against c-myc oncogene product in sera of colorectal cancer patients; Ben-Mahrez K et al.; We developed a Western blot assay using purified human c-myc protein expressed in E . coli in order to look for circulating anti-c-myc antibodies in human sera . The presence of IgG antibodies against c-myc was observed in 25 out of 44 sera from patients with colorectal cancer diagnosed and treated at the Hopital Curie in Paris, compared to the sera of 46 normal donors of which only 8 samples were positive (p = 0.001). J Biol Chem, 1990 Jul 15, 265(20), 11854 - 7 Site-directed mutagenesis of arginine 246, glutamic acid 247, and histidine 248 in the eukaryotic transcription factor S-II; Horikoshi N et al.; When His248 of the transcription factor S-II was replaced by alanine or tyrosine, the activity of the resulting mutants was less than 30% of that of the wild-type S-II . When Arg246, Glu247, and His248 were all replaced by leucine, the resulting mutant showed complete loss of activity . These results indicate that the amino acid sequence Arg-Glu-His at positions 246-248 of S-II is important for its stimulatory activity . The mutant S-II with no activity could not form a complex with RNA polymerase II, unlike wild-type S-II, but retained ability to interact with DNA. Science, 1990 Jul 13, 249(4965), 169 - 71 Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product; Frech M et al.; Krev-1 is known to suppress transformation by ras . However, the mechanism of the suppression is unclear . The protein product of Krev-1, Rap1A-p21, is identical to Ras-p21 proteins in the region where interaction with guanosine triphosphatase (GTPase) activating protein (GAP) is believed to occur . Therefore, the ability of GAP to interact with Rap1A-p21 was tested . Rap1A-p21 was not activated by GAP but bound tightly to GAP and was an effective competitive inhibitor of GAP-mediated Ras-GTPase activity . Binding of GAP to Rap1A-p21 was strictly guanosine triphosphate (GTP)-dependent . The ability of Rap1A-p21 to bind tightly to GAP may account for Krev-1 suppression of transformation by ras . This may occur by preventing interaction of GAP with Ras-p21 or with other cellular proteins necessary for GAP-mediated Ras GTPase activity. Cell, 1990 Jul 13, 62(1), 127 - 33 Role of DNA superhelicity in partitioning of the pSC101 plasmid; Miller CA et al.; Previous work has shown that a cis-acting locus (termed par for partitioning) on the pSC101 plasmid accomplishes its stable inheritance in dividing cell populations . We report here that the DNA of pSC101 derivatives lacking the par region shows a decrease in overall superhelical density as compared with DNA of wild-type pSC101 . Chemicals and bacterial mutations that reduce negative DNA supercoiling increase the rate of loss of par plasmids and convert normally stable plasmids that have minimal par region deletions into unstable replicons . topA gene mutations, which increase negative DNA supercoiling, reverse the instability of partition-defective plasmids that utilize the pSC101, p15A, F, or oriC replication systems . Our observations show that the extent of negative supercoiling of plasmid DNA has major effects on the plasmid's inheritance and suggest a mechanism by which the pSC101 par region may exert its stabilizing effects. Proc Natl Acad Sci U S A, 1990 Jul, 87(13), 4976 - 80 Cloning and expression of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats; Stanley SL Jr et al.; Entamoeba histolytica causes amebic dysentery and amebic liver abscess, major causes of morbidity and mortality worldwide . We have used differential hybridization screening to isolate an E . histolytica-specific cDNA clone . The cDNA was found to encode a serine-rich E . histolytica protein (SREHP) containing multiple tandem repeats . The structural motif of SREHP resembles some of the repetitive antigens of malarial species, especially the circumsporozoite proteins . A recombinant trpE fusion protein containing the tandem repeats of SREHP was recognized by immune serum from a patient with amebiasis, demonstrating that SREHP is a naturally immunogenic protein . An antiserum raised against the recombinant fusion protein specifically bound to two distinct bands with apparent molecular masses of 46 and 52 kDa in a crude preparation of E . histolytica trophozoite membranes . This antiserum also inhibited E . histolytica trophozoite adhesion to Chinese hamster ovary cells in vitro . The ability to isolate E . histolytica-specific genes, and to express those genes in Escherichia coli, may be important in studying the molecular basis of E . histolytica pathogenesis and for the future development of vaccines. Nucleic Acids Res, 1990 Jul 11, 18(13), 3893 - 901 The tL structure within the leader region of Escherichia coli ribosomal RNA operons has post-transcriptional functions; Theissen G et al.; We have investigated a series of mutations within a plasmid encoded E . coli ribosomal RNA leader region . The mutations are localized within a structure known as tL, which has been shown to mediate RNA polymerase pausing in vitro, and which is assumed to have a control function in rRNA transcription antitermination . The effects of the mutated plasmids were analyzed by in vivo and in vitro experiments . Some of the base change mutations led to severely reduced cell growth . As opposed to previous results obtained with mutants where the tL structure has been deleted in part or totally, the tL base change mutations did not result in polar transcription in vivo, rather they revealed a general reduction in the amount of the promoter proximal 16S versus the distal 23S RNA . The deficiency of the 16S RNA, which was most pronounced for some of the slowly growing transformant