Microb Pathog, 1990 Jul, 9(1), 1 - 11 Plasmid-encoded production of coli surface-associated antigen 1 (CS1) in a strain of Escherichia coli serotype O139.H28; Willshaw GA et al.; Production of coli surface-associated antigen 1 (CS1) by Escherichia coli strain E24377 of serotype O139.H28 was controlled by a plasmid that also encoded heat stable and heat labile enterotoxins and CS3 . The presence of a regulatory sequence was detected on this plasmid by hybridization with the cfaD gene that regulates expression of colonization factor antigen I fimbriae and is at least 96% homologous with the rns sequence controlling production of CS1 or CS2 fimbriae by strains of serotype O6.H16 of appropriate biotype . A separate plasmid, pDEP20, carrying the structural genes for CS1 synthesis was identified and transformed into E . coli strain HB101 or a derivative of strain E24377 without large plasmids . Transformants carrying pDEP20 did not produce CS1 fimbrial antigen, but antigen expression was obtained when a cloned cfaD gene or a wild-type plasmid carrying the rns sequence was introduced . Transposon mutagenesis with Tn1000 identified a 3.7 kbp region of pDEP20 essential for production of CS1 fimbriae . Genes encoding production of CS1 fimbriae were cloned on a 9.9 kbp BamHI fragment and were expressed in the presence of the cfaD sequence . A strain producing both CS1 and CS2 antigens was constructed by introduction of the cloned cfaD gene into a strain of serotype O6.H16 biotype C carrying plasmid pDEP20. Res Microbiol, 1990 Jul-Aug, 141(6), 621 - 31 Genetic analysis of the transfer region of the IncN plasmid N3; Glocker B et al.; Using lambda::Tn5 insertion mutagenesis and screening for conjugation, the boundaries of the IncN plasmid N3 transfer region were determined . Sensitivity to phage IKe infection was used to monitor that part of the N3 transfer region which harbours genes for pilus synthesis and assembly . We cloned this region, creating plasmid pBG21 . Escherichia coli cells transformed with pBG21 became sensitive to phage IKe and produced pili, as shown by electron microscopy . Various plasmid constructions containing parts of the pilus-encoding region were used for expression in a minicell system and for expression in an in vitro translation system, thus characterizing for the first time some of the gene products of domain I (Winans and Walker, 1985a) of the transfer region. Mol Gen Genet, 1990 Jul, 222(2-3), 317 - 22 Mapping of insertion element IS30 in the Escherichia coli K12 chromosome; Umeda M et al.; We identified seven phage clones containing the insertion element IS30 in a lambda phage library mini-set, which includes 476 clones carrying chromosomal segments that cover almost the entire chromosome of Escherichia coli K12 W3110 (Kohara et al . 1987) . We could assign locations and orientations to four copies of IS30 (named is30A to is30D) on the W3110 chromosome by restriction analysis of phage DNAs containing them . These IS30s were present at the same locations in chromosomes of both W3110 and another E . coli K12 strain JE5519, and thus are assumed to be present in other E . coli K12 derivatives, including early isolates . Among the IS30 copies found, one (is30B) contained a large deletion and possessed only a 181 bp stretch of the right terminal region of IS30. Mol Microbiol, 1990 Jul, 4(7), 1193 - 8 Immunocytochemical analysis of P-fimbrial structure: localization of minor subunits and the influence of the minor subunit FsoE on the biogenesis of the adhesin; Riegman N et al.; Antibodies recognizing the non-adhesive minor P-fimbral subunit protein E and the P-fimbrial adhesin were used in an immunocytochemical analysis of P-fimbrial structure . It was demonstrated that P-fimbriae of the serotypes F71, F72 and F11 carry their adhesin in a complex with protein E . These complexes are commonly found at the tip of the fimbrial structure . In P-fimbriae of serotype F9, expressed by the uropathogenic Escherichia coli strain 21086, adhesin-protein E complexes are localized at the tips as well as along the shafts of the fimbriae . Protein E of F71 fimbriae (FsoE) plays a catalysing role in the biogenesis of the adhesin, but has no effect on the eventual localization of the adhesin. Genes Dev, 1990 Jul, 4(7), 1079 - 93 Region-specific recombination and expression are directed by portions of the Drosophila engrailed promoter; Hama C et al.; The Drosophila engrailed gene is expressed in the cells of the posterior developmental compartments . To investigate how the engrailed gene is regulated, chimeric genes consisting of fragments of the engrailed promoter and Escherichia coli lacZ were incorporated into the Drosophila germ line by P-element-mediated recombination . Fusion constructs with 7.5 kb of 5'-flanking sequence contain sufficient information to promote expression in most of the embryonic, larval, and imaginal posterior compartments; transformants with smaller fragments of the 5' region do not . Remarkably, of 20 independent transformants with constructs containing more than 1 kb of 5'-flanking DNA, 7 integrated in or around the engrailed locus . These strains inactivate engrailed function to varying degrees, and some express lacZ with a position- and temporal-specific program that is indistinguishable from the normal engrailed gene . Presumably, in these strains, lacZ is expressed in the context of the engrailed promoter. Int J Radiat Biol, 1990 Jul, 58(1), 35 - 54 DNA base and strand damage in X-irradiated monkey CV-1 cells: influence of pretreatment using small doses of radiation; Bases R et al.; Base damage in alpha DNA from irradiated monkey CV-1 cells was determined by measuring release of 5'-32P-end labelled DNA fragments after digestion with endonuclease III of E . coli . The frequency and base sequence locations of the enzyme-sensitive sites were determined . Fragments were released from irradiated DNA at sequence sites of pyrimidines and guanines . The time for repair of half the single strand breaks was approximately 1.5 h . Repair of base damage as judged from loss of enzyme-sensitive sites in DNA was slower, with more than half of the damaged bases still detectable after 4 h of repair . Two important changes in the pattern of fragment release from DNA were produced when small radiation doses preceded the large ones needed to produce measurable DNA strand breaks and base damage . 5 Gy to cells incubated several hours before 320 Gy increased by five-fold the abundance of small DNA fragments with 3'-phosphoryl termini detected in high-resolution denaturing gels . These increases were detectable with doses as small as 0.2 Gy and were accompanied by the appearance of new species of DNA fragments of intermediate mobility at specific locations in the base sequence . The patterns resemble those produced by digesting DNA from heavily irradiated cells with endonuclease III. J Bacteriol, 1990 Jul, 172(7), 3631 - 6 Involvement of gamma-glutamyl peptides in osmoadaptation of Escherichia coli; McLaggan D et al.; Accumulation of K+ ions and glutamate plays a primary role in maintaining osmotic balance in Escherichia coli, as illustrated by the high concentrations of these ions present in cells growing in medium of high osmolality . We found that two gamma-glutamyl peptides and glutamine also accumulated during growth at high osmolarity . In a mutant unable to make trehalose growing in 1.3 osM medium, glutathione, gamma-glutamylglutamine, and glutamine accumulated to levels of 73, 33, and 140 mumol/g of protein, respectively . In such cells, K+ was present at 1,450 mumol/g of protein, indicating that glutathione and gamma-glutamylglutamine accounted for less than 10% of the low-molecular-weight anions accumulated with K+ . However, glutathione is needed for wild-type osmotolerance in this species . A mutant deficient in glutathione because of an insertion in the gshA gene was unable to grow above 1.4 osM, grew more slowly at intermediate osmolarities, and took longer to adapt to growth following osmotic upshock . The involvement of glutathione in osmoregulation was independent of the effect of glutathione on K+ retention. J Surg Res, 1990 Jul, 49(1), 37 - 44 LY171883 preserves mesenteric perfusion in porcine endotoxic shock; Cohn SM et al.; Superior mesenteric arterial perfusion (Q) decreases and gut intramucosal hydrogen ion concentration, {H+}, increases in resuscitated normodynamic endotoxic pigs . The present study tested the hypothesis that these adverse phenomena can be prevented by pretreatment with LY171883, a specific leukotriene (LT) D4/E4 receptor antagonist . Pentobarbital-anesthetized pigs (14-18 kg) were instrumented to permit measurement of Q (ultrasonic flow probe) and {H+} (tonometer) . Mesenteric O2 delivery (DO2) and consumption (VO2) were calculated from the O2 contents of arterial and superior mesenteric venous blood . At t = -20 min, groups (N = 6) of pigs were pretreated witH LY171883 (10 mg/kg) or vehicle . At t = 0 min, the pigs were infused over 20 min with lipopolysaccharide (LPS; 150 micrograms/kg) and resuscitated for 2 hr with saline (1.2 ml/kg min) . Irrespective of treatment group, mean arterial pressure and systemic vascular resistance index decreased significantly after infusion of LPS . In general, cardiac index (CI) was well preserved, although in controls at t = 20, 100, and 120 min, CI decreased significantly with respect to the t = 0 min value . Normal mesenteric Q and DO2 were maintained in the LY171883 group, whereas, in controls, these parameters decreased significantly . Mesenteric VO2 increased transiently but significantly in controls; this phenomenon was abrograted by the LT receptor antagonist . In controls, intramucosal {H+} increased by almost threefold; this adverse effect was significantly ameliorated by LY171883 . These data suggest that decreased mesenteric Q and increased intramucosal {H+} may be mediated by LT in this porcine endotoxic shock model. Mol Cell Biol, 1990 Jul, 10(7), 3551 - 61 Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate; Gangloff SP et al.; We have isolated genomic clones complementing the aconitase-deficient strain (glu1-1) of Saccharomyces cerevisiae . Identification of the aconitase gene was established by enzymatic assays and molecular analyses . The corresponding mRNA has been characterized, and its direction of transcription has been determined . The complete nucleotide sequence revealed strong amino acid homologies with the sequences of some peptides isolated from the mammalian protein . Disruption of the gene by deletion-insertion led to glutamate auxotrophy . Expression of the aconitase gene was sensitive to glucose repression and was synergistically down regulated by glucose and glutamate. AMB Rev Assoc Med Bras, 1990 Jul-Dec, 36(3-4), 153 - 6 {Malacoplakia of the large intestine, bladder and retroperitoneum: a case report}; Vattimo A et al.; The authors present a case of malakoplakia, involving colon, rectum, bladder and retroperitoneum . This rare pathology, generally associated to Escherichia coli infections, result in a granulomatous disease, that can involve one or more organs . Nowadays, it is believed that the illness is due to a failure in the bactericide activity of the macrophage . This case, the first reported in our country, was treated clinically with ascorbic acid and trimethoprim-sulfamethoxazole, and is also unique in the world literature. Mol Gen Genet, 1990 Jul, 222(2-3), 297 - 303 Expression of foreign epitopes in P-fimbriae of Escherichia coli; van Die I et al.; Hypervariable regions (HRs) of the major subunit of F11 fimbriae were exploited for insertion of foreign epitopes . Two insertion vectors were created that contain a unique cloning site in HR1 or HR4 respectively . Several oligonucleotides, coding for antigenic determinants derived from different pathogens, were cloned in both insertion vectors . Hybrid fimbrial subunits were generally shown to be assembled in fimbriae when the length of the inserted peptide did not exceed 14 amino acids . The inserted peptides appeared to be exposed in the fimbrial filament . One hybrid fimbrial protein induced detectable levels of antibodies against the inserted epitope if injected into mice. Mol Biol (Mosk), 1990 Jul-Aug, 24(4), 1100 - 8 {Fluorescent analogs of nucleoside-5'-phosphates for the study of nucleic acids by nonradioactive methods}; Aleksandrova LA et al.; The synthesis of 2'-deoxyuridine 5'-triphosphate analogues with fluorescent residues of fluorescein and rhodamine nature at C5 of the uracil base was performed . Reverse transcriptase of avian myeloblastosis virus, DNA polymerase beta of rat liver, terminal deoxynucleotidyl transferase of calf thymus and E . coli DNA polymerase I, Klenow fragment, were shown to be capable to incorporate a nucleotide residue with fluorescent label into 3'-terminus of oligonucleotide . These fluorescent labeled oligonucleotides were used as primers for synthesis of (-)-chain of M13mp10 phage . Fluorescently labeling template-primer complexes were used for DNA sequencing. Bioorg Khim, 1990 Jul, 16(7), 933 - 40 {Effective synthesis of oligo(poly)deoxyribonucleotides using an H-phosphonate method in plastic microcolumn}; Isaguliants MG et al.; A facile technique of manual oligonucleotide synthesis via H-phosphonate approach is developed . Syntheses carried out in pipette tips with siliconised glasswool filters take 3-3.5 min per cycle with 97-98% yields per condensation . The method was used to synthesize 12-55-mers: T7 and PL promoter regions, gene of the signal peptide of the E . coli OmpA protein, oligonucleotides coding for amino acid sequences 94-105 of preS1- and 133-143 of preS2-regions of hepatitis B virus, hybridisation probes, sequencing primers, oligonucleotides for site-directed mutagenesis, etc. Mol Microbiol, 1990 Jul, 4(7), 1083 - 90 Molecular aspects of phosphate transport in Escherichia coli; Rao NN et al.; Escherichia coli transports inorganic phosphate (Pi) by the low-affinity transport system, Pit . When the level of the external Pi is lower than 20 microM, another transport system, Pst, is induced with a Kt of 0.25 microM . An outer-membrane porin, PhoE, with a Km of about 1 microM is also induced . The outer membrane allows the intake of organic phosphates which are degraded to Pi by phosphatases in the periplasm . The Pi-binding protein will capture the free Pi produced in the periplasm and direct it to the transmembrane channel of the cytoplasmic membrane . The channel consists of two proteins, PstA and PstC, which have six and five transmembrane helices, respectively . On the cytoplasmic side of the membrane the channel is linked to the PstB protein, which carries a nucleotide (probably ATP)-binding site . PstB probably provides the energy required by the channel to free Pi . The Pst system has two functions in E . coli: (i) the transport of Pi, and (ii) the negative regulation of the phosphate regulon (a complex of 20 proteins mostly related to organic phosphate transport) . It is remarkable that these two functions are not related, since the repressibility of the regulon depends on the integral structure of Pst (PiBP + PstA + PstC + PstB) and not on the Pi transported . Another gene of the pst operon, phoU, produces a protein involved in the negative regulation of the Pho regulon, but the mechanism of this function has not been explained . Thus the regulatory function of the Pst system remains obscure . Its basal level, present when Pi is abundant, is sufficient to repress the Pho regulon but the negative regulatory function is lost upon Pi starvation. Mol Microbiol, 1990 Jul, 4(7), 1077 - 82 Signal transduction and gene regulation through the phosphorylation of two regulatory components: the molecular basis for the osmotic regulation of the porin genes; Mizuno T et al.; Expression of Escherichia coli outer-membrane porin proteins (OmpF and OmpC) is regulated by the osmolarity of the medium . EnvZ and OmpR, which are positive regulatory factors for the transcriptional osmotic regulation of the ompF and ompC genes, belong to a group of two-component regulatory factors that respond to a variety of environmental stimuli in bacteria . EnvZ-OmpR phosphotransfer was revealed to be involved in signal transduction in response to an osmotic stimulus, and to play a crucial physiological role in the consequent osmotic activation of the porin genes . Based on the various lines of experimental evidence, a model is proposed for the molecular mechanism underlying the osmotic regulation through phosphorylation of the activator (OmpR) by the membrane-located kinase (Env2). Mol Microbiol, 1990 Jul, 4(7), 1063 - 7 Translation initiation in Escherichia coli: old and new questions; Jacques N et al.; We discuss the features of Escherichia coli mRNAs which determine where and how efficiently translation is initiated . We have shown that DNA fragments comprising 60-80 nucleotides that bracket the initiation codon of real genes generally promote translation when inserted within a foreign mRNA, while those not corresponding to an authentic gene start do not do so even if they include a Shine-Dalgarno-like element followed by AUG or GUG . Therefore, the information that pinpoints the correct start sites, while extending beyond the mere presence of these elements, remains essentially local . The possible nature of this information is discussed . Next, we point out that, in order to remain accessible, translational starts must escape long-range base-pairing within large mRNAs, and we argue that the tight coupling between translation and transcription plays an important role in achieving this . Finally, we discuss two intriguing situations in which the initiation frequency should be dependent upon the rate of translation elongation. Protein Eng, 1990 Jul, 3(7), 591 - 8 Structural effects induced by removal of a disulfide-bridge: the X-ray structure of the C30A/C51A mutant of basic pancreatic trypsin inhibitor at 1.6 A; Eigenbrot C et al.; The X-ray structure of a variant of basic pancreatic trypsin inhibitor (BPTI) has been analyzed to determine the structural accommodation resulting from removal of a disulfide cross-link in a protein . The disulfide removed, Cys30-Cys51, has been implicated in both the folding pathway of the protein and its overall thermal stability . In the variant studied, C30A/C51A, the disulfide cysteines were replaced by less bulky alanines . The atomic displacements observed for C30A/C51A indicate a set of concerted shifts of two segments of chains, which together significantly diminish a packing defect at the site of the removed cysteine sulfur atoms . The observed structural changes are distributed asymmetrically around the sites of mutation, indicating that the adjacent beta-sheet is more resistant to the perturbation than the alpha-helix on the opposite side of the disulfide bond . The thermal parameters of groups involved in the structural accommodation are not significantly altered . A comparison of the X-ray structures reported for native BPTI determined in three different crystal forms indicates that the magnitude of its conformational variability exceeds that of the structural changes caused by the disulfide removal . This emphasizes the necessity of using isomorphous crystal systems to determine the relatively small effects due to mutation. Eur J Immunol, 1990 Jul, 20(7), 1541 - 5 Enhanced immunogenicity of recombinant peptide fusions containing multiple copies of a heterologous T helper epitope; Lowenadler B et al.; We have examined the immune response against the nonimmunogenic heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli using recombinant fusion proteins containing the STa-peptide linked to an IgG-binding analogue of protein A and varying numbers of the T helper epitope 323-339 from ovalbumin (ova) . By immunization of inbred strains of mice with a series of STa fusion proteins, containing up to four copies of ova tandemly multiplied, we demonstrated that the anti-STa antibody response is controlled by ova-specific T helper cells in a genetically restricted manner . In the responding mouse strains (2 out of 3 tested), the level of antibody production was increased by addition of multiple ova epitopes, the anti-STa response being considerably higher to fusion proteins containing four than one or two ova epitopes. Mutat Res, 1990 Jul, 236(1), 9 - 17 9-amino-ellipticine inhibits the apurinic site-dependent base excision-repair pathway; Lefrancois M et al.; The aromatic amine 9-amino-ellipticine is a synthetic DNA intercalating compound derived from the antitumor agent ellipticine, which cleaves at very low doses DNA containing apurinic sites by beta-elimination through formation of a Schiff base . This compound has been shown to potentiate the cytotoxic effect of alkylating drugs, such as dimethyl sulfate, in E . coli through a mechanism involving apurinic sites . We have studied the ability of 9-amino-ellipticine to inhibit an enzymatic repair system mimicking base-excision repair, in which E . coli exonuclease III only presents an endonuclease for apurinic/apyrimidinic site activity . 10 microM of 9-amino-ellipticine inhibits 70% of apurinic site repair . Other intercalating agents with similar affinities for DNA do not induce any inhibition . In another system designed for the direct assay of the exonuclease III-induced incisions 5' to AP sites 10 microM of 9-amino-ellipticine inhibits 65% of the endonuclease for apurinic/apyrimidinic site activity of E . coli exonuclease III . The 9-amino-ellipticine-induced formation of a 2',3'-unsaturated deoxyribose and cleavage at the 3' side of the apurinic site, and possible creation of an adduct, as suggested by Bertrand and coworkers (1989), on the 3' position of the deoxyribose seem to strongly inhibit the endonuclease for apurinic/apyrimidinic site activity . 9-Amino-ellipticine appears therefore to be the first small ligand which can inhibit, by an irreversible modification of the substrate, the repair of apurinic sites through the base excision-repair pathway at a pharmacological concentration. J Cell Biol, 1990 Jul, 111(1), 153 - 69 Elucidating the early stages of keratin filament assembly; Coulombe PA et al.; Because of extraordinarily tight coiled-coil associations of type I and type II keratins, the composition and structure of keratin subunits has been difficult to determine . We report here the use of novel genetic and biochemical methods to explore the early stages of keratin filament assembly . Using bacterially expressed humans K5 and K14, we show that remarkably, these keratins behave as 1:1 complexes even in 9 M urea and in the presence of a reducing agent . Gel filtration chromatography and chemical cross-linking were used to identify heterodimers and heterotetramers as the most stable building blocks of keratin filament assembly . EM suggested that the dimer consists of a coiled-coil of K5 and K14 aligned in register and in parallel fashion, and the tetramer consists of two dimers in antiparallel fashion, without polarity . In 4 M urea, both end-to-end and lateral packing of tetramers occurred, leading to a variety of larger heteromeric complexes . The coexistence of multiple, higher-ordered associations under strongly denaturing conditions suggests that there may not be a serial sequence of events leading to the assembly of keratin intermediate filaments, but rather a number of associations may take place in parallel. J Neuroimmunol, 1990 Jul, 28(2), 177 - 84 Recombinant human beta-galactoside binding lectin suppresses clinical and histological signs of experimental autoimmune encephalomyelitis; Offner H et al.; Human placental tissue contains regulatory molecules that may prevent allo-sensitization . Recently, a 14 kDa beta-galactoside binding protein with demonstrated immunoregulatory properties has been cloned using cDNA from human placenta and expressed in Escherichia coli . The present study assesses the ability of this recombinant immunomodulatory lectin (rIML-1), to prevent experimental autoimmune encephalomyelitis (EAE), a paralytic T cell-mediated disease directed against myelin basic protein (BP) . Injection of rIML-1 into Lewis rats inhibited the induction of both clinical and histological signs of EAE, apparently by blocking sensitization of encephalitogenic BP-specific T cells and inducing BP-dependent suppressor cells . Because it is neither immunogenic nor toxic, rIML-1 may have application in humans, and would have distinct advantages over unselective cytotoxic immunosuppressive agents used currently in the treatment of autoimmune diseases and transplantation. J Bacteriol, 1990 Jul, 172(7), 3952 - 8 The beta-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus; Kovacevic S et al.; Isopenicillin N isomerase (epimerase) has been purified from Streptomyces clavuligerus, and the amino acid sequence of the N-terminus has been determined . By using single oligonucleotide probes based on high GC codon bias ("guessmers"), the translation start codons were determined for two successive genes in the beta-lactam-biosynthetic pathway and mapped within a 3.6-kilobase-pair KpnI restriction fragment . The epimerase gene (cefD) was located immediately upstream of the deacetoxycephalosporin C synthetase (expandase) gene (cefE) that was characterized previously . cefD was sequenced and expressed in Escherichia coli; the resulting cell extracts contained epimerase activity . Western immunoblots demonstrated that a protein comigrated with purified S . clavuligerus epimerase at 44 kilodaltons . cefD and cefE were separated by an 81-base-pair segment . The DNA sequence upstream of the epimerase gene had a high AT content, suggestive of a promoter region . Primer extension analysis of S . clavuligerus mRNA showed that the start of transcription occurred approximately 130 base pairs upstream of the epimerase translation start site; Northern (RNA blot) analysis revealed a hybridization signal large enough to code for both epimerase and expandase, and nuclease S1 protection assays showed that a single message may code for epimerase, expandase, and another unknown protein . When cefD and cefE were placed in an expression vector, concomitant synthesis of both epimerase and expandase occurred in E . coli. J Bacteriol, 1990 Jul, 172(7), 3577 - 83 Effect of outer membrane permeability on chemotaxis in Escherichia coli; Ingham C et al.; The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC . Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC . A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays . AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II . Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate . Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability . These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E . coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane. EMBO J, 1990 Jul, 9(7), 2331 - 40 Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III; Blomberg P et al.; The replication frequency of IncFII plasmids is regulated through the availability of a rate-limiting protein, RepA . The synthesis of this protein is controlled post-transcriptionally by a small antisense RNA, CopA, which binds to the leader region of the RepA mRNA (CopT) . In this communication we report studies of the IncFII plasmid R1 . We show that the duplex between CopA and CopT is cleaved specifically in vivo . The in vivo cleavage maps to the same position as that resulting from in vitro cleavage of a CopA/CopT duplex by purified RNase III . By introducing plasmids carrying translational repA-lacZ fusions into cells deficient in RNase III we show that the expression of repA is elevated when RNase III activity is severely decreased . Hence, cleavage by RNase III seems to be a key event in the copy number control system of plasmid R1. EMBO J, 1990 Jul, 9(7), 2101 - 6 The human muscle nicotinic acetylcholine receptor alpha-subunit exist as two isoforms: a novel exon; Beeson D et al.; Analysis of acetylcholine receptor clones isolated from a human leg muscle cDNA library, revealed that the alpha-subunit existed as two isoforms . A novel exon, coding for 25 amino acids, was located in the human genomic DNA sequence; its insertion into the alpha-subunit gives the new isoform of 462 amino acids . In addition, mRNAs for the two isoforms were found in equal proportions in poly(A)+ RNA obtained from three further sources including partially denervated and innervated human muscle and the rhabdomyosarcoma cell line TE671 . Both protein isoforms can be expressed in E . coli . No evidence of a sequence related to that of the new exon was found in cDNA derived from poly(A)+ RNA isolated from fetal calf or embryonic chick muscle or Torpedo marmorata electric organ. Virology, 1990 Jul, 177(1), 124 - 30 Nucleotide sequence of the leader and nucleocapsid protein gene of mumps virus and epitope mapping with the in vitro expressed nucleocapsid protein; Tanabayashi K et al.; The nucleotide sequence of the leader and the gene encoding the nucleocapsid protein (NP) of mumps virus Miyahara strain have been determined . The leader sequence is 55 nucleotides in length and the NP gene is 1845 nucleotides in length, exclusive of poly(A) . The NP gene codes for a protein of 549 amino acids, with a calculated molecular weight of 61,365 . For epitope mapping, a series of NPs from which C-termini were serially deleted were expressed in vitro from five mRNA constructs and were examined by radioimmunoprecipitation assay (RIPA) with eight nonoverlapping monoclonal antibodies (MoAbs) against the mumps virus NP . It was found that seven out of eight MoAbs reacted with the NP synthesized in vitro . Five recognized the epitopes located within the C-terminal 74 amino acids region and one within the adjacent 64 amino acids upstream . The epitope of the remaining one was in the N-terminal half of the NP. Biotechnology (N Y), 1990 Jul, 8(7), 644 - 9 Expression in yeast of amino-terminal peptide fusions to hepatitis B core antigen and their immunological properties; Beesley KM et al.; Hepatitis B core protein (HBcAg) is a potent antigen that gives both a T-cell-dependent and a T-cell-independent antibody response . It has been shown that a foreign epitope can be fused to the amino terminus of HBcAg without affecting particle integrity, and that the resulting chimaeric cores retain the immunogenicity of the foreign epitope . Here we describe the efficient expression in yeast of two different chimaeric cores, carrying epitopes of Foot and Mouth Disease Virus (FMDV) or human chorionic gonadotrophin (hCG), which are candidates for FMD and contraceptive vaccines, respectively . These cores could not be produced in E . coli in soluble form but were expressed to high levels in yeast . We constructed a yeast expression vector that allows rapid production of different chimaeric cores by cloning in cassettes encoding foreign epitopes . Both FMDV and hCG-cores were shown to present the epitopes at the surface of the particles . The FMDV-cores produced in yeast were efficient inducers of neutralising antibodies in guinea-pigs after one low dose. Appl Microbiol Biotechnol, 1990 Jul, 33(4), 429 - 34 Production and characterization of human gamma interferon from Escherichia coli; Perez L et al.; The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, which simplified the purification process, is described . An expression plasmid carrying lipoprotein and the tryptophane promoters in tandem was used . Preparation of highly pure interferon was achieved using high resolution chromatography after denaturation and renaturation steps . Structural characteristics of this protein were verified by mass spectrometric analysis . Additional control tests have shown the suitability of the final product for clinical purposes. Biotechnol Prog, 1990 Jul-Aug, 6(4), 255 - 61 Sizing biological samples by photosedimentation techniques; Middelberg AP et al.; The performance of the Joyce-Loebl disk centrifuge in the sizing of Escherichia coli cells, protein inclusion bodies, and cell debris is evaluated . The need for a density gradient that extends throughout the entire spin fluid is highlighted, and a set of standard conditions that fulfill this requirement is defined . E . coli cells experience a reduction in their Stokes diameter when exposed to ethanol, indicating that a spin-buffer fluid combination such as glycerol-water is to be preferred for the sizing of bacteria . The instrument baseline is influenced by the presence of particles, and a method of estimating the baseline is described . The sizing of small particles is further complicated by baseline drift due to temperature sensitivity of the optical yoke . An analysis of diffusion in the spin fluid is conducted, and an expression for the sedimentation:diffusive flux ratio is derived . For the current samples, it is shown that diffusion within the spin fluid does not lead to significant errors for 0.15-microns particles, whereas the phenomenon may be significant at the manufacturer's size limit of 0.01 micron. Appl Microbiol Biotechnol, 1990 Jul, 33(4), 424 - 8 A constitutive expression vector system driven by the deo P1P2 promoters of Escherichia coli; Fischer M et al.; The P1P2 promoters of Escherichia coli K12 deo operon, residing on an AvaII restriction fragment, were used to construct a new expression vector . To evaluate the potential of the P1P2-driven expression system we have inserted the sequence of human superoxide dismutase (hSOD) downstream of the deo ribosome binding site . Expression of hSOD was evaluated by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzyme activity . In crude cell extracts hSOD expression levels were found to be high in hosts possessing no deoR or cytR repressors . Highest levels of hSOD expression were obtained with a high-copy-number plasmid regardless of the host used . Expressed hSOD can account for 35%-40% of total protein in E . coli. Science, 1990 Jun 29, 248(4963), 1625 - 30 Functional domains and upstream activation properties of cloned human TATA binding protein; Peterson MG et al.; The TATA binding protein, TFIID, plays a central role in the initiation of eukaryotic mRNA synthesis . Here, we present a human cDNA clone for this factor . Comparison of its predicted protein sequence with those from Drosophila and yeast reveals a highly conserved carboxyl-terminal 180 amino acids . By contrast, the amino-terminal region of TFIID has diverged in both sequence and length . A striking feature of the human protein is a stretch of 38 glutamine residues in the NH2-terminal region . Expression of human TFIID in both Escherichia coli and HeLa cells produces a protein that binds specifically to a TATA box and promotes basal transcription; the conserved COOH-terminal portion of the protein is sufficient for both of these activities . Recombinant TFIID forms a stable complex on a TATA box either alone or in combination with either of the general transcription factors, TFIIA or TFIIB . Full-length recombinant TFIID is able to support Sp1 activated transcription in a TFIID-depleted nuclear extract, while a deletion of the NH2-terminal half of the protein is not . These results indicate the importance of the NH2-terminal region for upstream activation functions and suggest that additional factors (co-activators) are required for mediating interactions with specific regulators. Biochem Biophys Res Commun, 1990 Jun 29, 169(3), 1129 - 37 Expression of simian virus 40 large T antigen in Escherichia coli using vectors based on the regulatable rac promoter; Pistillo JM et al.; Simian Virus 40 large T antigen is a multi-functional protein that is involved in the initiation of viral DNA replication, regulation of viral transcription and cell transformation . Bacterial expression vectors, pER23-1 and pER23-2, that are based on the regulatable rac promoter were used to produce T antigen either as a free protein or as a fusion protein . We have observed efficient transcription of the cloned T antigen gene in most of the recombinants . However, expression of the T antigen protein was inefficient and most of the expressed protein was truncated . This may be due to differences in codon usage in E . coli or to rapid protein degradation. Science, 1990 Jun 29, 248(4963), 1650 - 3 A cDNA for a protein that interacts with the human immunodeficiency virus Tat transactivator; Nelbock P et al.; The human immunodeficiency virus (HIV) tat protein (Tat) is a positive regulator of virus gene expression and replication . Biotinylated Tat was used as a probe to screen a lambda gt11 fusion protein library, and a complementary DNA encoding a protein that interacts with Tat was cloned . Expression of this protein, designated TBP-1 (for Tat binding protein-1), was observed in a variety of cell lines, with expression being highest in human cells . TBP-1 was localized predominantly in the nucleus, which is consistent with the nuclear localization of Tat . In cotransfection experiments, expression of TBP-1 was able to specifically suppress Tat-mediated transactivation . The strategy described may be useful for direct identification and cloning of genes encoding proteins that associate with other proteins to modulate their activity in a positive or negative fashion. Science, 1990 Jun 29, 248(4963), 1646 - 50 Cloning of a transcriptionally active human TATA binding factor; Kao CC et al.; Transcription factor IID (TFIID) binds to the TATA box promoter element and regulates the expression of most eukaryotic genes transcribed by RNA polymerase II . Complementary DNA (cDNA) encoding a human TFIID protein has been cloned . The human TFIID polypeptide has 339 amino acids and a molecular size of 37,745 daltons . The carboxyl-terminal 181 amino acids of the human TFIID protein shares 80% identity with the TFIID protein from Saccharomyces cerevisiae . The amino terminus contains an unusual repeat of 38 consecutive glutamine residues and an X-Thr-Pro repeat . Expression of DNA in reticulocyte lysates or in Escherichia coli yielded a protein that was competent for both DNA binding and transcription activation. Cell, 1990 Jun 29, 61(7), 1199 - 208 Selective inhibition of activated but not basal transcription by the acidic activation domain of VP16: evidence for transcriptional adaptors; Berger SL et al.; The interaction between the chimeric activator GAL4-VP16, consisting of the DNA binding domain of GAL4 and the acidic activation domain of VP16, and its target in the transcriptional machinery was studied in vitro . GAL4-VP16 stimulated transcription from a promoter bearing GAL4 sites, and greatly inhibited transcription from a promoter bearing binding sites for the dA:dT activator and from a basal promoter bearing only a TATA box . Mutations in the acidic domain that reduced activation from the GAL4 site promoter also reduced inhibition from the dA:dT promoter, indicating a similar interaction between VP16 and its target in both processes . Strikingly, if the DNA binding domain of GAL4-VP16 was occupied by a GAL4 site oligonucleotide, the protein inhibited activation by the dA:dT activator but did not inhibit basal transcription . We propose that, under these conditions, GAL4-VP16 acted to titrate an "adaptor" that bridges an interaction between the upstream activator and the basic transcriptional machinery at the TATA box. Biochemistry, 1990 Jun 26, 29(25), 5994 - 6002 Structural studies on the active site of Escherichia coli RNA polymerase . 2 . Geometrical relationship of the interacting substrates; Beal RB et al.; Since a major function of RNA polymerase must be to bring together substrates in the optimal configuration for internucleotide bond formation, studies have been undertaken to understand the geometrical relationship of the two substrates . A model has been constructed for the geometry of interaction of two ATP molecules poised on the active site of the Escherichia coli enzyme for the formation of the first bond in RNA synthesis . The model is based primarily on the distance, measured by EPR, between the two metals in the i and i + 1 subsites, as well as distances, measured by NMR, from each metal to points on the substrate in the same subsite, in the presence of a poly(dAdT).poly(dAdT) template . Both the Zn(II) in the i site and the Mg(II) in i + 1 are displaced by Mn(II) . The nucleotide bases are not parallel to each other, in line with the reaction of the ATP molecules with DNA within the transcription bubble . The metal in the i site appears too far removed from substrate to participate in catalysis, but the metal in i + 1 is in position to bind to the beta- and gamma-phosphate groups and probably is involved in cleavage of the triphosphate, as has been previously suggested. Biochemistry, 1990 Jun 26, 29(25), 5987 - 94 Structural studies on the active site of Escherichia coli RNA polymerase . 1 . Interaction of metals on the i and i + 1 sites; Chuknyisky PP et al.; The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion . These ions are therefore useful as probes of substrate interaction during RNA synthesis . We have studied interactions between the metals by EPR spectroscopy . The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II) . The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way . The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals . The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A. J Biol Chem, 1990 Jun 25, 265(18), 10189 - 92 Regulation of gene expression in vivo by liposome-mediated delivery of a purified transcription factor; Debs RJ et al.; We describe a procedure for assessing the functional activity in vivo of a glucocorticoid receptor derivative, T7X556, a mammalian transcriptional regulator that has been overexpressed in Escherichia coli and purified to homogeneity . The protein was assessed with DOTMA (N-{1-(2,3-dioleyloxy)propyl}-N,N,N-trimethyl-ammonium chloride) liposomes, which are internalized by cultured mammalian cells . T7X556 protein delivered in this manner localized rapidly to the nucleus and selectively enhanced expression from glucocorticoid response element-linked promoters, properties that are characteristic of this receptor derivative when it is synthesized endogenously in mammalian cells . Thus, in vivo activities of T7X556 were not disrupted by expression in bacteria or by biochemical purification . In general, liposome-mediated delivery may permit functional analyses of proteins that have been expressed in heterologous cells and manipulated in vitro. Nucleic Acids Res, 1990 Jun 25, 18(12), 3515 - 20 Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA; Weitzmann CJ et al.; The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase . At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence . In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence . The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively . Typical transcription reactions yielded 500-700 moles RNA per mole template . This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts . The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100) . Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed. Nucleic Acids Res, 1990 Jun 25, 18(12), 3479 - 87 Interaction of RNase P from Escherichia coli with pseudoknotted structures in viral RNAs; Mans RM et al.; In a previous study it was shown that RNase P from E . coli cleaves the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA in vitro (Guerrier-Takada et al . (1988) Cell, 53, 267-272) . Cleavage takes place at the 3' side of the loop that crosses the deep groove of the pseudoknot structure present in the aminoacyl acceptor domain . In the present study fragments of TYMV RNA with mutations in the pseudoknot, generated by transcription in vitro, were tested for susceptibility to cleavage by RNase P . Changes in the specificity with respect to the site of cleavage and decreases in the rate of cleavage were observed with most of these substrates . The behaviour of various mutants in the reaction catalyzed by RNase P is in agreement with the present model of the TYMV RNA pseudoknot (Dumas et al . (1987), J . Biomol . Struct . Dyn . 263, 652-657) . Base substitutions in the loop that crosses the shallow groove of the pseudoknot structure resulted, however, in an unexpected decrease in the rate of cleavage, probably due to conformational changes in the substrates . Studies on other tRNA-like structures revealed an important role in the reaction with RNase P for both the nucleotide at the 3' side of the loop that spans the deep groove and the nucleotide at position 4, which correspond to positions--1 and 73, respectively, in tRNA precursors. Nucleic Acids Res, 1990 Jun 25, 18(12), 3445 - 50 A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase; Mehrpouyan M et al.; A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated . Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min . on the bacterial chromosome . The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44 degrees C . The thermostability at 44 degrees C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme . Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation . A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about 4 fold at 37 degrees C . The role of the alpha dimer in the structure and function of RNA polymerase is discussed. Nucleic Acids Res, 1990 Jun 25, 18(12), 3439 - 43 Efficient site directed in vitro mutagenesis using ampicillin selection; Lewis MK et al.; A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis . The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance . Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template . The non-mutant DNA strand is selected against by growth in the presence of ampicillin . In tests of the vector, highly efficient (60-90%) mutagenesis was obtained. J Biol Chem, 1990 Jun 25, 265(18), 10666 - 73 Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid; Hama C et al.; The frequency of replication initiation of the ColIb-P9 plasmid depends on the level of repZ expression, which has been shown to be negatively regulated by inc RNA, the approximately 70-base-long product of the inc gene . To further understand the regulatory mechanism of repZ gene expression, we isolated mutants defective in ColIb-P9 replication using a lambda:ColIb-P9 hybrid phage . Among six mutants isolated, one amber mutant, rep57, failed to synthesize the RepZ protein . The mutation occurred in the repZ leader sequence that encodes a 29-amino-acid reading frame, designated as repY . We also isolated mutants that suppressed the rep57 phenotype . These mutations were single base insertions between the repY initiation codon and the rep57 mutation site and resulted not only in a frame shift of repY but also in the formation of repY-repZ fusions without changing the amino acid sequence of RepZ . Thus, repY is not directly involved in the replication reaction but rather functions as a positive regulator for repZ expression . We propose that repZ expression is coupled with repY translation, which acts to disrupt a secondary structure sequestering the repZ translation initiation signal . The positive and negative regulations of repZ expression were discussed . The other mutants were mapped in repZ, confirming that repZ is essential for ColIb-P9 replication. J Biol Chem, 1990 Jun 25, 265(18), 10637 - 44 Covalent association of the traI gene product of plasmid RP4 with the 5'-terminal nucleotide at the relaxation nick site; Pansegrau W et al.; Formation of relaxosomes is the first step in the initiation of transfer DNA replication during bacterial conjugation . This nucleoprotein complex contains all components capable of introducing a site- and strand-specific nick at a cognate transfer origin (oriT) on supercoiled plasmid DNA, thus providing the substrate for generation of the strand to be transferred . Characterization of the terminal nucleotides at the oriT nick site revealed that relaxation occurs by hydrolysis of a single phosphodiester bond between a 2'-deoxyguanosyl and a 2'-deoxycytidyl residue . The relaxation nick site and a 19-base pair invert repeat sequence that is recognized by asymmetric binding of the RP4 TraJ protein are interspaced by 8 base pairs . The nicking reaction results in covalent attachment of the RP4 TraI protein to the 5'-terminal 2'-deoxycytidyl residue of the cleaved strand . The arrangement of the TraJ binding site and the relaxation nick site on the same side of the DNA double helix suggests that protein-protein interactions between TraJ and TraI are a prerequisite for oriT specific nicking . In accordance with the current model of transfer DNA replication, the 3' end remains accessible for primer extension by DNA polymerase I, enabling replacement strand synthesis in the donor cell by a rolling circle-type mechanism. J Biol Chem, 1990 Jun 25, 265(18), 10631 - 6 Molecular cloning of a cDNA for rat hepatic glutaminase . Sequence similarity to kidney-type glutaminase; Smith EM et al.; Mammalian liver possesses a unique isozyme of phosphate-activated glutaminase which plays an important role in the regulation of glutamine catabolism . Antibodies to hepatic glutaminase were used to screen a lambda gt11 rat liver cDNA library . One cDNA to hepatic glutaminase was identified . Changes in the relative abundance of hepatic glutaminase mRNA were determined by hybridization to this cDNA . The mRNA is found only in liver; it is not present prior to birth but its abundance increases dramatically at birth . The abundance of the mRNA is increased approximately 4-fold in diabetes . The sequence of the cDNA was compared to that recently published for kidney (brain)-type glutaminase (Banner, C., Hwang, J.-J., Shapiro, R.A., Wenthold, R.J., Nakatani, Y., Lampel, K.A., Thomas, J.W., Huie, D., and Curthoys, N.P . (1988) Mol . Brain Res . 3, 247-254) . When the predicted amino acid sequences were compared a region of 123 amino acids with greater than 80% identity was found . The presence of scattered amino acid substitutions within stretches of identical amino acids suggests that the glutaminase isozymes are encoded by separate genes . This is the first demonstration of any similarity between the two glutaminases at the molecular level. J Biol Chem, 1990 Jun 25, 265(18), 10597 - 603 The basement membrane glycoprotein entactin promotes cell attachment and binds calcium ions; Chakravarti S et al.; Mouse entactin derived from the extracellular matrix of M1536-B3 cells and from insect cells infected with a recombinant virus containing entactin sequences were shown to promote the attachment of mouse mammary tumor, human melanoma, and other cells . The cell attachment was inhibited by antibodies against mouse entactin but not by anti-fibronectin or anti-laminin antibodies . On a weight basis entactin was as effective as laminin in promoting the attachment of mouse mammary tumor cells . The attachment of cells to entactin was in part mediated by the integrin recognition RGD peptide sequence . This was demonstrated by the cell attachment properties of peptides derived from entactin which contained this sequence . Furthermore, the peptide RGDS could inhibit the attachment of mouse mammary tumor cells to entactin to approximately 60% of control . It is suggested that additional cell recognition sequences may be present in entactin . The direct binding of calcium ions to entactin was observed . It is probable that the binding sites reside in peptide sequences located toward the NH2 terminus region of entactin . This conclusion was supported by the demonstration that synthetic peptides, containing potential calcium binding sequences derived from entactin, bound calcium . In addition, a recombinant peptide containing the amino-terminal 330 amino acids of entactin also bound calcium ions . The significance of these properties of entactin is discussed. J Biol Chem, 1990 Jun 25, 265(18), 10424 - 9 Nucleotide sequence of the fadA gene . Primary structure of 3-ketoacyl-coenzyme A thiolase from Escherichia coli and the structural organization of the fadAB operon; Yang SY et al.; The DNA insert of plasmid pK52 contains the fadAB operon coding for the Escherichia coli fatty acid oxidation complex . Studies on the operon's structure and organization revealed that the initiator codon (ATG) of the structural gene for 3-ketoacyl-CoA thiolase, the fadA gene, is located 109 nucleotides 3' to the stop codon (TGA) of the fadB gene that encodes the alpha-subunit, a multifunctional polypeptide . The direction of transcription of this operon is thus from fadB to fadA . The orientation of the fadA and fadB genes is the reverse of what had been published previously . The structural gene for thiolase is 1,164 nucleotides long and starts six nucleotides downstream from a Shine-Dalgarno sequence . 109 nucleotides of 5'-noncoding and 321 nucleotides of 3'-noncoding regions are also reported . The 3-ketoacyl-CoA thiolase beta-subunit is composed of 388 residues and has a calculated Mr of 40,889 . The alpha- and beta-subunits were separated by gel filtration in formic acid, and the sequence of the amino-terminal 10 amino acids of the beta-subunit coincided with that deduced from the nucleotide sequence data . Sequence comparisons suggest that Cys-91 of the E . coli enzyme is the active-site cysteine residue and that the consensus sequence of the active sites of 3-ketoacyl-CoA thiolases is Asn-Arg-X1-Cys-X2-Ser-X3-X4-Gln . Although the quaternary structure of E . coli 3-ketoacyl-CoA thiolase is different from that of other thiolases, the sequence is homologous to rat and human peroxisomal and rat mitochondrial 3-ketoacyl-CoA thiolases, to the extent of 42, 41, and 37%, identity, respectively . An evolutionary tree of thiolases was constructed; it suggests that the genes of E . coli and peroxisomal 3-ketoacyl-CoA thiolases diverged after the appearance of eukaryotic cells. J Biol Chem, 1990 Jun 25, 265(18), 10196 - 7 Crystallization and preliminary x-ray investigation of colicin E3 in complex with its immunity protein; Frolow F et al.; Crystals of the colicin E3-immunity protein complex have been grown from solutions of citrate at pH 5.6 . The crystals are monoclinic, space group P2(1), with unit cell dimensions a = 67.71, b = 196.67, c = 85.58 A, and beta = 113.67 degrees . The crystals diffract to 3-A resolution and are stable in the x-ray beam for at least a day . Although the stoichiometry of the complex in solution is 1:1 there are two, three, or four such binary complex molecules in the asymmetric unit. J Biol Chem, 1990 Jun 25, 265(18), 10327 - 33 Biotination of proteins in vivo . A post-translational modification to label, purify, and study proteins; Cronan JE Jr; Post-translational modification of proteins with biotin provides the means to specifically label proteins in vivo and to purify proteins from crude cell lysates . The carboxyl-terminal protein segments modified by reaction with biotin ligase are strongly conserved in nature . We have demonstrated that the proteins encoded by translational gene fusions of a number of heterologous proteins to these carboxyl-terminal sequences become biotinated in vivo . The minimum size of the protein segment needed to allow biotination of fusion proteins is 75 amino acids . This biotination sequence, although of bacterial origin, functions in Saccharomyces cerevisiae as well as in Escherichia coli . Fusion proteins are readily labeled with {3H}biotin in vivo and the labeling is highly specific due to the scarcity (less than 5) of biotinated protein species . Biotinated fusion proteins can be readily purified in native form by binding to columns of monomeric avidin followed by elution with buffers containing biotin . Alternatively, proteins can be purified in a denatured form in presence of 1% sodium dodecyl sulfate or 8 M urea . Thus, this technology allows purification by affinity chromatography of any protein to which a biotination sequence can be attached . The ability to specifically label a protein in vivo should have utility in studies such as intracellular protein trafficking and cytoskeletonal dynamics. J Biol Chem, 1990 Jun 25, 265(18), 10574 - 81 Transcription of osmB, a gene encoding an Escherichia coli lipoprotein, is regulated by dual signals . Osmotic stress and stationary phase; Jung JU et al.; The osmB gene, which encodes an outer membrane lipoprotein, can be induced by both osmotic and growth phase signals . Construction of two transcriptional fusions, an osmB-lacZ fusion in single copy on the bacterial chromosome and an osmB-cat fusion carried on a multicopy plasmid, demonstrated that induction of osmB by hyperosmolarity and during the stationary phase of growth occurred at the level of transcription . Two transcription initiation sites were identified by RNase protection of in vivo message . The downstream P2 promoter is the primary site for regulation; the basal level of expression is initiated at P2 and transcription from P2 is induced by elevated osmolarity or upon reaching stationary phase . Transcription from the P1 promoter, 150 base pairs (bp) upstream of the P2 promoter, occurred only when both osmotic and growth phase signals were present simultaneously; that is, when cells growing in high osmolarity medium have reached stationary phase . Deletion analysis narrowed the sequences necessary for P2 regulation to the 42-bp region upstream from the transcription start site . A 7-bp sequence just upstream from the -35 region was identified as a cis-acting regulatory element essential for osmotic stimulation of osmB expression . A hexanucleotide sequence within this segment could form the left arm of a region of dyad symmetry, flanking the -35 region of the promoter . Stationary phase induction at P2 does not require the 7-bp element. J Biol Chem, 1990 Jun 25, 265(18), 10565 - 73 Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase; Huber HE et al.; We have analyzed the processing of the RNA primer for (+) strand DNA synthesis by reverse transcriptase of the human immunodeficiency virus 1 . To test for specific RNA cleavage and primer usage, we constructed a 99-base pair RNA-DNA hybrid containing the viral polypurine tract and flanking viral sequences . Although the RNase H activity of reverse transcriptase cleaves the RNA strand into multiple fragments, only two primers are extended in the presence of nucleoside triphosphates . The major RNA primer includes the entire polypurine tract except for the last adenosine and has the sequence 5'-UUUUAAAAGAAAAGGGGGG-3' . The minor primer has the same 3' end but is two nucleotides shorter . In a subsequent processing step reverse transcriptase releases the primer intact via a cleavage at the RNA-DNA junction . RNA cleavage, primer extension, and primer removal can take place in a single reaction . However, specificity does not require coupling of the three steps and is preserved in the individual reactions . The polypurine primer is generated and removed after its elongation in the absence of DNA synthesis . Furthermore, the polypurine primer is selected among the several RNA fragments available and extended by reverse transcriptase as well as by p51, a short form of reverse transcriptase lacking RNase H activity. Science, 1990 Jun 22, 248(4962), 1550 - 3 An RNA polymerase II transcription factor shares functional properties with Escherichia coli sigma 70; Conaway JW et al.; A mammalian transcription factor, which, along with other factors, is essential for accurate initiation of transcription from promoters by RNA polymerase II, has been found to regulate the interaction of polymerase and DNA . This factor, designated beta gamma, drastically reduces the affinity of RNA polymerase II for free DNA containing either promoter or nonpromoter sequences . In this respect, beta gamma functions as does the bacterial transcription initiation factor sigma 70, which expedites the binding of Escherichia coli RNA polymerase to promoters in part by accelerating dissociation of the polymerase from nonpromoter sites in DNA. Biochim Biophys Acta, 1990 Jun 21, 1049(2), 223 - 6 Characterization of a cDNA clone encoding the complete amino acid sequence of cotton isocitrate lyase; Turley RB et al.; A cDNA clone encoding the glyoxysomal enzyme isocitrate lyase (ICL) (EC 4.1.3.1) was isolated from a library prepared from cotton (Gossypium hirsutum L.) cotyledon poly(A)+ RNA . The clone is 1893 basepairs (bp) in length and contains a 1728 bp open reading frame encoding a polypeptide of 576 residues (Mr = 64,741) . The deduced amino acid sequence of cotton ICL is 85.2%, 90.3% and 41.1% identical to ICL from rapeseed, castor bean and E . coli, respectively . Cotton ICL has a C-terminal tripeptide of A-R-M which is a putative trafficking signal for peroxisome (glyoxysome) proteins. Eur J Biochem, 1990 Jun 20, 190(2), 311 - 8 Protein-decorated micelle structure of sodium-dodecyl-sulfate--protein complexes as determined by neutron scattering; Ibel K et al.; The structure of the complex between sodium dodecyl sulfate (SDS) and a deuterated bifunctional enzyme, N-5'-phosphoribosylanthranilate isomerase/indole-3-glycerol-phosphate synthase (Mr 49,484), has been studied in dilute solution by small-angle neutron scattering . The complex nearly acquired its final size, as shown by molecular-sieve chromatography, at the chosen SDS concentration of 1.6 mM, which is slightly below the critical micelle concentration of 1.8 mM (at the ionic strength of 0.1 M) . The 452 amino-acid residues of the bifunctional enzyme were combined with 216 detergent molecules . The complex was found to be composed of three protein-decorated SDS micelles of unequal size, connected by short flexible polypeptide segments . The largest of the three micelles was the middle one . The SDS-protein complex contained the dodecyl hydrocarbon moieties in three globular cores . Each core was surrounded by a hydrophilic shell, formed by the hydrophilic and amphiphilic stretches of the polypeptide chain, and by the sulfate head groups of the detergent . The average thickness of these shells was 0.7-0.8 nm . The three-micelle complex was cleaved with trypsin at a single site, possibly in a micelle-connecting segment, into a single-micelle fragment at the carboxyl-terminal which comprised 73 SDS molecules and 163 amino-acid residues, and a dual-micelle fragment . One of the micelles within this larger fragment contained 42 SDS molecules and about 90 amino-acid residues; the other micelle contained 101 SDS molecules and about 190 amino-acid residues . The individual micelle sizes seemed to be determined by the amino-acid sequence. Biochim Biophys Acta, 1990 Jun 20, 1034(3), 253 - 9 Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichia coli; Falkenberg P et al.; The osmoregulatory NAD-dependent betaine aldehyde dehydrogenase (betaine aldehyde:NAD oxidoreductase, EC 1.2.1.8), of Escherichia coli, was purified to apparent homogeneity from an over-producing strain carrying the structural gene for the enzyme (betB) on the plasmid vector pBR322 . Purification was achieved by ammonium sulfate fractionation of disrupted cells, followed by affinity chromatography on 5'-AMP Sepharose, gel-filtration and ion-exchange chromatography . The amino acid composition was determined . The dehydrogenase was found to be a tetramer with identical 55 kDa subunits . Both NAD and NADP could be used as cofactor for the dehydrogenase, but NAD was preferred . The dehydrogenase was highly specific for betaine aldehyde . None of the analogs tested functioned as a substrate, but several inhibited the enzyme competitively . The enzyme was not activated by salts at concentrations encountered during osmotic upshock, but it was salt tolerant, retaining 50% of maximal activity at 1.2 M K+ . It is inferred that salt tolerance is an essential property for an enzyme participating in the cellular synthesis of an osmoprotectant. J Mol Biol, 1990 Jun 20, 213(4), 705 - 17 Construction of Escherichia coli amber suppressor tRNA genes . II . Synthesis of additional tRNA genes and improvement of suppressor efficiency; Kleina LG et al.; Using synthetic oligonucleotides, we have constructed 17 tRNA suppressor genes from Escherichia coli representing 13 species of tRNA . We have measured the levels of in vivo suppression resulting from introducing each tRNA gene into E . coli via a plasmid vector . The suppressors function at varying efficiencies . Some synthetic suppressors fail to yield detectable levels of suppression, whereas others insert amino acids with greater than 70% efficiency . Results reported in the accompanying paper demonstrate that some of these suppressors insert the original cognate amino acid, whereas others do not . We have altered some of the synthetic tRNA genes in order to improve the suppressor efficiency of the resulting tRNAs . Both tRNA(CUAHis) and tRNA(CUAGlu) were altered by single base changes, which generated -A-A- following the anticodon, resulting in a markedly improved efficiency of suppression . The tRNA(CUAPro) was inactive, but a hybrid suppressor tRNA consisting of the tRNA(CUAPhe) anticodon stem and loop together with the remainder of the tRNA(Pro) proved highly efficient at suppressing nonsense codons . Protein chemistry results reported in the accompanying paper show that the altered tRNA(CUAHis) and the hybrid tRNA(CUAPro) insert only histidine and proline, respectively, whereas the altered tRNA(CUAGlu) inserts principally glutamic acid but some glutamine . Also, a strain deficient in release factor I was employed to increase the efficiency of weak nonsense suppressors. J Mol Biol, 1990 Jun 20, 213(4), 777 - 88 IncN plasmid replicon . A deletion and subcloning analysis; Krishnan BR et al.; A DNA segment of approximately 2000 base-pairs bounded by restriction enzyme sites for PvuII and containing the minimal replicon of an N group plasmid was characterized . A natural derivative of this miniplasmid was found to have undergone a deletion within one of two tandem iteron families, the group I iterons . Further analysis showed that all plasmid-determined functions essential for stable maintenance in Escherichia coli were localized to a contiguous region of DNA of 1019 nucleotides that excludes entirely these iterons . However, the loss of these iterons led to an increase in plasmid copy number . This indicates that members of the group I iteron-family have a role in determining plasmid copy number perhaps by titrating a plasmid-specified trans-acting product . The 2000 base-pair segment contains six open reading frames of 40 or more amino acid residues . The essential segment contains a 368 nucleotide region that must be present in cis and within which there are three "GATC" sequences and a putative Escherichia coli DnaA protein-binding sequence (dnaA box) . An interesting feature is that the cis-acting region is present entirely within a presumptive rep gene . The essential segment contains four open reading frames, only one of which has an Escherichia coli canonical ribosome-binding site . The 2000 base-pair miniplasmid has two separable regions determining N group plasmid incompatibility. J Mol Biol, 1990 Jun 20, 213(4), 627 - 30 Induced-fit movements in adenylate kinases; Schulz GE et al.; The high-resolution crystal structures of three homologous adenylate kinases with zero, one and both ( = 2-substrate mimicking inhibitor) bound substrates have been compared . The comparisons are meaningful, because all structures occur in two or three different crystal contact environments indicating that they represent intrinsically stable conformations in solution . Molecular superimpositions revealed that two domains comprising 30 and 38 residues undergo large movements on substrate binding, which can be approximated by rigid-body rotations over 39 degrees and 92 degrees, respectively . Moreover, these movements can be subdivided into two steps: first, a change on binding substrate AMP, which involves only the 30 residue domain (C alpha shifts up to 8.2 A), and second, a change on additional binding of substrate ATP, which again involves the 30 residue domain (C alpha shifts up to 7.6 A) but also the 38 residue domain (C alpha shifts up to 32.3 A) . Taken together, these observations yield a three-picture "moving film" of the induced-fit. J Mol Biol, 1990 Jun 20, 213(4), 789 - 809 RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates . An ADP-resistant process; Rao BJ et al.; Efficient homologous pairing de novo of linear duplex DNA with a circular single strand (plus strand) coated with RecA protein requires saturation and extension of the single strand by the protein . However, strand exchange, the transfer of a strand from duplex DNA to the nucleoprotein filament, which follows homologous pairing, does not require the stable binding of RecA protein to single-stranded DNA . When RecA protein was added back to isolated protein-free DNA intermediates in the presence of sufficient ADP to inhibit strongly the binding of RecA protein to single-stranded DNA, strand exchange nonetheless resumed at the original rate and went to completion . Characterization of the protein-free DNA intermediate suggested that it has a special site or region to which RecA protein binds . Part of the nascent displaced plus strand of the deproteinized intermediate was unavailable as a cofactor for the ATPase activity of RecA protein, and about 30% resisted digestion by P1 endonuclease, which acts preferentially on single-stranded DNA . At the completion of strand exchange, when the distal 5' end of the linear minus strand had been fully incorporated into heteroduplex DNA, a nucleoprotein complex remained that contained all three strands of DNA from which the nascent displaced strand dissociated only over the next 50 to 60 minutes . Deproteinization of this intermediate yielded a complex that also contained three strands of DNA in which the nascent displaced strand was partially resistant to both Escherichia coli exonuclease I and P1 endonuclease . The deproteinized complex showed a broad melting transition between 37 degrees C and temperatures high enough to melt duplex DNA . These results show that strand exchange can be subdivided into two stages: (1) the exchange of base-pairs, which creates a new heteroduplex pair in place of a parental pair; and (2) strand separation, which is the physical displacement of the unpaired strand from the nucleoprotein filament . Between the creation of new heteroduplex DNA and the eventual separation of a third strand, there exists an unusual DNA intermediate that may contain three-stranded regions of natural DNA that are several thousand bases in length. J Mol Biol, 1990 Jun 20, 213(4), 719 - 26 Construction of Escherichia coli amber suppressor tRNA genes . III . Determination of tRNA specificity; Normanly J et al.; Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes . In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E . coli dihydrofolate reductase gene fol . The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon . The suppressors can be classified into three groups on the basis of the protein sequence information . Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid . The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase . The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine. J Mol Biol, 1990 Jun 20, 213(4), 617 - 9 Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme; Boulot G et al.; The associated heavy (VH) and light (VL) chain variable domains (Fv) of the monoclonal anti-lysozyme antibody D1.3, secreted from Escherichia coli, have been crystallized in their antigen-bound and free forms . FvD1.3 gives tetragonal crystals, space group P4(1)2(1)2 (or P4(3)2(1)2), with a = 90.6 A, c = 56.4 A . The FvD1.3-lysozyme complex crystallizes in space group C2, with a = 129.2 A, b = 60.8 A, c = 56.9 A and beta = 119.3 degrees . The crystals contain one molecule of Fv or of the Fv-lysozyme complex in their asymmetric units and diffract X-rays to high resolution, making them suitable for X-ray crystallographic studies. J Mol Biol, 1990 Jun 20, 213(4), 613 - 5 Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli; Glockshuber R et al.; The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0) . The crystals are hexagonal, space group P6(1)22 . The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit . The crystals diffract to 2.0 A . The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI. Eur J Biochem, 1990 Jun 20, 190(2), 257 - 61 Three human interferon-alpha 2 subvariants disclose structural and functional differences; von Gabain A et al.; The human interferon-alpha 2 subvariants 2a, 2b and 2c differ by only one or two amino acids at positions 23 and/or 34 of the mature protein . In this study, the coding regions of the three interferon-alpha 2 subvariants were derived from the cDNA of interferon-alpha 2c by site-directed in vitro mutagenesis . The interferon-alpha subvariants were synthesized using the same Escherichia coli strain for production and were subsequently purified . Comparative studies revealed that they differ significantly in their biological and antigenic properties . Therefore, amino acid positions 23 and 34 seem to be crucial for structure/function of human interferon-alpha . Furthermore, the study points to the importance of defining, whether such minor structural variants of naturally occurring polypeptides represent functional variants. J Mol Biol, 1990 Jun 20, 213(4), 607 - 11 Crystallization of genetically engineered active maltose-binding proteins, including an immunogenic viral epitope insertion; Rodseth LE et al.; Three mutants of the maltose- or maltodextrin-binding protein encoded by the malE gene of Escherichia coli, with extensive genetic changes, have been purified and crystallized in different crystal forms . Two of these mutant proteins, MalE178 and MalE341, carry net deletions of seven and 13 residues, respectively, near the surface of the molecule . These mutations have very little effect on either the transport activity of the mutant strains or the sugar-binding activity of the purified mutant proteins . The third mutant protein involves the insertion of an 11-residue peptide of the C3 epitope from type 1 poliovirus VP1 protein into the MalE178 deletion mutant, with retention of essentially all the biological properties of the wild-type and the immunological properties of the C3 epitope . We are undertaking three-dimensional structure analysis in order to understand how the protein accommodates these large changes in its surface structure and how the C3 epitope retains its immunological properties in this new environment . The same system could be used to determine easily the structures of other peptide epitopes, especially those in proteins with unknown structures. Biochemistry, 1990 Jun 19, 29(24), 5829 - 36 On the nature of the structural change of the colicin E1 channel peptide necessary for its translocation-competent state; Merrill AR et al.; Acidic pH conditions required in vitro for membrane binding and activity of the channel-forming colicin E1 resulted in an increased susceptibility to proteases of the 178-residue thermolytic channel peptide, an increased accessibility to acrylamide of a fluorescence probe linked to cysteine-505 of the peptide, and an increased partition into nonionic detergent . The structural change in the peptide sensed by the fluorescence probe caused by a transition from pH 6.0 to 3.5 occurred in less than 1 s . The presence of low concentrations of detergents (0.001% SDS or 0.44% octyl beta-D-glucoside) or urea (0.2 M) at pH 6 or 4 also increased the susceptibility of the channel peptide to proteases . The increase in protease susceptibility and acrylamide accessibility at low pH, as well as partition of the peptide into nonionic detergent, suggested that acidic pH or the detergents might cause peptide unfolding . However, the hydrodynamic radius of the channel peptide at pH 6, 21-23 A, was not changed at pH 3.5 or by detergents or urea under conditions that increased the susceptibility of the peptide to protease . The activity of the channel peptide at pH 6 measured with liposomes and planar bilayers, which was a factor of 10(3)-10(4) smaller than that at pH 4, was increased by 2-4 orders of magnitude by 0.001% SDS or 0.44% octyl beta-D-glucoside, with an additional small increment of activity on planar bilayers caused by 0.01% SDS . A small increase in Stokes radius of the peptide in the presence of SDS could be detected that was approximately correlated with increased activity. Biochemistry, 1990 Jun 19, 29(24), 5790 - 6 Glutathione reductase: comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes; Vanoni MA et al.; Kinetic parameters for NADPH and NADH have been determined at pH 8.1 for spinach, yeast, and E . coli glutathione reductases . NADPH exhibited low Km values for all enzymes (3-6 microM), while the Km values for NADH were 100 times higher (approximately 400 microM) . Under our experimental conditions, the percentage of maximal velocities with NADH versus those measured with NADPH were 18.4, 3.7, and 0.13% for the spinach, yeast, and E . coli enzymes, respectively . Primary deuterium kinetic isotope effects were independent of GSSG concentration between Km and 15Km levels, supporting a ping-pong kinetic mechanism . For each of the three enzymes, NADPH yielded primary deuterium kinetic isotope effects on Vmax only, while NADH exhibited primary deuterium kinetic isotope effects on both V and V/K . The magnitude of DV/KNADH at pH 8.1 is 4.3 for the spinach enzyme, 2.7 for the yeast enzyme, and 1.6 for the E . coli glutathione reductase . The experimentally determined values of TV/KNADH of 7.4, 4.2, and 2.2 for the spinach, yeast, and E . coli glutathione reductases agree well with those calculated from the corresponding DV/KNADH using the Swain-Schaad expression . This suggests that the intrinsic primary kinetic isotope effect on NADH oxidation is fully expressed . In order to confirm this conclusion, single-turnover experiments have been performed . The measured primary deuterium kinetic isotope effects on the enzyme reduction half-reaction using NADH match those measured in the steady state for each of the three glutathione reductases.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Jun 19, 29(24), 5752 - 61 1H NMR resonance assignments, secondary structure, and global fold of Apo bovine calbindin D9k; Skelton NJ et al.; The solution structure and dynamics of apo bovine calbindin D9k have been studied by a wide range of two-dimensional 1H nuclear magnetic resonance experiments . Due to the presence of conformational heterogeneity in the wild-type protein, the sequential resonance assignment was carried out on a Pro43----Gly mutant . By use of a combination of scalar correlation experiments acquired from H2O solution, 61 of the 76 1H spin systems could be assigned to particular amino acid types . The remaining resonances were assigned by a parallel series of experiments acquired from 2H2O solution . These spin system assignments provided a basis for complete sequential resonance assignments from interresidue backbone nuclear Overhauser effects (NOEs) . Elements of secondary structure were identified from sequential and medium-range NOEs, backbone spin-spin coupling constants, and slowly exchanging amide protons . Four sections of helix are delineated, together with a short antiparallel beta-sheet interaction between the peptide loops involved in Ca2+ binding . The global fold is provided by combining these elements of secondary structure with a subset of the long-range, interhelix NOEs . Comparison with similar studies on the Ca2(+)-saturated protein indicates that at this crude level the structures are very similar . However, removal of the Ca2+ does dramatically affect the dynamics of the protein, as judged by amide proton exchange rates and aromatic ring rotation . This is particularly evident in the increased flexibility of the residues in the hydrophobic core. Biochemistry, 1990 Jun 19, 29(24), 5711 - 8 Substrate overlap and functional competition between human nucleotide excision repair and Escherichia coli photolyase and (a)BC excision nuclease; Sibghat-Ullah et al.; Human cell free extract prepared by the method of Manley et al . (1980) carries out repair synthesis on UV-irradiated DNA . Removal of pyrimidine dimers by photoreactivation with DNA photolyase reduces repair synthesis by about 50% . With excess enzyme in the reaction mixture photolyase reduced the repair signal by the same amount even in the absence of photoreactivating light, presumably by binding to pyrimidine dimers and interfering with the binding of human damage recognition protein . Similarly, the UvrB subunit of Escherichia coli (A)BC excinuclease when loaded onto UV-irradiated or psoralen-adducted DNA inhibited repair synthesis by cell-free extract by 75-80% . The opposite was true also as HeLa cell free extract specifically inhibited the photorepair of a thymine dimer by DNA photolyase and its removal by (A)BC excinuclease . Cell-free extracts from xeroderma pigmentosum (XP) complementation groups A and C were equally effective in blocking the E . coli repair proteins, while extracts from complementation groups D and E were ineffective in blocking the E . coli enzyme . These results suggest that XP-D and XP-E cells are defective in the damage recognition subunit(s) of human excision nuclease. Biochemistry, 1990 Jun 19, 29(24), 5706 - 11 Reconstitution of Escherichia coli photolyase with flavins and flavin analogues; Payne G et al.; Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF) . A procedure was developed for reversible resolution of apophotolyase and its chromophores . To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted . Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase . The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD . Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity. Biochemistry, 1990 Jun 19, 29(24), 5698 - 706 Active site of Escherichia coli DNA photolyase: mutations at Trp277 alter the selectivity of the enzyme without affecting the quantum yield of photorepair; Li YF et al.; Escherichia coli DNA photolyase repairs pyrimidine dimers by a photoinduced electron-transfer reaction . The enzyme binds to UV-damaged DNA independent of light (the dark reaction) and upon absorbing a 300-500-nm photon breaks the cyclobutane ring of the dimer (the light reaction) and thus restores the DNA . No structural information on the enzyme is available at present . However, comparison of the sequences of photolyases from five different organisms has identified highly conserved regions of homology . These regions are presumably involved in chromophore (flavin and folate) and substrate binding or catalysis . Trp277 (W277) in E . coli photolyase is conserved in all photolyases sequenced to date . We replaced this residue with Arg, Glu, Gln, His, and Phe by site-specific mutagenesis . Properties of the mutant proteins indicate that W277 is involved in binding to DNA but not in chromophore binding or catalysis . Of particular significance is the finding that compared to wild type W277R and W277E mutants have about 300- and 1000-fold lower affinity, respectively, for substrate but were indistinguishable from wild-type enzyme in their photochemical and photocatalytic properties. Biochemistry, 1990 Jun 19, 29(24), 5694 - 8 Excited-state properties of Escherichia coli DNA photolyase in the picosecond to millisecond time scale; Heelis PF et al.; Escherichia coli DNA photolyase contains a stable flavin radical that is readily photoreduced in the presence of added electron donors . Picosecond, nanosecond, and conventional flash photolysis technique have been employed to investigate the events leading to photoreduction from 40 ps to tens of milliseconds following flash excitation . Direct light absorption by the flavin radical produces the first excited doublet state which undergoes rapid (within 100 ps) intersystem crossing to yield the lowest excited quartet (n pi*) state . In contrast, light absorption by the folate chromophore produces a new intermediate state via interaction of the folate excited singlet state with the ground-state flavin radical, leading to an enhanced yield of the excited radical doublet state and hence quartet state . Subsequent reaction of the excited quartet state involves hydrogen atom abstraction from a tryptophan residue . Secondary electron transfer from added electron donors occurs to the oxidized tryptophan radical with rate constants ranging from 10(4) (dithiothreitol) to 4 x 10(6) M-1 s-1 (n-propyl gallate) . The low value of the latter rate compared to reduction of the tryptophan radical in lysozyme suggests that the reactive tryptophan is highly buried in photolyase . A redox potential diagram has been constructed for the ground and excited states involved . It is concluded that the one-electron reduction potential of the excited quartet state of the flavin radical must be at least 1.23 V more positive than the ground state, in agreement with the value of delta E greater than 1.77 V calculated from spectroscopic data. Biochemistry, 1990 Jun 19, 29(24), 5761 - 6 A site-directed mutagenesis study on Escherichia coli inorganic pyrophosphatase . Glutamic acid-98 and lysine-104 are important for structural integrity, whereas aspartic acids-97 and -102 are essential for catalytic activity; Lahti R et al.; Analysis of the conservation of functional residues between yeast and Escherichia coli inorganic pyrophosphatases (PPases) suggested that Asp-97, Glu-98, Asp-102, and Lys-104 are important for the action of E . coli PPase {Lahti, R., Kolakowski, L . F., Heinonen, J., Vihinen, M., Pohjanoksa, K., & Cooperman, B . S . (1990) Biochim . Biophys . Acta 1038, 338-345} . We replaced these four residues by oligonucleotide-directed mutagenesis, giving variant PPases DV97, DE97, EV98, DV102, DE102, KI104, and KR104 . PPase variants DV97, DV102, and KI104 had no enzyme activity, whereas PPase variants DE97, EV98, DE102, and KR104 had 22%, 33%, 3%, and 3% of the wild-type PPase activity, respectively . This suggests that Asp-97, Asp-102, and Lys-104 are essential for the catalytic activity of E . coli PPase . PPase variants DV98 and KR104 also had an increased sensitivity to heat denaturation; incubation of these mutant PPases at 75 degrees C for 15 min in the presence of 5 mM magnesium ion decreased the activity to 20% and 1%, respectively, of the initial value while 74% of the activity was observed with wild-type PPase . Furthermore, these thermolabile mutant PPases displayed the most profound conformational changes of the PPase variants examined, as demonstrated by the binding of the fluorescent dye Nile red that monitors the hydrophobicity of protein surfaces . Accordingly, Glu-98 and Lys-104 seem to be important for the structural integrity of E . coli PPase. Biochemistry, 1990 Jun 19, 29(24), 5665 - 71 Chaperonin-facilitated refolding of ribulosebisphosphate carboxylase and ATP hydrolysis by chaperonin 60 (groEL) are K+ dependent; Viitanen PV et al.; Both the chaperonin- and MgATP-dependent reconstitution of unfolded ribulosebisphosphate carboxylase (Rubisco) and the uncoupled ATPase activity of chaperonin 60 (groEL) require ionic potassium . The spontaneous, chaperonin-independent reconstitution of Rubisco, observed at 15 but not at 25 degrees C, requires no K+ and is actually inhibited by chaperonin 60, with which the unfolded or partly folded Rubisco forms a stable binary complex . The chaperonin-dependent reconstitution of Rubisco involves the formation of a complex between chaperonin 60 and chaperonin 10 (groES) . Formation of this complex almost completely inhibits the uncoupled ATPase activity of chaperonin 60 . Furthermore, although the formation of the chaperonin 60-chaperonin 10 complex requires the presence of MgATP, hydrolysis of ATP may not be required, since complex formation occurs in the absence of K+ . The interaction of chaperonin 60 with unfolded or partly folded Rubisco does not require MgATP, K+, or chaperonin 10 . However, discharge of the complex of chaperonin 60-Rubisco, which leads to the formation of active Rubisco dimers, requires chaperonin 10 and a coupled, K(+)-dependent hydrolysis of ATP . We propose that a role of chaperonin 10 is to couple the K(+)-dependent hydrolysis of ATP to the release of the folded monomers of the target protein from chaperonin 60. Biochim Biophys Acta, 1990 Jun 19, 1039(2), 197 - 203 A mutational analysis of the epitopes of recombinant human H-ferritin; Arosio P et al.; Murine monoclonal antibodies were elicited by the recombinant human H-ferritin overexpressed in Escherichia coli . They had a specificity analogous to that of the antibodies elicited by natural human H-chain, and all of them showed low additivity in binding the recombinant ferritin . Four antibodies of each group were challenged with four H-ferritin mutants overexpressed in E . coli, altered in different accessible areas of the molecule . They consisted of deletions of the first 13 and last 22 amino acids, a duplication of an 18 amino acid sequence in the loop region, and a substitution of a 5 amino acid stretch in the three-fold symmetry axis region . Double diffusion, immunodot analyses and inhibition plots indicated that: (1) all the mutants were recognized by at least one antibody; (2) the deletion of the N-terminus and the duplication in the loop region had the strongest effect on antibody binding; and (3) epitope boundaries of the various antibodies could not be recognized . The antibodies were tested with H-containing ferritins from rat and hen hearts, and showed low or absent reactivities despite their high structural homology with human ferritin . Comparison of the amino acid sequences of human, mouse, rat and hen H-chains, together with mutational data, suggested that; (i) ferritin epitopes are large, probably encompassing a large portion of the subunit surface and (ii) Thr-5 and Cys-90 have a role in H-ferritin immunogenicity. Biochim Biophys Acta, 1990 Jun 19, 1039(2), 142 - 8 Substrate specificity for myelin basic protein-specific protein methylase I; Ghosh SK et al.; The substrate specificity of bovine brain myelin basic protein (MBP)-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), which methylates arginine residues of protein, has been studied using various MBPs, several synthetic peptides and heterogeneous nuclear ribonucleoprotein complex protein (hnRNP) . (1) Among MBPs from different species of brain, the carp MBP was found to be the best substrate for MBP-specific protein methylase I . This high degree of methyl acceptability is most likely due to the fact that carp MBP is not in vivo methylated at the arginine residue (Deibler, G.E . and Martenson, R.E . (1973) J . Biol . Chem . 248, 2387-2391) and that the methylatable amino acid sequence is present in this protein . (2) In order to study the minimum chain length of MBP polypeptide which functions as the methyl acceptor, several synthetic polypeptides whose sequences are identical to the region surrounding the residue 107 of bovine MBP (the in vivo methylation site) were synthesized . It was found that the hexapeptide, Gly-Lys-Gly-Arg-Gly-Leu (corresponding to residues 104-109 of bovine MBP), was the shortest methyl accepting peptide, while the tetrapeptide, Gly-Arg-Gly-Leu (corresponding to residues 106-109) was inactive as a substrate . (3) hnRNP protein is known to contain methylarginine at residue 193 (Williams, K.R., Stone, K.L., LoPresti, M.B., Merrill, B.M . and Plank, S.R . (1985) Proc . Natl . Acad . Sci . USA 82, 5666-5670) which is post-translationally modified . Thus, the RNP protein overproduced in Escherichia coli and therefore did not contain methylarginine was examined for its methyl acceptability . It was found that neither MBP-specific nor histone-specific protein methylase I could methylate this methylarginine-less RNP protein . This suggests a possible existence of a distinct protein methylase I specific for this nuclear protein. FEBS Lett, 1990 Jun 18, 266(1-2), 87 - 90 A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties; Boyot P et al.; We previously reported the production of a fused snake neurotoxin composed of protein A and erabutoxin a in E . coli . The hybrid had much lower toxicity and affinity for the acetylcholine nicotinic receptor than natural erabutoxin . By treating the hybrid with cyanogen bromide we generated a toxin which was purified in a single step by RP-HPLC . This compound, produced in a good yield, recovered all properties of native erabutoxin a, implying that the lower toxic activities of the hybrid were due to the bulky protein A and not to an incorrect folding of the toxin . This work serves as a basis for future studies of toxin-receptor interactions using engineered toxin mutants. FEBS Lett, 1990 Jun 18, 266(1-2), 67 - 71 Expression of catalytically active radish 3-hydroxy-3-methylglutaryl coenzyme A reductase in Escherichia coli; Ferrer A et al.; Two fragments of a cDNA encoding radish 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were cloned into the vector pET-8c and expressed in Escherichia coli . The large fragment, encoding both the membrane and the cytosolic domains, was expressed at low level, essentially as an insoluble protein without enzymatic activity . In contrast, the fragment encoding only the cytosolic domain was expressed at a high level in a catalytically active form . The amount of soluble active enzyme in cell-free extracts of E . coli dramatically increased when the temperature during the induction was lowered from 37 degrees C to 22 degrees C. FEBS Lett, 1990 Jun 18, 266(1-2), 63 - 6 Purification, some properties and nucleotide sequence of 5-carboxymethyl-2-hydroxymuconate isomerase of Escherichia coli C; Roper DI et al.; As part of an investigation into the evolution of catabolic pathway enzymes a cloned gene encoding the Escherichia coli C 5-carboxymethyl-2-hydroxymuconate (CHM) isomerase, an enzyme of the homoprotocatechuate catabolic pathway, was used to produce large amounts of the protein . The isomerase was purified to homogeneity and some of its properties determined . The reaction occurred optimally at pH 7.6 and the specificity constant was 5.8 x 10(5) M-1.s-1 with CHM and 6.0 x 10(2) M-1.s-1 with 2-hydroxyhepta-2,4-diene-1,7-dioate, the substrate of a second isomerase in the pathway . The pure protein showed one type of subunit of Mr 14,000 whilst the molecular mass of the native enzyme was 30,000, suggesting that it was a dimer of identical subunits . The first 19 N-terminal amino acids were sequenced and the data used to confirm that the open reading frame of 378 bp, identified from the nucleotide sequence, encoded the CHM isomerase. Biochem J, 1990 Jun 15, 268(3), 547 - 51 Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+; Cieplak W Jr et al.; The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins . Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+ . Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity . In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule . The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants . Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+ . The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity. Gene, 1990 Jun 15, 90(2), 215 - 20 Microbody phosphoglycerate kinase of Trypanosoma brucei: expression and complementation in Escherichia coli; Alexander K et al.; In the primitive eukaryotic parasite, Trypanosoma brucei, most of the enzymes of glycolysis are located within microbody organelles called glycosomes . Proteins destined for the glycosome are synthesized on free ribosomes and post-translationally translocated into the organelle . The gene, gPGK, encoding the glycosomal isozyme of phosphoglycerate kinase (gPGK), was cloned adjacent to a T7 promoter and cotransformed with a plasmid encoding T7 RNA polymerase into Escherichia coli Pgk-cells . Functional complementation occurred, but only after the creation of a ribosome-binding site by mutagenesis . This represents the first example of complementation of an E . coli mutant with a gene encoding a microbody protein . Enzymatically active recombinant gPGK was purified to near homogeneity by ion exchange chromatography from highly expressing E . coli . The recombinant protein will aid in studies of glycosomal biogenesis. FEMS Microbiol Lett, 1990 Jun 15, 58(1), 19 - 22 Cloning of the type VII trimethoprim-resistant dihydrofolate reductase gene and identification of a specific DNA probe; Towner KJ et al.; A 1.3 kb HindIII fragment encoding the type VII trimethoprim-resistant dihydrofolate reductase gene was cloned into pBR322 . Unidirectional deletion of this cloned fragment with exonuclease III identified the start of the dihydrofolate reductase gene . An internal 300bp EcoRV fragment was identified which could be used as a specific non-radioactive DNA probe to distinguish bacteria carrying the type VII gene from those carrying genes encoding other known dihydrofolate reductase types. J Biol Chem, 1990 Jun 15, 265(17), 9850 - 6 The presence and distribution of reduced folates in Escherichia coli dihydrofolate reductase mutants; Hamm-Alvarez SF et al.; Escherichia coli DNA photolyase was overproduced and purified from each of two mutant E . coli strains lacking dihydrofolate reductase . The extent of over-production in the mutants was comparable to that seen in the wild type strain . Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain . Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives . These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E . coli. J Biol Chem, 1990 Jun 15, 265(17), 9638 - 44 Identification of valine 177 as a mutation altering specificity for transport of sugars by the Escherichia coli lactose carrier . Enhanced specificity for sucrose and maltose; King SC et al.; A mutant of the Escherichia coli lactose carrier has been selected (in an invertase-positive strain) based on its ability to grow on 6 mM sucrose in a manner dependent upon lactose carrier induction by isopropyl-1-thio-beta-D-galactopyranoside . The mutant was cloned, and DNA sequencing revealed a point mutation in lacY which changed alanine 177 to valine . The valine 177 mutation increased the transport rate for both {14C}sucrose and the maltose analog 4-nitrophenyl-alpha-maltoside . The potency for inhibition of beta-ONPG transport by several sugars containing the glucopyranosyl moiety (maltose, cellobiose, or palatinose) was increased significantly relative to the parental carrier . Similar experiments showed that the mutation did not affect the affinity for such commonly studied substrates as 4-nitrophenyl-alpha-D-galactopyranoside and beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside . These data indicate that gross structural alteration of the galactoside binding site cannot account for increased transport of sucrose and maltose by the valine 177 mutant . We conclude that effects of the valine 177 mutation are not limited strictly to changes in observed sugar affinity and that sugar-specific changes in turnover number may be an important determinant of the altered spectrum of sugar specificities exhibited by the Val-177 carrier . These phenomena may be related to the effect of this mutation on proton recognition (described in King, S.C., and Wilson, T.H . (1990) J . Biol . Chem . 265, 9645-9651). J Biol Chem, 1990 Jun 15, 265(17), 10164 - 71 On RecA protein-mediated homologous alignment of two DNA molecules . Three strands versus four strands; Lindsley JE et al.; The recA protein from Escherichia coli can homologously align two duplex DNA molecules; however, this interaction is much less efficient than the alignment of a single strand and a duplex . Three strand paranemic joints are readily detected . In contrast, duplex-duplex pairing is detected only when the incoming (second) duplex is negatively supercoiled, and even here the pairing is inefficient . The recA protein-promoted four strand exchange reaction is initiated in a three strand region, with efficiency increasing with the length of potential three strand pairing available for initiation . This indicates that a paranemic joint involving three DNA strands may be an important intermediate in all recA protein-mediated DNA strand exchange reactions and that the presence of three strands rather than four is a fundamental structural parameter of paranemic joints. J Biol Chem, 1990 Jun 15, 265(17), 10055 - 60 Characterization of the integration host factor binding site in the ilvPG1 promoter region of the ilvGMEDA operon of Escherichia coli; Winkelman JW et al.; The ilvGMEDA operon of Escherichia coli, which encodes four of the five enzyme activities required for the biosynthesis of isoleucine and valine, is preceded by tandem promoters ilvPG1 and ilvPG2 which are separated by 72 base pairs . While both of these promoters are transcriptionally active in vitro, only the operon proximal promoter, ilvPG2, is transcriptionally active in vivo, and upstream DNA sequences encoding the ilvPG1 promoter region enhance the in vivo transcriptional activity of the ilvPG2 promoter 60-fold . The binding of the integration host factor protein (IHF) to this upstream region (Tsui, P., and Freundlich, M . (1989) J . Mol . Biol . 203, 817-820) has been shown to repress transcription from the ilvPG1 promoter both in vivo and in vitro (Pereira, R . F., Ortuno, M . J., and Lawther, R . P . (1988) Nucleic Acids Res . 16, 5972-5989) . Furthermore, E . coli strains deficient for IHF are compromised for isoleucine and valine biosynthesis (Friden, P., Voelkel, K., Sternglantz, R., and Freundlich, M . (1984) J . Mol . Biol . 172, 573-579) . Therefore, in order to further understand this repressor/activator role of IHF, we have undertaken a detailed analysis of the interaction of IHF with the DNA sequences in the ilvPG1 promoter region . The results of hydroxyl radical footprinting, dimethyl sulfate protection, and ethylation interference experiments show that IHF binds to a target site that overlaps the ilvPG1 promoter region . The results of these experiments also demonstrate that IHF interacts primarily with the minor groove of the DNA helix and that the IHF target site in the ilvPG1 promoter region shares a high degree of DNA sequence identity with other high affinity IHF target sites involved in DNA replication and site-specific recombination. Gene, 1990 Jun 15, 90(2), 207 - 14 Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences; Tsuge T et al.; An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter . Transformants arose on a selective medium containing 100 micrograms Hy/ml . There were two types of transformants, forming large and small colonies on the selective medium . Transformation with one microgram of the vector produced an average of 4.5 large colonies and 600 small ones . In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays . To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A . alternata were used to construct four new vectors for homologous recombination system . Use of these vectors gave higher transformation efficiency than the original plasmid . The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25 . Transformation events in A . alternata with pDH25r1a occurred by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA. J Biol Chem, 1990 Jun 15, 265(17), 9676 - 81 Organization and function of a dioxin-responsive enhancer; Fisher JM et al.; The dioxin-responsive enhancer upstream of the CYP1A1 gene contains four copies of the recognition motif for the liganded Ah receptor . The results of deletion analyses, linker-scanning analyses, and the analysis of individual enhancer subdomains reveal that each copy of the motif contributes to the response of the enhancer to 2,3,7,8-tetrachlorodibenzo-p-dioxin . In the context of the dioxin-responsive enhancer, a GC box, representing the DNA binding site for Sp1 (or a related transcription factor), has no detectable intrinsic activity but enhances gene expression when linked to a recognition motif for the liganded Ah receptor, thereby producing a synergistic effect on enhancer function. J Biol Chem, 1990 Jun 15, 265(17), 9645 - 51 Characterization of Escherichia coli lactose carrier mutants that transport protons without a cosubstrate . Probes for the energy barrier to uncoupled transport; King SC et al.; The Escherichia coli lactose carrier is an energy-transducing H+/galactoside cotransport protein which strictly couples sugar and proton transport in 1:1 stoichiometry . Here we describe five lactose carrier mutants which catalyze "uncoupled" sugar-independent H+ transport . Symptoms similar to uncoupling by a proton ionophore have been observed in cells expressing these mutant carriers . The mutations occur at two separate loci, encoding substitutions either for alanine 177 (valine) or tyrosine 236 (histidine, asparagine, phenylalanine, or serine) . Compared to the parent, cells expressing the valine 177 carrier grew slowly on minimal media with glucose as carbon source . When washed cells were incubated in the absence of added sugars the mutant showed a reduced protonmotive force compared with the parent . Addition of either thiodigalactoside or alpha-p-nitrophenylgalactoside reduced the defect in protonmotive force . Sugar-independent H+ entry rate into cells expressing either the normal carrier or the Val-177 mutant were measured directly using the pH electrode . Following sudden acidification of the external medium (by either oxygen-pulse or acid-pulse) protons entered more rapidly into cells expressing the Val-177 carrier . This novel sugar-independent mode of H+ transport probably depends on an acquired capa