Biopolymers, 1990 May-Jun, 29(6-7), 1105 - 19 The equilibrium partition function and base pair binding probabilities for RNA secondary structure; McCaskill JS; A novel application of dynamic programming to the folding problem for RNA enables one to calculate the full equilibrium partition function for secondary structure and the probabilities of various substructures . In particular, both the partition function and the probabilities of all base pairs are computed by a recursive scheme of polynomial order N3 in the sequence length N . The temperature dependence of the partition function gives information about melting behavior for the secondary structure . The pair binding probabilities, the computation of which depends on the partition function, are visually summarized in a "box matrix" display and this provides a useful tool for examining the full ensemble of probable alternative equilibrium structures . The calculation of this ensemble representation allows a proper application and assessment of the predictive power of the secondary structure method, and yields important information on alternatives and intermediates in addition to local information about base pair opening and slippage . The results are illustrated for representative tRNA, 5S RNA, and self-replicating and self-splicing RNA molecules, and allow a direct comparison with enzymatic structure probes . The effect of changes in the thermodynamic parameters on the equilibrium ensemble provides a further sensitivity check to the predictions. DNA Cell Biol, 1990 May, 9(4), 279 - 86 Reliable transient promoter assay using fluorescein-di-beta-D-galactopyranoside substrate; Ikenaka K et al.; The promoter region of the mouse myelin proteolipid protein (PLP) gene was cloned into a promoter testing vector, pIP111 . The pIP111 vector is a promoterless derivative of pCH110 (SV40 early region promoter-lacZ) and contains the Escherichia coli lpp transcription terminator sequence at the 5' end of the cloning site . The newly constructed PLP-lacZ fusion plasmid (pWP) was transfected into PLP-nonproducing NIH-3T3 fibroblasts or PLP-producing C6 cells . When the measured beta-galactosidase activity in the pWP-transfected cells was normalized to the pCH110-transfected cells (an appropriate control if the SV40 early region promoter functions constitutively in various cell lines), the results suggested that the promoter region of the PLP gene contains the information necessary for initiation of transcription in a C6 cell-specific manner . However, the beta-galactosidase produced in viable cells was also detected by fluorescein-di-beta-D-galactopyranoside (FDG) treatment followed by image analysis using inverted fluorescent microscopy, which allowed the transfection efficiency to be calculated, and the beta-galactosidase activity obtained by the regular ONPG method was normalized with the value obtained . This procedure indicated that the promoter region of the PLP gene did not show C6-specific expression, because the SV40 early-region promoter was 10 times more active in NIH-3T3 cells than in C6 cells . Thus, the standard experiment gave misleading results . As our detection method is simple and can be used to analyze the promoter activity in a single cell, many applications should be possible.(ABSTRACT TRUNCATED AT 250 WORDS) J Gen Virol, 1990 May, 71 ( Pt 5), 1141 - 51 Pseudorabies virus glycoprotein gI: in vitro and in vivo analysis of immunorelevant epitopes; Fuchs W et al.; Overlapping fragments of the gene encoding glycoprotein gI of pseudorabies virus (PRV; herpesvirus suis 1) were expressed in bacteria . Using the fusion proteins and a panel of monoclonal antibodies (MAbs) against gI as well as swine sera we found that the N-terminal part of gI (residues 33 to approximately 100) contains a highly antigenic and immunogenic domain . Transfer of antibodies binding to this region as well as vaccination with fusion proteins containing the N terminus of gI are able to confer protection to mice against a lethal challenge of virus . The results show that gI, which is non-essential for virus replication in tissue culture, can induce neutralizing and protective antibodies . The potential suitability of fusion proteins encompassing N-terminal parts of gI as diagnostic tools is demonstrated. J Gen Virol, 1990 May, 71 ( Pt 5), 1057 - 63 Production, purification and biological properties of an Escherichia coli-derived recombinant porcine alpha interferon; Lefevre F et al.; Recombinant plasmids for intracellular synthesis of mature porcine interferon alpha 1 (IFN-alpha 1) in Escherichia coli were constructed . High amounts of antiviral activity were obtained {up to 4 x 10(5) international units (IU) per ml of bacterial culture} . Recombinant porcine IFN-alpha 1 (rIFN-alpha 1) was purified to homogeneity by monoclonal antibody immunoaffinity and was found to have the expected Mr (17.5K) and N-terminal sequence (except for the apparent lack of an N-terminal methionine) . Its specific antiviral activity was 5 x 10(7) to 10 x 10(7) IU/mg MDBK cells . In vitro biological properties of this purified rIFN-alpha 1 were compared to those of virus-induced porcine leukocyte interferon: the two interferons shared similar antigenic determinants and had the same ability to induce a cytocidal effect on primary cultures of pig kidney epithelial cells . However, rIFN-alpha 1 was at least six times more active in inducing an antiviral state on homologous porcine cells . These properties are discussed in the light of a possible in vivo use of the purified recombinant molecule. Mutat Res, 1990 May, 238(3), 305 - 11 Repair of intrinsic DNA lesions; Lindahl T; The repair of apurinic/apyrimidinic (AP) sites is described . The major pathway involves hydrolysis of the stable phosphodiester bond on the 5' side of the lesion by an AP endonuclease . The 5' terminal deoxyribose-phosphate residue is excised by a separate phosphodiesterase which does not appear to be an exonuclease . Repair replication of the single missing nucleotide residue by a DNA polymerase and ligation complete the excision-repair process . The possibility that minor DNA lesions may accumulate with time in long-lived cells is considered . Such lesions should be chemically stable and should not be recognized by DNA-repair enzymes. Appl Environ Microbiol, 1990 May, 56(5), 1229 - 34 Antigenic nature of the chloride-stimulated cellobiosidase and other cellulases of Fibrobacter succinogenes subsp . succinogenes S85 and related fresh isolates; Huang L et al.; Polyclonal and monoclonal antibodies to the Cl-stimulated cellobiosidase of Fibrobacter succinogenes subsp . succinogenes S85 reacted with numerous proteins of both higher and lower molecular weights from F . succinogenes subsp . succinogenes S85, but not with Escherichia coli proteins, and only one protein each from Butyrivibrio fibrisolvens and Ruminococcus albus . Different profiles were observed for Western blots (immunoblots) of peptide digests of both the purified enzyme from F . succinogenes and immunoreactive proteins of higher and lower molecular weights, demonstrating that they were different proteins . Therefore, F . succinogenes appeared to produce numerous proteins with one or more common antigenic determinants . However, with the exception of Cl-stimulated cellobiosidase, none were cellulases that have been characterized . An affinity-purified polyclonal antibody to Cl-stimulated cellobiosidase reacted with numerous proteins in cells of each of three fresh isolates of F . succinogenes subsp . succinogenes and one of F . succinogenes subsp . elongata when analyzed by Western blotting . Antibodies to periplasmic cellodextrinase, endoglucanase 2 (EG2), and EG3, when reacted in Western blots with the various cellulases, including Cl-stimulated cellobiosidase, revealed limited antigenic similarity among the different proteins and none with either B . fibrisolvens or R . albus proteins . The periplasmic cellodextrinase antibody reacted with an antigen with a size corresponding to cellodextrinase in each of the three F . succinogenes subsp . succinogenes isolates but not with any antigens from the F . succinogenes subsp . elongata isolate . The anti-EG2 antibody reacted with single antigens in each of the four isolates, while the anti-EG3 antibody reacted with only one of the four isolates.(ABSTRACT TRUNCATED AT 250 WORDS) J Clin Invest, 1990 May, 85(5), 1566 - 74 Fine epitope mapping of the human SS-B/La protein . Identification of a distinct autoepitope homologous to a viral gag polyprotein; Kohsaka H et al.; To analyze the autoepitopes on the SS-B/La protein, a cDNA covering the entire region coding the protein was isolated from a human cDNA library . The cDNA was subcloned into an expression plasmid vector, pEX, to express its protein product as a fusion protein with cro-beta-galactosidase in Escherichia coli . A recombinant pEX plasmid expressing three-fourths of the protein (amino acid 112-408) was also constructed . The antigenicities of these recombinant proteins were confirmed with a patient's serum . Their various deletion mutants were produced with exonuclease III treatment from the 3' ends of the cDNAs without changing the proper translational frame . Immunoblot analysis and enzyme-linked immunosorbent assay were used to evaluate the reactivities of the recombinant proteins with patients' sera to determine the autoepitopes . A narrow segment (amino acid 88-101) and the region where several epitopes were located (amino acid 283-338) on the SS-B/La protein were universally recognized by all the sera with anti-SS-B/La antibodies examined . An additional epitope region (amino acid 179-220) was recognized by some patients' sera . Computer analysis revealed that the most distinct autoepitope, amino acid 88-101, had a striking homology to a retroviral gag polyprotein . These findings indicate that exogenous or endogenous retroviruses may play a role in initiation of the anti-SS-B/La autoimmunity. J Bacteriol, 1990 May, 172(5), 2336 - 42 Identification of a second promoter for the metY-nusA-infB operon of Escherichia coli; Granston AE et al.; The metY-nusA-infB operon of Escherichia coli encodes functions involved in both transcription and translation . Previous studies have identified a single promoter, P0, that directs transcription of the entire operon . We have identified a second promoter, P-1, that also is positioned to transcribe the complete operon . P-1 is located 50 base pairs upstream of and oriented in the same direction as P0 . Sequences associated with P-1 have features suggestive of regulatory elements . P-1 differs from any previously described naturally occurring E . coli promoter by having -35 and -10 sequences that perfectly match the procaryotic promoter consensus hexamer sequences, although the spacing between the two elements is 1 base pair more than optimal . We demonstrate that P-1 is active in vivo. J Immunol, 1990 May 1, 144(9), 3367 - 74 Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis . Implication for a conformational autoepitope; Surh CD et al.; The E2 component (acetyltransferase) of the pyruvate dehydrogenase (PDH) complex is the major mitochondrial autoantigen recognized by autoantibodies in patients with primary biliary cirrhosis (PBC) . Previous work, using only a partial length rat liver cDNA clone of PDH-E2, demonstrated that the immunodominant epitope was localized to the lipoic acid binding site . Human PDH-E2, in contrast to rat PDH-E2, has two lipoic acid binding sites . By using a full length human cDNA for PDH-E2, and by preparation of multiple overlapping recombinant fragments, we have determined that three autoreactive determinants are present on human PDH-E2: two cross-reactive lipoyl domains, and an area surrounding the E1/E3 binding region . The dominant epitope was localized to the inner lipoyl domain whereas the outer lipoyl domain only showed a weak cross-reactivity, and only 1/26 PBC sera reacted weakly to the E1/E3 binding region area . By probing recombinant fusion proteins expressed from small restriction fragments of the inner lipoyl domain, we have found that a minimum of 75 amino acids (residues 146-221) were required for detectable autoantibody binding, and that 93 amino acids (residues 128-221) were necessary for characteristically strong antimitochondrial autoantibody recognition . Such a requirement for a large region suggests the possibility that a conformational autoepitope may be recognized . In addition, we have found that absorption of PBC sera with the purified mammalian PDH complex does not remove reactivity against Escherichia coli Ag . The possible implications for such results are discussed. Endocrinology, 1990 May, 126(5), 2377 - 82 Characterization of a novel prolactin-related protein from bovine fetal placenta; Zieler CG et al.; The bovine fetal placenta transcribes a family of PRL-related genes that are distinct from the bovine placental lactogen gene . To demonstrate that one of these cDNAs, bovine PRL-related cDNA-I (bPRCI), is expressed at the protein level, a recombinant form of the bPRCI product was overexpressed in Escherichia coli . An antiserum raised against this recombinant product did not cross-react with the pituitary members of the PRL-GH family or with bovine placental lactogen, although cross-reactivity within the bPRC subfamily cannot be ruled out . The antiserum detected a doublet with apparent mol wt of 34,000 and 35,000 in bovine fetal placenta, but not in other fetal tissues . Treatment with several glycosidases revealed a glycoprotein with asparagine-linked carbohydrates of the biantennary complex or hybrid type . We conclude that the bovine fetal placenta expresses at least one of the novel members of this gene family at the protein level. Cancer Res, 1990 May 1, 50(9), 2704 - 7 Identification of an epitope region of the human proliferation-associated nucleolar antigen P120; Valdez BC et al.; An epitope region, located at amino acid residues 173-180 (EAAA-GIQW), of a human cell proliferation-associated nucleolar antigen, P120, has been defined by mutational analysis and competition assays . A synthetic peptide corresponding to this epitope region completely blocks the binding of the anti-P120 antibody to Escherichia coli-expressed P120 and the HeLa nucleolar P120 protein . Adjacent peptides lack inhibitory effects . The antigenic site includes a hydrophilic residue and a hydrophobic stretch . The glutamyl and tryptophanyl residues in this region make major contributions to the binding of P120 to its antibody, since peptides lacking either the glutamyl or the tryptophanyl residue do not block the antibody binding to the P120 antigen . This study provides a basis for drug design for specific binding to the epitope region of the P120 protein. J Invest Dermatol, 1990 May, 94(5), 617 - 23 Production of rabbit antibodies against carboxy-terminal epitopes encoded by bullous pemphigoid cDNA; Tanaka T et al.; A partial cDNA clone (called BP cDNA) with coding sequences for the carboxy-terminal region of bullous pemphigoid (BP) antigen has been recently isolated and sequenced . In order to determine whether specific peptides encoded by the cDNA could be used to raise antibodies against BP antigen, fusion proteins derived from fragments of the BP cDNA and 17-mer or 19-mer synthetic peptides, corresponding to its deduced amino acid sequence, were used to generate rabbit antibodies . Three restriction enzyme fragments, 1179 bp (5' end), 264 bp (middle), and 546 bp (3' end), of the 1992 open reading frame (ORF) of BP cDNA were subcloned in frame into pEX plasmids to make beta-galactosidase fusion proteins FP1, FP2, and FP3, respectively . Fusion proteins of the predicted molecular weight, and which bound anti-beta-galactosidase antibodies, were produced, confirming the length of the predicted ORF . Rabbits immunized with FP1, but not FP3, produced antibodies, similar to authentic antibodies from BP patients, which: 1) bound the epidermal basement membrane at titers over 10,000, as determined by indirect immunofluorescence; 2) bound the basement membrane on the roof of 1 M NaCl-split skin; 3) immunoprecipitated the 230-kD BP antigen; and 4) bound the hemidesmosome, as determined by immunoelectron microscopy . Rabbits immunized with FP2 also produced lower titer BP-like antibodies . We further showed that short hydrophilic synthetic peptides, contained in FP1, could induce similar BP-like antibodies in rabbits at immunofluorescence titers up to 2560 . These rabbit antibodies should prove useful for further studies on the function and structure of particular epitopes of BP antigen as well as on the pathophysiology of disease. Infect Immun, 1990 May, 58(5), 1261 - 8 Characterization of the immunodeficiency of RIIIS/J mice: immune response to polysaccharide antigens; Hiernaux JR et al.; RIIIS/J mice lack an autosomal dominant gene(s) that influences the magnitude of the antibody response to several polysaccharide antigens of bacterial origin . Low responsiveness is demonstrable whether polysaccharide is administered as a T-helper-cell-independent or -dependent antigen conjugated to an immunogenic carrier; however, RIIIS/J mice make good anti-hapten antibody responses to haptenated polysaccharides . The low antibody responses of RIIIS/J mice to type III pneumococcal polysaccharide do not appear to be the results of an imbalance in the activity of regulatory T lymphocytes . Compared with other strains of mice, RIIIS/J mice elicit low antibody responses to lipopolysaccharide (LPS) . They do not develop a cyclic primary or secondary antibody response to Escherichia coli O113 LPS; the latter is not due to a lack of mitogenic response to E . coli O113 LPS . They also produce auto-anti-idiotypic antibody after being immunized with trinitrophenyl-Ficoll. Biotechnol Prog, 1990 May-Jun, 6(3), 188 - 92 Chemical and biosynthetic approaches to the production of novel polypeptide materials; McGrath KP et al.; Three approaches to the synthesis of the repetitive copolypeptide {(GlyAla)3-GlyProGlu}n (1) are described . Direct chemical synthesis of 1 via classical solution methods required 18 steps and afforded a polydisperse product with an average molecular weight of less than 10,000 . Two alternative genetic strategies were also explored . In the first, chemically synthesized DNA oligomers were self-ligated to produce a population of multimers, which were fitted with translational start and stop signals and inserted into an expression plasmid containing the lambda PL promoter and a synthetic ribosome binding site . Transformation of E . coli led to the isolation of a stable recombinant plasmid carrying an insert encoding 12 repeats of sequence 1 . Attempts to identify polypeptide 1 after induction of transformed cultures were unsuccessful . A second strategy, generating a tripartite derivative of sequence 1 carrying short N- and C-terminal extensions, afforded excellent yields of product . The relative merits of chemical and genetic approaches to repetitive polypeptide materials are discussed. Biochem Biophys Res Commun, 1990 Apr 30, 168(2), 809 - 17 Identification, sequence determination, and expression of the flavodoxin gene from Desulfovibrio salexigens; Helms LR et al.; Restriction fragments of genomic DNA from Desulfovibrio salexigens (ATCC 14822) containing the structural gene coding for the flavodoxin protein were identified using the entire coding region of the gene for the Desulfovibrio vulgaris (Hildenborough) flavodoxin as a probe (Krey, G.D., Vanin, E.F., and Swenson, R.P . (1988) J . Biol . Chem . 263, 15436-15443) . A 1.4-kb PstI-HindIII fragment was ultimately identified which contains an open reading frame coding for a polypeptide of 146 amino acid residues that was highly homologous to the D . vulgaris flavodoxin, sharing a sequence identity of 55% . When compared to the X-ray crystal structure of the D . vulgaris protein, the homologous regions were largely confined to those portions of the protein which are in the immediate vicinity of the flavin mononucleotide cofactor binding site . Tryptophan-60 and tyrosine-98, which reside on either side of the isoalloxazine ring of the cofactor, are conserved, as are the sequences of the polypeptide loop that interacts with the phosphate moiety of the flavin . Acidic residues forming the interface of model electron-transfer complexes with certain cytochrome c proteins are retained . The flavodoxin holoprotein is over-expressed in E . coli from the cloned gene using its endogenous promoter. Gene, 1990 Apr 30, 89(1), 29 - 35 Identification of a seventh operon on plasmid RK2 regulated by the korA gene product; Thomas CM et al.; Broad-host-range IncP plasmids possess a series of operons involved in plasmid maintenance, whose expression is coordinated by a series of regulators, most of which are encoded in a central regulatory operon . The nucleotide sequence of a new monocistronic operon located between coordinates 55.0 and 56.0 kb on the genome of the IncP alpha plasmids RK2 and RP4 is presented . The operon encodes a 34 kDa protein which has a net negative charge . Transcription of the operon, designated by us kfrA (korF-regulated), is repressed not only by the product of the previously described korA gene but also by the product of a gene which we have designated korF and which has not been described previously . The korF gene is encoded downstream from korB within the key korA/korB regulatory operon . We propose that K or F binds to a novel inverted repeat overlapping the promoter for the kfrA operon. Gene, 1990 Apr 30, 89(1), 117 - 22 Blunt-end and single-strand ligations by Escherichia coli ligase: influence on an in vitro amplification scheme; Barringer KJ et al.; A ligase-based, in vitro DNA amplification system (LAR) has been described by Wu and Wallace {Genomics 4 (1989) 560-569} . This strategy is based on the ability of a DNA ligase to join the 5' phosphate of one DNA molecule to the 3' hydroxyl of a second during a nick-closing reaction . Escherichia coli DNA ligase has been used in place of the T4 DNA ligase in our study in order to limit template-independent ligation activities, which lower the sensitivity of this amplification procedure . The results of this study indicate that E . coli ligase also joins blunt-ended DNA molecules and some single-stranded oligodeoxyribonucleotides, in the absence of a complementary template, with an efficiency which is sensitive to both the concentrations of DNA substrate and enzyme. Gene, 1990 Apr 30, 89(1), 1 - 6 Symmetric lac operator derivatives: effects of half-operator sequence and spacing on repressor affinity; Sasmor HM et al.; We have analyzed lac repressor binding in vivo and in vitro to several symmetric lac operator sequences . Two features of the operator appear to be important for repressor binding: sequence, both of the operator and of its extended regions, and the spacing of the operator halves . Host mutations that alter DNA superhelical density (topA, gyrB) did not change the relative affinity of cloned symmetric operator sequences for repressor . Analysis by dimethylsulfate methylation and DNaseI digestion of repressor-operator complexes indicated that repressor makes symmetric contacts with the symmetric operator, in contrast to its contacts with the two halves of the natural operator. Biochim Biophys Acta, 1990 Apr 30, 1023(3), 383 - 8 Partition of parinaroylphosphatidylethanolamines and parinaroylphosphatidylglycerols in immiscible phospholipid mixtures; Martin LR et al.; Partitioning of two parinaroyl phosphatidylethanolamines and two parinaroyl phosphatidylglycerols between solid and fluid phase phospholipids was examined . Fluorescence quantum yields and fluorescence polarization measurements were used to calculate Ks/fp, the solid to fluid partition coefficient of each probe (Sklar, L.A., Miljanich, G.P . and Dratz, E.A . (1979) Biochemistry 18, 1707-1716) . In the immiscible mixture dipalmitoylphosphatidylcholine and dilinoleylphosphatidylcholine, both 1-palmitoyl-2-trans-parinaroylphosphatidylethanolamine and 1-palmitoyl-2-transparinaroylphosphatidylglycerol partitioned preferentially into solid phase lipid with mean Ks/fp values (calculated from quantum yields) of 3.4 +/- 1.5 and 2.1 +/- 0.7, respectively . In contrast, 1-oleoyl-2-cis-parinaroylphosphatidylethanolamine and 1-oleoyl-2-cis-parinaroylphosphatidylglycerol partitioned preferentially into fluid phase lipid in the same model system with mean Ks/fp values (calculated from quantum yields) of 0.44 +/- 0.26 and 0.16 +/- 0.07, respectively . Fluorescence polarization data on the same four parinaroyl phospholipids in mixtures of solid-phase dimyristoylphosphatidyl ethanolamine and fluid-phase dilinoleoylphosphatidylglycerol were similar to those obtained in the immiscible phosphatidylcholine system, demonstrating that the partitioning of these probes is not strongly dependent on head group . Knowledge of the partition properties of these fluorescent probes is relevant to use of these probes in investigation of the phase behavior of Escherichia coli inner membrane lipids, since phosphatidylethanolamine and phosphatidylglycerol species account for approximately 95% of these lipids. Biochim Biophys Acta, 1990 Apr 30, 1023(3), 357 - 64 Giant liposomes as model membranes for immunological studies: spontaneous insertion of purified K1-antigen (poly-alpha-2,8-NeuAc) of Escherichia coli; Decher G et al.; A flow chamber has been constructed to use giant liposomes (diameter 5-50 microns) as model membranes for immunological studies and other experiments involving the interaction with water-soluble compounds . As an example of immunological importance, the insertion of purified K-antigen from Escherichia coli K1 has been studied . Despite its large hydrophilic part (poly-alpha-2,8-NeuAc), which is capped at its potential reducing end with phosphatidic acid acting as a lipid anchor group, this water-soluble material is readily incorporated into liposomal membranes of dimyristoylphosphatidylcholine (DMPC) . The incorporation has been proven by immunofluorescence using a FITC-labeled monoclonal anti-K1-IgG . Without the lipid residue, however, no binding of poly-alpha-2,8-NeuAc to the liposomes has been observed . This could be shown by using colominic acid, an oligomeric form of alpha-2,8-NeuAc with free reducing ends instead of purified K1-antigen . The possibility for further manipulation of this model system has been shown by using a poly-alpha-2,8-NeuAc cleaving enzyme (endoneuraminidase) . The function of the endoneuraminidase has been proven by showing no binding of the antibody after enzyme treatment of K1-bearing liposomes as well as by rapid loss of fluorescence of a previously bound FITC-antibody. Gene, 1990 Apr 30, 89(1), 13 - 8 Primary structure of the deoxyguanosine triphosphate triphosphohydrolase-encoding gene (dgt) of Escherichia coli; Quirk S et al.; The complete nucleotide sequence has been determined for a 2027-bp region that encompasses the structural gene (dgt) encoding deoxyguanosine triphosphate triphosphohydrolase (dGTPase) from Escherichia coli . The gene resides between the htrA and dapD loci at 3.75-3.8' on the bacterial chromosome . Using homologous recombination in a recD recipient, a dgt- bacterial strain was constructed that was deficient in producing functional dGTPase . Comparison of dGTP pools in this and other strains revealed that dGTPase synthesized in vivo does to some degree modulate the level of dGTP in the bacterial cell, yet the magnitude of this modulation may be insufficient to explain the physiological function of dGTPase. Eur J Biochem, 1990 Apr 30, 189(2), 277 - 85 Electron microscopy of DNA.helicase-I complexes in the act of strand separation; Wessel R et al.; Electron microscopy was used to characterize the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase I . Linear DNA with 5'-protruding strands as well as single-stranded gaps was incubated, under unwinding assay conditions, with the helicase . E . coli single-stranded-DNA-binding protein (SSB) was added to order the denatured DNA . Up to 70% of the sites of SSB-complexed DNA were observed as forks . The position of the strand-separating enzyme was indicated by a gap in the complex between fork and SSB on that arm which initially provided the binding site . The complex between DNA and helicase varied in length although in all cases it was long enough to comprise several helicase I molecules . A mutant helicase I (helicase I del29) which, unlike the wild-type enzyme, fails to show cooperative DNA-binding behaviour was found to prevent an abnormally short stretch of DNA near the fork from binding SSB . Apparently, one or very few helicase molecules would be sufficient for the opening of a DNA duplex although, typically, the fork is shifted by a tract of helicase I molecules . SSB displaces helicase I from single-stranded DNA but fails to do so from a fork or a single-strand/double-strand junction . The difference is consistent with the observation that SSB does not inhibit the unwinding reaction despite its rapid association with the separated strands . Helicase I unwinds in the 5'-3' direction of the bound strand . Observations so far indicate that the enzyme exploits the single strand at the initial DNA-binding site for orienting its action, and not the complementary, completely base-paired strand. Eur J Biochem, 1990 Apr 30, 189(2), 267 - 76 Escherichia coli DNA helicase I . Characterization of the protein and of its DNA-binding properties; Benz I et al.; Gene traI of the Escherichia coli F sex factor which encodes DNA helicase I was subcloned in a lambda pL-based plasmid vector and expressed in a background of pL non-repressing cells . Neither the non-repressed pL promoter nor the production of a high level of functional helicase I are toxic . Enzyme purified from this source was studied in the electron microscope . The results show that helicase I binds cooperatively to single-stranded DNA . DNA covered with the helicase appears in fixed, negatively stained specimens as a smooth-contoured filament with a diameter of 12.5 +/- 0.4 nm and an axial periodicity of 7.0 +/- 0.2 nm . In unfixed specimens, discrete particles with axes of 12.7 +/- 0.5 nm and 7.2 +/- 0.5 nm are visible . They are consistent in size with helicase I monomers (Mr 180,000) suggesting that the molecule is almost isometric, despite a frictional ratio of 1.71 calculated from its diffusion coefficient . Helicase I free of DNA appears as aggregates . For comparison, a truncated traI, lacking coding for the amino-terminus of the product, was cloned by fusing it to an MS2 replicase gene fragment . The chimeric gene product (named helicase I del29) retains strand-separating activity although it fails to show cooperative DNA binding behavior . Judged from the length of the helicase-I-specific sequence of this polypeptide, traI is located 1.3 kb nearer to the distal end of the F transfer operon compared to the position proposed in a previous genetic map . The revised location of traI has implications for understanding distal functions of the transfer operon. Gene, 1990 Apr 30, 89(1), 19 - 27 Probing the function of individual amino acid residues in the DNA binding site of the EcoRI restriction endonuclease by analysing the toxicity of genetically engineered mutants; Oelgeschlager T et al.; We have developed an assay that allows analysis of the activity of EcoRI restriction endonuclease (ENase) and its mutants in vivo . This assay is based on the fact that wild type (wt) EcoRI ENase is toxic for Escherichia coli cells not expressing the EcoRI methyltransferase (MTase) . The viability factor defined by the ratio of the viable counts of E . coli cultures having or not having expressed the ecoRIR gene for a defined time is 10(-6) for wt EcoRI ENase and close to one for a totally inactive EcoRI ENase mutant . While the EcoRI MTase (M.EcoRI) provides substantial protection against the toxic effects of the wt EcoRI ENase and several of the mutants, some mutants become more toxic in the presence of M.EcoRI . Twenty-four different DNA-binding-site mutants of EcoRI ENase were characterized in their activity in vivo with this assay . The results obtained allow us to conclude that the structural integrity of the region at and around aa 200 seems to be very critical for the enzymatic function of EcoRI ENase: nonconservative replacements there lead to viability factors of 1-10(-2) . While our results indicate that the region around aa 144 and 145 is also involved in the EcoRI ENase-catalyzed reaction, it is also evident that the effects of mutation there are not as large: viability factors of approx . 10(-3) are obtained even for drastic replacements . These results are discussed in the light of the x-ray structure analysis of an EcoRI ENase-DNA recognition complex. Gene, 1990 Apr 30, 89(1), 109 - 15 The upstream region of the IPNS gene determines expression during secondary metabolism in Aspergillus nidulans; Gomez-Pardo E et al.; We have constructed a translational fusion between the isopenicillin-N-synthetase-encoding gene (IPNS) of Aspergillus nidulans and the lacZ gene of Escherichia coli . Recombinant strains carrying a single copy of the fusion integrated at the IPNS locus produced beta-galactosidase (beta Gal) during secondary metabolism . Integration of the fusion at the argB locus results in a situation in which the only 5'-flanking sequences of the IPNS gene upstream from the chimeric fused gene are those included in the transforming plasmid . Such a strain still expresses beta Gal activity during secondary metabolism, showing that a DNA fragment including sequences of the IPNS gene from nt -2000 to +35 (relative to the translation start codon) still contains sufficient information to drive expression of the fusion gene during secondary metabolism. Biochem Biophys Res Commun, 1990 Apr 30, 168(2), 771 - 7 Human acid beta-glucosidase: glycosylation is required for catalytic activity; Grace ME et al.; The role of oligosaccharide modification in human acid beta-glucosidase function was investigated . This lysosomal enzyme has five putative N-glycosylation sites, four of which are occupied . The unglycosylated human protein was stable when expressed in bacteria or in Spodoptera frugiperda cells in the presence of tunicamycin but lacked catalytic activity . Deglycosylation of purified acid beta-glucosidase from human placenta with N-Glycanase under native conditions resulted in the removal of an accessible oligosaccharide chain from a single site with no effect on activity, whereas complete deglycosylation resulted in proportionate loss of activity . These studies demonstrate that occupancy of at least one glycosylation site is required for the formation and maintenance of acid beta-glucosidase in an active conformation. Eur J Biochem, 1990 Apr 30, 189(2), 409 - 13 A 136-amino-acid-residue COOH-terminal fragment of colicin A is endowed with ionophoric activity; Baty D et al.; DNA regions encoding the various domains of a protein can be expressed as separate entities by inserting at appropriate sites a 'STOP-Shine-Dalgarno-sequence-ATG' cassette encoding a termination codon, a Shine-Dalgano sequence and an initiation codon within the structural gene . This technique has been used to obtain a 137-amino-acid-residue pore-forming protein designated DA70C comprising the final 136-amino-acid-residue COOH-terminal of colicin A preceded by an NH2-terminal methionine . Da70C was correctly expressed but poorly released to the extracellular medium . Its purification involved, as a final step, a partition in Triton X-114 thus demonstrating that hydrophobic regions are exposed in this protein . The ability of DA70C to form ion channels in planar lipid bilayers was investigated and pore properties were analyzed . The results indicate that helices 1-3 of the 204-amino-acid-residue colicin pore-forming domain (containing 10 alpha-helices) are not involved in ion conduction through the channel . However, they are important in maintaining the stability of the soluble state of the COOH-terminal domain. Science, 1990 Apr 27, 248(4954), 480 - 3 RNA polymerase II transcription blocked by Escherichia coli lac repressor; Deuschle U et al.; A reversible block to RNA polymerase II transcriptional elongation has been created with a lac operator sequence in the intron of the SV40 large T-antigen gene . When this transcription unit is injected into rabbit kidney cells expressing Escherichia coli lac repressor, T-antigen expression is reduced . This effect is not observed in cells lacking repressor or in the absence of the operator, and it is reversed by an inducer of the lac operon, namely isopropyl thiogalactoside (IPTG) . In an extract of HeLa nuclei supplemented with lac repressor, this and similar constructs give rise to shortened transcripts that map to the 5' boundary of the repressor-operator complex . These shorter RNAs are also sensitive to IPTG induction . This model system shows that a protein-DNA complex can block the passage of RNA polymerase II, and offers some insight into the control of eukaryotic gene expression during transcription elongation, a phenomenon observed in a variety of systems. Nature, 1990 Apr 26, 344(6269), 879 - 82 Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo- and a Cys-His domain; Karlsson O et al.; The activity of the rat insulin I gene enhancer is mainly dependent on two cis-acting protein-binding domains . Here we report the isolation of a complementary DNA encoding a protein, Isl-1, that binds to one of these domains . Isl-1 contains a homeodomain with greatest similarity to those of the Caenorhabditis elegans proteins encoded by mec-3 and lin-11 . In addition, Isl-1, like the lin-11 and mec-3 gene products, contains a novel Cys-His domain which is reminiscent of known metal-binding regions . Together these proteins define a novel class of proteins containing both a homeo- and a Cys His-domain . Isl-1 is preferentially expressed in cells of pancreatic endocrine origin . If the structural homologies between Isl-1 and the C . elegans gene products reflect functional similarities, a role for Isl-1 in the development of pancreatic endocrine cells could be envisaged. Cancer, 1990 Apr 15, 65(8), 1748 - 52 The effect of endotoxin on 1,2-dimethylhydrazine-induced colonic tumors in rats; Chen MF et al.; The effect of endotoxin on colon tumors was studied in male Sprague-Dawley rats . Colon tumors were induced in weanling rats by the administration of 20 weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH) . When colon tumors were detected by colonoscopy in 80% of the rats around week 24 after DMH injection, the animals were divided randomly into two groups . One group served as the control . The other group received six endotoxin (Escherichia coli) treatments every fifth day . The first dose was 50 micrograms/100 g (intraperitoneally); the remaining doses were 100 micrograms/100 g (subcutaneously) . Rats were killed 2 weeks after the last endotoxin injection . Endotoxin treatments resulted in larger colon tumors . The median tumor size was 71 mm2 for endotoxin-treated and 31 mm2 for untreated rats (P less than 0.02) . Endotoxin treatments also resulted in a significantly higher incidence (P less than 0.05) of ulcer development in the small intestine, that is 47% in the endotoxin-treated versus 23% in the untreated rats . After a single subcutaneous injection of endotoxin (100 micrograms/100 g), the colon mucosal reduced glutathione (GSH) level was raised by 21% at 16 hours, reached a peak on day 2, then decreased to baseline by day 4 . The increased GSH level in the colon mucosa was maintained up to the third endotoxin injection . By the fifth injection, no increase in the GSH level was observed . These results suggest that the growth of colon tumors in rats induced by DMH could be enhanced by endotoxin treatments . The enhanced tumor growth may be due to an increase in the colon GSH level and/or other mediators released by macrophages as a result of endotoxin treatments. Nucleic Acids Res, 1990 Apr 25, 18(8), 2087 - 92 Construction of a FRS1-FRS2 operon encoding the structural genes for the alpha and beta subunits of yeast phenylalanyl-tRNA synthetase and its use in deletion analysis; Sanni A et al.; FRS1 and FRS2, the structural genes encoding the large (alpha) and small (beta) subunits of yeast phenylalanyl-tRNA synthetase (PheRS) were placed under the control of the lacZ promoter by creating an artificial operon . The FRS2 gene was fused next to the promoter, followed by a 14 base pair intergenic sequence containing a translation reinitiation site in front of the FRS1 coding sequences . The engineered PheRS has 16 N-terminal amino acids from beta-galactosidase fused to the beta subunit . However, the purified protein shows a Km value for tRNA(Phe) that is indistinguishable from that of the the native enzyme . The product of the FRS2-FRS1 operon is not able to complement thermosensitive E . coli PheRS, indicating the lack of heterologous aminoacylation in vivo . We made a deletion in the FRS2 gene that removed about 150 amino terminal residues of the beta subunit . The truncated protein showed intact ATP-PPi exchange, whereas tRNA aminoacylation was lost . This result is similar to that of limited proteolysis performed on the native enzyme that yielded a tetrameric alpha 2 beta'2 structure, able to form aminoacyladenylate but unable to bind tRNA(Phe) . A deletion of 50 amino acids from the carboxyl terminus of the beta chain resulted in the loss of both enzyme activities; this suggests the participation of the C-terminal end of the beta subunit in the active site or in subunit assembly to yield a tetrameric functional enzyme. Nucleic Acids Res, 1990 Apr 25, 18(8), 2007 - 10 Excision of cytosine hydrates from Z-DNA; Duker NJ et al.; Ultraviolet irradiation of DNA produces cytosine hydrate, released as a free base by E . coli endonuclease III . Cytosine hydrate excision was investigated by assaying photoproduct release from cytosine-radiolabeled, irradiated poly(dG-dC):poly(dG-dC) . Conformational shifts between B-DNA and Z-DNA were affected by heating the polymer in either nickel chloride or cobaltous chloride, and were determined by circular dichroism . Rates of enzymic cytosine hydrate release did not differ between the different substrate conformations . Irradiation of left-handed poly(dG-dC):poly(dG-dC) resulted in cytosine hydrate formation . Therefore, neither formation nor enzymic excision of ultraviolet-induced cytosine hydrates are substantially affected by these DNA conformational states. J Biol Chem, 1990 Apr 25, 265(12), 6626 - 32 L-lactate 2-monooxygenase from Mycobacterium smegmatis . Cloning, nucleotide sequence, and primary structure homology within an enzyme family; Giegel DA et al.; L-Lactate 2-monooxygenase catalyzes the oxidation of L-lactate to acetate and carbon dioxide . The catalytic mechanism has been extensively investigated but very little is known about which amino acid residues may play a role in catalysis . As a first step toward this goal, the gene for this protein from Mycobacterium smegmatis has been cloned and sequenced . Peptide sequencing data for L-lactate 2-monooxygenase was used to construct three sets of fully redundant tetradecamer oligonucleotide probes, which were hybridized to restriction-digested M . smegmatis DNA . An approximately 3-kilobase pair PstI fragment hybridized with two of the probes . This region was subsequently isolated and cloned into Escherichia coli . From this size-fractionated gene bank, a 3.1-kilobase pair genomic DNA fragment was isolated by colony hybridization to two of the oligonucleotide probes . The complete gene for L-lactate 2-monooxygenase was contained on this fragment as shown by DNA sequencing of the whole insert . The DNA sequence codes for a mature protein that is 393 amino acids in length with a subunit molecular weight of 43,072 (including the FMN) . The protein sequence shows impressive homology with the primary structures of two mechanistically related proteins, yeast flavocytochrome b2 (Lederer, F., Cortial, S., Becam, A.-M., Haumont, P.-Y., and Perez, L . (1985) Eur . J . Biochem . 152, 419-428; Guiard, B . (1985) EMBO J . 4, 3265-3272) and spinach glycolate oxidase (Volkita, M., and Somerville, C . R . (1987) J . Biol . Chem . 262, 15825-15828; Cederlund, E., Lindqvist, Y., Soderlund, G., Branden, C.-I., and Jornvall, H . (1988) Eur . J . Biochem . 173, 523-530) . For each residue proposed from the crystal structure of glycolate oxidase to be involved in catalysis (Lindqvist, Y., and Branden, C.-I . (1989) J . Biol . Chem . 264, 3624-3628), an identical residue was found in a homologous position in lactate oxidase . Furthermore, most of these residues occur in regions whose sequences are highly conserved between lactate oxidase, flavocytochrome b2, and glycolate oxidase. Carbohydr Res, 1990 Apr 25, 200, 457 - 68 The structure of Escherichia coli K26 antigen; Beynon LM et al.; The structure of the capsular antigen of E . coli K26 has been found by a combination of chemical and spectroscopic techniques to be of the "5 + 1" type shown . An important step was the simultaneous separation and identification of a mixture of neutral and acidic oligosaccharides by g.l.c.-c.i.-m.s . {formula: see text} Carbohydr Res, 1990 Apr 25, 200, 449 - 56 A structural investigation of the capsular polysaccharide of Escherichia coli O9:K57:H32; Parolis H et al.; The primary structure of the acidic capsular polysaccharide of Escherichia coli K57 was elucidated by methylation analysis and 1D- and 2D-n.m.r . spectroscopy . The repeating unit was identified as a linear tetrasaccharide having the structure shown . ----2)-beta-D-Ribf-(1----4)-beta-D-Galp-(1----3)-alpha-D-GlcpNAc-( 1----4)-alpha - D-GalpA-(1----. Nucleic Acids Res, 1990 Apr 25, 18(8), 2153 - 7 Mutation frequency and spectrum resulting from a single abasic site in a single-stranded vector; Lawrence CW et al.; We have investigated the mutagenic properties of an abasic site in DNA by transfecting SOS-induced and uninduced cells of E . coli with a single-stranded M13mp7-based vector that carries a single example of this lesion at one or other of two unique and adjacent sites . Random samples of progeny phage were sequenced to determine the nature of the replication events that occurred at and around these locations . 5% to 7% of the vectors could be replicated in SOS-induced cells, but only 0.1% to 0.7% of them gave plaques in the absence of SOS induction . In SOS-induced cells, 93% and 96% of the phage replicated resulted from the insertion of a nucleotide opposite the abasic site, while the remainder resulted from a targeted omission of a single nucleotide . At one of the sites, nucleotide insertions were 54% dAMP, 25% dTMP, 20% dGMP and 1% dCMP . At the other site they were 80% dAMP, 4% dTMP, 15% dGMP and 1% dCMP . The sequence variation in all but two of the 204 sequences analyzed was restricted to the abasic site itself . In the remaining two, a change at the abasic site was accompanied by a mutation at an immediately flanking nucleotide. Nucleic Acids Res, 1990 Apr 25, 18(8), 2037 - 44 Identification of trans-dominant HIV-1 rev protein mutants by direct transfer of bacterially produced proteins into human cells; Mermer B et al.; A synthetic rev gene containing substitutions which introduced unique restriction sites but did not alter the deduced amino acid sequence was used as a vehicle to construct mutations in rev . Insertion or substitution mutations within a domain of Rev resulted in proteins able to inhibit the function of Rev protein in trans . Rev function was monitored in a cell line, HLfB, which contained a rev- mutant provirus . HLfB cells require the presence of rev for virus production, which was conveniently monitored by immunoblot detection of p24gag . Trans-dominant mutants were identified after expression in bacteria and delivery into HLfB cells by protoplast fusion . In addition, the trans-dominant phenotype was verified by expression of the mutant proteins in HLfB cells after cotransfection . These studies define a region between amino acid residues 81 and 88 of rev, in which different mutations result in proteins capable of inhibiting Rev function. J Biol Chem, 1990 Apr 25, 265(12), 6916 - 20 The preference for a 3' homologous end is intrinsic to RecA-promoted strand exchange; Konforti BB et al.; The recA protein (RecA) promotes DNA pairing and strand exchange optimally in the presence of single-stranded binding protein (SSB) . Under these conditions, 3' homologous ends are essential for stable joint molecule formation between linear single-stranded DNA (ssDNA) and supercoiled DNA (i.e . 3' ends are 50-60 times more reactive than 5' ends) . Linear ssDNAs with homology at the 5' end do not participate in pairing . In the absence of SSB, the strand exchange reaction is less efficient; however, linear ssDNAs with 3' end homology are still 5- to 10-fold more reactive than those with 5' end homology . The preference for a 3' homologous end in the absence of SSB suggests that this is an intrinsic property of RecA-promoted strand exchange . The preferential reactivity of 3' homologous ends is likely to be a consequence of the polarity of polymerization of RecA on ssDNA . Specifically, since RecA polymerizes in the 5'----3' direction, 3' ends are more likely to be coated with RecA and, hence, will be more reactive than 5' ends. J Biol Chem, 1990 Apr 25, 265(12), 6770 - 5 Site-directed mutagenesis of arginine 179 of thymidylate synthase . A nonessential substrate-binding residue; Santi DV et al.; X-ray structural studies have shown that Arg-179 of thymidylate synthase is complexed to bound inorganic phosphate or to the 5'-phosphate of the bound substrate dUMP . The importance of Arg-179 to the structure/function of thymidylate synthase is also indicated by its complete conservation among the 17 thymidylate synthases thus far sequenced . In the present work, Arg-179 has been replaced by Thr, Ala, Lys, and Glu using site-directed mutagenesis with a mixture of four synthetic oligonucleotides as primers . The mutant proteins complement thymidylate synthase-deficient Escherichia coli and show high enzyme activity . Each of these mutants has been purified to homogeneity, partially sequenced to verify the mutation, and has had its steady state kinetic parameters determined . The most significant effect of all mutations is localized to a decrease in the net rate of association of thymidylate synthase with dUMP; the Lys mutant also shows an apparent increase in the dissociation constant of the folate cofactor of the reaction . The high activity in the mutant enzymes is explained by "plasticity" of the enzyme and compensatory actions of the other Arg residues . Why the Arg-179 residue has been conserved during evolution remains an open question. J Biol Chem, 1990 Apr 25, 265(12), 6700 - 4 Mechanism of inactivation of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase by o-phthalaldehyde; Huynh QK; In order to identify the essential reactive amino acid residues of 5-enolpyruvoylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosphate (N-phosphonomethylglycine), chemical modification studies with o-phthalaldehyde were undertaken . Incubation of the enzyme with the reagent resulted in a time-dependent loss of enzyme activity . The inactivation followed first-order and saturation kinetics with a Kinact of 25 microM and a maximum rate constant of 0.34 min-1 . The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvoylshikimate 3-phosphate, or by a combination of shikimate 3-phosphate plus glyphosate, but not by phosphoenolpyruvate or glyphosate alone . Absorbance and fluorescence spectra studies indicate that complete inactivation of the enzyme resulted from the formation of two isoindole derivatives per molecule of enzyme . Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate resulted in the isolation of two peptides which were not found for the enzyme modified in the presence of shikimate 3-phosphate and glyphosate . Analyses of these two peptides indicate that Lys-22 and Lys-340 were the modified sites . The amino acid sequences around these residues are conserved in bacterial, fungal, as well as plant enzymes, suggesting that these regions may constitute part of the enzyme active site. J Biol Chem, 1990 Apr 25, 265(12), 6664 - 8 Pyridoxal 5'-phosphate-dependent histidine decarboxylase . Mechanism of inactivation by alpha-fluoromethylhistidine; Bhattacharjee MK et al.; Mechanism-based inactivation of pyridoxal phosphate-dependent histidine decarboxylase by (S)-alpha-(fluoromethyl)histidine was studied . The molar ratio of inactivator to enzyme subunit required for complete inactivation increased from 1.63 at 10 degrees C to 3.00 at 37 degrees C . Two inactivation products were isolated by chromatographic fractionation of the reaction mixture and identified by NMR spectroscopy as 1-(4-imidazolyl)-3(5'-P-pyridoxylidene) acetone (I), the adduct formed between pyridoxal phosphate and inactivator, and 1-(4-imidazolyl) acetone (II), an intermediate compound formed during inactivation . Formation of these two products supports a previously proposed mechanism of inactivation (Hayashi, H., Tanase, S., and Snell, E . E . (1986) J . Biol . Chem . 261, 11003-11009), with minor modifications . A precursor of I was linked covalently to the enzyme by NaBH4 reduction if the reaction was carried out immediately after inactivation, before development of the 403 nm peak of I . A mutant histidine decarboxylase (S322A) in which Ser-322 was changed to Ala was also inactivated by alpha-fluoromethylhistidine demonstrating that Ser-322 is not essential for inactivation even though it is close to the active site and is derivatized by borohydride reduction of the inactivated wild-type enzyme . Following inactivation, both the wild-type and the S322A mutant enzyme could be partially reactivated by prolonged dialysis against buffer. J Biol Chem, 1990 Apr 25, 265(12), 6624 - 5 Escherichia coli mutant trpA34 has an Asp----Asn change at active site residue 60 of the tryptophan synthetase alpha chain; Shirvanee L et al.; Asp-60 is believed to be a catalytically essential residue of the tryptophan synthetase alpha chain of Escherichia coli (Nagata, S., Hyde, C.C., and Miles, E.W . (1989) J . Biol . Chem . 264, 6288-6296) . Surprisingly, mutations altering Asp-60 were not observed in the many trpA missense mutants characterized in the 1960s . However, there was one genetic class of trpA missense mutants, represented by trpA34, for which protein structure analyses failed to detect an amino acid substitution . DNA sequence analyses have now shown that the trpA34 mutation was in codon 60 and that it resulted in replacement of Asp-60 by Asn . This finding provides additional support for the conclusion that the tryptophan synthetase alpha chain contains only a small number of absolutely essential residues. J Biol Chem, 1990 Apr 25, 265(12), 6548 - 51 The high Km glucose transporter of islets of Langerhans is functionally similar to the low affinity transporter of liver and has an identical primary sequence; Johnson JH et al.; The liver has been shown to contain a facilitated diffusion glucose transporter with high Km for glucose that is structurally distinct from the low Km glucose transporters found in most other tissues . We find that 3-O-methyl glucose is greater than 90% equilibrated across dispersed islet cells within 60 s, consistent with a facilitated diffusion transport mechanism . L-Glucose uptake was minimal throughout the time course, indicating stereospecificity . Measurement of glucose transport over a range of 3-O-methyl glucose concentrations from 0.05 to 60 mM revealed the presence of a component of glucose transport with an apparent Km of 17 mM, a value essentially identical to that previously reported for liver . Interestingly, a second component of glucose transport was also observed with an apparent Km of 1.4 mM, as has been reported for other tissues such as erythrocytes that are known to contain the "HepG2" or "erythroid/brain" type glucose transporter . Further evidence for the existence of two transport components is provided by the observation that a low concentration of cytochalasin B (0.4 microM) completely inhibits the low Km transport activity but has no effect on the high Km transporter . The kinetic similarity of high Km glucose transport in liver and islets is readily understood in light of our structural analysis . Sequence analysis of cDNA clones indicates that the liver and islet glucose transporters have identical sequences and, thus, are the products of the same gene. J Biol Chem, 1990 Apr 25, 265(12), 6633 - 7 Calcium- and magnesium-binding properties of oncomodulin . Direct binding studies and microcalorimetry; Cox JA et al.; Ca2+ binding to the wild type recombinant oncomodulin was studied by equilibrium flow dialysis in the absence and presence of 1, 2, and 10 mM Mg2+ . Direct Mg2(+)-binding experiments were carried out by the Hummel-Dryer gel filtration technique . These studies revealed that in the absence of Mg2+ oncomodulin binds two Ca2+ with KCa = 2.2 x 10(7) and 1.7 x 10(6) M-1, respectively . In the absence of Ca2+ the protein binds only one Mg2+ with KMg = 4.0 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding at the high affinity site according to the rule of direct competition . Ca2+ binding to the low affinity site is only slightly affected by Mg2+, so that in the presence of 2-3 mM Mg2+ the two sites have apparently an equal affinity for Ca2+ . Microcalorimetry showed that, in spite of the different affinities of the two Ca2(+)-binding sites, delta H0 for the binding of each Ca2+ is identical and exothermic for -18.9 kJ/site . It follows that the entropy gain upon binding of Ca2+ is +77.1 J K-1 site-1 for the high affinity Ca2(+)-Mg2+ site and +56.0 J K-1 site-1 for the low affinity Ca2(+)-specific site . Mg2+ binding is endothermic for +13 kJ/site with an entropy change of +111 J K-1 site-1 . The thermodynamic characteristics of the Ca2(+)-Mg2+ site resemble most those of site II (the so-called EF domain) of toad alpha-parvalbumin . The characteristics of Ca2+ binding to the specific site (likely the CD domain) are different from those of the Ca2+ specific sites in troponin C and in calmodulin and suggest that in oncomodulin hydrophobic forces do not play a predominant role in the binding process at the specific site. J Biol Chem, 1990 Apr 25, 265(12), 6984 - 91 Alteration of the pH-dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis; Jakes KS et al.; Colicin E1 is a soluble, bacteriocidal protein that forms voltage-gated channels in planar lipid bilayers . The channel-forming region of the 522-amino acid protein is near the COOH terminus, and contains a 35-amino acid hydrophobic segment which is presumed to be important in interacting with the membrane . We have used site-directed mutagenesis in the region immediately upstream from the hydrophobic segment to construct several functional colicin mutants in which a wild-type residue was replaced with a cysteine . We also replaced the only naturally occurring cysteine in the molecule, Cys-505, with alanine, so that synthetically introduced cysteines could unambiguously serve as targets for chemical modification . All of the replacements reported here (at positions 449, 459, 473, 505, and some combinations) resulted in a channel that had an ion selectivity (K+ versus Cl-) identical to wild type at low pH . At higher pH, however, one of these mutations, which replaced the negatively charged aspartate at position 473 (the upstream boundary of the hydrophobic segment), resulted in a channel that was less cation-selective than was wild type . When the introduced Cys-473 was reacted with iodoacetic acid, which inserted a COOH group close to the position of the missing aspartate COOH, wild-type ion selectivity was restored, suggesting that the greater cation selectivity of the wild-type channel was directly produced by the negative charge at Asp-473 . By comparing the ion selectivity of the Cys-473 mutant channel to that of the wild type as a function of the pH on the cis and trans sides of the membrane, it was possible to locate residue 473 close to the cis side . Locating in this manner the positions in the channel of particular residues places important constraints on channel model building. J Biol Chem, 1990 Apr 25, 265(12), 6936 - 43 Identification of the reverse transcriptase encoded by the Mauriceville and Varkud mitochondrial plasmids of Neurospora; Kuiper MT et al.; The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) that have characteristics of mtDNA introns and retroid elements . The plasmids contain a single long open reading frame (710 amino acids), whose amino-terminal half has structural similarity to reverse transcriptases . Using antibodies against synthetic peptides and trpE fusion proteins, we detected an 81-kDa protein encoded by this open reading frame in mitochondrial preparations from the plasmid-containing strains . This 81-kDa protein cosegregates with reverse transcriptase activity in sexual crosses and comigrates with reverse transcriptase activity in sodium dodecyl sulfate-polyacrylamide gels, where it can be assayed after renaturation of the protein . In glycerol gradients under nondenaturing conditions, the reverse transcriptase activity sediments at approximately 145 kDa, close to the value expected for a dimer of the 81-kDa protein . The 81-kDa protein represents most of the 710-amino acid open reading frame, but may be missing some amino acids at the amino terminus . The regions upstream and downstream of the putative reverse transcriptase domain lack sequences characteristic of gag, protease, RNase H, or integrase domains found in other retroid elements . The plasmid-encoded 81-kDa protein seems to be a novel type of reverse transcriptase that may provide insight into the evolution of these enzymes. J Biol Chem, 1990 Apr 25, 265(12), 6908 - 15 Human interferon-gamma receptor . Mapping of epitopes recognized by neutralizing antibodies using native and recombinant receptor proteins; Garotta G et al.; Monoclonal antibodies produced against native interferon-gamma receptor (IFN gamma-R) have been characterized for their capacity to react with purified receptor and receptor-positive cells, to inhibit the binding of IFN gamma to cellular receptor, to precipitate the receptor protein when cross-linked to IFN-gamma, and to recognize the recombinant interferon-gamma receptor and 19 overlapping fragments of this protein expressed in Escherichia coli . The results of this analysis showed that: (i) the extracellular portion of human IFN gamma-R is located between the N terminus and the transmembrane region (amino acids 18-246) . (ii) The intracellular domain is between the transmembrane region and the C terminus (amino acids 269-489) . (iii) The monoclonal antibodies that react with the IFN gamma-R intracellular domain recognize small linear epitopes . (iv) The human IFN gamma-R binding site is located between the N terminus and the transmembrane region . (v) The monoclonal antibodies that react with IFN gamma-R extracellular domain and inhibit the binding of IFN gamma recognize two different epitopes . One of these epitopes (included between amino acids 26 and 133) is very close to the binding site for IFN gamma . The second (included between amino acids 70 and 210) is related to the binding site for IFN gamma without including it . (vi) These two functional epitopes are conformational and need S-S bridges to maintain their architecture . (vii) These conformational epitopes are formed in receptor fragments expressed in E . coli. J Biol Chem, 1990 Apr 25, 265(12), 6800 - 10 Determinants of OmpF porin antigenicity and structure; Klebba PE et al.; Sixty-six murine hybridomas raised to Escherichia coli B/r porin were used to identify and differentiate the epitopes of this outer membrane protein . Anti-porin monoclonal antibodies (mAb) were raised against outer membrane fragments, purified native trimeric porin (trimer), and purified sodium dodecyl sulfate-denatured monomeric porin (monomer) . Immunochemical and flow cytometric methods identified five distinct cell surface-exposed determinants on OmpF . The peptide composition of porin epitopes was determined by analysis of mAb reactivity with cyanogen bromide-generated peptide fragments . Four of 43 anti-monomer mAb reacted with surface exposed sites on OmpF, defining epitopes that consist of residues within CNBr peptides d2, d3, and B . The anti-porin mAb panel was also used to evaluate changes in porin antigenic structure in strains with short ompF deletions . Flow cytometric experiments indicated that despite changes in porin permeability, little if any alteration of surface epitopes occurred in these strains . Western immunoblot analysis of the mutant porins showed loss of reactivity with numerous mAb, which was caused by changes in three spatially distinct epitopes at residues 108-111, 118-123, and 124-129 . Our findings indicate that in these ompF mutants the residues responsible for altering porin permeability are not exposed on the cell surface, but are buried within the tertiary structure of the protein . One of these regions, which is apparently involved in the determination of channel permeability characteristics, is conserved among 15 of 16 different porin molecules which were screened with the anti-OmpF mAb panel. Biochemistry, 1990 Apr 24, 29(16), 3821 - 7 Importance of residues Arg-167 and Gln-231 in both the allosteric and catalytic mechanisms of Escherichia coli aspartate transcarbamoylase; Stebbins JW et al.; Site-specific mutagenesis has been used to create two mutant versions of aspartate transcarbamoylase . Arg-167 and Gln-231, both previously identified as interacting with the portion of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate (PALA) that corresponds to aspartate {Krause, K . L., Voltz, K . W., & Lipscomb, W . N . (1987) J . Mol . Biol . 193, 527-553}, were replaced by glutamine and leucine, respectively . The Arg-167----Gln and the Gln-231----Leu enzymes show approximately 900-fold and 1500-fold reductions in the maximal observed specific activity, respectively . The aspartate concentration at half the maximal observed specific activity is increased 18-fold for the Gln-231----Leu enzyme compared to the value for the wild-type enzyme, but is altered little in the case of the Arg-167----Gln enzyme . The carbamoyl phosphate concentration at half the maximal activity is unchanged by either mutation, suggesting that these mutations result in only local changes to the aparatate binding site . Both mutations eliminate homotropic cooperativity; however, the Gln-231----Leu enzyme also has altered heterotropic interactions and no longer exhibits substrate inhibition . At relatively low concentrations of aspartate and saturating carbamoyl phosphate, PALA is able to activate the Gln-231----Leu enzyme, whereas the Arg-167----Gln enzyme is inhibited at PALA concentrations that normally activate the wild-type enzyme . Equilibrium binding measurements indicate that the Gln-231----Leu enzyme binds CTP approximately 10-fold more weakly than the wild-type enzyme, even though the mutation is some 70 A from the regulatory binding site.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1990 Apr 24, 263(2), 358 - 60 Eclosion hormone of the silkworm Bombyx mori . Expression in Escherichia coli and location of disulfide bonds; Kono T et al.; A gene encoding eclosion hormone (EH) from the silkworm, Bombyx mori was chemically synthesized, inserted into a secretion vector and expressed in Escherichia coli, leading to the production of biologically active EH . Sequence analysis of cystine-containing peptides in a thermolysin digest of this EH established the locations of 3 disulfide bonds in the molecule . Evidence was also obtained that the 6 residues at the NH2-terminal are dispensable but 4 residues at the COOH-terminal play an important role in EH activity. FEBS Lett, 1990 Apr 24, 263(2), 281 - 4 Osmium tetroxide, N,N,N',N'-tetramethylethylendiamine . A new probe of DNA structure in the cell; Boublikova P et al.; It was shown that the complex of osmium tetroxide with N,N,N',N'-tetramethylethylendiamine can be applied as a probe of DNA structure in the cell . This probe site-specifically recognized structural distortions at the B-Z junctions in plasmids pRW751 and pPK1 (containing (dC-dG)n segments) in E . coli cells. Biochemistry, 1990 Apr 24, 29(16), 3937 - 43 Calcium(II) site specificity: effect of size and charge on metal ion binding to an EF-hand-like site; Snyder EE et al.; The molecular mechanisms by which protein Ca(II) sites selectively bind Ca(II) even in the presence of high concentrations of other metals, particularly Na(I), K(I), and Mg(II), have not been fully described . The single Ca(II) site of the Escherichia coli receptor for D-galactose and D-glucose (GGR) is structurally related to the eukaryotic EF-hand Ca(II) sites and is ideally suited as a model for understanding the structural and electrostatic basis of Ca(II) specificity . Metal binding to the bacterial site was monitored by a Tb(III) phosphorescence assay: Ca(II) in the site was replaced with Tb(III), which was then selectively excited by energy transfer from protein tryptophans . Photons emitted from the bound Tb(III) enabled specific detection of this substrate; for other metals binding was detected by competitive displacement of Tb(III) . Representative spherical metal ions from groups IA, IIA, and IIIA and the lanthanides were chosen to study the effects of metal ion size and charge on the affinity of metal binding . A dissociation constant was measured for each metal, yielding a range of KD's spanning over 6 orders of magnitude . Monovalent metal ions of group IA exhibited very low affinities . Divalent group IIA metal ions exhibited affinities related to their size, with optimal binding at an effective ionic radius between those of Mg(II) (0.81 A) and Ca(II) (1.06 A) . Trivalent metal ions of group IIIA and the lanthanides also exhibited size-dependent affinities, with an optimal effective ionic radius between those of Sc(III) (0.81 A) and Yb(III) (0.925 A) . The results indicate that the GGR site selects metal ions on the basis of both charge and size.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1990 Apr 24, 263(2), 217 - 21 A rev/beta-galactosidase fusion protein binds in vitro transcripts spanning the rev-responsive element of human immunodeficiency virus type 1 (HIV-1); Aepinus C et al.; The rev protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 kDa apparent molecular mass, is essential to target the mRNA for virion polypeptides into the cytoplasm . This effect is mediated by a specific RNA stretch (rev-responsive element = RRE) localized within a 3'-terminal segment of the mRNA encoding virion proteins . We present evidence that rev expressed as a beta-galactosidase fusion protein in E . coli forms a complex with in vitro transcripts containing the RRE; it can be precipitated by monoclonal antibodies with rev or beta-galactosidase specificity . In addition, specific binding of rev protein to RNA could be demonstrated by Northwestern blotting. J Mol Biol, 1990 Apr 20, 212(4), 563 - 78 Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences; Bucher P; Optimized weight matrices defining four major eukaryotic promoter elements, the TATA-box, cap signal, CCAAT-, and GC-box, are presented; they were derived by comparative sequence analysis of 502 unrelated RNA polymerase II promoter regions . The new TATA-box and cap signal descriptions differ in several respects from the only hitherto available base frequency Tables . The CCAAT-box matrix, obtained with no prior assumption but CCAAT being the core of the motif, reflects precisely the sequence specificity of the recently discovered nuclear factor NY-I/CP1 but does not include typical recognition sequences of two other purported CCAAT-binding proteins, CTF and CBP . The GC-box description is longer than the previously proposed consensus sequences but is consistent with Sp1 protein-DNA binding data . The notion of a CACCC element distinct from the GC-box seems not to be justified any longer in view of the new weight matrix . Unlike the two fixed-distance elements, neither the CCAAT- nor the GC-box occurs at significantly high frequency in the upstream regions of non-vertebrate genes . Preliminary attempts to predict promoters with the aid of the new signal descriptions were unexpectedly successful . The new TATA-box matrix locates eukaryotic transcription initiation sites as reliably as do the best currently available methods to map Escherichia coli promoters . This analysis was made possible by the recently established Eukaryotic Promoter Database (EPD) of the EMBL Nucleotide Sequence Data Library . In order to derive the weight matrices, a novel algorithm has been devised that is generally applicable to sequence motifs positionally correlated with a biologically defined position in the sequences . The signal must be sufficiently over-represented in a particular region relative to the given site, but need not be present in all members of the input sequence collection . The algorithm iteratively redefines the set of putative motif representatives from which a weight matrix is derived, so as to maximize a quantitative measure of local over-representation, an optimization criterion that naturally combines structural and positional constancy . A comprehensive description of the technique is presented in Methods and Data. J Mol Biol, 1990 Apr 20, 212(4), 579 - 98 Genomic organization and physical mapping of the transfer RNA genes in Escherichia coli K12; Komine Y et al.; By using a set of 476 ordered DNA clones (in lambda phage vector) that covers the entire chromosome of Escherichia coli K12, we have made an exhaustive survey of tRNA genes in the E . coli genome . Ultraviolet-irradiated bacteria were separately infected with each of the 476 clones and the RNA molecules produced upon infection were labeled with 32P . The labeled tRNAs were separated by gel electrophoresis and then characterized by fingerprinting analysis . Fifty-nine of the 476 clones produced tRNAs, including adjacent overlapping ones that share the same tRNA genes . The products of all the previously mapped tRNA genes (about 60, to date) were detected according to their expected positions, and 19 more tRNA genes were newly elucidated . These new tRNA genes were identified by sequencing the DNA from relevant regions of the clones; the DNA sequences were scanned for the stretches that could be folded into the familiar cloverleaf structure and the transcription units were deduced by predicting the promoters and terminators . The total complement of the tRNA genes in E . coli K12 was 78 for 45 tRNA (or 41 anticodon) species, distributed in 40 different transcription units throughout the chromosome . In addition, a gene for selenocysteine tRNA was detected by hybridization and mapped to a specific DNA segment . A comprehensive tRNA gene map of E . coli was constructed, including the selenocysteine tRNA gene . All the tRNA genes encode the 3' CCA, and in several cases the terminal 19 nucleotides (including the 3' CCA) of a tRNA gene is repeated several times . Finally, in the present study the sites for a long inversion (approx . 800 x 10(3) base-pairs, around the oriC region) in Kohara's library was determined to be within the 23 S-5 S regions in rrnD and rrnE, revealing the exchange of combinations of spacer and distal tRNA genes between these two ribosomal RNA operons. J Mol Biol, 1990 Apr 20, 212(4), 557 - 9 Crystallization of the soluble lytic transglycosylase from Escherichia coli K12; Rozeboom HJ et al.; Lytic transglycosylases degrade the murein polymer of the bacterial cell wall to 1,6-anhydromuropeptides . These enzymes are of significant medical interest, not only because they are ideal targets for the development of new classes of antibiotics, but also because the low molecular weight products of their catalytic action can cause diverse biological activities in humans, which can be either beneficial or toxic . A soluble lytic transglycosylase was purified from an overproducing Escherichia coli strain and X-ray quality crystals were obtained at room temperature from hanging drops by vapor diffusion against 20 to 25% (NH4)2SO4, in 100 mM-sodium acetate buffer, pH 5.0 . The crystals diffract in the X-ray beam to 2.8 A resolution . Their space group is P2(1)2(1)2(1) with cell dimensions a = 81 A, b = 88 A and c = 135 A . Assuming one monomer (Mr 70,362) per asymmetric unit, the solvent content of these crystals is 63%. Eur J Biochem, 1990 Apr 20, 189(1), 89 - 94 Spinach cytosolic fructose-1,6-bisphosphatase . Purification, enzyme properties and structural comparisons; Ladror US et al.; Cytosolic fructose-1,6-bisphosphatase from spinach leaves was purified to homogeneity and characterized . The pure enzyme has a subunit mass of 38 kDa, its Km values for fructose 1,6-bisphosphate and Mg2+ are 1.5 microM and 260 mM, respectively, and its Vmax is 110-120 units/mg . It is inhibited by fructose 2,6-bisphosphate and AMP with Ki values of 0.07 microM and 120 microM, respectively . About 90% of the primary structure of the spinach cytosolic fructose-1,6-bisphosphatase has been determined by amino-acid sequencing . The sequence data demonstrate that the cytosolic enzyme lacks the sequence insert characteristic of chloroplast fructose-1,6-bisphosphatase . The data include also the sequences of peptides containing all seven cysteine residues . Only two of the seven cysteines are conserved between the two isozymes, none of which is believed to be involved with the light regulation of the chloroplast enzyme . Sequence comparisons between the spinach cytosolic enzyme and gluconeogenic fructose-1,6-bisphosphatases from other species reveal similarity ranging over 47-54%, which is higher than the 40-45% similarity between the chloroplast enzyme and gluconeogenic fructose-1,6-bisphosphatases . However, similarity between these isozymes and Escherichia coli fructose-1,6-bisphosphatase are 44% and 47% for the cytosolic and chloroplast enzymes, respectively . Similarity between the cytosolic and chloroplast counterparts is 52%, indicating wide divergence between these two fructose-1,6-bisphosphatases. J Mol Biol, 1990 Apr 20, 212(4), 669 - 82 Regulation of divergent transcription from the iron-responsive fepB-entC promoter-operator regions in Escherichia coli; Brickman TJ et al.; Transcriptional linkage of the enterobactin gene cluster entCEBA (P15) was confirmed by ent-lacZ gene fusion analysis . Control sequences directing iron-regulated expression of this polycistronic message were localized to the fepB-entC bidirectional promoter region . Transcriptional initiation sites defined by primer extension analysis were located 103 base-pairs apart for the divergent fepB and entC messages . Within this divergent regulatory region, strongly consensus -35 and -10 promoter determinants and potential Fur repressor-binding sequences were identified . A vector containing divergently oriented indicator gene fusions was constructed to monitor regulatory effects of mutations within this iron-responsive control region . The fepB-entC promoter-operator elements were confirmed by mutation, using the dual gene fusion system in multicopy and low copy number states . Mutations in the -35 and -10 regions of the fepB and entC promoters that decreased their similarity to consensus resulted in reduced promoter activity . Mutations in the Fur-controlled operators reduced induction ratios (iron-deficient levels/iron-rich levels) for the respective fusion gene activities by approximately sevenfold . Although operator mutants retained some degree of inducibility, complete relief of repression was observed for double operator mutants, suggesting that only minor regulatory influence is exerted by Fur occupation of the opposing operator site . DNase I footprinting experiments were performed to characterize the sequence-specific Fur interactions at the operator sequences . At the fepB operator, a 31 base-pair Fur-protected region was identified, corresponding to positions -19 to +12 with respect to the transcriptional start site . Similarly, Fur protected a 31 base-pair region in entC, corresponding to positions +1 to +31 in the message . A contiguous and sequentially occupied secondary Fur-binding site in entC was protected at higher Fur concentrations, extending the protected region to +49, and sequestering the putative Shine-Dalgarno sequence . Operator positional effects and co-operativity are discussed. J Mol Biol, 1990 Apr 20, 212(4), 695 - 708 Control of ColE1 plasmid replication . Interaction of Rom protein with an unstable complex formed by RNA I and RNA II; Tomizawa J; A transcript (RNA I) from ColE1 inhibits initiation of replication of the plasmid DNA by binding to the precursor of the primer RNA (RNA II) . The ability of RNA I to inhibit replication is altered by the presence of a plasmid-specified small protein, Rom . In vitro, RNA I binds to RNA II to form a very unstable complex, C* . Binding of a single molecule of Rom converts C* to a more stable complex, Cm* . Each of these complexes, C* or Cm*, transforms to a more stable complex, C** or Cm**, respectively . While formation of complex C* or Cm* is inferred from the inhibition of binding caused by a second RNA I species, that of complex C** or Cm** is detected by alteration of RNase sensitivity . Complex C* converts to complex Cm* very rapidly upon addition of Rom to the medium and complex Cm* converts to complex C* very rapidly by removal of Rom from the medium . On the other hand, complexes C** and Cm** do not rapidly interconvert, but can eventually transform to the same stable final product . Thus, Rom affects binding of RNA I to RNA II through conversion of a very unstable early intermediate to a more stable complex, creating a second pathway for their stable binding. J Mol Biol, 1990 Apr 20, 212(4), 683 - 94 Control of ColE1 plasmid replication . Intermediates in the binding of RNA I and RNA II; Tomizawa J; Replication of plasmid ColE1 is regulated by a plasmid-specified small RNA (RNA I) . RNA I binds to the precursor (RNA II) of the primer for DNA synthesis and inhibits primer formation . The process of binding of RNA I to RNA II that results in formation of a stably bound complex consists of a series of reactions forming complexes differing in the stability . Formation of a very unstable early intermediate that was previously inferred from the inhibition of stable binding caused by a second RNA I species was firmly established by more extensive studies . This complex is converted to a more stable yet reversible complex that was identified by its RNase sensitivity, which was altered from that of the earlier complex or from that of free RNA I or RNA II . In these complexes, most loops of RNA II interact with their complementary loops of RNA I . The kinetic and structural analyses of the binding process predict formation of a complex interacting at a single pair of complementary loops that precedes formation of these complexes . Thus the process of binding of RNA I to RNA II is seen to consist of a sequence of reactions producing a series of progressively more stable intermediates leading to the final product. Nature, 1990 Apr 19, 344(6268), 793 - 4 DNA mutagenesis and recombination; Jones DH et al.; The polymerase chain reaction is used for site-specific mutagenesis and for DNA recombination without any enzymatic reaction in vitro, apart from DNA amplification. Biochemistry, 1990 Apr 17, 29(15), 3724 - 31 Heterotropic effectors promote a global conformational change in aspartate transcarbamoylase; Eisenstein E et al.; The sigmoidal dependence of activity on substrate concentration exhibited by the regulatory enzyme aspartate transcarbamoylase (ATCase) of Escherichia coli is generally attributed to a ligand-promoted change in the quaternary structure of the enzyme . Although a global conformational change in ATCase upon the binding of ligands to some of the six active sites is well documented, a corresponding alteration in the structure of the wild-type enzyme upon the addition of the inhibitor, CTP, or the activator, ATP, has not been detected . Such evidence is essential for testing whether heterotropic, as well as homotropic, effects can be accounted for quantitatively in terms of coupled equilibria involving a conformational change in the enzyme and preferential binding of ligands to one conformation or the other . This evidence has now been obtained with a mutant form of ATCase in which Lys 143 in the regulatory chain was replaced by Ala, thereby perturbing interactions at the interface between the regulatory and catalytic chains in the enzyme and destabilizing the low-activity, compact (T) conformation relative to the high-activity, swollen (R) state . Difference sedimentation velocity experiments involving measurements of the changes caused by the binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate demonstrated that the sedimentation coefficient of the mutant enzyme was intermediate between that observed for the T and R states of wild-type ATCase . We interpret the results as indicating that the {T}/{R} ratio in phosphate buffer at pH 7.0 is reduced from about 2 X 10(2) for the wild-type enzyme to 2.7 for r143Ala ATCase.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Apr 17, 29(15), 3701 - 9 Characterization of Escherichia coli thioredoxins with altered active site residues; Gleason FK et al.; Escherichia coli thioredoxin is a small disulfide-containing redox protein with the active site sequence Cys-Gly-Pro-Cys-Lys . Mutations were made in this region of the thioredoxin gene and the mutant proteins expressed in E . coli strains lacking thioredoxin . Mutant proteins with a 17-membered or 11-membered disulfide ring were inactive in vivo . However, purified thioredoxin with the active site sequence Cys-Gly-Arg-Pro-Cys-Lys is still able to serve as a substrate for thioredoxin reductase and a reducing agent in the ribonucleotide reductase reaction, although with greatly reduced catalytic efficiency . A smaller disulfide ring, with the active site sequence Cys-Ala-Cys, does not turn over at a sufficient rate to be an effective reducing agent . Strain in the small ring favors the formation of intermolecular disulfide bonds . Alteration of the invariant proline to a serine has little effect on redox activity . The function of this residue may be in maintaining the stability of the active site region rather than participation in redox activity or protein-protein interactions . Mutation of the positively charged lysine in the active site to a glutamate residue raises the Km values with interacting enzymes . Although it has been proposed that the positive residue at position 36 is conserved to maintain the thiolate anion on Cys-32 (Kallis & Holmgren, 1985), the presence of the negative charge at this position does not alter the pH dependence of activity or fluorescence behavior . The lysine is most likely conserved to facilitate thioredoxin-protein interactions. Biochemistry, 1990 Apr 17, 29(15), 3612 - 21 Interaction of Escherichia coli DNA polymerase I with azidoDNA and fluorescent DNA probes: identification of protein-DNA contacts; Catalano CE et al.; The synthesis of an azidoDNA duplex and its use to photolabel DNA polymerases have been previously described (Gibson & Benkovic, 1987) . We now present detailed experiments utilizing this azidoDNA photoprobe as a substrate for Escherichia coli DNA polymerase I (Klenow fragment) and the photoaffinity labeling of the protein . The azidoDNA duplex is an efficient substrate for both the polymerase and 3'----5' exonuclease activities of the enzyme . However, the hydrolytic degradation of the azido-bearing base is dramatically impaired . On the basis of the ability of these duplexes to photolabel the enzyme, we have determined that the protein contacts between five and seven bases of duplex DNA . Incubation of azidoDNA with the Klenow fragment in the presence of magnesium results in the in situ formation of a template-primer with the azido-bearing base bound at the polymerase catalytic site of the enzyme . Photolysis of this complex followed by proteolytic digestion and isolation of DNA-labeled peptides results in the identification of a single residue modified by the photoreactive DNA substrate . We identify Tyr766 as the modified amino acid and thus localize the catalytic site for polymerization in the protein . A mansyl-labeled DNA duplex has been prepared as a fluorescent probe of protein structure . This has been utilized to determine the location of the primer terminus when bound to the Klenow fragment . When the duplex contains five unpaired bases in the primer strand of the duplex, the primer terminus resides predominantly at the exonuclease catalytic site of the enzyme . Removal of the mismatched bases by the exonuclease activity of the enzyme yields a binary complex with the primer terminus now bound predominantly at the polymerase active site . Data are presented which suggest that the rate-limiting step in the exonuclease activity of the enzyme is translocation of the primer terminus from polymerase to exonuclease catalytic sites. Mol Cell Endocrinol, 1990 Apr 17, 70(2), 175 - 84 Human c-erb A protein expressed in Escherichia coli: changes in hydrophobicity upon thyroid hormone binding; Ichikawa K et al.; The human c-erb A beta gene sequence was inserted in an Escherichia coli expression vector plasmid . The E . coli cells transformed with this plasmid produced proteins with molecular masses of 52 and 50 kDa . These products bound 3,5,3'-triiodo-L-thyronine (T3) with an affinity constant of 4.3 x 10(9) liter/mol . The order of affinity for iodothyronine analogs was triiodothyroacetic acid greater than T3 greater than 3,5,3'-triiodo-D-thyronine greater than L-thyroxine . Affinity labeling experiments showed that the 50 kDa protein was covalently labeled with {125I}T3, and this was competed by triiodothyroacetic acid, T3, and L-thyroxine (from potent to weaker competitor) . The c-erb A protein bound to calf thymus DNA-cellulose and the binding was inhibited by 0.3 M KCl or 10 mM pyridoxal 5'-phosphate . Aqueous two-phase partitioning studies showed that the c-erb A product became less hydrophobic upon T3 or triiodothyroacetic acid binding . The same finding was obtained when T3 bound to partially purified rat liver nuclear thyroid hormone receptor . However, thyroxine binding globulin became more hydrophobic upon T3 binding . Since the T3 molecule partitioned preferentially into the upper polyethylene glycol-rich phase, the alteration of partitioning behavior of thyroxine binding globulin was explained by a simple additive effect of T3 . In contrast, the alteration of partitioning behavior of the c-erb A product or receptor reflected a conformational transition upon T3 binding . The c-erb A protein expressed in E . coli showed various characteristics similar to classical thyroid hormone receptor and may be useful in studying the structure and function of the thyroid hormone receptor. Biochemistry, 1990 Apr 17, 29(15), 3765 - 71 Sequence determinants for H1 binding on Escherichia coli lac and gal promoters; Rimsky S et al.; The H1 protein is a likely candidate for structuring DNA in the bacterial nucleoid . We have studied determinants leading to its binding to DNA (and in particular to Escherichia coli lac and gal promoters) in vitro through the pattern of attack of both DNaseI and the copper-o-phenanthroline complex {(OP)2Cu+} . The binding of H1 depends on the primary sequence of DNA . H1 also associates with recognition sites for specific proteins, in particular with the Pribnow box and the CRP binding site . Binding of H1 to the Pribnow box of the wild-type lac promoter does not change the pattern of nucleolytic digestion with (OP)2Cu+ . In contrast, binding of H1 to the strong lac promoter mutants Ps and UV5 appears to change the conformational state of this DNA . Similar changes in accessibility of the minor groove surrounding the respective binding sites were observed for both H1-DNA and CRP-DNA complexes. Biochemistry, 1990 Apr 17, 29(15), 3716 - 23 Function of serine-171 in domain closure, cooperativity, and catalysis in Escherichia coli aspartate transcarbamoylase; Dembowski NJ et al.; Structural studies of Escherichia coli aspartate transcarbamoylase suggest that the R state of the enzyme is stabilized by an interaction between Ser-171 of the aspartate domain and both the backbone carbonyl of His-134 and the side chain of Gln-133 of the carbamoyl phosphate domain of a catalytic chain {Ke, H.-M., Lipscomb, W.N., Cho, Y., & Honzatko, R . B . (1988) J . Mol . Biol . 204, 725-747} . In the present study, site-specific mutagenesis is used to replace Ser-171 by alanine, thereby eliminating the interactions between Ser-171 and both Gln-133 and His-134 . The Ser-171----Ala holoenzyme exhibits no cooperativity, more than a 140-fold loss of activity, little change in the carbamoyl phosphate concentration at half the maximal observed specific activity, and a 7-fold increase in the aspartate concentration at half the maximal observed specific activity . Although the Ser-171----Ala enzyme exhibits no homotropic cooperativity, it is still activated by N-(phosphonacetyl)-L-aspartate (PALA), but not by succinate, in the presence of saturating carbamoyl phosphate and subsaturating aspartate . At subsaturating concentrations of aspartate, the Ser-171----Ala enzyme is still activated by ATP but is inhibited less by CTP than is the wild-type enzyme . At saturating concentrations of aspartate, the Ser-171----Ala enzyme is activated by ATP and inhibited by CTP to an even greater extent than at subsaturating concentrations of aspartate . At saturating aspartate, the wild-type enzyme is neither activated by ATP nor inhibited by CTP.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Apr 17, 29(15), 3668 - 76 Glucosamine-6-phosphate synthase from Escherichia coli: determination of the mechanism of inactivation by N3-fumaroyl-L-2,3-diaminopropionic derivatives; Kucharczyk N et al.; A mechanistic investigation of the inactivation of Escherichia coli glucosamine-6-phosphate synthase by N3-(4-methoxyfumaroyl)-L-2,3-diaminopropionate (FMDP) was undertaken . On the basis of the known participation of the N-terminal cysteine residue in this process {Chmara et al . (1986) Biochim . Biophys . Acta 870, 357; Badet et al . (1988) Biochemistry 27, 2282}, the model reactions between FMDP and L-cysteine and between FMDP and the synthetic decapeptide Cys-Gly-Ile-Val-Gly-Ala-Ile-Ala-Gln-Arg, corresponding to the amino-terminal protein sequence, were studied . The results allowed us to propose a pathway that is in perfect agreement with the biochemical results: enzyme inactivation arose from Michael addition of glutamine binding site cysteine-1 on the fumaroyl double bond at the beta-position of the ester group . Upon denaturation under slightly alkaline conditions, this adduct underwent cyclization to a transient succinimide adduct, which rearranged into the stable 2-substituted 1,4-thiazin-3-one-5-carboxylate involving participation of the cysteine amino group . The tryptic radiolabeled peptides purified from {3H}FMDP-treated enzyme and resistant to Edman degradation coeluted with the products resulting from the model reaction between the synthetic decapeptide and the inhibitor. Biochemistry, 1990 Apr 17, 29(15), 3621 - 6 A nucleotide that enhances the charging of RNA minihelix sequence variants with alanine; Shi JP et al.; We showed earlier that a single G3.U70 base pair within the amino acid acceptor helix is a major determinant of the identity of tRNA(Ala) . In addition, we demonstrated that an RNA hairpin minihelix that recreates the 12 base pair acceptor-T psi C stem of tRNA(Ala) is also aminoacylated in a G3.U70-dependent manner . Determinants for efficient aminoacylation at pH 7.5 have been further investigated with minihelix substrates that have sequence variations at 3.70 and other locations . Although a U,U mismatch and other 3.70 nucleotide alternatives to G.U were recently proposed by others as also important for alanine acceptance, neither that mismatch nor any of four other 3.70 nucleotide combinations confer aminoacylation in vitro with alanine, even with substrate levels of enzyme . In contrast, permutations of the so-called discriminator nucleotide N73 (at position 73) strongly modulate, but do not block, aminoacylation of those substrates that encode G3.U70 . In particular, the efficiency of G3.U70-dependent aminoacylation with alanine is strongly enhanced by having the wild-type A73 . The effect of N73 alone can explain most of the difference in aminoacylation efficiency of a G3.U70-containing tRNA and a minihelix substrate whose sequences vary significantly from their tRNA(Ala) counterparts . Comparison with earlier work suggests that the substantial modulating effect of N73 is partly or completely obscured when N73 tRNA variants are expressed as amber suppressors in vivo. Gene, 1990 Apr 16, 88(2), 269 - 73 Adenovirus proteinases: comparison of amino acid sequences and expression of the cloned cDNA in Escherichia coli; Houde A et al.; Adenoviruses (Ad) synthesize serine-center endoproteinases (AdEPs) responsible for maturation cleavages within the virus particle . Many questions regarding these enzymes remain unanswered because previous studies utilized crude cells or viral lysates as the enzyme source . Here, we report on the comparison of the amino acid (aa) sequences of several AdEPs and on the expression of the cDNA of the Ad2Ep in Escherichia coli . The AdEPs consist of about 200 aa and their size is around 23 kDa . Among the seven sequences known, 60% of aa were strictly conserved . The usual serine proteinase active site sequence, GDSGG, is absent . The recombinant Ad2EP, produced by an inducible vector as a protein-A fusion product is capable of autocatalytic cleavage, and of cleaving its natural viral substrates as well as foreign proteins . Therefore, other viral proteins or mammalian specific post-translational modifications are not required for enzyme activity. Gene, 1990 Apr 16, 88(2), 263 - 7 Synthesis of papain in Escherichia coli; Cohen LW et al.; We have transferred the cloned papain genetic information into an expression vector (pT7-7) regulated by the T7-promoter and have obtained in vitro expression as well as expression in Escherichia coli . In Western blots the proteins produced are immunologically recognizable as papain . Multiple forms of specific but differing sizes are detected, suggesting either that initiation can occur at more than one of the upstream methionines, or that the enzyme is processed after synthesis. Biochem Biophys Res Commun, 1990 Apr 16, 168(1), 288 - 94 Oxygen resistant strain of N2-fixing Escherichia coli; Iwahashi H et al.; Oxygen resistant N2-fixing Escherichia coli, which can grow at a high oxygen concentration in a nitrogen-free medium, were produced by several times of cultivation under a condition of 0.03% oxygen . Six isolated resistant strains could grow at an oxygen concentration ten times that of the parent strain . The nif-genes of these six strains were integrated into a chromosome . At low oxygen, they showed one third the nitrogenase activity to the parent strain, but more so at a high oxygen concentration . It is thus evident that the amount of nitrogenase protein in a cell is a factor determining the oxygen resistance of nitrogenase. Biochem J, 1990 Apr 15, 267(2), 517 - 25 Human carboxypeptidase E . Isolation and characterization of the cDNA, sequence conservation, expression and processing in vitro; Manser E et al.; Carboxypeptidase E (CPE), which cleaves C-terminal amino acid residues and is involved in neuropeptide processing, is itself subject to intracellular processing . Human CPE cDNA was isolated and sequence comparisons were made with those of a previously isolated brain cDNA (M1622) encoding rat CPE and of other human carboxypeptidases (M and N) . Human (2.5 kb) and rat (2.1 kb) CPE cDNAs approximated to the size of their respective mRNAs; additional sequences were located in putative 5' and 3' untranslated regions of human CPE mRNA . There is 79% sequence similarity between human and rat CPE cDNAs, with greater similarity (89%) over the coding region and short sections of the non-coding sequence . The predicted 476-amino acid-residue sequences of human and rat preproCPEs are highly conserved (96% identity), with lower degree of similarity of the N-terminal signal peptide (76%) . Human CPE showed 51% and 43% sequence similarity to human CPN and CPM respectively, with discrete regions of divergence dispersed between the highly conserved mechanistically implicated regions . Antiserum generated from a fusion protein, synthesized in Escherichia coli from constructs of the human cDNA, recognized an approx . 50 kDa membrane protein and a smaller soluble protein in rat and human brain preparations, corresponding to the two forms of native CPE . Human CPE mRNA transcripts directed the synthesis in reticulocyte lysate of a 54 kDa translation product, which in the presence of dog pancreas microsomal membranes was co-translationally processed with cleavage, insertion into membranes and glycosylation . Three processed forms were generated, the largest (56 kDa) and smallest (52 kDa) being equally glycosylated . The membrane association of the processed translation products and of native brain membrane CPE, detected immunologically, was resistant to moderate alkali but not pH 11.5 extraction . These results are consistent with secondary-structure predictions that CPE is a peripheral membrane protein . The dissimilar regions of human carboxypeptidases may provide information on sequences responsible for their different cellular disposition. J Biol Chem, 1990 Apr 15, 265(11), 6481 - 8 Glucose phosphorylation in tumor cells . Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase; Arora KK et al.; In rapidly growing tumor cells exhibiting high glucose catabolic rates, the enzyme hexokinase is markedly elevated and bound in large amounts (50-80% of the total cell activity) to the outer mitochondrial membrane (Arora, K.K., and Pedersen, P.L . (1988) J . Biol . Chem . 263, 17422-17428; Parry, D.M., and Pedersen, P.L . (1983) J . Biol . Chem . 258, 10904-10912) . In extending these studies, we have isolated a cDNA clone of hexokinase from a lambda gt11 library of the highly glycolytic, c37 mouse hepatoma cell line . This clone, comprising 4,198 base pairs, contains a single open reading frame of 2,754 nucleotides which encode a 918-amino acid hexokinase with a mass of 102,272 daltons . This enzyme exhibits, respectively, 68 and 32 amino acid differences, including several charge differences, from the recently sequenced human kidney and rat brain enzymes . The putative glucose and ATP binding domains present in the latter two enzymes and in rat liver glucokinase are conserved in the tumor enzyme . At its N-terminal region, tumor hexokinase has a 12-amino acid hydrophobic stretch which is present in the rat brain enzyme but absent in the rat liver glucokinase, a cytoplasmic enzyme . The mature tumor hexokinase protein has been overexpressed in active form in Escherichia coli and purified 9-fold . The overexpressed enzyme binds to rat liver mitochondria in the presence of MgCl2 . This is the first report describing the cloning and sequencing of a tumor hexokinase, and the first report documenting the overexpression of any hexokinase type in E . coli . Questions pertinent to the enzyme's mechanism, regulation, binding to mitochondria, and its marked elevation in tumor cells can now be addressed. J Biol Chem, 1990 Apr 15, 265(11), 6461 - 6 Specific binding of the TraY protein to oriT and the promoter region for the traY gene of plasmid R100; Inamoto S et al.; The traY gene product of plasmid R100 was purified as a hybrid protein, TraY-collagen-beta-galactosidase . The hybrid protein as well as the TraY' protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT . The oriT region consists of highly conserved and nonconserved regions among R100-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region . This supports the idea that the TraY protein has a role as a component of endonuclease in recognizing its own oriT sequence . Unexpectedly, however, the hybrid protein and the TraY' protein were also found to bind to two different AT-rich sequences (each 24 base pairs in length) in the promoter region preceding the traY gene (here called sbyB and sbyC) . This suggests that the TraY protein may have another role in regulating the expression of its own gene . The "TAA(A/T)T" sequence motif observed in these binding sites might constitute a core sequence recognized by the TraY protein . Mg2+ is not required for the specific binding of the TraY protein. J Biol Chem, 1990 Apr 15, 265(11), 6255 - 61 The reaction of acetyldithio-CoA, a readily enolized analog of acetyl-CoA with thiolase from Zoogloea ramigera; Anderson VE et al.; Acetyldithio-CoA has been shown to be a competent nucleophilic substrate but not an electrophilic substrate for the Claisen condensation catalyzed by thiolase, which normally dimerizes acetyl (Ac)-CoA to acetoacetyl-CoA . Acting as the nucleophile, the kcat/Km for dithioacetyl-CoA is comparable to that of Ac-CoA, the normal substrate . With acetoacetyl-pantetheine acetylating the thiolase to provide the electrophile, the kcat and kcat/Km for the Claisen condensation are 2.1 s-1 and 8.3 X 10(4) M-1 s-1, respectively . The product of the reaction is 3-ketobutyryldithio-CoA . The 3-ketobutyryldithio-CoA has a spectrally determined pKa of 6.55 and the enolate has a lambda max of 357 nm, epsilon 357 = 21,000 cm-1 M-1 . Product analysis indicates that acetyldithio-CoA does not serve as the electrophilic partner in the enzymic condensation . This failure is attributed to the inability demonstrated in this study of acetyldithio-CoA to thioacetylate the active site Cys89 of the Zoogloea ramigera thiolase . 1H NMR studies in D2O indicate that thiolase catalyzes the exchange of the alpha-hydrogens, without Cys89 being acetylated, with a rate of 0.63 +/- 0.25 s-1 . In the presence of a large excess of acetoacetyl-pantetheine, present to acetylate Cys89 and prevent the thiolytic back reaction, solvent exchange of the alpha-hydrogens can still be detected by observing the isotope-shifted 13C NMR spectrum of {2-13C}acetyldithio-CoA . The exchange of the acetyldithio-CoA alpha-hydrogens with solvent promoted by the acetylated enzyme, must proceed at a rate comparable to that of the condensation reaction. J Biol Chem, 1990 Apr 15, 265(11), 6086 - 91 Primary structure of a DNA (N6-adenine)-methyltransferase from Escherichia coli virus T1 . DNA sequence, genomic organization, and comparative analysis; Schneider-Scherzer E et al.; Escherichia coli virus T1 encodes a DNA (N6-adenine)-methyltransferase (M.T1) with the same sequence specificity as the E . coli DNA (N6-adenine)-methyltransferase (M.Eco dam) . This enzyme was purified to homogeneity and a partial amino acid sequence determined . Oligonucleotides were constructed and used not only as probes to map the gene on the T1 genome, but also as primers in sequencing reactions to establish the nucleotide sequence of the M.T1 locus by primer extension . These data represent the first analysis of the genomic organization of bacterial virus T1 on a molecular level . Significant homology to E . coli consensus transcription and translation-initiation signals suggest that the gene for M.T1 is most probably under control of its own promoter . It may be transcribed as a polycistronic mRNA, together with a downstream open reading frame which codes for a polypeptide containing 83 amino acids (HP 83) . Both the deduced primary and the secondary structure of the M.T1 were compared to those of other known DNA methyltransferases, especially those recognizing the sequence, GATC; there is little similarity of the T1 enzyme to the other members of this family. J Biol Chem, 1990 Apr 15, 265(11), 6441 - 7 Geometric arrangements of Tn3 resolvase sites; Benjamin HW et al.; Site-specific recombination by Tn3 resolvase normally occurs in vitro and in vivo only between directly repeated res sites on the same supercoiled DNA molecule . However, with multiply interlinked catenane substrates consisting of two DNA rings each containing a single res site, resolvase efficiently carried out intermolecular recombination . The topology of the knots produced by several rounds of this reaction proves that the DNA within the synaptic intermediate is coiled in an interwound (plectonemic) fashion rather than wrapped solenoidally around resolvase as in previously characterized supercoiled DNA-protein complexes . The synaptic intermediate can contain equivalently supercoil, catenane, or knot crossings as long as the res sites have a right-handed coiling and a particular relative orientation . The structure of the product knots and catenanes also shows the path the DNA takes during strand exchange . Intermolecular recombination within multiply linked catenanes required negative supercoiling, as does the standard intramolecular reaction. J Biol Chem, 1990 Apr 15, 265(11), 6339 - 45 cDNA-derived sequence of UMP-CMP kinase from Dictyostelium discoideum and expression of the enzyme in Escherichia coli; Wiesmuller L et al.; A cDNA coding for UMP-CMP kinase from Dictyostelium discoideum was isolated from a lambda gt11 expression library and sequenced . The corresponding mRNA has a size of 0.7 kilobase and is down-regulated during early development of D . discoideum . Southern blotting demonstrated that the UMP-CMP kinase is encoded by a single gene . The deduced amino acid sequence of UMP-CMP kinase shows a high degree of homology with adenylate kinases from different sources with the highest degree of homology to cytosolic adenylate kinase from vertebrate muscle (43%) . The enzyme expressed in Escherichia coli after cloning the cDNA into an ATG expression vector was purified and analyzed for its structural and kinetic properties . The UMP-CMP kinase uses preferentially ATP (Km,app = 25 microM) as phosphate donor and is specific for UMP (Km,app = 0.4 mM) and CMP (Km,app = 0.1 mM) . The enzyme is strongly inhibited by the substrate analogue P1-(adenosine-5')-P5-(uridine-5')-pentaphosphate (Ki between 0.05 and 0.1 microM) and is inactivated by modification of free thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid). J Biol Chem, 1990 Apr 15, 265(11), 6274 - 8 Activation of ATP hydrolysis by an uncoupler in mutant mitochondria lacking an intrinsic ATPase inhibitor in yeast; Ichikawa N et al.; An intrinsic ATPase inhibitor and 9-kDa protein are regulatory factors of mitochondrial ATP synthase in Saccharomyces cerevisiae . A gene encoding the ATPase inhibitor was isolated from a yeast genomic library with synthetic oligonucleotides as hybridization probes and was sequenced . The deduced amino acid sequence showed that the precursor protein contains an amino-terminal presequence of 22 amino acid residues . Mutant strains that did not contain the inhibitor and/or the 9-kDa protein were constructed by transformation of cells with their in vitro disrupted genes . The disruption of the chromosomal copy in recombinant cells was verified by Southern blot analysis, and the absence of the proteins in the mutant cells was confirmed by Western blot analysis . All the mutants could grow on a nonfermentable carbon source and the oxidative phosphorylation activities of their isolated mitochondria were the same as that of normal mitochondria . However, an uncoupler, carbonylcyanide-m-chlorophenylhydrazone, induced marked ATP hydrolysis in the inhibitor-deficient mitochondria, but not in normal mitochondria . These observations suggest that the ATPase inhibitor inhibits ATP hydrolysis by F1F0-ATPase only when the membrane potential is lost. J Photochem Photobiol B, 1990 Apr 15, 5(2), 167 - 78 Dark and photoreactivity of 4'-aminomethyl-4,5',8-trimethylpsoralen with T7 phage; Toth K et al.; The dark and photoreactions of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with T7 phage were investigated from biological and structural points of view . The dark reaction leads to the structural destabilization of the double helix of the DNA as is shown by optical melting measurements . The