EMBO J, 1990 Nov, 9(11), 3767 - 75 Control of replication of plasmid R1: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT; Persson C et al.; The replication frequency of plasmid R1 is determined by the availability of the RepA protein, which acts at the origin of replication to promote initiation . Synthesis of RepA is negatively regulated both at the transcriptional and post-transcriptional levels . Post-transcriptional control is exerted through the action of an antisense RNA, CopA RNA . The target of CopA RNA, CopT RNA, is located in the leader region of the RepA mRNA . Binding between CopA and CopT inhibits repA expression . We have previously presented an in vitro analysis of the binding reaction between CopA and CopT RNAs . In this communication, we extend the in vitro analysis by determining the regions of CopA required for binding, and also demonstrate that binding occurs in at least two steps . The first step is the formation of an initial, transient complex; stem-loop II is the structure in CopA necessary and sufficient for this step . The subsequent step(s), resulting in the formation of a complete duplex, requires a stretch of single-stranded nucleotides located 5' to stem-loop II in CopA, and its counterpart in CopT . We show that the single-stranded region can be positioned on either side of stem-loop II provided that there is a complementary stretch of nucleotides in CopT, indicating that the second step(s) is not sequence-specific . Furthermore, the effects of salt concentration and temperature on the binding reaction indicate that duplex formation occurs through a mechanism of gradual intra-strand breaking and inter-strand formation of hydrogen bonds. Biotechnology (N Y), 1990 Nov, 8(11), 1036 - 40 High-level direct expression of semi-synthetic human interleukin-6 in Escherichia coli and production of N-terminus met-free product; Yasueda H et al.; We have developed a direct expression system for high-level production of recombinant human interleukin-6 (rhIL-6) in Escherichia coli . In this system, (i) the natural N-terminal coding region of the hIL-6 gene was replaced by a synthetic sequence containing A-T rich codons, (ii) dual Shine-Dalgarno (SD) sequences were employed, (iii) an A-T rich segment was inserted in front of the initiation codon to avoid putative mRNA secondary structure in the region and (iv) the natural amber termination codon of the hIL-6 gene was changed to an ocher stop codon . The hIL-6 polypeptide, synthesized at a high level, formed cytoplasmic inclusion bodies . After refolding, the N-terminal methionine was removed by aminopeptidase-P in vitro . The purified recombinant hIL-6 had B-cell differentiation activity equivalent to natural IL-6 from a human T-cell culture. Biotechnology (N Y), 1990 Nov, 8(11), 1030 - 3 Protective surface antigen P69 of Bordetella pertussis: its characterization and very high level expression in Escherichia coli; Makoff AJ et al.; The surface antigen, P69 of Bordetella pertussis, an N-terminal fragment of the precursor protein, P93, is likely to be an important component of future subunit vaccines against whooping cough . We have expressed several defined N-terminal fragments of P93 in E . coli and compared their electrophoretic mobilities with that of purified P69 from B . pertussis . These experiments show that P69 is considerably smaller than the 69 kD originally estimated from its gel mobility and is probably 60.4 kD in size . Our initial plasmids expressed only very low levels of this antigen . We diagnosed the limiting factor to be a poor ribosome binding site (RBS) by demonstrating a large stimulation of expression on a two-cistron plasmid . The limitation of expression could be completely overcome by only two base changes close to the initiation codon, resulting in a further increase in expression of P69 at levels to 30-40% total cell protein . Although the protein accumulated as insoluble inclusion bodies, it could be solubilized by guanidinium chloride. Biotechnol Prog, 1990 Nov-Dec, 6(6), 494 - 7 Fractionation of recombinant tumor necrosis factor using hydrophobic and hydrophilic membranes; Dorin G et al.; Recombinant tumor necrosis factor (TNF) is expressed in Escherichia coli as a soluble intracellular protein . A purification process is described that incorporates a hydrophilic membrane (cellulosic) separation followed by a hydrophobic one (PTFE) . The hydrophilic step is a traditional one in that species are separated primarily on the basis of size . The hydrophobic step separates species on the basis of parameters apparently not related to size . By combining these two steps, an increase in TNF purity of 7-10-fold can be achieved with a yield of 50% . The effects of cellular debris and pH on selectivity and recovery of the hydrophobic filtration step are discussed. Gene, 1990 Oct 30, 95(1), 1 - 7 Cloning and sequencing the gene encoding Escherichia coli ribonuclease I: exact physical mapping using the genome library; Meador J 3rd et al.; The amino acid (aa) sequence of the N terminus of Escherichia coli RNase I was determined . A mixed oligodeoxynucleotide coding for that sequence was used to probe the 476 lambda clones of Kohara et al . {Cell 50 (1987) 495-508} . DNA from these clones carry almost the entire E . coli chromosome in overlapping segments . Two overlapping clones hybridized to the probe sequence . From one of them DNA containing the rna gene was subcloned and sequenced . The inferred protein contains 245 aa residues and has an Mr of 27,156, which agrees with earlier estimates from sodium dodecyl sulfate-polyacrylamide-gel electrophoresis . RNase I is close to twice the size of pancreatic RNase A, but both enzymes contain eight Cys and four His; those aa are important for structure and function of RNase A . Proximal to the rna gene is a sequence that would code for a 23-aa peptide which conforms to consensus rules for signal peptides, and thus should transport this periplasmic enzyme . Sites for eight restriction enzymes had been mapped on each lambda clone . By relating to the map for that specific region, it was possible to position the rna gene exactly at 659 kb from the thr locus (time zero on a time scale of 100 min) . This physical mapping gave a more precise (exact) map position based on distance than was possible using genetic mapping based on a time scale derived from conjugation, and should be applicable for mapping many other E . coli genes. Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 669 - 75 Glutathione S-transferase in yeast: induction of mRNA, cDNA cloning and expression in Escherichia coli; Tamaki H et al.; Glutathione S-transferase Y-2 mRNA synthesis was induced in yeast Issatchenkia orientalis approximately 37-fold by cultivation with o-dinitrobenzene . A cDNA library complementary to poly (A)+RNA of I . orientalis grown with o-dinitrobenzene was screened by colony hybridization . Twenty positive clones were obtained from 6,000 clones and seven of twenty positive clones expressed glutathione S-transferase activity in E . coli . One of the expressing clones harboring plasmid pHT108 had 28 times more glutathione S-transferase activity induced by Isopropyl-beta-D-thio-galactopyranoside than a strain harboring plasmid pUC118 . Expressed glutathione S-transferase Y-2 protein comigrated with yeast glutathione S-transferase Y-2 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as detected by immunoblot analysis. Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 432 - 8 The binding of ATP and AMP to Escherichia coli adenylate kinase investigated by 1H and 15N NMR spectroscopy; Glushka J et al.; {N6 15N}ATP and {N6 15N}AMP, complexed with E.coli adenylate kinase (AKe), were observed with 15N isotope-filtered NMR pulse sequences and 1H{15N} heterocorrelated experiments to determine differences between binding sites based on chemical shifts and competition by substrate analogs . The chemical shifts of the N6 amino proton and nitrogen signals changed significantly after mixing with adenylate kinase . Differences in chemical shifts between the bound ATP and AMP signals are slight . The response of these shifts to further addition of other substrates or Mg2+ supports the view that the unchelated nucleotides can bind to both the sites, whereas the metal complexed species are restricted to the MgATP/MgADP binding site. Biochemistry, 1990 Oct 30, 29(43), 10120 - 5 Subunit structure and activity of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system solubilized in detergent; Lolkema JS et al.; The original proposal of Saier stating that P-enolpyruvate-dependent mannitol phosphorylation is catalyzed by the monomeric form of the bacterial phosphotransferase enzyme IImtl, which would be the form predominantly existing in the phospholipid bilayer, whereas mannitol/mannitol-P exchange would depend on the transient formation of functional dimers, is refuted {Saier, M.H . (1980) J . Supramol . Struct . 14, 281-294} . The correct interpretation of the proportional relation between the rate of mannitol phosphorylation in the overall reaction and the enzyme concentration is that enzyme IImtl is dimeric under the conditions employed . Differences measured in the enzyme concentration dependency of the overall and exchange reactions were caused by different assay conditions . The dimer is favored over the monomer at high ionic strength and basic pH . Mg2+ ions bind specifically to enzyme IImtl, inducing dimerization . A complex formed by mixing inorganic phosphate, F-, and Mg2+ at sufficiently high concentrations inhibits enzyme IImtl, in part, by dissociation of the dimer . Enzyme IImtl was dimeric in 25 mM Tris, pH 7.6, and 5 mM Mg2+ over a large enzyme concentration range and under many different turnover conditions . The association/dissociation equilibrium was demonstrated in phosphate bufers, pH 6.3 . The dimer was the most active form both in the overall and in the exchange reaction under the conditions assayed . The monomer was virtually inactive in mannitol/mannitol-P exchange but retained 25% of the activity in the overall reaction. FEBS Lett, 1990 Oct 29, 273(1-2), 46 - 50 Time-resolved fluorescence studies on mutants of the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii; Schulze E et al.; Fluorescence anisotropy decays were measured for the wild-type dihydrolipoyl transacetylase (E2) component of pyruvate dehydrogenase complex from Azotobacter vinelandii and E . coli and for E2-mutants from A . vinelandii in which the alanine-proline-rich sequence between the binding domain and the catalytic domain is partially or completely deleted . In both E2-mutants the rotational mobility of the lipoyl domain and the overall activity after reconstitution of the complex are significantly decreased indicating the important role of the deleted sequence for the movement of the lipoyl domain and the transfer of substrates between the different active sites within the complex. FEBS Lett, 1990 Oct 29, 273(1-2), 23 - 6 Enhanced expression of group II phospholipase A2 gene in the tissues of endotoxin shock rats and its suppression by glucocorticoid; Nakano T et al.; We studied the regulation of group II phospholipase A2 (PLA2-II) gene in vivo, using endotoxin shock rat as a model for systemic inflammation . Administration of endotoxin into rats increased PLA2 activity in the plasma, as described by Vadas and Hay, using endotoxin-challenged rabbit . Specific absorption of this activity by anti-PLA2-II antibody indicated that the released PLA2 was PLA2-II . The levels of PLA2-II mRNA were elevated in the aorta, spleen, lung, and thymus but not in the liver and kidney . The tissues with high PLA2-II mRNA contents released a greater amount of PLA2-II than the tissues of control rats . These results suggest that in endotoxin shock rats, PLA2-II is synthesized de novo in the above tissues and released into circulation . Furthermore, our present study demonstrates that glucocorticoid suppresses the enhanced expression of the PLA2-II gene in the tissues of endotoxin shock rats. FEBS Lett, 1990 Oct 29, 273(1-2), 208 - 10 Influence of the aminoacyl-tRNA synthetase inhibitors and the diadenosine-5'-tetraphosphate phosphonate analogues on the catalysis of diadenosyl oligophosphates formation; Biryukov AI et al.; Well-known aminoacyl-tRNA synthetase (ARSase) inhibitors, namely the analogues of amino acids and aminoacyl adenylates (aminoalkyl- and aminophosphonyl adenylates with Ki congruent to 0.1 microM) as well as the diadenosine 5',5'''-p1,p4-tetraphosphate (Ap4A) phosphonoanalogues, were for the first time used for the Ap4A biosynthesis regulation . Effects of a set of such compounds on lysyl-, phenylalanyl- and alanyl-tRNA synthetases from E . coli, capable of synthesizing Ap4A in the presence of Zn2+ ions and pyrophosphatase, have been studied . The adenylate analogues were found to inhibit the Ap4A and Ap3A formation (I50 congruent to 6 mM) . Aminophosphonic and aminophosphonous acids are not involved in Ap3A and Ap4A biosynthesis and inhibited it at high concentrations . The Ap4A phosphoanalogues slightly inhibited the major reactions of ARSases, as well as the biosynthesis of Ap3A and Ap4A, at a concentration of 5 mM. FEBS Lett, 1990 Oct 29, 273(1-2), 147 - 9 Tight ATP and ADP binding in the noncatalytic sites of Escherichia coli F1-ATPase is not affected by mutation of bulky residues in the 'glycine-rich loop'; Pagan J et al.; It is shown that ATP dissociates very slowly (koff less than 6.4 x 10(5) s-1, t1/2 greater than 3 h) from the three noncatalytic sites of E . coli F1-ATPase and that ADP dissociates from these three sites in a homogeneous fashion with koff = 1.5 x 10(-4) s-1 (t1/2 = 1.35 h) . Mutagenesis of alpha-subunit residues R171 and Q172 in the 'glycine-rich loop' (Homology A) consensus region of the noncatalytic sites was carried out to test the hypothesis that unusually bulky residues at these positions are responsible wholly or partly for the observed tight binding of adenine nucleotides . The mutations alpha Q172G or alpha R171S,Q172G had no effects on ATP or ADP binding to or rates of dissociation from F1 noncatalytic sites . KdATP and KdADP of isolated alpha-subunit were weakened by approximately 1 order of magnitude in both mutants . The results suggest that neither residue alpha R171 nor alpha Q172 interacts directly with bound nucleotide, and show that the presence of bulky residues per se in the glycine-rich loop region of F1-alpha-subunit is not responsible for tight binding in the noncatalytic sites. Science, 1990 Oct 26, 250(4980), 553 - 6 Protection of mice against the Lyme disease agent by immunizing with recombinant OspA; Fikrig E et al.; Lyme borreliosis is a tick-borne illness caused by Borrelia burgdorferi . The gene for outer surface protein A (OspA) from B . burgdorferi strain N40 was cloned into an expression vector and expressed in Escherichia coli . C3H/HeJ mice actively immunized with live transformed E . coli or purified recombinant OspA protein produced antibodies to OspA and were protected from challenge with several strains of B . burgdorferi . Recombinant OspA is a candidate for a vaccine for Lyme borreliosis. Science, 1990 Oct 26, 250(4980), 528 - 32 DNA looping and unlooping by AraC protein; Lobell RB et al.; Expression of the L-arabinose BAD operon in Escherichia coli is regulated by AraC protein which acts both positively in the presence of arabinose to induce transcription and negatively in the absence of arabinose to repress transcription . The repression of the araBAD promoter is mediated by DNA looping between AraC protein bound at two sites near the promoter separated by 210 base pairs, araI and araO2 . In vivo and in vitro experiments presented here show that an AraC dimer, with binding to half of araI and to araO2, maintains the repressed state of the operon . The addition of arabinose, which induces the operon, breaks the loop, and shifts the interactions from the distal araO2 site to the previously unoccupied half of the araI site . The conversion between the two states does not require additional binding of AraC protein and appears to be driven largely by properties of the protein rather than being specified by the slightly different DNA sequences of the binding sites . Slight reorientation of the subunits of AraC could specify looping or unlooping by the protein . Such a mechanism could account for regulation of DNA looping in other systems. Nucleic Acids Res, 1990 Oct 25, 18(20), 6025 - 9 Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction; Ruffner DE et al.; A hammerhead domain of less than 50 nucleotides is responsible for a self-cleavage reaction in the replication of plant RNA pathogens . The hammerhead is composed of three helices joining at a central conserved core of 11 single stranded nucleotides . The core is believed to fold into a tertiary structure that provides functional groups for catalysis and to coordinate one or more divalent metal ions . In this study we use a phosphorothioate substitution interference assay to identify four phosphates in the conserved core which also play a role in the self-cleavage reaction. Nucleic Acids Res, 1990 Oct 25, 18(20), 5945 - 8 Sequence analysis of two temperature-sensitive mutations in the alpha subunit gene (rpoA) of Escherichia coli RNA polymerase; Igarashi K et al.; The rpoA gene of Escherichia coli encodes the alpha subunit of the DNA-dependent RNA polymerase . Two mutant alleles, rpoA101 and rpoA112, both of which produce RNA polymerase with altered thermostability and reduced fidelity of transcription in vitro (Ishihama et al . (1980) J . Mol . Biol . 137, 137-150), have been analyzed in details . The mutations were found to be responsible for the temperature-sensitive growth by complementation test using a rpoA-expression plasmid . Each mutant allele was amplified from total cell DNA by PCR (polymerase chain reaction) and directly sequenced . Both the mutant rpoA genes were found to carry a single base transition which leads to a substitution of Cys for Arg at the position 191 (rpoA101) or 45 (rpoA112), respectively . Since the rpoA112 mutation causes the defect in RNA polymerase assembly (Kawakami & Ishihama (1980) Biochemistry 19, 3491-3495), the amino-terminal region of alpha including the position 45 was considered to play an important role in subunit assembly. J Biol Chem, 1990 Oct 25, 265(30), 18656 - 62 The folate cofactor of Escherichia coli DNA photolyase acts catalytically; Hamm-Alvarez S et al.; Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm) . The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate . The function of the folate has been characterized in greater detail in this series of experiments . Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein . Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme . Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically . The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures . These results demonstrate that the folate of E . coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme. J Biol Chem, 1990 Oct 25, 265(30), 18213 - 8 Escherichia coli formate-hydrogen lyase . Purification and properties of the selenium-dependent formate dehydrogenase component; Axley MJ et al.; The formate-hydrogen lyase complex of Escherichia coli decomposes formic acid to hydrogen and carbon dioxide under anaerobic conditions in the absence of exogenous electron acceptors . The complex consists of two separable enzymatic activities: a formate dehydrogenase and a hydrogenase . The formate dehydrogenase component (FDHH) of the formate-hydrogen lyase complex was purified to near homogeneity in two column chromatographic steps . The purified enzyme was composed of a single polypeptide of molecular weight 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Metal analysis showed each mole of enzyme contained 3.3 g atoms of iron . Denaturation of FDHH released a compound which, when oxidized, displayed a fluorescence spectrum similar to that of the molybdopterin cofactor found in certain other enzymes . The enzyme contained selenium in the form of selenocysteine as determined by radioactive labeling of the enzyme with 75Se and amino acid analysis . FDHH activity was maximal between pH 7.5 and 8.5; however, the enzyme was maximally stable at pH 5.3-6.4 and highly unstable above pH 7.5 . Nitrate and nitrite salts caused a drastic reduction in activity . Although azide inhibited FDHH activity, it also protected the enzyme from inactivation by oxygen. J Biol Chem, 1990 Oct 25, 265(30), 18185 - 91 Identification of a region of Escherichia coli ribosomal protein L2 required for the assembly of L16 into the 50 S ribosomal subunit; Romero DP et al.; In vitro mutagenesis of rplB was used to generate changes in a conserved region of Escherichia coli ribosomal protein L2 between Gly221 and His231 . Mutants were selected by temperature sensitivity using an inducible expression system . A mutant L2 protein with the deletion of Thr222 to Asp228 was readily distinguishable from wild-type L2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ribosomes from the strain overexpressing this mutant protein were characterized by sucrose density gradient centrifugation and protein composition . In addition to 30 S and 50 S ribosomal subunits, cell lysates contained a new component that sedimented at 40 S in 1 mM Mg2+ and at 48 S in 10 mM Mg2+ . These particles contained mutant L2 protein exclusively, completely lacked L16, and had reduced amounts of L28, L33, and L34 . They did not reassociate with 30 S ribosomal subunits and were inactive in polyphenylalanine synthesis . Other mutants in the same conserved region, including the substitution of His229 by Gln229, produced similar aberrant 50 S particles that sedimented at 40 S and failed to associate with 30 S subunits. J Biol Chem, 1990 Oct 25, 265(30), 18154 - 60 The presence of both the signal sequence and a region of mature LamB protein is required for the interaction of LamB with the export factor SecB; Altman E et al.; In the accompanying paper (Altman, E., Bankaitis, V.A., and Emr, S.D . (1990) J . Biol . Chem . 265, 18148-18153) a putative SecB binding site was identified in the mature LamB protein . The export of wild-type LamB was unperturbed when this region was removed, however, suggesting the presence of a second site of interaction between SecB and LamB . In this paper we show that the interference caused by export-defective LamB proteins is influenced by the amount of signal sequence that is present . If a large portion of the signal sequence is deleted then the interference levels are significantly reduced . This result suggests that a region of the signal sequence contributes to the interaction of SecB with the LamB protein . Using anti-SecB affinity chromatography, we demonstrated directly that the association of SecB protein with precursor LamB is dependent on the presence of both the LamB signal sequence and the interfering region which maps to amino acids 320-380 of mature LamB . Although the interfering region is not necessary for the export of wild-type LamB under normal conditions, when the signal sequence is mutationally altered the interfering region is required to promote the efficient export of LamB protein . Also, deletion of the interfering region eliminates the ability of wild-type LamB precursor to be maintained in an export competent conformation in vivo . Collectively, our results indicate that efficient export of the LamB protein is achieved by an interaction with SecB that involves both the LamB signal sequence and the interfering region in mature LamB. J Biol Chem, 1990 Oct 25, 265(30), 18148 - 53 Characterization of a region in mature LamB protein that interacts with a component of the export machinery of Escherichia coli; Altman E et al.; It has been shown that the synthesis of an export-defective protein can interfere with the normal export process in Escherichia coli by limiting the availability of SecB protein, a component of the export apparatus (Collier, D.N., Bankaitis, V.A., Weiss, J.B., and Bassford, P.J . (1988) Cell 53, 273-283) . Consistent with this observation, we find that the interference elicited by an export-defective LamB protein is a titratable response resulting from the limitation of a single ligand . We have mapped the interfering region in LamB to between amino acids 320 and 380 of the mature protein . Expression of this sequence in the form of a LacZ-LamB-LacZ fusion protein elicits the export interference phenotype . Deletion of the sequence from an export-defective LamB protein eliminates the ability of this protein to interfere with the export of other secreted proteins . Together, these findings show that this sequence is both necessary and sufficient to cause export interference . Surprisingly, deletion of this sequence from an otherwise wild-type LamB protein does not cause the mutant LamB product to exhibit any obvious export defect . Based on our results, we propose that SecB interacts with both amino acids 320-380 of mature LamB and the LamB signal sequence during initiation of the export process. Nucleic Acids Res, 1990 Oct 25, 18(20), 6097 - 100 Sequence logos: a new way to display consensus sequences; Schneider TD et al.; A graphical method is presented for displaying the patterns in a set of aligned sequences . The characters representing the sequence are stacked on top of each other for each position in the aligned sequences . The height of each letter is made proportional to its frequency, and the letters are sorted so the most common one is on top . The height of the entire stack is then adjusted to signify the information content of the sequences at that position . From these 'sequence logos', one can determine not only the consensus sequence but also the relative frequency of bases and the information content (measured in bits) at every position in a site or sequence . The logo displays both significant residues and subtle sequence patterns. J Biol Chem, 1990 Oct 25, 265(30), 18366 - 71 Pyrophosphate-dependent phosphofructokinase . Conservation of protein sequence between the alpha- and beta-subunits and with the ATP-dependent phosphofructokinase; Carlisle SM et al.; Full-length cDNA clones for the alpha- and beta-subunits of pyrophosphate-fructose 6-phosphate 1-phosphotransferase have been isolated from a cDNA expression library derived from potato tuber poly(A)+ RNA . The nucleotide sequences indicate that the alpha- and beta-subunits are related with about 40% of amino acid residues being identical . A comparison of the deduced amino acid sequences of both subunits of this enzyme with that of the major ATP-dependent fructose 6-phosphate 1-phosphotransferase from Escherichia coli (Shirakihara, Y., and Evans, P . R . (1988) J . Mol . Biol . 204, 973-994) showed little homology between the proteins except for regions involved in the binding of fructose 6-phosphate/fructose, 1,6-bisphosphate and possibly between regions binding pyrophosphate and the beta- and gamma-phosphates of ADP/ATP . A comparison of the derived secondary structures of the two subunits of the PPi-dependent enzyme with the known secondary structure of the E . coli ATP-dependent enzyme indicated that the overall structure of these enzymes is similar . These data suggest that catalytic activity resides on the beta-subunit of the pyrophosphate-dependent enzyme. J Biol Chem, 1990 Oct 25, 265(30), 18095 - 7 Oxygen regulation of nitrate transport by diversion of electron flow in Escherichia coli; Denis KS et al.; Anaerobic nitrate respiration is regulated by oxygen at the level of nitrate transport; however, the mechanism of O2 inhibition is unknown . Potentially, oxygen could inhibit directly by causing conformational changes in the porter system or indirectly through diversion of electron flow from the nitrate reductase complex to oxygen reduction . Inhibition due to electron diversion implies that nitrate reduction is required for nitrate transport . In this regard, nitrate uptake and its regulation by oxygen were studied in mutants of Escherichia coli (strain AN386) deficient in cytochrome d (RG98), cytochrome o (RG101), and a mutant deficient in both cytochrome d and cytochrome o (RG99) . Respiratory nitrate uptake in RG99 was highly resistant to the effects of oxygen supporting the indirect mechanism of electron diversion in oxygen regulation . Nitrate transport in RG98 and RG101 is highly sensitive to oxygen; these mutants exhibited 81 and 85% inhibition, respectively, which is similar to inhibition in the wild type . These results indicate that during nitrate respiration, O2 inhibits transport by limiting the supply of electrons to the nitrate reductase complex. Nature, 1990 Oct 25, 347(6295), 780 - 2 The Drosophila claret segregation protein is a minus-end directed motor molecule; Walker RA et al.; A product encoded at the claret locus in Drosophila is needed for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo . The predicted amino-acid sequence of the segregation protein was shown recently to be strikingly similar to Drosophila kinesin heavy chain . We have expressed the claret segregation protein in bacteria and have found that the bacterially expressed protein has motor activity in vitro with several novel features . The claret motor is slow (4 microns min-1), unlike either kinesin or dyneins . It has the directionality, the ability to generate torque and the sensitivity to inhibitors reported previously for dyneins . The finding of minus-end directed motor activity for a protein with sequence similarity to kinesin suggests that the dynein and kinesin motor domains are ancestrally related . The minus-end directed motor activity of the claret motor is consistent with a role for this protein in producing chromosome movement along spindle microtubules during prometaphase and/or anaphase. J Biol Chem, 1990 Oct 25, 265(30), 18408 - 13 A short intervening structure can block rho factor helicase action at a distance; Steinmetz EJ et al.; We have characterized the helicase activity of transcription termination factor rho on a variety of substrates . Helicase activity requires specific recognition of a single-stranded region of RNA upstream (5') of the nucleic acid duplex on which rho acts . Spacer sequences of at least 450 nucleotides can be inserted between the rho-binding signals and the duplex region with little effect on activity . RNA-DNA helices of up to 120 base pairs, but not as long as 210 base pairs, can be disrupted efficiently by rho . The stoichiometry of release of substrates with long spacer sequences, as with the standard substrate, approaches a value of one RNA released per rho hexamer; thus cooperative binding by rho does not account for action at a distance . Instead, these results are consistent with a model in which a single rho hexamer binds initially to terminator sequences and then either loops out or tracks along the intervening RNA to reach the duplex region . Results with complex substrates are inconsistent with looping and support the tracking model: under conditions that allow disruption of RNA-DNA, but not RNA-RNA helices (0.4 mM Mg2+), the presence of a short RNA-RNA helix acts as a block to the disruption of an RNA-DNA helix downstream . These findings are discussed in relation to the mechanism of the helicase activity as well as its role in rho-dependent transcription termination. J Biol Chem, 1990 Oct 25, 265(30), 18379 - 85 Mutational and immunochemical analysis of plasminogen activator inhibitor 1; Shubeita HE et al.; We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry . Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells . Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects . The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied . Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators . However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity . Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators . Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region. Nucleic Acids Res, 1990 Oct 25, 18(20), 5969 - 73 Enzymatic recognition of DNA modifications induced by singlet oxygen and photosensitizers; Muller E et al.; DNA modifications induced either by photosensitization (illumination in the presence of methylene blue) or by chemically generated singlet oxygen (thermal decomposition of an 1,4-etheno-2,3-benzodioxin) are recognized and incised by repair endonucleases present in crude bacterial cell extracts . Only a small fraction of the incised modifications are sites of base loss (AP-sites) sensitive to exonuclease III, endonuclease IV from E . coli or to the UV-endonuclease from M . luteus . Cell extracts from E . coli strains overproducing or defective in endonuclease III recognize the modifications induced by illumination in the presence of methylene blue just as well as do those from wild-type E . coli strains . This indicates that dihydropyrimidine derivatives, which are characteristic of hydroxyl radical-induced DNA modifications, are absent . In contrast, most of the modifications induced are not recognized by a cell extract from a fpg strain defective in formamidopyrimidine-DNA glycosylase FPG protein) . Furthermore, incision by a cell extract from an E . coli strain overproducing FPG protein takes place at much lower protein concentration than with the wild-type strain . Experiments with purified FPG protein confirm that this enzyme is responsible for the recognition of singlet oxygen-induced DNA base modifications. Eur J Biochem, 1990 Oct 24, 193(2), 361 - 6 Distribution of the early light-inducible protein in the thylakoids of developing pea chloroplasts; Cronshagen U et al.; The distribution of the early light-inducible protein (ELIP) of pea (Pisum sativum) between grana and stroma thylakoids was studied . An antibody raised against a bacterial-expressed fusion protein containing ELIP sequences was used . Illumination of dark-grown pea seedlings causes an accumulation of the ELIP in the thylakoid membranes with a maximum level at 16 h . During continuous illumination exceeding 16 h the level decreases again . The fractionation of thylakoid membranes of 48-h-illuminated pea seedlings in grana and stroma thylakoids reveals that there is no uniform distribution of ELIP in the thylakoids . Rather 60-70% of ELIP was found in the stroma thylakoids and 30-40% in the grana thylakoids . This distribution is in accordance with that of photosystem I but not with that of photosystem II . After Triton-X-100 solubilization almost all ELIP is found in the photosystem-I-containing fraction . This also supports an association of ELIP with photosystem I. Eur J Biochem, 1990 Oct 24, 193(2), 325 - 30 Export and purification of a cytoplasmic dimeric protein by fusion to the maltose-binding protein of Escherichia coli; Blondel A et al.; A hybrid between the maltose-binding protein (MalE) of Escherichia coli and the gene 5 protein (G5P) of phage M13 was constructed at the genetic level . MalE is a monomeric and periplasmic protein while G5P is dimeric and cytoplasmic . The hybrid (MalE-G5P) was synthesized in large amounts from a multicopy plasmid and efficiently exported into the periplasmic space of E . coli . The export was dependent on the integrity of the signal peptide . MalE-G5P was purified from a periplasmic extract by affinity chromatography on cross-linked amylose, with a yield larger than 50,000 molecules/E . coli cell . The hybrid specifically bound denatured but not double-stranded DNA cellulose, as native G5P . Sedimentation velocity and gel-filtration experiments showed that MalE-G5P exists as a dimer . Thus, it was possible to efficiently translocate through the membrane a normally cytoplasmic and dimeric protein, by fusion to MalE . Moreover, the passenger protein kept its activity, specificity and quaternary structure in the purified hybrid . MalE-G5P will enable the study of mutant G5P that no longer binds single-stranded DNA and therefore cannot be purified by DNA-cellulose chromatography. Eur J Biochem, 1990 Oct 24, 193(2), 387 - 94 Overexpression in Escherichia coli of a methionine-free designed interleukin-2 receptor (Tac protein) based on a chemically cleavable fusion protein; Husken D et al.; Several fusion proteins of our previously chemically synthesized gene encoding the interleukin-2-receptor alpha subunit (IL-2R alpha or Tac protein) were constructed . They were designed in order to be cleavable by cyanogen bromide . Thus, the original internal methionines of the IL-2R alpha were replaced by either alanine, valine, leucine or isoleucine, based on secondary structure predictions . Additionally, aspartate at position 6 was substituted for glutamate in order to stabilize the acid-labile Asp-Pro bond . Direct C-terminal fusion of total beta-galactosidase and portions thereof did not result in substantial amounts of the expected construct . Ternary fusions consisting of beta-galactosidase domains N- and C-terminally fused to the mutant synthetic methionine-free interleukin-2 receptor alpha subunit (synIL-2R alpha) yielded inclusion bodies amounting to 4-7% of the total protein . This first overexpression of a type I membrane receptor can be rationalized by the known beta-galactosidase structure models . The fusion protein can be cleaved with cyanogen bromide, isolated and the resulting synIL-2R alpha detected by Western blot analysis. Eur J Biochem, 1990 Oct 24, 193(2), 319 - 24 Expression of three plant glutamine synthetase cDNA in Escherichia coli . Formation of catalytically active isoenzymes, and complementation of a glnA mutant; Bennett M et al.; Three cDNA clones encoding the closely related glutamine synthetase (GS) alpha, beta and gamma polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli . The GS expression plasmids correctly synthesised the recombinant alpha, beta and gamma polypeptides which then assembled into catalytically active homo-octameric isoenzymes . These isoenzymes behaved similarly to their native homologues on ion-exchange and gel-filtration chromatography . Furthermore, the alpha and gamma isoenzymes complemented a GS(glnA)-deficient mutant, thus demonstrating their physiological activity in E . coli . Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E . coli mutant . These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression . The production of active GS isoenzymes in E . coli facilitates the isolation and characterisation of the individual P . vulgaris homo-octameric GS isoenzymes. Biochemistry, 1990 Oct 23, 29(42), 9863 - 71 Engineering enzyme subsite specificity: preparation, kinetic characterization, and X-ray analysis at 2.0-A resolution of Val111Phe site-mutated calf chymosin; Strop P et al.; Comparison of the three-dimensional structure of bovine chymosin with the structures of homologous aspartic proteinases complexed with peptide inhibitors shows that Val111 in chymosin occupies a position between the specificity subsites S1 and S3 . A mutation corresponding to Val111 to Phe has been introduced in an intermediary plasmid construct of prochymosin by bridging its unique restriction sites by a synthetic mutant oligonucleotide duplex . A prochymosin fusion product was expressed in Escherichia coli in such a way that the extension and substitution of the propart does not interfere with the activation of the zymogen . After activation of the crude prochymosin, the enzyme was purified by affinity chromatography on Sepharose with V-dL-P-F-F-V-dL as ligand . This procedure provided large amounts of pure protein as judged by FPLC, the activity/protein ratio, and SDS-PAGE . The enzymatic properties were determined by using a variety of peptide substrates and inhibitors; KM values for the mutant enzyme were approximately twice those of the wild type, but the kcat values were little changed . The mutant enzyme was crystallized, X-ray data were collected to 2.0-A resolution by using a FAST area detector, and the structure was solved by using difference Fourier methods and refined to an R factor of 19.5% . The mutation leads to only local changes in conformation, with the phenylalanine side chain occupying part of the S1 and S3 pockets . This accounts for the increased KM of this mutant for a substrate with a large phenylalanine side chain at P1 . It is also consistent with the higher affinity of the mutant for an inhibitor with small side chains at P1 and P3 when compared with the wild-type enzyme. Biochim Biophys Acta, 1990 Oct 23, 1087(2), 219 - 25 Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor; Ribeiro A et al.; A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum . Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139 . Fusion protein specifically bound to human fibroblasts . The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell . Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts . It was demonstrated that partial fusion protein retained the functional activity of the native apo E . However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids . Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process. Biochim Biophys Acta, 1990 Oct 23, 1087(2), 205 - 11 Expression of active human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli and characterisation of the recombinant enzyme; Free ML et al.; A plasmid, pRG1, has been constructed by incorporating the coding sequence of human hypoxanthine-guanine phosphoribosyltransferase (HPRT) into the expression vector pT7-7 . Expression of human HPRT has been achieved in HPRT- Escherichia coli cells transformed with pRG1 and pGP1-2, as shown by: (1) exclusive labelling with {35S}methionine of a polypeptide with the same mobility as purified human HPRT on SDS-PAGE; and (2) measurement of HPRT activity after cell lysis . Although the majority of the recombinant HPRT was present in the particulate fraction after cell lysis and centrifugation, sufficient HPRT activity was present in the supernatant fraction to allow comparison with the HPRT purified from human erythrocytes and the activity in human haemolysates and lymphoblast lysates . Small differences in electrophoretic mobility on native gels were found between HPRT activity from these sources . The Km values of recombinant HPRT for the substrates 5-phospho-alpha-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT. Biochim Biophys Acta, 1990 Oct 23, 1087(2), 212 - 8 Nuclease S1 mapping of 16S ribosomal RNA in ribosomes; Rahman MA et al.; Escherichia coli 16S rRNA and 16S-like rRNAs from other species have several universally conserved sequences which are believed to be single-stranded in ribosomes . The quantitative disposition of these sequences within ribosomes is not known . Here we describe experiments designed to explore the availability of universal 16S rRNA sequences for hybridization with DNA probes in 30S particles and 70S ribosomes . Unlike previous investigations, quantitative data on the accessibility of DNA probes to the conserved portions of 16S rRNA within ribosomes was acquired . Uniquely, the experimental design also permitted investigation of cooperative interactions involving portions of conserved 16S rRNA . The basic strategy employed ribosomes, 30S subunits, and 16S rRNAs, which were quantitatively analyzed for hybridization efficiency with synthetic DNA in combination with nuclease S1 . In deproteinated E . coli 16S rRNA and 30S subunits, the regions 520-530, 1396-1404, 1493-1504, and 1533-1542 are all single-stranded and unrestricted for hybridization to short synthetic DNAs . However, the quantitative disposition of the sequences in 70S ribosomes varies with each position . In 30S subunits there appear to be no cooperative interactions between the 16S rRNA universal sequences investigated. Biochemistry, 1990 Oct 23, 29(42), 9956 - 62 Escherichia coli maltodextrin phosphorylase: contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of alpha-glucans; Schinzel R et al.; The role of Escherichia coli maltodextrin phosphorylase (EC 2.4.1.1) active site residues Glu637 and Tyr538 which line the sugar-phosphate contact region of the enzyme was investigated by site-directed mutagenesis . Substitution of Glu637 by an Asp or Gln residue reduced kcat to approximately 0.2% of wild-type activity, while the Km values were affected to a minor extent . This indicated participation of Glu637 in transition-state binding rather than in ground-state binding . 31P NMR analysis of the ionization state of enzyme-bound pyridoxal phosphate suggested that Glu637 is also involved in modulation of the protonation state of the coenzyme phosphate observed during catalysis . Despite loss of proposed hydrogen-bonded substrate contacts, the Tyr538Phe mutant enzyme retained more than 10% activity; the apparent affinity of all substrates was slightly decreased . Mutations at either site affected the error rate of the enzyme (ratio of hydrolysis/phosphorolysis) . Besides a role in substrate binding, the hydrogen-bond network of Tyr538 supports the exclusion of water from the active site. Mol Cell Endocrinol, 1990 Oct 22, 73(2-3), 83 - 91 Production of rat renin fusion protein in Escherichia coli and the preparation of renin-specific antisera; Campbell DJ et al.; Rat renin fused at the N-terminus with Sj26, a 26,000 Da glutathione S-transferase of Schistosoma japonicum, was expressed in Escherichia coli . The fusion protein was soluble and easily purified from crude bacterial lysates by affinity chromatography on immobilised glutathione . The fusion protein possessed no detectable renin activity . Antisera raised in rabbits against the fusion protein were specific for renin . These antisera did not bind soluble renin but bound immobilized renin . By immunoblotting, these antisera demonstrated rat renin to migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two broad bands of 33,000-34,000 and 35,000-37,000 Da . By immunocytochemistry of rat tissues, these antisera stained renin containing cells in the afferent arteriole of the glomerulus of the kidney, the zona glomerulosa of the adrenal and the corpus luteum of the ovary . However, apart from the afferent arteriole of the kidney, no immunoreactive renin was identified in blood vessels of the kidney, adrenal or ovary . These studies demonstrate that a recombinant renin fusion protein is a valuable alternative approach for the preparation of renin-specific antisera. J Mol Biol, 1990 Oct 20, 215(4), 549 - 57 Novel mutants of elongation factor G; Richter Dahlfors AA et al.; A novel mutant form of elongation factor G (EF-G) in Escherichia coli is described . This variant EF-G restricts reading frame errors by a factor of 2 to 3 in vivo at two different positions in a lacIZ fusion . In addition, a conventional fusidic acid resistant (fusR) mutant of EF-G was compared with the restrictive mutant . Both mutants were characterized in vitro in a steady-state poly(U) translating system . The data indicate that the restrictive EF-G variant has an altered interaction with the ribosome both in vivo and in vitro . In contrast, the conventional fusR variant is altered in its interaction with GTP, which is evident in vitro. J Mol Biol, 1990 Oct 20, 215(4), 497 - 510 Characterization of the Escherichia coli araFGH and araJ promoters; Hendrickson W et al.; The identities of two cloned, arabinose-inducible promoters were tested by hybridizing promoter DNA fragments with restriction digests of chromosomal DNA containing Mudlac phage inserted in either araFGH or in araE transport operons . One promoter, thought to be araE, is within 10(3) base-pairs of a Mudlac insertion in the araE gene . The second promoter was not found within several thousand base-pairs of either of the known transport genes . This promoter is now named araPJ (araJ) . The DNA sequence of the fragment containing the araFGH promoter was determined . The start site of transcription in vivo was located to within +/- 1 base-pair (bp) by S1 nuclease mapping . DNase 1 footprinting revealed that, in comparison with the araBAD and araE promoters, the locations of the AraC and cyclic AMP receptor protein (CRP) binding sites are reversed with CRP lying between AraC and RNA polymerase . The central location of the CRP binding site may explain why the araFGH promoter is more catabolite sensitive than the other ara promoters . AraC and CRP were both required for maximal transcription in vitro, although a low level of transcription was detected with CRP alone . S1 nuclease mapping of mRNA-DNA hybrids from the araJ promoter located the transcription start point to within #/- 3 bp, and demonstrates that the promoter is dependent upon AraC protein and CRP in vivo . DNase footprinting showed that the location of the AraC protein binding site on araJ is adjacent to the RNA polymerase site, as seen at the araBAD and araE promoters . Two CRP sites were observed; one is upstream from the AraC site and one is downstream from the transcription start site. J Mol Biol, 1990 Oct 20, 215(4), 493 - 5 Preliminary crystallographic analysis of the breakage-reunion domain of the Escherichia coli DNA gyrase A protein; Reece RJ et al.; The 64 x 10(3) Mr N-terminal breakage-reunion domain of the Escherichia coli DNA gyrase A protein was purified from an over-expressing strain . When complexed with the gyrase B protein, this truncated A protein has all of the enzymic properties of the full-length counterpart, although with reduced efficiency in some cases . The 64 x 10(3) Mr protein has been crystallized in several forms, a number of which were too small for crystallographic analysis . However, two forms grew to sufficient size for preliminary X-ray analysis . Both forms were tetragonal with a primitive lattice . One form (type I) had cell dimensions of a = b = 170 A, c = 145 A a space group of either P41212 (P43212) or P42212, and diffracted to 6 A resolution . The type II crystals had cell dimensions of a = b = 177 A, c = 175 A, a space group of P41212 (P43212) or P42212, and diffracted to at least 4.5 A resolution . Both crystal forms apparently contained four subunits (possibly a tetramer) in the asymmetric unit . We are attempting to increase the size and quality of these crystals. J Immunol Methods, 1990 Oct 19, 133(2), 227 - 33 A comparison of immunoblotting, flow cytometry and ELISA to monitor the binding of anti-lipopolysaccharide monoclonal antibodies; Nelson D et al.; This study was designed to assess the use of flow cytometry to observe the binding, under physiological conditions, of anti-lipopolysaccharide (LPS) monoclonal antibodies (mAbs) to whole bacteria, and to compare this with the more conventional whole cell ELISA and immunoblotting techniques . The bacteria consisted of two clinical isolates of E . coli 018:K1 and 06:K5 and two isogenic mutants of the 018 parent: a non-capsulate (018:K-) and a rough mutant (018rf) . Two cross-reactive anti-core mAbs and one 018 0-antigen-specific mAb were used . ELISA and flow cytometry showed that capsule and O-polysaccharide influenced the binding of mAbs to the bacteria, whilst the latter technique demonstrated that sub-populations existed . Immunoblotting showed the two anti-core mAbs to be different, one bound only to core which was not substituted with O-antigen, whilst the other bound both to substituted and unsubstituted core . This comparison for monitoring the binding of anti-LPS mAbs demonstrates the potential use of flow cytometry in bacterial cell surface research, and complements results obtained by ELISA and immunoblotting. Biochemistry, 1990 Oct 16, 29(41), 9667 - 77 Partial 1H NMR assignments of the Escherichia coli dihydrofolate reductase complex with folate: evidence for a unique conformation of bound folate; Falzone CJ et al.; Sequence-specific 1H assignments have been made for over 25% of the amino acid side chains of Escherichia coli dihydrofolate reductase complexed with folate by using a variety of two-dimensional techniques . Proton resonances were assigned by using a combination of site-directed mutagenesis and a knowledge of the X-ray crystal structure . Unique sets of NOE connectivities present in hydrophobic pockets were matched with the X-ray structure and used to assign many of the residues . Other residues, particularly those near or in the active site, were assigned by site-directed mutagenesis . The ability to assign unambiguously the proton resonances of these catalytically important residues allowed for extensive networks of NOE connectivities to follow from these assignments . As a consequence of these assignments, the orientation of the pterin ring of folate could be determined, and its conformation is similar to that of the productive dihydrofolate complex . Under these experimental conditions, only one bound form of the pterin ring could be detected. Biochemistry, 1990 Oct 16, 29(41), 9591 - 9 Folding of a predominantly beta-structure protein: rat intestinal fatty acid binding protein; Ropson IJ et al.; The equilibrium and kinetic properties of the unfolding-refolding transitions of Escherichia coli derived rat intestinal fatty acid binding protein have been examined using several different denaturants . This protein, which contains 2 tryptophans but no prolines or cysteines, has a predominantly beta-structure: its 10 antiparallel beta-strands are organized into 2 orthogonal sheets surrounding a large solvent-filled internal cavity . For urea and guanidine hydrochloride, the completely reversible transition was monitored by circular dichroism, absorbance, and fluorescence spectroscopy . Each of these data sets was best fit by a simple, two-state model involving only native and unfolded forms . However, linear extrapolation to determine the free energy of folding in the absence of denaturant resulted in different values for the free energy of folding depending upon which denaturant was used . When fluorescence was used to monitor the transition, the extrapolated free energy estimates for the two denaturants were markedly different: 10.03 +/- 0.24 kcal mol-1 for urea versus 5.22 +/- 0.33 kcal mol-1 for guanidine hydrochloride . The midpoints of these transitions were 5.51 and 1.36 M, respectively . The transition caused by either denaturant as monitored by circular dichroism and absorbance spectroscopy was virtually coincident with that monitored by fluorescence, further supporting the assignment of a two-state model for the equilibrium results . The addition of a 2-fold molar excess of ligand (oleate) increased the extrapolated estimates approximately 2.5 kcal mol-1 for both denaturants.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Oct 16, 29(41), 9522 - 31 Comparison of chemical reactivity, cytotoxicity, interstrand cross-linking and DNA sequence specificity of bis(platinum) complexes containing monodentate or bidentate coordination spheres with their monomeric analogues; Farrell N et al.; The properties of a new bis(platinum) complex containing two monodentate coordination spheres, {(trans-PtCl(NH3)2)2H2N(CH2)4NH2}Cl2 (1,1/t,t), are reported . Comparison is made with respect to chemical reactivity, in vitro biological activity in murine and tumor cells, DNA conformational changes, cross-linking efficiency, and sequence specificity between this complex and the previously reported complex containing two bidentate platinum atoms, {(Pt(mal)(NH3))2H2N(CH2)4NH2} (2,2/c,c), as well as with their respective monomeric analogues, {PtCl(dien)}Cl and cis-{PtCl2(NH3)2}(cis-DDP) . While both bis(platinum) complexes are active against cis-DDP-resistant cells, the monodentate bis(platinum) complex (1,1/t,t) has a lower resistance factor than the complex with bidentate coordination spheres (2,2/c,c) . More importantly, this property is repeated in a human ovarian carcinoma cell line . DNA-binding studies show that DNA interstrand cross-linking is more efficient for the 1,1/t,t complex . DNA sequencing studies employing the exonuclease activity of T4-polymerase demonstrate that there are a variety of binding sites; some are common to all complexes and some common to both bis(platinum) complexes, while the monodentate 1,1/t,t species also reacts at unique sites, not attacked by any of the other complexes studied . The circular dichroism of CT DNA modified by the 1,1/t,t complex is also unique and is not seen for any of the other agents. Biochemistry, 1990 Oct 16, 29(41), 9572 - 84 Activator-dependent preinduction binding of sigma-70 RNA polymerase at the metal-regulated mer promoter; Heltzel A et al.; Expression of the Tn21 mercury-resistance (mer) locus is controlled by the merR gene product, which represses mer structural gene (merTPCAD) transcription in the absence of mercuric ion {Hg(II)} and activates it in the presence of Hg(II) . In vivo DNA methylation of the mer regulatory region (merOP) shows that, with or without the inducer Hg(II), MerR strongly protects four guanine residues in a dyadic region located between the -10 and -35 hexamers of the structural gene promoter (PTPCAD) . Prior to induction by Hg(II), RNA polymerase is also bound at PTPCAD; occupancy of the uninduced promoter by RNA polymerase is dependent on MerR . Methylation and permanganate footprinting demonstrate that induction by Hg(II) results in MerR/Hg(II)-dependent promoter DNA melting in the -10 region of PTPCAD and in additional DNA structural distortions within the region of dyad symmetry . Thus, MerR fosters the binding of RNA polymerase to an inactive promoter, and upon induction, MerR/Hg(II) facilitates DNA distortions suitable for efficient formation of the active transcription complex. Biochemistry, 1990 Oct 16, 29(41), 9728 - 33 Purified internal G-domain of translational initiation factor IF-2 displays guanine nucleotide binding properties; Vachon G et al.; Translational initiation factor IF-2 is involved in a multistep pathway leading to the synthesis of the first peptide bond . IF-2 is a guanine nucleotide binding protein (G-protein) and catalyzes GTP hydrolysis in the presence of ribosomes . According to sequence homologies with other G-proteins, particularly EF-Tu, a theoretical model for the tertiary structure of the putative G-domain of IF-2 has been previously proposed {Cenatiempo, Y., Deville, F., Dondon, J., Grunberg-Manago, M., Hershey, J . W . B., Hansen, H . F., Petersen, H . U., Clark, B . F . C., Kjeldgaard, M., La Cour, T . F . M., Mortensen, K . K., & Nyborg, J . (1987) Biochemistry 26, 5070-5076} . A short fragment of IF-2 encompassing the putative G-domain was purified by limited proteolysis of a chimeric protein, synthesized from a gene fusion, between a segment of the IF-2 gene and lacZ . The N- and C-terminal sequences of this IF-2 peptide were characterized . Its calculated length is 181 amino acids and its molecular mass 19.4 kDa, whereas it migrates at 14 kDa in SDS-polyacrylamide gels . This segment of IF-2 can form binary complexes with GDP and can be cross-linked to GTP, therefore indicating that it really corresponds to the G-domain . However, in contrast to the situation described for the purified G-domain of EF-Tu, the IF-2 fragment did not hydrolyze GTP even in the presence of ribosomes . It is assumed that active centers of IF-2 located outside the G-domain are needed for the latter reaction.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1990 Oct 15, 272(1-2), 55 - 7 Importance of the G27-A43 mismatch at the anticodon stem of Escherichia coli tRNA(Thr2); Komine Y et al.; The tRNA(Thr2) isoacceptor of E . coli has a G-A mismatch at positions 27-43 . When the anticodon of this tRNA was converted to an amber anticodon (CUA), this tRNA showed suppressor activity in E . coli . Moreover, introduction of the base pair (G-C or U-A) at positions 27-43 of this suppressor tRNA reduced its suppressor activity . These results indicate that the G27-A43 mismatch is necessary for full function of tRNA(Thr2). Biochem Biophys Res Commun, 1990 Oct 15, 172(1), 357 - 63 An active site of growth hormone for eliciting the differentiation of preadipose 3T3-F442A cells to adipose cells; Uchida E et al.; In order to elucidate the active sites of growth hormone for eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes, four artificial mutant variants of human growth hormone (hGH) modified in the loop region of amino acid residues 54-74 were prepared in Escherichia coli by site-directed mutagenesis . Although the P59A (replacement of Pro59 with Ala) variant retained almost the same biological- and receptor binding-activity as hGH, the P61A (replacement of Pro61 with Ala) and the P59A-P61A (replacement of both Pro59 and Pro61 with Ala) both exhibited about half the activity, and the delta (62-67) variant (deletion of the residues 62-67) exhibited only about 0.1% the activity of those of intact hGH . The results suggest that Pro61 may be involved in formation of the active conformation of hGH, but Pro59 may not, and that the amino acid residues around 62-67 may be critical for the specific biological features of hGH. J Immunol, 1990 Oct 15, 145(8), 2440 - 7 Germinal center cells are a major IL-5-responsive B cell population in peripheral lymph nodes engaged in the immune response; Rabinowitz JL et al.; Germinal center formation and the development of B cell memory in lymphoid tissue is a T cell-dependent process . The specific B cell-T cell interactions, and/or cytokines, resulting in germinal center cell growth have not yet been identified . Germinal center B cells were separated from other lymph node (LN) B cells by panning on peanut agglutinin (PNA)-coated dishes . Resulting fractions enriched for PNA+ (germinal center) B cells, and the PNA- (other) LN B cells from immune SJL mice were assayed for proliferation in the presence of cytokines . PNA+ and PNA- B cells responded equally to IL-4 in the anti-mu co-stimulator assay . In contrast, PNA+ B cells responded to murine (r)IL-5 or human B cell growth factor in the dextran sulfate (DxS) co-stimulator assay, to a much greater degree than did PNA- B cells . The same results were obtained with PNA+ and PNA- cells from LAF1 mice . Unfractionated LN B cells from nonimmunized SJL or BALB/c mice did not respond to IL-5 with or without DxS . B cell populations from BALB/c mice such as from spleen and peritoneal cavity, which are known to be high in Ly-1+B cells, responded to IL-5 alone, and more dramatically, to IL-5 as a co-stimulator with DxS . Such populations of cells from SJL mice, which are known to contain low numbers of Ly-1+B cells, responded markedly less . These results are consistent with those of others which show that in nonimmunized mice, Ly-1+B cells are a major IL-5 responsive subpopulation . IL-1 enhanced the proliferation of PNA+ cells in response to rIL-5 and had no effect on PNA- cells . IL-4 and IL-5 did not enhance each other's effects as co-stimulators of proliferation . In contrast to PNA+ B cells from immune LN, B cells activated by Escherichia coli endotoxin exhibited no responses to rIL-5 . The present results indicate that in immune LN, PNA+, germinal center B cells constitute a prominent IL-5-responsive population. J Biol Chem, 1990 Oct 15, 265(29), 17980 - 7 Expression of actin in Escherichia coli . Aggregation, solubilization, and functional analysis; Frankel S et al.; Wild type Dictyostelium discoideum actin (42 kDa) and a truncated form of actin were expressed in Escherichia coli . Amino-terminal sequencing indicated that the truncated species was composed of two peptides, which were the result of internal translation initiation at Met-119 and Met-123 . After sonication or French press lysis, all of the actin was present in highly insoluble aggregates . When bacteria were lysed directly into Sarkosyl detergent, most of the actin was soluble, and greater than 50% remained soluble after Sarkosyl was removed . Full-length wild type actin was purified using DNase I affinity chromatography and gel filtration . This species was able both to polymerize and to bind myosin in an ATP-sensitive manner, indicating it was native . Affinity chromatography demonstrated that the truncated form of actin bound DNase I to the same extent as actin synthesized in eukaryotes, indicating the applicability of this approach to mutant forms of actin . Thus, lysis procedures utilizing Sarkosyl may prove useful in isolating some of the other proteins which are normally soluble but become insoluble after bacterial expression. J Biol Chem, 1990 Oct 15, 265(29), 17665 - 72 Variation of cofactor levels in Escherichia coli . Sequence analysis and expression of the pncB gene encoding nicotinic acid phosphoribosyltransferase; Wubbolts MG et al.; The pncB gene from Escherichia coli, which encodes nicotinic acid phosphoribosyltransferase (EC 2.4.2.11), was cloned on a 1.5-kilobase TaqI-EcoRI fragment . Its position on the E . coli chromosome was determined at 20.8 min between the asnS and pepN loci . The nucleotide sequence of the gene and the transcription and translation initiation sites were determined . Expression of pncB on a multicopy plasmid leads to a 25-fold increase in nicotinic acid phosphoribosyltransferase activity . Growth of E . coli in the presence of nicotinic acid leads to strong repression of nicotinic acid phosphoribosyltransferase activity, indicating that the cloned pncB sequence contains its own control sequences . It is shown that increased nicotinic acid phosphoribosyltransferase activity effects a 5-fold increase in the intracellular concentration of NAD . The cloned pncB gene can therefore be used as a tool to raise intracellular cofactor levels. J Biol Chem, 1990 Oct 15, 265(29), 17627 - 36 RNA primary sequence or secondary structure in the translational initiation region controls expression of two variant interferon-beta genes in Escherichia coli; Gross G et al.; Efficient expression in Escherichia coli (E . coli) of the human interferon-beta gene (IFN-beta) gene and of a chemically synthesized IFN-beta gene variant (506 base pairs; synIFN-beta) adapted to the E . coli codon usage, both fused to the E . coli atpE ribosome-binding site, is controlled either by primary sequence or by mRNA secondary-structure in the translational initiation region . High level expression of the natural human atpE/IFN-beta gene fusion is governed by the nucleotide composition preceding the initiator codon AUG . A single U----C exchange in the -2 or -1 position preceding the initiator codon AUG reduces the translational efficiency from 18% of total cellular protein to only 8% or 4%, respectively, while both U----C substitutions reduce IFN-beta expression below 1% . These sequence alterations interfere with efficient ribosome binding as revealed by toeprinting . They provide further evidence for the influence of the anticodon-flanking regions of tRNA(fMet) upon the initiation rate of translation . In contrast, translation of the synthetic variant atpE/synIFN-beta gene fusion is controlled by a moderately stable stem-loop structure (delta G = -4 kcal/mol; 37 degrees C) located within the coding region and overlapping the 30 S ribosomal subunit attachment site . That the stability of the hairpin interferes with the initiation of translation is inferred from site-directed mutagenesis and toeprint analyses . mRNA half-life in these variants is positively correlated with the rate of translation and involves two major endonucleolytic cleavage site 5'-upstream of the Shine-Dalgarno region. J Biol Chem, 1990 Oct 15, 265(29), 17493 - 8 Construction and properties of active chimeric enzymes between human aldolases A and B . Analysis of molecular regions which determine isozyme-specific functions; Kitajima Y et al.; To study the structure/function relationships of human aldolase isozymes, particularly isozyme-specific functions, we constructed Escherichia coli expression plasmids for six BA chimeric enzymes (BA34, BA108, BA137, BA212, BA306, and BA306*), each composed of the N-terminal side of isozyme B and the C-terminal side of isozyme A, and one BAB chimeric enzyme which contains a fragment of isozyme A (residues 213-306) inserted in between the N-terminal and the C-terminal fragments of isozyme B . They were transfected into E . coli, and the generated enzymes were characterized . This study reveals the following . (i) For isozyme A, the C-terminal Tyr-363 and the N-terminal region bearing isozyme group-specific sequences 1-3 and Lys-107 (the C-6 phosphate-binding site) are responsible for the higher catalytic activity toward fructose 1,6-bisphosphate, which is 7 times higher than that of aldolase B . Conversely, an internal region spanning positions 108-212 is required for the lower activity toward fructose 1-phosphate . (ii) For isozyme B, an internal sequence spanning positions 108-212 which includes some isozyme B-specific residues and a postulated C-1 phosphate-binding site (Lys-146 or Arg-148) is responsible for a higher catalytic activity toward fructose 1-phosphate, which is 8-10 times that of isozyme A . The more upstream sequence containing positions 1-107 is responsible for the lower catalytic activity toward fructose 1,6-bisphosphate . (iii) At least residues 212-306, composing a long stretch near the active-site Lys-229 and highly conserved among isozymes A, B, and C, may be required for the basal framework of the aldolase molecule to exhibit the activity common to the three isozymic forms. J Biol Chem, 1990 Oct 15, 265(29), 17451 - 5 Molecular cloning of wheat dihydrodipicolinate synthase; Kaneko T et al.; Four forms of dihydrodipicolinate synthase (DHDPS), which catalyzes the first reaction in the lysine-specific biosynthetic pathway in higher plants, were purified to homogeneity from a suspension culture of wheat (Triticum aestivum) . These polypeptides have similar N-terminal amino acid sequences . Two different cDNA clones were isolated by screening a wheat cDNA library with oligonucleotide probes based on these amino acid sequences . The predicted amino acid sequences indicate that both clones encode putative chloroplast transit peptides that have little homology to each other as well as the conserved mature protein portions of Mr 35,737 and 35,776 (94% identity) . Mature wheat DHDPS has 30% amino acid identity to Escherichia coli DHDPS . One of the cloned cDNAs, which had been fused to bacterial transcription/translation signals, genetically complemented a strain of E . coli that lacks endogenous DHDPS activity . Moreover, the expression of wheat DHDPS cDNA in wild-type E . coli increased the enzymatic activity 10-fold in the transformed bacteria. Gene, 1990 Oct 15, 94(2), 237 - 41 Expression in Escherichia coli of an immunoreactive visna virus transactivating protein; Watson G et al.; We have cloned the gene encoding the visna 1514 transactivating protein, Tat, into the Escherichia coli lambda pR expression plasmid, pRIT2T . Efficient synthesis of the protein A::Tat fusion protein was obtained in host strains which carried either wild-type or temperature-sensitive (ts) lambda repressors . However, constitutive synthesis of the fusion protein in these host strains resulted in selection against plasmids which synthesized the fusion protein . Efficient repression of the lambda pR promoter was obtained using a ts repressor gene carried on a multicopy plasmid . Synthesis of the fusion protein in this strain was efficient on induction, and reproducible after subculture . Antisera generated against the termini of visna Tat were used to demonstrate that the fusion protein retained the antigenicity of both the N and C termini of the transactivating protein. J Biol Chem, 1990 Oct 15, 265(29), 17826 - 36 Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I; Shuman S et al.; Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently . Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA . These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria . Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature . Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation . Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission . Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies . The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein . Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions . This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme . Noncovalent binding is therefore independent of competence in transesterification . It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes. J Biol Chem, 1990 Oct 15, 265(29), 17713 - 9 Identification of a critical cysteine in EcoRI DNA methyltransferase by mass spectrometry; Everett EA et al.; EcoRI DNA methyltransferase (MTase) is rapidly inactivated by N-ethylmaleimide with concomitant incorporation of 2 mol of N-ethyl{2-3H}maleimide/mol of functional monomer . Preincubation of the enzyme with either S-adenosylmethionine or DNA reduces the rate of activity loss, whereas preincubation with DNA and the S-adenosylmethionine analog sinefungin completely protects the enzyme from inactivation . An endo proteinase Glu-C digest of N-ethyl{2-3H}maleimide-modified enzyme was prepared and separated by high pressure liquid chromatography . Modified and unmodified cysteine-containing peptides were located and identified by radioactivity, mass spectrometry, and tandem mass spectrometry . In the absence of any ligands, cysteines 25, 116, and 223 are modified by N-ethylmaleimide; in the presence of DNA and sinefungin, Cys-223 is essentially unmodified . Thus, N-ethylmaleimide modification of Cys-223 in EcoRI DNA MTase is responsible for the loss of enzyme activity . Cys-223 is preceded by Asn, and this (or Cys-Asn) occurs with high frequency in adenine and cytosine (N-4) DNA MTases . Direct involvement of cysteine in methyl transfer reactions to adenine N-6 and cytosine N-4 is supported by the similarity of the reactions catalyzed by adenine N-6 and cytosine N-4 DNA MTases, the frequent presence of Asn-flanking Cys, and the importance of Cys-223 to EcoRI MTase function. J Biol Chem, 1990 Oct 15, 265(29), 17706 - 12 Carbohydrate-binding protein 35 . Isoelectric points of the polypeptide and a phosphorylated derivative; Cowles EA et al.; Purified carbohydrate-binding protein 35 (CBP35) and extracts of mouse cells containing CBP35 were analyzed by two-dimensional gel electrophoresis . Such an analysis on recombinant CBP35, obtained by expression of a cDNA clone in Escherichia coli, yielded a pI value of 8.7 . When extracts of mouse 3T3 cells were subjected to two-dimensional gel electrophoresis and immunoblotting, two spots were observed, corresponding to pI values of 8.7 and 8.2 . The pI 8.2 species represents post-translational modification of the CBP35 polypeptide (pI 8.7) by the addition of a single phosphate group . This conclusion is based on the finding that purified CBP35 contained a pI 8.2 species that was labeled with 32PO4 and could be converted to the unlabeled pI 8.7 species by alkaline phosphatase treatment . The phosphorylated (pI 8.2) form of CBP35 is found in both the cytosolic and nuclear fractions, whereas the unphosphorylated (pI 8.7) species is found exclusively in the nuclei . Quiescent populations of 3T3 fibroblasts (confluent monolayers or serum-starved sparse cultures) are characterized by the predominance of phosphorylated CBP35 . Stimulation of the same cells into the proliferative state resulted in an increase in the amount of the phosphorylated species; more dramatic, however, is the elevation of the level of the unphosphorylated form, which is barely detectable in quiescent cells. J Biol Chem, 1990 Oct 15, 265(29), 17680 - 7 A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis; Barra D et al.; An iron-containing superoxide dismutase has been purified from the protozoan Tetrahymena pyriformis . It has a molecular weight of 85,000 and is composed of four subunits of equal size . The tetramer contains 2.5 g atoms of ferric iron . Visible absorption and electron spin resonance spectra closely resemble those of other iron-containing superoxide dismutases . The amino acid sequence of the iron superoxide dismutase was determined . Each subunit is made up of 196 residues, corresponding to a molecular weight of 22,711 . Comparison of the primary structure with the known sequences of other iron-containing superoxide dismutases reveals a relatively low degree of identity (33-34%) . However, a higher percentage identity is found with mammalian manganese-containing superoxide dismutases (41-42%) . The amino acid sequence is discussed in consideration of residues that may distinguish iron from manganese or dimeric from tetrameric superoxide dismutases. Biochem J, 1990 Oct 15, 271(2), 415 - 20 cDNA structure and expression of calpactin, a peptide involved in Ca2(+)-dependent cell aggregation in sponges; Robitzki A et al.; Aggregation of cells of the marine sponge Geodia cydonium is mediated by an aggregation factor (AF) particle of Mr 1.3 X 10(8) . It is now reported that the AF particle is associated with calpactin, which was ascribed a role in the cell-adhesion process . In order to identify the sequence similarity to other members of the lipocortin family, the cDNA of sponge calpactin was cloned and found to display an 80% sequence similarity to vertebrate calpactin II but only a 47% similarity to calpactin I . The calpactin gene, which contains the consensus sequence coding for the amino acids G-T-D-E, was expressed in Escherichia coli and subsequently purified to a 37000-Mr polypeptide . Both the p32 and the p37 are provided with approximately two Ca2+ ions/molecule and the property to bind to phospholipids . The dissociation constant (calpactin-Ca2+) was in the absence of phospholipids in the range 500-700 microM-Ca2+ but in their presence about 20-30 microM-Ca2+ . On the basis of (i) inhibition studies with antibodies to calelectrin and (ii) competition experiments with soluble phospholipids (both chemically defined as well as total homologous membrane lipids) we conclude that the AF-associated calpactin and plasma-membrane-bound phospholipid(s) are involved in cell-cell aggregation in sponges. Biochem J, 1990 Oct 15, 271(2), 487 - 91 Investigation of putative active-site lysine residues in hydroxymethylbilane synthase . Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine; Hadener A et al.; A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx . 1000-fold over-expression of hydroxymethylbilane synthase (HMBS) . This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively) . All three modified enzymes are chromatographically separable from wild-type enzyme . Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp . cat./Kapp . m . Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5'-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues . Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q . The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS. Biochem Biophys Res Commun, 1990 Oct 15, 172(1), 211 - 6 Cloning and sequencing of the structural gene for the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1: evidence for two tryptophan residues involved in the active center; Chistoserdov AY et al.; In two independent clone libraries, clones were identified that hybridized with oligonucleotide probes based on N- or C-terminal polypeptide sequence of the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1 . Plasmids from all clones had in common a 5.2 kb Bam HI-HindIII DNA fragment . A 0.57 kb SacII-BclI subfragment that hybridized to the oligonucleotide probes was sequenced . Nucleotide sequence analysis coincided with polypeptide sequence data in the structural part of the small subunit with a single contradiction: amino acid 17 is Asp rather than Asn . The two amino acids that are involved in the active center which had not been determined from previous polypeptide sequencing proved to be tryptophans. Gene, 1990 Oct 15, 94(2), 195 - 9 Stem-loop structures at the 3' end of tobacco Rubisco large subunit mRNA; Akada S et al.; There are two inverted repeat nucleotide (nt) sequences, each capable of forming a stem-loop structure (sls) at the 3' end of the tobacco Rubisco large subunit mRNA (rbcL) . The smaller sls is followed by a larger sls . The in vivo functions of the 3' sls of the rbcL mRNA were characterized using the Escherichia coli system . S 1 mapping of the rbcL transcripts synthesized in E . coli revealed that the 3' end of a major transcript in the bacterial cell is almost identical to the 3' end of authentic chloroplast (cp) rbcL mRNA . This native 3' end is located 4 nt downstream from the larger sls for the cp mRNA and 6 nt for the bacterial transcript, respectively . Deletion experiments show that the larger sls is essential for producing the native 3' end of rbcL mRNA in E . coli . The sls do not function as an efficient transcription terminator but can stabilize upstream mRNA segments in vivo. J Biol Chem, 1990 Oct 15, 265(29), 17673 - 9 Osmotic control of proU transcription is mediated through direct action of potassium glutamate on the transcription complex; Prince WS et al.; Osmoregulated transcription from the proU promoter of Escherichia coli has been successfully reconstituted from purified components in a simple in vitro system consisting of plasmid DNA template, RNA polymerase, and nucleotides in the absence of any other protein factor . proU transcription is stimulated by addition of high concentrations of potassium glutamate, the ionic compound accumulated in vivo during hyperosmotic stress . Transcription from the nonosmoregulated promoters beta la, lac, and pepN is inhibited under the same conditions, demonstrating the specificity of potassium glutamate as an inducer of proU transcription . proU transcription requires a circular DNA template, but stable alterations in the degree of supercoiling are unnecessary for this potassium glutamate-dependent signaling . These results agree well with previous data obtained in an S-30 coupled transcription/translation system and suggest that physiological changes in the ionic composition of the intracellular millieu can regulate gene expression. Nucleic Acids Res, 1990 Oct 11, 18(19), 5787 - 92 Transcription of single-copy hybrid lacZ genes by T7 RNA polymerase in Escherichia coli: mRNA synthesis and degradation can be uncoupled from translation; Chevrier-Miller M et al.; In Escherichia coli transcription of individual genes generally requires concomitant translation, and thus the decay of mRNAs cannot be studied without the complication of translation . Here we have used T7 RNA polymerase to transcribe in vivo lacZ genes carrying ribosome binding sites of variable efficiency . We show that neither cell viability nor growth rate is affected by the T7-driven transcription of these genes, provided that they are present as single chromosomal copy . Furthermore, transcription is now completely uncoupled from translation, allowing large amounts of even completely untranslated mRNAs to be synthesized . Taking advantage of these features, we discuss the influence of the frequency of translation upon the processing and degradation of the lac message. Nucleic Acids Res, 1990 Oct 11, 18(19), 5713 - 6 A single-stranded DNA-binding protein promotes the binding of the purified oestrogen receptor to its responsive element; Mukherjee R et al.; The purified human oestrogen receptor (hER) does not form a detectable complex with an oestrogen responsive element (ERE) under conditions where hER-ERE complexes are readily formed with crude extracts from Hela or yeast cells expressing the hER . This indicates that other factor(s) are necessary for ER-ERE binding . Such a ER DNA binding stimulatory factor (DBSF) has been purified from the yeast Saccharomyces cerevisiae . It is a 45 kDa single-stranded DNA-binding protein (SSB) which cannot be substituted for by the purified E . coli SSB. Nucleic Acids Res, 1990 Oct 11, 18(19), 5673 - 6 Rat DNA polymerase beta gene can join in excision repair of Escherichia coli; Ohnishi T et al.; Though DNA polymerase I (poll) of Escherichia (E.) coli is understood to play a role in repair synthesis of excision repair, it is still obscure whether DNA polymerase beta (pol beta) plays a similar role in eukaryotic cells . To estimate the role of pol beta in excision repair processes, we inserted the rat pol beta gene into several mutant E . coli defective in a diverse set of enzymatic activities of poll . UV resistance was seen only when the 5'----3' exonuclease (exo) activity of poll molecules remained . Therefore it is suggested that 5'----3' exo activity as well as pol beta activity are essential for repair synthesis of excision repair in eukaryotic cells. Nucleic Acids Res, 1990 Oct 11, 18(19), 5659 - 65 In vivo and in vitro analyses of an intron-encoded DNA endonuclease from yeast mitochondria . Recognition site by site-directed mutagenesis; Sargueil B et al.; The pal 4 nuclease (termed I-Sce II) is encoded in the group I al 4 intron of the COX I gene of Saccharomyces cerevisiae . It introduces a specific double-strand break at the junction of the two exons A4-A5 and thus mediates the insertion of the intron into an intronless strain . To define the sequence recognized by pal 4 we introduced 35 single mutations in its target sequence and examined their cleavage properties either in vivo in E . coli (when different forms of the pal 4 proteins were artificially produced) or in vitro with mitochondrial extracts of a mutant yeast strain blocked in the splicing of the al 4 intron . We also detected the pal 4 DNA endonuclease activity in extracts of the wild type strain . The results suggest that 6 to 9 noncontiguous bases in the 17 base-pair region examined are necessary for pal 4 nuclease to bind and cleave its recognition site . We observed that the pal 4 nuclease specificity can be significantly different with the different forms of the protein thus explaining why only some forms are highly toxic in E . coli . This study shows that pal 4 recognition site is a complex phenomenon and this might have evolutionary implications on the transfer properties of the intron. Nucleic Acids Res, 1990 Oct 11, 18(19), 5633 - 6 T7 endonuclease I resolves Holliday junctions formed in vitro by RecA protein; Muller B et al.; T7 endonuclease I is known to bind and cleave four-way junctions in DNA . Since these junctions serve as analogues of Holliday junctions that arise during genetic recombination, we have investigated the action of T7 endonuclease I on recombination intermediates containing Holliday junctions . We find that addition of T7 endonuclease I to strand exchange reactions catalysed by RecA protein of Escherichia coli leads to the formation of duplex products that correspond to 'patch' and 'splice' type recombinants . Resolution of the recombination intermediates occurs by the introduction of nicks at the site of the Holliday junction . The recombinant molecules contain 5'-phosphate and 3'-hydroxyl termini which may be ligated to restore the integrity of the DNA. Nucleic Acids Res, 1990 Oct 11, 18(19), 5625 - 32 The involvement of base 1054 in 16S rRNA for UGA stop codon dependent translational termination; Hanfler A et al.; The deletion of the highly conserved cytidine nucleotide at position 1054 in E . coli 16S rRNA has been characterized to confer an UGA stop codon specific suppression activity which suggested a functional participation of small subunit rRNA in translational termination . Based on this structure-function correlation we constructed the three point mutations at site 1054, changing the wild-type C residue to an A, G or U base . The mutations were expressed from a complete plasmid encoded rRNA operon (rrnB) using a conditional expression system with the lambda PL-promoter . All three altered 16S rRNA molecules were expressed and incorporated into 70S ribosomal particles . Structural analysis of the protein and 16S rRNA moieties of the mutant ribosomes showed no differences when compared to wild-type particles . The phenotypic analysis revealed that only the 1054G base change led to a significantly reduced generation time of transformed cells, which could be correlated with the inability of the mutant ribosomes to specifically stop at UGA stop codons in vivo . The response towards UAA and UAG termination codons was not altered . Furthermore, in vitro RF-2 termination factor binding experiments indicated that the association behaviour of mutant ribosomes was not changed, enforcing the view that the UGA stop codon suppression is a direct consequence of the rRNA mutation . Taken together, these results argue for a direct participation of that 16S rRNA motif in UGA dependent translational termination and furthermore, suggest that termination factor binding and stop codon recognition are two separate steps of the termination event. Nature, 1990 Oct 11, 347(6293), 572 - 5 Altered protein conformation on DNA binding by Fos and Jun; Patel L et al.; The protein products of the c-fos and c-jun proto-oncogenes (Fos and Jun, respectively) form a heterodimeric protein complex that interacts with the activator protein-1 (AP-1) binding site and regulates gene transcription in response to extracellular stimuli . Protein dimerization is mediated primarily by a coiled-coil-like structure termed the leucine-zipper and DNA binding occurs primarily through regions of each protein rich in basic amino acids that contact both strands of the AP-1 site . The precise nature of the protein-DNA interaction is unknown as studies concerned with dimerization and DNA binding by Fos and Jun have relied on indirect methods to investigate protein-protein-DNA interactions . Here we have developed assay systems using fluorescence spectroscopy and circular dichroism to monitor dimerization and DNA binding directly . The results indicate that the interaction of Fos and Jun with DNA results in an altered conformation of the protein dimers and an increased alpha-helical content . These techniques may have general application in studies concerning the interaction of transcriptional regulatory proteins with specific DNA target sequences. Biochemistry, 1990 Oct 9, 29(40), 9395 - 402 Glycine to alanine substitutions in helices of glyceraldehyde-3-phosphate dehydrogenase: effects on stability; Ganter C et al.; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken was expressed in and purified from Escherichia coli . To investigate the physical basis of possible protein stabilization strategies, the effect of substitutions of glycine residues by alanine in helical regions was determined . One Gly to Ala substitution (G316A) located in the central core of the subunit was found to strongly stabilize the protein, while the other mutations are neutral or destabilize the protein . The effect seen for the stabilizing mutant in irreversible heat denaturation correlates with the first transition in folding equilibrium experiments that is observable by fluorescence, but not with the one detected by circular dichroism measurements or in dilution-induced dissociation experiments . The stabilizing effect of a Gly to Ala substitution therefore does not seem to be caused by an entropic effect on the unfolded state . Rather, an internal cavity is filled by the substitution G316A, probably stabilizing the native state . In large oligomeric proteins, imperfect packing may be a frequent cause of limited stability. Biochemistry, 1990 Oct 9, 29(40), 9352 - 7 Purification of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase large subunits suitable for reconstitution and assembly of active L8S8 enzyme; Lee B et al.; Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Anacystis nidulans was reconstituted in vitro from extracts of Escherichia coli strains that separately express large and small subunits . This reconstitution system was shown to be useful for monitoring the appearance of dissociated or fractionated subunit preparations . Recombinant large subunits were purified to a state of homogeneity and retained reconstitution capacity in the presence of added small subunits . The purified large subunits appeared to be in the form of an octamer, probably an L8 structure, and showed 0.15% of the carboxylase activity of the purified L8S8 enzyme . Purified large subunit octamers are disrupted by nondenaturing PAGE; however, the octamer is stable to electrophoresis in the presence of exogenous protein. Mol Cell Biochem, 1990 Oct 15-Nov 8, 98(1-2), 95 - 9 Crystal structure of chicken liver basic fatty acid-binding protein at 2.7 A resolution; Scapin G et al.; The three-dimensional structure of chicken liver basic fatty acid-binding protein has been determined at 2.7 A resolution by X-ray crystallography . Phases were calculated using the multiple isomorphous replacement procedure and a preliminary model was built . This model, with an initial R-factor of 0.57, was then improved by a cycle of refinement by simulated annealing which brought the R factor down to 0.32 . The protein is structured as a compact 10-stranded-beta-barrel which encapsulates a residual electron density that can be interpreted as a fatty acid molecule . The NH2-terminus portion of the molecule contains two short alpha-helices . The structure of this liver protein appears very similar to that of the Escherichia coli derived rat intestinal FABP recently determined by X-ray diffraction methods. Mol Cell Biochem, 1990 Oct 15-Nov 8, 98(1-2), 75 - 9 Expression of fatty acid-binding protein from bovine heart in Escherichia coli; Oudenampsen E et al.; The coding part of the cDNA of cardiac fatty acid-binding protein (cFABP) from bovine heart was cloned into the vector pKK233-2 . After induction with isopropyl-beta-D-thiogalactopyranoside cFABP was found in a soluble form in the cytosol of plasmid transformed E . coli amounting up to 5.7% of the soluble protein . cFABP was detected after SDS-polyacrylamide gelelectrophoresis and/or isoelectric focusing and Western blot by immuno-staining and was determined quantitatively by a solid phase enzyme-linked immuno sorbent assay . The cFABP produced by bacteria binds oleic acid with high affinity as shown by comigration of protein and ligand in both gelfiltration and isoelectric focusing . cFABP was purified from bacterial lysates to near homogeneity and resolved into four isoproteins. J Theor Biol, 1990 Oct 7, 146(3), 395 - 406 Analysis of a model for minichromosome segregation in Escherichia coli; Schurr T et al.; The present article contains a theoretical, quantitative analysis of the implications of the Helmstetter-Leonard model (1987, J . molec . Biol . 197, 195-204.) for the segregation of chromosomal DNA in Escherichia coli, on the expected copy-number distribution of minichromosomes in a culture in steady-state exponential growth . According to the model, two determinants are involved in the mechanism of chromosome segregation: a partition system that assures the equal allotment of chromosomes between daughter cells at cell division, and a locus within the minimal oriC region that specifies the attachment site of the chromosomes to the cell envelope at initiation of replication . There are many parameters that must be taken into account in such a study, and since some of them are probabilistic in nature, a strictly analytical approach is not feasible and we had to resort to computer simulation . A wide range of parameter values was tested, in all combinations . The minichromosome copy-number distributions obtained all had a prominent mode equal to the number of oriC binding sites and their main features were determined essentially by that and very little by any of the other parameters of the model . In order to avoid the unrealistic situation in which this one feature completely dominates the results, the original model was modified so that each individual minichromosome is no longer required to replicate during every cell generation, by introducing a limit to the number of unsuccessful attempts to locate a suitable binding site . The copy-number distributions predicted by this version of the model are quantitatively and qualitatively very different and depend on all the components of the model . The simulation results are sufficiently well-behaved to allow consideration as to whether a particular empirical minichromosome copy-number distribution--when such data become available--could in fact be governed by the proposed model; it may even be possible to get a rough estimate for the different parameters involved. J Biol Chem, 1990 Sep 25, 265(27), 16102 - 7 Site-directed mutagenesis of rat liver S-adenosylhomocysteinase . Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding; Gomi T et al.; Aspartic acid 244 that occurs at the putative NAD(+)-binding site of rat liver S-adenosylhomocysteinase was replaced by glutamic acid by oligonucleotide-directed mutagenesis . The mutant enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Gel permeation chromatography showed that the purified mutant enzyme was a tetramer as is the wild-type enzyme . In contrast to the wild-type enzyme, which possesses 1 mol of tightly bound NAD+ per mol of enzyme subunit, the mutant enzyme had only 0.05 mol of NAD+ but contained about 0.6 mol each of NADH and adenine per mol of subunit . The mutant enzyme, after removal of the bound compounds by acid-ammonium sulfate treatment, exhibited S-adenosylhomocysteinase activity when assayed in the presence of NAD+ . From the appearance of activity as a function of NAD+ concentration, the enzyme was shown to bind NAD+ with a Kd of 23.0 microM at 25 degrees C, a value greater than 280-fold greater than that of the wild-type enzyme . In the presence of a saturating concentration of NAD+, the mutant enzyme showed apparent Km values for substrates similar to those of the wild-type enzyme . Moderate decreases of 8- and 15-fold were observed in Vmax values for the synthetic and hydrolytic directions, respectively . These results indicate the importance of Asp-244 in binding NAD+, and are consistent with the idea that the region of S-adenosylhomocysteinase from residues 213 to 244 is part of the NAD+ binding site . This region has structural features characteristic of the dinucleotide-binding domains of NAD(+)- and FAD-binding proteins (Ogawa, H., Gomi, T., Mueckler, M . M., Fujioka, M., Backlund, P.S., Jr., Aksamit, R.R., Unson, C.G., and Cantoni, G.L . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 719-723). Eur J Biochem, 1990 Oct 5, 193(1), 91 - 5 Subcellular localisation of dihydrolipoamide dehydrogenase and detection of lipoic acid in bloodstream forms of Trypanosoma brucei; Jackman SA et al.; In the long-slender bloodstream form of Trypanosoma brucei, the enzyme dihydrolipoamide dehydrogenase exists in the absence of the 2-oxo-acid dehydrogenase complexes of which it is normally a component, and appears to be associated with the plasma membrane of the organism {Danson, M . J., Conroy, K., McQuattie, A . & Stevenson, K . J . (1987) Biochem . J . 243, 661-665} . In the present paper, a complete subcellular fractionation of T . brucei has been carried out and, by comparison with marker enzymes, it is confirmed that the dihydrolipoamide dehydrogenase is indeed associated with the plasma membrane . In addition, we now provide evidence that the distribution of the enzyme is over the whole surface of the membrane, including the flagellar pocket region, and that the enzyme is not found in any other cellular fraction . A study of the latency of the enzyme suggests that it is located on the cytoplasmic surface of the plasma membrane . The discovery of the presumed substrate of dihydrolipoamide dehydrogenase, lipoic acid, is reported for T . brucei . Using a biological assay involving a strain of Escherichia coli that requires lipoic acid for growth, we have found that acid-hydrolysed extracts of T . brucei contain 1.7 (+/- 0.2) ng of the cofactor/mg protein . The chemical nature of the lipoic acid was confirmed by gas chromatography/mass spectrometry. J Biol Chem, 1990 Oct 5, 265(28), 17348 - 54 Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease; DiIanni CL et al.; The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site . This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1) . To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP) . Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa . The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar . Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo . Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity. J Biol Chem, 1990 Oct 5, 265(28), 17341 - 7 Translocation of ProOmpA possessing an intramolecular disulfide bridge into membrane vesicles of Escherichia coli . Effect of membrane energization; Tani K et al.; In the absence of delta mu H+, the in vitro translocation of proOmpA resulted in the stable accumulation of a possible translocation intermediate in addition to a transiently accumulating one . The stable intermediate was detected on a polyacrylamide gel as two proteinase K-resistant bands corresponding to a molecular weight of about 28,000 . The appearance of the bands was appreciably enhanced when proOmpA was oxidized with ferricyanide . No mature OmpA appeared . When proOmpA reduced with dithiothreitol was used, on the other hand, the bands did not appear at all . Upon the replacement of Cys302 of OmpA with Gly, the intermediate accumulation was abolished . The proOmpA treated with dithiothreitol was labeled with N-{3H}-ethylmaleimide, whereas that treated with ferricyanide was not . The ferricyanide-treated proOmpA was translocated into membrane vesicles in the presence of delta mu H+ . The mature OmpA thus translocated and processed was not labeled with N-{3H}ethylmaleimide . It is concluded that proOmpA possessing the Cys290-Cys302 disulfide bridge can be translocated without cleavage of the bridge, when delta mu H+ is imposed . The accumulation of the disulfide bridge-containing intermediate was ATP-dependent, whereas its conversion to the translocated mature form was not blocked in the presence of adenosine 5'-(beta, gamma-imino)triphosphate . It is concluded that the early and late stages of the translocation reaction require ATP and delta mu H+ differently. J Biol Chem, 1990 Oct 5, 265(28), 17215 - 21 Characterization of Escherichia coli cells deficient in 1-acyl-sn-glycerol-3- phosphate acyltransferase activity; Coleman J; A mutant of Escherichia coli K-12 defective in 1-acyl-sn-glycerol-3-phosphate acyltransferase has been isolated . At the permissive temperature for growth, 30 degrees C, 20% of the total cellular glycerophospholipids is 1-acyl-sn-glycerol-3-phosphate, as identified by mass spectral analysis and proton NMR . This percentage of 1-acyl-sn-glycerol-3-phosphate rises to about 30% when the temperature of the culture is shifted to 42 degrees C . This increase is primarily at the expense of phosphatidylethanolamine . Extracts from cells harboring the plsC mutation have no detectable 1-acyl-sn-glycerol-3-phosphate acyltransferase activity . The fatty acid composition of the accumulated 1-acyl-sn-glycerol-3-phosphate is about 60% cis-vaccenate and 40% palmitate, with no detectable amounts of palmitoleate or other fatty acids, consistent with the known fatty acid composition of the sn-1 position of glycerophospholipids . The isolation of this gene, plsC, completes the list of genes known to be required for the synthesis of the major glycerophospholipids in E . coli. J Biol Chem, 1990 Oct 5, 265(28), 17050 - 4 recA protein filaments bind two molecules of single-stranded DNA with off rates regulated by nucleotide cofactor; Zlotnick A et al.; To probe the role of nucleotide cofactor in the binding of single-stranded DNA to recA protein, we have developed a sedimentation assay using 5'-labeled 32P-poly(dT).recA.poly(dT) complexes sediment quantitatively when centrifuged at 100,000 x g for 45 min, whereas free poly(dT) remains in the supernatant . In the presence of ATP, between 6 and 7 bases cosediment per recA monomer; but when ADP is present or in the absence of added nucleotide cofactor, only 3-3.5 bases/recA monomer cosediment . In competition experiments in which recA.32P-poly(dT) complexes are incubated with unlabeled poly(dT), we again find 3-3.5 bases of labeled poly(dT) cosedimenting per recA monomer when no nucleotide cofactor is present . However, when the same experiment is performed with ATP, only half of the expected 6-7 bases of labeled poly(dT) remain bound to the DNA, demonstrating that half of the poly(dT) in the complex exchanges rapidly with free poly(dT), whereas the other half equilibrates slowly, like poly(dT) in the absence of nucleotide . The rate of exchange of the second more tightly bound poly(dT) is accelerated when ADP is present . Our observations are rationalized by a model in which each recA protein helical filament binds two strands of poly(dT) with a stoichiometry of 3-3.5 bases/recA monomer/strand. Cell, 1990 Oct 5, 63(1), 203 - 11 Primary structure and functional expression of rat and human stem cell factor DNAs; Martin FH et al.; Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated . Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated . Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells, truncated forms of the rat and human proteins have been expressed in E . coli and mammalian cells and have been shown to possess biological activity . SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures . SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size. J Biol Chem, 1990 Oct 5, 265(28), 17167 - 73 The origin binding protein of herpes simplex virus 1 binds cooperatively to the viral origin of replication oris; Elias P et al.; The origin binding protein (OBP) or herpes simplex virus 1 has been expressed in Escherichia coli and used to study the role of multiple OBP binding sites in the herpes simplex virus #1 origin of replication, oris . Our results showed that the sequence CGTTCGCACTT was required for the binding of OBP to duplex DNA with high affinity . The minimal oris contains three repeats of this sequence or close derivatives thereof . Filter binding experiments have demonstrated that specific binding occurs to two of these repeats, box I and box II . An investigation using the DNase I footprinting technique revealed that the binding of OBP to box I and box II was cooperative and led to the formation of a highly organized complex in which the entire oris sequence was induced . We observed furthermore that the AT-rich sequence of the oris dyad was readily accessible to macromolecules even in the OBP.oris complex . The DNase I cleavage pattern of this sequence was, however, altered radically, indicating that a significant conformational change had occurred . A tentative structural model for the OBP-oris interaction is discussed on the basis of these observations. J Biol Chem, 1990 Oct 5, 265(28), 17078 - 83 Differential ATP requirements distinguish