Appl Parasitol, 1993 Nov, 34(4), 235 - 44 Multiple B-cell epitopes in a recombinant GRA2 secreted antigen of Toxoplasma gondii; Murray A et al.; Two cDNA clones encoding the GRA2 (G.P.28.5) secreted antigen of Toxoplasma gondii were expressed in Escherichia coli as glutathione-S-transferase fusion polypeptides . A high level of expression was obtained for the first clone expressing the 59C-terminal amino acids of GRA2 . The other one was an open-reading-frame of 212 amino acids containing the entire GRA2 cDNA . By ELISA, IgG antibodies directed against the 59aa recombinant polypeptide were detected in 33/44 (75%) sera from patients chronically infected with T . gondii and in 19/23 (82.6%) sera derived from patients with acute, primary toxoplasmosis . 10 of the 11 "chronic" sera which were negative by the 59aa ELISA were tested in a immunoblot against the 212aa open-reading-frame of GRA2: 8/10 were positive . A peptide representing the 15 C-terminal amino acids of GRA2 has been shown to contain the epitope recognized by a mouse monoclonal antibody (TG17-179) . The reactivity of human sera with the 59aa recombinant polypeptide was inhibited to varying degrees when the sera were co-incubated with this peptide . Twelve chronic sera showed a range of inhibition from 8 to 100% and twelve acute sera an inhibition range of 15 to 90% . This suggests that the 15aa C-terminal peptide contains an epitope recognized in both the acute and chronic phases of infection and that other major epitope(s) exist in the 59aa C-terminal region of GRA2 . As a conclusion, the recombinant GRA2 protein appears to contain at least three B-cell epitopes. Nippon Ronen Igakkai Zasshi, 1993 Nov, 30(11), 953 - 7 {Neutrophil functions during treatment with granulocyte colony-stimulating factor (G-CSF) in the elderly with non-Hodgkin's lymphoma: including two patients accompanied with interstitial pneumonitis during the treatment with G-CSF}; Katoh M et al.; We examined neutrophil functions in seven elderly patients with non-Hodgkin's lymphoma before and during treatment with granulocyte colony-stimulating factor (G-CSF) at the neutropenic stage after combination chemotherapy . Subcutaneous injection of 75 micrograms/d of G-CSF produced by E . coli was started when the neutrophil count decreased less than 1,500/microliter, and continued until the neutrophil count increased to about 10,000/microliter . The phagocytic activity of neutrophils from the elderly on day 3 of G-CSF treatment was markedly enhanced; 1,129.9 +/- 403 ps/100 PMNs, which was 185.7 +/- 31.4% (p < 0.001) as compared with that before G-CSF treatment . The neutrophil alkaline phosphatase (NAP) activity was also enhanced on day 3; 398.3 +/- 48 score, which was 135.2 +/- 5.1% (p < 0.001) as compared with that before G-CSF treatment . Two patients developed interstitial pneumonitis during or shortly after the treatment with G-CSF . Interstitial pneumonitis suddenly developed when their neutrophil count was increased, and the phagocytic activity and NAP activity recovered . The phagocytic activity of neutrophils from them was enhanced to 1,090 +/- 26 ps/100 PMNs and 772 ps/100 PMNs during the treatment with G-CSF, as compared with that before G-CSF treatment of 644 +/- 29 ps/100 PMNs and 465 +/- 69 ps/100 PMNs, respectively . The NAP activity was also enhanced to 372 from 264 . One patient suffered from transient pulmonary dysfunction during the treatment with G-CSF . His neutrophil count was more than 13,000/microliter, and the phagocytic activity enhanced to 949 +/- 105 ps/100 PMNs . Dyspnea with suppressed PaO2 recovered reversibly after cessation of G-CSF.(ABSTRACT TRUNCATED AT 250 WORDS) Hum Mol Genet, 1993 Nov, 2(11), 1857 - 60 Molecular basis of cystathionine beta-synthase deficiency in pyridoxine responsive and nonresponsive homocystinuria; Hu FL et al.; Cystathionine beta-synthase (CBS) deficiency is an autosomal recessive disorder associated with multisystem clinical disease . We analyzed PCR amplified products from patients' RNA and genomic DNA . Direct sequencing of the entire coding region of the CBS gene revealed a G-919 to A transition in exon 8, resulting in replacement of Gly 307 by Ser (G307S) in the protein . The mutation was detected in one allele of patient L171 of French/Scottish ancestry and in both alleles of patient L198 of Irish ancestry . Amplifying and sequencing exon 8 from the genomic DNA showed that both parents of L198 were heterozygotes for G307S . The pathogenicity of the mutation was demonstrated in an expression experiment . The mutant protein was apparently stable in E.coli extracts and lacked catalytic activity . Sequencing of exon 8 revealed the G307S mutation in five additional families . All patients have pyridoxine nonresponsive homocystinuria . We have now observed this mutation in 9 of 52 apparently unrelated alleles of varied ethnic backgrounds . All 9 are from patients with Celtic (Irish/English/Scottish/French) ancestry in either one or both parents . The G307S mutation was detected in 50% (9 of 18) of the Celtic alleles in our series . The second mutation found in exon 8 is the I278T mutation, which was described previously in one allele of a pyridoxine responsive patient . This missense mutation was detected in one allele of a pyridoxine nonresponsive patient and in both alleles of a pyridoxine responsive patient . The latter suggests that I278T is probably associated with pyridoxine responsiveness. J Gen Microbiol, 1993 Nov, 139 ( Pt 11), 2641 - 7 Antigenic and immunogenic differences in lipopolysaccharides of Escherichia coli J5 vaccine strains of different origins; Appelmelk BJ et al.; Escherichia coli strain J5 mutants of various origins have often been used as vaccines for induction of cross-reactive, cross-protective antibodies directed against the lipopolysaccharide (LPS) core region . The antigenic composition of LPS from J5 strains of different origin, i.e . strains J5(U), J5(UK), J5(2877) and J5(a), was investigated using monoclonal antibodies (mAbs) reactive only with LPS of a given chemotype, i.e . one specific for the incomplete E . coli core of the Rc chemotype, a second mAb reactive only with the E . coli R3 complete core, and a third specific for the O-antigen of E . coli serovar O111 . The LPS of strains J5(U) and J5(a) is almost exclusively composed of LPS of the Rc chemotype, LPS of the J5(UK) strain is composed of Rc LPS and R3 complete core, while LPS of the J5(2877) strain contains Rc, R3 complete core and O-antigen . Growth of the bacteria in medium supplemented with galactose led to increased expression of complete core . The immune responses to the various strains were investigated . Antiserum to the J5 strain expressing the largest amount of R3 core {J5(UK)} had much higher anti-R3 LPS antibody titres compared to antiserum to the other strains . mAb 53, representative of the anti-R3 response to J5 strains containing complete core, bound to those E . coli LPS expressing the R3 core . Thus, the R3 LPS, present in some J5 vaccine strains is at least partially responsible for some of the cross-reactivities exhibited by some anti-J5 antisera.(ABSTRACT TRUNCATED AT 250 WORDS) J Neurosci Res, 1993 Nov 1, 36(4), 472 - 9 Selective expression of foreign genes in glioma cells: use of the mouse myelin basic protein gene promoter to direct toxic gene expression; Miyao Y et al.; We have previously demonstrated that retrovirus-mediated genes were transferred to mouse glioma cells in a meningeal gliomatosis model (Yamada et al.: Japanese Journal of Cancer Research 83:1244-1247, 1992) . This retrovirus vector contains the Escherichia coli . beta-galactosidase (beta-gal) gene as a marker for integration of the lacZ gene, which is controlled by the SV40 early promoter . We investigated whether lacZ genes could be specifically controlled in mouse glioma cells by glial-specific promoters, including the 2.5 kb 5' flanking region of the mouse glial fibrillary acidic protein (GFAP) gene, the 1.3 kb 5' flanking region of the myelin basic protein (MBP) gene, and the 1.5 kb 5' flanking region of the myelin proteolipid protein (PLP) gene . Psi-2 packaging cells were transfected with each retrovirus vector (GFAP promoter-, MBP promoter-, and PLP promoter-lacZ) and the infectious virus particles were recovered from the supernatants . Blue staining for beta-gal was detected in various fibroblast, myeloma, and glioma cell lines transduced with the retrovirus BAG vector . On the other hand, blue staining was only detected in glioma cells after transduction with the lacZ gene-bearing retrovirus controlled by glial-specific promoters . The strongest promoter activity was detected after transduction with the retrovirus in which the MBP promoter controlled the lacZ gene . Mouse glioma cells transduced with retrovirus containing the MBP promoter directing the herpes simplex virus type 1 thymidine kinase (HTK) gene were extremely sensitive to ganciclovir, while the parental cells and cells transduced with retrovirus containing the lacZ gene were not sensitive to ganciclovir.(ABSTRACT TRUNCATED AT 250 WORDS) Cell Mol Biol (Noisy-le-grand), 1993 Nov, 39(7), 783 - 9 Lipopolysaccharide (LPS) binding in subpopulations of mouse peritoneal macrophages; Kriegsmann J et al.; Lipopolysaccharide-binding sites of mouse peritoneal macrophages were demonstrated by means of immunogold technique . Resident peritoneal macrophages identified by peroxidatic activity in the nuclear envelope and in the rough endoplasmic reticulum show moderate and constant specific binding of bacterial lipopolysaccharide from E . coli (026:B6) to cell surface structures . Monocyte-derived macrophages with peroxidatic activity in cytoplasmic granules are characterized by a broad binding pattern . A high percentage of monocyte-derived macrophages bind large amounts of LPS-gold particles whereas some others bind only less lipopolysaccharide . This is a further hint for the existence of monocyte subpopulations . The different binding patterns of LPS after fixation and the inhibitor-ability of this binding supports the hypothesis that LPS binding is at least partly receptor-mediated. Biochem Biophys Res Commun, 1993 Oct 29, 196(2), 773 - 9 CX-397, a novel recombinant hirudin analog having a hybrid sequence of hirudin variants-1 and -3; Komatsu Y et al.; To elucidate the differential roles of N- and C-terminal halves of hirudins in thrombin inhibition, we produced novel recombinant hirudin analogs, CX-397 and CX-397R, having a hybrid amino acid sequence of hirudin variants-1 (HV-1) and -3 (HV-3) . CX-397 is composed of the N-terminal half of HV-1 (HV-11-36) combined with the C-terminal half of HV-3 (HV-337-66) . CX-397R is the opposite combination . Their anti-thrombin activity was determined by a fluorogenic enzyme assay and compared with that of recombinant HV-1 (rHV-1) and rHV-3 . The order of the magnitude of dissociation constants (Ki) of these four hirudin analogs in thrombin inhibition was as follows: CX-397R (0.294 pM) > rHV-1 (0.148 pM) > rHV-3 (0.0593 pM) > CX-397 (0.0433 pM), indicating that CX-397 is the strongest inhibitor among them. Biochem Biophys Res Commun, 1993 Oct 29, 196(2), 729 - 36 Natural occurrence of Nuc in the sera of autoimmune-prone MRL/lpr mice; Kanai Y et al.; We previously established a clone of cells termed KML1-7 which produces a soluble factor that boosts anti-DNA antibody production both in vitro and in vivo across the H-2 barrier . By using the purified protein, termed nucleobindin (Nuc), we cloned cDNA and produced recombinant(r) Nuc in E.coli . Although the purified rNuc showed biological activities such as anti-DNA antibody boosting and DNA binding, there was no evidence that Nuc is really associated with autoimmune status in lupus-prone MRL/lpr mice . Here we report that identification of Nuc was successful from the sera of MRL/lpr mice, but not from those of the substrain MRL/n mice, which show no apparent autoimmune syndrome at the same age of MRL/lpr mice, by means of immunochemical as well as N-terminal amino-acid sequencing methods. Biochem Biophys Res Commun, 1993 Oct 29, 196(2), 583 - 8 Mercuric ion binding abilities of MerP variants containing only one cysteine; Sahlman L et al.; Using site-directed mutagenesis, Cys14 and Cys17 in MerP were replaced in turn by serine or alanine . All four variants were purified and partially characterized . The The mutant proteins all had one reactive thiol group left . In the absence of external thiols, the protein variants bound between two and four Hg2+, but unlike non-mutant MerP, none of the variants could bind Hg2+ when external thiol was added . This loss of the ability to specifically bind one Hg2+ per protein molecule shows that both cysteine residues 14 and 17 are necessary for binding of Hg2+ when there is competition from other thiol groups. Biochem Biophys Res Commun, 1993 Oct 29, 196(2), 553 - 60 Guanylin mRNA is expressed in villous enterocytes of the rat small intestine and superficial epithelia of the rat colon; Lewis LG et al.; Guanylin, and endogenous ligand for the Escherichia coli heat-stable enterotoxin receptor, is a recently characterized intestinal peptide . To understand the possible physiologic function of guanylin, we examined the cellular location of guanylin mRNA expression in the rat intestine . Intestinal cells were sequentially isolated from villous tip to crypt in rat jejunum and ileum . Northern blots of total RNA identified a single 0.65 kb guanylin transcript predominantly in the villous cell fractions . In situ hybridization studies demonstrated maximal signal intensity in villous cells in rat ileum and surface epithelial cells in the colon . In the ileum, the signal was nonuniform in distribution in the surface epithelial cells, with focal areas of intense signal in clusters of columnar absorptive cells . In both colon and ileum, signal intensity was near background level in deep crypt cells, lamina propria, and muscularis. Gene, 1993 Oct 29, 133(1), 79 - 84 C-terminal deletion mutants of the FokI restriction endonuclease; Li L et al.; We have constructed two C-terminal deletion mutants of the FokI restriction endonuclease by using the polymerase-chain-reaction technique and expressed them in Escherichia coli . The two mutant proteins (MP) of 41 and 30 kDa, were purified to homogeneity and their DNA-binding properties were characterized . The 41-kDa MP specifically binds the DNA sequence, 5'-GGATG/3'-CCTAC, like the wild-type (wt) FokI, but does not cleave DNA . The 30-kDa MP does not bind DNA . The affinity of the 41-kDa MP for the DNA substrate is comparable to that of wt FokI . The 41-kDa MP interacts with its substrate like the wt FokI, as revealed by hydroxyl radical footprinting experiments . In the presence of a DNA substrate, the 41-kDa MP is cleaved by trypsin into a 30-kDa N-terminal fragment and an 11-kDa C-terminal fragment . Addition of the HPLC-purified 11-kDa C-terminal fragment to the 30-kDa MP restores its sequence-specific DNA-binding property . These results confirm that the N-terminal 41-kDa fragment of the FokI ENase constitutes the DNA recognition domain of the ENase. Gene, 1993 Oct 29, 133(1), 17 - 22 Genome rearrangements by residual IS10 elements in strains of Escherichia coli K-12 which had undergone Tn10 mutagenesis and fusaric acid selection; Bogosian G et al.; Mutant strains selected as survivors of the lethal overexpression of a plasmid-encoded bovine somatotropin-beta-galactosidase fusion protein were found to include instances where an IS10 element had transposed from the chromosome into the fusion protein structural gene on the plasmid . Two distinct types of IS10 elements were found in these mutants, the well-known IS10R and a novel hybrid element composed of portions of both IS10R and IS10L . The strain in which the selection scheme was carried out had been constructed in a series of steps, including alteration of two loci by Tn10-mediated intramolecular transposition involving fusaric acid (FA) selection for loss of tetracycline resistance . Genetic dissection of this strain revealed that one of these altered loci was an origin for both types of IS10 elements, while the other locus was an origin for only IS10R elements . The finding that residual IS10 elements, left after FA selection for Tcs derivatives of Tn10-containing strains, can be a significant source of spontaneous mutation should be of interest to workers using strains that have been 'cured' of Tn10 in this way. Gene, 1993 Oct 29, 133(1), 109 - 13 Cloning and sequencing of the hemE gene encoding uroporphyrinogen III decarboxylase (UPD) from Escherichia coli K-12; Nishimura K et al.; Among the photoresistant revertants of the visA-deleted (hemH-deleted) strain of Escherichia coli K-12, three mutants defective in the hemE gene encoding uroporphyrinogen III decarboxylase (UPD) were identified . Using one of the mutants, we cloned and sequenced the hemE of E . coli . We found an open reading frame of 353 codons, which encoded a predicted amino acid (aa) sequence that exhibited a high degree of homology over its entire length to the aa sequence of UPD from humans and other organisms . This hemE was located at 90.3 min near the hupA gene on the linkage map of the E . coli chromosome. Biochem Biophys Res Commun, 1993 Oct 29, 196(2), 914 - 20 Expression and mutational analysis of the reverse transcriptase of the lentivirus equine infectious anemia virus; Shaharabany M et al.; The reverse transcriptase of equine infectious anemia virus (EIAV) shows sequence similarity with the reverse transcriptases of other lentiviruses, particularly with those of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) . We have constructed a plasmid that when introduced into E . coli induces the synthesis of substantial quantities of the nearly authentic EIAV reverse transcriptase . The viral and bacterially expressed reverse transcriptases are similar in their molecular weights . The bacterial expression clone was used to generate deletion mutants of the protein . Mutations in both amino and carboxyl terminal regions of the polypeptide strongly affect the DNA polymerase activity of the enzyme . Thus, EIAV reverse transcriptase resembles the reverse transcriptases of HIV-1 and HIV-2 and can serve as a suitable enzyme for studying the structure-function relationship in lentiviral reverse transcriptase. Biochem Biophys Res Commun, 1993 Oct 29, 196(2), 825 - 30 Dihydropyridine antagonists and agonists of calcium channels inhibit the induction of nitric oxide synthase by endotoxin in cultured macrophages; Szabo C et al.; Here we investigate the effects of the dihydropyridine-type antagonists of calcium channels nitrendipine, nimodipine, nisoldipine and the calcium channel agonist BAY K 8644 on the induction of nitric oxide synthase (NOS) by bacterial endotoxin (lipopolysaccharide; LPS) in J774.2 macrophages cultured in vitro . Pretreatment of J774.2 cells with these dihydropyridines (10(-8) -3 x 10(-6) M for 30 min) dose-dependently inhibited the LPS-stimulated (1 microgram/ml, 24 h) nitrite formation . For instance, at 10(-6) M, the inhibition was 59 +/- 3% for nitrendipine; 47 +/- 5% for nimodipine and 42 +/- 3% for nisoldipine (n = 9; p < 0.05) . BAY K 8644 caused a moderate, but significant inhibition of nitrite accumulation (by 16 +/- 3% at 10(-7) M, n = 9; p < 0.05) . The inhibition of LPS-stimulated nitrite accumulation produced by nitrendipine, nimodipine, and BAY K 8644 was significantly smaller when they were applied 2 or 4 h after LPS, indicating that these agents inhibit the induction, but not the activity of the induced NOS . At concentrations which caused a significant inhibition of the LPS-stimulated nitrite accumulation, the dihydropyridine calcium channel modulators did not inhibit mitochondrial respiration . Thus, dihydropyridine calcium channel modulators (antagonists and an agonist) inhibit the induction of the calcium-independent isoform of NOS produced by LPS in J774.2 macrophages . This effect is not related to the modulation of intracellular calcium levels. Nature, 1993 Oct 28, 365(6449), 852 - 5 Human xeroderma pigmentosum group D gene encodes a DNA helicase; Sung P et al.; Xeroderma pigmentosum (XP), a genetically heterogeneous human disease, results from a defect in nucleotide excision repair of ultraviolet-damaged DNA . XP patients are extremely sensitive to sunlight and suffer from a high incidence of skin cancers . Cell fusion studies have identified seven XP complementation groups, A-G . Group D is of particular interest as mutations in this gene can also cause Cockayne's syndrome and trichothiodystrophy . The XPD gene was initially named ERCC2 (excision repair cross complementing) as it was cloned using human DNA to complement the ultraviolet sensitivity of a rodent cell line . We have purified the XPD protein to near homogeneity and show that it possesses single-stranded DNA-dependent ATPase and DNA helicase activities . We tested whether XPD can substitute for its yeast counterpart RAD3, which is essential for excision repair and for cell viability . Expression of the XPD gene in Saccharomyces cerevisiae can complement the lethality defect of a mutation in the RAD3 gene, suggesting that XPD is an essential gene in humans. Biochemistry, 1993 Oct 26, 32(42), 11413 - 8 Intramolecular electron transfer in cytochrome o of Escherichia coli: events following the photolysis of fully and partially reduced CO-bound forms of the bo3 and oo3 enzymes; Morgan JE et al.; The events which follow photolysis of CO-inhibited fully reduced and CO-bound mixed-valence cytochrome o have been studied in two variants of the enzyme, one of which contains heme B at the low-spin site (bo3) and the other of which contains heme O (oo3) . For this, isolated enzyme was prepared from three different strains of Escherichia coli which produce these two variants in different relative amounts {Puustinen, A., Morgan, J . E., Verkhovsky, M., Thomas, J . W., Gennis, R . B., & Wikstrom, M . (1992) Biochemistry 31, 10363-10369} . In both types of enzyme microsecond electron redistribution was observed from the oxygen-binding heme to the low-spin heme . In the bo3 enzyme, the rate was similar to that in the bovine enzyme (3 microseconds), but in the oo3 enzyme, it was several times slower . However, in both types of cytochrome o, the same electron redistribution process was also apparently observed on other time scales, some faster and some slower . The rate of CO rebinding in the mixed-valence enzyme was found to be slower than in the fully reduced enzyme, apparently because of the subpopulation of oxidized oxygen-binding heme produced by the electron redistribution . The extent of this electron redistribution, and thus the inter-heme delta Em, can be calculated from this change in rate . The heme B and heme O containing low-spin sites have Em values about 20 and 50 mV lower, respectively, than the oxygen-binding heme. Biochemistry, 1993 Oct 26, 32(42), 11259 - 69 Effect of cavity-creating mutations in the hydrophobic core of chymotrypsin inhibitor 2; Jackson SE et al.; Hydrophobic residues in the core of a truncated form of chymotrypsin inhibitor 2 (CI2) have been mutated in order to measure their contribution to the stability of the protein . The free energy of unfolding of wild-type and mutants was measured by both guanidinium chloride-induced denaturation and differential scanning calorimetry . The two methods give results for the changes in free energy on mutation that agree to within 1% or 2% . The average change in the free energy of unfolding (+/- standard deviation) for an Ile-->Val mutation is 1.2 +/- 0.1 kcal mol-1, for a Val-->Ala mutation 3.4 +/- 1.5 kcal mol-1, and for either an Ile-->Ala or a Leu-->Ala mutation 3.6 +/- 0.6 kcal mol-1 . This gives an average change in the free energy of unfolding for deleting one methylene group of 1.3 +/- 0.5 kcal mol-1 . Two significant correlations were found between the change in the free energy of unfolding between wild-type and mutant, delta delta GU-F, and the environment of the mutated residue in the protein . The first is between delta delta GU-F and the difference in side-chain solvent-accessible area buried between wild-type and mutant (correlation coefficient = 0.81, 10 points) . The second and slightly better correlation was found between delta delta GU-F and N, the number of methyl/methylene groups within a 6-A radius of the hydrophobic group deleted (correlation coefficient = 0.84, 10 points) . The latter correlation is very similar to that found previously for barnase, suggesting that this relationship is general and applies to the hydrophobic cores of other globular proteins . The combined data for C12 and barnase clearly show a better correlation with N (correlation coefficient = 0.87, 30 points) than with the change in the solvent-accessible surface area (correlation coefficient = 0.82, 30 points) . This indicates that the packing density around a particular residue is important in determining the contribution the residue makes to protein stability . In one case, Ile-->Val76, a mutation which deletes the C delta 1 methyl group of a buried side chain, a surprising result was obtained . This mutant was found to be more stable than wild-type by 0.2 +/- 0.1 kcal mol-1 . We have solved and analyzed the crystal structure of this mutant and find that there are small movements of side chains in the core, the largest of which, 0.7 A, is a movement of the side chain that has been mutated.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1993 Oct 25, 268(30), 22900 - 7 Molecular characterization of avian muscle titin; Tan KO et al.; Titin is an approximately 3000-kDa polypeptide that constitutes a set of elastic filaments that connect thick filaments to the Z-line in vertebrate striated muscle myofibrils . To characterize the primary structure of titin, three overlapping cDNA clones comprising 2.4 kilobases of avian muscle titin coding sequence were obtained from a cDNA library constructed from embryonic chick cardiac muscle RNA size-selected for large transcripts . Expression of one cDNA clone in Escherichia coli produced a fusion protein that reacted specifically with titin antibodies, and titin antiserum affinity-purified against this fusion protein reacted specifically with titin on immunoblots of chicken cardiac and skeletal muscle myofibrils . Indirect immunofluorescence localization with the fusion protein-specific antibodies demonstrated that the cDNA sequence was from the region of titin located in the myofibrillar A-band adjacent to the A/I junction . Derived amino acid sequences demonstrated a repeating pattern of fibronectin type III and immunoglobulin C2 motifs, as shown previously for a portion of rabbit skeletal muscle titin located in the central region of the A-band and for other myofibrillar proteins that bind to myosin . Differences between the rabbit and chicken titin sequences included a unique, serine-rich region in one motif, which represents a potential phosphorylation site . This is the first report of sequence information for avian titin from a previously uncharacterized portion of the titin molecule. J Biol Chem, 1993 Oct 25, 268(30), 22771 - 6 Expression, purification, and characterization of SH2-containing protein tyrosine phosphatase, SH-PTP2; Sugimoto S et al.; A human protein tyrosine phosphatase containing two src homology 2 (SH2) domains (SH-PTP2) was expressed in Escherichia coli under T7 promoter control and purified to near homogeneity . The purified protein, with molecular mass of 68 kDa on SDS-polyacrylamide gel electrophoresis, was identified as SH-PTP2 by its protein tyrosine phosphatase activity and N-terminal amino acid sequence analysis . Its protein tyrosine phosphatase activity was sensitive to pH and salt concentration . Whereas its optimum pH for the low molecular weight substrate para-nitrophenyl phosphate is 5.6, the pH optima for peptide substrates were shifted toward neutral . With the artificial protein substrate reduced, carboxyamidomethylated, and maleylated lysozyme, it displays 2000-fold lower Km (1.7 microM) and 2.4-fold higher kcat (0.11 s-1) than with para-nitrophenyl phosphate . Among the phosphopeptides from autophosphorylation sites of receptors for epidermal growth factor and platelet-derived growth factor, SH-PTP2 displayed high activity toward phosphopeptides corresponding to pY992 of the epidermal growth factor receptor and pY1009 and pY1021 of the platelet-derived growth factor receptor . In further enzymatic studies with phosphopeptides corresponding to pY1009, SH-PTP2 showed nonlinear Line-weaver-Burk double-reciprocal plots, suggesting that the phosphopeptide corresponding to pY1009 may have a substrate and allosteric effect. J Biol Chem, 1993 Oct 25, 268(30), 22746 - 55 Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase beta by UV cross-linking; Prasad R et al.; Rat DNA polymerase beta (beta-pol) is a 39-kDa monomeric protein, organized in two structurally and functionally distinct domains . The 8-kDa NH2-terminal domain binds single-stranded (ss) DNA, whereas the 31-kDa COOH-terminal domain does not . To facilitate studies on ssDNA binding structure-function relationships of beta-pol, we overexpressed the 8-kDa domain in Escherichia coli, and purified the recombinant protein to homogeneity . Single-stranded nucleic acid binding of the recombinant 8-kDa domain was found to be similar to that previously reported for the 8-kDa fragment prepared by proteolysis of intact beta-pol (Kumar, A., Widen, S . G., Williams, K . R., Kedar, P . Karpel, R . L., and Wilson, S . H . (1990b) J . Biol . Chem . 265, 2124-2131; Casas-Finet, J . R., Kumar, A., Morris, G., Wilson, S . H., and Karpel, R . L . (1991) J . Biol . Chem . 266, 19618-19625) . Residues in or near the DNA-binding pocket of the recombinant 8-kDa domain were examined by photochemical cross-linking to {32P} p(dT)16 . Cross-linking was localized to a tryptic fragment spanning residues 28 through 35 and a V8 protease fragment spanning residues 27 through 58 . Sequence analysis of the various {32P}p(dT)16-labeled proteins indicated that Ser30 and His34 were modified by cross-linking to p(dT)16 . Therefore, these residues of the ssDNA-binding domain of beta-pol appear to be in close contact with this nucleic acid probe. J Biol Chem, 1993 Oct 25, 268(30), 22680 - 5 Cloning and functional expression of a Dictyostelium discoideum protein tyrosine phosphatase; Ramalingam R et al.; Using polymerase chain reaction methods, we cloned a 1.7-kilobase cDNA, denoted DdPTPa, that has high homology with other known eukaryotic protein tyrosine phosphatases . DdPTPa possess a 241-amino acid protein tyrosine phosphatase domain located in the C terminus, which exhibits a 39-43% amino acid sequence identity with published protein tyrosine phosphatases . Absence of a characteristic signal sequence and transmembrane domain suggests that DdPTPa is a nonreceptor type cytoplasmic protein tyrosine phosphatase . Southern blot analysis of genomic DNA indicates the presence of a multigene protein tyrosine phosphatase family in Dictyostelium . Northern blot analysis reveals four species of mRNA that hybridize to the DdPTPa probe, at least three of which are developmentally regulated . The entire coding sequence of DdPTPa was subcloned into the pET15-b vector and expressed in Escherichia coli . Affinity-purified DdPTPa protein efficiently dephosphorylates both p-nitrophenyl phosphate and tyrosine-phosphorylated reduced, carboxyamidomethylated, and maleylated lysozyme . A Dictyostelium transformant overexpressing DdPTPa does not develop normally . The overexpresser fails to aggregate, in contrast to the control transformant containing vector alone, and after 24 h gives rise to only a few abnormal slugs and small fruiting bodies. J Biol Chem, 1993 Oct 25, 268(30), 22618 - 26 ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli . Sequence and in vivo activities; Gottesman S et al.; The ATP-dependent Clp protease of Escherichia coli consists of two subunits, the ClpP subunit, which has the proteolytic active site, and ClpA, which possesses ATPase activity and activates the proteolytic activity of ClpP in vitro . Recently, Zylicz and co-workers (Wojtkowiak, D., Georgopoulos, C., and Zylicz, M . (1993) J . Biol . Chem . 268, 22609-22617) identified another E . coli protein that activated ATP-dependent degradation of lambda O protein in the presence of ClpP . The amino-terminal sequence of this protein corresponds to the translated amino-terminal sequence of a gene that we have named clpX . clpX encodes a protein with M(r) 46,300, similar to that observed for the protein purified by Wojtkowiak et al . clpX is an operon with clpP; both genes are cotranscribed in a single heat-inducible 2200-base mRNA, with clpP the promoter proximal gene . The sequence of ClpX includes a single consensus ATP-binding site motif and has limited homology to regions of ClpA and other members of the ClpA/B/C family . A third group of proteins, ClpY, closely related to ClpX, has been identified by sequence homology . Mutations in either clpX or clpP abolish degradation of the highly unstable lambda O protein in vivo . clpX mutants are not defective in degradation of previously identified ClpA/ClpP substrates such as a ClpA-beta-galactosidase fusion protein . It appears that selectivity of degradation by ClpP in vivo is determined by interaction of ClpP with different regulatory ATPase subunits. J Biol Chem, 1993 Oct 25, 268(30), 22525 - 30 Analysis of two distinct single-stranded DNA binding sites on the recA nucleoprotein filament; Zlotnick A et al.; The binding stoichiometry of Escherichia coli recA protein to single-stranded DNA (ssDNA) determined by two separate assays differs by a factor of 2.2-2.4 . Using the fluorescence of etheno-DNA (epsilon DNA), a chemically modified ssDNA, the stoichiometry was found to be 7.0 +/- 0.6 bases/recA protein monomer in a nucleo-protein filament . Using a competition assay, a similar stoichiometry, 7.5 bases/recA, is found for unmodified poly(dT) . Using the DNA-dependent ATPase of recA, which monitors bound protein rather than bound DNA, we find that each recA monomer needs to bind only 3.1 +/- 0.5 bases to fully activate the ATPase . The difference in site size determined by the two assays indicates that there are two DNA binding sites with differential effects on ATPase activation . When recA protein is mixed with ssDNA at a ratio of 7 bases/recA or greater, the complex that forms contains 7 bases/recA and acts as a kinetic trap for the ssDNA . Upon further addition of recA protein, no additional ATPase activity is observed . If, on the other hand, the ssDNA is initially mixed with excess recA (at a ratio of 3-3.5 bases/recA or less) the ATPase activity is twice as high . Analysis of the binding curves suggests that the first DNA strand binds recA to form a filament with a stoichiometry of 3-3.5 bases/protein monomer . The ATPase activity of recA is completely active in this complex . A second strand of DNA can then be bound to this filament yielding a final stoichiometry of approximately 7 bases/protein monomer . The presence of this second strand neither enhances nor inhibits ATP hydrolysis . This ternary complex may mimic the structures formed by recA in searching for homologous DNA sequences and/or in the strand exchange reaction. J Biol Chem, 1993 Oct 25, 268(30), 22502 - 7 ATP hydrolysis is not stoichiometrically linked with proteolysis in the ATP-dependent protease La from Escherichia coli; Fischer H et al.; Protein degradation in Escherichia coli involves the ATP-dependent serine protease La . Protease La is a homotetramer with one proteolytic and one ATP binding site per monomer . Its proteolytic activity has been shown to be highly increased by simultaneous hydrolysis of ATP, which is essential for the degradation of protein substrates by this enzyme . We have cloned and purified a proteolytically inactive La mutant, in which the catalytically active serine residue at position 679 was replaced by alanine . Fluorescence and circular dichroism spectra of the purified wild type and mutant enzyme revealed identical conformations of the proteins . Based on this observation, the catalytic properties of the wild type enzyme and the S679A mutant were compared . Although the S679A mutant lacks proteolytic activity toward both peptide and protein substrates under all conditions investigated, its ATPase activity is completely unaffected by the removal of the protease activity . Since protein substrates stimulate the ATP-dependent hydrolysis of peptides by protease La, it has been argued that this stimulation is due to interactions with a regulatory binding site on the enzyme . In accordance with this model, protein substrates such as alpha-casein and denatured bovine serum albumin stimulate the ATPase activity of the S679A mutant to the same degree as in the active protease . Therefore, the intrinsic ATPase activity of protease La as well as its stimulation is not dependent on the simultaneous hydrolysis of the protein substrate. J Biol Chem, 1993 Oct 25, 268(30), 22369 - 76 The inactivation of Fe-S cluster containing hydro-lyases by superoxide; Flint DH et al.; We report in this paper that highly purified Escherichia coli dihydroxy-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase are inactivated by O2- with second order rate constants in the range of 10(6) to 10(7) M-1 s-1 . Each of these enzymes belongs to the hydro-lyase class and contains catalytically active {4Fe-4S} clusters . Simultaneous with inactivation by O2- is the release of iron from their clusters . Our working hypothesis is O2- inactivates these enzymes by oxidizing their clusters to an unstable oxidation state, and cluster degradation follows . Consistent with this hypothesis is our observation that spinach dihydroxy-acid dehydratase, a member of the hydro-lyase class that has a catalytically active {2Fe-2S} cluster, is not inactivated and does not lose iron in the presence of O2- . Porcine fumarase, a member of the hydro-lyase class that does not contain an Fe-S cluster, is also not inactivated by O2- . We also report the rate constants for the inactivation of E . coli dihydroxy-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase by O2 are close to 2 x 10(2) M-1 s-1, and the rate constants for the inactivation of E . coli dihydroxy-acid dehydratase and mammalian aconitase by H2O2 are about 10(3) M-1 s-1 . E . coli dihydroxy-acid dehydratase has been reported previously to be inactivated in vivo when cells are grown in hyperbaric O2, presumably due to the increased O2- generated under these conditions . We report here that E . coli fumarase A, fumarase B, and aconitase are also inactivated in vivo by hyperbaric O2 . Thermodynamic parameters for the oxidation of the cluster of aconitase by O2- and O2 are calculated. J Biol Chem, 1993 Oct 25, 268(30), 22357 - 62 Rat liver heme oxygenase . High level expression of a truncated soluble form and nature of the meso-hydroxylating species; Wilks A et al.; A rat heme oxygenase (HO-1) gene without the sequence coding for the last 23 amino acids has been constructed and expressed behind the pho A promoter in Escherichia coli . The enzyme is expressed at high levels as a soluble catalytically active protein that causes the bacterial cells to accumulate biliverdin . The purified truncated heme-heme oxygenase complex is spectroscopically indistinguishable from the complex with the native enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase . Reaction of the recombinant heme-heme oxygenase complex with H2O2 produces a species with the spectroscopic properties of verdoheme . Unidentified products are obtained when this intermediate is directly extracted from the protein, but biliverdin is obtained if the verdoheme-protein complex is exposed to cytochrome P450 reductase and NADPH before the extraction step . In contrast, reaction of the heme-heme oxygenase complex with meta-chloroperbenzoic acid (mCPBA), tert-butylhydroperoxide, or cumene hydroperoxide yields a ferryl (FeIV = O) complex . Reaction of the heme-heme oxygenase complex with mCPBA also produces an EPR-detectable protein radical . In accord with formation of a ferryl intermediate, recombinant heme oxygenase catalyzes the mCPBA- and alkylhydroperoxide-dependent peroxidation of 2-methoxyphenol (guaiacol) . Guaiacol oxidation is not observed during turnover of the enzyme by cytochrome P450 reductase/NADPH or H2O2 . Conversely, biliverdin is not formed with tert-butylhydroperoxide or mCPBA . H2O2 thus supports the first step of the normal catalytic oxidation of heme by heme oxygenase, but alkyl and acyl hydroperoxides do not . These results suggest that the alpha-meso-hydroxylation required for biliverdin formation is mediated by the distal of the two oxygens in the iron-dioxygen intermediate (Fe-O-O) engendered by reaction with either cytochrome P450 reductase/NADPH or H2O2. J Biol Chem, 1993 Oct 25, 268(30), 22322 - 30 Mechanisms of camptothecin resistance in yeast DNA topoisomerase I mutants; Knab AM et al.; The anti-cancer drug camptothecin targets eukaryotic DNA topoisomerase I by trapping the covalent complex formed between the catalytically active enzyme and DNA . Saccharomyces cerevisiae cells expressing yeast DNA topoisomerase I mutant top1 vac (I725R, N726A) or top1N726L, in which the amino acid residues N terminus to the active site tyrosine Tyr-727 were changed as indicated, were found to be camptothecin-resistant even though the mutant proteins expressed in Escherichia coli were previously shown to be active . Assays of enzyme-catalyzed relaxation of supercoiled DNA in vitro and in vivo in yeast showed that the camptothecin resistance of these mutants arises by entirely different mechanisms . Top1N726L-catalyzed DNA relaxation was not detected in yeast . The Top1 vac protein was catalytically active; however, camptothecin was inefficient in trapping the covalent intermediate formed between the Top1 vac enzyme and DNA . Yeast cells expressing human mutant htop1 vac, with similar substitutions near the active site tyrosine Tyr-723, were also camptothecin-resistant . Surprisingly, in the absence of camptothecin, yeast rad52 mutants defective in the repair of double-stranded DNA breaks were nonviable when top1N726L or top1 vac was overexpressed but viable when htop1 vac was overexpressed . These results suggest differences between yeast and human enzyme function in vivo. J Biol Chem, 1993 Oct 25, 268(30), 22259 - 61 Rab3A GTPase-activating protein-inhibiting activity of Rabphilin-3A, a putative Rab3A target protein; Kishida S et al.; Rabphilin-3A is a putative target protein for Rab3A, a member of the small G protein superfamily that is implicated in regulated secretion, particularly in neurotransmitter release . Rabphilin-3A contains at least two functionally different domains: the N-terminal Rab3A-binding domain and the C-terminal C2 domain, which interacts with both Ca2+ and phospholipid . Because Rabphilin-3A interacts preferentially with GTP-Rab3A rather than with GDP-Rab3A, we have examined here whether Rabphilin-3A affects the GTPase activity of Rab3A . Rabphilin-3A and its N-terminal fragment, but not its C-terminal fragment, very weakly stimulated the basal GTPase activity of Rab3A . However, Rabphilin-3A and its N-terminal fragment strongly inhibited the Rab3A GAP-stimulated GTPase activity of Rab3A . Ca2+ and phospholipid showed no effect on these activities of Rabphilin-3A . The physiological significance of the GAP activity of Rabphilin-3A is obscure, but it is likely that Rabphilin-3A inhibits Rab3A GAP activity and keeps Rab3A in the GTP-bound active form during its action as a target molecule for Rab3A. FEBS Lett, 1993 Oct 25, 333(1-2), 89 - 95 Effects of a single intrastrand d(GpG) platinum adduct on the strand separating activity of the Escherichia coli proteins RecB and RecA; Villani G et al.; RecB and RecA proteins play key roles in the process of DNA recombination in Escherichia coli and both possess DNA unwinding activities which can displace short regions of duplex DNA in an ATP-dependent manner in vitro . We have examined the effect of the most abundant DNA adduct caused by the chemotherapeutic agent cis-diamminedichloroplatinum(II) on those activities . For this purpose, we have constructed a partially duplex synthetic oligonucleotide containing the intrastrand d(GpG) crosslink positioned at a specific site . We report here that both the DNA strand separating and DNA-dependent ATPase activities of the RecB protein are inhibited by the d(GpG) cis-DDP adduct . In contrast, neither the unwinding nor the ATPase activities of RecA protein appear to be perturbed by this lesion. FEBS Lett, 1993 Oct 25, 333(1-2), 197 - 202 Kinetic and structural characterization of an intermediate in the biomineralization of bacterioferritin; Le Brun NE et al.; The mechanism by which iron-storage proteins take up and oxidise iron(II) is not understood . We show by rapid-kinetic and EPR measurements that iron uptake, in vitro, by a bacterial iron-storage protein, bacterioferritin, involves at least three kinetically distinguishable phases: phase 1, the binding of Fe(II) ions, probably at a dimeric iron ferroxidase centre; phase 2, oxidation of the Fe(II) dimer and production of mononuclear Fe(III); and phase 3, iron core formation. FEBS Lett, 1993 Oct 25, 333(1-2), 183 - 7 The protein kinase mos activates MAP kinase kinase in vitro and stimulates the MAP kinase pathway in mammalian somatic cells in vivo; Nebreda AR et al.; The mos protooncogene encodes a serine/threonine protein kinase that is only expressed at significant levels in germ cells . Recombinant malE-mos protein (Xenopus mos protooncogene fused in frame to the maltose binding protein of E . coli) activates MAP kinase in cell-free extracts prepared from Xenopus oocytes and eggs . Here we show that malE-mos immunoprecipitates from Xenopus extracts phosphorylate and activate MAP kinase kinase in vitro, indicating that mos can function as a MAP kinase kinase kinase . Moreover, ectopic expression of mos in mammalian somatic cells, that lack any endogenous mos protein, triggers the activation of MAP kinase in vivo . These results identify the mos protooncogene as a direct activator of the MAP kinase pathway, with the potential to activate this kinase cascade even in cells where normally there is no expression of mos. Nucleic Acids Res, 1993 Oct 25, 21(21), 4948 - 53 Probing structural differences between native and in vitro transcribed Escherichia coli valine transfer RNA: evidence for stable base modification-dependent conformers; Derrick WB et al.; Structural differences between native (modified) and in vitro transcribed (unmodified) Escherichia coli tRNA(Val) were explored by comparing their temperature-absorbance profiles as a function of magnesium ion concentration and by probing their solution conformation with single- and double-strand-specific endonucleases . In vitro transcribed tRNA(Val) has a less ordered structure as monitored by thermal melting profiles; its Tm is appreciably lower than that of native tRNA(Val) at all Mg2+ concentrations . Structure probing experiments with nuclease S1 and ribonuclease V1 show that the unmodified tRNA(Val) transcript is more susceptible to nuclease attack at low Mg2+ concentrations, particularly in the D- and T-loops, indicative of at least a partial disruption of D-loop/T-loop interactions . These experiments also provide evidence for temperature-dependent alternative conformations of the anticodon loop of native tRNA(Val) . Modified nucleosides are essential for the stability of these conformers; they cannot be detected in the unmodified in vitro transcript . The observations suggest that post-transcriptional modifications in tRNA allow the adoption of unique conformations and act to stabilize those that are biologically active. Nucleic Acids Res, 1993 Oct 25, 21(21), 4929 - 35 Substrate recognition and selectivity in the type IC DNA modification methylase M.EcoR124I; Taylor I et al.; The type I DNA modification methylase M.EcoR124I binds sequence specifically to DNA and protects a 25bp fragment containing its cognate recognition sequence from digestion by exonuclease III . Using modified synthetic oligonucleotide duplexes we have investigated the catalytic properties of the methylase, and have established that a specific adenine on each strand of DNA is the site of methylation . We show that the rate of methylation of each adenine is increased at least 100 fold by prior methylation at the other site . However, this is accompanied by a significant decrease in the affinity of the methylase for these substrates according to competitive gel retardation assays . In contrast, methylation of an adenine in the recognition site which is not a target for the enzyme results in only a small decrease in both DNA binding affinity and rate of methylation by the enzyme. Nucleic Acids Res, 1993 Oct 25, 21(21), 4923 - 8 Rational design and PCR-based synthesis of an artificial Schizophyllum commune xylanase gene; Graham RW et al.; A synthetic gene encoding the Schizophyllum commune xylanase XynA was constructed by a novel PCR-based procedure . Three long oligonucleotides were synthesized and used in combination with flanking PCR primers to generate a 607 base pair gene which contained 31 unique locations for restriction enzyme cleavage . The amino acid sequence was tailored for expression in Escherichia coli by using only those codons found in highly expressed E . coli genes . The availability of the gene will facilitate analysis of the structure and function of this and other beta-(1,4) xylanases. J Biol Chem, 1993 Oct 25, 268(30), 22820 - 4 The nucleotide sequence of porcine formiminotransferase cyclodeaminase . Expression and purification from Escherichia coli; Murley LL et al.; We have isolated and characterized cDNA clones encoding the porcine liver octameric enzyme, 5-formiminotetrahydrofolate:L-glutamate N-formiminotransferase (EC 2.1.2.5)-formiminotetrahydrofolate cyclodeaminase (EC 4.3.1.4) . The cDNA encodes a novel amino acid sequence of 541 residues which contains exact matches to two sequences derived by automated sequence analysis of CNBr cleavage fragments isolated from the porcine enzyme . The recombinant enzyme has been expressed as a soluble protein in Escherichia coli at levels 4-fold higher than those observed in liver, and is bifunctional, displaying both transferase and deaminase activities . With a calculated subunit molecular mass of 58,926 Da, it is similar in size to the enzyme isolated from porcine liver . Purification of the enzyme from E . coli involves chromatography on a novel polyglutamate column which might interact with the folylpolyglutamate binding site of the protein . The purified recombinant enzyme has a transferase specific activity of 39-41 units/mg/min. J Biol Chem, 1993 Oct 25, 268(30), 22397 - 401 Processing and characterization of human proguanylin expressed in Escherichia coli; Garcia KC et al.; Guanylin is a 15-amino acid peptide hormone that was originally isolated from the jejunum of the rat small intestine and shown to be an endogenous activator of the intestinal heat-stable enterotoxin receptor-guanylyl cyclase . Guanylin is synthesized as a 115-amino acid prohormone, proguanylin, which is processed at a site yet to be determined, into a C-terminal bioactive fragment(s) . In order to examine the processing of proguanylin in vitro, we have generated large quantities of the properly folded prohormone by constructing an expression vector that directs its secretion into the periplasmic space of Escherichia coli . The bacterially expressed human proguanylin was then processed to smaller C-terminal fragments by protease digestion . Digestion with trypsin or lysine-C generated C-terminal peptides of different length, which have been purified and characterized . Guanylin-22 and guanylin-32 have binding affinities and biological activities similar to guanylin-15, while guanylin-63 and the entire proguanylin have only minimal bioactivity . Circular dichroism spectroscopy reveals that proguanylin is a stably folded protein containing mostly beta-sheet and beta-turn structure. Cell, 1993 Oct 22, 75(2), 341 - 50 Reverse branch migration of Holliday junctions by RecG protein: a new mechanism for resolution of intermediates in recombination and DNA repair; Whitby MC et al.; The RecG protein of E . coli is a junction-specific DNA helicase involved in recombination and DNA repair . The function of the protein was investigated using an in vitro recombination reaction catalyzed by RecA . We show that RecG counters RecA-driven strand exchange by catalyzing branch migration of the Holliday junction in the reverse direction . This activity represents a new mechanism for resolving recombination intermediates that is independent of junction cleavage . We discuss how reverse branch migration can facilitate DNA repair, promote recombination in conjugational crosses, and confine the distribution of Chi-stimulated cross-overs . We suggest that the RecG mechanism for resolution of junctions is universal and provides a simple system that allows gene conversion without associated crossing over. Int J Cancer, 1993 Oct 21, 55(4), 651 - 4 Production of monoclonal antibodies to human estrogen-receptor protein (ER) using recombinant ER (RER); al Saati T et al.; Two monoclonal antibodies (MAbs), IC5 and ID5, were produced using spleen cells from BALB/c mice immunized with recombinant estrogen-receptor protein (RER) . On immunoblotting, both MAbs reacted with the 67-kDa polypeptide chain obtained by transformation of E . coli and transfection of COS cells with plasmid vectors expressing ER . The epitopes of both MAbs were in the N-terminal domain (A/B region) of the receptor . In normal human tissues, IC5 and ID5 reacted with cells known to contain large amount of ER, such as cells of the mammary gland and the uterus . Staining was localized predominantly in nuclei with little or no cytoplasmic reactivity . IC5 and ID5 were unreactive with tissues usually considered to be negative for ER . The reactions of these 2 MAbs were further tested on different tumor types, using immunohistochemical (IHC) method on frozen sections . In breast cancer, a good correlation was found between the results obtained on frozen sections and those using the conventional radioligand dextran-coated charcoal (DCC) assay . Immunostaining with IC5 and ID5 MAbs was also assessed on routinely processed paraffin sections using the antigen-retrieval method . Staining was comparable to that obtained on frozen sections in virtually all the breast carcinomas . Negative reactions were consistently obtained with both antibodies on human neoplasms derived from other non-estrogen-dependent organs . IC5 and ID5 MAbs may thus be of value in routine diagnostic histopathology for assessment of the estrogen-receptor content in human carcinomas. J Mol Biol, 1993 Oct 20, 233(4), 766 - 80 How consistent are molecular dynamics simulations? Comparing structure and dynamics in reduced and oxidized Escherichia coli thioredoxin; Elofsson A et al.; In this study we have examined several parameters that can be used for checking the consistency and accuracy of protein structures and molecular dynamics simulations . This is done by comparing: (1) three X-ray structures of oxidized Escherichia coli thioredoxin (Trx-S2); (2) 14 NMR structures of reduced E . coli thioredoxin (Trx-(SH)2); and (3) 30 different simulations, 15 of Trx-S2 and 15 of Trx-(SH)2 . The energy, the agreement with NOE data, the root-mean-square deviation between structures, and the surface characteristics of all these structures are analyzed . The 30 simulations, four water simulations, 20 standard vacuum simulations and six alternative vacuum simulations, are examined with respect to mobility, temperature factors and aromatic side-chain mobility . It is shown that although vacuum simulations may reproduce some parameters, all the features of a water simulation cannot be reproduced in any of these simulations . Several of the parameters described above are shown to be good for discriminating between an accurate and an inaccurate simulation . It is also shown that 100 ps is too short a time to obtain statistically certain temperature factors and correlation functions of aromatic side-chain motions . The results also suggest that performing ten 100 ps simulations spans the conformation space better than one 1 ns simulation. J Mol Biol, 1993 Oct 20, 233(4), 659 - 70 Genetic analysis of periplasmic binding protein dependent transport in Escherichia coli . Each lobe of maltose-binding protein interacts with a different subunit of the MalFGK2 membrane transport complex; Hor LI et al.; Escherichia coli is able to accumulate maltose and maltodextrins by an ATP-binding cassette transporter known as the maltose transport system . This transport system is comprised of five proteins: the LamB protein in the outer membrane; the periplasmic maltose-binding protein (MBP); two integral inner membrane proteins, MalF and MalG; and MalK, which is associated with the cytoplasmic face of the inner membrane . It has been previously suggested that MBP interacts with MalF and MalG during sugar transport across the inner membrane . In two independent genetic studies, reported here, residue 210 of MBP has been identified as an important site for its interaction with MalF . In one study, allele-specific suppressors of a malF mutation, malF506, were isolated and yielded mutations which altered residue tyrosine 210 of MBP to aspartic acid . In the other study, dominant mutations in malE (the structural gene of MBP) were isolated; one of these altered the same tyrosine residue (210) to cysteine . It was shown that the Y210C MBP mutant is also an allele-specific suppressor malF506, and that of the suppressor MBP alleles also exhibited dominant-negative phenotypes . Previously it was shown that alterations at residues glycine 13 and aspartate 14 of MBP can result in suppression of a malG mutant . From these results and those described, it is possible to propose a simple model in which the amino-terminal lobe of MBP interacts with MalG and the carboxy-terminal lobe of MBP interacts with MalF . The locations of residues 13, 14 and 210 on the three-dimensional structure of MBP are in keeping with this model. J Mol Biol, 1993 Oct 20, 233(4), 615 - 28 Two acidic residues of Escherichia coli methionyl-tRNA synthetase act as negative discriminants towards the binding of non-cognate tRNA anticodons; Schmitt E et al.; Escherichia coli methionyl-tRNA synthetase recognizes its cognate tRNAs according to the sequence of the CAU anticodon . In order to identify residues of methionyl-tRNA synthetase involved in tRNA anticodon recognition, enzyme variants created by cassette mutagenesis were genetically screened for their acquired ability to charge tRNA(mMet) derivatives with an ochre or an amber anticodon and, consequently, to cause the suppression of a stop codon in an indicator gene . The selected enzymes are called suppressors . Mutations were firstly directed towards the region of the synthetase encompassing residues 451 to 467 . Several dozens of suppressor enzymes were compared . Statistical analysis of the mutations suggested that the substitution of an Asp side-chain at position 456 was sufficient to render possible the charging of the ochre or amber suppressor tRNAs . Point mutants at this position were therefore constructed . Their behaviour demonstrated that various tRNA(Met) derivatives having a non-Met anticodon could be aminoacylated in vitro provided only that the side-chain of residue 456 was no longer acidic . In turn, the Asp456 residue is not essential to the CAU anticodon recognition, since its substitution does not impair the aminoacylation of wild-type tRNA(Met) . The analysis was enlarged to a second region from residue 437 to residue 454 . The mutagenesis highlighted two other positions, one of which, Asn452, appeared involved in wild-type tRNA(Met) binding . The second position, Asp449, plays a role very similar to that of Asp456 . It is concluded that both Asp449 and 456 behave as "antideterminants", contributing together to the rejection by the enzyme of tRNAs carrying non-Met anticodons . Finally, it is shown that the activities of some particular methionyl-tRNA synthetase variants, which have been made indifferent to the sequence of the anticodon of a tRNA(Met), are tightly dependent on the presence of the nucleotide determinants specific to the acceptor stem of tRNA(Met). J Mol Biol, 1993 Oct 20, 233(4), 597 - 605 Two inactive fragments derived from the yeast mitochondrial ribosomal protein MrpS28 function in trans to support ribosome assembly and respiratory growth; Huff MO et al.; The mitochondrial ribosomal protein MrpS28 of Saccharomyces cerevisiae is one of several mitochondrial ribosomal proteins homologous to Escherichia coli ribosomal proteins within the context of a larger protein . Relative to a region of homology with E . coli ribosomal protein S15, the mature MrpS28 protein has unique sequence domains of 117 and 48 amino acids at its amino and carboxyl terminus, respectively . To better understand the role of the various sequence domains of the MrpS28 protein in vivo, truncated derivatives were expressed under conditions where they were the only potential source of functional MrpS28 protein . The results shown here demonstrate that the amino-terminal domain and the S15-like domain are both essential for respiratory growth . Interestingly an inactive amino-terminal fragment can be complemented in trans by a second inactive fragment comprising the S15-like domain and the carboxyl-terminal 48 amino acids . Consequently, the assembly of these fragments into ribosomal subunits can be examined when they are expressed individually or together . Results from these studies indicate that each of the MrpS28-derived fragments facilitates the incorporation of the other into 37 S ribosomal subunits. J Mol Biol, 1993 Oct 20, 233(4), 559 - 66 The redox properties of protein disulfide isomerase (DsbA) of Escherichia coli result from a tense conformation of its oxidized form; Wunderlich M et al.; Periplasmic protein disulfide isomerase (DsbA) from Escherichia coli is a strongly oxidizing thiol reagent with one catalytic disulfide bridge and an intrinsic redox potential of -0.089 V . Gel filtration experiments and analytical ultracentrifugation studies demonstrate that DsbA is a monomeric protein with a molecular mass of 21.1 kDa, independent of its redox state . In order to investigate the molecular basis of its redox properties, the guanidinium.chloride-induced folding/unfolding equilibrium of the reduced and the oxidized form of the enzyme were compared . The transitions at pH 7.0 and 30 degrees C were found to be fully reversible and allowed the calculation of the free energy of stabilization of oxidized and reduced DsbA according to a two-state model for the unfolding transition . The analysis reveals that reduced DsbA is 22.7 (+/- 4.0) kJ/mol more stable than oxidized DsbA . This energetic difference is essentially independent of temperature, although the overall free energies of stabilization of both oxidized and reduced DsbA vary strongly between 20 and 30 degrees C as a consequence of changes in the cooperativity of the transitions The conformational tension of 22.7 (+/- 4.0) kJ/mol in oxidized DsbA quantitatively explains the oxidizing properties of the protein, as it causes a change of redox equilibrium constants between DsbA and thiols of about four orders of magnitude, corresponding to an increase of the standard redox potential of 0.118 (+/- 0.021) V . We conclude that the oxidizing properties of DsbA mainly result from a tense conformation of its oxidized form, that is converted to the relaxed, reduced state upon oxidation of thiols by DsbA . The results are discussed in terms of a general principle underlying the oxidizing properties of protein disulfide isomerases. Biochim Biophys Acta, 1993 Oct 20, 1182(3), 283 - 90 Use of the hemagglutinating virus of Japan (HVJ)-liposome method for analysis of infiltrating lymphocytes induced by hepatitis B virus gene expression in liver tissue; Kato K et al.; We previously developed a method for introducing foreign genes into liver tissue using liposomes with incorporated hemagglutinating virus of Japan (HVJ, Sendai virus), and found that liver cells transfected with the E . coli beta-galactosidase gene or the gene for hepatitis B virus (HBV) surface protein (HBsAg) expressed these proteins in vivo . Here, we analyzed cellular reactions leading to hepatitis in the liver by expressing the genes of HBV in vivo . Lymphocytes were eluted directly from liver transfected with the HBsAg genes and shown to be cytotoxic only to cells expressing HBsAg in vitro . These lymphocytes were identified as cytotoxic T lymphocytes with the CD4- CD8+ phenotype . Transfer of these lymphocytes to transgenic mice with the whole HBV genome led to elevation of the serum glutamic-pyruvic transaminase (SGPT) level, indicating the induction of hepatitis due to the cytotoxic T lymphocytes in vivo . Similarly, direct transfer of the gene for the HBV secretory core protein (HBeAg) induced expression of HBeAg in hepatocytes and the appearance of antibody against HBeAg in the serum . However, using this system, we found that the lymphocytes infiltrating the transfected liver showed no cytotoxicity specific for HBeAg . These results indicate that expression of HBsAg, one of the components of virions, in animal liver induced hepatitis efficiently through generation of specific cytotoxic T lymphocytes (CTL) without any expression of the other viral components . This in vivo experimental system should be useful for evaluating how expression of a given gene induces cellular reactions and intrinsic functions in the living body. Biochim Biophys Acta, 1993 Oct 19, 1216(1), 31 - 42 Distribution and properties of major ribosome-inactivating proteins (28 S rRNA N-glycosidases) of the plant Saponaria officinalis L . (Caryophyllaceae); Ferreras JM et al.; We have studied the distribution of the protein synthesis inhibitory activity in the tissues of Saponaria officinalis L . (Caryophyllaceae) . Seven major saporins, ribosome-inactivating proteins, were purified to apparent homogeneity from leaves, roots and seeds using a new procedure of RIPs isolation including ion-exchange and hydrophobic chromatography . They all catalysed the depurination of rat liver ribosomes, which generate the Endo's diagnostic rRNA fragment upon treatment with acid aniline, thus indicating that A4324 from the 28S rRNA has been released (Endo et al . (1987) J . Biol . Chem . 262, 5908-5912) . The molecular mass of saporins by SDS-PAGE ranged between 30.2 and 31.6 kDa and by gel-filtration between 27.5 and 30.1 kDa . Amino acid composition and amino-terminal amino acid sequence indicate that all saporins may be considered isoforms . Only two saporins present in roots were glycosylated (SO-R1 and SO-R3) . All saporins are very active on cell-free translation systems derived from rabbit reticulocyte lysates, rat liver, Triticum aestivum L., Cucumis sativus L . and Vicia sativa L . However, they are poor inhibitors of an Escherichia coli translation system . They inhibit protein synthesis in HeLa, BeWo and NB 100 cells, HeLa cells being the most resistant . The enzymatic activity of at least one saporin isoform was dependent on magnesium concentration in the standard rat liver cell-free system. Biochemistry, 1993 Oct 19, 32(41), 11224 - 7 Cross-linking of Escherichia coli RNA polymerase subunits: identification of beta' as the binding site of omega; Gentry DR et al.; The omega protein is a peptide found in near-stoichiometric levels in highly purified Escherichia coli RNA polymerase . In order to determine the binding site of omega to RNA polymerase, we cross-linked omega to RNA polymerase with the hetero-bifunctional cross-linker N-hydroxysuccinimidyl 4-azidobenzoate and analyzed for cross-linked partners using antibodies raised against each of the subunits . Our analysis indicates that omega cross-links predominantly with the beta' subunit, while a very low level of cross-linking was detected to the alpha subunit . We did not detect cross-linking to either the sigma 70 or the beta subunits . This report demonstrates the utility of combining cross-linking and immunological techniques to determine interactions between RNA polymerase subunits. Biochemistry, 1993 Oct 19, 32(41), 11211 - 6 Role of a conserved histidine residue, His-195, in the activities of the Escherichia coli mannitol permease; Weng QP et al.; The mannitol permease, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) of Escherichia coli, carries out the transport and phosphorylation of D-mannitol in this organism . Previous studies have shown that His-554 and Cys-384 in the mannitol permease are sequentially phosphorylated in reactions necessary for the transport and phosphorylation of the substrate . These residues are located in a large cytoplasmic domain of the protein . Interaction of the permease with mannitol, and its membrane translocation, however, must involve the N-terminal, transmembrane domain (EIIC domain) of the protein . In this report, we use site-directed mutagenesis and mutant complementation to investigate the role of His-195 in the EIIC domain of the mannitol permease, a residue that is conserved in many PTS permeases . In a previous report {Weng, Q.-P., Elder, J., & Jacobson, G . R . (1992) J . Biol . Chem . 267, 19529-19535}, we inferred a role for His-195 that involves its hydrogen-bonding ability . Here we show that His-195 plays a role in high-affinity mannitol binding . Moreover, mutant complementation studies show that a functional His-195 must be on the same subunit as a functional Cys-384 in a permease dimer for phosphotransfer to mannitol to occur . These results and kinetic studies of His-195 mutant proteins imply that His-195 also may play an important role in this phosphotransfer reaction . His-195 is predicted to be in a cytoplasmic "loop" in the EIIC domain of the mannitol permease, in which several other residues have been shown to have roles in mannitol permease activity. Biochemistry, 1993 Oct 19, 32(41), 11173 - 80 Site-directed mutagenesis of highly conserved residues in helix VIII of subunit I of the cytochrome bo ubiquinol oxidase from Escherichia coli: an amphipathic transmembrane helix that may be important in conveying protons to the binuclear center; Thomas JW et al.; Cytochrome bo from Escherichia coli is a ubiquinol oxidase which is a member of the superfamily of heme-copper respiratory oxidases . This superfamily, which includes the eukaryotic cytochrome c oxidases, has in common a bimetallic center consisting of a high-spin heme component and a copper atom (CuB) which is the site where molecular oxygen is reduced to water . Subunit I, which contains all the amino acid ligands to the metal components of the binuclear center, has 15 putative transmembrane spanning helices, of which 12 are common to the entire superfamily . Transmembrane helix VIII has been noted to contain highly conserved polar residues that fall along one face of the helix . These residues could, in principle, be important components of a pathway providing a conduit for protons from the cytoplasm to gain access to the binuclear center . These conserved residues include Thr352, Thr359, and Lys362 . In addition, Pro358, in the middle of this transmembrane helix, is totally conserved in the superfamily . Some substitutions for Thr352 (Ala, Asn) result in major perturbations at the binuclear center as judged by the low-temperature Fourier transform infrared (FTIR) absorbance difference spectroscopy of the CO adducts . Whereas Thr352Ala is inactive enzymatically, both Thr352Asn and Thr352Ser have substantial activity . Substitutions for Thr359 (Ala or Ser) also do not perturb the spectroscopic properties of the binuclear metal center, but the Thr359Ala mutant is devoid of enzyme activity . Changing the neighboring Pro358 to Ala has no detectable effect on the properties of the oxidase . However, all substitutions for Lys362 (Leu, Met, Gln, or Arg) are inactive.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Oct 19, 32(41), 11112 - 7 In vitro functional characterization of overproduced Escherichia coli katF/rpoS gene product; Nguyen LH et al.; The katF/rpoS gene product (sigma s), a central regulator of stationary-phase gene expression in Escherichia coli, has been purified from an overproducing strain . sigma s was used as an immunogen for the production of monoclonal antibodies . Previous sequence analysis of sigma s strongly indicated homology to the sigma factor family . We show biochemically in this paper that sigma s is a sigma factor . This protein can bind to core RNA polymerase (E), and this binding can be competed effectively by the major E . coli transcription initiation factor, sigma 70 . Immunopurified sigma s holoenzyme (E sigma s) transcribes the promoters of the bolAp1 gene and the xthA gene . Interestingly, both promoters can also be transcribed by sigma 70 holoenzyme (E sigma 70). Biochemistry, 1993 Oct 19, 32(41), 10936 - 43 Amino acids of the recombinant kringle 1 domain of human plasminogen that stabilize its interaction with omega-amino acids; Hoover GJ et al.; A series of strategically designed recombinant (r) mutants of the kringle 1 region of human plasminogen ({K1HPg}) have been constructed and the resulting gene products employed to reveal the identities of the residues that contribute to stabilization of the binding of omega-amino acid ligands to this domain . On the basis of determinations of the binding constants of the ligands, 6-aminohexanoic acid and trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, to a variety of these mutants, we find that the anionic site of the polypeptide responsible for stabilization of the amino group of the ligands consists of both D54 and D56 and the cationic site of the polypeptide that interacts with the carboxylate group of the ligand is composed solely of R70 . The main hydrophobic interactions that stabilize binding of these ligands, likely by interactions with the ligand hydrophobic regions, are principally due to W61, Y63, and Y71 . The results obtained are consistent with conclusions that could be made from analysis of the X-ray crystal structure of r-{K1HPg} and from previous studies from this laboratory regarding the binding of ligands of this type to the kringle 2 region of tissue-type plasminogen activator ({K2tPA}) . It thus appears as though a common ligand binding site has evolved in different kringles with ligand specificity differences between r-{K2tPA} and r-{K1HPg} perhaps explainable by the different nature of the cationic sites on these polypeptides that are involved in coordination to the ligand carboxylate groups. Biochemistry, 1993 Oct 19, 32(41), 11100 - 11 Characterization of plant L-isoaspartyl methyltransferases that may be involved in seed survival: purification, cloning, and sequence analysis of the wheat germ enzyme; Mudgett MB et al.; Protein carboxyl methyltransferases (EC 2.1.1.77) that catalyze the transfer of a methyl group from S-adenosylmethionine to L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely, but not universally, distributed in nature . These enzymes can participate in the repair of damaged proteins by facilitating the conversion of abnormal L-isoaspartyl residues to normal L-aspartyl residues . In this work, we have identified L-isoaspartyl methyltransferase activity in a variety of higher plant species and a green alga . Interestingly, the highest levels of methyltransferase were located in seeds, where the problem of spontaneous protein degradation may become particularly severe upon aging . The wheat germ methyltransferase was purified as a monomeric 28,000-Da species by DEAE-cellulose chromatography, reverse ammonium sulfate gradient solubilization, and gel filtration chromatography . The purified enzyme recognized a variety of L-isoaspartyl-containing peptides, but did not recognize two D-aspartyl-containing peptides that are substrates for the mammalian enzyme . The partial amino acid sequence was utilized to design oligonucleotides to isolate a full-length cDNA clone, pMBM1 . Its nucleotide sequence demonstrated an open reading frame encoding a polypeptide of 230 amino acid residues with a calculated molecular weight of 24,710 . This sequence shares 31% identity with the L-isoaspartyl methyltransferase from Escherichia coli and 50% identity with the L-isoaspartyl/D-aspartyl methyltransferase from human erythrocytes . Such conservation in sequence is consistent with a fundamental role of this enzyme in the metabolism of spontaneously damaged polypeptides. Biochim Biophys Acta, 1993 Oct 19, 1216(1), 140 - 4 Dbp45A encodes a Drosophila DEAD box protein with similarity to a putative yeast helicase involved in ribosome assembly; Lavoie CA et al.; Proteins of the DEAD family of putative ATP-dependent RNA helicases have been implicated in translation initiation, ribosome assembly, and RNA processing in a variety of organisms from Escherichia coli to man . Among these proteins are eIF-4A, an essential component of the cap-binding complex, numerous yeast proteins required for pre-mRNA splicing, and proteins from yeast and E . coli necessary for ribosome assembly . We report the isolation of a new DEAD gene from Drosophila, Dbp45A, which is most abundantly expressed in 6-12 h embryos and adults . The predicted amino acid sequence of the Dbp45A product contains all eight highly conserved DEAD family motifs, and most closely resembles the Saccharomyces cerevisiae DRS1p among known DEAD box proteins . DRS1p has been implicated in ribosomal RNA processing. Biochim Biophys Acta, 1993 Oct 19, 1216(1), 1 - 8 Rapid determination of the affinity of 28- and 14-mer phosphorothioate oligonucleotides for HIV-1 reverse transcriptase by fluorescence spectroscopy; Maury G et al.; Intrinsic fluorescence of human immunodeficiency virus type 1 reverse transcriptase (E.C . 2.7.7.49) and displacement experiments of a fluorescent template.primer probe were used to study the interaction of the enzyme with several types of 28- and 14-mer normal or phosphorothioate oligodeoxycytidinylates and their duplexes with poly(rI) . The two methods gave convergent results and allowed in each case fast determinations of ligand affinities for the enzyme . The dissociation constants (Kd) obtained from intrinsic fluorescence changes were slightly lower than those determined from the less direct competitive displacement experiments . In all cases, the enzyme displayed better recognition of the hybrid than of the unannealed oligonucleotide . The Kd values of phosphorothioate oligomers and their hybrids were lower than those of the corresponding normal oligomers and hybrids, but the difference was not as significant as in the case of the Ki constants for (dC)28 and S(dC)28 (Majumdar et al . (1989) Biochemistry 28, 1340) . The affinities of the annealed phosphorothioate oligodeoxycytidinylates for the enzyme were found to be larger than for any other compounds in this series (Kd of poly(rI).S(dC)28: 0.28 nM at 25 degrees C) . Changing the beta stereochemistry of the oligomer bases to alpha did not alter the affinity of the oligodeoxycytidinylate and its hybrids for the enzyme. FEBS Lett, 1993 Oct 18, 332(3), 282 - 6 Molecular structure of ras-related small GTP-binding protein genes of rice plants and GTPase activities of gene products in Escherichia coli; Kidou S et al.; We isolated two rice cDNA clones (ric1 and ric2) encoding proteins homologous to the ras-related small GTP-binding protein . The amino acid sequences of ric1 and ric2 are conserved in four regions involved in GTP binding and hydrolysis which are characteristic in the ras and ras-related small GTP-binding protein genes . In addition, two consecutive cysteine residues near the carboxyl-terminal end required for membrane anchoring are also present in ric1 and ric2 . The ric1 and ric2 proteins synthesized in Escherichia coli possessed GTPase activity (i.e . hydrolysis of GTP to GDP). FEBS Lett, 1993 Oct 18, 332(3), 226 - 8 Mass spectrometric evidence for a disulfide bond in aequorin regeneration; Ohmiya Y et al.; Tryptic digests of purified recombinant apoaequorin were analyzed, before and after reduction with DTT, by fast atom bombardment mass spectrometry . The results showed that apoaequorin contains a disulfide bond between Cys145 and Cys152 and that the reduction of this bond is involved in the regeneration of aequorin. Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9591 - 5 Two tropinone reductases with different stereospecificities are short-chain dehydrogenases evolved from a common ancestor; Nakajima K et al.; In the biosynthetic pathway of tropane alkaloids, tropinone reductase (EC 1.1.1.236) (TR)-I and TR-II, respectively, reduce a common substrate, tropinone, stereospecifically to the stereoisomeric alkamines tropine and pseudotropine (psi-tropine) . cDNA clones coding for TR-I and TR-II, as well as a structurally related cDNA clone with an unknown function, were isolated from the solanaceous plant Datura stramonium . The cDNA clones for TR-I and TR-II encode polypeptides containing 273 and 260 amino acids, respectively, and when these clones were expressed in Escherichia coli, the recombinant TRs showed the same strict stereospecificity as that observed for the native TRs that had been isolated from plants . The deduced amino acid sequences of the two clones showed an overall identity of 64% in 260-amino acid residues and also shared significant similarities with enzymes in the short-chain, nonmetal dehydrogenase family . Genomic DNA-blot analysis detected the TR-encoding genes in three tropane alkaloid-producing solanaceous species but did not detect them in tobacco . We discuss how the two TRs may have evolved to catalyze the opposite stereospecific reductions. Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9350 - 4 Functional complementation of an Escherichia coli ribonuclease H mutation by a cloned genomic fragment from the trypanosomatid Crithidia fasciculata; Campbell AG et al.; A gene designated Cfa RNH1 has been cloned by complementation of an RNase H deficiency in an Escherichia coli rnhA mutant by using a genomic DNA library from the trypanosomatid Crithidia fasciculata . The encoded RNase H is predicted to have 494 amino acid residues and a molecular mass of 53.7 kDa . The carboxyl half of the protein is homologous to the 155-residue E . coli RNase HI (41% identity) and the 166-residue Saccharomyces cerevisiae RNase HI (33% identity) . The recombinant protein has been purified as a six-histidine-tagged fusion protein by metal chelate chromatography and was shown to have RNase H activity . Antibodies against the recombinant protein recognize proteins of approximately 65 kDa and 56 kDa on Western blots of C . fasciculata extracts . These results demonstrate the feasibility of cloning trypanosome genes by complementation of appropriate E . coli mutants with genomic DNA libraries. Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9325 - 9 Synergism in replication and translation of messenger RNA in a cell-free system; Morozov IY et al.; Combination of the Q beta replicase reaction with the Escherichia coli cell-free translation system markedly enhances replication of a recombinant RQ-DHFR RNA consisting of the dihydrofolate reductase (DHFR) mRNA sequence inserted into RQ135(-1) RNA, an efficient naturally occurring Q beta replicase template . The enhancement is associated with a replication asymmetry previously described for the replication of Q beta phage RNA in vivo; the sense (+)-strands are produced in large excess over the antisense (-)-strands . This, in turn, results in increased synthesis of the functionally active DHFR . These effects are not observed when DHFR mRNAs or RQ135(-1) RNAs are used as templates, if the translation system is not complete, or if it is inhibited by puromycin . The coupled replication-translation of nonviral mRNA recombinants can serve as a useful model for studying the fundamental aspects of virus amplification and can be implemented for large-scale protein synthesis in vitro. Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9290 - 4 A site required for termination of packaging of the phage lambda chromosome; Cue D et al.; Lambda chromosomes are cut and packaged from concatemeric DNA by phage enzyme terminase . Terminase initiates DNA packaging by binding at a site called cosB and introducing staggered nicks at an adjacent site, cosN, to generate the left cohesive end of the DNA molecule to be packaged . After DNA packaging terminase recognizes and cuts the terminal cosN, an event that does not require a wild-type cosB . In this work a site, called cosQ, has been identified that is required for termination of DNA packaging . cosQ, defined by mutations in a sequence called R4, is located approximately 30 bp upstream from cosN . The order of sites is cosQ-cosN-cosB . Helper packaging of repressed, tandem prophage chromosomes demonstrated that a cosQ point mutation affects DNA packaging only when placed at the terminal cos site, whereas cosB mutations only affect packaging initiation . In vitro packaging studies confirmed that cosQ mutations do not affect packaging initiation . In vivo studies indicated that cosQ mutations do not affect cutting of initial cos sites but do cause a defect in packaging termination . cosQ mutants accumulated expanded phage heads, indicating that cosQ mutations affect a step that occurs after packaging of a substantial length of phage DNA . These results show that cosQ mutations define a site required for use of cos sites present at the ends of lambda chromosomes undergoing packaging . Available evidence suggests that other viruses, including phages T3 and T7 and the herpesviruses, may ultimately prove to use cosQ-like sites for packaging termination. Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9280 - 4 Cloning, sequence determination, and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum: a target for antimalarial therapy; Rubin H et al.; Malaria remains a leading cause of morbidity and mortality worldwide, accounting for more than one million deaths annually . We have focused on the reduction of ribonucleotides to 2'-deoxyribonucleotides, catalyzed by ribonucleotide reductase, which represents the rate-determining step in DNA replication as a target for antimalarial agents . We report the full-length DNA sequence corresponding to the large (PfR1) and small (PfR2) subunits of Plasmodium falciparum ribonucleotide reductase . The small subunit (PfR2) contains the major catalytic motif consisting of a tyrosyl radical and a dinuclear Fe site . Whereas PfR2 shares 59% amino acid identity with human R2, a striking sequence divergence between human R2 and PfR2 at the C terminus may provide a selective target for inhibition of the malarial enzyme . A synthetic oligopeptide corresponding to the C-terminal 7 residues of PfR2 inhibits mammalian ribonucleotide reductase at concentrations approximately 10-fold higher than that predicted to inhibit malarial R2 . The gene encoding the large subunit (PfR1) contains a single intron . The cysteines thought to be involved in the reduction mechanism are conserved . In contrast to mammalian ribonucleotide reductase, the genes for PfR1 and PfR2 are located on the same chromosome and the accumulation of mRNAs for the two subunits follow different temporal patterns during the cell cycle. J Biol Chem, 1993 Oct 15, 268(29), 22105 - 11 Identification of regions in interleukin-1 alpha important for activity; Gayle RB 3rd et al.; Saturation mutagenesis of the mature human interleukin-1 alpha (IL-1 alpha) gene has been performed . Following expression in Escherichia coli, the biological and receptor binding activities of the mutant proteins were examined . Most of the molecule could be altered with little effect on either function . More than 3,500 mutants were examined, and only 23 unique amino acid sequences were identified which resulted in an altered ratio of biological to binding activity when compared with wild-type IL-1 alpha . These proteins possessed mutations at 38 of the 159 amino acid residues in IL-1 alpha . Random mutagenesis at several of these positions identified further substitutions that affected activity . Examination of a model for IL-1 alpha localized most of the residues which altered activity along one face of the molecule . This region appears to be distinct from areas of IL-1 which have been postulated to make contact with IL-1 receptor. J Biol Chem, 1993 Oct 15, 268(29), 22092 - 9 Crystal structures of ribonuclease HI active site mutants from Escherichia coli; Katayanagi K et al.; In order to investigate the relationships between the three-dimensional structure and the enzymic activity of E . coli RNase HI, three mutant proteins, which were completely inactivated by the replacements of three functional residues, Asp10 by Asn (D10N), Glu48 by Gln (E48Q), and Asp70 by Asn (D70N), were crystallized . Their three-dimensional structures were determined by x-ray crystallography . Although the entire backbone structures of these mutants were not affected by the replacements, very localized conformational changes were observed around the Mg(2+)-binding site . The substitution of an amide group for a negatively charged carboxyl group in common induces the formation of new hydrogen bond networks, presumably due to the cancellation of repulsive forces between carboxyl side chains with negative charges . These conformational changes can account for the loss of the enzymic activity in the mutants, and suggest a possible role for Mg2+ in the hydrolysis . Since the 3 replaced acidic residues are completely conserved in sequences of reverse transcriptases from retroviruses, including human immunodeficiency virus, the concepts of the catalytic mechanism deduced from this structural analysis can also be applied to RNase H activity in reverse transcriptases. J Biol Chem, 1993 Oct 15, 268(29), 21895 - 900 Overexpression in Escherichia coli and purification of an ATP-binding peptide from the yeast plasma membrane H(+)-ATPase; Capieaux E et al.; The two major hydrophilic domains from the Saccharomyces cerevisiae plasma membrane H(+)-ATPase fused to glutathione S-transferase have been expressed in Escherichia coli . The GST-L peptide contained the hydrophilic region from Ala340 to Ser660 . The GST-SL peptide contained in addition the hydrophilic region Glu162 to Val276 . After solubilization of the inclusion bodies with urea, renaturation, and affinity chromatography, 3 mg of highly purified peptides were recovered per liter of E . coli culture . The purified peptides interacted with 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), the fluorescence of which was enhanced identically upon binding of either GST-L or GST-SL . ATP competitively displaced the TNP-ATP binding . The observed dissociation constants for TNP-ATP (6.5 microM) and ATP (3 mM) are close to those found for the complete native H(+)-ATPase protein . The fluorescence of TNP-ATP was sensitive to Mg2+ indicating the existence of a Mg(2+)-binding site on the peptide . Apparent affinity for this Mg2+ site was found to vary from 50 microM at pH 7.5 to 400 microM at pH 5.5. J Biol Chem, 1993 Oct 15, 268(29), 21889 - 94 Random mutagenesis of G protein alpha subunit G(o)alpha . Mutations altering nucleotide binding; Slepak VZ et al.; Nucleotide binding properties of the G protein alpha subunit G(o)alpha were probed by mutational analysis in recombinant Escherichia coli . Thousands of random mutations generated by polymerase chain reaction were screened by in situ {35S}GTP gamma S (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G(o)alpha cDNA . Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts . A number of novel mutations reducing the affinity of G(o)alpha for GTP gamma S or Mg2+ were identified . Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21ras proteins . Other mutations show that previously unstudied residues also participate in the nucleotide binding . Several mutants lost GTP gamma S binding but retained the capacity to interact with the beta gamma subunit complex as determined by pertussis toxin-mediated ADP-ribosylation . One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor . Whereas wild-type G(o)alpha increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl- response, suggesting that this mutation resulted in a dominant negative phenotype. J Biol Chem, 1993 Oct 15, 268(29), 21854 - 61 Autoregulation-deficient mutant of the plasmid R6K-encoded pi protein distinguishes between palindromic and nonpalindromic binding sites; York D et al.; The autogenously regulated gene pir of Escherichia coli plasmid R6K encodes the replication protein pi . This protein binds to two sites in the operator region of the pir gene: a 22-base pair nonpalindromic sequence and a pair of palindromic 9-base pair sequences . These pi-binding sites are similar, suggesting that pi uses a single DNA-binding domain in recognizing them . We devised a plasmid system permitting isolation of mutants of the pi protein which are altered in autoregulation . A Ser87 to Asn substitution in one such mutant, designated pi 87, reduces the protein's ability to repress the pir gene promoter in vivo . DNase I protection and gel retardation assays were carried out with highly purified pi 87 protein . In these studies pi 87 exhibited altered binding to the palindromic but not to the nonpalindromic part of the operator of the pir gene . Chemical cross-linking and gel filtration analyses have shown that the dimerization properties of wild type pi and pi 87 proteins are similar in solution . We propose that the interaction of pi protein with the palindromic part of the pir operator is essential for autoregulation; we also propose that there is a fundamental difference in the mechanisms of pi protein recognition of palindromic and nonpalindromic sequences. J Biol Chem, 1993 Oct 15, 268(29), 21680 - 5 Regulation of folate and one-carbon metabolism in mammalian cells . IV . Role of folylpoly-gamma-glutamate synthetase in methotrexate metabolism and cytotoxicity; Kim JS et al.; Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpoly-gamma-glutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of methotrexate (MTX) . CHO cells expressing human FPGS metabolized MTX to polyglutamates characteristic of human cells . Cellular MTX accumulation and metabolism to polyglutamates were dependent on the level of FPGS activity and were unaffected by putative gamma-glutamyl hydrolase inhibitors . The sensitivity of cells continuously exposed to MTX was not influenced by FPGS activity . After short term exposure to MTX, cells expressing higher levels of FPGS were more sensitive to the drug . MTX was not transported into the mitochondria and MTX treatment had no effect on preexisting mitochondrial folates while cytosolic folates were converted to oxidized forms . Mitochondrial folate accumulation was significantly impaired by MTX treatment, suggesting that the mitochondrial folate transport system is specific for reduced folates. J Biol Chem, 1993 Oct 15, 268(29), 21674 - 9 Regulation of folate and one-carbon metabolism in mammalian cells . III . Role of mitochondrial folylpoly-gamma-glutamate synthetase; Lin BF et al.; Wild-type Chinese hamster ovary (CHO) cells and CHO cell transfectants expressing human folylpoly-gamma-glutamate synthetase (FPGS) activity contain mitochondrial FPGS activity of higher specific activity than the cytosolic isozyme . Expression of mitochondrial FPGS activity is required for folate accumulation by mitochondria . The mitochondrial folate pool in CHO cells is not in equilibrium with the cytosolic pool and contains folylpolyglutamates of longer glutamate chain length than cytosolic folates . The inability of AUX-coli, a CHO cell expressing high levels of Escherichia coli FPGS activity and containing pteroyltriglutamate, to support glycine synthesis is due to a lack of mitochondrial FPGS activity . AUX-coli cells lack mitochondrial folate despite containing high levels of cytosolic folate. J Biol Chem, 1993 Oct 15, 268(29), 21665 - 73 Regulation of folate and one-carbon metabolism in mammalian cells . II . Effect of folylpoly-gamma-glutamate synthetase substrate specificity and level on folate metabolism and folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism; Lowe KE et al.; The effect of folylpoly-gamma-glutamate synthetase (FPGS) levels on folate accumulation was investigated in Chinese hamster ovary cells expressing various levels of human and Escherichia coli FPGS activity . At low medium folate concentrations, folate accumulation was limited by influx and was independent of FPGS activity except in cells expressing extremely low levels of FPGS . Essentially all transported folate was metabolized to retained polyglutamate derivatives, the chain length of which varied with the level of FPGS activity . As medium folate concentration increased through the physiological to the pharmacological range, cellular folate accumulation became proportional to FPGS activity and the chain length of intracellular folates decreased . At high folate concentrations, competition between substrates for FPGS limited the extent of polyglutamylation and less than 5% of transported folate was retained by the cell . Pteroyltriglutamates functioned as effectively as the longer chain length polyglutamates normally found in mammalian cells in the metabolic cycles of de novo purine and thymidylate biosynthesis but were unable to support glycine and methionine synthesis . Transfectants expressing human FPGS and containing folates of glutamate chain length ranging from four to eight were equally effective at supporting glycine synthesis, and transfectants expressing higher levels of FPGS were able to grow in the absence of methionine . Growth in the absence of methionine required high (nonphysiological) intracellular folate levels and longer chain length polyglutamates. J Biol Chem, 1993 Oct 15, 268(29), 21657 - 64 Regulation of folate and one-carbon metabolism in mammalian cells . I . Folate metabolism in Chinese hamster ovary cells expressing Escherichia coli or human folylpoly-gamma-glutamate synthetase activity; Osborne CB et al.; Chinese hamster ovary (CHO) cell transfectants expressing various levels of human and Escherichia coli folylpoly-gamma-glutamate synthetase (FPGS) activity and possessing different folylpolyglutamate chain length distributions have been developed as models for folate and antifolate metabolism . The synthesis of pteroyltriglutamate was sufficient for normal cellular retention of folate and also overcame the phenotypic requirement for purines and thymidine of AUXB1, a CHO cell mutant lacking FPGS activity and lacking folylpolyglutamates . Only low levels of FPGS are required to enable cellular metabolism of folates to forms that are retained by mammalian cells . The higher levels found in mammalian cells are required for the synthesis of the long chain polyglutamate derivatives characteristic of mammalian cells . At low medium folate concentrations, folate accumulation by transfectants expressing human FPGS was not responsive to FPGS levels as the limiting step in metabolism was beyond the triglutamate, the chain length required for retention . The rate-limiting step in folate metabolism in cells expressing the E . coli enzyme was the conversion of diglutamate to triglutamate, and, at low FPGS levels, the E . coli enzyme was about 50-fold less effective than the human FPGS in enabling cellular folate accumulation . These data suggest that cellular accumulation of any folate analog whose mono- or diglutamate derivative is a poor substrate for FPGS would be very responsive to the level of FPGS activity. J Biol Chem, 1993 Oct 15, 268(29), 21645 - 9 Utility of polyhistidine-tagged ubiquitin in the purification of ubiquitin-protein conjugates and as an affinity ligand for the purification of ubiquitin-specific hydrolases; Beers EP et al.; The purification and biochemical characterization of protein substrates of the ubiquitin-dependent pathway of proteolysis is made difficult in part by the low steady state levels of ubiquitin-protein conjugates . We report here on the use of a polyhistidine-tagged ubiquitin molecule (HisUb) for the purification of ubiquitin-protein conjugates by metal chelate chromatography . When Escherichia coli extracts containing expressed HisUb were passed through a nitrilotriacetic acid-agarose column containing immobilized Ni2+ ions (Ni-NTA column), HisUb was retained . After washing to remove unbound and nonspecifically bound proteins, a pH 4.5 wash was used to elute highly purified HisUb . Purified HisUb and wild-type ubiquitin were tested for their ability to form Ni(2+)-binding ubiquitin-protein conjugates in a wheat germ in vitro conjugation reaction . In some experiments, wheat germ extracts were preincubated with iodoacetamide to inhibit ubiquitin activating and conjugating enzymes . Only those conjugation assays containing HisUb and an ATP-regenerating system not pretreated with iodoacetamide produced significant levels of multiple Ni(2+)-binding ubiquitin-protein conjugates . We also examined the potential of HisUb as an affinity ligand for the purification of higher plant ubiquitin-specific hydrolases . As a test, a crude lysate of E . coli expressing a yeast ubiquitin-specific hydrolase (Yuh1) was passed through a Ni-NTA column containing bound HisUb . Yuh1 was retained on the column and was specifically eluted when the column was equilibrated with buffer containing wild-type ubiquitin. J Biol Chem, 1993 Oct 15, 268(29), 21637 - 44 The dihydrofolate reductase domain of Plasmodium falciparum thymidylate synthase-dihydrofolate reductase . Gene synthesis, expression, and anti-folate-resistant mutants; Sirawaraporn W et al.; A 693-base pair gene coding for the 27,132-dalton dihydrofolate reductase (DHFR) domain of the thymidylate synthase-dihydrofolate reductase (TS-DHFR) bifunctional protein of Plasmodium falciparum was designed to have Escherichia coli codon preference and multiple unique restriction sites and was chemically synthesized . The gene was overexpressed (> 50% total cellular protein) in E . coli as insoluble inclusion bodies which could be unfolded and refolded to recover soluble enzyme activity . The refolded DHFR was purified by methotrexate-Sepharose affinity chromatography to give the homogeneous enzyme . Active site titration with methotrexate revealed that the purified protein was fully active . The purified DHFR migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent mass of approximately 30 kDa, and gel filtration showed that the protein is a monomer . The yield of purified enzyme was about 5-6 mg/liter of bacterial culture . Kinetic properties of the purified recombinant DHFR were similar to those reported for wild type bifunctional TS-DHFR . Cassette mutagenesis of the synthetic gene was performed to give the S108N and the N51I + S108N mutants which provided DHFRs analogous to pyrimethamine-resistant mutants found in nature. J Biol Chem, 1993 Oct 15, 268(29), 21545 - 52 Expression of cellular retinoic acid-binding protein (type II) in Escherichia coli . Characterization and comparison to cellular retinoic acid-binding protein (type I); Fiorella PD et al.; Cellular retinoic acid-binding protein type II, CRABP(II), has been expressed efficiently in Escherichia coli from the mouse cDNA and compared to E . coli-expressed cellular retinoic acid-binding protein type I, CRABP(I) . CRABP(II) had a molecular weight approximately 15,700, a pI of 5.46, an Amax of 350 nm with A350/A280 of 1.8, a fluorescence excitation maximum at 347 nm, and a fluorescence emission maximum at 465 nm (holoprotein) . All-trans-retinoic acid and 3,4-didehydro-, 4-hydroxy-, 4-oxo-, 16-hydroxy-4-oxo-, and 18-hydroxy-retinoic acids bind CRABP(II) and CRABP(I) stoichiometrically (Kd values no greater than approximately 10-20 nM) . 9-cis-Retinoic acid exhibited saturation binding to both CRABP(II) and CRABP(I) with similar affinities, Kd = approximately 50-70, as did 13-cis-retinoic acid, Kd = approximately 160-240 nM, demonstrating that CRABP(II) and CRABP(I) have similar orders of selectivity for known retinoids . HoloCRABP(II) transferred approximately 70% of its all-trans-retinoic acid to CRABP(I), however, indicating that CRABP(II) has approximately a 3-fold lower affinity for stoichiometrically binding retinoids than CRABP(I) . Stern-Volmer plots of both native and denatured CRABP(II) and CRABP(I) were consistent with comparable loci for the tryptophan residues and similar three-dimensional structures . These results suggest that CRABP(II) and CRABP(I), when expressed in vivo in excess of total retinoids, bind several ligands, and functional dissimilarities between the two proteins would not be related to unique preferences for known endogenous retinoids . Rather, CRABP(I) and CRABP(II) may modulate the steady-state concentrations of retinoids to different set points. J Biol Chem, 1993 Oct 15, 268(29), 21497 - 500 Conversion of a magnesium binding site into a zinc binding site by a single amino acid substitution in Escherichia coli alkaline phosphatase; Murphy JE et al.; The replacement of aspartic acid by histidine at position 153 in Escherichia coli alkaline phosphatase results in a mutant enzyme that is remarkably similar to certain mammalian alkaline phosphatases that are activated by magnesium in a time-dependent fashion . These mammalian alkaline phosphatases have histidine at the position corresponding to 153 of the E . coli sequence . Here we report the three-dimensional structure of the mutant E . coli alkaline phosphatase with histidine at position 153 . The structure reveals that the octahedral magnesium binding site has been converted to a tetrahedral zinc binding site with an imidazole ring nitrogen of His-153 as one of the ligands to the zinc . The alteration in metal binding caused by the mutation could explain the origin of the magnesium activation observed with the mammalian alkaline phosphatases . The structure also reveals differences in the mode of phosphate binding, explaining the enhanced phosphate affinity and the reduced activity of the mutant enzyme in the presence of zinc. Cancer Res, 1993 Oct 15, 53(20), 4920 - 6 Expression of a recombinant breast tumor-associated mucin fusion protein in Escherichia coli exposes the tumor-specific epitope; Hu P et al.; Mucins are highly immunogenic glycoproteins that are abundantly expressed by breast carcinomas and other carcinomas . The fact that deglycosylated normal mucin can induce tumor-specific monoclonal antibodies indicates that tumor-specific epitopes are hidden in the fully glycosylated form . Using recombinant DNA techniques, a fragment of mucin tandem repeats was inserted into pMal-p, an Escherichia coli expression vector, and resulted in the expression of an unglycosylated maltose-binding protein-mucin fusion protein . This fusion protein has been purified and showed strong affinity to breast tumor-specific monoclonal antibody SM3 . The antisera against this recombinant mucin fusion protein recognized all breast tumor cell lines we tested . Competition assay with monoclonal antibody SM3 shows that anti-recombinant mucin fusion protein binds the epitope that SM3 binds . These results confirm the hypothesis that unglycosylated mucin contains a tumor-specific epitope . This leads to the possibility that recombinant mucin may be used to develop vaccines against breast cancer and cytotoxic T-lymphocyte lines for immunotherapy of breast cancer. Cancer Res, 1993 Oct 15, 53(20), 4750 - 3 A single amino acid change in human O6-alkylguanine-DNA alkyltransferase decreasing sensitivity to inactivation by O6-benzylguanine; Crone TM et al.; Mammalian O6-alkylguanine-DNA alkyltransferases (AGTs) are readily inactivated by incubation with the pseudosubstrate, O6-benzylguanine, but the equivalent protein from the Escherichia coli ogt gene is much less sensitive and the Saccharomyces cerevisiae and E . coli ada gene product AGTs are completely resistant to this compound . We have expressed the normal human AGT and various point mutations (C145A, W100A, and P140A) in an ada- ogt- strain of E . coli and tested these proteins against DNA substrates containing O6-methylguanine, for inactivation by O6-benzylguanine and for the ability to produce guanine from O6-benzylguanine . The C145A mutation was inactive as expected since this residue forms the methyl acceptor site . Mutants W100A and P140A were fully active against methylated DNA substrates but the P140A mutant was much less sensitive to inactivation by O6-benzylguanine and failed to form significant amounts of {3H}guanine when incubated with O6-benzyl{8-3H}-guanine . The proline at position 140 in mammalian AGTs is replaced by alanine in the Ada and yeast AGTs and by serine in the Ogt AGT . These results suggest that this proline residue affects the configuration of the active site allowing the O6-benzylguanine to enter and react with the mammalian AGT . The production of resistance to O6-benzylguanine by a single base change raises the possibility that such resistance may arise quite readily in cells of tumors treated therapeutically with the combination of O6-benzylguanine and an alkylating agent. J Neurosci Res, 1993 Oct 15, 36(3), 305 - 14 Characterization of the neuroimmune protein F5: localization to the dendrites and perikarya of mature neurons and the basal aspect of choroid plexus epithelial cells; Arai M et al.; F5 was identified originally as an interleukin-2-regulated gene in L2 cells, a murine helper T-lymphocyte clone . In adult mouse, F5 mRNA was expressed at a modest level in lymphoid tissues, at a high level in mature neurons in the nervous system, but not in other tissues . Although the F5 sequence is highly conserved over evolution, the function of the F5 protein is unknown . In the present studies, the putative F5 protein-coding region was translated in vitro using a reticulocyte lysate system and in Escherichia coli, yielding a protein with the predicted molecular weight of 42 kDa . Polyclonal rabbit anti-F5 antibody was generated against a synthetic peptide corresponding to the C-terminus of the F5 protein . This antibody specifically recognized recombinant F5 protein . Western blot studies demonstrated a strongly-reactive 42-kDa band and a faint 39-kDa band in extracts of adult mouse brain regions, the levels of which paralleled F5 mRNA expression . Immunoperoxidase studies of adult mouse brain demonstrated F5 immunoreactivity in neuronal perikarya and dendrites but not axons . Neurons expressing the highest levels of F5 protein corresponded to those with the highest levels of F5 mRNA . Choroid plexus epithelial cells also exhibited strong reactivity localized to their basal aspect . These observations suggest that the F5 protein, expression of which appears to be regulated predominantly at the RNA level, may be involved in the maintenance of the functional or anatomic polarity of neurons and choroid plexus epithelial cells. FEMS Microbiol Lett, 1993 Oct 15, 113(2), 219 - 22 Activation of FNR-dependent transcription by iron: an in vitro switch for FNR; Green J et al.; FNR is an iron-binding transcriptional regulator of Escherichia coli which controls the expression of target genes in response to anaerobiosis . The mechanism used by FNR to sense and respond to anaerobiosis is unknown but it is thought to involve iron . In vitro transcription analyses have shown that iron-deficient FNR is unable to activate transcription from the FF-melR promoter, but activity could be restored by preincubation with Fe2+ and beta-mercaptoethanol . The reactivation of FNR was prevented and reversed by chelating agents, and this reactivation thus provides an artificial in vitro switch for FNR-dependent transcriptional activation. FEMS Microbiol Lett, 1993 Oct 15, 113(2), 197 - 200 Induction of cadmium tolerance in Escherichia coli K-12; Inbar O et al.; Cadmium ions are bacteriocidal, resulting in exponential